Muscle ankyrin repeat proteins: their role in striated muscle function in health and disease

269

Introduction

One of the amazing characteristics of our striated muscles that are constantly exposed to physical stresses

is their ability to remodel. Remodeling is an adaptive response to environmental demands with the aim of maintaining muscle performance. Adaptation includes

REVIEW ARTICLE

Muscle ankyrin repeat proteins: their role in striated muscle function in health and disease

Snezana Kojic1, Dragica Radojkovic1, and Georgine Faulkner2

1Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Serbia and 2CRIBI, University of Padova, Italy

AbstractRemodeling is a stringently controlled process that enables adequate response of muscle cells to constant physical stresses. In this process, different kinds of stimuli have to be sensed and converted into biochemical signals that ultimately lead to alterations of muscle phenotype. Several multiprotein complexes located in the sarcomere and organized on the titin molecular spring have been identified as stress sensors and signal transducers. In this review, we focus on Ankrd1/CARP and Ankrd2/Arpp proteins,which belong to the muscle ankyrin repeat protein family (MARP) involved in a mechano-signaling pathway that links myofibrillar stress response to muscle gene expression. Apart from the mechanosensory function, they have an important role in transcriptional regulation, myofibrillar assembly, cardiogenesis and myogenesis. Their altered expression has been demonstrated in neuromuscular disorders, cardiovascular diseases, as well as in tumors, suggesting a role in pathological processes. Although analyzed in a limited number of patients, there is a considerable body of evidence that MARP proteins could be suitable candidates for prognostic and diagnostic biomarkers.Keywords: Sarcomere, mechanosensors, Ankrd1/CARP, Ankrd2/Arpp, inherited myopathies, motor neuron diseases, cardiomyopathies, tumorsAbbreviations: aa, amino acid; ALS, amyotrophic lateral sclerosis; ANF, atrial natriuretic factor; Arg, arginine; BMD, Becker muscular dystrophy; BNP, brain natriuretic peptide; Btx, botulinum toxin; C/EBP, CCAAT/enhancer-binding protein; CASQ2, calsequestrin-2; CCD, central core disease; CFTD, congenital fiber type disproportion; ChRCC, chromophobe renal cell carcinoma; CM, congenital myopathies; CMD, congenital muscular dystrophies; cNLS, classical nuclear localization signal; DCM, dilated cardiomyopathy; DGC, dystrophin-associated glycoprotein complex; DMD, Duchenne muscular dystrophy; EMA, epithelial membrane antigen; FHL, four-and-a half LIM domain protein; FIGO, International Federation of Gynecology and Obstetrics; FKRP, fukutin-related protein; FSHMD, facioscapulohumeral muscular dystrophy; HCM, hypertrophic cardiomyopathy; HDAC, histone deacetylase; Ig, immunoglobulin; IS, insertion sequence; LDMD, laminin-α2-deficient muscular dystrophy; LGMD, limb-girdle muscular dystrophy; MAPK, mitogen-activated protein kinase; MARP, muscle ankyrin repeat protein family; MCK, muscle creatine kinase; MD, muscular dystrophies; Mdm, muscular dystrophy with myositis; MDM2, mouse double minute 2; MEF2, myocyte enhancer factor 2; MHC, myosin heavy chain; MLC-2v, ventricular myosin light chain 2; MLP, muscle LIM protein; M-PGAM, muscle-specific phosphoglycerate mutase; MRF, myogenic regulatory factor; m-Tor, mammalian target of rapamycin; MuRF, muscle-specific RING finger protein; NBR1, neighbor of BRCA1 gene-1; NFAT, nuclear factor of activated T cells; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; NLS, nuclear localization signal; PI, phosphoinositide; PKB, protein kinase B; PML, promyelocytic leukemia protein; pRB, retinoblastoma protein; RCC, renal cell carcinoma; RMS, rhabdomyosarcoma; RT PCR, reverse transcriptase polymerase chain reaction; RYR1, ryanodine receptor 1; SMA, spinal muscular atrophy; SMN1, survival motor neuron 1; SNB1, beta-1-syntrophin; SOD1, superoxide dismutase 1; SRF, serum response factor; TGF-β, transforming growth factor β; TMD, tibial muscular dystrophy; YB-1, Y-box protein 1; ZO1, zonula occludens protein 1

Address for Correspondence: Dr. Snezana Kojic, Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, PO Box 23, 11010 Belgrade, Serbia. E-mail: [email protected]

Referee Dr. Matteo Vatta, Department of Pediatrics, Baylor College of Medicine, Feigin Center, Houston, TX, USA

(Received 21 October 2011; revised 17 November 2011; accepted 20 November 2011)

Critical Reviews in Clinical Laboratory Sciences, 2011; 48(5-6): 269–294© 2011 Informa Healthcare USA, Inc.ISSN 1040-8363 print/ISSN 1549-781X onlineDOI: 10.3109/10408363.2011.643857

Critical Reviews in Clinical Laboratory Sciences

2011

48

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21 October 2011

17 November 2011

20 November 2011

1040-8363

1549-781X

© 2011 Informa Healthcare USA, Inc.

10.3109/10408363.2011.643857

643857

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270 S. Kojic et al.


changes in biochemical, morphological, and physiologi-cal features of individual muscle fibers (myofibers) that leads to alterations of muscle mass, contractile proper-ties, and metabolic status. This is accomplished through signal transduction, which starts at the cell surface where interaction between an extracellular signal and recep-tors occurs. Subsequently, activated factors in signaling pathways induce changes in gene expression, ultimately leading to remodeling of the myofiber1,2.

During striated muscle contractions, sarcomeric and sarcolemmal components receive mechanical stimuli that have to be converted into biochemical signals. This highly conserved process is defined as mechanosensa-tion and is found in a wide variety of different cell types, not only muscle cells3,4. Through mechanotransduction, mechanically induced signals are transmitted in order to initiate intracellular signaling pathways5,6. Signals are able to follow different paths both within and outside the muscle cell. One path is limited to the intracellular space, with the sarcomere and the nucleus as the final destina-tions, whereas the other is an inside/outside route to the sarcolemma and back to the nucleus through cell-surface receptor pathways7.

How striated muscle cells can sense mechanical stress and respond to dynamic changes in extracellular and intra-cellular conditions is not fully understood. Some insights into the molecular basis of stress response pathways and mechanosensors came from biochemical, immuno-histological and genetic studies related to striated muscle pathology. The sensing and propagation of mechanical forces involve many different components including pro-tein complexes associated with the sarcomere. So far, three mechanosensory “hotspots” along the titin molecule have been identified8–11. These signaling complexes located at the Z-disc, N2A or N2B region of the I-band and M-line (Figure 1) contain several proteins that can be found at multiple locations. In the sarcomere they bind titin and, in response to mechanical stimuli, they appear to relocalize to the nuclei where they participate in reprograming gene expression occurring during cell response to the structural or functional changes of contractile machinery12.

This review focuses on Ankrd1/CARP and Ankrd2/Arpp, MARP family members located in the I-band sig-nalosome and with the presumed function of messen-gers in stress response pathways9. Furthermore, in order to fully understand their function and their multi-tasking potential, we give a brief overview of signaling complexes in the sarcomere, with the emphasis on the proteins that are closely and functionally related to MARPs. We also discuss the role and potential diagnostic use of Ankrd1/CARP and Ankrd2/Arpp in neuromuscular disorders, cardiovascular diseases and tumors.

General overview of myofiber structure and organization

The contractile cells of our body can be classified into three major groups - cardiac, skeletal and smooth muscle

cells, according to their shape, number and position of the nuclei, presence of striation and whether they are under voluntary or involuntary control. Skeletal muscles are used to maintain posture and perform a wide range of movements and motions. They are able to produce contractions ranging from quick twitches to powerful sustained tension. Skeletal muscles contain elongated cells, multiple peripheral nuclei, visible striation and are under voluntary control. Cardiac muscles are exclusively localized in the heart and made of branching cells with a single central nucleus. They are also striated, but unlike skeletal muscle cells, they are under involuntary control. Spindle-shaped cells of smooth muscles possess a single central nucleus, but lack visible striations, and are under involuntary control.

A myofiber is an individual muscle cell. The myofiber content of a muscle determines its contraction speed and fatigue resistance. There are several types of myofi-bers which differ in their physiological, metabolic and biochemical parameters13. Classically, myofibers are classified into type I, type IIa, type IId/x, and type IIb according to the myosin heavy-chain isoform expression profile14,15. Type I myofibers or slow-twitch fibers are rich in mitochondria, have more capillaries surrounding each fiber, exhibit oxidative metabolism, have a low velocity of shortening and a high resistance to fatigue. The slow oxidative fibers are required to maintain posture and to perform tasks involving endurance. Type II fibers, also termed fast twitch myofibers, are glycolytic, exert quick contractions and fatigue rapidly.

Each muscle fiber contains many myofibrils, indi-vidual contractile subunits, extending from one end of the fiber to another. Myofibrils, composed of bundles of myofilaments, are organized in distinct repeating micro-anatomical units called sarcomeres. Sarcomere is defined as the region of myofilament structure between two Z-discs. It represents the fundamental contractile unit of striated muscle, a well organized cytoskeletal structure responsible for force generation and transmission.

Molecular components of myofilaments are precisely arranged in highly ordered, almost crystalline structures.The Z-disc serves as anchor site for thin filaments made of actin. The major component of thick filaments is myo-sin. These filaments are assembled in a very precise man-ner: thick myosin myofilaments are in between the thin actin myofilaments and are localized in the middle of the sarcomere. In vertebrates, in addition to thin and thick myofilaments, there is a third filament system formed by the giant elastic protein titin16–21.

As can be seen in Figure 1, the part of the sarcomere that contains only thin filaments is called I-band or light band. The A-band or dark zone contains thick filaments which may or may not overlap with the thin myofilaments. In the H-zone, localized at the center of the A-band, thick filaments do not overlap with thin filaments. The M-line is in the center of H-zone and serves to organize thick filaments. It is also the site where the C-terminal ends of titin overlap22.

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Muscle ankyrin proteins in muscle health and disease 271


The major protein components of the sarcomeric structures (actin, myosin, titin, α-actinin, tropomyosin, troponins, nebulin, M-protein and myomesin) have been known for a long time22. Recently, a number of previously unknown sarcomeric proteins were identified includ-ing: telethonin/T-cap, myotilin, obscurin, myopalladin, FATZ/myozenin/calsarcin and ZASP/cypher/oracle22,23. Besides structural roles, functional studies revealed their regulatory roles in signaling pathways24. Up to 2005, mutations in sarcomeric proteins were associated with around 20 different skeletal muscle diseases25.

The sarcomere was conventionally perceived as the most conserved molecular assembly in biology, with the localization of its constituent proteins precisely aligned, but now it is no longer considered as a static structure. In fact, the sarcomere is dynamic and undergoes constant changes while still maintaining its function. This contin-ual remodeling allows adaptation to stressors, including exercise, metabolic influences, or disuse, and must occur without affecting the integrity of the contractile force necessary for the muscle to continue to function10.

In this review, special emphasis is given to the emerg-ing role of sarcomeric components in mechanosensation and mechanotransduction. This topic will be discussed in the following section.

Mechanosensing complexes within the sarcomere

Three regions within the sarcomere (Z-disc, I-band and M-line) are recognized as localization sites of modules, composed of multiprotein complexes that function as a stress sensors and signal transducers (Figure 1). All sig-nalosomes include titin (as a scaffold for their organiza-tion), calpain 3 and their interacting proteins that have structural and signaling functions.

Titin can be considered as an important regulatory node implicated in integration, and possibly coordination of diverse signaling pathways26. This giant cytoskeletal

protein, known as the molecular spring in muscle cells, is exclusively expressed in striated muscle and represents a unique and essential constituent of sarcomeres. Titin is the largest protein in mammals; it is expressed in myri-ads of different isoforms with a maximal molecular mass of 4.2 MDa27. A single titin molecule extends over half of a sarcomere, with its N-terminus located in the Z-disc, and the C-terminus located in the M-line. One of titin’s classical roles in striated muscle is to provide elasticity to sarcomeres in order to maintain temporal and spa-tial assembly of the contractile filaments during muscle action28. This is achieved by generation of passive tension through molecular structures located in the I-band por-tion of the protein.

There are two principal spring elements with structur-ally distinct motifs: a tandemly arranged immunoglobulin-like (Ig-like) motif and the PEVK domain separated either by the N2B element expressed exclusively in cardiac variants or by the N2A element present in all skeletal and some cardiac isoforms8,29,30. The PEVK domain is rich in proline, glutamic acid, valine and lysine, lacks specific secondary structure, and belongs to the intrinsically unstructured protein family. As the length of the sarcom-ere extends, the tandem Ig-like modules are unfolded followed by extension of the PEVK domains. Different mutations in the titin gene (TTN) cause tibial muscular dystrophy (TMD) or Udd myopathy, an autosomal domi-nant distal myopathy with late onset and described in different populations31–34. Homozygous TMD mutations in TTN cause the more severe limb-girdle muscular dys-trophy (LGMD) type 2J (LGMD-2J)35.

Calpain 3 (also known as p94) is associated with all three regions of titin involved in the transmission of mechanical signals to signaling pathways. Calpain 3 is a skeletal muscle specific calcium dependent cysteine protease36–40. Generally, it is activated by auto-proteolytic removal of the internal propeptide from its active site38, but in muscle, it is normally inactive probably due to its binding to titin41. In contrast to other calpains, calpain

Figure 1. Schematic representation of the sarcomere structure in striated muscle. Localization sites of mechanosensor complexes are designated. Detailed explanation is given in the text.

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3 has two insertion sequences (IS1 and IS2) that have different functions42. The IS1 region has three autolytic sites, S1, S2, and S343, whereas the IS2 region has a nuclear localization signal (NLS), as well as a titin bind-ing site44. Recently, it was demonstrated that calpain 3 is activated in a physiological intracellular ionic environ-ment. Interestingly, its autolysis is Na+-dependent in the absence of Ca2+ and calpain 3 is the first example of an intracellular Na+-dependent enzyme45.

Calpain 3 substrates are structural proteins40,46,47, pro-teins involved in cell metabolism47–49, and in the regulation of gene and protein expression48,50–52 and include: titin40,53, the MARP family members54,55, filamin C46, filamin A40, talin40. As a consequence, it has been suggested that cal-pain 3 plays a role in three major physiological processes: the orchestration of sarcomere remodeling53,56,57, control of apoptosis52,58 and regulation of gene expression48,50–52. Mutations in the calpain 3 gene are responsible for a severe muscle disorder, LGMD-2A, characterized by selective atrophy and weakness of proximal muscles50.

The signaling module at the Z-disc

Z-discs are supra-molecular assemblies in eukaryotic muscle cells and represent the borders of individual sar-comeres in vertebrate striated muscle. They are specialized three-dimensional structures composed of multiprotein complexes, with the main function to align and anchor sarcomeric actin-containing thin filaments, as well as to anchor laterally the sarcomere to the sarcolemma. The Z-disc is well placed to monitor stretch and communicate strain through signal transduction pathways7,59. In fact, it serves as a scaffold for signaling complexes that contain a number of non-structural proteins such as protein kinases, protein phosphatases and transcriptional co-factors implicated in sarcomeric signaling to the nucleus. The N-terminus of the titin, calpain 3, telethonin/T-cap and muscle LIM protein (MLP) have all been suggested to be part of a putative mechanosensor complex at the Z-disc60.

Telethonin/T-cap is a 19 kDa sarcomeric protein, spe-cifically expressed in heart and skeletal muscle61. It links titin to the Z-disc, by binding to the N-terminal Ig-like domains, cross-linking two titin filaments in a very strong complex62–66. Telethonin/T-cap has been impli-cated in sensing mechanical strain through interaction with MLP60. Recently, it was demonstrated that loss of or mutations in telethonin/T-cap, combined with increased biomechanical stress, are associated with maladapta-tion, apoptosis and global heart failure67. Mutations in telethonin/T-cap are also linked to LGMD-2G68–70, as well as to hypertrophic and dilated forms of cardiomyopathy (HCM and DCM, respectively)60,71,72.

MLP is recruited to the Z-disc signalosome by its interaction with telethonin/T-cap60 and may have both structural and gene-regulatory roles. It is involved in the maintenance of normal muscle characteristics, as well as in early events during the recovery of skeletal muscle after injury73. MLP is indispensable for the Z-disc targeting of the

calcium-calmodulin activated protein phosphatase cal-cineurin74. Calcineurin is involved in control of fiber-type specific gene expression in skeletal muscles and its activa-tion in myocytes is correlated with selective up-regulation of slow-fiber specific gene promoters75. Together, MLP and calcineurin affect slow myosin heavy chain expression76.

After pressure overload or passive stretch, MLP can change subcellular localization from the Z-disc to the nucleus where it functions as a transcriptional co-activator60,77–79. Interaction of MLP with myogenic regulatory factors (MRFs) in the nucleus and its effect on expression of stretch response markers the brain natriuretic peptide (BNP) and the atrial natriuretic factor (ANF) make MLP a possible stretch-regulatory protein8.

The signaling module at the M-line

In striated muscle, in the centre of the sarcomere and in the middle of the A-band, there is the M-line, where the filaments of myosin and actin do not overlap80. In the M-line, the C-terminus of titin contains a catalytic serine-threonine kinase domain81, as well as binding sites for calpain 339 and muscle-specific RING finger proteins 1 and 2 (MuRF1 and MuRF2)82–85. MuRF isoforms can translocate to the nucleus in response to stress signals and mediate transcriptional repression via binding to the serum response factor (SRF)86.

Titin kinase regulates the transcriptional activity of the myofiber in response to a mechanical signal86 and has been proposed as a mechanosensor for stress-induced adaptation of muscle cell function87. When striated mus-cle is subjected to mechanical stress, the active site of titin kinase adopts an open conformation that allows the zinc-finger protein NBR1 (neighbor of BRCA1 gene-1) to bind. Subsequently, NBR1 forms a signaling complex with the ubiquitin-associated protein p62/SQSTM1, which in turn recruits a muscle-specific RING-B-box E3 ligase MuRF2 to the sarcomere86. In cardiomyocytes, in the absence of mechanical activity the complex unravels, p62/SQSTM1 moves to the intercalated disc and the periphery of the nucleus, whereas MuRF2 moves to the nucleus where it interacts with the nuclear SRF, inhibiting SRF-dependent gene expression86.

Signaling modules at the I-band

Signaling complexes at the I-band are assembled on the N2A and cardiac-specific N2B spring elements of titin9. Transcriptional co-activators, four-and-a half LIM domain proteins 1 and 2 (FHL1 and FHL2), interact with the unique sequence in the N2B region (N2-Bus) of the titin and translocate from the sarcomere and cytosol to the nucleus88,89. FHL1 forms a putative biomechanical stress sensor complex at N2-Bus with members of the MAPK family including MEK1/2, their activator Raf-1 and ERK2. This complex is thought to transform the mechanical stretch of N2-Bus to a hypertrophic response by shuttling activated ERK2 to the nucleus89.

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The N2A domain of titin, at position I80/I81, interacts with the three MARP proteins9, while the Ig-like domains I82/I83 bind another N2A ligand, muscle protease cal-pain 390. MARP proteins may provide a link between myo-fibrillar stress response and muscle gene expression since they are thought to shuttle to the nucleus in response to mechanical stress, thus implicating the N2A-domain in a mechano-signaling pathway. Calpain 3 possibly facili-tates coordinated signal transduction that originates from different sarcomeric regions. As noted above, it can be detected at the Z-disc, I-band and M-line of the sarcom-ere where other signaling complexes are found.

The importance of the signalosome composed of MARPs, calpain 3 and the N2A region of titin is observed in mdm (muscular dystrophy with myositis) mice, a model system for human TMD. Mdm is caused by a recessive mutation in the mouse titin gene91 and its prod-uct lacks 83 amino acids (aa) in the N2A region, a region which contains a putative binding site for calpain 3. Homozygous mdm mice show muscular dystrophy with severe and progressive muscular degeneration, and die within 3 months of birth91,92. Interestingly, MARP fam-ily members are more abundantly expressed in skeletal muscles of mdm than wild type mice92.

MARP proteins: structural characteristics and expression pattern

The MARP family includes three proteins: cardiac ankyrin repeat protein (Ankrd1/CARP)93–95, ankyrin repeat domain protein 2 (Ankrd2/Arpp)96–98 and diabe-tes-related ankyrin repeat protein (DARP)99. Their basic characteristics are given in Table 1.

Although expressed both in heart and skeletal muscle, their tissue expression patterns differ. Ankrd1/CARP is mainly expressed in the heart9,94,100,101, Ankrd2/Arpp in skeletal muscle9,96,97,102, and DARP is present in equivalent amounts in both tissues99. MARPs have dual intracellu-lar localization, found both in sarcomeric I-band and in nuclei. Their localization within the cell is subjected to change due to physiological (such as differentiation) and pathological (such as mechanical stress) conditions.

Three MARP proteins have similar structures, as revealed by in silico sequence analysis, sharing several common domains that define some of their functional features: tandem ankyrin repeats (four or five, depending on the analysis software), the coiled-coil domain, PEST motifs and NLS. The position of these motifs in the MARP proteins is schematically represented in Figure 2.

Ankyrin repeats are amongst the most frequent motifs found in proteins with different functions such as ion transport, regulation of cell cycle, initiation of transcrip-tion, cytoskeleton formation, signal transduction. They have been implicated in mediating the protein-protein interactions, are conserved among species and consist of 33 aa103. Proteins with ankyrin repeats have special importance in systems characterized by intensive com-munication between proteins. An excellent example is the muscle cell with its intensive metabolism, dynamic signal transduction and complex cellular network that regulates responses to different stimuli104. Binding sites for sarcomeric proteins titin9, calsequestrin-2 (CASQ2)105 and telethonin/T-cap106 are located in ankyrin repeat region of Ankrd1/CARP and/or Ankrd2/Arpp.

Coiled-coil structural motifs are able to mediate sub-unit oligomerization and protein–protein interactions. They typically consist of two to five α-helices wrapped around each other to form a supercoil107. Coiled-coil Table 1. Basic structural and functional features of the MARP

family members. Ankrd1 Ankrd2 DARPSynonym CARP,

MARP1Arpp, MARP2

Ankrd23, MARP3

Accession EAW50116.1(GenBank)

CAI14194.1(GenBank)

AAO24067.1(GenBank)

Location 10q23.31 10q23 2q11.2Size aa kDa

319 36.252

333 37.121

30534.297

Interactingproteins

Titin9

Calpain 355

p53143

YB-1100

Desmin110

MURFs180

Myopalladin167

CASQ2105

Titin9

Calpain 354

p53109

YB-1109

Telethonin/T-cap109

PML109

Akt2173

ZO1106

Titin9

Myopalladin9

Synonym: other names which have been used to describe the same protein. Accession: accession number for the protein (http://www.ncbi.nlm.nih.gov/protein/). Location: chromosomal localization of the human gene. Size: size of the protein is given as number of amino acids (aa) and molecular weight in kiloDaltons (kDa). Interacting proteins: list of the proteins that bind MARPs, superscripted numbers designate corresponding references.

Figure 2. Schematic representation of MARPs modular structure. Protein sequences of Ankrd1/CARP (319 aa, GenBank: EAW50116.1), Ankrd2/Arpp (333 aa, GenBank: CAI14194.1) and DARP (305 aa, GenBank: AAO24067.1) were analyzed in silico for the presence of ankyrin repeats, coil-coiled domains and NLS in ELM, The Eukaryotic Linear Motif Resource for functional sites in proteins (www.elm.eu.org299). PEST motif prediction was performed using the PESTfind program (http://emboss.bioinformatics.nl300).

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domains are identified in the N-terminus of MARPs and can contribute to their self-dimerization108–111.

The PEST sequences have been suggested to serve as signals for proteolytic degradation and are found in many short-lived, rapidly degraded proteins. These regions of protein instability are rich in proline (P), glutamic acid (E), serine (S), and threonine (T)112,113. Human Ankrd1/CARP possess two, while Ankrd2/Arpp is predicted to contain one PEST motif. Mutations or deletion of the PEST region increase Ankrd1/CARP protein stability in vivo114.

NLSs are specific aa sequences that mediate active nuclear transport of proteins from cytoplasm to nucleus. The nuclear import pathway is driven by the so-called classical NLS (cNLS), which contains a cluster of basic amino acids lysines (K) or arginines (R). This signal may be organized as a single-stretched monopartite NLS or in two small clusters, typically separated by ten to twelve aa, designated as a bipartite NLS. The SV40 large-T antigen (PKKKRKV) and nucleoplasmin (KRPAATKKAGQAKKKK) cNLS are the prototypical mono- and bipartite classical nuclear localization signals115,116. As seen in Figure 2, all three MARPs contain predicted nuclear localization signals.

Ankrd1/CARP is predominantly expressed in cardiac muscle93,94,100,101. During the early stages of mouse cardiac embryogenesis (E8–E10), as well as in 11–14-week-old human fetuses, it is uniformly expressed in all cardiac compartments100,101,117. As cardiogenesis proceeds, its expression levels decline in ventricular myocardium, but remain strong in the atrium and outflow tract100. Distinct atrial expression of Ankrd1/CARP is also observed in the heart of neonatal mouse101. The Ankrd1 gene is induc-ible in fetal heart in response to different forms of car-diovascular stress118. Ankrd1/CARP is also found in fetal skeletal muscle117, but barely detectable in adult skeletal muscle102,117,119. Its expression profile during development suggests potentially important functional roles in cardio-genesis and myogenesis.

In adult muscles, Ankrd1/CARP expression is gener-ally associated with pathological conditions and several studies have demonstrated its up-regulation in cardiac hypertrophy or heart failure with different etiologies9,120–124. Increased Ankrd1/CARP expression has been noted and studied in animal models of cardiac hypertrophy and heart failure caused by acute or chronic pressure overload including the aortic constriction model, spontaneously hypertensive rats, and Dahl salt sensitive rats121. Ankrd1 was also up-regulated in a canine model of the human heart failure with increased expression observed in left ventricles123. Although low under basal conditions, its expression in the skeletal muscle was induced under cer-tain conditions including exercise125–129, denervation94,102 and muscle pathologies119,130–133. In hypertrophic skeletal muscle, Ankrd1/CARP is up-regulated125–128, however, its expression can vary depending on the atrophic situation as it has been reported to be either up-regulated94 or down-regulated134,135.

During the regeneration process in rat muscles dam-aged by bupivacaine that induces muscle necrosis, Ankrd1/CARP expression was strongly up-regulated and peaked in the early stage of differentiation. Later on, it was down-reg-ulated to undetectable levels, accompanied by up-regula-tion of mature-type myosin heavy chains (MHC) indicating terminal differentiation of myofibers. An interesting point is that Ankrd1/CARP began to decrease before the onset of slow or fast MHC expression, pointing to its stage specific expression during differentiation of the regenerating mus-cle131. Ankrd1/CARP was also up-regulated during wound healing; its over-expression induced neovascularization and increased blood perfusion of experimental wounds, suggesting a role in angiogenesis136.

The Ankrd2/Arpp transcript and protein were detected in heart and kidney96,97, however, skeletal muscle is the predominant site of Ankrd2/Arpp expression96–98,117. During differentiation of myoblasts to myotubes, it is mainly localized in the nucleus of proliferating myoblasts. As differentiation proceeds, its expression increases and is more prominent in the cytoplasm97,137. Ankrd2 mRNA is present in the developing myotome of mouse embryos from as early as E9.5 and is also expressed during myo-blast fusion and maturation in vitro96. Its expression is fiber-type specific and in adult skeletal muscles it is mainly expressed in slow type I skeletal muscle fibers102. Nuclear accumulation of Ankrd2/Arpp is also observed in myocytes positioned next to damaged fibers after muscle injury of adult muscles, suggesting that Ankrd2/Arpp has a role in the regeneration process138.

Expression of Ankrd2/Arpp is induced in response to various forms of stress and is highly responsive to muscle mechanical status both in vivo and in vitro9,96, as well as to muscle plasticity induced by physical exercise127,128,139. Several lines of evidence strongly implicate Ankrd2/Arpp in muscle stress response. It is up-regulated in mouse muscles after chronic immobilization in a stretched position96,139, eccentric contraction of mouse127 and rat muscles128, as well as in injured muscles138. The level of Ankrd2/Arpp transcripts after eccentric contractions was sensitive to the mode of contraction (eccentric versus isocentric)127,128. Denervation exerted a dual effect on its expression, probably depending on the muscle type; decreased level of Ankrd2/Arpp was observed in den-ervated slow muscle (soleus)139, whereas denervation of fast muscle (gastrocnemius) increased its expression102. Ankrd2/Arpp expression is increased in type 1 diabetes, probably as a consequence of transition towards a slower fiber type, and it has been observed that physical activ-ity alleviates diabetes-induced Ankrd2/Arpp levels140. Finally, Lehti and colleagues have demonstrated that both Ankrd1/CARP and Ankrd2/Arpp were up-regulated in human muscles after fatiguing jumping exercise129.

Regulation of MARP expression

The control of muscle development and their postnatal function including stress response and corresponding

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muscle remodeling depends on the combined activity of the myogenic regulatory factors, pleiotropic transcrip-tion factors and epigenetic regulatory mechanisms141. Since MARP expression is not limited to only one type of muscle tissue (skeletal or cardiac), it is not surprising that MARPs share common tissue-specific regulators. To date, several transcriptional factors have been identi-fied as common regulators of MARP expression, namely MyoD, p53 and Nkx2.5.

Similar to the majority of skeletal muscle genes, transcription of the Ankrd1 and Ankrd2 genes is acti-vated by the muscle-specific transcription factor MyoD. It has been shown that Ankrd2 expression is regulated by MyoD in the early stages of myogenic differen-tiation142. MyoD also activates the Ankrd1 promoter in mouse myoblasts143 suggesting that Ankrd1/CARP expression in skeletal muscle cells may be governed by this tissue specific regulator. Since Ankrd1/CARP and Ankrd2/Arpp have been identified as downstream tar-gets of myogenic regulatory factors MyoD, myogenin and myocyte enhancer factor-2 (MEF2)144, it could be postulated that they are involved in propagation and amplification of signals initiated by MRFs during myo-genic differentiation.

Tumor suppressor p53 appears to regulate MARP expression since the promoter activity of the Ankrd1 and Ankrd2 genes was found to be up-regulated by p53 in mouse myoblasts106,143. The p53 is implicated in regu-lation of the muscle creatine kinase (MCK) gene145 and cooperates with MyoD in the induction of MCK tran-scription during muscle differentiation146. It activates the transcription of the muscle specific phosphoglycer-ate mutase gene (M-PGAM), a marker for cardiac and skeletal muscle differentiation, and also contributes to the control of M-PGAM cardiac expression in vivo147. However, the exact involvement and role of the p53 in the regulation of Ankrd1 and Ankrd2 expression during muscle differentiation still remains to be elucidated.

The Ankrd1 gene is a downstream target of the cardiac-specific transcription factor Nkx2.5 since its expression is significantly and selectively reduced in the fetal heart of Nkx2.5 knockout animals100 and up-regulated in the postnatal heart of transgenic mice over-expressing Nkx2.5148. Nkx2.5 controls Ankrd1 pro-moter activity in two ways: indirectly, through inter-action with the ubiquitous zinc finger transcription regulator GATA-4, which binds GATA-4 cis-element in Ankrd1 promoter95 and via direct binding to the 2.5-kb upstream regulatory region of the Ankrd1 gene essen-tial for its expression, as demonstrated in vivo149. This region also contains Sp3 binding sites, suggesting that Ankrd1 can be a direct target of the Sp3 transcription factor. In the Sp3−/– heart Ankrd1/CARP expression is down-regulated in ventricular, but not in atrial myo-cardium, although Nkx2.5 and GATA-4 expression is not affected149. Recently, we have demonstrated that Ankrd2 promoter activity is significantly up-regulated by Nkx2.5 in mouse myoblasts, indicating that cardiac

specific expression of Ankrd2/Arpp could be regulated by this transcription factor essential for cardiac devel-opment106. However, the functional significance of this finding needs further evaluation.

Since Ankrd1/CARP was identified as one of the fetal genes activated during cardiac myocyte hypertrophy95,121,148,150, several studies have investi-gated mechanisms of its transcriptional regulation in response to hypertrophic signals. Mitogen-activated protein kinase (MAPK) p38 and the small G protein Rac1 stimulate Ankrd1 promoter activity through the M-CAT element121. Stress-responsive p38 and Rac1 have been implicated in hypertrophy of ventricular myocytes151,152. Activation of Rac1 initiates a signaling cascade that leads to cardiac hypertrophy and involves downstream target proteins such as MAPK, p21WAF1/CIP1- activated kinase or focal adhesion kinase153,154. Similarly to MARPs, the MAPK pathway is involved in muscle growth and myogenesis, as well as activated by physi-cal training and stress155,156. Ankrd2/Arpp could also be involved in MAPK signaling since several genes (MEF2C, BDNF, FGF2, FLNB, GADD45B, MAP2K1, MAP3K7, MKNK2, NTF3, PDGFB, PDGFRB, TGFB2) from this pathway are affected on Ankrd2/Arpp silenc-ing. Notably, several transcription factors that interact with the Ankrd2/Arpp protein (CRK, JUN, p53, MEF2C, PAX6 and MeCP2) are also associated with the MAPK pathway106.

Ankrd1/CARP expression is induced by α- and β-adrenergic signaling157,158 known to control the rate and force of cardiac contractions, as well as to be associated with cardiac hypertrophy and failure159. Maeda and colleagues have demonstrated that the α1-adrenergic agonist phenylephrine elevated endogenous Ankrd1/CARP expression, while the antagonist prazosin low-ered it; these results were supported by finding that α1-adrenergic stimulation activates the Ankrd1 pro-moter. Since GATA-4 over-expression increases the α1- adrenergic response of the Ankrd1 promoter, GATA-4 was proposed as one of the mediators of α1-adrenergic-dependent Ankrd1/CARP activation157. Using the rat iso-prenaline infusion model it has been demonstrated that activation of the β-adrenergic signaling induces cardiac hypertrophy and significantly increases both Ankrd1/CARP mRNA and protein levels in the left ventricles158.

In vascular smooth muscle and endothelial cells, Ankrd1/CARP expression is directly regulated by trans-forming growth factor β (TGF-β) signaling, mediated through the binding of SMAD proteins to the CAGA motif within the Ankrd1 promoter160,161, thus linking Ankrd1/CARP to cell cycle inhibition.

Recently, it was suggested that Ankrd1/CARP could be involved in muscle disuse atrophy, since it was identi-fied as an indirect target gene of two transcription fac-tors (p50 and Bcl-3) associated with muscle wasting162. The expression of Ankrd1/CARP is transcriptionally down-regulated by GADD153 (CHOP-10), the CCAAT/enhancer-binding protein (C/EBP) family member,

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during apoptotic cell death induced by adriamycin (a cardiotoxic drug used in cancer treatment)163.

The molecular mechanisms of Ankrd2 gene regula-tion are less well understood in comparison to Ankrd1.Mohamed and colleagues164 showed anisotropic mecha-nosensitive regulation of Ankrd2 gene expression in skeletal muscles of both normal and mdm mouse dia-phragm. During each respiratory cycle, the diaphragm is exposed to biaxial loading, meaning that muscle fibers are stretched both in the transverse and longitudinal directions. Mechanical loads in both directions regulate Ankrd2 gene expression by two distinct mechanosensi-tive signaling pathways. Longitudinal stretch of the dia-phragm muscle fibers increases Ankrd2/Arpp expression due to the activation of Akt-dependent nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway, whereas Raf-1-dependent AP-1 sig-naling pathway increases Ankrd2/Arpp expression in response to transverse stretch.

Structural and regulatory roles of MARP proteins

Ankrd1/CARP and Ankrd2/Arpp have been studied for more than 15 years and their dual regulatory and struc-tural roles demonstrated165. Both proteins are active during muscle differentiation, controlling transcription of target genes as downstream targets of myogenic regu-latory factors. In adult muscles, MARPs are involved in cell stress management. They reside in mechanosensor complex of the I-band in striated muscle cells and shift to the nucleus upon mechanical stimuli, where they regu-late gene expression as transcriptional co-factors and also take part in signaling. It would appear that MARPs link stress sensing and stress response. Of note, knock-out mice for MARPs (single, double or triple), apart from slight differences in mechanical behavior, show no strik-ing changes in muscle phenotype165.

The structural role of MARPs in regulating mechani-cal behavior of myocytes emerged from the finding that skeletal muscle fibers were less stiff in their absence. Mice lacking all three MARPs express a longer isoform of titin than their wild-type counterparts, which proba-bly results in a longer resting sarcomere length in com-parison with control animals. Furthermore, it has been suggested that MARPs might also contribute to passive tension via interaction with titin through limitation of its flexibility165. Activation of MARPs is associated with the adaptation of the muscle towards a slower pheno-type upon mechanical activity such as stretch96,102,117,139 or botulinum toxin (Btx)-induced paralysis combined with exercise166.

Ankrd1/CARP and Ankrd2/Arpp are strongly respon-sive to the altered mechanical stimuli associated with Btx-induced paralysis or voluntary wheel running exer-cise training. Moreover, their expression is synergistically increased with the combination of Btx and exercise, implying that both genes are particularly sensitive to

passive stretch/strain of the muscle166. A role for MARPs in the maintenance of sarcomeric structure was suggested from the observation that altered interaction between Ankrd1/CARP and the I-band protein myopalladin led to destruction of the structure of the sarcomere167.

MARPs contain the nuclear localization signal (Figure 2) and can be detected in the nucleus where they possibly have a role of positive and negative co-factors able to act as transcriptional regulators of specific target genes97,100,101,109,138. A unique regulatory role of Ankrd1/CARP and Ankrd2/Arpp is anticipated during muscle morphogenesis. Precisely controlled myogenesis is essential for the proper development of the vertebrate embryo. Deregulated differentiation of muscle cells is able to cause several human congenital diseases, as well as cancer. A role for MARPs in myogenesis is supported by several findings. They were found to act as downstream targets of key muscle regulators MyoD, myogenin and MEF2144 with a proposed role in spreading signals initi-ated by MRFs during muscle morphogenesis and later in muscle remodeling.

Ankrd1/CARP is an early marker of the cardiac muscle cell lineage100 and several in vitro studies have shown its involvement in the negative regulation of cardiac gene expression during fetal heart development100,101. Its over-expression in cardiomyocytes inhibits cardiac troponin C and ANF gene expression101. As a transcriptional regu-lator, it participates in the Nkx2.5 pathway. The Nkx2.5 homeobox protein is essential for cardiogenesis and the Nkx2.5 pathway defines the early heart field in the devel-oping embryo168. In association with the transcription factor Y-box protein 1 (YB-1), Ankrd1/CARP represses its target, the ventricular myosin light chain-2 (MLC-2v) gene in the neonatal rat heart100. The MLC-2v protein is one of the earliest markers of ventricular regionalization during mammalian cardiogenesis and has been shown to play important roles in the maintenance of cardiac contractility and ventricular chamber morphogenesis169.

The Ankrd2/Arpp protein has a major role in skeletal muscle formation as a myogenic regulator, since its over-expression in C2C12 myoblasts significantly affects and down-regulates MyoD, myogenin, and their target gene Myh1137. It also contributes to the coordination of prolif-eration: Ankrd2/Arpp over-expression induces cell cycle arrest, whereas Ankrd2/Arpp silencing results in prolif-eration of mouse myoblasts137. Alteration in Ankrd2/Arpp expression promotes apoptosis, possibly via the p53 net-work, since many differentially expressed genes are either p53-target genes or p53-downstream effectors137. Ankrd2/Arpp is involved in cell to cell contacts and resultant sig-naling, since its silencing affects genes implicated in inter-cellular communication pathways: focal adhesion, tight junction and gap junction106. These processes play a role in myoblast fusion170,171 and in the coordination between gene expression and cell morphology172 during myogenesis.

Recently, it has been demonstrated that phosphoryla-tion of Ankrd2/Arpp by the serine/threonine kinase Akt2 is important for its function in muscle differentiation173.

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Akt2, also known as protein kinase B (PKB), is involved in cell cycle exit174 and Ankrd2/Arpp was identified as an Akt2 substrate, both in vitro and in vivo, in mouse muscle cells C2C12. The position of Ankrd2/Arpp phos-phorylation by Akt2 is Ser 72 which is in close proxim-ity of calpain 3 cleavage site (Figure 2). Oxidative stress induces Akt-dependent Ankrd2/Arpp phosphorylation and promotes its relocalization to the nucleus. It was proposed that Akt2 phosphorylation of Ankrd2/Arpp is implicated in preventing muscle differentiation follow-ing oxidative stress, which is in line with results of Bean and colleagues137, showing that Ankrd2/Arpp is a nega-tive regulator of muscle differentiation.

The proximity of two active sites, one for Ankrd2/Arpp phosphorylation and the other for its protease diges-tion, points towards competition between phosphory-lation, that leads to nuclear translocation, and calpain proteolysis that probably, similarly to Ankrd1/CARP, strengthens sarcomeric sequestration of Ankrd2/Arpp55. Post-translational modifications (phosphorylation and proteolysis in this case) are obviously able to substantially modulate MARP function and to regulate their intracel-lular localization.

Cardiac hypertrophy is observed in a number of patho-logical conditions caused by overwork of the heart, such as hypertension or cardiomyopathy. It is characterized by up-regulation of fetal genes and immediate-early genes, as well as genes encoding proteins involved in signaling pathways and energy metabolism175. It has been sug-gested that Ankrd1/CARP has a protective role in inhibi-tion of hypertrophic response since its levels increased in cardiac hypertrophy upon pressure overload121. Recently, the potential role of Ankrd2/Arpp in cardiac muscle has emerged. Cardiac specific transcription factors Nkx2.5, HAND2 and Ankrd1/CARP were able to regulate its expression, and Ankrd2/Arpp silencing in human myo-tubes affected pathways involved in HCM and DCM106. Studies on the role of Ankrd2/Arpp in heart muscle and to screening for the mutations in the Ankrd2 gene potentially linked to cardiomyopathies are awaited with interest.

Apart from its negative role in regulation of cardiac-specific genes, increased Ankrd1/CARP expression is associated with up-regulation of the p21WAF1/CIP1 inhibi-tor of the cell cycle in vascular smooth muscle cells160. We have recently demonstrated that Ankrd1/CARP acts as positive regulator and is able to modulate activities of transcriptional factors p53 and MyoD. The MyoD activity on the Ankrd2 promoter in mouse myoblasts106 and p53-mediated activation of the p21, Mdm2 and Ankrd2 pro-moters in SaOs2 cells, that are null for p53, are enhanced when Ankrd1/CARP is over-expressed143. The ability of Ankrd1/CARP to indirectly regulate the expression of another MARP family member warrants further investi-gation. It is possible that MyoD is implicated in the con-trol of Ankrd2/Arpp expression in ventricles of cardiac muscle where it can be detected98.

Recently, a master regulator of skeletal muscle differ-entiation MyoD was detected in Purkinje fibers localized

in the inner ventricular walls176. Similar to Ankrd1/CARP, Ankrd2/Arpp is also able to enhance the p53 up-reg-ulation of the p21 promoter in SaOs2 cells. Moreover, this effect is potentiated by its interaction with YB-1109.Another example of MARPs adjusting the action of tran-scription factors is the direct decrease of the DNA bind-ing activity of the NF-κB subunit p65 upon Ankrd1/CARP over-expression55. Taken together, these data support the regulatory role of MARPs as tissue specific transcrip-tional co-factors able to finely tune the performance of pleiotropic regulators.

Finally, there is strong evidence that MARPs are impli-cated in muscle adaptation. They are up-regulated after a variety of physical stresses imposed on muscle that trig-ger muscle adaptation9,96,125–129,138,139. Silencing of Ankrd2/Arpp in human myotubes106 affects signaling pathways known to be involved in skeletal muscle remodel-ing: MEF2/Histone deacetylase (HDAC), Calcineurin/Nuclear factor of activated T cells (NFAT), MAPK, Insulin-like growth factor, Akt and Mammalian target of rapamycin (m-Tor) pathways177. The role of Ankrd1/CARP in angiogenesis161, neovascularization136,178 and neurite outgrowth179 has also been demonstrated, but will not be discussed here.

Interacting partners of MARPs

One of the striking features of muscle, and particularly Z-disc proteins, is the high frequency of multiple pro-tein-protein interactions that have been detected. Most of the proteins have at least one binding partner and could not be considered isolated from the others23. This is extremely important for the study of newly identified muscle proteins, as implications regarding their func-tion can be deduced from the known functions of the interacting partner. In order to get a better insight into MARPs` functions and the cellular processes in which they actively participate, a brief overview of common MARP interacting partners with known biological func-tions will be presented.

As listed in Table 1, Ankrd1/CARP can interact with titin9, CASQ2105, YB-1100, myopalladin167, desmin110, MuRF1/MuRF2180, p53143 and calpain 355. The Ankrd2/Arpp can interact with titin9, telethonin/T-cap109, calpain 354, promyelocytic leukemia protein (PML)109, YB-1109, p53109, Akt2173 and zonula occludens 1 (ZO1)106. As the general structural and functional characteristics of titin and calpain 3 have been mentioned previously, we will only focus on their relationship with the MARPs. A brief outline of two other MARP common interacting partners, p53 and YB-1, with emphasis on their functional interac-tion, will be also addressed in following sections.

Titin and MARPs

All three MARPs have an ability to interact with titin. Ankrd1/CARP and Ankrd2/Arpp, localized in the central I-band region, co-localize with the titin-N2A

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region, more specifically with a tyrosine-rich motif lying between two Ig-like motifs (I80 and I81). Together with myopalladin, they constitute the I-band mechanosens-ing complex. Both Ankrd1/CARP and Ankrd2/Arpp proteins contain a binding site for titin within their ankyrin repeat region9. It has also been demonstrated that they have different binding affinities for the N2A region; the Ankrd1/CARP interaction is much stron-ger than the Ankrd2/Arpp interaction. The interaction between Ankrd2/Arpp and I80-PEVK region in normal or mdm muscle cells is also strongly suppressed by co-expression with Ankrd1/CARP, whereas Ankrd1/CARP is not affected54. The efficiency of binding between the titin and MARPs is affected by the mdm deletion54. An important question to be resolved is the time frame of these interactions and whether the binding partners bind simultaneously or competitively.

Calpain 3 and MARPs

In the sarcomeric I-band calpain 3 interacts with the same region of titin responsible for MARP binding (between Ig-like motifs I80 and I81)38. Both Ankrd1/CARP and Ankrd2/Arpp are the substrates of calpain 3; cleavage sites are at the N-terminus of MARPs, between aa 30 and 71 for Ankrd1/CARP54 and at Arg 77 for Ankrd2/Arpp55 (Figure 2). It has been suggested that this modulator protease might regulate the sarco-meric localization of MARPs and their interactions with other proteins of the I-band signaling complex. The calpain 3 is involved in strengthening the interaction between Ankrd1/CARP and titin. Furthermore, cleav-age of Ankrd1/CARP by calpain 3 theoretically dis-rupts the bipartite NLS potentially affecting its nuclear transport55. On the other hand, the calpain 3 cleavage of Ankrd2/Arpp (at Arg 77) does not interrupt its NLS motif. Calpain 3 modulates Ankrd2/Arpp binding to the titin “is” region by its competitive interaction with titin. Release of Ankrd2/Arpp from titin could provoke “desensitization” of the mechanosensor complex and its inability to respond to stretch signals54.

p53 and MARPs

The p53 protein is one of the most studied proteins of the living world181. The name “guardian of the genome” reflects its role in cell response to genotoxic agents and participation in processes that are crucial for maintenance of genome integrity. p53 exerts its role by influencing processes such as programmed cell death (apoptosis), repair of damaged DNA and cell cycle182–184. In non-stressed cells, p53 is localized in the cytoplasm, in complex with the E3 ligase MDM2 (mouse double minute-2)185 that mediate its ubiquitin dependent proteasomal degradation186. Different stimuli, such as radiation, viruses, oncogenes, or other types of stress, activate post-translational modifiers that phos-phorylate, acetylate and glycosylate p53 and cause

dissociation of the p53-MDM2 complex187. The released p53 is able to exert its function of transcriptional regu-lator of numerous downstream targets. Modulating the expression level of these genes, and depending on the severity of damage, cell type, environmental con-text, and the type of stress, p53 can promote cell cycle arrest, apoptosis or senescence in order to prevent the propagation of damaged or mutant cells that could potentially become cancerous188,189.

During skeletal muscle differentiation, both expres-sion and transcriptional activity of p53 increase146,190. Its involvement in the differentiation process is achieved through regulation of pRB (retinoblastoma protein) expression191. Retinoblastoma protein functions as a fundamental regulator to coordinate pathways of cellu-lar growth and differentiation, contributing to cell-cycle withdrawal and promoting the expression of late mark-ers of differentiation192. Experiments on p53−/–knockout mice demonstrated that p53 is not essential for normal muscle development, although the muscles of these ani-mals were susceptible to tumors193. During heart devel-opment, p53 transcripts decrease rapidly during early postnatal development, becoming almost undetectable in fully differentiated myocytes194.

Several lines of evidence support the role of p53 in muscle-specific stress response. In muscle hypertrophy caused by stretch195 and after resistance exercise126 an increase in nuclear p53 is evident. In atrophic conditions, both cytoplasmic and nuclear p53 are up-regulated196. Mechanical stretching of rat ventricular myocytes is able to induce p53-dependent apoptosis197,198 associated with up-regulation of p53 and p53-inducible genes as well as with the release of angiotensin II followed by enhance-ment of the cellular renin-angiotensin system197.

Cardiac fibroblasts are resistant to apoptosis and undergo cell cycle alteration by modulating expression of p21WAF1/CIP1 198. Functional association between stretch response and p53 was further confirmed by over-expres-sion of a dominant-negative p53 mutant in adult ventricu-lar myocytes that specifically inhibited renin-angiotensin activation and stretch-mediated up-regulation of genes that possess p53 regulatory sites (aogen, AT receptor and Bax)199.

A multilevel functional relationship between p53 and MARPs has been demonstrated106,109,143. As mentioned, p53 induces the activity of Ankrd1 and Ankrd2 promot-ers in vitro106,143, suggesting its potential role in regulation of MARP expression. On the other hand, both Ankrd1/CARP and Ankrd2/Arpp physically interact with p53 and modulate its transcriptional activity. p21WAF1/CIP1, MDM2 and Ankrd2/Arpp itself are indirect in vitro downstream targets of MARP proteins regulated via p53106,109,143. It is possible that MARPs act as muscle tissue specific regu-lators of p53 function during development and later in stress response pathways. They might exert their func-tion in myogenesis as signal transducers modulating p53 activity towards p21WAF1/CIP1, which plays a role in termi-nal withdrawal of myocytes from the cell cycle. Further

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studies are required to search for in vivo downstream target genes indirectly regulated by MARPs via p53 and implicated in signaling pathways affected by silencing or over-expression of MARPs.

YB-1 and MARPs

Y-box-binding protein, YB-1, encoded by the NESP1 gene, belongs to the family of cold shock proteins and contains a highly conserved nucleic acid binding motif very similar to the common CCAAT box200. It has multiple functions and it is implicated in RNA splicing, transla-tion, DNA repair, and transcription200,201. YB-1 is able to regulate gene expression both at transcriptional and translational levels202,203. The majority of YB-1 is located in the cytoplasm. Nuclear translocation of YB-1 occurs in response to a variety of stresses that include UV expo-sure, DNA damage, and hypothermia204–206. Once in the nucleus, YB-1 can stimulate cell proliferation and both positively and negatively regulate gene expression. It is also an important marker of and contributor to tumorigenesis.

YB-1 expression in clinical specimens correlates significantly with tumor stage and prognosis for many human tumors, including the common malignancies of the lung, breast, and colon200,207,208. YB-1 interacts with the tumor suppressor p53 and its nuclear translocation may also involve p53. It acts as a negative regulator of p53, repressing the p53 promoter and down-regulating endog-enous p53 expression209. Direct interaction between YB-1 and p53 influence their DNA binding affinities in oppo-site ways, it stimulates p53 sequence-specific DNA bind-ing and inhibits the binding of YB-1 to DNA210.

Recently, a new function of YB-1 was uncovered; nuclear Y-box binding protein 1 (YB-1/p32) is able to interact with the Msx1 homeoprotein and regulate C2C12 myoblast differentiation211. YB-1/p32 can inhibit the expression of endogenous MyoD, thus preventing termi-nal differentiation of C2C12 myoblasts. It was proposed that the biological function of YB-1/p32, in the context of myoblasts, is to inhibit cellular differentiation through interaction with cell type-specific regulators such as Msx1.

Ankrd1/CARP was identified as an interacting protein partner of the transcription regulator YB-1100. In this direct interaction, Ankrd1/CARP carries out a role of negative transcriptional co-factor of YB-1, which is implicated in regulation of the expression of chamber-specific genes during mammalian cardiogenesis. One of these genes is MLC-2v, the earliest marker of cardiac chambers region-alization212. YB-1 is one of the transcription factors that interact with Ankrd2/Arpp both in vitro and in vivo109. Similarly to Ankrd1/CARP, Ankrd2/Arpp could act as transcriptional co-factor of YB-1, but in an opposite way. Ankrd2/Arpp and YB-1 synergistically enhanced the p53-mediated up-regulation of the p21WAF1/CIP1 promoter109. The site of their common action could be the ventricles since Ankrd2/Arpp is mainly expressed there117 and YB-1

is a known negative regulator of ventricular-specific MLC-2v transcription95,100.

MARPs in diseases and their role as diagnostic and prognostic markers

In the last two decades, an increasing number of human muscle diseases have been shown to be caused by muta-tions in major components of the sarcomere. Signal complexes assembled on titin, important for normal muscle function, are also implicated in the mechanisms of muscle diseases. Mutations in molecular components of these complexes cause various types of muscular dys-trophies. For example, mutations in the telethonin/T-cap gene are responsible for LGMD-2G68, LGMD-2J is caused by mutations in calpain 350, whereas mutations in titin result in a TMD32.

Genetic studies of patients with cardiomyopathy indi-cate that many myocardial dysfunctions have a mechani-cal origin, in particular, those caused by mutations in the sarcomeric proteins such as actin, titin, α-tropomyosin, troponin and, most commonly, β-myosin heavy chain213. Of particular interest are mutations in genes for titin214–217 and the Z-disc proteins telethonin/T-cap71,72, MLP218,219, FATZ 2/myozenin 2/calsarcin 1220 and ZASP/cypher/oracle221 identified as a cause of HCM and DCM. As already mentioned, telethonin/T-cap and MLP have been implicated in the mechano-transduction process that promotes and propagates cell signaling responses.

So far, it has been demonstrated that MARP family members are altered in a number of neuromuscular and cardiovascular disorders, as well as in some tumors (Table 2) and that their immuno-histochemical evalua-tion is helpful for diagnostic purposes.

MARPs in neuromuscular disorders

In skeletal muscle, altered expression patterns of Ankrd1/CARP and Ankrd2/Arpp have been found in patients suf-fering from inherited myopathies (muscular dystrophies (MD), congenital myopathies (CM)) and motor neuron diseases (spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS)) (Table 2). It is worth noting that all these disorders have atrophy of muscle fibers as a common denominator. Evaluation of MARPs was gener-ally performed using immuno-histochemical analysis of skeletal muscle biopsy samples.

MARPs in inherited myopathies

Before the overview of MARP alterations in inherited myopathies, we would like to point out that the tradi-tional classification of these diseases (MD and non-dystrophic myopathies) will be used, and that we are aware of the imperfect nomenclature due to progress in the clinical and molecular understanding of these dis-eases. Dystrophies are characterized by the breakdown of muscle integrity and are often associated with mutations

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in sarcolemmal proteins leading to “dystrophic” features such as large hypertrophied myofibers and increased connective and fatty tissues. Congenital muscular dys-trophies (CMD) also have dystrophic attributes, but they are frequently present in infancy. Congenital myopathies have non-dystrophic pathological features, early onset and are relatively non-progressive. They are distinguished by muscle weakness and atrophy, and often character-ized by inclusions originating from abnormal sarcomeric components. However, there are exceptions among each group and symptoms may overlap. Some dystrophies do not have dystrophic features (e.g. some LGMDs and myotonic dystrophy types 1 and 2). Nemaline myopathy belongs to the congenital myopathies but may have adult onset and progression. Distal myopathy caused by dys-ferlin mutations is characterized by proximal weakness and may be classified as a LGMD222.

Muscular dystrophies represent a group of genetically inherited neuromuscular diseases marked by progres-sive wasting and weakness of the skeletal muscles, with occasional involvement of cardiac and smooth muscle or other tissues223. These diseases are the result of defects in genes that are responsible for normal muscle function. There are several subtypes of MD, the most common being Duchenne muscular dystrophy (DMD), affecting about 1 out of every 3500 boys and caused by mutations in the dystrophin gene resulting in no or an aberrant pro-tein product.

Dystrophin is a sarcolemmal protein and its loss causes muscle degeneration and necrosis224. It is a component of the dystrophin-associated glycoprotein complex (DGC) that forms a critical link between the cytoskeleton and the extra-cellular matrix. The DGC is an assembly of proteins spanning the sarcolemma of skeletal muscle fibers and forming a chain of links between the contractile actin in the cytoskeleton and the extracellular matrix225,226. Defects in the DGC can disrupt these links and alter the base-ment membrane organization, resulting in sarcolemmal

instability and muscle cell apoptosis. Apart from stabiliz-ing the sarcolemma, DCG is also essential in organiz-ing molecules involved in cellular signaling226. Becker muscular dystrophy (BMD) is similar to DMD, but is less common and progresses more slowly227. This form of MD, which affects approximately 1 in 30,000 boys, is caused by insufficient production of dystrophin.

LGMDs are grouped together based on common clinical features; they primarily and predominantly affect proximal muscles around the scapular and the pelvic girdles. About 20 different forms of LGMD are currently recognized. They are caused by deficiency in different muscle proteins such as dysferlin, caveolin 3, lamin A/C, calpain 3, α-, β- and δ-sarcoglycanes, telethonin/T-cap, and fukutin-related protein (FKRP)228. Estimated preva-lence for all forms of LGMD ranges from 1/14,500 to 1/123,000229,230. These figures should be accepted with reservation as until recently, diagnosis criteria for LGMDs were not accurate and were mimicked by other similar neurogenic and myogenic conditions229.

Facioscapulohumeral muscular dystrophy (FSHMD) is the third most common muscular dystrophy (4-10/100,000) and it is characterized by the progressive weakness and atrophy of the muscles of the face, scapula and upper arms231,232. It is caused by a reduction in the copy number of the D4Z4 macrosatellite repeat located at chromosome 4q35233,234. BMD, LGMD and FSHMD have a later onset, less severe clinical presentation and progress more slowly than DMD.

Congenital muscular dystrophies are a clinically and genetically heterogeneous group of inherited muscle disorders, distinguished by the presence of muscle weak-ness at birth or in infancy, and generally little or no pro-gression235. The incidence of all forms of CMD has been estimated at 1/21,500 with a prevalence of 1/125,000 in northeastern Italy and 1/16,000 in western Sweden236,237. Most genes implicated in these disorders code for proteins important for the function of the DGC in the sarcolemma

Table 2. A list of diseases in which Ankrd1/CARP and Ankrd2/Arpp expression profiles were determined (+). The corresponding data for DARP are not available.DISEASES Ankrd1/CARP Ankrd2/Arpp

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ies

Muscular dystrophies

Duchenne muscular dystrophy + +Becker muscular dystrophy + +Limb-girdle muscular dystrophies + +Facioscapulohumeral muscular dystrophy + +Congenital muscular dystrophies + +

Congenital myopathies

Myotubular myopathy + +Nemaline myopathy + +Central core disease + +Congenital fiber type disproportion + +

Motor neuron diseases Spinal muscular atrophy + +Amyotrophic lateral sclerosis + +

Cardiovascular diseases

Hypertrophic cardiomyopathy + –Dilated cardiomyopathy + –

Tumors Rhabdomyosarcomas + +Ovarian cancer + –Renal oncocytoma – +

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and extracellular matrix. The most common of the CMDs is the laminin-α2-deficient muscular dystrophy (LDMD), estimated to account for 50% of patients with congenital muscular dystrophies.

Evaluation of Ankrd1/CARP and Ankrd2/Arpp, per-formed in different types of muscular dystrophies (DMD, LGMD, FSHMD and CMD), has demonstrated non-uniform expression profiles. High expression of Ankrd1/CARP was detected in skeletal muscle samples of MD patients, but the population of Ankrd1/CARP-positive fibers varied among different MD types. Ankrd1/CARP-positive myofibers were detected more often in CMD than in DMD patients. It is interesting that in DMD patients, Ankrd1/CARP is highly expressed exclusively in small regenerating myofibers that co-express embryonic MHC. On the other hand, in CMD, Ankrd1/CARP expression is not limited to the regenerating fibers and was detected in fibers co-expressing embryonic, as well as mature-type MHC131.

In contrast, Ankrd2/Arpp expression was generally down-regulated in MD patients and its protein levels in the Ankrd2/Arpp-positive myofibers were lower than those in normal muscle. Ankrd2/Arpp expression was significantly down-regulated in slow MHC-positive type 1 myofibers. Although the expression levels were low in most fibers, some hypertrophic myofibers observed in MD muscles expressed Ankrd2/Arpp at comparable lev-els to normal control muscle fibers238.

The fact that expression of both MARP proteins is affected in DMD and CMD patients suggests that they might be involved in the molecular mechanisms under-lying the pathobiology of these diseases and further studies are required to evaluate their role. In our opinion, it is worth studying the role of Ankrd2/Arpp in CMD, since we have shown that Ankrd2/Arpp can interact with beta-1-syntrophin (SNB1), a peripheral membrane pro-tein associated with dystrophin and dystrophin-related proteins106.

Congenital myopathies are a molecularly, patho-logically and clinically heterogeneous group of neu-romuscular disorders that are often mild and slow or non-progressive. They usually present at birth or in early childhood with muscle weakness, hypotonia, atrophy and proximal muscle involvement being a frequent manifestation239. Based on genetic and morphological features CMs are divided into: myopathies with protein accumulations, myopathies with cores, myopathies with central nuclei and myopathies with fiber size variations240. They all have similar clinical presentation and accurate diagnosis of the subtypes is usually achieved by histo-pathological evaluation of biopsied muscle. Therefore, molecular markers that could identify specific subtypes of the disease would be beneficial for the differential diagnosis of congenital myopathies.

Alterations in expression of the members of the MARP family were studied in muscle tissues from patients with 4 different subtypes of CM: myotubular myopa-thy, nemaline myopathy, central core disease (CCD)

and congenital fiber type disproportion (CFTD)132,238. In brief, 8 out of the 14 cases were found to be positive for Ankrd1/CARP and its expression pattern was quite dif-ferent among the subtypes. The Ankrd2/Arpp expression was induced, particularly in atrophic or damaged myo-fibers, as both the numbers of Ankrd2/Arpp-positive myofibers and Ankrd2/Arpp expression levels in the muscle tissue were increased in comparison with those in normal muscle.

Myotubular myopathy belongs to myopathies with central nuclei and is caused by mutations in the myotu-bularin gene Mmt1241. Its product, myotubularin, is phos-phoinositide (PI) 3-phosphatase242. Myotubularin acts on signaling lipids that play important functions in the endocytic pathway by regulating the temporal recruit-ment of proteins required for endosome maturation, protein sorting, and lysosomal degradation243–245. In myo-tubularin-deficient mouse myofibers, an absence of this protein leads to abnormal distribution of triads and dys-regulation of genes involved in calcium homeostasis246.

Muscle samples from patients with myotubular myo-pathy were positive for Ankrd1/CARP. The expression level was not uniform among Ankrd1/CARP-positive fibers, as there were significantly more weakly positive fibers than the stronger ones132. Among markedly atro-phic myofibers with centrally placed nuclei, Ankrd2/Arpp positive and negative myofibers were randomly mixed similarly to normal control muscle. Nevertheless, both the number of Ankrd2/Arpp-positive myofibers and Ankrd2/Arpp expression level were higher than those observed in other subtypes of congenital myopathy as well as in normal muscle238.

Nemaline myopathy belongs to congenital myo-pathies with protein accumulation, and is caused by mutations in at least six distinct genes (α-tropomyosin, β-tropomyosin, nebulin, α-actin, cofilin 2 and troponin T)247. It is characterized by the presence of inclusions in myofibers, designated as nemaline rods or nema-line bodies. These rods are abnormal, electron-dense structures emanating from Z-discs, and composed of α-actinin and other Z-disc proteins239. In nemaline myo-pathy, both Ankrd1/CARP-positive and -negative cases have been described, with Ankrd1/CARP preferentially expressed in severely damaged myofibers. Levels of its expression may be correlated with the severity of the disease132. The Ankrd2/Arpp expression coincided with the slow MHC-positive myofibers, but the proportion of Ankrd2/Arpp positive myofibers differed among the patients. Ankrd2/Arpp was mainly expressed in small atrophic myofibers, and positive and negative myofi-bers were randomly admixed. It was also demonstrated that Ankrd2/Arpp positive myofibers tended to contain nemaline rods238. Rods were predominantly found in fiber type 1 muscles248, where Ankrd2/Arpp is also pref-erentially expressed. So far, the biological significance of the association between Ankrd2/Arpp expression and nemaline rod formation, as well as the pathogenesis of these rods is largely unknown.

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CCD belongs to myopathies with cores, and is char-acterized by the presence of amorphous-looking cen-tral areas in fibers lacking oxidative enzyme activities. This disorder is caused by mutations in the RYR1 gene encoding the ryanodine receptor 1, the principal Ca2+ release channel of sarcoplasmic reticulum247. Ankrd1/CARP was not detected in CCD132, while the expres-sion pattern of Ankrd2/Arpp differed from case to case. In one CCD patient, Ankrd2/Arpp-positive and negative fibers were arranged in a checkerboard-like pattern similar to normal muscle, whereas in another two patients almost all of the fibers were positive for Ankrd2/Arpp, although the expression level was rela-tively low238.

CFTD is a non-structural congenital myopathy belonging to myopathies with fiber size variation. It is characterized by the marked discrepancy in fiber sizes due to the abnormal hypotrophy of slow type 1 fibers relative to type 2 rapid contracting fibers239. The expres-sion pattern of Ankrd1/CARP and Ankrd2/Arpp was not consistent among CFTD patients. In muscles containing small and atrophic myofibers, Ankrd1/CARP-positive myofibers were scarcely detectable. However, Ankrd1/CARP was preferentially expressed in the large hyper-trophic fibers in admixtures of small atrophic and large hypertrophic myofibers. Hypertrophic Ankrd1/CARP-positive myofibers were negative for embryonic MHC132. In contrast, Ankrd2/Arpp was expressed in all analyzed specimens, but its expression pattern was diffuse or limited to small myofibers238.

The mechanisms responsible for the induction of MARP expression in patients with congenital myo-pathies are still not understood. It was suggested that the increased number of Ankrd2/Arpp positive myofibers might be associated with increased numbers of type 1 fibers as they predominate in patients with congenital myopathies, including all four analyzed subtypes; a concept supported by finding that Ankrd2/Arpp-positive myofibers expressed slow MHC238. Since Ankrd1/CARP was not detected in CCD, its evaluation may be helpful in the histopathological diagnosis of different CM subtypes.

MARPs in motor neuron diseases

Due to the affected expression of Ankrd1/CARP and Ankrd2/Arpp in denervated muscles, both proteins were investigated in SMA and ALS patients.

SMA is an autosomal recessive neurodegenerative disease, characterized by spinal motor neuron death and progressive muscle atrophy249. The prevalence of the disease is 3/100,000250. Based on the age of onset and clinical severity, SMA is classified into four types (SMA 1- SMA 4). SMA 1 is the most severe, with an onset during infancy, poor clinical prognosis and death before the age of two years. Individuals with other SMA types can live on into adulthood, suffering from varying degrees of muscle paralysis and atrophy. The vast majority of familial SMA is caused by deletion or homozygous mutation(s) in the

telomeric copy of the survival motor neuron 1 (SMN1) gene and subsequent reduction of the SMN protein levels251.

Expression of Ankrd1/CARP protein was induced in atrophic myofibers of SMA 1 and SMA 2 patients and barely detectable in the normal control muscle132. High levels of Ankrd1/CARP were found in large groups of severely atrophic myofibers, in comparison to mildly atrophic myofibers. Hypertrophic myofibers were nega-tive for Ankrd1/CARP, suggesting its selective expression in severely atrophic myofibers in SMA 1 and 2. In SMA 3, characterized with less severe atrophic changes than in SMA 1 and 2, Ankrd1/CARP-positive fibers were less common and they showed lower immuno-reactivity than in SMA 1 and 2. Based on the finding that Ankrd1/CARP is induced in denervated atrophic myofibers in the more severe cases of the disease, it is possible that its expres-sion reflects the status of muscle atrophy and might be used as useful monitoring tool.

In SMA patients with severe and intermediate sub-types, Ankrd2/Arpp was induced in hypertrophic myo-fibers238. Ankrd2/Arpp-positive myofibers were arranged in groups and Ankrd2/Arpp was expressed uniformly within the group. Among the slow MHC-positive myo-fibers there were both Ankrd2/Arpp-positive hypertro-phic and Ankrd2/Arpp-negative atrophic myofibers. In patients with SMA 3, Ankrd2/Arpp expression pattern was not consistent (Ankrd2/Arpp-positive myofibers were arranged either in a checkerboard-like pattern or in groups), but it was localized exclusively in slow MHC-positive myofibers238. Ankrd2/Arpp expression levels were higher in patients with severe rather than mild disease, suggesting possible association of Ankrd2/Arpp expression in SMA patients with disease severity. Since the grouped myofibers result from the process of denervation, innervation and subsequent denervation of re-innervated myofibers252 it was suggested that the Ankrd2/Arpp-positive myofibers specifically arranged in groups in SMA patients could be the consequence of motor unit denervation.

Based on our current knowledge, it seems that the expression pattern of MARPs in SMA could be a conse-quence of the atrophy caused by denervation and that their evaluation might be useful for monitoring disease status.

ALS is a progressive and fatal neurodegenerative motor neuron disease in which both upper and lower motor neurons are affected253. The prevalence of individuals with ALS in Europe is roughly 2-16/100,000254. ALS is epide-miologically classified as sporadic (90–95%) and familial (5-10%)255. Among the familial cases, approximately 20% are caused by dominantly inherited mutations in the Cu/Zn superoxide dismutase-1 (SOD1) protein256. Some of the cellular processes that occur after disease onset have been identified, but underlying cause is unknown.

The pathological characteristics of ALS include protein aggregation, proteasome inhibition, mito-chondria damage and apoptosis, oxidative stress,

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neuroinflammation, transcriptional dysfunction and generation of free radicals. The most characteristic fea-ture of pathologic changes in skeletal muscles of ALS is atrophy caused by denervation257. So far, the extensive research aimed to identify molecular markers in the cerebrospinal fluid and plasma of ALS patients has not found validated biomarkers for this disease.

Ankrd1/CARP was aberrantly expressed regardless of muscle fiber type.

Ankrd1/CARP-expressing muscle fibers were easily detected in ALS patients, opposed to the control muscles where they were hardly detectable. Of note, Ankrd1/CARP expression was increased in severely atrophic small fibers compared to less atrophic fibers or those resembling normal fibers. In addition, localization of Ankrd1/CARP in ALS muscle cells was altered; it was detected in the cytoplasm, but not in the nucleus119. In atrophic muscles of ALS patients, Ankrd2/Arpp-positive fibers were uni-formly distributed and the loss of the checkerboard-like pattern, characteristic for normal muscles, was observed. This aberrant phenotype was present in both severe and mild cases, and resembled the one observed in dener-vated gastrocnemius muscle fibers102,119. There was no specific correlation between the expression of Ankrd2/Arpp and slow MHC. Sub-cellular localization, Ankrd2/Arpp was highly expressed in the nuclei of muscle cells of ALS patients119.

Currently, the biological significance of altered MARP expression in ALS is largely unknown. Changes in MARP intracellular localization in ALS muscle cells are particu-larly interesting and in accordance with their distribu-tion in stressed muscle cells. Ankrd1/CARP is mainly expressed in the cytoplasm whereas Ankrd2/Arpp is found in the nucleus of ALS myofibers. Since approxi-mately 20% of familial ALS is caused by mutations in the SOD1 gene, oxidative stress, both in neurons and in muscle where antioxidant enzyme activity is altered258, is one of the potential pathogenic events associated with the disease. It has been recently demonstrated that oxidative stress, induced by hydrogen peroxide (H

2O

2),

triggers phosphorylation of Ankrd2/Arpp by Akt2 protein kinase leading to its nuclear translocation173. Similarly, in muscles stressed by cardiotoxin injections or dry ice, Ankrd2/Arpp accumulates in the nuclei of myofibers adjacent to damaged ones138.

Ankrd1/CARP expression is also induced in skeletal muscle under stress conditions such as response to acute resistance exercise126 and work-overload hypertrophy125.

Although the role of “sarcomeric Ankrd1/CARP” is not fully elucidated, a function in the maintenance of sarco-meric integrity via its interaction with titin and myopal-ladin has been proposed167. It could be hypothesized that in certain stress conditions (such as denervation and oxi-dative stress in ALS), muscle cells have to respond ade-quately to changes in environment. In order to complete this task, they need to activate cooperatively both MARP members: Ankrd2/Arpp in the nucleus to regulate tran-scription and Ankrd1/CARP in sarcomere to maintain

its integrity. The effect of MARP over-expression in ALS remains to be elucidated: it could influence disease pro-gression or might have a protective effect.

One promising line for future studies on MARP fam-ily members could be their further evaluation as specific prognostic and diagnostic markers in neuro-muscular disorders. To achieve this goal it is necessary to include more patients from different populations and to study Ankrd1/CARP and Ankrd2/Arpp in tandem.

MARPs in cardiovascular diseases

Cardiovascular diseases are the largest source of morbid-ity and mortality in Europe and are expected to become the leading cause of death worldwide by 2015259. In par-ticular, HCM (prevalence 1/500) and DCM (prevalence 1/2500) are very common primary diseases of the heart muscle and major causes of heart failure associated with high morbidity and mortality260. Although cardiomyo-pathies can be caused by a variety of different infections (myocarditis) or exposure to different toxins or drugs (such as alcohol), it is estimated that probably 80% of all HCM and at least 30% of all DCM cases are hereditary. A variety of different mutations in a wide range of differ-ent genes encoding mainly cytoskeletal or sarcomeric components have been identified as the cause of several familial cardiomyopathies261. Interestingly, cardiomyo-pathy and heart failure due to imperfect mechanosensa-tion is a relatively new hypothesis and, identification and functional characterization of mutations in MARPs impli-cated in HCM and DCM could contribute to the knowl-edge on the pathogenesis of these cardiac diseases.

The first report implicating Ankrd1/CARP in human cardiac disorders was that of Zolk and colleagues who found a significant increase of Ankrd1/CARP mRNA and protein levels in left ventricular myocardium in patients with end-stage heart failure suffering from dilated and ischemic cardiomyopathy123. Although Ankrd1/CARP expression was constitutively higher in atria, its expres-sion was induced selectively in ventricular myocardium during congestive heart failure. The Ankrd1/CARP pro-tein was predominantly localized in the nuclear fraction in non-failing as well as in failing hearts, showing that expression levels but not the subcellular distribution is changed in human heart failure.

Recently, several missense mutations in the Ankrd1 gene, associated with impaired protein-protein interac-tions and altered gene expression in response to mechan-ical stretch, have been reported in patients affected by HCM and DCM262–264. Some mutations in the Ankrd1 gene associated with DCM resulted in loss of binding of the Ankrd1/CARP protein with talin, a key interacting part-ner of the beta-integrin subunit of the integrin complex, thus linking Ankrd1/CARP not only via titin, but also via talin to mechanosensation. Impaired interactions could potentially lead to loss of stretch sensing and disruption of the link between the titin complex and cytoskeletal networks264.

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Ankrd1/CARP mutants identified by Duboscq-Bidot and colleagues263 showed reduced capability to repress transcription from the MLC-2v promoter, a known in vitro target of Ankrd1/CARP100. Over-expression of the same mutant proteins in rat cardiomyocytes treated with α1-adrenoceptor agonist phenylephrin-chloride abolished Ankrd1/CARP mediated limitation of hyper-trophic response measured as cardiomyocyte surface area263. Ankrd1/CARP mutations associated with HCM lead to an increased binding of Ankrd1/CARP to sar-comeric proteins titin and myopalladin, as well as its abnormal localization in cardiomyocytes262. Cellular distribution of the mutant Ankrd1/CARP proteins was not changed in immature cardiomyocytes. In mature cardiomyocytes, where Z-discs are well organized, mutant Ankrd1/CARP proteins displayed abnormal intra-nuclear accumulation and were localized either within the nuclei and/or at nuclear membrane. Wild type Ankrd1/CARP was assembled in a striated pattern at the Z/I-bands and co-localized with α-actinin, while most of the mature cardiomyocytes did not contain nuclear Ankrd1/CARP.

The role of Ankrd2/Arpp in the cardiac muscle is com-pletely unknown. However, our recent results regarding the functional interaction of Ankrd2/Arpp with cardiac specific regulatory factors Nkx2.5, HAND2 and Ankrd1/CARP, as well as alteration in genes implicated in HCM and DCM pathways upon Ankrd2/Arpp silencing106 have led us to speculate that Ankrd2/Arpp could have an important function in heart muscle. These findings open up a new field in Ankrd2/Arpp investigation and a future aim would be to decipher the molecular mechanisms of Ankrd2/Arpp, as well as Ankrd1/CARP action in heart and deregulation in cardiac diseases. One of the intrigu-ing possibilities is that they are part of the embryonic and fetal gene program of cardiac cytoskeletal proteins initi-ated during cardiac remodeling265,266, similar to their role in skeletal muscle remodeling and responding to muscle-specific stress (stretch or sarcomeric dysfunction).

MARPs in tumors

Tumors are diseases characterized by uncontrolled cell growth and strongly associated with changes in cellular signaling pathways that regulate their growth, survival, differentiation and invasion. To mention a few impli-cated in tumorigenesis and intensively studied: the p53 pathway267,268, JAK/STAT pathway269,270, NOTCH signal-ing pathway270,271, MAPK/ERK pathway270,272, PI3K/Akt pathway270,273, NFkB pathway270,274, Wnt pathway270,275 and TGF-β pathway270,276. In contrast to the slow progress in curing cancer, our understanding of signaling pathways that control normal cellular function, and how these pathways become altered in cancer, has substantially increased in recent years. This basic knowledge rep-resents a foundation for the development of targeted molecular therapies to kill tumor cells while leaving nor-mal cells unharmed.

Several findings indicate possible involvement of Ankrd1/CARP and Ankrd2/Arpp in some of these pathways, indicating a possible role of MARPs in the process of tumorigenesis. One indication is based on their complex functional relationship with the tumor suppressor p53, which is frequently mutated in human cancers. Disruption of normal p53 function is one of the factors that cause initiation and/or progression of tumors193,277. Over 50% of human tumors harbor DNA mutations in the p53 gene, and it is estimated that over 80% of tumors have dysfunctional p53 signaling188,278,279. We have identified p53 as a common downstream tar-get for both Ankrd1/CARP and Ankrd2/Arpp proteins. They bind p53 and enhance the p53-mediated up- regulation of the cell cycle inhibitor p21WAF1/CIP1 pro-moter. In turn, p53 can enhance Ankrd1 and Ankrd2 promoter activity implicating the role of p53 in regula-tion of its effectors109,143. Disrupted association between MARPs and p53 might affect the fine-tuning of p53 activity and, therefore, could be a potential mechanism by which Ankrd1/CARP and Ankrd2/Arpp are involved in tumorigenesis.

Ankrd1/CARP and Ankrd2/Arpp could also partici-pate in TGF-β and Wnt signaling pathways; both path-ways are implicated in tumorigenesis280,281. Ankrd1/CARP was found to be up-regulated in cells in which these two pathways were simultaneously induced. Those cells displayed an expression profile distinct from the one observed with single ligand treatments as dem-onstrated by a microarray-based approach282. Several genes involved in TGF-β and Wnt signaling, as well as in cancer pathways, were affected in Ankrd2/Arpp silenced myotubes, as revealed by gene expression profiling106. These results are in a favor of a potential role of Ankrd2/Arpp in tumorigenesis.

Activity of MARPs in fine modulation of p53 function, as well as their involvement in pathways known to par-ticipate in the mechanism of tumorigenesis, suggest that Ankrd1/CARP and Ankrd2/Arpp might display altered expression and/or function in tumors and promote their development. Both proteins have been investigated in rhabdomyosarcomas (RMS)117,283,284; Ankrd1/CARP expression was detected in ovarian adenocarcinomas285 and Ankrd2/Arpp observed in renal oncocytomas286 (Table 2).

Rhabdomyosarcomas and MARPs

RMS is the most common soft tissue sarcoma of child-hood and adolescence287. Tumor tissue contains skeletal muscle cells in various stages of muscle differentiation, from mononucleated myoblasts to multinucleated myo-tubes and rhabdomyoblasts that are dispersed among tumor cells288. The diagnosis of RMS is based on the detec-tion of specific phenotypic features of skeletal muscle such as cross-striation, but in cases where cross-striation is absent, immuno-histochemical analysis of muscle specific markers such as MyoD, myogenin and desmin

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is used to aid in differentiating RMS from other types of sarcomas289. The markers of early myogenic differentia-tion such as myogenin and MyoD are used routinely in clinical practice. They are not always reliable and can vary in sensitivity and specificity. For that reason, search for a more suitable disease marker is still in progress. Since cells positive for both Ankrd1/CARP and Ankrd2/Arpp proteins were observed in all tested RMS cases117, they were further evaluated as potential diagnostic mark-ers for RMS.

Ankrd1/CARP expression was immuno-histochem-icaly evaluated in 159 malignant tumors including 34 RMSs, 85 non-RMSs and 40 carcinomas. Intense Ankrd1/CARP staining was mainly observed in the cyto-plasm of tumor cells, in 85% of RMS cases and only in 4% of non-RMSs. Although Ankrd1/CARP is also localized in the nuclei where it participates in regulation of gene expression, it is interesting that nuclear immunoreactivity was rarely observed, and no RMS cases showed nuclear restricted expression of Ankrd1/CARP. The specificity and sensitivity of Ankrd1/CARP was similar to that observed for other myogenic markers283.

In the study of Ishiguro and colleagues284, tissue sec-tions of 37 RMS, 88 non-RMS sarcomas and 38 carci-nomas were analyzed for Ankrd2/Arpp expression. In contrast to non-RMS and other carcinomas, Ankrd2/Arpp was detected in 89.2% of RMS cases and in all samples when Western blot was used as the detection method284. Of note, Ankrd2/Arpp expressing cells did not always co-express other muscle specific markers such as desmin, fast MHC or muscle actin. These results indicate that Ankrd2/Arpp can be detected in those RMS cells in which other existing tumor markers are unde-tectable217. The sensitivity of Ankrd2/Arpp was shown to be higher than that of myoglobin and comparable with sensitivity of muscle-specific biomarkers such as actin, desmin, myogenin and MyoD284.

Taken together the results of these studies indicate that Ankrd1/CARP and Ankrd2/Arpp have significantly higher expression in RMS than in non-RMS tumors, thus representing sensitive and specific markers for RMS. In conclusion, MARP family members may have a potential use in diagnosis of RMS and furthermore may be useful for the differential diagnosis of RMS.

Ovarian cancer and Ankrd1/CARP

The incidence rates of ovarian cancer are highest in the Western world, where it is the leading cause of death from gynecological malignancies290. Ovarian cancer is associ-ated with high morbidity and mortality since approxi-mately 75% of patients are diagnosed with advanced disease (FIGO stages III and IV) when the prognosis is poor291. Another reason is the relapse of disease after initial response to first-line chemotherapy and overall treatment failure due to acquired resistance to further chemotherapy292. Epithelial ovarian cancer is the most common type of ovarian cancer and usually occurs in

women older than 50 years. Treatment of ovarian cancer is based on the integration of surgery and chemotherapy, with platinum-based chemotherapeutics as the standard cure. These agents are initially effective, but usually the disease relapses after a variable interval. So far, clinical prediction of platinum sensitivity and strategies to over-come platinum resistance are not possible.

Scurr and colleagues285 studied altered gene expres-sion associated with high sensitivity to cisplatin, in order to define novel targets to sensitize tumor cells to platinum compounds and ultimately improve the effectiveness of this widely used class of chemotherapeutics. They iden-tified Ankrd1/CARP as the most highly down-regulated gene in a cell line with unusual sensitivity to cisplatin. Similarly, the Ankrd1/CARP level was negatively corre-lated with cisplatin sensitivity in a panel of human can-cer cell lines. Furthermore, analysis of 71 ovarian cancer patients revealed that patients with a moderate to high level of Ankrd1/CARP had a worse outcome compared with patients whose tumors had low Ankrd1/CARP expression.

Since a significant correlation was observed between Ankrd1/CARP level and platinum response, its role in determining cellular response to platinum-based chemo-therapy in ovarian cancer treatment has been suggested. Ankrd1/CARP might represent a novel target for phar-macological inhibition, and decreasing its expression or function could be a potential strategy to sensitize tumors to platinum-based drugs, thus avoiding disease relapse and increasing the clinical effectiveness of the treatment. Ankrd1/CARP has a potential of a so-called molecularly targeted therapy predicted to make major improvements in ovarian cancer treatment. It is a new kind of therapy based on exploitation of biological pathways in tumor cells which differ from those in normal cells.

Renal oncocytoma and Ankrd2/Arpp

Renal oncocytoma is a benign tumor and belongs to the heterogeneous group of renal tumors. It is made of oncocytes, round shaped or polygonal, large, well-differ-entiated neoplastic cells with large number of mitochon-dria293–295 that originates from the renal collecting duct cells. Renal tumors include clear renal cell carcinoma (RCC), papillary RCC, chromophobe RCC (ChRCC), collecting duct carcinoma, and others296. These tumors frequently show complex and overlapping morphologi-cal characteristics, as well as histological heterogeneity within a single tumor morphology, making correct diag-nosis difficult. For example, oncocytomas may co-exist with primary renal carcinomas, clear RCC may present with oncocytic features or RCC may co-exist with onco-cytomas in the same or contra-lateral kidney297.

One of the most problematic differential diagnoses is to distinguish ChRCC from oncocytoma and this requires sophisticated microscopic, ultrastuctural and immuno-histochemical evaluation. Both tumors have different behavior and clinical outcomes despite their common

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origin in distal nephrons and similarities in pheno-typic features. Oncocytoma is a benign tumor, whereas ChRCC is malignant and the distinction between them is particularly relevant in patients with multiple, bilat-eral tumors and in patients with a solitary kidney and/or renal insufficiency, where surgical removal of tumors may render patients dialysis-dependent.

Although several immuno-markers, including vin-culin, paxillin, SHP2, parvalbumin and c-kit, are used to distinguish these two types of tumors, there is still no specific expression marker that reliably separates renal oncocytoma from eosinophilic variants of RCC, includ-ing ChRCC. For example, caveolin is expressed in roughly 90% of oncocytomas, but it was also detected in about 20% of RCC variants298. CD10, a marker for the proximal renal tubule was expressed in every subtype of renal tumor except for ChRCC286. A marker for distal renal tubule, EMA (epithelial membrane antigen) was highly expressed in oncocytomas, as well as in other subtypes286.

Shomori and colleagues286 investigated the Ankrd2/Arpp expression profile in normal kidney as well as in renal oncocytoma. Apart from striated muscle, Ankrd2/Arpp was also expressed in kidney, more specifically in the mitochondria and nuclei of cells located in part of the distal renal tubule, in both the renal cortex and medulla. Ankrd2/Arpp transcript and protein were also detected in the kidney by RT PCR and Western blot analysis97. Immuno-histochemical analysis of 100 renal tumors (14 oncocytomas and 86 different RCCs) revealed high expression of Ankrd2/Arpp in oncocytoma (12 of the 14 cases) and negative staining in all 11 ChRCC cases. Since Ankrd2/Arpp was selectively expressed in onco-cytomas but rarely in other types of renal tumor, its potential use as a specific marker for distinguishing oncocytomas from chromophobe RCC was suggested286. However, the clinical diagnostic usefulness of this marker for renal oncocytoma needs to be further validated by additional large-scale studies.

For the reasons given above, the MARP proteins represent potentially good candidates for tumor prog-nostic and/or diagnostic purposes. However, current knowledge about alterations in MARP expression and their role in tumorigenesis is limited since they have only been studied in a small number of patients from few populations. The involvement of MARPs in the pro-cesses of tumorigenesis in other tissues than muscle indicate pleiotropic function of MARPs and study of their expression and function in other tissues (such Ankrd1/CARP in angiogenesis) is a promising area for future research.

Conclusions and perspectives

This review summarizes the current understanding of the structure, expression pattern and function of two MARP family members Ankrd1/CARP and Ankrd2/Arpp in the remodeling processes (atrophy and hypertrophy),

stress response, cardio- and myogenesis and regula-tion of transcription factors. Their multi-tasking nature and activity in different developmental stages, during morphogenesis and later, in adulthood, during stress response, is emphasized.

The investigation of MARP family members is impor-tant for elucidating the signaling pathways that regulate skeletal muscle remodeling, which is pertinent to under-standing the pathology of neuro-muscular diseases. Unraveling the functional role of MARP proteins would help in understanding the molecular mechanisms of coordinated differential gene expression that occurs upon sensing mechanical signals and results in pheno-typic adaptation in striated muscle. It will be important to discover why both Ankrd1/CARP and Ankrd2/Arpp are expressed in the same location and at the same time. In order to do this it will be necessary to study the func-tion of both proteins in tandem and to use a combination of cellular and molecular approaches as well as live-cell imaging techniques.

There are several exciting areas for future research in this field, such as to identify downstream targets of MARPs in vivo and to study functional interdependence of post-translational modifications and their role in mod-ulation of MARPs function and intracellular localization. Further genetic screening of the Ankrd1 and eventually Ankrd2 genes would help to better understand the phys-iopathological role of MARP mutations in the etiology of HCM and DCM.

In contrast to the large set of fundamental data, infor-mation about alterations in MARP expression in diseases is rather scarce. Evaluation of their status was performed on a small set of patients. An interesting question to be resolved is whether alterations in MARP expression such as down-regulation of Ankrd2/Arpp in muscles of MD patients or up-regulation of MARP in denervated muscles of SMA patients, could be a useful marker for monitoring the clinical status of these diseases.

It would also be intriguing to investigate the role of Ankrd2/Arpp in the etiology of CMD in connection with its presumed interaction with DGC components. MARPs could be used as molecular markers for differential diag-nosis of CM, to distinguish among different subtypes of the disease or as monitoring tools for evaluation of disease severity in SMA. They also have a potential of sensitive and specific markers for differential diagnosis of tumors such as RMS and oncocytomas, or as pharma-cological target for ovarian cancer treatment. However, to explore these possibilities and to establish MARPs as prognostic and diagnostic markers in neuro-muscular disorders and tumors it would be essential to perform integrated studies on larger numbers of patients from diverse populations.

Declaration of interest

The experimental work of the authors was supported by grants from the Collaborative Research Programme,

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ICGEB, Italy (grant CRP/YUG-05-01), Ministry of Educa-tion and Science of Serbia (Projects 143051 and 173008), Telethon Foundation of Italy (grant GGP04088) and the Fondazione Cariparo, Italy (Progetto Eccellenza 2010 CROMUS). The authors declare no conflicts of interest.

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Muscle ankyrin repeat proteins: their role in striated muscle function in health and disease

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