Histochem Cell Biol (2010) 134:31–38

DOI 10.1007/s00418-010-0716-8

ORIGINAL PAPER

Increased P2X7R expression in atrial cardiomyocytes of caveolin-1 deWcient mice

K. Barth · C. PXeger · A. Linge · J. A. Sim · A. Surprenant · N. Steinbronn · R. H. Strasser · M. Kasper

Accepted: 26 May 2010 / Published online: 19 June 2010© Springer-Verlag 2010

Abstract It has recently been shown in epithelial cellsthat the ATP-gated ion channel P2X7R is in part, associ-ated with caveolae and colocalized with caveolin-1. In thepresent study of the mouse heart, we show for the Wrst time,using immunohistochemistry and cryoimmunoelectronmicroscopy, that P2X7R is expressed in atrial cardiomyo-cytes and in cardiac microvascular endothelial cells, but notin the ventricle cardiomyocytes. Furthermore, biochemicaldata indicate the presence of two forms of P2X7R, the clas-sical glycosylated 80 kDa isoform and a protein with themolecular weight of 56 kDa, in both cardiomyocytes andendothelial cells of the mouse heart. The functionality ofboth proteins in heart cells is still unclear. In cardiac tissuehomogenates derived from caveolin-1 deWcient mice(cav-1¡/¡), an increase of the P2Xrx7 mRNA and P2X7Rprotein (80 kDa) was found, particularly in atrial samples.In addition, P2rx7¡/¡ mice showed enhanced protein levelsof caveolin-1 in their atrial tissues. Although the details ofcellular mechanisms that underlie the relationship betweencaveolin-1 and P2X7R in atrial cardiomyocytes and the

electrophysiological consequences of the increased P2X7Rexpression in atrial cells of cav-1¡/¡ mice remain to be elu-cidated, the cardiomyopathy detectable in cav-1¡/¡ mice ispossibly related to a disturbed crosstalk between P2X7Rand caveolin-1 in diVerent heart cell populations.

Keywords P2X7R · Caveolin-1 · Cardiomyocytes · Mice

Introduction

Caveolin-1 is a main structural protein of caveolae and mightplay a role as a molecular organizer for membrane multipro-tein complexes, which are involved in diVerent cellular func-tions such as endocytosis, transcytosis, cell adhesion, lipidmetabolism, and signal transduction (Parton and Simons2007). Caveolae are considered to be speciWc lipid rafts,dynamic membrane microdomains of the plasma membranecontaining aggregates of cholesterol and sphingolipids andare enriched in the structural protein caveolin (Sonnino andPrinetti 2009). Caveolins are a family of 21- to 24-kDa inte-gral membrane proteins. Caveolin-1 (Cav-1) and caveolin-2(Cav-2) have a wide overlapping distribution: they are foundprimarily in endothelial cells, smooth muscle cells, type Ipneumocytes and Wbroblasts. Caveolin-3 (Cav-3) is involvedin the formation of caveolae in striated muscles, whereasCav-1 contributes to caveolae formation elsewhere. Cav-2 isnot necessary for the formation of caveolae, but heterooligo-merizes with Cav-1 (Razani et al. 2002).

Although expression of Cav-3 in ventricular cardiacmyocytes as well as Cav-1 in cardiac endothelial andsmooth muscle cells of blood vessels have been welldocumented (Gazzerro et al. 2010), expression of Cav-1 inventricular cardiac myocytes is still under debate (Cho et al.2007, 2010; Volonte et al. 2008). We have demonstrated that

K. Barth · C. PXeger · A. Linge · M. KasperDepartment of Anatomy, University of Technology Dresden, Fetscherstr. 76, 01307 Dresden, Germany

J. A. Sim · A. SurprenantFaculty of Medicine and Human Sciences, University of Manchester, Manchester M13 9PT, UK

N. Steinbronn · R. H. StrasserDepartment of Medicine and Cardiology, Medical Clinic, Fetscherstr. 76, 01307 Dresden, Germany

K. Barth (&)Institute of Anatomy, Medical Faculty, TU Dresden, Fetscherstr. 74, 01307 Dresden, Germanye-mail: kathrin.barth@tu-dresden.de

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32 Histochem Cell Biol (2010) 134:31–38

Cav-1 and Cav-3 are coexpressed in mouse and rat cardiacmyocytes of the atria but not of the ventricles (Volonteet al. 2008). The physiological importance of Cav-1 in car-diovascular regulation has recently been highlighted by thedescription of the heart phenotype in Cav-1 deWcient(cav-1¡/¡) mice (Drab et al. 2001; Razani et al. 2001)reviewed in Rahman and Swärd (2009). A number of stud-ies have now accumulated describing cardiomyopathy withsigns of systolic and diastolic severe heart failure in theseanimals (Wunderlich et al. 2008a, b). Morphologically,increased Wbrosis and hypertrophy of cardiomyocytes hasbeen noted (Drab et al. 2001).

Extracellular ATP and its analogues are important sig-nalling molecules that initiate potent eVects on the cardio-vascular system via cell surface purinergic (P2) receptors,called P2X and P2Y receptors (Burnstock and Knight2004). P2X receptors are non-selective cation channels per-meable to Na+, Ca2+, and K+ gated by ATP. Seven subtypesof P2X (P2X1–P2X7) receptor have been identiWed (Khakhand North 2006). Recent studies report that P2X receptorsare expressed in the heart and may play an important rolein cardiac function (Gurung et al. 2009; Musa et al. 2009;Ralevic and Burnstock 1998, 2003; Vassort 2001). It wasfurther shown that an increased expression or activation ofthe P2X4 receptor (P2X4R) may exert a salutary role incardiac hypertrophy and failure (Shen et al. 2006).

Previous studies, including our own, have shown that adiscrete population of P2X7 receptor (P2X7R) is associatedwith lipid rafts/caveolae (Barth et al. 2007; Garcia-Marcoset al. 2006). Lipid raft-associated caveolin proteins assem-ble as oligomeric aggregates to form a scaVolding complexthat gives caveolae their morphology and provide sites forprotein–protein interactions between caveolin and raft-associated proteins. Raft localization may have varyingfunctional consequences, for example as organizers ofreceptor/signalling complexes on the cell surface. In lungepithelial cells our previous results have been shown that apart of P2X7R is associated with Cav-1 (Barth et al. 2008).

The present study accordingly investigates the expres-sion of P2X7R in heart tissues of normal and cav-1¡/¡

mice, as well as the expression of Cav-1 in P2rx7¡/¡ mice.On one side we are interested to clarify if there is any evi-dence for a cell-type speciWc distribution of the P2X7R inthe mouse heart; on the other side we test the hypothesisthat the lack of Cav-1 inXuences the P2X7R distribution.

Materials and methods

Tissue protein extraction

For protein extraction tissue specimens, e.g. samples fromatria and ventricles of 3-month-old wild-type female mice

and cav-1¡/¡ female mice and P2rx7¡/¡ female mice werehomogenized in extraction buVer containing 0.02 M Tris,0.125 M NaCl, pH 8.5; 1% Triton X-100 and proteaseinhibitors using a homogenizer (VDI 25; VWR Interna-tional GmbH, Darmstadt, Germany). After solubilizationfor 3 h at 4°C with gentle rotation, sample was centrifugedat 15,000g for 15 min at 4°C (Allegra™ 64R, BeckmanF2402H rotor; Beckman Coulter, Fullerton, CA, USA).Finally, the supernatant was collected and subjected to fur-ther analysis.

Reverse transcription-PCR (RT-PCR)

For Reverse transcription-PCR (RT-PCR), total RNA fromfrozen tissue specimens of mice atria and ventricles wereisolated using the RNeasy Fibrous Tissue Kit (QiagenGmbH, Hilden, Germany) following the instructions of themanufacturer. Subsequently, DNase treatment was per-formed with DNase I (MBI Fermentas GmbH, St. Leon-Rot, Germany). From 2 to 4 �g total RNA were subjectedto cDNA synthesis using the Revert Aid™H Minus FirstStrand cDNA Synthesis Kit (MBI Fermentas GmbH).cDNA synthesized with oligo-dT primers was used as atemplate for the ampliWcation of the genes P2rx7 andGAPDH using the speciWc primers 5�- ATCCACTTCCCCGGCCACAA-3� (P2rx7-forward) and 5�-CCTCCAGTGCCGAAAACCAGGA-3� (P2rx7-reverse) and 5�-GGAAGGGCTCATGACCACAG-3� (GAPDH forward)and5�-TTGCTGTTGAAGTCGCAGGA-3� (GAPDH reverse).

The gene of the GAPDH was used as the housekeepinggene. For all primer sets PCR conditions were as follows:94°C for 5 min followed by 25–35 cycles of ampliWcation.Each cycle included 60 s denaturation at 94°C, 30 s anneal-ing at 60°C and 30 s synthesis at 72°C. To conWrm thespeciWcity of each primer set, the PCR product of each genewas subjected to direct cycle sequencing with the oligonu-cleotide primers used for ampliWcation.

Western blot analysis

Western blot analysis was performed as already described(Barth et al. 2007) with minor modiWcations. The followingprimary antibodies were used: monoclonal mouse anti-Cav-1(clone 2297, dilution 1:500, v/v; BD Biosciences Pharmingen,San Jose, CA, USA), and polyclonal rabbit anti-P2X7R(dilution 1:500, v/v; Alomone Labs Ltd., Jerusalem, Israel).Bound antibodies were detected using horseradish per-oxidase conjugated secondary antibodies (ECL anti-mouseIgG; dilution 1:2,000, v/v; Amersham Biosciences, LittleChalfont, Buckinghamshire, UK; or ECL donkey anti-rabbit IgG; dilution 1:4,000, v/v; Amersham Biosciences).Chemiluminescent signal was generated with ImmobilonWestern Chemiluminescent HRP Substrate (Millipore

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Histochem Cell Biol (2010) 134:31–38 33

Corporation, Billerica, MA, USA) and detected by ImageReader LAS-3000 (FujiWlm, Medical Systems USA Inc.,Stanford, CT, USA).

Immunohistochemistry

ParaYn sections of mouse hearts from wild-type (n = 2)and cav-1¡/¡ mice (n = 5) were used from previous studies(Volonte et al. 2008). Hearts of P2rx7¡/¡ mice wereembedded in paraYn as described (Volonte et al. 2008). Allimmunocytochemical techniques have been described pre-viously (Volonte et al. 2008). BrieXy, rabbit anti-Cav-1(dilution 1:50; Santa Cruz), rabbit anti-podocalyxin (kindgift from Dr. Marilyn G. Farquhar; La Jolla, CA; dilution1:3,000), goat anti-Cav-3 IgG (Santa Cruz; dilution 1:100),rabbit anti-P2X7R (a peptide corresponding to amino acidresidues 576–595 of rat P2X7R; intracellular C terminus),dilution 1:100; Sigma–Aldrich or rabbit anti P2X7R (thesame peptide), dilution 1:200, from Alomone, Jerusalem,Israel, have been used as primary antibodies after micro-wave pretreatment. For controls, primary antibodies werereplaced with non-speciWc, rabbit or goat immunoglobulinsor PBS. Blocking experiments were performed by incuba-tion of the primary anti-P2X7R antibody solution with thecorresponding immunizing peptide in excess for 30 min at37°C (data not shown). As secondary antibodies biotinyla-ted anti-rabbit or anti-goat antibodies (Vectastain ABC Kit;Vector Laboratories, Burlingame, CA) were used.

Cryoimmunoelectron microscopy

To determine the localization of P2X7R and Cav-1, smallpieces (1 £ 1 £ 1 mm) of cardiac tissues were Wxed in 2%(w/v) paraformaldehyde and 0.5% (w/v) glutaraldehyde in0.1 M phosphate buVer (PB), pH 7.4 for 2 min at 37°C and2 h at RT. The Wxed samples were mixed with 10% gelatinein 0.1 M PB at 37°C and incubated overnight in cold 2.3 Msucrose in 0.1 M PB at 4°C. Finally, the probes were frozenin Xuid nitrogen.

Ultrathin sections (65-70 nm) were cut on an Ultracut Smicrotome (Leica) with a cryoattachment (Leica EM-FCS)at ¡110°C and collected on formvar-coated nickel grids.They were then incubated in 0.1 M phosphate buVeredsaline (PBS), pH 7.4, containing 0.1% glycin for 10 minand washed in PBS for 10 min at RT. Before overnightincubation at 4°C with the primary antibody (anti-P2X7R,1:200, rabbit, Sigma), cells were incubated 2 £ 15 minwith PBG (0.1 M PBS with cold water Wsh skin gelatineand BSA-C, Aurion, Wageningen, Netherlands). Sectionswere then washed 5 £ 5 min in PBG and incubated withgoat anti-rabbit IgG, 12 nm gold (dilution 1:30) (dianova,Hamburg, Germany). Sections were then washed 6 £ 5 minin PBG, with a further 6 £ 5 min-washes in PBS, before

undergoing a 5-min incubation period with 2.5% (w/v) glu-taraldehyde in PBS at RT. Following brief washes (5 min)in distilled aqua dest., sections were stained by incubationwith 2% methyl cellulose-3% uranyl acetate (1:9) for10 min.

Statistics

All data were shown as means § SD of n observations. Sta-tistical comparison was performed as two-tailed, unpairedStudent’s t test (GraphPad Prism 5.03; GraphPad, SanDiego, CA). DiVerences were assumed to be signiWcantwith a probability value of less than 0.05.

Results

Immunohistochemical localization of Cav-1 and P2X7R in normal mouse hearts

To examine the immunohistochemical distribution ofCav-1 and P2X7R in mouse hearts, we prepared sagittalsections, which allow the direct examination of atria andventricles. As expected and already published (Volonte et al.2008), Cav-1 could be detected in the ventricle in endothe-lial cells, in smooth muscle cells of larger blood vessels(not shown) and in cardiomyocytes of the conducting sys-tem (not shown). In atrial tissue, additional immunostainingof cardiomyocytes could be observed (not shown). P2X7Rdistribution was similar in the normal mouse heart, exceptthat smooth muscle cells were devoid of immunoreactivity.Positive immunoreactivities of P2X7R using antibodiesagainst the extracellular domain of the receptor were seenin the atrial cardiomyocytes and in the endothelium of theentire heart (Fig. 1). Blocking of the antisera with the con-trol peptide abolished this immunoreactivity (not shown).Cryoimmunoelectron microscopy conWrmed the cell type-speciWc distribution of P2X7R (Fig. 2).

Cav-1¡/¡ animals show increased P2rx7 mRNA and P2X7R protein levels

To determine the expression of P2rx7, total RNA from cellhomogenates of atria and ventricles from wild-type andcav-1¡/¡ mice were prepared and subjected to RT-PCR.P2rx7 mRNA expression in cav-1¡/¡ mice demonstrated avery weak increase in the atrium and a strong decrease inthe ventricle compared to wild-type mice (Fig. 3).

These diVerences in the expression levels of P2X7Rproteins in the atria and ventricles from wild-type andcav-1¡/¡ mice were conWrmed by Western blot analysis.Biochemical analysis revealed the presence of two bandswith diVerent molecular weights, 80 and 56 kDa, respectively.

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34 Histochem Cell Biol (2010) 134:31–38

Both bands were removed in blocking experiments with thecorresponding blocking peptide (not shown).

The 80-kDa form of P2X7R protein, the functional gly-cosylated form, showed a strong increase in the atrium ofcav-1¡/¡ mice, in comparison to wild-type mice (Fig. 4).The 56-kDa form, however, decreased in lysates of atriaand ventricles. Accordingly, immunohistochemical evalua-tion revealed a slight increase of P2X7R immunoreactivityin atrial cardiomyocytes of cav-1¡/¡ mice but a slightdecrease in their endothelial cells (Fig. 1). Since the 80-kDaform of P2X7R does not show any biochemical diVerencesbetween the ventricles of wt and cav-1¡/¡ mice (Fig. 4),and the cardiomyocytes of the ventricles are lackingP2X7R (Fig. 1), the reduced immunoreactivity of P2X7R inthe endothelial cells of the ventricle can be addressed to the56-kDa form.

P2rx7¡/¡ animals show increased protein levels for cav-1

To determine the amount of Cav-1 proteins, cell homoge-nates of atria and ventricles from wild-type and P2rx7¡/¡

mice were prepared and subjected to Western blot analysis(Fig. 5). The Cav-1 protein content in the atrium of P2rx7¡/¡

mice was increased in comparison to wild-type mice. TheCav-1 protein content in the ventricle remained unchanged.However, immunohistochemical evaluation of heart sam-ples revealed no detectable diVerences in the caveolin-1immunoreactivity (not shown). Since unWxed samples werenot available for immunohistochemistry in this study, wehave to consider that immunohistochemical quantiWcationis diYcult after Wxation of tissues. To analyse other mor-phological abnormalities, we stained the cardiomyocytesand the endothelial cells with the corresponding marker

Fig. 1 ParaYn sections of mouse heart. Immunohisto-chemical demonstration of the P2X7R distribution in cardiac tissue of wild-type (upper panel) and cav-1¡/¡ (lower panel) mice. P2X7R is present in endo-thelial cells both of ventricle and atrium as well as in atrial cardio-myocytes of normal mice. Note the reduction of immunoreactiv-ity of P2X7R in endothelial cells and the increase in immunoreac-tivity of P2X7R in the atrial cardiomyocytes of cav-1¡/¡ mice. Bar 80 �m

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Histochem Cell Biol (2010) 134:31–38 35

proteins Cav-3 and podocalyxin, respectively (Fig. 6).There were no detectable abnormalities in either wild-typeor P2rx7¡/¡ mice.

Discussion

The exact cell-speciWc distribution of all members of theP2X receptor family in cardiac tissues is not yet known. Awidespread expression pattern of P2 receptors at mRNAlevel in diVerent regions of rat heart, however, have beenprovided by (Musa et al. 2009). The activation of P2 puri-nergic receptors causes an increased cardiac contractility inisolated mouse and rat hearts (Mei and Liang 2001). Fur-thermore, functionally active P2X1 receptors are involvedin the realization of the positive inotropic eVect of atria andventricles (Zverev et al. 2008).

The present study revealed a detectable increase in theexpression of P2X7R in samples of cardiac tissues fromcav-1¡/¡ mice. More importantly, the data demonstrate theclear existence of the purinergic receptor subtype P2X7R,in the atrial tissue homogenates of wild-type and cav-1¡/¡

mice. Their physiological role in this compartment has yetto be determined. There is only evidence that extracellularATP derived from cardiac ischaemia and hypoxia activatespurinergic receptors in the ventricular cardiomyocytes andinduces arrhythmic activity by inXuencing the Ca2+ homeo-stasis and by activation of depolarizing membrane currents(Gurung et al. 2009). The P2X4R has been found in cardio-myocytes by immunoblotting and immunohistochemistry(Bo et al. 2003; Hu et al. 2001). Electrophysiological inves-tigations conWrmed the importance of this receptor for theregulation of cardiac function by mediating a signiWcantvoltage-dependent current in cardiomyocytes (Shen et al.2006). In addition, P2X4R exerts the highest activity in thevascular endothelium (Erlinge and Burnstock 2008).

In our study, we provide clear evidence for the presenceof P2X7R in both endothelial cells and atrial cardiomyo-cytes in the mouse heart. P2X7R is known to be highlyexpressed in macrophages and other immune cells, inselected epithelial cells, glial and neuronal cells (Ferrariet al. 2006; Sperlagh et al. 2006). The functional relevanceof this receptor for the heart has been discussed in terms ofan involvement of macrophages, mast cells and otherinXammatory cells following cardiac injury (Ralevic andBurnstock 2003). P2X7Rs are ATP-gated ion channels thattrigger caspase-1 activation and subsequent IL-1� cleavage(Hewinson et al. 2008). Activation of P2X7Rs has severaleVects on immune cells, such as triggering the release ofpro-inXammatory cytokines, apoptosis and mycobacterialkilling (Surprenant and North 2009). The speciWc role forP2X7R in cardiomyocytes still has to be elucidated,although a recent study described the involvement of thisreceptor in the release of cardioprotective substances dur-ing ischemic pre- and post-conditioning (Vessey et al.2010). Importantly, cav-1¡/¡ as well as P2rx7¡/¡ miceshowed increased expression of either P2X7R or Cav-1 inatrial tissue homogenates. Western blot analysis revealedthe presence of two diVerent proteins with a molecularweight of 80 and 56 kDa in the cardiac tissue homogenates,although the change in receptor expression could be relatedto the classical P2X7R in the mouse with a molecularweight of 80 kDa. The second 56-kDa P2X7R-related pro-tein, however, was reduced in the microvascular endothe-lium of both the atrium and the ventricle. Preabsorption ofP2X7 receptor antibody with homologous peptide corre-sponding to amino acids 576–595 completely abolishedboth protein bands. The sequence of the 56-kDa protein hasyet to be characterized, although it may be identiWed as asplice variant of P2X7R. The recent report of 2 splice vari-

Fig. 2 Ultrathin cryosections of mouse heart. Immunogold labellingof a (ventricle) and b (atrial) tissue for P2X7R. Insets show lowermagniWcation. Note the immunostaining of endothelial (E) cells in aand b and the additional staining of the atrial cardiomyocyte (CM) inb. Bars in a 60 nm; in b 100 nm

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36 Histochem Cell Biol (2010) 134:31–38

ants in mouse P2X7 receptors (Cheewatrakoolpong et al.2005) cannot account for the observed 56-kDa protein inmouse heart: (1) the splice variant with deletions of the

carboxyl terminus, as the antibodies in use recognize theepitope in the C terminus, and (2) the other reported splicevariant lacking the transmembrane domain I has a higher

Fig. 3 For determination of the expression of P2X7R and GAP-DH, total RNA from cell homogenates of atrium and ven-tricle from wild-type and cav-1¡/¡ mice was prepared and subjected to RT-PCR. RT-PCR products were separated by aga-rose gel electrophoresis. A representative PCR (n = 3) is depicted. Data are presented as mean § SD

WT cav-1 -/- WT cav-1 -/-

atrium

GAPDH354 bp

P2rx7392 bp

WT cav-1 -/- WT cav-1 -/-

ventricle

GAPDH354 bp

P2rx7392 bp

WT

cav-

1 -/-

0

50

100

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250

atrium: P2rx7 expression

ns

n = 3

P2r

x7/G

APD

H-r

atio

[% o

f w

ildty

pe]

ventricle: P2rx7 expression

WT

cav-

1 -/ -

0

50

100

150

200

250

ns n = 3

P2r

x7/G

APD

H-r

atio

[% o

f w

ildty

pe]

Fig. 4 Analysis of P2X7R protein content in cell homogenates of atri-um and ventricle from wild-type and cav-1¡/¡ mice. 50 �g of proteinfrom each sample was loaded on the gel. Western blot analysis wasperformed using rabbit anti-P2X7 antibody (dilution 1:500) and

anti-�-tubulin antibody (1:1,000). �-tubulin served as a loadingcontrol. Representative data from four separate experiments areshown. Data are presented as mean § SD

P2X7R (56 kDa)

Cav-1

γ-Tubulin

WT cav-1 -/- WT cav-1 -/-

P2X7 R (80 kDa)

WT cav-1 -/- WT cav-1 -/-

Cav-1

γ-Tubulin

atrium ventricleatrium ventricle

WT

cav-

1 -/-

0

50

100

150

200

250atrium: P2X7R (80 kDa) protein content

*

p < 0,01, n = 4

P2X

7R

/γT

ub-r

atio

[% o

f w

ildty

pe]

WT

cav-

1 -/-

0

50

100

150

200

250atrium: P2X7R (56 kDa) protein content

*p < 0,001, n = 3

P2X

7R

/γT

ub-r

atio

[% o

f w

ildty

pe]

WT

cav-

1 -/-

0

50

100

150

200

250ventricle: P2X7R (56 kDa) protein content

*p < 0,001, n = 4

P2X

7R

/γT

ub-r

atio

[% o

f w

ildty

pe]

WT

cav-1

-/-

0

50

100

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250ventricle: P2X7R (80 kDa) protein content

ns

n = 4

P2X

7R

/γT

ub-r

atio

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Histochem Cell Biol (2010) 134:31–38 37

molecular weight (70 kDa). More experiments will berequired to clarify whether the 56-kDa band represents atruncated, intracellular P2X7R protein, as described byBarth et al. (2007), or a degradation of product of P2X7Runder our experimental conditions (Vonend et al. 2004).

Although there has not been any report in the literatureregarding cardiac dysfunction in P2rx7¡/¡ mice, our ownimmunohistochemical investigations did not reveal anyquantitative changes in morphology, such as cardiac hyper-trophy or blood vessel density in the cardiac tissue ofP2rx7¡/¡ mice. Nevertheless, the strong increase in the pro-tein content of P2X7R in the atria of cav-1¡/¡ mice as wellas the increase of Cav-1 in P2rx7¡/¡ mice needs further

experimental attention. Any suggestions about a directinteraction between Cav-1 and P2X7R are speculative,being based on preliminary data showing the presence ofP2X7R together with Cav-1 in raft-like membranes isolatedeither from rat submandibular gland cells (Garcia-Marcoset al. 2006) or from mouse alveolar epithelial lung cells(Barth et al. 2008). A better understanding of the functionalrole of Cav-1 on ion channels in contractile cells is essen-tial, as their role in other channels have been shown to beimportant. The role of Cav-1 has been studied on the tran-sient receptor potential channels (TRPCs) (Sundivakkamet al. 2009) and the calcium- and voltage-activated potas-sium channels (maxi-K channels) (Brainard et al. 2009)where the disruption of the Cav-1 binding sites leads toaltered current proWle in the cell.

Taken together, our data provide clear evidence that theATP-gated ion channel P2X7R, like other diverse cardiacion channels and exchangers (Balijepalli and Kamp 2008)can be detected in cardiomyocytes and that the expressionof this receptor is in part inXuenced by the presence of Cav-1.The detection of P2X7R in cardiomyocytes of mouse atriaas well as the increase in the expression of P2X7R insamples of cardiac tissues from cav-1¡/¡ mice requires fur-ther experimental eVort at the single cell level. In addition,further research in the P2rx7¡/¡ mice may demonstratethe functional consequences of the presence of this receptorin cardiomyocytes.

Fig. 5 For determination of the amount of Cav-1, cell homogenates ofatrium and ventricle from wild-type and P2rx7¡/¡ mice (n = 2) wereprepared and subjected to western blot analysis. 50 �g of protein fromeach sample was loaded on the gel. Western blot analysis was per-formed using rabbit anti-Cav-1 antibody (dilution 1:500) and anti-�-tubulin antibody (1:1,000). �-tubulin served as a loading control

P2X7R (80 kDa)

Cav-1

γ-Tubulin

WT P2rx7 –/– –/–WT P2rx7

atrium ventricle

Fig. 6 ParaYn sections of wild-type and P2rx7¡/¡ mice. Immunohistochemical staining of Cav-3 and podocalyxin. Bar 6 �m

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Acknowledgments The DFG research centre and cluster of excel-lence for Regenerative Therapies Dresden (CRTD) provided Wnancialsupport for this publication.

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