In Vivo Analysis of an Essential Myosin Light ChainMutation Linked to Familial Hypertrophic Cardiomyopathy

Atsushi Sanbe, David Nelson, James Gulick, Elizabeth Setser, Hanna Osinska, Xuejun Wang,Timothy E. Hewett, Raisa Klevitsky, Eric Hayes, David M. Warshaw, Jeffrey Robbins

Abstract—Mutations in cardiac motor protein genes are associated with familial hypertrophic cardiomyopathy. Mutationsin both the regulatory (Glu22Lys) and essential light chains (Met149Val) result in an unusual pattern of hypertrophy,leading to obstruction of the midventricular cavity. When a human genomic fragment containing the Met149Valessential myosin light chain was used to generate transgenic mice, the phenotype was recapitulated. To unambiguouslyestablish a causal relationship for the regulatory and essential light chain mutations in hypertrophic cardiomyopathy, wegenerated mice that expressed either the wild-type or mutated forms, using cDNA clones encompassing only the codingregions of the gene loci. Expression of the proteins did not lead to a hypertrophic response, even in senescent animals.Changes did occur at the myofilament and cellular levels, with the myofibrils showing increased Ca21 sensitivity andsignificant deficits in relaxation in a transgene dose–dependent manner. Clearly, mice do not always recapitulateimportant aspects of human hypertrophy. However, because of the discordance of these data with data obtained intransgenic mice containing the human genomic fragment, we believe that the concept that these point mutations bythemselves can cause hypertrophic cardiomyopathy should be revisited.(Circ Res. 2000;87:296-302.)

Key Words: myosinn heartn musclen mousen hypertrophy

Familial hypertrophic cardiomyopathy (FHC) is a domi-nant disease of the cardiac sarcomere.1 Multiple muta-

tions in the different thick- and thin-filament proteins havebeen linked to the disease, including theb-myosin heavychain (b-MHC), the essential and regulatory myosin lightchains (ELC and RLC), myosin binding protein C,a-tropomyosin, cardiac troponin T, cardiac troponin I, actin,and titin.2 The disease is clinically heterogeneous, with somemutant alleles resulting in a poor clinical prognosis and othersbeing relatively benign.3 That so many of the FHC mutationsare linked to genes encoding sarcomeric proteins suggeststhat the pathogenesis of the disease likely involves a commonmechanism of contractile dysfunction.

Mutations in the human ELC (Met149Val) and RLC(Glu22Lys, Pro94Arg), which result in midventricular cavityobstruction due to papillary muscle hypertrophy, have beendescribed,4 and the Met149Val mutation has been modeled inmice by inserting entire human mutant or wild-type geneticloci into transgenic (TG) mice.5 The authors hypothesizedthat the mutant light chain hypertrophic cardiomyopathyresulted from abnormal movement of the neck region andalterations of the myosin in a stretch-activation response.5

However, 2 novel missense mutations, Phe18Leu in exon 2and Arg58Gln in exon 4 of RLC, were later found in the

French population. These mutations presented a classic phe-notype of hypertrophic cardiomyopathy without mid leftventricular obstruction and papillary muscle hypertrophy.6

Considering the disparity in the 2 groups of data obtainedfrom the human populations concerning the occurrence ofhypertrophy resulting in a midventricular obstruction, wedecided to pursue the question of actual causality of the pointmutations. That is, are the point mutations located in themyosin light chains sufficient for the hypertrophy? Ratherthan using the genomic sequences of a heterologous species,the murine cDNA, free of all intronic DNA, was used toproduce the TG mouse lines to create mouse models in whichpartial replacements of cardiac ELC and RLC with proteinscarrying the Met149Val and Glu22Lys mutations, respec-tively, could be studied. Both epitope-tagged and untaggedconstructs were used to establish the degree of replacement,allowing us to determine dose-dependent effects of themutant transgenes. Unlike the murine model carrying thehuman ELCMet149Val gene locus, mice with the mutated murineELC cDNA or RLCGlu22Lys failed to exhibit either overthypertrophy or midventricular cavity obstruction.

Materials and MethodsAn expanded Materials and Methods section is available online athttp://www.circresaha.org.

Received April 28, 2000; revision received June 7, 2000; accepted June 20, 2000.From the Department of Pediatrics, Division of Molecular Cardiovascular Biology (A.S., D.N., J.G., E.S., H.O., X.W., T.E.H., R.K., J.R.), The

Children’s Hospital Research Foundation, Cincinnati, Ohio, and Department of Molecular Physiology and Biophysics (E.H., D.M.W.), University ofVermont, Burlington, Vt.

Correspondence to J. Robbins, PhD, Division of Molecular Cardiovascular Biology, 3333 Burnet Ave, Cincinnati, OH 45229-3039. E-mailjeff.robbins@chmcc.org

© 2000 American Heart Association, Inc.

Circulation Researchis available at http://www.circresaha.org

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ResultsGeneration of Myosin Light Chain TG MiceAn experimental strategy was devised that called for 4constructs to be used to generate ELC1v TG mice and 2constructs for the RLC2v analyses (Figure 1). Expression ofa cDNA encoding the full-length ELC1v transgene in linescarrying copy numbers of,90 leads to complete replacementof the endogenous isoform with TG protein but no overtchanges in heart function or structure.7 Line 66, whichdisplays these characteristics,7 was used to generate a controlcohort of mice to rule out any effects that might be due to TGoverexpression of a contractile protein. We modified thewild-type cDNA construct by adding a VSV tag at thecarboxyl terminus, which appears to be innocuous,8 but, as aprecaution, the TG lines generated with this construct werenot used for functional analyses because of the possibleconfounding effects of the epitope. These lines were usedboth for confirming that TG protein was incorporated nor-mally and to generate a curve in which transcript levels couldbe standardized and used to estimate the degree of replace-ment at the protein level. This strategy does, however,preclude us from directly assessing the degree of TG proteinsubstitution in animals used for many of the analyses,although strong inference remains. The third construct con-tained the Met3Val substitution at position 158 in the mousesequence, which corresponds to the human ELC1vMet149Val

FHC mutation (Figure 1). A fourth construct containing themutant protein with the VSV tag was used to generate anadditional set of TG lines. The wild-type RLC2v constructhas also been described, and TG expression leads to replace-ment of the endogenous isoforms (RLC2a3RLC2v) andRLC2vendogenous3RLC2vTG in the atria and ventricles, respec-tively. Ventricular function at the cardiomyocyte and fiberlevels is unaffected.9

Expression of ELC1vMet158Val

As the human genomic fragment used to generate TG micecontained the ELC1vMet149Val mutation, our initial analysesfocused on the lines generated with the analogous mousecDNA (Figure 2A). Overall protein stoichiometry of themyofibril was not affected by TG expression of a contractileprotein, and thus, TG expression led to a concomitantdecrease in the endogenous protein species such that normallight chain stoichiometry was maintained (Figure 2B). Dif-ferent lines showed varying levels of expression that corre-sponded to the number of TG copies; copy number variedfrom 2 to 15 (data not shown). TG transcript integrity wasconfirmed by sequencing (data not shown).

The degree of replacement could be estimated directly inthe atria (Figure 2B). We focused on lines 95 and 6, whichdisplayed TG expression levels of 3.8-fold and 4.2-foldrelative to the endogenous transcript, respectively, andwhich in the past resulted in 50% to 75% replacement.10

Quantification of atrial replacement showed between 60%and 80% substitution of the ELC1a with ELC1vMet158Val.Line 66 represents the wild-type ELC1v construct; this lineshows.90% replacement in the atrial compartment. Thecorresponding tagged lines, lines 83 and 55, respectively,show similar levels of replacement. As can be observed inthe atrial sample, the tag results in a slightly slowermobility under the electrophoresis conditions used (Figure

Figure 1. TG constructs. All DNAs were ligated into a cassetteconsisting of the a-MHC promoter and the human growth hor-mone polyadenylation signal (hGH). The following 3 constructswere made: clone 97 contained a single amino acid substitution;clone 38 contained the single amino acid change, as well as aVSV tag at the carboxyl terminus; and clone 21 consisted ofnormal ELC1v with the VSV tag at the carboxyl terminus. Line66, in which TG-encoded, wild-type ELC1v is expressed at lev-els sufficient for replacement of the endogenous protein, hasbeen described.7 The wild-type regulatory myosin light chainconstruct has also been described10; a Glu3Lys substitution atposition 22 in the cognate protein was made (RLC2v mu), reca-pitulating the human gene mutation.4 MyHC indicates myosinheavy chain.

Figure 2. A, Human and mouse homologies. A single basechange (ATG3GTG) was made in the mouse cDNA, causingthe methionine at position 158 (position 149 in the humansequence) to be substituted with a valine. The VSV tag, pres-ent in clones 21 and 38 (Figure 1), is underlined and in bold-face. B, Analysis of myofibrillar protein composition of ven-tricular tissues in ELC1v mutant TG lines. At least 3 animalsof mixed sex were used for each experiment after preliminaryexperiments showed no sex differences. In lines 55 and 83,the highest-expressing lines derived from clones 21 and 38,respectively, the epitope-tagged protein (1v Tag) can be dis-tinguished from endogenous ELC1v (1v) and was used toconstruct a standard curve, correlating mRNA expression (notshown) to the degree of protein substitution. wt indicates wildtype; mu, mutant; and 1a, ELC1a.

Sanbe et al Cardiac Myosin Mutations 297

2B). Four lines expressing varying amount of wild-typeprotein and an additional 4 lines expressing ELC1vMet158Val

tagged protein were used to generate a standard curve, asa linear relationship between the degree of atrial versusventricular replacement was apparent. Replacement wasmore complete in the ventricles than in the atria, possiblyas a result of the stronger affinity of ELC1v for itsendogenous contractile apparatus. Ventricular replacementwas estimated at'60% in line 95 and'80% in line 6. Allhistological analyses using the tagged proteins indicatedthat they were incorporated normally into the sarcomereand that the overall organization of the contractile appa-ratus was unperturbed by TG replacement. When tested at8 weeks for activation of the fetal transcriptional program,which serves as a sensitive marker for cardiac dysfunctionand/or hypertrophy,11 none could be detected (data notshown).

ELC1v Mutant Mice Exhibit Myocyte Disarrayand Interstitial FibrosisThe histopathological findings for FHC reflect the geneticheterogeneity of the disease and can be quite diverse. Com-mon findings are myocellular disarray, foci of disorganizedcells, and interstitial fibrosis. Trichrome staining of theELC1v mutant lines at 10 to 12 months showed varyingdegrees of histopathology (Figure 3). Sections from the apexof line 95 showed mild fibrosis, whereas line 6 shows a moresevere pathology. Line 6 also shows relatively more myocytedisarray, and interstitial spacing is obviously affected com-pared with nontransgenic (NTG) or line 66 mice, demonstrat-ing that the degree of pathology was dependent on transgenedose.

Transmission electron microscopy bore out the abnormalhistopathology. In the NTG animals, the regular arrangementof sarcomeres was obvious, with both A and I bands easilydistinguishable and Z bands aligned transversely. The inter-calated discs show a characteristic steplike structure (Figure4A). In contrast, sections taken from the LV (Figure 4C) orseptum (Figure 4D) of multiple 7-month-old hearts fromELC1vMet158Val (line 95) mice displayed obvious foci ofdegeneration, with the septum being most affected. Somecardiomyocytes showed a loss of sarcomeric organization inthe area of the intercalated discs, and necrosis was apparent.Ischemic cardiomyocytes with contracted sarcomeres andswollen mitochondria were also frequently found. Intercellu-lar spaces were widened and contained accumulations ofcollagen fibers in both male and female animals from lines 6and 95 (Figures 4D through 4F).

Figure 3. Histology of TG mouse hearts. Between 3 and 6 ani-mals of mixed sex were used for each experiment after prelimi-nary experiments showed no sex differences. Top, Longitudinalsections of whole hearts stained with trichrome. Bottom,Higher-magnification views of trichrome-stained sections. Adultmice (10 to 12 months old) from lines 95 and 6, which expressthe mutated protein (ELC1v-mu), and line 66, which expresseswild-type TG ELC1v(ELC1v-wt), as well as a NTG mouse, areshown. A section from the apex of line 95 shows mild fibrosis,whereas line 6 shows more severe pathology. Line 6 shows sig-nificant myocyte disarray and increased interstitial spacing com-pared with the NTG control. Wild-type ELC1v (wt) is indistin-guishable from the NTG adult mouse. Scale bars: top, 1 mm;bottom, 50 mm.

Figure 4. Transmission electron microscopy. Between 2 and 4animals of mixed sex were used for each experiment after pre-liminary experiments showed no sex differences. Multiple sec-tions were cut and .50 fields were observed; shown are fieldstypical for each example. A, Longitudinal sections through themyocardia were prepared from LVs derived from 6-month-oldNTG mice. ID indicates intercalated disks. B, Sections derivedfrom a line 66 animal expressing wild-type ELC1v. C and D,Section from the LV (C) and septum (D) of a 7-month-old heartderived from an ELC1vMet158Val animal (line 95). Some cardiomyo-cytes show loss of sarcomeric organization in the area of theintercalated disc (C). In the septum, ischemic cardiomyocyteswith contracted sarcomeres and swollen mitochondria are fre-quent. Intercellular spaces are widened and contain collagenfibers (arrows). E and F, Section from the LV (E) and septum (F)of a 7-month-old (line 6) heart shows numerous ultrastructuralabnormalities and cell vacuolization (F, asterisk). The interstitium(E) contains numerous collagen fibers (arrows).

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ELC1vMet158Val Mice Exhibit DecreasedVentricular MassThe histopathology of mice carrying the mutated transgenewas consistent with the clinical cellular pathology observedin a majority of FHC cases studied. However, as noted above,in young adult animals we were unable to detect activation ofthe fetal transcriptional programs characteristic of a hyper-trophic response. In light of those data, the heart weight/bodyweight ratios of the TG cohorts were examined (Table). Bothlines 95 and 6, which expressed the mutant protein, displayedreduced rather than increased values at 7 and 25 weeks whencompared with either the NTG or ELC1v wild type–express-ing cohorts. The decreases in heart mass were restricted to theLV (9%, line 95; 15%, line 6). A similar phenomenon wasobserved previously, when a mutated troponin T protein wasexpressed in mice. Decreased ventricular mass was due tosmaller myocyte size,12 consistent with the hypothesis that theprimary deficit in FHC is myocellular in nature. Cell size wasdetermined using isolated cardiomyocytes from line 6 andcompared with those derived from age- and sex-matchedwild-type animals. ELC1vMet158Val cardiomyocytes showed adecrease of 12% in cell volume as compared with the NTGcells (Figure 1 online, available at http://www.circresa-ha.org), a result consistent with the tissue weight data. Evenin senescent animals (1.5 years) derived from either line 95 or6 ELC1vMet158Val mice, no hypertrophy at any level wasobserved, nor were differences in chamber volume, as deter-mined by echocardiography, observed throughout the ani-mals’ lifetimes (data not shown).

ELC1vMet158Val Mutation Leads toFunctional DeficitsThe structural and ultrastructural deficits described aboveindicated that either a primary or secondary pathology wasoccurring. To determine whether the mutant protein substi-tution had functional consequences before any detectablepathology presented, skinned fibers were derived from thepapillary muscles of young adults (6 to 7 weeks) andsubjected to both mechanical and enzymatic analysis (Figure5). At this age, TG protein levels are at steady state, but noovert pathology is detected (data not shown). Thus, anychanges detected in the fiber kinetics should be due to the

primary isoform change and should not merely reflect sec-ondary compensatory processes such as fibrosis. TheELC1vMet158Val fibers exhibited leftward shifts in both ATPaseactivity (Figure 5A) and the pCa-force curve (Figure 5B).Significant decreases in the power-force relationships werealso noted (Figure 5C) and, although maximum shorteningvelocity and maximum force were unchanged (Figure 6),maximum power was decreased as a result of changes in theshape of the F:V curve (Figures 5C and 5D). The amplitudesof the shifts in myofibrillar Mg21ATPase activity, the pCa-

Normalized Heart Weights

Ventricular Weight/BodyWeight, mg/g

LV Weight/BodyWeight, mg/g

RV Weight/BodyWeight, mg/g

Atrial Weight/BodyWeight, mg/g

7 weeks

NTG (n56) 4.5460.05 3.6360.04 0.9160.03 0.4060.02

ELC1v TG (line 66), n55 4.4260.06 3.4060.06 1.0260.02 0.3560.02

ELC1vMet158Val (line 95), n55 4.1360.09*† 3.2460.07* 0.8960.04 0.4460.03†

ELC1vMet158Val (line 6), n55 4.0760.13*† 3.1060.09*† 0.9660.09 0.5660.02*†‡

25 weeks

NTG, n58 3.9760.06 3.1960.05 0.7760.02 0.3560.02

ELC1v TG (line 66), n55 3.9460.06 3.2560.04 0.7960.03 0.3860.02

ELC1vMet158Val (line 95), n55 3.6560.05*† 2.9460.04*† 0.7260.01 0.4860.02

ELC1vMet158Val (line 6), n55 3.5160.09*† 2.7160.10*†‡ 0.7960.03 0.7260.15*†‡

RV indicates right ventricle. Data are mean6SEM. *Versus NTG, †versus line 66, ‡versus line 95; P,0.05.

Figure 5. Enzyme and kinetic analyses. A through D, M, NTG;E, line 66; Œ, line 95; and f, line 6. A, Myofibrillar Mg21ATPaseactivity. Myofibrillar proteins were extracted from the LV freewall, and the Mg21ATPase activity was determined by standardcolorimetric procedures. Both lines 95 and 6 exhibit a leftwardshift in the enzymatic activity. B, pCa-force relationship. A left-ward shift in the pCa-force relationship was observed for bothlines 95 and 6. C, Force-power relationship. D, Maximumpower. Skinned fibers were isolated from the LVs of 7-week-oldNTG, line 66, line 95, and line 6 mice. Maximum force values ofNTG, ELC1v wild-type, and ELC1vMet158Val (lines 95 and 6) were8.260.3, 8.060.3, 7.660.2, and 7.0602 mN/mm2, respectively.P,0.05, compared with *NTG and #line 66 animals (A and D)and with *NTG, #line 66, and aline 95 animals (B). n55 for allgroups. No sex differences were observed.

Sanbe et al Cardiac Myosin Mutations 299

force relationship, and the decrease in maximum power fromline 6 hearts were all significantly greater than those observedfor line 95. Thus, the functional abnormalities of the fibersfrom line 6 are consistent with the more severe pathology(Figures 3 and 4) that eventually develops.

The initial studies using human-derived material showedthat the ELC1vMet149Val mutation led to an increase in thevelocity of actin translocation, as measured by the in vitromotility assay in human.4 Furthermore, alterations in cross-bridge cycling of ventricular skinned fibers derived from TGmice, which expressed the human mutation, were also detect-ed.5 To determine whether the point mutation led directly tochanges in crossbridge cycling rates, unloaded and maximumshortening velocities were measured.9 No significant differ-ences could be detected in either the unloaded shorteningvelocity (Figure 6A) or the maximum shortening velocity(Figure 6B) of either ELC1vMet158Val line, as compared with theNTG cohort. Finally, the in vitro motility assay was used todetermine the speed at which ELC1vMet158Val myosin derivedfrom line 6 was able to translocate actin; consistent with the fiberdata, no significant differences were observed (Figure 6C).

Are the functional changes in the fibers manifested at thewhole-organ level? On the basis of the leftward shifts

obtained for the pCa21-Mg21ATPase and pCa21-force rela-tionships, we hypothesized that the hearts might show nearnormal or even hypercontractility, but relaxation would beaffected. FHC patients commonly exhibit increased leftventricular ejection fractions, but this is usually attributed toeither the hypertrophy itself or to isoform shifts in thecontractile proteins.12,13 Ex vivo cardiac functional analysesusing the working heart preparation were carried out onmature adults (3 to 4 months old) from NTG, ELC1vwild-type (line 66), and ELC1vMet158Val (line 95 and line 6)cohorts (Table 1 online, available at http://www.circresa-ha.org). Both relaxation and contractile function were slightlyincreased in line 66 animals, compared with NTG controls.Hearts from line 95 showed a 25% increase in contractility asmeasured by1dP/dt, but a 22% deficit in relaxation (–dP/dt).Although 1dP/dt was unaffected in the experimental cohortderived from line 6 under conditions of moderate load, –dP/dtvalues were decreased by'50%. Thus, the ELC1vMet158Val

mutant hearts demonstrate a significant impairment in relax-ation and some hypercontractility (in line 95) as comparedwith NTG or line 66 (ELC1v wild-type) mice. This diver-gence between diastolic and systolic function in the differentlines probably reflects secondary epiphenomena occurringduring development of the pathogenesis that was observedvia light and electron microscopy.

RLC Glu22Lys MutationThe lack of an overt hypertrophic response in theELC1vMet158Val mice was puzzling, and we wished to confirmthese results with an independent mutation. Therefore, an-other myosin light chain mutation, the Glu3Lys substitutionfound at position 22 in the RLC,4 was also modeled in TGmice. The construct (Figure 1) was similarly linked to thea-MHC promoter and used to generate 4 TG lines thatshowed varying degrees of protein replacement in a copynumber–dependent fashion (data not shown). Initially, wefocused on lines 19, 79, and 32, which showed 27, 55, and.95% replacement of the corresponding endogenous isoformwith the TG species, respectively (Figure 7). However, whenpreliminary analyses showed no detectable phenotype, onlyline 32, in which essentially all of the RLC2v consists of theRLCGlu22Lys species as based on the degree of replacementobserved in the atria, was used. No hypertrophy could bedetected in mature adult animals either when chamberweights were determined (Table 2 online, available at http://www.circresaha.org) or at the cellular level (Figure 7B). Thehistology was also unremarkable, and at neither the grosscellular or molecular levels could any response be detected ineither sex (data not shown). Thus, even when replacementwith the TG species is much greater than 50%, a hypertrophicresponse is not elicited.

DiscussionThe function(s) of the cardiac myosin light chains is begin-ning to be elucidated. Both bind to a critical region of theheavy chain, and expression of a cardiac MHC lacking thelight chain binding site resulted in a hypertrophic cardiomy-opathy.14 With these data in mind, it is logical to suppose thatpoint mutations in either cardiac light chain could have

Figure 6. Velocity measurements. Material derived from 3 to 5mice 7 weeks old was used in each assay, the details of whichhave been published.9,17 A, Skinned fibers (papillary muscle)were used to determine the unloaded shortening velocity forNTG, line 66 (wild-type TG overexpressors), line 95, and line 6mice. B, Maximum shortening velocity. C, In vitro motilityassay18 was used to determine velocity of actin translocation inNTG and 8-week-old line 6 mice (n55). Mixed sexes were usedfor each experiment after preliminary experiments showed nosex differences. Values are mean6SEM.

300 Circulation Research August 18, 2000

detrimental effects that would result, eventually, in cardio-vascular disease.

Clearly the mice generated in the present study, in whichonly the single residue was altered, do not display an increasein chamber mass. The initial report describing theELC1vMet149Val mutation in the human population showed that,in contrast to other FHC mutations present in the force-producing proteins, this mutation led to hypervelocities ofactin translocation as measured using the in vitro motilityassay.4 Hypertrophy of the papillary muscles led to a leftventricular midcavity obstruction. When this mutation wasmodeled in TG mice, using the intact normal and mutanthuman gene loci, aspects of the phenotype were recapitulatedin the old (.1 year) adults expressing ELC1vMet149Val.5 Anexplanation for the different data resulting from placing thehuman locus in the mouse, versus expression of the mousesequence with the analogous mutation, is not readily appar-ent. Reconciliation of the data is particularly difficult, be-cause no data are available for the expression levels of thehuman transcript or protein in either the clinical population ormouse model. However, we modeled replacement levelsvarying from 25% to$95% and never detected papillarymuscle hypertrophy even when cohorts were carried out to 18months (data not shown), nor were hypervelocities observedin the motility assay (Figure 6C). One possible explanation

for the in vitro motility data discrepancies may lie in thestructural differences between the human and the mouseproteins. The MHC isoforms with which the light chainsnecessarily interact are also different; in the human theb-MHC is present, whereas in the adult mouse, onlya-MHCis expressed. The alanine- and proline-rich N-terminal do-main is significantly shorter in human ELC1v as comparedwith the mouse (a 9-amino acid deletion, Figure 2A), and thisdifferent sequence, when coupled with the Met149Val sub-stitution, might result in a phenotype unique to the humansequence. This region appears to be important in ELC andMHC interactions and/or interactions between ELC andactin.15 Although the normal human gene, when placed intothe mouse, did not yield a hypertrophic phenotype, the majordifferences in structure preclude an unambiguous assessmentof the phenotype, as differences were observed in the stretch-activated response of isolated fiber preparations. Mutations inother regions of the human locus or even aberrant splicingresulting in a protein containing additional mutations werenot ruled out.4,5 Other differences present in the 12-kb humangenomic fragment could also act synergistically with theELC1vMet149Val mutation. Those changes obviously would notbe present in the murine cDNAs used in our studies. Theeffects of ELC1vMet149Val might be within tolerable limits inthis particular mouse strain (FVB/N) as opposed to the micecarrying the human transgene (C57B/J6). Strain-specificvariation of phenotype does occur, and modifying factors canplay a major role in the hypertrophic response.16 The presentstudy does show, however, that in this genetic background,these mutations are not sufficient for induction ofhypertrophy.

The mutations are not completely benign. ELC1vMet158Val

fibers revealed a transgene dose–dependent decrease inpower output as measured by the force-velocity relationship.Isolated heart analyses were consistent with the fiber data, asrelaxation was impaired significantly in ELC1vMet158Val mice,whereas contractility was increased in line 95. The fiberstudies were performed at 7 weeks, minimizing the possibilitythat the functional defects observed reflected a developingsecondary pathology. It has been established that changes inthe light chain can have dramatic effects on the Ca21

sensitivity of a myofiber,11 and the most straightforwardinterpretation is that both the decrease in power output andthe increase in Ca21 sensitivity are directly due to thefunctional consequences of the altered ELC1v structure.

AcknowledgmentsThis work was supported by NIH Grants HL56370, HL41496,HL56620, HL52318, HL60546, and HL56620; by the MarionMerrell-Dow Foundation (to J.R.), HL59408 and HL51126 (toD.M.W.); and by the American Heart Association, Ohio ValleyAffiliate (to A.S.). We thank Lisa Martin for excellenttechnical assistance.

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Figure 7. RLC2vGlu22Lys mice. A, Analysis of myofibrillar proteincomposition of ventricular tissues in RLC2v mutant TG lines.Between 3 and 6 adult animals of mixed sex were used for eachexperiment after preliminary experiments showed no sex differ-ences. Lines 19, 79, and 32 (RLC2vGlu22Lys; 2v) and the line over-expressing TG wild-type RLC2v10 show the expected sizes; notruncated proteins or increases in the total amount of RLC2 pro-tein were detected. Atrial samples were used to estimate theamount of protein substitution in the ventricles; in line 32,essentially all of the RLC2 species are made up of the trans-genically encoded protein. 2a indicates RLC2a. B, Hematoxylinand eosin–stained left ventricular sections were prepared fromNTG, RLC2v wild-type (wt) overexpressors, and line 32RLC2vGlu22Lys mice (mu). No hypertrophy is apparent, consistentwith the heart/body weights obtained (Table 3 online, availableat http://www.circresaha.org).

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