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

Generation of Myosin Light Chain TG Mice

An experimental strategy was devised that called for 4 constructs to be used to generate ELC1v TG mice and 2 constructs for the RLC2v analyses (Figure 1⇓). Expression of a cDNA encoding the full-length ELC1v transgene in lines carrying copy numbers of <90 leads to complete replacement of the endogenous isoform with TG protein but no overt changes in heart function or structure.⁷ Line 66, which displays these characteristics,⁷ was used to generate a control cohort of mice to rule out any effects that might be due to TG overexpression of a contractile protein. We modified the wild-type cDNA construct by adding a VSV tag at the carboxyl terminus, which appears to be innocuous,⁸ but, as a precaution, the TG lines generated with this construct were not used for functional analyses because of the possible confounding effects of the epitope. These lines were used both for confirming that TG protein was incorporated normally and to generate a curve in which transcript levels could be standardized and used to estimate the degree of replacement at the protein level. This strategy does, however, preclude us from directly assessing the degree of TG protein substitution in animals used for many of the analyses, although strong inference remains. The third construct contained the Met→Val substitution at position 158 in the mouse sequence, which corresponds to the human ELC1v^Met149Val FHC mutation (Figure⇓ 1). A fourth construct containing the mutant protein with the VSV tag was used to generate an additional set of TG lines. The wild-type RLC2v construct has also been described, and TG expression leads to replacement of the endogenous isoforms (RLC2a→RLC2v) and RLC2v_endogenous→RLC2v_TG in the atria and ventricles, respectively. Ventricular function at the cardiomyocyte and fiber levels is unaffected.⁹

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Figure 1.

TG constructs. All DNAs were ligated into a cassette consisting of the α-MHC promoter and the human growth hormone polyadenylation signal (hGH). The following 3 constructs were made: clone 97 contained a single amino acid substitution; clone 38 contained the single amino acid change, as well as a VSV tag at the carboxyl terminus; and clone 21 consisted of normal ELC1v with the VSV tag at the carboxyl terminus. Line 66, in which TG-encoded, wild-type ELC1v is expressed at levels sufficient for replacement of the endogenous protein, has been described.⁷ The wild-type regulatory myosin light chain construct has also been described¹⁰ ; a Glu→Lys substitution at position 22 in the cognate protein was made (RLC2v mu), recapitulating the human gene mutation.⁴ MyHC indicates myosin heavy chain.

Expression of ELC1v^Met158Val

As the human genomic fragment used to generate TG mice contained the ELC1v^Met149Val mutation, our initial analyses focused on the lines generated with the analogous mouse cDNA (Figure 2A⇓). Overall protein stoichiometry of the myofibril was not affected by TG expression of a contractile protein, and thus, TG expression led to a concomitant decrease in the endogenous protein species such that normal light chain stoichiometry was maintained (Figure⇑ 2B). Different lines showed varying levels of expression that corresponded to the number of TG copies; copy number varied from 2 to 15 (data not shown). TG transcript integrity was confirmed by sequencing (data not shown).

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Figure 2.

A, Human and mouse homologies. A single base change (ATG→GTG) was made in the mouse cDNA, causing the methionine at position 158 (position 149 in the human sequence) to be substituted with a valine. The VSV tag, present in clones 21 and 38 (Figure 1), is underlined and in boldface. B, Analysis of myofibrillar protein composition of ventricular tissues in ELC1v mutant TG lines. At least 3 animals of mixed sex were used for each experiment after preliminary experiments 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 distinguished from endogenous ELC1v (1v) and was used to construct a standard curve, correlating mRNA expression (not shown) to the degree of protein substitution. wt indicates wild type; mu, mutant; and 1a, ELC1a.

The degree of replacement could be estimated directly in the atria (Figure 2B⇑). We focused on lines 95 and 6, which displayed TG expression levels of 3.8-fold and 4.2-fold relative to the endogenous transcript, respectively, and which in the past resulted in 50% to 75% replacement.¹⁰ Quantification of atrial replacement showed between 60% and 80% substitution of the ELC1a with ELC1v^Met158Val. Line 66 represents the wild-type ELC1v construct; this line shows >90% replacement in the atrial compartment. The corresponding tagged lines, lines 83 and 55, respectively, show similar levels of replacement. As can be observed in the atrial sample, the tag results in a slightly slower mobility under the electrophoresis conditions used (Figure 2B⇑). Four lines expressing varying amount of wild-type protein and an additional 4 lines expressing ELC1v^Met158Val tagged protein were used to generate a standard curve, as a linear relationship between the degree of atrial versus ventricular replacement was apparent. Replacement was more complete in the ventricles than in the atria, possibly as a result of the stronger affinity of ELC1v for its endogenous contractile apparatus. Ventricular replacement was estimated at ≈60% in line 95 and ≈80% in line 6. All histological analyses using the tagged proteins indicated that they were incorporated normally into the sarcomere and that the overall organization of the contractile apparatus was unperturbed by TG replacement. When tested at 8 weeks for activation of the fetal transcriptional program, which serves as a sensitive marker for cardiac dysfunction and/or hypertrophy,¹¹ none could be detected (data not shown).

ELC1v Mutant Mice Exhibit Myocyte Disarray and Interstitial Fibrosis

The histopathological findings for FHC reflect the genetic heterogeneity of the disease and can be quite diverse. Common findings are myocellular disarray, foci of disorganized cells, and interstitial fibrosis. Trichrome staining of the ELC1v mutant lines at 10 to 12 months showed varying degrees of histopathology (Figure 3⇓). Sections from the apex of line 95 showed mild fibrosis, whereas line 6 shows a more severe pathology. Line 6 also shows relatively more myocyte disarray, and interstitial spacing is obviously affected compared with nontransgenic (NTG) or line 66 mice, demonstrating that the degree of pathology was dependent on transgene dose.

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Figure 3.

Histology of TG mouse hearts. Between 3 and 6 animals of mixed sex were used for each experiment after preliminary experiments showed no sex differences. Top, Longitudinal sections of whole hearts stained with trichrome. Bottom, Higher-magnification views of trichrome-stained sections. Adult mice (10 to 12 months old) from lines 95 and 6, which express the mutated protein (ELC1v-mu), and line 66, which expresses wild-type TG ELC1v(ELC1v-wt), as well as a NTG mouse, are shown. A section from the apex of line 95 shows mild fibrosis, whereas line 6 shows more severe pathology. Line 6 shows significant myocyte disarray and increased interstitial spacing compared with the NTG control. Wild-type ELC1v (wt) is indistinguishable from the NTG adult mouse. Scale bars: top, 1 mm; bottom, 50 μm.

Transmission electron microscopy bore out the abnormal histopathology. In the NTG animals, the regular arrangement of sarcomeres was obvious, with both A and I bands easily distinguishable and Z bands aligned transversely. The intercalated discs show a characteristic steplike structure (Figure 4A⇓). In contrast, sections taken from the LV (Figure 4C⇓) or septum (Figure⇑ 4D) of multiple 7-month-old hearts from ELC1v^Met158Val (line 95) mice displayed obvious foci of degeneration, with the septum being most affected. Some cardiomyocytes showed a loss of sarcomeric organization in the area of the intercalated discs, and necrosis was apparent. Ischemic cardiomyocytes with contracted sarcomeres and swollen mitochondria were also frequently found. Intercellular spaces were widened and contained accumulations of collagen fibers in both male and female animals from lines 6 and 95 (Figures 4D⇓ through 4F).

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Figure 4.

Transmission electron microscopy. Between 2 and 4 animals of mixed sex were used for each experiment after preliminary experiments showed no sex differences. Multiple sections were cut and >50 fields were observed; shown are fields typical for each example. A, Longitudinal sections through the myocardia were prepared from LVs derived from 6-month-old NTG mice. ID indicates intercalated disks. B, Sections derived from a line 66 animal expressing wild-type ELC1v. C and D, Section from the LV (C) and septum (D) of a 7-month-old heart derived from an ELC1v^Met158Val animal (line 95). Some cardiomyocytes show loss of sarcomeric organization in the area of the intercalated disc (C). In the septum, ischemic cardiomyocytes with contracted sarcomeres and swollen mitochondria are frequent. Intercellular spaces are widened and contain collagen fibers (arrows). E and F, Section from the LV (E) and septum (F) of a 7-month-old (line 6) heart shows numerous ultrastructural abnormalities and cell vacuolization (F, asterisk). The interstitium (E) contains numerous collagen fibers (arrows).

ELC1v^Met158Val Mice Exhibit Decreased Ventricular Mass

The histopathology of mice carrying the mutated transgene was consistent with the clinical cellular pathology observed in a majority of FHC cases studied. However, as noted above, in young adult animals we were unable to detect activation of the fetal transcriptional programs characteristic of a hypertrophic response. In light of those data, the heart weight/body weight ratios of the TG cohorts were examined (Table⇓). Both lines 95 and 6, which expressed the mutant protein, displayed reduced rather than increased values at 7 and 25 weeks when compared with either the NTG or ELC1v wild type–expressing cohorts. The decreases in heart mass were restricted to the LV (9%, line 95; 15%, line 6). A similar phenomenon was observed previously, when a mutated troponin T protein was expressed in mice. Decreased ventricular mass was due to smaller myocyte size,¹² consistent with the hypothesis that the primary deficit in FHC is myocellular in nature. Cell size was determined using isolated cardiomyocytes from line 6 and compared with those derived from age- and sex-matched wild-type animals. ELC1v^Met158Val cardiomyocytes showed a decrease of 12% in cell volume as compared with the NTG cells (Figure 1⇑ online, available at http://www.circresaha.org), a result consistent with the tissue weight data. Even in senescent animals (1.5 years) derived from either line 95 or 6 ELC1v^Met158Val mice, no hypertrophy at any level was observed, nor were differences in chamber volume, as determined by echocardiography, observed throughout the animals’ lifetimes (data not shown).

ELC1v^Met158Val Mutation Leads to Functional Deficits

The structural and ultrastructural deficits described above indicated that either a primary or secondary pathology was occurring. To determine whether the mutant protein substitution had functional consequences before any detectable pathology presented, skinned fibers were derived from the papillary muscles of young adults (6 to 7 weeks) and subjected to both mechanical and enzymatic analysis (Figure 5⇓). At this age, TG protein levels are at steady state, but no overt pathology is detected (data not shown). Thus, any changes detected in the fiber kinetics should be due to the primary isoform change and should not merely reflect secondary compensatory processes such as fibrosis. The ELC1v^Met158Val fibers exhibited leftward shifts in both ATPase activity (Figure 5A⇓) and the pCa-force curve (Figure 5B⇓). Significant decreases in the power-force relationships were also noted (Figure 5C⇓) and, although maximum shortening velocity and maximum force were unchanged (Figure 6⇓), maximum power was decreased as a result of changes in the shape of the F:V curve (Figures 5C⇓ and 5D⇓). The amplitudes of the shifts in myofibrillar Mg²⁺ATPase activity, the pCa-force relationship, and the decrease in maximum power from line 6 hearts were all significantly greater than those observed for line 95. Thus, the functional abnormalities of the fibers from line 6 are consistent with the more severe pathology (Figures 3⇑ and 4) that eventually develops.

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Figure 5.

Enzyme and kinetic analyses. A through D, □, NTG; ○, line 66; ▴, line 95; and ▪, line 6. A, Myofibrillar Mg²⁺ATPase activity. Myofibrillar proteins were extracted from the LV free wall, and the Mg²⁺ATPase activity was determined by standard colorimetric procedures. Both lines 95 and 6 exhibit a leftward shift in the enzymatic activity. B, pCa-force relationship. A leftward shift in the pCa-force relationship was observed for both lines 95 and 6. C, Force-power relationship. D, Maximum power. Skinned fibers were isolated from the LVs of 7-week-old NTG, line 66, line 95, and line 6 mice. Maximum force values of NTG, ELC1v wild-type, and ELC1v^Met158Val (lines 95 and 6) were 8.2±0.3, 8.0±0.3, 7.6±0.2, and 7.0±02 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). n=5 for all groups. No sex differences were observed.

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Figure 6.

Velocity measurements. Material derived from 3 to 5 mice 7 weeks old was used in each assay, the details of which have been published.^{9 17} A, Skinned fibers (papillary muscle) were used to determine the unloaded shortening velocity for NTG, line 66 (wild-type TG overexpressors), line 95, and line 6 mice. B, Maximum shortening velocity. C, In vitro motility assay¹⁸ was used to determine velocity of actin translocation in NTG and 8-week-old line 6 mice (n=5). Mixed sexes were used for each experiment after preliminary experiments showed no sex differences. Values are mean±SEM.

The initial studies using human-derived material showed that the ELC1v^Met149Val mutation led to an increase in the velocity of actin translocation, as measured by the in vitro motility assay in human.⁴ Furthermore, alterations in crossbridge cycling of ventricular skinned fibers derived from TG mice, which expressed the human mutation, were also detected.⁵ To determine whether the point mutation led directly to changes in crossbridge cycling rates, unloaded and maximum shortening velocities were measured.⁹ No significant differences could be detected in either the unloaded shortening velocity (Figure 6A⇑) or the maximum shortening velocity (Figure 6B⇑) of either ELC1v^Met158Val line, as compared with the NTG cohort. Finally, the in vitro motility assay was used to determine the speed at which ELC1v^Met158Val myosin derived from line 6 was able to translocate actin; consistent with the fiber data, no significant differences were observed (Figure⇑ 6C).

Are the functional changes in the fibers manifested at the whole-organ level? On the basis of the leftward shifts obtained for the pCa²⁺-Mg²⁺ATPase and pCa²⁺-force relationships, we hypothesized that the hearts might show near normal or even hypercontractility, but relaxation would be affected. FHC patients commonly exhibit increased left ventricular ejection fractions, but this is usually attributed to either the hypertrophy itself or to isoform shifts in the contractile proteins.^{12 13} Ex vivo cardiac functional analyses using the working heart preparation were carried out on mature adults (3 to 4 months old) from NTG, ELC1v wild-type (line 66), and ELC1v^Met158Val (line 95 and line 6) cohorts (Table 1⇑ online, available at http://www.circresaha.org). Both relaxation and contractile function were slightly increased in line 66 animals, compared with NTG controls. Hearts from line 95 showed a 25% increase in contractility as measured by +dP/dt, but a 22% deficit in relaxation (–dP/dt). Although +dP/dt was unaffected in the experimental cohort derived from line 6 under conditions of moderate load, –dP/dt values were decreased by ≈50%. Thus, the ELC1v^Met158Val mutant hearts demonstrate a significant impairment in relaxation and some hypercontractility (in line 95) as compared with NTG or line 66 (ELC1v wild-type) mice. This divergence between diastolic and systolic function in the different lines probably reflects secondary epiphenomena occurring during development of the pathogenesis that was observed via light and electron microscopy.

RLC Glu22Lys Mutation

The lack of an overt hypertrophic response in the ELC1v^Met158Val mice was puzzling, and we wished to confirm these results with an independent mutation. Therefore, another myosin light chain mutation, the Glu→Lys substitution found at position 22 in the RLC,⁴ was also modeled in TG mice. The construct (Figure 1⇑) was similarly linked to the α-MHC promoter and used to generate 4 TG lines that showed varying degrees of protein replacement in a copy number–dependent fashion (data not shown). Initially, we focused on lines 19, 79, and 32, which showed 27, 55, and >95% replacement of the corresponding endogenous isoform with the TG species, respectively (Figure 7⇓). However, when preliminary analyses showed no detectable phenotype, only line 32, in which essentially all of the RLC2v consists of the RLC^Glu22Lys species as based on the degree of replacement observed in the atria, was used. No hypertrophy could be detected in mature adult animals either when chamber weights were determined (Table 2 online, available at http://www.circresaha.org) or at the cellular level (Figure 7B⇓). The histology was also unremarkable, and at neither the gross cellular or molecular levels could any response be detected in either sex (data not shown). Thus, even when replacement with the TG species is much greater than 50%, a hypertrophic response is not elicited.

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Figure 7.

RLC2v^Glu22Lys mice. A, Analysis of myofibrillar protein composition of ventricular tissues in RLC2v mutant TG lines. Between 3 and 6 adult animals of mixed sex were used for each experiment after preliminary experiments showed no sex differences. Lines 19, 79, and 32 (RLC2v^Glu22Lys; 2v) and the line overexpressing TG wild-type RLC2v¹⁰ show the expected sizes; no truncated proteins or increases in the total amount of RLC2 protein were detected. Atrial samples were used to estimate the amount of protein substitution in the ventricles; in line 32, essentially all of the RLC2 species are made up of the transgenically encoded protein. 2a indicates RLC2a. B, Hematoxylin and eosin–stained left ventricular sections were prepared from NTG, RLC2v wild-type (wt) overexpressors, and line 32 RLC2v^Glu22Lys mice (mu). No hypertrophy is apparent, consistent with the heart/body weights obtained (Table 3 online, available at http://www.circresaha.org).