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© 2000 American Heart Association, Inc.
Integrative Physiology |
In Vivo Analysis of an Essential Myosin Light Chain Mutation Linked to Familial Hypertrophic Cardiomyopathy
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 of Vermont, Burlington, Vt.
Correspondence to J. Robbins, PhD, Division of Molecular Cardiovascular Biology, 3333 Burnet Ave, Cincinnati, OH 45229-3039. E-mail jeff.robbins{at}chmcc.org
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Abstract—Mutations in cardiac motor protein genes are associated with familial hypertrophic cardiomyopathy. Mutations in 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 Met149Val essential myosin light chain was used to generate transgenic mice, the phenotype was recapitulated. To unambiguously establish a causal relationship for the regulatory and essential light chain mutations in hypertrophic cardiomyopathy, we generated mice that expressed either the wild-type or mutated forms, using cDNA clones encompassing only the coding regions 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 Ca2+ sensitivity and significant deficits in relaxation in a transgene dose–dependent manner. Clearly, mice do not always recapitulate important aspects of human hypertrophy. However, because of the discordance of these data with data obtained in transgenic mice containing the human genomic fragment, we believe that the concept that these point mutations by themselves can cause hypertrophic cardiomyopathy should be revisited.
Key Words: myosin • heart • muscle • mouse • hypertrophy
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Familial hypertrophic cardiomyopathy (FHC) is a dominant disease of the cardiac sarcomere.1 Multiple mutations in the different thick- and thin-filament proteins have been linked to the disease, including the ß-myosin heavy chain (ß-MHC), the essential and regulatory myosin light chains (ELC and RLC), myosin binding protein C,
-tropomyosin, cardiac troponin
T, cardiac troponin I, actin, and titin.2 The disease is
clinically heterogeneous, with some mutant alleles
resulting in a poor clinical prognosis and others being relatively
benign.3 That so many of the FHC mutations are linked to
genes encoding sarcomeric proteins suggests that the pathogenesis of
the disease likely involves a common mechanism of contractile
dysfunction. Mutations in the human ELC (Met149Val) and RLC (Glu22Lys, Pro94Arg), which result in midventricular cavity obstruction due to papillary muscle hypertrophy, have been described,4 and the Met149Val mutation has been modeled in mice by inserting entire human mutant or wild-type genetic loci into transgenic (TG) mice.5 The authors hypothesized that the mutant light chain hypertrophic cardiomyopathy resulted from abnormal movement of the neck region and alterations of the myosin in a stretch-activation response.5 However, 2 novel missense mutations, Phe18Leu in exon 2 and Arg58Gln in exon 4 of RLC, were later found in the French population. These mutations presented a classic phenotype of hypertrophic cardiomyopathy without mid left ventricular obstruction and papillary muscle hypertrophy.6
Considering the disparity in the 2 groups of data obtained from the human populations concerning the occurrence of hypertrophy resulting in a midventricular obstruction, we decided to pursue the question of actual causality of the point mutations. That is, are the point mutations located in the myosin light chains sufficient for the hypertrophy? Rather than using the genomic sequences of a heterologous species, the murine cDNA, free of all intronic DNA, was used to produce the TG mouse lines to create mouse models in which partial replacements of cardiac ELC and RLC with proteins carrying the Met149Val and Glu22Lys mutations, respectively, could be studied. Both epitope-tagged and untagged constructs were used to establish the degree of replacement, allowing us to determine dose-dependent effects of the mutant transgenes. Unlike the murine model carrying the human ELCMet149Val gene locus, mice with the mutated murine ELC cDNA or RLCGlu22Lys failed to exhibit either overt hypertrophy or midventricular cavity obstruction.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An expanded Materials and Methods section is available online at http://www.circresaha.org.
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Results |
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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 ELC1vMet149Val 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
(RLC2aRLC2v) and
RLC2vendogenousRLC2vTG
in the atria and ventricles, respectively. Ventricular
function at the cardiomyocyte and fiber levels is
unaffected.9
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Expression of ELC1vMet158Val
As the human genomic fragment used to generate TG mice contained
the ELC1vMet149Val 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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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 ELC1vMet158Val. 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
ELC1vMet158Val 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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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
ELC1vMet158Val (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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ELC1vMet158Val 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. ELC1vMet158Val
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 ELC1vMet158Val 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).
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ELC1vMet158Val 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
ELC1vMet158Val 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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, 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 ELC1vMet158Val (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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The initial studies using human-derived material showed that the
ELC1vMet149Val 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
ELC1vMet158Val line, as compared with the NTG
cohort. Finally, the in vitro motility assay was used to determine the
speed at which ELC1vMet158Val 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
ELC1vMet158Val (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
ELC1vMet158Val 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
ELC1vMet158Val mice was puzzling, and we wished
to confirm these results with an independent mutation. Therefore,
another myosin light chain mutation, the GluLys substitution found
at position 22 in the RLC,4 was also modeled in TG mice.
The construct (Figure 1) was similarly linked to the -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
RLCGlu22Lys 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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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The function(s) of the cardiac myosin light chains is beginning to be elucidated. Both bind to a critical region of the heavy chain, and expression of a cardiac MHC lacking the light chain binding site resulted in a hypertrophic cardiomyopathy.14 With these data in mind, it is logical to suppose that point mutations in either cardiac light chain could have detrimental effects that would result, eventually, in cardiovascular disease.
Clearly the mice generated in the present study, in which only the
single residue was altered, do not display an increase in chamber mass.
The initial report describing the ELC1vMet149Val
mutation in the human population showed that, in contrast to other FHC
mutations present in the force-producing proteins, this mutation
led to hypervelocities of actin translocation as measured using the in
vitro motility assay.4 Hypertrophy of the
papillary muscles led to a left ventricular midcavity
obstruction. When this mutation was modeled in TG mice, using the
intact normal and mutant human gene loci, aspects of the
phenotype were recapitulated in the old (>1 year) adults
expressing ELC1vMet149Val.5 An
explanation for the different data resulting from placing the human
locus in the mouse, versus expression of the mouse sequence with the
analogous mutation, is not readily apparent. Reconciliation of the data
is particularly difficult, because no data are available for the
expression levels of the human transcript or protein in either the
clinical population or mouse model. However, we modeled replacement
levels varying from 25% to 
95% and never detected papillary muscle
hypertrophy even when cohorts were carried out to 18 months
(data not shown), nor were hypervelocities observed in the motility
assay (Figure 6C). One possible explanation for the in vitro
motility data discrepancies may lie in the structural differences
between the human and the mouse proteins. The MHC isoforms with which
the light chains necessarily interact are also different; in the human
the ß-MHC is present, whereas in the adult mouse, only -MHC is
expressed. The alanine- and proline-rich N-terminal domain is
significantly shorter in human ELC1v as compared with the mouse (a
9-amino acid deletion, Figure 2A), and this different sequence,
when coupled with the Met149Val substitution, might result in a
phenotype unique to the human sequence. This region appears to
be important in ELC and MHC interactions and/or interactions between
ELC and actin.15 Although the normal human gene, when
placed into the mouse, did not yield a hypertrophic phenotype,
the major differences in structure preclude an unambiguous assessment
of the phenotype, as differences were observed in the
stretch-activated response of isolated fiber preparations.
Mutations in other regions of the human locus or even aberrant splicing
resulting in a protein containing additional mutations were not ruled
out.4 5 Other differences present in the 12-kb human
genomic fragment could also act synergistically with the
ELC1vMet149Val mutation. Those changes obviously
would not be present in the murine cDNAs used in our studies. The
effects of ELC1vMet149Val might be within
tolerable limits in this particular mouse strain (FVB/N) as opposed to
the mice carrying the human transgene (C57B/J6). Strain-specific
variation of phenotype does occur, and modifying factors can
play a major role in the hypertrophic response.16 The
present study does show, however, that in this genetic background,
these mutations are not sufficient for induction of
hypertrophy.
The mutations are not completely benign. ELC1vMet158Val fibers revealed a transgene dose–dependent decrease in power output as measured by the force-velocity relationship. Isolated heart analyses were consistent with the fiber data, as relaxation was impaired significantly in ELC1vMet158Val mice, whereas contractility was increased in line 95. The fiber studies were performed at 7 weeks, minimizing the possibility that the functional defects observed reflected a developing secondary pathology. It has been established that changes in the light chain can have dramatic effects on the Ca2+ sensitivity of a myofiber,11 and the most straightforward interpretation is that both the decrease in power output and the increase in Ca2+ sensitivity are directly due to the functional consequences of the altered ELC1v structure.
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Acknowledgments |
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This work was supported by NIH Grants HL56370, HL41496, HL56620, HL52318, HL60546, and HL56620; by the Marion Merrell-Dow Foundation (to J.R.), HL59408 and HL51126 (to D.M.W.); and by the American Heart Association, Ohio Valley Affiliate (to A.S.). We thank Lisa Martin for excellent technical assistance.
Received April 28, 2000; revision received June 7, 2000; accepted June 20, 2000.
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 A. M. Murphy Troponin I: In Sickness and In Health--and Normal Development Circ. Res., September 20, 2002; 91(6): 449 - 450. [Full Text] [PDF]
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T. M. Olson, M. L. Karst, F. G. Whitby, and D. J. Driscoll Myosin Light Chain Mutation Causes Autosomal Recessive Cardiomyopathy With Mid-Cavitary Hypertrophy and Restrictive Physiology Circulation, May 21, 2002; 105(20): 2337 - 2340. [Abstract] [Full Text] [PDF]
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B. M. Wolska, G. M. Arteaga, J. R. Pena, G. Nowak, R. M. Phillips, S. Sahai, P. P. de Tombe, A. F. Martin, E. G. Kranias, and R. J. Solaro Expression of Slow Skeletal Troponin I in Hearts of Phospholamban Knockout Mice Alters the Relaxant Effect of {beta}-Adrenergic Stimulation Circ. Res., May 3, 2002; 90(8): 882 - 888. [Abstract] [Full Text] [PDF]
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G. Chu, A. N. Carr, K. B. Young, J.W. Lester, A. Yatani, A. Sanbe, M. C. Colbert, S. M. Schwartz, K. F. Frank, P. D. Lampe, et al. Enhanced myocyte contractility and Ca2+ handling in a calcineurin transgenic model of heart failure Cardiovasc Res, April 1, 2002; 54(1): 105 - 116. [Abstract] [Full Text] [PDF]
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O. F Bueno, E. van Rooij, J. D Molkentin, P. A Doevendans, and L. J De Windt Calcineurin and hypertrophic heart disease: novel insights and remaining questions Cardiovasc Res, March 1, 2002; 53(4): 806 - 821. [Abstract] [Full Text] [PDF]
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A. Sanbe, J. Gulick, J. Fewell, and J. Robbins Examining the in Vivo Role of the Amino Terminus of the Essential Myosin Light Chain J. Biol. Chem., August 24, 2001; 276(35): 32682 - 32686. [Abstract] [Full Text] [PDF]
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B. M. Wolska, G. M. Arteaga, J. R. Pena, G. Nowak, R. M. Phillips, S. Sahai, P. P. de Tombe, A. F. Martin, E. G. Kranias, and R. J. Solaro Expression of Slow Skeletal Troponin I in Hearts of Phospholamban Knockout Mice Alters the Relaxant Effect of {beta}-Adrenergic Stimulation Circ. Res., May 3, 2002; 90(8): 882 - 888. [Abstract] [Full Text] [PDF]
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