Dilated Cardiomyopathy Mutant Tropomyosin Mice Develop Cardiac Dysfunction With Significantly Decreased Fractional Shortening and Myofilament Calcium Sensitivity
Mutations in striated muscle α-tropomyosin (α-TM), an essential thin filament protein, cause both dilated cardiomyopathy (DCM) and familial hypertrophic cardiomyopathy. Two distinct point mutations within α-tropomyosin are associated with the development of DCM in humans: Glu40Lys and Glu54Lys. To investigate the functional consequences of α-TM mutations associated with DCM, we generated transgenic mice that express mutant α-TM (Glu54Lys) in the adult heart. Results showed that an increase in transgenic protein expression led to a reciprocal decrease in endogenous α-TM levels, with total myofilament TM protein levels remaining unaltered. Histological and morphological analyses revealed development of DCM with progression to heart failure and frequently death by 6 months. Echocardiographic analyses confirmed the dilated phenotype of the heart with a significant decrease in the left ventricular fractional shortening. Work-performing heart analyses showed significantly impaired systolic, and diastolic functions and the force measurements of cardiac myofibers revealed that the myofilaments had significantly decreased Ca2+ sensitivity and tension generation. Real-time RT-PCR quantification demonstrated an increased expression of β-myosin heavy chain, brain natriuretic peptide, and skeletal actin and a decreased expression of the Ca2+ handling proteins sarcoplasmic reticulum Ca2+-ATPase and ryanodine receptor. Furthermore, our study also indicates that the α-TM54 mutation decreases tropomyosin flexibility, which may influence actin binding and myofilament Ca2+ sensitivity. The pathological and physiological phenotypes exhibited by these mice are consistent with those seen in human DCM and heart failure. As such, this is the first mouse model in which a mutation in a sarcomeric thin filament protein, specifically TM, leads to DCM.
Tropomyosin (TM) is an α helical coiled-coil fibrous protein that binds actin filaments providing structural stability and modulation of filament function. In striated muscle, TM along with the troponin complex regulates Ca2+-mediated actin–myosin crossbridges. Numerous mutations in many of the contractile proteins of the cardiac sarcomere have been associated with dilated and hypertrophic cardiomyopathy, where the myocardial performance is compromised. In humans, 2 dilated cardiomyopathy (DCM)-associated mutations (Glu54Lys and Glu40Lys) have been identified in α-tropomyosin (α-TM) (or TPM1),1 in contrast to the 8 distinct mutations in the same gene that are associated with familial hypertrophic cardiomyopathy (FHC).2 The DCM mutations in α-TM are located in a region (amino acids 40 to 100) where half of the reported human FHC mutations occur (Glu62Gln, Ala63Val, Lys70Thr, Val95Ala); this region does not interact with troponin (Tn)T.
Protein-modeling studies on the TM filaments harboring Glu54Lys and Glu40Lys substitutions show that both of them create a strong local increase in the positive charge in an otherwise negatively charged region of the molecule.1 The 2-Å crystal structure of TM indicates that Glu54 is linked to Lys49 and Glu40 is linked to Arg35.3 The substitution of glutamic acid for lysine would abolish this interaction and thus destabilize localized TM structure.4 Furthermore, this mutation occurs in the fifth (or e) position of a highly conserved heptad motif of repeating 7 amino acid units (a-b-c-d-e-f-g). Structural and molecular modeling studies suggest that the fifth and seventh position (e and g) amino acid side chains, which are typically of opposite charge, can contribute to the stability of the coiled-coil through formation of a salt bridge.1 Disruption of one salt bridging interaction through charge reversal of an amino acid might cause a local change in TM conformation or lability. Whether the primary effect of a single amino acid substitution is to alter protein stability or surface electrostatic charge characteristics, the integrity of the thin filament is likely to be compromised, and hence a defective force transmission has been attributed to be the cause of DCM.1 Two independent studies of the DCM-causing TM mutations involving an in vitro approach have demonstrated a decreased calcium sensitivity of myofilaments.4,5
In our previous studies using a transgenic (TG) mouse model approach, we extensively analyzed the functional consequences of FHC causing α-TM mutations.2,6–8 In the current study, we adopted a similar transgenic approach to address the effect of the DCM Glu54Lys α-TM mutation on cardiac development and function. We established 5 distinct TG lines that use a cardiac-specific α-myosin heavy chain (MHC) promoter to express the α-TM54 mutant protein in the heart. Western blotting and quantitative analyses demonstrated that, in all TG lines, an increase in transgenic protein led to reciprocal decreases in endogenous α-TM levels, and the total myofilament TM protein level remained unchanged.
Echocardiographic analyses of these mice showed that they exhibited a significant DCM phenotype with a marked decrease in the left ventricular (LV) fractional shortening. Histopathological and morphological analyses also revealed a slow progression from DCM to heart failure, with lethality frequently occurring within 6 months. Work-performing heart analyses showed significantly impaired systolic and diastolic functions concomitant with increased time to peak pressure and half-time to relaxation. Interestingly, with high concentrations of β-adrenergic stimulation (isoproterenol), a sudden steep increase in the performance is observed, restoring normal cardiac function. The force measurements of skinned myofiber preparations exhibit a significantly decreased Ca2+ sensitivity and tension generation with no alteration in sarcomere length dependence of activation. This mouse model provides an opportunity to delineate the pathophysiological mechanisms of the sarcomeric DCM mutation and to explore the relationship between TM mutations inducing DCM and FHC signal transduction pathways.
Materials and Methods
An expanded Materials and Methods section can be found in the online data supplement at http://circres.ahajournals.org.
Generation of DCM Mutant α-TM54 Transgenic Mice
Cardiac-restricted DCM mutant α-TM54 transgenic mice were generated using vectors containing the mouse α-MHC promoter and α-TM cDNA sequences, as described in the online data supplement. Experimental procedures describing the histologic, morphological, and molecular characterizations of those TG mice are also described in the online data supplement. The Institutional Animal Care and Use Committee approved the handling and maintenance of animals.
The cardiac performance of the DCM mutant α-TM54 TG mice was assessed by physiological studies, including echocardiography, work-performing heart model, and skinned fiber preparations, all of which are described in detail in the online data supplement. In these physiological experiments, the high expression mice refer to line 30. Initial studies found no functional differences in the results from the moderate expression mice (lines 67, 71, and 95); for this reason, the subsequent extensive studies concentrated on line 67.
Real-Time RT-PCR Analysis, Bacterial Recombinant Protein Expression, and Circular Dichroism Measurements
For details regarding the methods used, see the expanded Materials and Methods section in the online data supplement.
Generation of DCM Mutant α-TM54 TG Mice
The transgene construct used to generate the α-TM54 TG mice is shown in Figure 1A. Because there is 100% amino acid identity between the striated muscle α-TM proteins of the mouse and humans, the designed DCM mutation is reflective of the change found in human DCM. Work from our laboratory shows that overexpression of wild-type α-TM cDNA (WT-TG) using a similar construct in the heart does not lead to pathological changes nor functional alterations in cardiac or myofiber performance.7,9 Five different transgenic lines were established and genomic Southern blot analysis shows hybridizing bands at 1.7 kb and 3.4 kb (Figure 1B); the 1.7-kb band represents the endogenous α-TM gene (TPM1), which has a signal intensity that corresponds to a gene copy number of 2, whereas the 3.4-kb band indicates the incorporated transgene construct. Along with the nontransgenic (NTG) mice, 2 lines of WT-TG with varied copy number of wild-type transgene (29 and 35 copies) were included as controls. Quantification of the transgene by normalizing it to the 2-copy endogenous gene demonstrates that copy numbers of the transgene-overexpressing mutant α-TM54 protein range from 1 to multiple copies: line 67, 22 copies; line 71, 10 copies; line 76, 1 copy; line 95, 9 copies; and line 30, ≈174 copies. To negate the possibility that a deletion of the DCM α-TM54 DNA sequences occurred during transgenesis, we conducted nucleotide sequencing of TG mouse genomic DNA using construct specific primers. Results verified the presence of the designed mutation and indicated no additional changes in α-TM sequence. To further confirm the absence of any potential novel mutations in the transgene, cDNA synthesized from the total heart RNA of 2 TG lines (lines 67 and 30) that were used in the present study were PCR cloned with minimum cycle amplification (15 cycles). Direct forward and reverse sequencing of 15 independent clones from each line showed the presence of only the α-TM54 mutation.
Cardiac α-TM54 Mutant Transcript and Protein Expression in TG Mice
Message levels of control and transgene transcripts were assayed by Northern blot hybridization using a radiolabeled α-TM 5′ untranslated region probe and an α-MHC 1- to 2-exon probe, respectively, that were normalized to GAPDH expression. The expression levels of the endogenous α-TM transcript remain constant in all of the α-TM54 TG mouse lines, whereas the transgene expression levels correlate with their corresponding transgene copy numbers (Figure 1C). There is a slight decrease in endogenous α-TM mRNA levels in the WT-TG hearts.
To quantify mutant α-TM54 protein expression, myofibrillar proteins from the hearts of NTG and TG mice were subjected to 1D isoelectric focusing, followed by immunoblotting with a striated muscle-specific TM antibody. On a very narrow pH gradient (pI 4.2 to 4.9), the mutant α-TM54 protein focused at a higher pI when compared with the endogenous α-TM, which correlates with the theoretical pI values of endogenous α-TM protein (4.60) and mutant α-TM54 protein (4.65). Results show there are varying degrees of expression of the mutant TM protein in the hearts of the different lines of α-TM54 TG mice, which is coupled with a concomitant downregulation of endogenous α-TM (Figure 1D). Also, the ratio of the mutant α-TM54 to endogenous α-TM protein (Figure 1E) correlates with their corresponding transcript levels with the exception of TG line 30, which has a very high transgene copy number and mRNA expression but a low incorporation of mutant protein in the myofilaments. Further analysis of the cytosolic fraction of the TG mouse hearts revealed that only in TG line 30 was a major amount of mutant α-TM54 protein found in the cytoplasm (Figure 1D). (Transgenic expression of chimeric TMs or mutant transgenes does not lead to cytoplasmic accumulation of the exogenous protein.10) The significant accumulation of α-TM54 protein in the cytoplasm in TG line 30 was further confirmed by immunohistochemical analyses of TG heart sections using a TM-specific antibody (Figure 2A). Additional quantitative analysis shows that the amount of total striated muscle α-TM (generated from both endogenous and transgene sources) in total cell lysate remains unchanged when normalized to α-tubulin in the NTG, WT-TG, and α-TM54 TG hearts with the exception of α-TM54 TG line 30 (Figure IA in the online data supplement). The total amount of myofilament-incorporated striated muscle TM remains unchanged when normalized to striated muscle actin levels in the NTG, WT-TG and α-TM54 TG hearts including TG line 30 (supplemental Figure IB). Also, experiments demonstrated that there were no quantitative changes in the other non-TM cardiac contractile proteins in the TG hearts (data not shown).
Cardiac Morphology of DCM Mutant α-TM54 TG Mice
Cardiac structure was characterized in the transgenic mice at various time intervals from 1 to 8 months after birth. Results show that the high-copy TG mouse hearts develop a severe DCM phenotype by 2 weeks, and the dilation is seen in both the ventricles (Figure 2B, panels vi and viii). Morphological analyses of the ventricular wall show moderate myocyte hypertrophy with severe diffuse hyalinization of the myocyte cytoplasm. The cytoplasmic changes are characterized by loss of striation and a homogenous, ground-glass appearance. By 1 month, the high-copy TG animals had a significant increase in the heart weight-to-body weight ratio (Figure 3A) with most mice dying within 1.5 months (Figure 3B). In contrast, the moderate-copy mice show a tendency of developing DCM after two months of age with hearts showing mild myocyte hypertrophy and disorganization at the base of left ventricle and very mild interstitial fibrosis. By five months, they develop significant dilation of both ventricles (Figure 2B, panels ii and iv) with mild disorganization of the myocytes at the base of the ventricles. A moderate diffuse peribronchiolar neutrophil cuffing was seen in the lungs. Interestingly, the body weight of these TG mice show an increase of ≈50% (NTG, 24.83±1.8 g, n=8 and TG L67, 36.1±1.8 g, n=8); this increase in weight could be attributable to peripheral edema which was observed in these animals. These findings present a clinical feature associated with heart failure, but surprisingly the lung weight is normal and there was no ascites formation. In spite of the increase in body weight, they still show an increase in heart weight to body weight ratio (Figure 3A). The mice from all 3 moderate-expression lines start dying by 4 to 6 months of age (Figure 3B), and the survival data show that by 8 months, 38% of mice died in line 67, 18% in line 71, and 15% in line 95. The mice that survived beyond 8 months also showed an increased heart weight to body weight ratio and develop a DCM phenotype.
Cardiac Function of DCM Mutant α-TM54 TG Mice
To assess whether any functional changes in cardiac performance occur in the DCM mutant α-TM54 TG mice, we conducted several physiological studies by use of echocardiography, the work-performing heart model, and skinned fiber preparations. An in vivo physiologic assessment of cardiac function was conducted on 5-month-old moderate-copy mice, 1-month-old high-copy mice, and control littermate mice by Doppler echocardiographic analyses. In both moderate- and high-copy TG mice, the hearts demonstrated increased LV diastolic and systolic diameter as well as significant reduction in the LV fractional shortening (Figure 4A, Table 1, and supplemental Table I). The cardiac output was also significantly reduced, but the heart rate was not affected. In total, the echocardiographic results demonstrate the development of a DCM phenotype that can lead to heart failure.
Work-Performing Heart Model
The work-performing heart model was used to obtain an ex vivo assessment of cardiac performance. These measurements were conducted in moderate-copy TG (3 month-old) and high-copy TG (1-month-old) mouse hearts. As seen in Table 2 and supplemental Table II, the rates of relaxation and contraction were significantly reduced concomitant with increased time to peak pressure and half-time to relaxation. End-diastolic and diastolic pressures were significantly increased, whereas the systolic pressure was significantly decreased documenting their systolic and diastolic dysfunction. Previous work has demonstrated that transgenic mice that overexpress wild-type α-TM show no significant alterations in cardiac function.7,9
We also determined responses to isoproterenol to ascertain if the observed systolic and diastolic dysfunction was associated with impaired β-adrenergic responses. The reduced inotropic and lusitropic performance by hearts was assessed during stimulation with isoproterenol, a β-adrenergic agonist that augments muscle contraction and relaxation by cAMP/protein kinase A–dependent kinase. Interestingly, in the moderate-copy TG mouse hearts, whereas a blunted response was observed at lower concentrations of isoproterenol (10−11 to 10−8 mol/L), a sudden steep increase in the performance was observed at higher concentrations of isoproterenol (>10−8 mol/L), restoring normal cardiac function (Figure 4B). In contrast, in the high-copy TG mouse hearts, there was a blunting of β-adrenergic stimulation at all concentrations of isoproterenol (data not shown).
Ca2+–Force Measurements in Skinned Fiber Bundles
To examine the correlation between physiological results from the whole-heart and mutant α-TM54 expression at the sarcomere level, a series of experiments was conducted using detergent-extracted (skinned) fiber bundles. These experiments were conducted to compare the relation between Ca2+ and tension developed by myofilaments obtained from control versus mutant α-TM54 left ventricular fiber bundles of 5 month-old moderate-copy TG mice and 1 month-old high-copy TG mice. In the first set of experiments, we compared the pCa–tension relations for fiber bundles obtained from NTG (n=8) and TG (moderate-copy numbers; n=10) hearts at a sarcomeric length of 2.3 μm. As illustrated in Table 3 and supplemental Figure II, there is a significant reduction in maximum tension and pCa50 (−log of free [Ca2+]) in TG mice fiber bundles compared with NTG controls, with no significant differences in the Hill n values. Similar sets of experiments were completed with fiber bundles from the 1-month-old high-copy TG (n=8) hearts and their littermate NTG (n=8) hearts. Compared with fibers with a moderate-copy number, a more severely depressed maximum tension in the high-copy TG fibers was observed. There is also a significant reduction in the pCa50 in TG fibers compared with NTG and no significant difference between Hill n values. Furthermore, all the experiments when repeated at a sarcomere length of 1.9 μm presented a similar pattern, except that the DCM α-TM54 mutation had no effect on the length-dependent activation of tension development (supplemental Figure II).
Gene Expression Associated With Cardiomyopathy
Altered gene expression, a recognized feature of DCM,11 was examined in the α-TM54 moderate-copy TG mouse hearts at 5 months of age, a time point when the cardiomyopathy is already apparent. Real-time RT-PCR analysis of the RNA isolated from the ventricular tissue revealed that there was a significant upregulation of molecular markers of cardiomyopathy namely, β-MHC, brain natriuretic peptide, and skeletal actin (Figure 5A). To determine whether genes that regulate myocyte Ca2+ cycling were altered in the TG myocardium, we also measured mRNA levels of sarcoplasmic reticulum Ca2+-ATPase, ryanodine receptor, calsequestrin, L-type Ca2+ channel, and phospholamban. We observed significant downregulation only in the levels of sarcoplasmic reticulum Ca2+-ATPase and ryanodine receptor transcripts. All of these RNA levels were normalized to GAPDH values.
Effect of Glu54Lys Mutation on Tropomyosin Thermal Stability
Tropomyosin structure is weakened by FHC mutations, namely, Glu180Gly, Asp175Asn, Lys70Thr, and Ala63Val.12 Therefore, to investigate the effect of Glu54Lys mutation on TM structure, circular dichroism was used and thermal stability measurements were made by following the ellipticity (θ) of TM at 222 nm as a function of temperature. At intermediate temperatures, the mutation significantly altered θ 222, a measure of α-helical content. Results showed that the thermal denaturation curve of the mutant protein shifted toward the right when compared with the wild-type curve (Figure 5B), implying an increased stability conferred by the mutation. This change in amino acid causes a decrease in flexibility, which can influence actin binding13 as well as myofilament Ca2+ sensitivity.12
Two missense mutations (Glu54Lys and Glu40Lys) that alter the highly conserved residues of α-TM have been linked to DCM. The phenotypic severity associated with the sarcomeric mutations in human DCM patients and the altered sarcomeric function associated with the mutations is not well established. The family pedigree associated with the DCM α-TM54 mutation appears to be quite severe; these individuals all died at relatively early ages (26, 27, and 49 years).1 Cardiac phenotypic measurements of the proband are in agreement with those findings from both moderate and high-copy TM mice, namely increased LV internal diastolic dimension and LV internal systolic dimension, and decreased fractional shortening percentage (Table 1 and supplemental Table I). Because TM protein measurements were not conducted on the proband, the relative ratio of wild type: mutant TM is unknown. Nevertheless, the α-TM54 TG mice appear to be a good model system for studying DCM.
The results of this study show that expression of α-TM encoding an amino acid change of glutamic acid (negative side chain) to lysine (positive side chain) at codon 54 induces DCM. Physiological alterations include impairment in both cardiac contractile and relaxation functions, with hearts exhibiting a significantly reduced LV fractional shortening and a decrease in myofilament Ca2+ sensitivity. This is the first demonstration that exogenous expression of a sarcomeric thin filament protein encoding a known human DCM mutation in the mouse heart results in pathological and physiological defects associated with DCM and thus provides an excellent opportunity to understand the disease pathology. There has been a recent report of a DCM mouse model for a sarcomeric thick filament mutation in β-MHC that also exhibits the cardiomyopathic phenotype.14 Other reported murine models of sarcomeric DCM have not yet been shown to be related to human DCM, including cardiac overexpression of tropomodulin15 and homozygous expression in mice of a mutant form of the sarcomeric myosin-binding protein C.16
The altered cardiac structure and function of the DCM α-TM54 mutation are thought to be a consequence of impairment in actin-binding capability by TM coupled with a decreased Ca2+ sensitivity of the myofilaments.17 This hypothesis is reinforced by the results from measurements with skinned fiber preparations of the α-TM54 TG mouse hearts. As summarized in Table 3, at 5 months of age, compared with matched controls, moderate-copy TG myofilaments demonstrate significant desensitization to Ca2+ as well as a depression in maximum tension. Higher-copy TG myofilaments, at 1 month of age, also demonstrate a more severely depressed maximum tension and reduced Ca2+ sensitivity when compared with age-matched controls. Moreover, the reduced myofilament sensitivity to Ca2+ and depressed tension correlates with our finding of depressed cardiac function as determined by echocardiography and/or by studies in isolated working hearts. Studies using the in vitro motility assay also agree that alterations in the TM function and decreased myofilament Ca2+ sensitivity are associated with the Glu54Lys amino acid substitution.4,5
The altered expression of the Ca2+ handling proteins as observed by real-time RT-PCR quantification may contribute to a depressed Ca2+ transient, leading to impaired excitation–contraction coupling. The rescue of cardiac function, after high doses of isoproterenol, from the impaired adrenergic responsiveness in the work performing hearts of moderate-copy TG mice at 3 months of age indicates that they are in the early stages of heart failure. Furthermore, the result also suggests that apart from the altered expression of the Ca2+ handling proteins, there could also be an altered phosphorylation status of the Ca2+ handling proteins such as phospholamban, which is a key determinant of β-adrenergic stimulation in the heart.18 The blunted response of the high-copy TG mice would indicate that their hearts are in too severe of a pathological state to respond to isoproterenol.
Despite a low incorporation of the mutant protein in the high-copy TG myofilaments, we see a severe phenotype culminating in early death. This could be attributed to an overtly high transgene copy number in these mice and/or the excessive cytosolic presence of the mutant protein, which may interfere with cytoskeletal structures leading to a defective force transmission. The precise reason for significant cytosolic accumulation of mutant α-TM54 protein with decreased incorporation in the myofilaments is unclear but is an interesting area for future investigation. Interestingly, the muscle LIM protein associated with actin cytoskeleton at the Z-disc has been correlated to DCM in muscle LIM protein–null mice,19 and it has been proposed that defects in the cytoskeleton are primarily responsible for many human forms of DCM.20,21 Another possible mechanism of cardiac disease is the aggregate/amyloid formation by unfolded or misfolded proteins which has been reported in heart failure patients and also in desmin-related cardiomyopathic mice overexpressing mutant α-B-crystallin.22 This pathogenic process may be the cause of an early phenotype and death in the high-copy α-TM54 TG mice. Although we could not detect the cytosolic accumulation of the transgenic protein in the moderate expression lines, DCM still developed. This indicates that the α-TM54 mutation could be the primary cause for the development of DCM.
Circular dichroism titrations at 222 nm showed the temperature stability of the α-TM α helix with a Glu54Lys amino acid substitution is greater than wild-type α-TM. In contrast, all FHC-associated tropomyosin mutations (Glu180Gly, Asp175Asn, Lys70Thr, and Ala63Val) are reported to decrease the temperature stability of the α helical coiled-coil. Most FHC thin filament mutations lead to a variety of functional abnormalities in the sarcomere that include increased myofilament Ca2+ sensitivity.5,6,8,12 However, the DCM α-TM54 mutation is shown to decrease the myofilament Ca2+ sensitivity. These findings help to correlate FHC mutations with increased Ca2+ sensitivity and a destabilized α-TM helix, and DCM mutations with decreased myofilament Ca2+ sensitivity and a stabilized α-TM helix. It is interesting to consider whether these 2 properties are consistent with other TM mutations and whether they are linked to the disease causing pathways of FHC and DCM. Our data indicate that α-TM54 mutation in mice results in significantly decreased myofilament calcium sensitivity and significantly impaired systolic and diastolic functions. This observation, together with the absence of length dependent activation of tension development and the thermal stability data of the mutant α-TM54 protein favoring the defective actin binding capability, supports the notion that defects in force transmission rather than force generation may cause the observed DCM phenotype.1
We thank Maureen Bender for care of the animals. We acknowledge Dr Evangelia Kranias for critical reading of the manuscript.
Sources of Funding
This study was supported by National Heart, Lung, and Blood Institute grants HL-71952 (to D.F.W.), HL-79032 (to B.M.W.), K01 HL-67709-4 (to G.M.A.), HL-22231 (to R.J.S.), and HL-062426 (to R.J.S.).
↵*Both authors contributed equally to this study.
Original received January 10, 2007; revision received May 22, 2007; accepted May 24, 2007.
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