Published online before print June 7, 2007, doi: 10.1161/CIRCRESAHA.106.146670
© 2007 American Heart Association, Inc.
Integrative Physiology |
Knock-In Mouse Model of Dilated Cardiomyopathy Caused by Troponin Mutation
From the Departments of Clinical Pharmacology (C.-K.D., S.M., Q.-W. L., Y.-Y.W., D.-Y.Z., M.M., Y.M., F.T.-Y., T.S.) and Health Sciences (R.M.), Kyushu University Graduate School of Medicine, Fukuoka, Japan; Department of Cell Biology (K.N.), Tokyo Medical and Dental University; Clinical Genome Informatics Center (M.O.), Kobe University Graduate School of Medicine, Kobe, Japan; Research Laboratory (N.T.), Zenyaku Kogyo Co Ltd, Tokyo, Japan; Department of Pharmacology (S.K., T.I.), Fukuoka University School of Medicine, Fukuoka, Japan; and Department Physiology II (I.O.), Jikei University School of Medicine, Tokyo, Japan.
Correspondence to Sachio Morimoto, PhD, Department of Clinical Pharmacology, Kyushu University Graduate School of Medicine, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail morimoto{at}med.kyushu-u.ac.jp
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Abstract |
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We created knock-in mice in which a deletion of 3 base pairs coding for K210 in cardiac troponin (cTn)T found in familial dilated cardiomyopathy patients was introduced into endogenous genes. Membrane-permeabilized cardiac muscle fibers from mutant mice showed significantly lower Ca2+ sensitivity in force generation than those from wild-type mice. Peak amplitude of Ca2+ transient in cardiomyocytes was increased in mutant mice, and maximum isometric force produced by intact cardiac muscle fibers of mutant mice was not significantly different from that of wild-type mice, suggesting that Ca2+ transient was augmented to compensate for decreased myofilament Ca2+ sensitivity. Nevertheless, mutant mice developed marked cardiac enlargement, heart failure, and frequent sudden death recapitulating the phenotypes of dilated cardiomyopathy patients, indicating that global functional defect of the heart attributable to decreased myofilament Ca2+ sensitivity could not be fully compensated by only increasing the intracellular Ca2+ transient. We found that a positive inotropic agent, pimobendan, which directly increases myofilament Ca2+ sensitivity, had profound effects of preventing cardiac enlargement, heart failure, and sudden death. These results verify the hypothesis that Ca2+ desensitization of cardiac myofilament is the absolute cause of the pathogenesis of dilated cardiomyopathy associated with this mutation and strongly suggest that Ca2+ sensitizers are beneficial for the treatment of dilated cardiomyopathy patients affected by sarcomeric regulatory protein mutations.
Key Words: dilated cardiomyopathy • troponin • mutation • calcium sensitivity • knock-in mouse
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Introduction |
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Dilated cardiomyopathy (DCM) is a disorder of cardiac muscle characterized by cardiac enlargement and systolic dysfunction and accounts for more than 10 000 deaths annually by heart failure and sudden death in the United States.1–3 DCM is known to result from nongenetic insults, such as viruses, alcohol, toxins, and immunologic injury; however, recent genetic studies have revealed that mutations in genes for cytoskeletal (dystrophin, desmin,
-sarcoglycan), nuclear envelope (tafazzin and lamin A/C), and sarcomeric (cardiac actin, ß-cardiac myosin heavy chain, 
-tropomyosin, cardiac myosin-binding protein C, titin/connectin, cardiac troponin [cTn]T, cTnI, and cTnC) proteins are important causes of DCM,4 and the incidence of the inherited DCM is thought to be 20% to 35%.5–7
Cardiac muscle contraction is regulated through Ca2+ binding to cardiac troponin complex localized on the thin filaments,8 and DCM-causing mutations in troponin complex are associated with a malignant phenotype with a high incidence of premature cardiac death and heart transplantation.9 Cardiac troponin complex consists of 3 components of distinct structure and function, cTnT, cTnI, and cTnC. cTnT has a structural role in anchoring troponin complex to the thin filaments through its binding to tropomyosin, cTnI inhibits the interaction of myosin crossbridges with the thin filament at low Ca2+ concentration, and cTnC relieves the inhibitory action of cTnI on the thin filaments on Ca2+ binding. In a previous in vitro reconstitution experiment, selective displacement of endogenous cTnT in rabbit membrane-permeabilized (skinned) cardiac muscle fibers with human cTnT with a DCM-causing deletion mutation 
K210 was found to decrease the Ca2+ sensitivity of force generation, in direct opposition to Ca2+ sensitization caused by mutations associated with hypertrophic cardiomyopathy.10 This study led us to propose that decreased Ca2+ sensitivity of cardiac myofilaments might be a primary functional defect triggering the pathogenesis of DCM associated with the deletion mutation K210 in cTnT.
To test this hypothesis in vivo and to determine the exact cause of the pathogenesis of DCM associated with the deletion mutation K210 in cTnT, in the present study, we created mutant mice in which this mutation was just knocked in to the endogenous mouse TNNT2 gene using embryonic stem cell technology. Analyses of these knock-in mice demonstrated that decreased myofilament Ca2+ sensitivity is the absolute cause of DCM associated with the mutation K210 in cTnT and also suggested that Ca2+ sensitizers, such as pimobendan, might be beneficial for the treatment of DCM patients affected by sarcomeric regulatory protein mutations.
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Materials and Methods |
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An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Generation of a Knock-In Mouse Model by Gene Targeting
Gene targeting was performed using a targeting vector including a 13.8-kb cTnT gene fragment with the loxP-neo cassette11 on intron 12 and a deletion of 3 base pairs coding for K210 on exon 13. After germline transmission of the properly targeted ES cell clone, knock-in mice were obtained by crossing with a Cre transgenic mouse strain.12
The experimental protocol was reviewed by the Committee of Ethics on Animal Experiments in the Faculty of Medicine, Kyushu University, and performed in accordance with the Guideline for Animal Experiments of the Faculty of Medicine, Kyushu University, and the law (No. 105) and notification (No. 6) of the Japanese Government.
Preparation of Skinned Cardiac Muscle Fibers and Force Measurements
A small fiber (
200 µm in diameter) was dissected from the skinned left ventricular papillary muscle, and isometric force was measured as described previously.13
Preparation of Intact Cardiac Muscle Fibers and Force Measurements
The left ventricular papillary muscle was mounted horizontally in a thermostatically controlled chamber, and isometric force evoked by a bipolar electrical field stimulation was measured with a semiconductor strain gauge.
Fura-2 Loading and Simultaneous Measurements of Fluorescence and Sarcomere Length
Cardiomyocytes were loaded with fura-2 acetoxymethyl ester, and contraction was evoked by bipolar electrical field stimulation. [Ca2+]i and sarcomere length were simultaneously monitored using a fluorescence and contractility recording system (IonOptix) as described previously.14
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Results |
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We created knock-in mice in which 3 base pairs coding for the amino acid residue K210 in cTnT were deleted from their endogenous gene TNNT2 using gene-targeting technology (Figure 1). These knock-in mice have the same genomic gene condition as DCM patients affected by the deletion mutation
K210 in cTnT. Consistent with the previous study showing that this mutation in cTnT has a Ca2+-desensitizing effect on cardiac myofilaments in vitro,10 skinned cardiac muscle fibers prepared from mutant mice showed a decrease in Ca2+ sensitivity of force generation, as was evident from a rightward shift of the force/pCa relationship (Figure 2A). The pCa value at half-maximal force generation (pCa50, index of Ca2+ sensitivity) was statistically significantly lower in mutant mice than in wild-type mice, being smaller in homozygotes (TNNT2K210/K210) than in heterozygotes (TNNT2+/K210) (Figure 2B). On the other hand, the maximum force-generating capabilities (Figure 2C) and Hill coefficient values (an indicator of cooperativity or steepness of curve) (data not shown) were not significantly different between wild-type and mutant mice. Phosphorylation levels of cTnI at Ser22/23, known to influence myofilament Ca2+ sensitivity, were also not significantly different between wild-type and mutant mice (Figure 2D). No significant differences in the phosphorylation levels of serine and threonine residues in cTnI and cTnT, which were reported to affect the myofilament activation level and/or Ca2+ sensitivity,15 were detected between wild-type and mutant mice by using anti-phosphoserine and anti-phosphothreonine antibodies (data not shown). These results demonstrate that the deletion mutation K210 in cTnT in vivo does have a Ca2+-desensitizing effect on force generation in cardiac muscle without changing the maximum force-generating capability and cooperativity, confirming the results of previous in vitro reconstitution studies.10,16,17 It should be noted that the phosphorylation levels of cTnI and cTnT in skinned fibers could not reflect these levels in vivo, because skinned fibers were prepared and stored without denaturalization in ATP-containing relaxing solution, mimicking the intracellular environment. Further thorough analysis is necessary to reach a conclusion regarding alterations in sarcomeric protein phosphorylation in vivo.
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; 2 to 9 months old; n=16), TNNT2+/
; 2 to 9 months old; n=16), and TNNT2
; 2 to 3 months old; n=5) mice. B, Ca2+ sensitivities (pCa50) of force–pCa relationships in skinned cardiac muscle fibers. C, Maximum force-generating capabilities of skinned cardiac muscle fibers. D, Phosphorylation levels of cTnI in skinned cardiac muscle fibers. Data represent the means±SE for 3 fibers from different mice. Statistical significance was determined by ANOVA followed by post hoc Tukey’s multiple comparison test.
The decreased Ca2+ sensitivity of cardiac myofilament in knock-in mice is expected to lead to some reduction in the maximum force-generating capability of intact cardiac muscle; however, intact cardiac muscle fibers from mutant mice showed no significant decrease in maximum isometric force per cross-sectional area compared with those from wild-type mice (Figure 3A and 3B). Intact cardiac muscle fibers from TNNT2K210/K210 mice showed slightly higher rates of isometric force development and relaxation, as was evident from a decrease in the time to peak and a steeper rise and fall in force (Figure 3B). Figure 3C shows Ca2+ transients measured in fura-2–loaded cardiomyocytes. The peak amplitude was found to be significantly increased in mutant mice compared with wild-type mice, being greater in TNNT2K210/K210 than in TNNT2+/K210 mice (Figure 3D). The peak rates of increase and decrease in cytoplasmic Ca2+ in mutant mice are faster than those in wild-type mice, consistent with the faster kinetics of isometric force development and relaxation observed in TNNT2K210/K210 mice. Resting sarcomere length of cardiomyocytes in mutant mice was significantly longer than that in wild-type mice, consistent with decreased myofilament Ca2+ sensitivity (Figure 3E and 3F). No significant differences were observed in the number and shape of mitochondria in cardiomyocytes between wild-type and mutant mice, strongly suggesting that the change in peak amplitude of the Ca2+ transient was not caused by the altered distribution of Ca2+ among intracellular compartments (Figure I in the online data supplement). These findings indicate that the Ca2+ transient of cardiomyocytes was augmented in mutant mice, probably to compensate for decreased myofilament Ca2+ sensitivity and maintain the force-generating capability of cardiac muscle; however, fractional sarcomere shortening and peak velocity of sarcomere shortening of isolated cardiomyocytes were significantly decreased in TNNT2K210/K210 mice, indicating that a defect of dynamic contractile performance was still present in cardiomyocytes of these mutant mice (Figure 3F). Sarcoplasmic reticulum Ca2+ content assessed by caffeine-induced Ca2+ release was significantly increased in mutant mice, suggesting that sarcoplasmic reticulum Ca2+ pump (SERCA2a) activity was enhanced in mutant mice (supplemental Figure II). Whereas protein expression levels of ryanodine receptor, sarcoplasmic reticulum Ca2+ pump, and phospholamban (PLB) were not significantly different between wild-type and mutant mice, significant increases in the phosphorylation of ryanodine receptor and PLB catalyzed by cAMP-dependent protein kinase were observed in TNNT2K210/K210 mice, suggesting that an increase in the intracellular cAMP level might be responsible for the augmented Ca2+ transient in mutant mice (supplemental Figure III).
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Despite preserved maximum isometric force-generating capability in intact cardiac muscle, knock-in mice developed markedly enlarged hearts characteristic of DCM in a mutant-gene dosage-dependent manner, and histological examination of their cardiac sections showed significant interstitial fibrosis with normal cellular organization (Figure 4A). Cardiomyocyte apoptosis was significantly increased in the mutant mice, suggesting that apoptosis-inducing stress such as ischemia occurs in the myocardium of mutant mice (Figure 4B). Echocardiography demonstrated that left ventricular end-diastolic dimension was significantly increased in mutant mice, and left ventricular ejection fraction, an index of cardiac systolic function, was significantly reduced in mutant mice, with TNNT2K210/K210 being more severely affected than TNNT2+/K210 (Table 1). No significant differences were detected in ventricular wall thickness (interventricular septal thickness and left ventricular posterior wall thickness), heart rate, and blood pressure between wild-type and mutant mice (Table 1). There were no significant differences in in vivo basal hemodynamic parameters between wild-type and mutant mice, but left ventricular end-systolic pressure of TNNT2K210/K210 mice was significantly lower than that of wild-type mice after administration of isoproterenol (Table 2). Ex vivo analyses of isolated work-performing heart preparations showed that cardiac pump function was impaired in mutant mice (Figure 4C). These results indicate that the deletion mutation K210 in cTnT causes cardiac enlargement with marked ventricular dilation and systolic dysfunction in mice, closely recapitulating the phenotypes of human DCM patients.18,19
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Mutant mice showed high mortality in a mutant-gene, dosage-dependent manner (Figure 4D). TNNT2K210/K210 mice showed a particularly high incidence of sudden death in their growth periods from 1 to 3 months old. Surface ECG showed that TNNT2K210/K210 mice commonly had electrophysiological abnormality in the heart with long QT, which might be involved in their frequent sudden death (Table 1). Telemetric ECG recordings showed that mice died by abruptly developing repetitive Torsade de Pointes several hours before death, which ultimately degenerated into ventricular fibrillation (Figure 4E).
The expression of ß-cardiac myosin heavy chain (ß-MyHC) isoform in ventricular myocardium, known to be upregulated in heart failure,20–22 was found to be markedly increased in TNNT2K210/K210 mice at 2 months old (Figure 4F). A significant increase in the expression of ß-MyHC was also detected in TNNT2+/K210 mice at 3 and 5 months old but not at 2 months old, indicating that mutant mice undergo heart failure, with homozygous mice being affected from much earlier stages of life.
Oral administration of pimobendan, a Ca2+ sensitizer, known to directly increase the Ca2+ sensitivity of myofilament, was found to reduce the heart size of mutant mice, as was evident from a statistically significant decrease in the heart to body weight ratio (Table 3). Echocardiography showed that pimobendan also improved left ventricular systolic function while reducing the left ventricular end-diastolic dimension (Table 3). The expression of ß-MyHC polypeptide isoform in ventricular myocardium was also markedly decreased in mice treated with pimobendan (Figure 5A).
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Oral administration of pimobendan (30 mg/kg per day) was also found to markedly improve the life span of TNNT2K210/K210 mice (Figure 5B). Pimobendan is a so-called inodilator with inhibitory action on phosphodiesterase 3 as well as Ca2+-sensitizing action on cardiac myofilaments.23 Oral administration of a specific phosphodiesterase 3 inhibitor amrinone had no effect on the survival of TNNT2K210/K210 mice at 30 mg/kg per day and markedly increased the mortality of TNNT2K210/K210 mice at 100 mg/kg per day (Figure 5C); amrinone had no direct effect on the Ca2+ sensitivity of force generation in skinned cardiac muscle fibers (supplemental Figure IV). Echocardiography and blood pressure measurements indicated that amrinone had a weaker positive inotropic effect and a stronger vasodilating effect on TNNT2K210/K210 mice compared with pimobendan (Figure 5D). Amrinone also had a much stronger effect on the phosphorylation of PLB catalyzed by cAMP-dependent protein kinase (Figure 5E). These results strongly suggest that pimobendan exerts its preventive effects on cardiac enlargement, heart failure, and sudden death as a Ca2+ sensitizer. Intraperitoneal administration of ß-adrenergic agonist isoproterenol or antagonist propranolol increased the mortality of TNNT2K210/K210 mice even at a low dose (supplemental Figure V). The dramatic preventive effects of pimobendan on the development of pathology in TNNT2K210/K210 mice provide strong evidence that decreased myofilament Ca2+ sensitivity is the absolute cause of the pathogenesis of DCM associated with the mutation K210 in cTnT and suggest that Ca2+ sensitizers that antagonize the decrease in Ca2+ sensitivity of cardiac myofilaments are potential therapeutic drugs. Cardiomyocytes isolated from pimobendan-treated TNNT2K210/K210 mice no longer showed enhancement of the Ca2+ transient, indicating that pharmacological intervention with pimobendan can prevent or reverse functional remodeling involving the enhancement of intracellular Ca2+ handling (supplemental Figure VIA). Cardiomyocytes of pimobendan-treated TNNT2K210/K210 mice still had a defect of dynamic contractile performance (supplemental Figure VIB), and intact cardiac muscle fibers prepared from pimobendan-treated mice developed significantly lower isometric force than those from wild-type mice (supplemental Figure VIC). Skinned cardiac muscle fibers from pimobendan-treated mice still had decreased Ca2+ sensitivity of force generation, indicating that pharmacological intervention with pimobendan cannot remove the intrinsic defect from myofilaments (supplemental Figure VII).
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Discussion |
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The deletion mutation
K210 in the TNNT2 gene has so far been identified in 4 unrelated families.9,18,19 In a family reported by Kamisago et al,18 this mutation caused frequent sudden death in patients with mild heart failure before 30 years of age, including 2 infants and 3 young adults. In another family reported by Kamisago et al,18 this mutation caused frequent death attributable to congestive heart failure in young patients before 20 years of age, including 2 girls (17 and 19 years old), with marked dilation of ventricles and increased interstitial fibrosis and a boy (15 years old) with a markedly enlarged heart (400g). Early-onset phenotype with a high incidence of sudden death and/or heart failure was also reported in a family reported by Hanson et al.19 In a family with 4 affected individuals reported by Mogensen et al,9 3 patients died or underwent cardiac transplantation in their second or third decade of life; a patient with mild heart failure died suddenly at 36 years, a patient died of heart failure at 26 years, and a patient with very severe heart failure underwent cardiac transplantation at 22 years (weight of the explanted heart, >450 g). The knock-in mice developed enlarged hearts and heart failure and showed a high incidence of premature sudden death, closely recapitulating the clinical phenotypes of human patients; however, it should be noted that this mutation in knock-in mice causes a mutant-gene, dosage-dependent phenotype of DCM. This finding indicates that the DCM phenotype caused by the deletion mutation K210 in cTnT is inherited in a semidominant, additive manner, at least in mice, but not only in a true dominant manner, as implied by a limited number of human DCM patients, which strongly suggests that homozygous patients would exhibit a more severe phenotype than heterozygous patients.
Skinned cardiac muscle fibers prepared from mutant mice showed a significant decrease in the Ca2+ sensitivity of force generation with no change in maximum force-generating capability. Because intact cardiac muscle is known to be never activated beyond the half-maximal level,24 the decrease in Ca2+ sensitivity is expected to cause a reduction in the force generation of the myocardium even if there is no change in the maximum force-generating capability; however, intact cardiac muscle fibers showed no significant reduction in isometric force generated per cross-sectional area. Analyses using a Ca2+ indicator, fura-2, revealed a significant increase in the peak amplitude of Ca2+ transient in cardiomyocytes of mutant mice, which could account for the preserved isometric force-generating capability of intact cardiac muscle fibers. Despite the preserved isometric force-generating capability of intact cardiac muscle fibers, mutant mice developed marked cardiac enlargement, heart failure, and frequent sudden death, strongly suggesting that global functional defect of the heart caused by decreased myofilament Ca2+ sensitivity could not be fully compensated by augmentation of the Ca2+ transient. In this regard, it should be noted that the 2 isoforms of MyHC can produce a similar level of isometric force, but ß-MyHC produces much lower power output because of its much slower sliding velocity and lower ATPase activity compared with -MyHC.25,26 Therefore, the power output should be much lower in the myocardium of mutant mice, with ß-MyHC isoform expression being significantly increased (Figure 4C), consistent with the reduced dynamic contractile performance of cardiomyocytes in TNNT2K210/K210 mice (Figure 3F). Our hypothesis is that reduced power output of myocardium caused by decreased myofilament Ca2+ sensitivity leads to an augmentation of Ca2+ transient as a compensatory response, but an increase in intracellular Ca2+ will increase energy consumption through activating Ca2+-dependent metabolic processes such as the Ca2+ uptake of sarcoplasmic reticulum and cellular hypertrophic response, which in turn would induce the expression of ß-MyHC to save energy consumption. Therefore, the myocardium should repeat this compensatory response involving the augmentation of Ca2+ transient to offset the reduced power output of myocardium until it decompensates.
Mutant mice showed a high incidence of death, and two-thirds of TNNT2K210/K210 mice did not survive to 3 months of age (Figure 4D). In most cases, they died suddenly, without showing overt congestive heart failure symptoms, such as decreased spontaneous movement activity and dyspnea, to at least a day before their death. Instantaneous death was frequently observed by mild chest compression, suggesting that their hearts are very susceptible to fatal arrhythmias caused by mechanical stimuli. Preliminary experiments suggest that cardiomyocytes of mutant mice have a longer action potential duration and greater tendency to develop early afterdepolarization than wild-type mice, which might be responsible for their frequent sudden death (data not shown). Further studies are required to clarify the molecular mechanisms of sudden death, probably involving electrophysiological abnormality of the heart.
Finally, pimobendan had marked effects of preventing cardiac enlargement, heart failure, and sudden death of TNNT2K210/K210 mice exhibiting a particularly malignant phenotype. The results demonstrate that decreased myofilament Ca2+ sensitivity is the absolute causal functional defect triggering the pathogenesis of DCM associated with the mutation K210 in cTnT and suggest that Ca2+ sensitizers, such as pimobendan, might be beneficial for the treatment of DCM patients affected by mutations of the sarcomeric regulatory proteins troponin and -tropomyosin, which have been demonstrated to have a common functional consequence of decreasing myofilament Ca2+ sensitivity in vitro.10,16,17,27 The present study also demonstrated that cardiac myofilament Ca2+ sensitivity, and thus its primary regulator, troponin complex, has an extremely important role in maintaining the physiological structure and function of the heart.
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Acknowledgments |
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We thank Prof Jun-ichi Miyazaki at Osaka University School of Medicine for the kind donation of Cre transgenic mice.
Sources of Funding
This work was supported by Special Coordination Funds (to S.M. and I.O.) and a Grant-in-Aid for Scientific Research on Priority Areas (18059031 to T.I.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and Grants-in-Aid for Science Research (15300136 and 17300129) from the Japan Society for the Promotion of Science (to S.M.). D.-Y.Z. and C.-K.D. are Uehara Memorial Foundation Research Fellows, and Q.-W. L. is a recipient of the Society for the Promotion of Science Postdoctoral Fellowship for Foreign Researchers.
Disclosures
None.
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Footnotes |
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Original received December 13, 2006; revision received May 18, 2007; accepted May 24, 2007.
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References |
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S. Morimoto Sarcomeric proteins and inherited cardiomyopathies Cardiovasc Res, March 1, 2008; 77(4): 659 - 666. [Abstract] [Full Text] [PDF]
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J. P. Davis and S. B. Tikunova Ca2+ exchange with troponin C and cardiac muscle dynamics Cardiovasc Res, March 1, 2008; 77(4): 619 - 626. [Abstract] [Full Text] [PDF]
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P. Robinson, P. J. Griffiths, H. Watkins, and C. S. Redwood Dilated and Hypertrophic Cardiomyopathy Mutations in Troponin and {alpha}-Tropomyosin Have Opposing Effects on the Calcium Affinity of Cardiac Thin Filaments Circ. Res., December 7, 2007; 101(12): 1266 - 1273. [Abstract] [Full Text] [PDF]
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R. J. Solaro Translational Medicine With a Capital T, Troponin T, That Is Circ. Res., July 20, 2007; 101(2): 114 - 115. [Full Text] [PDF]
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