Knock-In Mouse Model of Dilated Cardiomyopathy Caused by Troponin Mutation

Abstract

Abstract-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 Ca 2ϩ sensitivity in force generation than those from wild-type mice. Peak amplitude of Ca 2ϩ 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 Ca 2ϩ transient was augmented to compensate for decreased myofilament Ca 2ϩ 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 Ca 2ϩ sensitivity could not be fully compensated by only increasing the intracellular Ca 2ϩ transient. We found that a positive inotropic agent, pimobendan, which directly increases myofilament Ca 2ϩ sensitivity, had profound effects of preventing cardiac enlargement, heart failure, and sudden death. These results verify the hypothesis that Ca 2ϩ desensitization of cardiac myofilament is the absolute cause of the pathogenesis of dilated cardiomyopathy associated with this mutation and strongly suggest that Ca 2ϩ sensitizers are beneficial for the treatment of dilated cardiomyopathy patients affected by sarcomeric regulatory protein mutations. (Circ Res. 2007;101:185-194.)Key Words: dilated cardiomyopathy Ⅲ troponin Ⅲ mutation Ⅲ calcium sensitivity Ⅲ knock-in mouse D ilated 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, ␤-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][6][7] Cardiac muscle contraction is regulated through Ca 2ϩ 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 ...

Generation and Functional Characterization of Knock-in Mice Harboring the Cardiac Troponin I-R21C Mutation Associated with Hypertrophic Cardiomyopathy

“…The severity of the HCM phenotype does not seem to be dependent on the R21C mutant gene dosage because ϳ25% of the total cTnI is mutated in the heterozygotes and is sufficient to promote a similar hypertrophic response to that of the homozygous mice. Nevertheless, previous studies have shown that an increase in the ratio of mutant to WT protein in vivo and in vitro within myofilaments can lead to larger phenotypic and/or lethal effects (43). Therefore, in vivo hemodynamic and echocardiographic parameters of the R21C KI mice will have to be examined to clearly determine whether the mutant gene dosage alters the severity of the R21C HCM phenotype in mice.…”

“…For example, Messer et al (45) demonstrated that in the donor heart samples, the bisphosphorylated species predominated (72.2%); whereas, in diseased hearts, the dephosphorylated species of cTnI predominated (ϳ77%). Notably, phosphorylation of Ser 43 and Thr…”

“…Ser 23 /Ser 24 to alanine) or lacking both the PKA and PKC phosphorylation sites (i.e. Ser 43 /Ser 45 and Thr 144 ) did not respond by decreasing the Ca 2ϩ sensitivity of contraction upon treatment with PKA; whereas, the WT mice decreased their pCa-tension relationship 0.28 pCa units (48,49). Furthermore, Kentish et al (17) have shown that overexpression of slow skeletal TnI, which lacks the N-terminal domain, did not decrease the myofilament Ca 2ϩ sensitivity upon PKA treatment.…”

Engineered Troponin C Constructs Correct Disease-related Cardiac Myofilament Calcium Sensitivity

“…RCM TnI R192H, DCM TnT ⌬K210, and ischemia-induced truncated TnI- were chosen to test the hypothesis because they represent different protein (TnI or TnT) modifications (missense mutation, deletion, or truncation) and disease subtypes (familial or acquired) that can afflict both mouse and humans (27,47,48). By engineering TnC with a wide, yet fine-tunable, range of Ca 2ϩ sensitivities, abnormally increased or decreased Ca 2ϩ binding associated with different cardiac dysfunctions can be corrected, and their altered Ca 2ϩ dissociation rates can be reversed.…”

“…Strikingly, for any particular class of inherited cardiomyopathies, the apparent Ca 2ϩ sensitivity of TnC and force development are typically altered in a qualitatively similar manner (20,22,25). Furthermore, pharmacological and genetic interventions that rectify the apparent Ca 2ϩ sensitivity of cardiac muscle in transgenic animal models harboring cardiomyopathic genes show promise of alleviating the disease symptoms (27)(28)(29). For instance, modulating TnI, TnT, or tropomyosin (each of which can indirectly tune the Ca 2ϩ sensitivity of TnC) counteracted the abnormal cardiac muscle Ca 2ϩ sensitivities and ameliorated the disease symptoms (28 -30).…”