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 ...

Over the last two decades, a large number of mutations have been identified in sarcomeric proteins as a cause of hypertrophic, dilated or restrictive cardiomyopathy. Functional analyses of mutant proteins in vitro have revealed several important functional changes in sarcomeric proteins that might be primarily involved in the pathogenesis of each cardiomyopathy. Creation of transgenic or knock-in animals expressing mutant proteins in their hearts confirmed that these mutations in genes for sarcomeric proteins induced distinct types of cardiomyopathies and provided useful animal models to explore the molecular pathogenic mechanisms and potential therapeutics of cardiomyopathy in vivo. In this review, I discuss the functional consequences of mutations in different sarcomeric proteins found in hypertrophic, dilated, and restrictive cardiomyopathies in conjunction with their effects on cardiac structure and function in vivo and their possible molecular and cellular mechanisms, which underlie the pathogenesis of these inherited cardiomyopathies.

A deletion mutation ⌬K210 in cardiac troponin T (cTnT) was recently found to cause familial dilated cardiomyopathy (DCM). To explore the effect of this mutation on cardiac muscle contraction under physiological conditions, we determined the Ca 2؉ -activated force generation in permeabilized rabbit cardiac muscle fibers into which the mutant and wild-type cTnTs were incorporated by using our TnT exchange technique. The free Ca 2؉ concentrations required for the force generation were higher in the mutant cTnTexchanged fibers than in the wild-type cTnT-exchanged ones, with no statistically significant differences in maximal force-generating capability and cooperativity. Exchanging the mutant cTnT into isolated cardiac myofibrils also increased the free Ca 2؉ concentrations required for the activation of ATPase. In contrast, a deletion mutation ⌬E160 in cTnT that causes familial hypertrophic cardiomyopathy (HCM) decreased the free Ca 2؉ concentrations required for force generation, just as in the case of the other HCM-causing mutations in cTnT. The results indicate that cTnT mutations found in the two distinct forms of cardiomyopathy (i.e., HCM and DCM) change the Ca 2؉ sensitivity of cardiac muscle contraction in opposite directions. The present study strongly suggests that Ca 2؉ desensitization of force generation in sarcomere is a primary mechanism for the pathogenesis of DCM associated with the deletion mutation ⌬K210 in cTnT.C ontraction of the vertebrate-striated muscles (i.e., skeletal and cardiac muscles) is regulated by Ca 2ϩ through its binding to a specific regulatory protein complex, troponin (Tn), which is distributed at regular intervals along the entire thin filament (1, 2). Tn is a complex of three different proteins, troponin T (TnT; tropomyosin-binding component), troponin I (TnI; inhibitory component), and troponin C (TnC; Ca 2ϩ -binding component). On Ca 2ϩ binding to TnC, a Ca 2ϩ -induced interaction of TnC with TnI relieves the inhibitory action of TnI exerted on the thin filament and enables the myosin head to cyclically interact with actin in the thin filament and generate force. The Ca 2ϩ sensitivity of muscle contraction is determined by the Ca 2ϩ -binding affinity of TnC, which is dynamically altered through interaction with TnI and TnT in the myofilament lattice (3-8).Mutations in genes for cardiac troponin T (cTnT) and cardiac troponin I (cTnI) have been found to cause familial hypertrophic cardiomyopathy (HCM), an autosomal dominant heart disease characterized by asymmetrical ventricular hypertrophy with a high incidence of sudden death in young adults (9). We have already examined the effects of eight HCM-linked cTnT mutations (I79N, R92Q, ⌬E160, E244D, R278C, and two truncated mutants produced by a splice donor site mutation Int15G 1 3A) and six HCM-linked cTnI mutations (R145G, R145Q, R162W, ⌬K183, G203S, and K206Q) on the contractile functions of cardiac muscle by using a technique for exchanging the exogenous Tn complex into skinned muscle fibers and isolated myofibrils. We found t...

We conclude that DCM-causing mutations in thin filament proteins abolish the relationship between myofilament Ca(2+) sensitivity and troponin I phosphorylation by PKA. We propose that this blunts the response to β-adrenergic stimulation and could be the cause of DCM in the long term.

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In search of chemical substances applicable for the treatment of cancer and other proliferative disorders, we studied the signal transduction of Dictyostelium differentiation-inducing factors (DIFs) in mammalian cells mainly using HeLa cells. Although DIF-1 and DIF-3 both strongly inhibited cell proliferation by inducing G 0 /G 1 arrest, DIF-3 was more effective than DIF-1. DIF-3 suppressed cyclin D1 expression at both mRNA and protein levels, whereas the overexpression of cyclin D1 overrode DIF-3-induced cell cycle arrest. The DIF-3-induced decrease in the amount of cyclin D1 protein preceded the reduction in the level of cyclin D1 mRNA. The decrease in cyclin D1 protein seemed to be caused by accelerated proteolysis, since it was abrogated by N-acetyl-Leu-Leu-norleucinal, a proteasome inhibitor. DIF-3-induced degradation of cyclin D1 was also prevented by treatment with lithium chloride, an inhibitor of glycogen synthase kinase-3␤ (GSK-3␤), suggesting that DIF-3 induced cyclin D1 proteolysis through the activation of GSK-3␤. Indeed, DIF-3 dephosphorylated Ser 9 and phosphorylated tyrosine on GSK-3␤, and it stimulated GSK-3␤ activity in an in vitro kinase assay. Moreover, DIF-3 was revealed to induce the nuclear translocation of GSK-3␤ by immunofluorescent microscopy and immunoblotting of subcellular protein fractions. These results suggested that DIF-3 activates GSK-3␤ to accelerate the proteolysis of cyclin D1 and that this mechanism is involved in the DIF-3-induced G 0 /G 1 arrest in mammalian cells.Differentiation-inducing factors (DIFs) 1 were identified in Dictyostelium discoideum as the morphogens required for stalk cell differentiation of Dictyostelium (1). In the DIF family, DIF-1 (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-1-hexanone) was the first to be identified, and DIF-3, the monochlorinated analogue of DIF-1, is a natural metabolite of DIF-1 in Dictyostelium (2). However, the actions of DIFs are not limited to Dictyostelium. They also have strong effects on mammalian cells. DIF-1 and/or DIF-3 strongly inhibit proliferation and induce differentiation in several leukemia cells, such as the murine erythroleukemia cell line B8, human leukemia cell line K562, and human myeloid leukemia cell line HL-60 (3, 4). DIF-3 has been reported to have the most potent antiproliferative effect on mammalian leukemia cells among DIF analogues examined to date (5). Recently, we found that DIF-1 strongly inhibits proliferation and induces differentiation in human vascular smooth muscle cells, indicating that cells sensitive to DIFs are not limited to transformed cells (6).However, the target molecule (receptor) of DIFs is unknown, and it is not clear even in Dictyostelium how DIFs induce an antiproliferative effect and cell differentiation. DIFs are small hydrophobic molecules and are therefore expected to be able to cross cell membranes without requiring channels or carriers. Also, the rapidity with which DIFs induce prestalk cell-specific gene expression suggests that they directly regulate gene expression. The...

BACKGROUND AND PURPOSE Catechins, biologically active polyphenols in green tea, are known to have a protective effect against cardiovascular diseases. In this study, we investigated direct actions of green tea catechins on cardiac muscle function to explore their uses as potential drugs for cardiac muscle disease. EXPERIMENTAL APPROACH The effects of catechins were systematically investigated on the force‐pCa relationship in skinned cardiac muscle fibres to determine their direct effects on cardiac myofilament contractility. The mechanisms of action of effective catechins were investigated using troponin exchange techniques, quartz crystal microbalance, nuclear magnetic resonance and a transgenic mouse model. KEY RESULTS (‐)‐Epicatechin‐3‐gallate (ECg) and (‐)‐epigallocatechin‐3‐gallate (EGCg), but not their stereoismers (‐)‐catechin‐3‐gallate and (‐)‐gallocatechin‐3‐gallate, decreased cardiac myofilament Ca2+ sensitivity probably through its interaction with cardiac troponin C. EGCg restored cardiac output in isolated working hearts by improving diastolic dysfunction caused by increased myofilament Ca2+ sensitivity in a mouse model of hypertrophic cardiomyopathy. CONCLUSIONS AND IMPLICATIONS The green tea catechins, ECg and EGCg, are Ca2+ desensitizers acting through binding to cardiac troponin C. These compounds might be useful compounds for the development of therapeutic agents to treat the hypertrophic cardiomyopathy caused by increased Ca2+ sensitivity of cardiac myofilaments.

Several mutations in human cardiac troponin T (TnT) gene have been reported to cause hypertrophic cardiomyopathy (HCM). To explore the effects of the mutations on cardiac muscle contractile function under physiological conditions, human cardiac TnT mutants, Ile79Asn and Arg92Gln, as well as wild type, were expressed in Escherichia coli and exchanged into permeabilized rabbit cardiac muscle fibers, and Ca2+-activated force was determined. The free Ca2+ concentrations required for tension generation were found to be significantly lower in the mutant TnT-exchanged fibers than in the wild-type TnT-exchanged fibers, whereas no significant differences were found in tension-generating capability under maximal activating conditions and in cooperativity. These results suggest that a heightened Ca2+ sensitivity of cardiac muscle contraction is one of the factors to cause HCM associated with these TnT mutations.