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Aims  Dilated cardiomyopathy (DCM) is associated with mutations in many genes encoding sarcomere proteins. Truncating mutations in the titin gene TTN are the most frequent. Proteomic and functional characterizations are required to elucidate the origin of the disease and the pathogenic mechanisms of TTN- truncating variants. Methods and results  We isolated myofibrils from DCM hearts carrying truncating TTN mutations and measured the Ca2+ sensitivity of force and its length dependence. Simultaneous measurement of force and adenosine triphosphate (ATP) consumption in skinned cardiomyocytes was also performed. Phosphorylation levels of troponin I (TnI) and myosin binding protein-C (MyBP-C) were manipulated using protein kinase A and λ phosphatase. mRNA sequencing was employed to overview gene expression profiles. We found that Ca2+ sensitivity of myofibrils carrying TTN mutations was significantly higher than in myofibrils from donor hearts. The length dependence of the Ca2+ sensitivity was absent in DCM myofibrils with TTN -truncating variants. No significant difference was found in the expression level of TTN mRNA between the DCM and donor groups. TTN exon usage and splicing were also similar. However, we identified down-regulation of genes encoding Z-disk proteins, while the atrial-specific regulatory myosin light chain gene, MYL7, was up-regulated in DCM patients with TTN -truncating variants. Conclusion  Titin - truncating mutations lead to decreased length-dependent activation and increased elasticity of myofibrils. Phosphorylation levels of TnI and MyBP-C seen in the left ventricles are essential for the length-dependent changes in Ca2+ sensitivity in healthy donors, but they are reduced in DCM patients with TTN -truncating variants. A decrease in expression of Z-disk proteins may explain the observed decrease in myofibril passive stiffness and length-dependent activation. [ABSTRACT FROM AUTHOR]

Aims: Heart muscle contraction is regulated via the β-adrenergic response that leads to phosphorylation of Troponin I (TnI) at Ser22/23, which changes the Ca2+ sensitivity of the cardiac myofilament. Mutations in thin filament proteins that cause dilated cardiomyopathy (DCM) and some mutations that cause hypertrophic cardiomyopathy (HCM) abolish the relationship between TnI phosphorylation and Ca2+ sensitivity (uncoupling). Small molecule Ca2+ sensitizers and Ca2+ desensitizers that act upon troponin alter the Ca2+ sensitivity of the thin filament, but their relationship with TnI phosphorylation has never been studied before. Methods and results: Quantitative in vitro motility assay showed that 30 µM EMD57033 and 100 µM Bepridil increase Ca2+ sensitivity of phosphorylated cardiac thin filaments by 3.1- and 2.8-fold, respectively. Additionally they uncoupled Ca2+ sensitivity from TnI phosphorylation, mimicking the effect of HCM mutations. Epigallocatechin-3-gallate (EGCG) decreased Ca2+ sensitivity of phosphorylated and unphosphorylated wild-type thin filaments equally (by 2.15 ± 0.45- and 2.80+0.48- fold, respectively), retaining the coupling. Moreover, EGCG also reduced Ca2+ sensitivity of phosphorylated but not unphosphorylated thin filaments containing DCM and HCM-causing mutations; thus, the dependence of Ca2+ sensitivity upon TnI phosphorylation of uncoupled mutant thin filaments was restored in every case. In single mouse heart myofibrils, EGCG reduced Ca2+ sensitivity of force and kACT and also preserved coupling. Myofibrils from the ACTC E361G (DCM) mouse were uncoupled; EGCG reduced Ca2+ sensitivity more for phosphorylated than for unphosphorylated myofibrils, thus restoring coupling. Conclusion: We conclude that it is possible to both mimic and reverse the pathological defects in troponin caused by cardiomyopathy mutations pharmacologically. Re-coupling by EGCG may be of potential therapeutic significance for treating cardiomyopathies. [ABSTRACT FROM AUTHOR]

We compared the contractile performance of papillary muscle from a mouse model of hypertrophic cardiomyopathy [α-cardiac actin (ACTC) E99K mutation] with nontransgenic (non-TG) littermates. In isometric twitches, ACTC E99K papillary muscle produced three to four times greater force than non-TG muscle under the same conditions independent of stimulation frequency and temperature, whereas maximum isometric force in myofibrils from these muscles was not significantly different. ACTC E99K muscle relaxed slower than non-TG muscle in both papillary muscle (1.4×) and myofibrils (1.7×), whereas the rate of force development after stimulation was the same as non-TG muscle for both electrical stimulation in intact muscle and after a Ca2+ jump in myofibrils. The EC50 for Ca2+ activation of force in myofibrils was 0.39 ± 0.33 μmol/l in ACTC E99K myofibrils and 0.80 ± 0.11 μmol/l in non-TG myofibrils. There were no significant differences in the amplitude and time course of the Ca2+ transient in myocytes from ACTC E99K and non-TG mice. We conclude that hypercontractility is caused by higher myofibrillar Ca2+ sensitivity in ACTC E99K muscles. Measurement of the energy (work + heat) released in actively cycling heart muscle showed that for both genotypes, the amount of energy turnover increased with work done but with decreasing efficiency as energy turnover increased. Thus, ACTC E99K mouse heart muscle produced on average 3.3-fold more work than non-TG muscle, and the cost in terms of energy turnover was disproportionately higher than in non-TG muscles. Efficiency for ACTC E99K muscle was in the range of 11-16% and for non-TG muscle was 15-18%. [ABSTRACT FROM AUTHOR]

About half of hypertrophic and dilated cardiomyopathies cases have been recognized as genetic diseases with mutations in sarcomeric proteins. The sarcomeric proteins are involved in cardiomyocyte contractility and its regulation, and play a structural role. Mutations in non-sarcomeric proteins may induce changes in cell signaling pathways that modify contractile response of heart muscle. These facts strongly suggest that contractile dysfunction plays a central role in initiation and progression of cardiomyopathies. In fact, abnormalities in contractile mechanics of myofibrils have been discovered. However, it has not been revealed how these mutations increase risk for cardiomyopathy and cause the disease. Much research has been done and still much is being done to understand how the mechanism works. Here, we review the facts of cardiac myofilament contractility in patients with cardiomyopathy and heart failure. [ABSTRACT FROM AUTHOR]