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To date, no compounds or interventions exist that treat or prevent sarcomeric cardiomyopathies. Established therapies currently improve the outcome, but novel therapies may be able to more fundamentally affect the disease process and course. Investigations of the pathomechanisms are generating molecular insights that can be useful for the design of novel specific drugs suitable for clinical use. As perturbations in the heart are stage-specific, proper timing of drug treatment is essential to prevent initiation and progression of cardiac disease in mutation carrier individuals. In this review, we emphasize potential novel therapies which may prevent, delay, or even reverse hypertrophic cardiomyopathy caused by sarcomeric gene mutations. These include corrections of genetic defects, altered sarcomere function, perturbations in intracellular ion homeostasis, and impaired myocardial energetics.

Aims Hypertrophic cardiomyopathy (HCM) has been associated with reduced β-adrenergic receptor (b-AR) signalling, leading downstream to a low protein kinase A (PKA)-mediated phosphorylation. It remained undefined whether all PKA targets will be affected similarly by diminished b-AR signalling in HCM. We aimed to investigate the role of b-AR signalling on regulating myofilament and calcium handling in an HCM mouse model harbouring a gene mutation (G . A transition on the last nucleotide of exon 6) in Mybpc3 encoding cardiac myosin-binding protein C. Methods and results Cardiomyocyte contractile properties and phosphorylation state were measured in left ventricular permeabilized and intact cardiomyocytes isolated from heterozygous (HET) or homozygous (KI) Mybpc3-targeted knock-in mice. Significantly higher myofilament Ca2+ sensitivity and passive tension were detected in KI mice, which were normalized after PKA treatment. Loaded intact cardiomyocyte force-sarcomere length relation was impaired in both HET and KI mice, suggesting a reduced length-dependent activation. Unloaded cardiomyocyte function revealed an impaired myofilament contractile response to isoprenaline (ISO) in KI, whereas the calcium-handling response to ISO was maintained. This disparity was explained by an attenuated increase in cardiac troponin I (cTnI) phosphorylation in KI, whereas the increase in phospholamban (PLN) phosphorylation was maintained to wild-type values. Conclusion These data provide evidence that in the KI HCM mouse model, b-AR stimulation leads to preferential PKA phosphorylation of PLN over cTnI, resulting in an impaired inotropic and lusitropic response. [ABSTRACT FROM AUTHOR]

Abstract: Objective: β-Adrenergic stimulation modulates cardiac contractility through protein kinase A (PKA), which phosphorylates proteins such as troponin I (cTnI) and C-protein (cMyBP-C). The relative contributions of cTnI and cMyBP-C to the regulation of myofilament Ca2+ sensitivity are still controversial because of difficulty in targeting specific protein phosphorylation. Recently, impaired relaxation was found in cMyBP-C-deficient mice (KO) in vivo under basal conditions and after β-adrenergic stimulation. The goal of this study was to analyse the length-dependent and PKA-dependent modulations of the cardiac contractile machinery in a mouse model lacking cMyBP-C. Methods: In the present work, we studied the PKA effect on myofilament Ca2+ sensitivity of left ventricular skinned myocytes isolated from 5-week- and 55-week-old wild-type (WT) and cMyBP-C knockout (KO) mice at 1.9 and 2.3 μm sarcomere lengths (SL). The cTnI content and phosphorylation status were examined by Western blot analysis. Results: Without PKA stimulation and at the shorter SL, Ca2+ sensitivity was higher in KO compared to WT. The difference disappeared at the longer SL. No difference in passive tension or maximal active tension was observed. PKA stimulation induced a desensitization of WT myofilaments at both SL but had almost no effect in KO myofilaments despite similar levels of cTnI phosphorylation. We also observed expression of slow skeletal TnI in KO animals that was not correlated with the PKA effects. Conclusion: The results suggest that cMyBP-C contributes to the regulation of cardiac contraction at short sarcomere length and that myofilament desensitization induced by PKA requires the presence of cMyBP-C and does not depend only upon TnI phosphorylation. [Copyright &y& Elsevier]

Abstract: Objective: Most cardiac myosin binding protein C (cMyBP-C) gene mutations causing familial hypertrophic cardiomyopathy (FHC) result in C-terminal truncated proteins. However, truncated cMyBP-Cs were undetectable in myocardial tissue of FHC patients. In the present study, we investigated whether truncated cMyBP-Cs are subject to accelerated degradation by the lysosome or ubiquitin–proteasome system (UPS). Methods and results: By using an adenovirus-based approach, we analyzed expression and localization of myc-tagged truncated proteins (M6t 3%, M7t 80% truncation, both mutations have been identified in FHC patients) compared to wild type (WT) in neonatal rat cardiomyocytes. Despite similar mRNA levels, protein expression of M6t and M7t was markedly lower than WT (70±4% and 11±5% of WT, respectively, p<0.05). M6t exhibited weak incorporation in the sarcomere, whereas M7t was mis-incorporated at the Z-disk and formed ubiquitin-positive aggregates. The lysosome inhibitor bafilomycin only slightly raised the protein level of M7t, whereas the UPS inhibitors lactacystin or MG132 markedly raised M6t and M7t to WT level. Using an adenovirus encoding a fluorescent reporter of UPS activity, we demonstrate that mutant cMyBP-Cs impair the proteolytic capacity of the UPS. Conclusion: Truncated cMyBP-Cs are preferentially degraded by the UPS, which, in turn, may competitively inhibit breakdown of other UPS substrates. Since the UPS plays an important role in a variety of fundamental cellular processes, we propose impairment of this system by mutant cMyBP-Cs as a contributing factor to the pathogenesis of FHC. [Copyright &y& Elsevier]

Objective: Cardiac myosin-binding protein C (cMyBP-C) gene mutations are involved in familial hypertrophic cardiomyopathy (FHC). Many of these mutations produce truncated proteins, which are unstable in the cardiac tissue of patients, suggesting that haploinsufficiency could account for the development of the phenotype. However, existing mouse models of cMyBP-C gene mutations have represented hypomorphic alleles without evidence of asymmetric septal hypertrophy, a key FHC phenotypic feature. In the present study, we generated a new model of cMyBP-C null mice and characterized the phenotype in both homozygotes and heterozygotes at different ages. Methods: The mouse model was based upon the targeted deletion of exons 1 and 2, which contain the transcription initiation site, and the phenotype was determined by molecular, functional and morphological analyses. Results: Herein, we demonstrate that inactivation of one or two mouse cMyBP-C alleles leads to different cardiac disorders at different post-natal time windows. The homozygous cMyBP-C null mice do not express the cMyBP-C gene, develop eccentric left ventricular hypertrophy with decreased fractional shortening at 3–4 months of age and a markedly impaired relaxation after 9 months. This is associated with myocardial disarray and an increase of interstitial fibrosis. The heterozygous cMyBP-C null mice present a slight but significant decrease of cMyBP-C amount and develop asymmetric septal hypertrophy associated with fibrosis at 10–11 months of age. Conclusion: These data provide evidence that heterozygous cMyBP-C null mice represent the first model with a key feature of human FHC that is asymmetric septal hypertrophy. [Copyright &y& Elsevier]

Objective: Cardiac myosin-binding protein C (cMyBP-C) is a component of sarcomere that contains at least three putative myosin-binding sites. Mutations in its gene are implicated in familial hypertrophic cardiomyopathy (FHC) and most of them are predicted to produce C-terminal truncated cMyBP-Cs. The aim of the present study was to analyze whether cMyBP-C truncated mutants resulting from FHC mutations interact in vitro with human β-MyHC. Methods: Recombinant proteins were produced using the baculovirus/insect cell system, and wild type and three truncated cMyBP-Cs were purified using metal affinity chromatography. The interaction between recombinant proteins was analyzed in real time using biosensor technology on immobilized anti-β-MyHC antibodies. Results: Biomolecular interaction with β-MyHC was detected for both wild type cMyBP-C and a truncated mutant lacking half of the C-terminal C10 domain. In contrast, no interaction with β-MyHC was found for two truncated cMyBP-Cs lacking at least the C5–C9 region. Conclusions: Biosensor technology allows in vitro analysis of the interaction between human β-MyHC and cMyBP-C mutants resulting from FHC mutations. The data show that the interaction depends on the size of the truncation. This suggests that, in the context of FHC, impairment of suitable interaction between β-MyHC and some of the truncated cMyBP-Cs may promote degradation of the truncated proteins and therefore contribute to the development of the disease. [Copyright &y& Elsevier]

Echocardiography is a reliable and reproducible method to assess non-invasively cardiac function in clinical and experimental research. Significant progress in the development of echocardiographic equipment and transducers has led to the successful translation of this methodology from humans to rodents, allowing for the scoring of disease severity and progression, testing of new drugs, and monitoring cardiac function in genetically modified or pharmacologically treated animals. However, as yet, there is no standardization in the procedure to acquire echocardiographic measurements in small animals. This position paper focuses on the appropriate acquisition and analysis of echocardiographic parameters in adult mice and rats, and provides reference values, representative images, and videos for the accurate and reproducible quantification of left ventricular function in healthy and pathological conditions., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.)

Objective: Cardiac myosin-binding protein C (cMyBP-C) is a component of sarcomere that contains at least three putative myosin-binding sites. Mutations in its gene are implicated in familial hypertrophic cardiomyopathy (FHC) and most of them are predicted to produce C-terminal truncated cMyBP-Cs. The aim of the present study was to analyze whether cMyBP-C truncated mutants resulting from FHC mutations interact in vitro with human beta-MyHC., Methods: Recombinant proteins were produced using the baculovirus/insect cell system, and wild type and three truncated cMyBP-Cs were purified using metal affinity chromatography. The interaction between recombinant proteins was analyzed in real time using biosensor technology on immobilized anti-beta-MyHC antibodies., Results: Biomolecular interaction with beta-MyHC was detected for both wild type cMyBP-C and a truncated mutant lacking half of the C-terminal C10 domain. In contrast, no interaction with beta-MyHC was found for two truncated cMyBP-Cs lacking at least the C5-C9 region., Conclusions: Biosensor technology allows in vitro analysis of the interaction between human beta-MyHC and cMyBP-C mutants resulting from FHC mutations. The data show that the interaction depends on the size of the truncation. This suggests that, in the context of FHC, impairment of suitable interaction between beta-MyHC and some of the truncated cMyBP-Cs may promote degradation of the truncated proteins and therefore contribute to the development of the disease.