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In cats, mutations in myosin binding protein C (encoded by the MYBPC3 gene) have been associated with hypertrophic cardiomyopathy (HCM). However, the molecular mechanisms linking these mutations to HCM remain unknown. Here, we establish Drosophila melanogaster as a model to understand this connection by generating flies harboring MYBPC3 missense mutations (A31P and R820W) associated with feline HCM. The A31P and R820W flies displayed cardiovascular defects in their heart rates and exercise endurance. We used RNA-seq to determine which processes are misregulated in the presence of mutant MYBPC3 alleles. Transcriptome analysis revealed significant downregulation of genes encoding small nucleolar RNA (snoRNAs) in exercised female flies harboring the mutant alleles compared to flies that harbor the wild-type allele. Other processes that were affected included the unfolded protein response and immune/defense responses. These data show that mutant MYBPC3 proteins have widespread effects on the transcriptome of co-regulated genes. Transcriptionally differentially expressed genes are also candidate genes for future evaluation as genetic modifiers of HCM as well as candidate genes for genotype by exercise environment interaction effects on the manifestation of HCM; in cats as well as humans.

Key points The Anrep effect represents the alteration of left ventricular (LV) contractility to acutely enhanced afterload in a few seconds, thereby preserving stroke volume (SV) at constant preload. As a result of the missing preload stretch in our model, the Anrep effect differs from the slow force response and has a different mechanism. The Anrep effect demonstrated two different phases. First, the sudden increased afterload was momentary equilibrated by the enhanced LV contractility as a result of higher power strokes of strongly-bound myosin cross-bridges. Second, the slightly delayed recovery of SV is perhaps dependent on Ca2+ /calmodulin-dependent protein kinase II activation caused by oxidation and myofilament phosphorylation (cardiac myosin-binding protein-C, myosin light chain 2), maximizing the recruitment of available strongly-bound myosin cross-bridges. Short-lived oxidative stress might present a new facet of subcellular signalling with respect to cardiovascular regulation. Relevance for human physiology was demonstrated by echocardiography disclosing the Anrep effect in humans during handgrip exercise. Abstract The present study investigated whether oxidative stress and Ca2+ /calmodulin-dependent protein kinase II (CaMKII) activity are involved in triggering the Anrep effect. LV pressure-volume (PV) analyses of isolated, preload controlled working hearts were performed at two afterload levels (60 and 100 mmHg) in C57BL/6N wild-type (WT) and CaMKII-double knockout mice (DKOCaMKII ). In snap-frozen WT hearts, force-pCa relationship, H2 O2 generation, CaMKII oxidation and phosphorylation of myofilament and Ca2+ handling proteins were assessed. Acutely raised afterload showed significantly increased wall stress, H2 O2 generation and LV contractility in the PV diagram with an initial decrease and recovery of stroke volume, whereas end-diastolic pressure and volume, as well as heart rate, remained constant. Afterload induced increase in LV contractility was blunted in DKOCaMKII -hearts. Force development of single WT cardiomyocytes was greater with elevated afterload at submaximal Ca2+ concentration and associated with increases in CaMKII oxidation and phosphorylation of cardiac-myosin binding protein-C, myosin light chain and Ca2+ handling proteins. CaMKII activity is involved in the regulation of the Anrep effect and associates with stimulation of oxidative stress, presumably starting a cascade of CaMKII oxidation with downstream phosphorylation of myofilament and Ca2+ handling proteins. These mechanisms improve LV inotropy and preserve stroke volume within few seconds.

Background: Dilated cardiomyopathy (DCM) is a relatively common clinical cardiac condition, and its progression will eventually lead to heart failure. Cardiac myosin-binding protein C (cMyBP-c) plays a vital role in the diastolic and contractile function of the heart muscle. At present, most of the relevant researches are focused on the genetic level. Our study discusses the expression of cMyBP-C in patients with DCM and its potential clinical application. Methodology: One hundred and twenty-two subjects were selected from the First Affiliated Hospital of Nanjing Medical University from August 2016 to October 2017. They were divided into two groups according to the left ventricular end-diastolic dimension (LVDd) as measured by echocardiography: normal (n = 34) and left ventricular enlargement (LVE) groups (n = 88). The LVE group was further divided into two subgroups according to the etiology: DCM (n = 57), and LVE due to hypertension (LVEH, n = 31). The left ventricular ejection fraction (EF%) was also defined in each group. Enzyme-linked immunosorbent assay (ELISA) was used for the determination of cMyBP-C in the serum. Results: (1) Serum cMyBP-C concentration was higher in DCM than in LVEH and control groups (P < 0.05) and (2) the LVDd, EF%, creatinine (Cr), urea (Ur), and uric acid (UA) in the DCM group were significantly higher than those in the LVEH group (P < 0.05). Conclusion: The serum level of cMyBP-C is expected to become a new biomarker for the diagnosis of DCM. Cr, Ur, and UA may be factors contributing to the development of DCM.

Las cardiomiopatías genéticas o adquiridas constituyen una importante causa de muerte en personas de todas las edades. En su etiología, juegan un papel relevante mutaciones en genes que codifican proteínas sarcoméricas y modificaciones postraduccionales de las mismas. En el presente estudio se evaluaron propiedades mecánicas de cardiomiocitos individuales permeabilizados, en el contexto de dos formas de cardiomiopatía. Se logró una parte de la optimización de los ensayos de sensibilidad al calcio para posteriormente ser trasladados a un modelo murino de miocardiopatía hipertrófica con mutación puntual en la proteína C de unión a miosina (cMyBP-C). Esta mutación puntual en cMyBP-C (R502W) es la mutación más frecuente en miocardiopatía hipertrófica. Dentro de las cardiomiopatías adquiridas, se conoce que la presencia de productos avanzados de glicación (AGEs) podrían ser un factor que contribuya al desarrollo de cardiomiopatía diabética. De esta forma, se evaluó la rigidez de cardiomiocitos murinos expuestos a procesos de glicación, encontrando que el agente metilglioxal parece no tener efecto sobre el desarrollo de fuerza pasiva generada en los cardiomiocitos en nuestras condiciones experimentales

© 2016 Elsevier Inc. All rights reserved. Mutations in MYBPC3, the gene encoding cardiac myosin binding protein C (cMyBP-C), are a major cause of hypertrophic cardiomyopathy (HCM). While most mutations encode premature stop codons, missense mutations causing single amino acid substitutions are also common. Here we investigated effects of a single proline for alanine substitution at amino acid 31 (A31P) in the C0 domain of cMyBP-C, which was identified as a natural cause of HCM in cats. Results using recombinant proteins showed that the mutation disrupted C0 structure, altered sensitivity to trypsin digestion, and reduced recognition by an antibody that preferentially recognizes N-terminal domains of cMyBP-C. Western blots detecting A31P cMyBP-C in myocardium of cats heterozygous for the mutation showed a reduced amount of A31P mutant protein relative to wild-type cMyBP-C, but the total amount of cMyBP-C was not different in myocardium from cats with or without the A31P mutation indicating altered rates of synthesis/degradation of A31P cMyBP-C. Also, the mutant A31P cMyBP-C was properly localized in cardiac sarcomeres. These results indicate that reduced protein expression (haploinsufficiency) cannot account for effects of the A31P cMyBP-C mutation and instead suggest that the A31P mutation causes HCM through a poison polypeptide mechanism that disrupts cMyBP-C or myocyte function.