Reduced Contraction and Altered Frequency Response of Isolated Ventricular Myocytes From Patients With Heart Failure

Background/Introduction: Mitochondria are dynamic regulators of cellular metabolism and homeostasis. The dysfunction of mitochondria has long been considered a major contributor to aging and age-related diseases. The prognosis of severe heart failure is still unacceptably poor and it is urgent to establish new therapies for this critical condition. Some patients with heart failure do not respond to established multidisciplinary treatment and they are classified as “non-responders”. The outcome is especially poor for non-responders, and underlying mechanisms are largely unknown. Purpose: Studies indicate mitochondrial dysfunction has causal roles for metabolic remodeling in the failing heart, but underlying mechanisms remain to be explored. This study tried to elucidate the role of Mitofusin-1 in a failing heart. Methods: We examined twenty-two heart failure patients who underwent endomyocardial biopsy of intraventricular septum. Patients were classified as non-responders when their left-ventricular (LV) ejection fraction did not show more than 10% improvement at remote phase after biopsy. Fourteen patients were classified as responders, and eight as non-responders. Electron microscopy, quantitative PCR, and immunofluorescence studies were performed to explore the biological processes or molecules involved in failure to respond. In addition to studies with cardiac tissue specific knockout mice, we also conducted functional in-vitro studies with neonatal rat ventricular myocytes. Results: Twenty-two patients with IDCM who underwent endomyocardial biopsy were enrolled in this study, including 14 responders and 8 non-responders. Transmission electron microscopy (EM) showed a significant reduction in mitochondrial size in cardiomyocytes of non-responders compared to responders. Quantitative PCR revealed that transcript of mitochondrial fusion protein, Mitofusin-1, was significantly reduced in non-responders. Studies with neonatal rat ventricular myocytes (NRVMs) indicated that the beta-1 adrenergic receptor-mediated signaling pathway induced microRNA-140 3p and 5p, which is negatively regulated Mitofusin-1 expression. Suppression of Mitofusin-1 resulted in a significant reduction in mitochondrial respiration of NRVMs. We generated left ventricular pressure overload model with thoracic aortic constriction (TAC) in cardiac specific Mitofusin-1 knockout model (c-Mfn1 KO). Systolic function was reduced in c-Mfn1 KO mice, and EM study showed an increase in dysfunctional mitochondria in the KO group subjected to TAC. Conclusions: Mitofusin-1 becomes a biomarker for non-responders with heart failure. In addition, our results suggest that therapies targeting mitochondrial dynamics and homeostasis would become next generation therapy for severe heart failure patients.

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Abstract 405: Unprocessed ProBNP Secretion is Regulated by miR30-GALNTs Axis Dependent O-glycosylation in the Pathological Myocardium

Circulation Research ◽

10.1161/res.119.suppl_1.405 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Yasuaki Nakagawa ◽

Koichiro Kuwahara ◽

Toshio Nishikimi ◽

Hideyuki Kinoshita ◽

Chinatsu Yamada ◽

...

Keyword(s):

Heart Failure ◽

Molecular Mechanisms ◽

Ventricular Myocytes ◽

Resistant Mutant ◽

Assay System ◽

Glycosylation Sites ◽

Patients With Heart Failure ◽

Lentivirus Vectors ◽

Underlying Mechanisms ◽

Essential Contribution

Background: It has been reported that biological inactive pro-brain natriuretic peptide(proBNP), precursor peptide of bioactive BNP, is increased in patients with heart failure, as well as mature BNP-32. However, underlying mechanisms of increased plasma proBNP levels in heart failure remain unclear. Methods: We used the conventional BNP assay system, which detects both proBNP and BNP-32, and our recently developed specific human proBNP assay system. We assessed cardiac production of proBNP and BNP(=proBNP+BNP-32) in patients with heart failure. To elucidate the molecular mechanisms underlying the processing and secretion of proBNP, we generated lentivirus vectors carrying several mutations in glycosylation sites of human proBNP. We evaluated the proBNP/BNP ratio, as an efficacy of processing of proBNP to BNP-32. Results: In coronary sinus, plasma proBNP/BNP ratios were significantly elevated in heart failure patients in accordance with the disease severity. In conditioned medium of cultured ventricular myocytes expressing wild type human proBNP, proBNP/BNP ratio was 37%, while in those of myocytes expressing glycosylation-resistant mutant of proBNP, proBNP/BNP ratio was only 3%, demonstrating the essential contribution of glycosylation to the secretion of proBNP. Among 7 glycosylation sites, we found that glycosylation of Thr48 and Thr71 in proBNP is critical for the secretion of proBNP from ventricular myocytes by attenuating proBNP processing. GalNAc-transferase(GALNT) is the largest glycosyltransferase enyme family, which controll O-glycosylasion. We identified GALNT 1 and 2 mediated the glycosylation-regulated increase in cardiac proBNP secretion by screening with siRNAs. Indeed, GALNT1 and 2-expression was increased in FCS-treated ventricular myocytes and failing hearts. We further found that GALNT1 and 2 expression is suppressed by miR-30 family, and that miR-30s expression is decreased in FCS-treated ventricular myocytes and failing hearts. Conclusions: We have elucidated novel molecular pathways that miR-30-GALNTs axis-mediated O -glycosylation in proBNP contributes to increased proportion of proBNP among secreted BNP in the pathological myocardium.

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