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Mechanism of contraction rhythm homeostasis for hyperthermal sarcomeric oscillations of neonatal cardiomyocytes.

ABSTRACT: The heart rhythm is maintained by oscillatory changes in [Ca²⁺]. However, it has been suggested that the rapid drop in blood pressure that occurs with a slow decrease in [Ca²⁺] preceding early diastolic filling is related to the mechanism of rapid sarcomere lengthening associated with spontaneous tension oscillation at constant intermediate [Ca²⁺]. Here, we analyzed a new type of oscillation called hyperthermal sarcomeric oscillation. Sarcomeres in rat neonatal cardiomyocytes that were warmed at 38-42 °C oscillated at both slow (~?1.4 Hz), Ca²⁺-dependent frequencies and fast (~?7 Hz), Ca²⁺-independent frequencies. Our high-precision experimental observations revealed that the fast sarcomeric oscillation had high and low peak-to-peak amplitude at low and high [Ca²⁺], respectively; nevertheless, the oscillation period remained constant. Our numerical simulations suggest that the regular and fast rthythm is maintained by the unchanged cooperative binding behavior of myosin molecules during slow oscillatory changes in [Ca²⁺].

SUBMITTER: Shintani SA

PROVIDER: S-EPMC7687892 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Mechanism of contraction rhythm homeostasis for hyperthermal sarcomeric oscillations of neonatal cardiomyocytes.

Shintani Seine A SA Washio Takumi T Higuchi Hideo H

Scientific reports 20201124 1

The heart rhythm is maintained by oscillatory changes in [Ca2+]. However, it has been suggested that the rapid drop in blood pressure that occurs with a slow decrease in [Ca2+] preceding early diastolic filling is related to the mechanism of rapid sarcomere lengthening associated with spontaneous tension oscillation at constant intermediate [Ca2+]. Here, we analyzed a new type of oscillation called hyperthermal sarcomeric oscillation. Sarcomeres in rat neonatal c ...[more]

PMID: 33235297

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Project description:Various types of stem cells have been shown to have beneficial effects on cardiac function. It is still debated whether fusion of injected stem cells with local resident cardiomyocytes is one of the mechanisms. To better understand the role of fusion in stem cell-based myocardial regeneration, the present study was designed to investigate the fate of human adipose tissue-derived stem cells (hASCs) fused with neonatal rat cardiomyocytes in vitro. hASCs labeled with the green fluorescent probe Vybrant DiO were cocultured with neonatal rat cardiomyocytes labeled with the red fluorescent probe Vybrant DiI and then treated with fusion-inducing hemagglutinating virus of Japan (HVJ). Cells that incorporated both red and green fluorescent signals were considered to be hASCs that had fused with rat cardiomyocytes. Fusion efficiency was 19.86 ± 4.84% at 5 d after treatment with HVJ. Most fused cells displayed cardiomyocyte-like morphology and exhibited spontaneous rhythmic contraction. Both immunofluorescence staining and lentiviral vector labeling showed that fused cells contained separate rat cardiomyocyte and hASC nuclei. Immunofluorescence staining assays demonstrated that human nuclei in fused cells still expressed the proliferation marker Ki67. In addition, hASCs fused with rat cardiomyocytes were positive for troponin I. Whole-cell voltage-clamp analysis demonstrated action potentials in beating fused cells. RT-PCR analysis using rat- or human-specific myosin heavy chain primers revealed that the myosin heavy-chain expression in fused cells was derived from rat cardiomyocytes. Real-time PCR identified expression of human troponin T in fused cells and the presence of rat cardiomyocytes induced a cardiomyogenic protein expression of troponin T in human ASCs. This study illustrates that hASCs exhibit both stem cell (proliferation) and cardiomyocyte properties (action potential and spontaneous rhythmic beating) after fusion with rat cardiomyocytes, supporting the theory that fusion, even if artificially induced in our study, could indeed be a mechanism for cardiomyocyte renewal in the heart.

Project description:RationaleImpaired myocardial relaxation is an intractable feature of several heart failure (HF) causes. In human HF, detyrosinated microtubules stiffen cardiomyocytes and impair relaxation. Yet the identity of detyrosinating enzymes have remained ambiguous, hindering mechanistic study and therapeutic development.ObjectiveWe aimed to determine if the recently identified complex of VASH1/2 (vasohibin 1/2) and SVBP (small vasohibin binding protein) is an active detyrosinase in cardiomyocytes and if genetic inhibition of VASH-SVBP is sufficient to lower stiffness and improve contractility in HF.Methods and resultsTranscriptional profiling revealed that VASH1 transcript is >10-fold more abundant than VASH2 in human hearts. Using short hairpin RNAs (shRNAs) against VASH1, VASH2, and SVBP, we showed that both VASH1- and VASH2-SVBP complexes function as tubulin carboxypeptidases in cardiomyocytes, with a predominant role for VASH1. We also generated a catalytically dead version of the tyrosinating enzyme TTL (TTL-E331Q) to separate the microtubule depolymerizing effects of TTL from its enzymatic activity. Assays of microtubule stability revealed that both TTL and TTL-E331Q depolymerize microtubules, while VASH1 and SVBP depletion reduce detyrosination independent of depolymerization. We next probed effects on human cardiomyocyte contractility. Contractile kinetics were slowed in HF, with dramatically slowed relaxation in cardiomyocytes from patients with HF with preserved ejection fraction. Knockdown of VASH1 conferred subtle kinetic improvements in nonfailing cardiomyocytes, while markedly improving kinetics in failing cardiomyocytes. Further, TTL, but not TTL-E331Q, robustly sped relaxation. Simultaneous measurements of calcium transients and contractility demonstrated that VASH1 depletion speeds kinetics independent from alterations to calcium cycling. Finally, atomic force microscopy confirmed that VASH1 depletion reduces the stiffness of failing human cardiomyocytes.ConclusionsVASH-SVBP complexes are active tubulin carboxypeptidases in cardiomyocytes. Inhibition of VASH1 or activation of TTL is sufficient to lower stiffness and speed relaxation in cardiomyocytes from patients with HF, supporting further pursuit of detyrosination as a therapeutic target for diastolic dysfunction.

Dataset Information

Mechanism of contraction rhythm homeostasis for hyperthermal sarcomeric oscillations of neonatal cardiomyocytes.

Publications

Mechanism of contraction rhythm homeostasis for hyperthermal sarcomeric oscillations of neonatal cardiomyocytes.

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