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Optimizing the Direction and Order of the Motion Unveiled the Ability of Conventional Monolayers of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes to Show Frequency-Dependent Enhancement of Contraction and Relaxation Motion.

ABSTRACT: Contractility of the human heart increases as its beating rate is elevated, so-called positive force-frequency relationship; however, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been reported to exert a negative force-frequency relationship. We tested the hypothesis that the regulation of motion directions by electrical pacing and/or oxygen supply may improve the electro-mechanical properties of hiPSC-CMs monolayers. To better evaluate the spatial and temporal relationship between electrical excitation and contractile motion, we simultaneously observed the field potential and motion vector of hiPSC-CMs sheets. Under spontaneous contraction, although an electrical excitation originating from a region propagated unidirectionally over the cell sheet, contraction wave started from multiple sites, and relaxation wave was initiated from a geometric center of hiPSC-CMs sheet. During electrical pacing, contraction and relaxation waves were propagated from the stimulated site. Interestingly, the maximum contraction speed was more increased when the hiPSC-CMs sheet was stimulated at an area relaxation initiated under spontaneous condition. Furthermore, motion vector analysis demonstrated that "positive contraction velocity-frequency relationship" in contraction and "frequency-dependent enhancement of relaxation" were produced in the cell sheet by optimizing the direction and order of the contractile motion with pacing at the relaxation-initiating area. A close analysis of motion vectors along with field potential recording demonstrated that relaxation process consists of fast and slow phases, and suggest that intracellular Ca2+ dynamics may be closely related to functions of Ca2+-ATPase pump and Na+-Ca2+ exchangers. Namely, the slow relaxation phase occurred after the second peak of field potential, suggesting that the slow phase may be associated with extrusion of Ca2+ by Na+-Ca2+ exchangers during repolarization. Increase of oxygen concentration from 20 to 95% as well as ?-adrenergic stimulation with isoproterenol accelerated the fast relaxation, suggesting that it could depend on Ca2+ uptake via Ca2+-ATPase during the depolarization phase. The ratio of maximum contraction speed to field potential duration was increased by the ?-adrenergic stimulation, indicating the elevated contraction efficiency per Ca2+-influx. Thus, these findings revealed potential ability of conventional monolayers of hiPSC-CMs, which will help apply them to translational study filling the gap between physiological as well as pharmacological studies and clinical practice.

SUBMITTER: Izumi-Nakaseko H

PROVIDER: S-EPMC7511828 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Optimizing the Direction and Order of the Motion Unveiled the Ability of Conventional Monolayers of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes to Show Frequency-Dependent Enhancement of Contraction and Relaxation Motion.

Izumi-Nakaseko Hiroko H Chiba Koki K Hagiwara-Nagasawa Mihoko M Satsuka Ayano A Goto Ai A Nunoi Yoshio Y Kambayashi Ryuichi R Matsumoto Akio A Takei Yoshinori Y Kanda Yasunari Y Naito Atsuhiko T AT Sugiyama Atsushi A

Frontiers in cell and developmental biology 20200910

Contractility of the human heart increases as its beating rate is elevated, so-called positive force-frequency relationship; however, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been reported to exert a negative force-frequency relationship. We tested the hypothesis that the regulation of motion directions by electrical pacing and/or oxygen supply may improve the electro-mechanical properties of hiPSC-CMs monolayers. To better evaluate the spatial and temporal rel ...[more]

PMID: 33015053

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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.

Project description:ObjectiveVenous thrombosis (VT) damages the vein wall, both physically by prolonged distension and from inflammation. These factors contribute to post-thrombotic syndrome (PTS). Interleukin (IL)-6 might play a role in experimental PTS and vein wall responses. Previous assessments of post-thrombotic vein wall injury used static measures such as histologic examination and immunologic assays. The purpose of the present study was to use myography to quantify the changes in contraction and relaxation of murine vessels exposed to an acute VT.MethodsWild-type (WT) C57BL/6 mice were used to determine the baseline vein wall passive tension on a DMT 610m myograph (DMT-USA, Inc., Ann Arbor, Mich), including dosing concentrations of phenylephrine (Phe) and acetylcholine (Ach). WT and IL-6-/- mice underwent VT using inferior vena cava (IVC) ligation (complete stasis) and stenosis (partial stasis), with no-surgery mice used as controls. The mice were harvested at 2 days (2D) and analyzed using a myograph. The vessels were stimulated with Phe and Ach to stimulate a contraction and relaxation response. The endothelial responses to VT were quantified by CD31 immunohistochemistry, Greiss assay, polymerase chain reaction, and Evans blue assay.ResultsOptimal passive tension was determined to be 2 mN, with an optimal concentration of Phe and Ach of 7E-3M and 1E-5M, respectively. No significant differences were found in the contractions when exposed to Phe between the WT control, WT 2D ligation, and WT 2D stenosis IVC segments and the IL-6-/- mice with and without thrombus (P > .05 for all). When treated with Ach, significantly more relaxation was found in the nonthrombosed control IVC segments than in those IVC segments that had had a 2D thrombus from either ligation- or stenosis-derived thrombotic mechanisms in both WT and IL-6-/- mice. CD31 staining showed ?20% less luminal endothelium after stasis thrombosis (P ? .01) but no loss in the controls (P > .05). Evans blue staining showed a trend toward increased leakiness in post-thrombotic vein walls. No significant difference in the endothelial gene markers or nitric oxide production was found.ConclusionsCompared with the controls, acute thrombosis in the total or partial stasis models did not impair IVC contractile responses, suggesting no effect on the medial vascular smooth muscle response. The relaxation response was significantly reduced in the post-thrombotic groups, likely from direct endothelial injury. These findings suggest, at acute points, that VT impairs the endothelial function of a vein wall while retaining the vascular smooth muscle cell function and might be a mechanism that promotes PTS.

Dataset Information

Optimizing the Direction and Order of the Motion Unveiled the Ability of Conventional Monolayers of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes to Show Frequency-Dependent Enhancement of Contraction and Relaxation Motion.

Publications

Optimizing the Direction and Order of the Motion Unveiled the Ability of Conventional Monolayers of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes to Show Frequency-Dependent Enhancement of Contraction and Relaxation Motion.

Similar Datasets

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