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Protocol to record and quantify the intracellular pH in contracting cardiomyocytes.

ABSTRACT: Intracellular pH (pH_i) plays critical roles in the regulation of cardiac function. Methods and techniques for cardiac pH_i measurement have continued to evolve since early 1960s. Fluorescent microscopy is the most recently developed technique with several advantages over other techniques including higher spatial and temporal resolutions, and feasibility for contracting cell measurement. Here, we describe detailed methods for mouse cardiomyocyte isolation, and simultaneous measurement and quantification of pH_i and sarcomere length in contracting cardiomyocytes. For complete details on the use and execution of this protocol, please refer to Lyu et al. (2022).

SUBMITTER: Lyu Y

PROVIDER: S-EPMC9026584 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Protocol to record and quantify the intracellular pH in contracting cardiomyocytes.

Lyu Yankun Y Timofeyev Valeriy V Overton James J Thai Phung N PN Yamoah Ebenezer N EN Chiamvimonvat Nipavan N Zhang Xiao-Dong XD

STAR protocols 20220412 2

Intracellular pH (pH<sub>i</sub>) plays critical roles in the regulation of cardiac function. Methods and techniques for cardiac pH<sub>i</sub> measurement have continued to evolve since early 1960s. Fluorescent microscopy is the most recently developed technique with several advantages over other techniques including higher spatial and temporal resolutions, and feasibility for contracting cell measurement. Here, we describe detailed methods for mouse cardiomyocyte isolation, and simultaneous me ...[more]

PMID: 35463464

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Project description:The heart pumps blood against the mechanical afterload from arterial resistance, and increased afterload may alter cardiac electrophysiology and contribute to life-threatening arrhythmias. However, the cellular and molecular mechanisms underlying mechanoelectric coupling in cardiomyocytes remain unclear. We developed an innovative patch-clamp-in-gel technology to embed cardiomyocytes in a three-dimensional (3D) viscoelastic hydrogel that imposes an afterload during regular myocyte contraction. Here, we investigated how afterload affects action potentials, ionic currents, intracellular Ca2+ transients, and cell contraction of adult rabbit ventricular cardiomyocytes. We found that afterload prolonged action potential duration (APD), increased transient outward K+ current, decreased inward rectifier K+ current, and increased L-type Ca2+ current. Increased Ca2+ entry caused enhanced Ca2+ transients and contractility. Moreover, elevated afterload led to discordant alternans in APD and Ca2+ transient. Ca2+ alternans persisted under action potential clamp, indicating that the alternans was Ca2+ dependent. Furthermore, all these afterload effects were significantly attenuated by inhibiting nitric oxide synthase 1 (NOS1). Taken together, our data reveal a mechano-chemo-electrotransduction (MCET) mechanism that acutely transduces afterload through NOS1-nitric oxide signaling to modulate the action potential, Ca2+ transient, and contractility. The MCET pathway provides a feedback loop in excitation-Ca2+ signaling-contraction coupling, enabling autoregulation of contractility in cardiomyocytes in response to afterload. This MCET mechanism is integral to the individual cardiomyocyte (and thus the heart) to intrinsically enhance its contractility in response to the load against which it has to do work. While this MCET is largely compensatory for physiological load changes, it may also increase susceptibility to arrhythmias under excessive pathological loading.

Dataset Information

Protocol to record and quantify the intracellular pH in contracting cardiomyocytes.

Publications

Protocol to record and quantify the intracellular pH in contracting cardiomyocytes.

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