Recording of Ionic Currents Under Physiological Conditions: Action Potential-Clamp and ‘Onion-Peeling’ Techniques

Proc. Natl. Acad. Sci. U.S.A.

Bossuyt

Griffiths

et al. 2018

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Heart failure (HF) following myocardial infarction (MI) is associated with high incidence of cardiac arrhythmias. Development of therapeutic strategy requires detailed understanding of electrophysiological remodeling. However, changes of ionic currents in ischemic HF remain incompletely understood, especially in translational large-animal models. Here, we systematically measure the major ionic currents in ventricular myocytes from the infarct border and remote zones in a porcine model of post-MI HF. We recorded eight ionic currents during the cell's action potential (AP) under physiologically relevant conditions using AP-clamp sequential dissection. Compared with healthy controls, HF-remote zone myocytes exhibited increased late Na current, Ca-activated K current, Ca-activated Cl current, decreased rapid delayed rectifier K current, and altered Na/Ca exchange current profile. In HF-border zone myocytes, the above changes also occurred but with additional decrease of L-type Ca current, decrease of inward rectifier K current, and Ca release-dependent delayed after-depolarizations. Our data reveal that the changes in any individual current are relatively small, but the integrated impacts shift the balance between the inward and outward currents to shorten AP in the border zone but prolong AP in the remote zone. This differential remodeling in post-MI HF increases the inhomogeneity of AP repolarization, which may enhance the arrhythmogenic substrate. Our comprehensive findings provide a mechanistic framework for understanding why single-channel blockers may fail to suppress arrhythmias, and highlight the need to consider the rich tableau and integration of many ionic currents in designing therapeutic strategies for treating arrhythmias in HF.

Section: Methodsmentioning

confidence: 99%

Complex electrophysiological remodeling in postinfarction ischemic heart failure

Proc. Natl. Acad. Sci. U.S.A.

Bossuyt

Griffiths

et al. 2018

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“…AP-clamp experiments were conducted as previously described [33, 35, 36]. Briefly, the steps are: (1) Recording the cell’s steady-state AP under I-clamp at given pacing frequency.…”

Section: Methodsmentioning

confidence: 99%

β-adrenergic regulation of late Na+ current during cardiac action potential is mediated by both PKA and CaMKII

Journal of Molecular and Cellular Cardiology

Bányász

Izu

et al. 2018

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Late Na current (I) significantly contributes to shaping cardiac action potentials (APs) and increased I is associated with cardiac arrhythmias. β-adrenergic receptor (βAR) stimulation and its downstream signaling via protein kinase A (PKA) and Ca/calmodulin-dependent protein kinase II (CaMKII) pathways are known to regulate I. However, it remains unclear how each of these pathways regulates I during the AP under physiological conditions. Here we performed AP-clamp experiments in rabbit ventricular myocytes to delineate the impact of each signaling pathway on I at different AP phases to understand the arrhythmogenic potential. During the physiological AP (2 Hz, 37 °C) we found that I had a basal level current independent of PKA, but partially dependent on CaMKII. βAR activation (10 nM isoproterenol, ISO) further enhanced I via both PKA and CaMKII pathways. However, PKA predominantly increased I early during the AP plateau, whereas CaMKII mainly increased I later in the plateau and during rapid repolarization. We also tested the role of key signaling pathways through exchange protein activated by cAMP (Epac), nitric oxide synthase (NOS) and reactive oxygen species (ROS). Direct Epac stimulation enhanced I similar to the βAR-induced CaMKII effect, while NOS inhibition prevented the βAR-induced CaMKII-dependent I enhancement. ROS generated by NADPH oxidase 2 (NOX2) also contributed to the ISO-induced I activation early in the AP. Taken together, our data reveal differential modulations of I by PKA and CaMKII signaling pathways at different AP phases. This nuanced and comprehensive view on the changes in I during AP deepens our understanding of the important role of I in reshaping the cardiac AP and arrhythmogenic potential under elevated sympathetic stimulation, which is relevant for designing therapeutic treatment of arrhythmias under pathological conditions.

“…That precludes the conventional voltage-clamp experiments that typically use exogenous Ca 2+ buffers to eliminate intracellular Ca 2+ transient and cell contraction. Therefore, we used our physiological self AP-clamp technique ( 22 ) to preserve Ca 2+ transient and contraction and recorded the total membrane current induced by afterload. First, we recorded the cell’s own steady-state AP under the load-free condition and then used it as the voltage command in self AP clamp ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Mechanoelectric coupling and arrhythmogenesis in cardiomyocytes contracting under mechanical afterload in a 3D viscoelastic hydrogel

Proc. Natl. Acad. Sci. U.S.A.

Shimkunas

Jian

et al. 2021

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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.