Sub-cellular Electrical Heterogeneity Revealed by Loose Patch Recording Reflects Differential Localization of Sarcolemmal Ion Channels in Intact Rat Hearts.

Igor V Kubasov,Andrei Stepanov,Maxim Dobretsov,Sandor Gyorke,Danila Bobkov,Przemysław B Radwanski,Maxim A Terpilowski

doi:10.3389/fphys.2018.00061

Abstract

The cardiac action potential (AP) is commonly recoded as an integral signal from isolated myocytes or ensembles of myocytes (with intracellular microelectrodes and extracellular macroelectrodes, respectively). These signals, however, do not provide a direct measure of activity of ion channels and transporters located in two major compartments of a cardiac myocyte: surface sarcolemma and the T-tubule system, which differentially contribute to impulse propagation and excitation-contraction (EC) coupling. In the present study we investigated electrical properties of myocytes within perfused intact rat heart employing loose patch recording with narrow-tip (2 μm diameter) extracellular electrodes. Using this approach, we demonstrated two distinct types of electric signals with distinct waveforms (single peak and multi-peak AP; AP1 and AP2, respectively) during intrinsic pacemaker activity. These two types of waveforms depend on the position of the electrode tip on the myocyte surface. Such heterogeneity of electrical signals was lost when electrodes of larger pipette diameter were used (5 or 10 μm), which indicates that the electric signal was assessed from a region of <5 μm. Importantly, both pharmacological and mathematical simulation based on transverse (T)-tubular distribution suggested that while the AP1 and the initial peak of AP2 are predominantly attributable to the fast, inward Na+ current in myocyte's surface sarcolemma, the late components of AP2 are likely representative of currents associated with L-type Ca2+ channel and Na+/Ca2+ exchanger (NCX) currents which are predominantly located in T-tubules. Thus, loose patch recording with narrow-tip pipette provides a valuable tool for studying cardiac electric activity on the subcellular level in the intact heart.

Highlights

In the heart, the action potential (AP) generated in the sinoatrial node propagates trough myocardial syncytium to induce coordinated contraction of myocytes composing the heart
In the present study we investigated electrical properties of myocytes within intact rat hearts employing loose patch recording with narrow-tip diameter (2–5 μm) extracellular electrodes
We demonstrated for the first time that propagating APs in cardiac myocytes exhibit significant subcellular heterogeneity as evidenced by presence of two distinct types of signals: single-peak (AP1) and multi-peak (AP2), respectively

Summary

Introduction

The action potential (AP) generated in the sinoatrial node propagates trough myocardial syncytium to induce coordinated contraction of myocytes composing the heart. There are two major compartments in cardiac myocyte membrane: the surface sarcolemma and T-tubules. These two compartments are populated by different sets of ion channels and transporters that contribute differently to AP propagation and myocyte contraction (Franzini-Armstrong et al, 1998; Brette and Orchard, 2007; Scriven et al, 2013). To further add to this complexity, the relative prominence of ion transport systems, such as NCX, in surface vs T-tubule sarcolemma may vary between different species (Gadeberg et al, 2017), different anatomical and histological areas of the heart (Gómez et al, 1997) and cardiac pathologies along with the stage of disease progression (Balijepalli et al, 2003; Banyasz et al, 2008; Dibb et al, 2009; Zima et al, 2014; Radwanski et al, 2016; Veeraraghavan et al, 2017; Yue et al, 2017). Developing a better appreciation for the contribution of ionic currents from the outer membrane and T-tubular regions to cardiac AP is a critical for understanding normal heart function as well as under pathologic conditions

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