Quantitative assessment of passive electrical properties of the cardiac T-tubular system by FRAP microscopy

Abstract

Well-coordinated activation of all cardiomyocytes must occur on every heartbeat. At the cell level, a complex network of sarcolemmal invaginations, called the transverse-axial tubular system (TATS), propagates membrane potential changes to the cell core, ensuring synchronous and uniform excitation-contraction coupling. Although myocardial conduction of excitation has been widely described, the electrical properties of the TATS remain mostly unknown. Here, we exploit the formal analogy between diffusion and electrical conductivity to link the latter with the diffusional properties of TATS. Fluorescence recovery after photobleaching (FRAP) microscopy is used to probe the diffusion properties of TATS in isolated rat cardiomyocytes: A fluorescent dextran inside TATS lumen is photobleached, and signal recovery by diffusion of unbleached dextran from the extracellular space is monitored. We designed a mathematical model to correlate the time constant of fluorescence recovery with the apparent diffusion coefficient of the fluorescent molecules. Then, apparent diffusion is linked to electrical conductivity and used to evaluate the efficiency of the passive spread of membrane depolarization along TATS. The method is first validated in cells where most TATS elements are acutely detached by osmotic shock and then applied to probe TATS electrical conductivity in failing heart cells. We find that acute and pathological tubular remodeling significantly affect TATS electrical conductivity. This may explain the occurrence of defects in action potential propagation at the level of single T-tubules, recently observed in diseased cardiomyocytes.cardiac disease | transverse-axial tubular system | diffusion | electrical conductivity | porous rock T he sequential and coherent recruitment of all cardiomyocytes that occurs on every heartbeat is fundamental for proper contraction of the heart. Every heartbeat is triggered by a depolarizing event, the action potential (AP), spontaneously generated in cardiac pacemaker cells, which propagates through the conductive tissue and the working myocardium. The well-coordinated activation of all cardiomyocytes involves a spreading AP wave, conducted from cell to cell throughout the lattice of interconnections called gap junctions. The 3D electrical network of cardiomyocytes includes a finer order of complexity that involves a system of deep sarcolemmal membrane invaginations within each cell. As a network within a network, these sarcolemmal invaginations occur transversely with a periodicity roughly corresponding to that of sarcomere z-lines (transverse tubules, or T-tubules) and branch in the longitudinal direction (axial tubules) to form a complex system in atrial and ventricular cells, named the transverse-axial tubular system (TATS) (1, 2). The TATS allows membrane potential changes to propagate rapidly into the cardiomyocyte core and is considered an ultimate structural player for excitation-contraction coupling. During rapid depolarization, such as seen during the AP, Ca 2+ enters the cytos...

Letter to the Editor

“…This work suggests that reactivation of the fetal gene programme is partly responsible for HF remodelling but cannot completely explain t-tubule functional defects in HF cardiomyocytes. The authors exclude the role of failure of action potential propagation in t-tubules of HF cardiomyocytes, as reported in our previous works (Sacconi et al 2012;Crocini et al 2014;Scardigli et al 2017). In particular, Lipsett and colleagues claim that there was no difference in action potential propagation in control and HF cardiomyocytes, observed using 2D confocal scanning of action potentials.…”

“…; Scardigli et al . ). In particular, Lipsett and colleagues claim that there was no difference in action potential propagation in control and HF cardiomyocytes, observed using 2D confocal scanning of action potentials.…”

“…; Scardigli et al . ). Because t‐tubules are on average 25 μm long (Soeller & Cannell, ), the voltage drop in the cardiac action potential from the surface sarcolemma to the end of the t‐tubule is only a few millivolts.…”

“…; Scardigli et al . ; Uchida & Lopatin, ) and could represent an additional pathological feature of HF cardiomyocytes.…”

“…Passive responses are only limited by the voltage drop of the current flowing from the surface sarcolemma along the t-tubule with a characteristic space constant . The space constant of t-tubules has been determined theoretically and experimentally to be of the order of 200 μm (Pásek et al 2008;Kong et al 2017;Scardigli et al 2017). Because t-tubules are on average 25 μm long (Soeller & Cannell, 1999), the voltage drop in the cardiac action potential from the surface sarcolemma to the end of the t-tubule is only a few millivolts.…”