Action potential propagation in transverse-axial tubular system is impaired in heart failure

Abstract

The plasma membrane of cardiac myocytes presents complex invaginations known as the transverse-axial tubular system (TATS). Despite TATS's crucial role in excitation-contraction coupling and morphological alterations found in pathological settings, TATS's electrical activity has never been directly investigated in remodeled tubular networks. Here we develop an ultrafast random access multiphoton microscope that, in combination with a customly synthesized voltage-sensitive dye, is used to simultaneously measure action potentials (APs) at multiple sites within the sarcolemma with submillisecond temporal and submicrometer spatial resolution in real time. We find that the tight electrical coupling between different sarcolemmal domains is guaranteed only within an intact tubular system. In fact, acute detachment by osmotic shock of most tubules from the surface sarcolemma prevents AP propagation not only in the disconnected tubules, but also in some of the tubules that remain connected with the surface. This indicates that a structural disorganization of the tubular system worsens the electrical coupling between the TATS and the surface. The pathological implications of this finding are investigated in failing hearts. We find that AP propagation into the pathologically remodeled TATS frequently fails and may be followed by local spontaneous electrical activity. Our findings provide insight on the relationship between abnormal TATS and asynchronous calcium release, a major determinant of cardiac contractile dysfunction and arrhythmias.cardiac disease | nonlinear microscopy | voltage imaging | t tubules T he transverse-axial tubular system (TATS) is a complex network characterized by transverse (t tubules, TT) and longitudinal (axial tubules, AT) components running from one transverse tubule to the next (1-3). The TATS of a myocyte rapidly conducts depolarization of the surface sarcolemma (SS) to the core of the cardiomyocyte (4). The coupling between sarcolemmal Ca 2+ entry during an action potential (AP) and Ca 2+ release from sarcoplasmic reticulum (SR) promotes synchronous myofibril activation throughout the myocyte (5). Recent studies highlight that AP-relevant ion channels and transporters are expressed in TATS membrane with different densities and isoforms from those in SS (6, 7). These findings, in combination with diffusional limitations in TATS's lumen (3,8), raise the possibility that AP may differ among membrane domains. Further interest in the TATS AP stems from the recent finding of loss and disorganization of tubules in several pathological conditions, including heart failure (9-14). Because correlation between morphological TATS alterations and Ca 2+ -release asynchronicity has been observed (14, 15), recording AP propagation in TATS can elucidate the fundamental electrophysiological mechanism linking structural and functional anomalies.Although the uniformity of the AP across the whole sarcolemma has been mathematically (16) and experimentally (17) proved, a potential alteration of the AP propagation i...

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

“…The K′ distribution highlights the presence of tubules experiencing an exceptionally low conductivity of the surrounding network. This finding is in line with previous observations where AP propagation failure across TATS was described in acutely detubulated cardiomyocytes (11).…”

“…Ensuring a good electrical coupling of the TATS is essential for propagation of membrane depolarization from the surface to the core of the cell. Measurement of AP propagation in the TATS is a quite recent possibility, achieved by combining random access two-photon microscopy and voltage imaging (11,35). In line with the above considerations, this methodology revealed that the majority of T-tubules properly propagate the AP, even in diseased cardiomyocytes (11)(12)(13)(14).…”

“…Loss and disorganization of the TATS (Fig. 5A and B) are common features of ventricular cardiomyocyte remodeling due to HF in different disease models, including aged SHR rats (5,11,26,27). Ultrastructural modifications, such as increased tubular radius, have been also observed in failing hearts using both electron (28) and superresolution optical microscopy (4).…”

“…All animal procedures performed conform to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes; experimental protocol is approved by the Italian Ministry of Health on July 6, 2015 (approved protocol 647/ 2015-PR). (11). Data from 87 T-tubules in n = 25 cells, isolated from N = 8 CTRL rats and 87 T-tubules in n = 23 cells, isolated from N = 8 SHR/HF rats.…”

“…Finally, in view of the formal analogy that exists between diffusion and electrical conduction, the apparent diffusion coefficient is linked to the effective electrical conductivity of the network. The methodology is first validated using an acutely detubulated cellular model (8,9) and then applied to assessing the effective electrical conductivity in diseased cardiomyocytes where structural (10) and functional (11)(12)(13)(14)(15) defects of the TATS have been described.…”