Diffusion Properties of Cardiac T-Tubular System

Abstract

Action potentials (APs), via the transverse axial tubular system (TATS), synchronously trigger uniform Ca2+ release throughout the cardiomyocyte. In cardiomyocytes from diseased hearts we previously documented the presence of TATS elements which failed to propagate AP (Sacconi et al. PNAS 2012). Using an imaging method to simultaneously assess TATS electrical activity and local Ca2+ release we recently demonstrated that failing tubular elements show a slower local Ca2+ transient compared with regions with electrically coupled elements (Crocini et al. PNAS 2014). The causes of AP propagation failure remained, however, unsolved. TATS structural remodelling that is generally associated with pathological settings may increase the tubular electrical resistance, augmenting the probability that the depolarization wavefront fails in reaching the AP threshold at the tubular membrane. TATS structural changes can modify the diffusion properties of T-tubule lumen. Here, we used fluorescence recovery after photo-bleaching (FRAP) microscopy to probe the diffusion properties of TATS lumen in cardiomyocytes. T-tubules of isolated cardiomyocytes from rodent models of cardiac diseases are labelled using fluorescent dextran that diffuses from extracellular space to TATS lumen. The fluorescent dextran present inside TATS lumen is photo-bleached and the diffusion of unbleached dextran from extracellular space to TATS is monitored using confocal imaging. Analysing the recovery of the fluorescence signal at pixel-by-pixel level, a diffusional map of the tubular system is obtained. Diffusion maps performed at different dextran molecular weight are acquired to probe structural features of the TATS lumen. Finally, diffusion maps in different pathological models are reconstructed to find potential mechanisms for the presence of AP failing TATS elements.