Determinants of Mechanical Dysfunction in Myocardium with Reduced Density of T-Tubules

Abstract

We have previously shown (Ferrantini et al 2010, Biophysical Journal98(3) pp. 5a) that formamide-induced osmotic shock is effective in achieving acute detachment of t-tubules from the surface in thin (< 200 µm thickness) rodent ventricular trabeculae, in which mechanical measurements can be performed in isometric conditions. Here, disconnection of t-tubules from the sarcolemma is verified by two-photon fluorescence imaging of labelled membrane and by directly probing the absence of tubular action potential by Random Access Multi-Photon (RAMP) microscopy. Formamide-induced detubulation in intact myocardium is incomplete and variable between different myocytes. This resembles t-tubule remodelling observed in disease settings. Simultaneous measurements of Force and Ca2+ transients in Fura-2 loaded trabeculae show that acute detachment of t-tubules leads to reduced twitch force and Ca2+ transient amplitude, impairment of the positive inotropic effect of high stimulus-rate, slower kinetics of contraction and prolonged Ca2+ transients that exhibit asynchronous time-course between different regions of the muscle. The DHPR blocker (Lacidipine) and the NCX blocker (SN-6) are used to prove that: (i) loss of tubular ICa-L accounts for the reduction of force and Ca2+-transient amplitude at both low and high pacing rates; (ii) loss of INCX contributes to the reduction in amplitude at high stimulation rates and plays a role in the prolongation of force relaxation and Ca2+ transient decay. Caffeine, applied at low doses (200 µM) to detubulated myocardium to increase RyR2 open probability, is able to re-synchronize Ca2+ transients within the muscle and partially reverse the loss of contractility at high stimulation rates. Simultaneous recordings of local Ca2+ release and action potential in the tubular network of single detubulated cardiomyocytes by RAMP microscopy indicate that non-uniform Ca2+release plays a major role in detubulation-induced mechanical dysfunction.