INTRASARCOMERE [CA2-SPEED DIGITAL IMAGING MICROSCOPY(] GRADIENTS IN VENTRICULAR MYOCYTES REVEALED BY HIGH)

Cardiac muscle contraction is triggered by a small and brief Ca2+ entr y across the t-tubular membranes, which is believed to be locally ampl ified by release of Ca2+ from the adjacent junctional sarcoplasmic ret iculum (SR). As Ca2+ diffusion is thought to be markedly attenuated in cells, it has been predicted that significant intrasarcomeric [Ca2+] gradients should exist during activation. To directly test for this, w e measured [Ca2+] distribution in single cardiac myocytes using fluore scent [Ca2+] indicators and high speed, three-dimensional digital imag ing microscopy and image deconvolution techniques. Steep cytosolic [Ca 2+] gradients from the t-tubule region to the center of the sarcomere developed during the first 15 ms of systole. The steepness of these [C a2+] gradients varied with treatments that altered Ca2+ release from i nternal stores. Electron probe microanalysis revealed a loss of Ca2+ f rom the junctional SR and an accumulation, principally in the A-band d uring activation. We propose that the prolonged existence of [Ca2+] gr adients within the sarcomere reflects the relatively long period of Ca 2+ release from the SR, the localization of Ca2+ binding sites and Ca2 + sinks remote from sites of release, and diffusion limitations within the sarcomere. The large [Ca2+] transient near the t-tubular/junction al SR membranes is postulated to explain numerous features of excitati on-contraction coupling in cardiac muscle.