Abstract 274: Physical Coupling Supports Local Ca2+ Coupling Between SR Subdomains and the Mitochondria in Heart Muscle

Abstract

Propagation of ryanodine receptor (RyR) dependent SR Ca2+ release to the mitochondria is locally supported by high [Ca2+] microdomains at close contacts between the organelles. The objective of this study was to clarify if the close associations of SR and mitochondria underlying the local Ca2+ communication were secured via a physical coupling. Crude and Percoll-purified mitochondrial fractions of rat heart homogenates were tested for the presence of mitochondria-associated SR fragments capable of RyR-dependent Ca2+ mobilization and Ca2+ delivery to the mitochondria. SR and mitochondrial marker proteins were analyzed by Western blot or visualized in situ by immunofluorescent labeling and imaged using confocal microscopy. Mitochondrial matrix [Ca2+] ([Ca2+]m) was monitored using fluorescence imaging of rhod-2-loaded adherent individual vesicles. Ample presence of SR markers (calsequestrin, SERCA2a, phospholamban) was detected in the crude mitochondria, whereas after percoll purification they were barely detectable in the ‘heavy’ fraction but remained present in the ‘light’ fraction. ‘Immunomitochemistry’ revealed various sizes of SR particles in the crude fraction, a large portion of which was colocalized with mitochondria, while showed less SR markers in the percoll-purified heavy fraction. Fluorescence Ca2+ imaging in the adherent particles revealed well-detectable [Ca2+]m responses to caffeine stimulation that was prevented by Ca2+ predepletion of the SR using the SERCA inhibitor thapsigargin (Tg), or by inhibitors of mitochondrial Ca2+ uptake. Because the Ca2+ released from SR was diluted in the large volume of the incubation medium without causing a change in the bulk [Ca2+], only local Ca2+ regulation could mediate the Ca2+ transfer to the mitochondria. Despite the hardly detectable presence of SR, the percoll-purified ‘heavy’ mitochondrial fraction still displayed a caffeine-induced [Ca2+]m rise that was abolished by Tg-pretreatment. Our data suggest that the SR-mitochondrial associations where the local Ca2+ coupling is established, are supported by physical links between the two organelles and the mitochondria bound SR surface represents a relatively small fraction of the total SR.