This research proposal aims to examine the fundamental molecular mechanisms that underlie the signaling of contraction in the mammalian heart. Previous studies have shown that cardiac signaling takes place in microdomains surrounding the dihydropyridine/ryanodine receptor complex, via interchange of Ca2+ signals criss-crossing between these two proteins and the Na+-Ca2+ exchanger. Such studies also show that the gain of ICa-gated release was voltage-dependent, suggesting that the Ca2+ release complex is either regulated by more than one mechanism or that there are different populations of Ca2+ release units with different gating modes. The central idea we propose to test is that the signaling of contraction in the heart might be multi-modally gated. Evaluation of this hypothesis requires not only precise identification of the duration, magnitude, and cellular location of such release sites, but also the ability to monitor many such sites simultaneously in order to differentiate between their gating, voltage dependence, and regulation by metabolic factors. Using a method that limits the diffusion of Ca2+ to about 50 nm, we propose to monitor 50-300 Ca2+ release sites simultaneously in areas of approximately 20 x 50 mum2 of a cardiac myocyte employing rapid two-dimensional confocal imaging. We shall specifically: a) characterize the kinetics of focal Ca2+ release sites as a function of voltage, ICa, Ca2+ load of the SR, phosphorylation, and pharmacological interventions; b) measure the distribution of focal Ca2+ release sites relative to ultrastructural determinants in both ventricular and atrial cells (i.e. cells with and without t-tubules); c) identify distribution of focal release sites relative to gating by voltage, ICa, INaCa, INa; d) measure the properties of focal release sites as a function of redox state, and temperature; e) explore whether the C-terminal tail of Ca2+ channel is the conduit to both Ca2+ and voltage signaling. It is our contention that techniques that can monitor 100s of release sites may be required to examine the multiplicity in the mechanisms of Ca2+ signaling in the heart. The proposed studies may provide a better insight into fundamental molecular mechanisms that regulate the signaling of contraction in normal and diseased heart.
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