We will investigate how the Na+/Ca2+ exchanger contributes to the intracellular calcium signal in heart muscle during the proposed work. We plan to use a new and powerful tool, the 'calcium spark', to examine Na+/Ca2+ exchanger function in heart muscle with unparalleled resolution and sensitivity. The 'calcium spark' was recently discovered by the PI and his co-workers (Cheng et al., 1993) and is the optical signal arising from the opening of a single sarcoplasmic reticulum (SR) Ca-release channel in single cells loaded with the calcium-sensitive indicator fluo-3. Whole-cell patch-clamp experiments will be carried out using single cardiac myocytes from rat and guinea pig with the simultaneous measurement of intracellular calcium. We use a confocal (and also a 'two photon') microscope to image [Ca2+]i with high spatial (sub-micron) and temporal (msec) resolution using the calcium-sensitive indicators fluo-3 and indo- 1. Rapid changes in [Ca2+]i will be produced by the photo-release of calcium from 'caged calcium' (e.g. DM-nitrophen or NP-EGTA) as an investigative tool. Similarly, rapid block and unblock of calcium channels will be carried out using photolabile dihydropyridine calcium channel blockers (e.g. nifedipine or nisoldipine) with rapid solution changes and flash photolysis of light-sensitive dihydropyridines. The proposed work address two questions. (1). What role does the Na+/Ca2+ exchanger play in triggering SR calcium release in heart muscle? Although work by others on this fundamental question in excitation-contraction(EC) coupling has been controversial, our preliminary data reveals that we have a unique approach that should provide a definitive answer. (2). How does the Na+/Ca2+ exchanger regulate [Ca2+]i in heart muscle? Our preliminary experiments reveal through calcium spark images the dynamic character of SR calcium release at all membrane potential and suggests that the Na+/Ca2+ exchanger actively contributes to this process. Furthermore, we find that the activation of calcium sparks occurs during all phases of the action potential and is under the control of two cellular processes: the Na+/Ca2+ exchanger and the sarcolemmal calcium channels We will investigate how the balance between these two regulators changes. The planned work on both questions will (a) identify Na+/Ca2+ exchanger contributions separated from the those of calcium channels, (b) determine the SR calcium content and the 'fuzzy space' contribution to Na+/Ca2+ exchanger function and (c) investigate how physiological and pharmacological interventions change Na+/Ca2+ exchanger function. The planned work will provide compelling quantitative information on the contributions made by the Na+/Ca2+ exchanger to cardiac contractility. The work should provide fundamental new information about how the calcium signal in the heart is regulated. The proposed experiments will broaden our understanding of normal and abnormal cardiac function and lay the foundation for medical treatments and drug development.
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