The overall goal is to assess if physiological changes in sarcoplasmic free calcium (Ca2+) control cardiac respiration. The applicant and others have shown a marked activation of mitochondrial dehydrogenases by physiological increases in extramitochondrial Ca2+. Most sensitive is a -ketoglutarate dehydrogenase, activation of which lowers steady-state levels of -ketoglutarate. The applicant proposes that upon an increase in cardiac work, the increased time-average sarcoplasmic free Ca2+ causes an increased mitochondrial free Ca2+ sufficient to activate this enzyme. Lowered levels of -ketoglutarate relieves its inhibition on oxalacetate transamination, thereby stimulating mitochondria aspartate formation which is rate limiting for the malate-aspartate shuttle. An increased shuttle flux and -ketoglutarate dehydrogenase would stimulate respiration to maintain ATP levels during increases in cardiac work. To test this hypothesis, the applicant would measure steady-state Ca2+ in isolated mitochondria by fura-2 fluorescence under conditions in which extramitochondrial Ca2+ would oscillate to simulate Ca kinetics during muscle contraction. The applicant would determine if the expected increase in mitochondrial Ca2+ would activate KGDH and stimulate M-A shuttle in isolated heart mitochondria. Finally, the applicant would study the relationship among cardiac O2 consumption, mitochondrial metabolism and cytosolic Ca2+ under conditions of varying cardiac work and different substrates using isolated myocytes.
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