Calcium Stimulation of Mitochondrial Respiration is Substrate Dependent and Tissue Specific

Abstract

Mitochondrial enzymes, such as pyruvate dehydrogenase, isocitrate dehydrogenase, and alpha-ketoglutarate dehydrogenase, have been shown to be stimulated by calcium ion in isolated enzyme experimental systems. However, the extent of Ca2+ stimulation of these enzymes and the resulting integrated stimulatory effect on mitochondrial energy metabolism in in-situ and in-vivo states in the heart and kidney remains to be clarified. In this study, we hypothesized that Ca2+ stimulation of mitochondrial respiration and ATP synthesis is substrate dependent and tissue-specific. To test this hypothesis, we isolated mitochondria from heart and kidney outer medulla from Sprague-Dawley rats. Oxygen consumption rates were determined under different respiratory states: leak state (energized mitochondria in absence of ADP) and the ADP-stimulated state (energized mitochondria in presence of saturating concentration of ADP) with/without bolus injection of different concentrations of CaCl2 using an Oroboros Oxygraph-2k Instrument with various respiratory substrates, including pyruvate+malate (PM), glutamate+malate (GM), alpha-ketoglutarate+malate (AM), palmitoyl-L-carnitine+malate (PCM), and succinate (SUC). The results demonstrated that the various respiratory substrates yielded differences in ADP consumption rates and hence, led to dramatically different respiratory rates in isolated mitochondria from the heart and kidney. Importantly, it was found that Ca2+ significantly increased mitochondrial state III respiration with GM and AM substrates compared to the other substrates in both organs. Moreover, Ca2+ effects on mitochondrial respiration were found to be biphasic, i.e. a small increases in [Ca2+] had a stimulatory effect while large increases had inhibitory effect. These results suggest that energized isolated mitochondria in presence of Ca2+ do not respond the same extent when different substrates are used. We conclude that isolated mitochondria require a suitable substrate combination, optimal [Ca2+], and specific tissue source of mitochondria to achieve a level of energy metabolism and ATP production comparable to in-vivo conditions.