Cytosolic, but not matrix, calcium is essential for adjustment of mitochondrial pyruvate supply

Mitochondrial oxidative phosphorylation (OXPHOS) and cellular workload are tightly balanced by the key cellular regulator, calcium (Ca2+). Current models assume that cytosolic Ca2+ regulates workload and that mitochondrial Ca2+ uptake precedes activation of matrix dehydrogenases, thereby matching OXPHOS substrate supply to ATP demand. Surprisingly, knockout (KO) of the mitochondrial Ca2+ uniporter (MCU) in mice results in only minimal phenotypic changes and does not alter OXPHOS. This implies that adaptive activation of mitochondrial dehydrogenases by intramitochondrial Ca2+ cannot be the exclusive mechanism for OXPHOS control. We hypothesized that cytosolic Ca2+, but not mitochondrial matrix Ca2+, may adapt OXPHOS to workload by adjusting the rate of pyruvate supply from the cytosol to the mitochondria. Here, we studied the role of malate-aspartate shuttle (MAS)-dependent substrate supply in OXPHOS responses to changing Ca2+ concentrations in isolated brain and heart mitochondria, synaptosomes, fibroblasts, and thymocytes from WT and MCU KO mice and the isolated working rat heart. Our results indicate that extramitochondrial Ca2+ controls up to 85% of maximal pyruvate-driven OXPHOS rates, mediated by the activity of the complete MAS, and that intramitochondrial Ca2+ accounts for the remaining 15%. Of note, the complete MAS, as applied here, included besides its classical NADH oxidation reaction the generation of cytosolic pyruvate. Part of this largely neglected mechanism has previously been described as the “mitochondrial gas pedal.” Its implementation into OXPHOS control models integrates seemingly contradictory results and warrants a critical reappraisal of metabolic control mechanisms in health and disease.

Abstract 467: Glucose Promotes M2-related Transcriptional Reprogramming of Resident Cardiac Macrophages in Response to Hypothermic Ischemic Arrest and Reperfusion

Stress hyperglycemia and inflammation frequently develop in open heart surgery patients. Although both factors independently contribute to increased peri-operative morbidity and mortality, the impact of high glucose levels on cardiac inflammatory response remains unknown. We investigated the isolated working rat heart as a model to study cardiac early stress response to surgery. Hearts of male Sprague Dawley rats were cold-arrested and subjected to 60 minutes normothermic reperfusion in the working mode with Krebs-Henseleit buffer supplemented with ketone bodies and propionate plus glucose (25 mM) or mannitol (25 mM; osmotic control). Alterations of gene expression in the left ventricle were determined by microarray and real-time PCR analyses. Compared to non-perfused hearts, perfused hearts displayed a more than twofold increased expression for 71 genes (mannitol group) and 103 genes (glucose group) connected to inflammation, cell proliferation, and apoptosis. The transcriptional changes were highly similar to gene alterations previously reported in the right atrium ( P < 2.34E-16) and left ventricle ( P < 4.83E-46) of patients who underwent cardiac surgery with cardiopulmonary bypass. Pathway analysis with Reactome revealed an up-regulation of metabolic processes associated with the proliferation and activation of immune cells, including glycolysis, glutaminolysis, fatty acid synthesis, polyamine synthesis, and hexosamine synthesis. Although the transcriptional remodeling occurred independently from the presence of glucose, glucose significantly increased further the expression of several transcription factors and markers associated with M2 polarization of macrophages, including Myc (1.6-fold), Nr4a1 (1.3-fold), Nr4a2 (1.8-fold), Zc3h12a (1.3-fold), Fosl2 (1.4-fold), Cebpb (1.2-fold), and Arg1 (1.7-fold). Interestingly, glucose failed to enhance the expression of M2-related genes in the heart of rats rendered insulin resistant by high-sucrose feeding. Besides demonstrating that the isolated working rat heart accurately reproduces the stress response associated with open heart surgery, the results also suggest that glucose promotes the alternative activation of resident cardiac macrophages in the stressed heart.