The Mitochondrial Calcium Uniporter Selectively Matches Metabolic Output to Acute Contractile Stress in the Heart

Abstract

SUMMARY In the heart, augmented Ca2+ fluxing drives contractility and ATP generation through mitochondrial Ca2+ loading. Pathologic mitochondrial Ca2+ overload with ischemic injury triggers mitochondrial permeability transition pore (MPTP) opening and cardiomyocyte death. Mitochondrial Ca2+ uptake is primarily mediated by the mitochondrial Ca2+ uniporter (MCU). Here we generated mice with adult and cardiomyocyte-specific deletion of Mcu, which produced mitochondria refractory to acute Ca2+ uptake, augmented ATP production and MPTP opening upon acute Ca2+ challenge. Mice lacking Mcu in the adult heart were also protected from acute ischemia-reperfusion injury. However, resting/basal mitochondrial Ca2+ levels were normal in hearts of Mcu-deleted mice and mitochondria lacking MCU eventually loaded with Ca2+ after stress stimulation. Indeed, Mcu-deleted mice were unable to immediately sprint on a treadmill unless warmed-up for 30 minutes. Hence, MCU is a dedicated regulator of short-term mitochondrial Ca2+ loading underlying a “fight-or-flight” response that acutely matches cardiac workload with ATP production.

Aberrant Mitochondrial Fission Is Maladaptive in Desmin Mutation–Induced Cardiac Proteotoxicity

“…Mitochondria in D7 Des Tg and Ntg hearts were isolated as described previously 22, 23. Briefly, hearts were harvested, homogenized in MS‐EGTA buffer (225 mmol/L mannitol, 75 mmol/L sucrose, 5 mmol/L HEPES, and 1 mmol/L EGTA, pH 7.4) and subjected to differential centrifugation.…”

“…Mitochondrial oxygen consumption rate was measured with an XF24 Extracellular Flux Analyzer (Seahorse Biosciences, North Billerica, MA) by methods as described previously 22, 23, 24. Heart mitochondria were isolated using MS‐EGTA buffer (225 mmol/L mannitol, 75 mmol/L sucrose, 5 mmol/L HEPES, and 1 mmol/L EGTA, pH 7.4) by differential centrifugation as described above.…”

“…Mitochondrial Ca 2+ retention capacity was measured with Calcium Green 5N (Molecular Probes) as previously described 22, 23. Cardiac mitochondria (100 μg) were suspended in KCl buffer containing 125 mmol/L KCl, 20 mmol/L HEPES, 2 mmol/L MgCl 2 , 2 mmol/L potassium phosphate, and 40 μmol/L EGTA, pH 7.2, 200 nmol/L Calcium Green‐5N (Molecular Probes), 7 mmol/L pyruvate, and 1 mmol/L malate.…”

“…Mitochondrial swelling was induced by challenging the mitochondria with 200 μmol/L CaCl 2 , and the absorbance at 540 nm was monitored 22, 23…”

Improving the physiological realism of experimental models

“…Indeed, additional tests using isolated mitochondria using a physiologically oxidized substrate showed only a modest contribution of Ca 2þ to the in vivo range of respiration [31,32], an observation explained by the finding cardiac function was unaffected in mouse models in which the mitochondrial calcium uniporter was genetically ablated [33,34]. However, conditional tissue-specific cardiac mitochondrial uniporter knockout models by Kwong et al [35] led to a more nuanced picture where exercising mice required a substantial 'warm up time' before being able to exercise at a level similar to control mice and that those changes were associated with changes in calcium content in mitochondria due to adrenergic stimulation. The physiological realism of the Beard mitochondrial C-model was recently revisited by Vinnakota et al [36], who showed that the near equilibrium behaviour of F 1 F O ATPase and the rapid transport of P i from extramitochondrial space to the matrix governed the kinetic feedback from P i to the TCA cycle [36].…”