The potent lipid mediator sphingosine‐1‐phosphate (S1P) regulates diverse physiological processes by binding to 5 specific GPCRs, although it also has intracellular targets. Here, we demonstrate that S1P, produced in the mitochondria mainly by sphin‐gosine kinase 2 (SphK2), binds with high affinity and specificity to prohibitin 2 (PHB2), a highly conserved protein that regulates mitochondrial assembly and function. In contrast, S1P did not bind to the closely related protein PHB1, which forms large, multimeric complexes with PHB2. In mitochondria from SphK2‐null mice, a new aberrant band of cytochrome‐c oxidase was detected by blue native PAGE, and interaction between subunit IV of cytochrome ‐c oxidase and PHB2 was greatly reduced. Moreover, depletion of SphK2 or PHB2 led to a dysfunction in mitochondrial respiration through cytochrome‐c oxidase. Our data point to a new action of S1P in mitochondria and suggest that interaction of S1P with homomeric PHB2 is important for cytochrome ‐c oxidase assembly and mitochondrial respiration.—Strub, G. M., Paillard, M., Liang, J., Gomez, L., Allegood, J. C., Hait, N. C., Maceyka, M., Price, M. M., Chen, Q., Simpson, D. C., Kordula, T., Milstien, S., Lesnefsky, E. J., Spiegel, S. Sphingosine‐1‐phos‐phate produced by sphingosine kinase 2 in mitochondria interacts with prohibitin 2 to regulate complex IV assembly and respiration. FASEB J. 25, 600–612 (2011). www.fasebj.org

Background— Opening of the mitochondrial permeability transition pore (mPTP) is a crucial event in lethal reperfusion injury. Phosphorylation (inhibition) of glycogen synthase kinase-3β (GSK3β) has been involved in cardioprotection. We investigated whether phosphorylated GSK3β may protect the heart via the inhibition of mPTP opening during postconditioning. Methods and Results— Wild-type and transgenic GSK3β-S9A mice (the cardiac GSK3β activity of which cannot be inactivated) underwent 60 minutes of ischemia and 24 hours of reperfusion. At reperfusion, wild-type and GSK3β-S9A mice received no intervention (control), postconditioning (3 cycles of 1 minute ischemia and 1 minute of reperfusion), the mPTP inhibitor cyclosporine A (CsA; 10 mg/kg IV), or the GSK3β inhibitor SB216763 (SB21; 70 μg/kg IV). Infarct size was assessed by triphenyltetrazolium chloride staining. The resistance of the mPTP to opening after Ca ²⁺ loading was assessed by spectrofluorometry on mitochondria isolated from the area at risk. In wild-type mice, infarct size was significantly reduced by postconditioning, CsA, and SB21, averaging 39±2%, 35±5%, and 37±4%, respectively, versus 58±5% of the area at risk in control mice ( P <0.05). In GSK3β-S9A mice, only CsA, but not postconditioning or SB21, reduced infarct size. Postconditioning, CsA, and SB21 all improved the resistance of the mPTP in wild-type mice, but only CsA did so in GSK3β-S9A mice. Conclusion— These results suggest that S9-phosphorylation of GSK3β is required for postconditioning and likely acts by inhibiting the opening of the mitochondrial permeability transition pore.

Background—: Under physiological conditions, Ca ²⁺ transfer from the endoplasmic reticulum (ER) to mitochondria might occur at least in part at contact points between the 2 organelles and involves the VDAC1/Grp75/IP3R1 complex. Accumulation of Ca ²⁺ into the mitochondrial matrix may activate the mitochondrial chaperone cyclophilin D (CypD) and trigger permeability transition pore opening, whose role in ischemia/reperfusion injury is well recognized. We questioned here whether the transfer of Ca ²⁺ from ER to mitochondria might play a role in cardiomyocyte death after hypoxia-reoxygenation. Methods and Results—: We report that CypD interacts with the VDAC1/Grp75/IP3R1 complex in cardiomyocytes. Genetic or pharmacological inhibition of CypD in both H9c2 cardiomyoblasts and adult cardiomyocytes decreased the Ca ²⁺ transfer from ER to mitochondria through IP3R under normoxic conditions. During hypoxia-reoxygenation, the interaction between CypD and the IP3R1 Ca ²⁺ channeling complex increased concomitantly with mitochondrial Ca ²⁺ content. Inhibition of either CypD, IP3R1, or Grp75 decreased protein interaction within the complex, attenuated mitochondrial Ca ²⁺ overload, and protected cells from hypoxia-reoxygenation. Genetic or pharmacological inhibition of CypD provided a similar effect in adult mice cardiomyocytes. Disruption of ER-mitochondria interaction via the downregulation of Mfn2 similarly reduced the interaction between CypD and the IP3R1 complex and protected against hypoxia-reoxygenation injury. Conclusions—: Our data (1) point to a new role of CypD at the ER-mitochondria interface and (2) suggest that decreasing ER-mitochondria interaction at reperfusion can protect cardiomyocytes against lethal reperfusion injury through the reduction of mitochondrial Ca ²⁺ overload via the CypD/VDAC1/Grp75/IP3R1 complex.

Mitochondrial Ca²⁺ uptake through the recently discovered Mitochondrial Calcium Uniporter (MCU) is controlled by its gatekeeper Mitochondrial Calcium Uptake 1 (MICU1). However, the physiological and pathological role of MICU1 remains unclear. Here we show that MICU1 is vital for adaptation to postnatal life and for tissue repair after injury. MICU1 knockout is perinatally lethal in mice without causing gross anatomical defects. We used liver regeneration after partial hepatectomy as a physiological stress response model. Upon MICU1 loss, early priming is unaffected, but the pro-inflammatory phase does not resolve and liver regeneration fails, with impaired cell cycle entry and extensive necrosis. Ca²⁺ overload-induced mitochondrial permeability transition pore (PTP) opening is accelerated in MICU1-deficient hepatocytes. PTP inhibition prevents necrosis and rescues regeneration. Thus, our study identifies an unanticipated dependence of liver regeneration on MICU1 and highlights the importance of regulating MCU under stress conditions when the risk of Ca²⁺ overload is elevated.

Mitochondrial Ca2+ uptake through the Ca2+ uniporter supports cell functions, including oxidative metabolism, while meeting tissue-specific calcium signaling patterns and energy needs. The molecular mechanisms underlying tissue-specific control of the uniporter are unknown. Here, we investigated a possible role for tissue-specific stoichiometry between the Ca2+-sensing regulators (MICUs) and pore unit (MCU) of the uniporter. Low MICU1:MCU protein ratio lowered the [Ca2+] threshold for Ca2+ uptake and activation of oxidative metabolism but decreased the cooperativity of uniporter activation in heart and skeletal muscle compared to liver. In MICU1-overexpressing cells, MICU1 was pulled down by MCU proportionally to MICU1 overexpression, suggesting that MICU1:MCU protein ratio directly reflected their association. Overexpressing MICU1 in the heart increased MICU1:MCU ratio, leading to liver-like mitochondrial Ca2+ uptake phenotype and cardiac contractile dysfunction. Thus, the proportion of MICU1-free and MICU1-associated MCU controls these tissue-specific uniporter phenotypes and downstream Ca2+ tuning of oxidative metabolism.

Type 2 diabetic cardiomyopathy features Ca²⁺ signaling abnormalities, notably an altered mitochondrial Ca²⁺ handling. We here aimed to study if it might be due to a dysregulation of either the whole Ca²⁺ homeostasis, the reticulum–mitochondrial Ca²⁺ coupling, and/or the mitochondrial Ca²⁺ entry through the uniporter. Following a 16-week high-fat high-sucrose diet (HFHSD), mice developed cardiac insulin resistance, fibrosis, hypertrophy, lipid accumulation, and diastolic dysfunction when compared to standard diet. Ultrastructural and proteomic analyses of cardiac reticulum–mitochondria interface revealed tighter interactions not compatible with Ca²⁺ transport in HFHSD cardiomyocytes. Intramyocardial adenoviral injections of Ca²⁺ sensors were performed to measure Ca²⁺ fluxes in freshly isolated adult cardiomyocytes and to analyze the direct effects of in vivo type 2 diabetes on cardiomyocyte function. HFHSD resulted in a decreased IP3R–VDAC interaction and a reduced IP3-stimulated Ca²⁺ transfer to mitochondria, with no changes in reticular Ca²⁺ level, cytosolic Ca²⁺ transients, and mitochondrial Ca²⁺ uniporter function. Disruption of organelle Ca²⁺ exchange was associated with decreased mitochondrial bioenergetics and reduced cell contraction, which was rescued by an adenovirus-mediated expression of a reticulum-mitochondria linker. An 8-week diet reversal was able to restore cardiac insulin signaling, Ca²⁺ transfer, and cardiac function in HFHSD mice. Therefore, our study demonstrates that the reticulum–mitochondria Ca²⁺ miscoupling may play an early and reversible role in the development of diabetic cardiomyopathy by disrupting primarily the mitochondrial bioenergetics. A diet reversal, by counteracting the MAM-induced mitochondrial Ca²⁺ dysfunction, might contribute to restore normal cardiac function and prevent the exacerbation of diabetic cardiomyopathy.