Endoplasmic reticulum-mitochondria miscommunication is an early and causal trigger of hepatic insulin resistance and steatosis.

BACKGROUND & AIMS: Hepatic insulin resistance in obesity and type 2 diabetes was recently associated with endoplasmic reticulum (ER)-mitochondria miscommunication. These contact sites (mitochondria-associated membranes: MAMs) are highly dynamic and involved in many functions; however, whether MAM dysfunction plays a causal role in hepatic insulin resistance and steatosis is not clear. Thus, we aimed to determine whether and how organelle miscommunication plays a role in the onset and progression of hepatic metabolic impairment. METHODS: We analyzed hepatic ER-mitochondria interactions and calcium exchange in a time-dependent and reversible manner in mice with diet-induced obesity. Additionally, we used recombinant adenovirus to express a specific organelle spacer or linker in mouse livers, to determine the causal impact of MAM dysfunction on hepatic metabolic alterations. RESULTS: Disruption of ER-mitochondria interactions and calcium exchange is an early event preceding hepatic insulin resistance and steatosis in mice with diet-induced obesity. Interestingly, an 8-week reversal diet concomitantly reversed hepatic organelle miscommunication and insulin resistance in obese mice. Mechanistically, disrupting structural and functional ER-mitochondria interactions through the hepatic overexpression of the organelle spacer FATE1 was sufficient to impair hepatic insulin action and glucose homeostasis. In addition, FATE1-mediated organelle miscommunication disrupted lipid-related mitochondrial oxidative metabolism and induced hepatic steatosis. Conversely, reinforcement of ER-mitochondria interactions through hepatic expression of a synthetic linker prevented diet-induced glucose intolerance after 4 weeks' overnutrition. Importantly, ER-mitochondria miscommunication was confirmed in the liver of obese patients with type 2 diabetes, and correlated with glycemia, HbA1c and HOMA-IR index. CONCLUSIONS: ER-mitochondria miscommunication is an early causal trigger of hepatic insulin resistance and steatosis, and can be reversed by switching to a healthy diet. Thus, targeting MAMs could help to restore metabolic homeostasis. LAY SUMMARY: The literature suggests that interactions between the endoplasmic reticulum and mitochondria could play a role in hepatic insulin resistance and steatosis during chronic obesity. In the present study, we reappraised the time-dependent regulation of hepatic endoplasmic reticulum-mitochondria interactions and calcium exchange, investigating reversibility and causality, in mice with diet-induced obesity. We also assessed the relevance of our findings to humans. We show that organelle miscommunication is an early causal trigger of hepatic insulin resistance and steatosis that can be improved by nutritional strategies.

Effect of Metformin on T2D-Induced MAM Ca2+ Uncoupling and Contractile Dysfunction in an Early Mouse Model of Diabetic HFpEF.

Diabetic cardiomyopathy (DCM) is a leading complication in type 2 diabetes patients. Recently, we have shown that the reticulum-mitochondria Ca2+ uncoupling is an early and reversible trigger of the cardiac dysfunction in a diet-induced mouse model of DCM. Metformin is a first-line antidiabetic drug with recognized cardioprotective effect in myocardial infarction. Whether metformin could prevent the progression of DCM remains not well understood. We therefore investigated the effect of a chronic 6-week metformin treatment on the reticulum-mitochondria Ca2+ coupling and the cardiac function in our high-fat high-sucrose diet (HFHSD) mouse model of DCM. Although metformin rescued the glycemic regulation in the HFHSD mice, it did not preserve the reticulum-mitochondria Ca2+ coupling either structurally or functionally. Metformin also did not prevent the progression towards cardiac dysfunction, i.e., cardiac hypertrophy and strain dysfunction. In summary, despite its cardioprotective role, metformin is not sufficient to delay the progression to early DCM.

An innovative sequence of hypoxia-reoxygenation on adult mouse cardiomyocytes in suspension to perform multilabeling analysis by flow cytometry.

Cardiovascular diseases remain the leading cause of death worldwide. Although major therapeutic progress has been made during the past decades, a better understanding of the underlying mechanisms will certainly help to improve patient's prognosis. In vitro models, particularly adult mouse cardiomyocytes, have been largely used; however, their fragility and large size are major obstacles to the use of flow cytometry. Conventional techniques, such as cell imaging, require the use of large numbers of animals and are time consuming. Here, we described a new, simple, and rapid one-day protocol using living adult mouse cardiomyocytes in suspension exposed to hypoxia-reoxygenation that allows a multilabeling analysis by flow cytometry. Several parameters can be measured by fluorescent probes labeling to assess cell viability (propidium iodide, calcein-AM, and Sytox Green), mitochondrial membrane potential [DilC1(5) and TMRM], reactive oxygen species production (MitoSOX Red), and mitochondrial mass (MitoTracker Deep Red). We address the robustness and sensitivity of our model using a cardioprotective agent, cyclosporine A. Overall, our new experimental set-up offers a high-speed quantitative multilabeling analysis of adult mouse cardiomyocytes exposed to hypoxia-reoxygenation. Our model might be interesting to investigate other cellular stresses (oxidative and inflammation) or to perform pharmacological screening.

Reduced reticulum-mitochondria Ca2+ transfer is an early and reversible trigger of mitochondrial dysfunctions in diabetic cardiomyopathy.

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.

Increased mitochondrial nanotunneling activity, induced by calcium imbalance, affects intermitochondrial matrix exchanges.

Exchanges of matrix contents are essential to the maintenance of mitochondria. Cardiac mitochondrial exchange matrix content in two ways: by direct contact with neighboring mitochondria and over longer distances. The latter mode is supported by thin tubular protrusions, called nanotunnels, that contact other mitochondria at relatively long distances. Here, we report that cardiac myocytes of heterozygous mice carrying a catecholaminergic polymorphic ventricular tachycardia-linked RyR2 mutation (A4860G) show a unique and unusual mitochondrial response: a significantly increased frequency of nanotunnel extensions. The mutation induces Ca2+ imbalance by depressing RyR2 channel activity during excitation-contraction coupling, resulting in random bursts of Ca2+ release probably due to Ca2+ overload in the sarcoplasmic reticulum. We took advantage of the increased nanotunnel frequency in RyR2A4860G+/- cardiomyocytes to investigate and accurately define the ultrastructure of these mitochondrial extensions and to reconstruct the overall 3D distribution of nanotunnels using electron tomography. Additionally, to define the effects of communication via nanotunnels, we evaluated the intermitochondrial exchanges of matrix-targeted soluble fluorescent proteins, mtDsRed and photoactivable mtPA-GFP, in isolated cardiomyocytes by confocal microscopy. A direct comparison between exchanges occurring at short and long distances directly demonstrates that communication via nanotunnels is slower.

Alpha-catenins control cardiomyocyte proliferation by regulating Yap activity.

RATIONALE: Shortly after birth, muscle cells of the mammalian heart lose their ability to divide. Thus, they are unable to effectively replace dying cells in the injured heart. The recent discovery that the transcriptional coactivator Yes-associated protein (Yap) is necessary and sufficient for cardiomyocyte proliferation has gained considerable attention. However, the upstream regulators and signaling pathways that control Yap activity in the heart are poorly understood. OBJECTIVE: To investigate the role of α-catenins in the heart using cardiac-specific αE- and αT-catenin double knockout mice. METHODS AND RESULTS: We used 2 cardiac-specific Cre transgenes to delete both αE-catenin (Ctnna1) and αT-catenin (Ctnna3) genes either in the perinatal or in the adult heart. Perinatal depletion of α-catenins increased cardiomyocyte number in the postnatal heart. Increased nuclear Yap and the cell cycle regulator cyclin D1 accompanied cardiomyocyte proliferation in the α-catenin double knockout hearts. Fetal genes were increased in the α-catenin double knockout hearts indicating a less mature cardiac gene expression profile. Knockdown of α-catenins in neonatal rat cardiomyocytes also resulted in increased proliferation, which could be blocked by knockdown of Yap. Finally, inactivation of α-catenins in the adult heart using an inducible Cre led to increased nuclear Yap and cardiomyocyte proliferation and improved contractility after myocardial infarction. CONCLUSIONS: These studies demonstrate that α-catenins are critical regulators of Yap, a transcriptional coactivator essential for cardiomyocyte proliferation. Furthermore, we provide proof of concept that inhibiting α-catenins might be a useful strategy to promote myocardial regeneration after injury.

Disruption of calcium transfer from ER to mitochondria links alterations of mitochondria-associated ER membrane integrity to hepatic insulin resistance.

AIMS/HYPOTHESIS: Mitochondria-associated endoplasmic reticulum membranes (MAMs) are regions of the endoplasmic reticulum (ER) tethered to mitochondria and controlling calcium (Ca(2+)) transfer between both organelles through the complex formed between the voltage-dependent anion channel, glucose-regulated protein 75 and inositol 1,4,5-triphosphate receptor (IP3R). We recently identified cyclophilin D (CYPD) as a new partner of this complex and demonstrated a new role for MAMs in the control of insulin's action in the liver. Here, we report on the mechanisms by which disruption of MAM integrity induces hepatic insulin resistance in CypD (also known as Ppif)-knockout (KO) mice. METHODS: We used either in vitro pharmacological and genetic inhibition of CYPD in HuH7 cells or in vivo loss of CYPD in mice to investigate ER-mitochondria interactions, inter-organelle Ca(2+) exchange, organelle homeostasis and insulin action. RESULTS: Pharmacological and genetic inhibition of CYPD concomitantly reduced ER-mitochondria interactions, inhibited inter-organelle Ca(2+) exchange, induced ER stress and altered insulin signalling in HuH7 cells. In addition, histamine-stimulated Ca(2+) transfer from ER to mitochondria was blunted in isolated hepatocytes of CypD-KO mice and this was associated with an increase in ER calcium store. Interestingly, disruption of inter-organelle Ca(2+) transfer was associated with ER stress, mitochondrial dysfunction, lipid accumulation, activation of c-Jun N-terminal kinase (JNK) and protein kinase C (PKC)ε and insulin resistance in liver of CypD-KO mice. Finally, CYPD-related alterations of insulin signalling were mediated by activation of PKCε rather than JNK in HuH7 cells. CONCLUSIONS/INTERPRETATION: Disruption of IP3R-mediated Ca(2+) signalling in the liver of CypD-KO mice leads to hepatic insulin resistance through disruption of organelle interaction and function, increase in lipid accumulation and activation of PKCε. Modulation of ER-mitochondria Ca(2+) exchange may thus provide an exciting new avenue for treating hepatic insulin resistance.

Depressing mitochondria-reticulum interactions protects cardiomyocytes from lethal hypoxia-reoxygenation injury.

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.

Exosome-Mediated Paracrine Signaling Unveils miR-1246 as a Driver of Aggressiveness in Fusion-Negative Rhabdomyosarcoma.

Rhabdomyosarcoma is a pediatric cancer associated with aggressiveness and a tendency to develop metastases. Fusion-negative rhabdomyosarcoma (FN-RMS) is the most commonly occurring subtype of RMS, where metastatic disease can hinder treatment success and decrease survival rates. RMS-derived exosomes were previously demonstrated to be enriched with miRNAs, including miR-1246, possibly contributing to disease aggressiveness. We aimed to decipher the functional impact of exosomal miR-1246 on recipient cells and its role in promoting aggressiveness. Treatment of normal fibroblasts with FN-RMS-derived exosomes resulted in a significant uptake of miR-1246 paired with an increase in cell proliferation, migration, and invasion. In turn, delivery of miR-1246-mimic lipoplexes promoted fibroblast proliferation, migration, and invasion in a similar manner. Conversely, when silencing miR-1246 in FN-RMS cells, the resulting derived exosomes demonstrated reversed effects on recipient cells' phenotype. Delivery of exosomal miR-1246 targets GSK3ß and promotes ß-catenin nuclear accumulation, suggesting a deregulation of the Wnt pathway, known to be important in tumor progression. Finally, a pilot clinical study highlighted, for the first time, the presence of high exosomal miR-1246 levels in RMS patients' sera. Altogether, our results demonstrate that exosomal miR-1246 has the potential to alter the tumor microenvironment of FN-RMS cells, suggesting its potential role in promoting oncogenesis.

High-sensitivity calcium biosensor on the mitochondrial surface reveals that IP3R channels participate in the reticular Ca2+ leak towards mitochondria.

Genetically encoded biosensors based on fluorescent proteins (FPs) are widely used to monitor dynamics and sub-cellular spatial distribution of calcium ion (Ca2+) fluxes and their role in intracellular signaling pathways. The development of different mutations in the Ca2+-sensitive elements of the cameleon probes has allowed sensitive range of Ca2+ measurements in almost all cellular compartments. Region of the endoplasmic reticulum (ER) tethered to mitochondria, named as the mitochondrial-associated membranes (MAMs), has received an extended attention since the last 5 years. Indeed, as MAMs are essential for calcium homeostasis and mitochondrial function, molecular tools have been developed to assess quantitatively Ca2+ levels in the MAMs. However, sensitivity of the first generation Ca2+ biosensors on the surface of the outer-mitochondrial membrane (OMM) do not allow to measure µM or sub-µM changes in Ca2+ concentration which prevents to measure the native activity (unstimulated exogenously) of endogenous channels. In this study, we assembled a new ratiometric highly sensitive Ca2+ biosensor expressed on the surface of the outer-mitochondrial membrane (OMM). It allows the detection of smaller differences than the previous biosensor in or at proximity of the MAMs. Noteworthy, we demonstrated that IP3-receptors have an endogenous activity which participate to the Ca2+ leak channel on the surface of the OMM during hypoxia or when SERCA activity is blocked.

SERCA2 phosphorylation at serine 663 is a key regulator of Ca2+ homeostasis in heart diseases.

Despite advances in cardioprotection, new therapeutic strategies capable of preventing ischemia-reperfusion injury of patients are still needed. Here, we discover that sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA2) phosphorylation at serine 663 is a clinical and pathophysiological event of cardiac function. Indeed, the phosphorylation level of SERCA2 at serine 663 is increased in ischemic hearts of patients and mouse. Analyses on different human cell lines indicate that preventing serine 663 phosphorylation significantly increases SERCA2 activity and protects against cell death, by counteracting cytosolic and mitochondrial Ca2+ overload. By identifying the phosphorylation level of SERCA2 at serine 663 as an essential regulator of SERCA2 activity, Ca2+ homeostasis and infarct size, these data contribute to a more comprehensive understanding of the excitation/contraction coupling of cardiomyocytes and establish the pathophysiological role and the therapeutic potential of SERCA2 modulation in acute myocardial infarction, based on the hotspot phosphorylation level of SERCA2 at serine 663 residue.

Opposite and tissue-specific effects of coenzyme Q2 on mPTP opening and ROS production between heart and liver mitochondria: role of complex I.

Coenzyme Q(2) (CoQ(2)) is known to inhibit mitochondrial permeability transition pore (mPTP) opening in isolated rat liver mitochondria. In this study, we investigated and compared the effects of CoQ(2) on mPTP opening and ROS production in isolated rabbit heart and rat liver mitochondria. Mitochondria were isolated from New Zealand White rabbit hearts and Wistar rat livers. Oxygen consumption, Ca(2+)-induced mPTP opening, ROS production and NADH DUb-reductase activity were measured. Rotenone was used to investigate the effect of CoQ(2) on respiratory complex I activity. CoQ(2) (23 µM) reduced the respiratory control index by 32% and 57% (p<0.01) in heart and liver mitochondria respectively, mainly through an increased oxygen consumption in state 4. CoQ(2) induced a 60% (p<0.05) decrease of calcium retention capacity (CRC) in heart mitochondria and inversely a 46% (p<0.05) increase in liver mitochondria. In basal condition, CoQ(2) induced a 170% (p<0.05) increase of H(2)O(2) production in heart mitochondria and 21% (ns) decrease of H(2)O(2) production in liver mitochondria. Because rotenone, a complex I inhibitor, increases H(2)O(2) production in heart but not in liver mitochondria we investigated the CoQ(2) effect in a dose-response assay of complex I inhibition by rotenone in both mitochondria. CoQ(2) antagonized the effect of rotenone on respiratory complex I activity in liver but not in heart mitochondria. CoQ(2) significantly reduced NADH DUb-reductase activity in liver (-47%) and heart (-37%) mitochondria. In conclusion, our data showed that on the contrary to what was observed in liver mitochondria, CoQ(2) favors mPTP opening and ROS production in heart mitochondria through an opposite effect on respiratory complex I activity.

Sphingosine-1-phosphate produced by sphingosine kinase 2 in mitochondria interacts with prohibitin 2 to regulate complex IV assembly and respiration.

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 sphingosine 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.

Postconditioning modulates ischemia-damaged mitochondria during reperfusion.

Cardiac ischemia damages the mitochondrial electron transport chain and the damage persists during reperfusion. Ischemic postconditioning (PC), applied during early reperfusion, decreases cardiac injury. This finding suggests that the ischemia-damaged mitochondria can be regulated to decrease cardiac injury. The reversible blockade of electron transport during ischemia prevents damage to mitochondria. We propose that the targets of PC cytoprotective signaling are mitochondria damaged by ischemia. Thus, if ischemia-mediated mitochondrial damage is prevented, PC at the onset of reperfusion will not result in additional protection. Isolated, Langendorff-perfused adult rat hearts underwent 25-minute global ischemia and 30-minute reperfusion. Amobarbital (2.5 mM) was used to reversibly inhibit electron transport during ischemia. PC (6 cycles of 10-second ischemia-reperfusion) was applied at the onset of reperfusion. Subsarcolemmal and interfibrillar mitochondria were isolated after reperfusion. Blockade of electron transport with amobarbital only during ischemia preserved oxidative phosphorylation and decreased myocardial injury. PC, after untreated ischemia, decreased cardiac injury without improvement of oxidative phosphorylation. Blockade of electron transport during ischemia or PC improved calcium tolerance and inner membrane potential in subsarcolemmal mitochondria after reperfusion. In hearts treated with amobarbital before ischemia, PC did not provide further protection. Thus, PC protects myocardium via the regulation of ischemia-damaged mitochondria during early reperfusion.

Cooling Uncouples Differentially ROS Production from Respiration and Ca2+ Homeostasis Dynamic in Brain and Heart Mitochondria.

Hypothermia provides an effective neuro and cardio-protection in clinical settings implying ischemia/reperfusion injury (I/R). At the onset of reperfusion, succinate-induced reactive oxygen species (ROS) production, impaired oxidative phosphorylation (OXPHOS), and decreased Ca2+ retention capacity (CRC) concur to mitochondrial damages. We explored the effects of temperature from 6 to 37 °C on OXPHOS, ROS production, and CRC, using isolated mitochondria from mouse brain and heart. Oxygen consumption and ROS production was gradually inhibited when cooling from 37 to 6 °C in brain mitochondria (BM) and heart mitochondria (HM). The decrease in ROS production was gradual in BM but steeper between 31 and 20 °C in HM. In respiring mitochondria, the gradual activation of complex II, in addition of complex I, dramatically enhanced ROS production at all temperatures without modifying respiration, likely because of ubiquinone over-reduction. Finally, CRC values were linearly increased by cooling in both BM and HM. In BM, the Ca2+ uptake rate by the mitochondrial calcium uniporter (MCU) decreased by 2.7-fold between 25 and 37 °C, but decreased by 5.7-fold between 25 and 37 °C in HM. In conclusion, mild cold (25-37 °C) exerts differential inhibitory effects by preventing ROS production, by reverse electron transfer (RET) in BM, and by reducing MCU-mediated Ca2+ uptake rate in BM and HM.

Activation of mitochondrial µ-calpain increases AIF cleavage in cardiac mitochondria during ischemia-reperfusion.

Ubiquitous calpains (calpain I and II) are generally recognized as cytosolic proteins. Recently, mitochondrial localized calpain I (µ-calpain) has been identified. Activation of mito-µ-calpain cleaves apoptosis inducing factor (AIF), a flavoprotein located within the mitochondrial intermembrane space, in liver mitochondria, but not in brain mitochondria. We first tested if activation of mito-µ-calpain cleaves AIF in isolated heart mitochondria. A decrease in AIF content within mitochondria increases cardiac injury during ischemia-reperfusion by augmenting oxidative stress. We hypothesize that the activation of mito-µ-calpain by calcium overload during ischemia-reperfusion results in decreased AIF content within mitochondria by cleaving AIF. The µ-calpain was present within mouse heart mitochondria, mostly in the intermembrane space. Exogenous calcium treatment induced a calpain-dependent decrease of mitochondrial AIF content in isolated mouse heart mitochondria. This process was blocked by a calpain inhibitor (MDL-28170). The Mitochondrial µ-calpain activity was increased by 160 ± 15% during ischemia-reperfusion compared to time control. In contrast, the mitochondrial AIF content was decreased by 52 ± 7% during reperfusion vs. time control in the buffer perfused mouse heart. Inhibition of mito-µ-calpain using MDL-28170 decreased cardiac injury by preserving AIF content within mitochondria during ischemia-reperfusion. Thus, activation of mito-µ-calpain is required to release AIF from cardiac mitochondria. Inhibition of calpains using MDL-28170 decreases cardiac injury by inhibiting both cytosolic calpains and mito-µ-calpain during ischemia-reperfusion.

A novel role for mitochondrial sphingosine-1-phosphate produced by sphingosine kinase-2 in PTP-mediated cell survival during cardioprotection.

Although mitochondria are key determinants of myocardial injury during ischemia-reperfusion (I/R), their interaction with critical cytoprotective signaling systems is not fully understood. Sphingosine-1-phosphate (S1P) produced by sphingosine kinase-1 protects the heart from I/R damage. Recently a new role for mitochondrial S1P produced by a second isoform of sphingosine kinase, SphK2, was described to regulate complex IV assembly and respiration via interaction with mitochondrial prohibitin-2. Here we investigated the role of SphK2 in cardioprotection by preconditioning. Littermate (WT) and sphk2 (-/-) mice underwent 45 min of in vivo ischemia and 24 h reperfusion. Mice received no intervention (I/R) or preconditioning (PC) via 5 min I/R before the index ischemia. Despite the activation of PC-cytoprotective signaling pathways in both groups, infarct size in sphk2 (-/-) mice was not reduced by PC (42 ± 3% PC vs. 43 ± 4% I/R, p = ns) versus WT (24 ± 3% PC vs. 43 ± 3% I/R, p < 0.05). sphk2 (-/-) mitochondria exhibited decreased oxidative phosphorylation and increased susceptibility to permeability transition (PTP). Unlike WT, PC did not prevent ischemic damage to electron transport or the increased susceptibility to PTP. To evaluate the direct contribution to the resistance of mitochondria to cytoprotection, SphK2, PHB2 or cytochrome oxidase subunit IV was depleted in cardiomyoblasts. PC protection was abolished by each knockdown concomitant with decreased PTP resistance. These results point to a new action of S1P in cardioprotection and suggest that the mitochondrial S1P produced by SphK2 is required for the downstream protective modulation of PTP as an effector of preconditioning protection.

Cardiomyocyte Protection by Hibernating Brown Bear Serum: Toward the Identification of New Protective Molecules Against Myocardial Infarction.

Ischemic heart disease remains one of the leading causes of death worldwide. Despite intensive research on the treatment of acute myocardial infarction, no effective therapy has shown clinical success. Therefore, novel therapeutic strategies are required to protect the heart from reperfusion injury. Interestingly, despite physical inactivity during hibernation, brown bears (Ursus arctos) cope with cardiovascular physiological conditions that would be detrimental to humans. We hypothesized that bear serum might contain circulating factors that could provide protection against cell injury. In this study, we sought to determine whether addition of bear serum might improve cardiomyocyte survival following hypoxia-reoxygenation. Isolated mouse cardiomyocytes underwent 45 min of hypoxia followed by reoxygenation. At the onset of reoxygenation, cells received fetal bovine serum (FBS; positive control), summer (SBS) or winter bear serum (WBS), or adult serums of other species, as indicated. After 2 h of reoxygenation, propidium iodide staining was used to evaluate cell viability by flow cytometry. Whereas, 0.5% SBS tended to decrease reperfusion injury, 0.5% WBS significantly reduced cell death, averaging 74.04 ± 7.06% vs. 79.20 ± 6.53% in the FBS group. This cardioprotective effect was lost at 0.1%, became toxic above 5%, and was specific to the bear. Our results showed that bear serum exerts a therapeutic effect with an efficacy threshold, an optimal dose, and a toxic effect on cardiomyocyte viability after hypoxia-reoxygenation. Therefore, the bear serum may be a potential source for identifying new therapeutic molecules to fight against myocardial reperfusion injury and cell death in general.

A Case-Control Study of Keratoconus Risk Factors.

PURPOSE: To evaluate risk factors associated with keratoconus in a case-control setting. METHODS: This single center, prospective, case-control study was carried out from May 2014 to November 2017 at the Rothschild Foundation (Paris, France). Two hundred two patients with keratoconus and 355 control patients were investigated and followed by a single ophthalmologist. Data regarding multiple variables were gathered, including eye rubbing, pattern of eye rubbing, dominant hand, allergies, history of dry eye, screen time, sleep position, and night-time work. RESULTS: After multivariable analysis, the following variables showed significant results: eye rubbing with knuckles [odds ratio (OR) = 8.29; 95% confidence interval (CI): 3.92-18.26, P < 0.001] or fingertips (OR = 5.34; 95% CI: 2.44-12.21, P < 0.001), a history of dry eye (OR = 4.16; 95% CI: 2.3-7.7; P < 0.001), male sex (OR = 4.16; 95% CI: 1.47-11.89; P < 0.001), screen time (OR = 1.02; 95% CI: 1.01-1.04; P < 0.001), prone sleep position (OR = 11.63; 95% CI: 3.88-38.16), and side sleep position (OR = 10.17, 95% CI 3.84-33.73). CONCLUSIONS: This study shows a strong correlation between eye rubbing and keratoconus, particularly when rubbing is performed with the knuckles. Additional associations were identified which may merit future investigation as risk factors, including sleep position, night-time work, and screen time.

Phosphorylation of cyclophilin D at serine 191 regulates mitochondrial permeability transition pore opening and cell death after ischemia-reperfusion.

The mitochondrial permeability transition pore (mPTP) plays a critical role in the pathogenesis of cardiovascular diseases, including ischemia/reperfusion injury. Although the pore structure is still unresolved, the mechanism through which cyclophilin D (CypD) regulates mPTP opening is the subject of intensive studies. While post-translational modifications of CypD have been shown to modulate pore opening, specific phosphorylation sites of CypD have not yet been identified. We hypothesized here that phosphorylation of CypD on a serine residue controls mPTP opening and subsequent cell death at reperfusion. We combined in silico analysis with in vitro and genetic manipulations to determine potential CypD phosphorylation sites and their effect on mitochondrial function and cell death. Importantly, we developed an in vivo intramyocardial adenoviral strategy to assess the effect of the CypD phosphorylation event on infarct size. Our results show that although CypD can potentially be phosphorylated at multiple serine residues, only the phosphorylation status at S191 directly impacts the ability of CypD to regulate the mPTP. Protein-protein interaction strategies showed that the interaction between CypD and oligomycin sensitivity-conferring protein (OSCP) was reduced by 45% in the phosphoresistant S191A mutant, whereas it was increased by 48% in the phosphomimetic S191E mutant cells. As a result, the phosphoresistant CypD S191A mutant was protected against 18 h starvation whereas cell death was significantly increased in phosphomimetic S191E group, associated with mitochondrial respiration alteration and ROS production. As in vivo proof of concept, in S191A phosphoresistant rescued CypD-KO mice developed significantly smaller infarct as compared to WT whereas infarct size was drastically increased in S191E phosphomimetic rescued mice. We conclude that CypD phosphorylation at S191 residue leads to its binding to OSCP and thus sensitizes mPTP opening for the subsequent cell death.