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SummaryGenetically 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.

International audience; 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.

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.

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.

Introduction Type 2 diabetic cardiomyopathy has been linked to Ca2+ signaling alterations, notably a decreased mitochondrial Ca2+ uptake. Recent discovery of Ca2+ microdomains between cardiac mitochondria and reticulum launched a new investigation avenue for cardiometabolic diseases. Objective Hereby, we aimed to investigate if the mitochondrial Ca2+ mishandling is due to a dysregulation of the reticulum-mitochondria interactions or of the mitochondrial Ca2+ uniporter in the diabetic mouse heart. Methods and results Mice fed for 16 weeks with a high fat high sucrose diet (HFHSD) displayed a cardiac insulin resistance combined to cardiac fibrosis, hypertrophy and contractile dysfunction (decreased strain rate) versus standard diet-fed mice (SD). Fractionation of HFHSD mice hearts revealed a decreased protein content of their mitochondria associated-reticulum membranes, together with a lower expression of tethering proteins quantified by mass spectrometry. On functional terms, reduced IP3R-VDAC proximity and co-immunoprecipitation were associated with a reduced IP3-stimulated Ca2+ transfer to mitochondria in isolated HFHSD cardiomyocytes, yet no changes in mitochondrial calcium uniporter protein expression and function were detected. Moreover, HFHSD cardiomyocytes displayed no significant changes in the reticular Ca2+ release level and the amplitude of cytosolic Ca2+ transients. Eight weeks of diet reversal restored the cardiac insulin signaling, MAM content, Ca2+ transfer and cardiac function in HFHSD mice. Conclusion Therefore, our data indicate that disruption of the cardiac reticulum-mitochondria interactions is an early and reversible event impairing mitochondrial Ca2+ handling and contributing to the cardiac contractile dysfunction in the diabetic mouse heart, which could be counteracted by diet reversal.

Ischaemic heart disease and stroke are the most common causes of death worldwide. Anoxia, defined as the lack of oxygen, is commonly seen in both these pathologies and triggers profound metabolic and cellular changes. Sphingolipids have been implicated in anoxia injury, but the pathomechanism is unknown. Here we show that anoxia-associated injury causes accumulation of the non-canonical sphingolipid 1-deoxydihydroceramide (DoxDHCer). Anoxia causes an imbalance between serine and alanine resulting in a switch from normal serine-derived sphinganine biosynthesis to non-canonical alanine-derived 1-deoxysphinganine. 1-Deoxysphinganine is incorporated into DoxDHCer, which impairs actin folding via the cytosolic chaperonin TRiC, leading to growth arrest in yeast, increased cell death upon anoxia-reoxygenation in worms and ischaemia-reperfusion injury in mouse hearts. Prevention of DoxDHCer accumulation in worms and in mouse hearts resulted in decreased anoxia-induced injury. These findings unravel key metabolic changes during oxygen deprivation and point to novel strategies to avoid tissue damage and death.

Introduction Because cardiovascular diseases still represent the leading cause of death worldwide, a better understanding of the underlying physiopathological mechanisms is therefore needed. In vitro cellular models participate to decipher the molecular mechanisms, especially adult mouse cardiomyocytes. Unfortunately, due to their high fragilitity as well as their size (150 μm), flow cytometry is usually not performed and mainly conventional techniques are used like cell imaging, which is time and animal-consuming and causes low statistical power. Methods and results Here, we described a new, simple and rapid one-day protocol in living adult mouse cardiomyocytes submitted to in suspension hypoxia-reoxygenation to allow multilabeling analysis by flow cytometry. Our method enables the measurement of several physiological parameters thanks to fluorescent probes labeling, assessing notably cell viability (Propidium Iodide, Calcein-AM and Sytox Green), mitochondrial membrane potential [DilC1(5), TMRM], reactive oxygen species (ROS) production (MitoSOX Red) and mitochondrial mass (MitoTracker Deep Red). Additionally, our model can be used to perform pharmacological screening illustrated here by the protective treatment via cyclosporine A (CsA) as a test to assess robustness and sensitivity of the utilized methods. Conclusion In summary, our new hypoxia-reoxygenation sequence in suspension will offer a high-speed quantitative multilabeling analysis of adult mouse cardiomyocytes, which can be extended to various cellular stress challenges (oxidative, inflammation) or pharmacological screening.

Ischaemic heart disease and stroke are the most common causes of death worldwide. Anoxia, defined as the lack of oxygen, is commonly seen in both these pathologies and triggers profound metabolic and cellular changes. Sphingolipids have been implicated in anoxia injury, but the pathomechanism is unknown. Here we show that anoxia-associated injury causes accumulation of the non-canonical sphingolipid 1-deoxydihydroceramide (DoxDHCer). Anoxia causes an imbalance between serine and alanine resulting in a switch from normal serine-derived sphinganine biosynthesis to non-canonical alanine-derived 1-deoxysphinganine. 1-Deoxysphinganine is incorporated into DoxDHCer, which impairs actin folding via the cytosolic chaperonin TRiC, leading to growth arrest in yeast, increased cell death upon anoxia-reoxygenation in worms and ischaemia-reperfusion injury in mouse hearts. Prevention of DoxDHCer accumulation in worms and in mouse hearts resulted in decreased anoxia-induced injury. These findings unravel key metabolic changes during oxygen deprivation and point to novel strategies to avoid tissue damage and death.

Background: Redox signaling plays an important role in the lives of cells. This signaling not only becomes apparent in pathologies but is also thought to be involved in maintaining physiolog-ical homeostasis. Reactive Oxygen Species (ROS) can activate protein kinases: CaMKII, PKG, PKA, ERK, PI3K, Akt, PKC, PDK, JNK, p38. It is unclear whether it is a direct interaction of ROS with these kinases or whether their activation is a consequence of inhibition of phosphatases. ROS have a biphasic effect on the transport of Ca2+ in the cell: on one hand, they activate the sarcoplasmic reticu-lum Ca2+-ATPase, which can reduce the level of Ca2+ in the cell, and on the other hand, they can inac-tivate Ca2+-ATPase of the plasma membrane and open the cation channels TRPM2, which promote Ca2+-loading and subsequent apoptosis. ROS inhibit the enzyme PHD2, which leads to the stabiliza-tion of HIF-α and the formation of the active transcription factor HIF. Conclusion: Activation of STAT3 and STAT5, induced by cytokines or growth factors, may include activation of NADPH oxidase and enhancement of ROS production. Normal physiological produc-tion of ROS under the action of cytokines activates the JAK/STAT while excessive ROS production leads to their inhibition. ROS cause the activation of the transcription factor NF-κB. Physiological levels of ROS control cell proliferation and angiogenesis. ROS signaling is also involved in beneficial adaptations to survive ischemia and hypoxia, while further increases in ROS can trigger programmed cell death by the mechanism of apoptosis or autophagy. ROS formation in the myocardium can be re-duced by moderate exercise

International audience; 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 RyR2(A4860G+/-) 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.

Cardiovascular diseases associated with metabolic syndrome remain frequent and still disabling for patients. The uncovering of Ca2+ microdomains between mitochondria and reticulum (SR) in the heart has launched a new area of investigation for cardiometabolic diseases. Here, we sought to determine the structural and functional role of the SR-mitochondria interactions in the mice heart during diabetes. We hypothesized that an alteration of the SR-mitochondria interactions would impair the Ca2+ signaling between the two organelles, thus leading to contractile dysfunction. Mice were either fed with a standard diet (SD: 16.9% proteins, 4.3% lipids) or a high-fat-high-sucrose diet (HFHSD:20% proteins, 36% lipids) for 16 weeks. In the HFHSD group, half of the mice were subjected to metformin gavage, an antidiabetic agent, for the last 4 weeks. Cardiac mitochondria associated membranes (MAM) isolation showed an increased protein amount of MAM/pure mitochondria in the HFHSD group vs. SD mice, which was prevented by metformin treatment (SD: 2.0 ± 0.4, HFHSD: 5.5 ± 0.9, HFHSD + MET: 2.8 ± 0.4, n = 5). After 70 min hypoxia-2 hrs reoxygenation, HFHSD isolated cardiomyocytes displayed higher propidium-iodide positive cells compared to SD and HFHSD + MET groups (60 ± 1 vs. SD: 46 ± 3 and HFHSD + MET: 50 ± 2%, n = 5). Interestingly, histamine-stimulated Ca2+ transfer to mitochondria was reduced by 50% in HFHSD ± MET cardiomyocytes. Concomitantly, IP3R-VDAC interactions assessed by proximity ligation assay were 30–40% reduced in HFHSD ± MET cardiomyocytes. Hence, our data indicate a role for the SR-mitochondria interactions during diabetes in the heart in hypoxia-reoxygenation-induced cell death and in the Ca2+ signaling dysfunction. Indeed, reduced formation of the IP3R-VDAC Ca2+ channeling complex and of the IP3R-stimulated Ca2+ transfer to mitochondria in the HFHSD ± MET cardiomyocytes suggest an inappropriate thickness of the extended MAM (too close or too far) for the IP3R complex formation.

Introduction Diabetic cardiomyopathy has been linked to Ca2 + signaling alterations, notably a decreased mitochondrial Ca2 + uptake. Uncovering the changes occurring at Ca2 + microdomains between reticulum and mitochondria in the heart has launched a new area of investigation for cardiometabolic diseases. Objective We here aimed to study if the impairment of mitochondrial Ca2 + handling could be due to a dysregulation of the reticulum-mitochondria interactions or of the mitochondrial Ca2 + uniporter in the diabetic mice heart. Methods Mice were either fed with a standard diet (SD: 16.9% proteins, 4.3% lipids) or a high-fat high sucrose diet (HFHSD: 20% proteins, 36% lipids) for 16 weeks. Cardiac mitochondria associated membranes (MAM) composition was analyzed by proteomics and immunoblotting. Proximity ligation assay, calcium imaging and hypoxia/reoxygenation were performed on isolated cardiomyocytes. Results Our HFHSD mice displayed a cardiac insulin resistance and hypertrophy. Decreased MAM/pure mitochondria content in the HFHSD vs SD heart was observed, with an elevated level of proteins involved in lipid metabolism and a decrease in tethering proteins. Decreased IP3R-VDAC proximity upon HFHSD was concomitant to a reduced IP3R-stimulated Ca2 + transfer to mitochondria, with no changes in mitochondrial calcium uniporter protein expression and function. Additionally, decreased amplitude of cytosolic Ca2 + transients was seen in the HFHSD cardiomyocytes, with no significant changes in the reticular Ca2 + release level. Besides, increased cell death susceptibility was measured after both in vitro hypoxia/reoxygenation and in vivo ischemia/reperfusion under HFHSD. Conclusion Our data, therefore, indicate that decreased reticulum-mitochondria interactions trigger an impaired mitochondrial Ca2 + handling in the diabetic mice heart, with no alteration of the mitochondrial Ca2 + uniporter, while the enhanced susceptibility to cell death could be referred to lipid toxicity.

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.

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.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.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.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.

Infarct size is determined not only by the severity of ischemia but also by pathological processes initiated at reperfusion. Accumulating experimental evidence indicates that lethal reperfusion injury might account for up to half of the final size of the myocardial infarct. Ischemic postconditioning (brief repeated periods of ischemia-reperfusion applied at the onset of coronary reflow) has been recently described as a powerful cardioprotection mechanism that prevents lethal reperfusion injury. This is the first method proven to reduce the final infarct size by about 50% in several in vivo models and to be confirmed in recent preliminary human studies. The molecular pathways are incompletely mapped but they probably converge to a mitochondrial key target: the mitochondrial permeability transition pore (PTP) which opening during early reperfusion is an event that promotes myocardial cell death. In different animal models and experimental settings, pharmacological PTP inhibition at the onset of reperfusion reproduces all the cardioprotective effects of ischemic postconditioning. In a recent proof-of-concept trial, the administration (just before percutaneous coronary intervention) of cyclosporine A, a potent PTP inhibitor, was associated with smaller infarct size. This review will focus on the physiological preclinical data on both ischemic and pharmacological postconditioning that are relevant to their translation to clinical therapeutics.

Among 15,000 forensic post-mortem examinations performed on the coroner's order over a 24-year period (January 1981-December 2004) in the area of Lyon, France (population: 2,000,000), 2250 cases of unexpected cardiac sudden death were identified retrospectively according to WHO criteria. Of these, 108 occurred during recreational sport and 12 occurred in athletes. In the latter category, a history of anabolic steroid abuse was found in 6 cases, whereas pre-existing ordinary cardiac lesions were observed in the 6 remaining cases. To shed light on the possible role of anabolic steroids in the induction of cardiac lesions, an experimental study was conducted in rabbits that were treated orally with norethandrolone 8mg/kg/day for 60 days, and sacrificed at day 90. The histopathological examination of the heart from treated animals showed coronary thrombosis associated with left ventricle hypertrophy in 3 cases, and lesions analogous to toxic or adrenergic myocarditis in all other treated animals. These findings were very similar to those observed after cardiac sudden death in the 6 athletes with a history of anabolic steroid abuse. In addition, elevated caspase-3 activity in the heart of treated rabbits as compared to controls suggests that apoptosis is involved in the induction of norethandrolone-induced cardiac lesions. These results confirm the cardiotoxic potential of anabolic steroid abuse.

Mitochondrial permeability transition pore (mPTP) inhibition plays a relevant role in postconditioning (PostC). Ischemia damages the electron transport chain, and the potential contribution of additional modifications in mitochondrial function caused by PostC remains unknown. We sought to determine which mitochondrial functions are involved in the inhibition of mPTP opening during the first minutes of reperfusion. Anesthetized New Zealand White rabbits underwent 30-min ischemia followed by 10-min reperfusion. At reperfusion, they received either no intervention (Control, C), PostC with 4 cycles of 1-min ischemia followed by 1-min reperfusion, or an IV injection of 5 mg/kg cyclosporine A (CsA: a powerful inhibitor of mPTP opening). Sham rabbits underwent no ischemia throughout the 40-min experiment. At the end of the 10-min reperfusion, mitochondria were isolated from the area at risk by differential centrifugations. Calcium retention capacity (CRC) and mitochondrial membrane potential (DeltaPsi(m)) were assessed by fluorimetry in subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria. Oxidative phosphorylation was assessed using a Clark-type electrode, and oxidative stress via protein carbonylation by Western blotting. PostC and CsA treatments improved CRC when compared to the C group. Control, PostC and CsA mitochondria exhibited a comparable significant dissipation of DeltaPsi(m), together with a comparable significant decrease in state 3 and an increase in state 4 respiration, in both SSM and IFM. However, PostC but not CsA treatment reduced total heart oxidative stress. These data suggest that during the early minutes of reperfusion, PostC reduces oxidative stress and inhibits mPTP opening, independent of alteration of oxidative phosphorylation or of DeltaPsi(m).

A large body of experimental evidence indicates that during an acute myocardial infarction (AMI), tissue injury occurring after reperfusion represents a significant amount of the whole, irreversible damage. It is now recognized that mitochondrial permeability transition pore opening plays a crucial role in this specific component of myocardial infarction. Ischaemic postconditioning and cyclosporine A (CsA) have been shown to dramatically reduce infarct size in many animal species. Recent proof-of-concept clinical trials support the idea that lethal myocardial reperfusion injury is also of significant importance in patients with ongoing AMI, and that targeting mitochondrial permeability transition by either percutaneous coronary intervention postconditioning or CsA can reduce infarct size and improve the recovery of contractile function after reperfusion. Large-scale trials are ongoing to address whether these new treatments may improve clinical outcome in reperfused AMI patients.

Mitochondrial permeability transition pore (mPTP) opening is a crucial event in cardiomyocyte death after I/R. We questioned whether preconditioning (PC) may inhibit mPTP opening during ischemia and/or during reperfusion and whether this effect would persist as reperfusion evolves. Anesthetized New Zealand white rabbits underwent a test ischemia followed by reperfusion. Ischemia lasted either 10 or 30 min, whereas reperfusion duration varied from 5 to 20, 60 and up to 240 min. For each duration of ischemia and reperfusion, animals were randomized as either control or PC. Preconditioning was induced by 5 min of ischemia followed by 5 min of reperfusion. Mitochondria were isolated from myocardium at risk for assessment of the calcium retention capacity (CRC) (potentiometric technique) used here as an index of sensitivity of the mPTP to Ca2+ loading. In controls, the CRC was moderately reduced after ischemia alone, but reperfusion severely and time-dependently accelerated further CRC reduction. Preconditioning failed to modify mPTP opening during ischemia alone, but significantly improved CRC during reperfusion. This protective effect persisted as reperfusion evolved. These data suggest that (a) reperfusion strikingly increases the susceptibility to Ca2+-induced mPTP opening, and that (b) PC inhibits mPTP opening at reflow and throughout the first hours of reperfusion.

Inhibition of mitochondrial permeability transition pore (mPTP) opening by cyclosporin A or ischemic postconditioning attenuates lethal reperfusion injury. Its impact on major post-myocardial infarction events, including worsening of left ventricular (LV) function and death, remains unknown. We sought to determine whether pharmacological or postconditioning-induced inhibition of mPTP opening might improve functional recovery and survival following myocardial infarction in mice. Anesthetized mice underwent 25 min of ischemia and 24 h ( protocol 1) or 30 days ( protocol 2) of reperfusion. At reperfusion, they received no intervention (control), postconditioning (3 cycles of 1 min ischemia-1 min reperfusion), or intravenous injection of the mPTP inhibitor Debio-025 (10 mg/kg). At 24 h of reperfusion, mitochondria were isolated from the region at risk for assessment of the Ca2+retention capacity (CRC). Infarct size was measured by triphenyltetrazolium chloride staining. At 30 days of reperfusion, mortality and LV contractile function (echocardiography) were evaluated. Postconditioning and Debio-025 significantly improved Ca2+retention capacity (132 ± 13 and 153 ± 31 vs. 53 ± 16 nmol Ca2+/mg protein in control) and reduced infarct size to 35 ± 4 and 32 ± 7% of area at risk vs. 61 ± 6% in control ( P < 0.05). At 30 days, ejection fraction averaged 74 ± 6 and 77 ± 6% in postconditioned and Debio-025 groups, respectively, vs. 62 ± 12% in the control group ( P < 0.05). At 30 days, survival was improved from 58% in the control group to 92 and 89% in postconditioned and Debio-025 groups, respectively. Inhibition of mitochondrial permeability transition at reperfusion improves functional recovery and mortality in mice.

In vivo evaluation of the transmural extension of myocardial infarction (TEI) is crucial to prediction of viability and prognosis. With the rise of transgenic technology, murine myocardial infarction (MI) models are increasingly used. Our study aimed to evaluate systolic strain rate (SR), a new parameter of regional function, to quantify TEI in a murine model of acute MI induced by various durations of ischemia followed by 24 h of reperfusion. Global and regional left ventricular (LV) function were assessed by echocardiography (13 MHz, Vivid 7, GE) in 4 groups of wild-type mice (C57BL/6, 2 mo old): a sham-treated group ( n = 10) and three MI groups [30 ( n = 11), 60 ( n = 10), and 90 ( n = 9) min of left coronary artery occlusion]. Conventional LV dimensions, anterior wall (AW) thickening, and peak systolic SR were measured before and 24 h after reperfusion. Area at risk (AR) was measured by blue dye and infarct size (area of necrosis, AN) and TEI by triphenyltetrazolium chloride staining. AN increased with ischemia duration (25 ± 2%, 56 ± 5%, 71 ± 6% of AR for 30, 60, and 90 min, respectively; P < 0.05). LV end-diastolic volume significantly increased with ischemia duration (30 ± 5, 34 ± 5, 43 ± 5 μl; P < 0.05), whereas LV ejection fraction decreased (63 ± 5%, 58 ± 6%, 46 ± 5%; P < 0.05). AW thickening decrease was not influenced by ischemia duration. Conversely, systolic SR decreased with ischemia duration (13 ± 5, 4 ± 3, −2 ± 6 s−1; P < 0.05) and was significantly correlated with TEI ( r = 0.89, P < 0.01). Receiver operating characteristic (ROC) curves identified systolic SR as the most accurate parameter to predict TEI. In conclusion, in a murine model of MI, SR imaging is superior to conventional echocardiography to predict TEI early after MI.

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.

Trimetazidine (TMZ) affects mitochondrial function during ischemia. Mitochondrial permeability transition is a pivotal event in cardiomyocyte death following acute ischemia. The aim of the present study was to determine whether the anti-ischemic agent TMZ might modulate mitochondrial permeability transition pore (mPTP) opening and limit lethal ischemia-reperfusion injury. Anesthetized NZW rabbits underwent 30 min of coronary artery occlusion followed by 4 hours of reperfusion. Prior to this, they underwent either no intervention (control, C), ischemic preconditioning (PC), or an IV injection of 5 mg kg(-1) TMZ 10 min before ischemia (TMZ). Additional rabbits (Sham group) underwent no ischemia/reperfusion throughout the experiment. Infarct size was assessed by triphenyltetrazolium staining, and apoptosis via measurement of caspase 3 activity. Ca(2+)-induced mPTP opening was assessed in mitochondria isolated from ischemic myocardium. TMZ and PC significantly reduced infarct size that averaged 34 +/- 4% and 21 +/- 4% of the risk region respectively, versus 63 +/- 6% in controls (P

The Fas/Fas ligand and mitochondria pathways have been involved in cell death in several cell types. We combined the genetic inactivation of the Fas receptor ( lpr mice), on the one hand, to the pharmacological inhibition of the mitochondrial permeability transition pore (mPTP), on the other hand, to investigate which of these pathways is predominantly activated during prolonged ischemia-reperfusion. Anesthetized C57BL/6JICO (control) and C57BL/6- lpr mice were pretreated with either saline or cyclosporin A (CsA; 40 mg/kg, 3 times a day), an inhibitor of the mPTP, and underwent 25 min of ischemia and 24 h of reperfusion. After 24 h of reperfusion, hearts were harvested: infarct size was assessed by 2,3,5-triphenyltetrazolium chloride staining, myocardial apoptosis by caspase 3 activity, and mitochondrial permeability transition by Ca2+-induced mPTP opening using a potentiometric approach. Infarct size was comparable in untreated control and lpr mice, ranging from 77 ± 5% to 83 ± 3% of the area at risk. CsA significantly reduced infarct size in control and lpr hearts. Control and lpr hearts exhibited comparable increase in caspase 3 activity that averaged 57 ± 18 and 49 ± 5 pmol·min−1·mg−1, respectively. CsA treatment significantly reduced caspase 3 activity in control and lpr hearts. The Ca2+overload required to open the mPTP was decreased to a similar extent in lpr and controls. CsA significantly attenuated Ca2+-induced mPTP opening in both groups. Our results suggest that the Fas pathway likely plays a minor role, whereas mitochondria are preferentially involved in mice cardiomyocyte death after a lethal ischemia-reperfusion injury.

International audience; Objective: We investigated whether ischemic preconditioning (PC) may modify mitochondrial permeability transition (MPT) pore opening. Methods: In protocol 1, New Zealand White rabbits underwent either no intervention (sham group) or 10 min of ischemia followed by 5 min of reperfusion, preceded (PC) or not (C; control) by one episode of 5 min of ischemia and 5 min of reperfusion. Rabbits were pretreated. by either saline or the MPT pore inhibitor cyclosporin A (CsA), or its non-immunosuppressive derivative Cs29 (10 mg/kg, IV bolus). Hearts were harvested and mitochondria isolated for further assessment of Ca2+-induced MPT using a Ca2+-sensitive micro-electrode. In protocol 2, C and PC hearts underwent 30 min of ischemia and 4 h of reperfusion. They were pretreated either by saline, CsA or Cs29, as in protocol 1. Infarct size was assessed by triphenyltetrazolium, and apoptosis by TUNEL staining. Results: In protocol 1, the Ca2+ overload required to induce MPT pore opening was significantly higher in PC than in C hearts. CsA and Cs29 significantly increased the Ca2+ overload required for MPT pore opening. In protocol 2, mean infarct size averaged 25% of the risk region in CsA/Cs29 treated hearts versus 15% in PC and 55% in controls (P

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.

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.

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.

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

Background— Systolic strain rate (SR) has been shown to correlate with the transmural extent of myocardial infarction (MI). Ischemic postconditioning (PC) decreases MI size. We aimed to determine whether SR may predict the reduction of left ventricular (LV) remodeling induced by PC in a murine model of ischemia-reperfusion. Methods and Results— C57BL6 mice underwent 60 minutes of left coronary artery occlusion followed by reperfusion. PC consisted of 3 cycles of 1 minute of reperfusion and 1 minute of ischemia performed immediately after reperfusion. After 24 hours of reperfusion, a first subset of mice was euthanized for determination of infarct size. An additional subset of mice underwent 3 months of reperfusion. Echocardiography and SR were serially assessed at baseline, 3 days, and 1, 2, 3 months after reperfusion. PC decreased the infarct size and increased SR values within infarcted segments as soon as 24 hours after reperfusion as compared with controls (14±1 versus 6±1 s −1 , P r =−0.88, P P P r =−0.75; P =0.001), and end-diastolic volumes ( r =−0.70; P Conclusions— In this murine model of MI, SR accurately assessed the reduction in MI size induced by PC early after reperfusion and the subsequent reduction of LV remodeling.

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

IntroductionL’arrêt cardio-circulatoire (ACC) réanimé est une modalité d’ischémie reperfusion (I/R) globale. L’ouverture du pore de transition de perméabilité mitochondrial (mPTP), inhibé par la ciclosporine A (CsA), est un événement majeur à l’origine des lésions cellulaires (nécrose et apoptose) et de la dysfonction cardio-circulatoire secondaires à l’I/R. Le but de l’étude a été de vérifier que l’inhibition pharmacologique du mPTP concomitante de la réanimation cardio pulmonaire (RCP) pouvait limiter in vivo les lésions cellulaires et leurs conséquences fonctionnelles.Matériels et MéthodesDes lapins NZW ont été soumis à 15min d’ACC hypoxique suivi d’une RCP (massage cardiaque et adrénaline) et de 120min de reperfusion. Trois groupes ont été étudiés (ACC sans autre intervention, CsA 5mg/kg administrée au début de la RCP, sham) dans deux protocoles expérimentaux (n=7 12/groupe) : (1) hémodynamique invasive (incluant sonomicrométrie, débit aortique et dP/dt) et mesure de la taille d’infarctus myocardique (triphényltétrazolium), (2) dosage de troponine Ic et mitochondries isolées avec mesure fluorimétrique de la capacité de rétention calcique (CRC) et oxygraphie.RésultatsLe succès de la RCP et la survie étaient améliorés de façon significative dans le groupe CsA avec des doses d’adrénaline plus faibles que dans le groupe ACC (p

Openers of K+(ATP) channels protect the myocardium from I/R injury. Sulfonylureas are known as potent blockers of K(ATP) channels. We investigated whether 1) mitochondrial permeability transition pore may be involved in the protection afforded by the mitoK+(ATP) opener nicorandil and 2) whether sulfonylureas may prevent this beneficial effect. Anesthetized New Zealand White rabbits underwent 30 min of coronary artery occlusion, followed by 60 (isolated mitochondria) or 240 min (infarct size) of reperfusion. They received an administration of either saline (control) or nicorandil (0.5 mg kg(-1), i.v.) 15 min before ischemia. Each control and nicorandil group was divided in four subgroups pretreated by either saline, glibenclamide (Glib; 1 mg kg(-1)), gliclazide (Glic; 1 mg kg(-1)), or glimepiride (Glim; 5 microg kg(-1)) 10 min before this. Infarct size was assessed by triphenyltetrazolium chloride staining. Mitochondria were isolated from the area at risk for further assessment of the calcium retention capacity. Glibenclamide (35 +/- 8), but neither Glic (61 +/- 9) nor Glim (48 +/- 7), reversed the improvement in calcium retention capacity due to nicorandil (58 +/- 10 vs. 27 +/- 8 nmoles CaCl2 mg(-1) proteins in control). Infarct size reduction by nicorandil (32% +/- 6% vs. 65% +/- 6% of area at risk) was abolished by Glib (55 +/- 5) but not by Glic (37 +/- 3) or Glim (31 +/- 5). These data suggest that 1) the protective effect of nicorandil involves the inhibition of the mitochondrial permeability transition pore and 2) that unlike Glib, second-generation sulfonylureas preserve this cardioprotection.

Mitochondrial permeability transition pore (mPTP) inhibition plays a crucial role in postconditioning (PostC). We sought to determine whether oxidative phosphorylation and mitochondrial membrane potential (ΔΨ m ), which both modulate mPTP opening, are involved in the inhibition of mPTP opening in the postconditioned heart. Anesthetized rabbits underwent 30 minutes of ischemia followed by 10 minutes of reperfusion. At the onset of reperfusion, they received either no intervention (Control, C), 4 cycles of 1 min ischemia followed by 1 min reperfusion (PostC), or an IV injection of 5mg/kg of the powerful inhibitor of mPTP opening, i.e. cyclosporine A (CsA). Sham rabbits underwent no ischemic insult throughout the 40 minute experiment. At the end of the 10 minute reperfusion period, the myocardial area at risk was excised, and mitochondria were isolated by differential centrifugations. Calcium retention capacity, an index of mPTP inhibition (CRC: nmol Ca 2+ /mg prot) and ΔΨ m (at state 4: % of FCCP-evoked maximum) were assessed by spectrofluorimetry in isolated subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria. Oxidative phosphorylation (at states 3 and 4: nmol O 2 /min/mg) was assessed using a Clark-type electrode (RCR: state 3 / state 4). As expected, PostC and CsA treatments improved CRC when compared to the C group. Control, PostC and CsA mitochondria exhibited a comparable significant dissipation of ΔΨ m , together with a comparable significant decrease of RCR in both SSM and IFM. In all three groups, this latter effect was related to a concomitant significant decrease in state 3 and to an increase in state 4 respiration. These data suggest that during the early minutes of reperfusion, postconditioning inhibits mPTP opening, independent of any specific modification of the oxidative phosphorylation or of ΔΨ m . Summarized data

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 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.

International audience; BACKGROUND: New therapeutic strategies aim to reduce the extent of myocardial infarction (MI) to decrease long-term left ventricular (LV) remodeling. These innovations are often developed on murine models of MI and have led to the need for a sensitive tool allowing follow-up. The aim of this study was to investigate by strain rate (SR) imaging early and long-term alteration in regional LV function occurring after MI in mice. METHODS: Echocardiography was serially performed during a 4-month follow-up period in 3 groups of C57BL6 male mice: 7 normal, 5 sham operated, and 27 with left coronary artery ligation (the MI group). In addition to conventional measurements, SR was obtained from short-axis views in the anterior wall and posterior wall (PW). Triphenyltetrazolium chloride staining allowed the localization and measurement of the transmural extent of MI. A transmural MI was defined as an extension > 75% of the wall thickness. RESULTS: In the MI group, LV ejection fractions significantly decreased, while LV dimensions and PW thicknesses increased from baseline to 4 months. On day 3, SR could differentiate transmural from nontransmural (1 +/- 1 vs 10 +/- 1 s(-1); P < .05) and noninfarcted myocardium (25 +/- 1 s(-1)). SR values did not significantly change between day 3 and month 4 and could still differentiate those segments at month 4. Wall thickening was not able to differentiate transmural from nontransmural infarcted segments at day 3 (16 +/- 3% vs 21 +/- 3%; P = NS) or at month 4. CONCLUSION: In this murine model of MI, SR was able to predict the transmural extent of MI as early as 3 days after MI, then remained stable and still differentiated them at 4 months.

Ca2+ is the main trigger for mitochondrial permeability transition pore opening, which plays a key role in cardiomyocyte death after ischemia-reperfusion. We investigated whether a reduced accumulation of mitochondrial Ca2+ might explain the attenuation of lethal reperfusion injury by postconditioning. Anesthetized New Zealand White rabbits underwent 30 min of ischemia, followed by either 240 (infarct size protocol) or 60 (mitochondria protocol) min of reperfusion. They received either no intervention (control), preconditioning by 5-min ischemia and 5-min reperfusion, postconditioning by four cycles of 1-min reperfusion and 1-min ischemia at the onset of reflow, or pharmacological inhibition of the transition pore opening by N-methyl-4-isoleucine-cyclosporin (NIM811; 5 mg/kg iv) given at reperfusion. Area at risk and infarct size were assessed by blue dye injection and triphenyltetrazolium chloride staining. Mitochondria were isolated from the risk region for measurement of 1) Ca2+ retention capacity (CRC), and 2) mitochondrial content of total (atomic absorption spectrometry) and ionized (potentiometric technique) calcium concentration. CRC averaged 0.73 ± 0.16 in control vs. 4.23 ± 0.17 μg Ca2+/mg proteins in shams ( P < 0.05). Postconditioning, preconditioning, or NIM811 significantly increased CRC ( P < 0.05 vs. control). In the control group, total and free mitochondrial calcium significantly increased to 2.39 ± 0.43 and 0.61 ± 0.10, respectively, vs. 1.42 ± 0.09 and 0.16 ± 0.01 μg Ca2+/mg in sham ( P < 0.05). Surprisingly, whereas total and ionized mitochondrial Ca2+ decreased in preconditioning, it significantly increased in postconditioning and NIM811 groups. These data suggest that retention of calcium within mitochondria may explain the decreased reperfusion injury in postconditioned (but not preconditioned) hearts.

Introduction: Activation of the PI3kinase pathway and subsequent Inhibition of GSK3β by phosphorylation at serine-9 residue has recently been shown to protect against myocardial lethal reperfusion injury. Hypothesis: We hypothesized that GSK3β may play a key role in protection afforded by postconditioning. We used transgenic mice (Tg) that express a constitutively active (i.e. that cannot be inactivated by phosphorylation at serine 9) form of GSK-3β. Methods: Anesthetized WT and Tg mice underwent 60 minutes of coronary artery occlusion followed by 24 hrs of reperfusion (n=9/gr). At reflow, mice received either no intervention (controls: C-WT and C-Tg), postconditioning (postC) by 3 cycles of [1 min ischemia / 1 min reperfusion], or an IV bolus injection (1 minute before reflow) of either 10 mg/kg of cyclosporine A (CsA), a potent inhibitor of the mitochondrial permeability transition pore (mPTP) opening, or 70μg/kg of SB 216763 (SB), a specific inhibitor of GSK3β. Infarct size (%of area at risk) was measured by TTC staining. CRC, an index of Ca 2+ -induced mPTP opening in isolated mitochondria was measured by a fluorimetric approach. Results: PostC, CsA and SB significantly reduced infarct to 29%, 36% and 34% respectively (p2+ /mg), but improved in postC, CsA and SB mice, averaging 155±31, 267±11 and 183±37 nmol Ca 2+ /mg respectively (p2+ /mg), but improved in CsA. Conclusion: These results suggest that inhibition of GSK3β is involved in postconditioning’s protection against infarction, likely by preventing mPTP opening during the first minutes of reperfusion.