Unacylated ghrelin analog prevents myocardial reperfusion injury independently of permeability transition pore

Reperfusion injury is responsible for an important part of myocardial infarct establishment due notably to triggering cardiomyocytes death at the first minutes of reperfusion. AZP-531 is an optimized analog of unacylated ghrelin currently in clinical development in several metabolic diseases. We investigated a potential cardioprotective effect of AZP-531 in ischemia/reperfusion (IR) and the molecular underlying mechanism(s) involved in this protection. In vivo postconditioning with AZP-531 in C57BL6 mouse IR model decreased infarct size. Western blot analysis on areas at risk from the different mouse groups showed that AZP-531 activates Akt, ERK1-2 as well as S6 and 4EBP1, mTORC1 effectors. We also showed an inhibition of caspase 3 cleavage and Bax translocation to the mitochondria. AZP-531 also stimulated the expression of antioxidants and was capable of decreasing mitochondrial H2O2 production, contributing to the reduction of ROS accumulation. AZP-531 exhibits cardioprotective effect when administrated for postconditioning in C57BL6 mouse IR model. Treatment with AZP-531 rescued the myocardium from cell death at early reperfusion by stimulating protein synthesis, inhibiting Bax/caspase 3-induced apoptosis as well as ROS accumulation and oxidative stressinduced necrosis. AZP-531 may prove useful in the treatment of IR injury

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

International audienceCardiovascular 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 mum), 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. 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, we validate our model by using the protective treatment : cyclosporine A (CsA) to illustrate robustness and sensitivity of the utilized methods for pharmacological screening. 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

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

International audienceHypothermia 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