Mitochondrial dynamic disorder is involved in myocardial ischemia/reperfusion (I/R) injury. To explore the effect of mitochondrial calcium uniporter (MCU) on mitochondrial dynamic imbalance under I/R and its related signal pathways, a mouse myocardial I/R model and hypoxia/reoxygenation model of mouse cardiomyocytes were established. The expression of MCU during I/R increased and related to myocardial injury, enhancement of mitochondrial fission, inhibition of mitochondrial fusion and mitophagy. Suppressing MCU functions by Ru360 during I/R could reduce myocardial infarction area and cardiomyocyte apoptosis, alleviate mitochondrial fission and restore mitochondrial fusion and mitophagy. However, spermine administration, which could enhance MCU function, deteriorated the above‐mentioned myocardial cell injury and mitochondrial dynamic imbalanced. In addition, up‐regulation of MCU promoted the expression and activation of calpain‐1/2 and down‐regulated the expression of Optic atrophy type 1 (OPA1). Meantime, in transgenic mice (overexpression calpastatin, the endogenous inhibitor of calpain) I/R model and OPA1 knock‐down cultured cell. In I/R models of transgenic mice over‐expressing calpastatin, which is the endogenous inhibitor of calpain, and in H/R models with siOPA1 transfection, inhibition of calpains could enhance mitochondrial fusion and mitophagy, and inhibit excessive mitochondrion fission and apoptosis through OPA1. Therefore, we conclude that during I/R, MCU up‐regulation induces calpain activation, which down‐regulates OPA1, consequently leading to mitochondrial dynamic imbalance.

Backgrounds Inflammation underlies the mechanism of different kinds of heart disease. Cytoplasmic membrane localized N‐terminal fragment of gasdermin‐D (GSDMD‐N) could induce inflammatory injury to cardiomyocyte. However, effects and dynamic changes of GSDMD during the process of lipopolysaccharide (LPS) related inflammatory stress induced cardiomyocyte injury are barely elucidated to date. In this study, LPS related cardiomyocyte injury was investigated based on potential interaction of GSDMD‐N induced mitochondrial injury and mitophagy mediated mitochondria quality control. Methods HL‐1 cardiomyocytes were treated with LPS and Nigericin to induce inflammatory stress. The dual‐fluorescence‐labelled GSDMD expressed HL‐1 cardiomyocytes were constructed to study the translocation of GSDMD. The mitochondrial membrane potential (MMP) was measured by JC‐1 staining. Mitophagy and autophagic flux were recorded by transmission electron microscopy and fluorescent image. Results GSDMD‐N showed a time‐dependent pattern of translocation from mitochondria to cytoplasmic membrane under LPS and Nigericin induced inflammatory stress in HL‐1 cardiomyocytes. GSDMD‐N preferred to localize to mitochondria to permeablize its membrane and dissipate the MMP. This effect couldn't be reversed by cyclosporine‐A (mPTP inhibitor), indicating GSDMD‐N pores as alternative mechanism underlying MMP regulation, in addition to mitochondrial permeability transition pore (mPTP). Moreover, the combination between GSDMD‐N and autophagy related Microtubule Associated Protein 1 Light Chain 3 Beta (LC3B) was verified by co‐immunoprecipitation. Besides, mitophagy alleviating GSDMD‐N induced mitochondrial injury was proved by pre‐treatment of autophagy antagonist or agonist in GSDMD‐knock out or GSDMD‐overexpression cells. A time‐dependent pattern of GSDMD translocation and mitochondrial GSDMD targeted mitophagy were verified. Conclusion Herein, our study confirmed a crosstalk between GSDMD‐N induced mitochondrial injury and mitophagy mediated mitochondria quality control during LPS related inflammation induced cardiomyocyte injury, which potentially facilitating the development of therapeutic target to myocardial inflammatory disease. Our findings support pharmaceutical intervention on enhancing autophagy or inhibiting GSDMD as potential target for inflammatory heart disease treatment.

To explore the role of autophagic flux in the increased susceptibility of the experimental diabetic heart to ischaemia‐reperfusion (I/R) injury, we established STZ‐induced diabetic mice and performed I/R. In vitro, neonatal mouse cardiomyocytes were subjected to high glucose and hypoxia/reoxygenation challenge to mimic diabetic I/R injury. We found that experimental diabetes aggravated I/R‐induced injury than compared with nondiabetic mice. Autophagic flux was impaired in I/R hearts, and the impairment was exacerbated in diabetic mice subjected to I/R with defective autophagosome formation and clearance. Calpains, calcium‐dependent thiol proteases, were upregulated and highly activated after I/R of diabetes, while calpain inhibition attenuated cardiac function and cell death and partially restored autophagic flux. The expression levels of Atg5 and LAMP2, two crucial autophagy‐related proteins, were significantly degraded in diabetic I/R hearts, alterations that were associated with calpain activation and could be reversed by calpain inhibition. Co‐overexpression of Atg5 and LAMP2 reduced myocardial injury and normalized autophagic flux. In conclusion, experimental diabetes exacerbates autophagic flux impairment of cardiomyocytes under I/R stress, resulting in worse I/R‐induced injury. Calpain activation and cleavage of Atg5 and LAMP2 at least partially account for the deterioration of autophagic flux impairment.