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Background: Activated protein C (APC) is a plasma serine protease with anticoagulant and anti-inflammatory activities. Endothelial protein C receptor (EPCR) is associated with APC's activity and mediates its downstream signaling events. APC exerts cardioprotective effects during ischemia and reperfusion (I/R). This study aims to characterize the role of the APC-EPCR axis in ischemic insults in aging. Methods: Young (3-4 months) and aged (24-26 months) wild type C57BL/6J mice, as well as EPCR point mutation (EPCR R84A/R84A ) knock-in C57BL/6J mice incapable of interaction with APC and its wild type of littermate C57BL/6J mice, were subjected to I/R. Wild type APC, signaling-selective APC-2Cys, or anticoagulant-selective APC-E170A were administrated before reperfusion. Results: The results demonstrated that cardiac I/R reduces APC activity, and the APC activity was impaired in the aged versus young hearts possibly attributable to the declined EPCR level with aging. Serum EPCR measurement showed that I/R triggered the shedding of membrane EPCR into circulation, while administration of APC attenuated the I/R-induced EPCR shedding in both young and aged hearts. Subsequent echocardiography showed that APC and APC-2Cys but not APC-E170A ameliorated cardiac dysfunction during I/R in both young and aged mice. Importantly, APC elevated the resistance of the aged heart to ischemic insults through stabilizing EPCR. However, all these cardioprotective effects of APC were blunted in the EPCR R84A/R84A mice versus its wild-type littermates. The ex vivo working heart and metabolomics results demonstrated that AMP-activated protein kinase (AMPK) mediates acute adaptive response while protein kinase B (AKT) is involved in chronic metabolic programming in the hearts with APC treatment. Conclusions: I/R stress causes shedding of the membrane EPCR in the heart, and administration of APC prevents I/R-induced cardiac EPCR shedding that is critical for limiting cardiac damage in aging.

A progressive defect in the energy generation pathway is implicated in multiple aging-related diseases, including cardiovascular conditions and Alzheimer's Disease (AD). However, evidence of the pathogenesis of cardiac dysfunction in AD and the associations between the two organ diseases need further elucidation. This study aims to characterize cellular defects resulting in decreased cardiac function in AD-model. 5XFAD mice, a strain expressing five mutations in human APP and PS1 that shows robust Aβ production with visible plaques at 2 months and were used in this study as a model of AD. 5XFAD mice and wild-type (WT) counterparts were subjected to echocardiography at 2-, 4-, and 6-month, and 5XFAD had a significant reduction in cardiac fractional shortening and ejection fraction compared to WT. Additionally, 5XFAD mice had decreased observed electrical signals demonstrated as decreased R, P, T wave amplitudes. In isolated cardiomyocytes, 5XFAD mice showed decreased fraction shortening, rate of shortening, as well as the degree of transient calcium influx. To reveal the mechanism by which AD leads to cardiac systolic dysfunction, the immunoblotting analysis showed increased activation of AMP-activated protein kinase (AMPK) in 5XFAD left ventricular and brain tissue, indicating altered energy metabolism. Mito Stress Assays examining mitochondrial function revealed decreased basal and maximal oxygen consumption rate, as well as defective pyruvate dehydrogenase activity in the 5XFAD heart and brain. Cellular inflammation was provoked in the 5XFAD heart and brain marked by the increase of reactive oxygen species accumulation and upregulation of inflammatory mediator activities. Finally, AD pathological phenotype with increased deposition of Aβ and defective cognitive function was observed in 6-month 5XFAD mice. In addition, elevated fibrosis was observed in the 6-month 5XFAD heart. The results implicated that AD led to defective mitochondrial function, and increased inflammation which caused the decrease in contractility of the heart.

Ischemic heart disease (IHD) is the leading cause of death, with age range being the primary factor for development. The mechanisms by which aging increases vulnerability to ischemic insult are not well understood. We aim to use single‐cell RNA sequencing to discover transcriptional differences in various cell types between aged and young mice, which may contribute to aged‐related vulnerability to ischemic insult. Utilizing 10× Genomics Single‐Cell RNA sequencing, we were able to complete bioinformatic analysis to identity novel differential gene expression. During the analysis of our collected samples, we detected Pyruvate Dehydrogenase Kinase 4 (Pdk4) expression to be remarkably differentially expressed. Particularly in cardiomyocyte cell populations, Pdk4 was found to be significantly upregulated in the young mouse population compared to the aged mice under ischemic/reperfusion conditions. Pdk4 is responsible for inhibiting the enzyme pyruvate dehydrogenase, resulting in the regulation of glucose metabolism. Due to decreased Pdk4 expression in aged cardiomyocytes, there may be an increased reliance on glucose oxidization for energy. Through biochemical metabolomics analysis, it was observed that there is a greater abundance of pyruvate in young hearts in contrast to their aged counterparts, indicating less glycolytic activity. We believe that Pdk4 response provides valuable insight towards mechanisms that allow for the young heart to handle ischemic insult stress more effectively than the aged heart.

Introduction: Metformin activates AMP-activated protein kinase (AMPK) to improve cardiac function during ischemia and reperfusion (I/R). We reported that Sestrin2 (Sesn2) is associated with AMPK and maintains oxidative phosphorylation (OXPHOS) under I/R stress. The role of age-related Sesn2-AMPK signaling in the beneficial actions of metformin on ischemic insults remains unknown. Hypothesis: Metformin maintains mitochondrial integrity and limits cardiac damage caused by ischemic insults through the Sesn2-AMPK signaling pathway. Methods: Young (3-6 months) and aged (22-24 months) C57BL/6J wild type mice, and 3 months of Sesn2 f/f and cardiomyocyte-specific Sesn2 knockout (cSesn2 -/- ) C57BL/6J mice were subjected to 45 minutes of ischemia followed by 2 mM metformin injection 5 minutes before 24-hour of reperfusion. Cardiac function and myocardial infarction were determined with echocardiography and 2,3,5-triphenyl tetrazolium chloride staining. Immunoblotting determines the mechanism of metformin in modulating Sesn2 to preserve mitochondrial OXPHOS components. The Seahorse XF Analyzer examined the mitochondrial respiratory functions. Results: Metformin administration can significantly improve cardiac function and reduce myocardial infarction size during I/R conditions in both young and aged wild-type C57BL/6J mice. Intriguingly, the beneficial effects of metformin administration on cardiac function and myocardial infarction were significantly blunted in the cSesn2 -/- versus Sesn2 f/f C57BL/6J mice. The immunoblotting showed metformin treatment augmented mitochondrial OXPHOS Complex II levels in young/aged wild type, and Sesn2 f/f but not cSesn2 -/- heart during I/R stress. Moreover, the mitochondrial respiration data displayed that metformin treatment improved the respiration rate of mitochondrial states 2 and 3μ in the isolated cardiomyocytes from Sens2 f/f but not from cSesn2 -/- mouse hearts under I/R stress conditions. Conclusions: Metformin can stabilize age-related Sesn2 levels in cardiomyocytes and improve cardiac function under I/R stress through maintaining mitochondrial integrity. Metformin is a potential therapeutic drug for ischemic heart disease in the elderly.