Abstract 4141340: Fbxo44 Alleviates Heart Aging and Mitochondrial Calcium Overload Injury by Modulating RyR2-Related Pathway

Background: Cardiac aging is a continuous pathological process. Imbalances in mitochondrial calcium homeostasis are prevalent in the aging myocardium. Fbxo44 is a member of the F-box family that has been shown to be involved in a variety of diseases. Here, we report an aging-associated mechanism in cardiomyocytes in which Fbxo44 disrupts mitochondrial calcium homeostasis by promoting phosphorylation of the calcium release channel protein RyR2. Hypothesis: Fbxo44 regulates cardiac aging through the RyR2-related pathway. Methods: The doxorubicin was used to established the animal models of cardiac aging in Cardiac myocyte-specific Fbxo44 knockout (CKO) and control mice. The age-related proteins and positive stained area were used to assess the senescence phenotypes. Cardiac function of mice was assessed using Doppler ultrasound. Expression levels of Fbxo44 and p-RyR2 were evaluated through Western blotting. Fbxo44 was knocked down using adenovirus in H9C2 cells, followed by the addition of doxorubicin to induce cellular senescence. The cell model was analyzed for senescence-related phenotypes by β-galactosidase staining and western blotting. Confocal laser scanning microscopy was employed to assess the expression of the Rhod-2 probe and RyR2 fluorescence. Western blotting was performed to detect the levels of Fbxo44 and phosphorylation of RyR2. Results: Myocardial tissue-specific knockdown of Fbxo44 resulted in improved cardiac function compared to the control group, along with a significant reduction in the degree of senescence and expression of associated pathway proteins. In the cellular model, down-regulation of Fbxo44 significantly reduced the proportion of positive β-galactosidase staining and decreased the expression of senescence markers and related pathway proteins. Furthermore, inhibition of Fbxo44 mitigated mitochondrial calcium damage, accompanied by a decrease in RyR2 fluorescence intensity. Conclusions: Downregulation of Fbxo44 regulates mitochondrial calcium overload and cardiac senescence via RyR2-related pathways.