Klotho/FGF23 Axis Regulates Cardiomyocyte Apoptosis and Cytokine Release through ERK/MAPK Pathway.

Coronary artery disease (CAD) as a major cardiovascular disease is the leading global cause of mortality, Klotho/FGF23 axis involved in development of cardiovascular disease, while the function and underlying mechanism of Klotho/FGF23 axis in CAD is unclear. Blood samples from 67 CAD patients with coronary artery bypass graft (CABG) surgery were collected, and the level of Klotho and FGF23 of those patients was measured by using an ELISA kit. Cardiomyocyte was isolated from 0 to 3 days Sprague Dawley (SD) rats. Expression of Klotho, FGF23 and the cardiomyocyte marker α-sarcomeric actin (α-SA), myosin heavy chain (MHC) and cardiac troponin I (cTnI) was assessed by immunofluorescence staining. Expression of Klotho and FGF23 mRNA was detected by qRT-PCR. Apoptosis and cell cycle were measured by flow cytometry. Cell viability was detected by using CCK-8. The protein expression of ERK/MAPK pathway related protein and cytokines production was measured by western blotting. The levels of Klotho in CAD patients increased after CABG surgery, while FGF23 decreased. Isolated cardiomyocyte morphology and structure were completed, and with stabilized beating within culture for 15 days, besides, α-SA, MHC, and cTnI proved positive. After transfected Lenti-Klotho and Lenti-FGF23 into isolated cardiomyocyte, fluorescence staining showed that the transfection was successful, and qRT-PCR results showed that the expression levels of Klotho and FGF23 mRNA significant increased compared with NEG (empty vector) group. Immunofluorescence staining results showed that compared with NEG group, there was a higher Klotho positive rate and lower FGF23 positive rate in Klotho overexpression (Klotho) group, while, there was a higher FGF23 positive rate and lower Klotho positive rate in FGF23 overexpression (FGF23) group. In addition, the expression of p-ERK1/2 and p-P38 increased in Klotho group but decreased in FGF23 group. Furthermore, overexpression of Klotho inhibited cardiomyocyte apoptosis, increased S phase fraction, promoted proliferation and elevated expression of transforming growth factor ß1 (TGF-ß1), nuclear factor-kappa B (NF-κB), angiotensin-II (AT-II), and activator protein-1 (AP-1), overexpression of FGF23 showed the opposite effect, however, ERK agonist (TPA) and inhibitor (U0126) reversed the effect caused by overexpression of Klotho and FGF23 separately. Klotho/FGF23 axis play a critical role in CAD progression through regulating ERK/MAPK pathway in Cardiomyocyte.

Levosimendan Postconditioning Attenuates Cardiomyocyte Apoptosis after Myocardial Infarction.

BACKGROUND: Levosimendan preconditioning has been shown to attenuate myocardial apoptosis in animal models. However, protective effects of levosimendan postconditioning against myocardial apoptosis following myocardial infarction (MI) have not been evaluated. Therefore, we investigated the effects of levosimendan postconditioning on myocardial apoptosis in MI rat models. METHODS: In an anoxia/reoxygenation (A/R) model, H9c2 cells were pretreated with or without levosimendan postconditioning after which their apoptosis rates were assessed by flow cytometry, RT-qPCR, and western blot analyses. Then, postconditioning was performed with or without levosimendan in MI rat models. Myocardiocyte apoptosis was evaluated by echocardiography, TTC staining, TUNEL staining, immunohistochemical staining, RT-qPCR, and western blot analysis. RESULTS: Levosimendan postconditioning inhibited H9c2 cell apoptosis in A/R models by elevating Bcl-2 while suppressing Caspase-3 and Bax at both mRNA and protein levels. Moreover, it improved cardiac functions and reduced the left ventricle infarction area in MI rat models. Compared to the MI control group, cardiomyocyte apoptosis rates in the levosimendan postconditioning group were low. The reduced cardiomyocyte apoptosis rates were associated with downregulation of Bax and Caspase-3 as well as with upregulation of Bcl-2 at mRNA and protein levels. CONCLUSIONS: Levosimendan postconditioning of MI rat models protected against cardiomyocyte apoptosis, implying that it is a potential strategy for preventing cardiomyocyte apoptosis in the treatment of cardiac dysfunction following MI.