Interaction between A-kinase anchoring protein 5 and protein kinase A mediates CaMKII/HDAC signaling to inhibit cardiomyocyte hypertrophy after hypoxic reoxygenation.

We reported that A-kinase anchoring protein 5 (AKAP5) played a role in cardiomyocyte apoptosis after hypoxia-reoxygenation (H/R). The role of AKAP5 in cardiomyocyte hypertrophy has not been fully elucidated. Herein we investigated whether AKAP5 regulates cardiomyocyte hypertrophy through calcium/calmodulin-dependent protein kinase II (CaMKII). After H/R, deficiency of AKAP5 in H9C2 cardiomyocytes and neonatal rat cardiac myocytes activated CaMKII and stimulated cardiomyocyte hypertrophy. AKAP5 upregulation limited this. Low expression of AKAP5 increased CaMKII interaction with histone deacetylases 4/5 (HDAC4/5) and increased nuclear export of HDAC4/5. In addition, AKAP5 interactions with protein kinase A (PKA) and phospholamban (PLN) were diminished. Moreover, the phosphorylation of PLN was decreased, and intracellular calcium increased. Interference of this process with St-Ht31 increased CaMKII signaling, decreased PLN phosphorylation and promoted post-H/R cell hypertrophy. And PKA-anchoring deficient AKAP5ΔPKA could not attenuate hypoxia-reoxygenation-induced cardiomyocyte hypertrophy, but AKAP5 could. Altogether, AKAP5 downregulation exacerbated H/R-induced hypertrophy in cardiomyocytes. This was due to, in part, to less in AKAP5-PKA interaction and the accumulation of intracellular Ca2+ with a subsequent increase in CaMKII activity.

Metoprolol Mitigates Ischemic Heart Remodeling and Fibrosis by Increasing the Expression of AKAP5 in Ischemic Heart.

The harm of heart failure mainly causes patients to develop dyspnea, fatigue, fluid retention, and other symptoms, which impair patients' activity tolerance and lead to a dramatic decrease in patients' quality of life. The purpose of this study was to verify whether metoprolol regulates AKAP5 expression and test the role of AKAP5 postinjury in mitigating cardiac infarction-associated tissue remodeling and fibrosis. Sprague-Dawley (SD) rats underwent coronary artery ligation (CAL), which was followed immediately with metoprolol daily. And western blot and coimmunoprecipitation experiments were performed to detect the expression of related proteins in the sham-operated group, model group, and drug-treated group. HW/BW ratio and cardiac expression of COL1 and COL3 were increased in rats following CAL compared with shams. Treatment with metoprolol postinjury was associated with a decrease in HW/BW ratio and COL1/COL3 expression compared to uncontrolled rats. CAL resulted in decreased cardiac AKAP5 expression compared to the control group, while metoprolol treatment restored levels compared to baseline shams. Cardiac expression levels of NFATc3/p-NFATc3 and GATA4 were modest at baseline and increased with injury, whereas metoprolol suppressed gene expression to below injury-associated changes. Immunoprecipitation indicated that AKAP5 could bind and regulate PP2B. In summary, we know that metoprolol alleviates ischemic cardiac remodeling and fibrosis, and the mechanism of alleviating remodeling may improve cardiac AKAP5 expression and AKAP5-PP2B interaction.

A-kinase anchoring protein 5-ancored calcineurin regulates the remodeling of H9c2 cardiomyocytes exposed to hypoxia and reoxygenation.

OBJECTIVE: A-kinase anchoring protein 5 (AKAP5) is involved in ventricular remodeling in rats with heart failure after myocardial infarction; however, the specific mechanism is not clear. This study investigated whether AKAP5 anchors calcineurin (CaN) to regulate the remodeling of H9c2 cardiomyocytes. METHODS: H9c2 cells were subjected to hypoxia stress for 3 h and reoxygenation for 24 h to create a hypoxia-reoxygenation (H/R) model. These cells were divided into three groups: H/R (model), empty vector +H/R (NC), and siRNA-AKAP5+H/R (siRNA-AKAP5) groups. The non-H/R H9c2 cells were used as normal controls. Western blotting was used to detect cardiac hypertrophy-related protein expression in the cells, including atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), beta myosin heavy chain (ß-MHC), and phosphorylated nuclear factor of activated T-cell 3 (p-NFATc3). Phalloidin staining was used to label the cytoskeleton and the cell area in different groups was measured. Immunofluorescence staining and coimmunoprecipitation were used to study the relationship between AKAP5 and CaN. H9c2 cells pretreated with the CaN inhibitor FK506 were used to further verify the relationship between AKAP5 and CaN. RESULTS: In the siRNA-AKAP5+H/R group, the expression level of cardiac hypertrophy-related proteins (ANP, BNP, and ß-MHC) and CaN and the area of cardiomyocytes were significantly increased, while the p-NFATc3/NFATc3 ratio was decreased in H9c2H/R cells. AKAP5 and CaN proteins were colocalized and interacted in the cells. The CaN inhibitor significantly suppressed the expression of CaN, increased the p-NFATc3/NFATc3 ratio, and reduced the expression levels of ANP, BNP, and ß-MHC proteins in the cells with low AKAP5 expression. CONCLUSIONS: AKAP5 downregulation aggravated the remodeling of cardiomyocytes after H/R. AKAP5 may anchor CaN to form a complex, which in turn activates NFATc3 dephosphorylation and expression of hypertrophy-related proteins.

A-kinase anchoring protein 5 anchors protein kinase A to mediate PLN/SERCA to reduce cardiomyocyte apoptosis induced by hypoxia and reoxygenation.

The A-kinase anchoring protein 5 (AKAP5) has a variety of biological activities. This study explored whether AKAP5 was involved in cardiomyocyte apoptosis induced by hypoxia and reoxygenation (H/R) and its possible mechanism. H9C2 cells were used to construct an H/R model in vitro, followed by AKAP5 overexpression. Flow cytometry was performed to determine the rate of cardiomyocyte apoptosis. Phosphorylation of phospholamban (PLN), sarcoplasmic/endoplasmic reticulum calcium ATPase 2a (SERCA2a), and apoptosis-related proteins was determined by western blotting. Immunofluorescence staining and immunoprecipitation were performed to detect the distribution and interaction between AKAP5, protein kinase A (PKA), and PLN. After H/R induction, H9C2 cells exhibited significantly reduced AKAP5 protein expression. Upregulation of AKAP5 promotes cell survival and significantly reduces lactate dehydrogenase (LDH) levels and apoptosis rates in H9C2 cells. In addition, the overexpression of AKAP5 was accompanied by the activation of the PLN/SERCA2a signaling pathway and a reduction in apoptosis. Immunofluorescence staining and immunoprecipitation revealed that AKAP5 co-localized and interacted with PLN and PKA. Interestingly, St-Ht31, an inhibitory peptide that disrupts AKAP interactions with regulatory subunits, inhibits the effect of AKAP5 overexpression on H/R-induced apoptosis in H9C2 cardiomyocytes. AKAP5 overexpression alleviated H/R-induced cardiomyocyte apoptosis possibly by anchoring PKA to mediate the PLN/SERCA pathway, suggesting that AKAP5 is a potential therapeutic target for the prevention and treatment of ischemia-reperfusion injury.