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| 180P London, UK Pharmacology 2016 |
The type 2A protein phosphatase regulatory subunit alpha4 is a novel inhibitor of apoptosis in cardiomyocytes
Introduction: Cardiomyocyte cell death associated with heart disease is thought to involve apoptosis1 and therefore it is of paramount importance to understand what drives apoptosis in the heart. In this study, we demonstrate that the type 2A protein phosphatase regulatory protein alpha4 behaves as a novel anti-apoptotic protein in cardiomyocytes.
Method: Alpha4 protein knockdown (alpha4 KD) in cultured H9c2 cardiomyocytes (1-8 days) was achieved using appropriate protein-specific short interfering RNA (siRNA). Western immunoblotting analysis using appropriate antibodies was used to determine a variety of apoptotic endpoints in response to alpha4 KD. Cell death associated with alpha4 KD was determined by light microscopy. Data are presented as mean ± SEM (n=3-5) relative to non-targeting control (CTR) and statistical analysis was performed by one-way ANOVA followed by Bonferroni’s multiple comparison test.
Results: Western immunoblotting analysis revealed differential regulation of the anti-apoptotic B-cell lymphoma 2 (BCL-2) protein and its pro-apoptotic binding partner BCL-2 associated X (BAX) protein. There was no significant difference in BAX expression 4 days after alpha4 KD, but after 6 days expression had significantly decreased by 72.7 ± 8.1% (p<0.05) compared to CTR cardiomyocytes. In contrast, alpha4 KD significantly (p<0.05) decreased expression of BCL-2 protein by 40.8 ± 8.9% and 61.2 ± 6.7% after 3 and 4 days, respectively, when compared to CTR cardiomyocytes. Therefore, the apoptotic index BAX:BCL-2 ratio significantly (p<0.05) peaked (4.4-fold) following 4 days alpha4 KD when compared to CTR cardiomyocytes. Expression of full length BH3-interacting domain (BID) protein was significantly (p<0.05) reduced by 58.7 ± 11.0% following 6 days alpha4 KD when compared to CTR cardiomyocytes. Alpha4 KD for 6 days not only significantly (p<0.05) reduced expression of the inactive 32kDa procaspase-3 by 58.8 ± 19.6%, but also significantly reduced expression of the caspase-3 proteolytic substrate poly ADP ribose polymerase 1 (PARP1) by 80.3 ± 16.6% (p<0.05) when compared to CTR cardiomyocytes. Cardiomyocyte cell death was confirmed by light microscopy following 8 days of alpha4 KD when compared to CTR cardiomyocytes.
Conclusion: Ablation of alpha4 expression disrupts the BAX/BCL-2 signalling pathway, induces proteolytic activation of BID and caspase-3 proteins, cleaves and inhibits a DNA repair protein PARP1 and induces cell death. In conclusion, this study illustrates the novel significance of alpha4 as an anti-apoptotic protein in cardiomyocytes.
References: 1. Olivetti G et al (1997). N Eng J Med 336: 1131-1141.