W. PP1␥ functionally augments the alternative splicing of CaMKII␦ through interaction with ASF. Am J Physiol Cell Physiol 306: C167-C177, 2014. First published November 6, 2013 doi:10.1152/ajpcell.00145.2013.-Protein phosphatase 1 (PP1) and Ca 2ϩ /calmodulin-dependent protein kinase ␦ (CaMKII␦) are upregulated in heart disorders. Alternative splicing factor (ASF), a major splice factor for CaMKII␦ splicing, can be regulated by both protein kinase and phosphatase. Here we determine the role of PP1 isoforms in ASF-mediated splicing of CaMKII␦ in cells. We found that 1) PP1␥, but not ␣ or ␤ isoform, enhanced the splicing of CaMKII␦ in HEK293T cells; 2) PP1␥ promoted the function of ASF, evidenced by the existence of ASF-PP1␥ association as well as the PP1␥ overexpression-or silencing-mediated change in CaMKII␦ splicing in ASFtransfected HEK293T cells; 3) CaMKII␦ splicing was promoted by overexpression of PP1␥ and impaired by application of PP1 inhibitor 1 (I1PP1) or pharmacological inhibitor tautomycetin in primary cardiomyocytes; 4) CaMKII␦ splicing and enhancement of ASF-PP1␥ association induced by oxygen-glucose deprivation followed by reperfusion (OGD/R) were potentiated by overexpression of PP1␥ and suppressed by inhibition of PP1␥ with I1PP1 or tautomycetin in primary cardiomyocytes; 5) functionally, overexpression and inhibition of PP1␥, respectively, potentiated or suppressed the apoptosis and Bax/Bcl-2 ratio, which were associated with the enhanced activity of CaMKII in OGD/R-stimulated cardiomyocytes; and 6) CaMKII was required for the OGD/R induced-and PP1␥ exacerbated-apoptosis of cardiomyocytes, evidenced by a specific inhibitor of CaMKII KN93, but not its structural analog KN92, attenuating the apoptosis and Bax/Bcl-2 ratio in OGD/R and PP1␥-treated cells. In conclusion, our results show that PP1␥ promotes the alternative splicing of CaMKII␦ through its interacting with ASF, exacerbating OGD/R-triggered apoptosis in primary cardiomyocytes.protein phosphatase 1␥; alternative splicing factor; CaMKII␦C; splicing; cardiomyocytes

Our previous studies showed that protein phosphatase 1γ (PP1γ) exacerbates cardiomyocyte apoptosis through promotion of Ca(2+)/calmodulin-dependent protein kinase δ (CaMKIIδ) splicing. Here we determine the role of PP1γ in abdominal aorta constriction-induced hypertrophy and remodelling in rat hearts. Systolic blood pressure and echocardiographic measurements were used to evaluate the model of cardiac hypertrophy. Sirius red staining and invasive haemodynamic/cardiac index measurements were used to evaluate the effects of PP1γ or inhibitor 1 of PP1 transfection. Western blot, reverse transcription polymerase chain reaction and co-immunoprecipitation were applied to investigate the molecular mechanisms. Transfection of PP1γ increased the value of the heart mass index, left ventricular mass index and cardiac fibrosis, and simultaneously decreased the value of maximal left ventricular pressure increase and decline rate, ejection fraction, fractional shortening, and left ventricular end-diastolic pressure, as well as left ventricular systolic pressure. Transfection of inhibitor 1 of PP1, however, showed opposite effects on the aforementioned indexes. Overexpression of PP1γ potentiated CaMKIIδC production and decreased CaMKIIδB production in the hypertrophic heart. In contrast, inhibition of PP1γ re-balanced the CaMKIIδ splicing. Furthermore, CaMKII activity was found to be augmented or attenuated by PP1γ overexpression or inhibition, respectively. Further mechanistic studies showed that abdominal aorta constriction stress specifically increased the association of alternative splicing factor with PP1γ, but not with PP1β. Overexpression of PP1γ, but not inhibitor 1 of PP1, further potentiated this association. These results suggest that PP1γ alters the cardiac hypertrophy and remodelling likely through promotion of the alternative splicing factor-mediated splicing of CaMKIIδ.