cAMP-dependent protein kinase (PKA) regulates the L-type calcium channel the ryanodine receptor and phospholamban (PLB) thereby increasing inotropy. enhanced in cardiomyocytes by chemical p38 inhibition by overexpression of dominating bad p38α and Rabbit Polyclonal to DIL-2. by p38α RNAi but not with dominating bad p38β. Treatment of cardiomyocytes with dominating negative p38α significantly decreased Ca2+-transient decay time indicating enhanced sarco/endoplasmic reticulum Ca2+-ATPase function and improved cardiomyocyte contractility. Analysis of signaling mechanisms involved showed LY2140023 that inhibition of p38 decreased the activity of protein phosphatase 2A which renders protein phosphatase inhibitor-1 phosphorylated and therefore inhibits PP1. In conclusion inhibition of p38α enhances PLB phosphorylation and diastolic Ca2+ uptake. Our findings provide evidence for novel mechanism regulating cardiac contractility upon p38 inhibition. test and for multiple experimental organizations one way analysis of variance followed by Bonferroni post hoc test was used. Variations were regarded as statistically significant at the level of p < 0.05. 3 Results 3.1 Inhibition of p38α enhances PLB phosphorylation in cardiomyocytes To better understand the part of p38 MAPKs in controlling cardiac calcium regulatory proteins we 1st studied if inhibition of p38 in isolated cardiomyocytes is sufficient to induce PLB phosphorylation. Treatment of neonatal rat ventricular cardiomyocytes (NRVM) by p38 inhibitor SB203580 (10 μM) improved phosphorylation of PLB at Ser16 (Fig. 1A). Phosphorylation of PLB Ser16 was also modestly enhanced when p38 inhibitor-treated cells were stimulated with the Gq agonist ET-1 (100 nM LY2140023 Fig. 1A). Inhibition of p38 however had no effect on PLB Thr17 phosphorylation (Fig. 1A). Phosphorylation status of heat-shock protein 27 (Hsp27) a p38 target confirms the inhibition of p38 (Fig. 1A). To determine if there is a difference between the cardiac p38 isoforms in regulating the response NRVMs were infected with adenoviruses encoding either for dominating bad p38α (dn-p38α) or dominating bad p38β (dn-p38β). Immunoblot analysis showed that overexpression of dn-p38α but not dn-p38β is sufficient to enhance PLB phosphorylation at Ser16 (Fig. 1B). dn-p38 adenoviruses experienced no significant effect on total PLB protein. Much like overexpression of dn-p38α p38α depletion by RNAi enhanced Ser16 PLB phosphorylation (Fig. 1C). p38α siRNA efficiently downregulated p38α mRNA levels but experienced no effect on p38β manifestation (Suppl. Fig. 1A). We then asked if pressured activation of p38α or p38β is sufficient to modulate PLB phosphorylation. As demonstrated in Fig. 1D pressured activation of p38α markedly reduced Ser16 PLB phosphorylation. Analysis of PLB protein levels however showed that pressured activation of either of the cardiac p38 isoforms caused significant reduction in total PLB protein (Fig. 1D). In summary inhibition of p38α but not p38β enhances PLB phosphorylation. Fig. 1 Inhibition ofp38 enhances PLB phosphorylation at Ser16. A Immunoblot analysis and quantification of Ser16 phosphorylated and total PLB and immunoblot analysis of PLB phosphorylation at Thr17 in NRVMs treated either with vehicle (DMSO) ET-1 (100 nM ... Much like data on protein level activation of either of the p38 isoforms resulted in almost total abolishment of PLB mRNA (Suppl. Fig. 1B). Pressured p38α and p38β activation also significantly suppressed SERCA2a and calsequestrin (CASQ2) mRNA levels whereas sodium-calcium exchanger LY2140023 (NCX) mRNA levels were not affected (Suppl. Fig. 1B). There LY2140023 is actually data from earlier studies showing that activation of p38 in vitro or in vivo suppresses SERCA2a manifestation [15 16 therefore worsening diastolic Ca2+ uptake and cardiomyocyte contractility. On the other hand overexpression of dn-p38α resulted in significant decrease in SERCA2a mRNA levels and overexpression of dn-p38β resulted in a decrease in PLB SERCA2a CASQ2 and NCX mRNA levels (Suppl. Fig. 1C). Depletion of p38α by siRNA modestly decreased PLB mRNA levels but experienced no effect on SERCA2a mRNA levels (Suppl. Fig. 1D). Pharmacological inhibition of p38 with SB203580 (10 μM) however had no effect on PLB LY2140023 SERCA2a CASQ2 or NCX mRNA levels (Suppl. Fig. 1E). In summary overexpression of either crazy type or dominating.