ild-type and mutant mice expressing AC3-I compared to mice expressing AC3-C. However, increased pCaMKII-T286/CaMKII ratio was not expected to enhance CaMKII activity in AC3-I mice, because autophosphorylated CaMKII is inhibited by AC3-I. To verify that AC3-I inhibited CaMKII activity in AC3-I mice, phosphorylation levels of RyR2 and phospholamban were determined by immunoblot analysis. RyR2 is phosphorylated at Ser-2809 by protein kinase A and at Ser-2815 by CaMKII. In agreement with previous studies, total RyR2 protein expression was PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19657297 reduced by,60% in homozygous mice compared to WT expressing inhibitory and control AC3 peptides. In R-115777 custom synthesis hearts expressing the control peptide, increased pRyR2-S2809/RyR2 and pRyR2-S2815/RyR2 ratios were observed in mutant hearts compared with total RyR2 protein. AC3-I reduced pRyR2-S2815/RyR2 phosphorylation ratios in wild-type and mutant hearts compared with hearts expressing the control peptide. One caveat is that we could not distinguish between mice carrying transgenes of AC3-I and AC3- C in one allele or both. It is therefore conceivable that in mice expressing fewer copies, the AC3-I concentration was suboptimal in inhibiting CaMKII associated with RyR2. PLN has two physiologically relevant phosphorylation sites, Ser16 phosphorylated by PKA and Thr-17 phosphorylated by CaMKII. 
Immunoblots in Fig. 6 show similar total PLN protein levels in Ryr2+/+ and Ryr2ADA/ADA mice harboring AC3-C or AC3-I peptides. Among PLN, pPLN-S16/PLN and pPLNT17/PLN panels a number of significant changes were observed. Comparable pPLN-T17/PLN phosphorylation ratios were present in Ryr2+/+ and Ryr2ADA/ADA hearts expressing the control peptide. In contrast, phosphorylation ratios of pPLN-T17/PLN decreased by 75% in Ryr2+/+ hearts compared to an 85% decrease in Ryr2ADA/ADA hearts expressing the inhibitory peptide. This suggests that AC3-I inhibited CaMKII activity in Ryr2+/+ and Ryr2ADA/ADA hearts. Protein kinase D is a member of the CaMK superfamily and has been reported to be inhibited by AC3-I. Fig. 7 compares protein levels of PKD and phosphorylation of PKD on Ser-744/Ser-748 and Ser-916 in wild-type and mutant hearts expressing AC3-I or AC3-C peptides. Similar PKD protein levels and pPKD/PKD phosphorylation ratios were observed in hearts of wild-type and Ryr2ADA/ADA mice. 6 Ryr2ADA/ADA Mice and AC3 Peptides determined. The significantly reduced Bmax of ryanodine binding in Ryr2ADA/ADA hearts compared to Ryr2+/+ was not altered further by AC3-I or AC3-C in Ryr2+/+ and Ryr2ADA/ADA mice. In mutant mice, reduced 45Ca2+ uptake rates were likely due to decreased SERCA2a protein content. In heart homogenates of the mutant mice, we measured decreased SERCA2a protein content; however, corresponding experiments with AC3 homogenates have not been done. 45Ca2+ uptake rates were reduced to a level similar in Ryr2ADA/ADA hearts expressing AC3-C or AC3-I compared to Ryr2+/+ hearts expressing AC3-I or AC3-C peptides. SR Ca2+ uptake was likely not reduced by AC3-I because PLN-S16 phosphorylation, which was less affected by AC3-I compared to PLN-17, was sufficient to mediate a maximal b-agonist-mediated cardiac response in perfused hearts. To compensate for the lower AC3-I concentration due to sample dilution, experiments were done in the presence of CaMKII inhibitor KN93. Taken together, the results suggest that the CaMKII inhibitory peptide did not improve depressed cardiac Ca2+ handling of Ryr2ADA/ADA mice. Discussion CaMKII and CaM modulate
