ermine whether CaMKII inhibition could prevent or reduce cardiac hypertrophy, we crossed mutant mice with mice transgenically expressing CaMKII autocamtide 3 inhibitory peptide or control peptide. Transgenic overexpression of AC3-I AZD-0530 cost protected mouse hearts against pathological remodeling in response to myocardial infarction and b-adrenergic stimulation. The present study shows that CaMKII inhibitory peptide AC3-I reduced phosphorylation of PLN at Thr-17 in Ryr2+/+ and Ryr2ADA/ADA mice without significantly altering life span, cardiac morphology and performance, or markers of cardiac hypertrophy relative to mice expressing the control peptide. The findings suggest that the pathological effects of the RyR2ADA mutation are independent of myocardial CaMKII. Materials and Methods Ethics Statement This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee. Materials Ryanodine was obtained from Perkin Elmer Life Sciences. Protease and phosphatase inhibitor cocktails were from Sigma. Rabbit polyclonal antibody F9221 against RyR2 amino acid sequence 13721387 was produced by New England Peptide. Rabbit polyclonal antibody pRyR2 on Ser-2809 was from Badrilla. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19657833 Rabbit polyclonal antibody to pRyR2 on Ser-2815 was the generous gift of Dr. Andrew Marks. Mouse monoclonal antibody PLN and rabbit polyclonal antibodies pPLN on Ser-16 and Thr-17 were from Badrilla. Rabbit polyclonal antibody pCaMKII on Thr-286 and 
two rabbit polyclonal antibodies against the conserved N-terminal and Cterminal regions of CaMKIIc and CaMKIId were from Abcam. Immunoblots were developed using enhanced chemiluminescence and quantified using ImageQuantTL Analysis Software. Quantitative RT-PCR Gene expression was measured by quantitative RT-PCR using the ABI Prism 7700 Sequence Detector . RNA was isolated from left ventricles of 10-day-old mice using the ABI Prism 6700 Automated Nucleic Acid Workstation according to the manufacturer’s protocol. Primers and fluorogenic probes for b-myosin heavy chain, atrial natriuretic peptide and brain natriuretic peptide were described. Levels of gene expression as a percentage of Ryr2+/+/AC3-C were determined relative to b-actin. Ryanodine binding Specific binding of ryanodine to RyRs was measured to determine the number of RyR high affinity binding sites. Cardiac muscle homogenates were incubated for 45 h at 24uC with a near saturating concentration of 20 nM ryanodine in 20 mM imidazole, pH 7.0, 0.6 M KCl, 0.1 mM Ca2+, and protease inhibitors as described. Nonspecific binding was determined using 1000-fold excess of unlabeled ryanodine. 45 Preparation of heart homogenates Hearts of 10-day old mice were homogenized using a Tekmar Tissumizer for 367 s at a setting of 13,500 rpm in 20 mM imidazole, pH 7.0, 0.3 M sucrose, 0.15 M NaCl, protease and phosphatase inhibitor cocktails, 25 mM b-glycerophosphate, 5 mM NaF and 2.5 mM NaVO4. Homogenates were stored in small aliquots at 280uC. Protein concentrations were determined using BCA assay. Ca2+ uptake rate ATP-dependent 45Ca2+ uptake rates by homogenates were determined using a filtration assay as described. 45Ca2+ uptake rates were determined in presence of 6 mM KN93, a CaMKII specific inhibitor. 4 Ryr2ADA/ADA Mice and AC3 Peptides Data analysis Results are expressed as mean 6 SEM.
