Abstract 20803: Skinned Myocytes From Children in Heart Failure Show Normal Responses to PKA Treatment
Dilated cardiomyopathy (DCM) is the most common reason for cardiac transplantation in children and adults. Although the beneficial effects of beta-blockade are well established in adults with DCM, a randomized clinical trial showed no beta-blockade effect on the pediatric heart. Protein kinase A (PKA), an effector kinase downstream of the beta-adrenergic receptor, is known to influence sarcomere function through phosphorylation of contractile proteins in adult hearts. We hypothesized that the lack of effect of beta-blockade in children might be due to differences in the sarcomeric response to PKA dependent phosphorylation. We tested this hypothesis by measuring the mechanical response of isolated skinned myocytes from control and failing pediatric hearts to PKA treatment. Skinned myocytes from explanted failing hearts (n=4) and unused donor hearts (n=4) were treated with buffer or PKA for one hour. Sarcomere length was set at 2.2 μm and force was measured for several pCa concentrations between 7 and 4.5. The Hill equation was fit to the force-pCa data to estimate maximum force, pCa50 and Hill coefficient. Remaining homogenates were separated by 1D-PAGE for phosphoprotein analysis. Sarcomeric protein phosphorylation was measured as the ratio of ProQ Diamond staining to Coomassie brilliant blue staining for each protein. There was no significant difference in the maximum force production (∼40 mN/mm2) between failing and non-failing groups nor with PKA treatment. The Hill coefficient was also unaffected by failure or treatment with PKA. The pCa50 was about 5.6 in failing and non-failing groups and PKA treatment decreased the pCa50 by about 0.2 in both groups. PKA treatment significantly increased the phosphorylation of TnI and myBPC to the same degree in both failing and non-failing groups. Failing adult myocytes show reduced maximum force and greater PKA-dependent changes in calcium sensitivity compared to controls. In contrast, failing pediatric myocytes show no difference in maximum force and have similar PKA dependent changes in phosphorylation and calcium sensitivity compared to controls. These results suggest that differences in sarcomeric responses to PKA may play a role in the lack of beta-blockade response in pediatric heart failure patients.
- © 2010 by American Heart Association, Inc.