Abstract 1443: Protein Kinase-A Phosphorylates Titin in Normal and Failing Human Heart and Reduces Passive Myocardial Stiffness
The passive stiffness of cardiomyocytes is lowered by activation of protein kinase-A (PKA), an effect putatively ascribed to phosphorylation of a cardiac-specific domain, the N2B-unique sequence, in the elastic region of titin. This PKA phosphorylation site is present in the cardiac-titin isoforms, N2B and N2BA, but it had been unknown whether titin is phosphorylated by PKA in human heart. We used 2% SDS-PAGE and autoradiography to study PKA-mediated titin phosphorylation in left ventricles of human control donor and end-stage failing non-ischemic DCM hearts (NYHA class IV, EF<30%). Force was measured in skinned fibers and isolated myofibrils to determine how titin phosphorylation affects passive stiffness. PKA phosphorylated both N2B and N2BA titin in human myocardium, and to a similar degree in control (n=5) and DCM (n=4) hearts. Moreover, PKA phosphorylated a proteolytic titin fragment, T2, which lacks most of the elastic I-band region. In control measurements with diaphragm muscle, PKA also phosphorylated the skeletal N2A-titin isoform, albeit to a lower degree than the cardiac isoforms. A recombinant construct comprising the cardiac-specific N2B domain was strongly phosphorylated by PKA in vitro, suggesting that this is the primary PKA-responsive element in human titin. Titin phosphorylation by PKA substantially reduced the passive stiffness of human cardiac-fiber bundles (n=6 per group) and the effect was confirmed in mechanical measurements on isolated cardiomyofibrils (n=5). The average stiffness drop was relatively higher at short than at long sarcomere lengths (~40±8% at 2.0μm versus 20±6% at 2.4μm). However, no difference in relative passive-stiffness decrease was detectable between control donor and DCM samples. PKA did not alter the passive stiffness of skinned diaphragm fibers, consistent with the absence of N2B-domain in N2A-titin.
Conclusion: PKA phosphorylates cardiac titin in normal and end-stage failing human hearts, thereby lowering titin-based stiffness. A similar stiffness decrease in control donor and diseased hearts suggests that altered passive stiffness in DCM is not caused by changes in titin phosphorylation. A potential target to lower pathologically increased passive stiffness may be phosphorylation of titin.