Abstract 444: Cardiac Troponin I Dephosphorylation in Human Heart Failure: The Role of Phosphatase Activation
We have isolated troponin (Tn) from non-failing (donor) and end-stage failing (explanted) human heart muscle. Using in vitro motility assays we have found an increase in Ca2+-sensitivity (EC50 decreased 2.6-fold) and a 12% reduction in crossbridge turnover rate. Measurement of total phosphorylation using Pro-Q Diamond showed TnT phosphorylation was 3.05±0.20 molsPi/ mol protein in non-failing and 3.11±0.42 in failing heart Tn. TnI phosphorylation level was 2.25±0.36 molsPi/mol protein in non-failing and 0.37±0.18 in failing heart Tn. Measurement of phosphorylation at TnI ser 23/24 was determined using a phospho-specific antibody and yielded 1.14±0.09 molPi/mol in non-failing and 0.19±0.06 in failing heart Tn. The difference between Pro-Q and Ser23/24 measurements is ascribed to phosphorylation at PKC sites on Tn. It was 1.11±0.34 molPi/mol protein in non-failing heart Tn and 0.18±0.17 in failing. Thus the level of phosphorylation of TnI in failing heart is substantially reduced at both PKA-specific and PKC-specific sites. Decreased TnI phosphorylation in failing heart was correlated with higher Ca2+-sensitivity and lower crossbridge turnover rate. Since PKC activity has been reported to be increased in failing hearts, it is possible that phosphatase activity may be increased. Using recombinant human cardiac TnI phosphorylated with PKA and [γ32P]ATP as substrate we found substantial TnI-specific phosphatase activity in washed myofibrils from human heart. Phosphatase activity was 95% inhibited by 2nM Okadaic acid and 30% inhibited by 1μM Inhibitor-2. The phosphatase was not activated by Ca2+, thus TnI dephosphorylating activity is predominantly type PP2a. We observed that the activity in myofibrils from failing heart was 14.8±5.0% higher than non-failing heart myofibrils (p=0.0104, paired students t-test). We measured the dephosphorylation of natively phosphorylated human cardiac Tn using Pro-Q Diamond and observed that the phosphatase activity in myofibrils specifically dephosphorylated TnI and not TnT. We conclude that activation of myofibril-associated phosphatase activity in failing heart could be responsible for the low levels of Tn I phosphorylation observed.