Abstract
The phosphorylation of cardiac muscle troponin I (CTnI) at two adjacent N-terminal serine residues by cAMP-dependent protein kinase (PKA) has been implicated in the inotropic response of the heart to beta-agonists. Phosphorylation of these residues has been shown to reduce the Ca2+ affinity of the single Ca(2+)-specific regulatory site of cardiac troponin C (CTnC) and to increase the rate of Ca2+ dissociation from this site (Robertson, S. P., Johnson, J. D., Holroyde, M. J., Kranias, E. G., Potter, J. D., and Solaro, R. J. (1982) J. Biol. Chem. 257, 260-263). Recent studies (Zhang, R., Zhao, J., and Potter, J. D. (1995) Circ. Res. 76, 1028-1035) have correlated this increase in Ca2+ dissociation with a reduced Ca2+ sensitivity of force development and a faster rate of cardiac muscle relaxation in a PKA phosphorylated skinned cardiac muscle preparation. To further determine the role of the two PKA phosphorylation sites in mouse CTnI (serine 22 and 23), serine 22 or 23, or both were mutated to alanine. The wild type and the mutated CTnIs were expressed in Escherichia coli and purified. Using these mutants, it was found that serine 23 was phosphorylated more rapidly than serine 22 and that both serines are required to be phosphorylated in order to observe the characteristic reduction in the Ca2+ sensitivity of force development seen in a skinned cardiac muscle preparation. The latter result confirms that PKA phosphorylation of CTnI, and not other proteins, is responsible for this change in Ca2+ sensitivity. The results also suggest that one of the serines (23) may be constitutively phosphorylated and that serine 22 may be functionally more important.
Highlights
Several lines of evidence have led to a general understanding of how -agonist stimulation leads to positive inotropic and chronotropic effects by phosphorylation of cellular substrates through cAMP-dependent protein kinase (PKA)1 [1, 2]
Since the affinity of cardiac troponin I (CTnI) for cardiac troponin C (CTnC) is quite high, the bound CTnI can only be eluted from the troponin C (TnC) affinity column in the presence of 6 M urea, 2 mM EDTA, and 1 M NaCl, or near the end of the described gradient, with other contaminating proteins eluted during the first half of the gradient
PKA phosphorylation of CTnI causes a decrease in the Ca2ϩ dependence of force development or of the myofibrillar ATPase of cardiac muscle and has been observed by many investigators [8, 9, 20, 21, 36]
Summary
Several lines of evidence have led to a general understanding of how -agonist stimulation leads to positive inotropic and chronotropic effects by phosphorylation of cellular substrates through cAMP-dependent protein kinase (PKA)1 [1, 2]. It has been shown that CTnI phosphorylation increases the rate of Ca2ϩ dissociation from a reconstituted troponin complex [23], suggesting the possible importance of this phosphorylation in modulating the rate of relaxation of cardiac muscle. Some reports have shown that CTnI remains phosphorylated even after removal of -agonists and the return of muscle contraction to prestimulated basal levels [4, 19] These results have suggested that phosphorylation of CTnI may not be as important as the phosphorylation of other proteins, such as phospholamban [4]. Using a reconstituted skinned cardiac muscle preparation (CSM), we have studied the effect of phosphorylation of each serine residue on the Ca2ϩ dependence of force development. Since the rate of phosphorylation is different, it is possible that the rate of dephosphorylation is different and may account for the slower dephosphorylation seen in intact systems [4] and suggests that one of the serines may be more or less constitutively phosphorylated with the other serine being functionally more important
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