Impact of site-specific phosphorylation of protein kinase A sites Ser²³and Ser²⁴of cardiac troponin I in human cardiomyocytes

Abstract

unphosphorylated, and cTnI-AA mimics both sites unphosphorylated. Force development was measured at various Ca 2ϩ concentrations in permeabilized cardiomyocytes in which the endogenous troponin complex was exchanged with these recombinant human troponin complexes. In donor cardiomyocytes, myofilament Ca 2ϩ sensitivity (pCa50) was significantly lower in cTnI-DD (pCa50: 5.39 Ϯ 0.01) compared with cTnI-AA (pCa50: 5.50 Ϯ 0.01), cTnI-AD (pCa50: 5.48 Ϯ 0.01), and cTnI-DA (pCa50: 5.51 Ϯ 0.01) at ϳ70% cTn exchange. No effects were observed on the rate of tension redevelopment. In cardiomyocytes from idiopathic dilated cardiomyopathic tissue, a linear decline in pCa50 with cTnI-DD content was observed, saturating at ϳ55% bisphosphorylation. Our data suggest that in the human myocardium, phosphorylation of both PKA sites on cTnI is required to reduce myofilament Ca 2ϩ sensitivity, which is maximal at ϳ55% bisphosphorylated cTnI. The implications for in vivo cardiac function in health and disease are detailed in the DISCUSSION in this article. myofilament function; protein phosphorylation; cardiomyocyte; troponin I DURING STRESS AND EXERCISE, sympathetic activation of the heart increases heart rate and stroke volume to meet the demands of the body. This is mediated via the stimulation of ␤ 1 -adrenergic receptors, which leads to the activation of a downstream kinase, PKA. PKA enhances cardiomyocyte contraction and relaxation by phosphorylation of proteins involved in Ca 2ϩ handling and myofilament proteins such as cardiac troponin (cTn)I, cardiac myosin-binding protein-C (cMyBP-C), and titin (for reviews, see Refs. 1 and 37).PKA-mediated phosphorylation of myofilament proteins is thought to exert a positive lusitropic effect, which enables the heart to relax more rapidly when heart rate increases. This positive lusitropic effect may be induced by a decrease in myofilament Ca 2ϩ sensitivity (32, 35, 51) and by enhanced cross-bridge cycling kinetics (11,20,33). It is well established (mainly from studies in rodents) that phosphorylation of cTnI at the PKA sites Ser 23 and Ser 24 leads to a decrease in myofilament Ca 2ϩ sensitivity, through a conformational change of the troponin complex. This structural change reduces the affinity of Ca 2ϩ binding to cTnC (16,30). The role of phosphorylation of cTnI at the PKA sites as a regulator of cross-bridge cycling is less clear. Some studies (11,20,36) have reported an increase in cross-bridge kinetics via phosphorylation of cTnI. However, others (7, 33) have attributed an increase in cross-bridge kinetics to phosphorylation of cMyBP-C independent of cTnI phosphorylation, whereas several studies (8,15,17,44) did not find an effect of PKA on cross-bridge kinetics at all. In the present study, we aimed to study the effect of site-specific phosphorylation of cTnI on myofilament Ca 2ϩ sensitivity and cross-bridge kinetics in human cardiomyocytes since insights into the functional effects of cTnI phosphorylation and the relation between the level of phosphorylation and the functional effec...

Cardiac Myosin-binding Protein C and Troponin-I Phosphorylation Independently Modulate Myofilament Length-dependent Activation

“…3, A and B). In addition, others have shown that ϳ50% cTnI phosphorylation is sufficient to induce a maximal response on myofilament calcium sensitivity (44). Fig.…”

Targeting the sarcomere to correct muscle function

“…Phosphorylation of L-type calcium channels and phospholamban increases contractility by enhancing calcium influx and pumping into the sarcoplasmic reticulum 127 . Sarcomeric proteins are phosphorylated to enhance relaxation in the rapidly contracting heart: myosin binding protein C (MyBP-C) phosphorylation increases actin-myosin crossbridge kinetics [128][129][130] , titin phosphorylation decreases passive stiffness 131 and troponin I phosphorylation decreases calcium sensitivity 132,133 . Dephosphorylation of MyBP-C, titin and troponin I is observed in a wide range of cardiac diseases, including both heart failure with reduced ejection fraction and heart failure with preserved ejection fraction 134 , as well as hypertrophic cardiomyopathy 134 and dilated cardiomyopathy 135 , suggesting that this may be a common adaptation to increased mechanical strain.…”

Length-dependent activation is modulated by cardiac troponin I bisphosphorylation at Ser23 and Ser24 but not by Thr143 phosphorylation

“…Recombinant human troponin complex was produced as described in detail previously (39). Shortly, six different cardiac troponin I (cTnI) forms were made via site-directed mutations of Thr143 and Ser23/ Ser24 into aspartic acid (D) to mimic phosphorylation or into alanine (A) to mimic dephosphorylation: 143D, 143A, 23D/24D, 23D/24A, 23A/24D, and 23A/24A.…”

“…Exchange of recombinant cTn in human cardiomyocytes was done as described previously (39). Briefly, single cardiomyocytes were mechanically isolated with a glass tissue homogenizer and permeabilized by Triton X-100 (0.5%; v/v) for 5 min.…”

“…Therefore, secondary experiments were performed with and without exogenous PKA incubation after cTn exchange in nonfailing donor cardiomyocytes. Also, as previous experiments showed that PKA-bisphosphorylation at Ser23 and Ser24 is required to decrease Ca 2ϩ sensitivity (39,44), we investigated effects of pseudo-mono-and pseudo-bisphosphorylation at cTnI-Ser23/24. To study the effect of Ser23/24 phosphorylation on LDA, experiments were performed in failing cardiomyocytes that have a low baseline level of cTnISer23/24 phosphorylation.…”