TY - JOUR
T1 - Phosphorylation of protein kinase C sites Ser42/44 decreases Ca 2+-sensitivity and blunts enhanced length-dependent activation in response to protein kinase A in human cardiomyocytes
AU - Wijnker, Paul J.M.
AU - Sequeira, Vasco
AU - Witjas-Paalberends, E. Rosalie
AU - Foster, D. Brian
AU - Dos Remedios, Cristobal G.
AU - Murphy, Anne M.
AU - Stienen, Ger J.M.
AU - Van Der Velden, Jolanda
PY - 2014/7/15
Y1 - 2014/7/15
N2 - Protein kinase C (PKC)-mediated phosphorylation of troponin I (cTnI) at Ser42/44 is increased in heart failure. While studies in rodents demonstrated that PKC-mediated Ser42/44 phosphorylation decreases maximal force and ATPase activity, PKC incubation of human cardiomyocytes did not affect maximal force. We investigated whether Ser42/44 pseudo-phosphorylation affects force development and ATPase activity using troponin exchange in human myocardium. Additionally, we studied if pseudo-phosphorylated Ser42/44 modulates length-dependent activation of force, which is regulated by protein kinase A (PKA)-mediated cTnI-Ser23/24 phosphorylation. Isometric force was measured in membrane-permeabilized cardiomyocytes exchanged with human recombinant wild-type troponin or troponin mutated at Ser42/44 or Ser23/24 into aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. In troponin-exchanged donor cardiomyocytes experiments were repeated after PKA incubation. ATPase activity was measured in troponin-exchanged cardiac muscle strips. Compared to wild-type, 42D/44D decreased Ca2+-sensitivity without affecting maximal force in failing and donor cardiomyocytes. In donor myocardium, 42D/44D did not affect maximal ATPase activity or tension cost. Interestingly, 42D/44D blunted the length-dependent increase in Ca 2+-sensitivity induced upon PKA-mediated phosphorylation. Since the drop in Ca2+-sensitivity at physiological Ca2+- concentrations is relatively large phosphorylation of Ser42/44 may result in a decrease of force and associated ATP utilization in the human heart.
AB - Protein kinase C (PKC)-mediated phosphorylation of troponin I (cTnI) at Ser42/44 is increased in heart failure. While studies in rodents demonstrated that PKC-mediated Ser42/44 phosphorylation decreases maximal force and ATPase activity, PKC incubation of human cardiomyocytes did not affect maximal force. We investigated whether Ser42/44 pseudo-phosphorylation affects force development and ATPase activity using troponin exchange in human myocardium. Additionally, we studied if pseudo-phosphorylated Ser42/44 modulates length-dependent activation of force, which is regulated by protein kinase A (PKA)-mediated cTnI-Ser23/24 phosphorylation. Isometric force was measured in membrane-permeabilized cardiomyocytes exchanged with human recombinant wild-type troponin or troponin mutated at Ser42/44 or Ser23/24 into aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. In troponin-exchanged donor cardiomyocytes experiments were repeated after PKA incubation. ATPase activity was measured in troponin-exchanged cardiac muscle strips. Compared to wild-type, 42D/44D decreased Ca2+-sensitivity without affecting maximal force in failing and donor cardiomyocytes. In donor myocardium, 42D/44D did not affect maximal ATPase activity or tension cost. Interestingly, 42D/44D blunted the length-dependent increase in Ca 2+-sensitivity induced upon PKA-mediated phosphorylation. Since the drop in Ca2+-sensitivity at physiological Ca2+- concentrations is relatively large phosphorylation of Ser42/44 may result in a decrease of force and associated ATP utilization in the human heart.
KW - Cardiomyocyte
KW - Myofilament function
KW - Protein kinase C
KW - Protein phosphorylation
KW - Troponin I
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U2 - 10.1016/j.abb.2014.04.017
DO - 10.1016/j.abb.2014.04.017
M3 - Article
C2 - 24814372
AN - SCOPUS:84901382124
SN - 0003-9861
VL - 554
SP - 11
EP - 21
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
ER -