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Protein kinase A-dependent modulation of Ca2+ sensitivity in cardiac and fast skeletal muscles after reconstitution with cardiac troponin.

Matsuba D, Terui T, O-Uchi J, Tanaka H, Ojima T, Ohtsuki I, Ishiwata S, Kurihara S, Fukuda N - J. Gen. Physiol. (2009)

Bottom Line: PKA enhanced phosphorylation of cTnI at Ser23/24 in skinned cardiac muscle and decreased Ca2+ sensitivity, of which the effects were confirmed after reconstitution with the cardiac Tn complex (cTn) or the hybrid Tn complex (designated as PCRF; fast skeletal TnT with cTnI and cTnC).The essentially same result was obtained when fast skeletal muscle was reconstituted with PCRF.It is therefore suggested that the PKA-dependent phosphorylation or dephosphorylation of cTnI universally modulates Ca2+ sensitivity associated with cTnC in the striated muscle sarcomere, independent of the TnT isoform.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology, Jikei University School of Medicine, Tokyo 105-8461, Japan.

ABSTRACT
Protein kinase A (PKA)-dependent phosphorylation of troponin (Tn)I represents a major physiological mechanism during beta-adrenergic stimulation in myocardium for the reduction of myofibrillar Ca2+ sensitivity via weakening of the interaction with TnC. By taking advantage of thin filament reconstitution, we directly investigated whether or not PKA-dependent phosphorylation of cardiac TnI (cTnI) decreases Ca2+ sensitivity in different types of muscle: cardiac (porcine ventricular) and fast skeletal (rabbit psoas) muscles. PKA enhanced phosphorylation of cTnI at Ser23/24 in skinned cardiac muscle and decreased Ca2+ sensitivity, of which the effects were confirmed after reconstitution with the cardiac Tn complex (cTn) or the hybrid Tn complex (designated as PCRF; fast skeletal TnT with cTnI and cTnC). Reconstitution of cardiac muscle with the fast skeletal Tn complex (sTn) not only increased Ca2+ sensitivity, but also abolished the Ca2+-desensitizing effect of PKA, supporting the view that the phosphorylation of cTnI, but not that of other myofibrillar proteins, such as myosin-binding protein C, primarily underlies the PKA-induced Ca2+ desensitization in cardiac muscle. Reconstitution of fast skeletal muscle with cTn decreased Ca2+ sensitivity, and PKA further decreased Ca2+ sensitivity, which was almost completely restored to the original level upon subsequent reconstitution with sTn. The essentially same result was obtained when fast skeletal muscle was reconstituted with PCRF. It is therefore suggested that the PKA-dependent phosphorylation or dephosphorylation of cTnI universally modulates Ca2+ sensitivity associated with cTnC in the striated muscle sarcomere, independent of the TnT isoform.

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Effects of PKA on cTnI phosphorylation and Ca2+ sensitivity in PP1-treated PLV. (A) Western blotting showing the effect of PP1 or PKA on the phosphorylation level of Ser23/24 in cTnI (12.5 µg protein/well). Cont., control with no treatment. Normalized intensity compared with control (n = 5): PP1, 40.16 ± 10.48%; PP1 + PKA (PKA phosphorylation after PP1 treatment), 207.76 ± 53.85% (P < 0.05 compared with PP1). (B) Force-pCa curves showing the effects of PP1 and subsequent PKA treatment on Ca2+ sensitivity. (Inset) Difference between the values of the PKA-induced shift of pCa50 (C, control minus PP1 + PKA; PP1, PP1 minus PP1 + PKA). *, P < 0.05. n = 7. PKA treatment did not significantly affect maximal force or nH (see Table II). (C; Left) Comparison of Ca2+ sensitization as indexed by the shift of pCa50 after PP1 treatment and cTn or PCRF reconstitution in PLV. (Right) Comparison of the PKA-induced shift of pCa50 in PP1-treated and cTn- or PCRF-reconstituted PLV. Statistical significance was not observed between groups in either graph.
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fig3: Effects of PKA on cTnI phosphorylation and Ca2+ sensitivity in PP1-treated PLV. (A) Western blotting showing the effect of PP1 or PKA on the phosphorylation level of Ser23/24 in cTnI (12.5 µg protein/well). Cont., control with no treatment. Normalized intensity compared with control (n = 5): PP1, 40.16 ± 10.48%; PP1 + PKA (PKA phosphorylation after PP1 treatment), 207.76 ± 53.85% (P < 0.05 compared with PP1). (B) Force-pCa curves showing the effects of PP1 and subsequent PKA treatment on Ca2+ sensitivity. (Inset) Difference between the values of the PKA-induced shift of pCa50 (C, control minus PP1 + PKA; PP1, PP1 minus PP1 + PKA). *, P < 0.05. n = 7. PKA treatment did not significantly affect maximal force or nH (see Table II). (C; Left) Comparison of Ca2+ sensitization as indexed by the shift of pCa50 after PP1 treatment and cTn or PCRF reconstitution in PLV. (Right) Comparison of the PKA-induced shift of pCa50 in PP1-treated and cTn- or PCRF-reconstituted PLV. Statistical significance was not observed between groups in either graph.

Mentions: Summary of the values of maximal active force, pCa50 and nH before and after PKA treatment in PLV after reconstitution with cTn or PCRF or treatment with PP1


Protein kinase A-dependent modulation of Ca2+ sensitivity in cardiac and fast skeletal muscles after reconstitution with cardiac troponin.

Matsuba D, Terui T, O-Uchi J, Tanaka H, Ojima T, Ohtsuki I, Ishiwata S, Kurihara S, Fukuda N - J. Gen. Physiol. (2009)

Effects of PKA on cTnI phosphorylation and Ca2+ sensitivity in PP1-treated PLV. (A) Western blotting showing the effect of PP1 or PKA on the phosphorylation level of Ser23/24 in cTnI (12.5 µg protein/well). Cont., control with no treatment. Normalized intensity compared with control (n = 5): PP1, 40.16 ± 10.48%; PP1 + PKA (PKA phosphorylation after PP1 treatment), 207.76 ± 53.85% (P < 0.05 compared with PP1). (B) Force-pCa curves showing the effects of PP1 and subsequent PKA treatment on Ca2+ sensitivity. (Inset) Difference between the values of the PKA-induced shift of pCa50 (C, control minus PP1 + PKA; PP1, PP1 minus PP1 + PKA). *, P < 0.05. n = 7. PKA treatment did not significantly affect maximal force or nH (see Table II). (C; Left) Comparison of Ca2+ sensitization as indexed by the shift of pCa50 after PP1 treatment and cTn or PCRF reconstitution in PLV. (Right) Comparison of the PKA-induced shift of pCa50 in PP1-treated and cTn- or PCRF-reconstituted PLV. Statistical significance was not observed between groups in either graph.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2713144&req=5

fig3: Effects of PKA on cTnI phosphorylation and Ca2+ sensitivity in PP1-treated PLV. (A) Western blotting showing the effect of PP1 or PKA on the phosphorylation level of Ser23/24 in cTnI (12.5 µg protein/well). Cont., control with no treatment. Normalized intensity compared with control (n = 5): PP1, 40.16 ± 10.48%; PP1 + PKA (PKA phosphorylation after PP1 treatment), 207.76 ± 53.85% (P < 0.05 compared with PP1). (B) Force-pCa curves showing the effects of PP1 and subsequent PKA treatment on Ca2+ sensitivity. (Inset) Difference between the values of the PKA-induced shift of pCa50 (C, control minus PP1 + PKA; PP1, PP1 minus PP1 + PKA). *, P < 0.05. n = 7. PKA treatment did not significantly affect maximal force or nH (see Table II). (C; Left) Comparison of Ca2+ sensitization as indexed by the shift of pCa50 after PP1 treatment and cTn or PCRF reconstitution in PLV. (Right) Comparison of the PKA-induced shift of pCa50 in PP1-treated and cTn- or PCRF-reconstituted PLV. Statistical significance was not observed between groups in either graph.
Mentions: Summary of the values of maximal active force, pCa50 and nH before and after PKA treatment in PLV after reconstitution with cTn or PCRF or treatment with PP1

Bottom Line: PKA enhanced phosphorylation of cTnI at Ser23/24 in skinned cardiac muscle and decreased Ca2+ sensitivity, of which the effects were confirmed after reconstitution with the cardiac Tn complex (cTn) or the hybrid Tn complex (designated as PCRF; fast skeletal TnT with cTnI and cTnC).The essentially same result was obtained when fast skeletal muscle was reconstituted with PCRF.It is therefore suggested that the PKA-dependent phosphorylation or dephosphorylation of cTnI universally modulates Ca2+ sensitivity associated with cTnC in the striated muscle sarcomere, independent of the TnT isoform.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology, Jikei University School of Medicine, Tokyo 105-8461, Japan.

ABSTRACT
Protein kinase A (PKA)-dependent phosphorylation of troponin (Tn)I represents a major physiological mechanism during beta-adrenergic stimulation in myocardium for the reduction of myofibrillar Ca2+ sensitivity via weakening of the interaction with TnC. By taking advantage of thin filament reconstitution, we directly investigated whether or not PKA-dependent phosphorylation of cardiac TnI (cTnI) decreases Ca2+ sensitivity in different types of muscle: cardiac (porcine ventricular) and fast skeletal (rabbit psoas) muscles. PKA enhanced phosphorylation of cTnI at Ser23/24 in skinned cardiac muscle and decreased Ca2+ sensitivity, of which the effects were confirmed after reconstitution with the cardiac Tn complex (cTn) or the hybrid Tn complex (designated as PCRF; fast skeletal TnT with cTnI and cTnC). Reconstitution of cardiac muscle with the fast skeletal Tn complex (sTn) not only increased Ca2+ sensitivity, but also abolished the Ca2+-desensitizing effect of PKA, supporting the view that the phosphorylation of cTnI, but not that of other myofibrillar proteins, such as myosin-binding protein C, primarily underlies the PKA-induced Ca2+ desensitization in cardiac muscle. Reconstitution of fast skeletal muscle with cTn decreased Ca2+ sensitivity, and PKA further decreased Ca2+ sensitivity, which was almost completely restored to the original level upon subsequent reconstitution with sTn. The essentially same result was obtained when fast skeletal muscle was reconstituted with PCRF. It is therefore suggested that the PKA-dependent phosphorylation or dephosphorylation of cTnI universally modulates Ca2+ sensitivity associated with cTnC in the striated muscle sarcomere, independent of the TnT isoform.

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