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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 PLV. (A; Top) Western blotting showing cTnI phosphorylation (12.5 µg protein/well). Cont., control without PKA treatment. (Bottom) Comparison of the intensity of cTnI Ser23/24 phosphorylation normalized with total cTnI content in control and PKA-treated PLV. *, P < 0.05. n = 3. (B) Force-pCa curves showing the effect of PKA on Ca2+ sensitivity. (Inset) pCa50 values before and after PKA treatment. *, P < 0.05. n = 7. (C) Force-pCa curves showing the effects of quasi-complete sTn reconstitution (shown in gray) and subsequent treatment with PKA on Ca2+ sensitivity. (Inset) pCa50 values. *, P < 0.05. n = 7. (D; Left) Plot of sTn treatment time versus sTn-induced shift of pCa50. (Middle) Plot of sTn treatment time versus PKA-induced shift of pCa50. (Right) Plot of residual cTnI ratio (i.e., cTnI/(cTnI + sTnI)) versus PKA-induced shift of pCa50. n = 7.
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fig1: Effects of PKA on cTnI phosphorylation and Ca2+ sensitivity in PLV. (A; Top) Western blotting showing cTnI phosphorylation (12.5 µg protein/well). Cont., control without PKA treatment. (Bottom) Comparison of the intensity of cTnI Ser23/24 phosphorylation normalized with total cTnI content in control and PKA-treated PLV. *, P < 0.05. n = 3. (B) Force-pCa curves showing the effect of PKA on Ca2+ sensitivity. (Inset) pCa50 values before and after PKA treatment. *, P < 0.05. n = 7. (C) Force-pCa curves showing the effects of quasi-complete sTn reconstitution (shown in gray) and subsequent treatment with PKA on Ca2+ sensitivity. (Inset) pCa50 values. *, P < 0.05. n = 7. (D; Left) Plot of sTn treatment time versus sTn-induced shift of pCa50. (Middle) Plot of sTn treatment time versus PKA-induced shift of pCa50. (Right) Plot of residual cTnI ratio (i.e., cTnI/(cTnI + sTnI)) versus PKA-induced shift of pCa50. n = 7.

Mentions: First, we tested how PKA-dependent phosphorylation of cTnI modulates Ca2+ sensitivity in PLV under the control condition without Tn reconstitution. We found that our PKA treatment nearly doubled Ser23/24 phosphorylation in cTnI (Fig. 1 A). Fig. 1 B shows the effect of PKA on myofibrillar Ca2+ sensitivity. Consistent with a previous study on porcine ventricular preparations (Zhang et al., 1995), the force-pCa curve was shifted to the right by 0.10 ± 0.01 pCa units (as indexed by pCa50), exhibiting a decrease in Ca2+ sensitivity. PKA did not affect maximal force or thin filament cooperative activation (as indexed by nH; Table I). Ca2+ desensitization was not observed when the preparation was treated with PKA in the presence of the PKI (pCa50: 5.57 ± 0.02 and 5.54 ± 0.03, respectively, before and after PKA+PKI; P > 0.05; n = 6).


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 PLV. (A; Top) Western blotting showing cTnI phosphorylation (12.5 µg protein/well). Cont., control without PKA treatment. (Bottom) Comparison of the intensity of cTnI Ser23/24 phosphorylation normalized with total cTnI content in control and PKA-treated PLV. *, P < 0.05. n = 3. (B) Force-pCa curves showing the effect of PKA on Ca2+ sensitivity. (Inset) pCa50 values before and after PKA treatment. *, P < 0.05. n = 7. (C) Force-pCa curves showing the effects of quasi-complete sTn reconstitution (shown in gray) and subsequent treatment with PKA on Ca2+ sensitivity. (Inset) pCa50 values. *, P < 0.05. n = 7. (D; Left) Plot of sTn treatment time versus sTn-induced shift of pCa50. (Middle) Plot of sTn treatment time versus PKA-induced shift of pCa50. (Right) Plot of residual cTnI ratio (i.e., cTnI/(cTnI + sTnI)) versus PKA-induced shift of pCa50. n = 7.
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Related In: Results  -  Collection

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

fig1: Effects of PKA on cTnI phosphorylation and Ca2+ sensitivity in PLV. (A; Top) Western blotting showing cTnI phosphorylation (12.5 µg protein/well). Cont., control without PKA treatment. (Bottom) Comparison of the intensity of cTnI Ser23/24 phosphorylation normalized with total cTnI content in control and PKA-treated PLV. *, P < 0.05. n = 3. (B) Force-pCa curves showing the effect of PKA on Ca2+ sensitivity. (Inset) pCa50 values before and after PKA treatment. *, P < 0.05. n = 7. (C) Force-pCa curves showing the effects of quasi-complete sTn reconstitution (shown in gray) and subsequent treatment with PKA on Ca2+ sensitivity. (Inset) pCa50 values. *, P < 0.05. n = 7. (D; Left) Plot of sTn treatment time versus sTn-induced shift of pCa50. (Middle) Plot of sTn treatment time versus PKA-induced shift of pCa50. (Right) Plot of residual cTnI ratio (i.e., cTnI/(cTnI + sTnI)) versus PKA-induced shift of pCa50. n = 7.
Mentions: First, we tested how PKA-dependent phosphorylation of cTnI modulates Ca2+ sensitivity in PLV under the control condition without Tn reconstitution. We found that our PKA treatment nearly doubled Ser23/24 phosphorylation in cTnI (Fig. 1 A). Fig. 1 B shows the effect of PKA on myofibrillar Ca2+ sensitivity. Consistent with a previous study on porcine ventricular preparations (Zhang et al., 1995), the force-pCa curve was shifted to the right by 0.10 ± 0.01 pCa units (as indexed by pCa50), exhibiting a decrease in Ca2+ sensitivity. PKA did not affect maximal force or thin filament cooperative activation (as indexed by nH; Table I). Ca2+ desensitization was not observed when the preparation was treated with PKA in the presence of the PKI (pCa50: 5.57 ± 0.02 and 5.54 ± 0.03, respectively, before and after PKA+PKI; P > 0.05; n = 6).

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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