Monophosphorylation of cardiac troponin-I at Ser23/24 is sufficient to regulate cardiac myofibrillar Ca2+ sensitivity and calpain-induced proteolysis

Abel Martin-garrido; Brandon J. Biesiadecki; Hussam E. Salhi; Yasin Shaifta; Cristobal G. Dos Remedios; Serife Ayaz-guner; Wenxuan Cai; Ying Ge; Metin Avkiran; Jonathan C. Kentish

doi:10.1074/jbc.RA117.001292

Monophosphorylation of cardiac troponin-I at Ser23/24 is sufficient to regulate cardiac myofibrillar Ca2+ sensitivity and calpain-induced proteolysis

Abel Martin-garrido, Brandon J. Biesiadecki, Hussam E. Salhi, Yasin Shaifta, Cristobal G. Dos Remedios, Serife Ayaz-guner, Wenxuan Cai, Ying Ge, Metin Avkiran, Jonathan C. Kentish

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Abstract

The acceleration of myocardial relaxation produced by β-adrenoreceptor stimulation is mediated in part by protein kinase A (PKA)-mediated phosphorylation of cardiac troponin-I (cTnI), which decreases myofibrillar Ca2+ sensitivity. Previous evidence suggests that phosphorylation of both Ser-23 and Ser-24 in cTnI is required for this Ca2+ desensitization. PKA-mediated phosphorylation also partially protects cTnI from proteolysis by calpain. Here we report that protein kinase D (PKD) phosphorylates only one serine of cTnI Ser-23/24. To explore the functional consequences of this monophosphorylation, we examined the Ca2+ sensitivity of force production and susceptibility of cTnI to calpain-mediated proteolysis when Ser-23/24 of cTnI in mouse cardiac myofibrils was nonphosphorylated, mono-phosphorylated, or bisphosphorylated (using sequential incubations in λ-phosphatase, PKD, and PKA, respectively). Phos-tag gels, Western blotting, and high-resolution MS revealed that PKD produced >90% monophosphorylation of cTnI, primarily at Ser-24, whereas PKA led to cTnI bisphosphorylation exclusively. PKD markedly decreased the Ca2+ sensitivity of force production in detergent-permeabilized ventricular trabeculae, whereas subsequent incubation with PKA produced only a small further fall of Ca2+ sensitivity. Unlike PKD, PKA also substantially phosphorylated myosin-binding protein-C and significantly accelerated cross-bridge kinetics (ktr). After phosphorylation by PKD or PKA, cTnI in isolated myofibrils was partially protected from calpain-mediated degradation. We conclude that cTnI monophosphorylation at Ser-23/24 decreases myofibrillar Ca2+ sensitivity and partially protects cTnI from calpain-induced proteolysis. In healthy cardiomyocytes, the basal monophosphorylation of cTnI may help tonically regulate myofibrillar Ca2+ sensitivity.

Original language	English
Pages (from-to)	8588-8599
Journal	Journal of Biological Chemistry
Volume	293
Issue number	22
Early online date	18 Apr 2018
DOIs	https://doi.org/10.1074/jbc.RA117.001292
Publication status	Published - 1 Jun 2018

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Martin-garrido, A., Biesiadecki, B. J., Salhi, H. E., Shaifta, Y., Dos Remedios, C. G., Ayaz-guner, S., Cai, W., Ge, Y., Avkiran, M., & Kentish, J. C. (2018). Monophosphorylation of cardiac troponin-I at Ser23/24 is sufficient to regulate cardiac myofibrillar Ca2+ sensitivity and calpain-induced proteolysis. Journal of Biological Chemistry, 293(22), 8588-8599. https://doi.org/10.1074/jbc.RA117.001292

@article{169d101fe6094d48accfa15cb0ee68c9,

title = "Monophosphorylation of cardiac troponin-I at Ser23/24 is sufficient to regulate cardiac myofibrillar Ca2+ sensitivity and calpain-induced proteolysis",

abstract = "The acceleration of myocardial relaxation produced by β-adrenoreceptor stimulation is mediated in part by protein kinase A (PKA)-mediated phosphorylation of cardiac troponin-I (cTnI), which decreases myofibrillar Ca2+ sensitivity. Previous evidence suggests that phosphorylation of both Ser-23 and Ser-24 in cTnI is required for this Ca2+ desensitization. PKA-mediated phosphorylation also partially protects cTnI from proteolysis by calpain. Here we report that protein kinase D (PKD) phosphorylates only one serine of cTnI Ser-23/24. To explore the functional consequences of this monophosphorylation, we examined the Ca2+ sensitivity of force production and susceptibility of cTnI to calpain-mediated proteolysis when Ser-23/24 of cTnI in mouse cardiac myofibrils was nonphosphorylated, mono-phosphorylated, or bisphosphorylated (using sequential incubations in λ-phosphatase, PKD, and PKA, respectively). Phos-tag gels, Western blotting, and high-resolution MS revealed that PKD produced >90% monophosphorylation of cTnI, primarily at Ser-24, whereas PKA led to cTnI bisphosphorylation exclusively. PKD markedly decreased the Ca2+ sensitivity of force production in detergent-permeabilized ventricular trabeculae, whereas subsequent incubation with PKA produced only a small further fall of Ca2+ sensitivity. Unlike PKD, PKA also substantially phosphorylated myosin-binding protein-C and significantly accelerated cross-bridge kinetics (ktr). After phosphorylation by PKD or PKA, cTnI in isolated myofibrils was partially protected from calpain-mediated degradation. We conclude that cTnI monophosphorylation at Ser-23/24 decreases myofibrillar Ca2+ sensitivity and partially protects cTnI from calpain-induced proteolysis. In healthy cardiomyocytes, the basal monophosphorylation of cTnI may help tonically regulate myofibrillar Ca2+ sensitivity.",

author = "Abel Martin-garrido and Biesiadecki, {Brandon J.} and Salhi, {Hussam E.} and Yasin Shaifta and {Dos Remedios}, {Cristobal G.} and Serife Ayaz-guner and Wenxuan Cai and Ying Ge and Metin Avkiran and Kentish, {Jonathan C.}",

year = "2018",

month = jun,

day = "1",

doi = "10.1074/jbc.RA117.001292",

language = "English",

volume = "293",

pages = "8588--8599",

journal = "Journal of Biological Chemistry",

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publisher = "American Society for Biochemistry and Molecular Biology Inc.",

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Martin-garrido, A, Biesiadecki, BJ, Salhi, HE, Shaifta, Y, Dos Remedios, CG, Ayaz-guner, S, Cai, W, Ge, Y, Avkiran, M & Kentish, JC 2018, 'Monophosphorylation of cardiac troponin-I at Ser23/24 is sufficient to regulate cardiac myofibrillar Ca2+ sensitivity and calpain-induced proteolysis', Journal of Biological Chemistry, vol. 293, no. 22, pp. 8588-8599. https://doi.org/10.1074/jbc.RA117.001292

Monophosphorylation of cardiac troponin-I at Ser23/24 is sufficient to regulate cardiac myofibrillar Ca2+ sensitivity and calpain-induced proteolysis. / Martin-garrido, Abel; Biesiadecki, Brandon J.; Salhi, Hussam E. et al.
In: Journal of Biological Chemistry, Vol. 293, No. 22, 01.06.2018, p. 8588-8599.

Research output: Contribution to journal › Article › peer-review

TY - JOUR

T1 - Monophosphorylation of cardiac troponin-I at Ser23/24 is sufficient to regulate cardiac myofibrillar Ca2+ sensitivity and calpain-induced proteolysis

AU - Martin-garrido, Abel

AU - Biesiadecki, Brandon J.

AU - Salhi, Hussam E.

AU - Shaifta, Yasin

AU - Dos Remedios, Cristobal G.

AU - Ayaz-guner, Serife

AU - Cai, Wenxuan

AU - Ge, Ying

AU - Avkiran, Metin

AU - Kentish, Jonathan C.

PY - 2018/6/1

Y1 - 2018/6/1

N2 - The acceleration of myocardial relaxation produced by β-adrenoreceptor stimulation is mediated in part by protein kinase A (PKA)-mediated phosphorylation of cardiac troponin-I (cTnI), which decreases myofibrillar Ca2+ sensitivity. Previous evidence suggests that phosphorylation of both Ser-23 and Ser-24 in cTnI is required for this Ca2+ desensitization. PKA-mediated phosphorylation also partially protects cTnI from proteolysis by calpain. Here we report that protein kinase D (PKD) phosphorylates only one serine of cTnI Ser-23/24. To explore the functional consequences of this monophosphorylation, we examined the Ca2+ sensitivity of force production and susceptibility of cTnI to calpain-mediated proteolysis when Ser-23/24 of cTnI in mouse cardiac myofibrils was nonphosphorylated, mono-phosphorylated, or bisphosphorylated (using sequential incubations in λ-phosphatase, PKD, and PKA, respectively). Phos-tag gels, Western blotting, and high-resolution MS revealed that PKD produced >90% monophosphorylation of cTnI, primarily at Ser-24, whereas PKA led to cTnI bisphosphorylation exclusively. PKD markedly decreased the Ca2+ sensitivity of force production in detergent-permeabilized ventricular trabeculae, whereas subsequent incubation with PKA produced only a small further fall of Ca2+ sensitivity. Unlike PKD, PKA also substantially phosphorylated myosin-binding protein-C and significantly accelerated cross-bridge kinetics (ktr). After phosphorylation by PKD or PKA, cTnI in isolated myofibrils was partially protected from calpain-mediated degradation. We conclude that cTnI monophosphorylation at Ser-23/24 decreases myofibrillar Ca2+ sensitivity and partially protects cTnI from calpain-induced proteolysis. In healthy cardiomyocytes, the basal monophosphorylation of cTnI may help tonically regulate myofibrillar Ca2+ sensitivity.

AB - The acceleration of myocardial relaxation produced by β-adrenoreceptor stimulation is mediated in part by protein kinase A (PKA)-mediated phosphorylation of cardiac troponin-I (cTnI), which decreases myofibrillar Ca2+ sensitivity. Previous evidence suggests that phosphorylation of both Ser-23 and Ser-24 in cTnI is required for this Ca2+ desensitization. PKA-mediated phosphorylation also partially protects cTnI from proteolysis by calpain. Here we report that protein kinase D (PKD) phosphorylates only one serine of cTnI Ser-23/24. To explore the functional consequences of this monophosphorylation, we examined the Ca2+ sensitivity of force production and susceptibility of cTnI to calpain-mediated proteolysis when Ser-23/24 of cTnI in mouse cardiac myofibrils was nonphosphorylated, mono-phosphorylated, or bisphosphorylated (using sequential incubations in λ-phosphatase, PKD, and PKA, respectively). Phos-tag gels, Western blotting, and high-resolution MS revealed that PKD produced >90% monophosphorylation of cTnI, primarily at Ser-24, whereas PKA led to cTnI bisphosphorylation exclusively. PKD markedly decreased the Ca2+ sensitivity of force production in detergent-permeabilized ventricular trabeculae, whereas subsequent incubation with PKA produced only a small further fall of Ca2+ sensitivity. Unlike PKD, PKA also substantially phosphorylated myosin-binding protein-C and significantly accelerated cross-bridge kinetics (ktr). After phosphorylation by PKD or PKA, cTnI in isolated myofibrils was partially protected from calpain-mediated degradation. We conclude that cTnI monophosphorylation at Ser-23/24 decreases myofibrillar Ca2+ sensitivity and partially protects cTnI from calpain-induced proteolysis. In healthy cardiomyocytes, the basal monophosphorylation of cTnI may help tonically regulate myofibrillar Ca2+ sensitivity.

U2 - 10.1074/jbc.RA117.001292

DO - 10.1074/jbc.RA117.001292

M3 - Article

SN - 0021-9258

VL - 293

SP - 8588

EP - 8599

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 22

ER -

Monophosphorylation of cardiac troponin-I at Ser23/24 is sufficient to regulate cardiac myofibrillar Ca2+ sensitivity and calpain-induced proteolysis

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