4015
J Physiol 594.14 (2016) pp 4015–4016
JOURNAL CLUB
The Journal of Physiology
Phosphorylation of cardiac myosin binding protein-C regulates heart contraction and dilatation in vivo during β-adrenergic receptor activation Eric R. Starr1 , Tupa Basuroy2 , Xiaoming Fan3 and Shengnan Du2 1 Biomedical Sciences Program in Neurosciences and Neurological Disorders, Department of Neurosciences, College of Medicine and Life Sciences, University of Toledo, 3000 Arlington Avenue, Toledo, OH 43614, USA 2 Biomedical Sciences Program in Cancer Biology, Department of Biochemistry and Cancer Biology, College of Medicine and Life Sciences, University of Toledo, 3000 Arlington Avenue, Toledo, OH 43614, USA 3 Biomedical Sciences Program in Molecular Medicine, Department of Medicine, University of Toledo, College of Medicine and Life Sciences, 3000 Arlington Avenue, Toledo, Ohio 43614, USA Email: [email protected] The heart is a muscular organ that pumps blood throughout the circulatory system via rhythmic contraction and dilatation. Dysfunction in heart contraction and dilatation can result in heart failure (HF), a syndrome characterized by an inability of the heart to pump enough blood to meet the metabolic demands of the body (Najafi et al. 2016). Currently no interventions have been developed that treat the progression of HF (Mamidi et al. 2014). Consequently, experiments investigating the role of myofilament proteins mediating heart contraction and relaxation are particularly exciting as they could illuminate potential therapeutic targets for treating HF. In a recently published article in The Journal of Physiology, Gresham & Steltzer (2016) examined phosphorylation of two myofilament proteins, cardiac myosin binding protein-C (MyBP-C) and cardiac troponin I (TnI), on heart contraction and relaxation. Their findings identify a critical role for phosphorylated MyBP-C in mediating normal heart functioning. Phosphorylation of signalling molecules in cardiomyocytes is primarily mediated by activation of β-adrenergic receptors (βRs). Under normal circumstances,
activation of the sympathetic nervous system (SNS) stimulates βRs. Stimulated βRs activate protein kinase A (PKA), which phosphorylates several key regulatory proteins on myofilaments to alter cardiac function (Najafi et al. 2016). MyBP-C and TnI are two such myofilament proteins. MyBP-C is a protein found on heavy filaments that regulates actin–myosin crossbridging during contraction. Phosphorylation of MyBP-C serine residues (Ser273, Ser282, Ser302) accelerates crossbridge formation, force generation, and crossbridge detachment and relaxation, an effect that corresponds to improved heart contraction and relaxation during activation of the SNS (Mamidi et al. 2014, Najafi et al. 2016). The second myofilament protein, TnI regulates myofilament Ca2+ sensitivity during heart contraction. Phosphorylation of TnI’s serine residues (Ser23, Ser24) during activation of the SNS reduces myofilament sensitivity to Ca2+ , an effect that corresponds to improved heart dilatation (Najafi et al. 2016). In HF patients, βR signalling becomes impaired resulting in decreased phosphorylation of downstream targets such as MyBP-C and TnI (Najafi et al. 2016). Dephosphorylation of these effectors can impair cardiac function in a way that resembles known deficits observed in HF. For example, constitutive dephosphorylation of MyBP-C induces cardiac hypertrophy and impairs heart contraction and dilatation. Similarly, phosphoablation of TnI has been reported to impair heart relaxation (Najafi et al. 2016). However, the relative contribution of MyBP-C phosphorylation and TnI phosphorylation on cardiac function during βR activation remains unknown. Therefore, in their recent publication, Gresham & Stelzer (2016) examined the role of MyBP-C and TnI on in vivo contractile and haemodynamic function. They showed that phosphorylation of MyBP-C is critical to changes in heart contractility observed during βR activation. To distinguish the roles of TnI and MyBP-C phosphorylation on cardiac function, Gresham & Stelzer (2016) conducted experiments in adult transgenic mice, replacing PKA-phosphorylatable serine residues with alanine in both TnI
C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
(TnIPKA− ; Ser23, Ser24) and MyBP-C (MyBP-CPKA− ; Ser273, Ser282, Ser302) or in transgenic mice expressing mutations in both proteins (DBLPKA− ). Analysis of the morphology of excised mouse hearts revealed that, in comparison to wild-type controls (WT), MyBP-CPKA− and DBLPKA− , but not TnIPKA− , mice showed significant increases in heart size with no indication of ventricular fibrosis. Additionally, the researchers observed that MyBP-C phosphoablation increased left ventricular wall thickness. These findings show that constitutive dephosphorylation of MyBP-C induces ventricular hypertrophy. To examine whether phosphoablation of MyBP-C and TnI was associated with disrupted cardiac activity, Gresham & Stelzer (2016) used echocardiography and a left ventricle catheter to measure changes in both the cardiac cycle and ventricular contractility. The cardiac cycle is the sequence of events that transition the heart from contraction to dilatation during a heartbeat. Within the ventricles, there are four phases of the cardiac cycle that can be measured. Ventricular filling is the first phase, in which the ventricle is relaxed and filling with blood. Isovolumetric contraction is the second phase, in which the ventricles begin to contract. Ejection is the third phase, in which the blood is pumped out of the heart. The final phase, isovolumetric relaxation, is characterized by a decrease in pressure as the ventricles relax. Examination of echocardiograms from both MyBP-CPKA− and DBLPKA− , but not TnIPKA− , mice exhibited a significantly increased duration of isovolumetric relaxation and disruptions in ejection fraction and fractional shortening in comparison to WT mice. By recording P–V loops, they confirmed that MyBPCPKA− and DBLPKA− mice displayed a significantly prolonged ventricular pressure decline during the early portion of isovolumetric relaxation. Such findings indicate that phosphorylation of MyBP-C is critical for maintaining the basal rate of relaxation and contractile force observed during ejection. Given that MyBP-C directly modifies actin–myosin association, it would be conceivable that these dysfunctions could arise from changes in actin–myosin crossbridging. Therefore, future experiments
DOI: 10.1113/JP272680
4016 should examine whether MyBP-CPKA− mice exhibit altered crossbridge formation, acceleration and detachment under basal conditions. To determine whether phosphorylation of MyBP-C or TnI is more critical for heart function during βR activation, Gresham & Steltzer (2016) examined the effects of MyBP-C and TnI phosphoablation on cardiac function by injecting mice with the βR agonist dobutamine. As expected, dobutamine administration mimicked the effects of βR activation on heart contractility in WT mice (Najafi et al. 2016). However, heart contraction and relaxation were impaired in MyBP-CPKA− and DBLPKA− mice following dobutamine administration. Both MyBP-CPKA− and DBLPKA− mice displayed significant reductions in systolic blood pressure as well as blunted elevations in ejection fraction, ventricular power and fractional shortening during βR activation. Additionally, MyBP-CPKA− and DBLPKA− mice exhibited a significant rise in the overall duration of contraction following dobutamine treatment, increasing the duration of isovolumetric contraction and ventricular ejection. Along with this increase in the duration of contraction, MyBP-CPKA− and DBLPKA− mice also exhibited significant reductions in the overall duration of dilatation, shortening the duration of isovolumetric refilling. TnIPKA− mice, however, exhibited no significant differences in pressure development or in the duration of contraction and dilatation, as compared to WT mice. Taken together, these findings suggest that phosphorylation of MyBP-C, but not TnI, during activation of the SNS regulates pressure development during contraction and pressure relaxation during dilatation. Gresham & Stelzer (2016) therefore conclude that PKA-induced phosphorylation of MyBP-C is critical for normal cardiac function during βR activation.
Journal Club The hallmark of HF is dysfunction in both heart contraction and relaxation. In light of their findings, Gresham & Stelzer (2016) propose that reduced MyBP-C phosphorylation could underlie these dysfunctions. This hypothesis is supported by studies conducted in the hearts of HF patients showing phosphorylation of MyBP-C to be drastically reduced (Gupta & Robbins 2014). Given that phosphorylation of MyBP-C plays a significant role in crossbridge formation and detachment during heart contraction, it is conceivable that gene delivery of pseudophosphorylated MyBP-C could restore contractile dysfunctions in patients with HF (Mamidi et al. 2014). However, recent studies have shown that phosphorylation of individual MyBP-C serine residues differentially regulates heart function. Transgenic mice expressing non-phosphorylatable alanine in place of serine at residue 282 on MyBP-C exhibit impaired crossbridge formation and pressure development during heart contraction (Gresham et al. 2014). Other studies examining phosphorylation of either Ser273 or Ser302 have indicated that phosphorylation of these residues also differentially regulates contractile function, cardiomyocyte survivability and heart structure (Gupta & Robbins, 2014). However, the role of phosphorylation on individual MyBP-C serine residues has not been fully characterized. Consequently, researchers should investigate the role of phosphorylation of individual MyBP-C serine residues on actin–myosin crossbridging during heart contraction and relaxation. In doing so, researchers could develop designer MyBP-Cs that could more precisely target contractile abnormalities in HF. The findings of Gresham & Stelzer (2016) highlight a critical role for MyBP-C phosphorylation in contractile function
J Physiol 594.14
during βR activation. Their results support the hypothesis that interventions targeting MyBP-C could have therapeutic benefits for treating HF. References Gresham KS, Mamidi R & Stelzer JE (2014). The contribution of cardiac myosin binding protein-c Ser282 phosphorylation to the rate of force generation and in vivo cardiac contractility. J Physiol 592, 3747–3765. Gresham KS & Stelzer JE (2016). The contributions of cardiac myosin binding protein C and troponin I phosphorylation to β-adrenergic enhancements of in vivo cardiac function. J Physiol 594, 669–686. Gupta MK & Robbins J (2014). Post-translational control of cardiac haemodynamics through myosin binding protein C. Pflugers Arch 466, 231–236. Mamidi R, Li J, Gresham KS & Stelzer JE (2014). Cardiac myosin binding protein-C: a novel sarcomeric target for gene therapy. Pflugers Arch 466, 225–230. Najafi A, Sequeira V, Kuster DWD & van der Velden J (2016). β-Adrenergic receptor signalling and its functional consequences in the diseased heart. Eur J Clin Invest 46, 362–374. Additional information Competing interests
None declared. Author contributions
All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. Acknowledgements
This project was a requirement for completion of the Scientific Communication Skills and Career Goals course offered at the University of Toledo. The authors would like to thank Dr. Jennifer Hill and Dr. Kandace Williams for their constructive feedback during the completion of this paper.
C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
J Physiol 594.14 (2016) pp 4015–4016
JOURNAL CLUB
The Journal of Physiology
Phosphorylation of cardiac myosin binding protein-C regulates heart contraction and dilatation in vivo during β-adrenergic receptor activation Eric R. Starr1 , Tupa Basuroy2 , Xiaoming Fan3 and Shengnan Du2 1 Biomedical Sciences Program in Neurosciences and Neurological Disorders, Department of Neurosciences, College of Medicine and Life Sciences, University of Toledo, 3000 Arlington Avenue, Toledo, OH 43614, USA 2 Biomedical Sciences Program in Cancer Biology, Department of Biochemistry and Cancer Biology, College of Medicine and Life Sciences, University of Toledo, 3000 Arlington Avenue, Toledo, OH 43614, USA 3 Biomedical Sciences Program in Molecular Medicine, Department of Medicine, University of Toledo, College of Medicine and Life Sciences, 3000 Arlington Avenue, Toledo, Ohio 43614, USA Email: [email protected] The heart is a muscular organ that pumps blood throughout the circulatory system via rhythmic contraction and dilatation. Dysfunction in heart contraction and dilatation can result in heart failure (HF), a syndrome characterized by an inability of the heart to pump enough blood to meet the metabolic demands of the body (Najafi et al. 2016). Currently no interventions have been developed that treat the progression of HF (Mamidi et al. 2014). Consequently, experiments investigating the role of myofilament proteins mediating heart contraction and relaxation are particularly exciting as they could illuminate potential therapeutic targets for treating HF. In a recently published article in The Journal of Physiology, Gresham & Steltzer (2016) examined phosphorylation of two myofilament proteins, cardiac myosin binding protein-C (MyBP-C) and cardiac troponin I (TnI), on heart contraction and relaxation. Their findings identify a critical role for phosphorylated MyBP-C in mediating normal heart functioning. Phosphorylation of signalling molecules in cardiomyocytes is primarily mediated by activation of β-adrenergic receptors (βRs). Under normal circumstances,
activation of the sympathetic nervous system (SNS) stimulates βRs. Stimulated βRs activate protein kinase A (PKA), which phosphorylates several key regulatory proteins on myofilaments to alter cardiac function (Najafi et al. 2016). MyBP-C and TnI are two such myofilament proteins. MyBP-C is a protein found on heavy filaments that regulates actin–myosin crossbridging during contraction. Phosphorylation of MyBP-C serine residues (Ser273, Ser282, Ser302) accelerates crossbridge formation, force generation, and crossbridge detachment and relaxation, an effect that corresponds to improved heart contraction and relaxation during activation of the SNS (Mamidi et al. 2014, Najafi et al. 2016). The second myofilament protein, TnI regulates myofilament Ca2+ sensitivity during heart contraction. Phosphorylation of TnI’s serine residues (Ser23, Ser24) during activation of the SNS reduces myofilament sensitivity to Ca2+ , an effect that corresponds to improved heart dilatation (Najafi et al. 2016). In HF patients, βR signalling becomes impaired resulting in decreased phosphorylation of downstream targets such as MyBP-C and TnI (Najafi et al. 2016). Dephosphorylation of these effectors can impair cardiac function in a way that resembles known deficits observed in HF. For example, constitutive dephosphorylation of MyBP-C induces cardiac hypertrophy and impairs heart contraction and dilatation. Similarly, phosphoablation of TnI has been reported to impair heart relaxation (Najafi et al. 2016). However, the relative contribution of MyBP-C phosphorylation and TnI phosphorylation on cardiac function during βR activation remains unknown. Therefore, in their recent publication, Gresham & Stelzer (2016) examined the role of MyBP-C and TnI on in vivo contractile and haemodynamic function. They showed that phosphorylation of MyBP-C is critical to changes in heart contractility observed during βR activation. To distinguish the roles of TnI and MyBP-C phosphorylation on cardiac function, Gresham & Stelzer (2016) conducted experiments in adult transgenic mice, replacing PKA-phosphorylatable serine residues with alanine in both TnI
C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
(TnIPKA− ; Ser23, Ser24) and MyBP-C (MyBP-CPKA− ; Ser273, Ser282, Ser302) or in transgenic mice expressing mutations in both proteins (DBLPKA− ). Analysis of the morphology of excised mouse hearts revealed that, in comparison to wild-type controls (WT), MyBP-CPKA− and DBLPKA− , but not TnIPKA− , mice showed significant increases in heart size with no indication of ventricular fibrosis. Additionally, the researchers observed that MyBP-C phosphoablation increased left ventricular wall thickness. These findings show that constitutive dephosphorylation of MyBP-C induces ventricular hypertrophy. To examine whether phosphoablation of MyBP-C and TnI was associated with disrupted cardiac activity, Gresham & Stelzer (2016) used echocardiography and a left ventricle catheter to measure changes in both the cardiac cycle and ventricular contractility. The cardiac cycle is the sequence of events that transition the heart from contraction to dilatation during a heartbeat. Within the ventricles, there are four phases of the cardiac cycle that can be measured. Ventricular filling is the first phase, in which the ventricle is relaxed and filling with blood. Isovolumetric contraction is the second phase, in which the ventricles begin to contract. Ejection is the third phase, in which the blood is pumped out of the heart. The final phase, isovolumetric relaxation, is characterized by a decrease in pressure as the ventricles relax. Examination of echocardiograms from both MyBP-CPKA− and DBLPKA− , but not TnIPKA− , mice exhibited a significantly increased duration of isovolumetric relaxation and disruptions in ejection fraction and fractional shortening in comparison to WT mice. By recording P–V loops, they confirmed that MyBPCPKA− and DBLPKA− mice displayed a significantly prolonged ventricular pressure decline during the early portion of isovolumetric relaxation. Such findings indicate that phosphorylation of MyBP-C is critical for maintaining the basal rate of relaxation and contractile force observed during ejection. Given that MyBP-C directly modifies actin–myosin association, it would be conceivable that these dysfunctions could arise from changes in actin–myosin crossbridging. Therefore, future experiments
DOI: 10.1113/JP272680
4016 should examine whether MyBP-CPKA− mice exhibit altered crossbridge formation, acceleration and detachment under basal conditions. To determine whether phosphorylation of MyBP-C or TnI is more critical for heart function during βR activation, Gresham & Steltzer (2016) examined the effects of MyBP-C and TnI phosphoablation on cardiac function by injecting mice with the βR agonist dobutamine. As expected, dobutamine administration mimicked the effects of βR activation on heart contractility in WT mice (Najafi et al. 2016). However, heart contraction and relaxation were impaired in MyBP-CPKA− and DBLPKA− mice following dobutamine administration. Both MyBP-CPKA− and DBLPKA− mice displayed significant reductions in systolic blood pressure as well as blunted elevations in ejection fraction, ventricular power and fractional shortening during βR activation. Additionally, MyBP-CPKA− and DBLPKA− mice exhibited a significant rise in the overall duration of contraction following dobutamine treatment, increasing the duration of isovolumetric contraction and ventricular ejection. Along with this increase in the duration of contraction, MyBP-CPKA− and DBLPKA− mice also exhibited significant reductions in the overall duration of dilatation, shortening the duration of isovolumetric refilling. TnIPKA− mice, however, exhibited no significant differences in pressure development or in the duration of contraction and dilatation, as compared to WT mice. Taken together, these findings suggest that phosphorylation of MyBP-C, but not TnI, during activation of the SNS regulates pressure development during contraction and pressure relaxation during dilatation. Gresham & Stelzer (2016) therefore conclude that PKA-induced phosphorylation of MyBP-C is critical for normal cardiac function during βR activation.
Journal Club The hallmark of HF is dysfunction in both heart contraction and relaxation. In light of their findings, Gresham & Stelzer (2016) propose that reduced MyBP-C phosphorylation could underlie these dysfunctions. This hypothesis is supported by studies conducted in the hearts of HF patients showing phosphorylation of MyBP-C to be drastically reduced (Gupta & Robbins 2014). Given that phosphorylation of MyBP-C plays a significant role in crossbridge formation and detachment during heart contraction, it is conceivable that gene delivery of pseudophosphorylated MyBP-C could restore contractile dysfunctions in patients with HF (Mamidi et al. 2014). However, recent studies have shown that phosphorylation of individual MyBP-C serine residues differentially regulates heart function. Transgenic mice expressing non-phosphorylatable alanine in place of serine at residue 282 on MyBP-C exhibit impaired crossbridge formation and pressure development during heart contraction (Gresham et al. 2014). Other studies examining phosphorylation of either Ser273 or Ser302 have indicated that phosphorylation of these residues also differentially regulates contractile function, cardiomyocyte survivability and heart structure (Gupta & Robbins, 2014). However, the role of phosphorylation on individual MyBP-C serine residues has not been fully characterized. Consequently, researchers should investigate the role of phosphorylation of individual MyBP-C serine residues on actin–myosin crossbridging during heart contraction and relaxation. In doing so, researchers could develop designer MyBP-Cs that could more precisely target contractile abnormalities in HF. The findings of Gresham & Stelzer (2016) highlight a critical role for MyBP-C phosphorylation in contractile function
J Physiol 594.14
during βR activation. Their results support the hypothesis that interventions targeting MyBP-C could have therapeutic benefits for treating HF. References Gresham KS, Mamidi R & Stelzer JE (2014). The contribution of cardiac myosin binding protein-c Ser282 phosphorylation to the rate of force generation and in vivo cardiac contractility. J Physiol 592, 3747–3765. Gresham KS & Stelzer JE (2016). The contributions of cardiac myosin binding protein C and troponin I phosphorylation to β-adrenergic enhancements of in vivo cardiac function. J Physiol 594, 669–686. Gupta MK & Robbins J (2014). Post-translational control of cardiac haemodynamics through myosin binding protein C. Pflugers Arch 466, 231–236. Mamidi R, Li J, Gresham KS & Stelzer JE (2014). Cardiac myosin binding protein-C: a novel sarcomeric target for gene therapy. Pflugers Arch 466, 225–230. Najafi A, Sequeira V, Kuster DWD & van der Velden J (2016). β-Adrenergic receptor signalling and its functional consequences in the diseased heart. Eur J Clin Invest 46, 362–374. Additional information Competing interests
None declared. Author contributions
All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. Acknowledgements
This project was a requirement for completion of the Scientific Communication Skills and Career Goals course offered at the University of Toledo. The authors would like to thank Dr. Jennifer Hill and Dr. Kandace Williams for their constructive feedback during the completion of this paper.
C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
