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Cardiac myosin binding protein C phosphorylation in cardiac disease.

Kuster DW, Bawazeer AC, Zaremba R, Goebel M, Boontje NM, van der Velden J - J. Muscle Res. Cell. Motil. (2011)

Bottom Line: Perturbations in sarcomeric function may in part underlie systolic and diastolic dysfunction of the failing heart.Sarcomeric dysfunction has been ascribed to changes in phosphorylation status of sarcomeric proteins caused by an altered balance between intracellular kinases and phosphatases during the development of cardiac disease.In the present review we discuss changes in phosphorylation of the thick filament protein myosin binding protein C (cMyBP-C) reported in failing myocardium, with emphasis on phosphorylation changes observed in familial hypertrophic cardiomyopathy caused by mutations in MYBPC3.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, VU University Medical Center, Amsterdam, The Netherlands. d.kuster@vumc.nl

ABSTRACT
Perturbations in sarcomeric function may in part underlie systolic and diastolic dysfunction of the failing heart. Sarcomeric dysfunction has been ascribed to changes in phosphorylation status of sarcomeric proteins caused by an altered balance between intracellular kinases and phosphatases during the development of cardiac disease. In the present review we discuss changes in phosphorylation of the thick filament protein myosin binding protein C (cMyBP-C) reported in failing myocardium, with emphasis on phosphorylation changes observed in familial hypertrophic cardiomyopathy caused by mutations in MYBPC3. Moreover, we will discuss assays which allow to distinguish between functional consequences of mutant sarcomeric proteins and (mal)adaptive changes in sarcomeric protein phosphorylation.

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Cardiac MyBP-C phosphorylation in end-stage heart failure. a Samples from donor, end-stage heart failure patients with IDCM or ISHD cardiomyopathy were analyzed for cMyBP-C and cTnI phosphorylation with ProQ Diamond stained gels and normalized to total SYPRO-stained cMyBP-C. Phosphorylation of cardiac cMyBP-C and cTnI is higher in donor compared to the ISHD and IDCM samples. Phosphorylation in the groups was normalized to donor, which was set to one. Figure adapted from Hamdani et al. (2010) with kind permission from Springer Science + Business Media. b Representative 2D-gels from donor and IDCM, showing a shift of cMyBP-C towards higher pI spots (left) in IDCM compared with donor, indicating less phosphorylation. Figure adapted from Copeland et al. (2010) with permission from Elsevier. c Quantification of the different spots from 2D gel analysis showing similar shifts in cMyBP-C phosphorylation in IDCM and ISHD compared to donor. *P < 0.05 versus donor in one-way ANOVA followed by post-test Bonferroni; #P < 0.05 versus IDCM in one-way ANOVA followed by post-test Bonferroni analysis
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Fig1: Cardiac MyBP-C phosphorylation in end-stage heart failure. a Samples from donor, end-stage heart failure patients with IDCM or ISHD cardiomyopathy were analyzed for cMyBP-C and cTnI phosphorylation with ProQ Diamond stained gels and normalized to total SYPRO-stained cMyBP-C. Phosphorylation of cardiac cMyBP-C and cTnI is higher in donor compared to the ISHD and IDCM samples. Phosphorylation in the groups was normalized to donor, which was set to one. Figure adapted from Hamdani et al. (2010) with kind permission from Springer Science + Business Media. b Representative 2D-gels from donor and IDCM, showing a shift of cMyBP-C towards higher pI spots (left) in IDCM compared with donor, indicating less phosphorylation. Figure adapted from Copeland et al. (2010) with permission from Elsevier. c Quantification of the different spots from 2D gel analysis showing similar shifts in cMyBP-C phosphorylation in IDCM and ISHD compared to donor. *P < 0.05 versus donor in one-way ANOVA followed by post-test Bonferroni; #P < 0.05 versus IDCM in one-way ANOVA followed by post-test Bonferroni analysis

Mentions: As indicated above, the other main target of PKA in the sarcomere is cMyBP-C and in cardiac tissue from end-stage heart failure patients its phosphorylation is also decreased (El-Armouche et al. 2007; Jacques et al. 2008; Zaremba et al. 2007), irrespective if the underlying cause of heart failure (Copeland et al. 2010; Hamdani et al. 2010). Using 1D gel electrophoresis and staining with the phospho-specific stain ProQ Diamond we observed reduced phosphorylation of cMyBP-C in end-stage heart failure patients with ischemic (ISHD) or idiopathic (IDCM) cardiomyopathy (Fig. 1a). Protein phosphorylation can be studied by a number of methods, one of which is 2D gel electrophoresis. This technique is based on the iso-electric point of a protein, which is reciprocally correlated to the amount of phosphorylation. With this method, it was shown that the cMyBP-C protein spots separated on the 2D gel (Fig. 1b) were shifted to the basic side (higher pI) in failing compared to donor cardiac samples in support for decreased cMyBP-C phosphorylation in both ischemic and idiopathic heart failure (Fig. 1c). Another method to quantitatively study protein phosphorylation is by phosphate affinity gel electrophoresis, in which the degree of phosphorylation is inversely related to the migration speed in the gel (Kinoshita et al. 2006). In human donor heart tissue, it was shown that most of the cMyBP-C exists in mono-, bi-, tri-, or tetra-phosphorylated forms with very little of the unphosphorylated form, while in hearts from end-stage heart failure patients the unphosphorylated form is predominant, with only some mono-phosphorylated cMyBP-C (Copeland et al. 2010). Protein phosphorylation can also be studied by using phosphorylation site-specific antibodies. cMyBP-C can be phosphorylated in vivo on at least three sites, all of which are located in the cardiac isoform specific M region, i.e., Ser273, Ser282, and Ser 302 (Barefield and Sadayappan 2010). At least one other site should exist in humans (Copeland et al. 2010) and multiple sites are predicted from studies in animal models (Yuan et al. 2006) or on the basis of in vitro studies (Jia et al. 2010). Using antibodies specific for these sites, it was shown that Ser282 phosphorylation was markedly reduced in end-stage failing heart tissue (El-Armouche et al. 2007), as was phosphorylation of Ser273 and Ser302 (Copeland et al. 2010).Fig. 1


Cardiac myosin binding protein C phosphorylation in cardiac disease.

Kuster DW, Bawazeer AC, Zaremba R, Goebel M, Boontje NM, van der Velden J - J. Muscle Res. Cell. Motil. (2011)

Cardiac MyBP-C phosphorylation in end-stage heart failure. a Samples from donor, end-stage heart failure patients with IDCM or ISHD cardiomyopathy were analyzed for cMyBP-C and cTnI phosphorylation with ProQ Diamond stained gels and normalized to total SYPRO-stained cMyBP-C. Phosphorylation of cardiac cMyBP-C and cTnI is higher in donor compared to the ISHD and IDCM samples. Phosphorylation in the groups was normalized to donor, which was set to one. Figure adapted from Hamdani et al. (2010) with kind permission from Springer Science + Business Media. b Representative 2D-gels from donor and IDCM, showing a shift of cMyBP-C towards higher pI spots (left) in IDCM compared with donor, indicating less phosphorylation. Figure adapted from Copeland et al. (2010) with permission from Elsevier. c Quantification of the different spots from 2D gel analysis showing similar shifts in cMyBP-C phosphorylation in IDCM and ISHD compared to donor. *P < 0.05 versus donor in one-way ANOVA followed by post-test Bonferroni; #P < 0.05 versus IDCM in one-way ANOVA followed by post-test Bonferroni analysis
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Related In: Results  -  Collection

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Fig1: Cardiac MyBP-C phosphorylation in end-stage heart failure. a Samples from donor, end-stage heart failure patients with IDCM or ISHD cardiomyopathy were analyzed for cMyBP-C and cTnI phosphorylation with ProQ Diamond stained gels and normalized to total SYPRO-stained cMyBP-C. Phosphorylation of cardiac cMyBP-C and cTnI is higher in donor compared to the ISHD and IDCM samples. Phosphorylation in the groups was normalized to donor, which was set to one. Figure adapted from Hamdani et al. (2010) with kind permission from Springer Science + Business Media. b Representative 2D-gels from donor and IDCM, showing a shift of cMyBP-C towards higher pI spots (left) in IDCM compared with donor, indicating less phosphorylation. Figure adapted from Copeland et al. (2010) with permission from Elsevier. c Quantification of the different spots from 2D gel analysis showing similar shifts in cMyBP-C phosphorylation in IDCM and ISHD compared to donor. *P < 0.05 versus donor in one-way ANOVA followed by post-test Bonferroni; #P < 0.05 versus IDCM in one-way ANOVA followed by post-test Bonferroni analysis
Mentions: As indicated above, the other main target of PKA in the sarcomere is cMyBP-C and in cardiac tissue from end-stage heart failure patients its phosphorylation is also decreased (El-Armouche et al. 2007; Jacques et al. 2008; Zaremba et al. 2007), irrespective if the underlying cause of heart failure (Copeland et al. 2010; Hamdani et al. 2010). Using 1D gel electrophoresis and staining with the phospho-specific stain ProQ Diamond we observed reduced phosphorylation of cMyBP-C in end-stage heart failure patients with ischemic (ISHD) or idiopathic (IDCM) cardiomyopathy (Fig. 1a). Protein phosphorylation can be studied by a number of methods, one of which is 2D gel electrophoresis. This technique is based on the iso-electric point of a protein, which is reciprocally correlated to the amount of phosphorylation. With this method, it was shown that the cMyBP-C protein spots separated on the 2D gel (Fig. 1b) were shifted to the basic side (higher pI) in failing compared to donor cardiac samples in support for decreased cMyBP-C phosphorylation in both ischemic and idiopathic heart failure (Fig. 1c). Another method to quantitatively study protein phosphorylation is by phosphate affinity gel electrophoresis, in which the degree of phosphorylation is inversely related to the migration speed in the gel (Kinoshita et al. 2006). In human donor heart tissue, it was shown that most of the cMyBP-C exists in mono-, bi-, tri-, or tetra-phosphorylated forms with very little of the unphosphorylated form, while in hearts from end-stage heart failure patients the unphosphorylated form is predominant, with only some mono-phosphorylated cMyBP-C (Copeland et al. 2010). Protein phosphorylation can also be studied by using phosphorylation site-specific antibodies. cMyBP-C can be phosphorylated in vivo on at least three sites, all of which are located in the cardiac isoform specific M region, i.e., Ser273, Ser282, and Ser 302 (Barefield and Sadayappan 2010). At least one other site should exist in humans (Copeland et al. 2010) and multiple sites are predicted from studies in animal models (Yuan et al. 2006) or on the basis of in vitro studies (Jia et al. 2010). Using antibodies specific for these sites, it was shown that Ser282 phosphorylation was markedly reduced in end-stage failing heart tissue (El-Armouche et al. 2007), as was phosphorylation of Ser273 and Ser302 (Copeland et al. 2010).Fig. 1

Bottom Line: Perturbations in sarcomeric function may in part underlie systolic and diastolic dysfunction of the failing heart.Sarcomeric dysfunction has been ascribed to changes in phosphorylation status of sarcomeric proteins caused by an altered balance between intracellular kinases and phosphatases during the development of cardiac disease.In the present review we discuss changes in phosphorylation of the thick filament protein myosin binding protein C (cMyBP-C) reported in failing myocardium, with emphasis on phosphorylation changes observed in familial hypertrophic cardiomyopathy caused by mutations in MYBPC3.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, VU University Medical Center, Amsterdam, The Netherlands. d.kuster@vumc.nl

ABSTRACT
Perturbations in sarcomeric function may in part underlie systolic and diastolic dysfunction of the failing heart. Sarcomeric dysfunction has been ascribed to changes in phosphorylation status of sarcomeric proteins caused by an altered balance between intracellular kinases and phosphatases during the development of cardiac disease. In the present review we discuss changes in phosphorylation of the thick filament protein myosin binding protein C (cMyBP-C) reported in failing myocardium, with emphasis on phosphorylation changes observed in familial hypertrophic cardiomyopathy caused by mutations in MYBPC3. Moreover, we will discuss assays which allow to distinguish between functional consequences of mutant sarcomeric proteins and (mal)adaptive changes in sarcomeric protein phosphorylation.

Show MeSH
Related in: MedlinePlus