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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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Dephosphorylation of cTnI and cMyBP-C by protein PP1 and AP. Incubations of skinned donor tissue with PP1 and AP were performed as described before (Zaremba et al. 2007). Briefly, human donor tissue (n = 3) was obtained during cardiac surgery and frozen and stored in liquid N2. Samples were homogenized in buffer containing 0.5% (v/v) Triton X-100. Samples were washed twice in buffer without Triton and 100 μl sample was subsequently incubated with 10 μl PP1 (catalytic subunit, Sigma) or 10 μl AP (calf intestinal; New England Biolabs). Phosphorylation was assessed by ProQ Diamond stained gels and normalized to total SYPRO-stained α-actinin. PP1 preferentially dephosphorylates cTnI, while AP also dephosphorylates cMyBP-C. *P<0.05 versus baseline. BL baseline
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Fig5: Dephosphorylation of cTnI and cMyBP-C by protein PP1 and AP. Incubations of skinned donor tissue with PP1 and AP were performed as described before (Zaremba et al. 2007). Briefly, human donor tissue (n = 3) was obtained during cardiac surgery and frozen and stored in liquid N2. Samples were homogenized in buffer containing 0.5% (v/v) Triton X-100. Samples were washed twice in buffer without Triton and 100 μl sample was subsequently incubated with 10 μl PP1 (catalytic subunit, Sigma) or 10 μl AP (calf intestinal; New England Biolabs). Phosphorylation was assessed by ProQ Diamond stained gels and normalized to total SYPRO-stained α-actinin. PP1 preferentially dephosphorylates cTnI, while AP also dephosphorylates cMyBP-C. *P<0.05 versus baseline. BL baseline

Mentions: In contrast to the extensive body of work published about kinase-mediated phosphorylation of cMyBP-C, only a limited number of studies have focused on the effects of phosphatases. As was mentioned above, cardiac PP1 expression and activity is increased in heart failure (Neumann et al. 1997). Purified cMyBP-C that was first phosphorylated by PKA, was subsequently dephosphorylated by 30–40% by incubation with the catalytic subunit of PP1 (Schlender et al. 1987). The same extent of dephosphorylation was seen after incubation of skinned donor heart tissue with PP1 (Yang et al. 2008; Zaremba et al. 2007) as well as in skinned mice cardiomyocytes (Yang et al. 2008). Incubations of donor tissue with a high concentration of PP1 resulted in almost complete dephosphorylation of cTnI, while cMyBP-C dephosphorylation was moderate (Fig. 5). PP1 incubations led to the decrease of the tri- and tetra-phosphorylated cMyBP-C and an increase in the unphosphorylated form (Copeland et al. 2010). Functionally, PP1 incubation in donor tissue resulted in a marked increase in the Ca2+-sensitivity of force development, which could be reversed by subsequent PKA incubation (Neulen et al. 2007). Although in another study, PP1 incubation had no effect on Ca2+-sensitivity in donor cells, but increased Ca2+-sensitivity after pre-incubation with PKC (Belin et al. 2007). The effect of PP1 on Ca2+-sensitivity is likely explained by the dephosphorylation of TnI, rather than cMyBP-C phosphorylation (Duncker et al. 2009).Fig. 5


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)

Dephosphorylation of cTnI and cMyBP-C by protein PP1 and AP. Incubations of skinned donor tissue with PP1 and AP were performed as described before (Zaremba et al. 2007). Briefly, human donor tissue (n = 3) was obtained during cardiac surgery and frozen and stored in liquid N2. Samples were homogenized in buffer containing 0.5% (v/v) Triton X-100. Samples were washed twice in buffer without Triton and 100 μl sample was subsequently incubated with 10 μl PP1 (catalytic subunit, Sigma) or 10 μl AP (calf intestinal; New England Biolabs). Phosphorylation was assessed by ProQ Diamond stained gels and normalized to total SYPRO-stained α-actinin. PP1 preferentially dephosphorylates cTnI, while AP also dephosphorylates cMyBP-C. *P<0.05 versus baseline. BL baseline
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351594&req=5

Fig5: Dephosphorylation of cTnI and cMyBP-C by protein PP1 and AP. Incubations of skinned donor tissue with PP1 and AP were performed as described before (Zaremba et al. 2007). Briefly, human donor tissue (n = 3) was obtained during cardiac surgery and frozen and stored in liquid N2. Samples were homogenized in buffer containing 0.5% (v/v) Triton X-100. Samples were washed twice in buffer without Triton and 100 μl sample was subsequently incubated with 10 μl PP1 (catalytic subunit, Sigma) or 10 μl AP (calf intestinal; New England Biolabs). Phosphorylation was assessed by ProQ Diamond stained gels and normalized to total SYPRO-stained α-actinin. PP1 preferentially dephosphorylates cTnI, while AP also dephosphorylates cMyBP-C. *P<0.05 versus baseline. BL baseline
Mentions: In contrast to the extensive body of work published about kinase-mediated phosphorylation of cMyBP-C, only a limited number of studies have focused on the effects of phosphatases. As was mentioned above, cardiac PP1 expression and activity is increased in heart failure (Neumann et al. 1997). Purified cMyBP-C that was first phosphorylated by PKA, was subsequently dephosphorylated by 30–40% by incubation with the catalytic subunit of PP1 (Schlender et al. 1987). The same extent of dephosphorylation was seen after incubation of skinned donor heart tissue with PP1 (Yang et al. 2008; Zaremba et al. 2007) as well as in skinned mice cardiomyocytes (Yang et al. 2008). Incubations of donor tissue with a high concentration of PP1 resulted in almost complete dephosphorylation of cTnI, while cMyBP-C dephosphorylation was moderate (Fig. 5). PP1 incubations led to the decrease of the tri- and tetra-phosphorylated cMyBP-C and an increase in the unphosphorylated form (Copeland et al. 2010). Functionally, PP1 incubation in donor tissue resulted in a marked increase in the Ca2+-sensitivity of force development, which could be reversed by subsequent PKA incubation (Neulen et al. 2007). Although in another study, PP1 incubation had no effect on Ca2+-sensitivity in donor cells, but increased Ca2+-sensitivity after pre-incubation with PKC (Belin et al. 2007). The effect of PP1 on Ca2+-sensitivity is likely explained by the dephosphorylation of TnI, rather than cMyBP-C phosphorylation (Duncker et al. 2009).Fig. 5

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