Cardiac myosin binding protein C phosphorylation in cardiac disease
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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.
KeywordsCardiac myosin binding protein C Phosphorylation Sarcomere Heart failure Familial hypertrophic cardiomyopathy
During recent years it has become increasingly evident that cardiac cMyBP-C exerts an important role in the regulation of sarcomere function with consequences for in vivo cardiac performance. The functional role of cMyBP-C is tightly regulated by kinase-mediated phosphorylation. The most important kinase which is known to phosphorylate cMyBP-C in vivo is protein kinase A (PKA), which is activated upon stimulation of the β-adrenergic receptors during increased stress as occurs during exercise. At the sarcomere level, β-adrenergic receptor activation reduces the sensitivity of myofilaments to calcium, reduces passive stiffness and increases the kinetics of cardiac contraction due to PKA-mediated phosphorylation of the three sarcomeric target proteins, cardiac troponin I (cTnI), titin, and cMyBP-C. Although cTnI exerts a “dominant” role in the reduction of myofilament Ca2+-sensitivity upon PKA-mediated phosphorylation, recent studies indicated a modulatory role for cMyBP-C in this process (Cazorla et al. 2006; Cuello et al. 2011; Kooij et al. 2010b). The most important regulatory role of cMyBP-C seems to be the effect on cross-bridge kinetics of sarcomere contraction (Stelzer et al. 2006a, 2006b). Involvement of cMyBP-C and its phosphorylation in stretch activation has been demonstrated in mice by Stelzer et al. (2006b, 2007). This stretch activation might play an important role in the development of systolic pressure (Steiger 1977; Stelzer et al. 2006a). It has been proposed that cMyBP-C acts as a structural constraint limiting cross-bridge formation and that phosphorylation of cMyBP-C accelerates cross-bridge kinetics which is required for enhanced rates of relaxation and force development in diastole and systole, respectively.
cMyBP-C phosphorylation in end-stage heart failure
Systolic heart failure (SHF or heart failure with reduced left ventricular ejection fraction) is the end-stage of various cardiac diseases (e.g., ischemic heart disease, valve defects) and is characterized by ineffective functioning of the heart which then cannot supply sufficient blood to meet the body’s demands. The body tries to compensate for the reduced cardiac output by sympathetic stimulation, in an attempt to maintain vital organ perfusion via an increase in heart rate and cardiac contractility. Because of the chronic nature of the disease, this leads to a prolonged increase in catecholamine levels in the serum. This in turn results in the down-regulation and desensitization of the beta-adrenergic receptor (Bristow et al. 1982) and reduced phosphorylation of downstream PKA target proteins in the end-stage failing heart (El-Armouche et al. 2004; Schwinger et al. 1999). In addition to reduced PKA signaling, increased activity and expression of protein phosphatase 1 (PP1) has been reported in a swine model of post-myocardial infarction (MI) cardiac remodeling (Duncker et al. 2009) as well as in patients with heart failure (Neumann et al. 1997). PP1 dephosphorylates many PKA target proteins and its activity is indirectly regulated by PKA via phosphorylation of the PP1 inhibitor protein. In addition to the changes in PP1 expression/activity, a decreased activity of this endogenous inhibitor of PP1 was observed in heart failure patients (El-Armouche et al. 2004).
In end-stage failing human myocardium, reduced phosphorylation of all PKA sarcomeric target proteins has been reported. Total phosphorylation of cTnI was decreased in end-stage failing myocardium (Bodor et al. 1997; van der Velden et al. 2003; Zaremba et al. 2007). This decrease could at least partially be attributed to reduced PKA-mediated phosphorylation, as phosphorylation of the PKA-specific sites Ser23/24 was lower in failing compared to non-failing donor heart tissue (Hamdani et al. 2010; Messer et al. 2007; van der Velden et al. 2006). In addition, phosphorylation of the giant protein titin is reduced in patients with systolic and diastolic heart failure (Borbely et al. 2009; Kruger et al. 2009).
cMyBP-C in familial hypertrophic cardiomyopathy (FHCM)
Another class of cardiac disease consists of the inherited cardiomyopathies (Watkins et al. 2011). In these forms of cardiomyopathy, cardiac dysfunction, and altered morphology are caused by genetic mutations rather than an external cause, such as coronary artery disease or hypertension. FHCM is the most prevalent inherited cardiac disease and has a prevalence of 1:500 (Maron 2004). FHCM is most frequently caused by mutations in genes coding for sarcomeric proteins (Richard et al. 2003). Interest in cMyBP-C grew when it became apparent that ~40% of the FHCM causing mutations were located in the MYBPC3 gene (Richard et al. 2003).
In both end-stage failing and most FHCM hearts phosphorylation was markedly lower than in donor hearts. It has been debated whether donor hearts truly represent the normal situation and can therefore be used as controls (Jweied et al. 2007; Marston and de Tombe 2008). Especially the fact that many donors receive positive inotropic support and may have brain damage leading to a catecholamine surge is of concern to studies of protein phosphorylation, as these conditions would lead to enhanced PKA activity. Furthermore, it has recently been shown that different tissue procurement strategies can affect the phosphorylation of sarcomeric proteins (Walker et al. 2011). Noteworthy, in the latter study cMyBP-C phosphorylation did not change with different procurement strategies.
The observation that cMyBP-C is less phosphorylated in a host of different cardiac disease states does not directly establish the functional consequences of lower phosphorylation of cMyBP-C. To study the functional consequences of cMyBP-C phosphorylation, either transgenic animal models can be used or studies can be performed by modulating phosphorylation in vivo or in vitro.
Transgenic animal models
The effects of cMyBP-C phosphorylation on its physiological function have been demonstrated with transgenic animal models. It has been established that cMyBP-C is vital for normal cardiac performance as complete knock-out of cMyBP-C (Carrier et al. 2004; Harris et al. 2002) or homozygous expression of a mutated MYBPC3 gene, resulting in less than 10% expression of truncated cMyBP-C (McConnell et al. 1999; Sadayappan et al. 2005), both led to a dilated and dysfunctional heart with cardiomyocyte disarray and fibrosis. Sadayappan and colleagues provided evidence that apart from cMyBP-C expression level also its phosphorylation is essential for cardiac performance. They showed that transgenic expression of wild-type cMyBP-C could rescue the phenotype of mice carrying homozygous mutated MYBPC3, while expression of cMyBP-C protein in which the three well-known phosphorylation sites (Ser273, 282, and 302) were replaced by unphosphorylatable alanines (cMyBP-CAllP−) did not correct dysfunction (Sadayappan et al. 2005). Furthermore, transgenic expression of cMyBP-C in which the known phosphorylation sites were replaced by the negatively charged aspartic acid (cMyBP-CAllP+), to mimic constitutive tri-phosphorylation, was able to rescue the phenotype of the null mutant (Sadayappan et al. 2006). Cardiomyocytes isolated from cMyBP-CAllP− mice showed a reduced stretch activation after PKA treatment compared to mice expressing wild-type cMyBP-C (Tong et al. 2008). In addition, the hearts of these animals showed an attenuated dobutamine-induced (Dob) contractile reserve compared with animals expressing the wild-type protein. To further elucidate the role of the individual phosphorylation sites in cMyBP-C, Sadayappan et al. (2011) used a transgenic mouse model in which the Ser282 site was either converted to an alanine or to an aspartic acid and bred into the cMyBP-C null mutant. This revealed that phosphorylation of the Ser302 depends on Ser282 phosphorylation, while the Dob-induced increase in cardiac contractility was dependent on all three sites being phosphorylated (Sadayappan et al. 2011). Taken together this illustrates the vital role of cMyBP-C phosphorylation for its physiological function and a proper cardiac performance.
Phosphorylation of cMyBP-C is not only important in contraction, but also seems to exert a protective effect against protein degradation as a canine model of low flow ischemia showed cMyBP-C dephosphorylation and degradation (Decker et al. 2005). Similar degradation was seen in a mouse model of ischemia–reperfusion, in which cMyBP-C was protected against degradation in mice with cMyBP-CAllP+ (Sadayappan et al. 2006). cMyBP-CAllP+ also protected against ischemia–reperfusion injury, as the ischemic area and apoptosis were reduced and fractional shortening was increased compared with wild-type cMyBP-C (Sadayappan et al. 2006, 2009).
Modulation of cMyBP-C phosphorylation
An area that has received a lot of attention in the last years, is the compartmentalization of PKA signaling, which enhances its specificity. This spatial regulation is mediated by so-called A-kinase anchoring proteins (AKAPs) that bind PKA and are able to localize to specific subcellular compartments (Fink et al. 2001; Ruehr et al. 2004). The thin filament protein troponin T has been identified as a sarcomeric AKAP, which provides a pool of PKA that can quickly phosphorylate myofilament proteins upon activation (Sumandea et al. 2011). Recently, myomegalin was shown to interact with cMyBP-C and act as an AKAP (Uys et al. 2011).
To test the role of PKA-mediated cMyBP-C phosphorylation in cardiac pathology, phosphorylation changes were studied in remodeled myocardium of swine 3 weeks after MI at baseline and upon in vivo administration of the beta-adrenergic receptor agonist Dob (Boontje et al. 2011; Duncker et al. 2009). At baseline there were no differences in cMyBP-C and cTnI phosphorylation in the post-MI animals compared with sham operated animals (Fig. 3c, d). Dob administration had a discordant effect on the PKA targets, as the increase in cTnI phosphorylation was markedly attenuated in the post-MI hearts compared to sham, while cMyBP-C was not different between post-MI and sham hearts (Fig. 3c, d). This might be explained by an increased activation of Ca2+-dependent calmodulin kinase II (CaMKII) in post-MI myocardium (Boontje et al. 2011).
Calmodulin kinase II was first found to phosphorylate cMyBP-C as a Ca2+-calmodulin dependent kinase co-purified with cMyBP-C isolated from chicken hearts (Hartzell and Glass 1984), which was subsequently identified as CaMKII (Schlender and Bean 1991). Whereas PKA could add 3 mol of phosphates per mole of cMyBP-C, CaMKII could add only one (Gautel et al. 1995). Furthermore, it seems that the site now identified as Ser282 is the target site for CaMKII (Gautel et al. 1995) and that this site needs to be phosphorylated first to facilitate phosphorylation of Ser302 (Sadayappan et al. 2011). Functionally, CaMKII phosphorylation seems to be important in the frequency-dependent increase in force as this was depressed in intact muscle treated with a CaMKII inhibitor (Tong et al. 2004).
Recently, Protein kinase D (PKD) and p90 ribosomal S6 kinase (p90 RSK or RSK) are added to the gamut of kinases able to phosphorylate cMyBP-C. PKD phosphorylates cTnI at the same sites as PKA (Haworth et al. 2004), while it phosphorylates cMyBP-C only at Ser302 (Bardswell et al. 2010). RSK on the other hand phosphorylates cMyBP-C at Ser-282 (Cuello et al. 2011). Phosphorylation of cMyBP-C with RSK was accompanied by a reduction in Ca2+-sensitivity of force development and acceleration of cross-bridge kinetics, independent from cTnI phosphorylation (Cuello et al. 2011).
Alkaline phosphatase (AP) is a widely available phosphatase that is commonly used for in vitro dephosphorylation assays. AP mainly dephosphorylates troponin T in the sarcomere, while cMyBP-C and TnI are significantly but to a lesser degree dephosphorylated (Kooij et al. 2010a; Zaremba et al. 2007) (Fig. 5). Functionally, incubation of myofilaments from donor hearts with AP leads to a slight, but significant increase in Ca2+-sensitivity of force development and passive force (Kooij et al. 2010a), but the role of cMyBP-C dephosphorylation herein is unclear.
Protein phosphatase 2a (PP2a) is able to dephosphorylate cMyBP-C (Schlender et al. 1987), although incubation of skinned cardiomyocytes with PP2a showed only a low degree of cMyBP-C dephosphorylation (Zaremba et al. 2007). A proteomic study on rat cardiomyocytes showed that the PP2a regulatory subunit B56α, is present in the myofilaments after skinning and that B56α level decreases after β-adrenergic receptor stimulation (Yin et al. 2010). PP2a’s sarcomeric localization is regulated via its interaction with P21-activated kinase-1 (PAK1) (Sheehan et al. 2007). Cultured cardiomyocytes with increased PAK1 activity and thus sarcomeric localization of PP2a, had lower phosphorylation of cMyBP-C and cTnI (Ke et al. 2004). Conflicting data is published about the effect of PP2a incubation on skinned cardiomyocytes. While Belin et al. (2007) found no changes on myofilament function in either non-failing or failing rat cardiac tissue incubated with PP2a, a recent paper by Wijnker et al. (2011) showed an increased Ca2+-sensitivity of force development in skinned cardiomyocytes isolated from human donor hearts, but not from end-stage failing hearts. This effect was attributed to dephosphorylation of cTnI, as PP2a did not dephosphorylate cMyBP-C (Wijnker et al. 2011). Further research is warranted to see if cMyBP-C is a target of PP2a in vivo.
Employing kinases and phosphatases to specifically phosphorylate or dephosphorylate cMyBP-C should help to distinguish between functional consequences of mutant sarcomere proteins and (mal)adaptive changes in sarcomeric protein phosphorylation. While a number of kinases have been identified that readily could phosphorylate cMyBP-C, no specific phosphatase has been found that can dephosphorylate cMyBP-C to a large extent. Whether dephosphorylation pathways play a role in modulating the (patho)physiological role of cMyBP-C warrants further study.
This work is supported by the 7th Framework Program of the European Union (“BIG-HEART,” grant agreement 241577). Human heart muscle samples were supplied by Prof C. dos Remedios, Sydney, Australia.
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
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