Abstract:In myocardium, the 90-kDa ribosomal S6 kinase (RSK) is activated by diverse stimuli and regulates the sarcolemmal Na ؉ /H ؉ exchanger through direct phosphorylation. Only limited information is available on other cardiac RSK substrates and functions. We evaluated cardiac myosin-binding protein C (cMyBP-C), a sarcomeric regulatory phosphoprotein, as a potential RSK substrate. In rat ventricular myocytes, RSK activation by endothelin 1 (ET1) increased cMyBP-C phosphorylation at Ser 282 , which was inhibited by t… Show more
“…The reason is that selective phosphorylation MBPC at serine 282 is accompanied by a significant reduction in Ca 2+ sensitivity and a significant acceleration of cross-bridge cycling kinetics [31], different from what we find here. We obtained some insight as to this mechanism when we discovered that demembranated trabeculae treated with the LKB1 complex (recombinant GST-LKB1/MO25/STRAD) retain the complex even after a stringent washout protocol to remove excess kinase complex ( Figure 3 ).…”
Contractile perturbations downstream of Ca2+ binding to troponin C, the so-called sarcomere-controlled mechanisms, represent the earliest indicators of energy remodeling in the diseased heart [1]. Central to cellular energy “sensing” is the adenosine monophosphate-activated kinase (AMPK) pathway, which is known to directly target myofilament proteins and alter contractility [2-6]. We previously showed that the upstream AMPK kinase, LKB1/MO25/STRAD, impacts myofilament function independently of AMPK [5]. Therefore, we hypothesized that the LKB1 complex associated with myofilament proteins and that alterations in energy signaling modulated targeting or localization of the LKB1 complex to the myofilament. Using an integrated strategy of myofilament mechanics, immunoblot analysis, co-immunoprecipitation, mass spectroscopy, and immunofluorescence, we showed that 1) LKB1 and MO25 associated with myofibrillar proteins, 2) cellular energy stress re-distributed AMPK/LKB1 complex proteins within the sarcomere, and 3) the LKB1 complex localized to the Z-Disk and interacted with cytoskeletal and energy-regulating proteins, including vinculin and ATP Synthase (Complex V). These data represent a novel role for LKB1 complex proteins in myofilament function and myocellular “energy” sensing in the heart.
“…The reason is that selective phosphorylation MBPC at serine 282 is accompanied by a significant reduction in Ca 2+ sensitivity and a significant acceleration of cross-bridge cycling kinetics [31], different from what we find here. We obtained some insight as to this mechanism when we discovered that demembranated trabeculae treated with the LKB1 complex (recombinant GST-LKB1/MO25/STRAD) retain the complex even after a stringent washout protocol to remove excess kinase complex ( Figure 3 ).…”
Contractile perturbations downstream of Ca2+ binding to troponin C, the so-called sarcomere-controlled mechanisms, represent the earliest indicators of energy remodeling in the diseased heart [1]. Central to cellular energy “sensing” is the adenosine monophosphate-activated kinase (AMPK) pathway, which is known to directly target myofilament proteins and alter contractility [2-6]. We previously showed that the upstream AMPK kinase, LKB1/MO25/STRAD, impacts myofilament function independently of AMPK [5]. Therefore, we hypothesized that the LKB1 complex associated with myofilament proteins and that alterations in energy signaling modulated targeting or localization of the LKB1 complex to the myofilament. Using an integrated strategy of myofilament mechanics, immunoblot analysis, co-immunoprecipitation, mass spectroscopy, and immunofluorescence, we showed that 1) LKB1 and MO25 associated with myofibrillar proteins, 2) cellular energy stress re-distributed AMPK/LKB1 complex proteins within the sarcomere, and 3) the LKB1 complex localized to the Z-Disk and interacted with cytoskeletal and energy-regulating proteins, including vinculin and ATP Synthase (Complex V). These data represent a novel role for LKB1 complex proteins in myofilament function and myocellular “energy” sensing in the heart.
“…PKC can phosphorylate Ser-273 and Ser-302, CaMKII can phosphorylate Ser-273, Ser-282, Ser-302, Ser 307 [28] and PKD can phosphorylate Ser-302 [1]. RSK can phosphorylate Ser-282, GSK3 can phosphorylate Ser-302, and CK2 can phosphorylate Ser-282 [5, 18, 20]. However, the actual kinases that actively phosphorylate these sites in vivo remain obscure and it is not clear which kinases act upon the different residues or even if multiple kinases can phosphorylate a single residue in vivo.…”
Section: Cmybp-c Phosphorylation Is Functionally Importantmentioning
confidence: 99%
“…Two important pathways activated by adrenergic signaling have, as their major players, protein kinase C (PKC) and protein kinase A (PKA). Although there are other critical targets for both PKC and PKA in modulating contractility [31], it is well established that multiple contractile proteins are important substrates [32], and cMyBP-C contains residues that are phosphorylatable and are phosphorylated in vivo by PKC, PKA, PKD, CaMKII, CK2, GSK3β, and RSK [20, 1, 5, 10, 23]. Thus, cMyBP-C phosphorylation represents a target at the contractile apparatus level for adrenergic activation and potentially, activation by other signaling pathways (eg SUMOylation) as well.…”
Cardiac myosin binding protein C (cMyBP-C) is an integral sarcomeric protein that associates with the thick, thin and titin filament systems in the contractile apparatus. Three different isoforms of MyBP-C exist in mammalian muscle: slow skeletal (MYBPC1), fast skeletal (MyBP-C2, with several variants), and cardiac (cMyBP-C). Genetic screening studies show that mutations in MYBPC3 occur frequently and are responsible for as many as 30–35% of identified cases of familial hypertrophic cardiomyopathy. The function of cMyBP-C is stringently regulated by its post-translational modification. In particular, the addition of phosphate groups occurs with high frequency on certain serine residues that are located in the cardiac-specific regulatory M domain. Phosphorylation of this domain has been extensively studied in vitro and in vivo. Phosphorylation of the M domain can regulate the manner in which actin and myosin interact, affecting the cross bridge cycle and ultimately, cardiac hemodynamics.
“…5, 6 cMyBP-C is readily phosphorylated by a host of kinases such as protein kinase A (PKA) 7 , PKC 8 , PKD 9 , calcium calmodulin kinase IIδ (CAMK2δ) 10 , glycogen synthase kinase 3β (GSK3β) 11 and ribosomal S6 kinase (RSK6) 12 . Unphosphorylated cMyBP-C appears to repress both cross-bridge attachment and detachment.…”
Background
Heart failure with preserved ejection fraction (HFpEF) accounts for approximately 50% of all cases of heart failure and currently has no effective treatment. Diastolic dysfunction underlies HFpEF; therefore, elucidation of the mechanisms that mediate relaxation can provide new potential targets for treatment. Cardiac myosin binding protein-C (cMyBP-C) is a thick filament protein that modulates cross-bridge cycling rates via alterations in its phosphorylation status. Thus, we hypothesize that phosphorylated cMyBP-C accelerates rate of cross-bridge detachment, thereby enhancing relaxation to mediate diastolic function.
Methods and Results
We compared mouse models expressing phosphorylation deficient cMyBP-C(S273A/S282A/S302A)-cMyBP-C(t3SA), phosphomimetic cMyBP-C(S273D/S282D/S302D)-cMyBP-C(t3SD), and WT-control cMyBP-C(tWT) to elucidate the functional effects of cMyBP-C phosphorylation. Decreased voluntary running distances, increased lung/body weight ratios, and increased brain natriuretic peptide (BNP) levels in cMyBP-C(t3SA) mice demonstrate that phosphorylation deficiency is associated with signs of heart failure. Echocardiography (ejection fraction, myocardial relaxation velocity) and pressure/volume measurements (−dP/dtmin, pressure decay time constant Tau-Glantz, passive filling stiffness) show that cMyBP-C phosphorylation enhances myocardial relaxation in cMyBP-C(t3SD) mice while deficient cMyBP-C phosphorylation causes diastolic dysfunction with preserved ejection fraction in cMyBP-C(t3SA) mice. Simultaneous force and [Ca2+]i measurements on intact papillary muscles show that enhancement of relaxation in cMyBP-C(t3SD) mice and impairment of relaxation in cMyBP-C(t3SA) mice are not due to altered [Ca2+]i handling, implicating that altered cross-bridge detachment rates mediate these changes in relaxation rates.
Conclusions
cMyBP-C phosphorylation enhances relaxation while deficient phosphorylation causes diastolic dysfunction and phenotypes resembling HFpEF. Thus, cMyBP-C is a potential target for treatment of HFpEF.
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