Analysis of cardiac myosin binding protein-C phosphorylation in human heart muscle

A unique feature of MyBP-C in cardiac muscle is that it has multiple phosphorylation sites. MyBP-C phosphorylation, predominantly by PKA, plays an essential role in modulating contractility as part of the cellular response to β-adrenergic stimulation. In vitro studies indicate MyBP-C can be phosphorylated at Serine 273, 282, 302 and 307 (mouse sequence) but little is known about the level of MyBP-C phosphorylation or the sites phosphorylated in heart muscle. Since current methodologies are limited in specificity and are not quantitative we have investigated the use of phosphate affinity SDS-PAGE together with a total anti MyBP-C antibody and a range of phosphorylation site-specific antibodies for the main sites (Ser-273, -282 and -302). With these newly developed methods we have been able to make a detailed quantitative analysis of MyBP-C phosphorylation in heart tissue in situ. We have found that MyBP-C is highly phosphorylated in non-failing human (donor) heart or mouse heart; tris and tetra-phosphorylated species predominate and less than 10% of MyBP-C is unphosphorylated (0, 9.3 ± 1%: 1P, 13.4 ± 2.7%: 2P, 10.5 ± 3.3%: 3P, 28.7 ± 3.7%: 4P, 36.4 ± 2.7%, n = 21). Total phosphorylation was 2.7 ± 0.07 molPi/mol MyBP-C. In contrast in failing heart and in myectomy samples from HCM patients the majority of MyBP-C was unphosphorylated. Total phosphorylation levels were 23% of normal in failing heart myofibrils (0, 60.1 ± 2.8%: 1P, 27.8 ± 2.8%: 2P, 4.8 ± 2.0%: 3P, 3.7 ± 1.2%: 4P, 2.8 ± 1.3%, n = 19) and 39% of normal in myectomy samples. The site-specific antibodies showed a distinctive distribution pattern of phosphorylation sites in the multiple phosphorylation level species. We found that phosphorylated Ser-273, Ser-282 and Ser-302 were all present in the 4P band of MyBP-C but none of them were significant in the 1P band, indicating that there must be at least one other site of MyBP-C phosphorylation in human heart. The pattern of phosphorylation at the three sites was not random, but indicated positive and negative interactions between the three sites. Phosphorylation at Ser-282 was not proportional to the number of sites available. The 2P band contained 302 but not 273; the 3P band contained 273 but not 302.     

Cardiac myosin binding protein-C restricts intrafilament torsional dynamics of actin in a phosphorylation-dependent manner

We have determined the effects of myosin binding protein-C (MyBP-C) and its domains on the microsecond rotational dynamics of actin, detected by time-resolved phosphorescence anisotropy (TPA). MyBP-C is a multidomain modulator of striated muscle contraction, interacting with myosin, titin, and possibly actin. Cardiac and slow skeletal MyBP-C are known substrates for protein kinase-A (PKA), and phosphorylation of the cardiac isoform alters contractile properties and myofilament structure. To determine the effects of MyBP-C on actin structural dynamics, we labeled actin at C374 with a phosphorescent dye and performed TPA experiments. The interaction of all three MyBP-C isoforms with actin increased the final anisotropy of the TPA decay, indicating restriction of the amplitude of actin torsional flexibility by 15–20° at saturation of the TPA effect. PKA phosphorylation of slow skeletal and cardiac MyBP-C relieved the restriction of torsional amplitude but also decreased the rate of torsional motion. In the case of fast skeletal MyBP-C, its effect on actin dynamics was unchanged by phosphorylation. The isolated C-terminal half of cardiac MyBP-C (C5–C10) had effects similar to those of the full-length protein, and it bound actin more tightly than the N-terminal half (C0–C4), which had smaller effects on actin dynamics that were independent of PKA phosphorylation. We propose that these MyBP-C-induced changes in actin dynamics play a role in the functional effects of MyBP-C on the actin–myosin interaction.     

心脏型肌球蛋白结合蛋白C基因c.2469-3_-4insAG和c.2469-5_-6insT突变与肥厚型心肌病的关系

目的 研究中国人群肥厚型心肌病(Hypertrophic cardiomyopathy,HCM)患者的致病基因突变位点,为遗传咨询提供证据。方法 对HCM先证者行26个HCM相关基因全部外显子及邻近区靶向高通量测序,对基因突变家系成员和80名健康志愿者行Sanger测序,以验证基因突变位点。采集分析HCM患者及其家系成员临床症状、体征、超声心动图、心电图等信息。结果 该家系两名成员的心脏型肌球蛋白结合蛋白C基因（cardiac myosin binding protein-C3,MYBPC3）内含子区域中均同时携带c.2469-3_-4insAG和c.2469-5_-6insT两个插入突变,该家系其余成员及80名健康志愿者中未检出异常突变基因。两名基因突变携带者均为HCM患者,且发病年龄晚,有心慌、胸闷症状,心脏超声提示室间隔肥厚。结论 基因突变功能预测,提示MYBPC3 c.2469-3_-4insAG和c.2469-5_-6insT基因突变可引起蛋白特性及剪接位点的改变,可能是家族性肥厚型心肌病的致病突变位点。     

Cardiac myosin activation part 1: from concept to clinic

Decreased cardiac contractility is a central feature of systolic heart failure and yet we have no effective drugs to improve cardiac contractility. Existing drugs that increase cardiac contractility do so indirectly through signaling cascades and their use is limited by their mechanism-related adverse effects. Direct activation of the cardiac sarcomere to increase cardiac contractility may provide a means to avoid these limitations. Using a reconstituted version of the cardiac sarcomere, we screened a small molecule library and identified several chemical classes that directly activate cardiac myosin. One compound class has been optimized extensively using an iterative process; omecamtiv mecarbil, a small-molecule, selective, cardiac myosin activator is the most advanced exemplar of this novel mechanistic class. It accelerates the transition of myosin into the force-generating state without affecting cardiac myocyte calcium homeostasis. In animal models, omecamtiv mecarbil increases cardiac function by increasing the duration of ejection without changing the rates of contraction. Initial clinical studies have demonstrated the translation of this mechanism into humans, and further clinical studies of its use in acute and chronic heart failure are planned. Cardiac myosin activation may provide a new therapeutic approach for systolic heart failure. This article is part of a special issue entitled “Key Signaling Molecules in Hypertrophy and Heart Failure.”     

Chronological effects of mild pressure overload on myosin ATPase activity in the canine right ventricle.

Mild pulmonic stenosis in the dog, performed by pulmonary artery banding, caused an increasing elevation in myosin ATPase activity which peaked in enzymatic V_max values by 5 weeks after surgery (35% above normal) and then steadily declined; activity fell 11% below normal by 16 weeks after operation. During the postoperative period there was a consistent elevation in right ventricular peak systolic pressure, which remained approximately 60% above that of normal animals. The free wall of the right ventricle reached maximum size in wet weight by 5 weeks after pulmonic banding.     

Effects of severe hemodynamic pressure overload on the properties of canine left ventricular myosin: mechanism by which myosin ATPase activity is lowered during chronic increased hemodynamic stress.

Left ventricular myosin ATPase activity, expressed as enzymatic V_max values, was analyzed in dogs subjected to severe left ventricular pressure overload (aortic stenosis). K⁺ and Ca²⁺ activated myosin ATPase activities in the left ventricle (LV) were significantly depressed (P < .01) in the experimental animals. For normal K⁺ activated myosin the V_max values in micromoles of Pi per mg per min were: right ventricle 2.10; left ventricle, 2.84. For Ca²⁺ activated myosin the V_max values were: right ventricle, 0.77; left ventricle 0.97, when assayed at 37°C. Myosin enzymatic activity in the left ventricle progressively declined following severe aortic banding, reaching a value similar to that observed for normal right ventricular myosin; NH₄⁺ activated left ventricular myosin ATPase activity remained unchanged (7.20 ± 0.4 μmol PO₄/mg.min). Left ventricular myosin from the hearts subject to severe stress simulated normal right ventricular myosin in ATPase activity, chain proportions and degree of calcium binding, Normal left ventricular myosin contained approximately 10% of the myosin protein concentration in the light chains; myosin from the left ventricles of the hemodynamically overloaded hearts contained 20% of the myosin protein concentration in the light chains (P < .001). With only one of the myosin light chains binding calcium left ventricular myosin from the stressed hypertrophied tissue bound approximately 2 mol Ca²⁺ mol^?1 myosin similar to myosin of the normal right ventricle; normal left ventricular myosin bound approximately 1 mol of Ca²⁺ mol^?1 myosin.     

Phosphorylation and function of cardiac myosin binding protein-C in health and disease

During the past 5 years there has been an increasing body of literature describing the roles cardiac myosin binding protein C (cMyBP-C) phosphorylation play in regulating cardiac function and heart failure. cMyBP-C is a sarcomeric thick filament protein that interacts with titin, myosin and actin to regulate sarcomeric assembly, structure and function. Elucidating the function of cMyBP-C is clinically important because mutations in this protein have been linked to cardiomyopathy in more than sixty million people worldwide. One function of cMyBP-C is to regulate cross-bridge formation through dynamic phosphorylation by protein kinase A, protein kinase C and Ca²⁺-calmodulin-activated kinase II, suggesting that cMyBP-C phosphorylation serves as a highly coordinated point of contractile regulation. Moreover, dephosphorylation of cMyBP-C, which accelerates its degradation, has been shown to associate with the development of heart failure in mouse models and in humans. Strikingly, cMyBP-C phosphorylation presents a potential target for therapeutic development as protection against ischemic-reperfusion injury, which has been demonstrated in mouse hearts. Also, emerging evidence suggests that cMyBP-C has the potential to be used as a biomarker for diagnosing myocardial infarction. Although many aspects of cMyBP-C phosphorylation and function remain poorly understood, cMyBP-C and its phosphorylation states have significant promise as a target for therapy and for providing a better understanding of the mechanics of heart function during health and disease. In this review we discuss the most recent findings with respect to cMyBP-C phosphorylation and function and determine potential future directions to better understand the functional role of cMyBP-C and phosphorylation in sarcomeric structure, myocardial contractility and cardioprotection.     

T445S:肥厚型心肌病的一个新的基因突变位点

目的调查中国人群肥厚型心肌病(HCM)患者心肌肌球蛋白结合蛋白C(MyBPC3)基因突变的发生情况并对基因型与表现型之间的关系进行分析。方法对92例肥厚型心肌病患者及100名正常对照的聚合酶链反应(PCR)扩增产物进行单链构象多态性分析(SSCP)分析,于MyBPC3基因第14-15、17、25、27、33号外显子范围内寻找突变位点,并了解基因型明确的HCM患者的临床特点。结果在一例HCM患者的MyBPC3基因的第14-15号外显子上发现了一个新的突变位点T445S。由于在100名正常对照中未见该错义突变,故我们认为此突变位点为该患者的致病基因位点。结论心肌肌球蛋白结合蛋白C的C3结构域对于其连接及调节功能起着至关重要的作用,该区域的基因突变可导致肥厚型心肌病的发生。     

Cardiac myosin binding protein C regulates postnatal myocyte cytokinesis

Homozygous cardiac myosin binding protein C-deficient (Mybpc^t/t) mice develop dramatic cardiac dilation shortly after birth; heart size increases almost twofold. We have investigated the mechanism of cardiac enlargement in these hearts. Throughout embryogenesis myocytes undergo cell division while maintaining the capacity to pump blood by rapidly disassembling and reforming myofibrillar components of the sarcomere throughout cell cycle progression. Shortly after birth, myocyte cell division ceases. Cardiac MYBPC is a thick filament protein that regulates sarcomere organization and rigidity. We demonstrate that many Mybpc^t/t myocytes undergo an additional round of cell division within 10 d postbirth compared with their wild-type counterparts, leading to increased numbers of mononuclear myocytes. Short-hairpin RNA knockdown of Mybpc3 mRNA in wild-type mice similarly extended the postnatal window of myocyte proliferation. However, adult Mybpc^t/t myocytes are unable to fully regenerate the myocardium after injury. MYBPC has unexpected inhibitory functions during postnatal myocyte cytokinesis and cell cycle progression. We suggest that human patients with homozygous MYBPC3-null mutations develop dilated cardiomyopathy, coupled with myocyte hyperplasia (increased cell number), as observed in Mybpc^t/t mice. Human patients, with heterozygous truncating MYBPC3 mutations, like mice with similar mutations, have hypertrophic cardiomyopathy. However, the mechanism leading to hypertrophic cardiomyopathy in heterozygous MYBPC3^+/− individuals is myocyte hypertrophy (increased cell size), whereas the mechanism leading to cardiac dilation in homozygous Mybpc3^−/− mice is primarily myocyte hyperplasia.Dilated cardiomyopathy (DCM) leads to heart failure and is a leading cause of morbidity and mortality (1, 2). DCM is generally diagnosed as left ventricular (LV) dilation with associated reduction in cardiac contraction measured as impaired fractional shortening (3). Hearts from affected individuals frequently demonstrate myocyte elongation, myocyte death, and fibrosis, in addition to LV dilation. DCM results from a variety of environmental factors, such as viral infection and alcohol abuse, as well as from mutations in a number of genes including titin, lamin A/C, cardiac actin, cardiac myosin heavy chain, and phospholamban (reviewed in refs. 4–6). Whether all of these DCM-inducing factors activate the same or different cellular pathways to produce similar clinical features remains uncertain. The mechanisms by which mutations in the cardiac myosin binding protein C (MYBPC3) gene and other sarcomere protein genes lead to cardiac dilatation are under investigation.MYBPC is a thick filament accessory protein component of the striated muscle sarcomere A band that constitutes 2–4% of the myofibril (discussed in ref. 7). Although there are four Mybpc genes in the mammalian genome, only cardiac Mybpc (Mybpc3) is expressed in embryonic, neonatal, and adult hearts (8, 9). Cardiac MYBPC interacts with at least four sarcomere components: myosin heavy chain, actin, myosin light chain 2, and titin (10–12). More than 400 cardiac MYBPC3 gene mutations have been identified in patients as a cause of hypertrophic cardiomyopathy (HCM), an autosomal dominant disorder resulting from defective sarcomeres (for reviews, see refs. 12, 13). Due to an ancient founder mutation, 4% of the population of India carries a truncating MYBPC3 mutation (14, 15). The majority of cardiac MYBPC3 mutations are predicted to encode truncated proteins that lack portions of either the carboxyl myosin and/or titin binding domains (7, 13). These truncating MYBPC3 mutations are thought to cause cardiac hypertrophy by inducing myocyte hypertrophy (increased cell size), rather than myocyte hyperplasia.We and other researchers have created mice that carry a mutant cardiac Mybpc3 gene to create murine HCM models (16–18). Heterozygous mice, designated Mybpc^t/+, like humans bearing the same mutation, develop adult onset HCM. Homozygous MYBPC3 mutations are a much rarer cause of human DCM than autosomal dominant mutations in other sarcomere protein genes. However, homozygous Mybpc^t/t mice that express two mutant alleles and no wild-type cardiac Mybpc3 develop LV dilation by 3 d postbirth and have all of the features of DCM, including LV chamber dilation, albeit mildly impaired fractional shortening (16). Unlike most humans with DCM, homozygous mutant cardiac Mybpc^t/t mice have normal survival despite their cardiac disease. Other homozygous null cardiac Mybpc3 mice develop an identical phenotype (7, 17, 18). Hence, for the studies described here, we assume that the phenotype of the Mybpc^t/t mice is due to lack of MYBPC protein, rather than to small amounts of truncated protein. Recently, two groups have demonstrated that delivery of MYBPC to Mybpc3-null hearts restores cardiac function and morphology (19, 20). Here, we have begun to dissect the mechanism by which homozygous Mybpc^t/t hearts develop DCM.Because Mybpc^t/t mice begin LV dilation within a few days postbirth (16), we hypothesized that this reflected abnormal development of neonatal myocytes. During fetal and early perinatal development in wild-type hearts, cardiomyocytes divide rapidly, producing hyperplastic cardiac growth (21). However, at 10 d postbirth, cardiomyocytes cease to divide and all subsequent increases in myocardial mass result from myocyte hypertrophy (22). Despite the importance of this phenomenon, little is known about the molecular basis for the transition from hyperplasic to hypertrophic-based myocardial growth. We hypothesized that abnormal cardiomyocyte growth, either hyperplastic or hypertrophic, in the perinatal period accounted for the LV dilation of Mybpc^t/t mouse hearts. To address this question, we have counted and measured cardiomyocytes from Mybpc^t/t and wild-type mice. We have also studied the consequences of reducing MYBPC levels by injecting Mybpc3-specific shRNA at birth. Neonatal cardiomyocytes lacking cardiac MYBPC, due to Mybpc3-specific shRNA knockdown, undergo an additional round of cytokinesis. We conclude that dramatic reductions in the amount of cardiac MYBPC leads to aberrant cell cycle regulation at the G1/S checkpoint, resulting in at least one extra round of myocyte division and DCM.     

Thick Filament Proteins and Performance in Human Heart Failure

Modifications in thick filament protein content and performance are thought to underlie contraction-relaxation dysfunction in human heart failure. It has been found that myofibrillar Mg.ATPase is reduced in failing myocardium, which may be due in part to the reduction in α-myosin heavy chain (MHC) isoform content from ∼5–10% in normal myocardium to <2% in failing myocardium. The physiological importance of this seemingly small amount of α-MHC appears substantiated by the development of cardiopathologies in humans with mutated α-MHC at normal abundance. Therefore, the replacement of α-MHC by β-MHC (possessing slower actomyosin enzymatic kinetics) may underlie to a significant degree the reduced myocardial shortening velocity and reduced relaxation function in human heart failure. The atrial isoform of myosin essential light chain (ELC) may replace up to 25% of the ventricular isoform in failing ventricles and in so doing promotes myocardial shortening velocity. An elevated accumulation of the higher performing atrial-ELC, unlike the reduced content of the higher performing α-MHC, is therefore considered a compensatory response in heart failure. Phosphorylation of the myofilament proteins myosin regulatory light chain and troponin-I are both reduced in heart failure and collectively result in an elevated myofilament sensitivity to calcium activation, which inhibits relaxation function. These and other modifications in thick filament proteins, as discussed in this review, directly affect mechanical power output and relaxation function of the myocardium and thereby may be considered to cause or in some cases to compensate for the otherwise ineffective myocardial performance in heart failure.     

Decreased phosphorylation levels of cardiac myosin-binding protein-C in human and experimental heart failure

Cardiac myosin-binding protein-C (cMyBP-C) is an important regulator of cardiac contractility, and its phosphorylation by PKA is a mechanism that contributes to increased cardiac output in response to beta-adrenergic stimulation. It is presently unknown whether heart failure alters cMyBP-C phosphorylation. The present study determined the level of phosphorylated cMyBP-C in failing human hearts and in a canine model of pacing-induced heart failure. A polyclonal antibody directed against the major phosphorylation site of cMyBP-C (Ser-282) was generated and its specificity was confirmed by PKA phosphorylation with isoprenaline in cardiomyocytes and Langendorff-perfused mouse hearts. Left ventricular myocardial tissue from (i) patients with terminal heart failure (hHF; n=12) and nonfailing donor hearts (hNF; n=6) and (ii) dogs with rapid-pacing-induced end-stage heart failure (dHF; n=10) and sham-operated controls (dNF; n=10) were used for quantification of total cMyBP-C and phospho-cMyBP-C by Western blotting. Total cMyBP-C protein levels were similar in hHF and hNF as well as in dHF and dNF. In contrast, the ratio of phospho-cMyBP-C to total cMyBP-C levels were >50% reduced in hHF and >40% reduced in dHF. In summary, cMyBP-C phosphorylation levels are markedly decreased in human and experimental heart failure. Thus, the compromised contractile function of the failing heart might be in part attributable to reduced cMyBP-C phosphorylation levels.     

Unique single molecule binding of cardiac myosin binding protein-C to actin and phosphorylation-dependent inhibition of actomyosin motility requires 17 amino acids of the motif domain

Cardiac myosin binding protein-C (cMyBP-C) has 11 immunoglobulin or fibronectin-like domains, C0 through C10, which bind sarcomeric proteins, including titin, myosin and actin. Using bacterial expressed mouse N-terminal fragments (C0 through C3) in an in vitro motility assay of myosin-generated actin movement and the laser trap assay to assess single molecule actin-binding capacity, we determined that the first N-terminal 17 amino acids of the cMyBP-C motif (the linker between C1 and C2) contain a strong, stereospecific actin-binding site that depends on positive charge due to a cluster of arginines. Phosphorylation of 4 serines within the motif decreases the fragments' actin-binding capacity and actomyosin inhibition. Using the laser trap assay, we observed individual cMyBP-C fragments transiently binding to a single actin filament with both short (~20 ms) and long (~300 ms) attached lifetimes, similar to that of a known actin-binding protein, α-actinin. These experiments suggest that cMyBP-C N-terminal domains containing the cMyBP-C motif tether actin filaments and provide one mechanism by which cMyBP-C modulates actomyosin motion generation, i.e. by imposing an effective viscous load within the sarcomere.     

Calmodulin regulates dimerization,motility, and lipid binding of Leishmania myosin XXI

Myosin XXI is the only myosin expressed in Leishmania parasites. Although it is assumed that it performs a variety of motile functions, the motor’s oligomerization states, cargo-binding, and motility are unknown. Here we show that binding of a single calmodulin causes the motor to adopt a monomeric state and to move actin filaments. In the absence of calmodulin, nonmotile dimers that cross-linked actin filaments were formed. Unexpectedly, structural analysis revealed that the dimerization domains include the calmodulin-binding neck region, essential for the generation of force and movement in myosins. Furthermore, monomeric myosin XXI bound to mixed liposomes, whereas the dimers did not. Lipid-binding sections overlapped with the dimerization domains, but also included a phox-homology domain in the converter region. We propose a mechanism of myosin regulation where dimerization, motility, and lipid binding are regulated by calmodulin. Although myosin-XXI dimers might act as nonmotile actin cross-linkers, the calmodulin-binding monomers might transport lipid cargo in the parasite.Over 12 million people worldwide are affected by leishmaniasis, which is caused by the flagellated protozoan parasite Leishmania. The disease manifests itself in a cutaneous form, which leaves disfiguring scars, or as visceral leishmaniasis, which is potentially fatal (1). The parasite has a life cycle in two stages. The elongated, flagellated, and motile promastigotes are found in the gut of the sand fly host. The egg-shaped, nonmotile amastigotes possess only a rudimentary flagellum, are found in mammalian macrophages, and are responsible for the human disease pathology (2, 3). In contrast to other eukaryotic cells that express at least 11 different isoforms (4), Leishmania donovani seems to express only a single isoform, myosin XXI (5). A later classification assigned myosin XXI to class XIII, a kinetoplastide-specific class of myosins (6). Previous attempts to identify and localize myosin Ib in L. donovani parasites using anti-Leishmania myosin Ib antibodies were unsuccessful (3). Intriguingly, only myosin XXI was shown to be expressed in both the motile promastigote and the nonmotile amastigote stages of the parasite’s life cycle (3). In the promastigote stage, this motor preferentially localized to the proximal part of the flagellum, although it was also found along the entire length of the flagellum and in other cell-body compartments (7). The myosin-XXI homozygous knockout is lethal. The heterozygous cells were unable to form the paraflagellar rod, a structure of unknown function that runs along the length of the flagellum and contains a variety of proteins including actin and myosin XXI (8). It has also been reported that reduced expression levels cause the loss of endocytosis within the flagellar pocket and affect other intracellular trafficking processes (7). This makes myosin XXI an intriguing candidate to study modes of structural adaptation of a single myosin isoform for a variety of cellular acto-myosin–based motile functions. Two distinct cellular fractions of myosin XXI have already been identified: a membrane-bound fraction that localized to the base of the flagellum and a cytosolic fraction possibly involved in transporting proteins within the flagellum (3). However, it is unknown in which way the motor is targeted to different cargo, which oligomerization states it can adopt, and how transitions between different functional states are regulated.The design of myosin XXI follows the general structure of myosin motors, which comprises a conserved N-terminal motor domain, followed by a neck region including IQ motifs for the binding of light chains of the calmodulin family, and finally a C-terminal cargo-binding tail domain (9) (Fig. 1A). Although the motor domain is responsible for the binding to actin and hydrolysis of ATP, it is usually the tail domain that determines its function within the cell by controlling dimerization or oligomerization, motor anchoring to membrane compartments, and selection and transport of specific cargo. The neck region, mechanically stabilized by binding calmodulins, is thought to serve as a lever arm to transduce force and movement to the cargo. In a previous study we identified six potential calmodulin-binding IQ motifs outside the motor domain of myosin XXI. The data suggested that only the motif closest to the motor domain bound Xenopus/human calcium-calmodulin (10). Intriguingly, sequence analysis indicated a coiled-coil domain (11, 12) with a strong propensity to dimerize in between the end of the motor domain and what we initially thought to be the first calmodulin-binding IQ motif. Closer inspection of this predicted coiled-coil domain, however, suggested a further potential calmodulin-binding IQ motif within the predicted dimerization site and close to the converter (yellow, Fig. 1A). This suggested that this myosin might be able to dimerize, but in doing so would lose its mechanically essential lever arm structure to the formation of a coiled coil and, as a consequence, make a transition from a motile monomeric form to a nonmotile dimer. Consistent with this, we found in a previous study that calcium-calmodulin binding was not required for the ATPase activity of the motor, but for myosin XXI in vitro motility (10).Open in a separate windowFig. 1.Calmodulin binding prevents the dimerization of myosin XXI. (A) Domain structure of full-length myosin XXI. By comparing the sequence with well-studied myosins, we identified the highly conserved regions of the motor domain (gray). In addition to the calmodulin-binding IQ motifs identified in a previous study (10), we found another IQ motif (yellow) immediately following the converter domain. Sequence analysis revealed four potential coiled-coil regions (in blue). The predicted propensity to oligomerize was scored between 0 and 10 using Scorer 2.0 (11); positive numbers predict dimer and negative numbers predict trimer formation. (B) Size exclusion chromatography using a Superose-6 column and silver staining of SDS/PAGE gels (C) of the elution fractions indicate that the motor is monomeric when expressed in high levels of calmodulin and dimeric or in a higher oligomeric state when expressed at low levels of calmodulin. Aggregated myosin XXI would appear in the void volume. (D) The Western blot does not resolve endogenous calmodulin in the purified myosin-XXI preparations that were expressed in the absence of added calmodulin virus (i.e., at low levels of calmodulin). By varying the amount of calmodulin virus added during expression, the amount of calmodulin bound to the expressed myosin XXI could be controlled. The apparent difference in molecular weight of calmodulin in C and D is due to the differences in buffers.To investigate the mechanism of dimerization and the mechanical properties of myosin XXI, we expressed L. donovani full-length myosin XXI, a truncated minimal motor domain, and a series of tail constructs. We also expressed L. donovani-specific calmodulin-like (CamL) proteins to characterize calcium-dependent binding to target peptides on the myosin-XXI heavy chain. Using size exclusion chromatography (SEC), analytical ultracentrifugation, Förster resonance energy transfer (FRET), motility assays, and electron microscopy (EM), we studied the effect of calmodulin binding on motor dimerization and motility. We used liposome pull-down experiments and phospholipid blot assays combined with sequence analysis to identify specific phospholipid-binding motifs in the head, neck, and tail domain of the motor. The data suggest a form of myosin regulation where dimerization, motility, and phospholipid binding of the motor are determined by binding of calmodulin.     

Myosin binding protein-C activates thin filaments and inhibits thick filaments in heart muscle cells

Myosin binding protein-C (MyBP-C) is a key regulatory protein in heart muscle, and mutations in the MYBPC3 gene are frequently associated with cardiomyopathy. However, the mechanism of action of MyBP-C remains poorly understood, and both activating and inhibitory effects of MyBP-C on contractility have been reported. To clarify the function of the regulatory N-terminal domains of MyBP-C, we determined their effects on the structure of thick (myosin-containing) and thin (actin-containing) filaments in intact sarcomeres of heart muscle. We used fluorescent probes on troponin C in the thin filaments and on myosin regulatory light chain in the thick filaments to monitor structural changes associated with activation of demembranated trabeculae from rat ventricle by the C1mC2 region of rat MyBP-C. C1mC2 induced larger structural changes in thin filaments than calcium activation, and these were still present when active force was blocked with blebbistatin, showing that C1mC2 directly activates the thin filaments. In contrast, structural changes in thick filaments induced by C1mC2 were smaller than those associated with calcium activation and were abolished or reversed by blebbistatin. Low concentrations of C1mC2 did not affect resting force but increased calcium sensitivity and reduced cooperativity of force and structural changes in both thin and thick filaments. These results show that the N-terminal region of MyBP-C stabilizes the ON state of thin filaments and the OFF state of thick filaments and lead to a novel hypothesis for the physiological role of MyBP-C in the regulation of cardiac contractility.Muscle contraction is driven by the relative sliding of the actin-containing thin filaments along the myosin-containing thick filaments arranged in a parallel array in the muscle sarcomere (Fig. 1A). Filament sliding in turn is driven by a structural change in the myosin head domains (Fig. 1B) while they are bound to actin, coupled to the hydrolysis of ATP (1). Contraction of skeletal and cardiac muscle is triggered by calcium binding to troponin in the thin filaments, accompanied by a change in the structure of the thin filaments that permits myosin head binding (2). However, the strength and dynamics of contraction are modulated by posttranslational modifications in other sarcomeric proteins, including the myosin regulatory light chain (RLC) (3), which is part of the myosin head, and myosin binding protein-C (4–6) (MyBP-C) (Fig. 1B). In an emerging concept of thick filament regulation in striated muscle that is analogous to myosin-linked regulation in smooth muscle (7–11), RLC and MyBP-C are thought to modulate contraction by controlling the conformation of the myosin heads.Open in a separate windowFig. 1.Sarcomere location and domain architecture of MyBP-C. (A) C-zone (green) of the thick filament in relation to its proximal (P) and distal (D) regions and the thin filament (gray). (B) Cartoon representation of MyBP-C (green) anchored to the thick filament backbone (purple) via its C-terminal domains; myosin heads are pink and troponin is yellow. (C) Domain organization and interactions of MyBP-C.According to this concept, the thick filament has an OFF state in which the myosin heads are folded back against its surface (Fig. 1B), rendering them unavailable for interaction with actin, and an ON state in which the heads are released from the thick filament surface and made available for actin binding. The physiological and pathological significance of thick filament regulation and its relationship to the well-studied thin filament mechanisms remain poorly understood, but much recent attention has focused on MyBP-C for two main reasons. First, mutations in the cardiac MYBPC3 gene are commonly associated with hypertrophic cardiomyopathy (12, 13), and this association has driven a wide range of studies at the molecular, cellular, and whole-animal levels aimed at understanding the etiology of MYBPC3-linked disease. Second, although MyBP-C is a constitutive component of the thick filament, there is a large body of evidence that it can also bind the thin filaments (14, 15), raising the possibility that one role of MyBP-C may be to synchronize the regulatory states of the thin and thick filaments (11, 15–17).MyBP-C is localized to the central region or “C-zone” of each half-thick filament (Fig. 1A), appearing in nine transverse stripes with a 43-nm periodicity closely matching that of the myosin heads (Fig. 1B) (10). MyBP-C has 11 Ig-like or fibronectin-like domains (Fig. 1C) denoted C0–C10, with additional linking sequences, notably the MyBP-C “motif” or “m” domain between C1 and C2 and the proline/alanine-rich (P/A) linker between C0 and C1. The m domain has multiple phosphorylation sites (4–6). Constitutive binding to the thick filament is mediated by interactions of domains C8–C10 with myosin and titin. The C1mC2 region binds to the coiled-coil subfragment-2 (S2) domain of myosin adjacent to the myosin heads, and this interaction is abolished by MyBP-C phosphorylation (5); the C0 domain binds to the RLC in the myosin head itself (18). The N-terminal domains of MyBP-C also bind to actin in a phosphorylation-dependent manner (14, 15) (Fig. 1B), and EM and X-ray studies on intact sarcomeres of skeletal muscle suggest that MyBP-C binds to thin filaments under relaxing conditions (10, 11).The function of MyBP-C and the mechanisms underlying its modulation in cardiomyopathy remain poorly understood, however. Ablation of MyBP-C in a knockout mouse model leads to a hypertrophic phenotype associated with impaired contractile function (19), but cardiomyocytes isolated from these mice exhibit increased power output during working contractions (20). A range of studies at the isolated protein and cellular levels have led to the concept that MyBP-C exerts a predominantly inhibitory effect on contractility mediated through two distinct mechanisms (15, 16, 21). MyBP-C may tether myosin heads to the surface of the thick filament, preventing their interaction with actin, and its N terminus may bind to thin filaments, inhibiting interfilament sliding at low load. Other studies, however, have demonstrated an activating effect of MyBP-C mediated by binding of its N-terminal domains to the thin filament. N-terminal fragments of MyBP-C enhance force production in skinned cardiac muscle cells and motility in isolated filament preparations at zero or submaximal calcium concentrations (22–25). The same effect is observed in cardiomyocytes from MyBP-C knockout mice (22), suggesting that the activating effect is not due to competitive removal of an inhibitory effect of native MyBP-C.To resolve these apparently contradictory hypotheses about the physiological function of the N-terminal domains of MyBP-C, we determined the structural changes in the thick and thin filaments of intact sarcomeres in heart muscle cells induced by N-terminal MyBP-C fragments using bifunctional rhodamine probes on RLC and troponin C (TnC) (26). These probes allowed the structural changes in both types of filament to be directly compared with those associated with calcium activation and myosin head binding in the native environment of the cardiac muscle sarcomere. The results lead to a model for the physiological function of MyBP-C that integrates the regulatory roles of the thin and thick filaments and the inhibitory and activating effects of MyBP-C at the level of the intact sarcomere.     

Myosin binding protein C phosphorylation in normal, hypertrophic and failing human heart muscle

Phosphorylation of myosin binding protein C (MyBP-C) was investigated in intraventricular septum samples taken from patients with hypertrophic cardiomyopathy undergoing surgical septal myectomy. These samples were compared with donor heart muscle, as a well-characterised control tissue, and with end-stage failing heart muscle. MyBP-C was partly purified from myofibrils using a modification of the phosphate-EDTA extraction of Hartzell and Glass. MyBP-C was separated by SDS-PAGE and stained for phosphoproteins using Pro-Q Diamond followed by total protein staining using Coomassie Blue. Relative phosphorylation level was determined from the ratio of Pro-Q Diamond to Coomassie Blue staining of MyBP-C bands as measured by densitometry. We compared 9 myectomy samples and 9 failing heart samples with 9 donor samples. MyBP-C phosphorylation in pathological muscle was lower than in donor (myectomy 40 ± 2% of donor, P < 0.0001; failing 45 ± 3% of donor, P < 0.0001). 6 myectomy samples were identified with MYBPC3 mutations, one with MYH7 mutation and two remained unknown, but there was no correlation between MYBPC3 mutation and MyBP-C phosphorylation level.In order to determine the number of phosphorylated sites in human cardiac MyBP-C samples, we phosphorylated the recombinant MyBP-C fragment, C0–C2 (1–453) with PKA using ^γ32P-ATP up to 3.5 mol Pi/mol C0–C2. This measurement of phosphorylation was used to calibrate measurements of phosphorylation in SDS-PAGE using Pro-Q Diamond stain. The level of phosphorylation in donor heart MyBP-C was calculated to be 4.6 ± 0.6 mol Pi/mol and 2.0 ± 0.3 mol Pi/mol in myectomy samples.We conclude that MyBP-C is a highly phosphorylated protein in vivo and that diminished MyBP-C phosphorylation is a feature of both end-stage heart failure and hypertrophic cardiomyopathy.     

Regional variations in myosin heavy chain concentration after healing of experimental myocardial infarction in cats

Densitometric scanning of SDS-polyacrylamide gels was used to measure myosin heavy chain concentration in left ventricular specimens obtained from cat hearts 3 to 12 months after healing of small experimental myocardial infarctions. The study was designed to test the hypothesis that myosin concentration varies as a function of anatomic proximity to the infarct scar. Myosin heavy chain concentration was elevated in non-scarred areas adjacent to a healed infarct and normal in areas remote from the scar. The scar itself had reduced concentrations, reflecting the loss of muscle mass in this area. The increased myosin heavy chain concentration in regions adjacent to the scar may be an attempt to regulate or compensate for the decrease in mechanical function of the scarred area.     

Alterations of subunit composition and ATPase activity of myosin in early hypertrophied right ventricles of dogs with mild experimental pulmonic stenosis.

Mild pulmonic stenosis was performed in dogs by banding of the pulmonary artery to evaluate the effect of systolic pressure overload on the enzymatic activity and subunit composition of myosin in early hypertrophied right ventricles. Three weeks following pulmonary constriction, 12 hypertrophied dogs were sacrificed and compared to 12 control animals. The weight of the hypertrophied right ventricles (HRV) relative to body weight was 46% greater than the weight of normal right ventricles (NRV) (P < .001) in dogs of similar body weights. Myosin ATPase activity (V_max values in μmol phosphate/mg min) was 25% higher in the stressed ventricles for both K⁺ and Ca²⁺ activated myosin (P < .001). Since the V_max values for the enzymatic activity of myosin with NH₄⁺ or Mn²⁺, as the activator cations, was the same in HRV and NRV, the augmented K⁺ and Ca²⁺ activity in HRV was not due to an increased concentration of more enzymatically active myosin. Associated with the increase in myosin activity there was a 33% decrease in the percent of light chains present in myosin from HRV as compared to myosin from NRV (P < .001). There was approximately 4 mol of myosin light chains/mol of myosin in NRV; in contrast, there was approximately 2 mol of myosin light chains/mole of myosin in HRV, similar to the proportion observed in NLV. The proportion of light chain C₁ to light chain C₂ did not change in myosin from HRV. Of the C₁ light chains analyzed on 2-dimensional gel electrophoresis, there was significantly less C_1d as compared to C_1c in HRV.     

Dissociation of myosin light chains and decreased myosin ATPase activity with acidification of synthetic myosin filaments: Possible clues to the fate of myosin in myocardial ischemia and infarction

The effect of mild acidification of synthetic (reconstituted) myosin filaments was studied in order to gain insight into some of the possible effects of ischemia-induced intracellular acidosis on the structure and function of myosin following myocardial infarction and myocardial ischemia. Degradation products of myosin that are soluble (at physiologic ionic strength and pH) would be of potential diagnostic value for myocardial infarction. Acidification of rabbit skeletal synthetic myosin filaments led to a pH dependent partial dissociation of the heaviest (LC₁) and lightest (LC₃) of the 3 light chains. Dissociation was detected from pH 5.0 to 6.5 and was maximal at pH 6.0, at which 30% of LC₁ was dissociated. Acidification of canine cardiac synthetic myosin filaments led to partial dissociation of both light chains; but more LC₁ than LC₂ was dissociated. Light chains reassociated with heavy chains upon return of the pH to 7. Light chains of myosin have recently been reported to appear in the peripheral blood after myocardial infarction but the small amount of free light chains in the heart is insufficient to account for the amount that appears in the blood. Acid-mediated dissociation of light chains in vitro suggests that circulating light chains after myocardial infarction may arise as a result of the intracellular acidosis of ischemic myocytes. The mechanisms responsible for the acidification-induced decrease in myofibrillar actomyosin adenosine triphosphatase (ATPase) activity are unclear. One possibility is that the decreased myofibrillar ATPase activity is due in part to an acid-induced decrease of the myosin ATPase of the myofibril irrespective of the effect of acid on the troponin-tropomyosin regulatory system. This possible mechanism is supported by the observations that acidification of rabbit skeletal and human and canine cardiac synthetic myosin filaments resulted in a reduction of ATPase activity (measured at pH 7.5) of the redissolved myosin which was progressive with greater acidification. The reduction in ATPase activity occurred whether the return of the myosin to pH 7.5 was accomplished in the presence or absence of dissociated light chains.     

心脏型肌球蛋白结合蛋白C基因18443A/G多态对肥厚型心肌病心肌肥厚程度的影响

目的探讨心脏型肌球蛋白结合蛋白C基因(Cardiac myosin binding protein c,MYBPC3)18443A/G多态对肥厚型心肌病(Hypertrophic cardiomyopathy,HCM)临床表型的影响.方法研究对象为在中国医学科学院阜外心血管病医院就诊的100例无血缘关系的HCM病人,120例性别、年龄匹配的健康人作为正常研究对照.设计特异引物,采用PCR扩增和直接测序法对MYB-PC3 18443A/G进行基因分型.结果携带MYBPC3 18443GG基因型的HCM患者肥厚心肌的厚度(24.3±7.3)mm大于携带AG基因型(20.0±5.3)mm(P<0.01)和AA基因型(17.4±2.8)mm(P<0.01)的HCM患者.未发现该多态与发病年龄、晕厥、心电图变化、左室舒张末平均内径、左房舒张末平均内径、收缩期二尖瓣叶前向运动等其他临床表型相关.结论MYBPC3不仅是HCM的主要致病基因,而且可能是影响心肌肥厚程度的修饰基因.     

Interaction of cardiac myosin and its light chains with calcium ions and regulation of binding by phosphorylation

The binding of calcium ions to cardiac myosin and to its isolated light chains has been measured and related to the state of phosphorylation. Isolated cardiac metal-binding light chains (LC₂) undergo a change in conformation when calcium or magnesium ions are added at physiological ionic strength. This can be followed by circular dichroism, which reveals that saturation with calcium under these conditions increases the α-helicity of the chain from about 27 to 34%. The profile is too steep to be accounted for in terms of a single titrating site, and there is evidently interaction between the primary binding site and weaker sites. The mid-point of the profile occurs at 0.6 μm and 1.25 μm free calcium concentration when the light chains are in the dephosphorylated and phosphorylated states respectively. In intact cardiac myosin the degree of saturation with calcium could be evaluated from the fractional protection of proteolytically labile sites. The level of protection conferred by bound calcium ions was critically dependent on the identity of the supporting electrolyte, ammonium acetate being particularly favourable. Calcium binding profiles were obtained at ionic strengths equivalent to physiological. They were consistent with the presence of independently titrating isolated sites. Again the affinity was appreciably perturbed by phosphorylation. Association constants of 5 × 10⁶ and 3 × 10⁶m^?1 were obtained for fully dephosphorylated and largely (but not completely) phosphorylated myosins respectively. These are compared with earlier published values, which vary over a range of 2 to 3 orders of magnitude. The circumstances under which the saturation of the metal binding sites could occur in the physiological milieu are considered.