Protein kinase A-mediated acceleration of the stretch activation response in murine skinned myocardium is eliminated by ablation of cMyBP-C

Beta-adrenergic agonists induce protein kinase A (PKA) phosphorylation of the cardiac myofilament proteins myosin binding protein C (cMyBP-C) and troponin I (cTnI), resulting in enhanced systolic function, but the relative contributions of cMyBP-C and cTnI to augmented contractility are not known. To investigate possible roles of cMyBP-C in this response, we examined the effects of PKA treatment on the rate of force redevelopment and the stretch activation response in skinned ventricular myocardium from both wild-type (WT) and cMyBP-C null (cMyBP-C(-/-)) myocardium. In WT myocardium, PKA treatment accelerated the rate of force redevelopment and the stretch activation response, resulting in a shorter time to the peak of delayed force development when the muscle was stretched to a new isometric length. Ablation of cMyBP-C accelerated the rate of force redevelopment and stretch activation response to a degree similar to that observed in PKA treatment of WT myocardium; however, PKA treatment had no effect on the rate of force development and the stretch activation response in null myocardium. These results indicate that ablation of cMyBP-C and PKA treatment of WT myocardium have similar effects on cross-bridge cycling kinetics and suggest that PKA phosphorylation of cMyBP-C accelerates the rate of force generation and thereby contributes to the accelerated twitch kinetics observed in living myocardium during beta-adrenergic stimulation.     

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.     

Molecular effects of the myosin activator omecamtiv mecarbil on contractile properties of skinned myocardium lacking cardiac myosin binding protein-C

Decreased expression of cardiac myosin binding protein-C (cMyBP-C) in the myocardium is thought to be a contributing factor to hypertrophic cardiomyopathy in humans, and the initial molecular defect is likely abnormal cross-bridge (XB) function which leads to impaired force generation, decreased contractile performance, and hypertrophy in vivo. The myosin activator omecamtiv mecarbil (OM) is a pharmacological drug that specifically targets the myosin XB and recent evidence suggests that OM induces a significant decrease in the in vivo motility velocity and an increase in the XB duty cycle. Thus, the molecular effects of OM maybe beneficial in improving contractile function in skinned myocardium lacking cMyBP-C because absence of cMyBP-C in the sarcomere accelerates XB kinetics and enhances XB turnover rate, which presumably reduces contractile efficiency. Therefore, parameters of XB function were measured in skinned myocardium lacking cMyBP-C prior to and following OM incubation. We measured k_tr, the rate of force redevelopment as an index of XB transition from both the weakly- to strongly-bound state and from the strongly- to weakly-bound states and performed stretch activation experiments to measure the rates of XB detachment (k_rel) and XB recruitment (k_df) in detergent-skinned ventricular preparations isolated from hearts of wild-type (WT) and cMyBP-C knockout (KO) mice. Samples from donor human hearts were also used to assess the effects of OM in cardiac muscle expressing a slow β-myosin heavy chain (β-MHC). Incubation of skinned myocardium with OM produced large enhancements in steady-state force generation which were most pronounced at low levels of [Ca^2 +] activations, suggesting that OM cooperatively recruits additional XB’s into force generating states. Despite a large increase in steady-state force generation following OM incubation, parallel accelerations in XB kinetics as measured by k_tr were not observed, and there was a significant OM-induced decrease in k_rel which was more pronounced in the KO skinned myocardium compared to WT skinned myocardium (58% in WT vs. 76% in KO at pCa 6.1), such that baseline differences in k_rel between KO and WT skinned myocardium were no longer apparent following OM-incubation. A significant decrease in the k_df was also observed following OM incubation in all groups, which may be related to the increase in the number of cooperatively recruited XB’s at low Ca^2 +-activations which slows the overall rate of force generation. Our results indicate that OM may be a useful pharmacological approach to normalize hypercontractile XB kinetics in myocardium with decreased cMyBP-C expression due to its molecular effects on XB behavior.     

Effect of cardiac myosin binding protein-C on mechanoenergetics in mouse myocardium

We examined the effect of cardiac myosin binding protein-C (cMyBP-C) on contractile efficiency in isovolumically contracting left ventricle (LV) and on internal viscosity and oscillatory work production in skinned myocardial strips. A 6-week diet of 0.15% 6-n-propyl-2-thiouracil (PTU) was fed to wild-type (+/+(PTU)) and homozygous-truncated cMyBP-C (t/t(PTU)) mice starting at age approximately 8 weeks and leading to a myosin heavy chain (MHC) isoform profile of 10% alpha-MHC and 90% beta-MHC in both groups. Western blot analysis confirmed that cMyBP-C was present in the +/+(PTU) and effectively absent in the t/t(PTU). Total LV mechanical energy per beat was quantified as pressure-volume area (PVA). O2 consumption (Vo2) per beat was plotted against PVA at varying LV volumes. The reciprocal of the slope of the linear Vo2-PVA relation represents the contractile efficiency of converting O2 to mechanical energy. Contractile efficiency was significantly enhanced in t/t(PTU) (26.1+/-2.6%) compared with +/+(PTU) (17.1+/-1.6%). In skinned myocardial strips, maximum isometric tension was similar in t/t(PTU) (18.7+/-2.1 mN x mm(-2)) and +/+(PTU) (21.9+/-4.0 mN x mm(-2)), but maximum oscillatory work induced by sinusoidal length perturbations occurred at higher frequencies in t/t(PTU) (7.31+/-1.17 Hz) compared with +/+(PTU) (4.48+/-0.60 Hz) and was significantly more sensitive to phosphate concentration in the t/t(PTU). Under rigor conditions, the internal viscous load was significantly lower in the t/t(PTU) compared with +/+(PTU), ie, approximately 40% lower at 1 Hz. These results collectively suggest that contractile efficiency is enhanced in the t/t(PTU), probably through a reduced loss of mechanical energy by a viscous load normally provided by cMyBP-C and through a gain of phosphate-dependent oscillatory work normally inhibited by cMyBP-C.     

The cardiac troponin C mutation Leu29Gln found in a patient with hypertrophic cardiomyopathy does not alter contractile parameters in skinned murine myocardium

The present study investigates the effects of the first mutation of troponin C (hcTnC^L29Q) found in a patient with hypertrophic cardiomyopathy (HCM) on force–pCa relations and the interplay with phosphorylation of sarcomeric PKA substrates. In triton-skinned murine cardiac fibers, the endogenous mcTnC was extracted and the fibers were subsequently reconstituted with recombinant wild-type and mutant hcTnC. Force–pCa relations of preparations containing hcTnC^L29Q or hcTnC^WT were similar. Incubation of fibers reconstituted with the recombinant proteins with phosphatase to dephosphorylate sarcomeric PKA substrates induced an increase in Ca²⁺ sensitivity, slightly more pronounced (0.04 pCa units) in hcTnC^L29Q-containing fibers. Incubation of the dephosphorylated fibers with PKA induced significant rightward shifts of force–pCa relations of similar magnitude with both, hcTnC^L29Q and hcTnC^WT. No significant effects of hcTnC^L29Q on the velocity of unloaded shortening were observed. In conclusion, no major differences in contractile parameters of preparations containing hcTnC^L29Q compared to hcTnC^WT were observed. Therefore, it appears unlikely that hcTnC^L29Q induces the development of HCM by affecting the regulation of Ca²⁺-activated force and interference with PKA-mediated modulation of the Ca²⁺ sensitivity of contraction.     

Effect of ionic strength on length-dependent Ca(2+) activation in skinned cardiac muscle

The length-dependence of myofilament Ca(2+) sensitivity is considered to be an important component of the steep force-length relationship in cardiac muscle (Frank-Starling relation). Recent studies suggest that Ca(2+) sensitivity is a function of the number of strong-binding cross-bridge interactions formed at a given sarcomere length. However, the length-dependent step in the thin filament activation process is still unknown. This study was designed to test the hypothesis that sarcomere length influences the transition of the thin filament from the unattached (blocked) state to the weakly bound (closed) state. This hypothesis was tested by determining the length-dependence of Ca(2+) sensitivity as a function of ionic strength in skinned bovine ventricular muscle. Previous studies have shown that reduction in ionic strength below a critical level, in the absence of Ca(2+), shifts the thin filament to the closed state. In this study normal Ca(2+) regulation was maintained at low ionic strength but the length-dependence of Ca(2+) sensitivity and the length-dependence of Ca(2+) binding were eliminated. These results are consistent with the hypothesis that the transition from the blocked to the closed state is a function of filament geometry as well as Ca(2+) and ionic strength.     

Mn2+-dependent protein phosphatase 1 enhances protein kinase A-induced Ca2+ desensitisation in skinned murine myocardium

OBJECTIVE: Phosphorylation of proteins in cardiac myofilaments is a major determinant in the regulation of the Ca(2+) sensitivity of contraction. Whereas most reports have focused on effects of phosphorylation, little is known about reverse effects of dephosphorylation in skinned myocardium. Here we studied the effect of the Mn(2+)-dependent catalytic subunit of protein phosphatase 1 (PP1c-alpha) on the Ca(2+) regulation of contraction. In particular, we tested the hypothesis that phosphorylation persists after the skinning procedure and thereby attenuates protein kinase A (PKA)-induced Ca(2+) desensitisation. METHODS: Effects of Mn(2+) and Mn(2+)-PP1c on the Ca(2+) sensitivity of contraction (pCa(50)) were investigated in triton-skinned cardiac fibres from mice and compared with those of PKA treatment. Phosphorylation of proteins was monitored by autoradiography. RESULTS: PKA treatment significantly decreased the pCa(50) by 0.04 pCa units. In contrast, treatment with PP1c or Mn(2+)-containing PP1c buffer significantly increased the pCa(50) by 0.26 units or 0.09 units, respectively. These Ca(2+) sensitisations were completely reversed by subsequent PKA treatment. Replacement of the endogenous cardiac troponin I (cTnI) in fibres with the phospho-mimicking mutant human cTnI(S22/23D) abolished the PP1c-induced Ca(2+) sensitisation. PP1c removed (32)P which had been incorporated into cTnI and cardiac myosin binding protein C by PKA treatment. PKA incorporated twofold more (32)P into cTnI in fibres pre-treated with PP1c. CONCLUSIONS: Mn(2+)-dependent PP1c increases the Ca(2+) sensitivity of contraction of skinned cardiac fibres. This can be ascribed to dephosphorylation of PKA-dependent phosphorylation sites. Hence PKA-dependent phosphorylation of sarcomeric proteins persists to a functionally relevant degree after the skinning procedure.     

Hypertrophic cardiomyopathy in cardiac myosin binding protein-C knockout mice

Familial hypertrophic cardiomyopathy (FHC) is an inherited autosomal dominant disease caused by mutations in sarcomeric proteins. Among these, mutations that affect myosin binding protein-C (MyBP-C), an abundant component of the thick filaments, account for 20% to 30% of all mutations linked to FHC. However, the mechanisms by which MyBP-C mutations cause disease and the function of MyBP-C are not well understood. Therefore, to assess deficits due to elimination of MyBP-C, we used gene targeting to produce a knockout mouse that lacks MyBP-C in the heart. Knockout mice were produced by deletion of exons 3 to 10 from the endogenous cardiac (c) MyBP-C gene in murine embryonic stem (ES) cells and subsequent breeding of chimeric founder mice to obtain mice heterozygous (+/-) and homozygous (-/-) for the knockout allele. Wild-type (+/+), cMyBP-C(+/-), and cMyBP-C(-/-) mice were born in accordance with Mendelian inheritance ratios, survived into adulthood, and were fertile. Western blot analyses confirmed that cMyBP-C was absent in hearts of homozygous knockout mice. Whereas cMyBP-C(+/-) mice were indistinguishable from wild-type littermates, cMyBP-C(-/-) mice exhibited significant cardiac hypertrophy. Cardiac function, assessed using 2-dimensionally guided M-mode echocardiography, showed significantly depressed indices of diastolic and systolic function only in cMyBP-C(-/-) mice. Ca2+ sensitivity of tension, measured in single skinned myocytes, was reduced in cMyBP-C(-/-) but not cMyBP-C(+/-) mice. These results establish that cMyBP-C is not essential for cardiac development but that the absence of cMyBP-C results in profound cardiac hypertrophy and impaired contractile function.     

Local activation or implantation of cardiac progenitor cells rescues scarred infarcted myocardium improving cardiac function

Ischemic heart disease is characterized chronically by a healed infarct, foci of myocardial scarring, cavitary dilation, and impaired ventricular performance. These alterations can only be reversed by replacement of scarred tissue with functionally competent myocardium. We tested whether cardiac progenitor cells (CPCs) implanted in proximity of healed infarcts or resident CPCs stimulated locally by hepatocyte growth factor and insulin-like growth factor-1 invade the scarred myocardium and generate myocytes and coronary vessels improving the hemodynamics of the infarcted heart. Hepatocyte growth factor is a powerful chemoattractant of CPCs, and insulin-like growth factor-1 promotes their proliferation and survival. Injection of CPCs or growth factors led to the replacement of approximately 42% of the scar with newly formed myocardium, attenuated ventricular dilation and prevented the chronic decline in function of the infarcted heart. Cardiac repair was mediated by the ability of CPCs to synthesize matrix metalloproteinases that degraded collagen proteins, forming tunnels within the fibrotic tissue during their migration across the scarred myocardium. New myocytes had a 2n karyotype and possessed 2 sex chromosomes, excluding cell fusion. Clinically, CPCs represent an ideal candidate cell for cardiac repair in patients with chronic heart failure. CPCs may be isolated from myocardial biopsies and, following their expansion in vitro, administered back to the same patients avoiding the adverse effects associated with the use of nonautologous cells. Alternatively, growth factors may be delivered locally to stimulate resident CPCs and promote myocardial regeneration. These forms of treatments could be repeated over time to reduce progressively tissue scarring and expand the working myocardium.     

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.     

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.     

Canine neutrophil activation by cardiac lymph obtained during reperfusion of ischemic myocardium

Cardiac lymph from a canine model of myocardial ischemia and reperfusion was examined for evidence of chemotactic activity. Lymph was continuously collected from awake animals before and during a 60-minute coronary artery occlusion and up to 6 hours after the initiation of reperfusion. It was assessed for the ability to activate the following proinflammatory functions in neutrophils isolated from the blood of healthy dogs: 1) morphological changes characteristic of chemotactic stimulation, which were assessed by phase contrast microscopy, 2) orientation of canine neutrophils in a gradient of cardiac lymph, which was assessed in Zigmond chambers, 3) the binding of monoclonal antibodies reactive with CD11b and CD18 adherence glycoproteins, which was assessed by flow cytometry, and 4) adherence of canine neutrophils to monolayers of canine jugular vein endothelium, which was assessed in vitro by a visual assay. Lymph samples collected after 1 hour of reperfusion in animals demonstrating ECG evidence of ischemia and histological evidence of infarction exhibited significant stimulatory activity for each of the functions tested. Shape change-inducing activity was evaluated at more frequent intervals than other functions and was found to peak at 1 hour after initiation of reperfusion and to disappear by 6 hours. In addition, the CD11b/CD18 levels on neutrophils isolated from cardiac lymph collected during reperfusion were significantly greater than neutrophils obtained before or during occlusion. Animals that failed to exhibit evidence of infarction also failed to exhibit increased stimulatory activity in lymph collected during reperfusion, and surface levels of CD11b/CD18 on neutrophils collected from reperfusion lymph were not elevated. This study provides direct evidence supporting the hypothesis that chemotactic activity is generated in ischemic and reperfused myocardium.     

The dynamic role of cardiac myosin binding protein-C during ischemia

Cardiac myosin binding protein C (cMyBP-C) is a myofibrillar protein important for normal myocardial contractility and stability. In mutated form it can cause cardiomyopathy and heart failure. cMyBP-C appears to have separate regions for different functions. Three phosphorylation sites near the N terminus modulate contractility by their effect on both the kinetics of contraction and the binding site of the N-terminus. The C terminal region binds to myosin rods and stabilizes thick filament structure. The aim of the study reported here was to test whether cMyBPC is important in producing the structural and functional changes that result from ischemia/reperfusion. In this study the sequential changes in cMyBP-C, contractility, and thick filament structure following dephosphorylation of cMyBP-C associated with ischemia and reperfusion have been studied in biopsied specimens from chronically instrumented dogs. One and two dimensional electrophoresis, electron microscopy and immunocytochemistry with multiple antibodies generated against different domains in cMyBP-C have been used to follow structural changes in cMyBP-C. Ischemia produced dephosphorylation of cMyBP-C. Subsequent reperfusion released the dephosphorylated cMyBP-C from myofibrils and activated proteolysis of the cytoplasmic cMyBP-C. This in turn leads to increased vulnerability of cMyBP-C to proteolysis and increased degradation of thick filaments. The state of cMyBP-C appears to be closely related to phosphorylation and dephosphorylation of serine 282. In the absence of the stabilizing action of cMyBP-C either as a consequence of genetic mutation or dephosphorylation, premature degradation of thick filaments occurs and is accompanied by persistent contractile dysfunction.     

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.     

Glutathione alters calcium responsiveness of cardiac skinned fibers

Summary The glutathione status of cardiac muscle, that is the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) changes in certain forms of cardiomyopathy and during reperfusion of ischemic heart. Here we show that it also affects the sensitivity of contractile proteins to calcium. GSH (4 mM) increased the pCa₅₀ for force development in skinned fibers by 0.2 pCa units and increased force ba 44%±5.4% at pCa 5.6 whereas GSSG (4 mM) decreased it by 54%±17.8% at pCa 5.6. Half maximal activations and inhibitions were seen with 4 mM GSH or GSSG, respectively.In contrast to GSH, the reducing agent dithiotreitol at 5 mM had no activating effect. Our results suggest that the loss of contractility observed after a reperfusion of the ischemic heart my, at least in part, be due to a decreased responsiveness of the contractile proteins due to an altered glutathione status.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 320).     

Effect of cardiac myosin-binding protein C on stability of the thick filament

In contrast to skeletal muscle isoforms of myosin-binding protein C (MyBP-C), the cardiac isoform has 11 rather than 10 modules (labeled C0-C10, N-C terminus), three phosphorylation sites between C1 and C2, and 28 additional amino acids in C5. Within the C5-C10 region of cardiac MyBP-C (cMyBP-C) there are interactions between C5 and C8 as well as C7 and C10. Isolated skinned cardiac trabeculae were incubated with one of three recombinant fragments of cMyBP-C to interfere with interactions of endogenous C5. 2-10 microM C5 or C5-containing peptide fragments of cMyBP-C reversibly reduced Ca sensitivity without extracting myofibrillar protein. C2-C4 fragments had no effect. This result indicated that the region of cMyBP-C that contains C5 maintains a specific structural arrangement of myosin that helps set its contractile properties. Greater than 10 microM C5 caused skinned trabeculae to lose a substantial amount of cMyBP-C and some myosin heavy chain, resulting in irreversible decline in maximum Ca-activated force. MyBP-C appears to stabilize the structure of the thick filament and modulate the way in which myosin heads extend to the thin filament.     

Evaluation of a new protein-C concentrate and comparison of protein-C assays in a child with congenital protein-C deficiency

Summary We investigated a new protein-C (PC) concentrate in a child with a type-II homozygous deficiency, concerning tolerance and safety. By means of various functional and antigen assays the in vivo recovery and the half-life were determined. In order to compare the results we reduced the measured values to the average halfrlife of 10.0 ± 0.5 h and to an optimal recovery of 96.6%. Considerable discrepancies observed in the response of functional (clotting) and both the amidolytic and antigen assays are characteristic for type II and anticoagulant treatment. The substituted protein C is activated by the endogenous system. Thus, the efficacy of activation can be determined in deficiency states. The antigen activity of one unit PC concentrate was found to be 120% (or 1.2 U/ml plasma), close to the 100% activity defined for endogenous PC.