Myosin Heavy Chain Fragments Research Articles

Myofibrillar 1-D fingerprints and myosin heavy chain MS analyses of beef loin at 36 h postmortem correlate with tenderness at 7 days

This study investigated the potential for relating changes in electrophoretic protein patterns derived from the longissimus dorsi of beef cattle 36 h postmortem with tenderness at 7 days. We report finding a significant correlation ( R 2=0.82) between electrophoretic l. dorsi myofibrillar fingerprints at 36 h postmortem and tenderness at 7 days, as determined by Warner–Bratzler shear analysis. In addition, we have used mass spectrometric analyses to identify fragments of bovine myosin heavy chain that are significantly correlated with tenderness. Furthermore, this method offers the potential to increase our understanding of the fundamental cellular mechanisms underlying the proteolytic breakdown of muscle proteins during the aging process.

Postmortem proteome changes of porcine muscle related to tenderness.

Proteome analysis was used to investigate the relation between changes in postmortem proteome of porcine muscle and tenderness development. Muscle samples were taken at slaughter and 72 h postmortem, and the registered changes in the proteome were related to Warner-Bratzler shear force. One hundred and three protein spots were found to change significantly (P < 0.01) over time, and of these the 27 most pronounced changes were identified. Eleven out of the 27 changes were fragments of actin. Other identified myofibril proteins or fragments included myosin heavy chain, titin, myosin light chain I, myosin light II, CapZ, and cofilin. Correlation analysis revealed significant correlations between three of the identified actin fragments and the myosin heavy chain fragment to shear force. Moreover, myosin light chain II and triose phosphate isomerase I were also found to correlate significantly to shear force. The results clearly demonstrate that postmortem degradation of actin and myosin heavy chain is related to meat tenderness.

A novel combination of promoter and enhancers increases transgene expression in vascular smooth muscle cells in vitro and coronary arteries in vivo after adenovirus-mediated gene transfer.

Recombinant adenoviruses are employed widely for vascular gene transfer. Vascular smooth muscle cells (SMCs) are a relatively poor target for transgene expression after adenovirus-mediated gene delivery, however, even when expression is regulated by powerful, constitutive viral promoters. The major immediate-early murine cytomegalovirus enhancer/promoter (MIEmCMV) elicits substantially greater transgene expression than the human cytomegalovirus promoter (MIEhCMV) in all cell types in which they have been compared. The Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) increases transgene expression in numerous cell lines, and fragments of the smooth muscle myosin heavy chain (SMMHC) promoter increase expression within SMC from heterologous promoters. We therefore, compared the expression of beta-galactosidase after adenovirus-mediated gene transfer of lacZ under the transcriptional regulation of a variety of combinations of the promoters and enhancers described, in vitro and in porcine coronary arteries. We demonstrate that inclusion of WPRE and a fragment of the rabbit SMMHC promoter along with MIEmCMV increases beta-galactosidase expression 90-fold in SMC in vitro and approximately 40-fold in coronary arteries, compared with vectors in which expression is regulated by MIEhCMV alone. Expression cassette modification represents a simple method of improving adenovirus-mediated vascular gene transfer efficiency and has important implications for the development of efficient cardiovascular gene therapy strategies.

Effects of a soy protein diet on exercise-induced muscle protein catabolism in rats

OBJECTIVE: We examined effects of dietary soy protein isolate on muscle calpain activity and myosin heavy chain (MHC) degradation in rats performing an acute running exercise. METHODS: In rats fed a 20% casein diet, the treadmill running exercise, fixed at 80 kg/m, transiently increased calpain activity in gastrocnemius muscles in parallel with the release of creatine kinase into plasma. The fixed running also caused an accumulation of immunoreactive degradation fragments of MHC in the muscle. Feeding a 20% soy protein isolate diet as opposed to the control casein diet to rats significantly suppressed the running-induced activation of μ- and m-calpains, fragmentation of MHC, and release of creatine kinase into plasma ( P < 0.05). RESULTS: Rats fed the soy protein isolate diet had significantly higher calpastatin activity in gastrocnemius muscle than did rats fed the casein diet ( P < 0.05), indicating that this increase ihibits the exercise-induced autoactivation of calpain. Activities of proteasome, cathepsin B + L, and antioxidant enzymes and the levels of glutathione and thiobarbituric acid-reactive substances in the muscle did not differ between the diet groups at the end of the exercise. CONCLUSIONS: Our results suggest that diets containing soy protein prevent exercise-induced protein degradation in skeletal muscle, possibly through inhibiting the calpain-mediated proteolysis.

Levels of Myosin Heavy Chain Fragments in Myoskeletal Injuries

Myosin heavy chain (MHC) is a structurally bound contractile protein of the thick filaments. The magnitude of myoskeletal injury may be estimated by serial determinations of these contractile proteins released into the circulation as a consequence of loss of cell membrane integrity. The polymorphism of MHC is believed to be responsible, at least in part, for the variable contraction velocity of muscles. It was the aim of the present study to confirm the utility of MHC for the assessment of skeletal muscle damage within 24 hr of injury in patients with myoskeletal injuries. Plasma MHC fragments was measured in 25 patients with muscle injuries. Samples were obtained immediately on hospital admission (A1) and 24 hr after admission (A2). These were compared with 15 noninjured controls. In addition, creatine kinase (CK), myoglobin and cardiac troponin I (cTnI) were measured in order to confirm the extent of the injury and to exclude protein released from the heart. Radioimmunoassay, enzyme immunoassay, and one-step immunoenzymometric assay (IEMA) techniques were used for measurements of plasma MHC fragments, CK plus myoglobin and cTnI, respectively. Mean (SD) uU/l of MHC was (94.1±116.1) p=NS and (673.3±1943) p<0.0001 on A1 and A2, respectively, compared to controls (96.8±96.35). CK (IU/l) was 226.0±231.0 on A1 and 451.0±678.0 on A2 compared to 104.0±51.0 on controls. Myoglobin was 200% higher at A1 and A2 than at controls. We substantiate previous study and conclude that MHC could be a useful tool in the study of myoskeletal injuries in human.

LEVELS OF MYOSIN HEAVY CHAIN FRAGMENTS IN MYOSKELETAL INJURIES

Myosin heavy chain (MHC) is a structurally bound contractile protein of the thick filaments. The magnitude of myoskeletal injury may be estimated by serial determinations of these contractile proteins released into the circulation as a consequence of loss of cell membrane integrity. The polymorphism of MHC is believed to be responsible, at least in part, for the variable contraction velocity of muscles. It was the aim of the present study to confirm the utility of MHC for the assessment of skeletal muscle damage within 24 hr of injury in patients with myoskeletal injuries. Plasma MHC fragments was measured in 25 patients with muscle injuries. Samples were obtained immediately on hospital admission (A1) and 24 hr after admission (A2). These were compared with 15 noninjured controls. In addition, creatine kinase (CK), myoglobin and cardiac troponin I (cTnI) were measured in order to confirm the extent of the injury and to exclude protein released from the heart. Radioimmunoassay, enzyme immunoassay, and one-step immunoenzymometric assay (IEMA) techniques were used for measurements of plasma MHC fragments, CK plus myoglobin and cTnI, respectively. Mean (SD) uU/l of MHC was (94.1±116.1) p=NS and (673.3±1943) p&lt;0.0001 on A1 and A2, respectively, compared to controls (96.8±96.35). CK (IU/l) was 226.0±231.0 on A1 and 451.0±678.0 on A2 compared to 104.0±51.0 on controls. Myoglobin was 200% higher at A1 and A2 than at controls. We substantiate previous study and conclude that MHC could be a useful tool in the study of myoskeletal injuries in human.

Markers of inflammation and myofibrillar proteins following eccentric exercise in humans.

The purpose of this study was to examine the time-course and relationships of technetium-99m (99mTc) neutrophils in muscle, interleukin-6 (IL-6), myosin heavy chain fragments (MHC), eccentric torque, and delayed onset muscle soreness (DOMS) following eccentric exercise in humans. Twelve male subjects completed a pre-test DOMS questionnaire, performed a strength test and had 100 ml blood withdrawn for analysis of plasma IL-6 and MHC content. The neutrophils were separated, labelled with 99mTc, and re-infused into the subjects immediately before the exercise. Following 300 eccentric repetitions of the right quadriceps muscles on an isokinetic dynamometer, the subjects had 10 ml of blood withdrawn with repeated the eccentric torque exercise tests and DOMS questionnaire at 0, 2, 4, 6, 20, 24, 48, 72 h, and 6 and 9 days. Bilateral images of the quadriceps muscles were taken at 2, 4, and 6 h. Computer analysis of regions of interest was used to determine the average count per pixel. The 99mTc neutrophils and IL-6 increased up to 6 h post-exercise (P < 0.05). The neutrophils were greater in the exercised muscle than the non-exercised muscle (P < 0.01). The DOMS was increased from 0 to 48 h, eccentric torque decreased from 2 to 24 h, and MHC peaked at 72 h post-exercise (P < 0.001). Significant relationships were found between IL-6 and 2 h and DOMS at 24 h post-exercise (r = 0.68) and assessment of the magnitude of change between IL-6 and MHC (r = 0.66). These findings suggest a relationship between damage to the contractile proteins and inflammation, and that DOMS is associated with inflammation but not with muscle damage.

Ultrastructural and molecular changes in the left and right ventricular myocardium associated with ascites syndrome in broiler chickens raised at low altitude.

The present study examines ultrastructural and molecular changes in ventricular myocardium associated with ascites cases in fast-growing broilers raised at low altitude. Extensive ultrastructural lesions were seen in the left and right ventricular myocardium of broilers with fulminant heart failure and ascites. Significant changes included lesions in the myofibril contractile apparatus, altered mitochondria, marked reduction in the myofibril component, and changes in the extracellular matrix (ECM) architecture. No lesions were observed in hearts of slow growing broilers, but mild to moderate changes (predominantly in the left ventriculum) were apparent in the hearts from some clinically normal, fast-growing broilers. SDS-PAGE profiles of washed myofibrils showed several distinctly different bands in preparations from left ventricular myocardium of ascitic birds. Western blot analysis of these samples revealed several fragments of myosin heavy chain, M-protein, and titin. Based on gelatinolytic activity, matrix metalloproteinases (MMP) in the cytosolic fraction of ventricular myocardium homogenates were identified as MMP-2. The relative activity of this enzyme appears to be considerably higher in preparations from broilers, particularly in the preparations from the left ventriculum of fast-growing broilers, in comparison to leghorns or slow growing broilers. The nature and distribution of the changes in the heart indicate that chronic cardiomyopathic process in the left ventricular myocardium occurs during the development of ascites. It is postulated that progressive deterioration of the left heart pump function caused by initial lesions in the left ventricular myocardium is a significant factor in the development of pulmonary hypertension and the pathogenesis of ascites in broilers raised at low altitude.

Levels of Myosin Heavy Chain Fragment in Patients with Tissue Damage

Background. Myosin heavy chain fragments (MHC) levels are observed to be higher in myoskeletal injuries after surgery. MHC could be a helpful supplementary tool in the study of myoskeletal injuries. Methods. Serum levels of myosin heavy chain fragments (MHC) were assessed in orthopedic patients before operation (OBO) and after operative (OAO) repairs and in the early phase of soft tissue injury (STI) using a radioimmunoassay involving monoclonal antibodies to the human beta-type MHC. Results. Mean (SD) μU/L of MHC in comparison with the control subjects (75.3 ± 47.1) was higher in OAO (305.8 ± 38.1) p <0.0001, and no significant changes in MHC were found in STI (67 ± 77.5). Myoglobin was notably higher in OBO (81.9 ± 95.0) compared to STI (43.9 ± 55.9) or controls p <0.05, but there was no further change in the protein after surgery. The mean proportional raised level of myoglobin in OBO was >twofold, and MHC increased by 27%. Neither myoglobin nor MHC increased in the plasma of the STI within 24 h of injury. Conclusions. These data suggest that the release of MHC could be a helpful supplementary tool in the study of tissue damage in humans.

Creatine kinase, myosin heavy chains and magnetic resonance imaging after eccentric exercise

The aim of this study was to examine the relationship between myosin heavy chain (MHC) release as a specific marker of slow-twitch muscle fibre breakdown and magnetic resonance imaging (MRI) of skeletal muscle injury after eccentric exercise. The effects of a single series of 70 high-intensity eccentric contractions of the quadriceps femoris muscle group (single leg) on plasma concentrations of creatine kinase and MHC fragments were assessed in 10 young male sport education trainees before and 1 and 4 days after exercise. To visualize muscle injury, MRI of the loaded thigh was performed before and 4 days after the eccentric exercise. All participants recorded an increase ( P ≪ 0.05) in creatine kinase after exercise. In five participants, T2 signal intensity was unchanged post-exercise compared with pre-exercise and MHC plasma concentration was normal; however, they showed an increase ( P ≪ 0.05) in creatine kinase after exercise. For the remaining five participants, there was an increase in T2 signal intensity of the loaded vastus intermedius and vastus lateralis. These changes in MRI were accompanied by an increase in MHC plasma concentration ( P ≪ 0.01) as well as an increase in creatine kinase ( P ≪ 0.01). We suggest that changes in MRI T2 signal intensity after muscle damage induced by eccentric exercise are closely related to damage to structurally bound contractile filaments of some muscle fibres. Additionally, MHC plasma release indicates that this damage affects not only fast-twitch fibres but also some slow-twitch fibres.

A Peptide Fragment of β Cardiac Myosin Heavy Chain (β-CMHC) Can Provoke Autoimmune Myocarditis as well as the Corresponding a Cardiac Myosin Heavy Chain (α-CMHC) Fragment

The validity of the general belief that a cardiac myosin heavy chain (α-CMHC) is primarily responsible for causing experimental autoimmune myocarditis because of the more profound tolerance induction to β-CMHC due to its expression during the embryonic stage has been examined. In order to completely avoid cross-contamination among components of the two myosin heavy chains, recombinant myosin fragments were synthesized in Escherichia coli using cDNA fragments of rat a- and (J-CMHC cloned by reverse transcription polymerase chain reaction (RT-PCR). Two fragments corresponding to amino acid residues 1107-1164 derived from a-and β-heavy chains were equally capable of provoking severe myocarditis in Lewis rats when immunized in complete Freunďs adjuvant. No significant differences in the severity, as judged from histological scoring, were observed between the diseases induced by the two different peptide fragments, indicating conclusively that β-CMHC is as pathogenic as α-CMHC.

Myosin heavy chain degradation during apoptosis in endothelial cells

The cytoskeleton undergoes dramatic changes during apoptosis and many cytoskeletal proteins are known to be degraded during this process. The number of proteases found to be involved in apoptosis is growing but the role of the proteolysis they cause remains poorly understood. This report describes for the first time that myosin heavy chain is cleaved in aortic endothelial cell apoptosis induced either by tumour necrosis factor-alpha or okadaic acid. The cleavage was specific since a well-defined major 97 kDa fragment of myosin heavy chain was produced. The intermediate filament component vimentin was also cleaved into well-defined fragments (31, 28 and 23 kDa). Kinetic studies showed that proteolysis occurred concomitantly with the morphological changes associated with apoptosis, i.e. cellular condensation and fragmentation in apoptotic bodies. These data suggest that the degradation of myosin and vimentin could be involved in the execution of the morphological alterations observed during apoptotic cell death.

Primary structure of myosin from the striated adductor muscle of the Atlantic scallop, Pecten maximus, and expression of the regulatory domain.

We have determined the complete cDNA and deduced amino acid sequences of the heavy chain, regulatory light chain and essential light chain which constitute the molecular structure of myosin from the striated adductor muscle of the scallop, Pecten maximus. The deduced amino acid sequences of P. maximus regulatory light chain, essential light chain and heavy chain comprise 156, 156 and 1940 amino acids, respectively. These myosin peptide sequences, obtained from the most common of the eastern Atlantic scallops, are compared with those from three other molluscan myosins: the striated adductor muscles of Argopecten irradians and Placopecten magellanicus, and myosin from the siphon retractor muscle of the squid, Loligo pealei. The Pecten heavy chain sequence resembles those of the other two scallop sequences to a much greater extent as compared with the squid sequence, amino acid identities being 97.5% (A. irradians), 95.6% (P. magellanicus) and 73.6% (L. pealei), respectively. Myosin heavy chain residues that are known to be important for regulation are conserved in Pecten maximus. Using these Pecten sequences, we have overexpressed the regulatory light chain, and a combination of essential light chain and myosin heavy chain fragment, separately, in E. coli BL21 (DE3) prior to recombination, thereby producing Pecten regulatory domains without recourse to proteolytic digestion. The expressed regulatory domain was shown to undergo a calcium-dependent increase (approximately 7%) in intrinsic tryptophan fluorescence with a mid-point at a pCa of 6.6.

Advantage of recombinant technology for the identification of cardiac myosin epitope of severe autoimmune myocarditis in Lewis rats.

Whole cardiac myosin induced experimental autoimmune myocarditis (EAM) in animals. The identification of the epitope causing EAM is expected to elucidate the mechanism leading to the onset of this autoimmune disease. Until now such studies have been done mostly with synthetic oligo-peptides. We employed recombinant technology to produce the immunogenic fragment of the self-cardiac myosin heavy chain (CMHC) for EAM in Lewis rats, and successfully induced severe myocarditis with short recombinant peptide fragment CMHC residues 1107 to 1186. The immunogenicity was completely abolished from the fragment with further excision of 12 amino acids from residues 1131 to 1143. This recombinant technology clearly has advantages in the ease of manipulation and the purity for the creation of immunogenic epitopes.

Isolation of T-Cell Antigens by Using a Recombinant Protein Library and Its Application to the Identification of Novel Vaccine Candidates against Schistosomiasis

We present here a novel approach to identify T-cell antigens from any infectious agent by use of a library of purified recombinant proteins. Essential features of this strategy include (i) a highly efficient cDNA cloning system which negatively selects against nonrecombinant transformants by making use of the bacterial EcoK restriction system, (ii) affinity staining of cDNA clones expressing recombinant proteins, and (iii) a procedure of simultaneous purification of recombinant proteins from large numbers of isolated clones (representing the protein library) in a single step from pools consisting of up to 24 individual clones. The feasibility of the screening system was confirmed by constructing a protein library of the human parasite Schistosoma mansoni. The recombinant antigens of this library were used to stimulate CD4(+) T cells derived from the axillary lymph nodes of mice vaccinated with irradiated cercariae. In initial screening experiments, we detected parasite-specific proliferation and gamma interferon (IFN-gamma) secretion in response to several pools of cDNA clones. Further analysis of one particular pool revealed that only one of its constituents stimulated considerable IFN-gamma secretion by CD4(+) T cells and that the expressed antigen is identical to a small fragment of myosin heavy chain.

Drug-Induced Rhabdomyolysis

The syndrome of rhabdomyolysis is the result of skeletalmuscle injury that alters the integrity of the sarcolemmaand leads to the eventual release of intracellular contentsinto the plasma. The causes are diverse, and includemuscle trauma (for instance, from vigorous exercise, crushinjuries, battering, or seizures), inadequate bloodperfusion, heat stroke, electrolyte imbalance, hereditaryenzyme deficiencies, infections and ingestion of drugs andtoxins. Various serious medical disorders, particularlythose resulting in states of disordered energy production,are also associated with rhabdomyolysis.In general, drug toxicity involves organs, such as thekidney, liver, gastrointestinal tract and the central nervoussystem, with skeletal muscle being usually less readilyaffected. Although drug-induced rhabdomyolysis wasuncommon in the past, it is no longer rare, due to theintroduction of more and increasingly potent drugs intoclinical practice.The incidence of drug-induced rhabdomyolysis isuncertain, largely because most of it is unreported.Similarly, the mortality rates are unknown. However, ithas been suggested that rhabdomyolysis from all causesleads to 5%-25% of cases of acute renal failure.

Two Nonmuscle Myosin II Heavy Chain Isoforms Expressed in Rabbit Brains: Filament Forming Properties, the Effects of Phosphorylation by Protein Kinase C and Casein Kinase II, and Location of the Phosphorylation Sites

During the course of the expression of a 47-kDa COOH-terminal fragment of brain-type nonmuscle myosin heavy chain (MIIBF47), we found two closely related forms of MIIB, designated MIIB alpha and MIIB beta, in rabbit brains. The B alpha form corresponded to SMemb, described by Kuro-o et al. [(1991) J. Biol. Chem. 266, 3768] and was the more abundant form in rabbit brain, while the B beta form was novel. MIIB beta F47 differed from MIIB alpha F47 at six positions, three of which were within the carboxyl-terminal nonhelical domain; in MIIB beta F47, Ser, Pro, and Lys replaced Pro, Ser, and Glu, respectively. MIIB alpha F47 and MIIB beta F47 differed in filament assembly properties in the presence of various concentrations of salt, and a chimera containing the helical domain of MIIB beta F47 and the nonhelical domain of MIIB alpha F47 behaved very much like MIIB beta F47. Protein kinase C (PK C) incorporated 1 and 2 mol of phosphate/mol peptide of MIIB alpha F47 and MIIB beta F47, respectively, and caused similar levels of inhibition of assembly for both isoforms. Casein kinase II (CK II) incorporated 4 and 2 mol of phosphate/mol of MIIB alpha F47 and MIIB beta F47 peptides, respectively, and this caused strong inhibition of assembly for MIIB alpha F47 but only slight inhibition for MIIB beta F47. PK C sites in MIIB alpha F47 were localized within a region containing a cluster of Ser residues near the predicted junction of the helical and nonhelical domains: P-I-S(PO4)-F-S(PO4)-S(PO4)-S(PO4)-R-S(PO4)-. Out of the five potential PK C sites, only one site seemed to be phosphorylated per peptide. The PK C sites in MIIB beta F47 were localized as S(PO4)-I-S-F-S-S-(PO4)-R-S(PO4)-, with total incorporation of about 2 mol/mol of peptide. In addition, PK C phosphorylated a Ser within the predicted helical domain, E-V-S(PO4)-T-L, in both MIIB alpha F47 and MIIB beta F47. For CK II, five sites were identified within the COOH end of MIIB alpha F47: S(PO4)-L-E-L-S(PO4)-D-D-D-T(PO4)-E-S-K-T-S(PO4)-D-V-N-E-T-Q-P-P-Q-S(PO4) -E. The same sites were phosphorylated in MIIB beta F47 except for the first Ser, which was replaced by Pro in MIIB beta F47. An average of about two of the four potential sites were phosphorylated in MIIB beta F47, while in MIIB alpha F47 all five sites could be fully phosphorylated by CK II. Our results demonstrate that (1) the helical domains dictate the intrinsic salt dependence of assembly for nonmuscle myosin, (2) the isoforms are phosphorylatable by different kinases in an isoform specific manner mostly within the COOH-terminal nonhelical domain, and (3) the effects of the phosphorylation on assembly are isoform specific.

Analysis of myosin heavy chain mRNA expression by RT-PCR.

An assay was developed for rapid and sensitive analysis of myosin heavy chain (MHC) mRNA expression in rodent skeletal muscle. Only 2 microg of total RNA were necessary for the simultaneous analysis of relative mRNA expression of six different MHC genes. We designed synthetic DNA fragments as internal standards, which contained the relevant primer sequences for the adult MHC mRNAs type I, IIa, IIx, IIb as well as the embryonic and neonatal MHC mRNAs. A known amount of the synthetic fragment was added to each polymerase chain reaction (PCR) and yielded a product of different size than the amplified MHC mRNA fragment. The ratio of amplified MHC fragment to synthetic fragment allowed us to calculate percentages of the gene expression of the different MHC genes in a given muscle sample. Comparison with the traditional Northern blot analysis demonstrated that our reverse transcriptase-PCR-based assay was reliable, fast, and quantitative over a wide range of relative MHC mRNA expression in a spectrum of adult and neonatal rat skeletal muscles. Furthermore, the high sensitivity of the assay made it very useful when only small quantities of tissue were available. Statistical analysis of the signals for each MHC isoform across the analyzed samples showed a highly significant correlation between the PCR and the Northern signals as Pearson correlation coefficients ranged between 0.77 and 0.96 (P < 0.005). This assay has potential use in analyzing small muscle samples such as biopsies and samples from pre- and/or neonatal stages of development.

5460959 Transduced fibroblasts

Recently we reported that the isolated 23 kDa N-terminal fragment of myosin heavy chain, which contains the ‘consencus’ ATP binding site, binds to actin in an ATP-sensitive manner (Muhlrad, A. (1989) Biochemistry 28, 4002). In order to determine whether the ‘consencus’ ATP site has a role in the ATP-dependent actin binding of the fragment, we isolated a shorter 21 kDa N-terminal fragment, which contains only a part of the ‘consencus’ site. The 21 kDa fragment was obtained by photocleavage of myosin subfragment-1 in the presence of vanadate (Mocz, G. (1989) Eur. J. Biochem. 179, 373); the cleavage was followed by dissociation of the S-1 heavy chain fragments with guanidine hydrochloride and renaturation. The isolated 21 kDa fragment binds to F-actin, since it cosediments with actin, inhibits the actin-activated ATPase activity of myosin subfragment-1 and shows increase in light scattering upon titration by actin. The affinity of the binding is rather high (Kassoc = 0.83 · 107M−1). The light scattering increase is reversed, e.g., the 21 kDa-actin complex is dissociated, upon addition of ATP both in the presence and absence of Mg, but less ATP is needed for dissociation when Mg is absent. Other polyphosphates, including inorganic triphosphate, pyrophosphate and ADP, also dissociate both the 21 kDa-actin and 23 kDa-actin complexes but the latter needs a higher concentration of polyphosphates for dissociation. However, these polyphosphates, except ATP, do not dissociate the (subfragment-1)-actin complex in the absence of Mg. The 21 kDa-actin and the 23 kDa-actin complexes are also dissociated by increasing ionic strength or by a low concentration of polyglutamate, which hardly affect the light scattering of the (subfragment-1)-actin complex. The results indicate that the binding of the N-terminal fragments of myosin to actin, unlike that of intact subfragment-1, is essentially of electrostatic nature. The polyanions dissociate the myosin fragment-actin complexes not by reacting with the ‘consencus’ ATP binding site, but by competing with actin for a positively charged binding site on the 21 kDa fragment. The only positively charged cluster in the amino acid sequence of this fragment is the 143–147 stretch, which may participate in forming the actin binding site.

Myocardial indium-111 antimyosin uptake after uncomplicated coronary artery bypass surgery

The prevalence of myocardial damage after coronary artery bypass grafting is related to the criteria of its evaluation. Indium-111 monoclonal antimyosin antibody scintigraphy has been shown to be highly sensitive and specific for even small areas of myocardial necrosis or injury like those of myocarditis or transplant rejection. Our purpose was to evaluate, by using this method, myocardial damage after uncomplicated coronary artery bypass grafting. Uptake of this radio tracer was evaluated after coronary artery bypass grafting in 14 informed and consenting consecutive patients without previous myocardial infarction, with no post-surgical complications and a favorable postoperative course, following coronary artery bypass grafting for stable angina pectoris. Monoclonal antimyosin antibody indium-111 74 MBq (Myoscint Centocor) was injected on the third postoperative day; planar images in the anterior, left anterior oblique 45 ° and 70 ° projections were obtained 24 and 48 h later and analyzed for myocardial uptake. Indium-111 antimyosin uptake was present in 10 out of 14 patients (71.4%); it was diffuse in 6 and localized in 4. The ratio of the maximal counts in the myocardium to the counts in the adjacent lung background was measured and found elevated: 1.94 ± 0.23, higher than the normal values reported in the literature. Indium-111 antimyosin uptake was clear in a group of patients after uncomplicated coronary artery bypass grafting. No correlation was observed between indium-111 antimyosin uptake or heart to lung ratio and creatine kinase, creatine kinase isoenzyme MB, glutamic oxalacetic transferase levels, duration of cardiopulmonary bypass or aortic cross-clamp time, while elevated serum β myosin heavy chain fragments (IRMA Pasteur) were observed (1378 ± 238 μU/1). This study suggests that some degree of myocardial damage, though silent, is common after coronary artery bypass grafting.