Pyrophosphate Gel Electrophoresis Research Articles

Myocardial contractility and left ventricular myosin isoenzyme pattern in cardiac hypertrophy due to chronic volume overload.

Chronic volume overloaded cardiac hypertrophy was induced by abdominal arteriovenous shunt in 10-week-old male Wistar rats. Ten weeks after the operation, myocardial mechanical examination was performed with isolated left ventricular papillary muscles. Left ventricular myosin isoenzyme pattern was also examined by pyrophosphate gel electrophoresis. In addition, morphological study by electron microscope was performed. In arteriovenous shunt rats, isometric developed tension (T) and its first derivative (dT/dt) were decreased, resting tension (RT) was increased and time to peak tension (TPT) was prolonged as compared to sham-operated control rats (T: 2.4 +/- 0.5 vs 2.8 +/- 0.8 g/mm2, NS. dT/dt: 23.9 +/- 4.8 vs 31.9 +/- 7.0 g/mm2.s, p less than 0.05. RT: 1.2 +/- 0.2 vs 0.9 +/- 0.1 g/mm2, p less than 0.05. TPT: 151.7 +/- 20.2 vs 128.3 +/- 10.3 msec, p less than 0.05). Myocardial mechanical responses to isoproterenol (10(-7) mol/l) and dibutyryl cyclic AMP (10(-5) mol/l) were both depressed in hypertrophied myocardium, although not significant statistically. Left ventricular myosin isoenzyme pattern was shifted towards VM-3 by chronic volume overload. Electron microscope study revealed increased collagen content in hypertrophied myocardium.

Myosin isozymes in rabbit and human smooth muscles.

Although multiple forms of myosin in cardiac and skeletal muscles have been identified, it has not been firmly established that myosin isozymes are present in adult smooth muscle. Myosin, extracted from human thoracic aorta and lower saphenous vein and rabbit aorta and uterus, was analyzed by pyrophosphate gel electrophoresis to determine if myosin isozymes are present in these tissues. In all smooth muscle tissues studied, two myosin isozymes were detected and labelled as smooth muscle 1 and smooth muscle 2, smooth muscle 2 being the faster migrating isozyme. Bovine cultured smooth muscle cells from the media of thoracic aorta also contained two forms of myosin. However, cultured fibroblasts contained only one form of myosin. Extracting myosin from either relaxed or contracting rabbit aortic smooth muscle did not influence the mobilities of smooth muscle 1 and smooth muscle 2 on pyrophosphate gels, suggesting that the degree of light chain phosphorylation did not significantly alter the electrophoretic mobility under our conditions. Smooth muscle 1 and smooth muscle 2 myosins each contain heavy chains (200,000 daltons) and light chains (20,000 and 17,000 daltons) in addition to filamin (235,000 daltons), which is closely associated with the native protein. Myosin peptide maps of rabbit aorta and uterus revealed areas of substantially different banding patterns between smooth muscle 1 and smooth muscle 2 from the same tissue. Similar peptide maps of smooth muscle 1 bands were produced from the different tissues, but the smooth muscle 2 maps were dissimilar.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of pH on calcium activation of myofilaments of adult and perinatal dog hearts. Evidence for developmental differences in thin filament regulation.

Our results show that calcium activation of myofilament preparations of dog heart in the perinatal period is unaffected by a reduction in pH from 7.0 to 6.5, which, in adult heart myofilaments, induces a 0.4 pCa unit (-log molar free calcium concentration) rightward shift in the relation between pCa and myofibrillar adenosine triphosphatase activity. Acidic pH also had no effect on calcium binding to myofibrillar troponin C of perinatal hearts. The stoichiometry of troponin C bound calcium at full myofilament activation (about 3 mol calcium/mol troponin C) was the same for adult and perinatal heart myofibrils, as was their myofibrillar troponin C content. Moreover, there were no differences in isoelectric pH of troponin C from adult and perinatal hearts. We tested whether variants of myofilament proteins other than troponin C could account for the differential effects of acidic pH. In adult and perinatal dog heart preparations, myosin heavy chain isoenzymes appeared the same as measured, using native pyrophosphate gel electrophoresis. No evidence for thick filament-related calcium regulation in the perinatal heart myofilaments was obtained, when tested in studies in which native thin filaments were displaced with a 10-fold molar excess of pure actin. In preparations in which native thick filaments were displaced with a 10-fold molar excess of pure skeletal muscle myosin, the effects of acidic pH on calcium activation were the same as in native adult and perinatal preparations. Our major conclusion from these results in that the perinatal heart myofilaments are likely to possess variations in thin filament activity and structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Histochemical and fatigue characteristics of conditioned canine latissimus dorsi muscle.

To induce fatigue resistance in the latissimus dorsi muscle of the dog in preparation for possible myocardial assistance, eight adult male beagles underwent unilateral electrical stimulation of the thoracodorsal nerve at a frequency of 2 Hz (120 stimuli/min) and 10 Hz (600 stimuli/min) for a 6-week period. The conditioned muscles were compared with their unconditioned contralateral controls by fiber typing, pyrophosphate gel electrophoresis, isometric characteristics, and fatigue rates. At the end of the period of stimulation, the conditioned muscles had a greater percentage of slow-twitch, fatigue-resistant fibers on acid and alkaline stains (100 +/- 0.7% and 83 +/- 15.3%), respectively, than did their contralateral controls (45 +/- 7.6% and 43 +/- 7.0). Pyrophosphate gel electrophoresis revealed an increase in the slow myosin and a decrease in the fast myosin content in the conditioned muscles; the stimulated muscles also demonstrated a slower contraction time (87 +/- 20 msec vs. 57 +/- 17.9 msec), a lower initial tension (4.4 +/- 1.45 kg vs. 7.2 +/- 2.11 kg), and a slower fatigue rate during a 30-minute fatigue test than did their contralateral controls. The muscles stimulated at 2 Hz had fatigue rates similar to those stimulated at 10 Hz, but generally had less diminution in muscle fiber diameters and less interfiber connective tissue. Thus, it is possible to make canine latissimus dorsi muscles more fatigue resistant, and, theoretically, more capable of myocardial assistance by electrical stimulation of the thoracodorsal nerve at a frequency as low as 2 Hz--the natural canine heart rate.

The Fiber Type of the Rabbits’ Striated External Urethral Sphincter: Electrophoretic Analysis of Myosin

The fiber type of the rabbits’ external urethral sphincter was investigated with O’Farrell’s 2-dimensional electrophoresis of constituent proteins of glycerinated muscle, peptide mapping of myosin heavy chains and non-denaturing pyrophosphate gel electrophoresis of myosin. It was determined as fast type by all three methods. The relative proportions of muscle fibers which were estimated from myosin light chain contents were 88 per cent fast type and 12 per cent slow type. On the basis of the results presented, it was shown to be fast twitch muscle, but not identical to the psoas muscle.

The mobility of gizzard myosin in pyrophosphate gel electrophoresis is increased by its light chain phosphorylation.

Phosphorylation of the 20,000 Mr light chain (L20) of gizzard myosin reversibly increased the mobility of myosin in pyrophosphate polyacrylamide gel electrophoresis (PP1 PAGE). Gizzard heavy meromyosin (HMM) with phosphorylated L20 also moved faster than that with unphosphorylated L20. This mobility increase of HMM is large enough to account for that of intact myosin. Scallop myosin, desensitized by removing its regulatory light chain, was combined with L20 and subjected to PPi PAGE. Hybrid myosin with the phosphorylated light chain moved faster than that with the unphosphorylated light chain. No such effect of light chain phosphorylation was observed with phosphorylatable light chain from breast or ventricular myosin. Thus, gizzard, but not breast or ventricular phosphorylatable light chain is furnished with the 'regulatory' property that is phosphorylation increases myosin mobility in PPi PAGE.

Influences of a long term treatment with antihypertensive drugs on left ventricular myosin isoenzyme pattern in spontaneously hypertensive rats

Rat’s left ventricular myosin is separated into three isoenzymes by pyrophosphate gel electrophoresis (1–3),i.e. VM-1,2,3, which have greater electrophoretic mobility and ATPase activity in order. Thyroid hormone’s administration and endurance swimming training can induce the shift of ventricular myosin isoenzyme pattern towards VM-1. Aging, chronic pressure overload and thyreostatic treatment, on the other hand, can shift the pattern towards VM-3 (4–12). The shift of myosin isoenzyme pattern towards VM-3 in pressure overloaded myocardium is thought to be an adaptational process to keep force development efficient with low oxygen and energy utilization (13,14). In spontaneously hypertensive rats (SHR) the concentration of VM-3 isomyosin in left ventricle is higher than that of age-matched normotensive rats. The aim of the present study is to examine to what extent alters myosin isoenzyme pattern with some antihypertensive drugs.

Myosin transitions in chronic stimulation do not involve embryonic isozymes.

Chronic low-frequency stimulation of a fast skeletal muscle effects a transition of myosin isozymes from those characteristic of a fast muscle to those characteristic of a slow muscle. This transformation involves changes in both myosin heavy and light chains. During development, muscles usually change directly from embryonic to neonatal to fast or from embryonic to neonatal to slow isozymes. We have questioned whether chronic stimulation of the adult fast muscle results in a direct fast-to-slow isozyme shift or whether transformation requires reexpression of the developmental isozymes prior to the synthesis of adult slow isozymes. We have examined these alternatives in the chronically stimulated dog diaphragm using adenosine triphosphatase (ATPase) histochemistry, pyrophosphate gel electrophoresis of native isozymes, peptide mapping of myosin heavy chains, immunoblotting with an antibody specific to embryonic myosin heavy chain, and solid-phase radioimmunoassay. We have demonstrated that a transition from adult fast to adult slow isozymes during chronic stimulation does not involve a recapitulation of embryonic isozymes.

Unusual fast myosin isozyme pattern in the lateral gastrocnemius of the chicken.

The myosin isozyme composition of the lateral gastrocnemius muscle of the chicken leg was investigated during various stages of development utilizing non-denaturing pyrophosphate gel electrophoresis, two-dimensional gel electrophoresis and peptide mapping techniques. An unusual isoform pattern for fast myosin in the lateral gastrocnemius muscle of the adult chicken leg was demonstrated which consisted of a predominance of myosin homodimers and lesser amounts of myosin heterodimer. In addition, a different myosin heavy chain isoform was present in the adult chicken lateral gastrocnemius muscle when compared to other adult fast-twitch muscles. While the adult lateral gastrocnemius muscle contained a different myosin heavy chain isoform from other adult fast-twitch muscles, the embryonic lateral gastrocnemius muscle contained a myosin heavy chain identical to that of the embryonic pectoralis major.

Myocardial catecholamine responsiveness of spontaneously hypertensive rats as influenced by swimming training.

Alterations of myocardial mechanical catecholamine responsiveness by swimming training (2 X 90 min/day, 4 weeks) were examined in 13-week-old spontaneously hypertensive males rats (SHR). The relationships between myocardial mechanical catecholamine responsiveness and ventricular beta-adrenoceptors as well as myosin isoenzyme pattern were also examined. Compared with sedentary controls, trained rats showed a greater responsiveness to isoproterenol (10(-6) mol/l) on isometric tension (T) and its first derivative (dT/dt) (delta T: 0.45 +/- 0.55 vs. -0.15 +/- 0.11 10(-2) N/mm2, p less than 0.01, delta dT/dt: 17.1 +/- 10.1 vs. 8.3 +/- 3.6 10(-2) N/mm2 X s, p less than 0.05). In sedentary SHR, dT/dtmax increased significantly, whereas developed tension decreased slightly, coupled with a decrease of time to peak tension by high dose (10(-6) mol/l) isoproterenol. Therefore, it can be stated that dT/dt is a better indicator for catecholamine sensitivity than isometric tension. beta-adrenoceptor density [( 3H]-dihydroalprenolol binding) decreased significantly in trained rats (68.7 +/- 7.62 vs. 102.4 +/- 4.37 fmol/mg protein, p less than 0.01) with no significant difference in KD values (4.61 +/- 2.26 vs. 6.11 +/- 1.94 nM, ns). In addition, myosin isoenzyme pattern revealed by pyrophosphate gel electrophoresis shifted towards VM-1 after swimming training. The increased catecholamine sensitivity of fast contracting myocardium is, in principle, compatible with the assumption of cAMP-dependent regulation of myofibrillar ATPase activity (21) or cross bridge kinetics (9), although other postreceptor processes should also be taken into consideration for the increased catecholamine sensitivity.

The influence of endurance training on mechanical catecholamine responsiveness, beta-adrenoceptor density and myosin isoenzyme pattern of rat ventricular myocardium.

An investigation was carried out on the effects of 4 weeks' swimming training (2 X 90 min/day) on myocardial isometric tension development and rate of tension rise, and also on the changes induced therein by in vitro application of isoproterenol. This was done in 9 isolated papillary muscles of 9-week-old male Wistar rats and the results were compared with the data of age-matched sedentary controls. Ventricular beta-adrenoceptors [( 3H]-dihydroalprenolol binding) and the isoenzyme pattern of myosin (pyrophosphate gel electrophoresis) were examined in the same individuals. Isometric tension (T) and its first derivative (dT/dt) measured at the optimum of the length-tension diagram were moderately increased by long-term swimming training. Isoproterenol (10(-5) mol/l) induced a greater absolute and relative increase of both mechanical parameters in specimens of trained animals than in age-matched controls (delta T: 3.6 +/- 1.6 vs. 1.9 +/- 0.6 X 10(-2) N/mm2, p less than 0.05. delta dT/dt: 43.4 +/- 14.0 vs. 30.4 +/- 9.5 X 10(-2) N/mm2 X s, p less than 0.05). KD decreased significantly (4.23 +/- 1.0 vs. 2.44 +/- 0.3 nM, p less than 0.02), indicating an increase in receptor affinity, whereas receptor density revealed a tendency to decrease (98.8 +/- 22.6 vs. 67.1 +/- 18.0 fmol/mg protein, p less than 0.1). In addition, there was a shift in the isoenzyme pattern of myosin towards VM-1 after swimming training. Thus, under the conditions of the present experiments, the mechanical response to isoproterenol does not correlate to beta-adrenoceptor density. It is probable that, apart from the altered sensitivity of the receptors, other membrane or post-membrane processes, are responsible for the increased mechanical responsiveness to catecholamines. Although a relationship between myosin isoenzyme pattern and mechanical responsiveness to catecholamines is apparent taking into account our results and the findings on hypertensive rats as reported in the literature, it cannot be accounted for simply by altered beta-adrenoceptor density.

Relationship between the myofibrillar ATPase activity of human biopsy material and hemodynamic parameters.

The myofibrillar ATPase activity and pyrophosphate gel electrophoretic pattern of native myosin of fresh human left ventricular papillary muscles were examined in 52 cases of mitral valve replacement. The myofibrillar ATPase activity of hypertrophied myocardium did not differ from that of non-hypertrophied myocardium (mean +/- SD, 36.2 +/- 8.7 vs 31.8 +/- 8.6 nmolPi/mg/min, ns) and there was no significant difference in myofibrillar ATPase activity as a function of left ventricular enddiastolic pressure. Pyrophosphate gel electrophoresis of myosin revealed the presence of two components. It is questionable whether the component of higher electrophoretic mobility (approximately 25-35% in concentration) is identical with rat ventricular myosin VM-1 because an increase in this component seems to correlate with a decrease of myofibrillar ATPase activity, its concentration was significantly higher in the hearts with left ventricular hypertrophy, high enddiastolic pressure, high aortic pressure or low cardiac index. From these results, it is not necessarily clear whether hemodynamic overload in valvular heart diseases can alter left ventricular myofibrillar ATPase activity, but it can be said that the overload influences the concentration of the two components of native myosin revealed by pyrophosphate gel electrophoresis.

Characterization of rabbit masseter muscle fibers

Myosins of histochemically distinguishable single fibers of rabbit masseter muscle--type 1, 2A, 2B, and slow fibers--have been characterized by gel electrophoresis under dissociating (sodium dodecyl sulfate) and nondissociating (inorganic pyrophosphate) conditions, and by analysis of peptide maps of the heavy chains following limited proteolytic degradation. Type 2B fibers contain more LC3 homodimer than type 2A fibers; peptide maps of their heavy chain are different although the two myosins comigrate on pyrophosphate gel electrophoresis. Slow fiber myosin migrates more slowly than fast myosin and has a distinct peptide map. Differences were also found among fibers of the same histochemical type but originating in different muscles. In adductor magnus 2B myosin the LC1 + LC3 heterodimer band is the strongest, while in masseter 2B myosin the heterodimer is the weakest. Statistical considerations suggest that in masseter there is a mechanism preferentially forming the homodimers. More work is needed to determine the mechanism by which phenotypical differences occur among various fiber types in the same muscle and between corresponding fiber types in different muscles.

Myosin transitions in developing fast and slow muscles of the rat hindlimb

Myosin isozymes from the slow soleus and fast EDL muscles of the rat hindlimb were analyzed by pyrophosphate gel electrophoresis, by peptide mapping of heavy chains, and by antibody staining. At the earliest stage examined, 20 days gestation, distinctions between the developing fast and slow muscles were seen by all these criteria; all fibers in the distal hindlimb reacted strongly with antibody to adult fast myosin. Some fibers also reacted with antibody to adult slow myosin; these fibers had a precise, axial distribution in the hindlimb. This pattern of staining which includes the entire soleus, foreshadows the adult distribution of slow fibers and may indicate that the specific pattern of innervation of the limb is already determined. In the early developing soleus there are four fetal and neonatal isozymes plus two isozymes present in equal proportions in the ‘slow’ area of the pyrophosphate gel. The mobility of these two slow isozymes decreases with maturity and the slowest moving isozyme gradually becomes the dominant species. Thus early diversity between the soleus and EDL is expressed by myosins which are distinct from the mature isozymes. The relative proportion of slow isozymes significantly increases with development and as this occurs the fetal and neonatal isozymes are progressively eliminated. Transiently at least one mature fast isozyme appears in the soleus. This is present at 15 days postpartum and probably correlates with the population of fast, type II fibers, which comprise 50% of this muscle cell population at 15 days. The EDL contained three fetal and neonatal isozymes and only one slow isozyme which does not change in mobility with age. Slow isozymes in the soleus and EDL are thus not identical. Each muscle underwent a unique series of changes until the adult pattern of isozymes and heavy chains was reached about one month postpartum.

6] Myosin light chain phosphorylation in smooth muscle and nonmuscle cells as a probe of calmodulin function

The function of Ca2+-calmodulin-dependent phosphorylation of phosphorylatable myosin light chain (P-light chain) is currently an area of immense interest and research. In the smooth muscle, P-light chain phosphorylation is a requisite for the interaction of myosin with actin. One major problem in working with intact smooth muscle or nonmuscle cells has been the lack of a sensitive technique for quantitating the extent of P-light chain phosphorylation in preparations that are routinely used in physiological and pharmacological investigations. This chapter discusses methodology for quantitating P-light chain phosphorylation during changes in isometric tension development of the bovine tracheal smooth muscle. The applicability of the methodology for quantitating P-light chain phosphorylation in various types of vascular smooth muscles, cardiac muscle, and skeletal muscles has also been determined in the chapter. This methodology should also be adaptable to other tissues or nonmuscle cells, such as platelets and macrophages, where myosin P-light chain phosphorylation may be involved in the regulation of motile processes. The combined use of pyrophosphate gel electrophoresis, isoelectric focusing, and ammoniacal-silver staining offers a relatively rapid, accurate, and sensitive technique for quantitating the extent of P-light chain phosphorylation during changes in contractile activity in the smooth muscle.

Quantitation of myosin light chain phosphorylation in small tissue samples.

A method to quantitate the extent of phosphorylation of the 20,000-dalton phosphorylatable myosin light chain (P-light chain) in cardiac, smooth, or skeletal muscle samples which have limited amounts of tissue and myosin is presented. Native myosin is isolated from other cellular proteins in crude homogenates prepared from a few milligrams of muscle by pyrophosphate-polyacrylamide gel electrophoresis. The extent of P-light chain phosphorylation is maintained throughout this procedure by the inclusion of myosin light chain kinase and phosphatase inhibitors. Myosin obtained by pyrophosphate-gel electrophoresis is subjected to isoelectric focusing on polyacrylamide gels to separate the phosphorylated from the nonphosphorylated forms of the P-light chain. Following staining with ammonial-silver phosphorylation is quantitated on a mole of phosphate incorporated per mol of P-light chain basis by densitometric scanning of the isoelectric focusing gels. Direct comparison of this methodology with another method used for quantitating phosphorylation revealed no difference between the techniques in measuring the extent of P-light chain phosphorylation in muscle biopsy samples. This methodology provides the means for examination of the possible regulatory roles of P-light chain phosphorylation in contraction of cardiac, smooth, skeletal muscle.

Alterations in cardiac oxygen consumption under chronic pressure overload. Significance of the isoenzyme pattern of myosin.

Hypertension and resulting left ventricular hypertrophy was induced in young male Wistar rats (60 to 70 days old) by narrowing of one renal artery (Goldblatt II). 8 and 24 weeks after operation, myocardial oxygen consumption was measured on a modified in situ heart-lung preparation with nearly isovolumetric left ventricular contractions. Measured myocardial oxygen consumption was related to left ventricular wall stress. The myosin isoenzyme pattern of each heart was determined with pyrophosphate gel electrophoresis. Oxygen consumption related to wall stress averaged over the entire heart cycle amounted to 15 mumoles O2/g X min 8 weeks after operation, and 24.4 mumoles O2/g X min in age-matched controls (delta 38%, p less than 0.0005). When wall stress was averaged over systole, oxygen consumption of the hypertrophied hearts amounted to 0.112 mumoles O2/g x beat, and 0.149 mumoles O2/g x beat in the controls (delta 25%, p less than 0.05). The proportion of VM-3 (the cardiac myosin isoenzyme of lowest ATPase activity) increased from 26.3% in the controls to 30.1% in the Goldblatt hearts (delta 14%, n.s.). 24 weeks after operation, oxygen consumption related to wall stress averaged over the entire heart cycle amounted to 16.1 mumoles O2/g x min, in age-matched controls 20.5 mumoles O2/g x min (delta 21%, p less than 0.05). When wall stress was averaged over systole, oxygen consumption of the Goldblatt hearts amounted to 0.080 mumoles O2/g x beat, and in the controls 0.107 mumoles O2/g x beat (delta 25%, p less than 0.0005). The proportion of VM-3 increased from 33.5% in the controls to 43.2% in the hypertrophied hearts (delta 29%, p less than 0.05). The present findings indicate that the reduced oxygen consumption of the pressure-loaded heart should be attributed to a redistribution of myosin isoenzymes. The transformation of myocardium into a slower, but more efficiently working muscle due to an increase in VM-3 can be interpreted as an adaptational process.

Electrophoretic analyses of atrial and ventricular cardiac myosins from foetal and adult rabbits

1. 1. Rabbit cardiac myosins from atrium and ventricle were found to differ in their native form in pyrophosphate gel electrophoresis as well as in their light chain pattern in one- and two-dimensional electrophoretic systems. 2. 2. The myosin light chain pattern in sodium dodecylsulfate gel electrophoresis differs between atrial and ventricular tissues in the mammalians (rabbit, dog, human, pig, sheep and rat) but not in the chicken. 3. 3. In foetal rabbit ventricle an additional light chain is observed which, in two-dimensional electrophoresis with adult cardiac light chains, was found to be the same as atrial light chain-1.

Isoenzymes of rabbit slow myosin

Pyrophosphate gel electrophoresis has been established as a sensitive and reliable method of resolving myosin isozymes. The existence of 3 isozymes of vertebrate fast myosin differing in their alkali light chain distribution is well documented. In order of increasing mobility they represent the (LCr)a(LC&, (LCr)(LC&(LCa) and (LC&(LC& species [1,2,3]. However, in rat ventricular myosin, where only one light chain is analogous to the alkali light chains of fast skeletal myosin, the isozymes differ with respect to the iso-forms of 2 distinct heavy chains [4,5]. Fast skeletal myosin isozymes differ in the heavy chain [6-91. In case of rabbit slow myosin no isozymes were distinguishable on pyrophosphate gel [IO]. The existence of 2 different light chains (LCI,,LCIb) analogous to the alkali light chains of fast myosin [ 111, and as reported here, the existence of 2 forms of slow subfragment-l (S-l) separated in pyrophosphate gel suggests the presence of the isozymes analogous to the fast myosin isozymes. On the basis of analysis in pyrophosphate gels of single fibers, some of which contain predominantly either LC,, or LCrb, there are at least 2 isozymes of rabbit slow myosin namely, (LC&(LC& and (LCrb)z(LC2)2 horndims.

Analysis of myosin light and heavy chain types in single human skeletal muscle fibers.

In this study, myosin types in human skeletal muscle fibers were investigated with electrophoretic techniques. Single fibers were dissected out of lyophilized surgical biopsies and typed by staining for myofibrillar ATPase after preincubation in acid or alkaline buffers. After 14C-labelling of the fiber proteins in vitro by reductive methylation, the myosin light chain pattern was analysed on two-dimensional gels and the myosin heavy chains were investigated by one-dimensional peptide mapping. Surprisingly, human type I fibers, which contained only the slow heavy chain, were found to contain variable amounts of fast myosin light chains in addition to the two slow light chains LC1s and LC2s. The majority of the type I fibers in normal human muscle showed the pattern LC1s, LC2s and LC1f. Further evidence for the existence in human muscle of a hybrid myosin composed of a slow heavy chain with fast and slow light chains comes from the analysis of purified human myosin in the native state by pyrophosphate gel electrophoresis. With this method, a single band corresponding to slow myosin was obtained; this slow myosin had the light chain composition LC1s, LC2s and LC1f. Type IIA and IIB fibers, on the other hand, revealed identical light chain patterns consisting of only the fast light chains LC1f, LC2f and LC3f but were found to have different myosin havy chains. On the basis of the results presented, we suggest that the histochemical ATPase normally used for fibre typing is determined by the myosin heavy chain type (and not by the light chains). Thus, in normal human muscle a number of 'hybrid' myosins were found to occur, namely two extreme forms of fast myosins which have the same light chains but different heavy chains (IIA and IIB) and a continuum of slow forms consisting of the same heavy chain and slow light chains with a variable fast light chain composition. This is consistent with the different physiological roles these fibers are thought to have in muscle contraction.