Luisa Gorza

Three myosin heavy chain isoforms in type 2 skeletal muscle fibres

SummaryMammalian skeletal muscles consist of three main fibre types, type 1, 2A and 2B fibres, with different myosin heavy chain (MHC) composition. We have now identified another fibre type, called type 2X fibre, characterized by a specific MHC isoform. Type 2X fibres, which are widely distributed in rat skeletal muscles, can be distinguished from 2A and 2B fibres by histochemical ATPase activity and by their unique staining pattern with seven anti-MHC monoclonal antibodies. The existence of the 2X-MHC isoform was confirmed by immunoblotting analysis using muscles containing 2X fibres as a major component, such as the normal and hyperthyroid diaphragm, and the soleus muscle after high frequency chronic stimulation. 2X-MHC contains one determinant common to 2B-MHC and another common to all type 2-MHCs, but lacks epitopes specific for 2A- and 2B-MHCs, as well as an epitope present on all other MHCs. By SDS-polyacrylamide gel electrophoresis 2X-MHC shows a lower mobility compared to 2B-MHC and appears to comigrate with 2A-MHC. Muscles containing predominantly 2X-MHC display a velocity of shortening intermediate between that of slow muscles and that of fast muscles composed predominantly of 2B fibres.

Myosin isoenzymes in normal and hypertrophied human ventricular myocardium.

We tested the hypothesis that hypertrophy of the human heart is associated with the redistribution of ventricular isomyosins. Human cardiac myosin was isolated from autopsy samples of left ventricular free wall of patients with cardiac hypertrophy and of fetal, young, and adult subjects without heart disease. The following parameters were studied: elecrrophoreric migration in denaturing and non-denaturing conditions; immunological cross-reactivities with three different types of antibodies; and early phosphate burst size and steady state ATPase activities stimulated by K+-EDTA, Ca++, μg++, and actin. The antibodies were chosen for their ability to recognize selectively the rat VI and V3 cardiac isomyosins. The first type was a monoclonal antibody, CCM-52, prepared against embryonic chick cardiac myosin, the second was an anti-beef atrial myosin, and the third was an anti-rat VI myosin. CCM-52 reacted with a greater affinity with rat V3 than with rat VI, and was thus a probe of mammalian V3. Anti-beef atrial myosin and anti-rat VI myosin both recognized specifically beef atrial and rat VI myosins, and were thus considered as probes of mammalian VI. Under non-denaturing conditions, human myosins migrated as rat V3 isomyosin; under denaturing conditions, no difference was observed in any of the elecrrophoreric parameters between all samples tested, except for the fetal hearts which contained a fetal type of light chain. The immunological studies indicated that human myosins were composed mostly of a V3 type (HV3), but contained also some VI isomyosin. A technique was developed to quantify the amount of human VI isomyosin which was found to range from almost 0 to 15% of total myosin, and to vary from one heart to the other, regardless of the origin of the heart. Enzymatic studies showed no significant difference between normal, hypertrophied, and fetal hearts in any of the activities tested. However, there was a significant correlation between Ca++-stimulated ATPase activities and HV1 amount (at 0.05 M KG, n = 18, r2 = 0.49, P < 0.01; at 0.5 M KC1, n = 18, r2 = 0.5, P < 0.01). These data demonstrate the heterogeneity of human ventricular myosin, which appears to be composed, as in other mammalian species, of VI and V3 isoforms of different ATPase activities (VI > V3). However it seems that VI to V3 shifts do not appear to be of physiological significance in the adaptation of human heart to chronic mechanical overloads.

Embryonic and neonatal myosin heavy chain in denervated and paralyzed rat skeletal muscle

Using immunofluorescence procedures with specific polyclonal and monoclonal antimyosin antibodies we have found that embryonic and neonatal myosin heavy chains (MHCs), which in rat skeletal muscle disappear during the first weeks after birth, are reexpressed in adult muscle after denervation. Reactivity for embryonic and neonatal MHCs was detected in some fibers as early as 3 days after denervation, became more evident by 7 days, and occurred exclusively in the type 2A fiber population. Paralysis of innervated muscles by tetrodotoxin block of the sciatic nerve also resulted in the reappearance of embryonic and neonatal MHCs in type 2A fibers. Significant variation in the degree of immunoreactivity was observed in different segments of the same muscle fiber, suggesting that coordination of muscle fiber nuclei in the control of myosin heavy chain gene expression is partially lost following denervation.

Slow-to-fast transformation of denervated soleus muscles by chronic high-frequency stimulation in the rat.

1. Adult soleus muscles were denervated and stimulated directly for 2‐130 days with ‘fast’ (short pulse trains at 100 Hz) or slow’ (continuously at 10 Hz, or long pulse trains at 15 Hz) stimulus patterns. 2. At the end of the period of stimulation isometric twitches and tetani and isotonic shortening velocities were measured. Frozen cross‐sections were later examined with antibodies against myosin heavy chains specific for adult fast, adult slow and fetal myosin. 3. Isometric twitch duration (twitch time‐to‐peak and half‐relaxation time) decreased during intermittent 100 Hz stimulation to values that were almost as fast as in the normal extensor digitorum longus (EDL) (95 and 94% transformation). The major part of the decrease occurred between 2 and 21 days after the onset of stimulation, and was accompanied by post‐tetanic potentiation of the twitch, sag’ in tension during an unfused tetanus, lower twitch/tetanus ratio and marked shifts to the right (higher frequencies) of the tension‐frequency curve of the muscle. In contrast, during 10 or 15 Hz stimulation the isometric twitch duration remained slow, the twitch continued to show post‐tetanic depression and absence of ‘sag’, while the twitch/tetanus ratio increased. 4. Denervation per se led to a slight increase and, then, after about a month, to a moderate and gradual decrease in twitch duration. The twitch/tetanus ratio increased markedly and post‐tetanic depression became less pronounced or disappeared. Muscle weight and particularly tetanic tension were markedly reduced and these reductions were to a large extent counteracted by electrical stimulation. 5. Implantation of sham electrodes had no effect on twitch duration of denervated or innervated control muscles, but reduced tetanic tension in the innervated control muscles. 6. Maximum isotonic shortening velocity of the whole muscle (mm/s) increased during intermittent 100 Hz stimulation to a value as fast as in the normal EDL (110% transformation). Since the muscle fibres also increased in length (35%) maximum intrinsic shortening velocity (fibre lengths/s) was only incompletely transformed (55%). The increase in Vmax occurred between 7 and 14 days after the onset of stimulation. 7. All the fibres stimulated intermittently at 100 Hz were strongly labelled with anti‐fast myosin and more than 90% were in addition weakly labelled by anti‐slow myosin. Weak and variable labelling with anti‐fast myosin was first detected 7 days after the onset of stimulation. In contrast, essentially all the fibres stimulated at 10 or 15 Hz showed no binding of anti‐fast but strong binding of anti‐slow myosin.(ABSTRACT TRUNCATED AT 400 WORDS)

Myosin types in the human heart. An immunofluorescence study of normal and hypertrophied atrial and ventricular myocardium.

Two distinct myosin heavy chain isoforms, referred to as a and ft, were identified in the human heart with specific antimyosin antibodies. By indirect immunofluorescence, myosin heavy chain a was found to be a major component of atrial myosin and a minor component of ventricular myosin, while heavy chain β was found to be a major component of ventricular myosin and a minor component of atrial myosin. In the normal heart, there was marked individual variability in the proportion of ventricular myocytes reactive for heavy chain α. Atrial myocytes staining for heavy chain β were rare in the left atrium and more numerous in the right atrium, especially in the crista terminalis and in the interatrial septum. Surgical and autoptic specimens from hypertrophied left ventricles of patients with mitral regurgitation showed a myosin immu-noreactivity pattern similar to that of normal specimens. Very rare muscle cells reactive for heavy chain a were seen in the hypertrophied left ventricles of subjects with hypertension and in the hypertrophied right ventricles of subjects with tetralogy of Fallot. A dramatic transformation of myosin heavy chain composition was observed in hypertrophied left atria of patients with mitral stenosis, with a shift to heavy chain β in a large proportion of atrial myocytes. The findings indicate that chronic exposure to hemodynamic overload can induce marked changes in the myosin heavy chain composition of human atria, whereas it affects only slightly that of the ventricles.

Fibre types in extraocular muscles: a new myosin isoform in the fast fibres

SummaryWe report on the existence of a myosin heavy chain (MHC) isoform with unique structural properties in extraocular (EO) muscles. Differences in MHC composition are apparent using a polyclonal antibody prepared against myosin isolated from bovine EO muscle myosin. In enzyme immunoassays and western blotting experiments, this anti-EO myosin antibody reacted specifically with the heavy chains of EO muscle myosin and not with the heavy chains of other myosins. The distribution of this new MHC isoform in the globe rotating muscles from different mammalian species was analysed using a panel of specific anti-myosin antibodies and comparing the histochemical myosin ATPase profile of muscle fibres with their isomyosin content. Most fibres which display a type 2 ATPase reaction pattern were selectively labelled by anti-EO antibodies. A few type 2 fibres were found to react with both anti-EO and anti-2A myosin antibodies and others, located almost exclusively in the orbital layers, reacted with anti-foetals as well as anti-EO antibodies.The presence of a distinct form of myosin in EO muscle fibres is probably related to the particular functional characteristics of these muscles, which are known to be exceptionally fast-contracting but display a very low tension output.

Heart conduction system: a neural crest derivative?

Using the anti-neurofilament monoclonal antibody iC8 we report here that muscle fibers of the conduction system of the adult and developing rabbit heart express a cytoskeletal protein antigenically and electrophoretically similar to the middle subunit of neurofilaments (NF-M). In the 11-day embryo a number of cardiac muscle cells also express a neural crest surface marker recognized by the monoclonal antibody HNK-1. Both markers are found in many cells of the 3rd and 4th branchial arches, which are populated by cells of neural crest origin. In the 11-day embryo cells of the 4th branchial arch are in close proximity to and intermingled with the atrial myocardium: cells co-expressing sarcomeric myosin heavy chain with iC8 and HNK-1 immunoreactivity are seen at these sites. The findings suggest that conduction tissue cells of the rabbit heart originate from a population of neural crest-derived cells migrating from the branchial arches into the developing heart.

Overexpression of the stress-protein Grp94 reduces cardiomyocyte necrosis due to calcium overload and simulated ischemia

Increase in free intracellular calcium [Ca 2+]i plays a crucial role in cardiomyocyte ischemic injury. Here we demonstrate that overexpression of the sarcoplasmic‐reticulum stress‐protein Grp94 reduces myocyte necrosis due to calcium overload or simulated ischemia. Selective three‐to eightfold Grp94 increase, with no change in Grp78 or calreticulin amount, was achieved by stable transfection of skeletal C2C12 and cardiac H9c2 muscle cells. After exposure to the calcium ionophore A23187, LDH release from five different Grp94‐overexpressing clones of either C2C12 and H9c2 origin was significantly lower than that of control ones and [Ca 2+]i increase was significantly delayed. The number of necrotic cells, evaluated by propidium iodide uptake, was reduced when cells from the Grp94‐overexpressing H9c2 clone were exposed to conditions simulating ischemia. Experiments performed in neonatal rat cardiomyocytes co‐transfected with grp94 and the green fluorescent protein (GFP) cDNAs validated the protective effect of Grp94 overexpression. A lower percentage of propidium‐iodide positive/GFP‐fluorescent myocytes co‐expressing exogenous Grp94, with respect to myocytes expressing GFP alone, was observed after exposure to either A23187 (6.6% vs. 14.0%, respectively) or simulated ischemia (8.5% vs. 17.7%, respectively). In conclusion, the selective increase in Grp94 protects cardiomyocytes from both ischemia and calcium overload counteracting [Ca 2+]i elevations.

Effects of age on physiological, immunohistochemical and biochemical properties of fast-twitch single motor units in the rat.

1. Physiological, enzyme‐histochemical, biochemical and morphometrical properties of fast‐twitch single motor units were compared between young (3‐6 months) and old rats (20‐24 months) using the glycogen depletion technique. Monoclonal antibodies (mAbs) were used to identify the myosin heavy chain (MHC) composition in the muscle fibres of the motor unit (motor unit fibres) in order to facilitate correlative physiological, histochemical, biochemical and morphometrical studies. 2. Earlier observations on effects of age on contractile properties of fast‐twitch motor units were confirmed and extended. That is, the duration of the isometric twitch, and the twitch and tetanus forces, were increased. Further, motor unit fibres were rearranged, occupying a larger territory and displaying an increased innervation ratio in old age, indicating a denervation‐reinnervation process. 3. Motor units with muscle fibres expressing the novel IIX myosin heavy chain (MHC) were observed in both young and old animals, and they constituted the predominant motor unity type identified in the old animals. In contrast to the type IIX MHC motor units in the young animals, the type IIX MHC units in old age often contained muscle fibres which expressed either the type IIA or type IIB MHC, although type IIX MHC fibres were in the majority (so called ‘IIX’ MHC motor units), but motor units containing all these three fibre types were never observed. There were also single fibres co‐expressing IIX and IIB MHCs in old age. 4. In the young animals the IIX MHC motor units had a higher (P less than 0.001) resistance to fatigue (fatigue ratio 0.45 +/‐ 0.11) than the type IIB MHC units (0.03 +/‐ 0.05), a succinate dehydrogenase (SDH) activity (0.62 +/‐ .007) intermediate (P less than 0.001) between those of type IIA muscle fibres classified according to myofibrillar ATPase activity after acid pre‐incubation, i.e. type IIA ATPase, (0.84 +/‐ 0.13) and type IIB MHC motor unit fibres (0.20 +/‐ 0.04), and cross‐sectional fibre areas (1650 +/‐ 320 microns 2) which were similar to those of type IIA ATPase muscle fibres (1460 +/‐ 150 microns 2) but smaller (P less than 0.001) than type IIB MHC motor unit fibres (4650 +/‐ 1180 microns 2).(ABSTRACT TRUNCATED AT 400 WORDS)

Isomyosin distribution in normal and pressure-overloaded rat ventricular myocardium. An immunohistochemical study.

We have used affinity-purified antibodies reacting with guinea pig soleus muscle and ventricular myosin heavy chains to analyze the distribution of specific isomyosin in the ventricular myocardium of normal and renal hypertensive rats. Immunofluorescent staining of cardiac tissue sections with the two antimyosins revealed striking variations in reactivity among ventricular muscle fibers, reactive fibers being more numerous in the left compared to the right ventricle and in subendocardial compared to subepicardial layers. The response of the ventricular myocardium changed during development: all fibers were stained in the newborn rat, whereas most fibers were unreactive in 1-month-old animals. The number of reactive fibers increased again in subsequent stages leading to a mixed pattern in adult animals. The normal mixed pattern of reactivity was transformed into a uniformly positive pattern in hypertensive rats 2 months after surgery. This complete transformation was observed in 20 out of 23 hypertensive animals examined. These findings indicate that the two antimyosins cross-react with a particular type of ventricular myosin heavy chain, whose distribution varies in different muscle cells and whose relative concentration changes during development and during cardiac hypertrophy Induced by systemic hypertension. We suggest that differences in pressure load may be responsible for both regional variations in isomyosin distribution and for isomyosin changes in hypertensive animals.