Spatial distribution of “Tissue-Specific” antigens in the developing human heart and skeletal muscle. II. An immunohistochemical analysis of myosin heavy chain isoform expression patterns in the embryonic heart

The spatial distribution of α- and β-myosin heavy chain isoforms (MHCs) was investigated immunohistochemically in the embryonic human heart between the 4th and the 8th week of development. The development of the overall MHC isoform expression pattern can be outlined as follows: (1) In all stages examined, β-MHC is the predominant isoform in the ventricles and outflow tract (OFT), while α-MHC is the main isoform in the atria. In addition, α-MHC is also expressed in the ventricles at stage 14 and in the OFT from stage 14 to stage 19. This expression pattern is very reminiscent of that found in chicken and rat. (2) In the early embryonic stages the entire atrioventricular canal (AVC) wall expresses α-MHC whereas only the lower part expresses β-MHC. The separation of atria and ventricles by the fibrous annulus takes place at the ventricular margin of the AVC wall. Hence, the β-MHC expressing part of the AVC wall, including the right atrioventricular ring bundle, is eventually incorporated in the atria. (3) In the late embryonic stages (approx. 8 weeks of development) areas of α-MHC reappear in the ventricular myocardium, in particular in the subendocardial region at the top of the interventricular septum. These coexpressing cells are topographically related to the developing ventricular conduction system. (4) In the sinoatrial junction of all hearts examined α- and β-MHC coexpressing cells are observed. In the older stages these cells are characteristically localized at the periphery of the SA node.     

Spatial distribution of "tissue-specific" antigens in the developing human heart and skeletal muscle. I. An immunohistochemical analysis of creatine kinase isoenzyme expression patterns

Using monoclonal antibodies against the M and B subunit isoforms of creatine kinase (CK) we have investigated their distribution in developing human skeletal and cardiac muscle immunohistochemically. It is demonstrated that in skeletal muscle, a switch from CK-B to CK-M takes place around the week 8 of development, whereas in the developing heart, CK-M is the predominant isoform from the earliest stage examined onward (i.e., 4 1/2 weeks of development). In all hearts examined, local differences in concentration of the CK isoforms are observed. The CK-M expression in the developing outflow tract (OFT) and conduction system is described in detail. Between the weeks 5 and 7 of development, the distal portion of the OFT is characterized by low CK-M expression, whereas around the week 8-10 of development the myocardium around the developing semilunar valves in the OFT expresses a very high level of CK-M. At all stages examined, a relatively low CK-M level is observed in those regions in which the "slow" components of the conduction system do develop (e.g., the sinoatrial junction and atrioventricular junction), whereas a relatively high concentration of CK-M is observed in those areas that are destined to become the "fast" components, i.e., the subendocardial myocardium of the ventricles. The high expression of CK-M in the developing "fast components" of the conduction system contrasts with the relatively low expression of CK-M in the force-producing myocardium of the interventricular septum and free ventricular wall.     

Spatial distribution of “tissue-specific” antigens in the developing human heart and skeletal muscle. I. An immunohistochemical analysis of creatine kinase isoenzyme expression patterns

Using monoclonal antibodies against the M and B subunit isoforms of creatine kinase (CK) we have investigated their distribution in developing human skeletal and cardiac muscle immunohistochemically. It is demonstrated that in skeletal muscle, a switch from CK-B to CK-M takes place around the week 8 of development, whereas in the developing heart, CK-M is the predominant isoform from the earliest stage examined onward (i.e., 4½ weeks of development). In all hearts examined, local differences in concentration of the CK isoforms are observed. The CK-M expression in the developing outflow tract (OFT) and conduction system is described in detail. Between the weeks 5 and 7 of development, the distal portion of the OFT is characterized by low CK-M expression, whereas around the week 8–10 of development the myocardium around the developing semilunar valves in the OFT expresses a very high level of CK-M. At all stages examined, a relatively low CK-M level is observed in those regions in which the “slow” components of the conduction system do develop (e.g., the sinoatrial junction and atrioventricular junction), whereas a relatively high concentration of CK-M is observed in those areas that are destined to become the “fast” components, i.e., the subendocardial myocardium of the ventricles. The high expression of CK-M in the developing “fast components” of the conduction system contrasts with the relatively low expression of CK-M in the force-producing myocardium of the interventricular septum and free ventricular wall.     

Spatial and temporal patterns of myosin heavy chain expression in developing rat extraocular muscle

Summary The present study describes transitions in myosin heavy chain expression in the extraocular muscles of rats between the ages of E17 and adult. The unique phenotype of the extraocular muscle is reflected in its fibre type composition, which is comprised by six distinct profiles, each defined by location (orbital versus global layer) and innervation pattern (single versus multiple terminals). During extraocular muscle myogenesis, developmental myosin heavy chains were expressed in both primary and secondary fibres from embryonic day E17 through the first postnatal week. At this time, the downregulation of developmental myosin heavy chain isoforms began in the global layer in a fibre type-specific manner, reaching completion only after the first postnatal month. By contrast, developmental isoforms were retained in the overwhelming majority of orbital layer fibres into adulthood and expressed differentially along the length of these fibres. Fast myosin heavy chain was detected pre- and postnatally in developing secondary fibres and in all of the singly innervated fibre types and one of the multiply innervated fibre types in the adult. As many as four fast isoforms were detected in maturing extraocular muscle, including the extraocular muscle-specific myosin heavy chain. Slow myosin heavy chain was expressed in primary fibres throughout development and in one of the multiply innervated fibre types in the adult. In contrast, the pure fast-twitch retractor bulbi initially expressed slow myosin heavy chain in fibres destined to switch to the fast myosin heavy chain developmental programme. Based upon spatial and temporal patterns of myosin heavy chain isoform transitions, we suggest that epigenetic influences, rather than purely myogenic stage-specific factors, are critical in determining the unique extraocular muscle phenotype.     

Spatial distribution of "tissue-specific" antigens in the developing human heart and skeletal muscle. III. An immunohistochemical analysis of the distribution of the neural tissue antigen G1N2 in the embryonic heart; implications for the development of the atrioventricular conduction system.

A monoclonal antibody raised against an extract from the Ganglion Nodosum of the chick and designated G1N2 proves to bind specifically to a subpopulation of cardiomyocytes in the embryonic human heart. In the youngest stage examined (Carnegie stage 14, i.e., 4 1/2 weeks of development) these G1N2-expressing cells are localized in the myocardium that surrounds the foramen between the embryonic left and right ventricle. In the lesser curvature of the cardiac loop this "primary" ring occupies the lower part of the wall of the atrioventricular canal. During subsequent development, G1N2-expressing cells continue to identify the entrance to the right ventricle, but the shape of the ring changes as a result of the tissue remodelling that underlies cardiac septation. During the initial phases of this process the staining remains recognizable as a continuous band of cells in the myocardium that surrounds the developing right portion of the atrioventricular canal, subendocardially in the developing interventricular septum and around the junction of the embryonic left ventricle with the subaortic portion of the outflow tract. During the later stages of cardiac septation, the latter part of the ring discontinues to express G1N2, while upon the completion of septation, no G1N2-expressing cardiomyocytes can be detected anymore. The topographic distribution pattern of G1N suggests that the definitive ventricular conduction system derives from a ring of cells that initially surrounds the "primary" interventricular foramen. The results indicate that the atrioventricular bundle and bundle branches develop from G1N2-expressing myocytes in the interventricular septum, while the "compact" atrioventricular node develops at the junction of the band of G1N2-positive cells in the right atrioventricular junction (the right atrioventricular ring bundle) and the ("penetrating") atrioventricular bundle. A "dead-end tract" represents remnants of conductive tissue in the anterior part of the top of the interventricular septum. The location of the various components of the avian conduction system is topographically homologous with that of the G1N2-ring in the human embryonic heart, indicating a phylogenetically conserved origin of the conduction system in vertebrates.     

Changes in skeletal muscle myosin heavy chain isoform content during congestive heart failure

Recent investigations have suggested that changes in contractile protein expression contribute to reductions in skeletal muscle function during congestive heart failure (CHF). Myosin heavy chain (MHC), a major contractile protein, has been shown to undergo alterations in protein isoform expression during CHF. The purpose of this investigation was twofold: (1) to determine whether muscles of the same functional group undergo similar changes in MHC expression, and (2) determine whether the magnitude of alterations in MHC is related to the severity of CHF. Using the rat coronary ligation model, mild and severe forms of CHF were produced and muscles of the plantar flexor group were analyzed. Whole-muscle MHC isoform proportions were not altered in the soleus and white gastrocnemius muscle, however significant increases in the percentage of fast MHC isoforms (7–9% increases in MHC IIx and IIb expression) were found in the red gastrocnemius muscle. In addition, there were significant proportional increases (8%) in MHC type IIb at the expense of MHC type IIx in the plantaris muscle. Many of the changes in the proportions of MHC isoforms were significantly correlated with indices of CHF severity. This indicates that changes in skeletal muscle MHC isoform expression are related to the severity of CHF and suggests that some peripheral skeletal muscles are more susceptible to shifts in MHC expression due to CHF. These changes in MHC isoform expression may contribute to alterations in the physiological performance of skeletal muscle and exercise capacity during CHF. Electronic Publication     

Changes in the distribution of slow skeletal myosin heavy chain SM1 in developing avian muscle fibres

Summary The use of monoclonal antibodies against fast skeletal and slow skeletal myosin heavy chains (MHC) has shown the presence of significant amounts of slow skeletal type MHC in embryonic skeletal muscles of white leghorn chickens. The presence of this slow skeletal myosin heavy chain (SMHC) was not restricted to presumptive slow muscles only, as it was also observed in presumptive fast skeletal muscles. As was the case for embryonic MHC reactive with the antibody against fast skeletal myosin heavy chain (FMHC), the presence of SMHC could be detected at the earliest stages of myogenesis. It appeared to be present in most muscle cells during early embryonic development. The changes in its cellular distribution during subsequent embryonic and post-hatch period indicated its suppression in a certain proportion of the cells in both presumptive fast and slow skeletal muscles. Its time course of suppression, however, was much prolonged, not synchronized, and varied in fast and slow skeletal muscles during both embryonic and post-hatch development.     

Ectopic expression of an embryonic skeletal myosin heavy chain in human fetal and Syrian hamster hearts

The mammalian heart is known to contain only two isoformic myosin heavy chain (MHC) genes, α and β. A previously uncharacterized MHC gene was isolated in Syrian hamster hearts (McCully et al., J Mol Biol 1991). We identified the novel MHC gene as a hamster embryonic skeletal MHC gene based on the developmental stage- and tissue-specific expression pattern: the restricted expression of mRNA to striated muscles was highest in embryonic skeletal muscle and was developmentally down-regulated. We confirmed that the embryonic skeletal MHC gene exhibited higher expression in cardiomyopathic than in normal hamster hearts, and was up-regulated during the development of cardiomyopathy. The sporadic expression was highly localized in the endocardium. The present study identified that a very small number of undifferentiated myogenic cells existed in adult hamster endocardium. Moreover, using RT-PCR, a homologue of embryonic skeletal MHC mRNA was also expressed in human embryonic, but not adult ventricles. Our data provide a new insight into the regulatory mechanisms of MHCs in the cardiomyopathic hamster heart. This revised version was published online in July 2006 with corrections to the Cover Date.     

Mechanical properties and myosin heavy chain isoform composition of skinned skeletal muscle fibres from a human biopsy sample

 Experiments were conducted to investigate the mechanics of contraction of chemically skinned muscle fibre segments of a biopsied sample of single human quadriceps muscle. Subsequently, the isoforms of the myosin heavy chain (MHC) were analysed by sodium dodecyl sulphate (SDS) gel electrophoresis.Of the 41 fibres, 26 contained MHCI (type I), 11 of the fibres contained MHCIIa (type IIA), and 4 of the fibres contained both MHCI and MHCIIa (of which MHCIIa was always slightly predominant (type IIC)). Distinct differences between fibre types were found in terms of the kinetics of force responses following stepwise length changes (order of velocity: IIA > IIC > I). The differences in maximal shortening velocity and in the kinetics of Ca²⁺-dependent activation were of the same order, but much less pronounced. Type I fibres had significantly greater fibre diameters than type IIA fibres. No significant differences were found among different fibre types in terms of isometric tension, resting sarcomere length or the length change needed to discharge the elasticity of maximally Ca²⁺-activated fibres (y _o value). The distribution of shortening velocity and kinetics of stretch activation values suggest that two muscle fibre subtypes may exist in human type I fibres. Received: 15 April 1997 / Received after revision 19 May 1997 / Accepted: 20 May 1997     

Evidence for the expression of neonatal skeletal myosin heavy chain in primary myocardium and cardiac conduction tissue in the developing chick heart.

We isolated a neonatal skeletal myosin heavy chain (MHC) cDNA clone, CV11E1, from a cDNA library of embryonic chick ventricle. At early cardiogenesis, diffuse expression of neonatal skeletal MHC mRNA was first detected in the heart tube at stage 10. During subsequent embryonic stages, the expression of the mRNA in the atrium was upregulated until shortly after birth. It then diminished, dramatically, and disappeared in the adult. On the other hand, in the ventricle, only a trace of the expression was detected throughout embryonic life and in the adult. However, transient expression of mRNA in the ventricle was observed, post-hatching. At the protein level, during the embryonic stage, the atrial myocardium was stained diffusely with monoclonal antibody 2E9, specific for chick neonatal skeletal MHC, whereas the ventricles showed weak reactivity with 2E9. At the late embryonic and newly hatched stages, 2E9-positive cells were located clearly in the subendocardial layer, and around the blood vessels of the atrial and ventricular myocardium. These results provide the first evidence that the neonatal skeletal MHC gene is expressed in developing chick hearts. This MHC appears during early cardiogenesis and is then localized in cardiac conduction cells. Dev Dyn 2000;217:37-49.     

Ageing alters the myosin heavy chain composition of single fibres from human skeletal muscle

The myosin heavy chain composition of single fibres (n = 1088) was analysed with an electrophoretic technique in biopsy material from m. vastus lateralis (n = 5) and m. biceps brachii (n = 4) of young (23-31 years old) and elderly men (68-70 years old). In m. vastus lateralis, elderly subjects had a higher proportion of fibres showing a coexistence of myosin heavy chain types I and IIa (20 +/- 3% vs 8 +/- 1%, P less than 0.05) and of myosin heavy chain types IIa and IIb (33 +/- 2% vs 12 +/- 4%, P less than 0.05). In contrast, the young subjects had a higher proportion of fibres containing only myosin heavy chain type I (50 +/- 5% vs 33 +/- %, P less than 0.05) and type IIa (26 +/- 3% vs 12 +/- 2%, P less than 0.05). A similar pattern of myosin heavy chain expression was found in single fibres from m. biceps brachii, with the exception that the elderly subjects had a lower proportion of fibres with coexistence of types IIa and IIb (23 +/- 1% vs 34 +/- 2%, P less than 0.05) and a higher proportion of fibres containing only myosin heavy chain type IIa (25 +/- 5% vs 12 +/- 2%, P less than 0.05). Three fibres from m. biceps brachii contained all three isoforms. These results indicate that coexistence of myosin heavy chain isoforms in single fibres is present in skeletal muscles of young adults, and that there is an increased occurrence of this phenomenon with ageing.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced embryonic nonmuscle myosin heavy chain isoform and matrix metalloproteinase expression in aortic abdominal aneurysm with rapid progression.

Abdominal aortic aneurysms (AAAs) are characterized by structural deterioration of aortic wall leading to progressive dilatation. The histopathological changes in AAAs are particularly evident within the elastic media, which is normally comprised mainly of vascular smooth muscle cells (SMCs). There are vascular myosin heavy chain (MHC) isoforms; SM2 is specifically expressed in differentiated SMCs and SMemb is a nonmuscle-type MHC abundantly expressed in SMCs of the fetal aorta with an immature phenotype. Although AAA altered expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), pathophysiological role of SMC phenotypic modulation in the AAA progression remains uncertain. To determine whether phenotypic modulation in vascular SMCs contributes to arterial medial degeneration, we examined MHC expression in SMCs of AAA. Aortic specimens were obtained from patients with slowly progressed AAA (n = 12) and rapidly progressed AAA (n = 5), and compared with normal aortic tissue (n = 3). Immunohistochemical staining was performed for detection of SMemb, SM2, MMP (types 2 and 9) and TIMP (types 1 and 2). Faint SMemb and abundant SM2 were observed in normal aorta, while the balance shifted to SMemb predominance in AAAs. Compared with slowly progressed AAA tissue, rapidly expanded AAA tissue demonstrated marked increases in SMemb expression with suppressed SM2. Predominant SMemb expression indicates presence of phenotypic modulated SMCs and enhanced MMP; while abundant TIMP was seen in mature SMCs expressing SM2. SMemb expression is markedly increased in AAA with MMP enhancement, and a significant imbalance between SMemb and SM2 results in rapid progression of AAA.     

An age-related type IIB to IIX myosin heavy chain switching in rat skeletal muscle

A novel fast-twitch motor unit type, called the IIX-myosin heavy chain (MHC) motor unit, identified by the glycogen depletion technique together with a series of monoclonal antibodies (mAbs) specific for MHCs, has been isolated recently in the rat tibialis anterior muscle. In young animals, this unit has physiological, biochemical and morphometrical properties which separate it from the IIA- and IIB-MHC motor units. In old age, on the other hand, the IIX-MHC units display physiological, biochemical and morphometrical properties resembling the IIB-MHC motor units. Based on these results it was proposed that a transition from IIB to IIX motor units occurs during ageing. In an attempt to clarify this point, the MHC composition was identified by 6% SDS-PAGE and immunoblotting analysis, using specific mAbs antibodies, in the same fast-twitch tibialis anterior muscles in young (3–6 months, n = 9) and old (20–24 months, n = 16) rats from which the single motor units had been identified previously. The IIX-MHC comigrates together with the HA-MHC band in 6% SDS-PAGE and only two MHC bands are observed in the rat tibialis anterior muscle, i.e. the IIA-IIX-and IIB-MHC bands. A significant increase (P < 0.001) in the average relative amount of the HA-IIX —MHC was observed in the old (45 ± 17%) as compared to the young (23 ± 4%) animals, accompanied by a corresponding decrease in IIB-MHC content. It was demonstrated in immunoblotting analysis that only trace amounts of IIA-MHC were detectable in the IIA-IIX-MHC band in both young and old TA muscles, indicating a substantial increase in the IIX-MHC content in old age. Thus, the present results together with previous observations at the motor unit level strongly support an age-related motor unit transition from type IIB- to IIX-MHC.     

Differences in sodium voltage-gated channel properties according to myosin heavy chain isoform expression in single muscle fibres

The myosin heavy chain (MHC) isoform determines the characteristics and shortening velocity of muscle fibres. The functional properties of the muscle fibre are also conditioned by its membrane excitability through the electrophysiological properties of sodium voltage-gated channels. Macropatch-clamp is used to study sodium channels in fibres from peroneus longus (PL) and soleus (Sol) muscles (Wistar rats, n = 8). After patch-clamp recordings, single fibres are identified by SDS-PAGE electrophoresis according to their myosin heavy chain isoform (slow type I and the three fast types IIa, IIx, IIb). Characteristics of sodium currents are compared (Student's t test) between fibres exhibiting only one MHC isoform. Four MHC isoforms are identified in PL and only type I in Sol single fibres. In PL, maximal sodium current ( I _max), maximal sodium conductance ( g _Na,max) and time constants of activation and inactivation (τ _m and τ _h ) increase according to the scheme I→IIa→IIx→IIb ( P < 0.05). τ _m values related to sodium channel type and/or function, are similar in Sol I and PL IIb fibres ( P = 0.97) despite different contractile properties. The voltage dependence of activation ( V _a,1/2) shows a shift towards positive potentials from Sol type I to IIa, IIx and finally IIb fibres from PL ( P < 0.05). These data are consistent with the earlier recruitment of slow fibres in a fast-mixed muscle like PL, while slow fibres of postural muscle such as soleus could be recruited in the same ways as IIb fibres in a fast muscle.     

Correlation between percentage fiber type area and myosin heavy chain content in human skeletal muscle

Histochemical methods are routinely used to delineate skeletal muscle fiber types. In the present investigation, this qualitative determination of fiber type composition was compared to the electrophoretically determined myosin heavy chain (MHC) content from a large number of human muscle biopsy samples. Biopsies were taken from the vastus lateralis muscle at the beginning and every 2 weeks during 8 weeks of highi-ntensity resistance training from men (n = 13) and woman (n = 8). Muscle was also extracted from nontraining men (n = 7) and women (n = 5) at the same periods. Six muscle fiber types (I, IC, IIAC, IIA, IIAB, and IIB) were determined using basic myofibrillar adenosine triphosphatase histochemistry. Cross-sectional areas were determined for the three major fiber types (I, IIA, and IIB) and used to calculate the percentage area of these types. Electrophoretic techniques were used to separate and quantify the percentage MHC content in these same biopsy samples, and these data were then used to compare with the percentage fiber type area. Correlation analyses suggest a relationship between the histochemically assessed percentage fiber type area and the electrophoretically assessed MHC content in human limb musculature. However, because of possible histochemical misclassification of some fibers (especially in trained muscle) both techniques may be essential in yielding important information about fiber type composition and possible fiber type transformations.     

An electrophoretic study of myosin heavy chain expression in skeletal muscles of the toad Bufo marinus

In this study we developed an SDS-PAGE protocol which for the first time separates effectively all myosin heavy chain (MHC) isoforms expected to be expressed in iliofibularis (IF), pyriformis (PYR), cruralis (CRU) and sartorius (SAR) muscles of the toad Bufo marinus on the basis of previously reported fibre type composition. The main feature of the method is the use of alanine instead of glycine both in the separating gel and in the running buffer. The correlation between the MHC isoform composition of IF, SAR and PYR muscles determined in this study and the previously reported fibre type composition of IF and SAR muscles in the toad and of PYR muscle in the frog was used to tentatively identify the MHC isoforms expressed by twitch fibre types 1, 2 and 3 and by tonic fibres. The alanine-SDS electrophoretic method was employed to examine changes in the MHC composition of IF, PYR, CRU and SAR muscles with the ontogenetic growth of the toad from post-natal life (body weight < 1 g) to late adulthood (body weight 200–450 g). The developmental changes in the MHC isoform composition of the toad IF muscle observed in this study are in very good agreement with those in the fibre type composition of the developing IF muscle reported in the literature.     

Electrophoretic separation of human skeletal muscle myosin heavy chain isoforms: the importance of reducing agents

An electrophoretic protocol previously used for the separation of rat myosin heavy chain (MHC) isoforms was slightly modified to improve the separation of human MHC isoforms in both large and minigel systems. The addition of reducing agents (beta-mercaptoethanol or dithiothreitol) to the top running buffer (TRB) radically improved separated MHC isoform resolution and the intensity of electrophoretic runs lasting longer than 5 h. In minigel systems, the MHC isoforms could be separated in as little as 5 h. The improved resolution of bands with the inclusion of reducing agents to the TRB facilitated the identification of clear boundaries for densitometric quantification of relative MHC isoform content, particularly for MHC IIa and MHC IIx. No significant effect of these reducing agents added to the TRB was observed for runs lasting only 100 min. Thus the inclusion of reducing agents in the TRB is essential for long electrophoretic runs, usually when separating large molecular mass proteins.