Anabolic-androgenic steroids induce apoptotic cell death in adult rat ventricular myocytes.

We tested whether exposure to anabolic-androgenic steroids (AASs) would induce apoptosis in adult rat ventricular myocytes in vitro. Myocytes were exposed to stanozolol (STZ), testosterone enanthate (TE) and testosterone (T) (0.1 micromol/L, 1 micromol/L, 10 micromol/L, and 100 micromol/L) for 20 h. The percentage of myocytes undergoing apoptosis was determined by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) and was found to be increased when compared to control myocytes at STZ 10 micromol/L 12 +/- 2% (mean +/- SD), STZ 100 micromol/L 42 +/- 3%; TE 1 micromol/L 11 +/- 2%, TE 10 micromol/L 21 +/- 3%, TE 100 micromol/L 62 +/- 2%; T 10 micromol/L 11 +/- 2%, T 100 micromol/L 40 +/- 3% (P < 0.001 vs. CTL 2 +/- 2%). The STZ-, TE- and T-induced dose-dependent apoptotic cell death was corroborated by a significantly increased DNA laddering in myocytes exposed to STZ and T > or = 10 micromol/L and TE > or = 1 micromol/L. Notably, STZ, TE, and T exposure markedly increased the expression of the pro-apoptotic oncogene Bax-alpha, as assessed by reverse transcription-polymerase chain reaction. Taken together, these results clearly show for the first time that AASs induce apoptotic cell death in a dose-dependent manner. This finding may have important implications in understanding the pathogenesis of ventricular remodeling, cardiomyopathy, and sudden cardiac death associated with AAS abuse.

Beta-adrenergic receptor subtypes differentially affect apoptosis in adult rat ventricular myocytes.

BACKGROUND-Catecholamine-induced apoptosis is mediated by activation of the beta-adrenergic signaling pathway. We tested the hypothesis that beta(1)- and beta(2)-adrenergic receptor (AR) subtypes differentially affect apoptosis in adult rat ventricular myocytes in vitro. METHODS AND RESULTS-Myocytes were first exposed to norepinephrine (NE) alone (10 mcmol/L) or NE+atenolol (AT) (10 mcmol/L) for 12 hours. AT, a beta(1)-selective AR antagonist, abolished the NE-induced increase in nick end-labeling (TUNEL)-positive cells compared with control (NE, 33+/-3% versus control, 3+/-1%, P<0.0001; NE+AT, 4+/-2% versus control, 3+/-1%, P=0. 98). Annexin V staining, DNA laddering, and caspase activity determinations corroborated these results. Subsequent experiments under prazosin treatment established the apoptosis dose-response curves for the increasingly beta(2)-selective AR agonists isoproterenol (ISO) (beta(1) approximately beta(2)) and albuterol (ALB) (beta(2)>beta(1)). ISO and ALB induced significantly less apoptosis than NE (beta(1)>beta(2)) at equimolar concentrations as assessed by TUNEL staining [1 mcmol/L: NE (8+/-2%) approximately ISO (7+/-1%)>ALB (2+/-1%); 10 mcmol/L: NE (35+/-2%)>ISO (23+/-1%)>ALB (3+/-1%); 100 mcmol/L: NE (50+/-2%)>ISO (29+/-2%)>ALB (14+/-1%), P<0.0001 except for NE versus ISO at 1 mcmol/L with P=0.62]. ALB-induced apoptosis at 100 mcmol/L was abolished by AT (10 mcmol/L), indicating a beta(1)AR-mediated effect. Importantly, ICI 118551 (0.1 mcmol/L), a highly selective beta(2)AR antagonist, did not decrease the percentage of NE-, ISO-, and ALB-induced apoptosis. Reverse transcription-polymerase chain reaction studies revealed that AT completely reversed the beta-adrenergic signaling-induced changes in the Bcl-2-to-Bax ratio. CONCLUSIONS-These observations provide evidence that beta AR-mediated apoptotic death signaling is largely dissociated from beta(2)ARs and selectively mediated by beta(1)ARs in adult rat ventricular myocytes.

Norepinephrine-induced apoptosis is inhibited in adult rat ventricular myocytes exposed to volatile anesthetics.

BACKGROUND: Volatile anesthetics are used to provide anesthesia to patients with heart disease under heightened adrenergic drive. The purpose of this study was to test whether volatile anesthetics can inhibit norepinephrine (NE)-induced apoptosis in cardiomyocytes. METHODS: Rat ventricular cardiomyocytes were exposed to NE (10 microm) alone or in the presence of increasing concentrations of isoflurane and halothane. RESULTS: Isoflurane at 1.6 minimum alveolar concentration (MAC) (4 +/- 2% [SD]) and halothane at 1.2 MAC (3 +/- 2%) abolished the percentage of cardiomyocytes undergoing NE-induced apoptosis (34 +/- 8%), as assessed by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) (P < 0.0001). Lower concentrations of isoflurane and halothane markedly decreased the number of TUNEL-positive cells. Similarly, isoflurane at 1.6 MAC (5 +/- 3%) and halothane at 1.2 MAC (6 +/- 3%) prevented the increase in annexinV-staining cardiomyocytes (38 +/- 7%; P < 0. 0001). These findings were corroborated with a decreased quantity of NE-induced DNA laddering by volatile anesthetics. Halothane at 1.2 MAC abolished the increase in TUNEL-positive cardiomyocytes exposed to the dihydropyridine Ca2+-channel agonist BAY K-8644 (1 microm) (BAY K-8644 + halothane: 3 +/- 2% vsBAY K-8644: 34 +/- 6%; P < 0. 0001) and the Ca2+-ionophore 4-bromo-A23187 (1 microm) (4-bromo-A23187 + halothane: 2 +/- 2% vs4-bromo-A23187: 13 +/- 4%; P = 0.03). NE treatment increased caspase-9 activity to 197 +/- 62% over control myocytes (P < 0.0001), whereas no caspase-8 activation was detectable. This increase in caspase-9 activity was blocked by isoflurane at 1.6 MAC and halothane at 1.2 MAC. CONCLUSIONS: Volatile anesthetics offer significant protection against beta-adrenergic apoptotic death signaling in ventricular cardiomyocytes. The authors present evidence that this protection is mainly mediated through modulation of cellular Ca2+ homeostasis and inhibition of the apoptosis initiator caspase-9.

(CTG)n repeats markedly inhibit differentiation of the C2C12 myoblast cell line: implications for congenital myotonic dystrophy.

Although the mutation for myotonic dystrophy has been identified as a (CTG)n repeat expansion located in the 3'-untranslated region of a gene located on chromosome 19, the mechanism of disease pathogenesis is not understood. The objective of this study was to assess the effect of (CTG)n repeats on the differentiation of myoblasts in cell culture. We report here that C2C12 myoblast cell lines permanently transfected with plasmid expressing 500 bases long CTG repeat sequences, exhibited a drastic reduction in their ability to fuse and differentiate into myotubes. The percentage of cells fused into myotubes in C2 C12 cells (53.4+/-4.4%) was strikingly different from those in the two CTG repeat carrying clones (1.8+/-0.4% and 3.3+/-0. 7%). Control C2C12 cells permanently transfected with vector alone did not show such an effect. This finding may have important implications in understanding the pathogenesis of congenital myotonic dystrophy.

Myotonic dystrophy: decreased levels of myotonin protein kinase (Mt-PK) leads to apoptosis in muscle cells.

The pathogenesis of myotonic dystrophy (DM) and the function of the product of the DM gene, myotonin protein kinase (Mt-PK), and its relationship to the disease are uncertain. To gain insight into the function of Mt-PK we studied the effect of decreasing the levels of Mt-PK in cultured human myoblasts. Myoblasts were transfected with an anti-sense oligonucleotide (ODN) targeted to the translation initiation site of DM mRNA which resulted in about 76% reduction in the levels of Mt-PK protein. A large percentage (about 48 to 90%) of myoblasts transfected with this oligonucleotide (but only about 2 to 23% of myoblasts transfected with a control oligonucleotide) underwent apoptosis within 24 h. To further substantiate these results we delivered a specific antibody to Mt-PK into the myoblast cells using a lipid carrier to inhibit its function and show that this resulted in apoptosis in 57 to 72% of the cells within 24 h. These results suggest that decreased levels of Mt-PK may contribute to muscle pathology in DM by leading to apoptosis of muscle cells.

In situ hybridization analysis for expression of myogenic regulatory factors in regenerating muscle of mdx mouse.

Precise temporal and spatial coordination of expression of the myogenic regulatory factors (MRF) plays a critical role in the development of skeletal muscle. Whether this pattern is recapitulated postnatally during regeneration of mature muscle after injury is not known. The aim of this study was to determine the cellular distribution of mRNA expression of the various myogenic regulatory factors (MyoD, Myogenin, Myf-5 and Myf-6) during regeneration in mature skeletal muscle. We used the mdx mouse, an animal model for Duchenne muscular dystrophy, which undergoes necrosis and vigorous regeneration of muscle fibers, as a natural model to study muscle fibers at various stages of maturity ranging from satellite cells to mature muscle fibers. In situ hybridization analysis revealed that MyoD expression was the most widespread and was expressed in satellite cells and small regenerating muscle fibers surrounding necrotic fibers. Myogenin, Myf5, and Myf6 were expressed weakly in some immature fibers and none of the MRF's could be detected in mature muscle. These findings, taken together with previous studies, suggests that the pattern of expression of the various MRF's in regenerating skeletal muscle differs from that in developing muscle in embryos and that MyoD may play a critical role in the initiation and progression of events which lead to regeneration in mature skeletal muscle.

Evidence for new isoform of fast myosin heavy chain in rat skeletal muscle.

The purpose of this study was to investigate whether the previously demonstrated heterogeneity of myosin heavy chain (MHC) in type 2B fibers of the superficial portion of the lateral gastrocnemius (SLG) muscle of the rat may be due to presence of type 2D/X fibers. Immunohistochemical identification of MHC heterogeneity, histochemical identification of appendicular 2D/X muscle fibers, and immunoblotting of myosins were used. It was found that some, but not all, of the heterogeneity of rat SLG is correlated with the presence of type 2D/X fibers. Immunoblots with MHC from several muscles revealed the presence of at least two antigenically distinct isoforms of MHC with the same electrophoretic mobility as the "2B" band. These results show that a previously undetected type of MHC is expressed in rat skeletal muscle and raise the possibility that there may be as yet undetected MHC isoforms in human muscle which may be clinically important.

Centronuclear myopathy heterogeneity: distinction of clinical types by myosin isoform patterns.

We studied muscles from 3 patients with centronuclear myopathy (CNM) by immunocytochemistry using myosin heavy chain (MHC)-specific monoclonal antibodies to determine whether subtypes of CNM express prenatal MHC and to assess if there is an arrest in development of these muscles. Muscle from a woman with childhood-onset CNM did not express prenatal MHC, yet this prenatal MHC was strongly expressed in the muscle fibers of 2 brothers with X-linked CNM. This finding represents the 1st immunocytochemical evidence of the expression of a prenatal myosin isoform in nonregenerating postnatal human muscle and suggests that the X-linked form of CNM differs from the other types because of a true arrest in maturation of the muscle.

Werdnig-Hoffmann disease: myosin isoform expression not arrested at prenatal stage of development.

It has been suggested, on the basis of mostly morphological and some biochemical evidence, that defective innervation of muscle of patients with Werdnig-Hoffmann (WH) disease results in maturational arrest of the fibers at a stage comparable to 20-week gestational muscle. Therefore, with the use of recently developed and characterized, myosin-isoform-specific monoclonal antibodies (McAbs), an immunocytochemical study of muscle of 6 children with Werdnig-Hoffmann disease was done to determine if the pattern of expression of myosin heavy chain isoforms (MHC) in these fibers was similar to that of 20-week gestation muscle. This work showed that the MHC isoform expression in the muscle of the children with WH did not mimic that seen in 20-week gestation muscle since only a few fibers (less than 1-11%) in each specimen expressed prenatal MHC as detected by reactivity to McAb, ALD 180 (specific for a prenatal MHC) whereas virtually all of the fibers from 20-week gestation muscle were strongly reactive with ALD 180. The majority of the fibers expressed either adult fast MHC or adult slow MHC similar to that seen in normal muscle, although some co-expressed multiple MHC isoforms. Our results indicate that the difference of adult MHC isoforms in the muscle fibers of WH patients either proceeds in the absence of innervation or that denervation of muscle fibers is subsequent to the neural input required to initiate myosin isoform transitions to the adult isoforms.

Monoclonal antibody ALD 180: a reagent specific for a human prenatal skeletal muscle myosin isoform.

We report the characterization of monoclonal antibody (MAb) ALD 180, prepared against the myosin of slow avian muscle, for studies of human muscle development and disease. With the use of radioimmunoassays, Western immunoblots of native and denatured myosins, and epifluorescent indirect immunocytochemistry, we show that ALD 180 is specific for an epitope in human prenatal skeletal muscle myosin heavy chain (MHC), which is expressed in diminishing abundance in fetal fibers from at least 19-22 weeks' gestation to term and also in regenerating muscle fibers seen in diseased muscles from both children and adults. ALD 180 recognizes an epitope apparently different from those reacting with anti-prenatal human myosin MAb previously described, and therefore affords a complementary reagent for use in future studies of human myosin isoform expression and regulation.

Coexpression of myosin isoforms in muscle of patients with neurogenic disease.

Three well-characterized antimyosin heavy chain monoclonal antibodies (McAbs) were used as immunocytochemical reagents to study myosin isoform expression in relationship to adenosine triphosphatase (ATPase) defined fiber types in human muscle. The biopsy specimens were from patients with neurogenic muscle disease whose muscle exhibited fiber type grouping and group atrophy. The use of McAbs revealed heretofore unrecognized coexpression of multiple myosin isoforms in selected fibers in the pathologic samples which was not apparent with ATPase reactions and not present in normal muscle. The fibers containing multiple myosin isoforms were probably undergoing neurally directed fiber type transformation. Furthermore, a small population of fibers in neurogenic specimens expressed a "prenatal" myosin signifying the presence of regenerating fibers. We also demonstrated immunocytochemical evidence of the persistence of adult slow myosin in denervated mature human skeletal muscle despite the reputed necessity of innervation for maintenance of expression of this myosin isoform proffered by others.

Heterogeneity of fast-oxidative muscle fibers of chicken demonstrated by anti-myosin monoclonal antibodies.

The fiber type composition of two fast muscles of the chicken, namely, adductor superficialis (AS) and pectoralis major (PM) was examined by the histochemical myosin ATPase staining and immunochemical techniques using monoclonal antibodies (McAbs). Two new McAbs produced against the myosin of the anterior latissimus dorsi (ALD) muscle of the chicken and named ALD-122 and ALD-83 were characterized to be specific for myosin heavy chain (MHC) and for myosin light chain-1 respectively. They were used in conjunction with previously reported McAbs specific for slow MHC (ALD-47), fast MHC (MF-14) and fast light chain-2 (MF-5). By the histochemical ATPase test most muscle fibers of AS and PM muscles reacted as IIA and IIB respectively. By immunofluorescent staining with the anti-MHC McAbs, ALD-122 and MF-14, the fibers of AS muscle showed remarkable heterogeneity whereas PM muscle fibers reacted uniformly. Differences in the myosin light chain composition of AS and PM muscles were also found by SDS-gel electrophoresis and immunoblot analysis with the anti-light chain McAb, ALD-83. The study clearly indicated that the histochemically homogenous (type IIA) AS muscle is composed of several subpopulations of fibers which differ in their myosin composition and that this heterogeneity of the muscle is not simply due to presence of variable amounts of slow myosin in its fibers.

Fiber types in normal and neonatally denervated fast muscles of the rat: immunocytochemical study with an antimyosin monoclonal antibody.

The differentiation of fiber types in normal and neonatally denervated gastrocnemius muscles of the rat was compared by myosin ATPase histochemistry and immunocytochemistry using a monoclonal antibody, HM-1.2. The specificity of HM-1.2 for the fast myosin heavy chain was determined by radioimmunoassay, immunoautoradiography, and indirect immunofluorescence techniques. In normal 1-month-old and adult rats, the type IIB (fast glycolytic) fibers of the gastrocnemius could be clearly divided into three subtypes by their graded immunofluorescence staining with the myosin heavy chain-specific monoclonal antibody. In the gastrocnemius muscle of the newborn rat, all fibers were negative with the monoclonal antibody. The transition from negative to three grades of immunoreactivity occurred 1 to 2 weeks postnatally. After neonatal denervation of the gastrocnemius muscle, however, uniformly positive monoclonal antibody immunofluorescence staining for the myosin heavy chain was observed without subtype differentiation. This study, thus, gave clear immunocytochemical evidence that the type IIB muscle fibers are heterogeneous with respect to their myosin isoform and that the expression of this heterogeneity is dependent on the normal developmental influence of motor innervation on the muscle fibers.

An immunocytochemical study of type I muscle fibres in developing human skeletal muscles.

An immunocytochemical study was done on the skeletal muscles of human fetuses (19-36 weeks gestation), infants and adults using a new monoclonal antibody (McAb) ALD-47. The antibody was generated against slow myosin of chicken and is specific for myosin heavy chain (MHC). In human infants and adults the type I muscle fibres are strongly reactive with this McAb and the type II fibres uniformly non-reactive. In the fetuses from 19-20 weeks gestation (in whom the fibre types are not distinguishable by the histochemical myosin ATPase test) a proportion of muscle fibres react specifically with ALD-47. Other muscle fibres at this stage react positively with a fast specific MHC McAb HM-1.2 or are negative to both ALD-47 and HM-1.2 antibodies. These McAbs, thus, identify three distinct fibre populations in the early fetal muscle which by histochemical staining appears homogeneous. The percentage of ALD-47 positive fibres increases in fetuses at later gestational periods; at all stages these fibres lack reactivity with the HM-1.2 antibody. Because of its selective fibre type reactivity in differentiating muscles, the McAb ALD-47 in conjunction with HM-1.2 should be useful in immunoaffinity fractionation and biochemical studies of myosin isoforms in developing human muscles.

Differentiation of the anterior latissimus dorsi muscle of the chicken examined by anti-myosin monoclonal antibodies.

Two new monoclonal antibodies (McAbs), ALD-180 and ALD-88, produced against the myosin of the slow anterior latissimus dorsi (ALD) muscle of the chicken are described. Their specificity for myosin heavy chain (MHC) was established by radioimmunoassay, immunoautoradiography, and immunofluorescence. They were used in conjunction with McAbs MF-14 and MF-30 (which have been characterized previously to be directed against MHC of the fast skeletal muscle) to examine the developmental changes of the chicken ALD muscle. At the 16-day embryonic, early posthatch, and adult stages the ALD muscle fibers differed in their reaction pattern with the McAbs; at the embryonic stage all fibers reacted strongly with ALD-180 and weakly with ALD-88 and MF-30; at the early posthatch stage there was a checkerboard pattern with many fibers not reacting with any of these three McAbs; and at the adult stage all fibers reacted strongly with ALD-180 and ALD-88 and weakly with MF-30. The MF-14 antibody did not react with ALD muscle at any developmental stage. The mature pattern of immunoreactivity of the ALD muscle fibers with the antibodies was established only after 9 weeks posthatch, and during this 9-week period the immunofluorescence changes were nonsynchronous. Based on immunocytochemical evidence of changes in myosin isoform expression, this study clearly demonstrates a distinctive neonatal (early posthatch) stage in the development of the chicken slow muscle.

Detection of a ventricular-specific myosin heavy chain in adult and developing chicken heart.

In the present study, a monoclonal antibody (McAb), ALD19, generated against myosin of slow tonic muscle, was shown to react with the heavy chain of ventricular myosin in the adult chicken heart. With this antibody, it was possible to detect a ventricular-specific myosin during myocardial differentiation and to show that the epitope recognized by ALD19 was present from the earliest stages of ventricular differentiation and maintained throughout development only in the ventricle. A second McAb, specific for atrial myosin heavy chain (MHC) (Gonzalez-Sanchez, A., and D. Bader, 1984, Dev. Biol., 103:151-158), was used as a control to detect an atrial-specific myosin in the caudal portion of the developing heart at Hamburger-Hamilton stage 15. It was found that the appearance of ventricular MHC predated the expression of atrial MHC by approximately 1 d in ovo and that specific MHCs were always differentially distributed. While a common primordial MHC may be present in the early heart, this study showed the tissue-specific expression of a ventricular MHC during the initial stages of heart development and its differential accumulation throughout development.

Characterization of a monoclonal antibody to myosin specific for mammalian and human type II muscle fibers.

We have characterized a monoclonal antibody (McAb), ALD-19, generated against slow myosin from chicken anterior latissimus dorsi (ALD) muscle for use in studies of human and animal muscle fiber types. This McAb bound selectively to the 200 kDa myosin heavy chain band in immunoblots against chicken, rat and human myosins and showed selective staining of A bands in the myofibrils. The reactivity of ALD-19 with various myosin types was quantitated by radioimmunoassays. Fiber type analysis revealed unexpected specificity of McAb ALD-19 for type II mammalian muscle fibers. This antibody should, therefore be useful for identification and quantification of normal type II fibers in human muscle biopsy specimens.

Avian adductor profundus muscle: characterization of a pure slow tonic region by histochemical, monoclonal antibody and peptide mapping studies.

The fibre type composition of the avian adductor profundus (AP) muscle which is composed of a thick white posterior part (Post. AP) and a thin red anterior part (Ant. AP) was investigated. Using the histochemical ATPase technique, monoclonal antibody analysis of myosin and C-protein isoforms, and electrophoretic and peptide mapping analyses of myosin, we have established that the Post. AP is composed of essentially pure slow tonic fibres similar to those of the anterior latissimus dorsi muscle (ALD). The Ant. AP, on the other hand, is shown to contain a mixture of slow and fast fibres, the latter giving immunocytochemical reactions atypical of the fast fibres. The larger size of the Post. AP in comparison with the ALD muscle should provide significantly more tissue for biochemical studies of tonic fibres than was previously available.

A freeze-fracture study of postembryonic differentiation of latissimus dorsi muscles of the chicken.

The differentiation of fiber type characteristics in the anterior (ALD) and posterior (PLD) latissimus dorsi muscles is examined by the freeze-fracture technique in 1-, 7- and 30-day-old chicks. Several characteristics of plasma membrane (caveolae, rectilinear arrays, intramembranous particles) and sarcoplasmic reticulum which show fiber type differences in the adult ALD and PLD muscles are compared in the developmental stages. The caveolar density in the ALD fibers is about 20/microns2 at 1 day increasing to about 37/microns2 at 30 days, whereas in the PLD fibers it remains at about 20/microns2 during this period. The distribution of the caveolae in the two muscles is different from the beginning; in the ALD fibers the caveolae are distributed throughout the plasma membrane and in PLD fibers they are patterned into clusters overlying the I band regions. The density of intramembranous particles of 1-day ALD and PLD plasma membranes appears similar, but by 7 days the particle counts in the sarcolemma of the ALD muscle are about twice as numerous as those in the PLD muscle. The rectilinear arrays are virtually absent in the ALD muscle, whereas in the PLD muscle their density is about 10/microns2 at 1 day and about 20/microns2 at 7 days. Already at 1 day posthatching the SR in ALD and PLD fibers has the adult configuration, i.e., an open irregular network in ALD fibers and periodically arranged tubules with triadic expansions in the PLD fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterogeneity of type 1 skeletal muscle fibers revealed by monoclonal antibody to slow myosin.

Monoclonal antibodies (McAbs) were generated against slow myosin from the chicken anterior latissimus dorsi (ALD) muscle and their reactivity was checked against fast (pectoralis), slow (ALD), cardiac (ventricular), and smooth (gizzard) myosins by radioimmunoassay (RIA), immunoautoradiography (immunoblots), and indirect immunofluorescence techniques. In RIAs, the McAb (ALD-58) described in this article reacted specifically with slow myosin, with only a weak cross-reactivity to cardiac myosin. In immunoblots against whole muscle homogenates and purified myosins, it bound selectively to the 200 Kd myosin heavy chain band. The ALD-58 antibody stained the fibers of the ALD muscle uniformly but gave three grades of reactions (strong, weak, and negative), with histochemically identified type 1 fibers of sartorius and gastrocnemius muscles demonstrating the immunological heterogeneity of myosins in type 1 skeletal muscle fibers.