Dystrophin lies beneath sarcolemma and links actin through the membrane-spanning dystrophin-associated glycoprotein complex (DAGC) to the extracellular matrix. Several human studies and animal models have indicated that destruction of this complex may lead to congenital cardiomyopathy (1-3) or acquired viral myocarditis (4). Furthermore, dystrophin-deficient myotubes from mdx mice were fragile to chemical injury and underwent apoptosis, which would normally be repaired in parental myotubes (5).
The sympathetic pathway increased apoptosis in adult rat ventricular myocytes by β-adrenergic activation both in vitro (6) and in vivo (7). In this study, we addressed the mechanism of myocardial cell disintegration in acute heart injury after isoproterenol stimulation, focusing on the structural alteration of dystrophin/DAGC and analysis of apoptosis progression.
A high dose of isoproterenol (10 mg/kg; Sigma, St. Louis, MO, U.S.A.) or physiological saline as a control was injected intraperitoneally into male Sprague-Dawley rats (n = 24; 250-300 g). These rats were then sacrificed through the time course of 4, 8, 12 and 16 h after the administration of isoproterenol or physiological saline.
Histological and immunohistological assessment
The hearts were sliced (2 mm thick) and fixed in 4% paraformaldehyde in Tris-buffered saline for 4 h at room temperature. These samples were then quickly frozen by liquid nitrogen, and cryostat sections (5 mm) were stained with hematoxylin and eosin (HE). Injured space was scored at lower magnification (40×) with HE staining, following the point-counting method described by Yaoita et al. (8). The remaining sections were permeabilized with 3% H2O2 in 80% methanol for 5 min at room temperature to quench the endogenous peroxidase activity. Nonspecific absorption of the antibodies to the sections was blocked by treatment in 5% normal rabbit serum for monoclonal antibody or 5% normal goat serum for polyclonal antibody, for 30 min at room temperature.
Sections were incubated overnight at 4°C with the primary antibodies to dystrophin (Novocastra Laboratories, Benton Lane, U.K.) or δ-sarcoglycan (SG) purified as described previously (2). These samples were then incubated with biotinylated rabbit anti-mouse immunoglobulin (Ig)G (Dako, Glostrap, Denmark) for monoclonal antibody or with biotinylated goat anti-rabbit IgG (Dako) for polyclonal antibody, then for 30 min at room temperature with horseradish peroxidase-conjugated avidin (ABC kit; Vector, Burlingame, CA, U.S.A.). At the end, all sections were visualized with diaminobenzidine (DAB) substrate.
Western blotting analysis
Heart as excised at the intervals of control, 4, 8, 12, 16 and 18 h (n = 6 at each time point). The apex of the heart was used for immunohistological analysis and the rest of the heart was homogenized in 4% sodium dodecyl sulfate sample buffer. After measuring protein concentration by Bradford's method (9), an equal amount of protein was applied on each well and separated by 5-20% gradient gel of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, according to the method we have published previously (10), all proteins were transferred overnight to a nitrocellulose membrane. After incubating with primary anti-dystrophin antibody, treated with peroxidase-linked specific anti-mouse IgG and with detection reagents (Amersham Life Science, Little Chalfont, Buckinghamshire, U.K.), the membrane was visualized for fluorescence development.
Immunohistological detection and the double staining
Consecutive sections were prepared in each time point and following the instruction stained by anti-dystrophin antibody or transferase-mediated dUTP-biotin nick end labeling-positive cardiomyocytes (TUNEL) (7). Double-staining was performed as described previously in detail (7). Briefly, we performed immunostaining for dystrophin using alkaline phosphatase-conjugated streptoavidin (Dako), visualized as blue color. The same section was treated according to the instructions for the apoptosis detection kit (Apop Tag Plus®; Intergen, Purchase, NY, U.S.A.) and visualized by DAB substrate, expressing brown color.
Myocardial injury by isoproterenol
Myocardial injury was detected in the subendocardium of the left ventricular wall in isoproterenol-administered animals. These injuries were confirmed by HE staining (data not shown). The lesions were focally detected in the subendocardium. They were characterized by degraded cardiomyocytes and many infiltrating cells. The score of injured space increased in a time-dependent manner (4 h, 5.67 ± 2.5; 8 h, 6.67 ± 4.7; 12 h, 9 ± 2.6; and 16 h, 17 ± 3.6). Some cardiomyocytes were deleted completely and interstitial edema was found from 22 h; the space of losing cells was replaced by fibroblasts at 40 h (data not shown).
Dystrophin and DAGC
Sarcolemma staining was homogeneous and the integrity was well preserved in control group. All sarcolemmae were circumferentially and clearly stained by both antibodies to dystrophin and β-SGs (Figs 1A and 2A). No staining was observed using nonimmunized mouse IgG (data not shown), indicating that the staining was specific to each protein.
To our surprise, dystrophin staining was heterogeneous in the subendocardium 16 h after the isoproterenol administration (Fig. 1B). This heterogeneity started at 4 h and increased thereafter (data not shown). Closer inspection at high magnification (400 ×) revealed that cardiomyocytes in the heterogeneous area were stained not only in the membrane, but also in the cytoplasm; some myoplasm positive for dystrophin had intact membranes but shrank in size. The caliber of these muscle cells were smaller than normal ones nearby (Fig. 1C).
Western blotting of dystrophin demonstrated that full-length proteins were preserved wholly in control heart. However, the samples performed by isoproterenol overdose presented three disrupted bands at 135-211 kD at 4 and 8 h, and then two shorter bands were also detected at 70-110 kD from 12 to 18 h (Fig. 1D).
In contrast, compared with control hearts treated by physiological saline, δ-SG staining in isoproterenol-treated hearts did not show significant pathological attenuation. The patterns of the staining were localized to the sarcolemma throughout the time course (Figs 2A and B).
Coincidence of dystrophin-disrupted cells and apoptotic cells
To distinguish exactly the locative position of dystrophin-degraded myocardial cells and apoptotic cells in the same field, we employed double-staining of dystrophin and TUNEL (Fig. 3). It should be emphasized that apoptotic myocardial cells were not detected at 4 h after the isoproterenol administration, although dystrophin was distinctly dislocated (Fig. 3A). All cells testing positive for TUNEL staining were restricted to macrophage and noncardiomyocytes at 4 h (data not shown).
However, at 8 h after the administration, TUNEL-positive cells were clearly observed (Fig. 3B). We noted that cardiomyocytes stained by anti-dystrophin in the cytoplasm completely matched the TUNEL-positive cells.
Dystrophin disruption in myocardial injury by isoproterenol
Gene mutation in F-actin, dystrophin, SGs and laminin-2 have been shown to occur in a family with a hereditary form of dilated cardiomyopathy (11). Dystrophin lies beneath the sarcolemma and links F-actin through transmembrane DAGC to the extracellular matrix, and may function as a bridge to delivery of mechanical force generated in the contractile element to the extracellular matrix or to transmit physical work to intracellular machinery. Another physiological function of dystrophin might be a "staple-like" action to prevent an excessive expansion of sarcolemma during the muscle contraction.
In this study, we have indicated that immunoproduct of dystrophin was present not only in the subsarcolemma, but also in cytoplasm after isoproterenol administration at 4 h (Fig. 3A). This phenomenon suggests that the dystrophin was disrupted and diffused from sarcolemma to cytoplasm. This scheme was confirmed by the Western blotting of dystrophin, which showed a time-matched cleavage fragment (Fig. 1D). It is difficult to simply attribute the cause of dystrophin disruption to a single reason. The isoproterenol overdose was attended tendentiously to contribution for mechanical stress mainly, rather than its agonist activation. Previous studies reported that dystrophin was disrupted under weak mechanical injuries, similar to downhill running or forced lengthening during the muscle contraction (12). Another evidence indicated that cardiac dystrophin was cleaved by an endogenous, gene-coded protease 2A during the infection of Coxsackie B3 virus (4).
The situation of dystrophin in dilated cardiomyopathy hamsters was quite different from that in our present experiment, because dystrophin was completely preserved in TO-2 strain heart, in which δ-SG gene was missing, and δ-SG protein was defective; accordingly, α, β, and δ-SGs were degraded sequentially (13). These results were in agreement with the fact that dystrophin was preserved in sarcolemma in mice lacking γ-SG and developing to progressive muscular dystrophy (14). In this study, δ-SG was also observed to maintain the structural integrity within myocardial sarcolemma after isoproterenol administration (Fig. 2B), and all of these results suggest that degradation process of dystrophin in myocardium is different from that of SG complex. Here, we show evidence that dystrophin could be disrupted in β-adrenergic stimulation.
Dystrophin disruption and apoptosis by isoproterenol stimulation
Double staining distinctly confirmed that both the dystrophin disruption and apoptosis occurred in the same cardiomyocytes. At 4 h after the isoproterenol administration, cardiomyocytes with disrupted dystrophin were detected without the apoptotic finding (Fig. 3A), meaning the onset of apoptosis detected by TUNEL staining was delayed from the beginning of dystrophin disruption. Then, adding the fact that TUNEL-positive cardiomyocytes were abundantly visible after 8 h, it denotes that dystrophin disruption in cardiomyocytes preceded the DNA fragmentation.
Recent studies have shown that numerous myotubes proceed degeneration via apoptosis in dystrophin-deficient (mdx) mouse cells, suggesting that dystrophin-deficient myotubes are fragile and easily subjected to apoptosis (5,15-17). We do not know the precise mechanism of apoptosis in the early-stage occurrence after the dystrophin disruption, but many possibilities have been reported that the destruction of dystrophin or DAGC may lead to progressive muscle degeneration like muscular dystrophy, through the following pathogenesis: (a) change in sarcolemma permeability; (b) defect in muscular intracellular free calcium homeostasis; (c) decreased mechanical stability of the sarcolemma and of the sarcomere (18-20). These data would denote that structural integrity of dystrophin and/or DAGC might be a physiological barrier to prevent the progression of apoptosis that contributes to cell loss in the common myocardial pathway, including acute heart injury induced by isoproterenol.
Acknowledgement: The authors thank Dr Takatoshi Ishikawa and Dr Yoko Nakatsuru for their kind suggestion on the immunostaining, and Ms. Chieko Hemmi for her technical assistance. This study was financially supported by grants-in-aid from the Ministry of Education, Science and Culture, the Ministry of Health and Welfare, Japan, the Research Foundation for Molecular Cardiology, Uehara Memorial Foundation, Sharyou Foundation and Suzuken Memorial Foundation.
1. Michels VV, Moll PP, Miller FA, et al. The frequency of familial dilated cardiomyopathy in a series of patients with idiopathic dilated cardiomyopathy. N Engl J Med
2. Sakamoto A, Ono K, Abe M, et al. Both hypertrophic and dilated cardiomyopathy are caused by mutation of the same gene, δ-SG, in hamster: a model of dystrophin
-associated glycoprotein complex. Proc Natl Sci Acad USA
3. Olson TM, Michels VV, Thibodeau SN, et al. Actin mutation in dilated cardiomyopathy, a heritable form of heart failure
4. Badorff C, Lee GH, Lampher BJ, et al. Enteroviral protease 2A cleaved dystrophin
: evidence of cytoskeletal disruption in an acquired cardiomyopathy. Nat Med
5. Sandri M, Massimino ML, Cantini M, et al. Dystrophin
deficient myotubes undergo apoptosis
in mouse primary muscle cell culture after DNA damage. Neurosci Lett
6. Communal C, Singh K, Pimental DR, et al. Norepinephrine stimulates apoptosis
in adult rat ventricular myocytes by activation of the beta-adrenergic pathway. Circulation
7. Kawaguchi H, Shin WS, Okai-Matsuo Y, et al. Apoptotic cell death in acute myocardial injury induced by isoproterenol
is mediated by nitric oxide. J Cardiovasc Pharmacol
8. Yaoita H, Ogawa K, Maehara K, et al. Attenuation of ischemia/reperfusion injury in rats by a caspase inhibitor. Circulation
9. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem
10. Okai-Matsuo Y, Takano-Ohmuro H, Toyo-oka H, et al. A novel myosin heavy chain isoform in vascular smooth muscle. Biochem Biophys Res Commun
11. Ervasti JM, Campbell KP. Dystrophin
and the membrane skeleton. Curr Opin Cell Biol
12. Komulainen J, Koskinen SO, Kalliokoski R, et al. Gender differences in skeletal muscle fiber damage after eccentrically biased downhill running in rats. Acta Physiol Scand
13. Kawada T, Nakatsuru Y, Sakamoto A, et al. Strain- and age-dependent loss of SG complex in cardiomyopathic hamster hearts and its reexpression by ?-SG gene transfer in vivo. FEBS Lett
14. Hack AA, Cordier L, Shoturma DI, et al. Muscle degeneration without mechanical injury in sarcoglycan
deficiency. Proc Natl Acad Sci USA
15. Tidball JG, Albrecht DE, Lokensgard BE, Spencer MJ. Apoptosis
precedes necrosis of dystrophin
-deficient muscle. J Cell Sci
16. Carraro U. Apoptotic death of dystrophic muscle fibers after exercise: a new hypothesis on the early events of muscle damage. Basic Appl Myol
17. Sandri M, Podhorska-Okolow M, Geromel V, et al. Exercise induces myonuclear ubiquitination and apoptosis
-deficient muscle of mice. J Neuropath Exp Neurol
18. Anderson MS, Kunkel LM. The molecular and biochemical basis of Duchenne musclar dystrophy. Trends Biochem Sci
19. Turner PR, Fong PY, Denetclaw WF, et al. Increased calcium influx in dystrophic muscle. J Cell Biol
20. Ervasti JM, Campbell KP. Dystrophin
-associated glycoproteins: their possible roles in the pathogenesis of Duchenne muscular dystrophy. In: Partridge T, ed. Molecular and cell biology of muscular dystrophy.
London: Chapman and Hall, 1993:139-66.
The symposium and the publication of this supplement were supported by an educational grant from Novartis Pharma K.K. Tokyo, Japan.