Anthracyclines Induce Calpain-dependent Titin Proteolysis and Necrosis in Cardiomyocytes
2004; Elsevier BV; Volume: 279; Issue: 9 Linguagem: Inglês
10.1074/jbc.m308033200
ISSN1083-351X
AutoresChee Chew Lim, Christian Zuppinger, Xinxin Guo, Gabriela M. Kuster, Michiel Helmes, Hans M. Eppenberger, Thomas M. Suter, Ronglih Liao, Douglas B. Sawyer,
Tópico(s)Viral Infections and Immunology Research
ResumoTitin, the largest myofilament protein, serves as a template for sarcomere assembly and acts as a molecular spring to contribute to diastolic function. Titin is known to be extremely susceptible to calcium-dependent protease degradation in vitro. We hypothesized that titin degradation is an early event in doxorubicin-induced cardiac injury and that titin degradation occurs by activation of the calcium-dependent proteases, the calpains. Treatment of cultured adult rat cardiomyocytes with 1 or 3 μmol/liter doxorubicin for 24 h resulted in degradation of titin in myocyte lysates, which was confirmed by a reduction in immunostaining of an antibody to the spring-like (PEVK) domain of titin at the I-band of the sarcomere. The elastic domain of titin appears to be most susceptible to proteolysis because co-immunostaining with an antibody to titin at the M-line was preserved, suggesting targeted proteolysis of the spring-like domain of titin. Doxorubicin treatment for 1 h resulted in ∼3-fold increase in calpain activity, which remained elevated at 48 h. Co-treatment with calpain inhibitors resulted in preservation of titin, reduction in myofibrillar disarray, and attenuation of cardiomyocyte necrosis but not apoptosis. Co-treatment with a caspase inhibitor did not prevent the degradation of titin, which precludes caspase-3 as an early mechanism of titin proteolysis. We conclude that calpain activation is an early event after doxorubicin treatment in cardiomyocytes and appears to target the degradation of titin. Proteolysis of the spring-like domain of titin may predispose cardiomyocytes to diastolic dysfunction, myofilament instability, and cell death by necrosis. Titin, the largest myofilament protein, serves as a template for sarcomere assembly and acts as a molecular spring to contribute to diastolic function. Titin is known to be extremely susceptible to calcium-dependent protease degradation in vitro. We hypothesized that titin degradation is an early event in doxorubicin-induced cardiac injury and that titin degradation occurs by activation of the calcium-dependent proteases, the calpains. Treatment of cultured adult rat cardiomyocytes with 1 or 3 μmol/liter doxorubicin for 24 h resulted in degradation of titin in myocyte lysates, which was confirmed by a reduction in immunostaining of an antibody to the spring-like (PEVK) domain of titin at the I-band of the sarcomere. The elastic domain of titin appears to be most susceptible to proteolysis because co-immunostaining with an antibody to titin at the M-line was preserved, suggesting targeted proteolysis of the spring-like domain of titin. Doxorubicin treatment for 1 h resulted in ∼3-fold increase in calpain activity, which remained elevated at 48 h. Co-treatment with calpain inhibitors resulted in preservation of titin, reduction in myofibrillar disarray, and attenuation of cardiomyocyte necrosis but not apoptosis. Co-treatment with a caspase inhibitor did not prevent the degradation of titin, which precludes caspase-3 as an early mechanism of titin proteolysis. We conclude that calpain activation is an early event after doxorubicin treatment in cardiomyocytes and appears to target the degradation of titin. Proteolysis of the spring-like domain of titin may predispose cardiomyocytes to diastolic dysfunction, myofilament instability, and cell death by necrosis. Titin is the largest known protein and is an integral part of the myofilament system in vertebrate striated muscle. Full-length titin extends the half-sarcomere from the Z-disk to the M-line, with the I-band region comprised of distinct elastic segments that account for the extensibility of titin. Titin has been implicated in the process of sarcomerogenesis, where it is thought to serve as a framework for ordered assembly of myofilament proteins (1Tokuyasu K.T. Maher P.A. J. Cell Biol. 1987; 105: 2781-2793Crossref PubMed Scopus (107) Google Scholar, 2Wang S.M. Greaser M.L. Schultz E. Bulinski J.C. Lin J.J. Lessard J.L. J. Cell Biol. 1988; 107: 1075-1083Crossref PubMed Scopus (132) Google Scholar, 3Gregorio C.C. Granzier H. Sorimachi H. Labeit S. Curr. Opin. Cell Biol. 1999; 11: 18-25Crossref PubMed Scopus (275) Google Scholar). The contribution of the elastic domain of titin to passive and restoring forces is well established (4Granzier H.L. Irving T.C. Biophys. J. 1995; 68: 1027-1044Abstract Full Text PDF PubMed Scopus (500) Google Scholar, 5Helmes M. Trombitas K. Granzier H. Circ. Res. 1996; 79: 619-626Crossref PubMed Scopus (183) Google Scholar, 6Linke W.A. Stockmeier M.R. Ivemeyer M. Hosser H. Mundel P. J. Cell Sci. 1998; 111: 1567-1574PubMed Google Scholar), and a physiological role is emerging where titin is an important determinant of the Frank-Starling relationship of the heart (7Cazorla O. Wu Y. Irving T.C. Granzier H. Circ. Res. 2001; 88: 1028-1035Crossref PubMed Scopus (196) Google Scholar, 8Fukuda N. Sasaki D. Ishiwata S. Kurihara S. Circulation. 2001; 104: 1639-1645Crossref PubMed Scopus (149) Google Scholar, 9Helmes M. Lim C.C. Liao R. Bharti A. Cui L. Sawyer D.B. J. Gen. Physiol. 2003; 121: 97-110Crossref PubMed Scopus (90) Google Scholar). Given the central role of titin in sarcomere formation as well as myocyte mechanics, pathological conditions leading to enhanced titin degradation may have profound consequences for myofilament stability and function. Indeed, there is evidence for disorganization and loss of titin in ischemic and failing human hearts (10Morano I. Hadicke K. Grom S. Koch A. Schwinger R.H. Bohm M. Bartel S. Erdmann E. Krause E.G. J. Mol. Cell. Cardiol. 1994; 26: 361-368Abstract Full Text PDF PubMed Scopus (78) Google Scholar, 11Hein S. Scholz D. Fujitani N. Rennollet H. Brand T. Friedl A. Schaper J. J. Mol. Cell. Cardiol. 1994; 26: 1291-1306Abstract Full Text PDF PubMed Scopus (104) Google Scholar, 12de Jonge N. van Wichen D.F. Schipper M.E. Lahpor J.R. Gmelig-Meyling F.H. Robles de Medina E.O. de Weger R.A. J. Am. Coll. Cardiol. 2002; 39: 963-969Crossref PubMed Scopus (63) Google Scholar). The mechanisms, however, for cardiac titin degradation under normal and pathological conditions have not been examined. Titin is known to be extremely sensitive to proteolysis, in vitro, which is preventable with calcium chelation in combination with leupeptin protease inhibition, conditions known to be necessary for inhibition of the calcium-dependent proteases, the calpains (13Wang K. Adv. Exp. Med. Biol. 1984; 170: 285-305Crossref PubMed Scopus (42) Google Scholar). The calpains are a family of calcium-dependent cysteine proteases that are believed to participate in basic calcium-mediated intracellular processes (14Sorimachi H. Ishiura S. Suzuki K. Biochem. J. 1997; 328: 721-732Crossref PubMed Scopus (621) Google Scholar). Of the calpain family, calpain isoenzymes I (μ-calpain) and II (m-calpain) are ubiquitously expressed and are activated in vitro in the presence of micro- and millimolar concentrations of calcium, respectively. Although the exact physiological role of calpains in the myocardium is not known, the ability of calpains to cleave cytoskeletal and myofilament proteins desmin, fodrin, filamin, C-protein, tropomyosin, troponin T, troponin I, nebulin, gelsolin, and vinculin in a variety of cell types, in vitro, suggest a regulatory role for calpains in remodeling of the myofibril (15Matsumura Y. Saeki E. Inoue M. Hori M. Kamada T. Kusuoka H. Circ. Res. 1996; 79: 447-454Crossref PubMed Scopus (75) Google Scholar, 16Huang J. Forsberg N.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12100-12105Crossref PubMed Scopus (268) Google Scholar, 17Papp Z. van der Velden J. Stienen G.J. Cardiovasc. Res. 2000; 45: 981-993Crossref PubMed Scopus (71) Google Scholar). Calpain activity is increased in a wide variety of pathological conditions associated with calcium overload including Alzheimer's disease (18Grynspan F. Griffin W.R. Cataldo A. Katayama S. Nixon R.A. Brain Res. 1997; 763: 145-158Crossref PubMed Scopus (130) Google Scholar, 19Tsuji T. Shimohama S. Kimura J. Shimizu K. Neurosci. Lett. 1998; 248: 109-112Crossref PubMed Scopus (83) Google Scholar), cataracts (20Andersson M. Sjostrand J. Karlsson J.O. Ophthalmic Res. 1996; 28: 51-54Crossref PubMed Scopus (16) Google Scholar), oxidative stress (21Andersson M. Sjostrand J. Petersen A. Karlsson J.O. Ophthalmic Res. 1998; 30: 157-167Crossref PubMed Scopus (15) Google Scholar), and ischemia reperfusion injury (22Yoshida K. Yamasaki Y. Kawashima S. Biochim. Biophys. Acta. 1993; 1182: 215-220Crossref PubMed Scopus (66) Google Scholar). In post-ischemic myocardium, the proteolytic activity of calpain may be linked to degradation of sarcomeric proteins troponin I and desmin (17Papp Z. van der Velden J. Stienen G.J. Cardiovasc. Res. 2000; 45: 981-993Crossref PubMed Scopus (71) Google Scholar, 23Gao W.D. Liu Y. Mellgren R. Marban E. Circ. Res. 1996; 78: 455-465Crossref PubMed Google Scholar). Interestingly, skeletal muscle-specific calpain-III (also known as p94) has been shown to bind to titin in skeletal muscle, and mutations in p94 have been associated with a severe form of limb-girdle muscular dystrophy (24Sorimachi H. Kinbara K. Kimura S. Takahashi M. Ishiura S. Sasagawa N. Sorimachi N. Shimada H. Tagawa K. Maruyama K. Suzuki K. J. Biol. Chem. 1995; 270: 31158-31162Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar, 25Kinbara K. Sorimachi H. Ishiura S. Suzuki K. Arch. Biochem. Biophys. 1997; 342: 99-107Crossref PubMed Scopus (94) Google Scholar, 26Richard I. Broux O. Allamand V. Fougerousse F. Chiannilkulchai N. Bourg N. Brenguier L. Devaud C. Pasturaud P. Roudaut C. Hillaire D. Passos-Bueno M. Zatz M. Tischfield J.A. Fardeau M. Jackson C.E. Cohen D. Beckmann J.S. Cell. 1995; 81: 27-40Abstract Full Text PDF PubMed Scopus (868) Google Scholar). The association between p94 and titin is of particular interest to researchers in food science, where the level of calpain activity and the degree of titin degradation have been related to postmortem meat tenderness (27Uytterhaegen L. Claeys E. Demeyer D. J. Anim. Sci. 1994; 72: 1209-1223Crossref PubMed Scopus (85) Google Scholar, 28Huff-Lonergan E. Parrish Jr., F.C. Robson R.M. J. Anim. Sci. 1995; 73: 1064-1073Crossref PubMed Scopus (154) Google Scholar). The extreme sensitivity of titin to calcium-dependent proteolysis as well as the known association between titin and p94 in skeletal muscle make titin a plausible target of the calpains in cardiac muscle. Doxorubicin cardiomyopathy is typically associated with myofibrillar deterioration; however, the early mechanisms responsible for this degenerative phenotype remain elusive (for review see Ref. 29Singal P.K. Deally C.M. Weinberg L.E. J. Mol. Cell. Cardiol. 1987; 19: 817-828Abstract Full Text PDF PubMed Scopus (446) Google Scholar). In addition to myofilament damage, doxorubicin is also known to cause intracellular calcium overload (29Singal P.K. Deally C.M. Weinberg L.E. J. Mol. Cell. Cardiol. 1987; 19: 817-828Abstract Full Text PDF PubMed Scopus (446) Google Scholar). These two aspects of doxorubicin cardiotoxicity may well be related, where a pathological increase in intracellular calcium may trigger indiscriminate activation of calcium-dependent proteases resulting in degradation of key myofibrillar proteins. We therefore hypothesized that titin degradation is an early process in doxorubicin-induced myofilament injury, with consequences for myofilament stability and perhaps cell viability. We further hypothesized that titin proteolysis occurs by the action of the calcium-dependent proteases, the calpains. To test the prospective role for calpains in pathologic titin degradation, we used a cell culture model of anthracycline-induced myofilament injury. Our results support a role for calpain in titin proteolysis early after doxorubicin treatment. The implications of these findings for doxorubicin and other forms of myocardial injury are discussed. Cell Culture—Adult rat ventricular myocytes were isolated from male Sprague-Dawley rats weighing 150-200 g, plated at medium to low density, and cultured according to a protocol described previously (30Sawyer D.B. Zuppinger C. Miller T.A. Eppenberger H.M. Suter T.M. Circulation. 2002; 105: 1551-1554Crossref PubMed Scopus (265) Google Scholar). Culture medium consisted of Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 5 mmol/liter creatine (Sigma), 2 mmol/liter l-carnitine (Sigma), 5 mmol/liter taurine (Sigma), 1% 100 units/ml penicillin/streptomycin (Invitrogen), 7% preselected fetal calf serum (Invitrogen), and 100 μmol/liter bromodeoxyuridine (Sigma). Culture media were replaced 2 h after plating and every 72 h. Myocytes were treated on day 10 in culture with 1 or 3 μmol/liter doxorubicin (Sigma) and incubated for 1, 6, 24, or 48 h. Calpain inhibitors ALLN 1The abbreviations used are: ALLN, N-acetyl-l-leucyl-l-leucyl-norleucinal; BDFMK, Boc-Asp(OMe)-fluoromethyl ketone; TRITC, tetramethylrhodamine isothiocyanate; PBS, phosphate-buffered saline; CK, creatine kinase; SR, sarcoplasmic reticulum. (Sigma), calpeptin (Calbiochem), calpastatin (Calbiochem), or leupeptin (Peptide International Institute) or caspase inhibitor BDFMK (Sigma) were added to myocytes (at concentrations indicated elsewhere) 1 h prior to doxorubicin treatment. Neonatal rat ventricular myocytes were isolated as described previously (31Sawyer D.B. Fukazawa R. Arstall M.A. Kelly R.A. Circ. Res. 1999; 84: 257-265Crossref PubMed Scopus (189) Google Scholar). Cells were cultured for 48 h in Dulbecco's modified Eagle's medium supplemented with 7% fetal calf serum and penicillin/streptomycin prior to treatment. Neonatal myocytes were treated with 0.5 μmol/liter doxorubicin without or with protease inhibitors (ALLN or BDFMK) and incubated for 24 h. Gel Electrophoresis—Following treatment, myocytes were solubilized by adding 1 volume of cell suspension to 9 volumes of solubilization buffer (50 mmol/liter Tris-HCl, 5% SDS, 10% glycerol, 80 mmol/liter dithiothreitol, pH 6.8 at 25 °C) preheated to 90-95 °C. The samples were solubilized for 60 s and allowed to cool down to room temperature. Protein concentration was determined by the Lowry method. Bromphenol blue was added (0.025%), and equal amounts of sample (200 μg) were analyzed by SDS-PAGE using a 2% acrylamide gel strengthened with 0.5% agarose (32Tatsumi R. Hattori A. Anal. Biochem. 1995; 224: 28-31Crossref PubMed Scopus (102) Google Scholar). Gels were run at 15 mA for 4 h at room temperature and fixed in 50% methanol and 7% acetic acid. Gels were stained with Coomassie overnight, destained with 50% methanol, 7% acetic acid followed by 5% methanol, 7% acetic acid. Wet gels were scanned at 400 or 900 dpi using a commercially available scanner (Molecular Analyst, Bio-Rad). T1 titin (both N2B and N2BA isoforms), titin degradation product T2, total titin (T1 isoforms + T2), and myosin heavy chain from the same samples were quantitated by densitometry. Total titin, T1, and T2 were normalized relative to myosin heavy chain to account for potential inaccuracies in protein loading. Calpain Assay—Calpain activity was determined using a fluorogenic assay as described by Sasaki et al. (33Sasaki T. Kikuchi T. Yumoto N. Yoshimura N. Murachi T. J. Biol. Chem. 1984; 259: 12489-12494Abstract Full Text PDF PubMed Google Scholar). At the end of the experimental protocol, myocytes were harvested and sonicated in calcium-free assay buffer containing 63.2 mmol/liter imidazole-HCl, 20 mmol/liter EGTA, 25 mmol/liter EDTA, and 10 mmol/liter 2-mercaptoethanol, pH 7.3. Samples were diluted to 1 mg/ml, and 25 μl of sample was combined with either 75 μl of calcium-free assay buffer or 75 μl of calcium buffer (containing 63.2 mmol/liter imidazole-HCl, 1.25 mmol/liter CaCl2, and 10 mmol/liter 2-mercaptoethanol, pH 7.3) in a 96-well fluorescent plate. Samples were incubated at 37 °C for 10 min, and N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin was added to the samples for a final concentration of 50 μmol/liter. After an additional 30 min of incubation, fluorescence was detected at 355 nm excitation and 460 nm emission. Calpain activity was determined as the difference between the calcium-dependent and the calcium-independent fluorescence. All experiments were run in duplicate. Caspase-3 Assay—The caspase-3 activity within the cells was measured with a caspase-3 fluorescent assay kit (Molecular Probes). Briefly, the caspase-3-specific substrate acetyl-Asp-Glu-Val-Asp-amido-4-methylcoumarin was incubated with 20 μg of myocyte lysates at room temperature for 30 min, and fluorescence of the cleaved product was detected at 355 nm excitation and 460 nm emission. All experiments were run in duplicate. The specificity of the cleavage reaction was verified by including 10 μmol/liter acetyl-Asp-Glu-Val-Asp-CHO (caspase-3 inhibitor) in the incubation, and 2 logs weaker propidium iodide fluorescence than G0 cells. Apoptosis was calculated as the percent of cells in the sub-G1/G0 peak. Statistics—Data are presented as mean ± S.E., unless indicated otherwise. Where appropriate, results were analyzed by Student's t test or analysis of variance with a post-hoc test of least significant differences. p < 0.05 was considered statistically significant. Doxorubicin Degrades Titin in Intact Cardiomyocytes—We examined the effect of doxorubicin on titin using 2% Coomassie-stained SDS-polyacrylamide gels (Fig. 1). As reported previously (36Cazorla O. Freiburg A. Helmes M. Centner T. McNabb M. Wu Y. Trombitas K. Labeit S. Granzier H. Circ. Res. 2000; 86: 59-67Crossref PubMed Scopus (319) Google Scholar), control rat myocytes express a predominant N2B titin band with a minor N2BA band compared with human and pig myocardium (see calibration gel). Incubation of myocytes with 1 μmol/liter doxorubicin resulted in partial degradation of titin, as evidenced by the appearance of titin degradation product T2 (Fig. 1, A and B). Table I shows that compared with the untreated control group, the T2/MHC ratio increased with doxorubicin incubation time and that pretreatment with calpain inhibitor ALLN, but not caspase inhibitor BDFMK, prevented the increase in the T2/MHC ratio. Interestingly, there was no difference in the total titin (T1 isoforms + T2)/MHC ratio, suggesting a very specific and limited degradation of titin to its T2 fragment at the time points studied (Table I). Higher concentrations of doxorubicin and longer incubation times, on the other hand, resulted in almost complete degradation of titin (Fig. 1, B and C). Pretreatment of myocytes with calpain inhibitors ALLN (100 μmol/liter), calpeptin (5 μmol/liter), or leupeptin (50 μmol/liter) for 1 h prior to doxorubicin treatment preserved titin similar to control levels, whereas caspase inhibitor BDFMK (100 μmol/liter) had no effect (Fig. 1, A-C).Table IComparison of sarcomere proteins in 1 μmol/liter doxorubicin-treated cardiomyocytesControl6 h24 h24 h + ALLN24 h + BDFMKT2/MHC0.015 ± 0.0020.018 ± 0.0020.026 ± 0.005ap < 0.005 versus control.bp < 0.05 versus 6 h.cp < 0.01 versus 24 h + ALLN.0.017 ± 0.0020.025 ± 0.006ap < 0.005 versus control.bp < 0.05 versus 6 h.cp < 0.01 versus 24 h + ALLN.T1/MHC0.195 ± 0.0170.193 ± 0.0140.189 ± 0.0130.194 ± 0.0140.187 ± 0.010Total titin/MHC0.211 ± 0.0190.211 ± 0.0160.214 ± 0.0180.211 ± 0.0160.213 ± 0.010MHC1.00 ± 0.030.99 ± 0.031.01 ± 0.021.00 ± 0.041.01 ± 0.03α-Actinin1.00 ± 0.160.98 ± 0.130.94 ± 0.131.11 ± 0.171.06 ± 0.07Actin1.00 ± 0.110.99 ± 0.141.04 ± 0.131.02 ± 0.071.00 ± 0.03Desmin1.00 ± 0.121.00 ± 0.130.98 ± 0.071.01 ± 0.130.97 ± 0.18a p < 0.005 versus control.b p < 0.05 versus 6 h.c p < 0.01 versus 24 h + ALLN. Open table in a new tab Western blot analysis and wet gel scans were performed to determine the effect of doxorubicin on other sarcomeric proteins. Treatment with 1 μmol/liter doxorubicin up to 24 h had no effect on protein levels of α-actinin, actin, desmin, or myosin heavy chain (Table I). Thus, doxorubicin treatment of myocytes results in early degradation of titin, which is preventable with calpain but not caspase inhibition. Doxorubicin Activates the Calpains and Caspase-3—The effect of doxorubicin on protease activity was examined in adult rat ventricular myocytes in culture at 1, 6, 24, and 48 h. Treatment with 1 μmol/liter doxorubicin resulted in a significant increase in calpain activity, which occurred as early as 1 h and remained elevated at 48 h (Fig. 2A). The increase in calpain activity was similar to that in cardiomyocytes treated with the calcium ionophore, ionomycin (data not shown). Furthermore, the doxorubicin-induced increase in calpain activity coincided with significant prolongation of the calcium transient and increase in total cytosolic calcium levels, assessed by time constant τ of the fura-2 transient decay and the area under the fura-2 transient curve, respectively (Table II). Caspase-3 activity, on the other hand, did not increase until 24 h of doxorubicin treatment (Fig. 2B). Pretreatment of myocytes with 100 μmol/liter ALLN prevented the doxorubicin-induced increase in calpain activity at 24 h but had no effect on caspase-3 activity. Conversely, pretreatment with 100 μmol/liter BDFMK prevented the increase in caspase-3 activity at 24 h but did not inhibit calpain activation.Table IIFura-2 calcium transients in cardiomyocytes treated with 1 μmol/liter doxorubicinControl1 h24 h48 hSystolic calcium (fura-2 ratio)1.074 ± 0.0571.082 ± 0.0411.124 ± 0.0491.153 ± 0.079ap < 0.05 versus control.bp < 0.05 versus 1 h.Diastolic calcium (fura-2 ratio)0.938 ± 0.0380.930 ± 0.0440.986 ± 0.078bp < 0.05 versus 1 h.0.955 ± 0.067τ (ms)61.2 ± 14.573.7 ± 14.9ap < 0.05 versus control.122.1 ± 32.2ap < 0.05 versus control.bp < 0.05 versus 1 h.73.8 ± 15.4ap < 0.05 versus control.cp < 0.01 versus 24 h.Total cytosolic calcium (area under the curve)0.0077 ± 0.00370.0115 ± 0.0052ap < 0.05 versus control.0.0150 ± 0.0040ap < 0.05 versus control.0.0231 ± 0.0048ap < 0.05 versus control.cp < 0.01 versus 24 h.a p < 0.05 versus control.b p < 0.05 versus 1 h.c p < 0.01 versus 24 h. Open table in a new tab Calpain-Calpastatin Ratio Is Increased with Doxorubicin—We examined the effect of 24 and 48 h of doxorubicin (1 μmol/liter) treatment on protein levels of calpains-I, -II, and calpastatin. By Western blot, calpain-I appeared as a single 80-kDa band that was significantly decreased at 24 and 48 h of doxorubicin (Fig. 3A). Doxorubicin treatment for 24 h resulted in significant autolysis of the 80-kDa calpain-II band to a higher mobility 78-kDa band, which is widely believed to be the more active form of calpain-II (for review see Ref. 37Goll D.E. Thompson V.F. Li H. Wei W. Cong J. Physiol. Rev. 2003; 83: 731-801Crossref PubMed Scopus (2378) Google Scholar). Calpastatin was significantly decreased at 24 and 48 h of doxorubicin treatment (Fig. 3A). Because the proteolytic activity of the calpains is negatively regulated by calpastatin, the calpain/calpastatin ratio provides a gauge to assess the proteolytic potential of the calpains (38Ray S.K. Patel S.J. Welsh C.T. Wilford G.G. Hogan E.L. Banik N.L. J. Neurosci. Res. 2002; 69: 197-206Crossref PubMed Scopus (58) Google Scholar, 39Enns D. Karmazyn M. Mair J. Lercher A. Kountchev J. Belcastro A. Mol. Cell. Biochem. 2002; 241: 29-35Crossref PubMed Scopus (16) Google Scholar). The ratio of calpain-II (combined 80- and 78-kDa products) to calpastatin was significantly elevated and the increase occurred earlier in comparison to the calpain-I/calpastatin ratio (Fig. 3B). Calpain Degrades Titin in Skinned Cardiomyocytes—To determine whether calpain can directly degrade titin, we incubated Triton-permeabilized myocytes with 10 units/ml of calpain-I (Calbiochem) with or without calpain inhibitor for 30 min. The skinning procedure resulted in some degradation of titin as shown by the T2 band in the control lane (Fig. 4A). Incubation with calpain-I resulted in a more prominent T2 band compared with control, and co-treatment with calpain inhibitor preserved titin to control levels. This effect of calpain on titin integrity was also visualized directly in skinned myocytes immunostained with 9D10, a monoclonal antibody to I-band titin at the PEVK domain (40Wang S.M. Greaser M.L. J. Muscle Res. Cell Motil. 1985; 6: 293-312Crossref PubMed Scopus (99) Google Scholar). Immunofluorescence microscopy revealed that 9D10 immunostaining was reduced in skinned myocytes treated with calpain compared with control myocytes, and this effect was prevented by calpain inhibition (Fig. 4B). Calpain Inhibition Prevents Doxorubicin-induced Myofibrillar Disarray—We examined the pattern of titin staining with 9D10 in myocytes treated with doxorubicin. High resolution images of myocytes stained with antibody 9D10 demonstrated a doublet pattern consistent with staining of the I-bands on either side of the Z-line (Fig. 5A). Myocytes treated with doxorubicin showed an overall decrease in 9D10 staining, along with loss of the doublet pattern (Fig. 5B). Co
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