Dystrophin, an important protein of the dystrophin-glycoprotein complex, has been implicated in the pathogenesis of experimental Chagas disease. It contributes to cell shape, mechanical resistance, contraction and force generation in cardiomyocytes. Dystrophin loss has been associated with end-stage cardiomyopathies and proposed as a common route for myocardial dysfunction and progression to advanced heart failure. One of the most intriguing aspects of chronic Chagasic cardiomyopathy is the long delay after the initial infection to the cardiac manifestations. This has been partially explained by our group in previous studies demonstrating loss/reduction of dystrophin in mice experimentally infected with T. cruzi. The analysis of dystrophin expression showed significant reduction in the acute phase, with the reduction maintained up to the chronic phase. Inflammatory mechanisms could be involved in the dystrophin loss since inflammation has been shown to play a key role in the activation of proteases responsible for dystrophin degradation.

•    Chagas disease
•    Trypanosoma cruzi
•    Dystrophin
•    Inflammation
•    Calpain-1

Malvestio LM, Prado CM (2016) The Role of Dystrophin Loss in the Trypanosoma cruzi Infection. J Vet Med Res 3(5): 1061

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), which is transmitted when the infected feces of the triatomine vector are inoculated through a bite site or through an intact mucous membrane of the mammalian host [1]. Originally confined to Latin American countries, it had been considered an exotic disease and received less attention from global health policy-makers and the scientific community than it could expect due to its high morbidity and mortality. The migration of infected persons to large urban cities and nonendemic countries changed the epidemiological profile of Chagas disease from a disease of poor rural areas to a globalized problem of large cities in Latin America as well as most of the developed world. By the early 1990s, the World Health Organization considered Chagas disease the most serious parasitic disease in Latin America and as having the greatest economic impact. The number of estimated infected people was approximately 18 million, with a further 100 million under risk. Now, the revised numbers are much reduced, with an estimate of about 10-13 million [2] or, even less, 8-10 million infected people [3].

Although Chagas disease was first described more than a century ago, the course of the disease and its clinical outcomes are still not totally understood. The clinical course of Chagas disease is usually divided into three phases: acute, indeterminate or latent, and chronic. In most cases, the initial infection is asymptomatic. However, a few cases will present acute symptoms and in some instances death may occur in 3%-5% of cases [4,5]. Infected individuals surviving the acute phase enter the indeterminate stage, characterized by a long asymptomatic period before the onset of clinical signs and symptoms. This phase can last 10-30 years or until the end of an individual’s life [6]. Approximately 30% of the infected individuals eventually develop late manifestations [5,7,8]. The symptomatic disease affects the heart in 94.5% of patients that are considered to have chronic Chagas cardiomyopathy (CCC), usually between 15 and 50 years of age. Congestive heart failure is the cause of death in 58% of these patients, whereas cardiac arrhythmias and unexpected death affects 36.5%. The remaining manifests as mega-syndromes of hollow viscera, usually megaesophagus and megacolon [9,10]. Different mechanisms have been proposed to explain the pathogenesis of CCC, such as, (1) direct tissue destruction by T. cruzi [11-16]; (2) autonomic abnormalities [9,17-22]; (3) microvascular changes [23-29] and (4) autoimmune mechanisms [13,31-38].

One of the most intriguing aspects of chronic Chagasic cardiomyopathy is the long delay after the initial infection to the cardiac manifestations. This has been partially explained by our group in previous studies demonstrating loss/reduction of dystrophin in mice experimentally infected with T. cruzi [39,40]. Similar to CCC in humans, cardiac complications due to cardiomyopathy also appear later in life in Duchenne muscular dystrophy and Becker muscular dystrophy, the most common X-linked recessive disorders resulting from mutations in the dystrophin gene that lead to an absence of or defect in the protein dystrophin in striated muscles. In this way, the development of cardiomyopathy in both Chagas disease and congenital dystrophinopathies occurs decades after infection or birth in humans, respectively. Dystrophin and associated glycoproteins form the so-called dystrophin glycoprotein complex (DGC), which contributes to cell shape, mechanical resistance, contraction and force generation in cardiomyocytes [41]. Dystrophin is localized beneath the sarcolemma and links cytoplasmic actin to the extracellular matrix through the membrane spanning glycoproteins (Figure 1) [42]. The reduction of dystrophin has been linked to end-stage cardiomyopathies and proposed as a common route to the induction of cardiomyopathy and heart failure [43,44]. Furthermore, dystrophin loss has been observed in different experimental models of cardiomyopathies, such as post-viral myocarditis caused by Coxsackie virus B [45], myocardial infarction [46], isoproterenol [44,47] and doxorubicin administration [48] and septic cardiomyopathy [49]. Based on these facts, our group was the first to study dystrophin expression in the hearts of mice experimentally infected by Trypanosoma cruzi in both acute and chronic stages of the disease.

Dystrophin Expression in Acute and Chronic Stages of Experimentally-Induced T. Cruzi Infection

Prado et al. [39], tested the hypothesis that cardiac dystrophin levels were decreased during the earlier phases of the experimental infection by T. cruzi in mice and maintained at low levels up to development of cardiomyopathy. Infection with specific strains of T. cruzi leads to a cardiomyopathy that evolves from the acute to the chronic phase. To experimentally reproduce this infection, male CD1 mice were infected with the Brazil strain of T. cruzi. Control and infected mice were killed 30 days post infection (dpi) (considered acute stage) and 100 dpi (considered chronic stage) and heart disturbances characterized. Densitometric analysis of Western blotting showed a significant reduction of dystrophin expression in the mice hearts at 30 dpi, with the reduction maintained up to 100 dpi. In addition to the quantification by Western blotting, the expression of dystrophin in the cardiac tissue was evaluated by immunofluorescence (IF). The IF revealed that dystrophin labeling occurred in a uniform pattern as a continuous rim at the periphery of most cardiomyocytes from control hearts (Figure 2). However, dystrophin was focally reduced or completely lost in cardiomyocytes of infected mouse hearts. At 30 dpi there were foci of myocytolysis associated with loss of the dystrophin fluorescent signal and infiltration of interstitial cells (Figure 2), asterisk). Additionally, foci of cardiomyocytes showing undamaged actin were observed with markedly reduced/loss of dystrophin fluorescent signal (Figure 2), arrows). The doublestained against dystrophin x actin was to detect cardiomyocyte cytoskeletal actin in order to determine whether dystrophin loss was a consequence of cardiomyocyte death. Spread blocks of cardiomyocytes lacking dystrophin distinctly showed the red fluorescent signal for actin, indicating an absence of correlation between myocyte necrosis and dystrophin loss. At 100 dpi the foci of loss/reduction of dystrophin fluorescent signal were evident (Figure 2), arrows). It has been demonstrated that dystrophin loss destabilizes the DGC present at the sarcolemma, disrupts the physical linkage of the subsarcolemmal cytoskeleton with the sarcolemma causing alterations in calcium homeostasis and increases the membrane permeability, thus impairing contractile transmission that results in contractile dysfunction [50].

Morphological and functional cardiac alteration: Since dystrophin loss has been linked to alterations of contractile force transmission, the next step was to characterize the morphological and functional alterations in these hearts.

At 30 dpi there was an intense and diffuse myocarditis characterized by lymphomononuclear interstitial infiltrate, disruption of myofibers, multiple pseudocysts of amastigotes, enlargement of the interstitial space and perivascular inflammatory infiltrate, mainly in the right ventricle (Figure 3), H&E). By 100 dpi, the number of lymphomononuclear inflammatory cells was reduced and no parasites were detected (Figure 3), H&E). The number of interstitial mononuclear cells was more pronounced at 30 dpi when compared to 100 dpi, especially in the right ventricle (Figure 3), graph). The analysis of picrosirius red-stained sections revealed mild myocardial fibrosis manifested by an increased amount of pericellular collagen (endomysial matrix) and mild perivascular fibrosis. These findings were more evident in the right ventricle. The increase in the volume fraction of fibrosis was significant only at 100 dpi in both ventricles (Figure 3), graph).

Magnetic resonance imaging (MRI) and echocardiography have been used with increasing frequency in experimental Chagas disease. There are many advantages related to the application of these imaging methodologies [51-55]. They are noninvasive and do not require exposure to radioactivity or contrast agents. They also permit the serial monitoring of individual mice, without the necessity of sacrifice. This reduces the number of mice required for the validation of results since each mouse can be imaged several times through the course of disease progression and heart alterations can be evaluated at multiple time points. The MRI study showed no significant difference in the left ventricular internal diameter in infected mice compared with uninfected controls. However, the inner dimension of the right ventricle was significantly dilated from 30 to 100 dpi (Figure 4), arrow). In the evaluation of systolic function by echocardiography, the left ventricle ejection fraction, evaluated by B-mode, did not show any change at 15 dpi in comparison with controls. However, there was a 23% reduction in the left ventricle ejection fraction at 30 dpi, 20% at 60 dpi and 20% at 100 dpi in comparison with respective controls (Figure 4), graph).

Dystrophin loss, inflammation and mortality rate: Although dystrophin gene mutations represent the primary cause in Duchenne and Becker muscular dystrophies, it has been demonstrated that the secondary processes, involving persistent inflammation with high levels of proinflammatory cytokines, likely sustain and exacerbate the progression of these diseases [56]. A previous study demonstrated that Duchenne patients with evidence of active myocarditis associated with myocyte damage and fibrosis had a faster progression to heart failure and death in comparison with Duchenne patients with evidence of healed myocarditis [57]. These observations indicate that inflammatory mechanisms could also be involved in the dystrophin changes observed in our studies. In addition, it has been suggested that inflammation plays a key role in the activation of proteases, mainly calpains. Many of the proteins linking the cytoskeleton to the plasma membrane are cleaved rapidly by the calpains, especially dystrophin [58,59]. To this end, tumor necrosis factor alpha (TNF-α), nuclear factor-kappa B (NF-kB) and calpain-1 levels were investigated in the peak of mortality at the acute phase.

The amount of TNF-α, quantified by Western blotting, was significantly increased, representing an increase of 110% in comparison with control mice. High TNF-α fluorescence expression was observed (Figure 5). Similarly, the amount of NF-KB increased 130% when compared with control animals. The immunofluorescence revealed that NF-kB peak was associated with increased number of inflammatory cells (Figure 5). Proinflammatory cytokines have been demonstrated to exert their actions on NF-kB, contributing to the dystrophic damage progression through activation of intracellular calcium dependent proteases, mainly calpain [43,60].

Calpains are calcium-activated neutral cysteine proteases with two major isoforms besides tissue specific forms: (a) calpain-1 or µ that requires micromolar Ca2+ concentrations for activity and (b) calpain-2 or m that requires millimolar Ca2+ concentrations [59]. The amount of calpain-1 was significantly increased, representing an increase of 70% compared to control mice. The fluorescent signal for calpain-1 was associated with an increased number of inflammatory cells (Figure 5). The detection of significantly increased expression of intracellular calpain implicates this protease, activated by increased intracellular calcium concentration, in the mechanism of dystrophin loss and proteolysis. Previous study demonstrated that calpains digest dystrophin very rapidly when the calcium concentration is compatible with their activation [61]. Increased calpain activity has often been reported as an aggravating factor in cardiovascular diseases and other pathophysiological conditions [62]. The activation of calpains following the elevated intracellular Ca2+ and proteolysis of dystrophin have been shown in different experimental models of cardiomyopathies such as myocardial infarction [46], isoproterenol [44,47] and doxorubicin administration [48] and sepsis [63].

Since proinflammatory cytokine response is associated with Chagas disease, the evaluation of the association among the high mortality rate observed in the acute infection, inflammation and loss/reduction of dystrophin was done. The levels of TNF-α and NF-kB were increased in mice infected with T. cruzi, but increased mortality was observed only when cardiac dystrophin expression was significantly decreased, highlighting the correlation among inflammation, dystrophin loss and mortality (Figure 6).

In vitro studies: In order to confirm our in vivo results, in vitro experiments were performed using cultured neonatal mouse cardiomyocytes. Cardiomyocytes were isolated from neonatal hearts, routinerally processed and when cardiomyocytes showed spontaneous contractility, serum (free of parasites) from either T. cruzi-infected or control mice were added to the cells. This serum was collected in the peak of cytokine production [64,65].

Following the experimental periods, the cardiac cells were processed for Western blotting and immunofluorescence labeling for dystrophin, calpain-1 and NF-kB.

he quantification of dystrophin by immunoblotting showed a reduction of 50% in cultured cardiomyocytes incubated with sera obtained from infected mice in comparison to those incubated with control sera. The IF analysis clearly showed decreased expression of dystrophin associated with formation of multiple blebs dispersed in the cytoplasm of cardiomyocytes. F-actin fluorescence staining revealed disruption and rearrangement of the filaments (Figure 7). Studies using renal tubule cells of rabbit and hepatocytes of rats exposed to highly toxic agents that act by increasing cytosolic calcium levels have shown that intracellular concentrations of calcium precede bleb formation. Pretreatment of these cells with protease inhibitor prevented bleb formation and decreased proteolysis rate [66-68]. The activation of calciumdependent proteases could be responsible for the rearrangement of F-actin filaments inducing the formation of bubbles [69]. These findings emphasize the role of proinflammatory cytokines present in the serum of animals experimentally infected with T. cruzi in the activation of calpain-1 observed in our studies. Immunoblotting results demonstrated that the amount of calpain-1 was increased by 33% in cardiomyocytes incubated with sera obtained from infected mice. This increase was clearly observed in the immunofluorescence (Figure 7).

Cultured cardiomyocytes incubated with sera obtained from infected mice displayed a marked increased fluorescence of NFkB in comparison with cardiomyocytes incubated with control sera. In addition, disruption and rearrangement of F-actin associated with reduced size of cardiomyocytes were observed after the addition of sera obtained from infected mice (Figure 7)

 

Chagas disease is one of the neglected tropical diseases now found in non-endemic areas of the world. In the past 100 years since the discovery of this disease by Carlos Chagas, the understanding of the pathology and pathogenesis of this disease has grown. There are many developments that are exciting and require further investigation. Among these, the long delay after the initial infection to the chronic cardiac manifestations deserves attention. Based on the evidences presented in the present review, these processes involving the decrease of dystrophin expression could be initiated in the acute stage of the disease and perpetuated to the chronic stage contributing to the late development of Chagas cardiomyopathy. Further studies with the use of specific calpain inhibitors could provide novel therapeutic targets to minimize cardiac damage during Chagas disease. This is a continuing interest of our laboratories.

This study was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 06/52882- 3; 06/59618-0; 09/17787-8; 10/19216-5; 09/54010-1).

CMP was supported by a Fogarty International Training Grant D43-TW007129.

1. Rassi A Jr, Rassi A, Marin-Neto JA. Chagas disease. Lancet. 2010; 375: 1388-1402.

2. Maguire JH. Chagas’ disease-can we stop the deaths? N Engl J Med. 2006; 355: 760-761.

3. Lee BY, Bacon KM, Bottazzi ME, Hotez PJ. Global economic burden of Chagas disease: a computational simulation model. Lancet Infect Dis. 2013; 13: 342-348.

4. Rassi A Jr, Rassi A, Marcondes de Rezende J. American trypanosomiasis (Chagas disease). Infect Dis Clin North Am. 2012; 26: 275-291.

5.Dias E, Laranja FS, Miranda A, Nobrega G. Chagas’ disease: a clinical, epidemiologic, and pathologic study. Circulation. 1956; 14: 1035- 1060.

6. Prata A. Clinical and epidemiological aspects of Chagas disease. Lancet Infect Dis. 2001; 1: 92-100.

7. Cunha-Neto E, Chevillard C. Chagas disease cardiomyopathy: immunopathology and genetics. Mediators Inflamm. 2014; 683230.

8. Bestetti RB, Muccillo G. Clinical course of Chagas’ heart disease: a comparison with dilated cardiomyopathy. Int J Cardiol. 1997; 60: 187- 193.

9. Köberle F. Chagas’ disease and Chagas’ syndromes: the pathology of American trypanosomiasis. Adv Parasitol. 1968; 6: 63-116.

10. Rossi MA, Ramos SG, Bestetti RB. Chagas’ heart disease: clinicalpathological correlation. Front Biosci. 2003; 8: e94-109.

11. Almeida HO, Teixeira VP, Gobbi H, Rocha A, Brandao MC. Inflammation associated with cardiac muscle cells parasitized by Trypanosoma cruzi, in chronic Chagas’ disease patients Arq Bras Cardiol. 1984; 42: 183-186.

12. Higuchi ML, Brito T, Reis MM, Barbosa A, Bellotti G, Pereira-Barreto AC, et al. Correlation between T. cruzi parasitism and myocardial inflammation in human chronic chagasic myocarditis. Light microscopy and immunohistochemical findings. Cardiovasc Pathol. 1993; 2: 101-106.

13. Higuchi M de L, Benvenuti LA, Martins Reis M, Metzger M. Pathophysiology of the heart in Chagas’ disease: current status and new developments. Cardiovasc Res. 2003; 60: 96-107.

14. Palomino SA, Aiello VD, Higuchi ML. Systematic mapping of hearts from chronic chagasic patients: the association between the occurrence of histopathological lesions and Trypanosoma cruzi antigens. Ann Trop Med Parasitol. 2000; 94: 571-579.

15. Teixeira V de P, Araujo MB, dos Reis MA, dos Reis L, Silveira SA, Rodrigues ML, et al. Possible role of an adrenal parasite reservoir in the pathogenesis of chronic Trypanosoma cruzi myocarditis. Trans R Soc Trop Med Hyg. 1993; 87: 552-554.

16. Vianna G. Contribuição para o estudo da anatomia patológica da “Moléstia de Carlos chagas”. Mem Inst Oswaldo Cruz. 1911; 3: 276- 293.

17. Arantes RM, Marche HH, Bahia MT, Cunha FQ, Rossi MA, Silva JS. Interferon-gamma-induced nitric oxide causes intrinsic intestinal denervation in Trypanosoma cruzi-infected mice. Am J Pathol. 2004; 164: 1361-1368.

18. Davila DF, Inglessis G, Mazzei de Davila CA. Chagas’ heart disease and the autonomic nervous system. Int J Cardiol. 1998; 66: 123-127.

19. Köberle F. The causation and importance of nervous lesions in American trypanosomiasis. Bull World Health Organ. 1970; 42: 739- 743.

20. Marin-Neto JA, Gallo L Jr, Manco JC, Rassi A, Amorim DS. Postural reflexes in chronic Chagas’s heart disease. Heart rate and arterial pressure responses. Cardiology. 1975; 60: 343-357.

21. Marin-Neto JA, Maciel BC, Gallo Junior L, Junqueira Junior LF, Amorim DS. Effect of parasympathetic impairment on the haemodynamic response to handgrip in Chagas’s heart disease. Br Heart J. 1986; 55: 204-210.

22. Ramos SG, Matturri L, Rossi L, Rossi MA. Sudden cardiac death in the indeterminate phase of Chagas’ disease associated with acute infarction of the right carotid body. Int J Cardiol. 1995; 52: 265-268.

23. Factor SM, Cho S, Wittner M, Tanowitz H. Abnormalities of the coronary microcirculation in acute murine Chagas’ disease. Am J Trop Med Hyg. 1985; 34: 246-253.

24. Morris SA, Tanowitz HB, Wittner M, Bilezikian JP. Pathophysiological insights into the cardiomyopathy of Chagas’ disease. Circulation. 1990; 82: 1900-1909.

25. Ramos SG, Rossi MA. Microcirculation and Chagas’ disease: hypothesis and recent results. Rev Inst Med Trop São Paulo. 1999; 41: 123-129.

26. Rossi MA. Microsvascular changes as a cause of chronic cardiomiopathy in Chagas’disease. Am Heart J. 1990; 120: 233-236.

27. Rossi MA, Carobrez SG. Experimental Trypanosoma cruzi cardiomyopathy in BALB/c mice: histochemical evidence of hypoxic changes in the myocardium. Br J Exp Pathol. 1985; 66: 155-160.

28. Rossi MA, Goncalves S, Ribeiro-dos-Santos R. Experimental Trypanosoma cruzi cardiomyopathy in BALB/c mice. The potential role of intravascular platelet aggregation in its genesis. Am J Pathol. 1984; 114: 209-216.

29. Tanowitz HB, Morris SA, Factor SM, Weiss LM, Wittner M. Parasitic diseases of the heart. Part I: Acute and chronic Chagas’disease. Cardiovasc Pathol. 1992; 1: 7-15.

30. Cossio PM, Diez C, Szarfman A, Kreutzer E, Candiolo B, Arana RM. Chagasic cardiopathy. Demonstration of a serum gamma globulin factor which reacts with endocardium and vascular structures. Circulation. 1974; 49: 13-21.

31. Cossio PM, Laguens RP, Kreutzer E, Diez C, Segal A, Arana RM. Chagasic cardiopathy. Immunopathologic and morphologic studies in myocardial biopsies. Am J Pathol. 1977; 86: 533-544.

32. Cunha-Neto E, Duranti M, Gruber A, Zingales B, De Messias I, Stolf N, et al. Autoimmunity in Chagas disease cardiopathy: biological relevance of a cardiac myosin-specific epitope crossreactive to an immunodominant Trypanosoma cruzi antigen. Proc Natl Acad Sci USA. 1995; 92: 3541-3545.

33. Cunha-Neto E, Coelho V, Guilherme L, Fiorelli A, Stolf N, Kalil J. Autoimmunity in Chagas’ disease. Identification of cardiac myosin-B13 Trypanosoma cruzi protein crossreactive T cell clones in heart lesions of a chronic Chagas’ cardiomyopathy patient. J Clin Invest. 1996; 98: 1709-1712.

34. Cunha-Neto E, Bilate AM, Hyland KV, Fonseca SG, Kalil J, Engman DM. Induction of cardiac autoimmunity in Chagas heart disease: a case for molecular mimicry. Autoimmunity. 2006; 39: 41-54.

35. Ribeiro-dos-Santos R, Rossi MA. Imunopatologia. In: Cançado JR, Chuster M, editors, Cardiopatia chagásica, Belo Horizonte: Fundação Carlos Chagas. 1985: 10-22.

36. Ribeiro dos Santos R, Rossi MA, Laus JL, Silva JS, Savino W, Mengel J. Anti-CD4 abrogates rejection and reestablishes long-term tolerance to syngeneic newborn hearts grafted in mice chronically infected with Trypanosoma cruzi. J Exp Med. 1992; 175: 29-39.

37. Santos-Buch CA, Teixeira AR. The immunology of experimental Chagas’ disease. III: Rejection of allogeneic heart cells in vitro. J Exp Med. 1974; 140: 38-53.

38. Teixeira AR, Santos-Buch CA. The immunology of experimental Chagas’ disease. II. Delayed hypersensitivity to Trypanosoma cruzi antigens. Immunology. 1975; 28: 401-410.

39. Prado CM, Celes MR, Malvestio LM, Campos EC, Silva JS, Jelicks LA, et al. Early dystrophin disruption in the pathogenesis of experimental chronic Chagas cardiomyopathy. Microbes Infect. 2012; 14: 59-68.

40. Malvestio LM, Celes MR, Milanezi C, Silva JS, Jelicks LA, Tanowitz HB, et al. Role of dystrophin in acute Trypanosoma cruzi infection. Microbes Infect. 2014; 16: 768-777.

41. Allikian MJ, McNally EM. Processing and assembly of the dystrophin glycoprotein complex. Traffic. 2007; 8: 177-183.

42. McNally E, Allikian M, Wheeler MT, Mislow JM, Heydemann A. Cytoskeletal defects in cardiomyopathy. J Mol Cell Cardiol. 2003; 35: 231-241.

43. Kumar A, Boriek AM. Mechanical stress activates the nuclear factor kappa B pathway in skeletal muscle fibers: a possible role in Duchenne muscular dystrophy. FASEB J. 2003; 17: 386-396.

44. Kawada T, Masui F, Tezuka A, Ebisawa T, Kumagai H, Nakazawa M, et al. A novel scheme of dystrophin disruption for the progression of advanced heart failure. Biochim Biophys Acta. 2005; 751: 73-81.

45. Badorff C, Lee GH, Lamphear BJ, Martone ME, Campbell KP, Rhoads RE, et al. Enteroviral protease 2A cleaves dystrophin: evidence of cytoskeletal disruption in an acquired cardiomyopathy. Nat Med. 1999; 5: 320-326.

46. Yoshida H, Takahashi M, Koshimizu M, Tanonaka K, Oikawa R, Toyooka T, et al. Decrease in sarcoglycans and dystrophin in failing heart following acute myocardial infarction. Cardiovasc Res. 2003; 59: 419- 427.

47. Campos EC, Romano MM, Prado CM, Rossi MA. Isoproterenol induces primary loss of dystrophin in rat hearts: correlation with myocardial injury. Int J Exp Pathol. 2008; 89: 367-681.

48. Campos EC, O’Connell JL, Malvestio LM, Romano MM, Ramos SG, Celes MR, et al. Calpain-mediated dystrophin disruption may be a potential structural culprit behind chronic doxorubicin-induced cardiomyopathy. Eur J Pharmacol. 2011; 670: 541-553.

49. Celes MR, Torres-Duenãs D, Malvestio LM, Blefari V, Campos EC, Ramos SG, et al. Disruption of sarcolemmal dystrophin and beta-dystroglycan may be a potential mechanism for myocardial dysfunction in severe sepsis. Lab Invest. 2010; 90: 531-542.

50. Danialou G, Comtois AS, Dudley R, Karpati G, Vincent G, Des Rosiers C, et al. Dystrophin-deficient cardiomyocytes are abnormally vulnerable to mechanical stress-induced contractile failure and injury. FASEB J. 2001; 15: 1655-1657.

51. Jelicks LA, Shirani J, Wittner M, Chandra M, Weiss LM, Factor SM, et al. Application of cardiac gated magnetic resonance imaging in murine Chagas’ disease. Am J Trop Med Hyg. 1999; 61: 207-214.

52. Chandra M, Shirani J, Shtutin V, Weiss LM, Factor SM, Petkova SB, et al. Cardioprotective effects of verapamil on myocardial structure and function in a murine model of chronic Trypanosoma cruzi infection (Brazil Strain): an echocardiographic study. Int J Parasitol. 2002; 32: 207-215.

53. Jelicks LA, Tanowitz HB. Advances in imaging of animal models of Chagas disease. Adv Parasitol. 2011; 75: 193-208.

54. Jelicks LA, Lisanti MP, Machado FS, Weiss LM, Tanowitz HB, Desruisseaux MS. Imaging of small-animal models of infectious diseases. Am J Pathol. 2013; 182: 296-304.

55. Jelicks LA, Tanowitz HB, Albanese C. Small animal imaging of human disease: from bench to bedside and back. Am J Pathol. 2013; 182: 294- 295.

56. Spencer MI, Tidball JG. Do immune cells promote the pathology of dystrophin-deficientmyopathies?. Neuromuscul Disord. 2001; 11: 556-564.

57. Mavrogeni S, Papavasiliou A, Spargias K, Constandoulakis P, Papadopoulos G, Karanasios E, et al. Myocardial inflammation in Duchenne Muscular Dystrophy as a precipitating factor for heart failure: a prospective study. BMC Neurol. 2010; 10: 33-40.

58. Zaidi SI, Narahara HT. Degradation of skeletal muscle plasma membrane proteins by calpain. J Membr Biol. 1989; 110: 209-216.

59. Goll DE, Thompson VF, Li H, Wei W, Cong J. The calpain system. Physiol Rev. 2003; 83: 731-801.

60. Tidball JG. Inflammatory processes in muscle injury and repair. Am J Physiol Regul Integr Comp Physiol. 2005; 288: 345-353.

61. Cottin P, Poussard S, Mornet D, Brustis JJ, Mohammadpour M, Leger J, et al. In vitro digestion of dystrophin by calcium-dependent proteases, calpains I and II. Biochimie. 1992; 74: 565-570.

62. Sorimachi H, Ono Y. Regulation and physiological roles of the calpain system in muscular disorders. Cardiovasc Res. 2012; 96: 11-22.

63. Celes MR, Malvestio LM, Suadicani SO, Prado CM, Figueiredo MJ, Campos EC, et al. Disruption of calcium homeostasis in cardiomyocytes underlies cardiac structural and functional changes in severe sepsis. PLoS One. 2013; 8: e68809.

64. Starobinas N, Russo M, Minoprio P, Hontebeyrie-Joskowicz M. Is TNF alpha involved in early susceptibility of Trypanosoma cruzi-infected C3H/He mice?. Res Immunol. 1991; 142: 117-122.

65. Panis C, Mazzuco TL, Costa CZF, Victorino VJ, Tatakihara VLH, Yamauchi LM, et al. Trypanosoma cruzi: effect of the absence of 5-lipoxygenase (5-LO)-derived leukotrienes on levels of cytokines, nitric oxide and iNOS expression in cardiac tissue in the acute phase of infection in mice. Exp Parasitol. 2011; 127: 58-65.

66. Armstrong SC, Shivell LC, Ganote CE. Sarcolemmal blebs and osmotic fragility as correlates of irreversible ischemic injury in preconditioned isolated rabbit cardiomyocytes. J Mol Cell Cardiol. 2001; 33: 149-160.

67. Smith MW, Phelps PC, Trump BF. Cytosolic Ca+2 deregulation and blebbing after HgCl2 injury to cultured rabbit proximal tubule cells as determined by digital imaging microscopy. Proc Natl Acad Sci U S A. 1991; 88: 4926-4930.

68. Nicotera P, Hartzell P, Davis G, Orrenius S. The formation of plasma membrane blebs in hepatocytes exposed to agents that increase cytosolic Ca+2 is mediated by the activation of a non-lysosomal proteolytic system. FEBS Lett. 1986; 209: 139-144.

69. Elliget KA, Phelps PC, Trump BF. HgCl2-induced alteration of actin filaments in cultured primary rat proximal tubule epithelial cells labeled with fluorescein phalloidin. Cell Biol Toxicol. 1991; 7: 263- 280