Dantrolene improves in vitro structural changes induced by serum from Trypanosoma cruzi-infected mice.

Dystrophin, an important protein of the dystrophin-glycoprotein complex, has been implicated in the pathogenesis of experimental Chagas disease. It is important for the maintenance of cell shape and contraction force transmission. Dystrophin loss has been related to end-stage cardiac myopathies and proposed as a common route for myocardial dysfunction and progression to advanced heart failure. Evidence suggests that calpains, calcium-dependent proteases, digest dystrophin when the calcium concentration is compatible with their activation. The objective of this in vitro study was to test the hypothesis that dantrolene, a calcium channel blocker, improves structural changes induced by serum from Trypanosoma cruzi-infected mice. Cultured neonatal cardiac myocytes were incubated with serum from T. cruzi-infected mice and treated with dantrolene for 24 h. Immunofluorescence and immunoblotting were performed to evaluate dystrophin and calpain-1 protein expression. The levels of dystrophin decreased 13 % and calpain increased 17 % after incubation of cultured neonatal cardiac myocytes with serum from T. cruzi-infected mice. The treatment with dantrolene restored the dystrophin and calpain levels near control levels. Our results demonstrate that alterations in calcium homeostasis in cardiac myocytes are responsible, in part, for cardiac structural changes in experimentally induced T. cruzi myocarditis and that calpain inhibitors may be beneficial in Chagasic heart disease.

Disruption of sarcolemmal dystrophin and beta-dystroglycan may be a potential mechanism for myocardial dysfunction in severe sepsis.

Evidence from our laboratory has shown alterations in myocardial structure in severe sepsis/septic shock. The morphological alterations are heralded by sarcolemmal damage, characterized by increased plasma membrane permeability caused by oxidative damage to lipids and proteins. The critical importance of the dystrophin-glycoprotein complex (DGC) in maintaining sarcolemmal stability led us to hypothesize that loss of dystrophin and associated glycoproteins could be involved in early increased sarcolemmal permeability in experimentally induced septic cardiomyopathy. Male C57Bl/6 mice were subjected to sham operation and moderate (MSI) or severe (SSI) septic injury induced by cecal ligation and puncture (CLP). Using western blot and immunofluorescence, a downregulation of dystrophin and beta-dystroglycan expression in both severe and moderate injury could be observed in septic hearts. The immunofluorescent and protein amount expressions of laminin-alpha2 were similar in SSI and sham-operated hearts. Consonantly, the evaluation of plasma membrane permeability by intracellular albumin staining provided evidence of severe injury of the sarcolemma in SSI hearts, whereas antioxidant treatment significantly attenuated the loss of sarcolemmal dystrophin expression and the increased membrane permeability. This study offers novel and mechanistic data to clarify subcellular events in the pathogenesis of cardiac dysfunction in severe sepsis. The main finding was that severe sepsis leads to a marked reduction in membrane localization of dystrophin and beta-dystroglycan in septic cardiomyocytes, a process that may constitute a structural basis of sepsis-induced cardiac depression. In addition, increased sarcolemmal permeability suggests functional impairment of the DGC complex in cardiac myofibers. In vivo observation that antioxidant treatment significantly abrogated the loss of dystrophin expression and plasma membrane increased permeability supports the hypothesis that oxidative damage may mediate the loss of dystrophin and beta-dystroglycan in septic mice. These abnormal parameters emerge as therapeutic targets and their modulation may provide beneficial effects on future cardiovascular outcomes and mortality in sepsis.

Mast cells in the pathophysiology of Duchenne muscular dystrophy in Golden Retriever dogs / O papel dos mastócitos na patogênese da distrofia muscular de Duchenne em cães Golden Retriever

The Golden Retriever muscular dystrophy (GRMD) is one of the best models of Duchenne muscular dystrophy (DMD), with similar genotypic and phenotypic manifestations. Progressive proliferation of connective tissue in the endomysium of the muscle fibers occurs in parallel with the clinical course of the disease in GRMD animals. Previous studies suggest a relationship between mast cells and the deposition of fibrous tissue due to the release of mediators that recruit fibroblasts. The aim of this study was to evaluate the presence of mast cells and their relationship with muscle injury and fibrosis in GRMD dogs of different ages. Samples of muscle groups from six GRMD and four control dogs, aged 2 to 8 months, were collected and analyzed. The samples were processed and stained with HE, toluidine blue, and Azan trichrome. Our results showed that there was a significant increase in infiltration of mast cells in all muscle groups of GRMD dogs compared to the control group. The average number of mast cells, as well as the deposition of fibrous tissue, decreased with age in GRMD dogs. In the control group, all muscle types showed a significant increase in the amount of collagenous tissue. This suggests increased mast cell degranulation occurred in younger GRMD dogs, resulting in increased interstitial space and fibrous tissue in muscle, which then gradually decreased over time as the dogs aged. However, further studies are needed to clarify the role of mast cells in the pathogenesis of fibrosis.(AU)

O cão Golden Retriever distrófico (Golden Retriever muscular dystrophy - GRMD) é um dos melhores modelos da distrofia muscular de Duchenne (DMD), com manifestações genotípicas e fenotípicas similares. A proliferação progressiva de tecido conjuntivo no endomísio das fibras musculares ocorre paralelamente ao curso clínico da doença em animais GRMD. Estudos anteriores sugerem uma relação entre os mastócitos e a deposição de tecido fibroso devido à liberação de mediadores que recrutam fibroblastos. O objetivo deste estudo foi avaliar a presença de mastócitos e sua relação com a lesão muscular e fibrose em cães GRMD de diferentes idades. Amostras de grupos musculares de seis GRMD e quatro controles, com idade entre 2 a 8 meses, foram coletadas e analisadas. As amostras foram processadas e coradas com HE, azul de toluidina e tricrômico de Azan. Nossos resultados mostraram que houve um aumento significativo na infiltração de mastócitos em todos os grupos musculares de cães GRMD em comparação com o grupo controle. O número médio de mastócitos, assim como a deposição de tecido fibroso, diminuiu com a idade em cães GRMD. No grupo controle, todos os tipos musculares mostraram um aumento significativo na quantidade de tecido colágeno. Isto sugere o aumento da degranulação de mastócitos em cães GRMD mais jovens, resultando em aumento do espaço intersticial e tecido fibroso no músculo, que então gradualmente diminuiu com o tempo à medida que os cães envelheceram. No entanto, mais estudos são necessários para esclarecer o papel dos mastócitos na patogênese da fibrose.(AU)

Role of dystrophin in acute Trypanosoma cruzi infection.

Previous studies have demonstrated loss/reduction of dystrophin in cardiomyocytes in both acute and chronic stages of experimental Trypanosoma cruzi (T. cruzi) infection in mice. The mechanisms responsible for dystrophin disruption in the hearts of mice acutely infected with T. cruzi are not completely understood. The present in vivo and in vitro studies were undertaken to evaluate the role of inflammation in dystrophin disruption and its correlation with the high mortality rate during acute infection. C57BL/6 mice were infected with T. cruzi and killed 14, 20 and 26 days post infection (dpi). The intensity of inflammation, cardiac expression of dystrophin, calpain-1, NF-κB, TNF-α, and sarcolemmal permeability were evaluated. Cultured neonatal murine cardiomyocytes were incubated with serum, collected at the peak of cytokine production and free of parasites, from T. cruzi-infected mice and dystrophin, calpain-1, and NF-κB expression analyzed. Dystrophin disruption occurs at the peak of mortality and inflammation and is associated with increased expression of calpain-1, TNF-α, NF-κB, and increased sarcolemmal permeability in the heart of T. cruzi-infected mice at 20 dpi confirmed by in vitro studies. The peak of mortality occurred only when significant loss of dystrophin in the hearts of infected animals occurred, highlighting the correlation between inflammation, dystrophin loss and mortality.

Disruption of calcium homeostasis in cardiomyocytes underlies cardiac structural and functional changes in severe sepsis.

Sepsis, a major cause of morbidity/mortality in intensive care units worldwide, is commonly associated with cardiac dysfunction, which worsens the prognosis dramatically for patients. Although in recent years the concept of septic cardiomyopathy has evolved, the importance of myocardial structural alterations in sepsis has not been fully explored. This study offers novel and mechanistic data to clarify subcellular events that occur in the pathogenesis of septic cardiomyopathy and myocardial dysfunction in severe sepsis. Cultured neonatal mice cardiomyocytes subjected to serum obtained from mice with severe sepsis presented striking increment of [Ca(2+)]i and calpain-1 levels associated with decreased expression of dystrophin and disruption and derangement of F-actin filaments and cytoplasmic bleb formation. Severe sepsis induced in mice led to an increased expression of calpain-1 in cardiomyocytes. Moreover, decreased myocardial amounts of dystrophin, sarcomeric actin, and myosin heavy chain were observed in septic hearts associated with depressed cardiac contractile dysfunction and a very low survival rate. Actin and myosin from the sarcomere are first disassembled by calpain and then ubiquitinated and degraded by proteasome or sequestered inside specialized vacuoles called autophagosomes, delivered to the lysosome for degradation forming autophagolysosomes. Verapamil and dantrolene prevented the increase of calpain-1 levels and preserved dystrophin, actin, and myosin loss/reduction as well cardiac contractile dysfunction associated with strikingly improved survival rate. These abnormal parameters emerge as therapeutic targets, which modulation may provide beneficial effects on future vascular outcomes and mortality in sepsis. Further studies are needed to shed light on this mechanism, mainly regarding specific calpain inhibitors.

Early dystrophin disruption in the pathogenesis of experimental chronic Chagas cardiomyopathy.

Chronic Chagas cardiomyopathy evolves over a long period of time after initial infection by Trypanosoma cruzi. Similarly, a cardiomyopathy appears later in life in muscular dystrophies. This study tested the hypothesis that dystrophin levels are decreased in the early stage of T. cruzi-infected mice that precedes the later development of a cardiomyopathy. CD1 mice were infected with T. cruzi (Brazil strain), killed at 30 and 100 days post infection (dpi), and the intensity of inflammation, percentage of interstitial fibrosis, and dystrophin levels evaluated. Echocardiography and magnetic resonance imaging data were evaluated from 15 to 100 dpi. At 30 dpi an intense acute myocarditis with ruptured or intact intracellular parasite nests was observed. At 100 dpi a mild chronic fibrosing myocarditis was detected without parasites in the myocardium. Dystrophin was focally reduced or completely lost in cardiomyocytes at 30 dpi, with the reduction maintained up to 100 dpi. Concurrently, ejection fraction was reduced and the right ventricle was dilated. These findings support the hypothesis that the initial parasitic infection-induced myocardial dystrophin reduction/loss, maintained over time, might be essential to the late development of a cardiomyopathy in mice.

Calpain-mediated dystrophin disruption may be a potential structural culprit behind chronic doxorubicin-induced cardiomyopathy.

The critical importance of dystrophin to cardiomyocyte contraction and sarcolemmal and myofibers integrity, led us to test the hypothesis that dystrophin reduction/loss could be involved in the pathogenesis of doxorubicin-induced cardiomyopathy, in order to determine a possible specific structural culprit behind heart failure. Rats received total cumulative doses of doxorubicin during 2 weeks: 3.75, 7.5, and 15 mg/kg. Controls rats received saline. Fourteen days after the last injection, hearts were collected for light and electron microscopy, immunofluorescence and western blot. The cardiac function was evaluated 7 and 14 days after drug or saline. Additionally, dantrolene (5 mg/kg), a calcium-blocking agent that binds to cardiac ryanodine receptors, was administered to controls and doxorubicin-treated rats (15 mg/kg). This study offers novel and mechanistic data to clarify molecular events that occur in the myocardium in doxorubicin-induced chronic cardiomyopathy. Doxorubicin led to a marked reduction/loss in dystrophin membrane localization in cardiomyocytes and left ventricular dysfunction, which might constitute, in association with sarcomeric actin/myosin proteins disruption, the structural basis of doxorubicin-induced cardiac depression. Moreover, increased sarcolemmal permeability suggests functional impairment of the dystrophin-glycoprotein complex in cardiac myofibers and/or oxidative damage. Increased expression of calpain, a calcium-dependent protease, was markedly increased in cardiomyocytes of doxorubicin-treated rats. Dantrolene improved survival rate and preserved myocardial dystrophin, calpain levels and cardiac function, which supports the opinion that calpain mediates dystrophin loss and myofibrils degradation in doxorubicin-treated rats. Studies are needed to further elucidate this mechanism, mainly regarding specific calpain inhibitors, which may provide new interventional pathways to prevent doxorubicin-induced cardiomyopathy.

Coronary microvascular disease in chronic Chagas cardiomyopathy including an overview on history, pathology, and other proposed pathogenic mechanisms.

This review focuses on the short and bewildered history of Brazilian scientist Carlos Chagas's discovery and subsequent developments, the anatomopathological features of chronic Chagas cardiomyopathy (CCC), an overview on the controversies surrounding theories concerning its pathogenesis, and studies that support the microvascular hypothesis to further explain the pathological features and clinical course of CCC. It is our belief that knowledge of this particular and remarkable cardiomyopathy will shed light not only on the microvascular involvement of its pathogenesis, but also on the pathogenetic processes of other cardiomyopathies, which will hopefully provide a better understanding of the various changes that may lead to an end-stage heart disease with similar features. This review is written to celebrate the 100th anniversary of the discovery of Chagas disease.