INCREASED SARCOLEMMAL PERMEABILITY AS AN EARLY EVENT IN EXPERIMENTAL SEPTIC CARDIOMYOPATHY: A POTENTIAL ROLE FOR OXIDATIVE DAMAGE TO LIPIDS AND PROTEINS

This study describes increased sarcolemmal permeability and myofilamentar damage that occur together with lipid peroxidation and protein nitration in the myocardium in severe sepsis induced by cecal ligation and puncture. Male C57BL/6 mice were submitted to moderate and severe septic injury and sham operation. Using light and laser confocal microscopy, diffuse foci of myocytolysis associated with focal disruption of the actin/myosin contractile apparatus could be seen in hearts with severe septic injury. The myocardial expressions of the sarcomeric proteins myosin and actin were downregulated by both severe and moderate injuries. The detection of albumin staining in the cytoplasm of myocytes to evaluate sarcolemmal permeability provided evidence of severe and mild injury of the plasma membrane in hearts with severe and moderate septic injury, respectively. The administration of a superoxide scavenger caused marked reduction of sarcolemmal permeability, indicating the involvement of free radicals in its genesis. On electron microscopy, these changes were seen to correspond to spread blocks of a few myocytes with fragmentation and dissolution of myofibrils, intracellular edema, and, occasionally, rupture of the sarcolemma. In addition, oxidative damage to lipids, using anti-4-hydroxynonenal, an indicator of oxidative stress and disruption of plasma membrane lipids, and to proteins, using antinitrotyrosine, a stable biomarker of peroxynitrite-mediated protein nitration, was demonstrated. These findings make plausible the hypothesis that increased sarcolemmal permeability might be a primary event in myocardial injury in severe sepsis possibly due to oxidative damage to lipids and proteins that could precede phenotypic changes that characterize a septic cardiomyopathy.FAPESP Fundacao de Amparo a Pesquisa do Estado de Sao Paulo[06/52882-3]FAPESP Fundacao de Amparo a Pesquisa do Estado de Sao Paulo[06/59618-0]FAPESP Fundacao de Amparo a Pesquisa do Estado de Sao Paulo[07/52556-1]FAPESP Fundacao de Amparo a Pesquisa do Estado de Sao Paulo[07/58843-2]FAPESP Fundacao de Amparo a Pesquisa do Estado de Sao Paulo[07/59448-0]Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, CNP

Modulation of the mTOR Pathway by Curcumin in the Heart of Septic Mice

mTOR is a signaling pathway involved in cell survival, cell stress response, and protein synthesis that may be a key point in sepsis-induced cardiac dysfunction. Curcumin has been reported in vitro as an mTOR inhibitor compound; however, there are no studies demonstrating this effect in experimental sepsis. Thus, this study aimed to evaluate the action of curcumin on the mTOR pathway in the heart of septic mice. Free curcumin (FC) and nanocurcumin (NC) were used, and samples were obtained at 24 and 120 h after sepsis. Histopathological and ultrastructural analysis showed that treatments with FC and NC reduced cardiac lesions caused by sepsis. Our main results demonstrated that curcumin reduced mTORC1 and Raptor mRNA at 24 and 120 h compared with the septic group; in contrast, mTORC2 mRNA increased at 24 h. Additionally, the total mTOR mRNA expression was reduced at 24 h compared with the septic group. Our results indicate that treatment with curcumin and nanocurcumin promoted a cardioprotective response that could be related to the modulation of the mTOR pathway

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. (C) 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [07/58843-2, 07/59448-0, 09/17787-8, 09/53544-2, 09/54010-1, 10/19216-5, 10/18629-4, 10/13199-1]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) [472419/2009-9, 150723/2010-5]National Institutes of Health [AI076248]National Institutes of HealthFogarty International Training Grant [D43-TW007129]Fogarty International Training Gran

Effect of Verapamil, an L-Type Calcium Channel Inhibitor, on Caveolin-3 Expression in Septic Mouse Hearts

Sepsis-induced myocardial dysfunction considerably increases mortality risk in patients with sepsis. Previous studies from our group have shown that sepsis alters the expression of structural proteins in cardiac cells, resulting in cardiomyocyte degeneration and impaired communication between cardiac cells. Caveolin-3 (CAV3) is a structural protein present in caveolae, located in the membrane of cardiac muscle cells, which regulates physiological processes such as calcium homeostasis. In sepsis, there is a disruption of calcium homeostasis, which increases the concentration of intracellular calcium, which can lead to the activation of potent cellular enzymes/proteases which cause severe cellular injury and death. The purpose of the present study was to test the hypotheses that sepsis induces CAV3 overexpression in the heart, and the regulation of L-type calcium channels directly relates to the regulation of CAV3 expression. Severe sepsis increases the expression of CAV3 in the heart, as immunostaining in our study showed CAV3 presence in the cardiomyocyte membrane and cytoplasm, in comparison with our control groups (without sepsis) that showed CAV3 presence predominantly in the plasma membrane. The administration of verapamil, an L-type calcium channel inhibitor, resulted in a decrease in mortality rates of septic mice. This effect was accompanied by a reduction in the expression of CAV3 and attenuation of cardiac lesions in septic mice treated with verapamil. Our results indicate that CAV3 has a vital role in cardiac dysfunction development in sepsis and that the regulation of L-type calcium channels may be related to its expression

Verapamil and dantrolene restrained ejection fraction (EF) and fractional shortening (FS) changes induced by severe sepsis in mice.

<p>(The left ventricular ejection fraction and fractional shortening in untreated mice (Sham) or in mice treated with either verapamil (SH+VP) or dantrolene (SH+DT) at 12 and 24 hours after sham operation were absolutely identical regarding each procedure and computed and represented as a single column). <b>A and B.</b> The left ventricular ejection fraction and fractional shortening in mice submitted to CLP-induced sepsis were similar to the values in mice submitted to sham operation at 12 hours after surgical procedure. But the left ventricular ejection fraction and fractional shortening in SSI mice was markedly reduced in comparison with the mean values observed in sham controls at 24 hours after surgery. In contrast, the treatment with either verapamil or dantrolene abrogated the left ventricular ejection fraction and fractional shortening in SSI mice that presented mean values similar to those observed sham-operated mice treated with either verapamil or dantrolene and sham-operated untreated mice. (n = 7 to 10 in each group; *, P<0.01).</p

Verapamil and dantrolene improved survival rate in mice submitted to CLP-induced sepsis.

<p>Sham-operated untreated mice presented a 100% survival rate throughout the analyzed period of 120 hours. SSI mice had a survival rate of 50% at 24 hours after surgery, decreasing to 10% at 96 hours and remaining steady at this rate until 120 hours after surgery. In contrast, SSI mice treated with verapamil showed a survival rate around 75% at 24 hours, decreasing to 50% at 96 hours and remaining steady at this rate until the end of the experimental period. Similarly, SSI mice treated with dantrolene disclosed a survival rate around 60% at 24 hours, decreasing to 50% at 96 hours and remaining steady at this rate until 120 hours after CLP. The bar graph illustrates the survival rate for each group at 5 days after the surgical procedure. (n = 8 animals per group).</p

Decreased expression and amount of dystrophin in cultured cardiomyocytes stimulated with septic serum.

<p>The fluorescent signal for dystrophin (green fluorescence) presented decreased expression (bottom panel) with bleb formation in cardiomyocytes stimulated with septic serum in comparison with the fluorescence expression in cardiomyocytes expose to serum from sham-operated mice (upper panel). F-actin was stained with phalloidin conjugated Alexa Fluor 594 dye (red fluorescence) and DNA with DAPI (blue fluorescence). Alexa Fluor 594 staining showed disruption and rearrangement of F-actin filaments. Western blotting demonstrated that dystrophin amount was markedly decreased, around 41%, in cardiomyocytes stimulated with serum from septic mice compared to amounts observed in cardiomyocytes stimulated with serum from sham-operated mice (*, P<0.01). α-Tubulin was used as a protein loading control. Data are expressed as mean ± S.D. Scale bars indicate 50 μm.</p

Electron microscopic study of the mice myocardium 24 hours after septic injury and sham-operation.

<p><b>A.</b> The ultrastructural examination revealed that the myocardium from control mice did not differ from that reported in the literature presenting continuous and regularly spaced myofibrils that alternate with serially arranged, elongated mitochondria (mi). Sarcomeres are clearly delimited by two neighboring well-defined Z-bands. Interfibrillar mitochondria are closely related to the sarcomere. Magnification bar represents 2 µm. <b>B.</b> The ultrastructure of the myocardium of mice with severe sepsis 24 hours after CLP showed ill-defined sarcomeres with abundant accumulation of autophagolysosomes within cardiomyocytes (arrows). Magnification bar represents 2 µm. <b>C.</b> Higher magnification showing autophagolysosomes (arrows), foci of ill-defined and disorganized/disrupted sarcomeres and blurring/breakage of Z-bands (Z). Magnification bar represents 1 µm. <b>D.</b> Increased number of electron-opaque calcium granules within mitochondria (arrow heads), breakage of Z band (Z) and autophagolysosome (arrow). Magnification bar represents 500 nm. M, M line; Nu, nucleus; pm, plasma membrane; IS, interstitial space; Cap, capillary.</p