Role of endothelin 1 in the pathogenesis of chronic chagasic heart disease.

On the basis of previous observations, endothelin 1 (ET-1) has been suggested as contributing to the pathogenesis of Chagasic cardiomyopathy. Therefore, ET-1flox/flox;alpha-MHC-Cre(+) mice in which the ET-1 gene was deleted from cardiac myocytes and ET-1flox/flox;Tie 2 Cre(+) mice in which the ET-1 gene was deleted from endothelial cells were infected with Trypanosoma cruzi. Genetic controls for these cell-specific ET-1 knockout mice were used. Ninety percentage of all mice survived acute infection with the Brazil strain and were evaluated 130 days postinfection. Inflammation and fibrosis were observed in all infected mice; however, fibrosis was reduced in ET-1flox/flox;alpha-MHC-Cre(+) mice. Cardiac magnetic resonance imaging revealed that infection resulted in a significant increase in right ventricular internal diameter (RVID) in all mice except ET-1flox/flox;alpha-MHC-Cre(+) mice; i.e., RVID was not changed in infected ET-1flox/flox;alpha-MHC-Cre(+) mice. Echocardiography of the left ventricle demonstrated increased left ventricular end-diastolic diameter, reduced fractional shortening, and decreased relative wall thickness in infected mice. However, the magnitude of the changes was significantly less in ET-1flox/flox;alpha-MHC-Cre(+) mice compared to other groups. These data provide further evidence of a role for ET-1, particularly cardiac myocyte-derived ET-1, in the pathogenesis of chronic Chagasic cardiomyopathy.

Effects of early and late verapamil administration on the development of cardiomyopathy in experimental chronic Trypanosoma cruzi (Brazil strain) infection.

Chagas' disease, caused by Trypanosoma cruzi, leads to acute myocarditis and chronic cardiomyopathy. Myocardial structure and function were evaluated in T. cruzi (Brazil strain)-infected CD1 mice by histopathology, cardiac gated magnetic resonance imaging (MRI) and transthoracic echocardiography. There was a significant reduction in inflammation and fibrosis in infected mice treated early in infection. In mice treated late in infection, echocardiography revealed a significant increase in the end diastolic diameter and a decrease in percent fractional shortening and relative wall thickness. MRI revealed an increase in the right ventricular internal dimension. These findings, consistent with a dilated cardiomyopathy, were ameliorated in the early but not in the late treatment group, demonstrating that late treatment with verapamil is ineffective in reversing the development of chagasic cardiomyopathy in chronically infected mice. Our data underscore the hypothesis that early events determine the progression to cardiomyopathy and that early treatment with verapamil can prevent such progression.

Caveolin-1 null (-/-) mice show dramatic reductions in life span.

Caveolae are 50-100 nm flask-shaped invaginations of the plasma membrane found in most cell types. Caveolin-1 is the principal protein component of caveolae membranes in nonmuscle cells. The recent development of Cav-1-deficient mice has allowed investigators to study the in vivo functional role of caveolae in the context of a whole animal model, as these mice lack morphologically detectable caveolae membrane domains. Surprisingly, Cav-1 null mice are both viable and fertile. However, it remains unknown whether loss of caveolin-1 significantly affects the overall life span of these animals. To quantitatively determine whether loss of Cav-1 gene expression confers any survival disadvantages with increasing age, we generated a large cohort of mice (n = 180), consisting of Cav-1 wild-type (+/+) (n = 53), Cav-1 heterozygous (+/-) (n = 70), and Cav-1 knockout (-/-) (n = 57) animals, and monitored their long-term survival over a 2 year period. Here, we show that Cav-1 null (-/-) mice exhibit an approximately 50% reduction in life span, with major declines in viability occurring between 27 and 65 weeks of age. However, Cav-1 heterozygous (+/-) mice did not show any changes in long-term survival, indicating that loss of both Cav-1 alleles is required to mediate a reduction in life span. Mechanistically, these dramatic reductions in life span appear to be secondary to a combination of pulmonary fibrosis, pulmonary hypertension, and cardiac hypertrophy in Cav-1 null mice. Taken together, our results provide the first demonstration that loss of Cav-1 gene expression and caveolae organelles dramatically affects the long-term survival of an organism. In addition, aged Cav-1 null mice may provide a new animal model to study the pathogenesis and treatment of progressive hypertrophic cardiomyopathy and sudden cardiac death syndrome.

Inhibition of cardiac myocyte apoptosis improves cardiac function and abolishes mortality in the peripartum cardiomyopathy of Galpha(q) transgenic mice.

BACKGROUND: Although the occurrence of cardiac myocyte apoptosis during heart failure has been documented, its importance in pathogenesis is unknown. Transgenic mice with cardiac-restricted overexpression of Galpha(q) exhibit a lethal, peripartum cardiomyopathy accompanied by apoptosis. To test whether apoptosis is causally linked to heart failure, we assessed whether inhibiting this cell death would improve left ventricular function and survival in the Galpha(q) peripartum cardiomyopathy model. METHODS AND RESULTS: The potent polycaspase inhibitor IDN-1965 or vehicle was administered subcutaneously to Galpha(q) mice by osmotic minipump beginning on day 12 of pregnancy and continuing through euthanasia at day 14 postpartum. As expected, IDN-1965 markedly suppressed cardiac caspase-3-like activity (86.5%; P<0.01), accompanied by reduction in the frequency of cardiac myocyte apoptosis from 1.9+/-0.3% to 0.2+/-0.1% (P<0.01). Animals receiving IDN-1965 exhibited significant improvements in left ventricular end-diastolic dimension (vehicle, 4.7+/-0.1 mm; IDN-1965, 4.2+/-0.1 mm; P<0.01), fractional shortening (vehicle, 30.7+/-1.2%; IDN-1965, 38.9+/-1.0%; P<0.01), positive (vehicle, 3972+/-412; IDN-1965, 5870+/-295; P<0.01) and negative (vehicle, 2365+/-213; IDN-1965, 3413+/-201; P<0.01) dP/dt, and complete suppression of mortality (vehicle, 6 of 20 died; IDN-1965, 0 of 14 died; P<0.05). CONCLUSIONS: Reduction in cardiac myocyte apoptosis by caspase inhibition improved left ventricular function and survival in pregnant Galpha(q) mice. These data indicate that cardiac myocyte apoptosis plays a causal role in the pathogenesis of cardiomyopathy in this model. Caspase inhibition may provide a novel therapeutic target for heart failure.

A mechanistic role for cardiac myocyte apoptosis in heart failure.

Heart failure is a common, lethal condition whose pathogenesis is poorly understood. Recent studies have identified low levels of myocyte apoptosis (80-250 myocytes per 10(5) nuclei) in failing human hearts. It remains unclear, however, whether this cell death is a coincidental finding, a protective process, or a causal component in pathogenesis. Using transgenic mice that express a conditionally active caspase exclusively in the myocardium, we demonstrate that very low levels of myocyte apoptosis (23 myocytes per 10(5) nuclei, compared with 1.5 myocytes per 10(5) nuclei in controls) are sufficient to cause a lethal, dilated cardiomyopathy. Interestingly, these levels are four- to tenfold lower than those observed in failing human hearts. Conversely, inhibition of cardiac myocyte death in this murine model largely prevents the development of cardiac dilation and contractile dysfunction, the hallmarks of heart failure. To our knowledge, these data provide the first direct evidence that myocyte apoptosis may be a causal mechanism of heart failure, and they suggest that inhibition of this cell death process may constitute the basis for novel therapies.

Cardiovascular imaging in clinical and experimental acute infectious myocarditis.

Acute myocarditis can be caused by a variety of organisms. Congestive heart failure and death may occur as a consequence of these infections. In recent years cardiac imaging, by echocardiography and magnetic resonance and nuclear imaging has become a useful adjunct in the diagnosis and management of infectious myocarditis. Specific examples of their usefulness will be given in the discussions of chagasic myocarditis, bacterial myocarditis, acquired immunodeficiency syndrome and peripartum cardiomyopathy. Finally, we explore the exciting area of cardiac imaging of small animals, such as mice infected with Trypanosoma cruzi.

Caveolin-1 null mice develop cardiac hypertrophy with hyperactivation of p42/44 MAP kinase in cardiac fibroblasts.

Recently, development of a caveolin-1-deficient (Cav-1 null) mouse model has allowed the detailed analysis of caveolin-1's function in the context of a whole animal. Interestingly, we now report that the hearts of Cav-1 null mice are markedly abnormal, despite the fact that caveolin-1 is not expressed in cardiac myocytes. However, caveolin-1 is abundantly expressed in the nonmyocytic cells of the heart, i.e., cardiac fibroblasts and endothelia. Quantitative imaging studies of Cav-1 null hearts demonstrate a significantly enlarged right ventricular cavity and a thickened left ventricular wall with decreased systolic function. Histological analysis reveals myocyte hypertrophy with interstitial/perivascular fibrosis. Because caveolin-1 is thought to act as a negative regulator of the p42/44 MAP kinase cascade, we performed Western blot analysis with phospho-specific antibodies that only recognize activated ERK1/2. As predicted, the p42/44 MAP kinase cascade is hyperactivated in Cav-1 null heart tissue (i.e., interstitial fibrotic lesions) and isolated cardiac fibroblasts. In addition, endothelial and inducible nitric oxide synthase levels are dramatically upregulated. Thus loss of caveolin-1 expression drives p42/44 MAP kinase activation and cardiac hypertrophy.

Cardioprotective effects of phosphoramidon on myocardial structure and function in murine Chagas' disease.

Chagas' disease is an important cause of cardiomyopathy. Endothelin-1, a vasoactive peptide has been implicated in the pathogenesis of chagasic cardiomyopathy. C57BL/6 x 129sv and CD1 mice were thus, infected with trypomastigotes of Trypanosoma cruzi (Brazil strain) and these infected mice were compared with infected mice treated with phosphoramidon. This compound inhibits endothelin-converting enzyme and neutral endopeptidases and does not affect the growth of the parasite in culture. Phosphoramidon was given in a dose of 10mg/kg for the initial 15 days post-infection None of the C57Bl/6 x 129sv mice died as a result of infection. However, there was marked myocardial inflammation and fibrosis in infected, untreated mice. The hearts of the infected, phosphoramidon-treated mice showed significantly less pathology. Cardiac magnetic resonance imaging of infected mice revealed right ventricular dilation that was less severe in those treated with phosphoramidon. Phosphoramidon-treated CD1 mice survived the acute infection. Transthoracic echocardiography demonstrated left ventricular dilation and reduced percent fractional shortening and relative wall thickness. These alterations were also attenuated as a result of phosphoramidon treatment. These data suggest that endothelin-1 contributes to the pathogenesis of chagasic cardiomyopathy and interventions that inhibit the synthesis of endothelin-1 and/or neutral endopeptidase might have a protective effect on myocardial structure and function in murine Chagas' disease.

Role of cardiac myocyte-derived endothelin-1 in chagasic cardiomyopathy: molecular genetic evidence.

Trypanosoma cruzi is the aetiological agent of Chagas' disease, an important cause of chronic cardiomyopathy. We previously demonstrated a role for endothelin-1 (ET-1) in the pathogenesis of chagasic heart disease. In order to explore further the significance of ET-1 in chagasic heart disease, we infected ET-1 (flox/flox); alpha-MHC-Cre(+) (ET-1KO) mice, in which the ET-1 gene has been deleted from cardiac myocytes, with 10(4) T. cruzi (Brazil strain) trypomastigotes. As controls, we used ET-1 (flox/flox);Cre(-) (FLOX) and C57BL/6x129sv (WT) mice. All mice survived and were evaluated 150-160 days post-infection. Cardiac magnetic resonance imaging revealed a significant increase in right ventricular internal diameter in all infected animals except ET-1KO mice (control WT, 1.6+/-0.10 mm; infected WT, 2.8+/-0.15 mm; control FLOX, 2.04+/-0.02 mm; infected FLOX, 2.76+/-0.28 mm). There was no significant difference in right ventricular internal diameter between infected and uninfected ET-1KO mice (control ET-1KO, 1.83+/-0.11 mm; infected ET-1KO, 2.14+/-0.20 mm). In another series of experiments, transthoracic echocardiography was performed on uninfected as well as infected ET-1KO mice, and uninfected and infected FLOX mice. Both infected groups had an increased left ventricular end-diastolic diameter and reduced fractional shortening. In addition, relative wall thickness was also decreased in infected animals. However, the magnitude of these changes was less in infected ET-1KO mice. These data provide further support for a role for ET-1 in the pathogenesis of chronic chagasic heart disease, and indicate that cardiac myocytes are an important source of ET-1 in this disease.

Caveolin-3 knock-out mice develop a progressive cardiomyopathy and show hyperactivation of the p42/44 MAPK cascade.

A growing body of evidence suggests that muscle cell caveolae may function as specialized membrane micro-domains in which the dystrophin-glycoprotein complex and cellular signaling molecules reside. Caveolin-3 (Cav-3) is the only caveolin family member expressed in striated muscle cell types (cardiac and skeletal). Interestingly, skeletal muscle fibers from Cav-3 (-/-) knock-out mice show a number of myopathic changes, consistent with a mild-to-moderate muscular dystrophy phenotype. However, it remains unknown whether a loss of Cav-3 affects the phenotypic behavior cardiac myocytes in vivo. Here, we present a detailed characterization of the hearts of Cav-3 knock-out mice. We show that these mice develop a progressive cardiomyopathic phenotype. At four months of age, Cav-3 knock-out hearts display significant hypertrophy, dilation, and reduced fractional shortening, as revealed by gated cardiac MRI and transthoracic echocardiography. Histological analysis reveals marked cardiac myocyte hypertrophy, with accompanying cellular infiltrates and progressive interstitial/peri-vascular fibrosis. Interestingly, loss of Cav-3 expression in the heart does not change the expression or the membrane association of the dystrophin-glycoprotein (DG) complex. However, a marker of the DG complex, alpha-sarcoglycan, was specifically excluded from lipid raft domains in the absence of Cav-3. Because activation of the Ras-p42/44 MAPK pathway in cardiac myocytes can drive cardiac hypertrophy, we next assessed the activation state of this pathway using a phospho-specific antibody probe. We show that p42/44 MAPK (ERK1/2) is hyperactivated in hearts derived from Cav-3 knock-out mice. These results are consistent with previous in vitro data demonstrating that caveolins may function as negative regulators of the p42/44 MAPK cascade. Taken together, our data argue that loss of Cav-3 expression is sufficient to induce a molecular program leading to cardiac myocyte hypertrophy and cardiomyopathy.

Caveolin-1/3 double-knockout mice are viable, but lack both muscle and non-muscle caveolae, and develop a severe cardiomyopathic phenotype.

The caveolin gene family consists of caveolins 1, 2, and 3. Caveolins 1 and 2 are co-expressed in many cell types, such as endothelial cells, fibroblasts, smooth muscle cells and adipocytes, where they form a heteroligomeric complex. In contrast, the expression of caveolin-3 is muscle-specific. Thus, the expression of caveolin-1 is required for caveolae formation in non-muscle cells, while the expression of caveolin-3 drives caveolae formation in striated muscle cell types (cardiac and skeletal). To create a truly caveolae-deficient mouse, we interbred Cav-1 null mice and Cav-3 null mice to generate Cav-1/Cav-3 double-knockout (Cav-1/3 dKO) mice. Here, we report that Cav-1/3 dKO mice are viable and fertile, despite the fact that they lack morphologically identifiable caveolae in endothelia, adipocytes, smooth muscle cells, skeletal muscle fibers, and cardiac myocytes. We also show that these mice are deficient in all three caveolin gene products, as caveolin-2 is unstable in the absence of caveolin-1. Interestingly, Cav-1/3 dKO mice develop a severe cardiomyopathy. At 2 months of age, analysis of Cav-1/3 dKO hearts via gated magnetic resonance imaging reveals a dramatic increase in left ventricular wall thickness, as compared with Cav-1-KO, Cav-3 KO, and wild-type mice. Further functional analysis of Cav-1/3 dKO hearts via transthoracic echocardiography demonstrates hypertrophy and dilation of the left ventricle, with a significant decrease in fractional shortening. As predicted, Northern analysis of RNA derived from the left ventricle of Cav-1/3 dKO mice shows a dramatic up-regulation of the atrial natriuretic factor message, a well-established biochemical marker of cardiac hypertrophy. Finally, histological analysis of Cav-1/3 dKO hearts reveals hypertrophy, disorganization, and degeneration of the cardiac myocytes, as well as chronic interstitial fibrosis and inflammation. Thus, dual ablation of both Cav-1 and Cav-3 genes in mice leads to a pleiotropic defect in caveolae formation and severe cardiomyopathy.

Significance of inducible nitric oxide synthase in acute myocarditis caused by Trypanosoma cruzi (Tulahuen strain).

Chagas' disease, caused by Trypanosoma cruzi, is associated with myocarditis and expression of myocardial cytokines and inducible nitric oxide synthase (NOS2). To assess the functional significance of NOS2 in murine Chagas' disease, we infected NOS2 knockout (NOS2(-/-)) and C57BL/6x129sv (wild type) mice with the Tulahuen strain of T. cruzi. Serial transthoracic echocardiography was performed to assess the progression of left and right ventricular dysfunction in infected mice. Uninfected wild type and NOS2(-/-) mice served as controls. At day 10 post-infection (p.i.), infected wild type mice had larger left ventricular end-diastolic diameter (2.52+/-0.14-vs-2.11+/-0.06 mm, P<0.02) and right ventricle (0.6+/-0.2-vs-0 visual grade, P<0.02) as compared with uninfected wild type mice. At day 19 p.i., compared with uninfected controls, infected wild type mice had larger left ventricular end-diastolic diameter (3.30+/-0.29-vs-2.11+/-0.07 mm), left ventricular end-systolic diameter (1.86+/-0.29-vs-0.88+/-0.05 mm), right ventricle (1.6+/-0.2-vs-0 visual grade), lower heart rate (413+/-27-vs-557+/-25 beats per min), left ventricular relative wall thickness (0.44+/-0.05-vs-0.64+/-0.03) and fractional shortening (45+/-4-vs-57+/-2%) [P<0.05 for all]. In contrast, no differences in left ventricular end-diastolic diameter or fractional shortening were noted among infected and uninfected NOS2(-/-) mice at day 19 p.i. Compared with uninfected controls, infected NOS2(-/-) mice had significantly lower heart rate (272+/-23-vs-512+/-31 beats per min, P<0.01) and larger right ventricle (0.6+/-0.2-vs-0, P<0.05 visual grade). The magnitude of right ventricular dilation in NOS2(-/-) mice was less than that observed in infected wild type mice. At necropsy, the heart weight was greater (129+/-16-vs-109+/-7 mg, P=0.02) and myocardial inflammation more severe in infected wild type compared with infected NOS2(-/-) mice. Myocardial interleukin (IL)-1beta, IL-6, tumour necrosis factor-alpha, and interferon-gamma were induced in all infected mice. These data indicate that nitric oxide derived from NOS2 plays an important role in the development and progression of ventricular dilation and systolic dysfunction in acute murine chagasic myocarditis caused by infection with the Tulahuen strain.

Cardioprotective effects of verapamil on myocardial structure and function in a murine model of chronic Trypanosoma cruzi infection (Brazil Strain): an echocardiographic study.

Verapamil has been shown to attenuate the extent of myocardial injury in murine models of chronic Trypanosoma cruzi infection. Infected mice treated with verapamil have significantly lower myocardial expression of inducible nitric oxide synthase and cytokines and substantially less inflammatory infiltrate and myocyte necrosis at necropsy. In the present study, we examined the cardiac structural and functional correlates of verapamil treatment in CD1 mice infected with the Brazil strain of T. cruzi using serial transthoracic echocardiography. There were four groups: uninfected- untreated control, uninfected-verapamil-treated, infected-untreated control, and infected-verapamil-treated. Verapamil was given in drinking water (1 gm/l) continuously from the day of infection for a total of 120 days. Mice were evaluated at baseline, 40 and 150 days p.i. Mice in the untreated-infected group compared with the mice in the infected-verapamil-treated group showed thinning of the left ventricular wall (0.84 +/- 0.02-vs-0.92 +/- 0.04, P<0.05 mm), increase in the left ventricular end-diastolic diameter (3.27 +/- 0.15-vs-2.74 +/- 0.05 mm, P<0.05) and reduction in percent fractional shortening (37 +/- 2-vs-53 +/- 4%, P<0.05). No differences in these parameters were noted among mice in the uninfected-untreated and uninfected-verapamil-treated groups. Furthermore, right ventricular dilation was more severe in mice from the infected-untreated group as compared with those in the infected- verapamil-treated group (visual grade 1.9 +/- 0.4-vs-1.0 +/- 0.2, P<0.05). At necropsy, the extent of myocardial injury, as determined histologically, was significantly greater in the infected-untreated mice. These data provide cardiac structural and functional correlates for the previously observed cardioprotective effects of verapamil in chronic chagasic cardiomyopathy.