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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.

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.

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.

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.

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.

Characterization of the severity of aortic stenosis relies on accurate measurement of the pressure gradient across the valve and the valve area. Pressure gradients measured by Doppler ultrasound based on the clinical form of the Bernoulli equation often overestimate pressure gradients by catheter as the result of pressure recovery. Doppler techniques measure the velocity of the vena contracta of the stenotic jet. This corresponds to the maximal pressure gradient and the minimal effective valve area. Pressure recovery can be characterized by analysis of the spread of the stenotic jet downstream of the valve as it fills the aorta and should be influenced by the shape of the velocity profile of the decaying jet. In this study, we addressed the hypothesis that the site of complete pressure recovery (the point at which the jet fully expands to the size of the aorta), the effective valve area, and the maximal pressure gradient are affected by jet eccentricity. To accomplish this, we developed a computational model of aortic stenosis that provides detailed velocity and pressure information in the vicinity of the valve. The results show that the width of the eccentric wall jet decreased and maximal velocity increased with greater jet eccentricity. Furthermore, for a constant anatomic area, the effective valve area decreased, the distance to complete pressure recovery increased, and the maximal pressure gradient increased with the degree of eccentricity. Failure to take this into account could fortuitously drive Doppler and catheter measurements toward agreement because the distal pressure sensor will not record the fully recovered pressure. Therefore the pressure gradient across a stenotic valve depends on jet eccentricity. The spread of the wall jet after attachment must be characterized to develop a robust method for the prediction of pressure recovery. (J Am Soc Echocardiogr 2001;14:853-62.)

Expression of Cardiac Cytokines and Inducible Form of Nitric Oxide Synthase (NOS2) in Trypanosoma cruzi-infected Mice. Journal of Molecular and Cellular Cardiology (1999) 31, 75-88. Both cardiac cytokine and inducible nitric oxide synthase (NOS2) expression have been implicated in the cardiac dysfunction associated with myocarditis and cardiomyopathy. Chagas' disease, caused by Trypanosoma cruzi, is an important cause of cardiomyopathy. We examined the effect of T. cruzi (Brazil strain) infection with or without verapamil treatment on the expression of cytokines and NOS2 in the heart. Messenger RNA for NOS2, IL-1beta, and TNF-alpha was induced in the myocardium of infected mice, and Western blot analysis as well as immunohistochemistry demonstrated a significant increase in NOS2 protein. Verapamil treatment reduced the expression of cardiac NOS2 protein and the mRNAs for NOS2, TNF-alpha, and IL-1beta. Infection-associated increases in cardiac L-citrulline were also reduced by verapamil treatment. Verapamil-treated infected mice that survived for 80 days exhibited less inflammation and fibrosis compared to untreated mice. Gated MRI and echocardiography revealed an increased right ventricular inner diameter (RVID) in untreated but not in verapamil-treated infected CD1 mice. This suggests that the infection-associated expression of cytokines and NOS2 in the heart correlate with the severity of myocarditis and the effect of verapamil. The RVID was significantly increased in infected wild-type (WT) compared to infected syngeneic NOS2 knockout (NOS2-/-) mice. Fractional shortening was decreased and myocardial L-citrulline was increased in infected WT mice. These data suggest that NO generated from cardiac NOS2 may participate in the pathogenesis of murine 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-1 flox/flox ;α-MHC-Cre(+) mice in which the ET-1 gene was deleted from cardiac myocytes and ET-1 flox/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-1 flox/flox ;α-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-1 flox/flox ;α-MHC-Cre(+) mice; i.e., RVID was not changed in infected ET-1 flox/flox ;α-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-1 flox/flox ;α-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.

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.

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 Gαq 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 Gαq peripartum cardiomyopathy model. Methods and Results— The potent polycaspase inhibitor IDN-1965 or vehicle was administered subcutaneously to Gαq 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 P P P P P P Conclusions— Reduction in cardiac myocyte apoptosis by caspase inhibition improved left ventricular function and survival in pregnant Gαq 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 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.

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, P0.02) and right ventricle (0.6+/-0.2-vs-0 visual grade, P0.02) 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%) [P0.05 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, P0.01) and larger right ventricle (0.6+/-0.2-vs-0, P0.05 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.

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, P0.05 mm), increase in the left ventricular end-diastolic diameter (3.27 +/- 0.15-vs-2.74 +/- 0.05 mm, P0.05) and reduction in percent fractional shortening (37 +/- 2-vs-53 +/- 4%, P0.05). 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, P0.05). 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.

To evaluate the role of gated cardiac magnetic imaging resonance (MRI) in Chagas' disease, we infected mice with Trypanosoma cruzi (Brazil strain). Two models were chosen for study, the CD1 and the inducible nitric oxide synthase knockout (NOS2-/-) mice. Infection of CD1 mice was associated with a significant increase in the right ventricular inner diameter (RVID) that was reversed in some mice by verapamil. Expression of cardiac NOS2 has been associated with myocardial dysfunction. Therefore, we evaluated chagasic cardiomyopathy in NOS2-/- and syngeneic wild type (WT) mice. Infected WT mice exhibited an increase in RVID in the acute phase (60 days postinfection) that was more marked during chronic infection (100 days postinfection). Chronically infected NOS2-/- mice had an increase in RVID. The RVID in infected WT mice was greater than in NOS2-/- mice. These data demonstrate that MRI is a useful tool in the serial evaluation of the heart in murine Chagas' disease. In addition, it supports the notion that the NOS2-/-/NO pathway may contribute to the pathogenesis of murine chagasic cardiomyopathy.

Electron microscopic cytochemistry was used to examine the distribution of the enzyme sodium potassium ATPase (Na+ K+-ATPase) on the plasma membrane of regenerating retinal pigment epithelium (RPE). Observations were made in rabbits that received intravenous sodium iodate, which elicits RPE degeneration followed by regeneration. A gradient in intensity and polarization of enzyme activity was observed from the least mature cells in the distal part of the regenerating epithelial sheet to more mature cells proximally. The least mature cells exhibited faint Na+ K+-ATPase activity over their entire plasma membrane except where it faced the basement membrane. As cells matured and re-expressed the cytologic characteristics of mature RPE, activity intensified and became localized to the apical plasma membrane, as in normal RPE. The observations show the ability of damaged RPE to recover its normal structural and functional polarity.

Testicular germ cell tumors rarely metastasize to the heart. We have seen a 26-year-old man with signs ofright-sided heart failure four years after a diagnosis of mixed germ cell testicular tumor. The tumor had components of yolk sac, embryonal carcinoma, seminoma, and teratoma. At the time of diagnosis, the patient undenvent left orchiectomy but refused chemotherapy and was lost to follow up. Transthoracic echocardiography showed a large, irregular, mobile mass within the right atrium (RA) that prolapsed through the tricuspid valve into the right ventricle (RV) in diastole (Fig. IA). The mass was further characterized by transesophageal echocardiography as a large, freely mobile, irregular, multilobulated echodensity occupying most of the RA in systole (Fig. IC) and much of the RA and RV (Fig. 1 D) in diastole. The mass was also visualized within the superior (SVC) and inferior vena cavas. A five-day course of chemotherapy with bleomycin, etoposide, and cisplatin was started. The patient subsequently underwent cardiac surgery with removal of an 8.0 X 6.0 X 1 .O cm mass from the RA consisting of multiple fragments of tan-yellow soft tissue with a grape-like appearance (Fig. IB). Tumor implants were also seen on chordae tendinae of the tricuspid valve, necessitating valve replacement. Histologic evaluation of the specimen showed a predominance of fibrin clot with islands of cartilage. Metastatic tumor cells with large and pleomorphic nuclei were seen scattered throughout the mass.