Different response of cellular DNA content to cardiac hypertrophy in human and rat heart myocytes

1. Rat and human heart myocytes adapt to overload-induced hypertrophy differently. 2. Human myocyte nuclei respond with polyploidization and multinucleation, thus increasing the DNA content per myocyte from 20 to 40 pg. As a result, nuclear DNA content per 10,000 microns3 of cell volume decreases from 12 to 10 pg. 3. In rat hearts with aortic constriction nuclear DNA content remains constant (13 pg), and the DNA content per 10,000 microns3 of myocyte volume falls from 9 to 6 pg. 4. We hypothesize that "dilution" of nuclear DNA in the hypertrophied rat heart myocyte limits the capacity to hypertrophy (much less than 100%). 5. The human heart myocyte, which is able to compensate for dilution of nuclear DNA, may increase in size more than three-fold. 6. The lower limit of DNA content per unit of myocyte volume is 6 pg/10,000 microns3 in both species.     

Expression of matrix metalloproteinase activity in idiopathic dilated cardiomyopathy: A marker of cardiac dilatation

BACKGROUND: Idiopathic dilated cardiomyopathy (DCM), ventricular systolic dysfunction and chamber dilatation are accompanied by architectural remodeling, wall thinning and cardiac myocyte slippage. Recent work has demonstrated an association between collagen degradation and an increased expression of matrix metalloproteinases (MMPs). Accordingly, we have sought to correlate (a) collagen degradation with MMP elevations and, (b) assay the neutralizing potential of a known inhibitor of MMP, tetracycline on MMPs in DCM. METHODS: Assessment of LV volume and shape by 2-D echocardiography was performed. Light microscopic assessment of histopathology in picrosirius red stained biopsy samples of 11 DCM patients and six post-transplant patients was performed. Zymographic estimation of MMP activity and influence of tetracycline on MMP activity was assessed. RESULTS: Small amount of interstitial collagen was noted in the control group, whereas in the DCM hearts, chamber dilatation was associated with areas of scanty myocyte necrosis, islands of excess collagen, and focal areas of absent or scanty collagen with intact myocytes. In cardiomyopathic tissue, collagenase activity was markedly elevated at 63% compared with 8% in post-transplant tissue. Tetracycline at a concentration of 285+/-10 microM (IC50) inhibited collagenase activity by 50% in cardiomyopathic tissue. CONCLUSIONS: Areas of focal interstitial collagen accumulation were accompanied by collagen fiber lysis and increased collagenase activity in dilated cardiomyopathy. This enhanced collagenolytic activity found in endomyocardial biopsy tissue was inhibited by tetracycline. The non-antibiotic property of tetracycline may be of potential value in the prevention of ventricular dilatation in idiopathic dilated cardiomyopathy.     

Expression of matrix metalloproteinase activity in idiopathic dilated cardiomyopathy: A marker of cardiac dilatation

Background: Idiopathic dilated cardiomyopathy (DCM), ventricular systolic dysfunction and chamber dilatation are accompanied by architectural remodeling, wall thinning and cardiac myocyte slippage. Recent work has demonstrated an association between collagen degradation and an increased expression of matrix metalloproteinases (MMPs). Accordingly, we have sought to correlate (a) collagen degradation with MMP elevations and, (b) assay the neutralizing potential of a known inhibitor of MMP, tetracycline on MMPs in DCM. Methods: Assessment of LV volume and shape by 2-D echocardiography was performed. Light microscopic assessment of histopathology in picrosirius red stained biopsy samples of 11 DCM patients and six post-transplant patients was performed. Zymographic estimation of MMP activity and influence of tetracycline on MMP activity was assessed. Results: Small amount of interstitial collagen was noted in the control group, whereas in the DCM hearts, chamber dilatation was associated with areas of scanty myocyte necrosis, islands of excess collagen, and focal areas of absent or scanty collagen with intact myocytes. In cardiomyopathic tissue, collagenase activity was markedly elevated at 63% compared with 8% in post-transplant tissue. Tetracycline at a concentration of 285 ± 10 μM (IC50) inhibited collagenase activity by 50% in cardiomyopathic tissue. Conclusions: Areas of focal interstitial collagen accumulation were accompanied by collagen fiber lysis and increased collagenase activity in dilated cardiomyopathy. This enhanced collagenolytic activity found in endomyocardial biopsy tissue was inhibited by tetracycline. The non-antibiotic property of tetracycline may be of potential value in the prevention of ventricular dilatation in idiopathic dilated cardiomyopathy. (Mol Cell Biochem 264: 183–191, 2004)     

Changes in binucleation and cellular dimensions of rat left atrial myocytes after induced left ventricular infarction

The left atrium of young rats has previously been demonstrated to respond with DNA synthesis and binucleation 11 days after left ventricular infarction. This investigation was designed to examine the hypertrophic response of the left atrial myocyte of the rat at 20 and 60 days after ventricular infarction. Male Sprague-Dawley rats were subjected to left coronary artery ligation (CAL) or sham operation. Following enzymatic separation, left atrial myocytes were examined at 20 and 60 days postoperation for number of nuclei and cellular dimensions (cell length, width and area, and nuclear area). Results demonstrated that the level of binucleation at 20 days (77.3%) and 60 days (71.3%) was nearly twice that observed in sham-operated animals, which were 33.1% binucleated at 20 days and 43.5% binucleated at 60 days. In both mononucleated and binucleated myocytes, the mean lengths, widths, and cell areas from CAL hearts were significantly greater than those of corresponding sham-operated animals. In all cases, these values were larger in binucleated myocytes than in mononucleated cells. The mean area of CAL cells was approximately twice that of sham-operated myocytes. With regard to mean lengths and widths, although both were greater in the CAL animals, there was a decrease in length and increase in width between 20 and 60 days in the CAL group. Mean nuclear areas were significantly greater in CAL myocytes than in those from the sham-operated group. These increases in nuclear number and cellular dimensions of the atrial myocyte are prominent features of the response to the stress imposed by left ventricular infarction.     

Nuclear characteristics of cardiac myocytes following the proliferative response to mincing of the myocardium in the adult newt,Notophthalmus viridescens

Summary Amphibian cardiac myocytes are predominantly mononucleated and have been demonstrated to respond to injury with DNA synthesis and mitosis. The nature of this response with regard to nuclear number and ploidy is unclear. In this study, the apex of the newt ventricle was minced and replaced, increasing the reactive area of the wound. At 45 days after mincing following multiple injections of tritiated thymidine (2.5-Ci/animal, 20 Ci/mM) 15 to 20 days after mincing, three ventricular zones were isolated and fixed: Zone 1, the minced area; Zone 2, extending approximately 500 m proximally from the amputation plane; and Zone 3, the portion proximal to Zone 2. Myocytes separated in 50% KOH were examined for DNA synthesis by autoradiography and for nuclear number and DNA content using a scanning microdensitometer on Feulgen-Naphthol yellow S-stained cells. No labeled myocyte nuclei were found in control hearts and 98.3% of the myocytes were 2C. At 45 days, 46.78% of myocyte nuclei within Zone 1 were labeled, while 13% were non-diploid. In Zone 2, 9.25% were labeled with 4.8% non-diploid. In Zone 3, 1.1% were labeled, with 2.8% non-diploid. The newt ventricle's response to injury apparently may involve complete mitosis and cytokinesis, resulting in mononucleated diploid cells.     

Global Intracoronary Infusion of Allogeneic Cardiosphere-Derived Cells Improves Ventricular Function and Stimulates Endogenous Myocyte Regeneration throughout the Heart in Swine with Hibernating Myocardium

Background

Cardiosphere-derived cells (CDCs) improve ventricular function and reduce fibrotic volume when administered via an infarct-related artery using the “stop-flow” technique. Unfortunately, myocyte loss and dysfunction occur globally in many patients with ischemic and non-ischemic cardiomyopathy, necessitating an approach to distribute CDCs throughout the entire heart. We therefore determined whether global intracoronary infusion of CDCs under continuous flow improves contractile function and stimulates new myocyte formation.

Methods and Results

Swine with hibernating myocardium from a chronic LAD occlusion were studied 3-months after instrumentation (n = 25). CDCs isolated from myocardial biopsies were infused into each major coronary artery (∼33×10⁶ icCDCs). Global icCDC infusion was safe and while ∼3% of injected CDCs were retained, they did not affect ventricular function or myocyte proliferation in normal animals. In contrast, four-weeks after icCDCs were administered to animals with hibernating myocardium, %LADWT increased from 23±6 to 51±5% (p<0.01). In diseased hearts, myocyte proliferation (phospho-histone-H3) increased in hibernating and remote regions with a concomitant increase in myocyte nuclear density. These effects were accompanied by reductions in myocyte diameter consistent with new myocyte formation. Only rare myocytes arose from sex-mismatched donor CDCs.

Conclusions

Global icCDC infusion under continuous flow is feasible and improves contractile function, regresses myocyte cellular hypertrophy and increases myocyte proliferation in diseased but not normal hearts. New myocytes arising via differentiation of injected cells are rare, implicating stimulation of endogenous myocyte regeneration as the primary mechanism of repair.     

Image and DNA analysis of hypertrophic myocytes in hypertensive heart disease and hypertrophic cardiomyopathy

OBJECTIVE: To investigate differences in the pathophysiology of cardiac hypertrophy between patients with hypertensive heart disease (HHD) and hypertrophic cardiomyopathy (HCM). STUDY DESIGN: The study group consisted of 30 autopsied heart disease patients (10 HHD, 10 HCM and 10 noncardiac heart disease). DNA synthesis by hypertrophic cardiac myocytes was examined, and three-dimensional myocyte structure image was investigated. DNA synthesis and the cell cycle were investigated by flow cytometry using autopsy material. Three-dimensional myocyte structure image was visualized. RESULTS: The percentage of cells in G2M phase of the cell cycle was significantly decreased in the myocardium of autopsied hearts with HCM as compared with hearts with HHD (HCM:HHD = 1.2 +/- 1.1%: 7.7 +/- 2.6%, mean +/- SD). Hypertrophic myocytes of HCM characteristically possessed myocardial disarray and irregular side-to-side branch connections between myocytes. No myocyte disarray or irregular connections could be observed in HHD. CONCLUSION: These results suggest that the mechanism of cardiac hypertrophy differs between patients with HHD and HCM and also suggest dissimilar cell vitality and latent proliferative viability of hypertrophic myocytes in a hypertrophic process between HHD and HCM. That is, hypertrophic myocytes may be called "restricted" myocytes in a morphologic and biochemical sense.     

Variability of the cardiomyocyte ploidy in normal human hearts.

We have performed cytophotometry for DNA in isolated myocytes of the left ventricle from 16 men, aged 19-39 years, who died from various non-cardiac or pulmonary causes. The mean ploidy of myocytes varied from 3.2-3.9 c to 6.6-7.3 c in different layers of the anterior wall of the left ventricle (where c is the haploid DNA content measured by cytophotometry in Feulgen-stained preparations). There was no correlation between the layers. The percentage of binuclear cells varied from 25 to 86% and correlated in every layer with the mean ploidy value of the whole myocyte population. Approximate calculation of total ploidy revealed low values in the ventricles of some individuals, and high values in others. Averaging the values for all the hearts studied obscures this variation. Mean myocyte ploidy in different layers of the anterior wall was similar: in the external layer it was 5.1 +/- 0.3 c, in the middle layer 5.5 +/- 0.3 c and in the inner layer 4.8 +/- 0.4 c. The mean percentage of binuclear myocytes in these three layers was also similar, being 61 +/- 3%, 63 +/- 4% and 54 +/- 5%, respectively. Myocyte ploidy in tissue from the posterior wall of the left ventricle also varied, but was always higher than for the same layer of the anterior wall in the same ventricle. We propose that high or low myocyte ploidy, as well as different proportions of mono- and binucleate cells, can be a factor affecting the course and result of cardiac pathology in the absence of any changes of myocyte genome determined during early ontogenesis and representing a stable characteristic of the individual.     

Inhibition of cyclooxygenase-2 improves cardiac function after myocardial infarction in the mouse

Cyclooxygenase (COX)-2 is expressed in the heart in animal models of ischemic injury. Recent studies have suggested that COX-2 products are involved in inflammatory cell infiltration and fibroblast proliferation in the heart. Using a mouse model, we questioned whether 1). myocardial infarction (MI) in vivo induces COX-2 expression chronically, and 2). COX-2 inhibition reduces collagen content and improves cardiac function in mice with MI. MI was produced by ligation of the left anterior descending coronary artery in mice. Two days later, mice were treated with 3 mg/kg NS-398, a selective COX-2 inhibitor, or vehicle in drinking water for 2 wk. After the treatment period, mice were subjected to two-dimensional M-mode echocardiography to determine cardiac function. Hearts were then analyzed for determination of infarct size, interstitial collagen content, brain natriuretic peptide (BNP) mRNA, myocyte cross-sectional area, and immunohistochemical staining for transforming growth factor (TGF)-beta and COX-2. COX-2 protein, detected by immunohistochemistry, was increased in MI versus sham hearts. MI resulted in increased left ventricular systolic and diastolic dimension and decreased ejection fraction, fractional shortening, and cardiac output. NS-398 treatment partly reversed these detrimental changes. Myocyte cross-sectional area, a measure of hypertrophy, was decreased by 30% in the NS-398 versus vehicle group, but there was no effect on BNP mRNA. The interstitial collagen fraction increased from 5.4 +/- 0.4% in sham hearts to 10.4 +/- 0.9% in MI hearts and was decreased to 7.9 +/- 0.6% in NS-398-treated hearts. A second COX-2 inhibitor, rofecoxib (MK-0966), also decreased myocyte cross-sectional area and interstitial collagen fraction. TGF-beta, a key regulator of collagen synthesis, was increased in MI hearts. NS-398 treatment reduced TGF-beta immunostaining by 40%. NS-398 treatment had no effect on infarct size. These results suggest that COX-2 products contribute to cardiac remodeling and functional deficits after MI. Thus selected inhibition of COX-2 may be a therapeutic target for reducing myocyte damage after MI.     

The capacity for reactive DNA synthesis of the myocytes in the heart conduction system in experimental and clinical myocardial pathology]

The DNA synthesis has been studied in the conductive system (CS) myocytes, compared to that in atrial and ventricular myocytes: 1) in the left ventricular myocardial infarction induced in two- and three-week-old and adult rats, 2) after isoproterenol injections to adult rats and mice, and 3) in the hypertrophied human heart. The extent of DNA synthesis reactivation was evaluated by the cumulative labeling indices in experiments with multiple 3HTdR injections to rats and mice. In the human cardiac myocyte nuclei, the DNA content was determined by the Feulgen-cytophotometry. The difference between the control and experimental mean values of the labeling indices for CS myocyte nuclei was statistically significant only for atrioventricular part of the CS in the infarcted hearts of adult rats. In the human heart CS the ability of myocytes to polyploidization varies from one cell type to another, the lowest being in nodal cells.     

Gap junction remodeling and altered connexin43 expression in the failing human heart

Gap junctions (GJ) are important determinants of cardiac conduction and the evidence has recently emerged that altered distribution of these junctions and changes in the expression of their constituent connexins (Cx) may lead to abnormal coupling between cardiomyocytes and likely contribute to arrhythmogenesis. However, it is largely unknown whether changes in the expression and distribution of the major cardiac GJ protein, Cx43, is a general feature of diverse chronic myocardial diseases or is confined to some particular pathophysiological settings. In the present study, we therefore set out to investigate qualitatively and quantitatively the distribution and expression of Cx43 in normal human myocardium and in patients with dilated (DCM), ischemic (ICM), and inflammatory cardiomyopathies (MYO). Left ventricular tissue samples were obtained at the time of cardiac transplantation and investigated with immunoconfocal and electron microscopy. As compared with the control group, Cx43 labeling in myocytes bordering regions of healed myocardial infarction (ICM), small areas of replacement fibrosis (DCM) and myocardial inflammation (MYO) was found to be highly disrupted instead of being confined to the intercalated discs. In all groups, myocardium distant from these regions showed an apparently normal Cx43 distribution at the intercalated discs. Quantitative immunoconfocal analyis of Cx43 in the latter myocytes revealed that the Cx43 area per myocyte area or per myocyte volume is significantly decreased by respectively 30 and 55% in DCM, 23 and 48% in ICM, and by 21 and 40% in MYO as compared with normal human myocardium. In conclusion, focal disorganization of GJ distribution and down-regulation of Cx43 are typical features of myocardial remodeling that may play an important role in the development of an arrhythmogenic substrate in human cardiomyopathies.     

Variability of the cardiomyocyte ploidy in normal human hearts

We have performed cytophotometry for DNA in isolated myocytes of the left ventricle from 16 men, aged 19–39 years, who died from various non-cardiac or pulmonary causes. The mean ploidy of myocytes varied from 3.2–3.9 c to 6.6–7.3 c in different layers of the anterior wall of the left ventricle (where c is the haploid DNA content measured by cytophotometry in Feulgenstained preparations). There was no correlation between the layers. The percentage of binuclear cells varied from 25 to 86% and correlated in every layer with the mean ploidy value of the whole myocyte population. Approximate calculation of total ploidy revealed low values in the ventricles of some individuals, and high values in others. Averaging the values for all the hearts studied obscures this variation. Mean myocyte ploidy in different layers of the anterior wall was similar: in the external layer it was 5.1±0.3 c, in the middle layer 5.5±0.3 c and in the inner layer 4.8±0.4 c. The mean percentage of binuclear myocytes in these three layers was also similar, being 61±3%, 63±4% and 54±5%, respectively. Myocyte ploidy in tissue from the posterior wall of the left ventricle also varied, but was always higher than for the same layer of the anterior wall in the same ventricle. We propose that high or low myocyte ploidy, as well as different proportions of mono- and binucleate cells, can be a factor affecting the course and result of cardiac pathology in the absence of any changes of myocyte genome determined during early ontogenesis and representing a stable characteristic of the individual.     

Myocardial cell death and regeneration during progression of cardiac hypertrophy to heart failure

Cardiac hypertrophy and ensuing heart failure are among the most common causes of mortality worldwide, yet the triggering mechanisms for progression of hypertrophy to failure are not fully understood. Tissue homeostasis depends on proper relationships between cell proliferation, differentiation, and death and any imbalance between them results in compromised cardiac function. Recently, we developed a transgenic (Tg) mouse model that overexpress myotrophin (a 12-kDa protein that stimulates myocyte growth) in heart resulting in hypertrophy that progresses to heart failure. This provided us an appropriate model to study the disease process at any point from initiation of hypertrophy end-stage heart failure. We studied detailed apoptotic signaling and regenerative pathways and found that the Tg mouse heart undergoes myocyte loss and regeneration, but only at a late stage (during transition to heart failure). Several apoptotic genes were up-regulated in 9-month-old Tg hearts compared with age-matched wild type or 4-week-old Tg hearts. Cardiac cell death during heart failure involved activation of Fas, tumor necrosis factor-alpha, and caspases 9, 8, and 3 and poly(ADP-ribose) polymerase cleavage. Tg mice with hypertrophy associated with compromised function showed significant up-regulation of cyclins,cyclin-dependent kinases (Cdks), and cell regeneration markers in myocytes. Furthermore, in human failing and nonfailing hearts, similar observations were documented including induction of active caspase 3 and Ki-67 proteins in dilated cardiomyopathic myocytes. Taken together, our data suggest that the stress of extensive myocardial damage from longstanding hypertrophy may cause myocytes to reenter the cell cycle. We demonstrate, for the first time in an animal model, that cell death and regeneration occur simultaneously in myocytes during end-stage heart failure, a phenomenon not observed at the onset of the disease process.     

Occurrence and distribution of atrial natriuretic peptide-containing cells in the left ventricle of hypertensive rats

In the ventricles of adult mammalian hearts, production of atrial natriuretic peptide (ANP) is negligible, restricted to the impulse-conducting cells, the papillary muscles, and a minority of subendocardial myocytes. ANP expression is reinduced in the ventricles of pressure-overloaded and failing hearts and is frequently used as a marker for myocyte hypertrophy. Using an immunohistochemical approach, we have characterized the size distribution of ANP-containing myocytes in the left ventricle of the spontaneously hypertensive rat (SHR) before and after chronic antihypertensive therapy and compared the results to age-matched normotensive Wistar rats (WR). Our findings show that in SHR the frequency of cells presenting ANP granularity is positively correlated with myocyte size (r=0.746, P<0.02). The highest proportion of ANP-positive myocytes (55-57%) was measured among cells of diameters 30-34 microm. In any corresponding cell size, the proportion of ANP-presenting myocytes was five- to tenfold higher in SHR than in the normotensive WR. We studied the effects of the antihypertensive drugs captopril, hydralazine, and nifedipine and found that, regardless of their effect on blood pressure or hypertrophy, all three eliminated ANP immunoproducts from the majority of the left ventricular myocytes and reduced the level of ANP mRNA, captopril being the most effective. The positive correlation between myocyte size and ANP expression was not maintained in the hearts of drug-treated SHR. Myocytes on the border of fibrotic areas or in regions of ANP presentation within the normal heart resisted the suppressive effect of the antihypertensive therapy, indicating that blood pressure or hypertrophy are not the sole correlates for ANP expression.     

TNF‐α‐mediated signal transduction pathway is a major determinant of apoptosis in dilated cardiomyopathy

Although J2N-k strain of cardiomyopathic hamsters is an excellent model of dilated cardiomyopathy, the presence and mechanisms of apoptosis in the hearts of these genetically modified animals have not been investigated. This study examined the hypothesis that cardiac dysfunction and apoptosis in the cardiomyopathic hamsters were associated with tumour necrosis factor-alpha (TNF-α)-mediated signalling pathway involving the activation of some pro-apoptotic proteins and/or deactivation of some antiapoptotic proteins. Echocardiographic assessment of 31-week-old hamsters indicated an increase in the internal dimension of the left ventricle as well as decreases in the ejection fraction, fractional shortening and cardiac output without any evidence of cardiac hypertrophy. Increased level of TNF-α and apoptosis in cardiomyopathic hearts were accompanied by increased protein content for protein kinase C (PKC) -α and -ɛ isozymes as well as caspases 3 and 9. Phosphorylated protein content for p38 MAPK and NFκB was increased whereas that for Erk1/2, BAD and Bcl-2 was decreased in cardiomyopathic hearts. These results support the view that TNF-α and PKC isozymes may promote apoptosis due to the activation of p38 MAPK and deactivation of Erk1/2 pathways, and these changes may contribute toward the development of cardiac dysfunction in dilated cardiomyopathy.     

Isolation,long-term culture,and ultrastructural characterization of adult cardiomyopathic cardiac muscle cells

Summary A long-term cell culture system for adult cardiomyopathic hamster cardiac muscle cells has been established. The diseased and control hearts were dissociated into single cell suspension with the modifications of our previous technique using collagenase and hyaluronidase as applied to the dissociation of the adult rat heart. The postperfusion of the diseased heart with Krebs-Ringer phosphate buffer and bovine serum albumin was very helpful in obtaining greater yield of viable diseased muscle cells; the cells were cultured for 4 wk. Approximately 60% of the myocytes from the diseased heart and 85% of the myocytes from the normal heart attached to the substrates and survived throughout the culture period. Approximately 60 to 70% of the cardiac myocytes from the diseased and control hearts were bi- or multinucleated; 30% of the diseased and 80% of the normal myocytes showed rhythmic contractility. Electron microscopy revealed the presence of two kinds of cardiac muscle cells in the diseased cell culture on the basis of their myofibril content: one with scanty myofibrils and another with abundant myofibrils. Myocytes with sparse myofibrils showed certain characteristic features that included autophagic vacuoles, amorphous matrix of fine filamentous texture, scattered strips of myofibrils, and abnormal organization of the Z-line. Cardiac muscle cells with abundant myofibrillar content contained unorganized myofibrils in certain sarcomeres. These studies demonstrate the feasibility of maintaining diseased cardiac muscle cells from adult cardiomyopathic hamsters for at least 4 wk in monolayer culture. This study was supported by a grant from the American Heart Association of Michigan, National Institutes of Health grant HL-25482, and by an Oakland University Biomedical Research Support Grant.     

Cardiac myocyte apoptosis provokes adverse cardiac remodeling in transgenic mice with targeted TNF overexpression

Although cardiac myocyte apoptosis has been detected in explanted hearts from patients with end-stage dilated and ischemic cardiomyopathy, the relative contribution of apoptotic cell death to left ventricular (LV) remodeling and cardiac decompensation is not known. To determine whether progressive cardiac myocyte apoptosis contributes to the transition from a hypertrophic to a dilated cardiac phenotype that is observed in transgenic myosin heavy chain secreted TNF (MHCsTNF) mice with cardiac restricted overexpression of tumor necrosis factor (TNF), we assessed cardiac myocyte apoptosis (using a DNA ligase technique) in MHCsTNF mice and littermate control mice in relation to serial changes in LV structure, which was assessed using MRI. The prevalence of cardiac myocyte apoptosis increased progressively from 4 to 12 wk as the hearts of the MHCsTNF mice underwent the transition from a concentric hypertrophic to a dilated cardiac phenotype. Treatment of the MHCsTNF mice with the broad-based caspase inhibitor N-[(1,3-dimethylindole-2-carbonyl)-valinyl]-3-amino4-oxo-5-fluoropentanoic acid significantly decreased cardiac myocyte apoptosis and significantly attenuated LV wall thinning and adverse cardiac remodeling. Additional studies suggested that the TNF-induced decrease in Bcl-2 expression and activation of the intrinsic mitochondrial death pathway were responsible for the cardiac myocyte apoptosis observed in the MHCsTNF mice. These studies show that progressive cardiac myocyte apoptosis is sufficient to contribute to adverse cardiac remodeling in the adult mammalian heart through progressive LV wall thinning.     

The ATP-binding cassette transporter ABCG2 (BCRP), a marker for side population stem cells, is expressed in human heart.

Efforts to improve severely impaired myocardial function include transplantation of autologous hematopoietic side population (SP) stem cells. The transmembrane ABC-type (ATP binding cassette) half-transporter ABCG2 (BCRP) serves as a marker protein for SP cell selection. We have recently shown that other ABC transport proteins such as ABCB1 and ABCC5 are differentially expressed in normal and diseased human heart. Here we investigated localization and individual ABCG2 expression in 15 ventricular (including 10 cardiomyopathic) and 51 auricular heart tissue samples using immunohistochemistry, confocal laser scanning fluorescence microscopy, and real-time RT-PCR. Individual genotypes were assigned using PCR-restriction fragment length polymorphism (RFLP) analysis and subsequently correlated to ABCG2 mRNA levels. ABCG2 was localized in endothelial cells of capillaries and arterioles of all samples. Ventricular samples from cardiomyopathic hearts exhibited significantly increased levels of ABCG2 mRNA (ABCG2/18S rRNA: 1.08 +/- 0.30 x 10(-7); p=0.028 (dilative cardiomyopathy) and 1.16 +/- 0.46 x 10(-7); p=0.009 (ischemic cardiomyopathy) compared with 0.44 +/- 0.26 x 10(-7) in nonfailing hearts). The individual haplotypes were not associated with altered mRNA expression. ABCG2 is variably expressed in endothelial cells of human heart, where it may function as a protective barrier against cardiotoxic drugs such as anthracyclines or mitoxantrone. ABCG2 expression is induced in dilative and ischemic cardiomyopathies.     

Immunofluorescent and immunogold replica studies of desmin distribution in cultured normal and cardiomyopathic hamster heart cells.

The architecture of desmin intermediate filament arrangements in cultured cardiomyocytes from heart of normal and cardiomyopathic hamsters was studied by immunofluorescent light microscopy and immunogold replica electron microscopy. Both polyclonal and monoclonal antidesmin antibodies were used in a biotin-streptavidin system. Immunofluorescent staining of normal and cardiomyopathic myocytes for desmin at 5 days in culture exhibited filamentous staining patterns with polyclonal antidesmin and a coarse punctate staining pattern with the monoclonal antibody. At 9 days in culture, most normal myocytes showed filamentous staining with the polyclonal antibody; many of the stained filaments were associated with Z lines. With the monoclonal antidesmin, these same cells exhibited a very fine 'spotty' staining pattern. These results suggest that the arrangements and immunoreactivities of intermediate filaments change during normal cardiac myocyte development. In cardiomyopathic cells, this pattern of rearrangement and immunoreactivity appears to be delayed or possibly nonexistent. The three-dimensional electron-microscopic observation of immunogold localization of desmin achieved by a deep-etching replica technique is made on both normal and cardiomyopathic cultured heart cells. Abnormalities of desmin filament arrangements in cardiomyopathic cells are confirmed.     

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.