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.     

Implications of protease activation in cardiac dysfunction and development of genetic cardiomyopathy in hamsters

It has become evident that protein degradation by proteolytic enzymes, known as proteases, is partly responsible for cardiovascular dysfunction in various types of heart disease. Both extracellular and intracellular alterations in proteolytic activities are invariably seen in heart failure associated with hypertrophic cardiomyopathy, dilated cardiomyopathy, hypertensive cardiomyopathy, diabetic cardiomyopathy, and ischemic cardiomyopathy. Genetic cardiomyopathy displayed in different strains of hamsters provides a useful model for studying heart failure due to either cardiac hypertrophy or cardiac dilation. Alterations in the function of several myocardial organelles such as sarcolemma, sarcoplasmic reticulum, myofibrils, mitochondria, as well as extracellular matrix have been shown to be due to subcellular remodeling as a consequence of changes in gene expression and protein content in failing hearts from cardiomyopathic hamsters. In view of the increased activities of various proteases, including calpains and matrix metalloproteinases in the hearts of genetically determined hamsters, it is proposed that the activation of different proteases may also represent an important determinant of subcellular remodeling and cardiac dysfunction associated with genetic cardiomyopathy.     

Chymase inhibition reduces the progression to heart failure after autoimmune myocarditis in rats

Chymase has been known as a local angiotensin II-generating enzyme in the cardiovascular system in dogs, monkeys, hamsters, and humans; however, recently it was reported that chymase also has various other functions. Therefore, we decided to examine whether the inhibition of chymase improves disease conditions associated with the pathophysiology of dilated cardiomyopathy in rats and its possible mechanism of action as rat chymase is unable to produce angiotensin II. We examined the effect of TY-51469, a novel chymase inhibitor (0.1 mg/kg/day [group CYI-0.1, n = 15] and 1 mg/kg/day [group CYI-1, n = 15]), in myosin-immunized postmyocarditis rats. Another group of myosin-immunized rats was treated with vehicle (group V, n = 15). Age-matched normal rats without immunization (group N, n = 10) were also included in the study. After 4 weeks of treatment, we evaluated cardiac function; area of fibrosis; fibrogenesis; levels of transforming growth factor (TGF)-beta1 and collagen III; hypertrophy and its marker, atrial natriuretic peptide (ANP); and mast cell activity. Survival rate and myocardial functions improved dose-dependently with chymase inhibitor treatment after myosin immunization. A reduction in the percent area of myocardial fibrosis, fibrogenesis, myocardial hypertrophy, and mast cell activity along with a reduction in TGF-beta1, collagen III, and ANP levels in the myocardium were noted in postmyocarditis rats that received chymase inhibitor treatment. The treatment also decreased myocardial aldosterone synthase levels in those animals. Inhibition of chymase reduces the pathogenesis of postmyocarditis dilated cardiomyopathy and progression to heart failure by preventing the pathological remodeling and residual inflammation in rats.     

Fourier transform infrared evaluation of microscopic scarring in the cardiomyopathic heart: effect of chronic AT1 suppression

Our primary aim was to investigate the use of Fourier transform infrared (FTIR) spectromicroscopy as an accurate assay of cardiac extracellular matrix remodeling. Abnormal rearrangement or remodeling of the cardiac extracellular matrix is known to contribute to cardiac dysfunction. The microscopic multifocal necrosis and scarring are modulated by chronic AT(1) receptor blockade in experimental cardiomyopathy; thus, we also wished to rationalize the spectromicroscopic differences among control, untreated cardiomyopathic (CMP), and losartan-treated cardiomyopathic (LOS) hearts according to the pathogenesis of experimental cardiomyopathy. Male UM-X7.1 cardiomyopathic Syrian hamsters at early and late (65 and 200 days) stages of cardiomyopathy were subjected to 4-week losartan (15 mg/kg/day continuous infusion) treatment. Focal collagen microdomain distribution was confirmed spectroscopically by observation of the collagen IR fingerprint in the 1000-1800 cm(-1) region. Synchrotron FTIR spectromicroscopic map data were obtained from control (F1-beta strain) hamsters, nontreated cardiomyopathic, and losartan-treated CMP animals and imaged with mapping software, according to intensity of collagen fingerprint. Compared to controls, untreated late-stage CMP myocardium was characterized by elevated levels of fibrillar collagens and this was partially normalized with a 4-week losartan treatment. FTIR spectromicroscopy revealed that elevated collagen expression in focal microdomains is present in late-stage cardiomyopathy, and 4-week AT(1) blockade is associated with attenuation of collagen absorption in these lesions.     

Alteration of extracellular matrix in dilated cardiomyopathic hamster heart

The purpose of this study was to characterize the collagen in hereditary dilated cardiomyopathic hamster hearts, and to examine the participation of the collagen in the occurrence and progression of cardiomyopathy.BIO 53.58 hamsters (5, 10, 20 weeks old) were used as the model of dilated cardiomyopathy. Flb hamsters were used as controls. The collagen content was almost constant at any age in the Flb hamsters, but increased with age in BIO 53.58 hamsters. Type III collagen increased significantly in BIO 53.58 hamsters at 10 weeks. The acetic acid solubility of collagen decreased in BIO 53.58 hamsters as the fibrosis progressed, but was unchanged in controls. Reducible crosslinks showed a tendency to decrease progressively in BIO 53.58 hamsters. There were no differences between Flb and BIO 53.58 hamsters at 5 weeks, but its expression in BIO 53.58 hamsters at 10 and 20 weeks of age increased compared to Flb controls.These findings indicate that in the early phase of cardiomyopathy the extracellular matrix of the myocardium is rich in type III collagen. In the later phase, the matrix resembles that of hard tissues, whose collagen is mainly of type I collagen and is insoluble. These data suggest that the increased collagen synthesis may impair the cardiac function in the development of cardiomyopathy.     

Chronic suppression of heart-failure progression by a pseudophosphorylated mutant of phospholamban via in vivo cardiac rAAV gene delivery

The feasibility of gene therapy for cardiomyopathy, heart failure and other chronic cardiac muscle diseases is so far unproven. Here, we developed an in vivo recombinant adeno-associated virus (rAAV) transcoronary delivery system that allows stable, high efficiency and relatively cardiac-selective gene expression. We used rAAV to express a pseudophosphorylated mutant of human phospholamban (PLN), a key regulator of cardiac sarcoplasmic reticulum (SR) Ca(2+) cycling in BIO14.6 cardiomyopathic hamsters. The rAAV/S16EPLN treatment enhanced myocardial SR Ca(2+) uptake and suppressed progressive impairment of left ventricular (LV) systolic function and contractility for 28-30 weeks, thereby protecting cardiac myocytes from cytopathic plasma-membrane disruption. Low LV systolic pressure and deterioration in LV relaxation were also largely prevented by rAAV/S16EPLN treatment. Thus, transcoronary gene transfer of S16EPLN via rAAV vector is a potential therapy for progressive dilated cardiomyopathy and associated heart failure.     

Pathological changes of myocardial cytoskeleton in cardiomyopathic hamster

Immunocytochemical investigation was performed on the cytoskeletal proteins in cardiac tissue of the cardiomyopathic hamster. Male cardiomyopathic UM-X7.1 hamsters at 180 days of age (n=8) and age- and sex-matched normal BIO-RB hamsters (n=8) were used in this study. Immunofluorescence microscopy using monoclonal antibodies against desmin, -actinin, titin, and vincullin was employed. The heart weight to body weight ratio was significantly increased in the heart of cardiomyopathic hamster compared with that of normal hamster. In cardiomyopathic hamster, the left ventricular cavity was markedly dilated. Light microscopically, hypertrophy and atrophy of myocytes and myocardial fibrosis were prominently observed in cardiomyopathic myocardium. Immunocytochemically, desmin, -actinin and titin showed the cross striations along the myofibers in normal myocardium. In contrast, in cardiomyopathic myocardium, desmin was irregularly distributed in myocytes and the amount of desmin was increased. Loss of cross striations of -actinin and titin were frequently observed. Immunofluorescence against vinculin was not significantly altered. We conclude that the alterations of cytoskeletal proteins in myocardial cells may relate to decreased myocardial function in cardiomyopathic hamster failing heart.     

Defective activity and isoform of the Na,K-ATPase in the dilated cardiomyopathic hamster.

The Na,K-ATPase function appears impaired in human heart failure with dilation; however little is known in animal model with idiopathic dilated cardiomyopathy. We studied Na,K-ATPase isoform composition and activity from cardiomyopathic hamsters of the MS 200 strain with pure dilated cardiomyopathy and compared them with those of healthy Syrian hamsters. 150-day-old male MS 200 Syrian hamsters (n = 16) and sex- and age-matched healthy Syrian hamsters (n = 15) were used. Antibodies specific for the three alpha-isoforms and against the beta1-isoform were used to study Na,K-ATPase isoform expression in ventricular myocardium. Na,K-ATPase activity was quantified in homogenate and membrane fractions. There was no significant change in left ventricular mass. Morphological examination revealed a decreased septum thickness in the dilated cardiomyopathy compared with control hamster. Idiopathic dilated cardiomyopathy in hamsters presented significantly reduced membrane alpha1 and beta1 abundances and reduced Na,K-ATPase activity (-35% vs. healthy control, p<0.05). Chronic heart failure had no effect on the Na,K-ATPase alpha2-subunit protein. We have demonstrated for the first time that dilated cardiomyopathy induces a specific reduction of both membrane alpha1- and beta1-isoform abundance and Na,K-ATPase activity in hamsters similar to those previously reported in human dilated heart failure.     

Rac-Induced Left Ventricular Dilation in Thyroxin-Treated ZmRacD Transgenic Mice: Role of Cardiomyocyte Apoptosis and Myocardial Fibrosis

The pathways inducing the critical transition from compensated hypertrophy to cardiac dilation and failure remain poorly understood. The goal of our study is to determine the role of Rac-induced signaling in this transition process. Our previous results showed that Thyroxin (T4) treatment resulted in increased myocardial Rac expression in wild-type mice and a higher level of expression in Zea maize RacD (ZmRacD) transgenic mice. Our current results showed that T4 treatment induced physiologic cardiac hypertrophy in wild-type mice, as demonstrated by echocardiography and histopathology analyses. This was associated with significant increases in myocardial Rac-GTP, superoxide and ERK1/2 activities. Conversely, echocardiography and histopathology analyses showed that T4 treatment induced dilated cardiomyopathy along with compensatory cardiac hypertrophy in ZmRacD mice. These were linked with further increases in myocardial Rac-GTP, superoxide and ERK1/2 activities. Additionally, there were significant increases in caspase-8 expression and caspase-3 activity. However, there was a significant decrease in p38-MAPK activity. Interestingly, inhibition of myocardial Rac-GTP activity and superoxide generation with pravastatin and carvedilol, respectively, attenuated all functional, structural, and molecular changes associated with the T4-induced cardiomyopathy in ZmRacD mice except the compensatory cardiac hypertrophy. Taken together, T4-induced ZmRacD is a novel mouse model of dilated cardiomyopathy that shares many characteristics with the human disease phenotype. To our knowledge, this is the first study to show graded Rac-mediated O(2)·(-) results in cardiac phenotype shift in-vivo. Moreover, Rac-mediated O(2)·(-) generation, cardiomyocyte apoptosis, and myocardial fibrosis seem to play a pivotal role in the transition from cardiac hypertrophy to cardiac dilation and failure. Targeting Rac signaling could represent valuable therapeutic strategy not only in saving the failing myocardium but also to prevent this transition process.     

Ameliorating effects of amlodipine on plasma and myocardial catecholamines in BIO 53.58 Syrian hamsters, a model of dilated cardiomyopathy

Our previous study has shown that the concentrations of norepinephrine, epinephrine and dopamine in the plasma of BIO 53.58 hamsters (a model of dilated cardiomyopathy: DCM) at 18 weeks of age (severe cardiomyopathic stage) were twice those of age-matched F1B control and conversely the myocardial norepinephrine level was decreased. The present study was undertaken to examine the effect of amlodipine on catecholamine concentration, myocardial receptors and histopathological changes in BIO 53.58 hamsters. Oral administration of amlodipine (10 mg/kg/day) for 7 weeks in 11 week-old-BIO 53.58 hamsters brought about marked decreases in the concentrations of norepinephrine, epinephrine and dopamine in the plasma, compared with those in vehicle-treated BIO 53.58 hamsters. This was accompanied by a concomitant increase in the concentration of myocardial catecholamine concentration. In other words, the concentrations of catecholamines in plasma and myocardium of amlodipine administered BIO 53.58 hamsters approximated to the control level in age-matched F1B. In addition, amlodipine administration caused a significant reduction of calcium deposition with a tendency toward a decrease in the myocardial necrosis, and it had little effect on the affinity and number of specific binding for (+)-[3H]PN 200-110, (-)-[125I]iodocyanopindolol (CYP) and [3H]prazosin in the myocardium. In conclusion, the present study shows that administration of amlodipine in BIO 53.58 hamsters may exhibit ameliorating effect on plasma and myocardial catecholamines with a significant reduction of calcium deposition. These data may offer further support for the use of amlodipine in patients with DCM.     

Effects of induced hyperthyroidism in normal and cardiomyopathic hamsters.

Thyroid hormones (TH) enhance cardiac function and reverse gene changes typical of pathological hypertrophy. However, reports in humans, but not animals, indicate that excess TH can cause heart failure. Also, the effects of TH on normal and cardiomyopathic hearts are likely to be different. The goal of this study was to characterize the effects of prolonged hyperthyroidism on cardiac function, chamber and cellular remodeling, and protein expression in both normal and cardiomyopathic hearts. Hyperthyroidism was induced in 3-mo-old normal BIO F1B and dilated cardiomyopathic BIO TO2 hamsters. After TH treatment for 10 days and 2 mo, hemodynamics, echos, myocyte length, histology, and protein expression were assessed. After 10 days and 2 mo, there were no differences between TO2-treated (Tx) and TO2-untreated (Untx) hamsters in chamber diameters or left ventricular function. After 2 mo of treatment, however, F1B-Tx showed evidence of dilated heart failure vs. F1B-Untx. Chamber diameters were increased, and ejection fraction and positive and negative changes in pressure over time were reduced. In F1B-Tx and TO2-Tx hamsters, beta-myosin isoform expression was reduced, whereas alpha-myosin increased significantly in F1B-Tx only. In TO2-Tx hamsters, the percent of viable myocardium was increased, and percent fibronecrosis was reduced vs. TO2-Untx. Myocyte length increased with TH treatment in both hamster strains. We conclude that 1) excess TH can induce heart failure in normal animals as observed in humans, 2) reversal of myosin heavy chain expression does not necessarily improve heart function, and 3) excess TH altered cellular remodeling but did not adversely affect chamber function or dimensions in TO2 hamsters.     

Alterations of beta-adrenergic signaling and cardiac hypertrophy in transgenic mice overexpressing TGF-beta(1)

Transforming growth factor-beta(1) (TGF-beta(1)) promotes or inhibits cell proliferation and induces fibrotic processes and extracellular matrix production in numerous cell types. Several cardiac diseases are associated with an increased expression of TGF-beta(1) mRNA, particularly during the transition from stable cardiac hypertrophy to heart failure. In vitro studies suggest a link between TGF-beta(1) signaling and the beta-adrenergic system. However, the in vivo effects of this growth factor on myocardial tissue have been poorly identified. In transgenic mice overexpressing TGF-beta(1) (TGF-beta), we investigated the in vivo effects on cardiac morphology, beta-adrenergic signaling, and contractile function. When compared with nontransgenic controls (NTG), TGF-beta mice revealed significant cardiac hypertrophy (heart weight, 164 +/- 7 vs. 130 +/- 3 mg, P < 0.01; heart weight-to-body weight ratio, 6.8 +/- 0.3 vs. 5.1 +/- 0.1 mg/g, P < 0.01), accompanied by interstitial fibrosis. These morphological changes correlated with an increased expression of hypertrophy-associated proteins such as atrial natriuretic factor (ANF). Furthermore, overexpression of TGF-beta(1) led to alterations of beta-adrenergic signaling as myocardial beta-adrenoceptor density increased from 7.3 +/- 0.3 to 11.2 +/- 1.1 fmol/mg protein (P < 0.05), whereas the expression of beta-adrenoceptor kinase-1 and inhibitory G proteins decreased by 56 +/- 9.7% and 58 +/- 7.6%, respectively (P < 0.05). As a consequence of altered beta-adrenergic signaling, hearts from TGF-beta showed enhanced contractile responsiveness to isoproterenol stimulation. In conclusion, we conclude that TGF-beta(1) induces cardiac hypertrophy and enhanced beta-adrenergic signaling in vivo. The morphological alterations are either induced by direct effects of TGF-beta(1) or may at least in part result from increased beta-adrenergic signaling, which may contribute to excessive catecholamine stimulation during the transition from compensated hypertrophy to heart failure.     

Brain natriuretic peptide appears to act locally as an antifibrotic factor in the heart

In addition to cardiac myocyte hypertrophy, proliferation and increased extracellular matrix production of cardiac fibroblasts occur in response to cardiac overload. This remodeling of the cardiac interstitium is a major determinant of pathologic hypertrophy leading to ventricular dysfunction and heart failure. Atrial and brain natriuretic peptides (ANP and BNP) are cardiac hormones produced primarily by the atrium and ventricle, respectively. Plasma ANP and BNP concentrations are elevated in patients with hypertension, cardiac hypertrophy, and acute myocardial infarction, suggesting their pathophysiologic roles in these disorders. ANP and BNP exhibit diuretic, natriuretic, and vasodilatory activities via a guanylyl cyclase-coupled natriuretic peptide receptor subtype (guanylyl cyclase-A or GC-A). Here we report the generation of mice with targeted disruption of BNP (BNP-/- mice). We observed focal fibrotic lesions in ventricles from BNP-/- mice with a remarkable increase in ventricular mRNA expression of ANP, angiotensin converting enzyme (ACE), transforming growth factor (TGF)-beta3, and pro-alpha1(I) collagen [Col alpha1(I)], which are implicated in the generation and progression of ventricular fibrosis. Electron microscopic examination revealed supercontraction of sarcomeres and disorganized myofibrils in some ventricular myocytes from BNP-/- mice. No signs of cardiac hypertrophy and systemic hypertension were noted in BNP-/- mice. In response to acute cardiac pressure overload induced by aortic constriction, massive fibrotic lesions were found in all the BNP-/- mice examined, accompanied by further increase of mRNA expression of TGF-beta3 and Col alpha1(I). We postulate that BNP acts as a cardiocyte-derived antifibrotic factor in the ventricle.     

A metabolic approach to the treatment of dilated cardiomyopathy in BIO T0-2 cardiomyopathic Syrian hamsters

Mechanisms underlying dilated cardiomyopathy (DCM) are poorly understood and effective therapy is still unavailable. The aim of this study was to examine the heart ultrastructure and dynamic of BIO T0-2 cardiomyopathic hamsters, an animal model of DCM, and to study in these animals, the effects of a co-formulation (HS12607) of propionyl-L-carnitine, coenzyme Q(10) and omega-3 fatty acids on cardiac mechanical parameters. Sarcomere length, Frank-Starling mechanism and force-frequency relations were studied on isolated ventricular papillary muscle from age-matched BIO F1B normal Syrian hamsters, BIO T0-2 control and BIO T0-2 HS12607-treated cardiomyopathic Syrian hamsters. At the optimum length to maximum active force, electron microscopy of left ventricular papillary muscle revealed that seven out of ten muscles studied showed shorter sarcomeres (1.20 +/- 0.29 microm), and the remaining three showed longer sarcomeres (2.80 +/- 0.13 microm), compared to those of normal hamsters (2.05 +/- 0.06 microm, n = 10). Severe alterations of the Frank-Starling mechanism, force-frequency relations and derivative parameters of contractile waves were also observed in vitro in the BIO T0-2 control hamsters. Long-term (8 weeks) treatment with HS12607 prevented alterations in sarcomere length in the BIO T0-2 cardiomyopathic hamsters; the Frank-Starling mechanism and force-frequency relations were also significantly (P < 0.05) improved in these hamsters. Therefore results of the present study strongly suggest the need for clinical studies on metabolic therapeutic intervention in the effort to stop the progression of dilated cardiomyopathy.     

Transforming growth factor beta1-regulated xylosyltransferase I activity in human cardiac fibroblasts and its impact for myocardial remodeling

In cardiac fibrosis remodeling of the failing myocardium is associated with a complex reorganization of the extracellular matrix (ECM). Xylosyltransferase I and Xylosyltransferase II (XT-I and XT-II) are the key enzymes in proteoglycan biosynthesis, which are an important fraction of the ECM. XT-I was shown to be a measure for the proteoglycan biosynthesis rate and a biochemical fibrosis marker. Here, we investigated the XT-I and XT-II expression in cardiac fibroblasts and in patients with dilated cardiomyopathy and compared our findings with nonfailing donor hearts. We analyzed XT-I and XT-II expression and the glycosaminoglycan (GAG) content in human cardiac fibroblasts incubated with transforming growth factor (TGF)-beta(1) or exposed to cyclic mechanical stretch. In vitro and in vivo no significant changes in the XT-II expression were detected. For XT-I we found an increased expression in parallel with an elevated chondroitin sulfate-GAG content after incubation with TGF-beta(1) and after mechanical stretch. XT-I expression and subsequently increased levels of GAGs could be reduced with neutralizing anti-TGF-beta(1) antibodies or by specific inhibition of the activin receptor-like kinase 5 or the p38 mitogen-activated protein kinase pathway. Usage of XT-I small interfering RNA could specifically block the increased XT-I expression under mechanical stress and resulted in a significantly reduced chondroitin sulfate-GAG content. In the left and right ventricular samples of dilated cardiomyopathy patients, our data show increased amounts of XT-I mRNA compared with nonfailing controls. Patients had raised levels of XT-I enzyme activity and an elevated proteoglycan content. Myocardial remodeling is characterized by increased XT-I expression and enhanced proteoglycan deposition. TGF-beta(1) and mechanical stress induce XT-I expression in cardiac fibroblasts and have impact for ECM remodeling in the dilated heart. Specific blocking of XT-I expression confirmed that XT-I catalyzes a rate-limiting step during fibrotic GAG biosynthesis.     

The involvement of transforming growth factor-beta1 secretion in urotensin II-induced collagen synthesis in neonatal cardiac fibroblasts

As the most potent vasoconstrictor in mammals, urotensin II (U II) has recently been demonstrated to play an important role in adverse cardiac remodeling and fibrosis. However, the mechanisms of U II-induced myocardial fibrosis remain to be clarified. We postulated that U II alters transforming growth factor-beta1 (TGF-beta1) expression, and thereby modulates cardiac fibroblast collagen metabolism. Experiments were conducted using cardiac fibroblast from neonatal Wistar rats to determine the expression of TGF-beta1, and the role of U II receptor UT in this process. The functional role of TGF-beta1 and UT in modulating U II effects on type I, III collagen mRNA expression and 3H-proline incorporation was also analyzed. TGF-beta1 gene and protein expression were consistently identified in quiescent cardiac fibroblasts. U II increased the expression of TGF-beta1 mRNA and protein in a time-dependent manner. This effect was UT mediated, because UT antagonist urantide abolished U II-induced TGF-beta1 expression. U II-induced increase in type I, III collagen mRNA expression and 3H-proline incorporation were both inhibited by a specific TGF-beta1 neutralizing antibody and UT receptor antagonist urantide. Hence, our results indicate that TGF-beta1 is upregulated in cardiac fibroblasts by U II via UT and modulates profibrotic effects of U II. These findings provide novel insights into U II-induced cardiac remodeling.     

Nuclear size of myocardial cells in end-stage cardiomyopathies

OBJECTIVE: To determine the alteration of nuclear size in myocardial cells and the relationship between nuclear size and DNA ploidy classes in normal and cardiomyopathic human hearts. STUDY DESIGN: The study group consisted of 46 hearts obtained at biopsy. These patients had undergone cardiac transplantation for intractable congestive heart failure (18 cases with ischemic cardiomyopathy and 28 cases with idiopathic dilated cardiomyopathy). Another 10 hearts were collected at autopsy and used as control hearts according to preautopsy, autopsy and histology criteria. One hundred fibroblasts and 200 myocytes were evaluated in each ventricle. The nuclear area and DNA content were estimated using image cytometry. RESULTS: End-stage ischemic and dilated cardiomyopathies were characterized by an increase in nuclear size of both the myocyte and nonmyocyte population. The nuclear area of interstitial cells increased about 30% in cardiomyopathic hearts. Augmentation of average nuclear area of myocytes was 1.2-fold in the ischemic group and about 1.5-fold in the dilated group as compared with the control group. Also, a tendency was found for the coefficient of variation of average nuclear area to decrease in the interstitial cell population and increased in the myocyte population in cardiomyopathic situations. Furthermore, the nuclear area of myocytes enlarged as augmentation of nuclear DNA content. The relative nuclear areas of myocytes can be presented as: 2c:4c:8c:16c :32c:64c = 1:1.65:2.75:4.60:7.25:9.18. CONCLUSION: The increase in nuclear size follows either one of two different processes: the first does not involve an increase in DNA content, whereas the second is concomitant with an incremental increase in DNA content. In the first instance, the enlargement of nuclear size is limited. In the second, augmentation of nuclear size can become very impressive. In end-stage ischemic and dilated cardiomyopathies, the nuclear growth of myocytes and interstitial cells may be due to different mechanisms. Enlargement of the nuclear area of myocytes represents a complex process, including simple nuclear hypertrophy, polyploidization and multinucleation. The main pattern of nuclear growth of interstitial cells is nuclear hypertrophy without an increase in DNA content.     

Cardiac contractile dysfunction in J2N-k cardiomyopathic hamsters is associated with impaired SR function and regulation

Although dilated cardiomyopathy (DCM) is known to result in cardiac contractile dysfunction, the underlying mechanisms are unclear. The sarcoplasmic reticulum (SR) is the main regulator of intracellular Ca²⁺ required for cardiac contraction and relaxation. We therefore hypothesized that abnormalities in both SR function and regulation will contribute to cardiac contractile dysfunction of the J2N-k cardiomyopathic hamster, an appropriate model of DCM. Echocardiographic assessment indicated contractile dysfunction, because the ejection fraction, fractional shortening, cardiac output, and heart rate were all significantly reduced in J2N-k hamsters compared with controls. Depressed cardiac function was associated with decreased cardiac SR Ca²⁺ uptake in the cardiomyopathic hamsters. Reduced SR Ca²⁺ uptake could be further linked to a decrease in the expression of the SR Ca²⁺-ATPase and cAMP-dependent protein kinase (PKA)-mediated phospholamban (PLB) phosphorylation at serine-16. Depressed PLB phosphorylation was paralleled with a reduction in the activity of SR-associated PKA, as well as an elevation in protein phosphatase activity in J2N-k hamster. The results of this study suggest that an alteration in SR function and its regulation contribute to cardiac contractile dysfunction in the J2N-k cardiomyopathic hamster. sarcoplasmic reticulum; cardiomyopathy; cAMP-dependent protein kinase; Ca²⁺/calmodulin-dependent protein kinase; sarco(endo)plasmic reticulum ATPase; phospholamban     

Cardiac carnitine deficiency and altered carnitine transport in cardiomyopathic hamsters

The Bio 14.6 hamster has a well-documented cardiomyopathy which leads to congestive heart failure. Previous work demonstrated that hearts from these hamsters have depressed fatty acid oxidation and depressed carnitine concentrations compared to those of normal hamsters. Analyses of tissue carnitine concentrations from 40 to 464 days of age demonstrate that the cardiomyopathic hamsters have a cardiac carnitine deficiency throughout life. Therefore, the carnitine deficiency is not a secondary effect of an advanced stage of the cardiomyopathy. Both the observation that other tissues of the cardiomyopathic hamster have normal or markedly elevated carnitine concentrations and the observation that oral carnitine treatment could not increase the cardiac carnitine concentrations to those of normal hamsters are consistent with the hypothesis that the cardiac carnitine deficiency is the result of a defective cardiac transport mechanism. Cardiac carnitine-binding protein (which may function in the cardiac carnitine transport mechanism) prepared from hearts of cardiomyopathic hamsters had a lower maximal carnitine binding and an increased dissociation constant for carnitine compared to the cardiac carnitine-binding protein prepared from normal hamsters. Thus, several types of data indicate that the cardiomyopathic hamster has an altered cardiac carnitine transport mechanism.     

Sustained-release prostacyclin analog ONO-1301 ameliorates tubulointerstitial alterations in a mouse obstructive nephropathy model

Tubulointerstitial injuries are crucial histological alterations that predict the deterioration of renal function in chronic kidney disease. ONO-1301, a novel sustained-release prostacyclin analog, accompanied by thromboxane synthase activity, exerts therapeutic effects on experimental pulmonary hypertension, lung fibrosis, cardiomyopathy, and myocardial ischemia, partly associated with the induction of hepatocyte growth factor (HGF). In the present study, we examined the therapeutic efficacies of ONO-1301 on tubulointerstitial alterations induced by unilateral ureteral obstruction (UUO). After inducing unilateral ureteral obstruction in C57/BL6J mice, a single injection of sustained-release ONO-1301 polymerized with poly (D,L-lactic-co-glycolic acid) sustained-release ONO-1301 (SR-ONO) significantly suppressed interstitial fibrosis, accumulation of types I and III collagen, increase in the number of interstitial fibroblast-specific protein-1 (FSP-1)(+) cells, and interstitial infiltration of monocytes/macrophages (F4/80(+)) in the obstructed kidneys (OBK; day 7). Treatment with SR-ONO significantly suppressed the increase of the renal levels of profibrotic factor TGF-β and phosphorylation of Smad2/3, and elevated the renal levels of HGF in the OBK. In cultured mouse proximal tubular epithelial cells (mProx24), ONO-1301 significantly ameliorated the expression of fibroblast-specific protein-1 and α-smooth muscle actin as well as phosphorylation of Smad3 and increased the expression of zonula occludens-1 and E-cadherin in the presence of TGF-β1 as detected by immunoblot and immunocytochemistry, partly dependent on PGI(2) receptor-mediated signaling. Administration of rabbit anti-HGF antibodies, but not the control IgG, partly reversed the suppressive effects of SR-ONO on tubulointerstitial injuries in the OBK. Taken together, our findings suggest the potential therapeutic efficacies of ONO-1301 in suppressing tubulointerstitial alterations partly mediated via inducing HGF, an antifibrotic factor counteracting TGF-β.