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)     

Developmental changes of calcium transients and contractility during the cultivation of rat neonatal cardiomyocytes

In the normal myocardium matrix metalloproteinases (MMP) are present in the latent form. To examine whether MMP are activated following infarction or idiopathic dilated cardiomyopathy (DCM), we extracted and measured MMP activity in tissue derived from 7 explanted, failing human hearts due to either previous myocardial infarction (MI) or DCM. MMP activity in infarcted left ventricle (LV), noninfarcted IV and right ventricle (RV) from MI patients, as well as tissue from either ventricle of DCM patients, were compared to the activity of donor heart tissue. SDS-PAGE and dye-binding assays were used to determine total protein concentration, while collagenase activity was measured by SDS-PAGE type substrate gels embedded with type I gelatin (zymography). Accuracy of the zymographic technique was shown for tissue samples as small as 0.05 mg and was comparable to results obtained by a spectrophotometric method.. After normalization for total protein concentration, we found 3 ± 1 % collagenase activity in normal atrial tissue which could be activated to 80–90% by trypsin or plasmin, indicating that collagenase is normally inactive or in a latent form in human heart. In endo- and epimyocardium of infarcted LV on the other hand, collagenase activity was 85–95% and 10–20%, respectively, while 5–10% and 3–5%, respectively, in noninfarcted LV In DCM, collagenolytic activity in the endo and epimyocardium was 75 ± 5 and 35 ± 5% in the LV and 35 ± 7 and 20 ± 5% in the RV, respectively. Thus, in dilated failing human hearts secondary to previous MI or DCM, MMP activity is increased. This is particularly the case within the endomyocardium of the infarcted and noninfarcted portions of either ventricle with MI and in both ventricles in DCM. This suggests that an activation of collagenase throughout the myocardium may contribute to its remodeling that includes ventricular dilatation and wall thinning.This work was supported in part by NIH grant GM-48595 and by a Grant-In-Aid from the American Heart Association, Missouri Affiliate (92-10517).     

Matrix remodeling in experimental and human heart failure: a possible regulatory role for TIMP-3

In the failing heart, an imbalance in matrix metalloproteinases (MMPs) and their biological regulators, the tissue inhibitors of MMPs (TIMPs), may result in cardiac dilatation from matrix degradation. We hypothesized that a reduction of myocardial TIMP-3 is associated with adverse matrix remodeling in both human and experimental heart failure. Cardiomyopathic hamsters at age 15 wk (normal), 25 wk (compensated stage), and 35 wk (overt failure) were compared with age-matched normal controls. MMP activity (gelatinase bioassay) was increased in cardiomyopathic hearts (P = 0.03) and peaked during the transition to overt heart failure. TIMP-3 content (immunoblot) was decreased compared with normal controls (74 +/- 5% at 25 wk, 69 +/- 10% at 35 wk; P = 0.001) and its reduction was associated with increased MMP activity (r = -0.6; P = 0.004). TIMP-1 increased progressively (P = 0.001), whereas TIMP-2, TIMP-4, and MMP protein levels were unchanged. Myocardial collagen (hydroxyproline content) increased with time during the progression to end-stage cardiac failure (P < 0.0001). Collagen synthesis ([(14)C]proline uptake) was elevated in cardiomyopathy at 15 and 25 wk (P < 0.05). The collagen cross-linking ratio (insoluble:soluble collagen) was reduced (P = 0.003) as the left ventricle dilated. By confocal microscopy restricted to viable myocardium, collagen content was reduced (P = 0.04) with fragmentation (P < 0.0001) and thinning (P = 0.003) of perimysial collagen fibers. Similarly, patients with end-stage congestive heart failure (n = 7) compared with nonfailing controls (n = 2) had elevated gelatinase MMP activity (P = 0.02) associated with isolated reductions in TIMP-3 (55 +/- 5% of normal; P = 0.003). Reductions of TIMP-3 parallel adverse matrix remodeling in the cardiomyopathic hamster and the failing human heart. TIMP-3 may contribute to the regulation of myocardial remodeling and its reduction may promote a transition from compensated to end-stage congestive heart failure.     

Cardiac matrix remodeling following intracoronary cell transplantation in dilated cardiomyopathic rabbits

Cellular cardiomyoplasty has been proposed as a promising therapeutic strategy for chronic heart failure. Previous studies focused on structural changes in cardiomyocytes to explain the potential benefits for contractile function. However, limited information is available about the cardiac matrix remodeling following cell transplantation in dilated cardiomyopathy (DCM). Here, we established a new animal model of intracoronary bone marrow mononuclear cells (BMMNCs) transplantation to explore extracellular matrix remodeling in adriamycin-induced cardiomyopathic rabbits. In vivo studies demonstrated that BMMNCs transplantation can dramatically delay the progress of collagen metabolism and decrease myocardial collagen volume fraction. The beneficial effects were mediated by attenuating stress-generated over-expression of matrix metalloproteinases (MMPs) in ventricular remodeling. Improved cardiac function may be contributed in part by stem-associated inhibition of extracellular matrix remodeling.     

Cardiac expression of urocortin (Ucn) in diseased heart; preliminary results on possible involvement of Ucn in pathophysiology of cardiac diseases

Recently, several studies reported that urocortin (Ucn) had beneficial effects on cardiovascular system and was expressed both in the normal heart and in the heart of dilated cardiomyopathy (DCM), yet the relationship between high expression of Ucn and pathophysiology of Ucn in diseased heart has been discussed. Thus, the present study was designed to elucidate the expression of Ucn in the diseased heart by immunohistochemical approach using endomyocardial biopsy specimens. The involvement of immunoreactive Ucn in pathophysiology of cardiac disease was evaluated using endomyocardial biopsy specimens obtained from the patients with some heart diseases, including DCM and hypertrophic cardiomyopathy (HCM). Ucn was detected in all endomyocardial biopsy specimens of ventricular tissue obtained from the patients with such cardiac diseases, a specimens of atrial tissue, and normal heart specimens obtained from autopsy cases. In DCM patients, left ventricular end-diastolic pressure significantly elevated in severely stained group. On the contrary, in HCM patients, left ventricular ejection fraction was higher in the severely stained group. Ucn was expressed more abundantly in the diseased heart, especially in HCM and DCM, than in the normal heart. In conclusion, such close relationship between Ucn expression in the heart and cardiac function indicated that clinical features of Ucn resembled those of norepinephrine and Ucn could play a certain pathophysiological roles in the cardiac diseases.     

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.     

Hepatocyte growth factor prevents tissue fibrosis, remodeling, and dysfunction in cardiomyopathic hamster hearts

Structural remodeling of the myocardium, including myocyte hypertrophy, myocardial fibrosis, and dilatation, drives functional impairment in various forms of acquired and hereditary cardiomyopathy. Using cardiomyopathic Syrian hamsters with a genetic defect in delta-sarcoglycan, we investigated the potential involvement of hepatocyte growth factor (HGF) in the pathophysiology and therapeutics related to dilated cardiomyopathy, because HGF has previously been shown to be cytoprotective and to have benefits in acute heart injury. Late-stage TO-2 cardiomyopathic hamsters showed severe cardiac dysfunction and fibrosis, accompanied by increases in myocardial expression of transforming growth factor-beta1 (TGF-beta1), a growth factor responsible for tissue fibrosis. Conversely, HGF was downregulated in late-stage myopathic hearts. Treatment with recombinant human HGF for 3 wk suppressed cardiac fibrosis, accompanied by a decreased expression of TGF-beta1 and type I collagen. Suppression of TGF-beta1 and type I collagen by HGF was also shown in cultured cardiac myofibroblasts. Likewise, HGF suppressed myocardial hypertrophy, apoptosis in cardiomyocytes, and expression of atrial natriuretic polypeptide, a molecular marker of hypertrophy. Importantly, downregulation of the fibrogenic and hypertrophic genes by HGF treatment was associated with improved cardiac function. Thus the decrease in endogenous HGF levels may participate in the susceptibility of cardiac tissue to hypertrophy and fibrosis, and exogenous HGF led to therapeutic benefits in case of dilated cardiomyopathy in this model, even at the late-stage treatment.     

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.     

Induction of oxidative stress and disintegrin metalloproteinase in human heart end-stage failure

Collagen degradation is required for the creation of new integrin binding sites necessary for cell survival. However, a complete separation between the matrix and the cell leads to apoptosis, dilatation, and failure. Previous studies have demonstrated increased metalloproteinase activity in the failing myocardium. To test the hypothesis that disintegrin metalloproteinase (DMP) is induced in human heart end-stage failure, left ventricle tissue from ischemic cardiomyopathic (ICM, n = 10) and dilated cardiomyopathic (DCM, n = 10) human hearts were obtained at the time of orthotopic cardiac transplant. Normal (n = 5) tissue specimens were obtained from unused hearts. The levels of reduced oxygen species (ROS) were 12 +/- 2, 25 +/- 3, and 16 +/- 2 nmol (means +/- SE, P < 0.005) in normal, ICM, and DCM, respectively, by spectrofluorometry. The percent levels of endothelial cells were 100 +/- 15, 35 +/- 19, and 55 +/- 11 in normal, ICM, and DCM, respectively, by CD31 labeling. The levels of nitrotyrosine by Western analysis were significantly increased, and endothelial nitric oxide (NO) by the Griess method was decreased in ICM and DCM compared with normal tissue. The synthesis and degradation of beta(1)-integrin and connexin 43 were significantly increased in ICM and DCM compared with normal hearts by Western analysis. Levels of DMP were increased, and levels of cardiac inhibitor of metalloproteinase (CIMP) were decreased. Aggrecanase activity of DMP was significantly increased in ICM and DCM hearts compared with normal. These results suggest that the occurrence of cardiomyopathy is significantly confounded by the increase in ROS, nitrotyrosine, and DMP activity. This increase is associated with decreased NO, endothelial cell density, and CIMP. In vitro, treatment of CIMP abrogated the DMP activity. The treatment with CIMP may prevent degradation of integrin and connexin and ameliorate heart failure.     

Pathological and biochemical analysis of dilated cardiomyopathy of broiler chickens--an animal model

Forty-seven birds (M/F = 33/14) with natural outbreak dilated cardiomyopathy (DCM) of right ventricle (RV) and 33 birds with artificially cool-induced DCM hearts were studied. Clinically, 20 severe and 13 mild DCM cases were induced during five weeks and the peak morbidity was in the 2nd week. The progressive dilatation of RV and hypokinesis of septum was shown by echocardiography. At autopsy, the ratio of heart weights to the body weights was increased, the ratio of RV weight to the total ventricle significantly increased, especially in the severe DCM cases (P < 0.05). The RV was dilated and the wall thickness was increased and finally both RV and left ventricle (LV) were markedly dilated and the septum became thinner. The struts, weave and coil demonstrated by silver impregnation stain were fragmented, dissociated and overstretched. The promatrix metalloproteinases (MMP)-2, -9 and active MMP-2 were markedly increased in natural outbreak DCM cases, especially in the RV (P < 0.05). The proMMP-2 and active MMP-2 was increased in the cool induced DCM cases, especially in the RV of severe DCM (P < 0.05). These indicated that both the natural outbreak and the artificially induced DCM of broiler chickens are ideal DCM animal models.     

Early induction of matrix metalloproteinase-9 transduces signaling in human heart end stage failure

Extracellular matrix (ECM) turnover is regulated by matrix metalloproteinases (MMPs) and plays an important role in cardiac remodeling. Previous studies from our lab demonstrated an increase in gelatinolytic-MMP-2 and -9 activities in endocardial tissue from ischemic cardiomyopathic (ICM) and idiopathic dilated cardiomyopathic (DCM) hearts. The signaling mechanism responsible for the left ventricular (LV) remodeling, however, is unclear. Administration of cardiac specific inhibitor of metalloproteinase (CIMP) prevented the activation of MMP-2 and -9 in ailing to failing myocardium. Activation of MMP-2 and -9 leads to induction of proteinase activated receptor-1 (PAR-1). We hypothesize that the early induction of MMP-9 is a key regulator for modulating intracellular signaling through activation of PAR and various downstream events which are implicated in development of cardiac fibrosis in an extracellular receptor mediated kinase-1 (ERK-1) and focal adhesion kinase (FAK) dependent manner. To test this hypothesis, explanted human heart tissues from ICM and DCM patients were obtained at the time of orthotopic cardiac transplants. Quantitative analysis of MMP-2 and -9 gelatinolytic activities was made by real-time quantitative zymography. Gel phosphorylation staining for PAR-1 showed a significant increase in ICM hearts. Western blot and RT-PCR analysis and in-situ labeling, showed significant increased expression of PAR-1, ERK-1and FAK in ICM and DCM. These observations suggest that the enhanced expression and potentially increased activity of LV myocardial MMP-9 triggers the signal cascade instigating cardiac remodeling. This early mechanism for the initiation of LV remodeling appears to have a role in end-stage human heart failure.     

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.     

Functional consequences of hypertrophic and dilated cardiomyopathy-causing mutations in alpha-tropomyosin

To study the functional consequences of various cardiomyopathic mutations in human cardiac alpha-tropomyosin (Tm), a method of depletion/reconstitution of native Tm and troponin (Tn) complex (Tm-Tn) in cardiac myofibril preparations has been developed. The endogenous Tm-Tn complex was selectively removed from myofibrils and replaced with recombinant wild-type or mutant proteins. Successful depletion and reconstitution steps were verified by SDS-gel electrophoresis and by the loss and regain of Ca2+-dependent regulation of ATPase activity. Five Tm mutations were chosen for this study: the hypertrophic cardiomyopathy (HCM) mutations E62Q, E180G, and L185R and the dilated cardiomyopathy (DCM) mutations E40K and E54K. Through the use of this new depletion/reconstitution method, the functional consequences of these mutations were determined utilizing myofibrillar ATPase measurements. The results of our studies showed that 1) depletion of >80% of Tm-Tn from myofibrils resulted in a complete loss of the Ca2+-regulated ATPase activity and a significant loss in the maximal ATPase activity, 2) reconstitution of exogenous wild-type Tm-Tn resulted in complete regain in the calcium regulation and in the maximal ATPase activity, and 3) all HCM-associated Tm mutations increased the Ca2+ sensitivity of ATPase activity and all had decreased abilities to inhibit ATPase activity. In contrast, the DCM-associated mutations both decreased the Ca2+ sensitivity of ATPase activity and had no effect on the inhibition of ATPase activity. These findings have demonstrated that the mutations which cause HCM and DCM disrupt discrete mechanisms, which may culminate in the distinct cardiomyopathic phenotypes.     

Tissue inhibitor of metalloproteinase-4 instigates apoptosis in transformed cardiac fibroblasts

Tumor cells become malignant, in part, because of their activation of matrix metalloproteinases (MMPs) and inactivation of tissue inhibitor of metalloproteinases (TIMPs). Myocardial tumors are rarely malignant. This raises the possibility that the MMPs and TIMPs are differentially regulated in the heart compared to other tissues. Therefore, we hypothesized that a tissue specific tumor suppressor exists in the heart. To test this hypothesis we prepared cardiac tissue extracts from normal (n = 4), ischemic cardiomypathic (ICM) [n = 5], and dilated cardiomyopathic (DCM) [n = 8] human heart end-stage explants. The level of cardiospecific TIMP-4 was determined by SDS-PAGE and Western-blot analysis. The results suggested reduced levels of TIMP-4 in ICM and DCM as compared to normal heart. TIMP-4 was purified by reverse phase HPLC and gelatin-sepharose affinity chromatography. Collagenase inhibitory activity of chromatographic peaks was determined using fluorescein-conjugated collagen as substrate and fluorescence spectroscopy. The activity of TIMP-4 (27 kDa) was characterized by reverse zymography. The role of TIMP-4 in cardiac fibroblast cell migration was examined using Boyden chamber analysis. The results suggested that TIMP-4 inhibited cardiac fibroblast cells migration and collagen gel invasion. To test whether TIMP-4 induces apoptosis, we cultured cardiac normal and polyomavirus transformed fibroblast cells in the presence and absence of TIMP-4. The number of cells were measured and DNA laddering was determined. The results suggested that TIMP-4 controlled normal cardiac fibroblast transformation and induced apoptosis in transformed cells. Cardiospecific TIMP-4 plays a significant role in regulating the normal cell phenotype. The reduced levels of TIMP-4 elicit cellular transformation and may lead to adverse extracellular matrix degradation (remodeling), cardiac hypertrophy and failure. This study suggests a possible protective role of TIMP-4 in other organs which are susceptible to malignancy.     

Identification of new biophysical markers for pathological ventricular remodelling in tachycardia‐induced dilated cardiomyopathy

Our aim was to identify biophysical biomarkers of ventricular remodelling in tachycardia‐induced dilated cardiomyopathy (DCM). Our study includes healthy controls (N = 7) and DCM pigs (N = 10). Molecular analysis showed global myocardial metabolic abnormalities, some of them related to myocardial hibernation in failing hearts, supporting the translationality of our model to study cardiac remodelling in dilated cardiomyopathy. Histological analysis showed unorganized and agglomerated collagen accumulation in the dilated ventricles and a higher percentage of fibrosis in the right (RV) than in the left (LV) ventricle (P = .016). The Fourier Transform Infrared Spectroscopy (FTIR) 1st and 2nd indicators, which are markers of the myofiber/collagen ratio, were reduced in dilated hearts, with the 1st indicator reduced by 45% and 53% in the RV and LV, respectively, and the 2nd indicator reduced by 25% in the RV. The 3rd FTIR indicator, a marker of the carbohydrate/lipid ratio, was up‐regulated in the right and left dilated ventricles but to a greater extent in the RV (2.60‐fold vs 1.61‐fold, P = .049). Differential scanning calorimetry (DSC) showed a depression of the freezable water melting point in DCM ventricles – indicating structural changes in the tissue architecture – and lower protein stability. Our results suggest that the 1st, 2nd and 3rd FTIR indicators are useful markers of cardiac remodelling. Moreover, the 2nd and 3rd FITR indicators, which are altered to a greater extent in the right ventricle, are associated with greater fibrosis.     

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.     

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     

Matrix metalloproteinases and collagen ultrastructure in moderate myocardial ischemia and reperfusion in vivo

Severe ischemic injury or infarction of myocardium may cause activation of matrix metalloproteinases (MMPs) and damage the interstitial matrix. However, it is unknown whether MMP activation and matrix damage occur after moderate ischemia and reperfusion that result in myocardial stunning without infarction, and if so whether such changes contribute to postischemic myocardial expansion and contractile dysfunction. To address these questions, open-chest anesthetized pigs underwent 90 min of regional ischemia (subendocardial blood flow 0.4 +/- 0.1 ml. g(-1). min(-1)) and 90 min of reperfusion. After ischemia plus reperfusion, histological and ultrastructural examination revealed no myocardial infarction or inflammatory cell infiltration. Myocardial MMP-9 content increased threefold with a fourfold increase in the active form (P < 0.001). Myocardial collagenase content doubled (P < 0.01) but remained in latent form. MMP-2 and tissue inhibitors of metalloproteinases were unaffected. Despite increases in MMPs, collagen ultrastructure (assessed by cell maceration scanning electron microscopy) was unaltered. Intracoronary administration of the MMP inhibitor GM-2487 did not prevent or attenuate myocardial expansion (assessed by regional diastolic dimensions at near-zero left ventricular pressure) or contractile dysfunction. We conclude that although moderate ischemia and reperfusion alter myocardial MMP content and activity, these effects do not result in damage to interstitial collagen, nor do they contribute to myocardial expansion or contractile dysfunction.     

Progression from hypertrophic to dilated cardiomyopathy in mice that express a mutant myosin transgene

A mouse model of hypertrophic cardiomyopathy (HCM) was created by expression of a cardiac alpha-myosin transgene including the R(403)Q mutation and a deletion of a segment of the actin-binding domain. HCM mice show early histopathology and hypertrophy, with progressive hypertrophy in females and ventricular dilation in older males. To test the hypothesis that dilated cardiomyopathy (DCM) is part of the pathological spectrum of HCM, we studied chamber morphology, exercise tolerance, hemodynamics, isolated heart function, adrenergic sensitivity, and embryonic gene expression in 8- to 11-mo-old male transgenic animals. Significantly impaired exercise tolerance and both systolic and diastolic dysfunction were seen in vivo. Contraction and relaxation parameters of isolated hearts were also decreased, and lusitropic responsiveness to the beta-adrenergic agonist isoproterenol was modestly reduced. Myocardial levels of the G protein-coupled beta-adrenergic receptor kinase 1 (beta-ARK1) were increased by more than twofold over controls, and total beta-ARK1 activity was also significantly elevated. Induction of fetal gene expression was also observed in transgenic hearts. We conclude that transgenic male animals have undergone cardiac decompensation resulting in a DCM phenotype. This supports the idea that HCM and DCM may be part of a pathological continuum rather than independent diseases.