Dynamic molecular and histopathological changes in the extracellular matrix and inflammation in the transition to heart failure in isolated volume overload

Left ventricular (LV) volume overload (VO) causes eccentric remodeling with inflammatory cell infiltration and extracellular matrix (ECM) degradation, for which there is currently no proven therapy. To uncover new pathways that connect inflammation and ECM homeostasis with cellular dysfunction, we determined the cardiac transciptome in subacute, compensated, and decompensated stages based on in vivo hemodynamics and echocardiography in the rat with aortocaval fistula (ACF). LV dilatation at 5 wk was associated with a normal LV end-diastolic dimension-to-posterior wall thickness ratio (LVEDD/PWT; compensated), whereas the early 2-wk (subacute) and late 15-wk (decompensated) ACF groups had significant increases in LVEDD/PWT. Subacute and decompensated stages had a significant upregulation of genes related to inflammation, the ECM, the cell cycle, and apoptosis. These changes were accompanied by neutrophil and macrophage infiltration, nonmyocyte apoptosis, and interstitial collagen loss. At 15 wk, there was a 40-fold increase in the matricellular protein periostin, which inhibits connections between collagen and cells, thereby potentially mediating a side-to-side slippage of cardiomyocytes and LV dilatation. The majority of downregulated genes was composed of mitochondrial enzymes whose suppression progressed from 5 to 15 wk concomitant with LV dilatation and systolic heart failure. The profound decrease in gene expression related to fatty acid, amino acid, and glucose metabolism was associated with the downregulation of peroxisome proliferator associated receptor (PPAR)-α-related and bioenergetic-related genes at 15 wk. In VO, an early phase of inflammation subsides at 5 wk but reappears at 15 wk with marked periostin production along with the suppression of genes related to PPAR-α and energy metabolism.     

Cardioprotection in female rats subjected to chronic volume overload: synergistic interaction of estrogen and phytoestrogens

Intact female rats fed a high-phytoestrogen diet are protected against adverse left ventricular (LV) remodeling induced by chronic volume overload. We hypothesized that both phytoestrogens and ovarian hormones, particularly estrogen, are necessary for this dietary-induced cardioprotection. To test this hypothesis, eight groups of female rats were studied; rats were fed either a high-phytoestrogen (+phyto) or phytoestrogen-free diet. Groups included sham-operated rats, intact rats with fistula (Fist), ovariectomized rats with fistula (Fist-OX), and Fist-OX rats treated with estrogen (EST). Myocardial function and remodeling were assessed after 8 wk of volume overload using a blood-perfused isolated heart apparatus. Fist-OX rats developed significant ventricular dilatation and increased compliance vs. intact Fist rats, which were associated with a significant decrease in contractility. Estrogen treatment prevented pulmonary edema and attenuated LV hypertrophy and dilatation but did not maintain contractility. However, dietary phytoestrogens completely prevented LV dilatation in both the Fist+phyto and Fist-OX+EST+phyto groups but had no effect on LV remodeling in the Fist-OX+phyto group. Contractility was significantly greater in the estrogen-treated rats fed the phytoestrogen diet than in those treated with estrogen alone. Dietary phytoestrogens did not affect LV or uterine mass, serum estrogen, LV estrogen receptor expression, or cardiac function in sham animals. These data indicate that estrogen is not solely responsible for the cardioprotection exhibited by intact females and that phytoestrogens can work synergistically with ovarian hormones to attenuate ventricular remodeling induced by chronic volume overload in female rats.     

Apoptosis in the left ventricle of chronic volume overload causes endocardial endothelial dysfunction in rats

The hypothesis is that chronic increases in left ventricular (LV) load induce oxidative stress and latent matrix metalloproteinase (MMP) is activated, allowing the heart to dilate in the absence of endothelial nitric oxide (NO) and thereby reduce filling pressure. To create volume overload, an arteriovenous (A-V) fistula was placed in male Sprague-Dawley rats. To decrease oxidative stress and apoptosis, 0.08 mg/ml nicotinamide (Nic) was administered in drinking water 2 days before surgery. The rats were divided into the following groups: 1) A-V fistula, 2) A-V fistula + Nic, 3) sham operated, 4) sham + Nic, and 5) control (unoperated); n = 6 rats/group. After 4 wk, hemodynamic parameters were measured in anesthetized rats. The heart was removed and weighed, and LV tissue homogeneates were prepared. A-V fistula caused an increase in heart weight, lung weight, and end-diastolic pressure compared with the sham group. The levels of malondialdehyde (MDA; a marker of oxidative stress) was 6.60 +/- 0.23 ng/mg protein and NO was 6.87 +/- 1.21 nmol/l in the LV of A-V fistula rats by spectrophometry. Nic treatment increased NO to 13.88 +/- 2.5 nmol/l and decreased MDA to 3.54 +/- 0.34 ng/mg protein (P = 0.005). Zymographic levels of MMP-2 were increased, as were protein levels of nitrotyrosine and collagen fragments by Western blot analysis. The inhibition of oxidative stress by Nic decreased nitrotyrosine content and MMP activity. The levels of tissue inhibitor of metalloproteinase-4 mRNA were decreased in A-V fistula rats and increased in A-V fistula rats treated with Nic by Northern blot analysis. TdT-mediated dUTP nick-end labeling-positive cells were increased in A-V fistula rats and decreased in fistula rats treated with Nic. Acetylcholine and nitroprusside responses in cardiac rings prepared from the above groups of rats suggest impaired endothelial-dependent cardiac relaxation. Treatment with Nic improves cardiac relaxation. The results suggest that an increase in the oxidative stress and generation of nitrotyrosine are, in part, responsible for the activation of metalloproteinase and decreased endocardial endothelial function in chronic LV volume overload.     

Effects of sex differences on constitutive nitric oxide synthase expression and activity in response to pressure overload in rats

Clinical studies have documented sex differences in left ventricular (LV) hypertrophy patterns, but the mechanisms are so far poorly defined. This study aimed to determine whether 1) severe pressure overload altered expression and/or activity of cardiac constitutive nitric oxide synthase (NOS1 and NOS3) and 2) these changes were modulated according to sex. Analyses were performed 0.4-20 wk after thoracic aortic constriction (TAC) in male and female Wistar rats. Male rats with TAC exhibited early signs of cardiac dysfunction, as shown by echocardiographic and LV end-diastolic pressure measurements, whereas females with TAC exhibited higher LV hypertrophy (+96% vs. males at 20 wk; P < 0.05). After TAC, cardiac NOS1 expression was rapidly induced (0.4 wk) and stable afterward in males (P < 0.05 vs. sham groups), whereas it was delayed in females. Accordingly, specific NOS1 activity was increased by 2 wk in male rats with TAC (+122%; P < 0.001 vs. sham groups) and only by 20 wk in females (+220%; P < 0.001 vs. sham groups). NOS1 activity was correlated with NOS1 level. Regarding cardiac NOS3, expression was unaffected by TAC, and the decrease in activity observed at early and late times in male and female rats with TAC, respectively, is shown to be related to NOS3 allosteric regulator caveolin-1 level. The data demonstrated a unique sex-dependent regulation of the constitutive NOSs in response to TAC in rats; such a difference might play a role in the sex-dependent adaptability of the heart in response to pressure overload.     

Dependence of changes in beta-adrenoceptor signal transduction on type and stage of cardiac hypertrophy.

To examine whether cardiac hypertrophy is associated with changes in beta-adrenoceptor signal transduction mechanisms, pressure overload (PO) was induced by occlusion of the abdominal aorta and volume overload (VO) by creation of an aortocaval shunt for 4 and 24 wk in rats. After hemodynamic assessment of the animals, the left ventricular (LV) particulate fraction was isolated for measurement of beta(1)-adrenoceptors and adenylyl cyclase activity, and cardiomyocytes were isolated for monitoring of the intracellular Ca(2+) concentration. Although PO and VO produced cardiac hypertrophy and increased LV end-diastolic pressure at 4 wk, cardiac function was increased in animals subjected to PO but remained unaltered in animals subjected to VO. Cardiac hypertrophy and increased LV end-diastolic pressure were associated with depressed cardiac function at 24 wk of PO or VO, but clinical signs of congestive heart failure were evident only in animals subjected to VO. Isoproterenol-induced increases in cardiac function, activation of adenylyl cyclase activity, and increase in intracellular Ca(2+) concentration, as well as beta(1)-adrenoceptor density, were unaltered by PO at 4 wk, augmented by VO at 4 wk, and attenuated by PO and VO at 24 wk. These results suggest that alterations in beta(1)-adrenoceptor signal transduction are dependent on the type and stage of cardiac hypertrophy.     

Chronic intermittent hypoxia exposure improves left ventricular contractility in transgenic mice with heart failure

We previously reported the unexpected finding that 4 wk of exposure to intermittent hypoxia (IH), which simulates the hypoxic stress of obstructive sleep apnea, improved LV cardiac function in healthy, lean C57BL/6J mice. The purpose of the present study was to assess the impact of 4 wk of IH on cardiac function in a transgenic murine model that exhibits a natural history of heart failure. We hypothesized that IH exposure would exacerbate cardiac decompensation in heart failure. Adult male FVB (wild type) and transgenic mice with cardiac overexpression of tumor necrosis factor α (TNF-αTG) at 10-12 wk of age were exposed to 4 wk of IH (nadir inspired oxygen 5-6% at 60 cycles/h for 12 h during light period) or intermittent air (IA) as control. Cardiac function was assessed by echocardiography and pressure-volume loop analyses, and mRNA and protein expression were performed on ventricular homogenates. TNF-αTG mice exposed to IA exhibited impaired LV contractility and increased LV dilation associated with markedly elevated cardiac expression of atrial natriuretic peptide and brain natriuretic peptide compared with wild-type mice. When wild-type FVB mice were exposed to IH, they exhibited increases in arterial pressure and dP/dt(max), consistent with our previous report in C57BL/6J mice. Surprisingly, we found that TNF-αTG mice exposed to IH showed a reduction in end-diastolic volume (38.7 ± 3.8 to 22.2 ± 2.1 ul; P < 0.01) and an increase in ejection fraction (29.4 ± 2.5 to 41.9 ± 3.1%; P < 0.05). In contrast to our previous study in C56Bl/6J mice, neither FVB nor TNF-αTG mice exhibited an upregulation in β-adrenergic expression or cAMP in response to IH exposure. We conclude that 4 wk of exposure to IH in mice induces adaptive responses that improve cardiac function in not only healthy animals but also in animals with underlying heart failure.     

Phospholipase C gene expression, protein content, and activities in cardiac hypertrophy and heart failure due to volume overload

Volume overload due to arteriovenous (AV) shunt results in cardiac hypertrophy followed by the progression to heart failure. The phosphoinositide phospholipase C (PLC) converts phosphatidylinositol 4,5-bisphosphate (PIP(2)) to 1,2-diacylglycerol (DAG) and inositol (1,4,5)-trisphosphate (IP(3)), which are known to influence cardiac function. Therefore, we examined the time course of changes in DAG and IP(3) as well as PLC isozyme gene expression, protein content, and activities in cardiac hypertrophy and heart failure induced by AV shunt in Sprague-Dawley rats by the needle technique. An increase in the left ventricle (LV)-to-body weight ratio demonstrated that LV hypertrophy was established at 4 wk after the induction of the shunt. PLC-beta(1) activity was increased two- and sevenfold at 3 days and 1 and 2 wk after the induction of volume overload, respectively. These changes were associated with increases in the mRNA and sarcolemmal (SL) protein content; however, no changes in PLC-beta(1) were detected at 4 wk. On the other hand, a significant increase in PLC-gamma(1) activity as well as mRNA and SL protein was seen at 3 days and 4 wk. A progressive decrease in PLC-delta(1) activity with concomitant reductions in the gene expression and SL protein abundance was detected during 1 to 4 wk. Activity of gamma(1)- and delta(1)-isozymes was significantly depressed during the 8- and 16-wk time points, whereas beta(1)-isozyme was increased significantly during these time points. A progressive decrease in the SL PIP(2) content was observed during cardiac hypertrophy and heart failure. Our findings indicate that PLC isozyme signaling processes are increased in hypertrophy and decreased in heart failure due to volume overload.     

Diastolic wall stress and ANG II in cardiac hypertrophy and gene expression induced by volume overload

We investigated the effects of diastolic wall stress (WS) and angiotensin II (ANG II) on the left ventricular (LV) hypertrophy (LVH) induced by volume overload and on the gene expression of LV adrenomedullin (AM) and atrial natriuretic peptide (ANP) in volume overload. Diastolic WS was pharmacologically manipulated with (candesartan) or without (calcium channel blocker manidipine) inhibition of ANG II type 1 receptors in aortocaval-shunted rats over 6 wk. Diastolic WS reached a plateau at 2 wk and subsequently declined regardless of further LVH. Although diastolic WS was decreased to a similar extent by both compounds, candesartan blunted LVH over 6 wk, whereas manidipine blunted LVH at 2 wk but not after 4 wk. Levels of AM and ANP gene expression increased as LVH developed but were completely suppressed by candesartan over 6 wk. ANP expression level was also attenuated by manidipine over 6 wk, whereas AM expression level was suppressed at 2 wk but not after 4 wk by manidipine. We concluded that diastolic WS and ANG II might be potent stimuli for the LVH and LV AM and ANP gene expression in volume overload and that diastolic WS could be relatively involved in the early LVH and in the gene expression of ANP rather than of AM.     

Contribution of ventricular remodeling to pathogenesis of heart failure in rats

We previously reported an approximately 50% incidence of rats with symptoms of congestive heart failure (CHF) at 8 wk postinfrarenal aorto-caval fistula. However, it was not clear whether compensatory ventricular remodeling could continue beyond 8 wk or whether the remaining animals would have developed CHF or died. Therefore, the intent of this study was to complete the characterization of this model of sustained volume overload by determining the morbidity and mortality and the temporal response of left ventricular (LV) remodeling and function beyond 8 wk. The findings demonstrate an upper limit to LV hypertrophy and substantial increases in LV volume and compliance, matrix metalloproteinase activity, and collagen volume fraction associated with the development of CHF. There was an 80% incidence of morbidity and mortality following 21 wk of chronic volume overload. These findings indicate that the development of CHF is triggered by marked ventricular dilatation and increased compliance occurring once the myocardial hypertrophic response is exhausted.     

Gender related alterations of β-adrenoceptor mechanisms in heart failure due to arteriovenous fistula

This study was undertaken to determine gender related changes in different components of β-adrenoceptor (β-AR) system in response to arteriovenous fistula (AV-shunt), which is known to produce heart failure due to volume overload. AV-shunt was induced in male and female rats for 16 weeks by the needle technique; ovariectomized (OVX) rats treated with or without estrogen were also used. Although AV-shunt for 16 weeks produced cardiac hypertrophy in both sexes, male animals showed cardiac dysfunction whereas cardiac performance was maintained in females. Both β(1) -AR and β(2) -AR protein content and mRNA levels were decreased in male and increased in female hearts post-AV-shunt. The basal adenylyl cyclase (AC) activity was lower in the female heart; however, AC protein content and the increase in epinephrine (EPi)-stimulated AC activity were greater in the female AV-shunt group as compared to males. While AC V/VI and β-arrestin 2 mRNA levels were decreased in males, mRNA level for GRK2 was increased in females post-AV-shunt. In contrast to intact females, AV-shunt OVX animals showed depressed cardiac function, decreased β(1) -AR, β(2) -AR, and AC protein content, as well as reduced EPi-stimulated AC activity. Treatment of OVX rats with 17-β estradiol attenuated the AV-shunt induced changes in β-AR and AC protein content as well as cardiac dysfunction. These results reveal that β-AR signal transduction system in response to AV-shunt is downregulated in males and upregulated in females. Furthermore, estrogen appears to play an important role in the upregulation of β-AR mechanisms and the maintenance of cardiac function in AV-shunt females.     

Tumor necrosis factor-α stimulation endothelial-to-mesenchymal transition during cardiac fibrosis via endothelin-1 signaling

Cardiac fibrosis is an important pathological change after myocardial infarction (MI). High concentration of tumor necrosis factor-α (TNF-α) contributes to cardiac fibrosis, and TNF-α has been demonstrated to be involved in transforming growth factor-β1-induced endothelial-to-mesenchymal transition (EndMT). However, the role and molecular mechanisms of TNF-α during cardiac fibrosis remain largely unexplored. In this study, we demonstrated that TNF-α and endothelin-1 (ET-1) were upregulated in cardiac fibrosis after MI, and genes associated with EndMT were also upregulated. An in vitro model of EndMT demonstrated that TNF-α promoted EndMT by upregulation of vimentin and α-smooth muscle actin, and which strongly increased ET-1 expression. ET-1 promoted TNF-α-induced expression of gene program through phosphorylation levels of SMAD family member 2, while subsequent inhibition of ET-1 almost abolished the effect of TNF-α during the process of EndMT. In summary, these findings demonstrated that ET-1 is involved in the EndMT induced by TNF-α during cardiac fibrosis.     

Regression of pressure overload-induced left ventricular hypertrophy in mice

As a prelude to investigating the mechanism of regression of pressure overload-induced left ventricular (LV) hypertrophy (LVH), we studied the time course for the development and subsequent regression of LVH as well as accompanying alterations in cardiac function, histology, and gene expression. Mice were subjected to aortic banding for 4 or 8 wk to establish LVH, and regression was initiated by release of aortic banding for 6 wk. Progressive increase in LV mass and gradual chamber dilatation and dysfunction occurred after aortic banding. LVH was also associated with myocyte enlargement, interstitial fibrosis, and enhanced expression of atrial natriuretic peptide, collagen I, collagen III, and matrix metalloproteinase-2 but suppressed expression of alpha-myosin heavy chain and sarcoplasmic reticulum Ca(2+)-ATPase. Aortic debanding completely or partially reversed LVH, chamber dilatation and dysfunction, myocyte size, interstitial fibrosis, and gene expression pattern, each with a distinct time course. The extent of LVH regression was dependent on the duration of pressure overload, evidenced by the fact that restoration of LV structure and function was complete in animals subjected to 4 wk of aortic banding but incomplete in animals subjected to 8 wk of aortic banding. In conclusion, LVH regression comprises a variety of morphological, functional, and genetic components that show distinct time courses. A longer period of pressure overload is associated with a slower rate of LVH regression.     

Infliximab substantially re-silenced Wnt/β-catenin signaling and ameliorated doxorubicin-induced cardiomyopathy in rats

The release of inflammatory cytokines, namely tumor necrosis factor-α (TNF-α), plays an important role in the pathogenesis of cardiomyopathy. TNF-α increases in plasma and in myocardium of heart failure patients. We aimed to investigate the role of TNF-α inhibitor (infliximab; IFX) in regulating dilated cardiomyopathy (DCM) induced in rats. DCM was induced in rats by doxorubicin (DOX; 3.5 mg. kg⁻¹, i.p) twice weekly for 3 weeks (21 mg. kg⁻¹ cumulative dose). DCM rats were treated with RPL (1 mg. kg⁻¹ orally, daily), IFX (5 mg. kg⁻¹; i.p. once) or their combination for 4 weeks starting next day of last DOX dose. Echocardiography was conducted followed by a collection of blood and left ventricle (LV) for biochemical and histological investigations. DCM rats revealed deteriorated cardiac function (increased CK-MB activity, LVIDs, LVIDd, ESV, and EDV, while decreased EF% and FS%), hypertrophy (increased HW/TL, β-MHC, and α-actin), inflammation (increased IL-1β, IL-6, and TNF-α). The activation of Wnt/β-catenin along with increased gene expression of RAS components (RENIN, ACE, and AT1) were evident. LV architecture also revealed abnormalities and some degree of fibrosis. Treatment with RPL and/or IFX suppressed TNF-α and consequently improved most of these parameters suppressing Wnt/β-catenin/RAS axis. Combined RPL and IFX treatment was the best among all treatments. In conclusion, Wnt/β-catenin/RAS axis is implicated in DOX-induced cardiomyopathy. The upstream TNF-α was proved for the first time in-vivo to stimulate this axis where its inhibition by RPL or IFX prevented DCM. Targeting this axis at two points using RPL and IFX showed better therapeutic efficacy.     

Effects of nonselective endothelin-1 receptor antagonism on cardiac mast cell-mediated ventricular remodeling in rats

The objective of this study was to investigate the effect a nonselective endothelin-1 (ET-1) receptor antagonist (bosentan) had on the acute myocardial remodeling process including left ventricular (LV) mast cells and matrix metalloproteinase (MMP) activity secondary to volume overload. Additionally, we investigated the overall functional outcome of preventative endothelin receptor antagonism during 14 days of chronic volume overload. LV tissue from sham-operated (Sham), untreated-fistula (Fist), and bosentan (100 mg.kg(-1).day(-1))-treated animals (Fist + Bos) was analyzed for mast cell density, MMP activity, and myocardial collagen volume fraction at 1 and 5 days after the creation of an aortocaval fistula. When compared with untreated fistulas, bosentan treatment prevented the marked increase in LV mast cell density at 1 day postfistula (3.1 +/- 0.3 vs. 1.3 +/- 0.3 LV mast cells/mm2, Fist vs. Fist + Bos, P 相似文献   

Extracellular high‐mobility group box 1 mediates pressure overload‐induced cardiac hypertrophy and heart failure

Inflammation plays a key role in pressure overload‐induced cardiac hypertrophy and heart failure, but the mechanisms have not been fully elucidated. High‐mobility group box 1 (HMGB1), which is increased in myocardium under pressure overload, may be involved in pressure overload‐induced cardiac injury. The objectives of this study are to determine the role of HMGB1 in cardiac hypertrophy and cardiac dysfunction under pressure overload. Pressure overload was imposed on the heart of male wild‐type mice by transverse aortic constriction (TAC), while recombinant HMGB1, HMGB1 box A (a competitive antagonist of HMGB1) or PBS was injected into the LV wall. Moreover, cardiac myocytes were cultured and given sustained mechanical stress. Transthoracic echocardiography was performed after the operation and sections for histological analyses were generated from paraffin‐embedded hearts. Relevant proteins and genes were detected. Cardiac HMGB1 expression was increased after TAC, which was accompanied by its translocation from nucleus to both cytoplasm and intercellular space. Exogenous HMGB1 aggravated TAC‐induced cardiac hypertrophy and cardiac dysfunction, as demonstrated by echocardiographic analyses, histological analyses and foetal cardiac genes detection. Nevertheless, the aforementioned pathological change induced by TAC could partially be reversed by HMGB1 inhibition. Consistent with the in vivo observations, mechanical stress evoked the release and synthesis of HMGB1 in cultured cardiac myocytes. This study indicates that the activated and up‐regulated HMGB1 in myocardium, which might partially be derived from cardiac myocytes under pressure overload, may be of crucial importance in pressure overload‐induced cardiac hypertrophy and cardiac dysfunction.     

The adipose triglyceride lipase,adiponectin and visfatin are downregulated by tumor necrosis factor-α (TNF-α) in vivo

Inflammatory cytokines have been linked to obesity-related insulin resistance. To investigate the effect of TNF-α, an inflammatory cytokine, on insulin action, C57BL/6J mice were treated with TNF-α for 7 days after which we examined the in vivo effects of TNF-α on glucose tolerance and insulin sensitivity with IV glucose tolerance tests and hyperinsulinemic-euglycemic clamps. In addition, we analyzed the in vivo effect of TNF-α on several metabolism-related genes and adipocytokines implicated in the development of insulin resistance. TNF-α treatment resulted in markedly increased fasting blood glucose, insulin and free fatty acids (FFA) levels and reduced glucose tolerance. During the clamps, the rates insulin-stimulated whole body (G_Rd) and skeletal muscle glucose uptake (MGU) and insulin’s ability to suppress hepatic glucose production (HGP) were decreased in TNF-α treated animals, indicating insulin resistance. In addition, both PPARγ and ATGL mRNA expression in adipose tissues as well as ATGL protein levels in plasma were downregulated. Moreover, adipose mRNA expression and plasma protein levels of adiponectin and visfatin were significantly down-regulated. We conclude that the alterations of PPARγ, ATGL, adiponectin and visfatin may contribute to the development of insulin resistance mediated by TNF-α.