Decreased Smad 7 expression contributes to cardiac fibrosis in the infarcted rat heart

We examined the role of the transforming growth factor (TGF)-beta(1) signaling inhibitor Smad 7 in cardiac fibrosis. TGF-beta(1) (10 ng/ml) was found to increase cytosolic Smad 7 expression in primary adult rat fibroblasts and induce rapid nuclear export of exogenous Smad 7 in COS-7 cells. Furthermore, overexpression of Smad 7 in primary adult fibroblasts was associated with suppressed collagen type I and III expression. We detected Smad 7, phosphorylated Smad 2, TGF-beta type I receptor (TbetaRI), and TGF-beta(1) proteins in postmyocardial infarct (MI) rat hearts. In 2 and 4 wk post-MI hearts, Smad 7 and TbetaRI expression were decreased in scar tissue, whereas TGF-beta(1) expression was increased in scar and viable tissue. In the 8 wk post-MI heart, Smad 7 expression was decreased in both scar tissue and myocardium remote to the infarct scar. Finally, we confirmed that these changes are paralleled by decreased expression of cytosolic phosphorylated receptor-regulated Smad 2 in 4-wk viable myocardium and in 2- and 4-wk infarct scar tissues. Taken together, our data imply that decreased inhibitory Smad 7 signal in cardiac fibroblasts may play a role in the pathogenesis of cardiac fibrosis in the post-MI heart.     

Cardiotrophin-1: Expression in experimental myocardial infarction and potential role in post-MI wound healing

Cardiotrophin-1 (CT-1), a member of the IL-6 family of cytokines, has been shown to be elevated in the serum of patients with ischemic heart disease and valvular heart disease, and induces cardiomyocyte hypertrophy in vitro. We investigated expression of CT-1 in post-MI rat heart and the effect of CT-1 on cultured primary adult rat cardiac fibroblasts. Elevated CT-1 expression was observed in the infarct zone at 24 h and continued through 2, 4 and 8 weeks post-MI, compared to sham-operated animals. CT-1 induced rapid phosphorylation of Jak1, Jak2, STAT1, STAT3, p42/44 MAPK and Akt in cultured adult cardiac fibroblasts. CT-1 induced cardiac fibroblast protein synthesis and proliferation. Protein and DNA synthesis were dependent on activation of Jak/STAT, MEK1/2, PI3K and Src pathways as evidenced by decreased ³H-leucine and ³H-thymidine incorporation after pretreatment with AG490, PD98059, LY294002 and genistein respectively. Furthermore, CT-1 treatment increased procollagen-1-carboxypropeptide (P1CP) synthesis, a marker of mature collagen synthesis. CT-1 induced cell migration of rat cardiac fibroblasts. Our results suggest that CT-1, as expressed in post-MI heart, may play an important role in infarct scar formation and ongoing remodeling of the scar. CT-1 was able to initiate each of the processes considered important in the formation of infarct scar including cardiac fibroblast migration as well as fibroblast proliferation and collagen synthesis. Further work is required to determine factors that induce CT-1 expression and interplay with other mediators of cardiac infarct wound healing in the setting of acute cardiac ischemia and chronic post-MI heart failure.     

Inhibition of Fas-associated apoptosis in granulation tissue cells accompanies attenuation of postinfarction left ventricular remodeling by olmesartan

Blockade of angiotensin II type 1 receptor (AT1) signaling attenuates heart failure following myocardial infarction (MI), perhaps through reduction of fibrosis in the noninfarcted myocardium. However, its specific effect on the infarct tissue itself has not been fully clarified, which we examined in the present study. After MI induction in mice, treatment with the AT1 blocker olmesartan, beginning on the 3rd day post-MI, significantly improved survival (94%) 4 wk post-MI, compared with saline (53%) and hydralazine (73%). Olmesartan-treated mice also showed significant attenuation of left ventricular dilatation and dysfunction, as well as significantly greater infarct wall thickness, although the absolute size of the infarct scar was unchanged. In addition, significantly greater numbers of nonmyocytes (mainly vascular cells and myofibroblasts) were present within the infarct scar in olmesartan-treated hearts. Ten days post-MI, apoptosis among granulation tissue cells was significantly suppressed in the olmesartan-treated hearts, where expression of Fas, Bax, procaspase-3, and Daxx and activation of caspase-3, c-Jun NH(2)-terminal kinase, and c-Jun were all significantly attenuated. By contrast, expression of Fas ligand, Bcl-2, and Fas-associated death domain and activation of caspase-8 were unaffected, suggesting olmesartan exerts a negative regulatory effect on the alternate pathway downstream of Fas receptor. In vitro, olmesartan dose-dependently inhibited Fas-mediated apoptosis in granulation tissue-derived myofibroblasts. The present study proposes this antiapoptotic effect as another important mechanism for an AT1 blocker in improving post-MI ventricular remodeling, as well as its antifibrotic effect, and also suggests a significant link between renin-angiotensin and Fas/Fas ligand systems in postinfarction hearts.     

Angiotensin II type 1 receptor blocker preserves tolerance to ischemia-reperfusion injury in Dahl salt-sensitive rat heart

Oxidative stress is involved in the tolerance to ischemia-reperfusion (I/R) injury. Because angiotensin II type 1 receptor blockers (ARBs) inhibit oxidative stress, there is concern that ARBs abolish the tolerance to I/R injury. Dahl salt-sensitive (DS) hypertensive and salt-resistant (DR) normotensive rats received an antioxidant, 2-mercaptopropionylglycine (MPG), or an ARB, losartan, for 7 days. Losartan and MPG significantly inhibited oxidative stress as determined by tissue malondialdehyde + 4-hydroxynoneal and increased expression of inducible nitric oxide synthase (iNOS) in the DS rat heart. However, losartan but not MPG activated endothelial nitric oxide synthase (eNOS) as assessed by phosphorylation of eNOS on Ser1177. Infarct size after 30-min left coronary artery occlusion followed by 2-h reperfusion was comparable between DS and DR rat hearts. Although MPG and losartan had no effect on infarct size in the DR rat heart, MPG but not losartan significantly increased infarct size in the DS rat heart. A selective iNOS inhibitor, 1400W, increased infarct size in the DS rat heart, but it had no effect on infarct size in the losartan-treated DS rat heart. However, a nonselective NOS inhibitor, Nomega-nitro-l-arginine methyl ester, increased infarct size in the losartan-treated DS rat heart. These results suggest that losartan preserves the tolerance to I/R injury by activating eNOS despite elimination of redox-sensitive upregulation of iNOS and iNOS-dependent cardioprotection in the DS rat heart.     

Angiotensin II upregulates Kv1.5 expression through ROS-dependent transforming growth factor-beta1 and extracellular signal-regulated kinase 1/2 signalings in neonatal rat atrial myocytes

Kv1.5 potassium channel represents a promising target for atrial fibrillation (AF) therapy. During AF, the renin–angiotensin system is markedly activated. Recent evidence indicates that angiotensin II (Ang II) can upregulate Kv1.5 channel, but the mechanism remains unknown. In this study, we report that Ang II-mediated transforming growth factor-beta1 (TGF-β₁)/Smad2/3 and extracellular signal-regulated kinase (ERK) 1/2 signalings are involved in atrial Kv1.5 expression. In neonatal rat atrial myocytes, quantitative PCR and Western blotting revealed that Ang II upregulated TGF-β₁, synapse-associated protein 97 (SAP97) and Kv1.5 expression in a time- and concentration-dependent manner. The Ang II-induced upregulation of Kv1.5, SAP97 and phosphorylated Smad2/3 (P-Smad2/3) were reversed by the Ang II type 1 (AT₁) receptor antagonist losartan, an anti-TGF-β₁ antibody and the ERK 1/2 inhibitor PD98059 but not by the AT₂ receptor antagonist PD123319. mRNA knockdown of either Smad2 or Smad3 blocked Ang II-induced expression of Kv1.5 and SAP97. These data suggest that AT₁ receptor/TGF-β₁/P-Smad2/3 and ERK 1/2 signalings are involved in Ang II-induced Kv1.5 and SAP97 expression. Flow cytometry and Western blotting revealed that losartan and the anti-TGF-β₁ antibody diminished Ang II-induced reactive oxygen species (ROS) generation and that the antioxidants diphenyleneiodonium and N-acetyl cysteine inhibited Ang II-induced expression of P-Smad2/3, phosphorylated ERK (P-ERK) 1/2, Kv1.5, SAP97, suggesting that ROS participate in Kv1.5 and SAP97 regulation by modulating Ang II-induced P-Smad2/3 and P-ERK 1/2 expression. In conclusion, we demonstrate that ROS-dependent Ang II/AT₁ receptor/TGF-β₁/P-Smad2/3 and Ang II/ERK 1/2 signalings are involved in atrial Kv1.5 and SAP97 expression. Antioxidants would be beneficial for AF treatment through inhibiting atrial Kv1.5 expression.     

ANG II type 1A receptor signaling causes unfavorable scar dynamics in the postinfarct heart

Blockade of ANG II type 1A receptor (AT(1A)) is known to attenuate postinfarction [postmyocardial infarction (post-MI)] heart failure, accompanying reduction in fibrosis of the noninfarcted area. In the present study, we investigated the influence of AT(1A) blockade on the infarcted tissue itself. Consistent with earlier reports, AT(1A) knockout (AT(1A)KO) mice showed significantly attenuated left ventricular (LV) remodeling (dilatation) and dysfunction compared with wild-type (WT) mice. Morphometry revealed that the infarcted wall was thicker and had a smaller circumferential length in AT(1A)KO than WT hearts. In addition, significantly greater numbers of cells were present within infarcts in AT(1A)KO hearts 4 wk post-MI; most notably, there was an abundance of vessels and myofibroblasts. One week post-MI, the incidence of apoptosis among granulation tissue cells was fewer (3.3 +/- 0.4 vs. 4.4 +/- 0.5% in WT, P < 0.05), whereas vessel proliferation was higher in AT(1A)KO hearts, which likely explains the later abundance of cells within the scar tissue. Insulin-like growth factor receptor-I was upregulated and its downstream signal protein kinase B (Akt) was significantly activated in infarcted AT(1A)KO hearts compared with WT hearts. Inactivation of Akt with wortmannin partially but significantly prevented the benefits observed in AT(1A)KO. Collectively, in AT(1A)KO hearts, Akt-mediated granulation tissue cell proliferation and preservation resulting from antiapoptosis likely contributed to an abundant cell population that altered the infarct scar structure, thereby reducing wall stress and attenuating LV dilatation and dysfunction at the chronic stage. In conclusion, altered structural dynamics of infarct scar and increasing myocardial fibrosis may be responsible for the deleterious effects of AT(1A) signaling following MI.     

Differential gene expression in infarct scar and viable myocardium from rat heart following coronary ligation

Post-myocardial infarction (MI) remodeling of cardiac myocytes and the myocardial interstitium results in alteration of gross ventricular geometry and ventricular dysfunction. To investigate the mechanisms of the remodeling process of the heart after large MI, the expression of various genes in viable left ventricle and infarct scar tissue were examined at 16 weeks post-MI. Steady-state expression of Na⁺-K⁺ATPase α-1 and −2, phospholamban (PLB), α-myosin heavy chain (α-MHC), ryanodine receptor (Rya) and Ca²⁺ ATPase (Serca2) mRNAs were decreased in the infarct scar vs noninfarcted sham-operated controls (P < 0.05). On the other hand, Giα2 and β-MHC mRNAs were upregulated (P < 0.05, respectively) in the infarct scar whereas Na⁺-K⁺ ATPase-β, Na⁺-Ca²⁺ exchanger and Gs mRNAs were not altered vs control values. In viable left ventricle, the a-1 subunit of Na⁺-K⁺ATPase, α-3, β-isoforms, Rya, β-MHC, Giα2, Gs and Na⁺-Ca²⁺ exchanger were significantly elevated while expression of the a-2 subunit of Na⁺-K⁺ ATPase, PLB and Serca2 were significantly decreased compared to controls. Expression of CK2α mRNA was elevated in noninfarcted heart (145 ± 15%) and diminished in the infarct scar (66 ± 13%) vs controls. Expression of β-MHC mRNA was elevated in both viable and infarct scar tissues of experimental hearts (140 ± 31% and 183 ± 30% vs. controls, respectively). These results suggest that cardiac genes in the infarcted tissue and viable left ventricle following MI are differentially regulated.     

The scar neovasculature after myocardial infarction in rats

A series of novel techniques, adapted from the field of tumor biology, were developed to quantify vascular structure and function and to explore the role of ANG II receptor AT1 in cardiac remodeling after myocardial infarction (MI). We examined the scar neovasculature at 1-4 wk post-MI in Sprague-Dawley rats with a view toward its ability to deliver and exchange oxygen. CD31 and DiOC7(3) staining was used to visualize anatomical vessels vs. those perfused. EF5/Cy3 immunohistochemical staining was used to quantify tissue hypoxia. We compared untreated controls with rats treated with losartan, an AT1 receptor antagonist. Our findings indicated that, at the infarct site, there was not only a 42-75% (1-4 wk post-MI) decrease in the number of anatomical vessels compared with controls but also a decrease in the fraction of perfused vessels from 70% in normal coronary vasculature to 48% at the infarct site. These changes were accompanied by progressive increases in diffusion distance and tissue hypoxia (100% increase in EF5/Cy3 staining at 4 wk post-MI). Losartan-treated rats exhibited a significantly less marked reduction in vascular perfusion and a significantly lesser extent of tissue hypoxia. Over the course of 4 wk post-MI, there is a reduction in coronary vasculature at the infarct site, the extent of which is attenuated by losartan. These findings implicate AT1 receptor upregulation, and perhaps angiotensin-related peptides, as being antiangiogenic.     

Dexamethasone treatment of post-MI rats attenuates sympathetic innervation of the infarct region.

Sympathetic fiber innervation of the damaged region following injury represents a conserved event of wound healing. The present study tested the hypothesis that impaired scar healing in post-myocardial infarction (post-MI) rats was associated with a reduction of sympathetic fibers innervating the infarct region. In 1-wk post-MI rats, neurofilament-M-immunoreactive fibers (1,116 +/- 250 microm(2)/mm(2)) were detected innervating the infarct region and observed in close proximity to a modest number of endothelial nitric oxide synthase-immunoreactive scar-residing vessels. Dexamethasone (Dex) treatment (6 days) of post-MI rats led to a significant reduction of scar weight (Dex + MI 38 +/- 4 mg vs. MI 63 +/- 2 mg) and a disproportionate nonsignificant decrease of scar surface area (Dex + MI 0.54 +/- 0.06 cm(2) vs. MI 0.68 +/- 0.06 cm(2)). In Dex-treated post-MI rats, the density of neurofilament-M-immunoreactive fibers (125 +/- 47 microm(2)/mm(2)) innervating the infarct region was significantly reduced and associated with a decreased expression of nerve growth factor (NGF) mRNA (Dex + MI 0.80 +/- 0.07 vs. MI 1.11 +/- 0.08; P < 0.05 vs. MI). Previous studies have demonstrated that scar myofibroblasts synthesize NGF and may represent a cellular target of Dex. The exposure of 1st passage scar myofibroblasts to Dex led to a dose-dependent suppression of [(3)H]thymidine uptake and a concomitant attenuation of NGF mRNA expression (untreated 3.47 +/- 0.35 vs. Dex treated 2.28 +/- 0.40; P < 0.05 vs. untreated). Thus the present study has demonstrated that impaired scar healing in Dex-treated post-MI rats was associated with a reduction of neurofilament-M-immunoreactive fibers innervating the infarct region. The attenuation of scar myofibroblast proliferation and NGF mRNA expression may represent underlying mechanisms contributing to the diminished neural response in the infarct region of Dex-treated post-MI rats.     

Upregulation of beta-adrenergic receptors in heart failure due to volume overload

To examine the mechanisms of changes in beta-adrenergic signal transduction in heart failing due to volume overload, we studied the status of beta-adrenoceptors (beta-ARs), G protein-coupled receptor kinase (GRK), and beta-arrestin in heart failure due to aortocaval shunt (AVS). Heart failure in rats was induced by creating AVS for 16 wk, and beta-AR binding, GRK activity, as well as their protein content, and mRNA levels were determined in both left and right ventricles. The density and protein content for beta1-ARs, unlike those for beta2-ARs, were increased in the failing hearts. Furthermore, protein contents for GRK isoforms and beta-arrestin-1 were decreased in membranous fractions and increased in cytosolic fractions from the failing hearts. On the other hand, steady-state mRNA levels for beta1-ARs and GRK2, as well as protein content for Gbetagamma-subunits, did not change in the failing heart. Basal cardiac function was depressed; however, both in vivo and ex vivo positive inotropic responses of the failing hearts to isoproterenol were augmented. Treatment of AVS animals with imidapril (1 mg.kg(-1).day(-1)) or losartan (20 mg.kg(-1).day(-1)) retarded the progression of heart failure; partially prevented changes in beta1-ARs, GRKs, and beta-arrestin-1 in the failing myocardium; and attenuated the increase in positive inotropic effect of isoproterenol. These results indicate that upregulation of beta1-ARs is associated with subcellular redistribution of GRKs and beta-arrestin-1 in the failing heart due to volume overload. Furthermore, attenuation of alterations in beta-adrenergic system by imidapril or losartan may be due to blockade of the renin-angiotensin system in the AVS model of heart failure.     

Effects of losartan on angiotensin receptors in the hypertrophic rat heart

The effects of losartan on angiotensin receptors in hypertrophic rat hearts were studied. The study was prompted by inconsistent findings of either an increase or decrease in the mRNA of the AT1 receptor in the hearts of cardiac hypertrophic rats treated with losartan, and a paucity of information on the effects of losartan on functional angiotensin receptors in the heart. Losartan, administered i.p. to aortic coarcted rats, dose-dependently attenuated the cardiac hypertrophy. Significant effect was observed with a dose of 2.72 micromol/kg/day for four days. Hypertrophy was accompanied by an increase in [125I]-Sar1-Ile8-angiotensin II binding sites (due mainly to an increase in AT2 binding) and AT2 receptor protein in cardiac ventricles of aortic coarcted rats. Treatment with effective anti-hypertrophic doses of losartan dose-dependently downregulated the [125I]-Sar1-Ile8-angiotensin II binding sites, constitutive AT1 receptor protein, and the over expressed AT2 receptor protein. It was suggested that the anti-cardiac hypertrophic action of losartan resulted from its ability to suppress the expression of both the basal and enhanced cardiac angiotensin receptors. This raises the question as to whether such drastic action could form the therapeutic basis for the use of losartan in cardiac pathologies.     

The role of Smad2 and Smad3 in regulating homeostatic functions of fibroblasts in vitro and in adult mice

The heart contains an abundant fibroblast population that may play a role in homeostasis, by maintaining the extracellular matrix (ECM) network, by regulating electrical impulse conduction, and by supporting survival and function of cardiomyocytes and vascular cells. Despite an explosion in our understanding of the role of fibroblasts in cardiac injury, the homeostatic functions of resident fibroblasts in adult hearts remain understudied. TGF-β-mediated signaling through the receptor-activated Smads, Smad2 and Smad3 critically regulates fibroblast function. We hypothesized that baseline expression of Smad2/3 in fibroblasts may play an important role in cardiac homeostasis. Smad2 and Smad3 were constitutively expressed in normal mouse hearts and in cardiac fibroblasts. In cultured cardiac fibroblasts, Smad2 and Smad3 played distinct roles in regulation of baseline ECM gene synthesis. Smad3 knockdown attenuated collagen I, collagen IV and fibronectin mRNA synthesis and reduced expression of the matricellular protein thrombospondin-1. Smad2 knockdown on the other hand attenuated expression of collagen V mRNA and reduced synthesis of fibronectin, periostin and versican. In vivo, inducible fibroblast-specific Smad2 knockout mice and fibroblast-specific Smad3 knockout mice had normal heart rate, preserved cardiac geometry, ventricular systolic and diastolic function, and normal myocardial structure. Fibroblast-specific Smad3, but not Smad2 loss modestly but significantly reduced collagen content. Our findings suggest that fibroblast-specific Smad3, but not Smad2, may play a role in regulation of baseline collagen synthesis in adult hearts. However, at least short term, these changes do not have any impact on homeostatic cardiac function.     

Modification of myosin protein and gene expression in failing hearts due to myocardial infarction by enalapril or losartan

The effects of enalapril, an angiotensin converting enzyme (ACE) inhibitor, and losartan, an angiotensin II receptor type I antagonist, were investigated on alterations in myofibrillar ATPase activity as well as myosin heavy chain (MHC) content and gene expression in failing hearts following myocardial infarction (MI). Three weeks after ligation of the left coronary artery, rats were treated with or without enalapril (10 mg/kg/day), and/or losartan (20 mg/kg/day) for 5 weeks. The infarcted animals exhibited an increase in left ventricle (LV) end diastolic pressure and depressed rates of LV pressure development as well as pressure decay. LV myofibrillar Ca2+ -stimulated ATPase activity was decreased in the infarcted hearts compared with controls, MHC alpha-isoform content was significantly decreased whereas that of MHC beta-isoform was markedly increased. The level of MHC alpha-isoform mRNA was decreased whereas that of MHC beta-isoform was increased in the viable infarcted LV. Treatment of animal with enalapril, losartan, or combination of enalapril and losartan partially prevented the MI induced changes in LV function, myofibrillar Ca2+ -stimulated ATPase activity, MHC protein expression and MHC gene expression. The results suggest that the beneficial effects of the renin-angiotensin system blockade in heart failure are associated with partial prevention of myofibrillar remodeling.     

Effects of captopril and angiotensin II receptor blockers (AT1, AT2) on myocardial ischemia-reperfusion induced infarct size

The renin-angiotensin system (RAS) plays a major role in regulating the cardiovascular system, and disorders of the RAS contribute largely to the cardiac pathophysiology, including myocardial ischemia-reperfusion (MI/R) injury. Two subtypes of angiotensin II (Ang II) receptors have been defined on the basis of their differential pharmacological properties. The current study was undertaken to address the question as to whether the inhibition of the angiotensin converting enzyme (ACE) by captopril and the AT1 and AT2 receptor blockers losartan and PD123319 modulate MI/R-induced infarct size in an in vivo rat model. To produce necrosis, a branch of the descending left coronary artery was occluded for 30 min followed by two hours of reperfusion. ECG changes, blood pressure, and heart rate were measured during the experiment. Captopril (3 mg/kg), losartan (2 mg/kg), and PD123319 (20 μg/kg/min) were given in an IV 10 min before ischemia and were continued during the ischemic period. The infarcted area was measured by TTC staining. The volume of infarct and the risk zone was determined by planimetry. Compared to the control group (55.62±4.00%) both captopril and losartan significantly reduced the myocardial infarct size (30.50±3.26% and 37.75±4.44%), whereas neither PD123319 nor PD123319+losartan affected the infarct size volume (46.50±3.72% and 54.62±2.43%). Our data indicates that captopril and losartan exert cardioprotective activity after an MI/R injury. Also, infarct size reduction by losartan was halted by a blockade of the AT2 receptor. Therefore, the activation of AT2 receptors may be potentially protective and appear to oppose the effects mediated by the AT1 receptors.     

Bradykinin metabolism in rat hearts with left-ventricular hypertrophy following myocardial infarction

The aim of the present study was to assess the contribution of angiotensin I converting enzyme (ACE)and neutral endopeptidase (NEP) in the coronary degradation of bradykinin (BK) after left-ventricular hypertrophy following myocardial infarction (MI) in rats. Myocardial infarction was induced by left descendant coronary artery ligation, and the contribution of ACE and NEP in the degradation of exogenous BK after a single passage through the coronary bed was assessed at 2, 5, and 36 days post-MI. BK degradation rate (V(max)/Km) was found to be significantly lower in hearts at 36 days (3.30 +/- 0.28 min(-1)) compared with 2 days (4.39 +/- 0.32 min(-1)) for noninfarcted hearts, but this reduction was just above the statistical level of significance for post-MI hearts. In infarcted hearts, V(max)/Km was increased significantly 5 days post-MI (4.91 +/- 0.28 min(-1)) compared with the 2 and 36 day-groups (3.43 +/- 0.20 and 2.78 +/- 0.16 min(-1), respectively). The difference between noninfarcted and MI was significant only 2 days post-MI. Treatment with the vasopeptidase inhibitor, omapatrilat, showed that the relative contribution of ACE and NEP combined increased over time in infarcted hearts and became significantly higher 36 versus 2 days post-MI. Finally, the treatment with an ACE inhibitor (enalaprilat) and a NEP inhibitor (retrothiorphan) in the 36-day infarcted and noninfarcted hearts showed that the relative contribution of ACE in infarcted hearts was comparable with that of noninfarcted hearts, whereas the relative contribution of NEP was increased significantly in infarcted hearts. In conclusion, experimental MI in rats induces complex changes in the metabolism of exogenous BK. The changes resulted in an increased relative contribution of NEP 36 days after infarction.     

Angiotensin II type 1 receptor regulation and differential trophic effects on rat cardiac myofibroblasts after acute myocardial infarction

Fibroblast growth in the scar and surviving tissue is a key element of the remodeling post myocardial infarction. The regulation of fibroblast growth after acute myocardial infarction remains to be determined. Recently, Angiotensin II has been demonstrated to be a mitogen for neonatal cardiac fibroblasts. In this study adult rat cardiac fibroblasts were isolated from different regions of the infarcted rat heart and Angiotensin II effects examined. Adult Wistar-rats were sham operated or left coronary artery ligated. After 4 days, hearts were removed and fibroblasts from sham operated, infarct- and non-infarct regions of the left ventricle isolated. Radioligand binding studies were performed and cell number, cell area, total protein, and AT(1) receptor mRNA after stimulation determined. Radioligand binding studies demonstrated that myofibroblasts expressed a single class of high affinity Angiotensin II AT(1) receptors. Myofibroblasts from the infarct area revealed a lower maximal binding capacity, compared to sham operated myocardium. Conversely, myofibroblasts from the non-infarct area had a higher expression of Angiotensin II AT(1) receptor mRNA compared to sham operated myofibroblasts. Angiotensin II (1 microM, 48 h) increased cell-number in sham operated and non-infarct, but not in infarct myofibroblasts. Angiotensin II elevated total protein in sham operated, non-infarct, and infarct myofibroblasts. In addition, Angiotensin II increased cell area in sham operated and infarct myofibroblasts. These data demonstrate that Angiotensin II acted as a mitogen in sham operated and non-infarct myofibroblasts and stimulated hypertrophy in infarct myofibroblasts. These regional different effects of Angiotensin II might participate in the remodeling post myocardial infarction.     

Reversal of subcellular remodelling by losartan in heart failure due to myocardial infarction

This study tested the reversal of subcellular remodelling in heart failure due to myocardial infarction (MI) upon treatment with losartan, an angiotensin II receptor antagonist. Twelve weeks after inducing MI, rats were treated with or without losartan (20 mg/kg; daily) for 8 weeks and assessed for cardiac function, cardiac remodelling, subcellular alterations and plasma catecholamines. Cardiac hypertrophy and lung congestion in 20 weeks MI‐induced heart failure were associated with increases in plasma catecholamine levels. Haemodynamic examination revealed depressed cardiac function, whereas echocardiographic analysis showed impaired cardiac performance and marked increases in left ventricle wall thickness and chamber dilatation at 20 weeks of inducing MI. These changes in cardiac function, cardiac remodelling and plasma dopamine levels in heart failure were partially or fully reversed by losartan. Sarcoplasmic reticular (SR) Ca²⁺‐pump activity and protein expression, protein and gene expression for phospholamban, as well as myofibrillar (MF) Ca²⁺‐stimulated ATPase activity and α‐myosin heavy chain mRNA levels were depressed, whereas β‐myosin heavy chain expression was increased in failing hearts; these alterations were partially reversed by losartan. Although SR Ca²⁺‐release activity and mRNA levels for SR Ca²⁺‐pump were decreased in failing heart, these changes were not reversed upon losartan treatment; no changes in mRNA levels for SR Ca²⁺‐release channels were observed in untreated or treated heart failure. These results suggest that the partial improvement of cardiac performance in heart failure due to MI by losartan treatment is associated with partial reversal of cardiac remodelling as well as partial recovery of SR and MF functions.     

Pharmacological concentration of angiotensin-(1-7) activates NADPH oxidase after ischemia-reperfusion in rat heart through AT1 receptor stimulation

The cardiovascular role of angiotensin-(1-7), especially in the functional and metabolic alterations associated with ischemia-reperfusion (IR), is still not clearly defined. Our objective was to evaluate the cardiac effects of angiotensin-(1-7), the receptors involved, and their relationships with NADPH oxidase activation under non-ischemic conditions and, during an ischemia-reperfusion sequence. Isolated perfused rat hearts underwent 45 min of non-ischemic perfusion, or 30 min of global ischemia followed by 30 min of reperfusion. Angiotensin-(1-7) and/or AT1 receptor blocker losartan or angiotensin-(1-7) receptor antagonist (D-Ala7)-angiotensin-(1-7) were perfused. Our results showed that angiotensin-(1-7) was without effect at low concentrations (10(-10) to 10(-7) M). At a pharmacological concentration, 0.5 microM angiotensin-(1-7) induced vasoconstriction, which was antagonised by losartan. After ischemia, we noted a partial recovery of functional parameters, which was not modified by any of the treatments. The expression of AT1 receptor mRNA was increased by ischemia-reperfusion, except in (D-Ala7)-angiotensin-(1-7) treated hearts. Angiotensin-(1-7) further increased the AT1 expression. NADPH oxidase activity was enhanced in 0.5 microM angiotensin-(1-7)-treated hearts subjected to ischemia-reperfusion, this effect was totally reversed by losartan. This is the first time that it has been shown that, in the heart, angiotensin-(1-7) at pharmacological concentration activates NADPH oxidase, an enzyme thought to be involved in several angiotensin II effects.     

Long-term effect of low energy laser irradiation on infarction and reperfusion injury in the rat heart.

Low-energy laser irradiation (LELI) has been found to modulate biological processes. The present study investigated the effect of LELI on infarct size after chronic myocardial infarction (MI) and ischemia-reperfusion injury in rats. The left anterior descending (LAD) coronary artery was ligated in 83 rats to create MI or ischemia-reperfusion injury. The hearts of the laser-irradiated (LI) rats received irradiation after LAD coronary artery occlusion and 3 days post-MI. At 14, 21, and 45 days post-LAD coronary artery permanent occlusion, infarct sizes (percentage of left ventricular volume) in the non-laser-irradiated (NLI) rats were 52 +/- 12 (SD), 47 +/- 11, and 34 +/- 7%, respectively, whereas in the LI rats they were significantly lower, being 20 +/- 8, 15 +/- 6, and 10 +/- 4%, respectively. Left ventricular dilatation (LVD) in the chronic infarcted rats was significantly reduced (50-60%) in LI compared with NLI rats. LVD in the ischemia-reperfusion-injured LI rats was significantly reduced to a value that did not differ from intact normal noninfarcted rats. Laser irradiation caused a significant 2.2-fold elevation in the content of inducible heat shock proteins (specifically HSP70i) and 3.1-fold elevation in newly formed blood vessels in the heart compared with NLI rats. It is concluded that LELI caused a profound reduction in infarct size and LVD in the rat heart after chronic MI and caused complete reduction of LVD in ischemic-reperfused heart. This phenomenon may be partially explained by the cardioprotective effect of the HSP70i and enhanced angiogenesis in the myocardium after laser irradiation.     

Metabolism of angiotensin I in the coronary circulation of normal and diabetic rats

Formation of metabolites from angiotensin I that passed the coronary vessels in the isolated working rat hearts of normal and streptozotocin-induced diabetes was evaluated. HPLC analysis showed that the levels of angiotensin II and angiotensin 1-7 were unaltered in the diabetic hearts, but the perfusates of the diabetic hearts contained smaller amounts of angiotensin 1-9. It is not clear why the perfusates of diabetic hearts contain less amount of angiotensin 1-9. It is possible that the peptide is metabolized faster or greater internalization takes place in the diabetic heart. The amount of angiotensin II in the perfusates of normal hearts was 5.8 times greater at the perfusion rate of 2 than at 10 ml/min/g wet heart weight. At such conditions, the amount of angiotensin 1-9 and angiotensin 1-7 in the perfusates were increased 2.4 and 1.5 times, respectively. A higher amount of angiotensin II during myocardial hypoperfusion may lead to constriction of the coronary vessels. As a result, myocardial damage may be facilitated.