Jiang Xu

Ac-SDKP Reverses Inflammation and Fibrosis in Rats With Heart Failure After Myocardial Infarction

Abstract—Inflammation may play an important role in the pathogenesis of cardiac fibrosis in heart failure (HF) after myocardial infarction (MI). N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a naturally occurring antifibrotic peptide whose plasma concentration is increased 4- to 5-fold by angiotensin-converting enzyme inhibitors. We tested the hypothesis that in rats with HF after MI, Ac-SDKP acts as an anti-inflammatory cytokine, preventing and also reversing cardiac fibrosis in the noninfarcted area (reactive fibrosis), and thus affording functional improvement. We found that Ac-SDKP significantly decreased total collagen content in the prevention group from 23.7±0.9 to 15.0±0.7 &mgr;g/mg and in the reversal group from 22.6±2.2 to 14.4±1.6 (P <0.01). Interstitial collagen volume fraction and perivascular collagen were likewise significantly reduced. We also found that infiltrating macrophages were reduced from 264.7±8.1 to 170.2±9.2/mm2, P <0.001 (prevention), and from 257.5±9.1 to 153.1±8.5 mm2, P <0.001 (reversal), while transforming growth factor (TGF)-&bgr;-positive cells were decreased from 195.6±8.4 to 129.6±5.7/mm2, P <0.01 (prevention), and from 195.6±8.4 to 130.7±10.8/mm2, P <0.01 (reversal). Ac-SDKP did not alter either blood pressure or left ventricular hypertrophy (LVH); however, it depressed systolic cardiac function in the prevention study while having no significant effect in the reversal group. We concluded that Ac-SDKP has an anti-inflammatory effect in HF that may contribute to its antifibrotic effect; however, this decrease in fibrosis without changes in LVH was not accompanied by an improvement in cardiac function.

Role of AT2 Receptors in the Cardioprotective Effect of AT1 Antagonists in Mice

Angiotensin II (Ang II) acts mainly on two receptor subtypes: AT1 and AT2. Most of the known biological actions of Ang II are mediated by AT1 receptors; however, the role of AT2 receptors remains unclear. We tested the hypothesis that the cardioprotective effects of AT1 receptor antagonists (AT1-ant) after myocardial infarction (MI) are partially mediated by activation of AT2 receptors; thus in AT2 receptor gene knockout mice (AT2−/Y), the effect of AT1-ant will be diminished or absent. MI was induced by ligating the left anterior descending coronary artery. Four weeks later, AT2−/Y and their wild-type littermates (AT2+/Y) were started on vehicle, AT1-ant (valsartan, 50 mg/kg per day), or ACE inhibitor (enalapril, 20 mg/kg per day) for 20 weeks. Basal blood pressure and cardiac function as well as remodeling after MI did not differ between AT2+/Y and AT2−/Y. AT1-ant increased ejection fraction and cardiac output and decreased left ventricular diastolic dimension, myocyte cross-sectional area, and interstitial collagen deposition in AT2+/Y, and these effects were significantly diminished in AT2−/Y. ACE inhibitors improved cardiac function and remodeling similarly in both strains. We concluded that (1) activation of AT2 during AT1 blockade plays an important role in the therapeutic effect of AT1-ant and (2) the AT2 receptor may not play an important role in regulation of cardiac function, either under basal conditions after MI remodeling or in the therapeutic effect of ACE inhibitors.

Role of the B1 Kinin Receptor in the Regulation of Cardiac Function and Remodeling After Myocardial Infarction

Kinins exert cardioprotective effects via 2 G-protein-coupled receptors, B1 and B2. Using B1 kinin receptor gene knockout mice (B1−/−), we tested the hypotheses that the B1 receptor plays an important role in preservation of cardiac function, whereas lack of B1 may accelerate cardiac remodeling and dysfunction after myocardial infarction, and that B2 receptors may compensate for lack of B1, whereas blockade of B2 receptors in B1−/− mice may cause further deterioration of cardiac function and remodeling. Female B1−/− mice and wild-type controls (C57BL/6J, B1+/+) underwent sham surgery or myocardial infarction and were treated with either vehicle or B2-antagonist (icatibant, 500 &mgr;g/kg per day, subcutaneous) for 8 weeks. We found that in sham myocardial infarction, B1−/− mice had a larger left ventricular diastolic chamber dimension both initially and at 4 to 8 weeks compared with B1+/+. Left ventricular mass and myocyte size were also larger in B1−/− with sham operation than in B1+/+, although cardiac function did not differ between strains. After myocardial infarction, cardiac remodeling and function were similar in both strains, although B1−/− mice tended to have lower blood pressure. Blockade of B2 receptors tended to worsen cardiac remodeling and dysfunction in B1−/− but not in B1+/+. These results may suggest that B2 receptors play an important role in compensating for lack of B1 receptors in mice with myocardial infarction. Dual blockade of both B1 and B2 eliminates this compensation, leading to further deterioration of cardiac dysfunction and remodeling after myocardial infarction.

Local angiotensin II aggravates cardiac remodeling in hypertension

Angiotensin II (ANG II) contributes to hypertension, cardiac hypertrophy, fibrosis, and dysfunction; however, it is difficult to separate the cardiac effect of ANG II from its hemodynamic action in vivo. To overcome the limitations, we used transgenic mice with cardiac-specific expression of a transgene fusion protein that releases ANG II from cardiomyocytes (Tg-ANG II) and treated them with deoxycorticosterone acetate (DOCA)-salt to suppress their systemic renin-angiotensin system. Using this unique model, we tested the hypothesis that cardiac ANG II, acting on the angiotensin type 1 receptor (AT(1)R), increases inflammation, oxidative stress, and apoptosis, accelerating cardiac hypertrophy and fibrosis. Male Tg-ANG II mice and their nontransgenic littermates (n-Tg) were uninephrectomized and divided into the following three groups: 1) vehicle-treated normotensive controls; 2) DOCA-salt; and 3) DOCA-salt + valsartan (AT(1)R blocker).Under basal conditions, systolic blood pressure (SBP) and cardiac phenotypes were similar between strains. In DOCA-salt hypertension, SBP increased similarly in both n-Tg and Tg-ANG II, and cardiac function did not differ between strains; however, Tg-ANG II had 1) greater ventricular hypertrophy as well as interstitial and perivascular fibrosis; 2) a higher number of deoxynucleotidyl-transferase-mediated dUTP nick end labeling-positive cells and infiltrating macrophages; 3) increased protein expression of NADPH oxidase 2 and transforming growth factor-β(1); and 4) downregulation of phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase B (Akt) phosphorylation. Valsartan partially reversed these effects in Tg-ANG II but not in n-Tg. We conclude that, when hemodynamic loading conditions remain unchanged, cardiac ANG II does not alter heart size or cardiac functions. However, in animals with hypertension, cardiac ANG II, acting via AT(1)R, enhances inflammation, oxidative stress, and cell death (most likely via downregulation of PI 3-kinase and Akt), contributing to cardiac hypertrophy and fibrosis.

The kinin B1 receptor contributes to the cardioprotective effect of angiotensin‐converting enzyme inhibitors and angiotensin receptor blockers in mice

Recent studies have shown that inhibition of angiotensin‐converting enzyme (ACE) or angiotensin II receptors causes upregulation of the B1 receptor (B1R). Here we tested the hypothesis that activation of the B1R partly contributes to the cardiac beneficial effect of ACE inhibitor (ACEi) and angiotensin II receptor blockers (ARB). B1R knockout mice (B1R−/−) and C57Bl/6J (wild‐type control animals, WT) were subjected to myocardial infarction (MI) by ligating the left anterior descending coronary artery. Three weeks after MI, each strain of mice was treated with vehicle, ACEi (ramipril, 2.5 mg kg−1 day−1 in drinking water) or ARB (valsartan, 40 mg kg−1 day−1 in drinking water) for 5 weeks. We found that: (1) compared with WT mice, B1R−/− mice that underwent sham surgery had slightly but significantly increased left ventricular (LV) diastolic dimension, LV mass and myocyte size, whereas systolic blood pressure, cardiac function and collagen deposition did not differ between strains; (2) MI leads to LV hypertrophy, chamber dilatation and dysfunction similarly in both WT and B1R−/− mice; and (3) ACEi and ARB improved cardiac function and remodelling in both strains; however, these benefits were significantly diminished in B1R−/− mice. Our data suggest that kinins, acting via the B1R, participate in the cardioprotective effects of ACEi and ARB.

Impairment of aldehyde dehydrogenase-2 by 4-hydroxy-2-nonenal adduct formation and cardiomyocyte hypertrophy in mice fed a high-fat diet and injected with low-dose streptozotocin

Reactive aldehydes such as 4-hydroxy-2-nonenal (4HNE) are generated in the myocardium in cardiac disease. 4HNE and other toxic aldehydes form adducts with proteins, leading to cell damage and organ dysfunction. Aldehyde dehydrogenases (ALDHs) metabolize toxic aldehydes such as 4HNE into nontoxic metabolites. Both ALDH levels and activity are reduced in cardiac disease. We examined whether reduced ALDH2 activity contributes to cardiomyocyte hypertrophy in mice fed a high-fat diet and injected with low-dose streptozotocin (STZ). These mice exhibited most of the characteristics of metabolic syndrome/type-2 diabetes mellitus (DM): increased blood glucose levels depicting hyperglycemia (415.2u2009±u200918.7u2009mg/dL vs. 265.2u2009±u20097.6u2009mg/dL; Pu2009<u20090.05), glucose intolerance with normal plasma insulin levels, suggesting insulin resistance and obesity as evident from increased weight (44u2009±u20093.1 vs. 34.50u2009±u20091.32u2009g; Pu2009<u20090.05) and body fat. Myocardial ALDH2 activity was 60% lower in these mice (0.1u2009±u20090.012 vs. 0.04u2009±u20090.015u2009µmol/min/mg protein; Pu2009<u20090.05). Myocardial 4HNE levels were also elevated in the hyperglycemic hearts. Co-immunoprecipitation study showed that 4HNE formed adducts on myocardial ALDH2 protein in the mice exhibiting metabolic syndrome/type-2 DM, and they had obvious cardiac hypertrophy compared with controls as evident from increased heart weight (HW), HW to tibial length ratio, left ventricular (LV) mass and cardiomyocyte hypertrophy. Cardiomyocyte hypertrophy was correlated inversely with ALDH2 activity (R2u2009=u20090.7; Pu2009<u20090.05). Finally, cardiac dysfunction was observed in mice with metabolic syndrome/type-2 DM. Therefore, we conclude that reduced ALDH2 activity may contribute to cardiac hypertrophy and dysfunction in mice presenting with some of the characteristics of metabolic syndrome/type-2 DM when on a high-fat diet and low-dose STZ injection.

Dose-dependent cardiac effect of oestrogen replacement in mice post-myocardial infarction

Hormonal replacement therapy (HRT) has recently been shown to increase the risk of cardiovascular events in women. However, it is not clear whether the adverse effect of HRT is related to dosage and/or the presence of progestin. Using a mouse model of myocardial infarction (MI), we studied the dose‐effect of oestrogen replacement on mortality and cardiac remodelling and dysfunction post‐MI in the absence of progestin. Six‐week‐old females were subjected to ovariectomy (OVX). A pellet containing a low, moderate or high dose of 17β‐oestradiol (E2; 0.42, 4.2 or 18.8 μg day−1) or placebo was implanted subcutaneously on the day of OVX. Myocardial infarction was induced 8 weeks later, and cardiac morphology and function were evaluated 8 weeks after MI. We found that E2 at moderate and high doses adversely affected mortality. A low dose of E2 that restored plasma oestrogen close to physiological levels had no significant effect on mortality but tended to improve cardiac function and remodelling, associated with reduced fibrosis and increased capillary density. At the moderate dose, E2 exacerbated cardiac fibrosis, hypertrophy, dysfunction and dilatation, associated with liver and kidney enlargement and ascites. Protein kinase C and extracellular signal‐regulated kinase were increased by MI but were not affected by E2. In summary, E2 at a low dose tended to be cardioprotective. At increased doses that raised plasma oestrogen far beyond the physiological level, E2 was detrimental to the heart. Our data suggest that dosage should be an important consideration when studying the effect of oestrogen replacement on the heart.

Angiotensin II‐induced dilated cardiomyopathy in Balb/c but not C57BL/6J mice

Balb/c mice, which are T‐helper lymphocyte 2 (Th2) responders, are highly susceptible to infectious and non‐infectious heart diseases, whereas C57BL/6 mice (Th1 responders) are not. Angiotensin II (Ang II) is not only a vasopressor but also a pro‐inflammatory factor that leads to cardiac hypertrophy, fibrosis and dysfunction. We hypothesized that Ang II exacerbates cardiac damage in Balb/c but not in C57BL/6 mice even though both strains have a similar level of hypertension. Twelve‐week‐old male C57BL/6J and Balb/c mice received either vehicle or Ang II (1.4 mg kg−1 day−1, s.c. via osmotic minipump) for 8 weeks. At baseline, Balb/c mice exhibited the following: (1) a lower heart rate; (2) an enlarged left ventricular chamber; (3) a lower ejection fraction and shortening fraction; and (4) twice the left ventricular collagen deposition of age‐matched C57BL/6J mice. Angiotensin II raised systolic blood pressure (to ∼150 mmHg) and induced cardiomyocyte hypertrophy in a similar manner in both strains. While C57BL/6J mice developed compensatory concentric hypertrophy and fibrosis in response to Ang II, Balb/c mice demonstrated severe left ventricular chamber dilatation, wall thinning and fibrosis, leading to congestive heart failure as evidenced by dramatically decreased ejection fraction and lung congestion (significant increase in lung weight), which are both characteristic of dilated cardiomyopathy. Our study suggests that the Th phenotype plays an active role in cardiac remodelling and function both in basal conditions and in hypertension. Angiotensin II‐induced dilated cardiomyopathy in Balb/c mice is an ideal animal model for studying the impact of the adaptive immune system on cardiac remodelling and function and for testing strategies to prevent or treat hypertension‐associated heart failure.

Effects of Cardiac Overexpression of the Angiotensin II Type 2 Receptor on Remodeling and Dysfunction in Mice Post–Myocardial Infarction

The activation of angiotensin II type 2 receptor (AT2R) has been considered cardioprotective. However, there are controversial findings regarding the role of overexpressing AT2R in the heart. Using transgenic mice with different levels of AT2R gene overexpression in the heart (1, 4, or 9 copies of the AT2R transgene: Tg1, Tg4, or Tg9), we studied the effect of AT2R overexpression on left ventricular remodeling and dysfunction post–myocardial infarction (MI). Tg1, Tg4, Tg9, and their wild-type littermates were divided into (1) sham MI, (2) MI plus vehicle, and (3) MI plus AT2R antagonist. Treatments were started 4 weeks after MI and continued for 8 weeks. AT2R protein and mRNA expression in the heart was significantly increased in transgenic mice, and the increase positively correlated with copies of the transgene. AT1R protein and mRNA expression remained unchanged in Tg1 and Tg4 but slightly increased in Tg9 mice. Systolic blood pressure and cardiac phenotypes did not differ among strains under basal conditions. MI caused myocardial hypertrophy, interstitial fibrosis, ventricular dilatation, and dysfunction associated with increased protein expression of Nox2 and transforming growth factor &bgr;1. These pathological responses were diminished in Tg1 and Tg4 mice. Moreover, the protective effects of AT2R were abolished by AT2R antagonist and also absent in Tg9 mice. We thus conclude that whether overexpression of AT2R is beneficial or detrimental to the heart is largely dependent on expression levels and possibly via regulations of Nox2 and transforming growth factor &bgr;1 signaling pathways.

Deletion of Inducible Nitric Oxide Synthase Provides Cardioprotection in Mice With 2-Kidney, 1-Clip Hypertension

Inducible NO synthase (iNOS) has been implicated in the pathogenesis of hypertension and target organ damage. We hypothesized that induction of iNOS contributes to left ventricular (LV) hypertrophy and dysfunction in mice with 2-kidney, 1-clip hypertension. Deletion of iNOS diminishes oxidative stress, thereby attenuating LV hypertrophy and enhancing cardiac performance. 2-Kidney, 1-clip hypertension was induced in mice lacking iNOS and wild-type controls (C57BL/6J). Sham-clipped mice served as controls. Systolic blood pressure was measured weekly by tail cuff. Left ventricular ejection fraction (by echocardiography) and cardiac response (maximum and minimum dP/dt, as well as an indicator of isovolumic contraction) to isoproterenol (50 ng per mouse, IV) were studied at the end of the experiment. 4-Hydroxy-2-nonenal (a byproduct of lipid peroxidation and an indicator of oxidative stress) was measured by immunohistochemical staining. gp91phox, endothelial NO synthase, and iNOS protein expression were determined by Western blot. We found that systolic blood pressure, LV weight, myocyte cross-sectional area, interstitial collagen fraction, ejection fraction, and cardiac response to isoproterenol did not differ between strains with sham clipping. 2-Kidney, 1-clip hypertension increased systolic blood pressure, LV weight, myocyte cross-sectional area, and interstitial collagen fraction similarly in both strains. However, in mice lacking iNOS, maximum and minimum dP/dt, as well as an indicator of isovolumic contraction, markedly increased in response to isoproterenol, associated with decreased cardiac 4-hydroxy-2-nonenal expression and urinary nitrate/nitrite. We concluded that deletion of iNOS does not seem to play a significant role in preventing 2-kidney, 1-clip hypertension-induced hypertension and cardiac hypertrophy; however, it does enhance preservation of cardiac function, probably because of a reduction of iNOS-induced oxidative stress.