Heart Weight/body Weight Research Articles

Sodium Houttuyfonate Alleviates Post-infarct Remodeling in Rats via AMP-Activated Protein Kinase Pathway.

With the chronic ischemia persisting after acute myocardial infarction, the accompanying low-degree inflammation and subsequent fibrosis result in progression of cardiac remodeling and heart failure. Recently, Sodium Houttuyfonate (SH), a pure compound extracted from Houttuynia cordata, has been confirmed exerting anti-inflammatory and anti-fibrotic effects under diseased situations. Here, we aimed to investigate whether SH could reverse the cardiac remodeling post-myocardial infarction by alleviating cardiac inflammation and fibrosis. Left anterior descending coronary artery of adult male Sprague-Dawley rats was ligated to elicit myocardial infarction. Low and high dose of SH was administered by oral gavage for four consecutive weeks post-myocardial infarction. Long-term SH treatment decreased heart rate, heart weight/ body weight (HW/BW), and left ventricle weight/body weight (LVW/BW), reduced cardiac expression of brain natriuretic peptide (BNP), improved left ventricular heart function, and ameliorated the histopathological changes caused by myocardial infarction. Western blotting revealed the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), transforming growth factor-β (TGF-β), collagen I, and collagen III of the infarcted ventricle were reduced by SH treatment. Meanwhile, we found that SH treatment post-myocardial infarction activated AMP-activated protein kinase (AMPK) and suppressed nuclear factor-κB p65 (NF-κB p65). Furthermore, on H9C2 cells induced hypoxic injury with cobalt chloride (CoCl2), the reduction of inflammatory cytokines (IL-6, TNF-α, and TGF-β), activation of AMPK, and suppression of NF-κB p65 were also observed by SH treatment. However, transfection of H9C2 with AMPKα siRNA blunted the suppression of NF-κB p65 and inflammatory cytokines (IL-6, TNF-α, and TGF-β) by SH post-hypoxia. Taken together, these findings suggested that long-term administration of SH post-myocardial infarction reduced cardiac inflammatory and fibrotic responses, and reversed cardiac remodeling process. The underlying mechanism may be activating AMPK and suppressing NF-κB pathway.

Abstract 101: Transforming Growth Factor-Beta-Receptor Antagonist Blocks the Cardiac Fibrosis and Remodeling in Guanylyl Cyclase/Natriuretic Peptide Receptor-A Gene-Disrupted Mice

Targeted-disruption of guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene ( Npr1 ) exhibits hypertension and provokes congestive heart failure in mice; however, the underlying mechanisms are not well clear. The objective of this study was to determine whether transforming growth factor-beta receptor (TGF-βR) antagonist, GW788388 inhibits the development of cardiac fibrosis and remodeling in Npr1 gene-disrupted mice. The adult male (16-20 weeks) Npr1 null mutant ( Npr1 -/- , 0-copy), heterozygous ( Npr1 +/- , 1-copy), and wild-type ( Npr1 +/+ , 2-copy) mice were orally administered with TGF-βR antagonist, GW788388 (1 mg/kg/day) for 28 days. The expression of cardiac fibrotic markers was analyzed using real-time PCR and Western blot. Heart weight-to-body weight (HW/BW) ratios were determined and heart functions were measured by echocardiographic analysis. The Npr1 -/- mice showed markedly increased cardiac fibrosis and HW/BW ratio with increased expression of collagen-1α (3.5-fold), monocyte chemoattractant protein (4-fold), connective tissue growth factor (CTGF, 5-fold), α-smooth muscle actin (α-SMA, 4-fold), TGF-βRI (4-fold), TGF-βRII (3.5-fold) and SMAD proteins (SMAD-2, 5-fold; SMAD-3, 3-fold) compared with Npr1 +/+ mice. The expression of phosphorylated extracellular-regulated kinase (pERK1/2) was also up-regulated by 68% ( P <0.001) in Npr1 -/- mice. The treatment of Npr1 -/- mice with GW788388 prevented the development of cardiac fibrosis and down-regulated the expression of fibrotic markers and SMAD proteins by 70-75% ( P <0.001) compared to vehicle-treated mice. In contrast, the expression of pERK1/2 proteins was unaffected in GW7885388-treated mice excluding the involvement of non-genomic pathway. The left ventricular dimensions (systole and diastole) and fractional shortening were significantly ( P <0.001) improved in the drug-treated Npr1 -/- mice. The results suggest that the cardiac fibrosis, remodeling, and dysfunction in Npr1 -/- mice are regulated through TGF-βR-mediated SMAD-dependent canonical pathway. The findings will be important for the development of new molecular therapeutic targets for the treatment of cardiac fibrosis, remodeling, and dysfunction in humans.

Anti-fibrotic Actions of Roselle Extract in Rat Model of Myocardial Infarction.

Heart failure-associated morbidity and mortality is largely attributable to extensive and unregulated cardiac remodelling. Roselle (Hibiscus sabdariffa) calyces are enriched with natural polyphenols known for antioxidant and anti-hypertensive effects, yet its effects on early cardiac remodelling in post myocardial infarction (MI) setting are still unclear. Thus, the aim of this study was to investigate the actions of roselle extract on cardiac remodelling in rat model of MI. Male Wistar rats (200-300g) were randomly allotted into three groups: Control, MI, and MI + Roselle. MI was induced with isoprenaline (ISO) (85mg/kg, s.c) for two consecutive days followed by roselle treatment (100mg/kg, orally) for 7days. Isoprenaline administration showed changes in heart weight to body weight (HW/BW) ratio. MI was especially evident by the elevated cardiac injury marker, troponin-T, and histological observation. Upregulation of plasma levels and cardiac gene expression levels of inflammatory cytokines such as interleukin (IL)-6 and IL-10 was seen in MI rats. A relatively high percentage of fibrosis was observed in rat heart tissues with over-expression of collagen (Col)-1 and Col-3 genes following isoprenaline-induced MI. On top of that, cardiomyocyte areas were larger in heart tissues of MI rats with upregulation of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) gene expression, indicating cardiac hypertrophy. Interestingly, roselle supplementation attenuated elevation of plasma troponin-T, IL-6, IL10, and gene expression level of IL-10. Furthermore, reduction of cardiac fibrosis and hypertrophy were observed. In conclusion, roselle treatment was able to limit early cardiac remodelling in MI rat model by alleviating inflammation, fibrosis, and hypertrophy; hence, the potential application of roselle in early adjunctive treatment to prevent heart failure.

Abstract 280: MCL-1 Matrix Promotes Mitochondrial Fusion and Protects Against TAC Induced Hypertrophy

The anti-apoptotic BCL-2 protein MCL-1 is highly expressed in the myocardium, and we have previously found that deletion in cardiomyocytes leads to mitochondrial dysfunction and rapid development of heart failure. MCL-1 exists both in the outer mitochondrial membrane (MCL-1 OM ) and in the mitochondrial matrix (MCL-1 Matrix ). Because Mcl-1 -/- mice lack both MCL-1 Matrix and MCL-1 OM , the functional role of each form remains unknown. While studies have implicated MCL-1 OM in regulating apoptosis, very little is known about the functional role of MCL-1 Matrix . Here, we show that mitochondria are responsive to alterations in cellular metabolism, and culturing mouse embryonic fibroblasts (MEFs) in “oxidative” media containing galactose or acetoacetate led to a significant increase in the MCL-1 Matrix /MCL-1 OM ratio and elongation of mitochondria. We confirmed that MCL-1 Matrix overexpression promoted mitochondrial fusion and reduced maximal mitochondrial respiration under baseline conditions. Interestingly, the elongated mitochondria were initially protected against FCCP-induced fission and mitophagy. However, prolonged exposure to FCCP resulted in abrogation of MCL-1 Matrix import. Accumulation of MCL-1 Matrix on the outer membrane led to its degradation and subsequent induction of mitophagy. Furthermore, to investigate its functional role in the heart, we generated cardiac specific MCL-1 Matrix (αMHC-MCL-1 Matrix ) transgenic mice. The mice show no differences in cardiac structure and function compared to wild-type litter mates at 3 months. However, ultrastructural analysis revealed the presence of enlarged mitochondria in myocytes from αMHC-MCL-1 Matrix mice. Finally, we subjected WT and αMHC-MCL-1 Matrix mice to transverse aortic constriction (TAC) and we found that αMHC-MCL-1 Matrix mice had reduced heart weight to body weight (HW/BW) ratio and decreased expression of hypertrophy markers ANF, BNP, and MHC-β at two weeks post-TAC compared to WT. Thus, our studies indicate that cardiac overexpression of MCL-1 Matrix promotes mitochondrial enlargement in myocytes and alleviates stress placed on the heart during pressure overload.

The involvement of AQP1 in myocardial edema induced by pressure overload in mice.

To investigate the effect of aquaporin-1 (AQP1) on heart edema induced by transverse aortic constriction (TAC) in mice, and to explore whether inhibiting the expression of AQP1 could attenuate myocardial edema and improve cardiac function. The C57BL/6 mice were divided into four groups: (1) the sham group; 2) the sham + acetazolamide group: mice were orally gavaged with acetazolamide (20 mg/kg/day) after sham operation; (3) the TAC group: a mouse model of pressure overload induced by TAC for two weeks; (4) the TAC + acetazolamide group: mice were orally gavaged with acetazolamide (20 mg/kg/day) after TAC. Cardiac function was detected by echocardiography after 2 weeks' TAC. The ratio of heart weight to body weight (HW/BW) and myocardial water content were calculated. The mRNA and protein expressions of AQP1 were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot, respectively. Significant myocardial hypertrophy and dysfunction were found in TAC mice. The ratio of HW/BW, myocardial water content, and the mRNA and protein expression of AQP1 of the TAC group were markedly higher than those of the sham group. By contrast, acetazolamide administration reduced the ratio of HW/BW and myocardial water content, whereas improved cardiac dysfunction induced by TAC. Moreover, acetazolamide reduced the mRNA and protein expression of AQP1 in TAC mice. The expression of AQP1 was closely related to myocardial edema induced by TAC. The inhibition of AQP1 could reduce myocardial edema and improve cardiac dysfunction.

A Reduction in ADAM17 Expression Is Involved in the Protective Effect of the PPAR-α Activator Fenofibrate on Pressure Overload-Induced Cardiac Hypertrophy.

The peroxisome proliferator-activated receptor-α (PPAR-α) agonist fenofibrate ameliorates cardiac hypertrophy; however, its mechanism of action has not been completely determined. Our previous study indicated that a disintegrin and metalloproteinase-17 (ADAM17) is required for angiotensin II-induced cardiac hypertrophy. This study aimed to determine whether ADAM17 is involved in the protective action of fenofibrate against cardiac hypertrophy. Abdominal artery constriction- (AAC-) induced hypertensive rats were used to observe the effects of fenofibrate on cardiac hypertrophy and ADAM17 expression. Primary cardiomyocytes were pretreated with fenofibrate (10 μM) for 1 hour before being stimulated with angiotensin II (100 nM) for another 24 hours. Fenofibrate reduced the ratios of left ventricular weight to body weight (LVW/BW) and heart weight to body weight (HW/BW), left ventricular anterior wall thickness (LVAW), left ventricular posterior wall thickness (LVPW), and ADAM17 mRNA and protein levels in left ventricle in AAC-induced hypertensive rats. Similarly, in vitro experiments showed that fenofibrate significantly attenuated angiotensin II-induced cardiac hypertrophy and diminished ADAM17 mRNA and protein levels in primary cardiomyocytes stimulated with angiotensin II. In summary, a reduction in ADAM17 expression is associated with the protective action of PPAR-α agonists against pressure overload-induced cardiac hypertrophy.

Prorenin independently causes hypertension and renal and cardiac fibrosis in cyp1a1-prorenin transgenic rats

Plasma prorenin is commonly elevated in diabetic patients and appears to predict the development of diabetic nephropathy. However, the pathological role of prorenin is unclear. In the present study, a transgenic, inducible, hepatic prorenin-overexpressing rat model was generated and the effect of prorenin in organ injury was examined. Four groups of rats (cyp1a1 prorenin transgenic male and female rats and non-transgenic littermates) were assigned to receive a diet containing 0.3% of the transgene inducer indole-3-carbinol (I3C) for 4 weeks. Plasma prorenin concentration was increased and mean arterial pressure (MAP) increased from 80 ± 18 to 138 ± 17 (mmHg), whereas renal prorenin/renin protein expression was unchanged, in transgenic rats fed with I3C diet. The intact prorenin, not renin, in plasma and urine samples was further observed by Western blot analysis. Importantly, transgenic rats with high levels of prorenin developed albuminuria, glomerular and tubulointerstitial fibrosis associated with increased expression of transforming growth factor β (TGFβ) 1 (TGFβ1), plasminogen activator inhibitor-1 (PAI-1), collagen, and fibronectin (FN). These rats also exhibited cardiac hypertrophy determined by echocardiography, with elevated ratio of heart weight to body weight (HW/BW). Cardiac collagen in interstitial and perivascular regions was prominent, accompanied by the increase in mRNA contents of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), β-myosin heavy chain (β-MHC), TGFβ1, PAI-1, and collagen in the heart tissue. Furthermore, renal protein levels of p-NF-κB-p65 and monocyte chemoattractant protein-1 (MCP-1), NAPDH oxidases, malondialdehyde (MDA) and 8-isoprostane (8-IP), p-ERK, p-β-catenin, and p-Akt were dramatically increased in prorenin overexpressing rats. These results indicate that prorenin, without being converted into renin, causes hypertension, renal and cardiac fibrosis via the induction of inflammation, oxidative stress and the ERK, β-catenin, and Akt-mediated signals.

MicroRNA Profile in Swimming Trained Female Rats and its Therapeutic Potential in Spontaneously Hypertensive Rats

IntroductionBoth aerobic exercise training (ET) and Hypertension (HY) induce different cardiac hypertrophy (CH) phenotypes which molecular differences and may lead to new targets for therapies in cardiovascular disease, as microRNAs (miRNAs).MethodsWe selected 15 miRNAs that were differentially expressed by ET from miRNAarray data and compared them with other from SHR (Spontaneously Hypertensive Rats) miRNAarray data. Four miRNAs were selected for qRT‐PCR confirmation: miRNA‐27a, 27b, 126 e 29c. Among then, miRNA 29c was chosen to be modulated by lentiviral vector due its role in fibrosis regulation. Intramuscular cardiac injection of the lentiviral vector particles 0.6×109 vp/was performed for two different times (7 and 14 days) (SHR7 and SHR14). The transduction efficiency was assessed by GFP expression by Western Blot. Blood pressure (BP) was measured by caudal plethysmography, CH was analyzed by ratio LVW/BW (left ventricle weight/body weight), HW/BW (heart weight/body weight) and by cardiomyocytes diameter (dCMO). qRT‐PCR was used to assess miRNA‐29c expression and its targets COLIAI and COLIIIAI gene expression. The LV collagen content was assessed by histology (Picrossirius red), by collagen volume fraction, and by hydroxiproline concentration.ResultsBoth groups that received the low doses were GFP positive in the heart and liver tissue. BP was increased to SHR7, but it was prevented by miR‐29c lentivirus treatment to SHR14. The miRNA‐29c expression increased in all treated groups versus their control (CSI). COLIAI expression decreased in treated groups, while COLIIIAI did not change. Collagen volume fraction decreased in all treated groups, which shows that the treatment was efficient to decrease the cardiac collagen. HW/BW ratio decreased 7–11% in SHR14, while the dCMO was increased in all treated groups.ConclusionsThese results suggest that miR‐29c lentivirus treatment induces beneficial cardiac remodeling in SHR, increasing dCMO and decreasing cardiac fibrosis. These results open perspective for further investigation of antifibrotic therapies for cardiovascular diseases.Support or Funding InformationFAPESP, CNPq, CAPES and NAPmirThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Secretoneurin suppresses cardiac hypertrophy through suppression of oxidant stress

The neuropeptide secretoneurin (SN) plays protective roles in myocardial ischemia. In the present study, the effect of SN in cardiac hypertrophy was investigated. We observed that, in isoproterenol (ISO) treatment induced cardiac or cardiomyocytes hypertrophy, a marked increase in the expression of endogenous SN in mouse plasma, myocardium and primary-cultured cardiomyocytes occurs. In hypertrophic mice, the heart size, heart weight/body weight (HW/BW) ratio, cardiomyocyte size, and atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) expression were significantly higher than those in controls but were effectively suppressed by SN gene therapy. Similarly, the protective effects of SN were also observed in cultured cardiomyocytes following ISO treatment. SN significantly increased the activity of catalase and superoxide dismutase (SOD) in parallel with the decrease in reactive oxygen species levels in cardiomyocytes. We observed that SN evoked the activation of all of the AMPK, P38/MAPK and ERK/MAPK pathways in cardiomyocytes, but pretreatment with only AMPK inhibitor (compound C) and ERK1/2/MAPK inhibitor (PD98059) counteracted the protective effects of SN against cardiomyocyte hypertrophy and the suppressive effects of SN on oxidant stress in cardiomyocytes. These results indicated that endogenous SN is induced in hypertrophic cardiomyocytes, and may play a protective role in the pathogenesis of cardiac hypertrophy. These results suggest that exogenous SN supplementation protects the cardiac hypertrophy induced by ISO treatment through the activation of AMPK and ERK/MAPK pathways, thus upregulating antioxidants and suppressing oxidative stress.

Hydrogen inhibits isoproterenol‑induced autophagy in cardiomyocytes invitro and invivo.

A previous study from our group has demonstrated that hydrogen administration can attenuate cardiovascular hypertrophy invivo by targeting reactive oxygen species‑dependent mitogen‑activated protein kinase signaling. The aim of the present study is to determine the effect of hydrogen on cardiomyocyte autophagy during β‑adrenoceptor activation invivo and invitro. We prepared hydrogen‑rich medium, and the concentration of hydrogen was measured by using the MB‑Pt reagent method. For the invitro study, H9c2 cardiomyocytes were stimulated with isoproterenol (ISO; 10µM) for 5, 15 and 30min, and then the protein expression levels of the autophagy marker microtubule‑associated protein 1 light chain 3β II (LC3B II) were examined by western blotting. The effect of hydrogen‑rich medium was then tested by pretreating the H9c2 cardiomyocytes with hydrogen‑rich medium for 30min, then stimulating with ISO, and examining the protein expression levels of the autophagy marker LC3B II. For the invivo study, mice received hydrogen (1ml/100g/day, by intraperitoneal injection) for 7days prior to ISO administration (0.5mg/100g/day, by subcutaneous injection), and subsequently received hydrogen with or without ISO for another 7days. Hypertrophic responses were examined by heart weight (HW) and heart weight/body weight (HW/BW) measurements. The protein expression of autophagy markers Beclin1, autophagy‑related protein 7 (Atg7) and LC3B II were examined. The results demonstrated that excessive autophagy occurred following 5min of ISO stimulation invitro. This enhanced autophagy was blocked by pretreatment with hydrogen‑rich medium. Furthermore, hydrogen improved the deteriorated hypertrophic responses and inhibited the enhanced autophagic activity mediated by ISO administration invivo, as indicated by decreasing HW and HW/BW, and suppressing the protein expression levels of Beclin1, Atg7 and LC3B II. Therefore, the results of the present study demonstrated that hydrogen inhibited ISO‑induced excessive autophagy in cardiomyocyte hypertrophy models invitro and invivo.

Metastasis-Associated Protein 1 Deficiency Results in Compromised Pulmonary Alveolar Capillary Angiogenesis in Mice.

BackgroundThe aim of this study was to investigate the effects of metastasis-associated protein 1 (MTA1) deficiency during angiogenesis of pulmonary alveolar capillaries in mice and to determine the molecular mechanisms involved.Material/MethodsThe expressions of MTA1, CD34, vascular endothelial growth factor (VEGF), alpha smooth muscle actin (α-SMA), and HIF-1α were analyzed in the lungs of MTA1-knockout (KO) and wild-type mice at embryonic day 18.5 and 2 months by quantitative PCR, immunoblotting, and immunohistochemistry. The morphological changes were investigated during pulmonary alveolar capillary formation. The heart weight/body weight (HW/BW) ratio and the size of the right ventricular wall cardiomyocytes were also measured. Regulation of MTA1 on HIF-1α was determined in vitro.ResultsMTA1 deficiency reduced the number of pulmonary alveolar capillaries compared to the wild-type mice. MTA1-KO mice exhibited a decreased expression of HIF-1α and VEGF in the lungs. The retarded growth of the MTA1-KO mice was also noticed during the first week after birth. Accordingly, MTA1 deficiency resulted in increased infant mortality. In surviving adult mice, MTA1 deficiency induced myocardial hypertrophy, highlighted by an increased heart weight/body weight ratio and larger cardiomyocytes. In cultured cells, HIF-1α and VEGF levels were significantly upregulated upon MTA1 overexpression, suggesting a close relationship between all 3 molecules.ConclusionsMTA1 participates in the formation of pulmonary capillaries via stabilization of HIF-1α. This finding sheds new light on the function of MTA1 in lung development, opening new avenues for the diagnosis/treatment of related pulmonary diseases.

Effects of hydrogen sulfide on inducible nitric oxide synthase activity and expression of cardiomyocytes in diabetic rats.

The aim of the present study was to investigate the effects of hydrogen sulfide (H2S) on the activity and expression of inducible nitric oxide synthase (iNOS) in the myocardial tissue of type 1 diabetic rats. Rats were divided randomly into four groups: Normal control (NC), diabetes mellitus (DM), DM+DL-Proparglygylcine (DM+PAG) and DM+sodium hydrosulfide (DM+NaHS) groups. Type 1 diabetes was induced in the respective groups by a single intraperitoneal (i.p.) injection of streptozotocin. Rats in the DM+PAG and DM+NaHS groups were injected with PAG and NaHS (i.p.) once a day, respectively. The level of fasting blood glucose (FBG), the heart-weight to body-weight (HW/BW) ratio and the ventricular hemodynamic parameters were measured. The activities of serum total NOS (tNOS), iNOS, lactate dehydrogenase (LDH), creatine kinase (CK) and creatine kinase MB isozyme (CK-MB), and the content of nitric oxide (NO) were detected. The contents of myocardial malondialdehyde (MDA) and NO, and the activities of superoxide dismutase (SOD), tNOS and iNOS were determined. The myocardial tissue was examined for histological and ultrastructural alterations. The expression level of iNOS at the transcriptional and translational levels in the myocardial tissue was estimated. The level of FBG was increased in the DM group compared with the NC group, verifying the diabetic condition of the rats. The function of the left ventricle, the myocardial histological alterations and ultrastructures were damaged in the DM group. The DM group additionally demonstrated an increase in the serum NO content and tNOS, iNOS, LDH, CK and CK-MB activities. The myocardial MDA, NO content and tNOS levels were additionally increased in this group. The iNOS activity was increased significantly whereas the myocardial SOD activity was decreased. The increase in the iNOS activity was supported by an enhanced expression level of myocardial iNOS mRNA and protein in the DM group. In the DM+PAG group, in the absence of H2S, the dysfunction of the left ventricle and the oxidative stress injury were increased compared with the DM group. The activity and the expression of tNOS and iNOS were increased significantly. However, the rats in the DM+PAG group demonstrated the opposite effects. In conclusion, H2S exhibits a protective effect on the myocardium in type 1 diabetic rats, which may be associated with the suppression of iNOS activity and expression, a decrease in the NO content and the inhibition of oxidative stress injury.

Protective effects of Xinji′erkang on myocardial infarction induced cardiac injury in mice

BackgroundMyocardial infarction (MI) is a major risk factor responsible for morbidity and mortality. Xinji′erkang (XJEK) has been clinically used as an effective medication in the treatment of coronary heart disease and myocarditis. The purpose of this study was to investigate the cardioprotective effect of Xinji′erkang on MI mice.MethodsForty male mice were randomly assigned into four groups as follows (n = 10): sham, model, MI with administration of XJEK and fosinopril for four weeks. At the end of studies, hemodynamic parameters and electrocardiography (ECG) were recorded. Heart and body mass were measured and heart weight/body weight (HW/BW) ratio was calculated as index of hypertrophy. The hypertrophy of heart and aorta was examined using the hematoxylin and eosin (HE) staining, and the collagen deposition was evaluated using Van Gieson (VG) staining. Serum nitric oxide level (NO), superoxide dismutase (SOD) activity and malondialdehyde (MDA) concentration were assayed by colorimetric analysis. The expressions of endothelial NO synthetase (eNOS) expression in serum and cardiac tissues were determined using ELISA assay and immunohistochemistry. Angiotensin II (Ang II) in serum and cardiac tissues was measured using ELISA assay. Besides, tumor necrosis factor-α (TNF-α), interleukin1β (IL-1β) and interleukin10 (IL-10) were observed in cardiac tissues with ELISA assay as well.ResultsThe administration of XJEK significantly improved cardiac dysfunction and abnormal ECG with reduced HW/BW ratio and ameliorated cardiomyocyte hypertrophy and collagen deposition compared to MI, which was partly due to the decreased SOD and increased MDA in serum. Moreover, XJEK treatment also improved endothelial dysfunction (ED) with not only enhanced eNOS activities in serum and cardiac tissues and elevated NO levels in serum, but also decreased Ang II content in serum and cardiac tissues. Finally, protein expressions of pro-inflammation cytokines, TNF-α and IL-1β in the cardiac tissues with XJEK treatment were significantly decreased compared to model. On the contrary, IL-10, an anti-inflammatory cytokine concentrated in cardiac tissues was significantly enhanced compared to model.ConclusionXinji′erkang exerts cardioprotective effect on myocardial infarction in mice, which may be due to the improvement of endothelial dysfunction and the reduction of oxidative stress and inflammation response.

Ginsenoside Rh2 Improves Cardiac Fibrosis via PPARδ-STAT3 Signaling in Type 1-Like Diabetic Rats.

Ginsenoside Rh2 (Rh2) is an active principal ingredient contained in ginseng (Panax ginseng Meyer), a medicinal herb used to enhance health worldwide. The present study is designed to investigate the effect of Rh2 on myocardial fibrosis in diabetic rats. In a streptozotocin-induced model of type-1 diabetic rats (STZ-diabetic rats), the increased fasting blood glucose levels and heart weight/body weight (HW/BW) ratio were substantially alleviated by Rh2. Moreover, Rh2 improved cardiac performance in STZ-diabetic rats. Histological results from Masson staining showed that Rh2 attenuated cardiac fibrosis in STZ-diabetic rats. The effects of Rh2 were reversed by GSK0660 at a dose sufficient to inhibit peroxisome proliferator-activated receptor δ (PPARδ) in STZ-diabetic rats. The role of PPARδ was subsequently investigated in vitro. Rh2 restored the decreased PPARδ expression level in high glucose-cultured cardiomyocytes. Moreover, increased protein levels of fibrotic signals, including signal transducer and activator of transcription 3 (STAT3), connective tissue growth factor (CCN2) and fibronectin, were reduced by Rh2 in high glucose-cultured cardiomyocytes. These effects of Rh2 were reversed by GSK0660 or siRNA specific for PPARδ Taken together, PPARδ activation may inhibit STAT3 activation to reduce CCN2 and fibronectin expression in diabetic rats with cardiac fibrosis. Moreover, Rh2 improves cardiac function and fibrosis by increasing PPARδ signaling. Therefore, Rh2 is suitable to develop as an alternative remedy for cardiac fibrosis.

Wild-type p53-induced Phosphatase 1 Deficiency Exacerbates Myocardial Infarction-induced Ischemic Injury.

Background:Myocardial infarction (MI) is a major disease burden. Wild-type p53-induced phosphatase 1 (Wip1) has been studied extensively in the context of cancer and the regulation of different types of stem cells, but the role of Wip1 in cardiac adaptation to MI is unknown. We investigated the significance of Wip1 in a mouse model of MI.Methods:The study began in June 2014 and was completed in July 2016. We compared Wip1-knockout (Wip1-KO) mice and wild-type (WT) mice to determine changes in cardiac function and survival in response to MI. The heart weight/body weight (HW/BW) ratio and cardiac function were measured before MI. Mouse MI was established by ligating the left anterior descending (LAD) coronary artery under 1.5% isoflurane anesthesia. After MI, survival of the mice was observed for 4 weeks. Cardiac function was examined by echocardiography. The HW/BW ratio was analyzed, and cardiac hypertrophy was measured by wheat germ agglutinin staining. Hematoxylin and eosin (H&E) staining was used to determine the infarct size. Gene expression of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) was assessed by quantitative real-time polymerase chain reaction (qPCR), and the levels of signal transducers and activators of transcription 3 (stat3) and phosphor-stat3 (p-stat3) were also analyzed by Western blotting. Kaplan-Meier survival analysis, log-rank test, unpaired t-test, and one-way analysis of variance (ANOVA) were used for statistical analyses.Results:Wip1-KO mice had a marginally increased HW/BW ratio and slightly impaired cardiac function before LAD ligation. After MI, Wip1-deficient mice exhibited increased mortality (57.14% vs. 29.17%; n = 24 [WT], n = 35 [Wip1-KO], P < 0.05), increased cardiac hypertrophy (HW/BW ratio: 7 days: 7.25 ± 0.36 vs. 5.84 ± 0.18, n = 10, P < 0.01, and 4 weeks: 6.05 ± 0.17 vs. 5.87 ± 0.24, n = 10, P > 0.05; cross-sectional area: 7 days: 311.80 ± 8.29 vs. 268.90 ± 11.15, n = 6, P < 0.05, and 4 weeks: 308.80 ± 11.26 vs. 317.00 ± 13.55, n = 6, P > 0.05), and reduced cardiac function (ejection fraction: 7 days: 29.37 ± 1.38 vs. 34.72 ± 1.81, P < 0.05, and 4 weeks: 19.06 ± 2.07 vs. 26.37 ± 2.95, P < 0.05; fractional shortening: 7 days: 13.72 ± 0.71 vs. 16.50 ± 0.94, P < 0.05, and 4 weeks: 8.79 ± 1.00 vs. 12.48 ± 1.48, P < 0.05; n = 10 [WT], n = 15 [Wip1-KO]). H&E staining revealed a larger infarct size in Wip1-KO mice than in WT mice (34.79% ± 2.44% vs. 19.55% ± 1.48%, n = 6, P < 0.01). The expression of IL-6 and p-stat3 was downregulated in Wip1-KO mice (IL-6: 1.71 ± 0.27 vs. 4.46 ± 0.79, n = 6, P < 0.01; and p-stat3/stat3: 1.15 ± 0.15 vs. 1.97 ± 0.23, n = 6, P < 0.05).Conclusion:The results suggest that Wip1 could protect the heart from MI-induced ischemic injury.

Knockout of immunoproteasome subunit β2i ameliorates cardiac fibrosis and inflammation in DOCA/Salt hypertensive mice

The immunoproteasome is a multicatalytic protease complex in all eukaryotic cells, which plays a key role in regulating essential cellular processes. However, the role of immunoproteasome subunit β2i in regulation of cardiac fibrosis and inflammation in deoxycorticosterone-acetate (DOCA)/salt mice remains unknown. Wild-type (WT) and β2i knockout (KO) mice were subjected to uninephrectomy and DOCA/salt treatment for 21 days. Blood pressure was measured by the tail-cuff system. Cardiac function and remodeling were examined by echocardiography, hematoxylin-eosin (H&E) and Masson's trichrome staining. The gene and protein expressions were detected by quantitative real-time PCR, and Western blot analysis. After 21 days, DOCA/salt treatment significantly up-regulated the expression of β2i mRNA and protein in the hearts. Moreover, systolic blood pressure and heart weight/body weight (HW/BW) ratio were significantly higher in DOCA/salt mice than in sham groups, and these effects were markedly reversed in β2i knockout mice. Importantly, DOCA/salt-induced cardiac fibrosis, inflammation and the expression of collagen I, collagen III, α-SMA, IL-1β, IL-6 and TNF-α in the wild-type hearts, which were markedly attenuated by β2i knockout. These beneficial effects were due, at least in part, to the inhibition of IκBα/NF-κB and TGF-β1/Smad2/3 signaling pathways. Collectively, these findings indicate that knockout of β2i ameliorates DOCA/salt-induced cardiac fibrosis and inflammation, and may be a novel potential therapeutic target for hypertensive heart diseases.

Xinfuli Granule improves post-myocardial infarction ventricular remodeling and myocardial fibrosis in rats by regulating TGF-β/Smads signaling pathway.

BackgroundRecent clinical and experimental studies have confirmed the effects of Xinfuli Granule (XG), a compound Chinese medicine in the prevention and treatment of heart failure (HF). This study aimed to investigate the effects and the mechanisms of XG on ventricular reconstruction in rats with acute myocardial infarction (AMI).MethodsSprague-Dawley rats were subjected to left anterior descending branch ligation. The rats that survived 24 h were randomly assigned to five groups: medium-dose of XG group (MI+XGM), high-dose of XG group (MI+XGH), carvedilol group (MI+C), medium-dose of XG + carvedilol group (MI+C+XGM). Fourteen rats underwent identical surgical procedures without artery ligation, serving as sham controls. At 28 days, left ventricular weight to body weight (LVW/BW) and heart weight to body weight (HW/BW) were calculated; left ventricular ejection fraction (LVEF), left ventricular shortening fraction (LVFS), left ventricular internal diameter at systole (LVIDS) were measured by ultrasound; HE staining, Masson staining, and Sirius red staining were used to assess the myocardial pathological and physiological changes as well as myocardial fibrosis area and non-infarct zone I/III collagen ratio. Expression of Smad3 were detected and analyzed by Western blot, immunohistochemistry and immunofluorescence. P-Smad3, Smad2 and Smad7 in the TGF-β/Smads signaling pathway were also analyzed by Western blot.ResultsThe LVIDS (P < 0.01), HW/BW (P < 0.05), type I/III collagen ratio (P < 0.01) and myocardial collagen (P < 0.01) decreased significantly while the LVW/BW, LVFS (P < 0.05) increased significantly in MI+XGM group as compared with those in other groups. The expression of key signal molecules of the TGF-β/Smads signaling pathway, including Smad3, P-Smad3 and Smad2 protein were decreased, while the expression of Smad7 increased in both XG and carvedilol treatment groups as compared to those of the MI group (all P < 0.01). Immunohistochemistry and immunofluorescence further confirmed the down-regulated Smad3 expression.ConclusionXG can improve ventricular reconstruction and inhibit myocardial fibrosis in rats with AMI by regulating TGF-β/Smads signaling pathway.

Inhibition of inducible nitric oxide synthase (iNOS) by simvastatin attenuates cardiac hypertrophy in rats.

The left ventricular hypertrophy (LVH) occurs in response to the haemodynamic overload in some physiological and pathological conditions. This study was designed to investigate the possible cardioprotective effect of simvastatin (SIM) treatment against isoproterenol (ISO)-induced LVH and the probable underlying mechanism in adult male Wistar rats. Animals were allocated into four groups. Rats of control group received normal saline orally for 30 days and intraperitoneally for the last 7 days. Rats of SIM group received SIM orally (10 mg/kg/day in saline) for 30 days. Rats of ISO group received normal saline orally for 30 days and ISO intraperitoneally (5 mg/kg) for the last 7 days to induce LVH. Rats of ISO/SIM group received SIM for 30 days and ISO intraperitoneally for the last 7 days. At the end of the experiment, all animals were sacrificed by cervical decapitation under anaesthesia. Truncal blood was collected and serum was separated and used for biochemical assay. The heart was dissected and processed for histological and immunohistochemical studies. The results of the present study confirmed the ISO-induced myocardial lesions including significant increase of heart weight (HW), heart weight/body weight (HW/BW) ratio, LVH, interstitial myocardial fibrosis (increased collagen types I and III), inflammatory cellular infiltration, necrosis of cardiomyocytes, and increased expression of inducible nitric oxide synthase (iNOS) and thioredoxin in cardiomyocytes. These changes were accompanied by significant increase in serum levels of troponin-T, creatine phosphokinase-MB (CPK-MB), tumour necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). Co-administration of SIM to ISO-injected rats significantly reduced all these cardiac changes and serum biochemical markers in addition to marked depletion of iNOS and thioredoxin expression in cardiomyocytes. It is concluded that SIM co-administration attenuated ISO-induced cardiac lesions including LVH by inhibiting iNOS expression in cardiomyocytes.

Prevention of doxorubicin induced cardiomyopathy after delivery of modified acidic fibroblast growth factor by using ultrasound-targeted microbubble destruction: a preliminary experimental study

Objective To observe the preventive effects of doxorubicin-induced cardiomyopathy (DOX-CM) rats after delivery of modified acidic fibroblast growth factor (MaFGF) by using ultrasound-targeted microbubble destruction (UTMD). Methods Fifty male SD rats were randomly divided into five groups: ①Control group; ②DOX-CM group; ③MaFGF group; ④UTMD group; ⑤MaFGF+ UTMD group. ②~⑤groups were intraperitoneally injected with doxorubicin to induce DOX-CM and given the corresponding interventions. Six weeks after intervention, all rats were underwent conventional echocardiography and velocity vector imaging (VVI) exams. Left ventricular internal dimension-diastole (LVIDd), left ventricular internal dimension-systole (LVIDs), left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were measured using conventional echocardiography. The segmental mean peak systolic radial velocity (Vs), systolic radial strain (Sr), and systolic radial strain rate (SRr) of six walls at the papillary muscle level were measured in left ventricular short-axis view by VVI. At last, myocardial tissues of all rats were stained with HE to observe the myocardial tissue morphology and with Van Gieson (VG) to calculate the collagen volume fractions (CVF). Results Six weeks after intervention, heart weight/body weight (HW/BW), LVIDs and LVIDd of MaFGF+ UTMD group were significantly decreased compared with those of DOX-CM group(P<0.05), while the LVEF, LVFS, Vs, Sc and Sr were significantly increased (P<0.05). Van Gieson stains showed that the collagen volume fractions (CVF) of DOX-CM groups significantly increased compared with that of the control group (P<0.01), while the CVF of MaFGF+ UTMD groups significantly decreased (P<0.01). Conclusions Delivery of MaFGF to myocardium in DOX-CM rats by UTMD could retard the formation of myocardial fibrosis and effectively protect the left ventricular function of DOX-CM rats. Key words: Echocardiography; Microbubbles; Acidic fibroblast growth factor; Cardiomyopathy; Doxorubicin

Changes in endogenous sulfur dioxide pathway in angiotensin II- induced myocardial hypertrophy in mice

Objective To explore the changes in the endogenous sulfur dioxide (SO2) pathway in the myocardial hypertrophy induced by the angiotensin Ⅱ(Ang Ⅱ) in mice. Methods Fourteen healthy C57BL mice, 9 weeks old, were randomly divided into control group(n=7) and Ang Ⅱ group(n=7), and capsule osmotic pump with pre-loaded 9 g/L saline and Ang Ⅱ was implanted into the back of each mouse subcutaneously.After 2 weeks, the mice were executed.The heart weight/body weight (HW/BW) and the left heart weight/full heart weight (LVW/HW) of the mice were measured.The microstructure of the cardiac myocyte was observed by hematoxylin-eosin (HE) staining under the microscope.The expression of myocardial alpha myosin heavy chain (α-MHC) was detected by immunohistochemistry and Western blot methods.SO2 enzymes aspartate aminotransferase 1 (AAT1) and AAT2 protein expression were detected by Western blot method.Myocardial SO2 content and AAT activity were measured by high performance liquid chromatography with fluorimetric detection and colometric method. Results Compared with control group, the HW/BW and LVW/HW in mice of Ang Ⅱ group were significantly increased(all P<0.01), the cardiac myocytes were hypertrophy, and α-MHC positive staining in the cytoplasm of myocardium was weakened.Moreover, Western blot data showed that α-MHC protein expression in heart tissue of Ang Ⅱ-treated mice was decreased significantly (all P<0.05). Simultaneously, the data showed that AAT2 protein expression, SO2 content and AAT activity in heart tissue of Ang Ⅱ-treated mice were also decreased markedly[(1.093±0.131) μmol/g protein vs.(0.737±0.233) μmol/g protein, P<0.05; (7.979±1.317) U/mg protein vs.(6.470±0.516) U/mg protein, P<0.01]. Furthermore, there was a negative correlation between LVW/HW and cardiac SO2 content in heart tissue (r=-0.56, P<0.05). Conclusions Myocardial endogenous SO2/AAT2 pathway is down-regulated in the development of myocardial hypertrophy induced by Ang Ⅱ in mice. Key words: Myocardial hypertrophy; Sulfur dioxide; Angiotensin Ⅱ