Abstract 337: Activation of Myocardial AMP-activated Protein Kinase by Metformin Prevents Progression of Heart Failure in Dogs; Involvement of eNOS Activation

Background; Several studies have shown that metformin activates AMP-activated protein kinase (AMPK), which mediates potent cardioprotection against ischemia-reperfusion injury. AMPK is also activated in experimental failing myocardium, suggesting that activation of AMPK is beneficial for the pathophysiology of heart failure. We investigated whether metformin prevents oxidative stress-induced cell death in rat cardiomyocytes and attenuates the progression of heart failure in dogs. Methods and Results; The treatment with metformin (10 μmol/L) protected the rat cultured cardiomyocytes against cell death due to H 2 O 2 exposure (50 μmol/L) as indicated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), TUNEL staining, and flow cytometry. These effects were blunted by an AMPK inhibitor, compound-C (20 μmol/L), suggesting that the activation of AMPK decreased the extent of apoptosis-induced cell death due to H 2 O 2 exposure. Continuous rapid ventricular pacing (230/min for 4 weeks) in dogs caused heart failure and the treatment with metformin (100 mg/kg/day PO, n=8) decreased left ventricular (LV) end-diastolic dimension (32.8±0.4 vs. 36.5±1.0 mm, p< 0.01) and pressure (11.8±1.1 vs. 22±0.9 mmHg, p< 0.01), and increased LV fractional shortening (18.6±1.8 vs. 9.6±0.7 %, p< 0.01) along with enhanced phosphorylation of AMPK and the decreased the number of TUNEL-positive cells of the LV myocardium compared with the vehicle group (n=8). Interestingly, metformin increased the protein and mRNA levels of endothelial nitric oxide synthase of the LV myocardium and plasma nitric oxide levels. Metformin improved the plasma insulin resistance without increased myocardial GLUT-4 translocation. Furthermore, the subcutaneous administration of AICAR (50 mg/kg/every other day), another AMPK activator mediated the equivalent effects to metformin, strengthening the pivotal role of AMPK in reduction of apoptosis and prevention of heart failure. Conclusions; Activation of myocardial AMPK attenuated the oxidative stress-induced cardiomyocyte apoptosis and prevented the progression of heart failure in dogs, along with eNOS activation. Thus, metformin or AICAR may be applicable as a novel therapy for heart failure.

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Abstract 565: The Activation of AMP-Activated Protein Kinase Ameliorates the Severity of Heart Failure in Dogs

Circulation ◽

10.1161/circ.116.suppl_16.ii_102-a ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Hideyuki Sasaki ◽

Hiroshi Asanuma ◽

Masashi Fujita ◽

Hiroyuki Takahama ◽

Masakatsu Wakeno ◽

...

Keyword(s):

Heart Failure ◽

Protein Kinase ◽

Cardiac Myocytes ◽

Left Ventricular ◽

Neonatal Rat ◽

Cellular Damage ◽

Control Group ◽

Compound C ◽

Rapid Pacing ◽

Amp Activated Protein Kinase

Backgrounds; Since AMP-activated protein kinase (AMPK) is activated in the pressure-overloaded hypertrophic hearts, we investigated whether the activation of AMPK caused by metformin attenuates the progression of heart failure induced by rapid pacing in dogs and decreases cellular damage caused by oxidative stress in neonatal rat cardiac myocytes. Methods and Results; Heart failure was induced by right ventricular (RV) pacing at 230 bpm for 4 weeks in dogs. Treatment of dogs with metformin (100mg/kg/day, orally, n=8, Met group) for 4 weeks prevented significantly the progression of pacing-induced heart failure evaluated by echocardiographical and hemodynamic measurement compared with the control group (n=8). Left ventricular (LV) diastolic and systolic dimension (LVDd and LVDs) were smaller (32.8±0.4 and 26.7±0.9 mm, respectively) and fractional shortening (FS) and ejection fraction (EF) were preserved in Met group (18.6±1.8 and 45.5±3.5 %, respectively) compared with the control group (LVDd and LVDs; 36.5±1.0 and 33.0±1.0 mm, FS and EF; 9.6±0.7 and 27.0±1.9 %, p<0.05 vs. Met group each). Furthermore, both pulmonary capillary wedge pressure (PCWP) and mean pulmonary arterial pressure (mPA) were significantly lower in Met group (11.1±0.9 and 18.1±1.4 mmHg, respectively) compared with the control group (21.0±2.2 and 26.8±2.8 mmHg, respectively). Treatment of cultured cardiac myocytes with a maximal physiological concentration of metformin (10μmol/L) attenuated the cellular damage against H 2 O 2 exposure (50μmol/L). These effects were blunted by an AMPK inhibitor, compound-C (20μmol/L), suggesting that the activation of AMPK increased the cellular viability during H 2 O 2 exposure. Conclusions; Metformin that activates AMPK prevented the progression of heart failure induced by rapid pacing in dogs and attenuated the cellular damage against H 2 O 2 exposure in cardiac myocytes. AMPK may be one of new targets for preventing heart failure in clinical settings.

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Abstract 186: AMP-Activated Protein Kinase Mediates Cardioprotection in the Regenerative MRL Mouse Strain

Circulation Research ◽

10.1161/res.111.suppl_1.a186 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Susan T Varghese ◽

Jenan Holley-Cuthrell ◽

Chad M Warren ◽

Ann Kuenster ◽

Ahlke Heydemann

Keyword(s):

Oxidative Stress ◽

Protein Kinase ◽

Systolic Function ◽

Fold Increase ◽

Atp Depletion ◽

Mrna Levels ◽

Post Exercise ◽

Protein Levels ◽

Compound C ◽

Amp Activated Protein Kinase

Introduction: The Murphy Roths Large (MRL/MpJ) mouse model is well-known for its healing and regenerative properties. The actual mechanisms promoting regeneration are still unknown. AMP activated protein kinase (AMPK) has been lauded for its role as a cardioprotective sensor of cellular metabolic stress, cell growth, and ATP depletion. Based on our preliminary data, we hypothesize that increased levels of AMPK in the MRL hearts is primarily responsible for its altered mitochondrial biogenesis and metabolism, reduced oxidative stress, and altered cardiac function in the MRL mice. Methods: Adult C57BL/6 (control) and MRL mice (n=5/group) were studied pre- and post-7 days of exercise, and post-exercise with 3 days injection of either 5-amino -imidazole carboxamide riboside (AICAR, AMPK stimulator, 250ug/g, IP), or Compound C (AMPK inhibitor, 20ug/g, IP). Results: Compared to control at baseline, the MRL hearts had higher AMPK protein levels (9 fold increase, p<0.001), equivalent AMPK mRNA levels by qRT-PCR, and higher AMP to ATP ratios by HPLC (3 fold increase, p>0.05, n.s.). The MRL hearts also had increased mitochondria by electron microscopy (by 65 ± 4.5%, p<0.05), reduced HW to BW ratio (48% decrease, p<0.001), reduced cellular and mitochondrial reactive oxygen species (by DHE and Mitosox stains respectively), and increased glycolysis (Glut-4 sarcolemmal translocation). Hypertrophic response to exercise was greater in the MRL mice (HW/BW increased by 40% vs. 2% in control, p<0.001). Mimicking injury, Compound C reduced active phosphorylated AMPK protein levels in control (by 26%, p<0.05), while increased it in MRL mice (by 30%, p<0.05). AICAR injection significantly increased pAMPK protein in both strains (p=0.008). Baseline MRL mice echocardiographic analysis (vs. control) indicated higher systolic function, but increased diastolic E/A and E/E’ ratios (p<0.05). In the MRL hearts, Compound C treatment reduced %EF and %FS, while AICAR reduced post-exercise hypertrophy (p=0.02). Conclusions: This study highlights the cardioprotective role AMPK plays in the MRL mice, through its modulation of mitochondrial function and reduction of oxidative stress. This can in turn be used to develop new therapeutic treatment for heart failure.

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Abstract 814: Atorvastatin Attenuates Oxidative Stress And Improves Neuronal Nitric Oxide Synthase In The Brain Stem Of Heart Failure Mice

Circulation ◽

10.1161/circ.116.suppl_16.ii_157-d ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Joseph Francis ◽

Li Yu ◽

Anuradha Guggilam ◽

Srinivas Sriramula ◽

Irving H Zucker

Keyword(s):

Oxidative Stress ◽

Nitric Oxide ◽

Myocardial Infarction ◽

Heart Failure ◽

Brain Stem ◽

Mrna Expression ◽

Natriuretic Peptide ◽

Left Ventricular ◽

Neuronal Nos ◽

The Brain

3-Hydroxyl-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been shown to reduce the incidence of myocardial infarction independent of their lipid-lowering effects. Nitric oxide (NO) in the central nervous system contributes to cardiovascular regulatory mechanisms. Imbalance between nitric oxide (NO) and superoxide anion (O 2 . − ) in the brain may contribute to enhanced sympathetic drive in heart failure (HF). This study was done to determine whether treatment with atorvastatin (ATS) ameliorates the imbalance between NO and O 2 . − production in the brain stem and contributes to improvement of left ventricular (LV) function. Methods and Results: Myocardial infarction (MI) was induced by ligation of the left coronary artery or sham surgery. Subsequently, mice were treated with ATS (10 μg/kg) (MI + ATS), or vehicle (MI + V). After 5 weeks, echocardiography revealed left ventricular dilatation in MI mice. Realtime RT-PCR indicated an increase in the mRNA expression of the LV hypertrophy markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). Neuronal NOS (nNOS) and endothelial NOS (eNOS) mRNA expression were significantly reduced, while that of NAD(P)H oxidase subunit (gp91phox) expression was elevated in the brain stem of MI mice. Compared with sham-operated mice, ATS-treated mice showed reduced cardiac dilatation, decreased ANP and BNP in the LV. ATS also reduced gp91phox expression and increased nNOS mRNA expression in the brain stem, while no changes in eNOS and iNOS were observed. Conclusion: These findings suggest that ATS reduces oxidative stress and increases neuronal NOS in the brain stem, and improves left ventricular function in heart failure.

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Role of inducible nitric oxide synthase in cardiac function and remodeling in mice with heart failure due to myocardial infarction

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00546.2005 ◽

2005 ◽

Vol 289 (6) ◽

pp. H2616-H2623 ◽

Cited By ~ 77

Author(s):

Yun-He Liu ◽

Oscar A. Carretero ◽

Oscar H. Cingolani ◽

Tang-Dong Liao ◽

Ying Sun ◽

...

Keyword(s):

Oxidative Stress ◽

Nitric Oxide ◽

Heart Failure ◽

Cardiac Function ◽

Systolic Function ◽

Left Ventricular ◽

Cross Sectional ◽

Cardiac Reserve ◽

Inos Knockout Mice ◽

Inducible Nitric Oxide

Using inducible nitric oxide (NO) synthase (iNOS) knockout mice (iNOS−/−), we tested the hypotheses that 1) lack of iNOS attenuates cardiac remodeling and dysfunction and improves cardiac reserve postmyocardial infarction (MI), an effect that is partially mediated by reduction of oxidative stress due to reduced interaction between NO and reactive oxygen species (ROS); and 2) the cardioprotection afforded by iNOS deletion is eliminated by Nω-nitro-l-arginine methyl ester (l-NAME) due to inhibition of endothelial NOS (eNOS) and neuronal NOS (nNOS). MI was induced by ligating the left anterior descending coronary artery. Male iNOS−/− mice and wild-type controls (WT, C57BL/6J) were divided into sham MI, MI+vehicle, and MI+l-NAME (100 mg·kg−1·day−1 in drinking water for 8 wk). Cardiac function was evaluated by echocardiography. Left ventricular (LV) maximum rate of rise of ventricular pressure divided by pressure at the moment such maximum occurs (dP/d t/instant pressure) in response to isoproterenol (100 ng·kg−1·min−1 iv) was measured with a Millar catheter. Collagen deposition, myocyte cross-sectional area, and expression of nitrotyrosine and 4-hydroxy-2-nonenal (4-HNE), markers for ROS, were determined by histopathological and immunohistochemical staining. We found that the MI-induced increase in LV chamber dimension and the decrease in ejection fraction, an index of systolic function, were less severe in iNOS−/− compared with WT mice. l-NAME worsened LV remodeling and dysfunction further, and these detrimental effects were also attenuated in iNOS−/− mice, associated with better preservation of cardiac function. Lack of iNOS also reduced nitrotyrosine and 4-HNE expression after MI, indicating reduced oxidative stress. We conclude that iNOS does not seem to be a pathological mediator of heart failure; however, the lack of iNOS improves cardiac reserve post-MI, particularly when constitutive NOS isoforms are blocked. Decreased oxidative stress and other adaptive mechanisms independent of NOS may be partially responsible for such an effect, which needs to be studied further.

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Cardioprotective role of myocardial endothelial nitric oxide synthase and serum nitric oxide induced by hexarelin in rats with myocardial infarction-induced heart failure

10.32592/ircmj.2021.23.6.395 ◽

2021 ◽

Keyword(s):

Oxidative Stress ◽

Nitric Oxide ◽

Myocardial Infarction ◽

Heart Failure ◽

Nitric Oxide Synthase ◽

Endothelial Nitric Oxide Synthase ◽

Left Ventricular ◽

Nitric Oxide Level ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

Background: Growth hormone-releasing peptides (GHRP) have been reported to possess cardioprotective properties; nonetheless, their mechanisms of action are still not very clear. Objectives: Some studies have suggested that modulation of endothelial nitric oxide synthase (eNOS) and the upregulation of nitric oxide (NO) are cardioprotective. Therefore, the present study strived to test the hypothesis that a potent GHRP analog (hexarelin) could increase serum nitric oxide level and regulate myocardial eNOS to alleviate the development of heart failure. Methods: Myocardial infarction-induced heart failure in rats was established by permanent coronary artery ligation. The sham group, control group, and heart failure group all received normal saline (100 µg/kg; SC BID; 30days), while the rats in the hexarelin treatment group were treated with hexarelin (100 µg/kg, SC BID, 30 days). The rats were tested for myocardial apoptosis, oxidative stress, left ventricular function, various molecular analyses, as well as pathological and structural myocardial changes. Results: Hexarelin treatment improved contractile function and attenuated myocardial histopathological damages, oxidative stress, ﬁbrosis, as well as apoptosis. All these were accompanied by the upregulation of myocardial eNOS and an increase in serum NO concentration. Conclusion: As evidenced by the obtained results, the anti-cardiac failure capacity of hexarelinin in a rat model is mediated by an increase in serum nitric oxide level and the up-modulation of myocardial eNOS; therefore, they can be considered therapeutic targets against heart failure.

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Abstract P381: Ecklonia Stolonifera Okamura Extract Suppresses Hypertrophic Responses In Cardiomyocytes And Development Of Heart Failure By Inhibiting P300-HAT Activity

Circulation Research ◽

10.1161/res.129.suppl_1.p381 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

Takahiro Katagiri ◽

Yoichi Sunagawa ◽

Masafumi Funamoto ◽

Yasufumi Katanasaka ◽

Yusuke Miyazaki ◽

...

Keyword(s):

Heart Failure ◽

Gene Transcription ◽

Left Ventricular ◽

Vehicle Group ◽

Cardiomyocyte Hypertrophy ◽

Cell Diameter ◽

Mrna Levels ◽

Ecklonia Stolonifera ◽

Hypertrophic Response ◽

Cultured Cardiomyocytes

Introduction: Heart failure is the leading cause of death in the world. Cardiomyocyte hypertrophy is observed during the development of heart failure, suggesting that its inhibition is a potential target for the prevention and treatment of heart failure. In this study, we screened a natural compound library using cultured cardiomyocytes and found that Ecklonia stolonifera Okamura extract (ESE) suppressed cardiomyocyte hypertrophy. ESE, a perennial brown alga, has been reported to have various bioactive effects, such as antioxidant and anti-inflammatory activity, but its effect on heart failure is still unclear. Therefore, we investigated whether ESE has an inhibitory effect on cardiomyocyte hypertrophic response and on the progression of heart failure in post-myocardial infarction (MI) rats. Methods and Results: First, primary cultured cardiomyocytes from neonatal rats were treated with ESE and then stimulated with phenylephrine (PE) for 48 hours. ESE (1000 μg/mL) significantly suppressed PE-induced increases in cardiomyocyte surface area, hypertrophic response gene transcription, and acetylation of histone H3K9. An in vitro p300-HAT assay indicated that ESE directly inhibited p300-HAT activity (IC50: 505 μg/mL). Next, one week after the ligation of the left anterior descending artery, rats with moderate MI (left ventricular fractioning shorting (LVFS) <40%) were randomly assigned to three groups: vehicle (saline) (n=9), ESE (0.3 g/kg) (n=10), or ESE (1 g/kg) (n=10). Daily oral administration was repeated for 8 weeks. After treatment, LVFS was significantly higher in the ESE (1 g/kg) group (23.3 ± 0.7%, p<0.05) than in the vehicle group (16.6 ± 1.3%). Next, the hearts were isolated and histological analysis, evaluation of gene transcription, and measurement of histone H3K9 acetylation. were performed. ESE treatment significantly suppressed MI-induced increases both in myocardial cell diameter and in the mRNA levels of hypertrophic response genes. ESE also inhibited MI-induced perivascular fibrosis and the acetylation of histone H3K9. Conclusion: These results suggest that ESE suppresses both hypertrophic responses in cardiomyocytes and the development of heart failure by inhibiting p300-HAT activity. Further studies are needed to clarify the effectiveness of ESE for heart failure therapy.

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Activation of AMP-Activated Protein Kinase by Metformin Improves Left Ventricular Function and Survival in Heart Failure

Circulation Research ◽

10.1161/circresaha.108.190918 ◽

2009 ◽

Vol 104 (3) ◽

pp. 403-411 ◽

Cited By ~ 245

Author(s):

Susheel Gundewar ◽

John W. Calvert ◽

Saurabh Jha ◽

Iris Toedt-Pingel ◽

Sang Yong Ji ◽

...

Keyword(s):

Heart Failure ◽

Protein Kinase ◽

Left Ventricular Function ◽

Ventricular Function ◽

Left Ventricular ◽

Amp Activated Protein Kinase

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MicroRNA-137-3p Improves Nonalcoholic Fatty Liver Disease through Activating AMPKα

Analytical Cellular Pathology ◽

10.1155/2021/4853355 ◽

2021 ◽

Vol 2021 ◽

pp. 1-13

Author(s):

Yuanjie Yu ◽

Chunping He ◽

Shiyun Tan ◽

Mengjun Huang ◽

Yitian Guo ◽

...

Keyword(s):

Oxidative Stress ◽

Liver Disease ◽

Protein Kinase ◽

Fatty Liver ◽

Nonalcoholic Fatty Liver Disease ◽

Fatty Liver Disease ◽

Nonalcoholic Fatty Liver ◽

Compound C ◽

High Prevalence ◽

Amp Activated Protein Kinase

Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases worldwide and can develop to nonalcoholic steatohepatitis and later hepatic cirrhosis with a high prevalence to hepatocellular carcinoma. Oxidative stress and chronic hepatic inflammation are implicated in the pathogenesis of NAFLD. MicroRNA-137-3p (miR-137-3p) are associated with oxidative stress and inflammation; however, its role and mechanism in NAFLD remain unclear. Mice were fed with a high-fat diet (HFD) for 24 weeks to establish the NAFLD model. To overexpress or suppress hepatic miR-137-3p expression, mice were intraperitoneally injected with the agomir, antagomir, or respective controls of miR-137-3p at a dose of 100 mg/kg weekly for 6 consecutive weeks before the mice were sacrificed. To validate the involvement of AMP-activated protein kinase alpha (AMPKα) or cAMP-specific phosphodiesterase 4D (PDE4D), HFD mice were intraperitoneally injected with 20 mg/kg compound C or 0.5 mg/kg rolipram every other day for 8 consecutive weeks before the mice were sacrificed. Hepatic miR-137-3p expression was significantly decreased in mice upon HFD stimulation. miR-137-3p agomir alleviated, while miR-137-3p antagomir facilitated HFD-induced oxidative stress, inflammation, and hepatic dysfunction in mice. Mechanistically, we revealed that miR-137-3p is directly bound to the 3 ′ -untranslated region of PDE4D and subsequently increased hepatic cAMP level and protein kinase A activity, thereby activating the downstream AMPKα pathway. In summary, miR-137-3p improves NAFLD through activating AMPKα and it is a promising therapeutic candidate to treat NAFLD.

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Abstract 259: Dysregulation of AMP-activated Protein Kinase-alpha Isoforms in Left Ventricular Myocardium of Dogs With Chronic Heart Failure

Circulation Research ◽

10.1161/res.127.suppl_1.259 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Ramesh C Gupta ◽

Vinita Singh-Gupta ◽

Hani N Sabbah

Keyword(s):

Heart Failure ◽

Protein Kinase ◽

Chronic Heart Failure ◽

Protein Expression ◽

Mrna Expression ◽

Left Ventricular ◽

Nuclear Fraction ◽

Α Subunit ◽

Protein Levels ◽

Amp Activated Protein Kinase

Background: The adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) plays critical roles in regulating cellular growth and metabolism and is activated when cellular ATP levels decrease. The heterotrimeric structure of AMPK consists of a catalytic alpha (α) subunit and regulatory beta (β) and gamma (γ) subunits. The two isoforms of the catalytic α subunit have different cellular localization: AMPKα1 is predominantly found in the non-nuclear fraction and AMPKα2 is found in both the nuclear and the non-nuclear fractions. Both AMPKα isoforms reside in cardiomyocytes, increased expression of AMPKα1 during stress can trigger an increased inflammatory state whereas AMPKα2 has recently been recognized as a regulator of mitophagy, the selective degradation of damaged mitochondria by autophagy. Dysregulation in AMPKα1 and AMPKα2 has been reported in explanted failed human hearts. This study tested the hypothesis that mRNA and protein expression levels of AMPKα1 increase and that of AMPKα2 decrease in LV myocardium of dogs with chronic heart failure (HF) produced by microembolization. Methods: RNA was extracted and sodium dodecyl sulfate (SDS)-extract homogenate prepared from LV tissue of 6 dogs with microembolization-induced HF and 6 normal (NL) dogs. mRNA expression of AMPKα1 and AMPKα2 was measured using real-time PCR and normalized to GAPDH. Protein expression of AMPKα1 and AMPKα2 was measured by Western blotting and normalized to GAPDH. Bands were quantified in densitometric units (du). Results: mRNA and protein levels of GAPDH, used as internal control, were similar between NL and HF dogs. In HF dogs, mRNA expression of AMPKα2 was reduced by 5.09 fold and protein levels were reduced by 2.08 fold (1.02 ± 0.09 vs. 2.12 ± 0.18 du, p<0.05) compared to NL dogs. In HF dogs, AMPKa1 mRNA and protein levels were increased 5.67 fold and 2.09 fold (0.67 ± 0.10 vs. 0.32 ± 0.03 du, p<0.05), respectively compared to NL dogs. Conclusions: The results indicate that in LV myocardium of dogs with chronic HF, mRNA and protein levels for AMPKα1 are upregulated while AMPKα2 levels are downregulated compared to NL dogs. This AMPK isoform shift can trigger abnormalities of mitochondrial respiration and turnover that contribute to an abnormal energetic state characteristic of the failing heart.

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