This study aimed to explore the mechanism of resveratrol(RES) pretreatment in improving mitochondrial function and alleviating myocardial ischemia-reperfusion(IR) injury by inhibiting stromal interaction molecule 2(STIM2) through microRNA-20 b-5 p(miR-20 b-5 p). Ninety rats were randomly assigned into sham group, IR group, IR+RES(50 mg·kg~(-1) RES) group, IR+RES+antagomir NC(50 mg·kg~(-1) RES+80 mg·kg~(-1) antagomir NC) group, and IR+RES+miR-20 b-5 p antagomir(50 mg·kg~(-1) RES+80 mg·kg~(-1) miR-20 b-5 p antagomir) group, with 18 rats/group. The IR rat model was established by ligation of the left anterior descending coronary artery. Two weeks before the operation, rats in the IR+RES group were intraperitoneally injected with 50 mg·kg~(-1) RES, and those in the sham and IR groups were injected with the same dose of normal saline, once a day. Ultrasonic instrument was used to detect the left ventricular internal diameter at end-diastole(LVIDd) and left ventricular internal diameter at end-systole(LVIDs) of rats in each group. The 2,3,5-triphenyte-trazoliumchloride(TTC) method and hematoxylin-eosin(HE) staining were employed to detect the myocardial infarction area and histopathology, respectively. Real-time quantitative PCR(qRT-PCR) was carried out to detect the expression of miR-20 b-5 p in myocardial tissue. Oxygen glucose deprivation/reoxygenation(OGD/R) was performed to establish an OGD/R model of H9 c2 cardiomyocytes. CCK-8 assay was employed to detect H9 c2 cell viability. H9 c2 cells were assigned into the control group, OGD/R group, OGD/R+RES group(25 μmol·L~(-1)), OGD/R+RES+inhibitor NC group, OGD/R+RES+miR-20 b-5 p inhibitor group, mimic NC group, miR-20 b-5 p mimic group, inhibitor NC group, and miR-20 b-5 p inhibitor group. Flow cytometry was employed to detect cell apoptosis. Western blot was employed to detect the expression of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), cleaved-cysteine proteinase 3(cleaved-caspase-3), and STIM2 in cells. The mitochondrial membrane potential(MMP) assay kit, reactive oxygen species(ROS) assay kit, and adenosine triphosphate(ATP) assay kit were used to detect the MMP, ROS, and ATP levels, respectively. Dual luciferase reporter gene assay was adopted to verify the targeting relationship between miR-20 b-5 p and STIM2. Compared with the sham group, the modeling of IR increased the myocardial infarction area, LVIDd, LVIDs, and myocardial pathology and down-regulated the expression of miR-20 b-5 p(P<0.05). These changes were alleviated in the IR+RES group(P<0.05). The IR+RES+miR-20 b-5 p antagomir group had higher myocardial infarction area, LVIDd, LVIDs, and myocardial pathology and lower expression of miR-20 b-5 p than the IR+RES group(P<0.05). The OGD/R group had lower viability of H9 c2 cells than the control group(P<0.05) and the OGD/R+RES groups(25, 50, and 100 μmol·L~(-1))(P<0.05). Additionally, the OGD/R group had higher H9 c2 cell apoptosis rate, protein levels of Bax and cleaved caspase-3, and ROS level and lower Bcl-2 protein, MMP, and ATP levels than the control group(P<0.05) and the OGD/R+RES group(P<0.05). The OGD/R+RES+miR-20 b-5 p inhibitor group had higher H9 c2 cell apoptosis rate, protein levels of Bax and cleaved-caspase 3, and ROS level and lower Bcl-2 protein, MMP, and ATP levels than the OGD/R+RES group(P<0.05). miR-20 b-5 p had a targeting relationship with STIM2. The expression of STIM2 was lower in the miR-20 b-5 p mimic group than in the mimic NC group(P<0.05) and lower in the inhibitor NC group than in the miR-20 b-5 p inhibitor group(P<0.05). RES pretreatment can inhibit the expression of STIM2 by promoting the expression of miR-20 b-5 p, thereby improving the function of mitochondria and alleviating myocardial IR damage.
Animals ; Rats ; Adenosine Triphosphate ; Antagomirs/metabolism* ; bcl-2-Associated X Protein/metabolism* ; Caspase 3/metabolism* ; Glucose/metabolism* ; MicroRNAs/metabolism* ; Mitochondria, Heart/drug effects* ; Myocardial Infarction/drug therapy* ; Myocardial Reperfusion Injury/drug therapy* ; Myocytes, Cardiac ; Oxygen/metabolism* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Rats, Sprague-Dawley ; Reactive Oxygen Species/metabolism* ; Resveratrol/therapeutic use* ; Stromal Interaction Molecule 2/metabolism*

OBJECTIVE: To investigate the effects of salvianolic acid A (SAA) on cardiomyocyte apoptosis and mitochondrial dysfunction in response to hypoxia/reoxygenation (H/R) injury and to determine whether the Akt signaling pathway might play a role. METHODS: An in vitro model of H/R injury was used to study outcomes on primary cultured neonatal rat cardiomyocytes. The cardiomyocytes were treated with 12.5, 25, 50 μg/mL SAA at the beginning of hypoxia and reoxygenation, respectively. Adenosine triphospate (ATP) and reactive oxygen species (ROS) levels were assayed. Cell apoptosis was evaluated by flow cytometry and the expression of cleaved-caspase 3, Bax and Bcl-2 were detected by Western blotting. The effects of SAA on mitochondrial dysfunction were examined by determining the mitochondrial membrane potential (△Ψm) and mitochondrial permeability transition pore (mPTP), followed by the phosphorylation of Akt (p-Akt) and GSK-3β (p-GSK-3β), which were measured by Western blotting. RESULTS: SAA significantly preserved ATP levels and reduced ROS production. Importantly, SAA markedly reduced the number of apoptotic cells and decreased cleaved-caspase 3 expression levels, while also reducing the ratio of Bax/Bcl-2. Furthermore, SAA prevented the loss of △Ψm and inhibited the activation of mPTP. Western blotting experiments further revealed that SAA significantly increased the expression of p-Akt and p-GSK-3β, and the increase in p-GSK-3β expression was attenuated after inhibition of the Akt signaling pathway with LY294002. CONCLUSION SAA has a protective effect on cardiomyocyte H/R injury; the underlying mechanism may be related to the preservation of mitochondrial function and the activation of the Akt/GSK-3β signaling pathway.
Adenosine Triphosphate ; analysis ; Animals ; Animals, Newborn ; Caffeic Acids ; pharmacology ; Cell Hypoxia ; Cells, Cultured ; Glycogen Synthase Kinase 3 beta ; physiology ; Lactates ; pharmacology ; Mitochondria, Heart ; drug effects ; physiology ; Mitochondrial Membrane Transport Proteins ; drug effects ; Myocytes, Cardiac ; drug effects ; Proto-Oncogene Proteins c-akt ; physiology ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; physiology

OBJECTIVE: To investigate whether CaN-NFAT3 pathway mediates the protective effects of aldehyde dehydrogenase (ALDH) 2 in high glucose-treated neonatal rat ventricular myocytes. METHODS: The ventricular myocytes were isolated from the heart of neonatal (within 3 days) SD rats by enzyme digestion and cultured in the presence of 5-Brdu. After reaching confluence, the cultured ventricular myocytes were identified using immunofluorescence assay for -SA protein. The cells were then cultured in either normal (5 mmol/L) or high glucose (30 mmol/L) medium in the presence of ALDH2 agonist Alda-1, ALDH 2 inhibitor Daidzin, or Alda-1 and NFAT3 inhibitor (11R-VIVIT). Fluorescent probe and ELISA were used to detect intracellular Ca concentration and CaN content, respectively; ALDH2, CaN and NFAT3 protein expressions in the cells were detected using Western blotting. RESULTS: Compared with cells cultured in normal glucose, the cells exposed to high glucose showed a significantly decreased expression of ALDH2 protein ( < 0.05) and increased expressions of CaN ( < 0.05) and NFAT3 proteins with also increased intracellular CaN and Ca concentrations ( < 0.01). Alda-1 treatment significantly lowered Ca concentration ( < 0.05), intracellular CaN content ( < 0.01), and CaN and NFAT3 protein expressions ( < 0.05), and increased ALDH2 protein expression ( < 0.05) in high glucose- exposed cells; Daidzin treatment significantly increased Ca concentration ( < 0.01) and intracellular CaN content ( < 0.05) in the exposed cells. Compared with Alda-1 alone, treatment of the high glucose-exposed cells with both Alda-1 and 11R-VIVIT did not produce significant changes in the expression of ALDH2 protein (>0.05) but significantly reduced the expression of NFAT3 protein ( < 0.05). CONCLUSIONS Mitochondrial ALDH2 protects neonatal rat cardiomyocytes against high glucose-induced injury possibly by negatively regulating Ca-CaN-NFAT3 signaling pathway.
Aldehyde Dehydrogenase, Mitochondrial ; antagonists & inhibitors ; metabolism ; Animals ; Animals, Newborn ; Benzamides ; pharmacology ; Benzodioxoles ; pharmacology ; Calcium ; metabolism ; Cells, Cultured ; Culture Media ; Enzyme Inhibitors ; pharmacology ; Glucose ; administration & dosage ; pharmacology ; Isoflavones ; pharmacology ; Mitochondria, Heart ; enzymology ; Myocytes, Cardiac ; drug effects ; metabolism ; NFATC Transcription Factors ; metabolism ; Nuclear Pore Complex Proteins ; metabolism ; Rats ; Rats, Sprague-Dawley

Sheng-Mai-San (SMS), a well-known Chinese medicinal plant formula, is widely used for the treatment of cardiac diseases characterized by deficiency of Qi and Yin syndrome. A mouse chronic intermittent hypoxia (CIH) model was established to mimic the primary clinical features of deficiency of Qi and Yin syndrome. Mice experienced CIH for 28 days (nadir 7% to peak 8% oxygen, 20 min per day), resulting in left ventricle (LV) dysfunction and structure abnormalities. After administration of SMS (0.55, 1.1, and 5.5 g·kg(-1)·d(-1)) for four weeks, improved cardiac function was observed, as indicated by the increase in the ejection fraction from the LV on echocardiography. SMS also preserved the structural integrity of the LV against eccentric hypotrophy, tissue vacuolization, and mitochondrial injury as measured by histology, electron microscopy, and ultrasound assessments. Mechanistically, the antioxidant effects of SMS were demonstrated; SMS was able to suppress mitochondrial apoptosis as indicated by the reduction of several pro-apoptotic factors (Bax, cytochrome c, and cleaved caspase-3) and up-regulation of the anti-apoptosis factor Bcl-2. In conclusion, these results demonstrate that SMS treatment can protect the structure and function of the LV and that the protective effects of this formula are associated with the regulation of the mitochondrial apoptosis pathway.
Animals ; Antioxidants ; pharmacology ; therapeutic use ; Apoptosis ; Cardiomyopathies ; drug therapy ; etiology ; Caspase 3 ; metabolism ; Cytochromes c ; metabolism ; Disease Models, Animal ; Drug Combinations ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Heart Ventricles ; drug effects ; pathology ; physiopathology ; Hypoxia ; Male ; Mice, Inbred ICR ; Mitochondria ; drug effects ; metabolism ; Myocardium ; pathology ; Oxygen ; metabolism ; Phytotherapy ; Qi ; Up-Regulation ; Ventricular Dysfunction, Left ; drug therapy ; etiology ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo investigate the influence of total flavonoids of Elsholtzia splendens (TFES) on isolated ischemia/reperfusion rat hearts and its underlying mechanisms.

METHODSHearts isolated from male SD rats were perfused on the Langendorff apparatus and subjected to global ischemia for 30 min followed by 120 min of reperfusion. The cardiac infarct size was measured by TTC staining. Hemodynamic parameters and the level of lactate dehydrogenase (LDH) in the coronary effluent were measured. Absorbance at 520 nm was determined in isolated cardiac mitochondria exposed to 200 micromol/L CaCl2 to detect the opening of the mitochondrial permeability transition pore.

RESULTSPretreatment with TFES (1, 10, 100 microg/ml) for 5 min decreased infarct size and LDH release and improved the recovery of the left ventricular developed pressure. In mitochondria, the decrease of absorbance at 520 nm evoked by CaCl2 was greatly inhibited by TFES.

CONCLUSIONTFES prevents myocardial ischemia/reperfusion injury, and this cardioprotective effect is probably via inhibiting mitochondrial permeability transition pore opening.

Animals ; Cardiotonic Agents ; pharmacology ; Disease Models, Animal ; Flavones ; pharmacology ; In Vitro Techniques ; Lamiaceae ; chemistry ; Male ; Mitochondria, Heart ; drug effects ; Mitochondrial Membrane Transport Proteins ; drug effects ; Myocardial Reperfusion ; Myocardial Reperfusion Injury ; prevention & control ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study the phosphorylation activity of mitochondrial signal transducer and activator of transcription 3 (STAT3) in the myocardium of rats with selenium deficiency and its association with myocardial injury.

METHODSThirty-six rats were randomized into normal control group (n=18) and selenium deficiency model group (n=18) for feeding with normal and low-selenium chow, respectively, for 20, 30 and 40 weeks. The cardiac function of the rats was evaluated by carotid artery intubation, and the damage of cardiac mitochondria was observed under electron microscopy. The cardiac mitochondria were extracted for assessing succinate dehydrogenase and cytochrome C oxidase activities, and the protein expressions of phosphorylated and total STAT3 were detected.

RESULTSCompared with the corresponding control groups, the rats in the model group showed significantly decreased cardiac function with obvious structural and functional damage of the cardiac mitochondria (P<0.05), which aggravated as the low-selenium feeding time extended (P<0.05). The rats in the model group also showed significantly decreased mitochondrial STAT3 activity (p-STAT3/STAT3) in the myocardium as the low-selenium feeding time prolonged (P<0.05). Pearson linear correlation analysis showed that the activity of cardiac mitochondrial STAT3 had positive correlations with the left ventricular systolic pressure, maximal increased rate of the left ventricular pressure, and the activities of succinate dehydrogenase and cytochrome C oxidase (P<0.01).

CONCLUSIONSelenium deficiency down-regulates the activity of mitochondrial STAT3 in rat heart to contribute to cardiac mitochondrial injury and the progression of heart failure.

Animals ; Diet ; Electron Transport Complex IV ; metabolism ; Female ; Heart Injuries ; metabolism ; Male ; Mitochondria, Heart ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley ; STAT3 Transcription Factor ; metabolism ; Selenium ; deficiency ; pharmacology ; Signal Transduction ; Succinate Dehydrogenase ; metabolism

Animals ; Codonopsis ; Drugs, Chinese Herbal ; pharmacology ; Fatigue ; Male ; Mitochondria, Heart ; drug effects ; metabolism ; Oxidative Stress ; Physical Conditioning, Animal ; Rats ; Rats, Sprague-Dawley ; Selenium

OBJECTIVETo explore changes of mitochondrial structure and functions, as well as the protection of ligustrazine in the process of myocardial hypertrophy.

METHODSNeonatal myocardial cells were isolated and cultured with angiotensin II (Ang II) for 72 or 96 h. The total protein content was detected using BCA method. The cell diameter was measured by inverted microscope, by which to reflect the proliferation situation of cardiomyocytes. The mitochondrial membrane potential (MMP) was measured by fluorescence microscope. The mitochondrial monoamine oxidase (MAO) activity was detected by spectrophotometer. The mitochondrial cytochrome oxidase (COX) activity and the mitochondrial damage percentage were detected by microplate reader, by which to reflect the damage of mitochondrial outer membrane's structure and the membranes' function. Also, cells were treated with ligustrazine and losartan and then the pharmacological effects on the mitochondrial structure and functions in the myocardial cells treated with Ang II were observed.

RESULTSAt 72 h and 96 h, when compared with the blank group, cells treated with Ang II had increased total protein content (P < 0.01) and enlarged diameter (P < 0.01). Treated with Ang II, the MAO activity and the outer membrane damage percentage of myocardial cells significantly increased (P < 0.01), and mitochondrial COX activity and the mitochondrial MMP significantly decreased (P < 0.01). Compared with the model group at the same time period, ligustrazine significantly reduced myocardial cells' total protein content and myocardial cell diameter, and significantly decreased myocardial cells' MAO activity, increased mitochondrial COX activity, improved the outer membrane damage percentage and inner membrane MMP at 72 and 96 h, all showing statistical difference (P < 0.01, P < 0.05).

CONCLUSIONSDuring the process of myocardial hypertrophy existed the damage to the mitochondrial structure and functions. Ligustrazine protected the mitochondrial structure and functions of the myocardial cells in reversing Ang II induced myocardial cell hypertrophy.

Angiotensin II ; adverse effects ; Animals ; Cardiomyopathy, Hypertrophic ; chemically induced ; metabolism ; pathology ; Cells, Cultured ; Electron Transport Complex IV ; metabolism ; Mitochondria, Heart ; drug effects ; enzymology ; Monoamine Oxidase ; metabolism ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; Pyrazines ; pharmacology ; Rats ; Rats, Sprague-Dawley

BACKGROUNDMitochondrial dysfunction plays a pivotal role in the progression of left ventricular (LV) remodeling and heart failure (HF). Recombinant human neuregulin-1 (rhNRG-1) improves cardiac function in models of experimental HF and in clinical trials; however, its impact on mitochondrial function during chronic HF remains largely unknown. The purpose of this study was to investigate whether rhNRG-1 could attenuate the functional and structural changes that occur in cardiac mitochondria in a rat model of HF induced by myocardial infarction.

METHODSSixty adult rats underwent sham or coronary ligation to induce HF. Four weeks after ligation, 29 animals with LV ejective fraction ≤ 50% were randomized to receive either vehicle or rhNRG-1 (10 µg×kg(-1)×d(-1), I.V.) for 10 days, another 12 sham-operated animals were given no treatment. Echocardiography was used to determine physiological changes. Mitochondrial membrane potential (MMP), respiratory function and tissue adenosine triphosphate (ATP) production were analyzed. Cytochrome c expression and cardiomyocyte apoptosis were determined. Oxidative stress was evaluated by reactive oxygen species production using fluorescence assays and gene expression of glutathione peroxidase measured by real-time quantitative PCR.

RESULTSCompared with sham-operated animals, vehicle treated HF rats exhibited severe LV remodeling and dysfunction, significant mitochondrial dysfunction, increased mitochondrial cytochrome c release, increased myocyte apoptosis and enhanced oxidative stress. Short-term treatment with rhNRG-1 significantly attenuated LV remodeling and cardiac function. Concomitant with this change, mitochondrial dysfunction was significantly attenuated; with ATP production, MMP and respiratory function restored, cytochrome c release and apoptosis inhibited, and oxidative stress reduced.

CONCLUSIONThe present study demonstrated that rhNRG-1 can significantly improve LV remodeling and cardiac function in the failing heart, this beneficial effect is related to reducing mitochondrial dysfunction, myocyte apoptosis and oxidative stress.

Animals ; Apoptosis ; drug effects ; Blotting, Western ; Echocardiography ; Heart Failure ; Mitochondria ; drug effects ; metabolism ; Myocardial Infarction ; drug therapy ; metabolism ; pathology ; Neuregulin-1 ; therapeutic use ; Rats ; Rats, Wistar ; Reactive Oxygen Species ; metabolism ; Real-Time Polymerase Chain Reaction

OBJECTIVETo explore the intervention effect and the possibly mechanism of the glutamine (Gln) on the opening change of the permeability transition pore (PTP) in the myocardial mitochondrial membrane under the overtraining state.

METHODS30 SD rats were randomly divided into 3 groups (n = 10): control group (CG group), overtraining group (OG group) and supplementary (Gln) + overtraining group group). Spectrophotometry was used to test the openness of the permeability transition pore in the myocardial mitochondrial membrane. Electrochemistry was used to test the malondialdehyde (MDA) and the glutathione (GSH) content and the phospholipase A2 (PLA2) activity.

RESULTSOG group compared with the GOG group, the absorbance (A0) and the absorbance change (Delta A) were decreased significantly (P < 0.05). Rh123 fluorescence (F0) intensity was significantly increased (P < 0.05). Rhodamine123 (Rh123) fluorescence change (delta F) was significantly decreased (P < 0.05). Compared with the GOG, the mitochondrial GSH was significantly decreased (P < 0.05), the PLA2 activity and the content of MDA were significantly increased (P <0.05).

CONCLUSIONOvertraining could lead to opening increase of permeability transition pore in the myocardial mitochondrial membrane, after overtraining, the production of the reactive oxygen species (ROS) and PLA2 activity were increased, GSH content was decreased. But added exogenous Gln had a significant intervention effect for these changes.

Animals ; Glutamine ; pharmacology ; Glutathione ; metabolism ; Male ; Mitochondria, Heart ; drug effects ; physiology ; Mitochondrial Membrane Transport Proteins ; metabolism ; Mitochondrial Membranes ; drug effects ; physiology ; Myocardium ; metabolism ; Permeability ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism