The Effects And Mechanisms Of Autophagy And Mitochondrial Coenzyme Q On Cardiac Function Of Rats With Sepsis

Backgroud&ObjectiveDespite major advances in critical care management and antibiotic therapies, Sepsis remains the leading cause of death in ICU patients, from which the mortality rate have been estimated to range between 40% to 60% in some developed countries.The death with sepsis mainly results from the multiple organ dysfunction syndrome(MODS). Cardiac dysfunction is an important part of MODS with sepsis, from whom with cardiac dysfunction will gain more 50% death rate. Sepsis has gained such a high rate results from its complex pathophysiological changes. Current study results suggest that oxidative stress, mitochondrial damage, excessive reactive oxygen species (ROS) generation is the main sources of the pathogenesis of sepsis, from which the heart mitochondria covers 30% from where will be easily affected by ROS, so the mitochondrial damage, ROS is the main cause of sepsis with cardiac dysfunction, but the exact pathogenesis remains incompletely understood.The main function of autophagy is for recycling of intracellular macromolecules, removing the damaged cellular organelles, intracellular pathogen clearance, from which can maintain cellular homeostasis, enough supply of cellular energy and at the end protect the cell homeostasis. However, excessive autophagy may also cause the death of autophagic cell. Some studies have reported that the increased cardiac autophagy have been observed in animal models and clinical sepsis patients, but the cardiac dysfunction in sepsis would be protected or damaged by the autophagy is still needed to be demonstrated.Since oxidative stress, mitochondrial injury, excessive ROS generation is the major factor in cardiac dysfunction in sepsis, Antioxidants are considered to be the most promising treatment to the cardiac dysfunction in sepsis in theory, but antioxidants did not achieve satisfactory results in clinical experiments and animal studies which may be associated with these antioxidants metabolized in the body can not reach the main site of the mitochondrial oxidative stress occurred. On the basis of this logic, mitochondrial coenzyme Q (MitoQ) will be more protective to the ROS-induced damage to organs and tissues through the mitochondrial antioxidants.The aim of the present study was to investigate the effects and changes of autophagy on cardiac dysfunction in rats with sepsis through LPS-induced rats in vivo and LPS-treated cardiomyocytes in vitro, and whether MitoQ would prevent cardiomyocytes systolic and diastolic dysfunction induced by LPS via autophagy alleviated endoplasmic reticulum stress. In addition, in order to elucidate the protective mechanisms, whether MitoQ administration would decreased the function of autophagy by reduced ROS generation.Methods1. The effects of autophagy and MitoQ on the survival rate in rats with sepsisSprague Dawley (SD) rats were randomly divided into five groups (n=10 in each group):lipopolysaccharide(LPS) group, LPS + Wortmannin group, LPS + MitoQ group, Wortmannin group, and MitoQ group. Rats in LPS group, LPS + Wortmannin group and LPS + MitoQ group were intraperitoneally administered LPS(10 mg/kg) and followed by an injection of Wortmannin(2 mg/kg) and MitoQ(6.5 μmol/kg) via tail vein 1 hour later, respectively. Rats in each group were given same amount of normal sodium in addition to different interventional drugs. Animal survival and the presence were observed every 1 h for 12 h after the intraperitoneally injection.2. The effects of autophagy and MitoQ on cardiac function in rats with sepsis2.1 Animal model and groupingSD rats were randomly were divided into ten groups (n = 10 each):the control 4 h and 6 h groups, LPS 4 h,6 h,8 h and 12 h groups, LPS + Wortmannin 4 h group, Wortmannin 4 h group, LPS + MitoQ 6 h group and MitoQ 6 h group. Rats in all group including LPS were intraperitoneally administered LPS(10 mg/kg) and followed by an injection of Wortmannin(2 mg/kg) and MitoQ(6.5 μmol/kg) via tail vein 1 hour later, respectively. Rats in other group were given same amount of normal sodium in addition to different interventional drugs.2.2 Inserted tube of left ventricle and data recordingThe rats in each group were anesthetized with 10% chloralhydrate (0.3 ml/kg) by intraperitoneal injection at the appropriate time points. Separation of the right common carotid artery, Inserted PE50 tube. The cardiac function parameters were measured by a BL-420E+ biosignal collection system.2.3 The collection of plasma samples and observation of myocardial tissue pathologyAfter the left ventricular pressure measurement, blood samples from abdominal aorta were taken at each time point. The plasma of separation were placed in a-70℃ freezer as backup. The myocardial tissues were removed and rinsed thoroughly with cold phosphate-buffered saline (PBS). One part was treated for biopsy, another part was fixed for the production of electron microscope specimens, and the remaining part was frozen in liquid nitrogen and placed in a-70℃ freezer until analysis.The pathological changes of myocardial tissues were observed by light and electronic microscopy.2.4 Detection of creatinine kinase MB isoenzyme (CK-MB) and lactate dehydrogenase (LDH)A Beckman automatic biochemical analyzer from the United States was used to measure plasma CK-MB and LDH levels.2.5 Determination of IL-1β, TNF-a and ROS in plasma and myocardial tissuesThe IL-1β, TNF-a and ROS contents were measured with an enzyme-linked immunosorbent assay (ELISA), according to the kit instructions.2.6 Determination of activity of Ca2+-Mg2+-ATPase in myocardial tissuesActivity of Ca+-Mg+-ATPase in myocardial tissues was assayed by spectrophotometric method.2.7 Determination of LC3 and GRP78 expression in myocardial tissues with immunoblotting3. Autophagy improve LPS-challenged cardiomyocytes contraction and diastolic dysfunction via alleviating endoplasmic reticulum stress3.1 Isolation of cardiomyocytes from ratsSD male Rats were intraperitoneally administered heparin (2500 U/kg). After 30 minutes, rats were anesthetized with 10% chloralhydrate (0.4 ml/kg) by intraperitoneal injection. Cardiomyocytes were isolated with Enzymatic hydrolysis, using a Langendorff perfusion rig. Isolated cardiomyocytes were used later experimention within 8 h. According to differented objective, aliquots of myocardial cells were pretreated with LPS(100 ng/ml、200 ng/mh、1 μg/ml、4 μg/ml), tauroursodeoxycholic acid (TUDCA,500 μM), MitoQ(1μmol/L),Wortmannin (200nM) at 37℃ for 2 h before cardiomyocytes mechanical function, cardiomyocytes intracellular calcium balance, activity of Ca2+-Mg2+-ATPase, intracellular ROS levels and expression of LC3 and GRP78 were detected.3.2 Assessment of cardiomyocytes contraction and diastolic functionPeak shortening and ±dL/dt of the isolated myocardial cell was measured by video based motion edge detection system (IonOptix).3.3 Assessment of cardiomyocytes diastolic calcium and calcium transientsIsolated cardiomyocytes were loaded with Fluo-4/AM. Cardiomyocytes diastolic calcium and calcium transients were assessed by the video based motioned detection system.3.4 Determination of activity of Ca2+-Mg2+-ATPase in supernatant of cardiomyocytesActivity of Ca+-Mg+-ATPase in myocardial tissues was assayed by spectrophotometric method. BCA method was used to measure protein concentration.3.5 Determination of concentrations of CK-MB and LDH in supernatant of cardiomyocytesConcentrations of CK-MB and LDH in supernatant in cardiomyocytes were assayed by spectrophotometric method. The manufacturer’s instructions were followed when performing these assays.3.6 Determination of levels of intracellular ROS in cardiomyocytesLevels of intracellular ROS in cardiomyocytes were monitored by the fluorescent DCFH-DA. The changes of fluorescence intensity determinated the levels of intracellular ROS in cardiomyocytes.3.7 Determination of LC3 and GRP78 expression in cardiomyocytes with immunoblottingResults1. General performance and survivalAbout 1 h after the intraperitoneal injection of LPS, typical clinical symptoms of endotoxemia were observed in the rats, including listlessness, diarrhea, piloerection, incontinence, reduced activity, reduced eating, reduced drinking, and so on. The symptoms in the LPS + MitoQ group were not alleviated relative to those of the LPS group, whereas those in the LPS+Wortmannin group were more pronounced and hematuric symptoms appeared. At 12 h after the LPS injection, two rats had died in the LPS group, four rats had died in the LPS + MitoQ group, and all the rats had died in the LPS + Wortmannin group. The survive rate of the rats in LPS + MitoQ group, LPS + Wortmannin group and LPS group were 60%,0%, and 80%, respectively. The difference between group were significantly ( X2=14.882, P=0.001).The survive rate of rats in LPS + Wortmannin group was significantly lower than that in LPS group (X2=10.208, P=0.001). The survival time in the LPS + Wortmannin group was significantly shorter than that in the LPS group ((7.5±0.64) h vs (11.9±0.13) h, X2=19.847, P=0.001). The survival time of the LPS + MitoQ and LPS groups did not differ significantly ((11.6±0.24) h vs (11.9±0.13) h,X2=1.055, P=0.137).2. The effects of autophagy and MitoQ on cardiac function in rats with sepsis2.1 Detection of creatinine kinase MB isoenzyme (CK-MB) and lactate dehydrogenase (LDH)The plasma CK-MB and LDH levels in the rats of the LPS 6 h group were all higher than that of the same time control group (all P<0.05). The levels of plasma CK-MB and LDH in LPS + Wortmannin 4 h group were all higher than that of the LPS 4 h group (all P<0.05), whereas that of the LPS + MitQ group were still higher than that of the control group (all P<0.05).2.2 Determination of activity of Ca2+-Mg2+-ATPase in myocardial tissuesThe Ca2+-Mg2+-ATPase activity of myocardial tissues in LPS 6 h group was significantly lower than that in control (F=3.049, P=0.016). Compared with LPS 4 h group, Ca2+-Mg2+-ATPase activity in LPS + Wortmannin 4 h group was significantly decreased (F=10.221, P=0.001). The Ca2+-Mg2+-ATPase activity in LPS + MitQ group was significantly lower than that in control group(F=4.438, P=0.009), whereas Compared with LPS 6 h group, there was no significantly difference (F=4.438, P=0.698).2.3 Assessment of cardiac function in vivoCompared with control group, LVSP, ±dp/dt max were significantly decreased in 6 h LPS group,6 h LPS + MitoQ group and 4 h LPS + Wortmannin group (all P<0.05), LVEDP was significantly increased in these 3 groups (all P<0.05). LVSP and ±dp/dt max in LPS + Wortmannin 4 h group was significantly lower than that in LPS 4 h group(all P<0.05), and LVEDP was significantly increased(P<0.05). LVSP, ±dp/dt and LVEDP in LPS + MitQ group were no significantly difference than these in LPS 6 h group(all P>0.05).2.4 Histopathological changes in the myocardial tissuesUnder light microscopy, significant pathological changes were observed in the myocardial tissues of the LPS 6 h group, LPS + MitoQ 6 h group, and LPS + Wortmannin 4 h group. Electron microscopy showed that the number of autophagic vacuoles increased 6 h after LPS was administered, but did not increase significantly thereafter to 12 h. There was no difference at any time point in the number of autophagic vacuoles in the group given MitoQ and LPS.2.5 Determination of IL-1β, TNF-α and ROS in plasma and myocardial tissuesThe levels of TNF-a in plasma and myocardial tissues in the rats of the LPS 4 h group were all higher than that of the control group (all P<0.05). Compared with LPS 4 h group, the levels of TNF-α in plasma and myocardial tissues in LPS + Wortmannin 4 h group was significantly increased(all P<0.05). The levels of TNF-a in plasma and myocardial tissues in LPS + MitoQ 6 h group were all higher than that of control, whereas Compared with LPS 6 h group, there was no significantly difference(all P>0.05).The levels of IL-1βin plasma and myocardial tissues in the rats of the LPS 6 h group were all higher than that of the control group (all P<0.05). Compared with LPS 4 h group, the levels of IL-1β in plasma and myocardial tissues in LPS + Wortmannin 4 h group was significantly increased(all P<0.05). The levels of IL-1β in plasma and myocardial tissues in LPS + MitoQ group were all higher than that of control, whereas Compared with LPS 6 h group, there was no significantly difference(all P>0.05).The levels of ROS in plasma and myocardial tissues in the rats of the LPS 6 h group were all higher than that of the control group (all P<0.05). Compared with LPS 4 h group, the levels of ROS in plasma and myocardial tissues in LPS + Wortmannin 4 h group was significantly increased(all P<0.05). The levels of ROS in plasma and myocardial tissues in LPS + MitoQ group were all higher than that of control, whereas Compared with LPS 6 h group, there was no significantly difference(all P>0.05).2.6 Determination of LC3 and GRP78 expression in myocardial tissuesThe levels of LC3-II and GRP78 expression in the LPS 6 h group were all higher than that of the control group (all P<0.05), whereas those in the LPS 12 h group had no significantly difference compared with the LPS 6 h group (all P>0.05). The levels of LC3-Ⅱ in LPS + MitoQ 6 h group was lower than that of the LPS 6 h group(P< 0.05). The levels of GRP78 expression in LPS + Wortmannin 4 h group was higher than that of the LPS 4 h group(P<0.05).3. Autophagy improve LPS-challenged cardiomyocytes contraction and diastolic dysfunction via alleviating endoplasmic reticulum stress3.1 The effect of different concentration LPS on autophagy function of cardiomyocytesWith increased as the concentration of LPS, autophagy marker LC3-Ⅱ protein expression gradually increases, but along with further increased concentration of LPS, LC3-Ⅱ protein expression has a downward trend.3.2 The effect of Wortmannin on the levels of CK-MB, LDH and Ca2+-Mg2+-ATP in LPS(100 ng/ml)-treated cardiomyocytesCompared with LPS group, the levels of CK-MB and LDH were significantly increased, whereas the levels of Ca2+-Mg2+-ATP was significantly decreased (all P< 0.05). The levels of CK-MB and LDH in LPS + Wortmannin + TUDCA group were lower than that of the LPS+Wortmannin group, whereas the levels of Ca2+-Mg2+-ATP were significantly increased(all P<0.05).3.3 The effect of Wortmannin on the concentration of intracellular ROS in LPS(100 ng/ml)-treated cardiomyocytesThe concentration of intracellular ROS in LPS + Wortmannin group was significantly higher than that of the LPS group(F=63.278, P=0.000). Compared with LPS + Wortmannin group, the concentration of intracellular ROS in LPS + Wortmannin + TUDCA group was significantly decreased (F=63.278, P=0.000).3.4 The effect of Wortmannin on the LPS(100ng/ml)-challenged cardiomyocytes contraction and diastolic functionThe PS and ±dL/dt of cardiomyocytes in LPS + Wortmannin group were significantly lower than those of the control group(all P<0.05). Compared with LPS + Wortmannin group, the Ps and ±dL/dt in LPS + Wortmannin + TUDCA group were significantly decreased (all P<0.05).3.5 The effect of Wortmannin on diastolic calcium and calcium transients in the LPS(100ng/ml)-challenged cardiomyocytesCompared with LPS group, the concentration of diastolic calcium in cardiomyocytes in LPS + Wortmannin group was significantly increased, whereas calcium transients was significantly decreased (all P<0.05). Diastolic calcium and calcium transients of cardiomyocytes were significantly difference between LPS + Wortmannin + TUDCA group and LPS + Wortmannin group (all P<0.05).3.6 The effect of Wortmannin on GRP78 expression in the LPS(100ng/ml)-challenged cardiomyocytesThe level of GRP78 expression in LPS + Wortmannin group was significantly higher than that of the LPS group(F=5.886, P=0.001). Compared with LPS + Wortmannin group, the level of GRP78 expression in LPS + Wortmannin + TUDCA group were significantly decreased(F=5.886, P=0.043).3.7 The effect of MitoQ on the levels of CK-MB, LDH and Ca2+-Mg2+-ATP in LPS(4 μg/ml)-treated cardiomyocytesCompared with the control group, the levels of CK-MB and LDH were significzntly increased, whereas the levels of Ca2+-Mg2+-ATP was significantly decreased (all P< 0.05). The levels of CK-MB, LDH and Ca2+-Mg2+-ATP in LPS + MitoQ group were significantly difference compared with the control group(all P< 0.05), whereas those were no significantly difference compared with the LPS group(all P>0.05).3.8 The effect of MitoQ on the concentration of intracellular ROS in LPS(4 μg/ml)-treated cardiomyocytesThe concentrations of intracellular ROS in LPS + MitoQ group and LPS group were significantly higher than that of the control group(all P<0.05). Compared with LPS group, the concentration of intracellular ROS in LPS + MitoQ group was significantly decreased (F=22.753,P=0.003).3.9 The effect of MitoQ on LC3-II expression in the LPS(4 μg/ml)-challenged cardiomyocytesThe levels of LC3-Ⅱ expression in LPS + MitoQ group and LPS group were significantly higher than that of the control group(all P<0.05). Compared with LPS group, the level of LC3-II expression in LPS + MitoQ group were significantly decreased (F= 15.019, P=0.011).3.10 The effect of MitoQ on the LPS(4 μg/ml)-challenged cardiomyocytes contraction and diastolic functionThe PS and ±dL/dt of cardiomyocytes in LPS + MitoQ group and LPS group were significantly lower than those of the control group(all P<0.05). There were no significantly difference between LPS + MitoQ group with the LPS group(all P>0.05).3.11 The effect of MitoQ on diastolic calcium and calcium transients in the LPS(4 μg/ml)-challenged cardiomyocytesCompared with the control group, the concentration of diastolic calcium in cardiomyocytes in LPS group was significantly increased, whereas calcium transients was significantly decreased (all P<0.05). Diastolic calcium and calcium transients of cardiomyocytes were no significantly difference between LPS + MitoQ group and LPS (all P>0.05). There were significantly difference between LPS + MitoQ group and the control group(all P<0.05).Conclusions1. Inhibition of autophagy reduces the survival rate and survival time of septic rats, MitoQ has no effect on the survival rate and survival time of septic rats.2. Autophagy function in Myocardial tissue of septic rats is limited. Inhibition of autophagy can lead to sepsis-induced cardiac dysfunction. Autophagy may improve cardiac dysfunction by limiting the production of proinflammatory cytokine IL-1β, TNF-α, inflammatory mediators ROS and reducing the endoplasmic reticulum stress and preventing Ca2+-Mg2+-ATPase inactivation in septic rats. MitoQ could reduce autophagy function of the myocardial tissue of sepsis rats, but has no protective effect on cardiac dysfunction in septic rats.3. The protective role of autophagy on LPS-challenged myocardial cells is very limited. Inhibition of autophagy can cause LPS-challenged myocardial cells structure and function injury, affect myocardial intracellular calcium balance, leading to myocardial contractile and diastolic dysfunction. Therefore, autophagy may has protective effect of LPS-challenged myocardial cells contraction and diastolic dysfunction. Autophagy can alleviate endoplasmic reticulum stress and the production of ROS of myocardial cells, suggesting that autophagy may plays a protective role in myocardial cells contraction and diastolic dysfunction by improving endoplasmic reticulum stress and decreasing the production of ROS. MitoQ could reduce the expression of LC3-II and the production of ROS, suggesting that ROS may be one of the autophagy activator. MitoQ may has no protective effect on LPS-challenged myocardial cells contraction and diastolic dysfunction in an autophagy-mediated way.