| Background and objectiveSepsis is defined as a systemic inflammatory response occurring in the presence of microbial pathogens, eventually leading to septic shock and multiple organ failure. According to the American epidemiological investigation,40%?50% of the patients suffering with sepsis were accompanied with cardiac dysfunction. Moreover,7% of them finally developed to severe heart failure. Thus, septic myocardial dysfunction is a leading cause of death in severe sepsis. It has been reported that sepsis is provoked by an overwhelming inflammatory response initiated by the innate immune system. Specially, the excessive production of proinflammatory cytokines and reactive oxygen triggered by activated macrophages can produce irreversible damage to cardiomyocytes, such as impairment of contractile efficiency and cell apoptosis, which is the main cause of septic myocardial dysfunction.Trimetazidine (TMZ), clinically used as an antianginal agent, makes energy production shift from free fatty acids to glucose oxidation by inhibiting long-chain 3-ketoacylco enzyme Athiolase activity to relieve angina in pharmacology. In the normal physiological condition,60%-70% of myocardial energy metabolism is dependent on fatty acid beta oxidation, whereas 20%-25% is dependent on the oxidative metabolism of glucose, and 5%-10%is from glycolysis. However, to produce the same amount of ATP, fatty acid oxidation requires higher oxygen consumption than glucose metabolism. In the ischemic and hypoxic conditions, TMZ improves the energy producing efficiency of cardiomyocytes by increasing the use of glucose and inhibiting fatty acid ?-oxidation. Recently, it has been reported that TMZ protects left ventricular remodeling by attenuating oxidative stress and inflammation. However, whether TMZ protects against septic myocardial dysfunction has not been reported. Therefore, in the present study, we investigate whether TMZ attenuates septic myocardial dysfunction in LPS-induced septic mice models. In addition, the effects of TMZ on the macrophages mediated proinflammatory response and cardiomyocytes apoptosis, as well as the potential molecular mechanisms were explored in vitro.Methods and results1. Firstly, C57BL/6 mice were challenged with LPS by intraperitoneal injection. And TMZ was administered in two different treatment regimens according to the different treatment time. In the first pretreatment model, One of them was pre-treatment model:mice were pre-treated with TMZ for 3 consecutive days, and then challenged with LPS. Another was post-treatment model, mice were firstly injected with LPS for 6 hours and then treated with TMZ. Cardiac function was evaluated by echocardiography and hemodynamic analysis with a Millar catheter. Results revealed that both TMZ pre-treatment and post-treatment improved cardiac function after LPS challenge, including increased ejection fraction (EF), fractional shortening (FS) and LV dP/dt, as well as decreased left ventricular internal diameter (LVID) and left ventricular end-diastolic pressure (LVEDP). Meanwhile, MAP measurements and survival analysis indicated that TMZ post-treatment ameliorated hypotension and improved survival rate in LPS-challenged mice.2. In the TMZ pretreatment model, the heart and blood were collected 6 hours after LPS injection. Cardiomyocytes apoptosis and myocardial macrophages infiltration were detected by TUNEL staining and F4/80 immunohistochemical staining, respectively. Real-time RT-PCR and ELISA were used to detect the expression of proinflammatory cytokines in cardiac tissue and plasma. Results revealed that cardiomyocytes apoptosis, the levels of proinflammatory cytokines (TNFa, MCP-1, IL-1?, and IL-6) in plasma and heart tissues, as well as myocardial macrophages infiltration were all significantly increased after LPS stimulation. However, TMZ pretreatment attenuated cardiomyocytes apoptosis, levels of proinflammatory cytokines and myocardial macrophages infiltration in LPS-induced mice.3. To further explore whether bone marrow or myocardium TMZ pre-treatment is responsible for decreasing macrophage infiltration, we performed reciprocal bone marrow transplantation between TMZ pre-treated mice and WT mice. TMZ pre-treated mice were pretreatment with TMZ (20 mg/kg, t.i.d.) for 3 consecutive days before the bone marrow transplantation. Four types of bone marrow chimeric mice were generated as below. (1) Firstly, recipient mice (WT) received sub-lethal dosage of y-irradiation. Then, donor bone marrow cells isolated from a separate cohort of nonirradiated WT mice were injected into irradiated WT mice through the tail vein (WT> WT group). (2) Recipient mice (TMZ pre-treated) received sub-lethal dosage of y-irradiation. Then, donor bone marrow cells isolated from a separate cohort of nonirradiated WT mice were injected into irradiated TMZ pre-treated mice through the tail vein (WT> TMZ group). (3) Recipient mice (WT) received sub-lethal dosage of y-irradiation. Then, donor bone marrow cells isolated from nonirradiated TMZ pre-treated mice were injected into irradiated WT mice through the tail vein (TMZ> WT group). (4) Recipient mice (TMZ pre-treated) received sub-lethal dosage of ?-irradiation. Then, donor bone marrow cells isolated from nonirradiated TMZ pre-treated mice were injected into irradiated TMZ pre-treated mice through the tail vein (TMZ> TMZ group). After recovery from transplantation, mice in the four bone marrow transplanted groups (WT> WT,WT> TMZ,TMZ> WTand TMZ> TMZ) were all subjected with LPS.6 hours after LPS injection, cardiac function was evaluated by echocardiography and hemodynamic analysis with a Millar catheter. Then the heart tissues were collected. H&E staining was used to evaluate myocardial structure. Myocardial macrophages infiltration were detected by F4/80 immunohistochemical staining and flow cytometry.Results depicted that the mice receiving the bone marrow from TMZ pretreated mice (TMZ> WT and TMZ> TMZ groups) increased EF, FS, LVESP and LV dP/dt, as well as decreased LVID and LVEDP. Furthermore, these mice exhibited normal myocardial structures with few myocardial macrophage infiltration. Moreover, the numbers of macrophages in the myocardium of TMZ> WT and TMZ> TMZ mice were paralleled by the numbers of their precursors, monocytes. These indicated that bone marrow rather than myocardium or other parechymal cells from TMZ pre-treated mice, improved the LPS-induced myocardial dysfunction, and attenuated the infiltration of monocytes and macrophages in the myocardium of TMZ> WT and TMZ> TMZ mice. Furthermore, we made a correlation analysis between the numbers of macrophages detected by F4/80 staining and the cardiac function parameters. These results indicated a significant negative correlation between the numbers of myocardial macrophage and cardiac function in recipient mice, which evaluated by LVEF, LVFS, dP/dt max and dP/dt min.4. Then we sorted cardiac macrophages by FACS from LPS-induced mice which pretreated with or without TMZ. At the same time, we isolated primary peritoneal macrophages, which were firstly treated with TMZ and then subjected to LPS stimulation. Real-time RT-PCR was used to detect the expression of proinflammatory cytokines. Results showed that TMZ pre-treatment reduced the expression of proinflammatory cytokines in sorted cardiac macrophages of LPS-induced mice, as well as in LPS-treated peritoneal macrophages. These results suggested that the general effects of LPS and TMZ on cardiac macrophages and peritoneal macrophages were consistent. As the sorted of cardiac macrophages was not enough for more examinations, so the peritoneal macrophages were used in the following experiments.5. To further investigate the relationship between macrophages and cardiomyocytes, we co-cultured peritoneal macrophages with neonatal cardiomyocytes using Transwell chamber. Pretreated peritoneal macrophages (LPS, TMZ, LPS+TMZ and LPS+ proinflammatory cytokine neutralizing antibody) were seeded onto transwell inserts, and placed over cardiomyocytes. FACS was used to detect the apoptosis level of cardiomyocytes. The results showed that LPS-activated macrophages significantly induced apoptosis of cardiomyocytes, but TMZ pretreatment decreased apoptosis of co-cultured cardiomyocytes. Proinflammatory cytokine neutralizing antibody also decreased apoptosis of co-cultured cardiomyocytes. These results revealed that TMZ pre-treatment decreased macrophages proinflammatory response, which mediated the cardiomyocyte apoptosis.6. In the cultured peritoneal macrophages, the fluorescence probe DHE was used to detect the levels of ROS after different stimulations (LPS, TMZ and ROS scavenger NAC). Additionally, the levels of proinflammatory cytokines were determined by real-time RT-PCR. Our results showed that TMZ attenuated the LPS-induced ROS production and the increase of proinflammatory cytokines, and these effects were comparable with NAC, an effective ROS scavenger.7. Further, we explored the underlying molecular mechanism. In the TMZ pre-treatment model, western blotting was used to detect the expression of nuclear and cytosolic Sirtl in the myocardium. Our results showed that TMZ treatment induced a translocation of Sirtl from cytoplasm to nucleus, preventing LPS-induced effects on Sirtl. In the cultured peritoneal macrophages, western blotting was used to detect the protein levels of Sirtl, NADPH subunits (gp91phox and p47phox) after different stimulations (LPS, TMZ and Sirtl inhibitor NAM or Sirtl si-RNA). Additionally, the fluorescence probe DHE was used to detect the levels of ROS, while the levels of proinflammatory cytokines were determined by real-time RT-PCR. The results showed that LPS markedly up-regulated the levels of ROS and proinflammatory cytokines, increased NADPH subunits (gp91phox and p47phox) and expression of cytosolic Sirtl in primary macrophages. However, TMZ clearly suppressed LPS-induced levels of ROS and proinflammatory cytokines. TMZ also decreased LPS-induced expression of NADPH subunits (gp91phox and p47phox), and induced a translocation of Sirtl from cytoplasm to nucleus. In the presence of Sirtl specific inhibiter NAM or Sirtl siRNA, these anti-oxidation and anti-inflammation effects of TMZ were reversed. These results indicated that TMZ attenuated ROS production and then decreased the inflammatory response via up-regulation of nuclear Sirtl in peritoneal macrophages.8. Next, in the cultured peritoneal macrophages, AMPK phosphorylation and AMPK expression were tested after different stimulations (LPS, TMZ and Sirtl inhibitor NAM or Sirtl siRNA) by western blotting. The results showed that TMZ significantly increased the phosphorylation of AMPK in LPS-induced peritoneal macrophages. In the presence of the Sirtl inhibitor NAM or Sirtl siRNA, the effect of TMZ on AMPK phosphorylation within LPS-stimulated peritoneal macrophages was blocked. These results indicated AMPK phosphorylation was modulated by TMZ via Sirtl. Furthermore, in the cultured peritoneal macrophages, Nrf2 and HO-1 protein levels were tested after different stimulations (LPS, TMZ and AMPK inhibitor Compound C) by western blotting. Meanwhile, the fluorescence probe DHE was used to detect the levels of ROS, and the expression of proinflammatory cytokines were determined by real-time RT-PCR. Results showed that TMZ increased the expression of Nrf2 and HO-1 which were suppressed by LPS. TMZ significantly decreased LPS-induced the ROS generation and proinflammatory cytokines mRNA expression. However, in the presence of AMPK inhibitor Compound C, the effects of TMZ on Nrf2 and HO-1, ROS generation and proinflammatory cytokines mRNA expression were all reversed. Together, Sirtl/AMPK was also involved in decreasing ROS production via Nrf2 and HO-1 activation, finally attenuated the expression of proinflammatory cytokines.9. On the other hand, peritoneal macrophages were stimulated with LPS alone or LPS+TMZ or LPS+TMZ+Sirtl inhibitor NAM, nuclear PPARa protein levels were determined by western blotting. Results indicated that LPS significantly reduced the expression of nuclear PPARa, but TMZ increased expression of nuclear PPARa compared with LPS. However,the effects of TMZ on nuclear PPARa was partially blocked in the presence of NAM, indicating TMZ increased nuclear PPARa via Sirtl. Futhermore, phosphorylated IkBo and nuclear p65 were examined by western blotting after different stimulations (LPS, TMZ and PPARa antagonist GW6471). Meanwhile, the fluorescence probe DHE was used to detect the levels of ROS, and the expression of proinflammatory cytokines were determined by real-time RT-PCR. Results showed that TMZ decreased LPS-induced phosphorylation of I?B? and expression of nuclear NF-KB-p65. And TMZ also obviously reduced LPS-increased ROS generation and proinflammatory cytokines mRNA expression. Additionally, the effects of TMZ in anti-oxidative stress and attenuation of inflammatory response were partially inhibited by adding PPARa antagonist GW6471. Taken together, TMZ attenuated LPS-induced ROS production through Sirtl/PPARa pathway activation, which was accompanied with p-I?B?/NF-?B inhibition and ultimately suppressed macrophage proinflammatory response.10. Finally, peritoneal macrophages were stimulated with LPS alone or LPS+TMZ or LPS+TMZ+inhibitors (Sirtl inhibitor NAM, AMPK inhibitor Compound C and PPARa antagonist GW6471). The culture medium were collected and then added to cardiomyocytes. ELISA was used to detect the levels of proinflammatory cytokines in the culture medium, and cardiomyocytes apoptosis were determined by FACS. The ELISA results revealed that TMZ attenuated LPS-induced proinflammatory cytokines. However, in the presence of Sirtl inhibitor NAM or AMPK inhibitor Compound C or PPARa antagonist GW6471, the levels of proinflammatory cytokines were all increased. Flow cytometry analysis showed that the culture medium from LPS-induced peritoneal macrophages significantly increased cardiomyocytes apoptosis, whereas TMZ pretreatment attenuated LPS-stimulated apoptosis, the effects of which were partially prevented by adding Sirtl inhibitor NAM, AMPK inhibitor Compound C and PPARa antagonist GW6471, respectively. Therefore, TMZ attenuated LPS-induced macrophages mediated cardiomyocytes apoptosis through Sirt1/AMPK or Sirt1/PPARa pathway.ConclusionOur results demonstrated that TMZ attenuated LPS-induced myocardial dysfunction. The effect of TMZ is dependent on decrease of macrophage infiltration and proinflammatory responses mediated cardiomyocytes apoptosis. These findings may provide an innovative therapeutic strategy for septic myocardial dysfunction. |
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