| BackgroundAs a serious manifestation and late stage of various heart diseases,the incidence of heart failure is increasing.In developed countries,the incidence of heart failure is 1%～2%,and over 70 years old is 10%.In China,the prevalence of heart failure in adults≥35 years old is 1.3%,about 13.7 million people.Decreased exercise tolerance is a typical characteristic of heart failure patients,which seriously affects the quality of life and prognosis of patients.In the late stages of chronic heart failure,loss of skeletal muscle mass(sarcopenia)is usually observed.After adjusting for cardiac function,skeletal muscle wasting remained an independent predictor of decreased exercise tolerance in patients with heart failure.Studies have shown that the incidence of sarcopenia is 20%higher in patients with heart failure than that in healthy people,and the decline in exercise tolerance is more severe in patients with heart failure and sarcopenia than in patients with heart failure alone.As a common complication of heart failure,sarcopenia reduces patients’ exercise ability,increases the rate of rehospitalization and mortality,and is an important indicator of poor prognosis.However,in the state of heart failure,the occurrence and development mechanism of sarcopenia is unclear,and there is a lack of targeted therapy based on mechanism.The establishment of animal models suitable for clinical practice is the basis of disease research and can really provide effective evidence for clinical diagnosis and treatment.However,animal models of heart failure and sarcopenia are still lacking.Muscle is a high energy consuming organ and mitochondria is an important organelle in regulating energy metabolism of skeletal muscle.Mitochondria respond to disease and aging by adjusting their size,structure and function.Mitochondrial dysfunction usually leads to apoptosis,but due to the multinucleate nature of skeletal muscle cells,apoptosis leads to muscle fiber atrophy rather than whole cell death,and apoptosis occurs before skeletal muscle protein catabolism.Mitophagy is highly selective autophagy,in which damaged or redundant mitochondria are degraded by the autophagolysosome system to achieve mitochondrial quality control,and protect cells from harmful effects caused by mitochondrial damage.When mitophagy is weakened,damaged mitochondria accumulate in the cell,producing ROS and activating the ubiquitin-proteasome system.Therefore,mitochondria-related apoptosis and mitochondrial autophagy of skeletal muscle may be one of the mechanisms of sarcopenia in heart failure.In order to find effective intervention to improve the reduced exercise tolerance and reduce the occurrence of sarcopenia in patients with heart failure,we conducted relevant exploration form drug and gene levels.In the treatment of heart failure,levosimendan has been proved to have mitochondrial protective effect.A series of experimental and clinical studies have found that levosimendan can activate mito-K+ATP channels of cardiomyocytes,and this effect is related to cardiac protection,infarction size reduction and reduction of ischemia/reperfusion injury.However,whether levosimendan can inhibit skeletal muscle atrophy through mito-K+ATP opening remains unclear.Studies have shown that TRIB3 knockout can promote autophagy and improve skeletal muscle atrophy in aging mice.However,whether TRIB3 is involved in mitophagy and thus affects skeletal muscle atrophy remains unclear.In this study,we intended to investigate the role of mitochondria-associated skeletal muscle cell apoptosis and mitochondrial autophagy in ischemic heart failure and sarcopenia and the intervention effect of levosimendan at animal and cellular levels.Objectives(1)To investigate the feasibility of establishing mouse model of heart failure and sarcopenia by ligation of anterior descending branch combined with hind limb unloading;(2)To investigate the role of mitochondria-induced apoptosis of skeletal muscle cells in heart failure and sarcopenia;(3)To investigate the possible mechanism of levosimendan in improving heart failure and sarcopenia;(4)To investigate whether TRIB3 mediated mitophagy is associated with the occurrence and development of heart failure and sarcopenia;(5)To investigate the possible mechanisms of TRIB3 mediated mitophagy in heart failure and sarcopenia.Methods(1)Animals:male C57BL/6 mice aged about 10～12 weeks were divided into control group,sham operation group,left anterior descending branch(LAD)ligation group,LAD ligation+hind limb unloading(HU)group;male C57BL/6 mice aged about 10～12 weeks were divided into control group,heart failure group,heart failure+solvent group,heart failure+levosimendan group,heart failure+sarcopenia group,heart failure+sarcopenia+solvent group,heart failure+sarcopenia+levosimendan group;male C57BL/6 and Trib3-/-mice aged about 10～12 weeks were divided into two groups:control group and heart failure+sarcopenia group;(2)In vitro groups:Cultured C2C12 cells were divided into control group,dexamethasone group,dexamethasone+levosimendan group,dexamethasone+levosimendan+5-HD group,and dexamethasone+5-HD group;C2C12 cells were cultured and differentiated into control group,shTRIB3 group,TRIB3 adv group,dexamethasone group,dexamethasone+shTRIB3 group,and dexamethasone+TRIB3 adv group;C2C12 cells were cultured and differentiated into control group,shTRIB3 group,dexamethasone group,dexamethasone+TRIB3-/-group,dexamethasone+shTRIB3+EX527 group;(3)The changes of cardiac structure and function were detected by echocardiography;(4)The exercise ability of mice was tested by forelimb grip force test,cage wire test and treadmill exhaustion test;(5)The exercise ability of mice was tested by forelimb grip force test,cage wire test and treadmill exhaustion test;(6)Histopathological staining was used to detect the changes of skeletal muscle morphology,muscle fiber typing,inflammatory index expression level and apoptosis level;(7)Molecular biology was used to detect the changes of oxidative stress,mitochondrial function,apoptosis level and mitophagy level in tissues and cells.Results(1)Compared with the sham group,LVEF and LVFS of LAD ligation+HU group decreased significantly,indicating that the cardiac function of LAD ligation+HU group decreased.Compared with the sham group,the forelimb grip strength,hanging impulse,the running time and running distance of LAD ligation+HU group decreased,indicating that the exercise ability of LAD ligation+HU group decreased.Compared with the sham group,the levels of serum LDH and CK increased,the length of gastrocnemius weight/tibia length decreased,and the cross-sectional area of muscle fibers decreased in LAD ligation+HU group,indicating skeletal muscle injury and muscle fiber atrophy in LAD ligation+HU group.Compared with the sham group,the ratio of slow muscle fiber decreased,the ratio of fast muscle fiber increased,and the ratio of slow muscle fiber/fast muscle fiber decreased in LAD ligation+HU group;(2)Compared with the control group,the LVEF and LVFS of the HF+sarcopenia group were significantly decreased,the forelimb grip strength and hanging impulse were decreased,running time was shortened,running distance was decreased,and the gastrocnemius weight/tibia length and muscle fiber cross-sectional area were decreased in HF+sarcopenia group.The content of mitochondria and mitochondrial membrane potential decreased,the level of apoptosis increased,and the levels of oxidative stress and inflammation increased in skeletal muscle of HF+sarcopenia group.Compared with the HF+sarcopenia group,the LVEF and LVFS of the HF+sarcopenia+levosimendan group were significantly increased,the forelimb grip strength and hanging impulse were increased,running time was prolonged,running distance was increased,and the gastrocnemius weight/tibia length and muscle fiber crosssectional area were increased in HF+sarcopenia+levosimendan group.The ratio of slow muscle fiber increased,the ratio of fast muscle fiber decreased,the ratio of slow muscle fiber/fast muscle fiber increased HF+sarcopenia+levosimendan group.The content of mitochondria and mitochondrial membrane potential of skeletal muscle increased,and the level of apoptosis,oxidative stress and inflammation decreased HF+sarcopenia+levosimendan group;(3)Compared with the control group,the expression of atrophy index,mitochondrial content and mitochondrial membrane potential decreased,oxidative stress and apoptosis levels increased in dexamethasone group.Compared with dexamethasone group,the expression of atrophy index,mitochondrial content and mitochondrial membrane potential,oxidative stress and apoptosis were decreased in dexamethasone+levosimendan group.Compared with the dexamethasone+levosimendan group,the expression of atrophy index,mitochondrial content and mitochondrial membrane potential decreased,oxidative stress and apoptosis levels increased in the dexamethasone+levosimendan+5-HD group;(4)Compared with HF+sarcopenia group,the difference of LVEF and LVFS in TRIB3-/+HF+sarcopenia group was not statistically significant,the forelimb grip strength and hanging impulse were increased,the running time was prolonged,the running distance was increased,the gastrocnemius weight/tibia length was not statistically significant,and the muscle fiber cross-sectional area was increased.The ratio of slow muscle fiber increased,the ratio of fast muscle fiber decreased,and the ratio of slow muscle fiber/fast muscle fiber increased in the TRIB3-/-+HF+sarcopenia group.The content of skeletal muscle mitochondria and mitochondrial membrane potential increased,the level of oxidative stress decreased,the level of mitochondrial autophagy increased,and the expression level of Sirtl/Mfn2 increased in TRIB3-/-+HF+sarcopenia group:(5)Compared with dexamethasone group,the expression of atrophy index,mitochondrial content and mitochondrial membrane potential,oxidative stress level,mitophagy level and Sirt1/Mfn2 expression level were decreased in dexamethasone+shTRIB3 group.Compared with dexamethasone+TRIB3 silencing group,myocytes in dexamethasone+shTRIB3+EX527 group showed increased expression of atrophy index,decreased mitochondrial content and mitochondrial membrane potential,increased oxidative stress level,decreased mitophagy level,and decreased Sirt1/Mfn2 expression.Conclusions(1)In this study,an animal model of heart failure and sarcopenia was established by ligation of left anterior descending coronary artery combined with hind limb unloading;(2)Mitochondrial dysfunction of skeletal muscle in mice with ischemic heart failure and sarcopenia further led to increased apoptosis of muscle cells,skeletal muscle atrophy,and decreased exercise ability;(3)Levosimendan could increase mitochondrial function,inhibited apoptosis of skeletal muscle,improved skeletal muscle atrophy and exercise ability in mice with heart failure and sarcopenia through the activation of mito-K+ATP,providing a new option for the treatment of heart failure and sarcopenia;(4)TRIB3 knockout improved skeletal muscle mitochondrial dysfunction,promoted mitophagy,inhibited skeletal muscle atrophy,and improved exercise ability in mice with heart failure and sarcopenia;(5)TRIB3 silencing improved mitochondrial function,reduced oxidative stress level,promoted mitochondrial autophagy,and inhibited myocyte atrophy by up-regulating Sirtl/Mfn2 expression. |
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