| BackgroundHeart failure is a life-threatening syndrome,with high incidence rate and mortality,low quality of life of patients,and great burden on the medical and health system.Pathological cardiac hypertrophy is the pathological basis of heart failure and is closely related to the incidence and development of heart failure.It is an independent risk factor for heart failure and sudden cardiac death.Preventing and treating myocardial hypertrophy is crucial for reducing the incidence of heart failure and improving patient prognosis.However,there is still a lack of effective intervention methods for pathological cardiac hypertrophy,and there is an urgent need to find new treatment strategies and targets.Previous studies generally believed that cold exposure was an adverse factor for the incidence and development of cardiovascular diseases.Long term cold exposure increased the incidence rate and mortality of cardiovascular diseases,while recent studies showed that short-term cold exposure had protective effects on many diseases.Shortterm cold exposure improves obesity induced liver inflammation by MaR2 secreted by activated brown adipose tissue,or significantly reduces blood glucose by activated brown adipose tissue,thereby inhibiting glycolytic metabolism in cancer cells and the growth of tumors.However,whether cold exposure regulates cardiovascular diseases and its mechanisms are still unclear,and further exploration is urgently needed.Brown adipose tissue is mainly present in the neck,above the clavicle,around the spine,mediastinum,armpits,and abdomen.Based on the endocrine function of brown adipose tissue,an increasing number of studies have found that brown adipose tissue plays an inter organ communication role in various diseases through endocrine pathways,regulating the pathological and physiological processes of other organs.The active molecule secreted by brown adipose tissue is called Batokine,which includes proteins,metabolites,and extracellular vesicles.The research shows that the content of brown adipose tissue is negatively related to the incidence rate of cardiovascular diseases,and brown adipose tissue can regulate cardiovascular related pathophysiological processes such as atherosclerosis,cardiovascular metabolism,myocardial ischemia and reperfusion through Nrg4,12,13diHOME and small extracellular vesicles.The preliminary research of the research group found that brown adipose tissue activated by moderate cold exposure mainly regulates pathological cardiac hypertrophy through the endocrine pathway mediated by small extracellular vesicles.However,there is still no in-depth report on how activated brown adipose tissue regulates pathological cardiac hypertrophy through small extracellular vesicles.The mechanism by which further moderate cold exposure regulates pathological cardiac hypertrophy through organ-organ communication mediated by activated brown adipose tissue remains unclear.The preliminary experimental results of the research group found that EHD2 protein is a differential protein between small extracellular vesicles from moderate cold exposure mediated activated brown adipose tissue and room temperature(25℃)mediated nonactivated brown adipose tissue.EHD2(Eps15 homology domain containing protein 2)is a protein located in the neck region of Caveolae,belonging to the EHD ATPase family related to motor proteins.Caveolae is a small pit like invagination structure on the cell membrane,with a size of 30-100nm,present in many cell types,including adipocytes,endothelial cells,and cardiomyocytes,which are abundant in content.Caveolae is involved in various important biological processes,including signal transduction,endocytosis,cell cycle regulation,and cellular mechanical stress response.Caveolae mainly exists on the cell membrane in two forms:adhering to the cell membrane or detached from it,and the balance between these two states is called Caveolae dynamics.The deficiency of EHD2 protein leads to the destruction of the circular oligomer structure in the neck region of Caveolae,causing Caveolae to detach from the cell membrane and free from it.Upregulation of EHD2 protein can maintain the dynamic homeostasis of Caveolae.However,the role of Caveolae dynamics in pathological cardiac hypertrophy is currently unclear.In summary,moderate cold exposure may alleviate pathological cardiac hypertrophy and cardiac dysfunction through activated small extracellular vesicles derived from brown adipose tissue,and EHD2 protein may be a key protein in small extracellular vesicles.Therefore,this research applies proteomic analysis,specific gene knockout and overexpression techniques to further investigate the protective effects and mechanisms of moderate cold exposure on pathological cardiac hypertrophy,which can provide potential therapeutic strategies and targets for pathological cardiac hypertrophy.Based on the above research ideas,we have developed the following experimental plan:Methods1.Moderate cold exposure time to improve pathological cardiac hypertrophyMale C57BL/6 mice aged 6-8 weeks were acclimated at 18℃ for one week,then kept at 4℃ for 3,7 and 14 days respectively,and kept at 25℃ for 3 days,then underwent transverse aortic constriction(TAC operation model).After 6 weeks,the cardiac function and the degree of cardiac hypertrophy were observed.Furthermore,mice exposed to cold at 4℃ for 7 days were raised at room temperature of 25℃ for 3 days,and micropump containing angiotensin Ⅱ was buried subcutaneously.After 4 weeks,the cardiac function and cardiac hypertrophy were observed.2.Moderate cold exposure reduces pathological cardiac hypertrophy through activated brown adipose tissueMale C57BL/6 mice aged 6-8 weeks were housed in moderate cold,and then the brown adipose under the scapula was removed by operation.TAC operation model was performed,and the cardiac function and cardiac hypertrophy were observed after 6 weeks.Furthermore,male C57BL/6 mice aged 6-8 weeks were injected with β3-adrenergic receptor agonists CL316,243,and TAC operation model was performed.After 6 weeks,the cardiac function and the degree of cardiac hypertrophy were observed.3.Moderate cold exposure activated small extracellular vesicles derived from brown adipose tissue alleviate pathological cardiac hypertrophyFirstly,male C57BL/6 mice of 6-8 weeks were injected with PBS,moderate cold exposure activated brown adipose conditioned medium,conditioned medium without small extracellular vesicles and conditioned medium with small extracellular vesicles respectively,and the cardiac function and the degree of cardiac hypertrophy were observed after 6 weeks.Secondly,male C57BL/6 mice of 6-8 weeks were injected with PBS,mouse plasma after moderate cold exposure,plasma without small extracellular vesicles and plasma small extracellular vesicles respectively,and the cardiac function and the degree of cardiac hypertrophy were observed after 6 weeks.Furthermore,male C57BL/6 mice of 6-8 weeks were injected with AAV-shRab27a locally,then exposed to moderate cold,followed by TAC operation model,and the cardiac function and the degree of cardiac hypertrophy were observed after 6 weeks.In addition,male C57BL/6 mice aged 6-8 weeks were injected with PBS,non-activated brown adipose-derived small extracellular vesicles at room temperature of 25℃ and activated brown adipose-derived small extracellular vesicles after TAC operation,and the cardiac function and cardiac hypertrophy were observed after 6 weeks.4.Moderate cold exposure activated brown adipose tissue derived small extracellular vesicles rich in EHD2 protein alleviate pathological cardiac hypertrophyFirstly,the differential proteins in the non-activated brown adipose-derived small extracellular vesicles and the activated brown adipose-derived small extracellular vesicles under moderate cold exposure were detected by proteomic sequencing.The content of EHD2 protein in inactive brown fat-derived small extracellular vesicles and activated brown fat-derived small cell extracellular vesicles,plasma extracellular vesicles,heart tissues and cardiomyocytes at room temperature of 25℃ was detected by WB experiment.Secondly,brown adipocytes specific EHD2 knockout mice were constructed.After moderate cold exposure,TAC operation model was performed,and the cardiac function and the degree of cardiac hypertrophy were observed after 6 weeks.Subsequently,mice with cardiomyocyte-specific EHD2 knockout were established,and mice with cardiomyocyte-specific EHD2 overexpression were established by AAV injection.TAC operation model was performed,and cardiac function and cardiac hypertrophy were observed after 6 weeks.In addition,after the mice with myocardial cell-specific overexpression of EHD2 were established by AAV inj ection,a micropump containing angiotensin Ⅱ was buried subcutaneously,and the cardiac function and the degree of cardiac hypertrophy were observed after 4 weeks.After transfection plasmid overexpressed and knocked down EHD2 gene in cardiomyocytes,after 24 hours of PE induction,the ratio of width/length,cell size and mRNA expression level of cardiomyocyte hypertrophy-related genes(ANP,BNP)were detected.5.Moderate cold exposure activated brown adipose tissue small extracellular vesicles myocardial cell axis regulates pressure overload induced Caveolae dynamics in myocardial cells through EHD2 protein regulationObserving the regulatory effect of the moderate cold exposure activated brown adipose tissue small extracellular vesicle EHD2 gene axis on pressure induced abnormal caveolae dynamics in cardiomyocytes through transmission electron microscopy.In addition,the regulatory effect of EHD2 gene on the abnormal dynamics of caveolae induced by PE in adult mouse cardiomyocytes was further observed through transmission electron microscopy.Results1.Mice exposed to 4℃ cold for 7 days(EF:53.45±4.197%,LV mass:112.2±5.531mg)showed significant reduction in pathological cardiac hypertrophy(LV mass:P<0.01)and cardiac dysfunction(EF:P<0.001)after 6 weeks of pressure load induced pathological cardiac hypertrophy model(TAC surgical model)compared to mice fed continuously at 25℃room temperature environment(EF:39.97±2.050%,LV mass:142.5±14.72mg).However,mice exposed to cold for 3 days(EF:45.16±2.719%,LV mass:127.5±10.68mg)and 14 days(EF:42.48±5.433%,LV mass:142.0±18.16mg)at 4℃ showed no significant differences in pathological cardiac hypertrophy and cardiac dysfunction indicators after 6 weeks of TAC surgery compared to mice housed in a continuous room temperature environment(25℃)(EF:P>0.05,LV mass:P>0.05).Similarly,mice exposed to moderate cold(4℃,7 days)(EF:60.82±8.154%,LV mass:101.8±13.39mg)showed reduced pathological cardiac hypertrophy(LV mass:P<0.05)and cardiac dysfunction(EF:P<0.01)in the Ang Ⅱ model induced by angiotensin Ⅱ,compared to mice fed continuously at room temperature at 25℃(EF:45.56±4.400%,LV mass:130.7±17.95mg).2.After moderate cold exposure(4℃,7 days),mice(EF:43.91±7.512%,LV mass:149.9±19.14 mg)who underwent surgical removal of brown adipose tissue under the scapula showed aggravated pathological cardiac hypertrophy(LV mass:P<0.001)and cardiac dysfunction(EF:P<0.01)compared to mice(EF:55.37±5.266%,LV mass:107.7±6.878 mg)who did not remove brown adipose tissue after moderate cold exposure(4℃,7 days).In addition,mice injected with CL316,243(EF:57.15±5.903%,LV mass:111.7±12.63mg)showed reduced pathological cardiac hypertrophy(LV mass:P<0.01)and cardiac dysfunction(EF:P<0.001)after 6 weeks of TAC surgery compared to mice injected with DMSO control group(EF:42.42±2.443%,LV mass:141.0±15.06mg).3.Mice injected with small extracellular vesicles derived from moderate cold exposure(4℃,7 days)activated brown adipose tissue conditioned medium(EF:54.32±4.809%,LV mass:108.2±11.74mg),compared with mice injected with PBS control group(EF:41.67±3.309%,LV mass:150.2±16.39mg),showed reduced pathological cardiac hypertrophy(LV mass:P<0.05)and cardiac dysfunction(EF:P<0.001)after 6 weeks of TAC surgery in the model.Mice inj ected with conditioned medium supernatant without small extracellular vesicles(EF:43.05±3.335%,LV mass:138.2±18.49mg)showed no significant differences in pathological cardiac hypertrophy and cardiac dysfunction indicators after 6 weeks of TAC surgery compared to mice injected with PBS control group(EF:41.67±3.309%,LV mass:150.2±16.39mg)(EF:P>0.05,LV mass:P>0.05).In addition,mice injected with moderate cold exposure(4℃,7 days)plasma derived small extracellular vesicles(EF:51.26±3.140%,LV mass:108.2±11.74mg),compared with mice injected with PBS control group(EF:39.44±6.066%,LV mass:148.8±15.99mg),showed reduced pathological cardiac hypertrophy(LV mass:P<0.001)and cardiac dysfunction(EF:P<0.01)after 6 weeks of TAC surgery.Mice injected with plasma supernatant without small extracellular vesicles(EF:42.53±6.963%,LV mass:145.9±14.64mg)showed no significant differences in pathological cardiac hypertrophy and cardiac dysfunction indicators after 6 weeks of TAC surgery compared to mice injected with PBS control group(EF:39.44±6.066%,LV mass:148.8±15.99mg)(EF:P>0.05,LV mass:P>0.05).Subsequently,moderate cold exposure mice(EF:44.20±4.547%,LV mass:138.6±11.22mg)after local injection of AAV-shRab27a were compared with moderate cold exposure mice(EF:54.59±4.833%,LV mass:114.9±8.39mg)after injection of control AAV-shNC.After 6 weeks of TAC surgery,pathological cardiac hypertrophy(LV mass:P<0.05)and cardiac dysfunction(EF:P<0.01)were significantly aggravated in mice with AAV-shRab27 injection.Furthermore,mice injected with small extracellular vesicles from moderate cold exposure mediated activated brown adipose tissue(EF:53.44±2.068%,LV mass:110.5±6.599mg)showed reduced pathological cardiac hypertrophy(LV mass:P<0.001)and cardiac dysfunction(EF:P<0.001)after 6 weeks of TAC surgery compared to mice injected with PBS control group(EF:41.16±3.415%,LV mass:137.7±11.66mg).Mice injected with small extracellular vesicles derived from non-activated brown adipose tissue at room temperature(EF:43.33±5.252%,LV mass:144.0±17.03mg)showed no significant differences in pathological cardiac hypertrophy and cardiac dysfunction indicators after 6 weeks of TAC surgery compared to mice injected with PBS control group(EF:41.16±3.415%,LV mass:137.7±11.66mg),with no statistical significance(EF:P>0.05,LV mass:P>0.05).4.Proteomics and WB detection revealed that EHD2 protein was significantly upregulated in small extracellular vesicles derived from activated brown adipose tissue by moderate cold exposure compared to small extracellular vesicles derived from non-activated brown adipose tissue at room temperature of 25℃(P<0.01),and the expression levels were significantly downregulated in pressure overload induced myocardial hypertrophy cardiac tissue and PE induced cardiomyocytes(both P<0.01).Brown adipocyte specific EHD2 knockout mice exposed to moderate cold(EF:43.88±2.089%,LV mass:134.9±8.060mg)showed more serious pathological cardiac hypertrophy(LV mass:P<0.01)and cardiac dysfunction(EF:P<0.01)after 6 weeks of TAC surgery compared to flox/flox control mice exposed to moderate cold(EF:51.36±3.052%,LV mass:105.2±9.496mg).Cardiomyocytes specific EHD2 knockout mice(EF:31.87±6.725%,LV mass:196.2±23.92mg),compared with flox/flox control mice(EF:41.06±4.249%,LV mass:140.0±19.49mg),showed aggravated pathological cardiac hypertrophy(LV mass:P<0.001)and cardiac dysfunction(EF:P<0.01)after 6 weeks of TAC surgery in the model 05).Subsequently,mice injected AAV virus with cardiomyocyte specific EHD2 overexpression(EF:53.73±4.43%,LV mass:119.0±8.680mg)compared with mice injected with control virus(EF:43.96±4.185%,LV mass:146.2±2.927mg),after 6 weeks of TAC surgery,pathological cardiac hypertrophy(LV mass:P<0.001)and cardiac dysfunction(EF:P<0.01)were significantly reduced.Furthermore,cardiomyocytes transfected with pcDNA3.1-EHD2 plasmid(width/length:0.222±0.046,cell size:2709±636.4μm2,ANP mRNA:0.777±0.065,BNP mRNA:2.595±0.286),compared with cardiomyocytes transfected with control plasmids(width/length:0.296±0.054,cell size:3306±679.3μm2,ANP mRNA:1.872±0.527,BNP mRNA:4.437±0.340),the cell width/length(P<0.001)and cell size(P<0.001)were significantly reduced,and the mRNA expression levels of ANP(P<0.01)and BNP(P<0.001)were significantly reduced.Cardiomyocytes transfected with shEHD2 plasmid(width/length:0.337±0.089,cell size:4369±882.8μm2,ANP mRNA:2.467±0.128,BNP mRNA:7.540±1.087),compared with cardiomyocytes transfected with control plasmids(width/length:0.236±0.050,cell size:3283±688.8μm2,ANP mRNA:2.031±0.053,BNP mRNA:5.467±0.694),the cell width/length(P<0.01)and cell size(P<0.001)significantly increased,and the mRNA expression levels of ANP(P<0.01)and BNP(P<0.05)significantly increased.5.Compared with the sham surgery group,the expression of EHD2 in membrane proteins was significantly reduced(P<0.001)in the heart tissue of TAC surgery model mice,while there was no significant difference(P>0.05)in the expression of EHD2 in cytoplasm proteins,indicating no statistical significance.In the heart tissue of Ang Ⅱ model mice,compared with the control group of heart tissue,the expression level of EHD2 in membrane protein was significantly reduced(P<0.05),and there was no significant difference in the expression level of EHD2 in cytoplasm protein(P>0.05),indicating no statistical significance.Firstly,Caveolae was observed in the cardiomyocytes of the TAC surgical model mouse heart tissue through transmission electron microscopy.Compared with the cardiomyocytes of the control group mouse heart tissue,after 6 weeks of TAC surgery,the Caveolae dynamics in TAC mice showed a significant abnormal state(P<0.01).In moderate cold exposure mice,the abnormal dynamics of Caveolae were significantly reduced compared to mice in a room temperature environment(P<0.01),while in mice with brown adipose tissue removed after moderate cold exposure,the abnormal dynamics of Caveolae were significantly aggravated after TAC surgery for 6 weeks(P<0.001).After injecting AAV-shRab27a virus into moderately cold exposed mice,compared with moderately cold exposed mice injected with the control virus,after 6 weeks of TAC surgery in the model,the abnormal dynamics of Caveolae significantly worsened(P<0.001).After injecting AAVcTnT-EHD2 virus into mice,compared with mice injected with the control virus,the abnormal dynamics of Caveolae were significantly reduced(P<0.001)after 6 weeks of TAC surgery in the model.In addition,adult mouse cardiomyocytes transfected with shEHD2 plasmid showed significantly more serious abnormal Caveolae dynamics after PE induction compared to adult mouse cardiomyocytes transfected with control plasmid(P<0.001).Compared with adult mouse cardiomyocytes transfected with control plasmid,adult mouse cardiomyocytes transfected with pcDNA3.1-EHD2 plasmid showed a significant reduction in Caveolae dynamics abnormalities after PE induction(P<0.05).ConclusionBased on the above research results,we reveal that moderate cold exposure can improve pathological cardiac hypertrophy and cardiac dysfunction by small extracellular vesicles derived from activated brown adipose tissue.Further research has confirmed that the EHD2 protein in small extracellular vesicles derived from activated brown adipose tissue regulates the abnormal Caveolae dynamics induced by pressure overload in cardiomyocytes,alleviating pathological cardiac hypertrophy and cardiac dysfunction.This study preliminarily elucidates the moderate cold exposure conditions that can improve pathological cardiac hypertrophy,explores new potential preventive and therapeutic strategies for improving pathological cardiac hypertrophy,and proves that small extracellular vesicles derived from moderate cold exposure mediated activated brown adipose tissue resist pathological cardiac hypertrophy,and that EHD2 protein in small extracellular vesicles regulates pressure overload induced abnormal Caveolae dynamics in cardiomyocytes.We also clarify the dynamic changes of the EHD2-Caveolae axis in cardiomyocytes induced by pressure overload,in order to protect the incidence and development of pathological cardiac hypertrophy. |
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