| BackgroundIschemic myocardial disease is one of the leading causes of death in human.The main pathogenesis is inadequate blood supply to heart cause by narrowing or blockage of the coronary arteries.Hypoxia and nutrient deficiency in acute or persistent cardiac ischemia could induce apoptosis of cardiomyocytes,which in turn leads to pathological heart remodeling and systolic dysfunction.At present,there is no effective way to protect the heart from ischemic damage in clinic.As an essential trace element,copper(Cu)is closely linked to the occurrence and development of ischemic heart disease.As a key cofactor of various proteins,Cu is involved in various life activities such as mitochondrial energy metabolism,oxidative stress response,angiogenesis and the formation of connective tissue.Clinical studies have found a significant reduction in cardiac Cu concentrations of patients with myocardial infarction(MI).And previous studies showed that the Cu concentrations were decreased in the ischemic heart of mice and rhesus monkeys which were subjected to MI induced by left anterior descending in coronary artery(LAD)permanent ligation.The Cu efflux from heart after ischemic injury was accompanied with impaired myocardial systolic function,inhibited angiogenesis,and enhanced cardiac fibrosis.Is it possible to protect the heart from ischemic injury by inhibiting the reduction of Cu in ischemic myocardial tissue?However,the mechanism of Cu loss in ischemic myocardial tissue is still unknown.Cu homeostasis is precisely regulated by copper transporters.Abnormal expression of Cu transporters can lead to disorders of Cu homeostasis,and then promote the occurrence and development of diseases.Recent studies have found that there were no significant changes in CTR1 and CTR2,which related to cellular uptake of Cu,in myocardial tissue after ischemic injury,suggesting that ischemic injury does not affect cardiac copper uptake.In our research,we found that the protein level of Cu chaperone COMMD1 was significantly increased in ischemic myocardial tissue.And it has been reported that COMMD1 is mainly involved in the excretion of copper from liver to bile,and the deletion of Commdl gene leads to copper toxicosis in liver of Wellington dogs and mice.Based on the studies above,the following hypothesis were proposed in this study:1.Elevated expression of Cu chaperone COMMD1 promotes copper efflux in the ischemic myocardial tissues.2.Specific deletion of COMMD1 in cardiomyocytes can inhibit copper loss in ischemic myocardial tissue,thereby protecting the structure and function of the ischemic infarcted heart.Objective1.To investigate the relationship between Cu loss and disordered expression of Cu chaperon COMMD1 in the ischemic myocardial tissues.2.To further investigate whether decreased COMMD1 protein expression by gene modification can inhibit the Cu loss in the ischemic myocardial tissues,and protect the structure and function of the ischemic infarcted heart,and then explore its possible mechanisms.Methods1.Study on the correlation between Cu loss and disordered expression of Cu transporters in the ischemic myocardial tissues of rhesus monkeys and miceMale rhesus monkeys(2-3 years old)and male C57BL/6 mice(8 weeks old)were subjected to LAD permanent ligation to induce MI,while the Sham operated control group was subjected to the same operations without LAD ligation.Heart samples were collected 8 weeks after surgery in rhesus monkeys and 1,4,7 days after surgery in mice,and the hearts of MI group were divided into ischemic infarcted area(IA)and remote area(RA).The Cu concentrations of myocardial tissues were detected by atomic absorption spectroscopy(AAS),and the proteins expression and cell localization of CCS,COMMD1 and ATP7B were detected by western blotting(WB),immunohistochemistry(IHC)and immunofluorescence IF).2.Construction and evaluation of mice model with cardiomyocytes specific deletion of COMMD1Construction of cardiomyocyte-specific knockout of COMMD1 mice model(COMMD1CMC▲/▲):We crossed Commd1/loxp/loxp mice,with the well-characterized transgenic knock-in mouse line,and Myh6-CreER mice,expression Cre-recombinase under the cardiomyocyte-specific Myh6 promoter and tamoxifen induction,to generate Myh6-CreER;Commd1loxp/loxp mice and Commd1/loxp/loxp mice.Myh6-CreER;Commd1loxp/loxp mice and Commd1loxp/loxp mice(6-7 weeks old)were induced by tamoxifen for 2-3 weeks to obtain the experimental group(COMMD1CMC▲/▲)and control group(WT)mice.Evaluation of COMMD1CMC▲/▲ mice:The changes in body temperature,body weight,and heart function of mice were dynamically detected during tamoxifen induction.Blood and organ tissues of mice were collected three weeks after tamoxifen induction.The mRNA levels of COMMD1 in all organs were detected by qRT-PCR to access COMMD1 deletion.The Cu concentrations of all organs and serum were detected by AAS and the histopathological changes of all organs were detected by HE and SR staining.3.To investigate the effect of cardiomyocyte-specific knockout of COMMD1 on Cu loss in the ischemic myocardial tissuesMyh6-CreER;Commd1loxp/loxp mice and Commd1loxp/loxp mice(6-7 weeks old)were induced by tamoxifen for two weeks to obtain the experimental group(COMMD1CMC▲/▲)and control group(WT)mice,and then the mice were constructed MI or Sham operation.Therefore,the experiments were divided into four groups:WT-Sham,COMMD1CMC▲/▲-Sham,WT-MI and COMMD1CMC▲/▲-MI.Blood and all organs were collected 7 days after the operation,and the Cu concentrations in serum and organs were detected by AAS.4.To investigate the effect of cardiomyocyte-specific knockout of COMMD1 on cardiac structure,cardiac functions and survival rate in MI miceThe operations were same as the above steps.The heart functions of mice were detected by echocardiography 7 days after LAD surgery.Subsequently,the mice were deeply anesthetized and sacrificed for cardiac samples.The hearts were sectioned at an interval of 200 μm,and then histopathological staining(SR and HE)were performed to calculate the infarcted size of heart.The survival rate of mice was dynamically monitored within 7 days after surgery.5.To investigate the effects of cardiomyocyte-specific knockout of COMMD1 on angiogenesis,cardiomyocytes survival and collagen deposition in the ischemic myocardial tissuesFollowing the same operations as the above steps,frozen sections and total RNA were obtained from the heart tissues of the four groups’ mice 7 days after LAD surgery.Angiogenesis detection:The genes expression of Vegf,Vegfr-1 and Vegfr-2 in the ischemic myocardial tissues were detected by qRT-PCR,and the vessel density was detected by IF of CD31.Surviving cardiomyocytes detection:The cardiomyocytes in the ischemic myocardial tissues sections were labelled by IF of TNNI3 and WGA.Collagen deposition detection:SR staining was used to detect collagen deposition in the ischemic myocardial tissues.The genes expression of Ⅰ/Ⅲcollagen,MMP9/12/13 and LOX were detected by qRT-PCR in the ischemic myocardial tissues.Results1.In the ischemic myocardial tissues,Cu loss was associated with the accumulation of COMMD1 protein in cardiomyocytesIn Rhesus monkeys:Compared with Sham group,the Cu concentrations were significantly decreased and the protein levels of CCS,COMMD1 and ATP7B were significantly increased in the IA of MI group.The increased CCS and COMMD1 were located in cardiomyocytes and the increased ATP7B was located in cardiac fibroblasts.In mice:Compared with Sham group,the Cu concentrations were significantly decreased in the IA of MI group at 7 days after the operation,and protein level of COMMD1 was significantly increased in the IA of MI group at 1,4,and 7 days after the operation.The increased COMMD1was also located in cardiomyocytes in the ischemic myocardial tissues.2.Successful construction and physiological characteristics of COMMD1CMC▲/▲ mice modelCompared with WT mice,the mRNA levels of COMMD1 in COMMD1CMC▲/▲ mice were only reduced about 67%in heart tissues,but there were no significant changes in liver,spleen,lung,intestine and brain tissue,indicating the successful construction of the COMMD1CMC▲/▲ mice model.Compared with WT mice,the body temperature experienced a decrease and then returned to normal,and the body weight experienced a decrease and then reached a plateau in COMMD1CMC▲/▲ mice during 3 weeks of tamoxifen induction.After 3 weeks of tamoxifen induction,there were no significant changes in the histopathological structures,functions and Cu concentrations of hearts,but the HW/BW(heart weight/body weight)was a slightly increased in COMMD1CMC▲/▲ mice.3.During MI,cardiomyocyte-specific deletion of COMMD1 increased Cuconcentrations in the ischemic myocardial tissues and decreased Cu concentrations in the serumCu concentrations of heart tissues:Compared with the WT-Sham group,the Cu concentrations in the WT-IA group were significantly reduced about 64%.Compared with the WT-IA group,the Cu concentrations in the COMMD1CMC▲/▲-IA group were significantly increased about 53%.The WT-Sham group was 51.3±2.74 g/g,the WT-IA group was 18.3±4.7 g/g,the COMMD1CMC▲/▲-IA group was 28.0±5.5 g/g.Cu concentrations of serum:Compared with the WT-Sham group,the Cu concentrations in the WT-MI group were significantly increased about 109.1%.Compared with the WT-MI group,the Cu concentrations in the COMMD1CMC▲/▲-MI group were significantly reduced about 30.4%.The WT-Sham group was 796.7±137.2 g/L,the WT-MI group was 1666.3±703.2 g/L,the COMMD1CMC▲/▲-MI group was 1159.5 ± 311.0 g/L.Cu concentrations of non-cardiac tissue:there were no significant difference in Cu concentrations between the four groups.4.During MI,cardiomyocyte-specific deletion of COMMD1 effectively improved cardiac functions,reduced infarcted size of hearts and improved survival rate of miceCardiac functions:Compared with the WT-Sham group,the cardiac functions in the WT-MI group were significantly reduced.Compared with the WT-MI group,the cardiac function in the COMMD1CMC▲/▲-MI group was significantly increased.The WT-Sham group:EF was 78.3 ± 7.8%,FS was 41.7 ± 7.2%;the WT-MI group:EF was 46.1± 9.6%,FS was 19.7 ± 4.8%;the COMMD1CMC▲/▲-MI group:EF was 58.1 ± 11.9%,FS was 26.8 ± 7.5%.Infarcted size of hearts:Compared with the WT-Sham group,the infarcted size of heart in the WT-MI group was significantly increased about 72.3%.Compared with the WT-MI group,the infarcted size of heart in the COMMD1CMC▲/▲-MI group was significantly reduced about 17.2%.The WT-Sham group was 0%,the WT-MI group was 72.3±13.2%,the COMMD1CMC▲/▲-MI group was 55.1 ± 7.9%.Survival rate of mice:The survival rate of WT mice was 83.9%at 1 day after MI and 77.4%at 7 days after ML The survival rate of COMMD1CMC▲/▲mice was 85%at 1 to 7 days after MI.5.In the ischemic myocardial tissues,cardiomyocyte-specific deletion of COMMD1 promoted angiogenesis,increased the number of alive cardiomyocytes and decreased collagen depositionAngiogenesis:Compared with the WT-Sham group,the genes’ expression of Vegf,Vegfr-1 and Vegfr-2 and vessel density in the WT-IA group were significantly decreased.Compared with the WT-IA group,the genes’ expression of Vegf and Vegfr-1 and vessel density in the COMMD1CMC▲/▲-IA group were significantly increased,and the gene’s expression of vegfr-2 was no change.The number of surviving cardiomyocytes:Compared with the WT-Sham group,the number of surviving cardiomyocytes in the WT-IA group was significantly decreased.Compared with the WT-IA group,the number of surviving cardiomyocytes in the COMMD1CMC▲/▲-IA group was significantly increased.Collagen deposition:Compared with the WT-Sham group,the genes’expression of Ⅰ/Ⅲ type Collagen,Mmp12/13 and Lox in the WT-IA group were significantly increased.Compared with the WT-IA group,the genes’ expression of Ⅰ/Ⅲ type Collagen in the COMMD1CMC▲/▲-IA group were significantly decreased,but the genes’ expression of Mmp12/13 and Lox were no change.Conclusion:1.In the ischemic myocardial tissues,the protein level of COMMD1 in cardiomyocytes increased significantly,and promoted the reduction of Cu concentration.2.In the ischemic myocardial tissues,the cardiomyocyte-specific knockout of COMMD1 can partially prevent the reduction of Cu concentrations.The recovery of Cu concentrations promoting angiogenesis,maintains the number of cardiomyocytes and reduces the deposition of collagen in the ischemic area of the heart,ultimately resulting in reduced infarcted size of hearts and improved cardiac functions. |
PDF Full Text Request