Role Of Bim In Abnormal Temperature Stress Induced Effect On Heart Disease And Stem Cells Transplantation Intervention

Background:In recent years, extreme climate events occurred around the world frequently, such as severe snow or freezing weather disaster and the extraordinary summer heat. Extreme climate events can lead to aggravation of cardiovascular disease and higher morbidity and mortality. About the specific effects of cold and heat stress on heart disease, and the role of cold and heat stress in damage procedure in myocardial tissue, including relevant cellular biologic and molecular mechanisms, still lack of systematic research report yet. As to individuals with heart disease under cold and heat stress conditions, the deterioration of cardiac function, pathologic and anatomical parameters of myocardium damage, and related biochemical alterations, also require quantitative analysis, so as to provide data and information about the effects of cold and heat stress on cardiovascular disease.Cardiomyocytes injury can be manifested in many ways, including the damage of cell structural integrity, mitochondrial energy metabolism disorder, intracellular reactive oxygen species (ROS) outbreak, calcium overload and apoptosis. Myocardial cell injury caused by cold/heat stimulation may be reflected in all aspects of the above. Analysis of myocardial cell damage caused by cold/heat stimulation in various ways of observation may help to demonstrate characteristics of aggravation of cardiac disease induced by cold/heat stimulation at the cellular level.Cardiomyocyte apoptosis is one of important pathological processes of myocardial injury. Decrease of mitochondrial membrane potentialΔψm is the initiative event of apoptosis cascade procedure. Collapse ofΔψm leads to interruption of electron transport chain, arrest of oxidative phosphorylation, and eventually changes the electrochemical redox state of cells, and induces apoptosis protease-caspase activator release, then activation of Caspase-9, Caspase-3 lead to cell death or apoptosis. Study is needed to clarify that whether there is some relationship between cold/heat stimulation induced myocardial injury and the mitochondrial pathway. The outbreak of intracellular ROS is important mechanism in cardiomyocyte apoptosis. But in what way cold/heat stress to cause the outbreak of intracellular ROS is remained unclear. Reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex is major source of ROS in vivo. NADPH is composed by a number of different subunits. Most important ones among those subunits are nox-1 and p22-phox. Whether cold/heat stress causes expression alternations of nox-1 and p22-phox in cardiac cells, and the subsequence ROS activation, also needs experimental confirm.Bim is member of Bcl-2 family that with only BH3 domain (BH3-only), and it's considered as important pro-apoptotic protein. Bim is widely distributed through the body in various organizations, and performs the function as apoptosis regulator. In different cells and different apoptosis-inducing conditions, the regulation mechanism of Bim protein expression may be different. Currently, role of Bim protein in cardiomyocytes apoptosis hasn't been reported frequently yet. As one of the key protein that regulating cell apoptosis, Bim may plays significant role in cold/heat stimulation-induced myocardial cell apoptosis, and likely to regulate maintenance of cellular structure integrity, mitochondrial energy metabolism, intracellular ROS generation, calcium overload, and many other aspects in myocardial cell. To explore the role of Bim in cold/hot stimulation induced myocardial cell injury may shed some new light on the understanding of this pathologic procedure from in-depth apoptotic attitude, and may generate important theoretical significance.Bim may be closely associated with a variety of classic apoptosis-related signaling pathway. To investigate signal transduction pathway of Bim-mediated cold/ heat stimulation induced myocardial injury at level of molecular biology may suggest novel and effective therapeutic intervention target. Regulation to myocardial apoptosis performed by Bim may involve a number of signaling molecules, including PI3K/Akt/GSK-3β, ERK and other pathway. Whether these signaling pathways involved in the regulation of cold/heat stimulation induced myocardial injury and their relationship with the expression of Bim, has not been reported yet. Therefore, the role of Bim protein in the cold/heat stress induced myocardial injury and relative molecular mechanism, as well as interaction between PI3K/Akt/GSK-3βsignal pathway, ERK signaling pathway, will be established as topic of this current research.Many studies have confirmed the beneficial effects of stem cells transplantation on repair of myocardial injury. Mesenchymal stem cells (BM-MSCs) were commonly used in cell transplantation therapy and tissue engineering. BM-MSCs can improve and restore the cardiac function under state of myocardial ischemia or infarction in many ways. In extreme weather conditions, whether pretreatment by BM-MSCs transplantation intervention can enhance the tolerance of individuals suffering from myocardial infarction (MI) to cold/heat stress, as well as what possible specific mechanisms may be involved, have not been reported yet and also as one of the aspects to be investigated in this study.Methods:Based on the RXZ-300A artificial mimic climate chamber, we established model unit of cold/heat environmental stress stimulation. Animal models of myocardial infarction were given the cold/heat stress. Experimental groups were designed as:Ⅰ. Normal temperature group (MI),Ⅱ. Cold group (MI+Cold),Ⅲ. Heat group (MI+Heat). Size of myocardial infarct area was determined by standard staining method; Cardiac function was assessed by echocardiography; Parameters of hemodynamic were recorded via physiological recorder; Pathological characteristics of myocardium was evaluated by hematoxylin-eosin (HE) staining of myocardial tissue section; iNOS and eNOS mRNA expression in myocardial tissue were detected via real-time PCR; Myocardial expression of Bim protein was detected by western blot. In addition, animal model of cardiac hypertrophy were given the cold/heat stress as well. Groups were designed as:Ⅰ. Normal temperature group (H),Ⅱ. Cold group (H+Cold),Ⅲ. Heat group (H+Heat). Echocardiography and detection by multi-channel physiological recorder were performed after the cold/heat stress intervention, to evaluated cardiac function and parameters of hemodynamic.We established cellular cold/heat stimulation model in vitro. Neonatal rat myocardial cells were cultured to accept cold/heat stimulation, combined with Bim siRNA pretreatment. Experimental groups were designed as:Ⅰ. Control group (Control),Ⅱ. Cold stimulation group (Cold),Ⅲ. Heat stimulation (Heat),Ⅳ. Bim siRNA+Cold stimulation group (Bim siRNA+Cold),Ⅴ. Bim siRNA+Heat stimulation (Bim siRNA+Heat),Ⅵ. Non-coding siRNA+Cold stimulation (Non-coding siRNA+Cold),Ⅶ. Non-coding siRNA+Heat stimulation (Non-coding siRNA+Heat). Lactate dehydrogenase (LDH) activity in cell culture medium was detected; viability of cardiomyocytes was evaluated by MTS assay; levels of intracellular calcium, reactive oxygen species, mitochondrial membrane potential were detected via flow cytometry; TUNEL and AnnexinⅤPI fluorescence probe detection were performed to evaluate myocardial cell apoptosis; Bim protein expression in cardiomyocytes was analyzed by western blot detection.Neonatal rat myocardial cells were cultured to accept cold/heat stimulation, combined with Bim siRNA pretreatment. Experimental groups were designed as:Ⅰ. Control group (Control),Ⅱ. Cold stimulation group (Cold),Ⅲ. Heat stimulation (Heat),Ⅳ.Bim siRNA+Cold stimulation group (Bim siRNA+Cold),Ⅴ. Bim siRNA+Heat stimulation (Bim siRNA+Heat),Ⅵ. Non-coding siRNA+Cold stimulation (Non-coding siRNA+Cold),Ⅶ. Non-coding siRNA+Heat stimulation (Non-coding siRNA+Heat). Protein expression of Caspase-9, nox-1, p22-phox, ERK and phosphorylated ERK, PI3K and phosphorylated PI3K, GSK-3βand phosphorylated GSK-3βwere detected by western blot.Neonatal rat myocardial cells were cultured to accept cold/heat stimulation, combined with ERK pathway inhibitor-PD98059 or PI3K/Akt pathway inhibitor-LY294002 intervention. Experimental groups were designed as:Ⅰ. Control group (Control),Ⅱ. Cold stimulation group (Cold),Ⅲ. Heat stimulation group (Heat),Ⅳ. Cold stimulation+LY294002 group (Cold+LY294002),Ⅴ. Heat stimulation+LY294002 group (Heat+LY294002),Ⅵ. Cold stimulation+PD98059 group (Cold+PD98059),Ⅶ. Heat stimulation+PD98059 group (Heat+PD98059). Expression of Bim protein was detected by western blot.Autologous bone marrow mesenchymal stem cells (BM-MSCs) were cultured from bone marrow of rabbit. Stem cell transplantation was performed via direct injection into infarcted area in heart of rabbit MI model. Then animals underwent cold/heat stress. Groups were designed as:Ⅰ. MI+Cold intervention group (MIC), Ⅱ. MI+Heat intervention group (MIH),Ⅲ. MI+cell culture medium injection (control)+Cold intervention group (MIC+M),Ⅳ. MI+cell culture medium injection (control)+Heat intervention group (MIH+M),Ⅴ. MI+BM-MSCs transplantation+ Cold intervention group (MIC+SCs),Ⅵ. MI+BM-MSCs transplantation+Heat intervention group (MIH+SCs). Size of myocardial infarct area was determined by standard staining method; cardiac function was assessed by echocardiography; expression of Bim protein was detected by western blot.Results:After animal model of myocardial infarction accepted cold/heat stress intervention, size of myocardial infarcted area in MI+Cold group and MI+Heat group were expanded compared with MI group (p<0.01, MI:36.27±4.43%vs. MI+Cold: 46.74±3.21% & MI+Heat:42.62±2.80%). After the cold/heat stress intervention, MI rabbit's left ventricular end diastolic diameter (LVEDD) and left ventricular end systolic diameter (LVESD) were increased compared with normal temperature group; left ventricular posterior wall thickness (LVWT) and systolic interventricular septal thickness (IVST) were decreased compared with normal temperature group; left ventricular ejection fraction (LVEF) was significantly decreased compared with normal temperature group. Left ventricular end systolic pressure (LVESP) in Cold/ Heat stress group was lower than normal temperature group; left ventricular end diastolic pressure (LVEDP) was higher than normal temperature group; maximal rate of left ventricular pressure rise (+dp/dtmax) and left maximal rate of ventricular pressure decline (-dp/dtmax) were significantly lower than normal temperature group. Tissue sections HE staining showed that the fibrosis of infarcted area and pathological changes of myocardial structure in Cold/Heat group were more serious than that in normal temperature group. After Cold/Heat stress intervention, the iNOS mRNA expression in infarcted myocardium in MI+Cold group was about 2 times of that in MI group; eNOS mRNA expression was about 60% of that in MI group. The iNOS mRNA expression in infarcted myocardium in MI+Cold group was about 1.5 times of that in MI group; eNOS mRNA expression was about 40% of that in MI group. After Cold/Heat stress intervention, Bim protein expression level in infarcted myocardium was increased (MI+Cold:0.59±0.07; MI+Heat:0.46±0.01; vs. MI: 0.34±0.04, p<0.05).After animal model of cardiac hypertrophy accepted cold/heat stress intervention, end-diastolic left ventricular posterior wall thickness (LVPWTd), end-diastolic interventricular septal thickness (IVSTd), left ventricular end diastolic diameter (LVDd) in Cold/Heat group were higher than that in normal temperature group, suggested increased cardiac hypertrophy. Cold/Heat group's left ventricular systolic pressure (LVSP), maximum rate of left ventricular pressure (±dp/dtmax), were increased compared with normal temperature group, suggested further increase of the systolic pressure and deterioration of high-pressure in left ventricular caused by cardiac hypertrophy.After Cold/Heat stimulation, LDH activity in myocardial cell culture medium increased significantly (Cold:105.15±6.98 IU/L; Heat:96.49±5.94 IU/L vs. Control:41.09±4.59 IU/L, p<0.01). In Bim RNA interference groups, LDH activity increasing in myocardial cell culture were alleviated than that in the pure Cold/Heat stimulation groups (p<0.01, Bim siRNA+Cold:81.56±5.38 IU/L; Bim siRNA+Heat: 62.79±3.90 IU/L). After Cold/Heat stimulation, cardiomyocytes viability was decreased significantly (Cold:65.46±4.56%; Heat:68.56±3.92% vs. Control: 90.13±5.01%, p<0.01). Bim RNA interference induced improved survival rate of cardiomyocytes under cold stimulation (p<0.01, Bim siRNA+Cold:80.65±3.05%), while with no effect on survival rate of cardiomyocytes in Heat condition (p> 0.05, Bim siRNA+Heat:76.37±5.61%). After Cold/Heat stimulation, accumulation of intracellular calcium in cardiomyocytes was increase (Cold:625±27 nmol/L; Heat: 588±49 nmol/L vs. Control:361±52 nmol/L, p<0.01). Bim RNA interference can alleviate intracellular calcium increasing under Cold/Heat condition (p<0.01, Bim siRNA+Cold:619±33 nmol/L; Bim siRNA+Heat:591±61 nmol/L). After Cold/ Heat stimulation, the level of intracellular reactive oxygen species was significantly increased (Cold:10.8±0.85; Heat:9.92±0.93 vs. Control:3.61±0.71, p<0.01). While accepted Bim RNA interference pretreatment, intracellular ROS was decreased significantly compared with pure Cold/Heat stimulation group (p<0.01, Bim siRNA+Cold:4.82±0.48; Bim siRNA+Heat:4.80±0.51).Mitochondrial membrane potentialΔΨm level detection by flow cytometry resulted as:after Cold/Heat stimulation,ΔΨm was significantly lower (Cold: 51.32±5.57; Heat:47.18±5.11 vs. Control:83.29±7.20, p<0.01). Bim RNA interference can alleviateΔΨm decreasing when compared with pure Cold/Heat stimulation group (p<0.05, Bim siRNA+Cold:69.26±7.13; Bim siRNA+Heat: 70.08±6.56). Mitochondrial membrane potential detection via fluorescence microscopy resulted as:cardiomyocytes under Cold/Heat stimulation with higher green fluorescence intensity than control group, while the red fluorescence intensity was decreased, which indicated mitochondrial membrane potential damage. Bim RNA interference preconditioning weaken the intensity of green fluorescence, enhanced red fluorescence intensity in cardiomyocytes under Cold/Heat stimulation, suggested that reduced Bim by RNA interference can alleviate Cold/Heat stimulation induced decline in cardiac mitochondrialΔψm. Results of TUNEL detection and flow cytometry AnnexinⅤPI detection showed that the Cold/Heat stimulation significantly increased myocardial apoptosis rate. Bim RNA interference decreased cardiomyocytes apoptosis caused by Cold/Heat stimulation.Cold/Heat stimulation significantly increased the expression of Bim protein in cardiomyocytes (Cold:1.14±0.08; Heat:1.05±0.08 vs. Control:0.20±0.03, p<0.01). Bim RNA interference significantly down-regulated high expression of Bim protein in cardiomyocytes induced by Cold/Heat stimulation, compared with pure Cold/ Heat stimulation group (p<0.01, Bim siRNA+Cold:0.47±0.05; Bim siRNA+Heat: 0.32±0.05).After Cold/Heat stimulation, Caspase-9 protein level in cardiomyocytes increased significantly (Cold:1.05±0.09; Heat:1.38±0.13 vs. Control:0.11±0.01, p <0.01). Bim siRNA pretreatment alleviated intracellular Caspase-9 expression increasing in cardiomyocytes under Cold/Heat stimulation (p<0.01, Bim siRNA+Cold:0.22±0.01; Bim siRNA+Heat:0.32±0.01). After Cold/Heat stimulation, nox-1, p22-phox protein expression were increased (nox-1:Cold:1.05±0.09; Heat:1.38±0.13 vs. Control:0.11±0.01, p<0.01; p22-phox:Cold:1.35±0.06; Heat:1.58±0.03 vs. Control:0.39±0.01, p<0.01). Bim RNA interference attenuated heat stimulation caused high expression of nox-1(p<0.01, Bim siRNA+Heat:0.32±0.01 vs. Heat:1.04±0.01) and cold stimulation caused p22-phox high expression (p <0.01, Bim siRNA+Cold:0.87±0.03; Bim siRNA+Heat:1.05±0.06).After Cold/Heat stimulation, ERK5 and p-ERK5 protein expression in cardiomyocytes were significantly increased. Bim siRNA alleviated intracellular high expression of ERK5 and p-ERK5 induced by cold stimulation and high expression of p-ERK5 induced by heat stimulation, but performed no significant change to heat stimulation induced intracellular ERK5 high expression. After Cold/Heat stimulation, PI3K and p-PI3K protein expression in cardiomyocytes tended to increase slightly, but no significant changes were observed (p> 0.05). Bim siRNA didn't make significant change to PI3K and p-PI3K protein expression in cardiomyocytes under the Cold/Heat stimulation (p> 0.05). Cold/Hot stimulation did not cause significantly change in GSK-3βprotein expression in cardiomyocytes (p> 0.05). Bim siRNA performed no significant effects on GSK-3βprotein expression in cardiomyocytes under Cold/Heat stimulation. Cold/Heat stimulation induced increasing of p-GSK-3βprotein in cardiomyocytes (p<0.01, vs. Control group). Bim siRNA alleviated increasing of p-GSK-3βprotein in cardiomyocytes under cold/heat stimulation (p<0.01, Cold:1.15±0.06 vs. Bim siRNA+Cold:0.89±0.06; Heat: 1.05±0.09 vs. Bim siRNA+Heat:0.83±0.03).LY294002 intervention performed no effect on Bim expression in cardiomyocytes under Cold/Heat stimulation. PD98059 intervention furthered Bim expression increasing in cardiomyocytes under cold stimulation (p<0.05, Cold: 0.57±0.03 vs. Cold+PD98059:0.71±0.04) and caused more obvious higher expression of Bim protein under heat stimulation (p<0.01, Heat:0.59±0.05 vs. Heat+PD98059:0.87±0.03).BM-MSCs transplantation pretreatment performed no effect on the size of infarcted area of MI in vivo. But the LVEF of MI individuals under Cold/Heat stress improved significantly by BM-MSCs transplantation pretreatment. BM-MSCs transplantation pretreatment also attenuated Cold/Heat stress induced high expression of pro-apoptotic protein Bim in infarcted myocardium (p<0.01, MIC+SCs: 0.52±0.02 vs. MIC:0.83±0.02; MIH+SCs:0.76±0.01 vs. MIH:0.85±0.02). Conclusion:Cold/Heat stress can lead to developed size of infarcted area of MI in vivo, aggravate cardiac systolic and diastolic function disorder, and further aggravate myocardial necrosis and apoptosis; Cold/Heat stress can induce increased expression of iNOS mRNA and decreased eNOS mRNA expression in infarcted myocardium to perform detrimental effect; Cold/Heat stress can lead to aggravation of pathologic structural change and hemodynamic disorder in cardiac hypertrophy situation; Cold/ Heat stimulation cause intracellular calcium overload, increased ROS level, and reduced mitochondrial membrane potential, resulting in decreased survival rate and increased apoptosis rate of cardiomyocytes in vitro; Intracellular pro-apoptotic protein Bim expression is associated with damage that Cold/Heat stimulation caused in cardiomyocytes. Bim may mediate the Cold/Heat stimulation induced myocardial cell injury; Bim can induce increased expression of Caspase-9, nox-1 and p22-phox protein expression, increase intracellular ROS level, to mediate apoptotic effect of Cold/Heat on cardiomyocytes; ERK5 signaling pathway suppresses Cold/Heat stimulation caused expression of Bim protein in feedback style, and plays a protective role against cardiomyocytes apoptosis. PI3K/GSK-3P signaling pathway may regulate the Cold/Heat stimulation caused expression of Bim protein; BM-MSCs transplantation pretreatment can alleviate deterioration of cardiac function disorder in MI individuals under Cold/Heat stress, and possibly play a protective role via down-regulation of pro-apoptotic protein Bim expression.