Ablation Of C/EBP Homologous Protein Increases The Acute Phase Mortality And Doesn’t Attenuate Cardiac Remodeling In Mice With Myocardial Infarction

Background and ObjectiveMyocardial infarction (MI) is a leading cause of morbidity and mortality of coronary heart disease in the world. ST segment elevation myocardial infarction (STEMI),a common type of acute myocardial infarction, seriously threats to human health and increases medical costs of the patients. Early reperfusion therapy including thrombolysis and primary percutaneous coronary intervention is the most practical and widely practiced approach for MI in clinic. In fact, a considerable part of myocardial infarction patients can’t receive timely reperfusion therapy for restriction of technical, economic and environmental factors. For MI patients who have no opportunity to receive reperfusion therapy, attenuating post-infarction cardiac remodeling is a reasonable strategy.The endoplasmic reticulum (ER) is classically characterized as an organelle that participates in the folding of membrane proteins and secretory proteins. Various cellular stresses, including ischemia, hypoxia, heat shock, genetic mutation, oxidative stress, and increased protein synthesis, can lead to impairment of ER function. Stimuli that cause ER dysfunction are collectively known as ER stress. When ER stress is excessive and/or prolonged, however, apoptotic signals are initiated by the ER, including induction of C/EBP homologous protein (CHOP), activation of Jun N-terminal kinase (JNK), and cleavage of caspase-12. If the period of acute myocardial ischemia is prolonged (more than 20 minutes), a "wave front" of cardiomyocyte death begins in the subendocardium and extends transmurally over time toward the epicardium. The deprivation of oxygen and nutrient supply results in a series of abrupt biochemical and metabolic changes within the myocardium. The absence of oxygen halts oxidative phosphorylation, leading to mitochondrial membrane depolarization, ATP depletion, and inhibition of myocardial contractile function. This process is exacerbated by the breakdown of any available ATP, as the ATPase functions in reverse to maintain the mitochondrial membrane potential, resulting in ATP hydrolysis and an increase in mitochondrial inorganic phosphate. In the absence of oxygen, cellular metabolism switches to anaerobic glycolysis, resulting in the accumulation of lactate, which reduces intracellular pH (to<7.0). The intracellular accumulation of protons activates the Na+-H+ ion exchanger, which extrudes protons from the cell in exchange for Na+ entry. The lack of ATP during ischemia ceases function of the 3Na+-2K+ ATPase, thereby exacerbating the intracellular Na+ overload. In response, the reverse activation of the Na+-Ca2+ ion exchanger results in intracellular Ca2+ overloading as the cell tries to extrude Na+. Consequently, myocardial intracellular Ca2+ overloading leads to the initiation of the ER stress.Myocardial apoptosis and fibrosis greatly contribute to cardiac remodeling and dysfunction. Acute myocardial ischemia is caused mostly by the interruption and a sharp reduction of coronary ischemia. As a result, myocardial necrosis happens which was replaced by scar tissue and leads to impairment of left ventricular contractility. There is a high risk of death in the acute phase of myocardial infarction, while the chronic phase is featured with ventricular remodeling and heart failure. Pathological manifestations of ventricular remodeling are the expansion of left ventricular, thinning of wall-thickness, decrease in the systolic and diastolic functions. Ventricular remodeling is a pathological process which is closely related with hemodynamic disorder and if it goes for a long time, chronic heart failure will be developed.Myocardial apoptosis and fibrosis greatly contribute to cardiac remodeling and dysfunction. ER stress has been demonstrated to be closely associated with apoptosis and fibrosis. ER stress is prolonged and aggravated in hearts subjected to pressure overload, ischemia/reperfusion injury or MI, whereas CHOP is up-regulated and contributes to cardiac dysfunction. Ablation of CHOP can attenuate cardiac ER stress in mice subjected to pressure overload, attenuate myocardial reperfusion injury by inhibiting myocardial apoptosis and inflammation. However, the role of CHOP deficiency in the heart suffering from MI without reperfusion remains unknown. Considering the clues aforementioned, we hypothesized that CHOP ablation would inhibit post-MI cardiac remodeling.ER stress is a proapoptotic and profibrotic stimulus. Ablation of CHOP is reported to reverse cardiac dysfunction by attenuating cardiac ER stress in mice with pressure overload or ischemia/reperfusion, but it is unclear whether loss of CHOP also inhibits permanent-infarction induced cardiac remodeling.Materials and methods1. MI models8-12 weeks,20-25g weight CHOP-/-mice (KO) and CHOP+/+littermates (WT) were used to generate MI models by left coronary artery ligation as described elsewhere. In brief, mice were anaesthetized with a mixture of xylazine (5mg-kg-1, ip) and ketamine (100 mg-kg-1, ip), and the depth of anesthesia was assessed by monitoring the pedal withdrawal reflex. Mice were then ventilated and subjected to a left-sided thoracotomy and the left coronary artery ligation to induce MI with subsequent development of heart failure. Ischemia was judged by myocardial blanching and electrocardiogram ST-segment elevation. Sham operated mice underwent the same procedure without ligation of left coronary artery.2. Myocardial ischemia/reperfusion models (IR)Both KO and WT mice (aged 8-12 weeks, weighing 20-25g) were used to generate IR models by left coronary artery ligation as described elsewhere. In brief, mice were anaesthetized with a mixture of xylazine (5mg·kg-1, ip) and ketamine (100 mg·kg-1, ip), and the depth of anesthesia was assessed by monitoring the pedal withdrawal reflex. Mice were then ventilated and the fourth left intercostal thoracotomy was performed to expose the heart. The left coronary artery was ligated using an 8-0 silk suture, and a plastic bead was placed between the ligature and the artery. The ligature was tightened around the plastic bead to induce myocardial ischemia. After 45 min, plastic bead was removed for reperfusion for 7 days. An increasing ST segment and a high T segment indicated myocardial ischemia, and the decreasing ST segment and the recovered T segment after loosening the ligature indicated the successful reperfusion. Sham operated mice underwent the same procedure without ligation of left coronary artery.3. Survival analysis8-12 weeks,20-25g weight KO and WT mice were used to make MI models, which were divided into WT+ sham group, WT+MI group, KO+ sham group and KO+MI group. The survival of mice was checked out twice a day to ensure that once a dead mouse is found, autopsy should be performed immediately to confirm the reason of death.4. Myocardial infarct size measurementsWe measured myocardial infarct size by using TTC (triphenyltetrazolium chloride) staining to evaluate the effect of CHOP knockout on the degree of myocardial infarction. Experimental groups were designed as WT+ MI group and KO+MI group. We sacrificed the mice 3-12 hours after MI surgery, and the harvested hearts were frozen and sliced for TTC staining. The infarct area (grey white), the area at risk (red) and the remote area (blue) could be distinguished, myocardial infarct size (%)=infarct area/left ventricular area x 100%.5. Echocardiographic measurementBoth left ventricular dimensions and function were evaluated by using echocardiography at 28 days after MI models. Mice were divided into WT+sham group, WT+MI group, KO+sham group and KO+MI group. Mice were fixed in waking state to avoid the influence of anesthesia on the parameter measurements. Two-dimensional echocardiographic views of the mid-ventricular short axis were obtained at the level of the papillary muscle tips below the mitral valve. From M-mode tracings, left ventricular anterior and posterior wall thickness at diastole (LVAWd and LVPWd), the left ventricular end-diastolic diameter (LVEDd) and left ventricular end-systolic diameter (LVESd) were measured, while left ventricular fractional shortening (LVFS) and ejection fraction (LVEF) were calculated by the following equations:LVFS (%)= (LVEDd-LVESd)/LVEDd x 100%; LVEF (%) = [(LV end-diastolic volume - LV systolic volume)/LV end-diastolic volume] *100%. AH animals were sacrificed by overdose anesthesia with pentobarbital sodium (150 mg.kg-1, ip) and cervical dislocation, and their hearts and lungs were extracted for calculation of heart weight to body weight ratio (HW/BW) and lung weight to BW ratio (LW/BW).6. Left ventricular hemodynamicsLeft ventricular hemodynamics was evaluated at 28 days after MI models. Briefly, mice from each group were anesthetized with a combination of xylazine and ketamine (light anesthesia for MI mice), and were ventilated. A Millar catheter was inserted via the right carotid artery and carefully introduced into the left ventricular to measure the heart rate (HR), systolic pressure (LVSP), end-diastolic pressure (LVEDP), maximum and minimum rates of change in the left ventricular pressure (dp/dt max and dp/dt min, respectively). Both the contractility index (max dp/dt divided by the pressure at the time of max dp/dt) and the exponential time constant of relaxation (τ) were calculated using a software program. As well as echocardiographic measurements, Animals were divided into WT+sham group, WT+MI group, KO+ sham group and KO+MI group.7. TUNEL assay and Masson stainingWe evaluated the effect of CHOP knockout on apoptosis in myocardium and myocardial fibrosis in mice with MI by TUNEL staining and Masson staining. We sacrificed the mice 3-12 hours followed MI surgery or 24 hours after successful reperfusion by overdose anesthesia and cervical dislocation. The hearts were embedded with Paraffin and slice for TUNEL assay and Masson staining.8. Statistical analysisStatistical significance was calculated using SPSS 16.0. Numerical data were expressed as mean±standard error of mean. Statistical differences were analyzed with ANOVA followed by Boneferron’s correction for post hoc multiple comparisons. The overall survival of MI mice for 4 weeks was evaluated using Kaplan-Meier survival analysis and groups were compared by log-rank test. P values lower than 0.05 were considered to be statistically significant.Results1. Ablation of CHOP increased MI-induced mortalityThe survival of mice was checked twice a day to ensure once a dead mouse is found, and autopsy should be performed immediately to confirm the reason of death. We found that the overall mortality of CHOP mice with MI was significantly higher than in wildtype group (68% vs 38.5%, P<0.05). Most of them died in the first week. These results indicate that ablation of CHOP increased post-MI rupture in mice without prompt reperfusion.2. Ablation of CHOP failed to decrease myocardial infarct size of MI miceSince knockout CHOP increased post-MI rupture in mice. We measured myocardial infarct size by using TTC staining to evaluate the effect of CHOP knockout on the degree of myocardial infarction. Mice were sacrificed 3-12 hours after MI surgery by overdose anesthesia and cervical dislocation. The harvested hearts were frozen and sliced for TTC staining. We found no significant difference between KO group and WT group (45.7% vs 47.3%, P=0.54), which indicate that ablation of CHOP doesn’t decrease myocardial infarct size of MI mice.3. Ablation of CHOP didn’t ameliorate cardiac remodeling and cardiac dysfunction of MI miceWe checked cardiac remodeling and cardiac dysfunction by echocardiographic measurements four weeks after MI models, no significant differences were noted on left ventricular diameter and systolic function except that LVAWd (infarcted wall) was significantly thicker in CHOP MI mice than in wildtype ones. When compared with the corresponding sham group, it was similar to wildtype mice that CHOP-deficient mice with MI showed enlarged left ventricle (increased LVESD and LVEDD), decreased LVFS and LVEF. Besides, at 4 weeks after MI, both CHOP knockout mice and their wild-type littermates exhibited increased heart weight to body weight ratio (HW/BW). However, there was no significant difference on HW/BW or LW/BW. These findings revealed that deficiency of CHOP can’t significantly ameliorate post-MI cardiac remodeling.4. Ablation of CHOP didn’t improve post-MI left ventricular hemodynamicThe left ventricular hemodynamic assessment showed that MI for 4 weeks resulted in a significant reduction of LVSP, max dp/dt, min dp/dt, and contractility as well as a significant increase of LVEDP and τ when compared with the corresponding sham groups, but depletion of CHOP didn’t exert significant influences on those indexes.5. Ablation of CHOP had no benefit to cardiomyocyte apoptosis and myocardial fibrosis of MI miceThe effect of CHOP deletion on myocardial fibrosis and cardiomyocyte apoptosis was detected by TUNEL assay and Masson staining after MI for 4 weeks. Infarct area was replaced by fibrotic scar tissues, there is no significant difference on old myocardial infarct size (fibrosis) and myocardial fibrosis in the remote area between CHOP knockout mice and wildtype ones and apoptosis in this area was similar between CHOP knockout mice and wildtype ones. These findings once indicated that CHOP deletion has no benefit to myocardial infraction.6. Effect of CHOP ablation on myocardial infraction area, cardiomyocyte apoptosis and myocardial fibrosis of ischemic/reperfusion injury miceSince ablation of CHOP increased MI-induced mortality, and had no effect on myocardial infarct size, cardiac remodeling, cardiac dysfunction or cardiomyocyte apoptosis and myocardial fibrosis of MI mice, which is far from our expectations. We checked the effect of CHOP ablation on MI size, cardiomyocyte apoptosis and myocardial fibrosis in ischemia/reperfusion models. As expected, in mice with ischemia for 45 min and reperfusion for 24 hours, myocardial infarct size was significantly smaller in CHOP knockout mice than in their wildtype littermates (29.83% VS 17.34%, P<0.05). Fibrosis was checked 1 week after reperfusion. We noted that fibrosis in infarct area was significantly less in CHOP knockout mice than in wildtype ones (23.0% VS 16.0%, P<0.05), while it was similar in remote area. In response to ischemia for 45 minutes and reperfusion for 1 week, apoptosis in infarct area was significantly less in CHOP knockout mice than in wildtype ones (0.84% VS 0.56%, P<0.05). These findings indicates that CHOP deletion benefits to ischemic/reperfusion injury.ConclusionInhibition of CHOP is known to exert anti-apoptotic and anti-fibrotic role in heart, howerver, deletion of CHOP increases the incidence of post-MI cardiac rupture and doesn’t prevent cardiac remodeling in the case of permanent coronary ligation without early reperfusion, but knockout of CHOP attenuates myocardial reperfusion injury, which indicates early reperfusion therapy a basic prerequisite for patients suffering from MI benefited from ablation of CHOP.