Effects And Mechanism Of Differential Tumor Necrosis Factor Alpha And Tumor Necrosis Factor Receptors In Mice With Cardiac Hypertrophy

BackgroundHeart failure occurs by a variety of causes that ventricular contraction (diastolic) function decline, ejection dysfunction, so that cardiac output can not meet the needs of metabolism, organ, tissue blood perfusion with a scries of clinical syndrome. In developed countries, there are nearly1-2%of adults suffer from heart failure; the prevalence rate can be as high as10%in the population over the age of70.5-year mortality of late period heart failure is up to50%, which is even higher than cancer and AIDS. Cardiac hypertrophy is the early compensatory critical stage in the process of heart failure, which adapted to a variety of stimuli myocardial cells, such as increasing myocardial cell size and weight. Studies suggest that the primary mechanism of cardiac hypertrophy is mechanical activated, neuroendocrine system and chronic inflammation activation. Realizing the mechanism of heart failure, especially the process of cardiac hypertrophy to heart failure is very important to learn heart failure as well as providing new therapeutic strategies in clinical.Process of heart failure is associated with chronic inflammatory reaction. Inflammatory cytokines that highly expressed in patients with heart failure could promote cardiac hypertrophy, myocardial fibrosis, ventricular remodeling and myocardial cell apoptosis with process of heart failure. Tumor necrosis factor (TNF-α) is one of the key factors in many inflammatory factors. Levine et al found an inflammatory factor TNF-alpha was significantly higher in patients with heart failure in vivo in1990. After that, a large number of in-depth studies focus on TNF-alpha in heart failure and it has been confirmed that TNF-alpha and its receptors play an important role in cardiac hypertrophy and heart failure process. The mechanism currently accepted TNF-alpha on myocardial effects includes:interference of calcium influx, activation of NOS, and up-regulation of local NO level, suppression of myocardium contraction; activation of MMP and TIMPs, which involved in reconstruction of extracellular matrix. Therefore, more experiments and clinical studies focused on the anti-TNF-alpha therapy. Ten years’ studies proved the advantage of TNF antagonist on rat or mouse heart failure models. However, clinical studies revealed increased mortality of heart failure patients after treatment with TNF-αantagonist. It is meaningful to study the mechanism of TNF-αin heart failure.Divergent TNF-α Receptors Modulate Different Effect in Mice Cardiac Hypertrophy induced by Pressure-OverloadResearch aims:The effects and possible molecular mechanism of TNF receptors in Mice Cardiac Hypertrophy induced by Pressure-OverloadMethod:1. Performing pressure-overload cardiac hypertrophy models in TNFR1knockout (TNFR1KO) mice, TNFR2knockout (TNFR2KO) mice, TNFRs double knockout (TNFDKO) mice and wild type (WT) mice by Transverse Aortic Constriction (TAC) for14days. We characterized models by using morphological, hemodynamic and echocardiographic analysis. Inflammatory factors and key signalling pathway were examined to confirm the differences between different TNF receptor knockout mice.2. The embryonic rat heart-derived H9c2cells were transfected with siRNA of respective TNF receptors to downregulate the expression of TNF receptors. Cardiomyocytes were induced hypertrophy by Angiotensin Ⅱ. Testing and blocking the critical signalling pathway to confirm its effect of improving cardiac hypertrophy in vitro.Results:1. Different TNF-alpha receptors mediated different effects in the thoracic aortic coarctation-induced pressure overload cardiac hypertrophy. TNFR1increased the degree of cardiac hypertrophy, aggravated systolic diastolic dysfunction; TNFR2reduced the degree of cardiac hypertrophy and improve cardiac systolic and diastolic function.2. Expression of both TNF-alpha receptors was increased in thoracic aortic coarctation induced pressure overload cardiac hypertrophy, but the TNFR1elevation was higher than TNFR2.3. TNF-alpha receptors mediated different inflammatory reaction in thoracic aortic coarctation-induced pressure overload cardiac hypertrophy. TNFR1promoted expression of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) and downregulated anti-inflammatory cytokine (IL-10) expression. Conversely, TNFR2reduced the expression of pro-inflammatory cytokines, and enhance the anti-inflammatory cytokine expression.4. Different TNF-alpha receptor mediated different cardiac hypertrophy signalling pathway.5. Downregulation of TNFR1expression with the siRNA could reduce Angiotensin Ⅱ induced cardiomyocyte hypertrophy; silencing TNFR2increased Angiotensin Ⅱ induced cardiomyocyte hypertrophy.6. TNFR1induced cardiomyocyte hypertrophy by activation of NFκB, TNFR2protected myocardial cells through activation of the AKT signalling pathway. Effect and mechanism that caused by different forms of TNF-alpha in pressure overload-induced cardiac hypertrophy in miceResearch aims:Approaching relationship and mechanism of two forms TNF-alpha and its receptor in pressure overload-induced cardiac hypertrophy in miceMethod:1. Alzet micro-injection pumps filled with TACE inhibitor TAPI-1were used after thoracic aortic coarctation operation to reduce the transform from tmTNF to sTNF. Find the funtion and effect with upregulated tmTNF in TNF receptors knockout mice with hypertrophy. And identify the relevant signalling pathways.2. STNF was used in H9c2(2-1) cardiac myocytes after relative receptor siRNA transfection, simulating high levels of sTNF with different receptor relationship and function. H9c2(2-1) cells that transfected with relative receptors siRNA were cocultured with NIH-3T3cells that stable transfected of tmTNF. Cell proliferation and apoptosis pathway were tested, confirming the relationship and function of different types of TNF-alpha and TNF-alpha receptor.Results:1. Expression of tmTNF and secretion of sTNF were evaluated in mice cardiac hypertrophy that induced by thoracic aortic coarctation operation.2. Micro-injection pumps filled with TACE inhibitor TAPI-1could reduce the myocardial hypertrophy in mice that induced by TAC. The effect could be detected in wild-type, TNFR1and TNFR2knockout mice, but more obvious in TNFR2knock mice.3. sTNF mediated cardiomyocyte apoptosis and cytotoxicity mainly through TNFR1. tmTNF could reduce apoptosis and played a protective role in myocardial hypertrophy through TNFR2. Function of MDSC in pressure-overload induced cardiac hypertrophy mice modelResearch aims:Identify the relationship between MDSC and heart failure, and its function in pressure overload-induced cardiac hypertrophy mice model.Method:1. Using Flow Cytometry to detect the MDSC ratio in peripheral blood of patients with heart failure and identify relationship between MDSC and heart failure. Cardiac hypertrophy was constructed using thoracic aortic coarctation operation in different TNFR knockout mice. Flow Cytometry was applied to detect alteration of MDSC in peripheral blood, spleen and myocardium.2. Separated and purified MDSC were directly or indirectly cultured with myocardial cells, which was used to confirm the effect and mechanism between MDSC and Ang II induced myocardial cell hypertrophy.Results:1. The number of MDSC was elevated with advanced heart failure classification in peripheral blood of patients.2. MDSC ratio was increased in peripheral blood, spleen and myocardium of different TNFR knockout mouse with thoracic aortic coarctation operation induced cardiac hypertrophy, especially in TNFR1knockout mice.3. Co-culture of MDSC and myocardial cells revealed Angll induced myocardial cell hypertrophy was decreased by up-regulated IL-10expression during direct action of MDSC and myocardial cells. IL-10expression was higher in TNFR2expressed MDSC, which also was more useful for anti-cardiac hypertrophy.Statistical AnalysisContinuous data are summarized as mean±SD. SPSS13.0software is performed for statistical analysis. For2-group comparisons, we used the unpaired2-sample t-test. For comparisons of2groups, we used1-way ANOVA if there was1independent variable. Statistical significance was defined as P<0.05.Conclusion1. TNF-alpha and its receptors mediated different effects in the thoracic aortic coarctation-induced pressure overload cardiac hypertrophy. TNFR1increased the degree of cardiac hypertrophy, aggravated systolic diastolic dysfunction; TNFR2reduced the degree of cardiac hypertrophy and improve cardiac systolic and diastolic function.2. Expression of both TNF-alpha receptors was increased in thoracic aortic coarctation induced pressure overload cardiac hypertrophy, but the TNFR1elevation was higher than TNFR2.3. TNF-alpha receptors mediated different inflammatory reaction in thoracic aortic coarctation-induced pressure overload cardiac hypertrophy. TNFR1promoted expression of pro-inflammatory cytokines (TNF-a, IL-1β and IL-6) and downregulated anti-inflammatory cytokine (IL-10) expression. Conversely, TNFR2reduced the expression of pro-inflammatory cytokines, and enhance the anti-inflammatory cytokine expression.4. Different TNF-alpha receptor mediated different cardiac hypertrophy signalling pathway.5. Downregulation of TNFR1expression with the siRNA could reduce Angiotensin II induced cardiomyocyte hypertrophy; silencing TNFR2increased Angiotensin II induced cardiomyocyte hypertrophy.6. TNFR1induced cardiomyocyte hypertrophy by activation of NFκB, TNFR2protected myocardial cells through activation of the AKT signalling pathway.7. Inhibit transform of tmTNF to sTNF could reduce the myocardial hypertrophy in mice that induced by TAC, especially in TNFR2knock mice.8. sTNF mediated cardiomyocyte apoptosis and cytotoxicity mainly through TNFR1. tmTNF could reduce apoptosis and played a protective role in myocardial hypertrophy through TNFR2. 9. Number of MDSC increased with the severity of heart failure in peripheral blood of patients with heart failure.