Regulation Of LRP6 Played A Critical Role During The Development Of Heart Failure

We built pressure overload model by transverse aorta constriction (TAC) to induce cardiac hypertrophy and heart failure, and observed the expression profiles of LRP6 and p-LRP6 during the process of cardiac hypertrophy and heart failure induced by TAC. The results showed that LRP6 was up-regulated after TAC, p-LRP6 was up-regulated at 3d, 1w and 2w after TAC but was down-regulated at 4w after TAC. In cultured cardiomyocytes subjected to mechanical stretch or treated with angiotensin Ⅱ, the expression profiles of LRP6 and p-LRP6 were consistent with those in vivo. In addition, the present data revealed that TAC induced the proliferation of cardiac side population cells (CSPs) was associated with the up-regulation of p-LRP6 in CSPs mediated by mechanical stretch.We constructed shLRP6 lentivirus and LRP6 overexpression lentivirus to down-regulate the expression of LRP6 or overexpression LRP6 in cardiomyocytes subjected to mechanical stretch. The results showed that mechanical stretch activated MAPK signaling, up-regulated the mRNA expressions of ANP, BNP, SAA and induced cardiomyocytes hypertrophy, while shLRP6 greatly promoted these effects induced by mechanical stretch. However, LRP6 overexpression partly inhibited the up-regulation of p-ERK, p-P38 and p-JNK induced by mechanical stretch. Furthermore, shLRP6 greatly up-regulated the protein expression of AT1 in cultured cardiomyocytes. CO-IP results exhibited the binding of LRP6 with AT1 at physiological condition, but mechanical stretch inhibited the association of LRP6 with AT1.To explore the potential mechanisms in vivo, we generated Tamoxifen-inducible Cre-mediated cardiaomyocyte-specific LRP6 excision mice. Surprisingly, we observed that tamoxifen induced LRP6 knockout in cardiomyocytes quickly resulted in heart failure and death in mice. We observed the serious damage in cardiac contractility, enlargement of the heart, mitochondrial dysfunction and mitophagy. Further analysis showed the cardiac-specific LRP6 knockout resulted in the down-regulation of p-AMPK, the activation of mTOR, the translocation of DRP1 to mitochondria. These results suggested that these pathways or signaling may be involved in the process of heart failure after LRP6 knockout in cardiomyocytes of mice.So we can draw a conclusion that, LRP6 was involved in pressure overload-induced cardiac hypertrophy and played a critical role in cardiac hypertrophy and heart failure.Part 1 The expression profile of LRP6 during the development of heart failure induced by pressure overloadObjective:To elucidate whether LRP6 was involved in the pressure overload-induced cardiac hypertrophy or heart failure, we detected the expression of LRP6 during the process.Methods:8-10 weeks old C57B/L6 mice were subjected to sham or TAC operation for 3d, 1w,2w,4w respectively. Transthoracic echocardiographic analysis and invasive hemodynamics measurement were performed after TAC, the expression of LRP6, p-LRP6 were detected by western blot analysis. Cardiomyocytes subjected to mechanical stretch or treated with angiotensin Ⅱ were collected to extract protein for the following experiments. CSPs were isolated from sham or TAC mice by fluorescence-activated cell sorting (FACS) to detect p-LRP6 by NIA. Neonatal rat CSPs were subjected to mechanical stretch, then detected p-LRP6 by immunofluorescence analysis or NIA.Results:Pressure overload model for mice established. LRP6 was up-regulated after TAC, p-LRP6 was up-regulated at 3 days after TAC but down-regulated at 4 weeks. For cardiomyocytes subjected to mechanical stretch, LRP6 was up-regulated after 6 hours, whereas, p-LRP6 was up-regulated after 10 minutes, and reached peak value in 1 hour, then gradually down-regulated. For cardiomyocytes treated with angiotensin II, LRP6 was up-regulated after 6 hours, p-LRP6 was up-regulated in 5 minutes then gradually down-regulated after 6 hours. IWP2 had no observable effect on mechanical stretch or angiotensin II induced p-LRP6 up-regulation. Pressure overload model resulted in CSPs proliferation, TAC and mechanical stretch greatly up-regulated p-LRP6 in CSPs.Conclusion:The expression of LRP6 and p-LRP6 changed obviously during pressure overload. It suggested that LRP6 may be involved in the development of heart failure induced by pressure overload.The expression profile of LRP6 during the development of heart failure induced by pressure overloadPart 2 The investigation of underlying mechanism of LRP6 involved in pressure overload-induced cardiac hypertrophyObjective:To investigate the effect of LRP6 on pressure overload-induced cardiac hypertrophy.Methods:We packaged shLRP6 lentivirus to down-regulate the expression of LRP6 in cardiomyocytes. After interfering LRP6, cardiomyocytes were subjected to mechanical stretch for 8 minutes, then detected p-ERK, p-P38 and p-JNK. Total RNA was extracted and Real-Time PCR was performed to detect the expression of ANP, BNP and SAA after mechanical stretch for 1 hour,6 hours,12 hours and 24 hours respectively. The surface areas of cardiomyocytes were measured by immunofluorescence staining with a-MHC after mechanical stretch for 24 hours. We packaged overexpression lentivirus to up-regulate the expression of LRP6 in cardiomyocytes. Cardiomyocytes were subjected to mechanical stretch for 8 minutes or 30 minutes to detect p-ERK, p-P38 and p-JNK. The protein and mRNA expression level of ATI were detected after interfering LRP6 in cardiomyocytes. The binding of LRP6 and ATI in cardiomyocytes was analyzed by Co-immuneprecipitation.Results:Mechanical stretch induced the activation of p-ERK, p-P38 and p-JNK, the up-regulation of ANP, BNP and SAA expressions, the enlargement of surface areas of cardiomyocytes compared to those in control group. Down-regulation of LRP6 by shLRP6 grealty promoted these effects induced by mechanical stretch, while LRP6 overexpression partly inhibited the up-regulation of p-ERK, p-P38 and p-JNK induced by mechanical stretch. Insterestingly, the protein expression level of ATI was up-regulated in LRP6 interfered cardiomyocytes than that in control group, while the mRNA expression level of ATI was down-regulated slightly. CO-IP analysis exhibited the binding of LRP6 and ATI in cardiomyocytes, but mechanical stretch inhibited the combination.Conclusion:LRP6 down-regulation promoted mechanical stretch-induced cardiac hypertrophy, and this effect might partly result from AT1 up-regulation. LRP6 might regulate ATI protein expression at posttranscriptional level. CO-IP results exhibited the combination of LRP6 and ATI in cardiomyocytes, but mechanical stretch inhibited the effect.Part 3 Tamoxifen-inducible Cre-mediated cardiac-specific LRP6 excision mice exhibited failing heart phenotype and the investigation of mechanismObjective:To investigate the effect of LRP6 on cardiac function in adult mice in vivo.Methods:We generated Tamoxifen-inducible Cre-mediated cardiac-specific LRP6 excision mice.8 weeks old mice were treated with Tamoxifen by intraperitoneal injection 3 days(0.1mg/g/d) to induce cardiac-specific LRP6 excision. Transthoracic echocardiographic analysis and invasive hemodynamics measurement were performed, paraffin embedded hearts were sectioned and stained with hematoxylin and eosin (HE), electron microscopy was also performed. Mitochondria were isolated from heart. Total protein, mitochondrial protein and cytoplasmic protein were extracted for Western Blot analysis.Results:cardiac-specific LRP6 excision mice exhibited heart failure:left ventricular wall thickness was decreased, left ventricular internal diameter was enlarged, EF was decreased. Mitophagy and destruction of structure were detected by electron microscopy. Mitochondrial membrane potential was decreased. p-DRP1(ser616) was significantly increased, DRP1 was translocated to mitochondria, cytoplasmic β-catenin level was not significantly decreased.Conclusions:Cardiac-specific LRP6 excision mice exhibited failing heart phenotype, this might partly result from mitochondrial dysfunction, and was independent of Wnt/β-Catenin signaling.