时间限制性进食对HFpEF小鼠心肌损伤改善及血清和心肌组织FGF21水平调节作用

Effect of time-restricted feeding on myocardial injury and its regulatory effect on FGF21 level in serum and myocardial tissues in mice with heart failure with preserved ejection fraction

目的观察时间限制性进食(TRF)对射血分数保留性心力衰竭(HFpEF)小鼠心功能障碍、心肌重构、心肌线粒体结构及功能、心肌氧化应激反应、心肌能量代谢的改善及对血清和心肌组织FGF21水平的调节作用,以探讨TRF对HFpEF心肌损伤的改善作用及机制。方法30只成纤维细胞生长因子21(FGF21)敲除(Fgf21-/-)小鼠均分为FGF21敲除自由进食(ALb)组和FGF21敲除TRF组,30只野生型(WT)小鼠均分为野生ALb组、野生TRF组。上述四组均给予60%高脂饲料,并且在每日饮水中加入Nω-硝基-L-精氨酸甲酯盐酸盐(L-NAME,0.5 g/L)建立HFpEF模型。野生TRF组和FGF21敲除TRF组小鼠接受TRF,每晚21:00至次日5:00进食,其余时间禁食;野生ALb组和FGF21敲除ALb组小鼠24 h均开放进食。四组均喂养8周,观察小鼠心肌损伤相关指标:心脏功能(LVEF、E/A、E/E'、BNP)、心肌重构情况(心肌肥厚程度相关指标心脏指数HW/TL、心肌细胞平均横截面积,心肌纤维化程度相关指标心肌胶原沉积程度及心肌纤维化相关基因Col1a1、Col3a1、Fn1和CTGF mRNA)、心肌线粒体结构及功能(损伤线粒体比、ATP生成量、心肌ATP含量、复合物Ⅰ活性、复合物Ⅴ活性)、心肌氧化应激水平(ROS、SOD活性,MDA含量)、心肌能量代谢情况(脂肪酸氧化关键分子CD36、CPT1b及13C Ac-CoA百分比,葡萄糖代谢相关基因Glut1、Glut4、PFK1 mRNA表达水平)以及血清、心肌组织FGF21表达水平。结果各组喂养8周时,与野生ALb组相比,野生TRF组小鼠E/A、E/E′值及血清BNP水平降低(P均<0.01);心脏质量、HW/TL值及心肌细胞平均横截面积降低(P均<0.01);心脏左室胶原占比及Col1a1、Col3a1、Fn1、CTGF mRNA表达降低(P均<0.01);心肌损伤线粒体占比减小,线粒体ATP生成量、线粒体复合物Ⅰ和Ⅴ活性升高(P均<0.01);心肌ROS生成及MDA含量减少,SOD活性升高(P均<0.01);心肌CD36和CPT1b表达升高,13C Ac-CoA百分比上升(P均<0.01);心肌Glut 1、Glut 4和PFK 1 mRNA表达降低(P均<0.01);血清FGF21水平、心肌FGF21蛋白及mRNA表达均增加(P均<0.01)。与野生TRF组相比,FGF21敲除TRF组小鼠E/A、E/E′值及血清BNP水平升高(P均<0.01);心脏质量、HW/TL值及心肌细胞平均横截面积增加(P均<0.01);心脏左室胶原占比及Col1a1、Col3a1、Fn1、CTGF mRNA表达增加(P均<0.01);心肌损伤线粒体占比增加,ATP生成量、复合物Ⅰ和Ⅴ活性降低(P均<0.01);心肌ROS生成及MDA含量增多,SOD活性下降(P均<0.01);心肌CD36和CPT1b表达减少,13C Ac-CoA百分比下降(P均<0.01);心肌Glut 1、Glut 4和PFK 1 mRNA表达增加(P均<0.01)。结论TRF对HFpEF小鼠的心脏功能及心肌重构、心肌线粒体结构及功能、心肌氧化应激反应、心肌能量代谢均具有改善作用,且可促进FGF21的表达;TRF可通过提高FGF21表达水平,进而增强心肌脂肪酸代谢、改善心肌线粒体结构功能并抑制氧化应激水平,改善HFpEF小鼠的心功能障碍及心肌重构,从而改善心肌损伤。

Objective To observe the effects of time-restricted feeding(TRF)on cardiac dysfunction,myocardial remodeling,myocardial mitochondrial structure and function,myocardial oxidative stress response,and myocardial energy metabolism in mice with heart failure with preserved ejection fraction(HFpEF),and to investigate the regulatory role of TRF on the levels of fibroblast growth factor 21(FGF21)in serum and myocardial tissues,in order to explore the role and mechanism of TRF in ameliorating myocardial injury in HFpEF.Methods Thirty FGF21 knockout(Fgf21-/-)mice were divided into the Fgf21-/-+ALb group and Fgf21-/-+TRF group.Thirty wild-type(WT)mice were divided into the WT+ALb group and WT+TRF group.Mice in all the four groups were fed a 60%high-fat diet,and Nω-nitro-L-arginine methyl ester hydrochloride(L-NAME,0.5 g/L)was added to daily drinking water to establish the HFpEF models.Mice in the WT+TRF group and Fgf21-/-+TRF group underwent TRF,with feeding allowed from 21:00 to 05:00 the next day and fasting during the rest of the time.Mice in the WT+ALb group and Fgf21-/-+ALb group had access to food 24 h a day.After 8 weeks,the myocardial injury-related indicators were observed,including cardiac function(LVEF,E/A,E/E',BNP),myocardial remodeling(HW/TL,mean cross-sectional area of cardiomyocyte,myocardial collagen deposition,and the mRNA levels of fibrosis-related genes Col1a1,Col3a1,Fn1,and CTGF),myocardial mitochondrial structure and function(damaged mitochondria ratio,ATP production,myocardial ATP content,Complex I activity,Complex V activity),myo‐cardial oxidative stress levels(ROS production,SOD activity,MDA content),myocardial energy metabolism(protein ex‐pression levels of CD36,CPT1b,the percentage of 13C Ac-CoA,and the mRNA levels of glucose metabolism-related genes Glut1,Glut4,PFK1),as well as FGF21 expression levels in serum and myocardial tissues.Results After 8 weeks of feeding,compared with the WT+ALb group,the E/A,E/E'values and serum BNP level decreased(all P<0.01),heart weight,HW/TL value and mean cross-sectional area of cardiomyocyte decreased(all P<0.01),the collagen deposition and the mRNA expression levels of Col1a1,Col3a1,Fn1,CTGF decreased(all P<0.01),the proportion of damaged mito‐chondria decreased,mitochondrial ATP production and activities of mitochondrial complexⅠandⅤincreased(all P<0.01),myocardial ROS production and MDA content decreased,SOD activity increased(all P<0.01),the protein ex‐pression levels of CD36 and CPT1b increased,the percentage of 13C Ac-CoA increased(all P<0.01),the mRNA expres‐sion levels of Glut1,Glut4 and PFK1 decreased(all P<0.01),and the serum FGF21 level and the myocardial FGF21 pro‐tein and mRNA expression levels increased in the WT+TRF group(all P<0.01).Compared with the WT+TRF group,the E/A,E/E'values and serum BNP levels increased(all P<0.01),heart weight,HW/TL value and mean cross-sectional area of cardiomyocyte increased(all P<0.01),the collagen deposition and mRNA expression levels of Col1a1,Col3a1,Fn1,CTGF increased(all P<0.01),the proportion of damaged mitochondria increased,mitochondrial ATP production and the activities of mitochondrial complexⅠandⅤdecreased(all P<0.01),myocardial ROS production and MDA con‐tent increased,SOD activity decreased(all P<0.01),the protein expression levels of CD36 and CPT1b decreased,the percentage of 13C Ac-CoA decreased(all P<0.01),and the mRNA expression levels of Glut1,Glut4 and PFK1 increased in Fgf21-/-+TRF group(all P<0.01).Conclusions TRF has beneficial effects on cardiac function,myocardial remodel‐ing,myocardial mitochondrial structure and function,myocardial oxidative stress response,and myocardial energy metabo‐lism in mice with HFpEF.Additionally,it promotes the expression of FGF21.By elevating the expression level of FGF21,TRF can enhance myocardial fatty acid metabolism,improve myocardial mitochondrial structural and function,and inhibit oxidative stress levels,thereby ameliorating cardiac dysfunction and myocardial remodeling in HFpEF mice,and ultimate‐ly mitigating myocardial injury.