CAJ | 학술논문

目的分析心肌肌浆网Ca2+-ATP酶(sarcoplasmic reticulum Ca2+ ATPase 2a,SERCA2a)基因转导对慢性心力衰竭(HF)犬心肌蛋白质组的影响,探讨SERCA2a基因转导改善心功能的机制.方法快速右心室起搏建立HF犬模型并随机分为HF组、HF+绿色荧光蛋白(enhanced green fluorescent pmtein,EGFP)组、HF+SERCA2a组.后两组分别向心肌内注射携带EGFP和SERCA2a基因的rAAV载体.于基因转导30 d时停止起搏后进行超声心动图和血流动力学检查并制备心室肌双向电泳蛋白样品和心肌双向电泳图谱,图像分析软件分析蛋白表达差异点,MALDI-TOF-MS数据库搜索鉴定蛋白质.结果基因转导30 d时,HF+SERCA2a组犬的症状、超声心动图和血流动力学指标与HF+EGFP组相比有显著好转(P＜0.05);与对照组相比差异无统计学意义(P＞0.05).挑选SERCA2a基因转导后表达量发生明显改变的10个蛋白点进行分析,经质谱鉴定分别为心肌收缩相关蛋白、线粒体能量代谢酶类和应激相关蛋白.结论以rAAV为载体介导SERCA2a基因转导能够改善HF犬心脏的收缩和舒张功能,其可能的机制是恢复了心肌收缩相关蛋白正常表型或正常表达量,增加了心肌能量的产生,改变了应激相关蛋白的表达.
목적 분석심기기장망Ca2+-ATP매(sarcoplasmic reticulum Ca2+ ATPase 2a,SERCA2a)기인전도대만성심력쇠갈(HF)견심기단백질조적영향,탐토SERCA2a기인전도개선심공능적궤제.방법 쾌속우심실기박건립HF견모형병수궤분위HF조、HF+록색형광단백(enhanced green fluorescent pmtein,EGFP)조、HF+SERCA2a조.후량조분별향심기내주사휴대EGFP화SERCA2a기인적rAAV재체.우기인전도30 d시정지기박후진행초성심동도화혈류동역학검사병제비심실기쌍향전영단백양품화심기쌍향전영도보,도상분석연건분석단백표체차이점,MALDI-TOF-MS수거고수색감정단백질.결과 기인전도30 d시,HF+SERCA2a조견적증상、초성심동도화혈류동역학지표여HF+EGFP조상비유현저호전(P＜0.05);여대조조상비차이무통계학의의(P＞0.05).도선SERCA2a기인전도후표체량발생명현개변적10개단백점진행분석,경질보감정분별위심기수축상관단백、선립체능량대사매류화응격상관단백.결론 이rAAV위재체개도SERCA2a기인전도능구개선HF견심장적수축화서장공능,기가능적궤제시회복료심기수축상관단백정상표형혹정상표체량,증가료심기능량적산생,개변료응격상관단백적표체.
Objective Overexpression of SERCA2a could improve cardiac function in human and experimental heart failure(HF)models.We observed the proteomics changes post SERCA2a overexpression in a pacing induced HF model in dogs.Methods Beagles were divided into four groups:control group,HF group(230 beats/min for 4 weeks),HF+EGFP group(myocardial injection of 1 × 1012 v.g recombinant adeno-associated virus carrying enhanced green fluorescent protein gene,rAAV2/1-EGFP)and HF+ SERCA2a group ( myocardial injection of 1 × 1012 v.g recombinant adeno-associated virus carrying SERCA2a gene,rAAV2/1-SERCA2a).Thirty days after gene transduction,left ventficular systolic and diastolic functions were measured by echoeardiography and invasive hemodynamics in all animals.By use of 2-dimensional gel electrophoresis(2-DE),-500 distinct protein spots were detected in myocardium of all animals.Protein spots observed to be altered between failing and SERCA2a overexpressed hearts were subjected to tryptic peptide mass fingerprinting for identification by MALDI-TOF mass spectrometry in combination with LC/MS/MS analysis.Results At 30 day after gene transfer,HF signs were significantly reduced,cardiac function[LVSP:(214.72±31.74)mm Hg(1 mm Hg=0.133 kPa)vs.(139.32±36.79)mm Hg,+dp/dtmax:(6779.43±217.58)mm Hg/s vs.(2746.85±931.23)mm Hg/s and -dp/dtmax:(-4341.42±322.02)mm Hg/s vs.(r-2531.14 ±616.15)mm Hg/s,LVEDP:(21.86±6.95)mm Hg vs.(59.78±6.92)mm Hg]significantly improved in HF+SERCA2a dogs than those in HF+ EGFP group(all P＜0.05)and parameters were comparable between HF+SERCA2a and control groups.We identified alterations in the expression level of more than 10 proteins in myocardium.These protein changes were observed mainly in two subcellular compartments:the cardiac contractile apparatus and metabolism/energetics.Conclusion These results showed that overexpression of SERCA2a could improve cardiac function accompanied with numerous alterations in protein expressions involved in calcium handling,myofibrils,and energy production in this dog model of chronic heart failure.