靶向干预线粒体NAD+/NADH比率对心力衰竭的影响及机制

NAD+/NADH比率是线粒体功能重要标志，线粒体功能损伤与心力衰竭密切相关。但由于缺少相关技术，线粒体NAD+/NADH比率的实时监控和调节作用尚不清楚。在前期研究中，我们开发了一种动态追踪线粒体NAD+/NADH比率的遗传编码荧光探针，揭示了线粒体与胞浆NAD(H)代谢表型差异，通过靶向调控线粒体与胞浆NAD+/NADH比率，明确了维持线粒体NAD+/NADH比率能抑制小鼠心力衰竭。我们拟利用此探针研究病理生理条件下线粒体NAD(H)代谢表型变化，构建探针转基因鼠，在体呈现心衰不同时期NAD+/NADH比率变化，综合运用多种生物学手段研究NAD+/NADH比率变化的机制，解析线粒体和胞浆NAD+/NADH比率的差异调控。本项目的实施将不仅开发一种高时空检测线粒体NAD+/NADH比率的遗传编码荧光探针及工具鼠，还完善线粒体NAD(H)代谢表型的研究空白，并且为心力衰竭的治疗提供新思路。

Nicotinamide adenine dinucleotide (NAD) exists in oxidized (NAD+) or reduced (NADH) form and the NAD+/NADH ratio dose not only set intracellular redox balance, but also affects mitochondrial function by regulating protein post-translational modification. Impairments of mitochondrial function in the heart are linked intricately to the development of heart failure, however, it remains unknown about mitochondrial NAD+/NADH ratio measurement, the change of mitochondrial NAD+/NADH ratio under physiological or pathological conditions, or the influence of regulating mitochondrial NAD+/NADH ratio on heart failure. Here, we developed a genetically encoded sensor (mito-SoNar) for monitoring mitochondrial NAD+/NADH ratio in real time. NAD+/NADH ratios in cytosol versus mitochondria responded promptly, but differently, to acute metabolic perturbations, indicating segregated NAD(H) pools. Mitochondrial and cytosolic NAD+/NADH ratios were also distinctly regulated under physiological (electric pacing or pathological (ischemia reperfusion or phenylephrine) conditions, reflecting their unique roles in the cell. Targeting mitochondrial NAD+/NADH ratio could prevent cardiomyocyte hypertrophy and heart failure. We will further map the NAD(H) metabolism using this indicator and measure the signal changes of subcellular NAD+/NADH ratio in vivo. We will also investigate the molecular mechanism for the NAD+/NADH ratio changes and determine distinct regulation of NAD+/NADH ratio in cytosol and mitochondrial compartments. Our aim is not only to develop a compartment-targeted biosensor for real-time imaging, but also provide a novel strategy for preventing heart failure development by maintaining mitochondrial function.