Control Mechanisms for Matching ATP Supply and Demand in Heart Mitochondria

不能提供能量以满足身体的需求限制了功能储备能力，并且在某些压力时期，如缺血，可能导致不可逆的细胞和组织损伤。这种匹配在具有高且快速波动的代谢率的组织（如心脏）中至关重要。线粒体是满足细胞需求的主要ATP供应者。线粒体使用的燃料通过线粒体内膜运输到基质，并产生电子源，电子源的氧化还原势能反过来又被电子运输链利用。电子的通量反映在氧的消耗上。从该电子流释放的能量用于将质子运输出基质穿过线粒体内膜，形成梯度，其质子动力驱动ATP合酶产生ATP。这种“上游”调控被称为“推动”机制。ATP合成酶的控制机制的完整描述仍然缺乏。已经提出了两种一般机制作为关键调节器：1）ADP和Pi浓度：胞质溶胶中的ATP利用/水解增加ADP和Pi向线粒体的通量，从而增加ATP产生的可用底物的量; 2）Ca 2+浓度; ATP利用/水解与游离胞质钙和线粒体Ca 2+的变化相关联，后者控制参与ATP产生的某些线粒体反应速率决定酶的Ca 2+依赖性激活。在高水平的能源需求的问题是否平行的“推”机制信号作用于ATP合酶也可以促进电子传递链氧化还原通量，提高ATP生产的效率。这种效应模拟了对上游通量的明显的额外“拉动”，其导致呼吸的特定比例增加。迄今为止，尚未证明这种由Ca 2+及其靶点调节的“拉动”机制。 心肌细胞响应于电刺激引起的胞质Ca 2+的周期性“增加”而收缩。作为收缩功水平的结果，ATP被收缩成分成比例地利用。因此，从需求的角度来看，Ca 2+是一个直接的效应器，可以很好地定位在能量匹配调节机制中发挥作用。细胞质和线粒体Ca 2+之间也存在相关性。Ca 2+通过线粒体单向转运体进入线粒体，并被线粒体Na+/Ca 2+交换器挤出。这导致响应于刺激频率或Ca 2+瞬时振幅的增加的线粒体Ca 2+积累。因此，线粒体中的Ca 2+水平反映了心肌做功和ATP消耗的变化，从而反映了对ATP的需求。研究表明，线粒体Ca 2+可以激活参与ATP产生的线粒体酶。 因此，电刺激过程中线粒体Ca 2+的变化与ATP供需的变化有关。 我们和其他人已经表明，线粒体体积的微小变化可以调节呼吸，进而调节能量的产生。还已知Ca 2+环境可以调节分离的线粒体悬浮液中的线粒体体积，这提出了通过增加电刺激的Ca 2+循环的Ca 2+生理变化是否会以这种方式起作用的问题。我们发现，虽然增加电刺激，生理Ca 2+循环不会检测到改变“舒张”线粒体长轴和短轴尺寸（即体积）后不久（2.5分钟）从休息过渡到低或更高的工作负荷，但它仍然导致细胞呼吸增加（反过来促进能量产生）在这两种情况下。这些结果与其他人在分离的线粒体模型中观察到的结果相反。此外，我们发现控制心脏线粒体ATP供应的机制包括“推”和“拉”机制，“拉”机制直接靶向ATP合酶。我们发现，“拉”的机制是由线粒体Ca 2+控制，并可以进一步促进线粒体体积的调节。在低心脏工作负荷下，“推”机制足以匹配ATP供给和需求，并且线粒体跨膜ADP/Pi梯度大概足以驱动“推”和“拉”机制。然而，在相同的实验条件下，药理学诱导的调节线粒体体积增加被发现，以促进线粒体Ca 2+进入负责进一步推动呼吸，而在较高的工作量，线粒体Ca 2+进入不需要这样的促进，反过来是足够的和必不可少的驱动“推”和“拉”的呼吸作用。此外，药理学增强的线粒体Ca 2+积累（不改变胞质Ca 2+）也被发现推动呼吸。 促进这些“推”和“拉”机制正在被研究作为一种潜在的治疗方法，以逆转ATP供需匹配中的信号传导缺陷，例如发生在困扰数百万人，特别是老年人的心力衰竭中。

Failure to supply energy to match the body's demands limits the functional reserve capacity, and under certain periods of stress, such as ischemia, can lead to irreversible cell and tissue damage. This matching is critical in tissues with high and rapidly fluctuating metabolic rates such as the heart. Mitochondria are the main ATP suppliers to meet cellular demands. The fuel used by mitochondria is transported across the inner mitochondrial membrane to the matrix and produces a source of electrons whose redox-potential energy is, in turn, harnessed by the electron transport chain. The flux of electrons is reflected in oxygen consumption. The energy released from this electron flow is used to transport protons out of the matrix across the inner mitochondrial membrane forming a gradient whose proton-motive force drives ATP synthase to make ATP. This "upstream" regulation is known as the "push" mechanism. A complete description of the ATP synthase control mechanisms is still lacking. Two general mechanisms have been suggested to serve as key regulators: 1) ADP and Pi concentrations; ATP utilization/hydrolysis in the cytosol increases ADP and Pi fluxes to mitochondria and hence the amount of available substrates for ATP production increases; 2) Ca2+ concentration; ATP utilization/hydrolysis is coupled to changes in free cytosolic calcium and mitochondrial Ca2+, the latter controlling Ca2+-dependent activation of certain mitochondrial reaction-rate-determining enzymes taking part in ATP production. At high levels of energy demand the question arises whether parallel to the "push" mechanism signals acting on ATP synthase could also facilitate the electron transport chain redox flux, enhancing the efficiency of ATP production. This effect simulates an apparent additional "pull" on the upstream flux, which causes as a specific proportionate increase in respiration. Proof of such a "pull" mechanism regulated by Ca2+ and its target has not been demonstrated to-date. Cardiomyocytes contract in response to driven cyclic 'increases' in cytosolic Ca2+ in a response to electrically stimulation. As a consequence of the levels of contractile work, ATP is proportionately utilized by the contractile elements. Therefore, from the demand point of view Ca2+ is a direct effector that might be well positioned to play a role in the energy matching regulatory mechanisms. A correlation has also been shown between cytosolic and mitochondrial Ca2+. Ca2+ enters the mitochondria through the mitochondrial uniporter and is extruded by the mitochondrial Na+/Ca2+ exchanger. This results in mitochondrial Ca2+ accumulation in response to an increase in stimulation frequency or Ca2+ transient amplitude. Therefore, Ca2+ levels in the mitochondria reflect changes in both myocardial work and ATP consumption and, hence, the demand for ATP. It was shown that mitochondrial Ca2+ can activate the mitochondrial enzymes taking part in ATP production. Therefore, changes in mitochondrial Ca2+ during electrical stimulation are linked to changes in ATP supply and demand. We and others have shown that small changes in mitochondrial volume can regulate respiration and in turn energy production. It is also known that the Ca2+ environment may regulate mitochondrial volume in isolated mitochondrial suspension raising the question whether physiological changes in Ca2+ via increasing electrically stimulated Ca2+ cycling would act in this way. We found that while increasing electrically stimulated, physiological Ca2+ cycling does not detectibly change the 'diastolic' mitochondrial long- and short-axis dimensions (i.e, volume) shortly (2.5 min) after the transition from rest to low or higher workloads, it nevertheless caused an increase in cell respiration (and in turn facilitated energy production) in both conditions. These results were in contrast to that observed by others in the isolated mitochondria models. Additionally, we found that the mechanisms that control ATP supply from the hearts mitochondria consist of both 'push' and 'pull' mechanisms and that 'pull' mechanism directly targets ATP synthase. We identified that the 'pull' mechanism is controlled by mitochondrial Ca2+ and can be further facilitated by pharmacologically regulating mitochondrial volume. At low cardiac workload, the 'push' mechanism is sufficient to match ATP supply and demand, and the mitochondrial transmembrane ADP/Pi gradient is presumably sufficient to drive the 'push' and 'pull' mechanisms. However, under the same experimental conditions, pharmacological induction of a regulatory mitochondrial volume increase was found to facilitate mitochondrial Ca2+ entry responsible for further pushing respiration, whereas at higher workloads, mitochondrial Ca2+ entry did not require such facilitation, and in turn was sufficient and essential to drive both "push" and 'pull' effects on respiration. Moreover, pharmacologically-enhanced mitochondrial Ca2+ accumulation (without changing cytosolic Ca2+) was also found to push respiration. Facilitation of these 'push' and 'pull' mechanisms is being examined as a potential treatment to reverse signaling defects in matching ATP supply and demand, such as occurs in heart failure which afflicts millions of people, especially the elderly population.