Comprehensive Heart Failure Center (CHFC), University Clinic Würzburg, Würzburg, Germany.
Affiliation when/at which experiments were performed: Clinic III for Internal Medicine, University Clinic Homburg, Homburg, Germany.
J Physiol. 2020 Apr;598(7):1361-1376. doi: 10.1113/JP276766. Epub 2019 Mar 27.
Mitochondrial Ca uptake stimulates the Krebs cycle to regenerate the reduced forms of pyridine nucleotides (NADH, NADPH and FADH ) required for ATP production and reactive oxygen species (ROS) elimination. Ca /calmodulin-dependent protein kinase II (CaMKII) has been proposed to regulate mitochondrial Ca uptake via mitochondrial Ca uniporter phosphorylation. We used two mouse models with either global deletion of CaMKIIδ (CaMKIIδ knockout) or cardiomyocyte-specific deletion of CaMKIIδ and γ (CaMKIIδ/γ double knockout) to interrogate whether CaMKII controls mitochondrial Ca uptake in isolated mitochondria and during β-adrenergic stimulation in cardiac myocytes. CaMKIIδ/γ did not control Ca uptake, respiration or ROS emission in isolated cardiac mitochondria, nor in isolated cardiac myocytes, during β-adrenergic stimulation and pacing. The results of the present study do not support a relevant role of CaMKII for mitochondrial Ca uptake in cardiac myocytes under physiological conditions.
Mitochondria are the main source of ATP and reactive oxygen species (ROS) in cardiac myocytes. Furthermore, activation of the mitochondrial permeability transition pore (mPTP) induces programmed cell death. These processes are essentially controlled by Ca , which is taken up into mitochondria via the mitochondrial Ca uniporter (MCU). It was recently proposed that Ca /calmodulin-dependent protein kinase II (CaMKII) regulates Ca uptake by interacting with the MCU, thereby affecting mPTP activation and programmed cell death. In the present study, we investigated the role of CaMKII under physiological conditions in which mitochondrial Ca uptake matches energy supply to the demand of cardiac myocytes. Accordingly, we measured mitochondrial Ca uptake in isolated mitochondria and cardiac myocytes harvested from cardiomyocyte-specific CaMKII δ and γ double knockout (KO) (CaMKIIδ/γ DKO) and global CaMKIIδ KO mice. To simulate a physiological workload increase, cardiac myocytes were subjected to β-adrenergic stimulation (by isoproterenol superfusion) and an increase in stimulation frequency (from 0.5 to 5 Hz). No differences in mitochondrial Ca accumulation were detected in isolated mitochondria or cardiac myocytes from both CaMKII KO models compared to wild-type littermates. Mitochondrial redox state and ROS production were unchanged in CaMKIIδ/γ DKO, whereas we observed a mild oxidation of mitochondrial redox state and an increase in H O emission from CaMKIIδ KO cardiac myocytes exposed to an increase in workload. In conclusion, the results obtained in the present study do not support the regulation of mitochondrial Ca uptake via the MCU or mPTP activation by CaMKII in cardiac myocytes under physiological conditions.
线粒体 Ca 摄取可刺激三羧酸循环，从而再生产生 ATP 和消除活性氧（ROS）所需的还原型吡啶核苷酸（NADH、NADPH 和 FADH）。钙/钙调蛋白依赖性蛋白激酶 II（CaMKII）通过线粒体 Ca 单向转运蛋白磷酸化被提出可调节线粒体 Ca 摄取。我们使用两种小鼠模型，即全局 CaMKIIδ 缺失（CaMKIIδ 敲除）或心肌细胞特异性 CaMKIIδ 和 γ 缺失（CaMKIIδ/γ 双重敲除），以探究 CaMKII 是否在分离的线粒体中和在心脏肌细胞的β-肾上腺素能刺激期间控制线粒体 Ca 摄取。CaMKIIδ/γ 并未在分离的心脏线粒体和β-肾上腺素能刺激和起搏期间的分离的心脏肌细胞中控制 Ca 摄取、呼吸或 ROS 发射。本研究的结果不支持 CaMKII 在生理条件下对心脏肌细胞中线粒体 Ca 摄取的重要作用。
线粒体是心脏肌细胞中 ATP 和活性氧（ROS）的主要来源。此外，线粒体通透性转换孔（mPTP）的激活诱导程序性细胞死亡。这些过程主要受 Ca 控制，Ca 通过线粒体 Ca 单向转运蛋白（MCU）摄取到线粒体中。最近提出，钙/钙调蛋白依赖性蛋白激酶 II（CaMKII）通过与 MCU 相互作用来调节 Ca 摄取，从而影响 mPTP 的激活和程序性细胞死亡。在本研究中，我们在生理条件下研究了 CaMKII 的作用，其中线粒体 Ca 摄取与心脏肌细胞的能量供应相匹配。相应地，我们测量了从心肌细胞特异性 CaMKII δ 和 γ 双重敲除（CaMKIIδ/γ DKO）和全局 CaMKIIδ 敲除（CaMKIIδ KO）小鼠中分离的线粒体和心脏肌细胞中的线粒体 Ca 摄取。为了模拟生理性工作负荷增加，对心脏肌细胞进行β-肾上腺素能刺激（异丙肾上腺素灌流）和刺激频率增加（从 0.5 到 5 Hz）。与野生型同窝仔相比，在两种 CaMKII KO 模型的分离线粒体或心脏肌细胞中均未检测到线粒体 Ca 积累的差异。在 CaMKIIδ/γ DKO 中，线粒体氧化还原状态和 ROS 产生没有变化，而在 CaMKIIδ KO 心脏肌细胞中观察到线粒体氧化还原状态轻度氧化和 H2O2 发射增加，这些细胞暴露于增加的工作负荷下。总之，本研究中的结果不支持 CaMKII 通过 MCU 调节线粒体 Ca 摄取或 mPTP 在生理条件下激活的假说。
