Ahmed Rafeeq P H, Kanisicak Onur, Alam Perwez
Department of Pathology and Laboratory Medicine, College of Medicine, University of Cincinnati, Cincinnati, OH 45267, USA.
Department of Emergency Medicine, The Ohio State University Wexner Medical Center, Columbus, OH 43210, USA.
Cells. 2025 Jun 6;14(12):853. doi: 10.3390/cells14120853.
: The limited regenerative capacity of adult mammalian cardiomyocytes (CMs) poses a significant challenge for cardiac repair following myocardial infarction. In contrast to adult mammals, CMs in zebrafish and newt hearts retain a lifelong capacity for proliferation and cardiac regeneration. Likewise, neonatal mice exhibit a brief postnatal period, during which CMs retain the ability to proliferate and contribute to myocardial repair, which markedly diminishes within the first week of life. Emerging evidence indicates that adult CM cell cycle progression is critically influenced by oxidative stress. Adult mammalian CMs possess a high mitochondrial content to meet their substantial energy demands. However, this also leads to elevated reactive oxygen species (ROS) production, resulting in DNA damage and subsequent cell cycle arrest. We hypothesize that reducing the mitochondrial content in adult CMs will mitigate ROS production, thereby facilitating cell cycle progression. : Adult CMs were isolated from adult rats (≥12 weeks old). To induce mitophagy, adult CMs were transfected with parkin-expressing plasmid and then treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a mitochondrial protonophore, for 7 days. Post-treatment assessments included the quantification of adult CM proliferation, mitochondrial content, and ROS levels. : CCCP-treated adult CMs exhibited a significant increase in proliferation markers, including EdU incorporation, KI67, phospho-histone H3, and Aurora B. Furthermore, CCCP treatment significantly reduced the mitochondrial content, as evidenced by decreased MitoTracker, TMRM, and Tom20 staining compared to controls. This was accompanied by electron microscopy analysis, which showed a significant reduction in the mitochondrial number in the adult CM after CCCP treatment. Moreover, our results also demonstrate a marked reduction in oxidative stress, demonstrated by lower 123-dihydro-rhodamine (123-DHR), CellROX signals, and VDAC. : Our findings demonstrate that CCCP-mediated mitochondrial depletion reduces oxidative stress and promotes cell cycle re-entry in adult CM. This study provides direct experimental evidence and substantiates the role of elevated mitochondria and ROS levels in adult CM cell cycle exit.
成年哺乳动物心肌细胞（CMs）有限的再生能力对心肌梗死后的心脏修复构成了重大挑战。与成年哺乳动物不同，斑马鱼和蝾螈心脏中的CMs终生保持增殖和心脏再生的能力。同样，新生小鼠在出生后有一段短暂的时期，在此期间CMs保留增殖能力并有助于心肌修复，但这种能力在出生后的第一周内会显著下降。新出现的证据表明，成年CM细胞周期进程受到氧化应激的关键影响。成年哺乳动物CMs具有高线粒体含量以满足其大量的能量需求。然而，这也导致活性氧（ROS）生成增加，从而导致DNA损伤和随后的细胞周期停滞。我们假设减少成年CMs中的线粒体含量将减轻ROS生成，从而促进细胞周期进程。
成年CMs从成年大鼠（≥12周龄）中分离出来。为了诱导线粒体自噬，将表达帕金蛋白的质粒转染到成年CMs中，然后用线粒体质子载体羰基氰化物3-氯苯腙（CCCP）处理7天。处理后的评估包括成年CM增殖、线粒体含量和ROS水平的定量。
经CCCP处理的成年CMs增殖标志物显著增加，包括EdU掺入、KI67、磷酸化组蛋白H3和极光激酶B。此外，CCCP处理显著降低了线粒体含量，与对照组相比，MitoTracker、TMRM和Tom20染色减少证明了这一点。这伴随着电子显微镜分析，显示CCCP处理后成年CM中线粒体数量显著减少。此外，我们的结果还表明氧化应激显著降低，123-二氢罗丹明（123-DHR）、CellROX信号和电压依赖性阴离子通道（VDAC）降低证明了这一点。
我们的研究结果表明，CCCP介导的线粒体耗竭可降低氧化应激并促进成年CM重新进入细胞周期。本研究提供了直接的实验证据，并证实了线粒体水平升高和ROS水平在成年CM细胞周期退出中的作用。
