Mastoor Yusuf, Harata Mikako, Silva Kavisha, Liu Chengyu, Combs Christian A, Roman Barbara, Murphy Elizabeth
Laboratory of Cardiac Physiology, Cardiovascular Branch, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD 20892.
These authors contributed equally.
J Mol Cell Cardiol Plus. 2024 Jun;8. doi: 10.1016/j.jmccpl.2024.100074. Epub 2024 Apr 7.
An increase in mitochondrial calcium via the mitochondrial calcium uniporter (MCU) has been implicated in initiating cell death in the heart during ischemia-reperfusion (I/R) injury. Measurement of calcium during I/R has been challenging due to the pH sensitivity of indicators coupled with the fall in pH during I/R. The development of a pH-insensitive indicator, mitochondrial localized Turquoise Calcium fluorescence Lifetime Sensor (mito-TqFLITS), allows for quantifying mitochondrial calcium during I/R via fluorescent lifetime imaging. Mitochondrial calcium was monitored using mito-TqFLITS, in neonatal mouse ventricular myocytes (NMVM) isolated from germline MCU-KO mice and MCU treated with CRE-recombinase to acutely knockout MCU. To simulate ischemia, a coverslip was placed on a monolayer of NMVMs to prevent access to oxygen and nutrients. Reperfusion was induced by removing the coverslip. Mitochondrial calcium increases threefold during coverslip hypoxia in MCU-WT. There is a significant increase in mitochondrial calcium during coverslip hypoxia in germline MCU-KO, but it is significantly lower than in MCU-WT. We also found that compared to WT, acute MCU-KO resulted in no difference in mitochondrial calcium during coverslip hypoxia and reoxygenation. To determine the role of mitochondrial calcium uptake via MCU in initiating cell death, we used propidium iodide to measure cell death. We found a significant increase in cell death in both the germline MCU-KO and acute MCU-KO, but this was similar to their respective WTs. These data demonstrate the utility of mito-TqFLITS to monitor mitochondrial calcium during simulated I/R and further show that germline loss of MCU attenuates the rise in mitochondrial calcium during ischemia but does not reduce cell death.
通过线粒体钙单向转运体(MCU)增加线粒体钙已被认为与缺血再灌注(I/R)损伤期间心脏细胞死亡的启动有关。由于指示剂对pH敏感以及I/R期间pH下降,在I/R期间测量钙具有挑战性。一种对pH不敏感的指示剂——线粒体定位的绿松石钙荧光寿命传感器(mito-TqFLITS)的开发,使得通过荧光寿命成像来定量I/R期间的线粒体钙成为可能。使用mito-TqFLITS监测从种系MCU基因敲除小鼠分离的新生小鼠心室肌细胞(NMVM)以及用CRE重组酶处理以急性敲除MCU的NMVM中的线粒体钙。为了模拟缺血,将盖玻片放置在NMVM单层上以阻止氧气和营养物质的进入。通过移除盖玻片诱导再灌注。在MCU野生型(MCU-WT)中,盖玻片缺氧期间线粒体钙增加三倍。在种系MCU基因敲除小鼠的盖玻片缺氧期间,线粒体钙有显著增加,但明显低于MCU-WT。我们还发现,与野生型相比,急性MCU基因敲除在盖玻片缺氧和复氧期间线粒体钙没有差异。为了确定通过MCU摄取线粒体钙在启动细胞死亡中的作用,我们使用碘化丙啶来测量细胞死亡。我们发现在种系MCU基因敲除小鼠和急性MCU基因敲除小鼠中细胞死亡均显著增加,但这与它们各自的野生型相似。这些数据证明了mito-TqFLITS在模拟I/R期间监测线粒体钙的实用性,并进一步表明MCU的种系缺失减弱了缺血期间线粒体钙的升高,但并未降低细胞死亡。