key: cord-0908516-6azkxkzl authors: Manohar, Kuruba; Gupta, Rishikesh Kumar; Gupta, Parth; Saha, Debasmita; Gare, Suman; Sarkar, Rahuldeb; Misra, Ashish; Giri, Lopamudra title: FDA approved L-type channel blocker Nifedipine reduces cell death in hypoxic A549 cells through modulation of mitochondrial calcium and superoxide generation date: 2021-10-16 journal: Free Radic Biol Med DOI: 10.1016/j.freeradbiomed.2021.08.245 sha: fd5dddc5e5e1e2a46eb36abfccf3594128c9b229 doc_id: 908516 cord_uid: 6azkxkzl As hypoxia is a major driver for the pathophysiology of COVID-19, it is crucial to characterize the hypoxic response at the cellular and molecular levels. In order to augment drug repurposing with the identification of appropriate molecular targets, investigations on therapeutics preventing hypoxic cell damage is required. In this work, we propose a hypoxia model based on alveolar lung epithelial cells line using chemical inducer, CoCl(2) that can be used for testing calcium channel blockers (CCBs). Since recent studies suggested that CCBs may reduce the infectivity of SARS-Cov-2, we specifically select FDA approved calcium channel blocker, nifedipine for the study. First, we examined hypoxia-induced cell morphology and found a significant increase in cytosolic calcium levels, mitochondrial calcium overload as well as ROS production in hypoxic A549 cells. Secondly, we demonstrate the protective behaviour of nifedipine for cells that are already subjected to hypoxia through measurement of cell viability as well as 4D imaging of cellular morphology and nuclear condensation. Thirdly, we show that the protective effect of nifedipine is achieved through the reduction of cytosolic calcium, mitochondrial calcium, and ROS generation. Overall, we outline a framework for quantitative analysis of mitochondrial calcium and ROS using 3D imaging in laser scanning confocal microscopy and the open-source image analysis platform ImageJ. The proposed pipeline was used to visualize mitochondrial calcium and ROS level in individual cells that provide an understanding of molecular targets. Our findings suggest that the therapeutic value of nifedipine may potentially be evaluated in the context of COVID-19 therapeutic trials. Imaging hypoxia-induced ROS formation 215 During hypoxia exposure, as mentioned in the above experiments, cells were treated 216 with Nifedipine and melatonin, and Intracellular ROS were detected using MitoSOX dye 217 (Invitrogen, USA). In addition, 3D imaging was performed using LSCM (Leica, Germany) at 218 excitation at 561 nm using 63X oil objective. 120 z-stacks were obtained in a span of 5 µm and 219 were merged to obtain a 2D image. The region of interest (n=10) in each cell was drawn 220 manually, and the average intensity per cell was obtained in a pseudo-color scale from 0 to 255 221 units using LASX software. Multiple z-stacks corresponding to different focal planes were scanned to construct 3D 234 images within the cell. Rhod-2 and MitoSOX intensity was captured by photomultiplier tube 235 (PMT) scanning for a total of 120 stacks (designated as one complete z-scan), which 236 corresponds to a thin disk-like section of thickness 5 μm. In order to obtain the spatial 237 distribution of mitochondrial calcium and ROS generation, a 3D reconstruction was obtained 238 from the z-stacks using LASX software. To account for the Rhod-2 intensity from all z-stacks, 239 individual stack images were first merged using LASX software. Then we obtained compressed Table S1 shows All experiments were repeated at least three times, and representative results are shown. The differences between the two treatment groups were compared with a t-test and Kruskal- Wallis test (MATLAB). A two-tailed P<0.05 was considered to indicate statistical significance. Data are presented as the mean ± standard deviation (SD). Figures 1a and 1b) . The result clearly shows that nifedipine has a protective effect on cells (Figure 3d, e) . 374 Sequestration of calcium in granular structure and mitochondrial Ca 2+ load is significantly 375 lower in the case of nifedipine treatment compared to CoCl2 treated cells (Figure 4d, e) . Figure 376 3e also shows no significant difference between cytosolic calcium levels between treatment 377 with nifedipine and treatment with CoCl2 + nifedipine. The qRT-PCR result shows that there is a more than 2-fold increase in the Nrf2 expression 430 (Figure 5f) . Also, it is observed that that the higher level of Nrf2 expression is maintained at 431 12 and 24 hours starting from 6 hours post-treatment with nifedipine. Additionally, from 432 immunocytochemistry assay, localization of Nrf2 was observed in CoCl2 + nifedipine treated 433 cells between 6 and 12 hours (Figure 5g ). Severe COVID-19 patients had vasoconstrictive, pro-inflammatory, and pro-oxidative effects 564 in lung cells [52] . As nifedipine is known to reduce pulmonary vasoconstriction in general [32] , 565 it is speculated that nifedipine may also inhibit hypoxic pulmonary vasoconstriction during 566 COVID-19. 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