FDA approved L-type channel blocker Nifedipine reduces cell death in hypoxic A549 cells through modulation of mitochondrial calcium and superoxide generation

• Published in: Free radical biology & medicine Vol. 177; pp. 189 - 200
• Main Authors: Manohar, Kuruba, Gupta, Rishikesh Kumar, Gupta, Parth, Saha, Debasmita, Gare, Suman, Sarkar, Rahuldeb, Misra, Ashish, Giri, Lopamudra
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.12.2021; Published by Elsevier Inc
• Subjects: A549 Cells; Calcium; Calcium channel blocker; Calcium Channel Blockers - pharmacology; Calcium Channel Blockers - therapeutic use; Cell Death; COVID-19; Humans; Hypoxia; Hypoxia - drug therapy; Mitochondrial calcium; Nifedipine; Nifedipine - pharmacology; Reactive oxygen species (ROS); SARS-CoV-2; Superoxides; Hypoxia; SARS-Cov-2; Calcium channel blocker; Mitochondrial calcium; Reactive oxygen species (ROS); Nifedipine
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2021.08.245

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, CoCl2 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. [Display omitted] •There is significant increase in cytosolic and mitochondrial calcium along with ROS generation in hypoxic A549 cells.•We demonstrate that L-type calcium channel blocker, nifedipine has protective effcet on hypoxic A549 cells.•The protective effect of nifedipine is achieved through increase in Nrf2 expression and reduction of ROS generation.•We outline a framework for quantification of subcellular responses using 3D imaging in laser scanning confocal microscopy.