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.

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Live imaging based testing of ligands and identification of toxic dose range during in vitro studies is a time-intensive process due to the inherent heterogeneity present in cell responses. In this context, high magnification imaging and large-scale data visualization remains challenging for analysis of toxicity during the drug screening as well as selection of dose-range. To address this challenge, we propose the measurement of cytosolic calcium ion (Ca2+) using spinning disk confocal microscopy at a higher resolution for generation of imaging data that can be visualized using uniform manifold and projection (UMAP). First, we performed large scale experiments and showed norepinephrine induced increase in Ca2+ flux in HeLa cells for a large range of doses. Secondly, the time-series dataset was mapped in 2D plane using UMAP. We also show that the proposed framework can be used to depict the relative distribution of various responses corresponding to a range of drug doses. To the best of our knowledge, this is the first attempt for UMAP visualization of time-series dose-response and identification of Ca2+ signature in the toxic dose-range. Such quantitative microscopy can be used for prediction of toxic drug dose range, and identification of drugs that lead to lytic cell death.

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