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

Abstract

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.

A novel transient receptor potential C3/C6 selective activator induces the cellular uptake of antisense oligonucleotides

“…Moreover, SKF96365 abolished the ASO uptake increased by CBD, which is known to activate TRPVs and TRPA1 in A549 cells expressing TRPV1 and TRPA1, as well as TRPC3 and TRPC6 ( 53 , 54 ). In addition, ASO uptake was not affected by nifedipine, a selective blocker of L-type voltage dependent calcium channels, that are widely expressed in various cells as a major source of calcium entry ( 55 , 56 ). These results suggest that Ca 2+ influx, particularly via TRPC, is required to promote ASO uptake.…”

Evaluation of Biological Activity Exerted by Dibenzo[b,e]Thiophene-11(6H)-One on Left Ventricular Pressure Using an Isolated Rat Heart Model

“…Analyzing these data and other reports which suggests that some dibenzo derivatives can exert changes in intracellular calcium levels 45 46 . Besides, it is important to mention that in the literature there are some studies indicating that nifedipine can block the positive inotropic activity of some compounds 47 48 49 ; for this reason in this study the biological activity produced by Dibenzo[b,e]thiophene-11(6H)-one on left ventricular pressure was evaluated en the absence or presence of nifedipine drug ( type-L calcium antagonist) 50 . The results showed that Dibenzo[b,e]thiophene-11(6H)-one increases left ventricular pressure in a dose-dependent manner and this effect was inhibited in the presence of nifedipine.…”

Three-Dimensional Tracking of Intracellular Calcium and Redox State during Real-Time Control in a Hypoxic Gradient in Microglia Culture: Comparison of the Channel Blocker and Reoxygenation under Ischemic Shock

“…Here, we studied the effect of nifedipine treatment, reoxygenation, and "reoxygenation + nifedipine treatment" using imaging and gene expression studies. Although nifedipine has been used to protect cells through Nrf2 translocation under long-term hypoxic conditions with prophylactic treatment, 19 whether the CCB can be used nonprophylactically to protect molecules in a shorter time scale is not known. In the current study, we compared the drug efficacy to reoxygenation in the case of nonprophylactic intervention.…”

“…In our previous work, we attempted to create a CoCl 2 -based hypoxia model for understanding the hypoxic markers for lung epithelial cells in the context of COVID-19. 19 However, such an approach neither allows the implementation of acute ischemic shock nor permits reoxygenation without media reperfusion. Therefore, a gas chamber-based hypoxia model is better suited for creating acute ischemic shock.…”

“…Apart from stroke-specific models, other disease models, including neurodegeneration, chronic obstructive pulmonary disease, and tumor microenvironment, heavily depend on the CoCl 2 -based hypoxia model because it allows the experimentalist to open the culture plate many times while maintaining the stabilization of HIF. In our previous work, we attempted to create a CoCl 2 -based hypoxia model for understanding the hypoxic markers for lung epithelial cells in the context of COVID-19 . However, such an approach neither allows the implementation of acute ischemic shock nor permits reoxygenation without media reperfusion.…”