Bmal1 modulates mitochondrial morphology and functioning in 661W cells

Abstract

Purpose : Cone photoreceptors contain a circadian clock that regulates many aspects of their biology. Previous studies have shown that removal of the clock gene, Bmal1, from the mouse retina affects cone photoreceptor functioning and viability. Using 661W cells—a cone-like cell line—we have previously shown that Bmal1 mediates sensitivity to oxidative stress by regulating the antioxidant activity of these cells. Several studies indicate oxidative stress alters mitochondrial function and structure. Our results may imply that Bmal1 also mediates mitochondrial functioning. In this study, we investigated mitochondrial structure and functioning in 661W cells and in 661W cells without Bmal1 (BKO).

Methods : Mitochondrial morphology was investigated in 661W and BKO cells using transmission electron microscopy (JEOL1200EX). The number, size, and shape of mitochondria in each cell were analyzed using ImageJ (NIH). Mitochondrial functioning was determined in 661W and BKO cells using Seahorse XF Analyzer with a Cell Mito Stress Test Kit (Agilent). The level of Mitofilin (a protein implicated in mitochondrial architecture) was determined by western blotting using a Mitofilin antibody (Proteintech).

Results : Our data indicated that BKO cells contained larger mitochondria than 661W cells (10 µm² vs. 6 µm², t-test, p<0.05), although the number of mitochondria did not differ between the two groups. We observed more onion-shaped mitochondria in BKO cells than 661W cells (20% vs. 10% of the total mitochondria count within the cell, t-test, p<0.05). We also observed vesicular-shaped mitochondria in cells, but the number of such mitochondria did not differ between the two groups. Analysis of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) revealed that BKO cells showed a significant decrease in basal respiration, proton leak, maximal respiration capacity and ATP production (t-tests, p<0.001 in all cases). The amount of Mitofilin was also decreased in BKO cells (t-test, p<0.05).

Conclusions : Our results indicate that removal of Bmal1 from 661W cells significantly affects mitochondrial morphology and reduces the functioning of these organelles. These results may help to explain the mechanism(s) by which removal of Bmal1 may decrease the capability of these cells to respond to oxidative stress and may also provide an explanation for the reduced viability of cones observed in the Bmal1 KO mouse during aging.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.