Fig. 4: Micropatterning impacts mitochondrial distribution independently of mitochondrial mass.

A Mitochondrial distribution stained by MitoTracker^TM Orange (red) and DAPI (blue) and analysed by confocal microscopy. The insert represents a zoomed view of mitochondrial network. Global scale bar 50 µm, insert scale bar 10 µm. B Quantitative analysis of mitochondrial direction measured with Fiji software. n = 18 non-patterned cells, n = 16 micropatterned cells; N = 2 differentiations. C Representative pictures obtained with MitoTracker^TM Orange and IMARIS software. Each mitochondrion is identified by a color in accordance with its volume (the smallest mitochondria are blue and the largest are red). D Total mitochondrial volume quantified by IMARIS. n = 28,675 non-patterned cells, n = 64,919 micropatterned cells; N = 3 differentiations. E Scatter plots show the number of mitochondria per cell. n = 18 non-patterned cells, n = 20 micropatterned cells; N = 3 differentiations. F Mitochondrial DNA (mtDNA) content in non-patterned (n = 17) and micropatterned (n = 11) hiPSC-CMs, quantified by amplification of the mitochondrial Mttl1 gene relative to nuclear gene b-Actin. N = 3 differentiations. G Cardiac level of ATP5A, UQCRC2, SDHB, I-NDUFB8, TOMM40, VDAC, OPA1, and SDHA were analysed by western blot in non-patterned and micropatterned hiPSC-CMs. Total Histone3 was used as loading control (n = 6). Results, mean ± standard deviation. Statistical analyses: Unpaired t-test was used for C, Mann–Whitney was used for D through G. *p < 0.05, ns: not significant for the indicated comparisons.