Figure 3.

Circadian Clock Δ19 mutation impairs mitochondrial function, mitophagy and cell viability in cardiac myocytes. Epifluorescence microscopy of cardiac myocytes under normoxic (NMX) or hypoxic (HPX) conditions in the absence and presence of Clock ∆19 expression vector; (A), mitochondrial reactive oxygen species (ROS) by dihydroethidine (red fluorescence), bar: 100 μm; (B), mitochondrial membrane potential (ΔΨm) by TMRM (red fluorescence), bar: 20 μm; (C), mitochondrial permeability transition pore opening (mPTP), (green fluorescence) bar: 20 μm; see Materials and Methods for details; (D), Upper, representative images for MitoKeima staining, as an index of mitophagy in cardiac myocytes under NMX and HPX conditions in the absence and presence of Clock ∆19 expression vector, bars: 10 μm. Magnified regions are depicted by the white boxes, green fluorescent puncta demark mitochondria that are unfused with lysosomes (neutral pH), red/yellow fluorescent puncta demark mitochondria that have fused with lysosomes (acidic pH) indicative of mitophagy. Bottom histogram, quantitative analysis for conditions shown above; (E), Cell viability as assessed with vital dyes calcein-AM and ethidium-homodimer-1 to identify the number of live (green) and dead (red) cells, respectively, bar: 100 μm, see methods for details; data are expressed as mean ± SEM. *p < 0.05; **p < 0.01, n = 3–4 independent myocyte isolations, counting >200 cells for each condition tested