Phenotype and swimming capacity of bcl2l13 knockout fish

(A) Representative image of adult male fish.

(B) Length.

(C) Weight.

(D) Mean MO₂ during the incremental exercise test.

(E–H) MO₂max and Ucrit in absolute values and normalized by length.

(I–K) Outcomes of the spontaneous locomotion test. For all panels, n = 16 fish per group from three independent cohorts. Error bars are mean ± SEM, ^∗p < 0.05, ^∗∗p < 0.01 (unpaired t test), ^#p < 0.01 (Mann-Whitney). WT: wild type and KO bcl2l13. MO₂ is oxygen consumption rate, Ucrit is critical swimming speed.

See also Figure S1.

Skeletal muscle structure of WT and KO fish

(A) Representative immunohistochemistry of transverse muscle sections of WT and KO fish stained with phalloidin (red). Slow fibers are marked with an antibody against MYH7B (green, merge is yellow).

(B) Mean CSA of fast muscle fibers per fish.

(C) Mean number of fast fibers per ROI per fish.

(D) Mean CSA of slow muscle fibers per fish.

(E) Mean number of slow fibers per ROI per fish.

(F) Representative electron micrographs of fast muscle fibers of WT and KO fish.

(G) Contingency graph for healthy fibers vs. fibers with SR accumulation. For (C–E) n = 6 fish per group. Error bars are mean ± SEM, ^∗p < 0.05 (unpaired t test). For (G) n = 8 fish per group. p = 0.0007 (Fisher’s exact test). Squares (white in A, black in F) represent the regions that are zoomed out. WT: wild type and KO: Mutant bcl2l13. CSA is cross-sectional area; ROI is region of interest.

Skeletal muscle proteomics of KO and WT zebrafish

(A–D) Fold change heat maps in selected proteins associated with different cellular functions.

(E) Enrichment score KO/WT of significantly modified protein groups related to gene ontology molecular function (GOMF). The score is derived from 1D annotation enrichment analysis.⁴⁰ For all panels, n = 8 fish per group. ^∗p < 0.05 (t test with multiple testing correction: permutation-based FDR at 0.05).

See also Figures S2 and S4, and Tables S2 and S3.

Mitochondrial content, morphology and respiration in bcl2l13 KO muscle

(A) Representative immunohistochemistry of transverse muscle sections of WT and KO fish stained with phalloidin (red). Mitochondria are marked with an antibody against MCTO1 (green, merge is yellow).

(B) Mitochondrial area in fast muscle fibers.

(C) Representative micrographs with ROI and mitochondria circling (yellow overlays) and zoomed section (black squares).

(D–F) Analyses of IMF mitochondrial parameters.

(G–K) Oxygen consumption rates normalized to mitochondrial content with Mitotracker Deep Red (OCR/MTDR). Black vertical lines indicate addition of substrates and electron transport chain (ETC) complexes inhibitors. (H–J) Mean ETC complex I activity (CI), complex II activity (CII), and complex IV (CIV) activity. For (D–F), n = 5 WT and 6 bcl2l13 KO zebrafish. For (G–K), n = 8 biological replicates per group. For all panels, error bars are mean ± SEM. ^∗p < 0.05, ^∗∗∗∗p < 0.0001 (unpaired t test). WT: Wild type and KO: Mutant bcl2l13. IMF is intermyofibrillar, SS is subsarcolemmal, CI–CIV are ETC complexes I to IV, NADH is beta-nicotinamide adenine dinucleotide, Succ is succinate, ROT is rotenone, AA is antimycin A, TMPD is N,N,N′,N′-tetramethyl-1,4-phenylendiamin, Asc is ascorbic acid.

See also Figure S3.

Subcellular localization of BCL2L13 and ERMCs quantifications

(A) Representative western blot of isolated subcellular fractions. Expression of BCL2L13, FACL4, NDUFS3, and SERCA2 in crude mitochondria (CM), pure mitochondria (PM), EMRCs, ER, and cytosol (Cyt). Experiments were repeated 4 times.

(B) Representative confocal microscopy images of HeLa cells expressing SPLICS_S/L probes (green) and BFP from P2A constructs (blue).

(C) Quantification of SPLICS_S and (D) SPLICS_L contacts in HeLa cells expressing control-P2A-BFP (Control) or BCL2L13-P2A-BFP (BCL2L13). n = 30 cells from four independent transfections.

(E) Number of ERMCs per mitochondria and (F) percentage of the mitochondrial membrane in contact with ERMCs (% mito mb in contact) measured in electron micrographs from siControl and siBCL2L13 transfected C2C12 myotubes.

(G) Representative electron micrographs of C2C12 myotubes in siControl and siBCL2L13 transfected cells. Orange arrows indicate examples of ERMCs. n = 27 micrographs (4.95 × 4.95 μm) per condition from four independent experiments. For all panels, the median is shown by a white full line while quartiles are dotted lines.

See also Figures S5 and S6.

Ca²⁺ dynamics in siBCL2L13 and siControl C2C12 myotubes

(A) 300 s confocal time-lapse recording of cytosolic Ca²⁺ levels.

(B) Quantification of normalized Fluo-4 fluorescence in response to 2.5 mM caffeine.

(C) 300 s confocal time-lapse recording of mitochondrial Ca²⁺ levels.

(D) Normalized Rhod-2 fluorescence in response to 1 μM thapsigargin. (A–D) n = 4 independent transfections per condition. (B and D) peak fluorescence was normalized to baseline. Error bars are mean ± SEM, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗∗p < 0.0001 (unpaired t test).

See also Figure S5.