Induction of VDAC1 and VDAC2 but not VDAC3 expression restores rhythmic cardiac contraction in transgenic tremblor/ncx1h lines. (A) Schematic diagram of VDAC transgenic construct. The cardiomyocytes-specific promoter myl7 drives Gal4-ecdysone receptor fusion protein (Gal4EcR), which becomes transcriptionally activated in response to tebufenozide (TBF), an ecdysone receptor agonist and binds to the upstream activating sequence (UAS), resulting in the simultaneous expression of both FLAG-tagged VDAC and EGFP. Transgenic lines were bred in the tremblor/ncx1h background. (B) Whole-mount in situ hybridization analysis demonstrating that VDAC expression is induced specifically in the heart upon TBF treatment in transgenic zebrafish (Tg:VDAC). Embryos were treated with either DMSO or TBF from 24 hpf until they were fixed for in situ hybridization at 48 hpf. (C) Western blotting of 32 hpf transgenic embryo lysate with an anti-FLAG antibody showing that VDAC protein expression is induced after embryos are treated with TBF. β-actin was used as a loading control. (D) While only 13.5 ± 3.6% of DMSO-treated Tg:VDAC1; tremblor/ncx1h embryos exhibit rhythmic cardiac contraction (N = 3, n = 233), 56.72 ± 3.5% of TBF-treated Tg:VDAC1; tremblor/ncx1h embryos established rhythmic contraction (N = 3, n = 238). Similarly, as opposed to only 16.8 ± 5.7% of DMSO-treated Tg:VDAC2; tremblor/ncx1h embryos (N = 3, n = 161), 49.6 ± 3.1% of TBF-treated Tg:VDAC2; tremblor/ncx1h embryos showed cardiac contraction (N = 3, n = 227). In contrast, the effect of TBF-induced overexpression is minimal in Tg:VDAC3; tremblor/ncx1h (13.7 ± 2.9%, N = 3, n = 283 in DMSO-treated embryos compared to 17.9 ± 4.0%, N = 3, n = 373 in TBF-treated embryos). Overall rescue percentages represent the mean rescue percentage ± s.e.m. from N independent experiments, using a total of n embryos.
|
