A. Timeline of the experimentation with zebrafish embryos (hpf, hours post fertilization).B. Representative video captures of the beating heart of a zebrafish embryo. The yellow lines show the small diameter of the ventricle in systole (left) and in diastole (right).

A. Impact of empagliflozin on zebrafish embryos? hearts under hypoxic exposure. Fractional shortening (FS) of the beating hearts of zebrafish embryos was measured in normoxia and hypoxia, as well as in hypoxia after treatment with empagliflozin (50 and 100 µM). **** P<0.0001, *** P<0.001, **P<0.01, *P<0.05. Capped bars on top of the columns indicate standard error of the means.B. Relative expression of Bnp determined with RT-qPCR of hypoxia-exposed zebrafish embryos treated with empagliflozin (50 µM or 100 µM). Bnp expression was downregulated in response to empagliflozin treatment. *** P<0.001, **P<0.01, *P<0.05. Capped bars on top of the columns indicate standard error of the means.

Relative expression of Mmp13a (A) and Vtg7 (B) mRNAs derived from transcriptome analysis. Zebrafish embryos exposed to hypoxia were treated with empagliflozin (100 µM). Empagliflozin eliminated the increased expression of mmp13a and vtg7 caused by hypoxia. Statistical analysis criteria used were: fold change > 2; false discovery rate < 0.05. Capped bars on top of the columns indicate standard deviations.

Effect of MMP13 inhibitor (MMP13 I) on fractional shortening of hypoxia-exposed zebrafish embryos? hearts. The concentration of the MMP13 inhibitor was 40 µM. *** P<0.001, **P<0.01, *P<0.05. Capped bars on top of the columns indicate standard error of the means.

Measurement of apoptosis in zebrafish embryo hearts using acridine orange. Embryos subjected to hypoxia and treated for 6 h with DMSO, empagliflozin (empa 50, 50 µM) or MMP13 inhibitor (MMP13 I, 40 µM) were stained with acridine orange (AO) and imaged with fluorescence microscope. A) Reflected white light (RL) and fluorescence images (AO) of zebrafish embryos. B) Quantitation of mean AO fluorescence intensity in the heart region. normoxia, n=11; hypoxia, n=9; empa 50 + hypoxia, n=7; MMP13 I + hypoxia, n=6. Scale bars 100 µm. **P<0.01, *P<0.05. Capped bars on top of the columns indicate standard error of the means.

Measurement of glutathione content in zebrafish embryo hearts using monochlorobimane. Embryos subjected to hypoxia and treated for 24 h with DMSO, 50 µM empagliflozin (empa 50) or 40 µM MMP13 inhibitor (MMP13 I) were stained with monochlorobimane (mBCI) and imaged with fluorescence microscope. A) Reflected white light and fluorescence images of mBCI in zebrafish embryos. B) Quantitation of integrated mBCI fluorescence signal intensity in the heart region. normoxia, n=14; hypoxia, n=12; empa 50 + hypoxia, n=11; MMP13 I + hypoxia, n=13. Scale bars 100 µm. **P<0.01, *P<0.05. Capped bars on top of the columns indicate standard error of the means.