Dhcr24 activates the PI3K/Akt/HKII pathway and protects against dilated cardiomyopathy in mice

Figures

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  Figure 1. Detection of Dhcr24 expression in heart tissue. (A), The expression of Dhcr24 in the heart tissues from WT mice at 1, 3 and 6 mo of age was detected by Western blot. (B), Quantitative analysis of Dhcr24 in the WT mice used GAPDH for normalization (n = 3 mice per group). (C), Dhcr24 expression in heart tissues of the cTnTR 141W transgenic mice at 3 mo of age was detected by Western blot. (D), Quantitative analysis of Dhcr24 in the cTnTR 141W mice used GAPDH for normalization (n = 3 mice per group, **P < .01). (E), Immunodetection of Dhcr24 in the heart from the cTnTR 141W and the NTG mice at 5 mo of age. Scale bar = 20 μm. (F), The cholesterol level in the heart tissues of the cTnTR 141W and the NTG mice (n = 3 mice per group, **P < .01)

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  Figure 2. Generation of the transgenic mice. (A), The transgenic construct was generated by inserting the Dhcr24 gene under the control of the α‐MHC cardiac‐specific promoter, and the transgenic mice were created following microinjection. (B), The mouse line with Dhcr24 over‐expression was identified by Western blot. (C), Quantitative analysis of Dhcr24 in the transgenic mouse line used GAPDH for normalization. (D), Immunodetection of Dhcr24 in the heart tissue of the NTG and the Dhcr24 transgenic mice. Scale bar = 20 μm. (E), The cholesterol level in heart tissues of the NTG and the Dhcr24 transgenic mice (n = 6 mice per group, **P < .01)

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  Figure 3. The effects of Dhcr24 on heart dimensions and survival rate. (A), The representative M‐mode echocardiographic images of the LV long‐axis from the NTG, Dhcr24, cTnTR 141W and DTG mice at 5 mo of age. (B), The left ventricle (LV) posterior wall at end‐systole (LVPW in the NTG, Dhcr24, cTnTR 141W and DTG mice at 1, 3, 5, and 7 mo of age. (C), The LV fractional shortening (LVFS) in the NTG, Dhcr24, cTnTR 141W and DTG mice at the 1, 3, 5, and 7 mo of age. (D), The cumulative survival data were recorded until 7 mo of age

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  Figure 4. The histopathological profile of the heart tissue from mice at 5 mo of age. (A), H&E staining patterns of the whole‐heart longitudinal sections. (B), Magnification of the H&E‐stained sections of the left ventricle. (C), Magnification of the Masson trichrome stained sections of left ventricle. Scale bar = 20 μm. (D), The expression of Col1α1, Col3α1 and Itgα8 was detected by RT‐PCR. (E), Quantitative analysis of the expression of Col1α1, Col3α1 and Itgα8 using GAPDH for normalization

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  Figure 5. The measurement of signalling proteins of the PI3K and the mitochondrial pathways of apoptosis. The heart tissues were sampled from the NTG, Dhcr24, cTnTR 141W and DTG mice at 5 mo of age. (A), The levels of Dhcr24, PI3K, phosphorylated AKT and HKII were detected by Western blot in the heart tissues. (B), Quantitatively analyzed using GAPDH for normalization. (C), The fraction of Bax and cytochrome c in the cytoplasm and the mitochondria was detected in the heart tissues by Western blot. (D), (E), Quantitatively analysis using COX4 and GAPDH for normalization, respectively. (F), The activation of caspase‐9 and ‐3 was measured by Western blot. (G), Quantitatively analyzed using GAPDH for normalization

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  Figure 6. Observation of the mitochondria and TUNEL assay. (A), The morphology of the mitochondria was observed by TEM from the LV free wall in the NTG, Dhcr24, cTnTR 141W and DTG mice at 5 mo of age. (B), Photomicrographs of the heart tissue used for the TUNEL assay. The arrows indicate TUNEL‐positive cells. Scale bar = 10 μm. (C), Quantitative analysis of apoptotic cells in the hearts of mice (n = 3, *P < .05, **P < .01)

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  Figure 7. The measurement of the signalling proteins of the PI3K pathway and apoptosis in H9c2 cells. The H9c2 cells, Dhcr24 over‐expressing (Dhcr24‐ov) cells and Dhcr24 knockdown (Dhcr24‐kd) cells were sampled, and the signalling proteins of the PI3K pathway and the mitochondrial pathway of apoptosis were detected by Western blot and TUNEL assay. (A), The levels of Dhcr24, PI3K, phosphorylated AKT and HKII were measured by Western blot. (B), Quantitative analysis using GAPDH for normalization (n = 3 independent experiments, *P < .05, **P < .01). (C), The fraction of Bax and cytochrome c in the cytoplasm and the mitochondria was detected by Western blot in the heart tissues. (D), (E), Quantitative analysis of the level of Bax and cytochrome c using COX4 and GAPDH for normalization, respectively, (n = 3 independent experiments, **P < .01). (F), Photomicrographs of the H9c2 cells and the Dhcr24‐ov and Dhcr24‐kd cells used for the TUNEL assay. The arrows indicate TUNEL‐positive cells. (G), Quantitative analysis of the apoptotic cells (magnification × 400, n = 3 independent experiments, *P < .05)

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  Figure 8. The measurement of apoptosis of the Dhcr24‐ov cells in the presence of inhibitors of PI3K and HKII. The Dhcr24‐ov cells were treated with LY294002 (PI3K inhibitor) or 3‐BrPA (HKII inhibitor), and the signalling proteins of the PI3K and the mitochondrial pathways of apoptosis were detected by Western blot and TUNEL assay. (A), The levels of PI3K, phosphorylated AKT and HKII were measured by Western blot. (B), Quantitative analysis using GAPDH for normalization. (C), The fractions of Bax and cytochrome c in the cytoplasm and the mitochondria were detected by Western blot in the heart tissues. (D), (E), Quantitative analysis using COX4 and GAPDH for normalization, respectively (n = 3 independent experiments, **P < .01). (F), Photomicrographs of Dhcr24‐ov cells treated with LY294002 (PI3K inhibitor) or 3‐BrPA (HKII inhibitor) used for the TUNEL assay. The arrows indicate TUNEL‐positive cells. (G), Quantitative analysis of apoptotic cells (magnification × 400, n = 3 independent experiments, *P < .05, **P < .01)