Activation of the M3AChR and Notch1/HSF1 Signaling Pathway by Choline Alleviates Angiotensin II‐Induced Cardiomyocyte Apoptosis

Figures

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  Figure 1. Choline attenuated angiotensin II- (Ang II) induced stimulation of the mitochondrial death pathway and cardiomyocyte apoptosis. (a) 4-Diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) administration blocked the effect of choline on terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling- (TUNEL-) positive neonatal rat ventricular myocytes (NRVMs). Scale bar: 50 μm. (b) The ratio of TUNEL-positive cells to total cells. Data are presented as means ± SD (n = 5 independent experiments). (c) Choline treatment increased the Bcl-2/Bax ratio and inhibited caspase-3 activation. The protective effects of choline were reversed by 4-DAMP. (d, e) Quantification of Bcl-2/Bax and cleaved-caspase-3. Data are presented as means ± SD (n = 5 independent experiments). (f) Representative image and graph showing levels of Omi/high-temperature requirement protein A2 (HtrA2), the second mitochondrial activator of caspases/direct inhibitor of apoptosis-binding protein with low P (Smac/DIABLO), and cytochrome c in the cytosol fraction. Data are presented as means ± SD (n = 5 independent experiments). (g) Representative western blot images and data of Omi/HtrA2, Smac/DIABLO,…

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  Figure 2. Choline counteracted the Ang II-induced increase in mitochondrial reactive oxygen species (mtROS) levels and surface area. (a–c) Representative images and data showing mtROS levels. MitoSOX Red fluorescence colocalized with MitoTracker Green. Data are presented as means ± SD (n = 4 independent experiments). Scale bar: 50 μm. (d) Representative immunofluorescence staining of NRVMs with α-actinin antibodies (red); the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bar: 100 μm. (e) Statistical analysis of the cardiomyocyte surface area. n = 5 independent experiments. Data are presented as means ± SD. ∗∗∗P < 0.001 versus control (CTR); ##P < 0.01 and ###P < 0.001 versus Ang II; &P < 0.05 and &&P < 0.01 versus the choline-treated Ang II group.

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  Figure 3. Choline improved Notch1 intracellular domain (NICD) expression and promoted nuclear translocation of heat shock transcription factor 1 (HSF1). (a–c) NICD protein levels (red) in NRVMs were quantified via immunostaining with the indicated antibodies; nuclei were stained with DAPI (blue). Scale bar: 50 μm. Data are presented as means ± SD; n = 5 independent experiments. (d) HSF1 immunofluorescence staining. Scale bar: 50 μm. n = 5 independent experiments. (e, f) Representative images and graph of HSF1 protein levels in the nuclear fraction. Data are presented as means ± SD (n = 5 independent experiments). ∗P < 0.05 and ∗∗P < 0.01 versus control (CTR); #P < 0.05 and ###P < 0.001 versus Ang II; &&P < 0.01 and &&&P < 0.001 versus the choline-treated Ang II group.

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  Figure 4. Downregulation of type 3 muscarinic acetylcholine receptors (M3AChR) blocked the protective effect of choline on Ang II-induced cardiomyocyte apoptosis. (a) Choline increased the Bcl-2/Bax ratio and inhibited cleaved-caspase-3 expression; these effects were abolished by M3AChR small interfering ribonucleic acids (siRNAs). (b, c) Quantification of Bcl-2/Bax and cleaved-caspase-3. Data are presented as means ± SD (n = 5 independent experiments). (d) Representative image and graph of Omi/HtrA2, Smac/DIABLO, and cytochrome c protein levels in the cytosol fraction. Data are presented as means ± SD (n = 5 independent experiments). (e) Representative western blot images and data of Omi/HtrA2, Smac/DIABLO, and cytochrome c levels in the mitochondrial fraction. Data are presented as means ± SD (n = 5 independent experiments). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 vs. the negative control (NC) siRNA group; #P < 0.05, ##P < 0.01, and ###P < 0.001 versus the choline-treated NC siRNA group.

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  Figure 5. M3AChR siRNA blocked choline-mediated activation of the Notch1/HSF1 pathway. (a–c) The protein levels of NICD (red) in NRVMs were quantified via immunostaining with the indicated antibodies; nuclei were stained with DAPI (blue). Scale bar: 50 μm; n = 5 independent experiments. (d) HSF1 immunofluorescence. Results represent five independent experiments. Scale bar: 50 μm. (e, f) Representative images and graph of HSF1 protein levels in the nuclear fraction. Data are presented as means ± SD (n = 5 independent experiments). ∗∗∗P < 0.001 versus the negative control (NC) siRNA group; ###P < 0.001 versus the choline-treated NC siRNA group.

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  Figure 6. Downregulation of Notch1 blocked the protective effect of choline on Ang II-induced apoptosis. (a) Representative image and graph of Bcl-2, Bax, and cleaved-caspase-3 protein levels. (b, c) Quantification of Bcl-2/Bax and cleaved-caspase-3. Data are presented as means ± SD (n = 5 independent experiments). (d) Representative image and graph of Omi/HtrA2, Smac/DIABLO, and cytochrome c protein levels in the cytosol fraction. Data are presented as means ± SD (n = 5 independent experiments). (e) Representative western blot images and data of Omi/HtrA2, Smac/DIABLO, and cytochrome c protein levels in the mitochondrial fraction. Data are presented as means ± SD (n = 5 independent experiments). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus the NC siRNA group; #P < 0.05, ##P < 0.01, and ###P < 0.001 versus the choline-treated negative control (NC) siRNA group.

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  Figure 7. Downregulation of Notch1 blocked the inhibiting effect of choline on Ang II-induced mtROS production and promotion of HSF1 nuclear translocation. (a–c) Representative images and data showing mitochondrial (mt) ROS levels. MitoSOX Red fluorescence was colocalized with MitoTracker Green. Data are presented as means ± SD (n = 5 independent experiments). Scale bar: 50 μm. (d) HSF1 immunofluorescence staining. Results represent five independent experiments. Scale bar: 50 μm. (e, f) Representative images and graph of HSF1 protein levels in the nuclear fraction. Data are presented as means ± SD (n = 5 independent experiments). ∗∗∗P < 0.001 versus the negative control (NC) siRNA group; ###P < 0.001 versus the choline-treated NC siRNA group.

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  Figure 8. Choline inhibited cardiac remodeling and apoptosis in Ang II-treated rats. (a) Representative left ventricular sections were stained with H&E and Masson's trichrome to assess cardiomyocyte hypertrophy and fibrosis, respectively; scale bars = 50 μm. (b) Cross section of cardiac myocytes in the left ventricle; n = 8 rats for each group with three random fields. (c) The level of fibrosis (%) stained by Masson's trichrome; n = 8 rats for each group. (d) Representative image and graph of Bcl-2, Bax, and cleaved-caspase-3 protein levels. (e, f) Quantification of Bcl-2/Bax and cleaved-caspase-3; n = 8 rats for each group. (g) Representative image and graph of NICD and nuclear HSF1 protein levels. (h, i) Quantification of NICD and nuclear HSF1. Data are presented as means ± SD. ∗P < 0.05 and ∗∗∗P < 0.001 versus sham; ##P < 0.01 and ###P < 0.001 versus the Ang II group.

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  Figure 9. Choline likely attenuates Ang II-induced cardiomyocyte apoptosis by inhibiting mtROS generation and the release of proapoptotic proteins from the mitochondria via the M3AChR and Notch1/HSF1 pathway. Ang II: angiotensin II; M3AChR: type 3 muscarinic acetylcholine receptor; HSF1: heat shock transcription factor 1; Omi/HtrA2: Omi/high-temperature requirement protein A2; Smac/DIABLO: second mitochondrial activator of caspases/direct inhibitor of apoptosis-binding protein with low P; mtROS: mitochondrial reactive oxygen species.