Insulin-Like Growth Factor-II Receptor Mediates Angiotensin II-Induced Rab9-Dependent Macroautophagy and Apoptosis in Cardiomyocytes

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Ding-Yu Lin (林町餘) 1_{, Wei-Wen Kuo (郭薇雯)}2_{, Fu-Jen Tsai (蔡輔仁)}3_{, Chang-Hai Tsai (蔡長海)}4_{, Chih-Yang Huang (黃志揚)}1,5

1Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan; 2 Department of Biological Science and Technology, China Medical University, Taichung, Taiwan 3 Department of Pediatrics, Medical Research and Medical Genetics, China Medical University Hospital, Taichung, Taiwan 4 Department of Healthcare Administration Asia University, Taichung, Taiwan 5Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan.

Macroautophagy (hereafter referred to as autophagy) has emerged as an important process in the pathogenesis of cardiovascular diseases.

Angiotensin II (Ang II) plays an important role in pathogenesis of heart disease. Our previous studies demonstrated that up-regulation of igf2r (Insulin-like growth factor -II receptor) gene and the subsequent activation of IGF2R-Gαq signaling contributed to AngII-induced myocardial cell hypertrophy, and apoptosis.

Insulin-Like Growth Factor-II Receptor Mediates Angiotensin II-Induced

Rab9-Dependent Macroautophagy and Apoptosis in Cardiomyocytes

第二型類胰島素生長因子受體調控血管收縮素

第二型類胰島素生長因子受體調控血管收縮素II在心肌細胞所誘導的

在心肌細胞所誘導的

在心肌細胞所誘導的Rab9依賴型自噬作用

在心肌細胞所誘導的

依賴型自噬作用

與細胞凋亡

AU

Figure 2. Induction of Autophagy by IGF2R activation. (A).Typical autophagic vacuoles were observed in Leu27-IGFII treated myocardial cells by transmission electron microscopy (TEM). AU: autophagosome; AV: Autolysosome. (B). Increase of autophagy levels by IGF2R activation.

Fig2(A) Fig2 (B)

Control

Figure 6. Rab9-dependent autophagy contributes to IGF2R-induced apoptosis in cardiomyocytes.

Figure 8. Rab9-depedent autophagy is associated with Ang II-induced myocardial cell apoptosis. (A).Time-course induction of Rab9-depedent autophagy and apoptosis in Ang treated H9c2 cells. (B).Suppressed Ang II-induced apoptosis by inhibition of Rab9-depedent autophagy in NRVMs. **P<0.01.

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Fig 6 (A). Pro-apoptotic proteins induced by IGF2R activation were attenuated markedly by autophagy

inhibition or knockdown of Rab9-dependet autophagy required ATGs (Autophagy related genes) in H9c2 cells and NRVMs. Autophagy levels

BACKGROUND

AL Fig7 (A) Study design

Cells: H9c2 rat cardiomyoblast cells were obtained from the American Type Culture Collection (ATCC) and primary neonatal rat ventricular myocytes (NRVMs) were harvested from1 to 3 days old Sprague Dawley rats. To investigate whether autophagy is involved in AngII-induced IGF2R signaling and further clarify whether the induced autophagy is associated with IGF2R-mediated apoptosis in myocardial cells.

Figure 1. (A.) Illustrations of Ang II-mediated IGF2/IGF2R signals contributing to cardiac remodeling (Chu et al., Endocrinology, 2009; Am J Physiol Endocrinol Metab, 2006 ). (B).Comparison of canonical and non-canonical autophagy (Nishida Y et al., Nature. 2009).

α -Tubulin LC3 I LC3 II BafA1(10-8M) －－－－－－－－－－－－－－－－－－－－＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋ Hours 0 4 6 8 24 4 4 6 6 8 8 24 24 Leu27IGF-II (10-8 M) －－－－＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋－－－－＋＋＋＋－－－－＋＋＋＋－－－－＋＋＋＋－－－－＋＋＋＋ 16 kDa 14 55

Figure 3. Canonical autophagy was suppressed by IGF2R activation. LC3 turnover assay indicated that canonical autophagy was inhibited after Leu27IGF-II 24hrs incubation compared with no-stimuli H9c2 cells .

Fig 1 (A) Fig 1 (B)

Figure 4. Rab9-dependet autophagy is mediated through IGF2R-Gαq signaling in cardiomyocytes. (A).Up-regulation of rab9 gene by IGF2R activating. (B).Rab9-depednet autophagy was induced by IGF2R-Gαq signaling. (C). Determine autophagy levels by Lysotracker Red stain. siRNA-scramble(ctrl) were used as the control si-RNA. *P<0.05; **P<0.01.

Leu27IGF-II + 3MA Leu27IGF-II

Fig 4 (A) Western blotting SYBR Green Real-Time PCR

TUNEL DAPI si-Ctrl Leu27IGF- II + si-Beclin I Leu27IGF-II + si-Rab9 Leu27IGF-II + si-Ctrl Leu27IGF- II + si-ATG5 Leu27IGF-II + si-Gαq TUNEL DAPI

Figure 8. Rab9-depedent autophagy may contribute to IGF2R mediated mitochondria-dependent apoptosis through mitophagy. (A). Reduction of IGF2R-inudced depolarized mitochondria by autophagy inhibition. (B). Increase of mitophagy was observed in IGF2R-activated H9c2 cells.

Fig 6 (B). Apoptosis levels were detected by TUNEL assay. Suppressed IGF2R-induced apoptosis

by inhibition of Rab9-depedent autophagy. (Cells: NVRMs; *P<0.05; **P<0.01)

Fig 6 (C). Annexin V/PI staining assay of cell apoptosis induced by Leu27IGF-II. The total apoptotic

cells (early and late-stage apoptosis) are represented by the right side of the panel (Annexin V staining alone or together with PI) in which the total cell death number. Results indicated that Rab9-depedent autophagy contributes to IGF2R-Gαq mediated apoptosis. (Cells: H9c2; *P<0.05; **P<0.01)

Fig7 (B)

OBJECTIVES

METHODS

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* **

Fig8 (A). JC-1 Mitochondrial membrane potential

assay

Fig8 (B). Immunofluorescence

staining with mitochondria and lysosomes. Increase of mitochondria-containing lysosomes in IGF2R-activated H9c2 cells. ** ** 17 55 H9c2 cells NRVMs

Cells were incubated with Ang II(10-7 M) or Leu27IGF-II (10-8 M) for the subsequent analysis. Leu27IGF-II is an analog of IGF-II which interacts selectively with the IGF-IIR.

Autophagy assessments

si-RNAs (small interference RNAs) were transfected into myocardial cells to specifically knockdown the target genes; Gene silencing efficiency were over 60% in all experiment groups.

3-Methyladenine(3-MA;10mM) was used for autophagy inhibitor and Bafilomycin A1(BafA1,100nM) was used for autolysosome inhibitor. Autophagy levels were quantified by flow cytometr with LysoTracker Red dye.

Ang II-induced Rab9-depentdent autophagy is mediated by IGF-IIR and this non-canonical autophagy contributes to Ang II-induced apoptosis in cardiomyocytes.

Selective activation of IGF-IIR by Leu27IGF-II confirmed that the induction of Rab9-depedent autophagy is mediated by IGF-IIR-Gαq signaling in cardiomyocytes, which may in turn contribute to mitochondria-mediated apoptosis.

Control Leu27IGF-II si-ctrl Leu27IGF-II + si-Ctrl Leu27IGF-II + si-Rab9

Fig 4 (B)

Fig 4 (C)

Figure 5. Rab9-depedent autophagy contributes to IGF2R-induced myocardial cells death. (A). H9c2 cells. (B). NVRMs. Cell viability were assessed by Trypan Blue Exclusion Test.

si-Ctrl Leu27IGF-II + si-Gαq

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* *

RESULTS

CONCLUSIONS

Leu27IGF-II + 3MA

Leu27IGF-II + si-Ctrl Leu27IGF-II + si-Rab9

Figure 7. Rab9-depedent autophagy induced by Ang II is mediated by IGF2R in myocardial cells. (A). Increase of igf2r gene expression by Ang II in a time dependent manner. (B-C). Ang II mediated up-regulation of igf2 gene via AT-1 receptor. Transcription levels were analyzed by RT-PCR. ; Losartan: AT-1 receptor antagonist; PD 123319: AT-2 receptor antagonist.

Fig7 (A) Fig7 (B)

Fig7 (C)

Arrows indicated the image colorization with Tom20 and Lamp2.

H9c2 cells.

Schematic IGF2R death signal in cardiomyocytes

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