Journal: Journal of Advanced Research

Article Title: ORP5 promotes cardiac hypertrophy by regulating the activation of mTORC1 on lysosome

doi: 10.1016/j.jare.2024.12.014

Figure Lengend Snippet: Oxysterol binding protein (OSBP)-related protein 5 (ORP5) exhibited upregulation in pathological cardiac hypertrophy. (A) mRNA levels of OSBPL5 in human hypertrophic cardiomyopathy (HCM) and heart failure (HF) from datasets GSE36961 and GSE57338 . (B) Immunoblotting and relative mRNA expression of ORP5 in normal and HCM hearts (n = 3 per group). (C) Immunoblotting and semi-quantification of ORP5 in NRVMs at various times after phosphate-buffered saline (PBS) or AngII treatment (n = 6 per group). (D) mRNA levels of Osbpl5 and Nppa in NRVMs 24 h post PBS or AngII treatment (n = 6 per group). (E) Fluorescence images and ORP5 quantification in NRVMs 24 h post-PBS or AngII treatment (n ≥ 20 cells per group). (F) Fluorescence images of α-actinin (green) and ORP5 (red) in hearts post-sham surgery or TAC treatment (n ≥ 20 visual fields from 5 mice per group). The data are shown as mean ± SEM and were analyzed using Student’s t-tests (A, B, D, E, F), and one-way ANOVA followed by Bonferroni’s post hoc test (C). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. ns, no significance. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: After 24 h of lentivirus transfection, cells were cultured in serum-free DMEM/F12 for another 24 h. Cardiomyocyte hypertrophy was induced with 1 mM angiotensin II (AngII) for 24 h. Rapamycin (100 nM, MCE #HY-10219) was used to inhibit mTORC1, and Lonafarnib (2 μM, MCE #HY-15136) was used to inhibit Rheb, as previously described .

Techniques: Binding Assay, Western Blot, Expressing, Saline, Fluorescence

Journal: Journal of Advanced Research

Article Title: ORP5 promotes cardiac hypertrophy by regulating the activation of mTORC1 on lysosome

doi: 10.1016/j.jare.2024.12.014

Figure Lengend Snippet: The knockdown of ORP5 mitigated pathological cardiac hypertrophy in both in vitro and in vivo models. (A) Protocol for administering AAV-9 and performing TAC. (B) Immunoblotting and semi-quantification of ORP5 in AAV9-Veh and AAV9-sh-ORP5 mice 4 weeks post-sham or TAC surgery (n = 6 per group). (C) Echocardiography images (left) and measurements (right) of heart weight to body weight (HW/BW), fraction ejection (EF), fractional shortening (FS), and heart rate (HR) in AAV9-Veh and AAV9-sh-ORP5 mice 4 weeks post-sham or TAC surgery (n = 6 per group). (D) Representative images showing gross view and WGA staining of AAV9-Veh and AAV9-sh-ORP5 mice 4 weeks post-sham or TAC surgery (n = 6 per group). (E) Cardiomyocyte cross-sectional area measurements from (D) (n ≥ 20 cells from 6 mice per group). (F) Representative Masson staining images of AAV9-Veh and AAV9-sh-ORP5 mice 4 weeks post-sham or TAC surgery (n = 6 per group). (G) Left ventricular collagen volume measurements from (F) (n ≥ 20 fields from 6 mice per group). (H) Relative mRNA levels of Nppa, Nppb, and Myh7 in AAV9-Veh and AAV9-sh-ORP5 mice 4 weeks post-sham or TAC surgery (n = 4 per group). (I) Immunoblotting and semi-quantification of ORP5 in Lenti-Veh and Lenti-shORP5 infected NRVMs 24 h post-PBS or AngII treatment (n = 6 per group). (J) Fluorescence of α-actinin (left) and cell surface measurement (right) in Lenti-Veh and Lenti-shORP5 infected NRVMs 24 h post-PBS or AngII treatment (n ≥ 20 cells per group). (K) Relative mRNA levels of Nppa, Nppb, and Myh7 in Lenti-Veh and Lenti-shORP5 infected NRVMs 24 h post-PBS or AngII treatment (n = 4 per group). The data are shown as mean ± SEM and were analyzed using two-way ANOVA with Bonferroni’s post hoc test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. ns, no significance.

Article Snippet: After 24 h of lentivirus transfection, cells were cultured in serum-free DMEM/F12 for another 24 h. Cardiomyocyte hypertrophy was induced with 1 mM angiotensin II (AngII) for 24 h. Rapamycin (100 nM, MCE #HY-10219) was used to inhibit mTORC1, and Lonafarnib (2 μM, MCE #HY-15136) was used to inhibit Rheb, as previously described .

Techniques: Knockdown, In Vitro, In Vivo, Western Blot, Staining, Infection, Fluorescence

Journal: Journal of Advanced Research

Article Title: ORP5 promotes cardiac hypertrophy by regulating the activation of mTORC1 on lysosome

doi: 10.1016/j.jare.2024.12.014

Figure Lengend Snippet: ORP5 alleviates pathological cardiac hypertrophy via mTORC1 pathway. (A) ORP5 and MTOR protein binding conformation: cartoon diagram (left) and surface diagram (right) with yellow indicating the binding area. (B) Immunoprecipitation and Western Blot (WB) analysis of proteins from cultured NRVMs transfected with vector or Flag-ORP5 plasmids using Flag magnetic beads. (C, E) Immunoblotting and semi-quantification of the mTORC1 pathway in LentiNC- and LentishORP5-infected NRVMs, 24 h post-PBS or AngII treatment (n = 6 per group). (D, F) Immunoblotting and semi-quantification of the mTORC1 pathway in AAV9-Veh and AAV9-sh-ORP5 mice, 4 weeks post-sham or TAC surgery (n = 6 per group). The data are shown as mean ± SEM and were analyzed using two-way ANOVA with Bonferroni’s post hoc test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. ns, no significance. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: After 24 h of lentivirus transfection, cells were cultured in serum-free DMEM/F12 for another 24 h. Cardiomyocyte hypertrophy was induced with 1 mM angiotensin II (AngII) for 24 h. Rapamycin (100 nM, MCE #HY-10219) was used to inhibit mTORC1, and Lonafarnib (2 μM, MCE #HY-15136) was used to inhibit Rheb, as previously described .

Techniques: Protein Binding, Binding Assay, Immunoprecipitation, Western Blot, Cell Culture, Transfection, Plasmid Preparation, Magnetic Beads, Infection

Journal: Journal of Advanced Research

Article Title: ORP5 promotes cardiac hypertrophy by regulating the activation of mTORC1 on lysosome

doi: 10.1016/j.jare.2024.12.014

Figure Lengend Snippet: Pathological cardiac hypertrophy was exaggerated by ORP5 overexpression and reversed by rapamycin. (A, B) Immunoblotting and semi-quantification of the mTORC1 pathway in NRVMs infected with Lenti-Veh or Lenti-OE-ORP5, 24 h post PBS or AngII treatment (n = 6 per group). (C, D) Fluorescence imaging of α-actinin and cell surface area measurement in the same infected NRVMs, 24 h post PBS or AngII treatment (n ≥ 20 cells per group). (E) Relative mRNA levels of Nppa, Nppb, and Myh7 in NRVMs infected with Lenti-Veh or Lenti-OEORP5, 24 h post PBS, AngII, or Rapamycin treatment (n = 4 per group). (F) Experimental protocol for AAV9 administration, TAC, and rapamycin treatment. (G) Echocardiography images (left) and measurements of HW/BW, EF, FS and HR of AAV9-Veh and AAV9-OE-ORP5 mice 4 weeks post-sham, TAC surgery, or rapamycin treatment (n = 6 per group). (H) Representative images of gross view and WGA staining of AAV9-Veh and AAV9-OE-ORP5 mice 4 weeks post-sham, TAC surgery, or rapamycin administration (n = 6 per group). (I) Cardiomyocyte cross-sectional area measurement from (H) (n ≥ 20 fields from 6 mice per group). (J) Representative Masson staining images of AAV9-Veh and AAV9-OE-ORP5 mice 4 weeks post-sham, TAC surgery, or rapamycin administration (n = 6 per group). (K) Left ventricular collagen volume measurement from (J) (n ≥ 20 fields from 6 mice per group). (L, M) Immunoblotting and semi-quantification of the mTORC1 pathway in AAV9-Veh and AAV9-OE-ORP5 mice 4 weeks post-sham, TAC surgery, or rapamycin treatment (n = 6 per group). (N) Relative mRNA levels of Nppa, Nppb, and Myh7 in these mice under the same conditions (n = 4 per group). The data are shown as mean ± SEM and were analyzed using two-way ANOVA with Bonferroni’s post hoc test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. ns, no significance.

Article Snippet: After 24 h of lentivirus transfection, cells were cultured in serum-free DMEM/F12 for another 24 h. Cardiomyocyte hypertrophy was induced with 1 mM angiotensin II (AngII) for 24 h. Rapamycin (100 nM, MCE #HY-10219) was used to inhibit mTORC1, and Lonafarnib (2 μM, MCE #HY-15136) was used to inhibit Rheb, as previously described .

Techniques: Over Expression, Western Blot, Infection, Fluorescence, Imaging, Staining

Journal: Journal of Advanced Research

Article Title: ORP5 promotes cardiac hypertrophy by regulating the activation of mTORC1 on lysosome

doi: 10.1016/j.jare.2024.12.014

Figure Lengend Snippet: ORP5 enhances mTORC1-mediated cardiac hypertrophy by promoting its translocation to the lysosome. (A, C) Immunoblotting and semi-quantification of the mTORC1 pathway in Lenti-Veh, Lenti-OEORP5, and si-mTOR infected NRVMs 24 h post-PBS or AngII (n = 6 per group). (B, D) Fluorescence of α-actinin and cell surface measurement in the same groups (n ≥ 20 cells per group). (E) Immunoblotting and semi-quantification of mTOR and ORP5 in plasma and lysosomes of Lenti-Veh and Lenti-OE-ORP5 infected NRVMs, 24 h post PBS or AngII treatment, using GAPDH and Lamp-2 as housekeeping genes for cytoplasm and lysosome, respectively (n = 3 per group). (F) Immunofluorescence and intensity analysis of mTOR and Lamp-2 in Lenti-Veh and Lenti-OE-ORP5 infected HL-1 cells, 24 h post PBS or AngII treatment (n ≥ 20 cells per group). (G, I) Immunoblotting and semi-quantification of the mTORC1 pathway in Lenti-Veh and Lenti-OEORP5 infected NRVMs 24 h post PBS, AngII, or lonafanib treatment (n = 6 per group). (H, J) Fluorescence of α-actinin and cell surface measurement in Lenti-Veh and Lenti-OEORP5 infected NRVMs 24 h post PBS, AngII, or lonafanib treatment (n ≥ 20 cells per group). The data are shown as mean ± SEM and were analyzed using two-way ANOVA with Bonferroni’s post hoc test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. ns, no significance.

Article Snippet: After 24 h of lentivirus transfection, cells were cultured in serum-free DMEM/F12 for another 24 h. Cardiomyocyte hypertrophy was induced with 1 mM angiotensin II (AngII) for 24 h. Rapamycin (100 nM, MCE #HY-10219) was used to inhibit mTORC1, and Lonafarnib (2 μM, MCE #HY-15136) was used to inhibit Rheb, as previously described .

Techniques: Translocation Assay, Western Blot, Infection, Fluorescence, Clinical Proteomics, Immunofluorescence

Journal: Journal of Advanced Research

Article Title: ORP5 promotes cardiac hypertrophy by regulating the activation of mTORC1 on lysosome

doi: 10.1016/j.jare.2024.12.014

Figure Lengend Snippet: The ORD domain of ORP5 is indispensable for ORP5-mediated mTORC1-dependent cardiac hypertrophy (A) Diagram showing ORP5 and its deletion mutants missing PH, ORD, and Tm domains. (B) Proteins from NRVMs transfected with vector, ORP5-Flag, ΔORD, ΔPH, and ΔTm plasmids were immunoprecipitated using Flag magnetic beads, followed by Western blot. (C, D) Representative Western blots and semi-quantification of the mTORC1 pathway in wild-type and sh-ORP5 NRVMs infected with vector, ΔORD, ΔPH, and ΔTm plasmids 24 h after PBS or AngII treatment (n = 6 per group). (E) Representative fluorescence of α-actinin (left) and measurement (right) of cell surface of the same cells 24 h after PBS or AngII treatment (n ≥ 20 cells per group). (F) Protocol for AAV-9 administration and TAC. (G) Echocardiography images (left) and measurements of HW/BW, EF, FS and HR of AAV9-Veh and AAV9-sh-ORP5 plus AAV9-ΔORD or AAV9-OE-ORP5 mice 4 weeks post-sham or TAC surgery (n = 6 per group). (H) Gross view and WGA staining images of the same mice 4 weeks post-sham or TAC surgery (n = 6 per group). (I) Measurement of the cardiomyocyte cross-sectional area in (H) (n ≥ 20 cells per group). (J) Masson staining images of the same mice 4 weeks post-sham or TAC surgery (n = 6 per group). (K) Measurement of left ventricular collagen volume in (I) (n ≥ 20 cells per group). (L, M) Immunoblotting (L) and semi-quantification (M) of mTORC1 pathway in the same mice 4 weeks post-sham or TAC surgery (n = 6 per group). (N) Relative mRNA levels of Nppa, Nppb, and Myh7 in the same mice 4 weeks post-sham or TAC surgery (n = 4 mice per group). The data are shown as mean ± SEM and were analyzed using two-way ANOVA with Bonferroni’s post hoc test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. ns, no significance.

Article Snippet: After 24 h of lentivirus transfection, cells were cultured in serum-free DMEM/F12 for another 24 h. Cardiomyocyte hypertrophy was induced with 1 mM angiotensin II (AngII) for 24 h. Rapamycin (100 nM, MCE #HY-10219) was used to inhibit mTORC1, and Lonafarnib (2 μM, MCE #HY-15136) was used to inhibit Rheb, as previously described .

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Magnetic Beads, Western Blot, Infection, Fluorescence, Staining