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

Journal: International Journal of Molecular Sciences

Article Title: Cardiovascular Damage in COVID-19: Therapeutic Approaches Targeting the Renin-Angiotensin-Aldosterone System

doi: 10.3390/ijms21186471

Figure Lengend Snippet: Role of sACE2 and MasR activators in COVID-19. Infusion of the soluble isoform of ACE2 could serve as a decoy receptor for SARS-CoV-2 particles, reducing infectivity while conserving cardioprotective actions of the plasma membrane isoform (via Ang-(1–9) and Ang-(1–7)). On the other hand, some Ang-(1–7) mimetics (cyclic Ang-(1–7), HPβCD-Ang-(1–7), NPA7, TXA127) and MasR agonists (AVE 0991, CGEN-856, CGEN-857) could also trigger protective actions for the CV system in COVID-19 subjects. Ang and ACE stand for angiotensin and angiotensin converting enzyme, respectively. sACE2 and NEP stand for soluble ACE2 and neprilysin, respectively. MasR and AT2R stand for Mas receptor and angiotensin receptor type-II, respectively. Finally, HPβCD-Ang-(1–7) means hydroxypropyl β-cyclodextrin-Ang-(1–7).

Article Snippet: Since Ang-(1–7) possesses a short half-life in plasma, alternative Ang-(1–7) mimetics (cyclic Ang-(1–7), HPβCD-Ang-(1–7), NPA7, TXA127) and MasR agonists (AVE 0991, CGEN-856, CGEN-857) have been successfully tested in experimental models against oxidation, thrombogenesis, fibrosis, inflammation, endothelial function, and high blood pressure [ , ] ( ).

Techniques: Infection, Clinical Proteomics, Membrane