RMC-6272, a selective third-generation bi-steric mTORC1 inhibitor, improves cardiac function in pressure overload-induced cardiac hypertrophy

Figures

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  Fig. 1. RMC-6272 selectively inhibits mTORC1 signaling in cardiomyocytes. (A) Schematic of the mTOR pathway highlighting mTORC1/2 functions and the mechanisms of rapamycin, Torin1, and RMC-6272. (B) Immunoblots blot showing dose-dependent effects of RMC-6272 (1–100 nM, 6 h) on mTORC1 (p-S6S235/236, p-4EBP1T37/46) and mTORC2 (p-AktS473)-phosphorylation targets and quantification of p-AktS473, p-S6S235/236 and p-4EBP1T37/46 normalized to alpha-Tubulin in response to RMC-treatment. (C) Immunoblots showing phosphorylation changes over time (6, 24, 48 h) for Rapamycin (5 nM), Torin1 (100 nM), and RMC-6272 (5 nM) and time-course quantifications of p-S6S235/236 and p-4EBP1T37/46 in response to indicated inhibitors. Data are presented as mean ± SEM. (B–C) n = 2 independent cardiomyocyte isolation per group with two technical replicates each. Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc test. *p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001.

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  Fig. 2. RMC-6272 counteracts PE-induced cardiomyocyte hypertrophy by inhibiting mTORC1 signaling in vitro. (A) Immunoblots of canonical mTORC1 (p-S6S235/236, p-4EBP1T37/46) and mTORC2 (p-AktS473) targets in NRCMs treated with control, RMC-6272 (5 nM), PE (50 μM), or PE + RMC-6272 (24 h) and quantifications (B–D) of p-AktS473, p-S6S235/236 and p-4EBP1T37/46 (C) Quantification of cell size in NRCMs treated with PE or PE + RMC-6272 and (F) representative immunofluorescence images of NRCMs showing alpha- sarcomeric-Actin (SMA) (green), P-4EBP1T37/46 (red), and DAPI (blue). Data are presented as mean ± SEM. (A–D)n = 2 independent cardiomyocyte isolations with 3 technical replicates each. (E–F)n = 3, Cardiomyocytes from independent isolations. Each condition was analyzed in 24 technical replicates (12 wells per plate, two plates). Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc test. ****p < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 3. RMC-6272 selectively inhibits mTORC1 activity without impairing cardiomyocyte contractility (A) Percent sarcomere shortening in ARCMs treated with vehicle, RMC-6272, Iso, or RMC-6272 + Iso and time decay constant (Tau) in seconds under the indicated conditions. (B) Representative immunoblots of p-AktS473, p-S6S235/236, p-4EBP1T37/46 and p-Troponin IS23/24 in adult rat cardiomyocytes (ARCMs) treated with vehicle, RMC 6272, isoprenaline (Iso), or RMC-6272 + Iso. (C–F) Quantifications of phosphorylation levels for P-AktS473, P-S6S235/236, P4EBP1T37/46 and P-Troponin IS23/24 normalized to Alpha-Tubulin. Data are presented as mean ± SEM. (A) n = 2 independent cardiomyocyte isolations from individual animals. Multiple cells were isolated from each animal for each condition. (B–F)n = 1 independent cardiomyocyte isolation group with 4 technical replicates each. Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc test. *p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001.

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  Fig. 4. RMC-6272 selectively suppresses mTORC1-dependent -mRNA translation in cardiomyocytes in vivo. (A) Schematic illustration of the experimental workflow. Ribo-tag mice (αMHC-Cre;Rpl22) were treated with vehicle or RMC-6272 (10 mg/kg intraperitoneally). Ribosome-associated mRNAs were isolated from left ventricles for ribosome profiling. (B) Volcano plot showing differentially translated mRNAs in cardiomyocytes after RMC-6272 treatment. Downregulated mRNAs are highlighted in blue, and upregulated mRNAs in red. (C) Dot plots representing the most enriched pathways (GO-Term biological processes) involved among the downregulated hits in RMC treated animals. (D) Puromycin incorporation assays confirming decreased overall protein synthesis after RMC-6272 treatment and decreased expression of identified targets after RMC-6272 treatment. (E) Representative Western blot analysis of eEF2 (90 kDa) and Rps5 (25 kDa) protein levels in cardiomyocytes treated with vehicle (Veh) or RMC-6272. Total protein straining is shown as loading control. (F) Unchanged transcripts levels of eEF2 and Rps5 after RMC-6272 treatment assessed by qRT-PCRs. Data are presented as mean ± SEM. (D–E) n = 1 independent…

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  Fig. 5. RMC-6272 treatment preserves cardiac function during pressure overload but has minimal impact on hypertrophic remodeling. (A) Representative immunoblots of p-S6(Ser235/236) and p-4EBP1(Thr37/46) in ventricular tissue from sham, ORAB-vehicle and ORAB-RMC-6272 mice. Mice received 20 mg/kg RMC-6272 intraperitoneally on postoperative day 1 and were euthanized 5 h after treatment. (B–C) Quantification of p-S6(Ser235/236) and p-4EBP1(Thr37/46) normalized to total protein per lane confirming mTORC1 activation in the ORAB model and effective pathway inhibition by RMC-6272. (D) Experimental schema illustrating the ORAB model and treatment protocol. Mice underwent ORAB or sham surgery and received 10 mg/kg RMC-6272 or vehicle by intraperitoneal injection on days 1, 3, 7, 14, and 21. (E) Quantification of heart weight-to-body weight ratio (HW/BW), showing increased ratios in ORAB groups compared to sham, with no significant reduction by RMC-6272 treatment. (F) Representative WGA-stained of cardiac sections 28 days post-surgery. (G) Quantification of cardiomyocyte cross-sectional area based on WGA staining. (H-J) Echocardiographic measurements of ejection fraction (EF) at days 14 (H), 21…

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  Fig. 6. RMC-6272 improves cardiac function in established hypertrophy. (A) Modified treatment schema for delayed intervention. Mice underwent ORAB surgery and received 10 mg/kg RMC-6272 or vehicle through intraperitoneal injection on days 7 and 14. (B) Quantification of heart weight-to-body weight ratio (HW/BW) after 28 days, showing no significant reduction in RMC-6272-treated mice compared to vehicle controls. (C) Representative WGA-stained of cardiac sections 28 days post-surgery. (D) Quantification of cardiomyocyte cross-sectional area based on WGA staining, showing no significant reduction in RMC-6272-treated groups. (E–G) Echocardiographic measurements of ejection fraction (EF) at days 14 (E), 21 (F), and 28 (G) post-surgery and the left ventricular wall dimension (H), showing preserved cardiac function in RMC-6272-treated ORAB mice compared to vehicle controls. (I) Representative Sirius Red-stained cardiac sections from sham, ORAB-vehicle, and ORAB-RMC-6272 groups. (J) Relative Col1A1 RNA expression levels in left ventricular tissue from Sham, ORAB-Vehicle and ORAB-RMC-6272 mice assessed by quantitative RT-PCR. Gene expression was normalized to GAPDH and expressed as fold of…