Fig. 6: Effects of the GDP-bound RalAS28N protein on TAC-induced cardiomyopathy.
From: The RalGAPα1–RalA signal module protects cardiac function through regulating calcium homeostasis
A Ca2+ transients upon field stimulation in RalGAPα1-depleted neonatal rat cardiomyocytes that were transfected with or without RalAS28N. Amplitude, FDHM and Tau of Ca2+ transients were quantified from 13 (siNC), 44 (siRalGAPα1), and 10 (siRalGAPα1 + RalAS28N) cells. FDHM: p = 0.012 (siNC vs siRalGAPα1) and 0.0006 (siRalGAPα1 vs siRalGAPα1 + RalAS28N). Tau: p = 0.024 (siNC vs siRalGAPα1), and p < 0.0001 (siRalGAPα1 vs siRalGAPα1 + RalAS28N). Amplitude: p = 0.0041 (siNC vs siRalGAPα1) and 0.0071 (siRalGAPα1 vs siRalGAPα1 + RalAS28N). B Experimental design for AAV-mediated gene therapy of TAC-induced cardiomyopathy. Male C57BL/6 J mice (12-week-old) were subjected to TAC or sham surgery. AAV9-GFP or AAV9-RalAS28N were administered into mice via vein injection on 2 days after surgery. Cardiac function was monitored before and after treatment. C Flag-RalAS28N mutant protein expression in the heart of AAV9-GFP or AAV9-RalAS28N administered male mice (20-week-old). D, E Oligomerization of SERCA2a in the heart of AAV9-GFP or AAV9-RalAS28N administered male mice (20-week-old) that were subjected to TAC or sham surgery. D, representative blots. E quantitation of SERCA2a oligomerization. Data were presented as fold changes of the ratio of oligomer SERCA2a to total SERCA2a. n = 3 (Sham and TAC AAV9-Control) and 4 (TAC AAV9-RalAS28N). p = 0.0063 (Sham vs TAC AAV9-Control) and 0.014 (TAC AAV9-Control vs TAC AAV9-RalAS28N). F Ca2+ transients in primary cardiomyocytes isolated from AAV9-GFP or AAV9-RalAS28N administered male mice (16-week-old) that were subjected to TAC or sham surgery upon field stimulation. Amplitudes, FDHM and Tau of Ca2+ transients were measured in 28 Sham cells, 54 TAC-AAV-Control cells and 51 TAC-AAV-RalAS28N cells, respectively. FDHM: p = 0.0001 (Sham vs TAC AAV9-Control) and 0.0037 (TAC AAV9-Control vs TAC AAV9-RalAS28N). Tau: p = 0.0012 (Sham vs TAC AAV9-Control) and 0.0014 (TAC AAV9-Control vs TAC AAV9-RalAS28N). Amplitude: p = 0.314 (Sham vs TAC AAV9-Control) and 0.533 (TAC AAV9-Control vs TAC AAV9-RalAS28N). G. Cardiac function in AAV9-GFP or AAV9-RalAS28N administered male mice that were subjected to TAC or sham surgery. n = 6. Ejection fraction: p = 0.793 (0 W/Sham vs 0 W/TAC AAV9-Control), p = 0.162 (0 W/TAC AAV9-Control vs 0 W/TAC AAV9-RalAS28N), p < 0.0001 (6 W/Sham vs 6 W/TAC AAV9-Control), p = 0.0005 (0 W/TAC AAV9-Control vs 6 W/TAC AAV9-Control), and p = 0.0027 (6 W/TAC AAV9-Control vs 6 W/TAC AAV9-RalAS28N). Fraction shortening: p = 0.718 (0 W/Sham vs 0 W/TAC AAV9-Control), p = 0.164 (0 W/TAC AAV9-Control vs 0 W/TAC AAV9-RalAS28N), p < 0.0001 (6 W/Sham vs 6 W/TAC AAV9-Control), p = 0.0008 (0 W/TAC AAV9-Control vs 6 W/TAC AAV9-Control), and p = 0.0034 (6 W/TAC AAV9-Control vs 6 W/TAC AAV9-RalAS28N). The data are given as the mean ± SEM. Statistical analyses were carried out using one-way ANOVA for A, E and F, and two-way ANOVA for G. One-asterisk indicates p < 0.05, two-asterisk indicates p < 0.01 and three-asterisk indicates p < 0.001. One-dagger indicates p < 0.001 (TAC AAV-control 0 W vs 6 W). Source data are provided as a Source Data file.
