Orai3 knockdown at the onset of EACH limits cardiac hypertrophy, accelerates alteration of function and dilation and promotes fibrosis. (A) Schematic representation of the protocol where mice implanted with an iso-pump at day 0 were subjected to a unique ultrasound-guided intramyocardial transthoracic injection of Scramble or Orai3 siRNA at day 8 and submitted to an echocardiographic follow-up. (B) In some experiments, siRNA was added with sulforhodamine: typical in-vivo visualization of intracardiac localization of sulforhodamine at day 1 post-injection using an IVIS spectrum in-vivo imaging system. (C) Echocardiographic parameters (mean ± SEM of mice, n = 8–22 mice/group, see Table 3, ANOVA for repeated measures followed by Dunn-Sidak post-hoc tests). (D,E) Efficient knockdown of Orai3 mRNA and protein levels in cardiac homogenates at d12, d15 and d28 following injection (mean ± SEM of mice, n = 7 mice/group, Kruskal-Wallis followed by Dunn’s post-hoc tests). Full length blots were included in SI. (F,G) SiOrai3 injection induced a decrease in Heart Weight/Body Weight ratio, myocyte area and Myh7 mRNA level, an increase in fibrosis, and an elevation of Col1a1, Col3a1 and Tgfβ mRNA levels (mean ± SEM of mice, n = 7 mice/group, Mann-Whitney U tests). (H) SiOrai3 injection induced a decrease in the ratio phospho-GSK3β/GSK3β at day 12 and day 15 following injection (mean ± SEM of mice, n = 7 mice/group, Kruskal-Wallis followed by Dunn’s post-hoc test). Full length blots were included in SI. *p < 0.05, **p < 0.01.