Fig 2: Expression levels of mitochondrial dynamics and calcium handling pathway-related proteins in a hypertrophic hearts of ZDF rats.A Western blotting images and summarized data showing phospho-Drp1 (S616) and total Drp1 protein levels in the ventricular tissue of ZL and ZDF rats (n = 6). B Western blotting images and summarized data showing phospho-Drp1 (S637) and total Drp1 protein levels in the ventricular tissue of ZL and ZDF rats (n = 6). C Western blotting images and summarized data showing Mfn2 and Opa1 protein levels in the ventricular tissue of ZL and ZDF rats (n = 6). D–F Western blotting images and summarized data showing Orai1, phospho-ERK1/2, total ERK1/2, and CnA protein levels in the ventricular tissue of ZL and ZDF rats (n = 6). Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 vs ZL group.

Fig 3: TRAF6-mediated ubiquitination of CRTC2 disrupts its dephosphorylation via CnA. (a) Diagram and sequence of CRTC2 CnA-binding sites and ubiquitination site. (b) Effect of IL-1ß (10 µg l-1) on CnA binding of wild type or K628R mutant CRTC2. (c) Effect of IL-1ß (10 µg l-1) on dephosphorylation of wild type or K628R mutant CRTC2 in primary hepatocytes exposed to glucagon (20 nm). (d) Effect of IL-1ß (10 µg l-1) on CnA binding of wild-type CRTC2 or mutant CRTC2 containing a high affinity CnA-binding site (PVIVIT). (e) Effect of IL-1ß (10 µg l-1) on dephosphorylation of wild type or PVIVIT CRTC2 in primary hepatocytes exposed to glucagon (20 nm). (f, g) Effect of wild type and PVIVIT CRTC2 on G6pase mRNA amounts (f) and glucose output (g) in hepatocytes exposed to IL-1ß (10 µg l-1) and glucagon (20 nm) (*P<0.05). (h) Schematic of proposed mechanism. In response to septic shock, pro-inflammatory cytokines such as IL-1ß stimulates CRTC2 ubiquitination at Lys628 through TRAF6, which in turn blocks CRTC2 activation by disrupting CnA binding.

Fig 4: Schematic diagram depicting proposed Orai1–Drp1 signaling pathway in cardiomyocyte hypertrophy induced by HG.HG stimulation induces upregulation of Orai1. Then, Orai1-mediated Ca2+ entry targets CnA to inhibit Drp1 phosphorylation at S637, inducing cardiac hypertrophy. Orai1-mediated Ca2+ entry also targets p-ERK1/2 to induce Drp1 phosphorylation at S616. Phosphorylation of Drp1 at S616 is increased, while phosphorylation of Drp1 at S637 is decreased, promoting mitochondrial fission and accelerating HG-induced cardiac hypertrophy. Inhibition of the Orai1–Drp1 signaling pathway could prevent cardiac hypertrophy induced by HG.

Fig 5: RNAi activity of a self-delivering NPPA:PPP3CA Cond-siRNA in a rat cardiomyoblast model(A) qRT-PCR results show that NPPA mRNA was overexpressed in phenylephrine (PE)-exposed cells but not in PBS-exposed cells. (B) The Cond-siRNA-activated RNAi activity against PPP3CA (calcineurin) in PE-exposed cells and reduced overexpression of PPP3CA in a dose-dependent manner up to 5 nM. In cells exposed to PBS, there was no significant RNAi silencing of PPP3CA. The negative control was scrambled siRNA. The positive control was a commercial PPP3CA siRNA (A and B). The activated Cond-siRNA had similar silencing activity as the commercial siRNA. Significance was calculated by two-way ANOVA. *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001. #, p value comparison between PBS and PE; *, comparison with the control. Error bars denote one standard deviation. (C) Quantification of protein levels of calcineurin (Cell Signaling Technology, 2614S) in the cytoplasm of NRVMs treated with 1 nM Cond-siRNA constructs or the scrambled control siRNA. 24 h after isolation, primary rat cardiomyocytes were transfected with the Cond-siRNA; after 24 h, cells were treated with 50 µM PE for 48 h, and protein expression was determined 72 h after transfection. Data are shown as fold versus scramble (scrm) PBS. Statistics: unpaired t test; *p = 0.05, **p = 0.01.