Fig. 1

BCAAs inhibit Ptpn6/SHP1 to maintain ACTC1_Y55 phosphorylation, thereby preserving cardiac filament stability against SIMD. a Analysis of cardiac function, troponin I level, rate of death and cardiac Ptpn6/SHP1 activity of SIMD mice with BCAA supplementation and Ptpn6/SHP1 modulation. Eight-week-old mice were fed a BCAAe diet for 0, 2, or 6 weeks prior to LPS injection. SC-43 (Ptpn6/SHP1 agonist, 30 mg/kg) or NSC87877 (Ptpn6/SHP1 inhibitor, 5 mg/Kg) was administered 30 min after LPS injection. AAV9 (5 × 10¹¹ vector genomes per mouse) was injected 4 weeks before LPS injection. Representative images of echocardiography (scale bar: 4 mm, 0.1 s). Left ventricle systolic function, expressed as ejection fraction (EF) (%), fractional shortening (FS) (%), and cardiac output (mL/min), was evaluated 24 h after LPS injection (n = 3~21 per group). Troponin I in serum (n = 3~5 per group), rate of death (n = 4~24 per group) and cardiac Ptpn6/SHP1 activity (n = 3 ~ 6 per group) were also assessed at 24 h after LPS injection. b Bioinformatics analysis of RNA sequencing and phosphorylation mass spectrometry data. Left panel: volcano plot from RNA sequencing illustrates differentially expressed genes (DEGs, shown in blue) between healthy and SIMD mouse hearts, using a threshold of |log2 (foldchange)| > 0.58 and an adjusted P-value < 0.05 (n = 3 per group). DEGs that were restored by 6 w (but not 2 w) of BCAA supplementation are highlighted in pink, with Ptpn6/SHP1 ranking fourth. Middle panel: Gene Ontology (GO) analysis of DEGs restored by BCAA 6 w (but not 2 w) revealed significantly enriched biological process terms. The Term “Actin cytoskeleton organization” is associated with ACTC1, while “Peptidyl−tyrosine phosphorylation” pertains to Tyr phosphorylation. Other enriched terms reflect known changes during sepsis development. Right panel: Venn diagram and heatmap from phospho-MS illustrate differentially phosphorylated sites between healthy and SIMD mouse hearts, using a threshold of |log2 (foldchange)| > 0.263 and an adjusted P-value < 0.05 (n = 3 per group). Nine differentially phosphorylated sites induced by LPS are shown in purple. Among these, eight were reversed by BCAA supplementation (displayed in yellow), and three were reversed by NSC87877 (displayed in green). c Representative immunoblot (IB) images. Mouse hearts were homogenized, lysed, and subjected to immunoprecipitation (IP). Upper panel: analysis of the interaction between Ptpn6/SHP1 and ACTC1. Lower panel: analysis of ACTC1_Y55 phosphorylation in response to LPS and NSC87877 treatment. The blots shown are representative of three independent experiments. d Representative images and percentage of NRCMs with clear filament striations in response to LPS (1 μg/mL), BCAA (60 or 150 mg/L), NSC87877 (0.5 μg/mL) and Ad-ACTC1_Y55E. Immunofluorescence staining with anti‑α‑Actinin was used to visualize NRCM filament striations. Left panel: representative images of NRCM filaments (scale bar, 20 μm). Right panel: bar plot showing the percentage of NRCMs with clear striations. All cell experiments were repeated three times, and values are expressed as mean ± SEM. Statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. A Chi-square test was used to analyze the rate of death data in a. Two-way analysis of variance (ANOVA) with a Bonferroni post hoc test was performed for statistical analysis in a, d. PBS phosphate-buffered saline, LPS lipopolysaccharide, BCAAe BCAA-enriched diet, 0/2/6 w BCAA supplementation through diet for 0, 2 or 6 weeks, AAV9 adeno associated virus 9, phospho-MS phosphorylation mass spectrometry, NRCM neonatal rat cardiomyocytes, Ad adenovirus, Tyr tyrosine, Y tyrosine, E glutamic acid, P phosphorylation