NOD1 is a key mediator of atrial myopathy in heart failure

Figures

• 

  Figure 1. NOD1 signaling is upregulated in atrial myocardium of patients and swine models of heart failure. (A) Representative hematoxylin-eosin (H&E) and immunohistochemical (IHC) staining for NOD1 in atrial tissue, with isotype controls from non-failing (NF) and heart failure (HF) patients. (B) Western blot and quantification of NOD1 (NF, n = 24; HF, n = 25), its adaptor RIP2 (NF, n = 20; HF, n = 17), IL-1β (NF, n = 13; HF, n = 18), and IL6 (NF, n = 17; HF, n = 18) expression in atrial tissue, normalized to GAPDH. (C) Prevalence of AF (%) in both groups. (D) Subgroup comparison of NOD1 protein levels in HF patients in sinus rhythm (HF-SR, n = 18) and atrial fibrillation (HF-AF, n = 7). (E) Representative echocardiographic images and correlations between atrial NOD1 protein levels and maximal and minimal atrial volumes and reservoir strain (NF, n = 29; HF, n = 26). (F) Representative H&E, NOD1 IHC staining and isotype controls obtained in LA tissue from aortic banding (AoB) and left-atrial infarction (LAI) porcine models and Control animals. (G-I) Western blot and quantification of NOD1 and its adaptor RIP2 expression in left atrial (LA) tissue from (G-H) Control (n = 12) and AoB (n = 10)…

• 

  Figure 2. NOD1 deficiency prevents atrial remodeling and proteomic alterations in mice subjected to pressure overload. (A) Representative long-axis four-chamber CMR images illustrating differences in left atrial size across groups. (B) Maximal and minimal LA volumes and reservoir function obtained in WT-Sham (n = 13), WT-TAC (n = 17), Nod1-/--Sham (n = 11) and Nod1⁻/⁻-TAC (n = 18). (C) Representative H&E staining illustrating atrial remodeling in all groups. (D) Representative images and quantification of LA cardiomyocyte cross-sectional area in WT-Sham (n = 57cells/6 mice), WT-TAC (n = 33 cells/5 mice), Nod1-/--Sham (n = 53 cells/3 mice) and Nod1⁻/⁻-TAC (n = 35 cells/6 mice). (E) Representative IHC staining for NOD1 in LA tissue, with isotype controls. (F) LA mRNA expression of Nod1 and Rip2 normalized to 36B4 and relative to WT-Sham. (G) LA mRNA expression of Il1b and Il6 normalized to 36B4 and relative to WT-Sham. (H) Functional enrichment analysis of murine LA tissue showing differential enrichment of inflammation- and Ca²⁺-related pathways across the indicated comparisons. Bar plots represent differential pathway enrichment for the following comparisons (n = 4 per group). Zc values…

• 

  Figure 3. NOD1 deficiency or pharmacological inhibition prevents TAC-induced intracellular Ca²⁺ dysregulation. WT-Sham and WT-TAC mice were treated with the selective NOD1 inhibitor ML-130 (ML; 2 mg/kg, i.p., every other day for 4 weeks). (A-B) Quantification of RyR2 protein abundance (A; Zq values, log₂ fold-change relative to WT-Sham) and RyR2 phosphorylation at Ser2814 (B; Zpq values). Zpq values represent log₂ fold-change standardized to units of standard deviation. (C) Western blot validation of RyR2 phosphorylation at Ser2814 in LA tissue, normalized to total RyR2 (WT-Sham, n = 9; WT-TAC, n = 11; Nod1-/--Sham, n = 8; Nod1-/--TAC, n = 7). (D-F) Representative confocal line-scan images and quantification of (D) sparks-mediated Ca²⁺ leak indicating diastolic Ca²⁺ leak in quiescent cardiomyocytes, (E) caffeine-evoked Ca²⁺ transients reflecting sarcoplasmic reticulum (SR) Ca²⁺ load and (F) electrically evoked Ca²⁺ transients in LA cardiomyocytes from WT-Sham (n = 27-41 cells/6 mice), WT-TAC (n = 19-31 cells/5 mice), Nod1-/--Sham (n = 22-42 cells/3 mice), Nod1⁻/⁻-TAC (n = 12-22 cells/6 mice), WT-Sham ML (n = 21-39 cells/3 mice), and WT-TAC ML (n = 22-40 cells/3 mice). Data are mean ± SEM;…

• 

  Figure 4. NOD1 activation induces CaMKII-dependent Ca²⁺ mishandling in atrial cardiomyocytes. RNAseq analysis of human atrial myocardium. WT, Nod1⁻/⁻, and RyR2-S2814A mice were used to assess the effects of genetic and pharmacological NOD1 modulation. (A) Representative echocardiographic images (left panel) and LA area (right panel) in WT-Vehicle (WT-Veh) and WT-C12-iE-DAP (WT-iE). (B-E) Representative confocal line-scan images and quantification of Ca²⁺ transients amplitude (C and E, left panel), SR Ca²⁺ load (C and E, central panel), and sparks-mediated diastolic Ca²⁺ leak (D and E, right panel) in WT-Veh (n = 17-47 cells/6 mice), WT-iE (n = 18-26 cells/3 mice), Nod1-/--Veh (n = 13-35 cells/3 mice), Nod1-/--iE (n = 9-10 cells/2 mice), WT-KN (n = 17-27 cells/4 mice), WT-iE-KN (n = 15-18 cells/3 mice), RyR2 S2814A-Veh (n = 12-13 cells/3 mice) and RyR2 S2814A-iE (n = 9-17 cells/3 mice). (F) Transcriptomic gene set enrichment analysis of human atrial tissue showing upregulation of inflammation- and Ca²⁺-related pathways in HF vs. NF patients. * indicates significantly enriched pathways at FDR < 0.05. (G) Representative Western blot (upper panel) and quantification (lower panel) of CaMKII…