TRPC channels blockade abolishes endotoxemic cardiac dysfunction by hampering intracellular inflammation and Ca2+ leakage

Abstract

Intracellular Ca²⁺ dysregulation is a key marker in septic cardiac dysfunction; however, regulation of the classic Ca²⁺ regulatory modules cannot successfully abolish this symptom. Here we show that the knockout of transient receptor potential canonical (TRPC) channel isoforms TRPC1 and TRPC6 can ameliorate LPS-challenged heart failure and prolong survival in mice. The LPS-triggered Ca²⁺ release from the endoplasmic reticulum both in cardiomyocytes and macrophages is significantly inhibited by Trpc1 or Trpc6 knockout. Meanwhile, TRPC's molecular partner - calmodulin - is uncoupled during Trpc1 or Trpc6 deficiency and binds to TLR4's Pococurante site and atypical isoleucine-glutamine-like motif to block the inflammation cascade. Blocking the C-terminal CaM/IP3R binding domain in TRPC with chemical inhibitor could obstruct the Ca²⁺ leak and TLR4-mediated inflammation burst, demonstrating a cardioprotective effect in endotoxemia and polymicrobial sepsis. Our findings provide insight into the pathogenesis of endotoxemic cardiac dysfunction and suggest a novel approach for its treatment.

Fig. 1. The Trpc1 or Trpc6 knockout protects endotoxemic hearts.

a The protein expressions of TRPCs in the ventricles of LPS-challenged mice (pooled tissues from 3 male mice per sample, n = 3 biological independent experiments). b Kaplan-Meier survival curves of the WT, Trpc1^−/−, and Trpc6^−/− mice (n = 10 male mice per group). Statistical significance was determined using the log-rank test. Exact P value = 6.5 × 10⁻⁵ (WT + LPS vs Trpc1^−/− + LPS), 9.1 × 10⁻⁵ (WT + LPS vs Trpc6^−/− + LPS). c The mean arterial blood pressure (MAP) and heart rate of the LPS-challenged WT, Trpc1^−/−, and Trpc6^−/− mice during 6 h (mean ± SEM, n = 8 male mice per group). d Representative M-mode echocardiography still and the statistical analysis of ejection fraction in WT, Trpc1^−/−, and Trpc6^−/− mice 6 h after LPS challenge (mean ± SEM, n = 6 male mice per group). Statistical significance was determined using the one-way ANOVA with Tukey’s multiple comparisons test. Exact P value = 8.4 × 10⁻⁵ (WT + LPS vs Trpc1^−/− + LPS), 1.3 × 10⁻⁵ (WT + LPS vs Trpc6^−/− + LPS). e Representative photomicrographs of ventricular tissues stained with hematoxylin and eosin. Black arrows indicate the myocardial interstitial edema associated with the mononuclear inflammatory cells infiltration (n = 6 images from 3 male mice per group). f The levels of serum cardiac troponin-T, troponin-I, and creatine kinase-MB in the mice challenged with LPS (mean ± SEM, n = 6 male mice samples per group). Statistical significance was determined using the one-way ANOVA with Tukey’s multiple comparisons test. Troponin-I, exact P value = 7.8 × 10⁻⁹ (WT + LPS vs Trpc1^−/− + LPS), 4.5 × 10⁻⁷ (WT + LPS vs Trpc6^−/− + LPS). Troponin-T, exact P value = 9.3 × 10⁻⁷ (WT + LPS vs Trpc1^−/− + LPS), 3.8 × 10⁻⁷ (WT + LPS vs Trpc6^−/− + LPS). Creatine kinase-MB, exact P value = 1.9 × 10⁻¹⁰ (WT + LPS vs Trpc1^−/− + LPS), 2.7 × 10⁻⁹ (WT + LPS vs Trpc6^−/− + LPS). g The effects of TRPC1 and TRPC6 on IP3Rs and RyRs-regulated Ca²⁺ release in LPS-induced mice cardiomyocytes. Neonatal and adult mice cardiomyocytes in Ca²⁺-free extracellular solution were pre-incubated with ryanodine (Rya.), tetracaine (Tet.), and/or low molecular weight heparin (LMWH), and then treated with LPS to measure [Ca²⁺]_i levels using a digital wide-field fluorescence imaging system. Typical trace recordings (left panel) and the statistical analysis (right panel) are shown (mean ± SEM, n = 15–20 cells from 3 mice per group). Statistical significance was determined using the one-way ANOVA with Games Howell’s multiple comparisons test. In neonatal mice cardiomyocytes, exact P value = 8.6 × 10⁻¹² (WT vs Trpc1^−/−), 1.5 × 10⁻¹² (WT vs WT + LMWH), 9.5 × 10⁻¹³ (WT vs WT + Rya.), 9.0 × 10⁻¹³ (WT vs WT + Tet.), 3.0 × 10⁻⁶ (WT + Rya. vs Trpc1^−/− + Rya.), 4.1 × 10⁻⁸ (WT + Rya. vs Trpc1^−/− + Rya. + LMWH), 6.1 × 10⁻⁸ (WT + Tet. vs Trpc1^−/− + Tet.), 9.8 × 10⁻⁹ (WT + Tet. vs Trpc1^−/− + Tet. + LMWH), 8.0 × 10⁻⁶ (WT vs Trpc6^−/−), 1.3 × 10⁻¹² (WT vs WT + LMWH), 9.0 × 10⁻¹³ (WT vs WT + Rya.), 8.7 × 10⁻¹³ (WT vs WT + Tet.), 3.0 × 10⁻⁶ (WT + Rya. vs Trpc6^−/− + Rya.), 4.3 × 10⁻⁹ (WT + Rya. vs Trpc6^−/−+Rya.+LMWH), 8.6 × 10⁻⁵ (WT + Tet. vs Trpc6^−/−+Tet.), and 3.2 × 10⁻⁸ (WT + Tet. vs Trpc6^−/−+Tet.+LMWH). In adult mice cardiomyocytes, exact P value = 4.1 × 10⁻¹¹ (WT vs Trpc1^−/−), 1.1 × 10⁻¹² (WT vs WT + LMWH), 8.5 × 10⁻¹³ (WT vs WT + Rya.), 9.4 × 10⁻¹³ (WT vs WT + Tet.), 2.7 × 10⁻⁵ (WT + Rya. vs Trpc1^−/−+Rya.), 5.2 × 10⁻⁷ (WT + Rya. vs Trpc1^−/−+Rya.+LMWH), 1.2 × 10⁻⁷ (WT + Tet. vs Trpc1^−/−+Tet.), 7.8 × 10⁻¹⁰ (WT + Tet. vs Trpc1^−/−+Tet.+LMWH), 1.3 × 10⁻⁵ (WT vs Trpc6^−/−), 1.2 × 10⁻¹² (WT vs WT + LMWH), 8.9 × 10⁻¹³ (WT vs WT + Rya.), 1.1 × 10⁻¹² (WT vs WT + Tet.), 8.3 × 10⁻⁵ (WT + Rya. vs Trpc6^−/−+Rya.), 8.5 × 10⁻⁹ (WT + Rya. vs Trpc6^−/−+Rya.+LMWH), 2.7 × 10⁻⁷ (WT + Tet. vs Trpc6^−/−+Tet.), and 1.5 × 10⁻¹⁰ (WT + Tet. vs Trpc6^−/−+Tet.+LMWH). Source data are provided as a Source Data file.

Fig. 2. The Trpc1 or Trpc6 knockout inhibits TLR signaling pathway.

a Pearson’s correlation test of all the detected genes of RNA-seq in the LPS-challenged mice compared to control. b Volcano plot of the changed genes in RNA-seq between Trpc1^−/− and Trpc6^−/− mice after LPS challenge. c Counting pie charts depicting the top-ranked biological process classification of the differentially expressed genes in RNA-seq using Gene Ontology terms. d The top 20 down-regulated pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database of LPS-challenged Trpc1^−/− or Trpc6^−/− mice compared to the WT mice. e The heatmap of the genes in the TLR signaling pathway from the KEGG database based on RNA-seq analysis. The values from RNA-seq in a–e are obtained from 3 male mice per group. f Serum levels of TNF-α, IFN-β, IL-6, and IL-1β in mice 6 h after LPS challenge (mean ± SEM, n = 6 male mice samples per group). Statistical significance was determined using the one-way ANOVA with Tukey’s multiple comparisons test. TNF-α, exact P value = 2.4 × 10⁻¹² (WT + LPS vs Trpc1^−/−+LPS), 3.7 × 10⁻¹¹ (WT + LPS vs Trpc6^−/−+LPS); IFN-β, exact P value = 9.9 × 10⁻¹³ (WT + LPS vs Trpc1^−/−+LPS), 3.1 × 10⁻¹² (WT + LPS vs Trpc6^−/−+LPS); IL-6, exact P value = 5.4 × 10⁻¹¹ (WT + LPS vs Trpc1^−/−+LPS), 8.3 × 10⁻¹³ (WT + LPS vs Trpc6^−/−+LPS); IL-1β, exact P value = 8.0 × 10⁻⁸ (WT + LPS vs Trpc1^−/−+LPS), 3.4 × 10⁻⁵ (WT + LPS vs Trpc6^−/−+LPS). g Representative photomicrographs (left panel) and quantitative data (right panel) of MIP-1α immunohistochemical staining on ventricular tissues. Arrows show MIP-1α positive cells (mean ± SEM, n = 12 images from 4 male mice per group). Statistical significance was determined using the one-way ANOVA with Tukey’s multiple comparisons test. Source data are provided as a Source Data file.

Fig. 3. TRPC1 and TRPC6 associate with TLR4 and Ca²⁺ signaling pathways.

a-b The effects of Trpc1 or Trpc6 knockout on nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPK) signaling pathways in the hearts of mice 4 h after LPS challenge (pooled tissues from 3 male mice per sample, n = 3 biological independent experiments). c The effects of Trpc1 or Trpc6 knockout on Toll like receptor 4 (TLR4)‐mediated myeloid differentiation primary response protein 88 (MyD88)- and TIR domain-containing adaptor inducing IFN-β (TRIF)-dependent signaling pathways in the hearts of mice 4 h after LPS challenge (pooled tissues from 3 male mice per sample, n = 3 biological independent experiments). d Heatmap depicting the genes involved in the Ca²⁺ signaling pathway from the KEGG pathway database based on RNA-seq analysis (n = 3 male mice per group). e-f The effects of Trpc1 or Trpc6 deletion on Calm2 mRNA (n = 3 male mice with triplicate measurements taken, mean ± SEM) and calmodulin (CaM) protein expressions (pooled tissues from 3 male mice per sample, n = 3 biological independent experiments) in the hearts of mice 4 h after LPS challenge. g-h, The activity of calcineurin (mean ± SEM, n = 6 male mice samples per group) and NFAT3 nuclear translocation (pooled tissues from 3 male mice per sample, n = 3 biological independent experiments) in the hearts of mice 4 h after LPS challenge. Statistical significance was determined using the one-way ANOVA with Tukey’s multiple comparisons test. Source data are provided as a Source Data file.

Fig. 4. CaM interacts with TLR4 in Trpc1^−/− or Trpc6^−/− hearts.

a Co-immunoprecipitated (Co-IP) analysis of TRPC1 or TRPC6 interaction with CaM in the hearts of mice 4 h after LPS challenge (pooled tissues from 3 male mice per sample, n = 2 biological independent experiments). b Co-IP analysis of CaM interaction with TLR4 and its adaptor proteins in the hearts of WT, Trpc1^−/−, and Trpc6^−/− mice 4 h after LPS challenge (pooled tissues from 3 male mice per sample, n = 2 biological independent experiments). c The protein interactions between recombinant human CALM2 protein and TLR4, MyD88, or TRIF-related adaptor molecule (TRAM) using microscale thermophoresis (MST) assay (mean ± SEM, n = 3 biological independent experiments). d Immunofluorescence microscopy analysis of CaM and TLR4 in adult mice cardiomyocytes (n = 6 images from 3 biological independent experiments). Source data are provided as a Source Data file.

Fig. 5. CaM interacts with TLR4 to inhibit the inflammation cascades.

a The effects of CALP1 (20 μM) pretreatment on the interaction between CaM and TLR4 in the LPS-stimulated neonatal mice cardiomyocytes (n = 2 biological independent experiments). b The effects of CALP1 on phosphorylated ERK, JNK, and P38 MAPK expressions in the LPS-stimulated neonatal mice cardiomyocytes (n = 3 biological independent experiments). c-d The effects of CaM inhibitor W-7 (30 μM) pretreatment on the interaction of CaM and TLR4, and MAPK activation in neonatal mice cardiomyocytes stimulated with LPS for 4 h (n = 2 biological independent experiments for c and n = 3 biological independent experiments for d). e The effects of W-7 pretreatment on MAPK activation in mice bone marrow-derived macrophages stimulated with LPS for 4 h (n = 3 biological independent experiments). f–h The effects of W-7 pretreatment on TNF-α and IFN-β levels in neonatal mice cardiomyocytes and macrophages stimulated with LPS for 6 h (mean ± SEM, n = 6 samples per group). Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test. IFN-β in f, exact P value = 8.6 × 10⁻⁹ (Trpc1^−/−+LPS vs Trpc1^−/−+LPS + W-7) and 5.3 × 10⁻¹⁰ (Trpc6^−/−+LPS vs Trpc6^−/−+LPS + W-7); IFN-β in g, exact P value = 6.7 × 10⁻⁹ (WT + LPS vs WT + LPS + W-7) and 7.5 × 10^-5 (Trpc6^−/−+LPS vs Trpc6^−/−+LPS + W-7). Source data are provided as a Source Data file.

Fig. 6. CaM binds to both the Poc site and the atypical IQ motif of TLR4.

a The interacted domain of TLR4 (Myc-tagged) with CaM (FLAG-tagged) in co-transfected HEK293T cells measured using Co-IP (n = 2 biological independent experiments). b Representative immunofluorescent photomicrographs (left panel) and traces of fluorescence intensity spatial profiles (right panel) of TLR4 (Myc-tagged) with CaM (FLAG-tagged) localization in co-transfected HEK293T cells (n = 6 images from 3 biological independent experiments). c The CD spectroscopies of the synthesized peptides. d The interaction of CaM with the peptides encompassing nonclassical IQ motifs detected by a non-denaturing gel (n = 2 biological independent experiments). e The dissociation constants of CaM and peptide TLIQ2 measured using MST (mean ± SEM, n = 3 biological independent experiments). f Structural model of signaling complex formed by TLR4 and CaM. Secondary structure elements and position of mutational sites in the human TLR4 sequence (upper panel) and a homologous modeling of TLR4 (TLR2, PDB 1Fyx) and TLIQ2 contacting with CaM (PDB 1QX5) (lower panel) are shown. Source data are provided as a Source Data file.

Fig. 7. The pleiotropic roles of TRPCs in regulating CaM and IP3R1.

a The Co-IP analysis of TRPC1 or TRPC6 binding with IP3R1 in mice heart tissues (pooled tissues from 3 male mice per sample, n = 2 biological independent experiments). b The interaction between CaM and IP3R1 in the hearts of WT, Trpc1^−/−, and Trpc6^−/− mice 4 h after LPS challenge (pooled tissues from 3 male mice per sample, n = 2 biological independent experiments). c TRPC1-IP3R1 and TRPC6-IP3R1 interactions in LPS-challenged neonatal WT mice cardiomyocytes. Representative PLA photomicrographs (left panel) and the statistical analysis (right panel) are shown (mean ± SEM, n = 6 images from 3 biological independent experiments). Statistical significance was determined using the two-tailed Student’s t-test. Exact P value = 2.0 × 10⁻⁵ (Trpc1^−/− vs Trpc1^−/−+LPS) and 3.5 × 10⁻⁵ (Trpc6^−/− vs Trpc6^−/−+LPS). d IP3R1-CaM interactions in LPS-challenged neonatal WT, Trpc1^−/−, and Trpc6^−/− mice cardiomyocytes. Representative PLA photomicrographs (upper panel) and the statistical analysis (lower panel) are shown (mean ± SEM, n = 6 images from 3 biological independent experiments). Statistical significance was determined using the one-way ANOVA with Tukey’s multiple comparisons test. Exact P value = 8.3 × 10⁻¹³ (WT + LPS vs Trpc1^−/−+LPS) and 9.6 × 10⁻¹³ (WT + LPS vs Trpc6^−/−+LPS). e Representative immunofluorescent photomicrographs of TRPC1, CaM, IP3R1, and PDI (upper panel) and traces of fluorescence intensity spatial profiles of IP3R1, TRPC1, and PDI (lower panel) in LPS-stimulated neonatal mice cardiomyocytes (n = 6 images from 3 biological independent experiments). f The effects of W-7 on the LPS-triggered intracellular Ca²⁺ influx in adult mice cardiomyocytes (left panel) and mice bone marrow-derived macrophages (right panel) are shown (mean ± SEM, n = 15–20 cells from 3 male mice per group). Statistical significance was determined using the one-way ANOVA with Game Howell’s multiple comparisons test. In cardiomyocytes, exact P value = 8.4 × 10⁻¹⁰ (Trpc1^−/− vs Trpc1^−/−+W-7) and 1.3 × 10⁻⁸ (Trpc6^−/− vs Trpc6^−/−+W-7. Source data are provided as a Source Data file.

Fig. 8. SKF96365 (SKF) blocks TRPC to obstruct the Ca²⁺ release and TLR4-mediated inflammation burst.

a Structural insights into the SKF binding pocket in human TRPC6 (PDB code 5YX9) and its interaction with the CIRB domain of TRPC6. b The interaction between the C-terminal TRPC1 fusion protein and potential inhibitors, SKF, Larixyl acetate (LA), Pyr10 (Pyr), and BI-749327 (BI), measured by MST assay (mean ± SEM, n = 3 biological independent experiments). c The effects of TRPC inhibitors on TNF-α and IFN-β productions in LPS-stimulated neonatal mice cardiomyocytes (mean ± SEM, n = 6 samples per group). Statistical significance was determined using the one-way ANOVA with Tukey’s multiple comparisons test. TNF-α, exact P value = 4.1 × 10⁻¹² (WT vs WT + LPS), 4.1 × 10⁻¹² (WT + LPS vs LPS + SKF 20 μM), 4.1 × 10⁻¹² (WT + LPS vs LPS + SKF 30 μM), 1.4 × 10⁻⁵ (WT + LPS vs LPS + LA 30 μM), 7.5 × 10⁻¹² (WT + LPS vs LPS + Pyr 25 μM), and 8.0 × 10⁻⁸ (WT + LPS vs LPS + BI 30 μM); IFN-β, exact P value = 4.1 × 10⁻¹² (WT vs WT + LPS), 4.1 × 10⁻¹² (WT + LPS vs LPS + SKF 20 μM), 4.1 × 10⁻¹² (WT + LPS vs LPS + SKF 30 μM), 6.7 × 10⁻⁵ (WT + LPS vs LPS + LA 30 μM), 3.1 × 10⁻⁸ (WT + LPS vs LPS + Pyr 25 μM), and 8.0 × 10⁻⁶ (WT + LPS vs LPS + BI 30 μM). d SKF inhibits the LPS-triggered intracellular Ca²⁺ release in adult mice cardiomyocytes in Ca²⁺-containing extracellular solution (mean ± SEM, n = 15–20 cells from 3 male mice per group). e The effects of SKF on the interactions between TRPC1 and CaM/IP3R1 and between CaM and TLR4/IP3R1 in LPS-challenged neonatal mice cardiomyocytes (n = 2 biological independent experiments). Source data are provided as a Source Data file.

Fig. 9. SKF cures septic cardiac dysfunction.

a Representative M-mode echocardiography still and the statistical analysis of ejection fraction in vehicle- or SKF-treated mice at 6 h after LPS challenge (mean ± SEM, n = 6 male mice per group). Statistical significance was determined using the one-way ANOVA with Tukey’s multiple comparisons test. Exact P value = 4.5 × 10⁻⁷ (LPS vs LPS + SKF 10 mg/kg) and 2.9 × 10⁻¹⁰ (LPS vs LPS + SKF 20 mg/kg). b SKF treatment on the serum levels of TNF-α and IFN-β in LPS-challenged mice (mean ± SEM, n = 6 male mice samples per group). Statistical significance was determined using the one-way ANOVA with Tukey’s multiple comparisons test. TNF-α, exact P value = 2.0 × 10⁻⁶ (LPS vs LPS + SKF 10 mg/kg) and 1.2 × 10⁻⁹ (LPS vs LPS + SKF 20 mg/kg); IFN-β, exact P value = 8.7 × 10⁻⁵ (LPS vs LPS + SKF 5 mg/kg), 1.0 × 10⁻¹² (LPS vs LPS + SKF 10 mg/kg), and 8.3 × 10⁻¹³ (LPS vs LPS + SKF 20 mg/kg). c SKF treatment on the serum markers of myocardial damage in LPS-challenged mice (mean ± SEM, n = 6 male mice samples per group). Statistical significance was determined using the one-way ANOVA with Tukey’s multiple comparisons test. Troponin-I, exact P value = 7.0 × 10⁻⁶ (LPS vs LPS + SKF 20 mg/kg); Troponin-T, exact P value = 1.0 × 10⁻⁶ (LPS vs LPS + SKF 20 mg/kg). d Kaplan-Meier survival curves of single-dose SKF treatment (n = 10 male mice per group). e Survival curves of 10 mg/kg SKF multiple-dosing (every 12 h, n = 10 male mice per group). Statistical significances in d and e were determined using the log-rank test. Exact P value = 3.0 × 10⁻⁶ (SKF vs LPS). f Echocardiographic assessment of left ventricular function of SKF-treated mice at 6 h after CLP surgery. Typical heart M-mode echocardiography still (left), ejection fraction (right panel) are shown (mean ± SEM, n = 6 male mice per group). Statistical significance was determined using the one-way ANOVA with Game Howell’s multiple comparisons test. Exact P value = 5.7 × 10⁻⁵ (WT vs CLP) and 7.0 × 10⁻⁶ (CLP vs CLP + SKF). g The serum levels of TNF-α and IFN-β in the SKF-treated CLP mice (mean ± SEM, n = 6 male mice samples per group). Statistical significance was determined using the one-way ANOVA with Tukey’s multiple comparisons test. TNF-α, exact P value = 5.8 × 10⁻⁹ (WT vs CLP) and 1.2 × 10⁻⁵ (CLP vs CLP + SKF); IFN-β, exact P value = 5.8 × 10⁻⁹ (WT vs CLP) and 2.3 × 10⁻⁷ (CLP vs CLP + SKF). h SKF treatment on the serum markers of myocardial damage in CLP mice (mean ± SEM, n = 6 male mice samples per group). Statistical significance was determined using the one-way ANOVA with Tukey’s multiple comparisons test. Troponin-I, exact P value = 5.8 × 10⁻⁹ (WT vs CLP) and 1.3 × 10⁻⁷ (CLP vs CLP + SKF); Troponin-T, exact P value = 5.8 × 10⁻⁹ (WT vs CLP) and 2.5 × 10⁻⁸ (CLP vs CLP + SKF); Creatine kinase-MB, exact P value = 5.8 × 10⁻⁹ (WT vs CLP) and 2.2 × 10⁻⁷ (CLP vs CLP + SKF). i Kaplan-Meier survival curves of multiple-dosing SKF treatment on CLP mice (n = 10 male mice per group). Statistical significance was determined using the log-rank test. Exact P value = 3.0 × 10⁻⁶ (WT vs CLP). Source data are provided as a Source Data file.