The complement C3-complement factor D-C3a receptor signalling axis regulates cardiac remodelling in right ventricular failure

Abstract

Failure of the right ventricle plays a critical role in any type of heart failure. However, the mechanism remains unclear, and there is no specific therapy. Here, we show that the right ventricle predominantly expresses alternative complement pathway-related genes, including Cfd and C3aR1. Complement 3 (C3)-knockout attenuates right ventricular dysfunction and fibrosis in a mouse model of right ventricular failure. C3a is produced from C3 by the C3 convertase complex, which includes the essential component complement factor D (Cfd). Cfd-knockout mice also show attenuation of right ventricular failure. Moreover, the plasma concentration of CFD correlates with the severity of right ventricular failure in patients with chronic right ventricular failure. A C3a receptor (C3aR) antagonist dramatically improves right ventricular dysfunction in mice. In summary, we demonstrate the crucial role of the C3-Cfd-C3aR axis in right ventricular failure and highlight potential therapeutic targets for right ventricular failure.

Fig. 1. Global depletion of complement factor C3 attenuates right ventricular remodelling in pulmonary artery constriction (PAC) mouse model.

a Global gene expression heatmap for differentially expressed genes in the left ventricle (LV), ventricular septum, and right ventricle (RV) (n = 3–4). b, c Ingenuity pathway analysis of differentially expressed genes. Significance of the association between the dataset and the canonical pathway (−log (p-value) and fold change), and the z-score prediction are shown. The significance values are calculated by the right-tailed Fisher’s exact test. d–f mRNA expression of CFD, C3, and C3AR1 in human RV and LV (n = 3, p = 0.0004, p < 0.0001, p = 0.001). Data are presented as mean ± standard error of the mean (SEM). g–i mRNA expression of Cfd, C3, and C3ar1 in sham-operated (n = 4–5, p = 0.0006, p < 0.0001, p = 0.003) and PAC model mice (n = 4–5, p = 0.024, p = 0.1798, p = 0.0207). Data are presented as mean ± SEM. j, k Measured values of the echocardiogram in sham and PAC models of wild type (WT) and C3 knockout (C3KO) mice (n = 3–5, j p = 0.001, p < 0.0001. k p < 0.0001, p = 0.002). The RV contractile function (right ventricular fractional shortening [RV FS]) and RV size (right ventricular end-diastolic diameter [RVDd]) were evaluated. Data are presented as mean ± standard deviation (SD). l Representative immunostaining images for C3d in the RV of PAC model and sham-operated mice (n = 3). m Measured values of the catheter analysis in sham and PAC models of WT and C3KO mice (n = 3, p = 0.0174, p = 0.0059). RV end-diastolic pressure was evaluated. n, o Representative Azan staining images of the heart in WT and C3KO mice with PAC (n = 3). p–s mRNA expression of Nppb, Myh7, Col3a1, and Ccn2 in the LV, ventricular septum, and RV of WT and C3KO mice with PAC (n = 3–4, p = 0.00194, p = 0.0010, p = 0.0029, p = 0.0017). Data are presented as mean ± SEM. mRNA expression of target genes was normalised to that of Gapdh. Significance was assessed using a two-tailed unpaired Student’s t-test. *p < 0.05; **p < 0.01.

Fig. 2. The liver-specific C3 deletion rescues pulmonary artery constriction (PAC)-induced right ventricular dysfunction in mice.

a, b Measured values obtained from the echocardiogram in α-myosin heavy chain promoter-driven Cre (αMHC-Cre) PAC, and albumin promoter-driven Cre (Alb-Cre) PAC, C3 floxed αMHC-Cre (C3^fl/fl αMHC-Cre) PAC, and C3 floxed Alb-Cre (C3^fl/fl Alb-Cre) PAC mice (n = 3, a p = 0.0002, p < 0.0001, b p = 0.0015, p = 0.0147). The right ventricle (RV) contractile function (right ventricular fractional shortening [RV FS]) and RV size (right ventricular end-diastolic diameter [RVDd]) were evaluated. Data are presented as mean ± standard deviation (SD). c Representative images of Azan staining of the heart in C3^fl/fl αMHC-Cre PAC mice (n = 3). d Representative images of Azan staining of the heart in C3^fl/fl Alb-Cre PAC mice. LV, left ventricle (n = 3). e, f Quantified fibrotic area of the RV and LV in C3^fl/fl αMHC-Cre and C3^fl/fl Alb-Cre PAC model mice (n = 3, p = 0.0005, p = 0.039). Data are presented as mean ± SD. g–j qRT-PCR analysis of the expression of heart failure markers (Nppb and Myh7) and fibrotic markers (Col3a1 and Ccn2) in the LV, ventricular septum, and RV of αMHC-Cre PAC, and Alb-Cre PAC, C3^fl/fl αMHC-Cre PAC, and C3^fl/fl Alb-Cre PAC mice (n = 3, g p < 0.0001, p = 0.0003, h p < 0.0001, p < 0.0001, i p < 0.0001, p < 0.0001, j p < 0.0001, p = 0.0012). Data are presented as mean ± standard error of the mean. In qRT-PCR analysis, expression of target genes was normalised to that of Gapdh. Significance was assessed using a two-tailed unpaired Student’s t-test. *p < 0.05; **p < 0.01.

Fig. 3. Complement factor D (Cfd) plays an important role in mice with pulmonary artery constriction (PAC)-induced right ventricular (RV) dysfunction and patients with RV failure.

a, b Measured values obtained from the echocardiogram in wild type (WT) sham, WT PAC mice, and Cfd knockout (Cfd KO) PAC mice (n = 4, 5, 3, p < 0.0001, p = 0.0017). The right ventricle (RV) contractile function (right ventricular fractional shortening [RV FS]) and RV size (right ventricular end-diastolic diameter [RVDd]) were evaluated. Data are presented as mean ± standard deviation (SD). c Representative images of hematoxylin-eosin (HE) staining and Azan staining of the heart in Cfd KO PAC mice. LV, left ventricle (n = 4). d, e Quantified fibrotic area of the RV and LV in WT PAC and Cfd KO PAC model mice (n = 4). Data are presented as mean ± SD. f, g qRT-PCR analysis of the expression of heart failure markers (Nppb and Myh7) in the LV, ventricular septum, and RV of WT sham, WT PAC, and Cfd KO PAC mice (n = 3–5, p = 0.0135, p = 0.0005). Data are presented as mean ± standard error of the mean (SEM). In qRT-PCR analysis, expression of target genes was normalised to that of Gapdh. Significance was assessed using a two-tailed unpaired Student’s t-test. *p < 0.05; **p < 0.01. h Scatter plots showing the correlation between the CFD concentration and B-type natriuretic peptide (BNP) concentration in the overall cohort (n = 129; mean age = 66.5 ± 15.1 years; 69.8% women). Spearman correlation coefficient and two-tailed p-value are shown. Linear regression line (blue line) with 95% confidence intervals (grey area) is represented. i Scatter plots showing the correlation between the CFD concentration and mean pulmonary artery (PA) pressure in the overall cohort (n = 133; mean age = 66.1 ± 15.3 years; 70.7% women). Spearman correlation coefficient and two-tailed p-value are shown. Linear regression line with 95% confidence intervals is represented.

Fig. 4. C3a receptor blockade attenuates right ventricular (RV) failure and ventricular arrhythmia.

a Western blotting of phospho-ERK (pERK), total ERK (ERK), phospho-p38 (pp38), total p38, phospho-JNK (p-JNK), and total JNK (n = 3) in neonatal rat ventricular cardiomyocytes (NRVCs) treated with C3a in a time-dependent manner. Endothelin 1 (ET1) was used as a positive control. b Western blotting of pERK and ERK in the presence of C3ar1 siRNA (n = 3). c Global gene expression heatmap of NRVCs in the presence of C3a (n = 3). d Pathway enrichment analysis using genes differentially expressed under recombinant C3a protein stimulation. e, f Measured values of the echocardiogram in wild type (WT) sham, WT pulmonary artery constriction (PAC), and WT PAC mice treated with SB290157 (n = 4–5, p = 0.007, p = 0.004). The Right ventricular (RV) fractional shortening [RV FS] and RV end-diastolic diameter [RVDd] were evaluated. Data are presented as mean ± standard deviation (SD). g, h Representative Azan staining images of WT PAC mice under placebo or SB290157 treatment (n = 3). LV, left ventricle. i, j Quantified fibrotic area of the RV and LV in WT PAC mice under placebo or SB290157 treatment (n = 3, p = 0.0156, p = 0.6217). Data are presented as mean ± SD. k Representative electrocardiogram showing ventricular tachycardia (VT) after electrical program stimulation (red arrowhead) in WT PAC mice under SB290157 treatment. Black arrowhead shows normal ventricular beats. l, m The percentages of VT induction and VT duration after electrical program stimulation in WT sham, WT PAC, C3 knockout (C3KO) PAC, and WT PAC mice treated with SB290157 (n = 4–9, p < 0.001, p < 0.001, p = 0.044). Data are presented as mean ± SD. n–q Measured values of Ca²⁺ transients in singled cardiomyocytes from RV and LV of sham and PAC models of WT and C3KO mice. Time to peak and Ca²⁺ wave frequency (n = 15–26, n p < 0.001, p = 0.0017, o p = 0.0054, p p = 0.020, p = 0.0197) were evaluated. Data are presented as mean ± standard error of the mean. Significance was assessed using a two-tailed unpaired Student’s t-test. *p < 0.05; **p < 0.01.