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Paracrine signalling by cardiac calcitonin controls atrial fibrogenesis and arrhythmia

Abstract

Atrial fibrillation, the most common cardiac arrhythmia, is an important contributor to mortality and morbidity, and particularly to the risk of stroke in humans1. Atrial-tissue fibrosis is a central pathophysiological feature of atrial fibrillation that also hampers its treatment; the underlying molecular mechanisms are poorly understood and warrant investigation given the inadequacy of present therapies2. Here we show that calcitonin, a hormone product of the thyroid gland involved in bone metabolism3, is also produced by atrial cardiomyocytes in substantial quantities and acts as a paracrine signal that affects neighbouring collagen-producing fibroblasts to control their proliferation and secretion of extracellular matrix proteins. Global disruption of calcitonin receptor signalling in mice causes atrial fibrosis and increases susceptibility to atrial fibrillation. In mice in which liver kinase B1 is knocked down specifically in the atria, atrial-specific knockdown of calcitonin promotes atrial fibrosis and increases and prolongs spontaneous episodes of atrial fibrillation, whereas atrial-specific overexpression of calcitonin prevents both atrial fibrosis and fibrillation. Human patients with persistent atrial fibrillation show sixfold lower levels of myocardial calcitonin compared to control individuals with normal heart rhythm, with loss of calcitonin receptors in the fibroblast membrane. Although transcriptome analysis of human atrial fibroblasts reveals little change after exposure to calcitonin, proteomic analysis shows extensive alterations in extracellular matrix proteins and pathways related to fibrogenesis, infection and immune responses, and transcriptional regulation. Strategies to restore disrupted myocardial calcitonin signalling thus may offer therapeutic avenues for patients with atrial fibrillation.

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Fig. 1: Myocardial production of CT.
Fig. 2: CT regulates ACFs.
Fig. 3: CT and physiology of ACFs.
Fig. 4: CT–CTR signalling, atrial fibrosis and AF inducibility.

Data availability

All data generated or analysed during this study are included in the Article and its Supplementary Information. The scRNA-seq data are deposited in the GEO database (accession codes GSE148506, GSE148507 and GSE148504). Source data are provided with this paper.

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Acknowledgements

We thank the Oxford Genomics Centre at the Wellcome Centre for Human Genetics (funded by Wellcome Trust grant reference 203141/Z/16/Z) for the generation and initial processing of the ACF microarrays data; M. Farrall for the help with statistics; K. Clark in the WIMM Flow Cytometry Facility for his help; J. Digby for assistance with analysis and detection of CT by ELISA in human ACFs and ACMs; C. St-Cyr for managing, handling and genotyping mouse colonies at the Montreal site; R. Hiram for initial help with EP analysis in mice; J. Dewing for creating an artistic sketch summary of the main findings; L. E. Schmidt and X. Yin for help with the proteomic experiments; S. Farid and V. Srivastava for help with collection of some human atrial specimens during revision; P. Wookey for advice on CTR protein detection; R. Wijesurendra and P. Gajendragadkar for initial help in obtaining patient consent for the study; and A. Recalde and M. C. Carena for initial help with optimizing the fibroblast isolation protocol. Funded by the British Heart Foundation (BHF) Intermediate Fellowship in Basic Science, the Oxford BHF Centre of Research Excellence (CRE; RG/13/1/30181) Transitional Fellowship, a BHF CRE Overseas Collaboration Travel award, the Medical Science Division Internal Fund, the Wellcome Trust Institutional Strategic Support Fund, the Oxfordshire Health Services Research Committee, the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre and LAB282 grants (to S.R.); BHF Chair award CH/16/1/32013 (to K.M.C.); the Canadian Institutes of Health Research (CIHR) and Heart and Stroke Foundation of Canada (to S.N.); and Fonds de Recherche en Santé de Québec (FRQS) and CIHR postdoctoral fellowships to A.T.

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Authors

Contributions

S.R. and S.N. conceived the study, designed the experiments and wrote and edited the manuscript. L.M.M. and A.T. wrote parts of the manuscript and carried out and analysed most of the experimental work. K.M.C. provided intellectual input on experiments using clinical samples. N.E., P.R., A.J.S. and C.R. performed some PCR, ELISAs and HPA measurements in human samples. Imaging and analysis of the CTR cellular localization in human ACFs and HEK293 cells were performed by D.A.M. and L.M.M. All in vivo work in mice was carried out by A.T., M.H. and S.K.L., supervised by X.H., T.W. and S.N. Staining, imaging and analysis of fibrosis in mouse heart sections were done by A.T., M.H., S.K.L. and C.P.; M.S. provided full access and supervision of the histological and imaging experiments at the Montreal site. Primer design and gene expression assays in mice were carried out by P.N. and M.H.; primer design for human transcripts was performed by N.E., L.M.M. and C.P. Functional electrophysiological studies in CTR-KO mice were carried out by M.-A.G. and A.T., supervised by S.N.; echocardiography in CTR-KO mice was supported by J.C.T. The CTR-KO mice were generated and provided by J.D.Z. and R.A.D., who also supervised mouse tissue collection at the Melbourne site, genotyping and analysis of the selected morphologic parameters carried out by M.V.C. and P.K.R. in Melbourne. All experimental work and data analysis in the LKB1-based mice was carried out by M.H. and S.K.L., supervised by X.H. and T.W. Experiments in human cells were done by L.M.M., N.M. and N.E. The proteomic study was designed, executed and analysed by J.B.B. and K.T. under the supervision of M.M. Transcriptome profiling by scRNA-seq, performed by N.A., L.M.M. and N.M., was designed and supervised by A.M. and S.R. Bioinformatics analysis of scRNA-seq results was carried out by A.A., supervised by A.J.S. Scar-in-a-jar assay and analysis was carried out by A.L., supervised by S.B. Patient consent was obtained by N.M., L.M.M. and M.N.; human atrial biopsies were collected by cardiac surgeons R.S. and G.K. under ethical approval granted to B.C. and S.R. All authors discussed the results and had the opportunity to comment on the manuscript.

Corresponding author

Correspondence to Svetlana Reilly.

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The authors declare no competing interests.

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Peer review information Nature thanks Igor R. Efimov, Jeffery D. Molkentin, Andrew F. Russo and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Effects of human αCGRP on human ACF function.

a, Secretion of αCGRP (ELISA) by human ACMs vs TT cells. b, αCGRP protein (immunoblot) in human right atrial tissue lysates obtained from patients with SR or AF. ce, mRNA of human ACM CT, αCGRP or CT/αCGRP ratio between SR and AF groups. f, Correlation between donors’ age and ACM-CT secretion (ELISA over 4–6 h); 95% CI = –0.7912 to 0.01258, R = –0.4862, R2 = 0.236, P = 0.056 by Pearson correlation test. gi, Human atrial myocardium (g) expresses CTR isoform 1a, but not 1b (PCR using specific isoform primers) and CTR protein (h; TT cells, positive control; see Extended Data Fig. 8a); CTR protein content in ACFs shown in i. j, Representative traces (real-time impedance assay) showing CT-induced concentration-dependent increase of the baseline-normalized cell index (CI). k, Total and phosphorylated ERK were not altered by CT (immunoblotting). l, m, CTR mRNA (qRT–PCR) and protein content (immunoblotting) were decreased in human ACFs with CTR knockdown due to LNA antisense oligonucleotides (designs LNA-aCTR1 and LNA-aCTR2); fc, fold change versus the CTR-NC control. n, Effect of 10 and 100 nM αCGRP on 72-h collagen accumulation (by Sirius red) in ACF secretomes. oq, Effect of 500 nM CT in human ACFs stimulated with TGFβ1 (10 ng/ml) on cell migration (o; fc, fold change versus vehicle at 0 h), collagen content in conditioned medium (p) and cell proliferation (q). r, Effect of 10 nM αCGRP on 72-h collagen 1 accumulation in human ACF cell lysates and secretomes; representative blots (left) and quantification (right); n, individual participants; fc, fold change versus control. Data are presented as mean ± s.e.m., except in a, b (pro-αCGRP), ce, k, m, panels 2, 3 of r (medians and interquartile ranges), n (mean with paired scattered dots), o (mean ± s.d.). P values were determined by two-sided tests: unpaired t-test (b, αCGRP, pq), Mann–Whitney U-test (b, Pro-αCGRP, c, e), Friedman test (n, panels 2, 3 of r), Kruskal–Wallis test with Dunn’s correction (a, k) and repeated-measures one-way ANOVA with Sidak correction (l, o, panels 1, 4 of r). Data are pooled from individual donors assessed in single replicates (a, b, f, gk, m, or) or duplicates (ce, l, n); all results were reproduced independently twice. For gel source data, see Supplementary Fig. 1. Source data

Extended Data Fig. 2 Effect of CT on collagen 1 processing and single-cell transcriptome (10× scRNA-seq) of cultured human ACFs.

ad, Effect of CT on synthesis of collagen 1 (a) and 3 (b) (by qRT–PCR) and on extracellular (c) and intracellular (d) content of collagen 1 C-terminal telopeptide (ICTP). e, Representative blots (left) and quantification (right) of unprocessed (pro-Col, pro-collagen; pc-Col, pC collagen) and processed collagen 1 (Col 1) in human ACFs treated with 100 nM CT (fc, fold change versus vehicle). fh, Effect of exogenous CT on bone morphogenetic protein 1 (f; BMP1, immunoblotting), BPM1 gene expression (g; qRT–PCR) and BMP1 activity (h) in the presence or absence of BMP1 inhibitor (BMP1 inh; RFU, relative fluorescence units). i, j, Effect of 24-h 500 nM CT on collagen-1 (Col1A1) mRNA (qRT–PCR) and C-terminal telopeptide (ICTP by ELISA). Data are mean ± s.e.m., except in b, g, j (medians with interquartile ranges); n, individual participants. Two-sided tests: unpaired t-test (a, cf, i), Mann–Whitney U-test (b, g, j) and one-way ANOVA with Sidak correction (h). Data are pooled from individual donor cells assessed in single replicates, or duplicates in a, b, g, i, on the same day in one batch. Results were reproduced twice (ac, fh) in different donors. For gel source data, see Supplementary Fig. 1. kn, Unbiased transcriptional clustering of scRNA-seq data from human ACFs cultured with 100-nM CT for 24 h or vehicle; demultiplexed by final cell count per hash-tag in (k), transcriptional clusters in (l), pharmacological intervention in (m) and by each donor in (n); D1–D6 indicate individual donors. Active cycling cells are pointed by arrow. All data are colour-coded within the figure. Data are pooled from 6 individual donors in SR assessed in 14,742 cells (after quality control after filtering the initial 18,466 total cellular barcodes) on the same day in one batch. tSNE, t-distributed stochastic neighbour embedding; UMAP, Uniform Manifold Approximation and Projection. Source data for kn have been deposited in the GEO database. Source data

Extended Data Fig. 3 CTR expression and CT-mediated changes in ACF.

ad, Effects of 72-h treatment with 100 nM CT on IGF-II, CCL2, CTGF and TNFα in human ACF conditioned medium. Data are pooled from individual donor cells assessed on the same day in technical duplicates, repeated twice; n, individual donors. P values were calculated by two-sided tests: paired t-test (ac) and Wilcoxon test (d). e, GO enrichment analysis (David 6.8 web-tool) of the differentially expressed ACF proteins under the above GO terms stratified by adjusted P values. The bold number next to each GO term represents the number of genes under each term. The original data used for this analysis were pooled from 6 individual donors treated with vehicle or 72-h 100 nM CT assessed in single replicates on the same day in one batch. fk, Representative blots of the CTR protein (f, h, j) and gene expression (qPCR, g, i, k) in human ACFs from patients with AF or SR. ln, Effects of CT treatment of ACFs with persistent AF on fibronectin (l), α-SMA protein (m) and cell migration (n) by scratch wound assay (fc, fold change). Data are mean ± s.e.m., except in l, n (medians with interquartile ranges), ad (means and linked paired samples); n, individual donors. P values were determined by two-sided tests: paired t-test (ac), unpaired t-test (fk, m), Wilcoxon test (d), Mann–Whitney U-test (n), and Kruskal–Wallis test with Dunn’s correction (l). Data were pooled from individual donors (l) or separate days (m, n) and are assessed in single replicates on the same day in one batch apart from n (single replicates on two different days), or in duplicates in assessed on the same day (g, i, k). Findings in ad, j were validated by another method (Fig. 3a, b, g, Extended Data Fig. 6b). All except e were reproduced twice in cells from different donors. For gel source data, see Supplementary Fig. 1. Source data

Extended Data Fig. 4 Single-cell transcriptome of freshly isolated human ACFs (scRNA-seq SMART-seq2).

a, b, Transcriptional clustering (a) of freshly isolated human ACFs stratified by donors in b labelled on the graph as SR1–SR4 or AF1–AF4. cf, Differentially expressed genes (DEGs) in transcriptional cluster 1 (c, d, f) and volcano plots for clusters 2–5 (e; also see Source Data for cf). P values for DEGs were calculated by a log-likelihood ratio test on a hurdle model (MAST framework tool) and corrected for multiple testing using Benjamini–Hochberg correction (see Supplementary Methods sections 1.12 and 1.16). Data are pooled from 268 single cells isolated from 8 individual donors; scRNA-seq workflow was performed on the same day in one batch.

Extended Data Fig. 5 Cluster comparison of single-cell transcriptomes (SMART-seq2) of freshly isolated human ACFs.

a, Transcriptional clustering of human ACFs (after quality control) pooled from 4 individual donors in SR and 4 individual donors in AF; figure shows the top 10 most abundant genes in each cluster. b, Gene Ontology (GO) functional enrichment analysis for human ACF transcriptional clusters. The number of significantly enriched genes is shown within the figure. The P values for GO panels are generated from a hypergeometric distribution with a Benjamini–Hochberg correction. The original data were pooled from 268 single cells isolated from 8 individual donors; scRNA-seq workflow was carried out on the same day in one batch.

Extended Data Fig. 6 Protein profiling of the selected CT–CTR downstream targets.

a, Representative blots showing atrial protein content of BMP1, PKA subunit C (PKAC), PKA subunit R2 (PKAR2), EPAC2, EPAC1, CREB and cAMP in AF (4 individual donors) versus 5 individual control donors in the SR group. All proteins but CREB were assessed in the same membrane after protein stripping; all proteins are normalized to GAPDH and expressed as fold of SR control (fc); the red dotted line indicates y axis value of 1; n, individual donors. Data are presented as medians with interquartile ranges. P values were determined by two-sided Mann–Whitney U-test between the SR and AF groups for each protein. Data are pooled from individual donors assessed in single replicate on the same day; results were reproduced in the same donors twice. For gel source data, see Supplementary Fig. 1. b, Immunofluorescence staining shows predominantly intracellular localization of the CTRs (green) in ACFs obtained from patients with persistent AF. By contrast, in SR-ACFs, the CTR is localized to the cell surface. Cells were counterstained with filamin A (red) and nuclei (DAPI). Data are pooled from the individual donors (a few cells in each field as shown in the figure) collected over 2-year period, assessed on separate days and validated by 3 independent experimenters. For source data, see Supplementary Fig. 1. Source data

Extended Data Fig. 7 Atrial gene expression, morphological parameters and AF-duration in mice.

ad, Global CTR gene deletion does not alter atrial expression of the genes for collagen 1 (Col1A1), collagen 3 (Col3A1), fibronectin (Fn) and alpha-smooth muscle actin (ACTA2) in male and female mice. el, Selected morphological parameters in the CTR-KO males and females. m, n, Mean duration of AF in CTR-KO and control mice expressed as ‘mean of all AF episodes in mice that experienced AF’ (m) or ‘mean of all AF episodes in all mice’ (n). o, Schematic representation of the constructs used to generate atrial-specific LKB1-KD, LKB1/CT-dKD and LKB1-KD+CT mice. The Lkb1fl/fl mice were injected with AAV9-ANF-CRE. Because the ANF promoter drives expression of CRE exclusively in the atria, LKB1 was downregulated only in the atria of these LKB1-KD mice. The LKB1-KD+CT cDNA mice received AAV9-ANF-CRE + AAV9-ANF-CT cDNA injections. Under the control of the ANF promoter, CT was overexpressed exclusively in the atria of these mice. The LKB1/CT dKD mice received AAV9-ANF-CRE + AAV9-loxP-STOP-loxP-shCT injections. Both LKB1 and LoxP-STOP-LoxP were deleted by atrial-specifically expressed Cre enzyme, allowing the expression of CT shRNA, which selectively targets the CT/pro-CT but not the αCGRP sequence and hence resulted in the downregulation of both LKB1 and CT. Data are presented as mean values ± s.e.m., except in d (females), g (males), medians with interquartile ranges. P values were determined by two-sided tests: unpaired t-test in all apart from d (females), g (males), j, m, n, which were analysed by Mann–Whitney U-test; n denotes individual animals. Data are pooled from individual animals assessed in single replicates on the same day and reproduced in two centres in a, c, d. Results in m, n were obtained from individual animals over ~2.5 years. Source data

Extended Data Fig. 8 Validation of anti-CTR antibody and study summary.

a, Data summary: under physiological conditions in SR (left), human atrial cardiomyocytes produce and excrete endogenous CT, which binds to the CTR of atrial cardiofibroblasts (ACFs). Increased Gs-mediated cAMP inhibits multiple steps of fibrogenesis including, but not limited to, BMP1 activity and collagen processing by ACFs, thus keeping atrial fibrosis in check. In persistent AF (right), atrial cardiomyocytes secrete less CT, and ACFs show abnormal intracellular CTR localization; the consequent reduced CT–CTR activation enables unchecked structural remodelling and fibrosis in the atria that promotes AF maintenance and inducibility. b, Immunostaining with anti-CTR antibody shows barely detectable signal for CTR (green) in human kidney embryonic cell line (HEK293) and adult human dermal fibroblasts (HDF), and prominent positive CTR staining in human medullary carcinoma (TT) cells; red, filamin A; blue (DAPI), nuclei. Negative control for secondary antibodies (with primary antibodies omitted) in human ACFs is shown. c, Detection of positive immunofluorescence staining (green) with anti-CTR antibody in control ACFs, but not in CTR-KD ACFs, using anti-CTR LNA antisense oligonucleotides. df, The same antibody was used to detect CTR in HEK293 cells stably overexpressing (confirmed by qRT–PCR) human CTR protein (+hCTR; d) by flow cytometry (e; control cells negative for CTR (left plot) were used to determine the position of the P2 gate and the CTR+ cells (right plot) were sorted based on this gate and an antibody for CTR bound to AF647) and by immunofluorescence (f; CTR+ cells are stained in green and nuclei with DAPI, in blue). Gating strategy shown (bottom 3 panels): cells were first gated by general size and granularity (left plot), and then doublets were excluded using a standard plot of forward scatter height versus area (middle plot), eliminating cells with a large area for any given signal height, and then plotted on a log scale for mean fluorescent intensity of AF647 (right plot, gate P2) for CTR+ cells. The P2 gate was set based on unstained cells and shows events from the sample with a mean fluorescent intensity higher than the control in the P2 gate. g, Validation of the antibody for human pro-CT in human atrial tissue by immunoblotting. Representative example of the blot performed on 4 individual donors assessed on one day; this antibody was also tested in another 4 individual donors on a different day with the same result; recombinant human pro-CT was used as a positive control. h, i, CT ELISA kit confirms detection of human recombinant (in black) or synthetic CT (in green) in concentration-dependent manner with no cross-reactivity with recombinant human αCGRP or recombinant human pro-CT (in magenta) at serial dilutions. j, Cellular pellets in proteomic experiments were processed in duplicates to validate reproducibility. Data in e are presented as medians with interquartile ranges, as analysed by two-sided unpaired t-test after log transformation. FSC-A, forward scatter area. Data in bd are representative images of cells stained on the same day and reproduced three times on three separate days. Data were pooled from individual cultures assessed in duplicates (e) or from technical triplicates (h, i) and technical duplicates (j) analysed on the same day. For gel source data, see Supplementary Fig. 1. Source data

Extended Data Table 1 Clinical characteristics of the study participants
Extended Data Table 2 In vivo echocardiographic and haemodynamic parameters in mice

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Moreira, L.M., Takawale, A., Hulsurkar, M. et al. Paracrine signalling by cardiac calcitonin controls atrial fibrogenesis and arrhythmia. Nature 587, 460–465 (2020). https://doi.org/10.1038/s41586-020-2890-8

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