Complement Receptor C5aR1 Plays an Evolutionarily Conserved Role in Successful Cardiac Regeneration

Abstract

Background: Defining conserved molecular pathways in animal models of successful cardiac regeneration could yield insight into why adult mammals have inadequate cardiac regeneration after injury. Insight into the transcriptomic landscape of early cardiac regeneration from model organisms will shed light on evolutionarily conserved pathways in successful cardiac regeneration.

Methods: Here we describe a cross-species transcriptomic screen in 3 model organisms for cardiac regeneration: axolotl, neonatal mice, and zebrafish. Apical resection to remove ≈10% to 20% of ventricular mass was carried out in these model organisms. RNA-sequencing analysis was performed on the hearts harvested at 3 time points: 12, 24, and 48 hours after resection. Sham surgery was used as internal control.

Results: Genes associated with inflammatory processes were found to be upregulated in a conserved manner. Complement receptors (activated by complement components, part of the innate immune system) were found to be highly upregulated in all 3 species. This approach revealed induction of gene expression for complement 5a receptor 1 in the regenerating hearts of zebrafish, axolotls, and mice. Inhibition of complement 5a receptor 1 significantly attenuated the cardiomyocyte proliferative response to heart injury in all 3 species. Furthermore, after left ventricular apical resection, the cardiomyocyte proliferative response was diminished in mice with genetic deletion of complement 5a receptor 1.

Conclusions: These data reveal that activation of complement 5a receptor 1 mediates an evolutionarily conserved response that promotes cardiomyocyte proliferation after cardiac injury and identify complement pathway activation as a common pathway of successful heart regeneration.

Keywords: C5aR1; axolotl; cardiac regeneration; complement system; cross-species; mice; zebrafish.

Figure 1. RNA-seq analysis pipeline to identify evolutionarily conserved genes involved in early heart regeneration

Ventricular myocardium was resected, and RNA-sequencing was performed at 12, 24, and 48 hours post-injury in neonatal mice, axolotl, and zebrafish. Each time point was internally controlled with sham surgery. (A) Schematic of transcriptomic approach for identifying genes whose expression was conserved during early heart regeneration. Black dots represent genes, and gray squares represent computational filtration steps. (B) Intersection of genes that were upregulated at 12, 24, or 48 hours relative to sham controls in neonatal mice and zebrafish. (C) Intersection of genes that are downregulated at 12, 24, or 48 hours relative to sham controls in neonatal mice and zebrafish. (D) Gene ontology (GO) analysis of commonly upregulated neonatal mouse and zebrafish genes. (E) Gene ontology (GO) analysis of commonly downregulated neonatal mouse and zebrafish genes.

Figure 2. Schematic of analysis pipeline used to identify upregulated inflammatory genes in mouse, zebrafish an axolotl

(A). Inflammatory response genes including complement components and associated proteins that are upregulated in early cardiac regeneration in an evolutionarily conserved manner. The list is ordered according to fold change; genes with higher fold change in mouse are first, followed by genes in decreasing order. Note that the top two genes are receptors for activated complement components, C5aR1 and C3aR1. (B). Complement 5a receptor 1 (C5aR1) is upregulated in early cardiac regeneration in zebrafish, axolotl and mouse at the timepoints shown, in hours (C), expression of C5aR1 is normalized to sham levels (dotted line).

Figure 3. Inhibition of C5aR1 decreases proliferating cardiomyocytes after apical resection in zebrafish

(A) resected zebrafish treated with vehicle, representative image, (B) resected zebrafish treated with PMX205, representative image, cardiomyocyte marker tropomyosin (red), PCNA (green), cardiomyocyte nuclei (MEF2, blue); scale bar - 50µm (C) quantification of proliferating cardiomyocytes in vehicle and PMX205 treated hearts of zebrafish, * p<0.01, n=5 (vehicle), 8 (PMX205).

Figure 4. Inhibition of C5aR1 in axolotl and mouse

Inhibition of C5aR1 results in a reduction of proliferating cardiomyocytes in axolotl hearts. cardiomyocyte marker MF20 is shown in red, phospho Histone 3 (pH3) is shown in green and nuclei are in blue. (A) Sham surgery of axolotl hearts, (B) resected – vehicle treatment and (C) resected – PMX205 treatment; scale bar - 100µm. Quantification of proliferating cardiomyocytes is summarized in (D), * p<0.05, n=4. Effect of C5aR1 inhibition on cardiomyocyte proliferation in mouse (E–H). Cardiac Troponin T (cTnT, cardiomyocyte marker) is shown in red, phosphoHistone 3 staining is in green and nuclei are in blue. (E) sham surgery of mouse hearts, (F) resected – vehicle treatment and (G) resected – PMX205 treatment, scale bar - 20µm. Quantification of cardiomyocyte proliferation is summarized in (H), * p<0.05, n=4. Effect of C5aR1 inhibition on scar formation after apical resection (I–L). Masson trichrome images of hearts 21 days post-resection show an increase in scar formation in PMX205 treatment after apical resection. Representative images of sham surgery (I), resected – vehicle treatment, resected – PMX205 treatment. Scar infiltration is quantified in (L). * p<0.05, n≥6, scale bar 100 µm.

Figure 5. Localization of C5aR1 to cardiomyocytes and endothelial cells after apical resection in neonatal mice

Localization of C5aR1 to cardiomyocytes and endothelial cells after apical resection in neonatal mice. C5aR1 (red) localizes with cardiomyocyte marker Troponin T (green) in neonatal mouse hearts, 48 hours after apical resection (nuclei are in blue), scale bar 20µm (A). C5aR1 upregulation is absent in hearts from littermate sham controls (B). C5aR1 (red) expression localizes with endothelial marker CD-31 (green) (C), scale bar - 20µm. C5aR1 (red) does not localize with macrophage marker CD68 (green)in resected hearts (D), scale bar - 20µm.

Figure 6. Mice lacking C5aR1 mice have a reduction in cardiomyocyte proliferation after apical resection compared to wild-type littermate controls

Representative images from C5aR1 wild-type sham surgery (A), C5aR1 wild-type resected (B), C5aR1 knock-out sham surgery (C) and C5aR1 knock-out resected (D). Cardiac troponin T (green), phosphoHistone 3 (red) and nuclei (blue), scale bar - 20µm. Quantification of proliferating cardiomyocytes shows the absence of proliferative response upon resection in C5aR1 knock-out hearts (E). *p <0.05, n≥4. Measurement of fibrosis 21 days after apical resection (F–J). Representative Masson Trichrome stained images from C5aR1 wild-type sham surgery (F), C5aR1 wild-type resected (G), C5aR1 knock-out sham surgery (H) and C5aR1 knock-out resected (I), scale bars, 100µm. Quantification of fibrosis in cardiac apex after resection (J), *p<0.05, n=8.