Cardiac Repair after Myocardial Infarction is Controlled by a Complement C5a Receptor 1-Driven Signaling Cascade

Heart failure (HF) is a chronic medical condition characterized by the heart's inability to efficiently pump blood, often resulting from recurrent myocardial infarctions (MIs).[1] [2] Despite improvements in risk management and interventional strategies, HF presents significant health care challenges and leads to increased health care expenditure due to gaps in HF therapy targets.[3] Therefore, it is essential to identify treatable traits.[4] Cardiac repair is a highly regulated process consisting of inflammatory, proliferation, and remodeling phases overlapping each other. This follows necrotic loss of cardiomyocytes after MI in an attempt to repair the damage and restore heart function. Mechanisms in the repair process following MI include activation of the complement system as part of the innate immune response. The complement anaphylatoxin C5a interacts primarily with its two receptors, the classical proinflammatory C5a receptor 1 (C5aR1, CD88) and C5a receptor 2 (C5aR2, C5L2 [C5a receptor like 2]). Previous studies have implicated C5aR1 in cardiac regeneration after left ventricular apical resection[5] and cardiac inflammation.[6] [7] However, its role in cardiac repair processes following MI is poorly defined and the role of C5aR2 in MI is largely unknown. Our current study focused on late phases after MI up to 4 weeks after the infarction insult, a time window that specifically reflects the outcome of the repair process and that so far has not been investigated. Using C5ar1- and C5ar2-deficient mice, we studied the role of both C5aR1 and C5aR2 in cardiac function and repair processes following MI. We combined functional analyses and mechanistic studies in vivo and in vitro to demonstrate that targeting C5aR1 may provide a potent lever to improve cardiac repair after MI.

To systematically study the role of C5a receptors in cardiac repair processes following MI, wild-type C57Bl/6J, C5ar1^−/− and C5ar2^−/− mice were investigated after chronically ligating the left anterior descending artery (LAD). Histological analysis revealed a significantly reduced MI size ([Fig. 1A, B]) as well as lower collagen content in the infarcted area ([Fig. 1C] and [Supplementary Fig. S1A], available in online version the online version) in C5ar1^−/− mice compared with wild-type mice 4 weeks after MI. Infarct size and collagen content were not reduced in C5ar2^−/− mice. This indicated a protective effect of C5ar1 deficiency in MI. Consistent with the reduced MI size, C5ar1^−/− mice showed a significantly increased ejection fraction and reduced end-diastolic volume when compared with wild-type mice 4 weeks after MI, indicating an improved ventricular function and contractility ([Supplementary Table S1], available in the online version). Cardiac repair after MI relies on an intense inflammatory response that provides molecular cues for activation of reparative cells.[8] As recruitment of inflammatory cells and the proliferation of tissue-resident macrophages in later stages are pivotal in this process, we analyzed the content of monocytes/macrophages and neutrophils in the infarcted areas. One week after induction of MI, the content of Mac3-positive monocytes/macrophages in the infarcted area peaked in wild-type mice and was significantly reduced in C5ar1^−/− mice by 43% ([Fig. 1D, E] and [Supplementary Fig. S1B], available in the online version). This peak of monocyte/macrophage accumulation drastically lowered at 4 weeks after MI without significant differences between groups ([Fig. 1E]). In contrast, the content of MPO+ cardiac neutrophils transiently increased 24 hour after MI in wild-type mice and this increase was significantly reduced in both C5ar1^−/− and C5ar2^−/− mice ([Supplementary Fig. S2], available in the online version). The reduced neutrophil accumulation in the infarcted heart could provide some cardioprotective effects in both C5ar1^−/− and C5ar2^−/− mice. However, the observation that infarction size was only reduced in C5ar1^−/− mice suggested a predominant involvement of another C5ar1-mediated mechanism in cardiac repair after MI, urging us to also examine effects on cell proliferation versus apoptosis and the expression of genes implicated in the late phase of cardiac repair. At 1 week, cell proliferation in the infarcted heart increased and the number of proliferating cells was significantly further increased in both C5ar1^−/− and C5ar2^−/− mice compared with control wild-type mice ([Supplementary Fig. S3A, B], available in the online version). Myocardial necrosis resulting from improper blood perfusion to the cardiac tissue after MI is a detrimental event, and cardiac repair mechanisms aim at removing necrotic tissue while inducing neovascularization. We therefore also examined the effect of C5ar1 deficiency on myocardial necrosis in vivo and found reduced necrosis in C5ar1-deficient hearts 24 hour after MI ([Supplementary Fig. S3C–E], available in the online version). Likewise, C5ar1 deficiency reduced apoptotic cells in the infarcted hearts ([Fig. 1F, G] and [Supplementary Fig. S3F, G], available in the online version).

Fig. 1 C5aR1 controls cardiac repair mechanisms following myocardial infarction (MI). (A–E) MI was induced by chronically ligating the left anterior descending artery (LAD). (A, B) Histomorphometrical analysis of the infarcted myocardium 4 weeks after MI in wild-type (WT), C5ar1^−/− and C5ar2^−/− mice (n = 7–9 per group) as measured by planimetry. Shown are representative Gomori's one-step trichrome-stained sections (A) and the quantification of infarcted areas (B). (C) Quantification of collagen content in infarcted area. (D) Representative immunostaining of MAC3⁺ monocytes/macrophages in infarcted myocardium analyzed 1 week after MI. (E) Quantification of myocardial infiltration of Mac3⁺ monocytes/macrophages analyzed 1 day, 1 week, and 4 weeks after MI. Cells were visualized by immunostaining and the quantification is presented as the percentage of positively stained cells of total cell count in infarcted area in field of view (FOV). n = 4–6 per group. (F, G) Apoptosis rate in infarcted myocardium 24 hours after MI induction in WT and C5ar1 ^−/− mice detected by TUNEL staining. Shown is representative immunostaining (F). The quantification of cells is presented as the percentage of positively stained cells per FOV-infarcted area (G). A complete set of these data are presented in [Supplementary Fig. S3C, D] (available in the online version). (H–J) MI was performed in WT and C5ar1^−/− mice, thereafter, infarcted heart areas were isolated 4 weeks after infarction. Representative immunoblot (H) and corresponding quantification of Tgf-β1 (I) and Vegf-A (J) normalized to actin. Representative staining (K) and quantification (L) of SMA⁺/CD31⁻ myofibroblasts in the infarcted myocardium. White arrows indicate SMA⁺ myofibroblasts (green fluorescence). Scale bars 100 µm. (M) Cardiac fibroblasts isolated from WT and C5ar1^−/− mice were stimulated with TGF-β1 alone or together with C5a. Quantification of protein expression of Vegf-A normalized to actin. (N, O) Supernatant from TGF-β1-stimulated WT or C5ar1^−/− cardiac fibroblasts were incubated on WT-ECs or C5ar1^−/− -ECs respectively, in a coculture, and tube formation was determined in WT-ECs and C5ar1^−/− -ECs. Shown are representative images (N) and quantification of tube formation (O). (P, Q) Neoangiogenesis was assessed by CD31⁺/α-SMA⁻ staining of WT, and C5ar1^−/− infarcted heart areas 4 weeks after infarction. (P) Representative CD31 staining of infarcted heart areas and (Q) corresponding quantification. N = 7 mice per group. Data are presented as mean ± standard error of mean.

To further assess the mechanisms underlying the overall improved heart function upon C5ar1 deficiency, we determined gene expression of key repair cytokines in infarcted hearts from wild-type and C5ar1^−/− mice that underwent MI. Four weeks after MI (complete healing and maturation of the scar), we found significantly increased expression of Tgf-β1 and Vegf-A both on protein and mRNA levels, in C5ar1^−/− infarcted hearts, when compared with corresponding control wild-type hearts, whereas the levels 1 week after MI were not significantly altered ([Fig. 1H–J] and [Supplementary Fig. S4A–D], available in the online version). We further observed an increased expression of Vegf-A in TGF-β1-stimulated cardiac fibroblasts ([Supplementary Fig. S4E], available in the online version). TGF-β1 was described as having a transient role in macrophage polarization and myofibroblasts differentiation during healing after MI.[9] Given the increased expression of Tgf-β1 and Vegf-A, a highly potent angiogenic agent, during the maturation of the scar, we reasoned that C5ar1 deficiency may promote a transient myofibroblast differentiation response resulting in improved cardiac repair. Surprisingly though, while the number of cardiac myofibroblasts remained unchanged in both genotypes 1 week after MI ([Supplementary Fig. S4F, G], available in the online version), the number of α-SMA⁺ cells in the infarcted myocardium was significantly reduced in C5ar1^−/− mice compared with wild-type controls 4 weeks after MI ([Fig. 1K, L]). To further scrutinize the effect of the increased expression of Tgf-β1 in C5ar1^−/− mice, we isolated cardiac fibroblasts from wild-type mice and quantified the expression of C5ar1. We found considerable expression of C5ar1 in cardiac fibroblasts, which was not altered upon TGF-β1 stimulation ([Supplementary Fig. S5A-D], available in the online version). We then asked whether C5a/C5aR1 signaling affects the TGF-β1-driven conversion of cardiac fibroblasts to myofibroblasts. Exposure of wild-type cardiac fibroblasts to TGF-β1 was able to induce transdifferentiation as determined by α-SMA expression, but this was independent of C5a/C5aR1 signaling ([Supplementary Fig. S5E, F], available in the online version). As overall myofibroblast transdifferentiation was not affected upon C5ar1 deficiency, we hypothesized that the regulatory response of these cells was skewed with consequences for myofibroblast function. To test this notion mechanistically, cardiac fibroblasts isolated from wild-type and C5ar1^−/− mice were stimulated with TGF-β1. Analysis of mRNA and protein expression revealed a TGF-β1-induced upregulation of Vegf-A levels in wild-type myofibroblasts, which was significantly increased in C5ar1^−/− myofibroblasts ([Fig. 1M] and [Supplementary Fig. S5G], available in the online version). This is an indication that C5aR1-deficient myofibroblasts may contribute to the VEGF pool in the myocardium, by increasing the balance toward a proangiogenic phenotype of cardiac fibroblasts. After MI, neovascularization in the border zone adjacent to the ischemic region helps to preserve cardiac function and attenuate adverse left ventricular remodeling.[10] Our observation that the expression of Vegf-A is significantly upregulated in C5ar1^−/− myofibroblasts and in infarcted heart areas from C5ar1^−/− mice, potentially in part through effects on intracellular signaling including ERK and p38 MAPKs,[11] led us to reason that upon stimulation, cardiac myofibroblasts may be a source of Vegf-A for endothelial cells (ECs) to support neoangiogenesis. To investigate this, we performed matrigel tube formation assays, where wild type-ECs were incubated with supernatants derived from stimulated wild type-fibroblasts and C5ar1^−/− -ECs were exposed to C5ar1^−/− -myofibroblast supernatant, mimicking the in vivo microenvironment in our model. We found increased tube formation in C5ar1^−/− -ECs/C5ar1^−/− -myofibroblast cocultures compared with cell responses elicited in wild-type cells ([Fig. 1N, O]) indicating, to our knowledge, for the first time a C5a/C5aR1-axis-mediated EC–fibroblast interaction in neovessel formation during the cardiac repair process following MI. This was consistent with significantly improved neovascularization in infarcted heart areas from C5ar1^−/− mice as revealed by increased newly formed CD31⁺ blood vessels 4 weeks after MI ([Fig. 1P, Q]). Hence, the C5a/C5aR1-axis may mediate EC–fibroblast interactions in cardiac repair processes and contribute to improved heart function observed in C5ar1^−/− mice 4 weeks after MI. The identified EC–fibroblast interaction requires additional studies to further scrutinize the role of C5ar1 in cardiac fibroblast activation and related mechanisms.

Collectively, the results presented here show that C5ar1 deficiency (1) reduces infarct size following MI and enhances overall cardiac function; (2) attenuates myocardial necrosis; and (3) enhances VEGF production by myofibroblasts and EC–fibroblast interactions to promote neovascularization in the infarcted heart. Although the exact contribution of C5aR1 on ECs versus fibroblasts in this EC–fibroblast interaction remains to be further clarified, our findings overall demonstrate an important role for the C5a/C5aR1-axis in the late endogenous repair mechanism following MI. This could complement cardioprotective effects provided by C5ar1 deficiency on other cell types including platelets, where C5ar1 deficiency promotes tissue neovascularization by reducing C5a-triggered secretion of the anti-angiogenic factor CXCL4.[12] Also, other immune cells including dendritic cells and CD4⁺ T cells have been implicated in left ventricular remodeling and the progression of cardiac dysfunction following MI.[13] [14] As C5aR1 has been shown to regulate the function of both dendritic cells and CD4⁺ T cells,[15] [16] we cannot exclude effects of C5aR1 on these cells in the current study. Considering the pleiotropic role of C5aR1 in various cell types and signaling pathways, it is crucial to dissect in future studies the precise cell-specific C5aR1-dependent mechanisms involved in all phases of healing after MI: the acute inflammatory phase, the intermediate proliferation phase, and the late fibrosis phase. Unveiling the distinct, cell type- and time-dependent functions of C5aR1 signaling could further support the design of personalized therapeutic strategies to improve cardiac repair after MI.

Conflict of Interest

None declared.

Acknowledgment

We thank Roya Soltan, Yuan Kong, Tanja Vajen, Adelina Curaj, Melanie Garbe, and Stefanie Elbin for excellent technical assistance.

Ethical Approval Statement

All animal experiments were approved by local authorities (Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Germany) and complied with the German animal protection law (AZ: 8.87-50.10.35.09.088).

Data Availability Statement

All data and materials are presented in the main manuscript or supplementary materials and are available on reasonable request.

Authors' Contribution

Y.A. and E.S. designed research with input from E.L; Y.A., S.S., E.L., J.K., G.S., M.S., H.N. performed research, Y.A., E.S., E.L., S.S., A.K., A.Z., J.B. analyzed data; Y.A., and E.S. wrote the paper. All authors reviewed and edited the manuscript.

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Publication History

Received: 27 June 2024

Accepted: 17 August 2024

Accepted Manuscript online:
04 October 2024

Article published online:
29 October 2024