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. 2023 Jan 26;33:101434. doi: 10.1016/j.bbrep.2023.101434

The role of complement C3 in the outcome of regional myocardial infarction

Zhou Fang a, Xiang Li a, Junying Liu a, Haekyung Lee a, Louis Salciccioli c, Jason Lazar c, Ming Zhang a,b,∗
PMCID: PMC9898614  PMID: 36748063

Abstract

Coronary heart disease leading to myocardial ischemia is a major cause of heart failure. A hallmark of heart failure is myocardial fibrosis. Using a murine model of myocardial ischemia/reperfusion injury (IRI), we showed that, following IRI, in mice genetically deficient in the central factor of complement system, C3, myocardial necrosis was reduced compared with WT mice. Four weeks after the ischemic period, the C3−/− mice had significantly less cardiac fibrosis and better cardiac function than the WT controls. Overall, our results suggest that innate immune response through complement C3 plays an important role in necrotic cell death, which contributes to the cardiac fibrosis that underlies post-infarction heart failure.

Keywords: Ischemia/reperfusion injury (IRI), Complement C3, Necrosis, Cardiac fibrosis. 1

Highlights

  • •

    Following IRI, in mice genetically deficient in the central factor of complement system, C3, myocardial necrosis was reduced compared with WT mice.

  • •

    Four weeks after the ischemic period, the C3−/− mice had significantly less cardiac fibrosis and better cardiac function than the WT controls.

  • •

    Innate immune response via C3 is critical in necrotic cell death.

1. Introduction

Ischemia/reperfusion injury (IRI) underlies critical clinical scenarios involving major organs, e.g. myocardial infarction, transplantation, trauma and perioperative organ injury. IR elicits an acute inflammatory response involving complement factors of the innate immune system [[1], [2], [3]]. During IR, the local tissues undergo drastic intracellular changes: mitochondria become progressively dysfunctional and excess free radicals are generated, leading to lipid and protein oxidation and DNA damage [4]. At the cell signaling level, factors of various cell death programs are activated [5,6]. During the ensuing necrosis, cytoplasmic membranes become unstable, resulting in the dispersal or exposure of intracellular contents to the extracellular space [7]. This triggers an immune response, as evidenced by release of pro-inflammatory cytokines [8], activation of the complement system [[1], [2], [3]] and infiltration of inflammatory cells [9]. Together with the intracellular changes, these inflammatory responses result in the cell death of ischemic tissues and subsequent long-term consequences; e.g. in the heart, myocardial IR related post-infarction heart failure with the hallmarks of cardiac fibrosis [10,11] and heart dysfunction [12,13].

It has long been suggested that limiting inflammation during cardiac IR injury, for instance via complement inhibitors, would reduce myocardial-IR related cell death and thus prevent post-infarction heart failure [[14], [15], [16], [17], [18]]. Previous pre-clinical studies supported such a hypothesis, particularly those using anti-complement C5 [16,[18], [19], [20], [21]]. However, limited positive results were obtained with the few inhibitors studied in clinical trials [3]. In particular, anti-complement C5 (Pexelizumab) failed to meet the primary endpoints in acute myocardial infarction patients [22].

The earliest work that established the role of C5 in a rat myocardial IR injury model revealed that, while anti-C5 reduced infarct size and apoptosis, it did not inhibit C3 deposition in the injured myocardium [18]. Similar results obtained using inhibition of the C5a receptor [[23], [24], [25]] supported the conclusion that activated C5 was pro-apoptotic. However, anti-C5 failed to inhibit assembly of the terminal complement complex (TCC) in patients with ST-elevation myocardial infarction undergoing primary percutaneous coronary intervention [26], possibly due to intense complement activity or different mechanisms leading to TCC activation.

Our view of these basic science and clinical results is that using anti-C5 targets a downstream factor in the common complement pathway, leaving earlier factors, e.g. C3, unaffected. Complement activation in general can be initiated via three pathways: the classical pathway (CRP, antibodies, C1q, C4, C2), the alternative pathway (spontaneous C3 hydrolysis, Factor B (FB)) and the lectin pathway (mannose binding lectin (MBL), ficolins) [[27], [28], [29], [30], [31], [32], [33], [34]]. These pathways converge at the formation of C3 convertase (C3b•Bb). Amplification via a loop involving C3 convertases occurs only through the FB-dependent alternative pathway [35,36]. C3 convertases yield C3a and additional C3b, the latter forming C5 convertases (C4b•C2a•C3b and C3b•Bb•C3b) and that cleave C5 to C5a and C5b. C5b initiates formation of the cell membrane attack complex (C5b-C9) which binds to and destroys targets. The C3 and C5 convertases are regulated by membrane-bound proteins (e.g. CD46, CD55) and soluble factors (e.g. FH, C4BP and FI) [35,37]. Thus, it is possible that in the cited studies, activation of factors prior to C5, e.g. C3, would initiate upstream complement signaling pathways leading to acute cell death and chronic inflammation, thus contributing to post-infarction heart failure. Supporting this hypothesis, a clinical study found that coronary artery disease patients with lower C3 had less worse outcome than those higher levels of C3 [38].

Previously we used a clinically-relevant, myocardial IR injury mouse model (60 min ischemia resembling clinical door-to-balloon time (DTBT) time [[39], [40], [41]]) to show that myocardial necrosis, which increased upon reperfusion and was maintained through 24 h, was accompanied by deposition of activated C3 fragments in reperfused tissues [42]. Here we used this model to investigate the mechanisms by which C3 contributes to cell death in the acute phase of infarction (the first 24 h of reperfusion). In addition, we examined the long-term effect of C3 on post-infarction heart failure, as indicated by cardiac fibrosis and dysfunction.

2. Materials and Methods

2.1. Mouse model of surgically induced myocardial IR injury

Complement C3 knockout (C3−/−) mice and WT (C57BL/6) mouse strains were obtained from the Jackson Laboratory (Bar Harbor, ME) and maintained at the SUNY Downstate Health Science University Department of Laboratory Animal Resources. Genotyping was provided by GeneTyper (New York, NY). Male mice were used at 10–12 weeks of age (weight 26–30 g) in accordance with the requirements of the NIH and the Institutional Animal Care and Use Committee (IACUC) of SUNY Downstate Health Science University. The protocol was approved by the IACUC of SUNY Downstate Health Science University (Approval #11–10276).

We employed an established model of myocardial IR injury model [42,43]. Mice were anesthetized using sodium pentobarbital (60 mg/kg, i.p.), intubated and ventilated with a mouse ventilator (Harvard Apparatus, MA). Following sternotomy, the left anterior descending artery (LAD) was ligated for 1 h; occlusion of the LAD was confirmed by the appropriate color change of myocardial tissue and the ST elevation on ECG; reperfusion was verified by the reversed color change of the left ventricle and the appropriate ECG changes. Postoperative management included fluid replacement with normal saline and pain relief with the analgesic buprenorphine (0.1 mg/kg, intramuscularly). The mice were sacrificed after 24 h of reperfusion; the hearts were harvested for histopathologic analyses.

2.2. Evaluation of murine myocardial necrosis by fluorescence using two probes delivered in vivo

A fluorescent method for tracking necrosis (developed by others [44,45] and further refined by us [42]) was used. Shortly before the end of the reperfusion period described above and before tissue harvesting, mice were anesthetized, intubated again as described above, and injected i.v. with propidium iodide (PI), which enters damaged cells, intercalates with DNA and fluoresces, thus identifying necrotic tissue. The LAD was then re-occluded and blue fluorescent microspheres (BFM, ThermoFisher, PA) were injected through the aortic arch to delineate the non-ischemic region of the heart. The mice were sacrificed, the hearts harvested and the atria removed. Each ventricle was sectioned into four slices (∼1 mm thickness) which were weighed and imaged under a fluorescent microscope (Olympus, PA) using the red fluorescent channel for PI, the blue channel for BFM.

The percentage of the tissue in a heart which was at risk for necrosis (negative for blue fluorescence) and which became necrotic (positive for red fluorescence) was determined by computerized planimetry (Image J, NIH, Bethesda, MD) and by the following equations:

Weight of necrotic tissue = (A1 x Wt1) + (A2 x Wt2) + (A3 x Wt3) + (A4 x Wt4), where A was the percentage of the area of a slice staining for necrosis (red fluorescence) measured by planimetry (average of both sides of a slice) and Wt was the weight of that slice of ventricle.

2.2.1. Weight of tissue at risk for necrosis (weight at risk, WAR) =

(R1 x Wt1) + (R2 x Wt2) + (R3 x Wt3) + (R4 x Wt4), where R is the percentage of the area of a slice which lacked the blue fluorescence of BFM, determined by planimetry (average of both sides of a slice used). In all cases, the tissue with red fluorescence was within the boundary of the tissue which lacked blue fluorescence. Blue = no blood flow. Red – necrosis tissue.

Percentage of the weight of a ventricle at risk for necrosis which became necrotic = (weight of necrotic tissue/WAR) x 100.

2.3. Echocardiography

Echocardiography was performed as previously described [46]. Briefly, mice chests were shaved and allowed to rest for at least 1 h before echocardiography. Echocardiography, using the Phillips SONOS 5500 with a 15 MHz linear probe, was performed on conscious mice to avoid any cardiodepression produced by anesthesia. Imaging employed the M mode short axis view, measuring systolic and diastolic cardiac dimensions. Images were digitized for analysis. Left ventricular systolic (LVESD), diastolic (LVEDD), septal (SW), and posterior wall (PW) thicknesses were measured. Left ventricular fractional shortening (FS) was calculated from the following formula: FS (%) = ((LVEDD − LVESD)/LVEDD)) × 100. The ejection fraction (EF) is calculated from the following formula: EF = ((LVEDD2 − LVESD2)/LVEDD2)) × 100. Left ventricular mass is calculated from the following formula: LV mass = 1.05 ((LVEDD + SW + PW)3 − (LVEDD3)).

2.4. Cardiac fibrosis analysis

For analysis of cardiac fibrosis, mice were sacrificed 4 weeks after 1 h of heart ischemia produced as described in Material and Methods, (Section 1). The cardiac fibrosis present in sections from each of 4 slices/heart obtained as in Materials and Methods, were assessed by the Masson Trichrome stain kit (VWR, PA), according to the manufacturer's instructions.

2.5. Statistical analysis

Statistical analyses were performed using IBM SPSS Software version 20 (IBM Corp., NY). For animal studies, an independent t-test with two tails and unequal variances was used to determine the statistical significance of differences between the results of experimental and control groups. Descriptive data were summarized as mean ± standard error of mean. Power analyses were performed using G*Power 3.1 [47] showed >99% power for detection of differences in infarct size with 6–8 animals per group.

3. Results

3.1. Myocardial necrosis is reduced in C3−/− mice after 60 min of heart ischemia followed by 24 h of reperfusion

Loss of plasma membrane integrity (LPMI) is a hallmark of necrotic cell death [5,7]. We have developed an in vivo fluorescent method [41] modified from earlier reports [44,45] to track LPMI in necrotic myocardial death. The method permits subsequent analysis of fluorescently tagged tissue for other pathological events, a significant advantage over the traditional evaluation of myocardial necrosis using triphenyl tetrazolium chloride. After 1 h ischemia/24 h reperfusion, necrosis in C3−/− mice was significantly reduced compared with WT mice (Fig. 1a and b; post hoc power analysis>90% power; similar post hoc statistical powers were identified for the other pilot experiments).

Fig. 1.

Fig. 1

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Necrosis is decreased while apoptosis is increased in the hearts of C3−/− mice after IR.

(a) C3−/− and WT mice (n = 6–8/group) were subjected to occlusion of the left anterior descending (LAD) artery for 1 h followed by reperfusion for 24 h. Next, propidium iodide (PI - enters cells through damaged cell membranes and binds DNA in necrotic cells) and blue fluorescent microspheres (BFM – present in un-occluded blood vessels) (the latter after re-occlusion of the LAD) were injected in vivo just prior to heart harvesting to delineate the infarcted area, and the area at risk (AAR) for necrosis (i.e., lacking BFM), respectively. After animal sacrifice, each ventricle was divided into four slices (top and bottom of each slice are adjacent). LPMI-positive necrotic tissue (bright red) was visualized immediately under a fluorescent microscope with a 2× objective lens. Non-ischemic tissue was defined by the blue fluorescence of BFM, the non-fluorescing tissue constituting ischemic tissue, the area at risk (AAR) for necrosis. The necrotic area was traced with dotted lines as an example.

(b) The LPMI-positive necrotic area expressed as % of AAR.

(c) C3−/− mice had less cardiac fibrosis after IR. C3−/− mice and control WT mice (n = 4 per group) underwent 1 h myocardial ischemia followed by reperfusion. 4 weeks after initial heart ischemia, mice were sacrificed and cardiac fibrosis in heart sections was assessed by the Masson Trichrome stain (VWR, PA). Blue arrows indicated the areas of positive staining for fibrosis.

(d) Fibrotic areas were quantified and expressed as a percentage of the total area of heart sections.

(e) C3−/− mice had better LVEDD after IR. Cardiac functions were followed by echocardiography during the 4-week post-ischemia period. *indicates P < 0.05 between the C3−/− and WT groups at 4 weeks' post-ischemia.

These results indicate that when WT hearts experience regional ischemia in a 1 h time period resembling the length of clinical DTBT, their cardiomyocytes are directed by C3 towards necrosis during the acute 24 h long phase of reperfusion. How C3 regulates this switch is unknown.

3.2. Cardiac fibrosis and dysfunction are reduced in C3−/− mice 4 weeks after myocardial IR injury

After a clinically relevant period of myocardial ischemia, C3−/− mice had a significant reduction in necrosis accompanied by an increase in apoptosis during the acute phase of reperfusion. Whether such a change in cell death type could lead to a different long term outcome in heart structure and function is not known. Published work has established that following ischemia, a reparative cardiac fibrosis occurs resulting from cardiomyocyte death, to replace the void left by dead myocytes [9]. Its hallmark is the excessive deposition of extracellular matrix, leading to tissue scarring and organ dysfunction. This is either manifested as myocardial stiffness (diastolic dysfunction) or may impact the entire left ventricle causing dilatation and systolic dysfunction, eventually resulting in heart failure [10,48]. The extent of fibrosis is important for prognosis of heart failure [49,50].

Using C3−/− and WT mice, we examined the long term effect of C3 on post-infarction heart failure, as indicated by cardiac fibrosis and heart dysfunction. Four weeks after the initial 1 h of ischemia, C3−/− mice had significantly less cardiac fibrosis than WT mice (Fig. 1c and d). When cardiac function was evaluated by echocardiography at this time, C3−/− mice showed significantly better LVEDD than WT control mice (Fig. 1 e).

4. Discussion

We used a clinically relevant myocardial IRI model (60 min of ischemia resembling DTBT time followed by 24 h reperfusion) to investigate the mechanisms of cell death involving C3 in the acute phase of infarction. In mice genetically deficient in C3 which is the central molecule in all complement pathways, myocardial necrosis was significantly reduced compared with WT mice (Fig. 1a–b). The results imply that in WT mice during IR, C3 acts to promote necrosis.

Our studies showed that 4 weeks after IR injury, C3−/− mice had significantly less post-ischemia cardiac fibrosis and improved cardiac function (Fig. 1 c-e). These results indicate that the myocardial IR inflammatory response in WT mice involving C3 results in increased cardiac fibrosis and adverse tissue remodeling. Our results were in alignment with an earlier report by Weisman et al. that using sCR1 as an inhibitor of C3 activation in WT mice significantly reduced IRI in a rat model [51]. It is of note that sCR1 was administered to the animals before ischemia in Weisman's study, suggesting that pre-emptive inhibition of C3 may be necessary to protect heart before ischemia occurs. Similarly, our model used C3−/− mice which lack C3 before ischemia took place. Thus, the effect of C3 on IRI of WT animals may start at the ischemia phase (by C3 remaining in the area of ischemia), then exacerbate at reperfusion phase when circulation bring more C3 once the blood flow is re-established in the ischemic area. Whether C3 acted directly at extracellular or intracellular target(s) in IRI was not determined in this study but could be an interesting line of future studies. Nevertheless, our previous studies suggested that intracellular target(s), such as non-muscle myosin, may be exposed to natural antibodies during the IRI and subsequently activate complement system [43,52,53]. Therefore, it is possible that C3 participates in the recognition of intracellular targets being exposed in IRI.

It is of note that in our study, cardiac function of WT mice had sharply deteriorated at 4 weeks after ischemia, which is in agreement with others using rodent models of IRI [[54], [55], [56]]. One possible explanation is that cardiac function was initially restored by reperfusion, but cardiac fibrosis gradually evolved and reached the threshold at 4 weeks after ischemia. Thus, the over-threshold level of cardiac fibrosis overweighted the capacity of heart compensation and pronounced the failing of cardiac function at 4 weeks.

A recent study by Torf et al. using a Langendorff heart perfusion model found that C3 knockout mouse heart had larger infarct size than WT heart [57]. Although it seemed contrary to our findings, the main differences are: 1) Langendorff model uses an isolated heart with global ischemia which rarely happens in human acute myocardial infarction (AMI). In contrast, our in vivo IRI model mimics the human AMI scenario of regional ischemia. 2) The reperfusion time in Torf's study was only 60 min, while our study examined the long term effects of cardiac fibrosis and function (4 weeks). Thus, Torf's findings may have certain value at the first hour immediately after ischemia, our findings imply the long term effects which are more resemble to clinical scenario.

Our results suggest that targeting C3 has the potential to reduce significantly post-infarction heart failure. In AMI, shortening ischemia by hastening reperfusion of occluded coronary vessels, especially a DTBT of ≤90 min for primary percutaneous coronary intervention (∼60 min in the U.S.), results in smaller infarct size and lower mortality [[39], [40], [41],58]. Thus, the research focus has thus shifted from reducing mortality to tackling the consequence of survival: post-infarction heart failure [59,60]. This process, which has increased globally in the past 3 decades, places a substantial burden on health-care systems [61,62].

The main determinant of post-infarction heart failure is infarct size, which results not only from ischemia but from reperfusion injury. Currently, clinicians and basic scientists are focusing on therapies to reduce the latter [63]. However, to date, large-scale clinical trials, i.e. antagonism of the renin-angiotensin- aldosterone system, have provided limited evidence of clinical benefit to heart failure and few interventions have successfully passed the proof-of-concept stage [12,64]. Our results provide new insights into the mechanisms of reperfusion injury, and thus may offer potential therapeutic strategies. For instance, targeting complement C3 during reperfusion following AMI may significantly benefit patients and reduce long term healthcare costs. As several inhibitors for complement C3 are being tested in various clinical trials of rare diseases [65,66], it is possible that some inhibitors may be effective in cardiovascular diseases as well.

In summary, this study has identified a mechanism during IR involving C3 that modulates the nature of cell death relevant to the pathological outcome of IR injury. As a long-term consequence of IR injury, C3- plays an important role in necrotic cell death which contributes to the cardiac fibrosis that underlies post-infarction heart failure. The study provides new insights into the mechanisms of IR injury and suggests a potential approach for intervention in post-infarction heart failure.

Declaration of competing interest

No conflict of interest for all.

Acknowledgements

The authors thank Drs. James Cottrell and David Wlody for their continued support, and Dr. Julie Rushbrook for helping to edit this manuscript.

Data availability

Data will be made available on request.

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