Monocyte-Derived Macrophages Aggravate Cardiac Dysfunction After Ischemic Stroke in Mice

Abstract

Background: Cardiac damage induced by ischemic stroke, such as arrhythmia, cardiac dysfunction, and even cardiac arrest, is referred to as cerebral-cardiac syndrome (CCS). Cardiac macrophages are reported to be closely associated with stroke-induced cardiac damage. However, the role of macrophage subsets in CCS is still unclear due to their heterogeneity. Sympathetic nerves play a significant role in regulating macrophages in cardiovascular disease. However, the role of macrophage subsets and sympathetic nerves in CCS is still unclear.

Methods and results: In this study, a middle cerebral artery occlusion mouse model was used to simulate ischemic stroke. ECG and echocardiography were used to assess cardiac function. We used Cx3cr1^GFPCcr2^RFP mice and NLRP3-deficient mice in combination with Smart-seq2 RNA sequencing to confirm the role of macrophage subsets in CCS. We demonstrated that ischemic stroke-induced cardiac damage is characterized by severe cardiac dysfunction and robust infiltration of monocyte-derived macrophages into the heart. Subsequently, we identified that cardiac monocyte-derived macrophages displayed a proinflammatory profile. We also observed that cardiac dysfunction was rescued in ischemic stroke mice by blocking macrophage infiltration using a CCR2 antagonist and NLRP3-deficient mice. In addition, a cardiac sympathetic nerve retrograde tracer and a sympathectomy method were used to explore the relationship between sympathetic nerves and cardiac macrophages. We found that cardiac sympathetic nerves are significantly activated after ischemic stroke, which contributes to the infiltration of monocyte-derived macrophages and subsequent cardiac dysfunction.

Conclusions: Our findings suggest a potential pathogenesis of CCS involving the cardiac sympathetic nerve-monocyte-derived macrophage axis.

Keywords: NLRP3 inflammasome; cardiac dysfunction; ischemic stroke; monocyte‐derived macrophage; sympathetic nerve.

Figure 1. Abnormal cardiac systolic function and electrocardiogram presented in MCAO mice.

A, Representative CBF imaging indicates the right MCA blood supply area (white frame) before and after occlusion as well as reperfusion in the sham or MCAO mice. Scale bar: 1 mm. B, Quantification of the CBF fractional changes in the sham group (n=8) and MCAO group (n=13). C, Representative brain slices from mice with triphenyl tetrazolium chloride staining at 3 days after surgery. The white area refers to the infarcted area. Scale bar: 5 mm. D, Representative images of right hemispheric atrophy in the MCAO groups at 28 days after surgery. Scale bar: 2 mm. E, ECG monitoring on day 3 and day 28 after the sham and MCAO procedure, with black lines representing sham mice and red lines representing MCAO mice. F, Quantification of the QT (P=0.0231 and P=0.0157) and QTc intervals (P=0.0105 and P=0.0001) on day 3 (n=8 in the sham group and n=13 in the MCAO group) and day 28 (n=5 in the sham group and n=9 in the MCAO group) based on the distance between the Q‐wave and T‐wave. G, Representative echocardiography frames on day 3 and day 28 after the sham or MCAO procedure. LVIDd and LVIDs are indicated by yellow lines. H, Quantification of the left ventricular ejection fraction (P=0.5423 and P=0.0032) and fractional shortening (P=0.1768 and P=0.0027) on day 3 (n=8 in the sham group and n=13 in the MCAO group) and day 28 (n=5 in the sham group and n=9 in the MCAO group). CBF indicates cerebral blood flow; LVIDd, left ventricular internal diameter at end diastole; LVIDs, left ventricular internal diameter at end systole; MCA, middle cerebral artery; MCAO, middle cerebral artery occlusion; and n.s., not significant. *P<0.05, **P<0.01, ***P<0.001.

Figure 2. Cardiac macrophage subset altered after ischemic stroke.

A, Increased cardiac macrophages (blue arrow) in cardiac cross‐sectional sections are indicated by IBA1 immunohistochemical staining. Scale bar: 1 mm and 100 μm. B, Quantification of cardiac macrophages in the sham and MCAO groups (n=5 in each group, P=0.0007); the y axis represents the IBA1‐positive cell number. Each dot represents the mean IBA1‐positive cell number of 4 different fields of view in each slice of the heart. C, Representative flow cytometry plots of resident macrophages (green frame) and monocyte‐driven macrophages (red frame). D, Quantification of resident‐(CX3CR1⁺, P=0.9306) and monocyte‐derived macrophages (CCR2⁺, P=0.0369) in the sham and MCAO groups (n=4 in each group). E, Representative immunofluorescence images of resident‐ (yellow arrow) and monocyte‐driven macrophages (white arrow) in cross‐sectional sections of the heart. Green: CX3CR1; Red: CCR2. Scale bar: 100 μm. F, Quantification of CX3CR1⁺ (P=0.3908) and CCR2⁺ (P=0.0025) in the sham and MCAO groups (n=5 in each group). The y axis represents the number of CX3CR1⁺‐ or CCR2⁺‐positive cells. Each dot represents the mean number of positive cells in 3 different fields of view in each slice of the heart. CCR2 indicates C‐C chemokine receptor type 2; CX3CR1, C‐X3‐C motif chemokine receptor 1; IBA1, ionized calcium binding adaptor molecule 1; MCAO, middle cerebral artery occlusion; and n.s., not significant. *P<0.05, **P<0.01.

Figure 3. Pharmacological inhibition of CCR2⁺ monocyte‐derived macrophages ameliorated ischemic stroke‐induced cardiac dysfunction.

A, Quantification of cardiac CCL2 mRNA expression level in the sham and MCAO groups (n=5 in each group, P=0.0110). B, CCL2 protein of myocardial tissue is indicated by Western blot and quantification in the sham and MCAO groups (n=6 in each group, P=0.0303). C, Schematic diagram of RS‐504394 treatment to inhibit CCR2⁺ monocyte‐derived macrophages in vivo. Mice received RS‐504394 (1 mg/kg) once a day 24 hours before the MCAO or sham procedure. D, Representative IBA1 immunohistochemical staining of cardiac macrophages (blue arrow) in cardiac cross‐sectional section. Scale bar: 100 μm. Quantification of cardiac macrophages in the sham and MCAO groups with vehicle or RS‐504394 treatment (n=4 in each group, P<0.0001 and P<0.0001). The y axis represents the IBA1‐positive cell number. Each dot represents the mean IBA1‐positive cell number of four different fields of view in each slice of the heart. E, Representative echocardiography frames and quantification of the LVEF and LVFS on day 3 (n=8–13 in each group) and day 28 (n=4–6 in each group) in the sham and MCAO groups with vehicle or RS‐504394 treatment. F, Representative ECG monitoring image and quantification of the QT and QTc intervals on day 3 (n=8–10 in each group) and day 28 (n=4–6 in each group) in the sham and MCAO groups with vehicle or RS‐504394 treatment. CCL2 indicates chemokine ligand 2; CCR2, C‐C chemokine receptor type 2; Echo, echocardiography; IBA1, ionized calcium binding adaptor molecule 1; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; MCAO, middle cerebral artery occlusion; n.s., not significant; and s.c., subcutaneous injection. *P<0.05, **P<0.01, ***P<0.001.

Figure 4. RNA sequencing of the cardiac macrophage subset during CCS progression.

A, The schematic diagram indicates that the mice were subjected to the MCAO procedure, and then hearts were collected for FACS sorting. All resident macrophages and monocyte‐derived macrophages of each mouse heart were collected for Smart‐seq2 RNA sequencing. B, The cell number of FACS‐sorted cardiac macrophages. Each dot represents the cardiac macrophages from a mouse. Red: monocyte‐derived macrophage; Green: resident macrophage. C, Volcano plot showing the differentially expressed genes between cardiac monocyte‐derived macrophages and resident macrophages in MCAO mice. Red: upregulated genes, blue: downregulated genes, gray: unchanged genes. RNA sequencing analysis with the parameters of P<0.05 and absolute fold change ≥1.2 were considered differentially expressed genes. D, Heat map of differentially expressed anti‐ and proinflammatory genes between different cardiac macrophage subsets in MCAO mice. E, GO enrichment analysis of cardiac monocyte‐derived macrophages and resident macrophages. The y axis represents the GO pathways, and the x axis represents the rich factor. CCS indicates cerebral‐cardiac syndrome; FACS, fluorescence‐activated cell sorting; GO, Gene Ontology; and MCAO, middle cerebral artery occlusion.

Figure 5. NLRP3 inflammasome activation in the heart contributed to CCS.

A, Heat map of NLRP3 inflammasome activation‐related genes expression from Smart‐seq2 RNA sequencing in MCAO mice. Red: upregulated genes, blue: downregulated genes. B, Quantification of NLRP3, ASC, Caspase1, and IL1β mRNA expression levels in the heart in the sham and MCAO groups (n=5 in each group, P=0.0026, P=0.0080, P=0.0014, and P=0.0001). C, Representative echocardiography frames and quantification of the LVEF and LVFS on day 3 and day 28 in the sham and MCAO groups of WT or Nlrp3 ^−/− mice (n=5–9 in each group). D, Representative ECG monitoring image and quantification of the QT and QTc intervals on day 3 and day 28 in the sham and MCAO groups of WT or Nlrp3 ^−/− mice (n=5–8 in each group). E, Representative IBA1 immunohistochemical staining of cardiac macrophages (blue arrow) in cardiac cross‐sectional section. Scale bar: 100 μm. Quantification of cardiac macrophages in the sham and MCAO groups in WT or Nlrp3 ^−/− mice (n=5 in each group, P<0.0001 and P=0.0036). CCS indicates cerebral‐cardiac syndrome; IBA1, ionized calcium binding adaptor molecule 1; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; MCAO, middle cerebral artery occlusion; NLRP3, NOD‐like receptor thermal protein domain associated protein; n.s., not significant; and WT, wild type. *P<0.05, **P<0.01, ***P<0.001.

Figure 6. Cardiac sympathetic nerves from the superior cervical ganglion were activated after ischemic stroke.

A, Schematic diagram showing the generation of Th ^tdTomato mice by crossing Th ^Cre/+ mice and ROSA26 ^tdTomato/+ (Ai9) mice. B, Schematic diagram of intramyocardial WGA‐488 injection for retrograde neural tracers in Th ^tdTomato mice. Representative images of intramyocardial injections by using an ultrasound‐guided transthoracic procedure. The red arrow indicates the needle. The red dashed area indicates the outer wall of the left ventricle, and the white dashed area indicates the inner wall of the left ventricle. C, Representative images of cardiac slices after intramyocardial WGA‐488 injection. WGA‐488 in the heart indicates a successful injection. Green: WGA‐488; Red: tdTomato; Blue: DAPI. Scale bar: 100 μm and 1 mm. D, Representative images of retrograde WGA‐488 (green) in TH‐positive cells (red, white arrow) in the SCG. Scale bar: 100 μm. E, Representative images of activating neurons (white arrow) in the SCG with C‐fos staining and quantification of the number of positive cells in sham and MCAO mice (n=5 in each group, P=0.0001). The y axis represents the number of C‐fos‐positive cells in the SCG. Each dot represents the mean number of positive cells in 3 different fields of view of each slice. Green: C‐fos; Blue: DAPI. Scale bar: 50 μm. F, Quantification of the level of norepinephrine in serum in sham (n=8) and MCAO mice (n=9, P=0.0347). G, Quantification of the ratio of low‐frequency power to high‐frequency power in heart rate variability in sham and MCAO mice (n=6 in each group, P=0.0021). MCAO indicates middle cerebral artery occlusion; SCG, superior cervical ganglion; and TH, tyrosine hydroxylase. *P<0.05, **P<0.01, ***P<0.001.

Figure 7. Cardiac sympathetic nerve activation recruited cardiac macrophage and induced cardiac dysfunction.

A, Representative images refer to the spatial relationship between cardiac macrophages (yellow arrow) and sympathetic nerve fibers (white arrow). Green: IBA1; Red: TH; Blue: DAPI. Scale bar: 50 μm. B, Schematic diagram indicating that SCG resection was performed at 7 days before the MCAO or sham procedure in mice. C, Representative IBA1 immunohistochemical staining of cardiac macrophages (blue arrow) in cardiac cross‐section. Scale bar: 100 μm. Quantification of cardiac macrophages in the sham and MCAO groups with and without SCG resection (n=5 in each group, P<0.0001 and P<0.0001). The y axis represents the IBA1‐positive cell number. Each dot represents the mean IBA1‐positive cell number of 4 different fields of view in each slice of the heart. D, Representative echocardiography frames and quantification of the LVEF and LVFS on day 3 and day 28 in the sham and MCAO groups with and without SCG resection (n=6–10 in each group). E, Representative ECG monitoring image and quantification of the QT and QTc intervals on day 3 and day 28 in the sham and MCAO groups with and without SCG resection (n=6–10 in each group). IBA1 indicates ionized calcium binding adaptor molecule 1; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; MCAO, middle cerebral artery occlusion; n.s., not significant; and SCG, superior cervical ganglion. *P<0.05, **P<0.01, ***P<0.001.