In this investigation, 4 individuals afflicted with HF (with patients 2–4 specifically presenting with DCM) were subjected to a trans jugular interventricular septum myocardial biopsy. The cardiac biopsy samples were utilized for pathological evaluation, whereas biopsy samples of HF patients with non-DCM were employed as a control group. Table 1 provides the demographic characteristics of the 4 DCM patients. Incorporated into this investigation of cell sorting in vitro were 23 individuals who had been clinically diagnosed with DCM, as well as 14 healthy individuals who served as controls. Table 2 provides an overview of the patients’ general information. The exclusion criteria were as follows: myocarditis, pericardial disease, acute cerebrovascular disease, moderate or severe liver dysfunction, severe infection, severe lung disease, severe renal dysfunction (estimated glomerular filtration rate [eGFR] 15 mL/min/1.73 m${}^2$, calculated by using the CKD-EPI formula), history of malignant tumors, thyroid disorders, autoimmune diseases, hemopathy and recently experienced trauma or surgery. The study was approved by the Ethics Committee of Tianjin Chest Hospital and written informed consent was obtained from all participants.

Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy and DCM donors using density centrifugation with Lymphoprep (STEMCELL Technologies, Vancouver, Canada). From the PBMCs fraction, CCR2${}^{+}$ monocytes were isolated by magnetic-activated cell sorting (MACS) using a magnetic pole (EasySepTM #18000, Miltenyi, Cologne, Germany), EasySep™ Release Human PE Positive Selection Kit (#17654, STEMCELL Technologies, Vancouver, Canada) and CD192 (CCR2) and Antibody #130-118-338 (Miltenyi, Cologne, Germany). In order to generate CCR2${}^{+}$ bone marrow-derived macrophages (BMDMs), 10${}^7$/mL CCR2${}^{+}$ PBMCs were seeded into a six well plate and cultured for a period of 5 days in RPMI 1640 medium (#31870074, Life Technologies, Carlsbad, CA, USA) supplemented with 20 ng/ml of M-CSF (#14-8789-80, eBioscience, San Diego, CA, USA) and 10% of FBS (Lonza, Basel, Switzerland). On the third day, the medium was replaced to ensure optimal growth conditions. CCR2${}^{+}$ BMDMs attached to plates were detached using StemPro Accutase Cell Dissociation Reagent (Lonza, Basel, Switzerland). Cell activity was assessed using MTT assay (MTT kit, #C0009S, Beyotime Biotech. Inc., Beijing, China).

The Seahorse XF24 analyzer (Seahorse Bioscience, USA) was utilized to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in accordance with our previously described method [13]. CCR2${}^{+}$ BMDMs (5 $\times$ 10${}^6$ cells/mL) were grown in 24-well plates with 1 µM oligomycin, 1 µM trifluoromethoxy carbonyl cyanide phenylhydrazone, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and 1 µM rotenone together with 1 µM antimycin A were added in sequence. The Seahorse analyzer software was utilized to calculate OCR and ECAR.

CCR2${}^{+}$ BMDMs (5 $\times$ 10${}^6$ cells/mL) were placed in a glucose-free RPMI medium with 5 µM of fluorescent D-glucose analogue 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (Cayman Chemical, Ann Arbor, MI, USA) and incubated for an hour at 37 °C. The Lionheart FX automated imaging system (Bio Tek, Winooski, VT, USA) was employed to analyze the fluorescent intensities.

Isolation of intact mitochondria from macrophages was conducted in accordance a commercially available mitochondrial extraction kit (Solarbio, Beijing, China) as per the manufacturer’s instructions. For the purpose of measuring intramitochondrial ROS level, isolated mitochondria were transferred to a 96-well flat-bottomed plate and a 2${}^{\prime }$, 7${}^{\prime }$-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe detection kit (#C2938, Thermo Fisher Scientific, Waltham, CA, USA) together with a Lionheart FX automated imaging system (Bio Tek, USA) was used.

Quantitative real-time polymerase chain reaction (RT-PCR) was conducted using the mRNA reverse transcription kit (Roche, Basle, Switzerland) as per the manufacturer’s instructions. SYBR Green PCR master mix (Roche, Basle, Switzerland) was employed in RT-PCR, which was conducted using a CFX96TM PCR detection system (BioRad, Redmond, WA, USA). The primer sequence is provided in Table 3.

CCR2${}^{+}$ PBMCs and BMDMs were transfected with GLUT1 inhibitory sequence (GLUT1${}^{-/-}$) containing lentivirus (Genechem, Shanghai, China) or lentivirus containing scrambled control sequences. Then, 5 $\times$ 10${}^6$ cells/mL CCR2${}^{+}$ PBMCs and BMDMs were inoculated into a 6-well plate. The infection reagent and lentivirus were added to CCR2${}^{+}$ PBMCs and BMDMs according to the manufacturer’s instructions. Following a 72-hour infection period, the identification of GLUT1 mRNA expression in CCR2${}^{+}$ PBMCs and BMDMs was conducted through the utilization of RT-PCR.

The heart tissues were fixed in 4% paraformaldehyde for 72 hours, following which they were embedded in paraffin and cut into 4 µm thick sections. The sections were then subjected to hematoxylin/eosin staining (H&E). The tyramide signal amplification plus multiplex fluorescence staining kit (#G1236-100T, Servicebio, Wuhan, Hubei, China) was used for staining CCR2 (#ab254375, Abcam, Cambridge, UK), CX3CR1 (#ab167571, Abcam, Cambridge, UK) and $\alpha$-Actin (#ab11003, Abcam, Cambridge, UK) according to the manufacturer’s protocol. Following washing with PBS, the sections were counterstained with DAPI and observed through a fluorescence microscope and digital camera (Axio Observer Al, Carl Zeiss, Germany). Immunohistochemical staining of CD3 (total T cells), CD4 (helper T cells), CD8 (cytotoxic T cells), CD68 (macrophages), BCL-2 (proteins marker of apoptosis), CD19 (B cells) and CD20 (B cells) was entrusted to the Pathology Department of Tianjin Chest Hospital.

Western blot analysis was performed to determine the NLRP3 (#ab263899, Abcam, Cambridge, UK) expression in CCR2${}^{+}$ macrophages. The relative values were adjusted to GAPDH expression levels and normalized relative to the baseline controls.

Data analysis was conducted using SPSS software version 24 (v24, IBM Corp., Chicago, IL, USA). The Shapiro-Wilk test was performed to determine the normality of continuous variables. Normally distributed continuous variables were presented as mean and standard deviation ($\overline{x}$$\pm$ s), and Intergroup comparisons were done using independent two-tailed Student’s t-tests. For differences across multiple groups with one variable, one-way analysis of variance (ANOVA) was utilized, and for groups with multiple variables, a two-way ANOVA was applied. Non-normally distributed continuous variables were presented as median and interquartile interval (M(Q)), and the Wilcoxon Mann-Whitney test was used to compare different groups. Categorical variables were expressed as frequencies and compared using Fisher’s exact test. A p value 0.05 was considered statistically significant. All experimental n numbers are provided in the figure legends.

From July to October 2021, the Cardiac intensive care unit of Tianjin Chest Hospital administered cardiac biopsies on four patients that had been clinically diagnosed with cardiomyopathy; these results showed that three of the cases correlated with the clinical manifestations of heart failure resulting from dilated cardiomyopathy, and they were consequently included in the study. Patients 2–4 were clinically identified as having DCM, Patients 3 and 4 were identified as having heart failure (DCM/HF), as demonstrated by the clinical information. Relevant examinations and tests are provided in Table 2. Fig. 1A provides typical cardiac magnetic resonance (CMR) images of these patients.

Fig. 1.

Patient 1-4 CMR histological examination. (A) Typical CMR images of patient 1–4. (B) H&E staining of patient 1–4 (scale bar = 100 µm). (C) Immunohistochemical staining of CD3, CD4, CD8, CD68, BCL-2, CD19 and CD20 (scale bar = 100 µm). (D) Patient 1-4 immunostaining of CX3CR1 (Red), $\alpha$- Actin (green), CCR2 (Pink) (scale bar = 20 µm).

Histological examination (H&E staining) revealed that the myocardium of DCM patients suffered from severe edema and vacuolar degeneration, with leukocyte infiltration between the myocardium The degree of injury increased as the left ventricular ejection fraction decreased (Fig. 1B). Immunohistochemical staining revealed that CD19 and CD20 were not present in the myocardium of the patients, whereas CD68, CD3, CD4, CD8 and BCL2 were expressed (Fig. 1C).

The connection between macrophages and myocardial injury was assessed through co-localization immunofluorescence staining (Fig. 1D, CX3CR1: cardiac resident macrophage marker, CCR2: myeloid proinflammatory macrophage marker, $\alpha$- Actin: myocardial skeleton protein). Results indicated that CX3CR1${}^{+}$ cells (i.e., cardiac resident macrophages, Red) were exhausted in both DCM and DCM/HF patients, and as DCM progressed to HF, the number of proinflammatory macrophages (i.e., circulating infiltrating macrophages, CCR2${}^{+}$ cells, purple) significantly increased (Fig. 1D). The findings revealed that the increased number of CCR2${}^{+}$ macrophages was associated with the myocardial injury of DCM. This was further substantiated by the CMR examination results (CMR, Fig. 1A), which confirmed the positive correlation between the degree of CCR2${}^{+}$ macrophage cardiac injury and DCM heart.

To verify the link between CCR2${}^{+}$ macrophages and DCM, a study was conducted on CCR2${}^{+}$ monocytes isolated from peripheral blood of 23 DCM patients and 14 control patients. The patient information is provided in Table 3. Briefly, the DCM group and the control group exhibited considerable differences in assessing the primary indicators of heart failure such as NYHA classification, LVEF%, and the level of NT-proBNP and hs-TnT; No substantial divergence was observed between the two groups in terms of the prevalence of hypertension and coronary artery stenosis; The usage of anti-heart-failure-related drugs such as angiotensin converting enzyme inhibitor (ACEI)/ angiotensin receptor antagonist (ARB)/ angiotensin receptor enkephalinase inhibition (ARNI), $\beta$ blockers, spironolactone, diuretics, and digoxin in the DCM group was significantly higher than that in the control group; and the proportion of patients in the two groups who had ingested ivabradine, statins, nitrates and anticoagulants was not significantly different.

Isolated CCR2${}^{+}$ monocytes were induced into macrophages in vitro and the mRNA expression analyzed for inflammation-related genes (IL-1$\beta$, IL-6 and TNF-$\alpha$) in CCR2${}^{+}$ monocytes and CCR2${}^{+}$ macrophages by RT-PCR. The results revealed that the mRNA expression of inflammation-associated genes IL-1$\beta$, IL-6 and TNF-$\alpha$ in CCR2${}^{+}$ monocytes and macrophages from the DCM group was significantly higher than that of the control group (Fig. 2A–C). There was a positive correlation to the New York Heart Association (NYHA) classification (Fig. 2D–F). It was observed that with the deterioration of cardiac function, the expression of inflammatory related mRNA in CCR2${}^{+}$ monocytes and macrophages from peripheral blood of patients went higher (Fig. 2D–F).

Fig. 2.

The level of inflammation in CCR2${}^{\mathrm{+}}$ monocytes and macrophages of patients with dilated cardiomyopathy. mRNA expression of inflammation-associated genes IL-1$\beta$ (A), IL-6 (B) and TNF-$\alpha$ (C) in CCR2${}^{+}$ monocytes and macrophages. NYHA classification is associated with IL-1$\beta$ (D), IL-6 (E) and TNF-$\alpha$ (F) mRNA expression. mRNA expression of inflammation-associated genes TGF-$\beta$ (G), MMP2 (H) and MMP9 (I) in CCR2${}^{+}$ macrophages. NYHA classification is associated with TGF-$\beta$ (J), MMP2 (K) and MMP9 (L) mRNA expression. (M) Relationship between statins used by DCM and IL-1$\beta$, IL-6 and TNF-$\alpha$. (N) Relationship between $\beta$-receptor blockers, ARNI, and Rivaroxaban/Dabigatran used by DCM and IL-1$\beta$. (O) Relationship between $\beta$-receptor blockers, ARNI, and Rivaroxaban/Dabigatran used by DCM and TGF-$\beta$. All data are presented as the mean $\pm$ SD. Statistical significance is indicated as: *p 0.05, **p 0.01 compared with the control group.

The mRNA expression of the related genes of extracellular matrix remodeling such as transforming growth factor-$\beta$ (TGF-$\beta$), matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) in CCR2${}^{+}$ macrophages were examined using RT-PCR. Compared to the control group, the mRNA expression of TGF-$\beta$, MMP2 and MMP9 of CCR2${}^{+}$ macrophages in the DCM group were significantly higher (Fig. 2G–I) . There was a positive correlation between the mRNA expression and the worsening of NYHA cardiac function grading (Fig. 2J–L).

Utilizing the prior medication history of patients in the DCM group, consideration was given to possible effects of statins, $\beta$-receptor blockers, ARNI, and direct oral anticoagulants (Rivaroxaban/Dabigatran) — all of which are commonly prescribed for patients with heart failure. Statins had a slight effect on decreasing the IL-1$\beta$ and IL-6 mRNA expression of CCR2${}^{+}$ monocytes, though without any statistical significance (Fig. 2M–N). On the other hand, neither $\beta$ receptor blockers, ARNI, nor rivaroxaban/dabigatran had any effect on IL-1$\beta$ mRNA expression (Fig. 2N). Furthermore, statins, ARNI, and rivaroxaban/dabigatran had a slight effect on reducing the TGF-$\beta$ of CCR2${}^{+}$ macrophages mRNA expression, though without any statistical significance, while $\beta$ receptor blockers had no effect (Fig. 2O).

CCR2${}^{+}$ monocyte and macrophage oxygen consumption rate (OCR) did not differ statistically between the two groups (Fig. 3A,C), but extraceullular acidification rate (ECAR) was significantly higher between the two groups (Fig. 3B,D).

Fig. 3.

Metabolic reprogramming of CCR2${}^{\mathrm{+}}$ monocytes and macrophages in DCM patients. oxygen consumption rate (OCR) (A,C) and extracellular acidification rate (ECAR) (B,D) in CCR2${}^{+}$ monocytes and macrophages measured using a Seahorse Bioscience XF24 analyzer (n = 4). Probes were done with serial addition of A: oligomycin, B: FCCP, and C: antimycin A/rotenone as indicated (E,F) are heat maps displaying the mRNA expression of genes related to glycolysis in CCR2${}^{+}$ monocytes and macrophages (n = 5). Also shown are the mRNA expression of GLUT 1–4 in CCR2${}^{+}$monocytes (G) and macrophages (H), n = 5. (I) mRNA expression of PKM2 in CCR2${}^{+}$ monocytes and macrophages, n = 5. (J) Glucose uptake in CCR2${}^{+}$ monocytes and macrophages were measured using the fluorescence-labeled glucose analogue (2-NBDG) by mean fluorescence intensity (MFI). (K) reactive oxygen species (ROS) levels in CCR2${}^{+}$ monocytes and macrophages using MitoSOX fluorescent probe. (L) ROS levels in CCR2${}^{+}$ macrophages exposed to different concentrations of glucose. NYHA classification is associated with 2-NBDG MFI (M) and mtROS MFI (N). (O) the activity of CCR2${}^{+}$ monocytes and macrophages, n = 5. IL-1$\beta$ (P), TNF-$\alpha$ (Q) and IL-6 (R) mRNA expression level after administering 2-DG, n = 5. All data are presented as the mean $\pm$ SD. Statistical significance is indicated as: *p 0.05, **p 0.01 compared with the control group.

RT-PCR analysis revealed an upregulation of CCR2${}^{+}$ monocyte and macrophage glycolysis related genes, such as pyruvate kinase isoform M1 (PKM1), pyruvate kinase isoform M2 (PKM2), GLUT1, GLUT2, GLUT3, GLUT4, phosphoinositol dependent protein kinase 1 (PDK1), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases 3 (PFKFB3), phosphofructokinase 1 (PFK1), and hexokinase 2 (HK2), in DCM patients (Fig. 3E,F). Quantitative analysis further revealed that the expression of GLUT1 in CCR2${}^{+}$ monocytes and macrophages of DCM patients was significantly higher than that of the control group, with statistical significance, while the expression of GLUT2, GLUT3 and GLUT4 had no statistical difference between the two groups (Fig. 3G,H).The enzyme PKM2, a pivotal component of glycolysis, displayed a similar up-regulation trend as the other enzymes, with a statistically significant variation between the two groups (Fig. 3I).

CCR2${}^{+}$ monocytes and macrophages of DCM patients had high glucose uptake and ROS levels in comparison to the control group (Fig. 3J,K). This increase correlated with a worsening of cardiac function (NYHA classification, Fig. 3M,N). The mitochondrial ROS level of CCR2${}^{+}$ monocytes and macrophages of DCM patients was found to be positively correlated with glucose concentration (Fig. 3L).

CCR2${}^{+}$ monocytes and macrophages from DCM patients were exposed to 2-DG, an artificial glucose analogue that can simulate a lack of glucose for 6 hours. The activity of CCR2${}^{+}$ monocytes and macrophages remained unchanged (Fig. 3O), but IL-6 and IL-1$\beta$ mRNA expression level decreased significantly (Fig. 3P,R). There was no major fluctuation in the TNF-$\alpha$ mRNA expression (Fig. 3Q).

To further investigate the potential mechanism of CCR2${}^{+}$ monocytes and macrophages inflammation and glucose uptake, shRNA was employed to suppress the mRNA expression of GLUT1 in CCR2${}^{+}$ monocytes and macrophages from DCM patients. Immunofluorescence staining revealed that CCR2${}^{+}$ macrophages in DCM patients expressed GLUT1 at a higher level than those in the control group (Fig. 4A). Furthermore, silencing CCR2${}^{+}$ monocytes and macrophages in DCM patients with shRNA resulted in a decrease in the mRNA expression of GLUT1 (GLUT1${}^{-/-}$, Fig. 4B). Following the silencing of GLUT1, the glucose uptake capacity of CCR2${}^{+}$ monocytes and macrophages in the DCM group was significantly decreased (Fig. 4C), the level of mitochondrial ROS was significantly decreased (Fig. 4D), and the expression of the inflammatory factor IL-1$\beta$ was significantly reduced (Fig. 4E). However, silencing GLUT1 had no significant effect on the mRNA expression of IL-6 and TNF-$\alpha$ (Fig. 4F,G). In addition, for NOD-like receptor protein 3 (NLRP3) under GLUT1${}^{-/-}$, the results revealed that the NLRP3 expression in CCR2${}^{+}$ macrophages from the peripheral blood of DCM group were significantly higher than that of the control group. Also, the NLRP3 expression significantly decreased after GLUT1 was silenced (Fig. 4H,I). Furthermore, NLRP3 immunofluorescence staining also confirmed this result (Fig. 4J).

Fig. 4.

GLUT1 regulates the metabolism reprogramming of CCR2${}^{\mathrm{+}}$ macrophages in DCM patients to promote inflammatory response. (A) Immunostaining of GLUT1 in CCR2${}^{+}$ macrophages. (B) mRNA expression of GLUT1 in DCM CCR2${}^{+}$ monocytes and macrophages under GLUT1${}^{-/-}$, n = 5. The level of 2NBDG MFI (C) and mtROS MFI (D) in DCM CCR2${}^{+}$ monocytes and macrophages under GLUT1${}^{-/-}$, n = 5. IL-1$\beta$ (E), IL-6 (F) and TGF-$\beta$ (G) mRNA expression level in DCM CCR2${}^{+}$ monocytes and macrophages under GLUT1${}^{-/-}$, n = 5. (H,I) Protein expression of NLRP3 in CCR2${}^{+}$ macrophages under GLUT1${}^{-/-}$, n = 3. (J) Immunostaining of NLRP3 in DCM CCR2${}^{+}$ macrophages. All data are presented as the mean $\pm$ SD. Statistical significance is indicated as: *p 0.05, **p 0.01 compared with the control group.