All compounds, including ISO (Cat No. 420355) and SLU-PP-332 (Cat No. SML3908), were commercially sourced from Sigma-Aldrich (St. Louis, MO, USA). Mouse IgG was sourced from Bioss Biotechnology Company (Beijing, China, Cat No. bs-0296P). DRm217 antibody, a particular antibody targeting the DR-region (⁸⁹⁷DVEDSYGQQWTYEQR⁹¹¹) of the NKA$\alpha$1 subunit, was prepared in our lab [15]. The primary antibodies against $\alpha$-smooth muscle actin ($\alpha$-SMA, Cat No. GB111364-50), CD68 (Cat No. GB113109-50), LY6G (Cat No. GB11229-50), CD3 (Cat No. GB13014-50), and Horseradish Peroxidase (HRP)-labeled rabbit antibodies (Cat No. GB23303) were sourced from Servicebio Technology Company (Wuhan, Hubei, China). Primary antibody targeting estrogen-related receptor $\alpha$ (ERR$\alpha$, Cat No. CY5617) was sourced from Abways company (Shanghai, China), and primary antibody targeting $\beta$-actin (Cat No. 66009-1-Ig) was sourced from Proteintech Biotechnology Company (Wuhan, Hubei, China).

All animal procedures were approved by the Animal Care and Use Committee of Xi’an Jiaotong University (Ethics numbers: IAUC/765/2019, #19765) and adhered to guidelines established by the National Health and Medical Research Council of China. Eight-week-old male wild-type (WT) and NKA$\alpha$1^+⁣/- mice were subcutaneously injected with ISO (30 mg/kg) daily for 14 consecutive days. Following euthanasia via cervical dislocation, hearts were harvested for subsequent analyses. To evaluate the effects of DRm217, 18 male C57BL/6 mice were randomly assigned to saline, ISO+IgG, or ISO+DRm217 groups. IgG or DRm217 was administered intraperitoneally at a dose of 10 mg/kg every 5 days.

To evaluate histopathological changes, the hearts were rinsed with 0.9% saline and subsequently fixed in 4% paraformaldehyde. Each heart was divided into three segments and embedded in paraffin. Serial sections of 5 µm thickness were prepared from the basal, mid, and apical levels of the heart. The sections were stained with hematoxylin and eosin (H&E). A pathologist who was blinded to the identify of experimental groups evaluated the histopathological scores based on hyper-eosinophilic bundles, leukocyte infiltration, and cardiomyocyte necrosis. At least 10 fields per slide were examined, with the severity of changes graded as severe (++++), moderate (+++), mild (++), minimal (+), or nil (–). The extent of myocardial fibrosis was assessed using Masson’s trichrome staining, while collagen content was quantified after staining with Sirius red. Histopathological images were captured and analyzed using a digital camera attached to a microscope (Nikon, Tokyo, Japan). The percentage of fibrotic area in each image was measured using ImageJ software (VERSION 1.53t, National Institutes of Health, Bethesda, MD, USA), and calculated as the ratio of positively stained areas to the total field area.

For antigen retrieval, paraffin-embedded sections were deparaffinized, rehydrated, and immersed in 0.01 M citric acid buffer (pH 10.0). Sections were treated with 3% hydrogen peroxide for 10 minutes to quench endogenous peroxidase activity, then blocked with rabbit serum for 30 minutes at room temperature. Subsequently, the slides were incubated overnight at 4 °C with primary antibodies against CD68 (1:100), Ly6G (1:100), CD3 (1:100), or $\alpha$-SMA (1:100). Next, slides were incubated with HRP-labeled goat anti-rabbit secondary antibody for 1 h. Sections were then washed, stained with 3,3’-Diaminobenzidine (DAB) (Cat No. G1212-200T, Servicebio Technology Company, Wuhan, Hubei, China), and counterstained with hematoxylin. Histopathological images were captured and analyzed using a digital camera linked to a microscope (Nikon, Tokyo, Japan). To quantify immunohistochemistry results, a pathologist blinded to the experimental groups counted cells with positive immunostaining for CD68, Ly6G, and CD3. For alpha-smooth muscle actin ($\alpha$-SMA) staining, the positively stained area was analyzed using ImageJ software and expressed as the percentage of stained cortical area relative to the total area.

Heart tissues were homogenized in RIPA lysis buffer containing a protease inhibitor cocktail (Roche, Basel, Switzerland) and a phosphatase inhibitor cocktail (Sigma, St. Louis, MO, USA). After incubation on ice for 30 minutes, the supernatant was collected by centrifugation for 20 minutes at 4 °C and 12,000 rpm. Protein samples were separated by 10% Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE) and transferred onto a Polyvinylidene Fluoride (PVDF) membrane (Thermo Fisher Scientific, Waltham, MA, USA). The membrane was blocked with 10% milk in TBST buffer (10 mM Tris-HCl, 120 mM NaCl, and 0.1% Tween 20, pH 7.4) for 1 h at room temperature and then incubated with ERR$\alpha$ primary antibody (1:1000) overnight at 4 °C. Membranes were washed three times with TBST buffer and incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:5000) for 1 h at room temperature. After washing, visualization was performed using an enhanced chemiluminescence kit (GE Healthcare, Chicago, IL, USA). Protein bands were captured using ImageQuant LAS 400 (GE Healthcare, Chicago, IL, USA) and band intensity was quantified by densitometry analysis using ImageJ (version 1.53t, National Institutes of Health, Bethesda, MD, USA).

Cardiac tissues were fixed in 3% glutaraldehyde for 24 h and subsequently fixed in 1% osmium tetroxide for 1 h. Following dehydration in an ethanol gradient and 2% uranyl acetate staining, samples were embedded in Embed 812 resin for transmission electron microscopy (JEM-1400PLUS, JEOL, Tokyo, Japan) ultrastructural assessment. The images were reviewed in a blinded fashion by two radiologists.

The levels of interleukin-6 (IL-6, BGK08505), tumor necrosis factor-$\alpha$(TNF-$\alpha$, BGK06804), interleukin-1$\beta$ (IL-1$\beta$, BGK10749), and interleukin-18 (IL-18, ab216165) were measured using corresponding ELISA kits (Peprotech Company, Rocky Hill, NJ, USA for IL-6, TNF-$\alpha$, and IL-1$\beta$, and Abcam Company, Shanghai, China for IL-18) according to the manufacturer’s instructions.

Ventricular cardiomyocytes, macrophages, and fibroblasts were isolated from WT or NKA$\alpha$1^+⁣/- mice as previously described [16]. Cardiomyocytes were identified based on their characteristic rod-shaped morphology under light microscopy. Fibroblasts were recognized by their characteristic spindle-shaped or stellate morphology with cytoplasmic processes, along with relatively large oval-to-fusiform nuclei, observed under microscopy. Macrophages were confirmed using flow cytometry with antibodies against F4/80. To ensure uniformity across treatments and comparisons, isolation of cardiomyocytes, fibroblasts, and macrophages followed a strictly standardized protocol performed consistently by the same technician for all groups. Isolated cardiomyocytes were cultured on 0.1% gelatin-precoated 6-well plates in Minimum Essential Medium (MEM, Thermo Fisher Scientific, Waltham, MA, USA, Cat No. 11095080) supplemented with 5% fetal bovine serum (FBS, Lonsa Science SRL, Suzhou, China, Cat No, S711-050S), 100 U/mL penicillin (Thermo Fisher Scientific, Waltham, MA, USA, Cat No. 15070063), and 100 mg/mL streptomycin (Thermo Fisher Scientific, Waltham, MA, USA, Cat No. 15070063). Isolated macrophages were quantified and seeded in RPMI-1640 (Thermo Fisher Scientific, Waltham, MA, USA, Cat No. 11875093) medium supplemented with 10% FBS, 100 mg/mL streptomycin, and 100 U/mL penicillin. Isolated fibroblasts were quantified and seeded in DMEM (Thermo Fisher Scientific, Waltham, MA, USA, Cat No. 11965092). The isolated ventricular cardiomyocytes, macrophages, and fibroblast have undergone mycoplasma infection testing. To detect the effects of ISO on cardiomyocytes, macrophages and fibroblasts, 10 µM ISO was added to the culture medium for 48 h. For DRm217 treatment, IgG or DRm217 was suppled 15 minutes after ISO addition at a final dose of 1 µM concentration. To detect the effects of SLU-PP-332, a specific ERR agonist, it was suppled in cell culture medium at a final dose of 1 µM concentration.

For the co-culture models of cardiomyocytes with macrophages or fibroblasts, cardiomyocytes were pre-plated in 6-well plates at a density of 5 $\times$ 10⁵ cells/mL. Macrophages or fibroblasts were seeded onto cell culture inserts at a density of 1.0 $\times$ 10⁵ cells/cm². The inserts containing macrophages or fibroblasts were then placed above the wells containing cardiomyocytes. These cells were co-cultured in MEM supplemented with 10 µM ISO or vehicle for 48 h. For the co-culture model of macrophages and fibroblasts, fibroblasts were pre-plated in 6-well plates at a density of 5 $\times$ 10⁵ cells/mL. Inserts containing previously activated macrophages were placed above the wells containing fibroblasts. These cells were co-cultured in DMEM medium for 48 h.

Cellular injury was assessed by measuring LDH release. Following 48 h treatment with ISO (10 µM), LDH activity in the culture medium was measured using a Roche LDH assay kit (Cat No. 04744926001, Mannheim, Germany) according to the manufacturer’s protocol and a microplate reader.

Total RNA was isolated from heart tissue and cells using Trizol reagent (Cat No. B610409-0100, Sangon, Shanghai, China) and reverse-transcribed into cDNA using the MightyScript™ RT reagent kit (Cat No. B639252, Sangon, Shanghai, China). Quantitative polymerase chain reaction (qPCR) was performed using the SYBR Green reagent kit (Cat No. B532955, Sangon, Shanghai, China) on a QuantStudio™ 3 Real-Time PCR System (Bio-Rad, Hercules, CA, USA). Primer sequences used for qRT-PCR are listed in Table 1. All amplifications were normalized to $\beta$-actin. Data were analyzed using the comparative Ct (2^{-Δ⁢Δ⁢C⁢t}) method and expressed as fold change relative to the respective control.

Data are presented as the mean $\pm$ standard error. Statistical analysis was performed using SPSS software (version 22.0; IBM Corporation, Armonk, NY, USA). Student’s t-test was used for comparisons between two groups. Multiple group comparisons were analyzed by one-way ANOVA followed by Tukey’s post hoc test. A p-value 0.05 was considered statistically significant.

Since animals completely lacking the $\alpha$1 gene suffer from embryonic lethality, heterozygous mice lacking only one copy of the allele ($\alpha$1^+⁣/-) are a commonly used experimental model. H&E staining revealed myocardial cell rupture, cellular vacuolization, and inflammatory cell infiltration in WT mice treated with ISO. These pathological alterations were more pronounced in NKA$\alpha$1^+⁣/- mice treated with ISO (Fig. 1A,B). NKA$\alpha$1 haploinsufficiency also increased interstitial collagen deposition under ISO treatment, as evidenced by Masson trichrome staining and Sirius red staining (28.13 $\pm$ 2.54% vs. 8.72 $\pm$ 1.26% for Masson staining; 28.46 $\pm$ 2.24% vs. 9.61 $\pm$ 1.15% for Sirius red staining, p 0.05) (Fig. 1C,D). These results demonstrate that NKA$\alpha$1 haploinsufficiency exacerbates heart lesions and fibrosis under ISO challenge.

Fig. 1.

Na⁺, K⁺-ATPase (NKA) $\alpha$1 haploinsufficiency exacerbated isoproterenol (ISO)-induced heart fibrosis. (A) Representative images of hematoxylin and eosin (H&E) staining, Masson staining, and Sirius red staining in the different groups (scale bar = 100 µm). (B) Quantitative assessment of the myocardial lesion area. (C) Quantitative analysis of fibrotic (Masson blue) areas in cardiac sections. (D) Quantitative analysis of collagen (Sirius red) areas in cardiac sections. Data are presented as means $\pm$ SD; n = 6. ***p 0.001 for WT+ISO vs wild-type (WT) group and NKA$\alpha$1^+⁣/- + ISO vs NKA$\alpha$1^+⁣/- group; ###p 0.001, vs WT+ISO group.

Our previous unbiased proteomic analysis of cardiac tissues from WT and NKA$\alpha$1^+⁣/- mice challenged with ISO revealed differential protein expression patterns [14]. Re-analysis of our pre-proteomics data showed that proteins related to organization of the ECM, including collagen types III, VIII, XII, XIV, fibronectin 1 (Fn1), periostin (Postn), and metallopeptidase 2 (MMP2), were significantly upregulated (Fig. 2A). qPCR results confirmed the upregulation of collagen 1a1, collagen 3a1 (Col3a1), and Fn1 in cardiac tissue from ISO-treated NKA$\alpha$1^+⁣/- mice (Fig. 2B–D). Myofibroblast differentiation, characterized by upregulation of $\alpha$-SMA, represents a pivotal event in cardiac fibrogenesis [17]. Immunostaining results showed markedly increased expression of $\alpha$-SMA in hearts from NKA$\alpha$1^+⁣/- mice (Fig. 2E,F). These findings suggest that NKA$\alpha$1 haploinsufficiency leads to aberrant ECM deposition and myofibroblast differentiation.

Fig. 2.

NKA$\alpha$1 haploinsufficiency induced the aberrant expression of extracellular matrix (ECM) genes and myofibroblast differentiation. (A) Proteomics analysis of cardiac tissue from ISO-treated WT mice and NKA$\alpha$1^+⁣/- mice. Enrichment map of proteins associated with ECM organization. (B–D) qRT-PCR analysis of Fn1 (B), Col1a1 (C) and Col3a1 (D) expression in cardiac tissues from WT and NKA$\alpha$1^+⁣/- mice treated with saline or ISO. (E) Representative images of $\alpha$-SMA immunostaining in different groups (scale bar = 50 µm). (F) Quantitative analysis of $\alpha$-SMA-positive staining areas in cardiac sections. Data are presented as means $\pm$ SD; n = 6. **p 0.01, ***p 0.001, for WT+ISO vs wild-type (WT) group and NKA$\alpha$1^+⁣/- + ISO vs NKA$\alpha$1^+⁣/- group; ###p 0.001, vs WT+ISO group.

Analysis of our pre-proteomics data revealed increased levels of monocyte and macrophage-related proteins, including CD14, CD68, macrophage-capping protein (Capg), and Galectin-3 (Fig. 3A). We then further detected immune cell infiltration in heart tissues. There was a significantly increased macrophage infiltration in the hearts of ISO-NKA$\alpha$1^+⁣/- mice. The increased macrophages were predominantly localized to fibrotic areas. No significant changes were observed for neutrophil infiltration. Although the number of T cells infiltrating the heart is lower than that of macrophages, it also exhibited significant variations across groups (Fig. 3B–E). Furthermore, NKA$\alpha$1 haploinsufficiency increased the levels of IL-6, TNF-$\alpha$, and IL-1$\beta$ in the heart under ISO-induced conditions compared to controls (Fig. 3F–H).

Fig. 3.

NKA$\alpha$1 haploinsufficiency promoted macrophage accumulation and inflammatory factor expression in the ISO-challenged heart. (A) Enrichment map of proteins related to immune cells. (B) Representative images of immunostaining for CD68, LY6G, and CD3 in different groups (scale bar = 50 µm). (C) Quantitative analysis of CD68-positive cells in cardiac sections. (D) Quantitative analysis of Ly6G-positive cells in cardiac sections. (E) Quantitative analysis of CD3-positive cells in cardiac sections. (F–H) ELISA results for interleukin-6 (IL-6), tumor necrosis factor-$\alpha$ (TNF-$\alpha$), and IL-1$\beta$ in cardiac tissues from different groups. Data are presented as the mean $\pm$ SD; n = 6. **p 0.01, ***p 0.001, for WT+ISO vs wild-type (WT) group and NKA$\alpha$1^+⁣/- + ISO vs NKA$\alpha$1^+⁣/- group; #p 0.05, ###p 0.001, vs WT+ISO group.

Since whole-body NKA$\alpha$1 haploid knockout mice were used in this research, the increased deposition of ECM proteins and macrophage infiltration might result from the direct effects of ISO on NKA$\alpha$1 haplo-insufficient fibroblast cells or macrophages. Therefore, we first isolated cardiomyocytes, fibroblasts, and macrophages from WT and NKA$\alpha$1-deficient heterozygous mice and treated them with ISO. NKA$\alpha$1 haploinsufficiency had no significant effect on the activation of macrophages or fibroblasts (Fig. 4A–C), but exacerbated cardiomyocyte injury, as evidenced by the release of LDH (Fig. 4D). Subsequently, we investigated whether damaged NKA$\alpha$1^+⁣/- cardiomyocytes influenced macrophage activation or fibroblast differentiation. Macrophages or fibroblasts from WT mice were exposed to the primary cultured cardiomyocytes isolated from WT or NKA$\alpha$1^+⁣/- mice and co-cultured them in MEM medium supplemented with 10 µM ISO. Co-culture of cardiomyocytes with macrophages significantly enhanced cytokine secretion by the latter cells (Fig. 4E,F). Moreover, co-culture of cardiomyocytes with fibroblasts also increased the expression of $\alpha$-SMA, an indicator of fibroblast-to-myofibroblast conversion (Fig. 4G). Interestingly, when activated macrophages were co-cultured with fibroblasts, this interaction also increased $\alpha$-SMA expression (Fig. 4H).

Fig. 4.

Impaired NKA$\alpha$1^+⁣/- cardiomyocytes enhanced macrophages activation and fibroblasts differentiation. (A,B) ELISA results for IL-6 and TNF-$\alpha$ in the culture media of macrophages challenged with ISO or vehicle control. (C) qRT-PCR analysis of $\alpha$-SMA expression in fibroblasts challenged with ISO or vehicle control. (D) Lactate dehydrogenase (LDH) activity in the culture media of cardiomyocytes challenged with ISO or vehicle control. (E,F) ELISA results for IL-6 and TNF-$\alpha$ in normal macrophages co-cultured with different cardiomyocytes under ISO-challenged conditions. (G) qRT-PCR analysis of $\alpha$-SMA expression in normal fibroblasts co-cultured with different cardiomyocytes under ISO-challenged conditions. (H) qRT-PCR analysis of $\alpha$-SMA expression in normal fibroblasts co-cultured with macrophages activated by co-culturing with different cardiomyocytes. Data are presented as the mean $\pm$ SEM; n = 3. **p 0.01, ***p 0.001, for: (A–D) WT+ISO vs WT and NKA$\alpha$1^+⁣/-+ISO vs NKA$\alpha$1^+⁣/- and (E–H) NKA$\alpha$1^+⁣/- vs WT; ###p 0.001, vs WT+ISO group.

Subcellular enrichment analysis of our pre-proteomics results revealed significant downregulation of mitochondrial proteins (Fig. 5A). Morphological analysis of mitochondria in heart tissues demonstrated a large number of abnormal mitochondria in the hearts of ISO-NKA$\alpha$1^+⁣/- mice, characterized by uneven size, disordered arrangement, unclear structure, and disrupted cristae (Fig. 5B). ERR$\alpha$ regulates a large number of genes involved in mitochondrial function [18]. Reanalysis of our pre-proteomics data revealed that ERR$\alpha$ was downregulated in ISO-challenged NKA$\alpha$1^+⁣/- mice (Fig. 5C). This finding was confirmed by Western blotting (Fig. 5D). SLU-PP-332 is an ERR agonist that has the highest potency for ERR$\alpha$. Treatment with SLU-PP-332 partially alleviated ISO-induced cell damage in NKA$\alpha$1^+⁣/- cardiomyocytes (Fig. 5E). It has been reported that ISO induces macrophage infiltration into the heart in an IL-18-dependent manner [19]. ERR$\alpha$ agonists SLU-PP-332 also reduced the release of IL-18 from NKA$\alpha$1^+⁣/- cardiomyocytes under ISO conditions (Fig. 5F).

Fig. 5.

NKA$\alpha$1 regulated mitochondria-related myocardial cell injury under ISO-challenged conditions. (A) Subcellular enrichment analysis of differential proteins from proteomics results. (B) Mitochondrial morphology observed by transmission electron microscope (upper panel scale bar, 2 µm; lower panel scale bar, 500 nm). (C) ERR$\alpha$ expression level determined from proteomics results. (D) Western blotting analysis of ERR$\alpha$ in cardiac tissues. (E) LDH content in the culture media of cardiomyocytes treated under different conditions. (F) IL-18 expression in cardiomyocytes treated under different conditions. Data are presented as the mean $\pm$ SEM; n = 3. #p 0.05, vs WT+ISO group; &&&, p 0.001, vs NKA$\alpha$1^+⁣/-+ISO group.

The DR region (⁸⁹⁷DVEDSYGQQWTYEQR⁹¹¹) of NKA$\alpha$1 has been identified as an activation site for NKA [20]. Our group and others reported that DR-region-specific antibodies enhance the activity and level of membrane expression of NKA$\alpha$1 [15, 21]. We subsequently evaluated the effects of DRm217, a specific DR-region monoclonal antibody, on ISO-treated mice. Histopathological analyses revealed that DRm217 attenuated heart lesions, fibrosis, and macrophage accumulation under ISO-challenged conditions (Fig. 6A–D). The levels of inflammatory cytokines, including IL-6, TNF-$\alpha$, and IL-1$\beta$, were also downregulated by DRm217 treatment under ISO-insulted conditions (Fig. 6E–G).

Fig. 6.

DRm217 treatment mitigates ISO-induced fibrosis and macrophage activation under ISO-challenged condition. (A) Representative images of H&E, Masson staining, and CD68 immunostaining in different groups (scale bar, 100 µm). (B) Quantitative analysis of the myocardial lesion area. (C) Quantitative analysis of fibrotic (Masson blue) areas in cardiac sections. (D) Quantitative analysis of CD68-positive cells in cardiac sections. (E–G) ELISA results for IL-6, TNF-$\alpha$, and IL-1$\beta$ in cardiac tissues from different groups. n = 6. Data are presented as the mean $\pm$ SD; *p 0.05, **p 0.01, ***p 0.001, vs saline group; ##p 0.01, ###p 0.001, vs ISO+IgG group.