Congenic MKP-5-deficient (Mkp-5^-/- ) mice were generated and characterized as previously described (21). Briefly, an Mkp-5 gene targeting vector was transfected into TC-1 mouse embryonic stem cells to replace exon 1 that encodes the first 250 amino acids and its 5’ flanking sequences with a neomycin-resistance gene through homologous recombination. Two homologous recombinants were then injected into C57BL/6 blastocysts. The homozygous Mkp-5^-/- mice were generated using male chimaera mice (21). Wild-type (Mkp-5^+/+ ) mice were derived from MKP-5 heterozygous mating and propagated subsequently. Eight-week-old male mice were used for all studies. The mice were housed in temperature-controlled rooms with a 12-hour light/12-hour dark cycle and provided free access to food and water throughout the experimental period. All experimental animal procedures were conducted in accordance with guidelines approved by the Institutional Animal Care and Use Committee of Temple University and Yale University.

TAC surgery was performed as previously described to generate pressure overload in a mouse heart (28). Mice were anesthetized with 3% isoflurane inhalation. The mice were then confirmed to be at a surgical level of anesthesia by the absence of a toe pinch reflex. An incision was made on the center of the chest above the first rib. Muscle layers and thymus tissue are carefully pulled aside to expose the aorta arch. A 7-0 nylon suture was placed around the transverse aorta between the brachiocephalic artery and the left common carotid artery. The aorta is constricted by tightening the suture around a 27-gauge needle, which is then pulled free. The muscle closure was made in one layer with a suture (6-0), followed by skin closure with a non-absorbable polypropylene suture (5-0). Sham-operated control mice were subjected to the identical surgical procedure, including isolation of the aorta, but without suture placement. A pressure gradient over 40 mmHg determined by echocardiography one day after surgery was used as a standard for animals included in the study.

The mouse MI model was performed as previously described (29). Briefly, mice were anesthetized with 2% isoflurane inhalation. A small skin cut (1.2 cm) was made over the left chest and a purse suture was made. After dissection and retraction, a small hole was made at the fourth intercostal space to open the pleural membrane and pericardium. With the clamp slightly open, the heart was smoothly and gently “popped out” through the hole. The left coronary artery was located, sutured and ligated at a site about 3 mm from its origin using a 6-0 silk suture. After ligation, the heart was immediately placed back into the intra-thoracic space followed by manual evacuation of air and closure of muscle and the skin. The mouse was then allowed to breathe room air and monitored during the recovery period.

As previously described, transthoracic echocardiography was performed to assess mouse cardiac function at different time points after surgery (13). Mice were anesthetized with 2% isoflurane inhalation and placed on a heated ECG platform. Cardiac function was analyzed by B-mode and M-mode echocardiography using a high-resolution ultrasound system (Vevo3100, Visual Sonics) equipped with an ultrahigh-frequency (38 MHz) linear array transducer. The heart at the left ventricle papillary muscle level was imaged on the left parasternal long and short axis views with a depth setting of 2 cm. Left ventricle posterior wall (LVPW) thickness in diastole/systole, ejection fraction (EF), and fractional shortening (FS) were measured and calculated.

Mouse hearts were perfused through the left ventricle with ice-cold PBS, followed by perfusion with a 10% KCl solution. The hearts were then perfusion-fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich) for 15 min followed by a further incubation with 4% PFA overnight at 4°C, and subsequently processed for paraffin sections (5 μm in thickness). Heart sections were deparaffinized and hydrated through 100%, 95%, 75%, and 50% ethanol solutions, and then they were used for Sirius Red staining or Masson’s trichrome staining to visualize fibrosis, as previously described (30).

Mouse hearts were dissected and cut into small pieces following 10 ml ice-cold PBS perfusion. Tissues were then processed for enzymatic digestion with 450 U/ml collagenase I, 125 U/ml collagenase XI, 60 U/ml DNase I, and 60 U/ml hyaluronidase (all Sigma-Aldrich) for 1 h at 37°C with agitation. Subsequently, the heart tissues were triturated and passed through 70 μm cell strainers (BD Falcon), washed, and centrifuged to obtain single-cell suspensions. Next, cells were first enriched for CD11b⁺ cells by using Miltenyi CD11b microbeads and MACS columns according to the manufacturer’s instructions. Then the enriched CD11⁺ cells were stained with antibodies for FACS analysis. The antibodies used were as follows: CD45-APC/Cyanine7 (BioLegend), Ly6G-APC (BioLegend), F4/80-PE (Invitrogen), and Ly6C-BV421 (BioLegend) at 4°C for 30 min. DAPI (ThermoFisher Scientific) was used as a cell viability marker. Flow cytometric analysis and cell sorting were conducted by using a FACSAria™ cell sorter (BD Biosciences). Data were analyzed with FlowJo software.

Bone marrow cells were isolated from femurs and tibias of 8-week-old Mkp-5^+/+ and Mkp-5^-/- mice as previously described (31). The isolated bone marrow cells were cultured in macrophage growing media (RMPI 1640 supplemented with 10% FBS, 20% L929 fibroblast conditioned media as a source of macrophage colony-stimulating factor, 2 mM L-glutamine, and 1% penicillin/streptomycin) overnight. The next day, non-adherent cells were collected, resuspended, and then cultured with macrophage growing media. Culture media was changed every 2-3 days. Fully differentiated BMDMs were used for studies on day 7.

Total RNA from mouse hearts and cultured cells was isolated using the RNeasy Mini kit (Qiagen) and Trizol reagent (Invitrogen), respectively, according to the manufacturer’s instructions. One microgram of total RNA was used to perform reverse transcription with the iScript™ cDNA synthesis kit (Bio-Rad). qRT-PCR was then conducted using SYBR Green PCR Master Mix (Bio-Rad). cDNA samples were amplified using a CFX96 Touch Real-Time PCR system (Bio-Rad). The relative mRNA levels normalized to 18S rRNA expression were determined using the comparative ΔΔCt method (32). All the primers used for qRT-PCR are shown in Supplementary Table 1 .

For fibrotic gene profiling, total RNA from FACS-purified cells from 3 mice in each group was extracted using the RNeasy Micro kit (Qiagen) following the manufacturer’s instructions. A mouse Fibrosis RT² Profiler array (Qiagen) was then used to investigate the expression profile of fibrosis-related genes in FACS-purified Ly6C^low cardiac macrophages. First-strand cDNA synthesis, qRT-PCR, and data analysis were performed according to the manufacturer’s protocol as previously described (31).

Mouse hearts were homogenized and lysed on ice in RIPA lysis buffer supplemented with protease and phosphatase inhibitors (1 mM Na₃VO₄, 10 mM NaF, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A, 5 μg/ml aprotinin, 5 μg/ml leupeptin). Cultured cell samples were homogenized in RIPA lysis buffer containing protease and phosphatase inhibitor cocktail (Santa Cruz). The protein concentration was determined using the bicinchoninic acid (BCA) reagent according to the manufacturer’s instructions (Thermo Fisher Scientific). 30 μg of protein was separated by 10-15% SDS-PAGE gel and then transferred to nitrocellulose membranes (Bio-Rad). After blocking with 5% BSA in Tris-buffered saline/Tween-20 (TBST) for 1 h at room temperature, blots were incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: MMP-9 (1:500, Abcam), p-p38 MAPK (1:1000, Cell Signaling), p38 MAPK (1:1000, Cell signaling), p-JNK (1:1000, Cell Signaling), JNK (1:1000, Santa Cruz), p-ERK (1:1000, Cell Signaling), ERK (1:1000, Cell Signaling), HSP90 (1:3000, BD Biosciences). Immunoblots were then incubated with corresponding secondary antibodies (1:5000, LI-COR) for 1 h at room temperature and developed using ODYSSEY Infrared Imaging System (LI-COR). Hsp90 was used as a loading control. Data were quantified with Image Studio software (LI-COR, Version 5.2).

Gelatin zymography was performed as previously described (33, 34). Briefly, protein lysates were obtained using ice-cold NP-40 lysis buffer containing protease inhibitors. Equal amounts of protein sample were loaded onto a 10% SDS-PAGE gel supplemented with 0.1% gelatin. After electrophoresis, gels were washed with 0.25% Triton X-100 and incubated with developing buffer (50 mmol/L Tris-HCl, pH 7.4; 5 mM CaCl₂; 0.02% Brij-35; and 200 mmol/L NaCl) at 37°C for 20 h. Subsequently, the gels were stained using 0.5% Coomassie Brilliant Blue R-250 in 25% isopropanol and 10% glacial acetic acid for 30 min, followed by incubation in 50% methanol and 10% glacial acetic acid for 5-10 min. MMP-digested regions were visualized as unstained, transparent bands against a dark background.

Data are expressed as mean ± standard errors of the means (SEM). The statistical significance of differences between groups was analyzed using the 2-tailed, unpaired Student’s t-test, one-way or two-way analysis of variance (ANOVA) followed by post-hoc analysis. A p-value < 0.05 was considered as statistically significant. Prism 8 software (GraphPad, CA, USA) was used for graphing and statistical analyses.

To examine the potential contribution of MKP-5 in cardiac injury, the expression level of MKP-5 during pathologic remodeling of the heart was examined in mice that underwent sham or TAC surgery. As determined by qRT-PCR analysis, gene expression of Mkp-5 in the heart (left ventricle) was significantly increased at 4 weeks after TAC-induced pressure overload, compared with those from sham-operated controls ( Figure 1A ). Given that MKPs inactivate the MAPKs through direct dephosphorylation (21), we assessed whether MKP-5 regulates MAPK activity in the mouse left ventricle by measuring the phosphorylation of the MAPKs in cardiac tissues of Mkp-5^+/+ and Mkp-5^-/- mice after either sham or TAC surgery. Our data show that the phosphorylation of p38 MAPK and JNK, but not ERK, was significantly increased in the hearts of Mkp-5^-/- mice as compared with Mkp-5^+/+ mice in both sham and TAC groups at 4 weeks after surgery ( Figures 1B–E ). These results are consistent with our previous observations showing the effects of MKP-5 on MAPK activity in skeletal muscle (23). Taken together, these data suggest that MKP-5 is activated in the hearts that undergo pressure overload and may be involved in the pathogenesis of heart failure.

TAC-induced cardiac pressure overload leads to impaired cardiac function and a robust cardiac hypertrophy response in addition to the development of cardiac fibrotic remodeling (35). To evaluate the effects of Mkp-5 deficiency on cardiac function and cardiac hypertrophy after pressure overload injury, Mkp-5^+/+ and Mkp-5^-/- mice were subjected to either TAC or sham surgery. First, we examined cardiac function and remodeling using M-mode echocardiography in sham and TAC mice at 4 and 12 weeks after surgery. As demonstrated by representative echocardiographic images ( Figure 2A ) and analyses ( Figures 2B–D ), MKP-5 deficiency protected against TAC surgery-induced deterioration of left ventricle function as measured by the ejection fraction (EF) and fractional shortening (FS). In addition, left ventricle posterior wall (LVPW) thickening was attenuated by 12 weeks in Mkp-5^-/- mice as compared with those in Mkp-5^+/+ pressure overload-induced mice ( Figure 2D ). The echocardiographic parameters in Mkp-5^-/- mice were comparable to those in Mkp-5^+/+ controls in sham-operated animals ( Figure 2 ) or at baseline level ( Supplementary Table 2 ). These results suggest that MKP-5 deficiency protects against cardiac dysfunction induced by pressure overload injury.

Next, cardiac hypertrophy was analyzed in sham- and TAC-operated hearts from Mkp-5^+/+ and Mkp-5^-/- mice at 4 and 12 weeks after surgery. As expected, Mkp-5^+/+ hearts that underwent 4 weeks of TAC exhibited an increased heart size, indicative of cardiac hypertrophy, as compared with sham-operated controls. However, the heart size of Mkp-5^-/- mice was comparable to sham-operated mice at 4 weeks post TAC ( Figure 2E ). The extent of cardiac hypertrophy was also determined by both the heart-to-body weight ratio and heart weight-to-femur length ratio. At both 4 and 12 weeks after TAC, hearts from Mkp-5^-/- mice showed decreased heart weight-to-body weight ratio as compared with Mkp-5^+/+ controls ( Figure 2F ). Similarly, the heart weight-to-femur length ratio was also reduced in Mkp-5^-/- mice ( Figure 2G ). In addition, gene expression levels of cardiac hypertrophy markers Myh7/Myh6, Nppa, and Nppb were significantly lower in the hearts from Mkp-5^-/- mice as compared with Mkp-5^+/+ controls at 4 weeks after TAC ( Figures 2H–J ). Together, these results demonstrate that MKP-5 deficiency attenuates cardiac hypertrophy after TAC-induced pressure overload injury.

To investigate the effects of MKP-5 on cardiac fibrotic remodeling, collagen deposition, a hallmark of cardiac fibrosis, was visualized by Sirius Red staining. As expected, TAC-induced pressure overload resulted in significant collagen deposition and myocardial fibrosis as compared with sham-operated Mkp-5^+/+ mice ( Supplementary Figure 1 ). However, Mkp-5^-/- mice exhibited reduced collagen deposition in cardiac tissues as compared with Mkp-5^+/+ controls at 12 weeks post-TAC ( Figures 3A, B ). We further examined the expression of fibrotic ECM genes in the hearts of Mkp-5^+/+ and Mkp-5^-/- mice at 4 weeks after TAC. Pressure overload induced a marked increase in the expression of Col1a1, Col3a1, and Fn1 in Mkp-5^+/+ hearts compared with those from sham-operated mice ( Figures 3C–E ). However, the expression of these fibrogenic genes remained at basal levels in TAC-induced Mkp-5^-/- mice as compared with Mkp-5^+/+ controls ( Figures 3C–E ). These findings demonstrate that MKP-5 deficiency attenuates cardiac fibrosis in response to TAC-induced pressure overload. In addition, we confirmed the protective role of MKP-5 deficiency in myocardial fibrosis by using a mouse model of MI. We observed that loss of MKP-5 leads to decreased cardiac fibrosis compared to Mkp-5^+/+ controls at 4 weeks after MI ( Supplementary Figure 2 ). Thus, MKP-5 deficiency protects against cardiac fibrosis in both pressure overload and an acute ischemic injury model, suggesting that MKP-5 acts as a regulator of myocardial fibrosis.

Although cardiac fibroblasts and cardiomyocytes contribute to pressure overload induced cardiac fibrosis (1, 2, 6), tissue macrophages also play prominent roles in the regulation of fibrotic remodeling in many different tissues (16, 36, 37) and have been shown to prevent cardiac fibrosis (38). In the current study, we focused on cardiac macrophages and hypothesized that MKP-5 modulates macrophage responses in the heart following pressure overload. Two major macrophage populations Ly6C^high and Ly6C^low are involved in cardiac remodeling, similar to pro-inflammatory M1- and anti-inflammatory M2-type macrophages, respectively (13). We investigated macrophage responses as a direct consequence of MKP-5 deficiency in mice after TAC. CD45⁺; CD11b⁺; F4/80⁺; Ly6G^- Ly6C⁺ macrophages were sorted from hearts derived from Mkp-5^+/+ and Mkp-5^-/- mice at 1-week post-TAC ( Figure 4A ), a time point that cardiac macrophage number peaks, as previously described (13, 39). TAC-induced pressure overload increased the number of cardiac macrophages compared with sham-operated controls in Mkp-5^+/+ control mice ( Figure 4B ). Loss of MKP-5 showed no significant change in the total number of cardiac macrophages ( Figure 4C ) or Ly6C^low (CD45⁺; CD11b⁺; F4/80⁺; Ly6G^-; Ly6C^low) cardiac macrophage subset but resulted in a marked decrease in the number of Ly6C^high (CD45⁺; CD11b⁺; F4/80⁺; Ly6G^-; Ly6C^high) macrophage subset in comparison with the Mkp-5^+/+ control mice after TAC ( Figures 4D, E ). Furthermore, MKP-5 deficiency did not influence either Ly6C^low or Ly6C^high monocyte subsets in the blood of Mkp-5^+/+ and Mkp-5^-/- mice in response to TAC ( Supplementary Figure 3 ).

Functionally, Ly6C^low macrophages are similar to M2-type macrophages, which have been suggested to orchestrate cardiac fibrotic remodeling in response to cardiac injuries such as myocardial infarction and pressure overload (13, 39). To investigate whether MKP-5 is involved in regulating Ly6C^low macrophages in response to pressure overload-induced cardiac fibrosis, we examined the expression of MKP-5 in FACS-sorted Ly6C^low cardiac macrophages from Mkp-5^+/+ and Mkp-5^-/- mice after TAC. We found a significant increase in Mkp-5 mRNA expression levels in Ly6C^low macrophages isolated from Mkp-5^+/+ mice subjected to TAC compared to the sham group ( Figure 5A ). Given that MKP-5 deficiency did not affect the overall number of Ly6C^low cardiac macrophages, regulation of fibrosis-related gene expression in Ly6C^low macrophages was then examined. We compared the expression profile of fibrosis-related genes in FACS-sorted Ly6C^low macrophages from Mkp-5^+/+ and Mkp-5^-/- myocardium at 1 week post-TAC using a fibrosis qRT-PCR array. We identified up- and down-regulated fibrosis-modulating genes regulated by MKP-5 in cardiac macrophages ( Supplementary Table 3 ). Among these, we found that Mmp-9 is the most upregulated fibrosis-related gene in Ly6C^low cardiac macrophages from Mkp-5^-/- mice after TAC, suggesting the involvement of MMPs in MKP-5-mediated fibrosis. Consistent with this notion, we also observed Mmp-13 being upregulated in Ly6C^low macrophages from Mkp-5^-/- TAC mice. This result was confirmed by qRT-PCR using Ly6C^low cardiac macrophages sorted from the second cohort of Mkp-5^+/+ and Mkp-5^-/- mice that underwent 1 week of TAC ( Figures 5B, C and Supplementary Figure 4 ). MMP-9 belongs to the family of MMPs that encode proteolytic enzymes responsible for ECM degradation (17, 18), and importantly, macrophages have been documented as the key source of MMPs (36). Thus, it is conceivable that increased MMP expression in cardiac macrophages could contribute to an enhanced matrix-degrading phenotype and thus, attenuated pressure overload-induced cardiac fibrosis in MKP-5 deficient mice.

To investigate the mechanisms by which MKP-5 regulates the expression of MMP-9 in macrophages, IL-4-stimulated BMDMs were used as a pro-fibrotic polarization model (13). Treatment with IL-4 for 10 h significantly induced the mRNA expression of Mkp-5 in BMDMs ( Figure 6A ). Consistent with the in vivo findings that TAC upregulates Mkp-5 in the heart ( Figure 1A ) and Ly6C^low cardiac macrophages ( Figure 5A ), these results suggest that MKP-5 is responsive to pro-fibrotic stimuli. IL-4 stimulation increased Mmp-9 mRNA level in a time-dependent manner with a peak expression at 12 h in BMDMs ( Figure 6B ). Consistent with our qRT-PCR array data using FACS-sorted Ly6C^low cardiac macrophages, significant upregulation of MMP-9 expression at both mRNA and protein levels was observed in Mkp-5^-/- BMDMs as compared with Mkp-5^+/+ controls after IL-4 stimulation ( Figures 6B, C ). In agreement with these observations, the gelatin zymography assay showed a marked increase of MMP-9 enzymatic ECM-degrading activity in cell lysates derived from Mkp-5^-/- BMDMs compared with Mkp-5^+/+ controls in response to IL-4 ( Figure 6D ).

MKP-5 has been shown to dephosphorylate p38 MAPK, JNK, and ERK to a lesser extent (40, 41). The effects of MKP-5 deficiency on the activation of the MAPKs were examined in IL-4-treated BMDMs. In response to IL-4 stimulation, Mkp-5^-/- BMDMs exhibited enhanced phosphorylation of p38 MAPK, JNK, and to a lesser extent ERK as compared with Mkp-5^+/+ controls ( Figures 6E, F ). These results demonstrate that MKP-5 negatively regulates p38 MAPK and JNK activities in IL-4-stimulated M2-type macrophages and results in upregulation of Mmp-9 expression. Previous studies have shown that the activated MAPKs (p38 MAPK, JNK, and ERK) can promote MMP-9 expression in macrophages (42). We therefore examined whether MKP-5 regulates MMP-9 in a MAPK-dependent manner in BMDM. After treatment with p38 MAPK-, JNK-, or ERK-specific inhibitors SB203580, SP600125, or PD98059, respectively, the increase of MMP-9 expression induced by IL-4 in Mkp-5^-/- BMDMs relative to Mkp-5^+/+ controls was blocked at both mRNA and protein levels ( Figures 6G–I ). Collectively, these data indicate that MAPK signaling is required for MKP-5 mediated regulation of MMP-9 in pro-fibrotic macrophages. Together with the in vivo MKP-5 loss of function studies, these data reveal a regulatory pathway by which MKP-5 inhibits the expression of MMP-9 by inactivating MAPK signaling in Ly6C^low M2-type macrophages, which subsequently leads to an attenuated ECM-degrading activity and thus, at least in part, development of cardiac fibrosis induced by pressure overload.