The Downregulation of ADAM17 Exerts Protective Effects against Cardiac Fibrosis by Regulating Endoplasmic Reticulum Stress and Mitophagy

2.1. Induction of Myocardial Infarction (MI) and Treatment

All animal care and experimental protocols were performed in accordance with the Institutional Animal Care and were approved by the Ethics Committee of the Sun Yat-sen University. Male C57BL/6 mice (6–8 weeks old, 20–25 g) were subjected to the induction of MI by LAD ligation, as described previously [32]. Briefly, the mice were anesthetized with sodium pentobarbital (intraperitoneal injection, 50 mg/kg; Merck) and mechanically ventilated using the HX-101E ventilator (Chengdu Tai Meng Software Ltd.). Their chests were opened through the 4th left intercostal space, and then, the LAD was ligated with 8-0 polyester sutures. Mice in the sham group were subjected to the same experimental procedures but without LAD constriction. The mice were randomly divided into four groups (n = 6 per group): control/MI groups receiving vehicle buffer (2% Tween 80 and 0.5% methylcellulose) by oral gavage twice daily and TMI-005/MI+TMI-005 groups receiving 10 mg/kg ADAM17 inhibitor (TMI-005, Glpbio, GC35377) by oral gavage twice daily. After 4 weeks of intragastric administration (endpoint), cardiac function was assessed by echocardiography analysis, and then, the hearts were harvested.

2.2. Isolation and Culture of CFs

First, mouse cardiac fibroblasts (mCFs) were isolated and cultured as described in a previous study [33]. mCFs were isolated from 1- to 3-day-old neonatal mice obtained from the Animal Centre of the Sun Yat-sen University. Briefly, the hearts of mice were quickly excised and digested with 0.1% trypsin/D-Hanks solution at 4°C with gentle rotation for 12 hours. Then, the hearts were digested with 0.8 mg/mL collagenase II/D-Hanks solution at 37°C for 10 minutes and were repeated 2–3 times. Cells were collected and suspended in Dulbecco's modified Eagle medium: Nutrient Mixture F12 (DMEM/F12, Gibco, Thermo Fisher Scientific) containing 10% fetal bovine serum. After plating at 37°C for 0.5 h, mCFs adhered onto the culture plates, and the nonadherent cells in the supernatant were removed by washing with PBS. Then, the mCFs were cultured with DMEM/F12 containing 10% FBS (Invitrogen, Carlsbad) and 1% penicillin/streptomycin (Gibco, Thermo Fisher Scientific) at 37°C in humid air containing 5% CO₂, until they reached confluence; then, they were passaged further. The identity of mCFs was confirmed by immunofluorescence staining with vimentin. mCFs at the second or third passages were used in the subsequent experiments. After starvation in the serum-free medium for 12 h, the mCFs were treated with 5 ng/mL recombinant mouse TGF-β1 protein (R&D Systems, #7666-MB) for 48 h.

2.3. Transfection Procedure and TGF-β1 Administration

ADAM17-specific siRNAs were purchased from RiboBio (Guangzhou, China). mCFs were seeded on 6-well plates at 50–60% confluence before transfection. Each siADAM17 (50 nM) (Table s1) and Lipofectamine® RNAiMAX (Invitrogen) were mixed, incubated at room temperature for 10–15 min, and then added to the cell cultures. Twenty-four hours after transfection, the mCFs were treated with TGF-β1 (5 ng/mL) for 48 h or thapsigargin (TG, 1 μM, Sigma-Aldrich, T9033) for 24 h.

2.4. Wound Healing Assay

To verify the migration ability of the mCFs, a wound healing assay was performed. Transfected mCFs (2 × 10⁵ cells/well) left untreated or those treated with TGF-β1 or thapsigargin were seeded into 6-well plates and grown until subconfluence. A scratch was then created in each well using a 200 μL pipette tip, and the wounded monolayers were washed twice with PBS to remove the cell debris and floating cells. The wounds were photographed at the beginning (time 0 h) and then after 24 h under an inverted microscope, and the width of the wound area was measured at the reference points ImageJ software.

2.5. CCK-8 Assay

The CCK-8 method was used to analyze the proliferation of mCFs according to the manufacturer's instructions. The mCFs, transfected with siADAM17 as described above, were seeded into 96-well plates at a density of 5 × 10³ cells/well and treated with TGF-β1 or thapsigargin for a total of 24, 48, and 72 hours, respectively. Then, 100 μL of fresh medium containing 10 μL CCK-8 (DOJINDO) was added into the wells, followed by incubation in the presence of 5% CO₂ at 37°C for 2 h; next, the absorbance readings of the samples were obtained at 450 nm using a microplate reader (Invitrogen).

2.6. Quantitative Real-Time RT-PCR

Total RNA was extracted from the mouse heart tissues or mCFs using the Trizol reagent (Invitrogen). A PrimeScript™ RT Master Mix Kit (Takara Bio, RR036A) was used to reverse-transcribe 1 μg of RNA into cDNA, as described previously [4]. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the SYBR method (Takara Bio, RR420A) in a LightCycler® 96 Real-Time PCR System (Roche). The qRT-PCR analysis was performed using the primers listed in Table s2. Normalization of gene expression was achieved by comparing the expression of the target genes to that GAPDH in the corresponding samples.

2.7. Western Blotting

Total proteins were extracted from the mCFs or mouse heart tissues using a radio immunoprecipitation assay (RIPA) buffer (CST) containing protease and phosphatase inhibitors (Roche, 04906845001, 04693124001). The mitochondrial and cytosolic fractions were isolated from cells using a commercially available kit (Thermo Fisher Scientific, #89874) according to the manufacturer's instructions. Total protein or mitochondrial protein concentrations were measured using a BCA Protein Assay Kit (Pierce, Thermo Fisher Scientific, 23227). Equal amounts of the total protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis; the resultant bands were transferred to 0.2 μm PVDF membranes (Millipore). Then, the membranes were blocked with 5% BSA diluted with tris-buffered saline tween-20 at room temperature for 1 h and incubated overnight at 4°C with the following primary antibodies: antibodies against collagen I (1 : 1000, Abcam), α-SMA (1 : 1000, Abcam), ADAM17 (1 : 1000, Abcam), Smad2/3 (1 : 1000, CST), p-Smad2/3 (1 : 1000, CST), p-PERK (1 : 1000, CST), ATF4 (1 : 1000, Abcam), p-IRE1 (1 : 1000, Abcam), XBP1s (1 : 1000, Abcam), ATF6 (1 : 1000, Proteintech), GRP78 (1 : 1000, Abcam), and GAPDH (1 : 1000, CST). They were then washed with TBST (×3) and incubated with horseradish peroxidase-conjugated secondary antibody (1 : 5000, CST) for 1 h at room temperature. Next, the membranes were washed with TBST (×3), followed by the detection of the protein bands using ECL reagents (Merck Millipore, WBULS050). The intensity of the protein bands was quantified using the ImageJ software and normalized to the intensity of GAPDH.

2.8. ADAM17 Enzymatic Activity Assay

The ADAM17 activity assay was performed using the InnoZyme TACE Activity Kit (Sigma-Aldrich, CBA042) following the manufacturer's protocol. The activity of ADAM17 was measured using an internally quenched fluorescent substrate, MCA-KPLGL-Dpa-AR-NH2. The resultant fluorescence was measured at an excitation wavelength of 320 nm and an emission wavelength of 405 nm. The ADAM17 activity was expressed as relative fluorescence units per milligrams of protein.

2.9. Echocardiography

Echocardiography was performed to assess the left ventricular end systolic diameter (LVESD), left ventricular end diastolic diameter (LVEDD), left ventricular ejection fraction (LVEF), and left ventricular fractional shortening (LVFS) using a VisualSonics Echo System (Vevo 2100, VisualSonics, Inc.) equipped with a MicroScan Transduce (MS-400, 30 MHz, VisualSonics, Inc.). The mice were slightly anesthetized via inhalation of 1% isoflurane, and their heart rate was maintained at >400 bpm. The hearts' short axis views were obtained in B-mode and M-mode. The LVESD, LVEDD, LVEF, and LVFS were calculated from the M-mode measurements; the measurements represented the mean of three successive cardiac cycles, as described previously [34].

2.10. Masson's Trichrome Staining

The mouse hearts were dissected and fixed in 4% paraformaldehyde for 24 h; then, they were embedded in paraffin and stained with Masson's trichrome stain, as previously described [3]. The area occupied by collagen was calculated using the ImageJ software. The percentage of fibrosis was measured using the following formula: (fibrotic areas/total left ventricular areas) × 100%.

2.11. Statistical Analysis

All data are presented as the mean ± SEM. Statistical analysis was performed using Student's t-test for the comparison of two groups and one-way ANOVA followed by Bonferroni test for the comparison of multiple groups, and the significance was set at P < 0.05. The statistical graphs were prepared using GraphPad Prism Software (version 7).

3.1. ADAM17 is Upregulated in Fibrosis Heart Tissues and MCFs Treated with TGF-β1

To determine the potential mechanistic link between ADAM17 and cardiac fibrosis in vivo, a murine model of MI was established using a previously described protocol. The expression levels of fibrotic proteins, such as collagen I and α-SMA, were increased in the MI group. Meanwhile, ADAM17 mRNA and protein levels were significantly higher in fibrosis hearts than in normal hearts (Figures 1(a) and 1(b)). In vitro, mCFs isolated from neonatal mice hearts were incubated with TGF-β1 (5 ng/mL, for 48 h). The mRNA and protein levels of collagen I and α-SMA were increased, indicating that the mCFs were effectively differentiated into myofibroblasts. Consistent with our results in the fibrosis heart tissues, the ADAM17 expression levels were higher in TGF-β1-treated mCFs than in those not subjected to TGF-β1 stimulation (Figures 1(c) and 1(d)). Taken together, these data indicate that ADAM17 may be involved in the regulation of cardiac fibrosis and activation of mCFs.

3.2. ADAM17 Knockdown Attenuated TGF-β1-Induced MCF Activation and the Upregulation of ECM Proteins

To further identify the important role of ADAM17 in activation of mCFs, the cells were transfected with ADAM17-specific siRNA or scramble siRNA as a negative control (NC) for 24 h before treatment; qRT-PCR was used to confirm the knockdown efficiency of ADAM17. We found that transfection with siRNA3 reduced the ADAM17 levels by nearly 80%, compared to those in the NC groups (Figure 2(a)). Following the TGF-β1-induced activation of mCFs, the transcript and protein levels of the ECM protein collagen I, as well as those of the myofibroblast marker α-SMA, were increased. Further, siRNA3 successfully inhibited the expression of ADAM17, α-SMA, and collagen I (Figures 2(b) and 2(c)). Our data revealed that knockdown of ADAM17 not only inhibited mCF differentiation but also reduced the synthesis and secretion of collagen I. Next, we assessed the mCF migration and proliferation abilities using wound healing and CCK-8 assays, respectively. We found that TGF-β1 significantly increased the proliferation and migration abilities of mCFs; the knockdown of ADAM17 partially reversed these phenomena (Figures 2(d) and 2(e)). Thus, our results suggested that ADAM17 enhances the collagen synthesis and secretion, migration, and proliferation abilities of mCFs; that is, it promotes the activation of mCFs.

3.3. ADAM17 Regulates ER Stress and Mitophagy to Promote the Activation of MCFs

It is well-known that TGF-β1 signal through the activation of Smad2/3 (canonical pathway) to promote mCF differentiation, migration, and proliferation and the deposition of ECM proteins during the formation of pathological cardiac fibrosis [35, 36]. Therefore, we investigated the effects of ADAM17 on the TGF-β1/Smad2/3 pathway in mCFs. Western blotting analysis revealed that the phosphorylation levels of Smad2/3 were increased significantly after stimulation with TGF-β1, but they were unaltered between the NC and ADAM17 knockdown groups. These data indicated that ADAM17 had no effect on the canonical Smad2/3 pathway. In addition, TGF-β1 is known to trigger an increase in ER stress and the unfolded protein response. After the TGF-β1-induced activation of mCFs, the downstream signaling molecules associated with ER stress, phosphorylated PERK, and IRE1 (p-PERK and p-IRE1), ATF4, XBP1s, ATF6, and GRP78 were markedly upregulated. Then, we found that the activity of the ATF6 was further reduced after the silencing of ADAM17, compared to the case in the NC groups (Figure 3(a)). These results indicate that the ATF6, but not the PERK/ATF4 and IRE1/XBP1 pathways associated with ER stress, may mediate ADAM17-induced activation of mCFs.

To further investigate whether the ATF6 is involved in the activation of mCFs, we employ thapsigargin (TG), a key stimulator of acute ER stress. Compared with those in the TGF-β1 treatment groups, the administration of the TGF-β1 and TG groups not only increased the expression levels of ATF6 and GRP78, as expected, but also upregulated the expression levels of collagen I and α-SMA. However, after adding TG, there was no significant difference in the expression levels of collagen I, α-SMA, ATF6, and GRP78 between the siRNA3 group and the NC group (Figure 3(b)). Next, migration and proliferation abilities of mCFs also showed similar changes, according to the results of the wound healing and CCK-8 assays, respectively. Knockdown of ADAM17 inhibited mCF migration and proliferation abilities, but this difference disappeared after using TG (Figures 3(c) and 3(d)). Collectively, these data suggested that the decreased expression of ADAM17 protects against mCF activation via the inhibition of the ATF6 in ER stress.

A recent study reported that ADAM17 and mitophagy are involved in the occurrence and development of tumors [31]. To clarify whether mitophagy is involved in the regulation of mCF activation by ADAM17, we observed the mitophagy response in the process of mCF activation. PINK1/Parkin pathway acts as a classical regulation pathway in mitophagy and plays an important role in cardiac fibrosis [26, 37]. We found that the protein levels of PINK1 and Parkin were enhanced after TGF-β1 stimulation, and their protein levels were further increased after the knocking down ADAM17 (Figure 3(e)). These data showed that ADAM17 may regulate mCF activation by affecting mitophagy.

3.4. Inhibition of ADAM17 with TMI-005 Reduced the Degree of Post-MI Fibrosis and Enhanced Cardiac Function in Vivo

To assess whether ADAM17 mediates post-MI cardiac fibrosis in vivo, we employed TMI-005, a pharmacological selective inhibitor of ADAM17. The 8-week-old mice were subjected to MI via permanent LAD ligation and then administered 10 mg/kg TMI-005 by oral gavage twice daily for 4 weeks, as depicted in Figure 4(a). We performed echocardiography and Masson's trichrome staining analyses to assess collagen deposition and cardiac function, respectively. After TMI-005 treatment alone for 4 weeks, ADAM17 activity was remarkably inhibited in the heart, and there was no significant difference in collagen deposition and cardiac function, compared to those in the control group (Figures 4(b)–4(d)). Then, we observed the heart function and degree of fibrosis in the mice post-MI after they were treated with TMI-005 for 28 days. The Masson's trichrome staining results showed that the ADAM17 inhibitor prevented MI-associated increase in the area of cardiac fibrosis (Figure 4(e)). In Figure 4(f), compared with the MI group, although the TMI-005 administration group had no obvious difference in LVESD and LVEDD, the LVEF and LVFS were improved. And the western blotting results also showed that the levels of the fibrotic proteins collagen I and α-SMA were reduced in the hearts of the mice treated with the ADAM17 inhibitor (Figure 4(g)). Thus, the ADAM17 inhibitor TMI-005 notably improved cardiac function after MI. These findings support the speculation that ADAM17 inhibition exerts protective effects on fibrosis post-MI and cardiac function.