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Introduction

Coronary heart disease is the leading cause of mortality worldwide (1). Regardless of rapid progress in the management of coronary heart disease and acute myocardial infarction (MI), heart failure following MI remains a great challenge in clinical practice (2). Thus, novel therapies are required to improve the long-term prognosis, and to reduce the morbidity and mortality following MI.

During post-MI remodeling, compensatory eccentric hypertrophy of the viable myocardium occurs and progressive dilatation of the left ventricle (LV) results in heart failure, which may lead to mortality (2). A crucial mediator in the pathogenesis of cardiac remodeling and the fibrogenic pathways in the healing infarct is interleukin 1 (IL-1) signaling. Interventions targeting the IL-1 system may therefore prevent remodeling following MI (3). Previous studies have demonstrated that NLRP3-IL-1β signaling is involved in cardiac dysfunction. One such study revealed that NLRP3 promoted myocardial inflammation and systolic dysfunction through the production of proinflammatory IL-1β (4). Another study demonstrated that NLRP3 was upregulated in the heart in an experimental mouse model of MI. Moreover, the inhibition of the NLRP3 pathway prevented the formation of the inflammasome, and limited the infarct size and cardiac enlargement following MI (5).

Modulation of the inflammasome may therefore represent a novel strategy to prevent cardiac dysfunction following MI. It had remained unclear whether blocking cathepsin B, which is proposed to be upstream of NLRP3 activation (6), results in beneficial effects on post-MI remodeling. In the present study, we aimed to investigate the effects of the novel cathepsin B inhibitor, CA-074Me, on cardiac dysfunction, remodeling and fibrosis following MI in a rat model, and to explore the underlying mechanisms of action.

Materials and methods

Induction of rat MI

Male Sprague-Dawley rats were purchased from Vital River Laboratories Co., Ltd. (Beijing, China). Animals were housed and maintained under standard conditions in the Experimental Animal Center of Shandong University. All experiments conformed to the Guide for the Care and Use of Laboratory Animals, provided by our institute. The study was approved by the ethics committee of Shandong University. MI was induced in male rats (age, 10 weeks; weight, 180–320 g) by ligating the left anterior descending coronary artery, as previously described (7). Briefly, rats were anesthetized with isoflurane (5% induction, 2% maintenance) prior to intubation and ventilation. The adequacy of the anesthesia was monitored by the loss of reflexes and the degree of muscle relaxation. A left-sided thoracotomy was performed between the fifth and sixth ribs, the pericardium was opened and the heart was exteriorized. The coronary artery was localized 1–2 mm below the junction of the pulmonary conus and the left atrial appendage. A 5.0 silk suture (Sigma-Aldrich, St. Louis, MO, USA) was used to permanently constrict the artery from the left border of the pulmonary conus to the right border of the left atrial appendage. The heart was returned to the chest cavity, and the lungs were re-expanded prior to closure of the chest wall with a 4.0 silk suture. Sham animals underwent the aforementioned procedures, with the exception of the coronary artery ligation.

Treatment

Stock solutions of the cathepsin B inhibitor, CA-074Me, were prepared at a concentration of 10 mg/ml in dimethyl sulfoxide (DMSO). This was diluted at a ratio of 1:10 in saline, and administered at a dose of 10 mg/kg by intraperitoneal injection, according to previously validated protocols (8–9). All rats were randomly assigned to three groups: Group I, sham-operated rats as the normal control (n=10); Group II, rats with MI treated with vehicle (10% DMSO) for 4 weeks (n=15); Group III, rats with MI treated with CA-074Me treatment at a dosage of 10 mg/kg/day for 4 weeks (n=15).

Cathepsin B activity assay

Cathepsin B activity was measured using the synthetic fluorometric substrate, Z-Arg-Arg-NHMec, as previously described (10). Briefly, 150 μM Z-Arg-Arg-NHMec (pH 6.0) was added to the assay buffer and the fluorescence was measured in triplicate, at 1-min intervals for 30 min, and at an excitation of 360 nm and an emission of 465 nm. Data are represented as relative fluorescent units, and presented as the mean ± standard deviation (SD) of three independent experiments.

Western blot analysis

Total protein extracts were prepared by homogenizing the heart tissues in lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA), before storage at −20°C. The protein content was determined using the bicinchoninic assay method (Pierce Biotechnology, Inc., Rockford, IL, USA). Equal amounts of protein were boiled with sodium dodecyl sulfate (SDS) buffer, loaded onto 8–12% denaturing polyacrylamide gels and blotted onto a polyvinylidene fluoride (PVDF) membrane. Subsequent to blocking with 5% skimmed milk, the following specific primary antibodies were added: Anti-NLRP3 (dilution, 1:2,000; BD Biosciences, Franklin Lakes, NJ, USA); anti-caspase-1p20, anti-pro-IL-1β/-IL-1β and anti-pro-IL-18/-IL-18 (all with dilution, 1:2,000; all from Cell Signaling Technology, Inc.). The immunoblots were visualized by enhanced chemiluminescence, and quantified for the specific protein content by densitometry with normalization for the housekeeping gene, β-actin (dilution, 1:2,000; Cell Signaling Technology, Inc.).

Enzyme-linked immunosorbent assay (ELISA)

The serum IL-1β and IL-18 levels were determined by ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Cardiac function assessed by echocardiography

The cardiac function of all rats was evaluated by noninvasive echocardiography, as described previously (11). Briefly, images were recorded using a 10- to 12-MHz phased-array transducer (model 21380A with HP SONOS 5500 imaging system; Agilent Technologies, Inc., Santa Clara, CA, USA). Diastolic and systolic left ventricle internal dimensions (LVIDD and LVIDS, respectively) and LV fractional shortening (LVFS) were measured with M-mode tracing from the short-axis view of the LV at the papillary muscle level. All measurements were performed in a blinded manner according to the guidelines of the American Society for Echocardiology, and were averaged over three consecutive cardiac cycles. All data were acquired and analyzed by a single blinded observer, using EchoPac (GE Vingmed, USA) off-line processing.

Infarct size

The Masson’s trichrome-stained slides were examined by light microscopy, digitized and analyzed using image analysis software (Analytical Imaging Station, AIS, Version 6.0; Imaging Research, Inc., St. Catherines, ON, Canada). The infarct size was assessed morphologically and calculated as the ratio of the average scar circumferences of the endocardium and the epicardium to the average LV circumferences of the endocardium and the epicardium, as previously described (12).

Extracellular matrix (ECM) deposition

Sections were stained with Masson’s trichrome stain to examine the ECM deposition, as previously described (13). All tissues were assessed with the examiner blinded to the experimental groups. The accumulation of matrix within the non-infarct zone (NIZ) was then quantified as described previously (14). Briefly, stained sections from the mid-left ventricle were digitally captured in their entirety with a standard polarizing filter, and loaded onto a Pentium III computer (IBM Corporation, Armonk, NY, USA). To isolate the NIZ from the infarct and the peri-infarct zone, the infarct and a 2-mm zone on either side of it were excluded from the analysis. The remaining myocardium comprised the NIZ and was analyzed using computer-assisted image analysis (15–16) with the AIS software. The whole NIZ was used for quantification of the ECM in order to prevent possible bias from using selected fields. An area of blue on a trichrome-stained section, representing the ECM, was selected for its color range. For sham animals, the ECM content of the entire LV was quantitated by the same method.

Histological and immunofluorescence analysis

The rat hearts were collected 4 weeks following MI and fixed with buffered 10% PFA for 1 h, followed by a 1-h incubation in 0.1 M glycine and overnight incubation at 4°C in 0.6 M sucrose. Samples were embedded in optimal cutting temperature (OCT) media, and sections (4 μm) were cut and stored at −20°C. Sections for Masson’s trichrome staining were processed according to the manufacturer’s instructions (Sigma-Aldrich). In a separate set of immunohistochemistry (IHC) experiments, rat heart sections were permeabilized and blocked for 1 h in 1% bovine serum albumin (BSA) and probed with primary polyclonal antibodies to α-sarcomeric actin (dilution, 1:50; Sigma-Aldrich), wheat germ hemagglutinin (WGA), and anti-His (both with a dilution of 1:50; both from Invitrogen Life Technologies, Carlsbad, CA, USA). The secondary antibodies (1:100; Invitrogen Life Technologies) were used at the recommended dilution and incubated with sections for 1 h at room temperature. The nuclei were counterstained with DAPI. Images were acquired on an inverted epifluorescent microscope (Zeiss; Leica, Germany).

Statistical analysis

Data are presented as the mean values ± standard deviation. Statistical analyses included Student’s t-test and one-way analysis of variance (ANOVA) followed by the Tukey and Bonferroni multiple comparison tests. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using SPSS 16.0 (SPSS, Inc., Chicago, IL, USA).

Results

CA-074Me inhibits the activation of the cathepsin B-NLRP3-IL-1β pathway

We initially confirmed that cathepsin B activity was inhibited by CA-074Me treatment (Fig. 1). Furthermore, we identified, by ELISA, whether CA-074Me treatment affected the serum levels of the proinflammatory cytokines, IL-1β and IL-18. As demonstrated in Fig. 2A and B, a significant increase in the serum levels of these cytokines was observed in the vehicle-treated rats compared with the low baseline levels observed in the sham control rats (P<0.01 and P<0.001 for IL-1β and IL-18, respectively). In addition, the serum levels in the CA-074Me-treated rats were significantly lower than those in the vehicle-treated animals (P<0.05 and P<0.01, for IL-1β and IL-18, respectively).

Figure 1 Effect of CA-074Me on cathepsin B activity following MI. Cathepsin B activity in the hearts was measured as described in Materials and methods. Data are represented as relative fluorescent units and presented as the mean ± SD of three independent experiments. #P<0.001 vs. sham group and ##P<0.01 vs. vehicle (10% DMSO)-treated group. MI, myocardial infarction.

Figure 2 CA-074Me inhibits NLRP3 inflammasome activation. The serum levels of (A) IL-1β and (B) IL-18 4 weeks following MI, as measured by ELISA. (C) Representative western blot analysis of NLRP3, IL-1β, IL-18 and Casp1 in the ventricular tissue 4 weeks following MI. The appearance of mature 17-kDa IL-1β, 18-kDa IL-18 and the Casp1 p20 subunit indicates activation. β-actin was used as an internal control. (D) Quantification of the NLRP3/β-actin, mature IL-1β/β-actin, mature IL-18/β-actin and Casp1/β-actin ratios. In the histograms, data are presented as the mean ± SEM for 10 mice per group. Sham (white bars), vehicle (10% DMSO)-treated (black bars) and CA-074Me-treated (hatched bars) mice. *P<0.05, **P<0.01 and ***P<0.001. MI, myocardial infarction; ELISA, enzyme-linked immunosorbent assay; Casp1, caspase-1; IL, interleukin.

As demonstrated by western blot analysis, increasing levels of mature IL-1β (Fig. 2C and D) were observed in the lysates of vehicle-treated hearts, but not in those of the CA-074Me-treated or sham control hearts. We then measured whether inflammasome activation was suppressed by CA-074Me administration. The NLRP3 protein levels in the heart tissues were observed using western blot analysis. In the vehicle-treated rats, the levels of NLRP3 protein (Fig. 2C and D) were significantly increased compared with that of the sham controls, and these effects were inhibited by CA-074Me treatment. Consistent with cytokine maturation, caspase-1 activation (Fig. 2C and D) was also observed in the vehicle-treated hearts, as demonstrated by the appearance of the p20 subunit. This effect was significantly inhibited by CA-074Me treatment.

Treatment with CA-074Me improves cardiac function

Echocardiography was used to examine the cardiac structure and function of the rats. At the baseline, no differences were observed between the three groups (Fig. 3A). Treatment with CA-074Me for 4 weeks contributed to significant improvements in the LVIDD, LVIDS, LVFS and ejection fraction (EF) (Fig. 3A and Table I).

Figure 3 CA-074Me limits cardiac dysfunction, infarct size and hypertrophy of cardiomyocytes. (A) Representative images of echocardiography tracing in the rats with MI treated with either vehicle (10% DMSO) or CA-074Me in each group at weeks 0, 1 and 4. (B) Rat heart cross-sections were obtained at week 4 for sham-treated rats and rats with MI treated with either vehicle or CA-074Me. (C) Mean cell circumference and (D) cross-sectional area of >100 cardiomyocytes. #P<0.001 vs. sham-treated group and ##P<0.01 vs. vehicle-treated group. (E) Representative images of cardiomyocytes from each group stained for α-sarcomeric actin (red), [wheat germ agglutinin (WGA)-fluorescein for surface membranes, green] and nuclei (DAPI, blue); white bar, 20 μm. MI, myocardial infarction.

Table I Echocardiography of each group.

Table I

Echocardiography of each group.

	Sham	MI + vehicle	MI + CA-074Me
HR (beats/min)	254±39	243±15	251±18
LVIDD (cm)	0.86±0.02	0.98±0.03a	0.84±0.05b
LVIDS (cm)	0.54±0.04	0.79±0.04a	0.67±0.06b
LVFS (%)	38.24±2.10	21.15±1.10a	24.82±2.90b
EF (%)	73.25±2.80	46.08±3.40a	53.81±2.90b

{ label (or @symbol) needed for fn[@id='tfn1-mmr-08-02-0361'] } Values are the mean ± SEM.

a P<0.05 vs. sham and

b P<0.05 vs. vehicle.

{ label (or @symbol) needed for fn[@id='tfn4-mmr-08-02-0361'] } MI, myocardial infarction; HR, heart rate; LVIDD and LVIDS, left ventricular internal diameters in diastole and systole, respectively; LVFS, left ventricular fractional shortening; EF, ejection fraction.

Infarct size and LV cardiomyocyte size were reduced by CA-074Me

Treatment with CA-074Me significantly reduced the infarct size compared with that of the vehicle-treated MI groups (22±2.8 vs. 38±5.4%, respectively; P<0.01; Fig. 3B). The cardiomyocyte size was assessed using high magnification microscopy to quantify the circumference (Fig. 3C) and the cross-sectional area (Fig. 3D and E). These data demonstrated that treatment with CA-074Me prevented cardiac remodeling when compared with the vehicle-treated and sham groups (P<0.01 for all comparisons, n=15).

CA-074Me treatment reduced cardiac fibrosis

The extent of the cardiac fibrosis was evaluated by Masson’s trichrome staining 4 weeks following MI. In the trichrome-stained sections, ECM deposition in the NIZ was >7-fold higher in MI animals compared with sham animals. The ECM deposition was particularly pronounced in the subendocardial region of the NIZ (Figs. 4 and 5). Treatment with CA-074Me resulted in a reduction in the ECM throughout the NIZ, to levels similar to those observed in the sham animals (Figs. 4 and 5). Non-infarcted animals treated with CA-074Me exhibited similar levels of ECM compared with the untreated sham-operated animals (0.26±0.07 vs. 0.19±0.07%, respectively).

Figure 4 Quantitation of collagenous matrix in the NIZ, expressed as the proportional area of blue staining on trichrome-stained sections of the rat hearts. &P<0.01 vs. sham and &&P<0.01 vs. vehicle (10% DMSO)-treated post-MI rats. NIZ, non-infarct zone; MI, myocardial infarction.

Figure 5 Representative Masson’s trichrome-stained sections demonstrating fibrosis in a (A) sham infarcted rat heart; (C) 4 weeks following MI in animals treated with vehicle (10% DMSO) and (E) with CA-074Me. Magnification, ×4. A reduced level of fibrosis is present throughout the viable myocardium of the NIZ. The right panel shows high power images (magnification, ×200) of (B) the subendocardial region of the NIZ in sham infarcted rat hearts; (D) rats treated with vehicle and (F) with CA-074Me 4 weeks following MI. (B and D) When compared with vehicle-treated post-MI animals, CA-074Me-treated rats demonstrated less fibrosis in the subendocardium of the NIZ. MI, myocardial infarction; NIZ, non-infarct zone.

Discussion

The present study indicated that CA-074Me attenuated cardiac dysfunction, cardiomyocyte hypertrophy and fibrosis following MI. This resulted in improved systolic function in a rat model of MI, which was associated with inhibition of the NLRP3-IL-1β signaling pathway.

The significance of the proinflammatory cytokine, IL-1β, in the pathogenesis of heart disease has been demonstrated in animal experiments, and exogenous administration in vivo and in vitro has led to structural remodeling with reduced cardiac function (17–18). Additional studies in animals have validated these results (19). Previous studies have suggested that IL-1β may induce systolic dysfunction in patients with heart failure, further supporting a negative role for this cytokine in heart disease (20). In post-MI clinical trials, administration of the IL-1-receptor antagonist (anakinra) reduced adverse remodeling and improved cardiac function (21). In addition, previous studies have indicated that upstream of IL-1β processing, the NLRP3 inflammasome promotes adverse cardiac remodeling following MI in mice (5). Cathepsin B release is proposed to be upstream of NLRP3 activation (22). However, it remains unknown as to whether the inhibition of cathepsin B improves systolic dysfunction in post-MI heart failure, by affecting NLRP3 activation. In the present study, we identified a critical role for cathepsin B-NLRP3 in proinflammatory cytokine production, and in the progression of systolic dysfunction.

The inflammatory response during heart failure has severe consequences on cardiac contractility. Dying cells release danger signals within the injured tissue microenvironment, which are subsequently recognized by danger-sensing systems. NLRP3 oligomerization with pro-caspase-1 and apoptosis-associated speck-like protein containing a CARD domain (ASC) leads to the activation of caspase-1, which processes pro-IL-1β for secretion, inducing sterile inflammation (23–24). A previous study, in which cardiac contractility was improved via NLRP3 siRNA in mice following MI (5), demonstrated that inflammasome/IL-1β antagonism is involved in acutely evolving injuries, including ischemia/reperfusion and infarction, as well as in the progression of heart disease, where ongoing myocardial stress yields chronic tissue damage.

Cathepsin B is a prominent lysosomal protease and is highly abundant in the left ventricular myocardium of patients with hypertensive heart failure. Thus, it has been implicated in cardiac remodeling in this disease (25).

It has been demonstrated that cathepsin B is involved in apoptosis, as well as in the degradation of myofibrillar proteins in MI (26). The activity of cathepsin B has been demonstrated to significantly increase in the serum and the hearts of isoproterenol (ISO)-induced myocardial infarcted rats (27). Increased myocardial expression of cathepsin B in failing human hearts suggests that cathepsin B may be involved in the development of heart failure (26).

In addition, although there has been substantial investigation into the roles of hypertrophy and dilatation, certain studies have demonstrated the importance of fibrosis, remote from the site of infarction, in the pathogenesis of post-MI cardiac dysfunction (28). The predilection for fibrosis in the subendocardium of the NIZ is viewed as a significant contributory factor to mechanical dysfunction (29) and propensity for dysrhythmia (30) following MI. In the present study, in contrast to its neutral effect on fibrosis in the infarct zone, CA-074Me treatment significantly reduced ECM deposition in the areas remote from the site of infarction, particularly the subendocardial region, which was associated with a reduction in the level of in LV function. Notably, studies have revealed that NLRP3 is closely associated with organ fibrosis in the lung, liver and kidney (31–33). We speculated that the reduced cardiac fibrosis due to CA-074Me treatment was associated with inhibited activation of the NLRP3 pathway.

Therefore, our study has provided support for the hypothesis that the inhibition of cathepsin B induces a significant decrease in NLRP3-IL-1β activation, resulting in improved cardiac function and reduced levels of fibrosis. In addition, complex signaling pathways are involved in myocyte hypertrophy, and our results demonstrated that the increase in cardiomyocyte size was also attenuated by such inhibition. This supports the hypothesis that the cathepsin B-NLRP3-IL-1β pathway has an adverse effect on cardiac contractility and function.

References