The Effects Of Cathepsin B Inhibitor CA-074Me On Cardiac Dysfunction And Remodeling In Post-myocardial Infaction Rat

Objective:This study aimed to elucidate the effects of a specific cathepsin B inhibitor CA-074Me on cardiac dysfunction and remodeling as well as fibrosis following myocardial infarction using an MI rat model, and to investigate its potential mechanisms of action.Methods:1) Induction of Rat MIMale Sprague-Dawley rats were purchased from Vital River Laboratories (Beijing, China). Animals were housed and maintained under standard conditions in our experimental animal center. All experiments conformed to the Guide for the Care and Use of Laboratory Animals by our Institute. MI was induced in male10-week old rats (weighing280-320g) by ligating the left anterior descending coronary artery as previously described. Briefly, anaesthesia with isoflurane (5%induction,2%maintenance) preceded intubation and ventilation. Adequacy of anaesthesia was monitored by loss of reflexes, and degree of muscle relaxation. Left-sided thoracotomy was performed between the fifth and sixth ribs. The pericardium was opened, and the heart exteriorized. The coronary artery was localized1-2mm below the junction of the pulmonary conus and the left atrial appendage. A5.0silk suture 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, and lungs were re-expanded before closing the chest wall with4.0silk. Sham animals underwent every procedure except coronary artery ligation.2) Treatment Stock solutions of the cathepsin B inhibitor CA-074Me were made at a concentration of10mg/ml in dimethyl sulfoxide (DMSO). This was diluted1:10in saline and administered at a dose of10mg/kg by intraperitoneal injection according to previously validated protocols. All rats were randomly assigned to3groups. Group I (n=10):sham-operated rats as the normal control; Group II:MI rats with vehicle (10%DMSO)(n=15) for4weeks; Group III:MI rats with CA-074Me treatment at a dosage of10mg/kg/day (n=15) for4weeks.3) Cathepsin B activity assayCathepsin B activity was measured using the synthetic fluorometric substrate Z-Arg-Arg-NHMec. Briefly,150μM Z-Arg-Arg-NHMec (pH6.0) was added to the assay buffer and fluorescence was measured in triplicate, at one-minute intervals for30minutes, at an excitation of360nm and an emission of465nm. Data are represented as relative fluorescent units and presented as mean±standard deviation (SD) of three independent experiments.4) Western blotTotal protein extracts were prepared by homogenizing heart tissues in lysis buffer (Cell Signaling Technology) and stored at-20C. The protein content was determined using the bicinchoninic assay method (Pierce). Equal amounts of protein were boiled with SDS buffer, loaded on8-12%denaturing polyacrylamide gels, blotted onto PVDF membrane. After blocking with5%skimmed milk, specific primary antibodies were used as follows:anti-NLRP3(1:2000; BD); anti-caspase-lp20(1:2000), anti-pro-IL-1β/IL-1β (1:2000), anti-pro-IL-18/IL-18(1:2000)(all from Cell Signaling Technology). Immunoblots were visualized by enhanced chemiluminescence, and quantified for specific protein content by densitometry with normalization for housekeeping gene β-actin (1:2000; Cell Signaling Technology).5) ELISASerum IL-1β and IL-18levels were determined by ELISA kits (R&D Systems) according to the manufacturer’s instructions.6) Cardiac function assessed by echocardiographyCardiac function of all rats was evaluated by noninvasive echocardiography. Briefly, images were recorded using a10-12MHz phased-array transducer (model21380A with HP SONOS5500imaging system; Agilent Technologies). Diastolic and systolic LV internal dimensions (LVIDD and LVIDS) and LV fractional shortening (LVFS) were measured with M-mode tracings from the short-axis view of the LV at the papillary muscle level. All measurements were performed in a blinded fashion according to the guidelines of the American Society for Echocardiology and averaged over3consecutive cardiac cycles. All data were acquired and analyzed by a single blinded observer using EchoPAC (GE Vingmed) off-line processing.7) Infarct sizeThe Masson’s trichrome-stained slides were examined under light microscopy and digitized, then analyzed using image analysis (AIS, Analytical imaging Station Version6.0, Ontario, Canada). Infarct size was assessed morphologically and calculated as the ratio of scar average circumferences of the endocardium and the epicardium to LV average circumferences of the endocardium and the epicardium.8) Extracellular matrix depositionSections were stained with Masson’s Trichrome to examine extracellular matrix (ECM) deposition as previously described. All tissues were assessed with the examiner masked to the experimental groups. The accumulation of matrix within the non-infarct zone (NIZ) was then quantified. Briefly, stained sections from the mid left ventricle were digitally captured in their entirety with a standard polarizing filter, and then loaded onto a Pentium III IBM computer. To isolate the NIZ from the infarct and the peri-infarct zone, the infarct and a2mm zone on either side of it were excluded from analysis. The remaining myocardium composed the NIZ, and was analyzed using computer-assisted image analysis using image analysis software (Analytical imaging Station Version6.0, Canada). The whole NIZ was used for quantification of ECM in order to prevent possible bias from using selected fields. An area of blue on a trichrome-stained section, representing ECM, was selected for its color range. For sham animals, the ECM content of the entire LV was quantitated by the same method, as described above.9) Histological and immunofluorescence analysis Rat hearts were collected at4weeks post-MI fixed with buffered10%PFA for1h, followed by1h incubation in0.1M glycine and overnight incubation at4℃in0.6M sucrose. Samples were embedded in OCT and sections (4μm) were cut and stored in-20℃. Sections for Masson-trichrome were processed as per manufacturer instructions (Sigma).In a separate set of IHC experiments, rat heart sections were permeabilized and blocked for1h in1%BSA and probed with primary polyclonal antibodies to a-sarcomeric actin (1:50; Sigma), wheat germ hemagglutinin (WGA)(1:50), and anti-His (1:50)(both from Invitrogen). Secondary antibodies (1:100; Invitrogen) were used at the recommended dilution and incubated on sections for1h at room temperature. Nuclei counterstained with (DAPI). Images were acquired on an inverted epifluorescent microscope (Zeiss).Results:1. CA-074Me inhibited activation of cathepsin B-NLRP3-IL-1β pathwayFirst of all, we confirmed that cathepsin B activity was inhibited by CA-074Me treatment. Further, we examined whether CA-074Me treatment affected serum levels of the proinflammatory cytokines IL-1β and IL-18by ELISA. A marked increase in serum levels of these cytokines was seen in vehicle-treated rats compared to the low baseline levels observed in sham control rats, and levels in CA-074Me-treated rats were significantly lower than those in vehicle-treated animals.As demonstrated by Western blot analysis, increasing amounts of mature IL-1β were observed in lysates of vehicle-treated hearts, but not CA-074Me-treated or sham control hearts. We then measured whether inflammasome activation was suppressed by CA-074Me administration. We then measured NLRP3protein levels in heart tissues using. In vehicle-treated rats, levels of NLRP3protein were significantly increased compared to sham controls and these effects were inhibited by CA-074Me treatment. Consistent with cytokine maturation, caspase-1activation was also seen in vehicle-treated hearts, as demonstrated by the appearance of the p20subunit, and this effect was significantly inhibited by CA-074Me treatment.2. Treatment with CA-074Me improves cardiac function Echocardiography was used to examine cardiac structure and function in rats. At baseline, no differences between the3groups were observed. CA-074Me treatment for4weeks contributed to significant improvements in LVIDD, LVIDS, LVFS and ejection fraction (EF).3. Infarct size and LV cardiomyocyte size were reduced by CA-074MeTreatment with CA-074Me significantly reduced the infarct size compared with vehicle-treated MI groups (22±2.8%vs38±5.4%, p<0.01). The cardiomyocyte size was assessed using high magnification microscopy to quantify circumference and the cross-sectional area. These data showed that treatment with CA-074Me prevented cardiac remodeling when compared with vehicle and sham (P<0.01for all comparisons, n=15).4. CA-074Me treatment reduced cardiac fibrosisThe extent of cardiac fibrosis was evaluated by Masson-trichrome staining at4weeks post-MI. On trichrome-stained sections, ECM deposition in the NIZ was more than sevenfold higher in MI animals compared with sham animals that were especially pronounced in the subendocardial region of the NIZ. Treatment with CA-074Me resulted in a reduction in ECM throughout the NIZ to levels similar to those seen in sham animals. Non-infarcted animals treated with CA-074Me had similar levels of ECM compared to the untreated sham-operated animals (0.26±0.07%versus0.19±0.07%, respectively).Conclusion:Our study provides additional data that inhibition of cathepsin B induced a significant decrease in NLRP3-IL-1β activation, resulting in improved cardiac function and less fibrosis. In addition, complex signaling pathways are involved in myocyte hypertrophy, and our findings showed that cardiomyocyte size was as also attenuated by such inhibition, which supports an adverse effect of the cathepsin B-NLRP3-IL-1β pathway on cardiac contractility and function.