Cathepsin A inhibition attenuates myocardial infarction-induced heart failure on the functional and proteomic levels

Abstract

Background: Myocardial infarction (MI) is a major cause of heart failure. The carboxypeptidase cathepsin A is a novel target in the treatment of cardiac failure. We aim to show that recently developed inhibitors of the protease cathepsin A attenuate post-MI heart failure.

Methods: Mice were subjected to permanent left anterior descending artery (LAD) ligation or sham operation. 24 h post-surgery, LAD-ligated animals were treated with daily doses of the cathepsin A inhibitor SAR1 or placebo. After 4 weeks, the three groups (sham, MI-placebo, MI-SAR1) were evaluated.

Results: Compared to sham-operated animals, placebo-treated mice showed significantly impaired cardiac function and increased plasma BNP levels. Cathepsin A inhibition prevented the increase of plasma BNP levels and displayed a trend towards improved cardiac functionality. Proteomic profiling was performed for the three groups (sham, MI-placebo, MI-SAR1). More than 100 proteins were significantly altered in placebo-treated LAD ligation compared to the sham operation, including known markers of cardiac failure as well as extracellular/matricellular proteins. This ensemble constitutes a proteome fingerprint of myocardial infarction induced by LAD ligation in mice. Cathepsin A inhibitor treatment normalized the marked increase of the muscle stress marker CA3 as well as of Igγ 2b and fatty acid synthase. For numerous further proteins, cathepsin A inhibition partially dampened the LAD ligation-induced proteome alterations.

Conclusions: Our proteomic and functional data suggest that cathepsin A inhibition has cardioprotective properties and support a beneficial effect of cathepsin A inhibition in the treatment of heart failure after myocardial infarction.

Keywords: Cardiovascular diseases; Drug therapy; Heart failure; Mouse model; Myocardial infarction.

Fig. 1

Experimental design of the study. Sham operation or permanent LAD ligation were performed in mice. LAD ligation was either treated with placebo or with the cathepsin A inhibitor SAR1. After 28 days cardiac functionality was evaluated and a quantitative proteome study was conducted

Fig. 2

Functional parameters in the murine MI model. Cardiac parameters were assessed 4 weeks after permanent ligation of the LAD. Three groups have been compared: sham (n = 10), MI-placebo (n = 15), MI-SAR1 (n = 10). Results are expressed as mean ± SEM. Differences significant at p < 0.05 are marked with an asterisk (*). LVESV left ventricular end-systolic volume, LVEDV left ventricular end-diastolic volume, LVEDP left ventricular end-diastolic pressure, CO cardiac output, EF ejection fraction, SV stroke volume, MAP mean arterial pressure, HR heart rate, dP/dt peak positive or negative first derivative of LV pressure, BNP plasma brain natriuretic peptide

Fig. 3

Protein identification and quantification overview for the proteome analysis of murine hearts. a Venn diagram representing the number of identified proteins in each replicate and their overlap. 1074 proteins were found in at least three replicates. b Distribution and geometric mean (horizontal bar) of fold change values (log₂ of relative protein ratios) of proteins from the four biological replicates. Each replicate contains an animal from each group (sham, MI-placebo, MI-SAR1)

Fig. 4

Cardiac proteome fingerprint of cathepsin A inhibition upon LAD ligation. a Scatter plot and linear regression analysis of protein abundance alterations observed for placebo-treated LAD ligation (as compared to sham operation) and SAR1-treated LAD ligation (as compared to sham operation). b Heatmap comparison of fold changes MI-placebo/sham and MI-SAR1/sham of the 104 significantly affected proteins upon LAD ligation. Fold changes in the first column (MI-placebo/sham) are placed in descending order

Fig. 5

Effect of cathepsin A inhibition on proteome alterations in LAD ligation. a Bar chart depiction of proteins that are significantly increased in placebo-treated LAD ligation (as compared to sham operation) and for which SAR1 treatment decreased this effect by a fold-change value of more than 50 % [difference of log₂ ratios (MI-placebo/sham)–(MI-SAR1/sham) > 0.58]. b Same as (a) but for significantly decreased proteins in LAD ligation. Results are expressed as mean ± SEM. Differences significant at p < 0.05 are marked with an asterisk (*). c Western blot analysis of selected proteins. Shown here is a representative blot. GAPDH is used as loading control

Fig. 6

Effect of cathepsin A inhibition in an in vitro model of ischemia. Cells were incubated for 24 h with 10 μM SAR1 or DMSO as a solvent-only control in simulated ischemia culture conditions (hypoxia and serum deprivation, see Methods section for details). a Quantitative analysis of caspase-3 activity in the total cell lysate of cells treated with SAR1 in standard conditions (normoxia; DMEM supplemented with FCS) and simulated ischemia (hypoxia; DMEM serum-free). b Cells in simulated ischemia were stained with YO-PRO-1 or PI and evaluated by flow cytometry. The percentage of cells positive to YO-PRO-1 or to PI was calculated. Results are expressed as mean ± SEM. Differences significant at p < 0.05 are marked with an asterisk (*)