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. 2023 Nov 9;21(1):799.
doi: 10.1186/s12967-023-04659-6.

Pharmacological inhibition of the cysteine protease cathepsin C improves graft function after heart transplantation in rats

Baoer Liu  1   2 , Brice Korkmaz  3 , Patricia Kraft  1 , Tobias Mayer  1 , Alex A Sayour  4 , Marc A Grundl  5 , Roxane Domain  3 , Matthias Karck  1 , Gábor Szabó  1   2 , Sevil Korkmaz-Icöz  6   7
Affiliations

Pharmacological inhibition of the cysteine protease cathepsin C improves graft function after heart transplantation in rats

Baoer Liu et al. J Transl Med. .

Abstract

Background: Heart transplantation (HTX) is the standard treatment for end-stage heart failure. However, reperfusion following an ischemic period can contribute to myocardial injury. Neutrophil infiltration, along with the subsequent release of tissue-degrading neutrophil elastase (NE)-related serine proteases and oxygen-derived radicals, is associated with adverse graft outcomes. The inhibition of cathepsin C (CatC) has been shown to block NE-related protease activation. We hypothesized that the CatC inhibitor BI-9740 improves graft function after HTX.

Methods: In a rat model of HTX, the recipient Lewis rats were orally administered with either a placebo (n = 12) or BI-9740 (n = 11, 20 mg/kg) once daily for 12 days. Donor hearts from untreated Lewis rats were explanted, preserved in a cardioplegic solution, and subsequently heterotopically implanted. In vivo left-ventricular (LV) graft function was assessed after 1 h of reperfusion. The proteolytic activity of neutrophil serine proteases was determined in bone marrow lysates from BI-9740-treated and control rats. Additionally, myocardial morphological changes were examined, and heart samples underwent immunohistochemistry and western blot analysis.

Results: The NE-related proteolytic activity in bone marrow cell lysates was markedly decreased in the BI-9740-treated rats compared to those of the placebo group. Histopathological lesions, elevated CatC and myeloperoxidase-positive cell infiltration, and nitrotyrosine immunoreactivity with an increased number of poly(ADP-ribose) polymerase (PARP)-1-positive cells were lowered in the hearts of animals treated with BI-9740 compared to placebo groups. Regarding the functional parameters of the implanted graft, improvements were observed in both systolic function (LV systolic pressure 110 ± 6 vs 74 ± 6 mmHg; dP/dtmax 2782 ± 149 vs 2076 ± 167 mmHg/s, LV developed pressure, at an intraventricular volume of 200 µl, p < 0.05) and diastolic function in the hearts of BI-9740 treated animals compared with those receiving the only placebo. Furthermore, the administration of BI-9740 resulted in a shorter graft re-beating time compared to the placebo group. However, this study did not provide evidence of DNA fragmentation, the generation of both superoxide anions and hydrogen peroxide, correlating with the absence of protein alterations related to apoptosis, as evidenced by western blot in grafts after HTX.

Conclusions: We provided experimental evidence that pharmacological inhibition of CatC improves graft function following HTX in rats.

Keywords: Cathepsin C; Heart transplantation; Ischemia; Neutrophil elastase; Reperfusion injury.

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Conflict of interest statement

Dr. M. A. Grundl is employed by Boehringer Ingelheim Pharma GmbH & Co KG, Biberach a.d. Riss, Germany. Dr B. Korkmaz has been paid for the time spent as a committee member for advisory boards (Brensocatib Advisory Board (BRAB) INSMED, USA), other forms of consulting (Boehringer Ingelheim (Germany), Neuprozyme Therapeutics Aps (Denmark), Santhera Pharmaceuticals (Switzerland), Chiesi (Italy), Gerson Lehrman Group (GLG) (USA), symposium organisation (INSMED) and travel support, lectures or presentations, outside the submitted work. Other authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
Effect of BI-9740 on bone marrow neutrophil serine protease activation. A Activities of neutrophil elastase (NE) and cathepsin G (CatG) and B Western blotting analysis of NE in bone marrows from placebo and BI-9740-treated rats. The upper blot was incubated with anti-NE, and the lower blot was incubated with anti-myeloperoxidase (MPO) for use as the loading control. Values are presented as mean ± SEM. *p < 0.05 vs. placebo
Fig. 2
Fig. 2
Effect of BI-9740 (BI) on myocardial morphology in grafts after heart transplantation (HTX). A Representative images of hematoxylin and eosin (HE) staining (× 200, scale bar: 200 µm) on cardiac tissue samples from the control group (41 pictures from 7 rats), HTX group (66 pictures from 11 rats) and HTX + BI group (44 pictures from 8 rats). In the HE staining images, nuclei are stained blue, cytoplasm is stained red, and collagen fibers display varying shades of red. B The degree of histopathological changes was assessed by examiners who were blinded to the experimental groups. The evaluation was conducted across 3–8 randomly selected, non-overlapping fields within each rat’s myocardial tissue. A indicates no changes; + , mild changes; +  + , moderate changes; and +  +  + , marked changes
Fig. 3
Fig. 3
Effect of BI-9740 (BI) on cathepsin C (CatC) and myeloperoxidase (MPO) expression in grafts after heart transplantation (HTX). Representative images of A CatC (× 200, scale bar: 200 µm) and B MPO (× 400, scale bar: 100 µm) immunostaining followed by quantification based on the percentage of CatC-positive cells over the total cell count (control: 56 pictures from 7 rats; HTX: 80 pictures from 10 rats; HTX + BI: 76 pictures from 10 rats) and the total number of MPO-positive cells in the tissue section (control: 47 pictures from 6 rats; HTX: 72 pictures from 9 rats; HTX + BI: 88 pictures from 11 rats). The samples were assessed by two examiners who were blinded to the experimental groups. The evaluation was carried out on 4–8 randomly selected non-overlapping fields within each rat’s myocardial tissue. Values are expressed as mean ± SEM and correspond to the mean of all pictures. *p < 0.05 vs. control, #p < 0.05 vs HTX
Fig. 4
Fig. 4
Effect of BI-9740 (BI) on nitrotyrosine and poly(ADP-ribose) polymerase (PARP)-1 expression in grafts after heart transplantation (HTX). Representative images of A nitrotyrosine (× 100, scale bar: 500 µm) and B PARP-1 (× 400, scale bar: 100 µm) immunostaining are shown, along with semi-quantitative analysis of nitrotyrosine immunoreactivity (control: 8 rats, HTX: 10 rats, HTX + BI: 10 rats) and quantification of PARP-1-positive cells (control: 10 rats, HTX: 11 rats, HTX + BI: 10 rats). The samples were assessed by two examiners who were blinded to the experimental groups. The evaluation was conducted on 4–8 randomly selected non-overlapping fields per rat’s myocardial tissue. Values are expressed as mean ± SEM. *p < 0.05 vs. control, #p < 0.05 vs HTX
Fig. 5
Fig. 5
Effect of BI-9740 on protein expression of bcl-2, bax, and caspase-3 (casp-3) in grafts after heart transplantation (HTX). Quantification of the protein bands showing A bax-to-bcl-2 ratio, and B cleaved (cl.) caspase-3-to-caspase-3 ratio. Values are expressed as mean ± SEM
Fig. 6
Fig. 6
Effect of BI-9740 on left-ventricular (LV) graft function after heart transplantation (HTX). A Left-ventricular systolic pressure (LVSP), B developed pressure, C maximal slope of systolic pressure increment (dP/dtmax), D rate pressure product, and E maximal slope of diastolic pressure decrement (dP/dtmin) were measured at different LV volumes. Values are expressed as mean ± SEM. *p < 0.05 vs. HTX. n = 11–12 rats/group
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