Cathepsin A Mediates Ventricular Remote Remodeling and Atrial Cardiomyopathy in Rats With Ventricular Ischemia/Reperfusion

Figure 1. Differential Expression Pattern of CatA in Human ICM and in Rats With Permanent Ligation and in Rats With Ventricular I/R (A and B) Human ischemic cardiomyopathy (ICM). (A) Messenger ribonucleic acid (mRNA) expression of cathepsin A (CatA) and atrial natriuretic peptide (ANP) in left ventricular tissue from healthy nonfailing donor hearts (Ctr, n = 8) and from patients with end-stage ICM (n = 8). (B) Representative Western blot and quantification of CatA protein levels in the left ventricle of Ctr and ICM patients (n = 8 per group). (C and D) Rats with 30 min of ventricular ischemia followed by 10 weeks of reperfusion (ischemia/reperfusion [I/R]). (C) CatA and ANP mRNA content in the left ventricle (I/R-Sham, n = 9-8; I/R-Placebo, n = 14-13). (D) Left atrial tissue 10 weeks after I/R in sham-operated rats (I/R-Sham, n = 9) and rats receiving placebo (I/R-Placebo; n = 15-9). Values are mean ± SEM. GAPDH = glyceraldehyde 3-phosphate dehydrogenase; IOD = integrated optical density.

Figure 2. Regional Left Ventricular Function Using magnetic resonance imaging, 16 short-axis cine imaging slices of ischemia/reperfusion (I/R)-Sham (n = 9), I/R-rats given placebo (n = 15), and I/R-rats treated with ramipril (n = 14) or (S)-3-{[1-(2-Fluoro-phenyl)-5-methoxy-1H-pyrazole-3-carbonyl]-amino}-3-o-tolyl-propionic-acid (SAR; n = 14) were performed; slices were divided into 97 cords. (A) Every cord was analyzed for left ventricular wall motion (WM) at rest and (B) during dobutamine stress testing. Analysis of left ventricular systolic wall thickness (SWTS) for every cord (C) at rest and (D) during dobutamine stress testing. Values are mean ± SEM. #I/R-SAR vs. I/R-Ramipril. ψI/R-SAR vs. I/R-Placebo. §I/R-Ramipril vs. I/R-Placebo.

Figure 3. Atrial Electrophysiological Measurements in I/R-Rats (A) Atrial fibrillation (AF) inducibility and duration induced by burst-stimulation in I/R-Sham (n = 8), I/R-rats given placebo (n = 11), and I/R-rats treated with ramipril (n = 14) or SAR (n = 13). (B) Areas of slow conduction. (C) Total atrial activation time (I/R-Sham, n = 7; I/R-Placebo, n = 9; I/R-Ramipril, n = 9; I/R-SAR, n = 9). (D) Left atrial effective refractory period (AERP) at a stimulation frequency of 400/min (basic cycle lengths: 150 ms). Values are mean ± SEM. Abbreviations as in Figures 1 and 2.

Figure 4. Atrial Extracellular Matrix Remodeling Representative histological images of (A) conventional total collagen (red fibers) and (B) polarized light microscopy (collagen type I [red-yellow fibers] and collagen type III [green fibers]) of Picro-Sirius Red–stained left atrial preparations in I/R-Sham (n = 9), I/R-rats given placebo (n = 13), and I/R-rats treated with ramipril (n = 12) or SAR (n = 11). (C) Quantification of left atrial (LA) fibrosis amount. (D) Collagen type I/type III ratio and (E) mRNA expression of collagen 1a (Col1a1), Col1a2, Col3a1, and Col4a1 normalized to expression of GAPDH in I/R-Sham (n = 9-7), I/R-Placebo (n = 14), I/R-Ramipril (n = 11-10), and I/R-SAR (n = 14-12). Values are mean ± SEM. Abbreviations as in Figures 1 and 2.

Figure 5. Atrial Hypertrophy Representative histological images of (A) hematoxylin and eosin–stained LA tissue preparations in I/R-Sham rats, I/R-rats given placebo, and I/R-rats treated with ramipril or SAR. (B) Ratio of LA weight per 100 g body weight of I/R-Sham (n = 9), I/R-Placebo (n = 12), I/R-Ramipril (n = 12), and I/R-SAR (n = 13) rats. (C) Quantification of LA myocyte diameter and (D) brain natriuretic peptide (BNP) mRNA level in the left atrium in I/R-Sham (n = 9), I/R-rats given placebo (n = 11-12) and I/R-rats treated with ramipril (n = 11-12) or SAR (n = 11-14). Values are mean ± SEM. Abbreviations as in Figures 1, 2, and 4.

Figure 6. Atrial Cx43 Distribution (A) Representative immunohistochemical staining for connexin 43 (Cx43) in left atrial tissue preparations in I/R-Sham (n = 6), I/R-rats given placebo (n = 7), and I/R-rats treated with ramipril (n = 7) or SAR (n = 7). (B) Quantification of Cx43-positively stained lateral and (C) polar cardiomyocyte membrane. Values are mean ± SEM. Other abbreviations as in Figure 1.