Rapamycin Attenuates Cardiac Fibrosis in Experimental Uremic Cardiomyopathy by Reducing Marinobufagenin Levels and Inhibiting Downstream Pro-Fibrotic Signaling

Abstract

Background: Experimental uremic cardiomyopathy causes cardiac fibrosis and is causally related to the increased circulating levels of the cardiotonic steroid, marinobufagenin (MBG), which signals through Na/K-ATPase. Rapamycin is an inhibitor of the serine/threonine kinase mammalian target of rapamycin (mTOR) implicated in the progression of many different forms of renal disease. Given that Na/K-ATPase signaling is known to stimulate the mTOR system, we speculated that the ameliorative effects of rapamycin might influence this pathway.

Methods and results: Biosynthesis of MBG by cultured human JEG-3 cells is initiated by CYP27A1, which is also a target for rapamycin. It was demonstrated that 1 μmol/L of rapamycin inhibited production of MBG in human JEG-2 cells. Male Sprague-Dawley rats were subjected to either partial nephrectomy (PNx), infusion of MBG, and/or infusion of rapamycin through osmotic minipumps. PNx animals showed marked increase in plasma MBG levels (1025±60 vs 377±53 pmol/L; P<0.01), systolic blood pressure (169±1 vs 111±1 mm Hg; P<0.01), and cardiac fibrosis compared to controls. Plasma MBG levels were significantly decreased in PNx-rapamycin animals compared to PNx (373±46 vs 1025±60 pmol/L; P<0.01), and cardiac fibrosis was substantially attenuated by rapamycin treatment.

Conclusions: Rapamycin treatment in combination with MBG infusion significantly attenuated cardiac fibrosis. Our results suggest that rapamycin may have a dual effect on cardiac fibrosis through (1) mTOR inhibition and (2) inhibiting MBG-mediated profibrotic signaling and provide support for beneficial effect of a novel therapy for uremic cardiomyopathy.

Keywords: cardiac fibrosis; cardiomyopathy; cardiovascular diseases; fibrosis; heart failure.

Figure 1

A, Chemical structure of marinobufagenin (MBG), 3β,5β‐dihydroxy‐14,15‐epoxy bufadienolide, PubChem CID: 11969465. B, MBG production in human placental chorionic epithelial cells (JEG‐3 cells) after incubation with 1 μmol/L of rapamycin (Rapa) for 3 and 6 hours. *P<0.05 versus control; **P<0.01 versus control.

Figure 2

A, Representative (top) and quantitative analysis of collagen 1 (mean±SEM) western blots performed on cardiac tissue from the different groups. Actin was used as a loading control. B, Representative Sirius red and Fast green stained photomicrographs obtained from cardiac tissue derived from the different experimental groups. Scale bar=100 μmol/L. Amount of fibrosis expressed as mean±SEM measured using computer‐assisted morphometry, as we have previously described.5 Sham refers to animals subject to sham surgery (n=8); PNx refers to partial nephrectomy (n=10); PNx+Rapa refers to PNx surgery and rapamycin infusion using minipumps (n=6); Rapa refers to rapamycin infusion using minipumps (n=8); MBG+Rapa refers to coadministration of marinobufagenin (MBG) and rapamycin using minipumps (n=8); and MBG refers to MBG infusion using minipumps (n=8). *P<0.01 versus sham and Rapa; **P<0.01 versus PNx and MBG; ^# P<0.05 versus sham.

Figure 3

Representative (top) and quantitative analysis of phosphoribosomal S6 protein western blots derived from cardiac fibroblasts treated with marinobufagenin (MBG; 1 or 100 nmol/L), rapamycin (Rapa; 0.01 or 0.1 μmol/L), or in combination with the corresponding quantitative data shown as the mean±SEM of 5 experiments. Total ribosomal S6 protein was used as loading control. *P<0.01 versus control; **P<0.01 versus MBG 100 nmol/L, MBG 100 nmol/L+Rapa 0.01 μmol/L, MBG 1 nmol/L, and MBG 1 nmol/L+Rapa 0.01 μmol/L.

Figure 4

Representative (top) and quantitative analysis of procollagen‐1 western blots derived from cardiac fibroblasts treated with marinobufagenin (MBG) 100 nmol/L with or without rapamycin (Rapa; 0.01 or 0.1 μmol/L) with the corresponding quantitative data shown as the mean±SEM of 5 experiments. *P<0.01 versus control; ^# P<0.05 versus control; **P<0.01 versus MBG 100 nmol/L.

Figure 5

Representative and quantitative analysis of carbonylated protein western blots derived from cardiac fibroblasts treated with marinobufagenin (MBG; 100 or 1 nmol/L), rapamycin (Rapa; 0.1 μmol/L) or in combination with the corresponding quantitative data shown as the mean±SEM of 5 experiments. Ponceau S staining of the membrane was used to control for loading. A; *P=0.01 versus control; **P<0.01 versus control; ^## P=0.01 versus MBG 100 nmol/L; ^# P<0.05 versus MBG 1‐nmol/L treatment.