sirolimus and marinobufagenin

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.. 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.. 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.

Topics: Animals; Blood Pressure; Bufanolides; Cardiomyopathies; Cells, Cultured; Enzyme Inhibitors; Fibroblasts; Fibrosis; Heart; Humans; Immunosuppressive Agents; Male; Myocardium; Nephrectomy; Rats; Rats, Sprague-Dawley; Sirolimus; Sodium-Potassium-Exchanging ATPase; Uremia

Decreases in cardiac Na/K-ATPase have been documented in patients with heart failure. Reduction of Na/K-ATPase α1 also contributes to the deficiency in cardiac contractility in animal models. Our previous studies demonstrate that reduction of cellular Na/K-ATPase causes cell growth inhibition and cell death in renal proximal tubule cells. To test whether reduction of Na/K-ATPase in combination with increased cardiotonic steroids causes cardiac myocyte death and cardiac dysfunction, we examined heart function in Na/K-ATPase α1 heterozygote knock-out mice (α1(+/-)) in comparison to wild type (WT) littermates after infusion of marinobufagenin (MBG). Adult cardiac myocytes were also isolated from both WT and α1(+/-) mice for in vitro experiments. The results demonstrated that MBG infusion increased myocyte apoptosis and induced significant left ventricle dilation in α1(+/-) mice but not in their WT littermates. Mechanistically, it was found that in WT myocytes MBG activated the Src/Akt/mTOR signaling pathway, which further increased phosphorylation of ribosome S6 kinase (S6K) and BAD (Bcl-2-associated death promoter) and protected cells from apoptosis. In α1(+/-) myocytes, the basal level of phospho-BAD is higher compared with WT myocytes, but MBG failed to induce further activation of the mTOR pathway. Reduction of Na/K-ATPase also caused the activation of caspase 9 but not caspase 8 in these cells. Using cultures of neonatal cardiac myocytes, we demonstrated that inhibition of the mTOR pathway by rapamycin also enabled MBG to activate caspase 9 and induce myocyte apoptosis.

Topics: Animals; Anti-Bacterial Agents; Apoptosis; Bufanolides; Caspase 8; Caspase 9; Cells, Cultured; Enzyme Activation; Enzyme Inhibitors; Heart Diseases; Mice; Mice, Mutant Strains; Muscle Proteins; Myocytes, Cardiac; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; Sodium-Potassium-Exchanging ATPase; src-Family Kinases; TOR Serine-Threonine Kinases