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

View Article: PubMed Central - PubMed

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.

No MeSH data available.


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.
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jah31799-fig-0003: 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.

Mentions: Activation of the mTOR pathway results in phosphorylation of ribosomal S6, which is commonly used as an indicator of mTOR activation.9, 10, 12 Cultured cardiac fibroblasts treated with 1 and 100 nmol/L of MBG resulted in a significant increase in phosphorylation of ribosomal S6 protein expression determined by western blot compared to controls, indicating activation of the mTOR pathway (P<0.01; Figure 3). Treatment with rapamycin at 0.1 μmol/L alone and in combination with MBG at 1 and 100 nmol/L caused a significant reduction in phospho‐S6 expression compared to MBG treatment (P<0.01; Figure 3). Treatment with low‐dose rapamycin (0.01 μmol/L) in combination with MBG resulted in a less‐profound reduction in phospho‐S6 expression (Figure 3; P=NS). MBG treatment at 1 and 100 nmol/L resulted in a significant increase in procollagen‐1 protein expression compared to controls (both P<0.01; only 100 nmol/L data shown in Figure 4). Both the high‐ (0.1 μmol/L) and low‐dose (0.01 μmol/L) rapamycin treatments significantly attenuated MBG‐induced procollagen‐1 expression (P<0.01; Figure 4).


Rapamycin Attenuates Cardiac Fibrosis in Experimental Uremic Cardiomyopathy by Reducing Marinobufagenin Levels and Inhibiting Downstream Pro ‐ Fibrotic Signaling
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.
© Copyright Policy - creativeCommonsBy-nc-nd
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5121507&req=5

jah31799-fig-0003: 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.
Mentions: Activation of the mTOR pathway results in phosphorylation of ribosomal S6, which is commonly used as an indicator of mTOR activation.9, 10, 12 Cultured cardiac fibroblasts treated with 1 and 100 nmol/L of MBG resulted in a significant increase in phosphorylation of ribosomal S6 protein expression determined by western blot compared to controls, indicating activation of the mTOR pathway (P<0.01; Figure 3). Treatment with rapamycin at 0.1 μmol/L alone and in combination with MBG at 1 and 100 nmol/L caused a significant reduction in phospho‐S6 expression compared to MBG treatment (P<0.01; Figure 3). Treatment with low‐dose rapamycin (0.01 μmol/L) in combination with MBG resulted in a less‐profound reduction in phospho‐S6 expression (Figure 3; P=NS). MBG treatment at 1 and 100 nmol/L resulted in a significant increase in procollagen‐1 protein expression compared to controls (both P<0.01; only 100 nmol/L data shown in Figure 4). Both the high‐ (0.1 μmol/L) and low‐dose (0.01 μmol/L) rapamycin treatments significantly attenuated MBG‐induced procollagen‐1 expression (P<0.01; Figure 4).

View Article: PubMed Central - PubMed

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&#8208;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&#8208;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&#8208;3 cells is initiated by CYP27A1, which is also a target for rapamycin. It was demonstrated that 1&nbsp;&mu;mol/L of rapamycin inhibited production of MBG in human JEG&#8208;2 cells. Male Sprague&#8208;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&plusmn;60 vs 377&plusmn;53&nbsp;pmol/L; P&lt;0.01), systolic blood pressure (169&plusmn;1 vs 111&plusmn;1&nbsp;mm&nbsp;Hg; P&lt;0.01), and cardiac fibrosis compared to controls. Plasma MBG levels were significantly decreased in PNx&#8208;rapamycin animals compared to PNx (373&plusmn;46 vs 1025&plusmn;60&nbsp;pmol/L; P&lt;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&#8208;mediated profibrotic signaling and provide support for beneficial effect of a novel therapy for uremic cardiomyopathy.

No MeSH data available.