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. 2014 Jul 11;3(4):e000965.
doi: 10.1161/JAHA.114.000965.

Reducing endoglin activity limits calcineurin and TRPC-6 expression and improves survival in a mouse model of right ventricular pressure overload

Navin K Kapur  1 , Xiaoying Qiao  1 , Vikram Paruchuri  1 , Emily E Mackey  1 , Gerard H Daly  1 , Kishan Ughreja  1 , Kevin J Morine  1 , Jonathan Levine  1 , Mark J Aronovitz  1 , Nicholas S Hill  1 , Iris Z Jaffe  1 , Michelle Letarte  2 , Richard H Karas  1
Affiliations

Reducing endoglin activity limits calcineurin and TRPC-6 expression and improves survival in a mouse model of right ventricular pressure overload

Navin K Kapur et al. J Am Heart Assoc. .

Erratum in

  • J Am Heart Assoc. 2014 Aug;3(4):e000419. Ughreja, Kesahn [Corrected to Ughreja, Kishan]

Abstract

Background: Right ventricular (RV) failure is a major cause of mortality worldwide and is often a consequence of RV pressure overload (RVPO). Endoglin is a coreceptor for the profibrogenic cytokine, transforming growth factor beta 1 (TGF-β1). TGF-β1 signaling by the canonical transient receptor protein channel 6 (TRPC-6) was recently reported to stimulate calcineurin-mediated myofibroblast transformation, a critical component of cardiac fibrosis. We hypothesized that reduced activity of the TGF-β1 coreceptor, endoglin, limits RV calcineurin expression and improves survival in RVPO.

Methods and results: We first demonstrate that endoglin is required for TGF-β1-mediated calcineurin/TRPC-6 expression and up-regulation of alpha-smooth muscle antigen (α-SMA), a marker of myofibroblast transformation, in human RV fibroblasts. Using endoglin haploinsufficient mice (Eng(+/-)) we show that reduced endoglin activity preserves RV function, limits RV fibrosis, and attenuates activation of the calcineurin/TRPC-6/α-SMA pathway in a model of angio-obliterative pulmonary hypertension. Next, using Eng(+/-) mice or a neutralizing antibody (Ab) against endoglin (N-Eng) in wild-type mice, we show that reduced endoglin activity improves survival and attenuates RV fibrosis in models of RVPO induced by pulmonary artery constriction. To explore the utility of targeting endoglin, we observed a reversal of RV fibrosis and calcineurin levels in wild-type mice treated with a N-Eng Ab, compared to an immunoglobulin G control.

Conclusion: These data establish endoglin as a regulator of TGF-β1 signaling by calcineurin and TRPC-6 in the RV and identify it as a potential therapeutic target to limit RV fibrosis and improve survival in RVPO, a common cause of death in cardiac and pulmonary disease.

Keywords: cardiac remodeling; fibrosis; heart failure; pulmonary hypertension; right ventricle.

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Figures

Figure 1.
Figure 1.
Calcineurin regulates myofibroblast transformation and TRPC‐6 expression in right ventricular fibroblasts. A, Western blots showing calcineurin, α‐SMA, pSmad3, total Smad3, and GAPDH expression in human right ventricular fibroblasts (RVFB) after stimulation with TGF‐β1 (10 ng/mL for 16 to 24 hours) in the presence and absence of cyclosporine (CS). B and D, mRNA levels of calcineurin, α‐SMA, and TRPC‐6 in human RVFB after stimulation with TGF‐β1 in the presence and absence of CS (n=3/group). E, Western blot showing silencing of TRPC‐6 in human RVFB. F, Western blot showing calcineurin and α‐SMA levels in human RVFB after TGF‐β1 stimulation in the presence and absence of a siRNA against TRPC‐6 (siTRPC‐6). *P<0.05 versus vehicle; †P<0.05 versus TGF‐β1 stimulation; ‡P<0.05 versus WT+TGF‐β1 stimulation. α‐SMA indicates α‐smooth muscle antigen; TGF‐β1, transforming growth factor beta 1; TRPC‐6, transient receptor protein channel 6.
Figure 2.
Figure 2.
Reduced endoglin activity limits calcineurin expression and myofibroblast transformation in right ventricular fibroblasts. A and B, Western blots showing calcineurin, α‐SMA, pSmad3, and GAPDH levels in fibroblasts from human right (RVFB) and left (LVFB) ventricular fibroblasts after TGF‐β1 stimulation in the presence and absence of increasing concentrations of a neutralizing endoglin antibody (N‐Eng Ab). Quantification of calcineurin and a‐SMA levels in human RVFB and LVFB (n=3/group). C, mRNA levels of calcineurin and α‐SMA in right (RVFB) and left (LVFB) ventricular fibroblasts derived from WT and Eng+/− mice after TGF‐β1 stimulation (n=6/group). D, Western blots showing calcineurin and α‐SMA levels after TGF‐β1 stimulation in RVFB and LVFB from WT and Eng+/− mice. D and E, Quantification of calcineurin and α‐SMA protein levels in RVFB and LVFB from WT and Eng+/− mice stimulated with TGF‐β1. *P<0.05 versus vehicle; †P<0.05 versus TGF‐β1 stimulation; ‡P<0.05 versus LVFB+TGF‐β1 stimulation. α‐SMA indicates α‐smooth muscle antigen; TGF‐β1, transforming growth factor beta 1; WT, wild type.
Figure 3.
Figure 3.
Reduced endoglin expression limits fibrosis and calcineurin expression in a murine model of angio‐obliterative pulmonary hypertension. A through C, Right ventricular systolic pressure, tau, and RV compliance in Eng+/+ and Eng+/− mice after 5 weeks of treatment with Sugen compound under normoxic (Su‐Norm) or hypoxic (Su‐Hypox) conditions (n=6/group). D, mRNA levels of type I collagen in WT and Eng+/− mice under Su‐Norm or Su‐Hypox conditions (n=6/group). E and F, Representative histologic staining for RV collagen abundance in Eng+/+ and Eng+/− mice under Su‐Norm or Su‐Hypox conditions. Quantification of percent RV fibrosis is shown (n=6/group). G, mRNA levels of calcineurin, TRPC‐6, and a‐SMA in RV tissue from WT and Eng+/− mice under Su‐Norm or Su‐Hypox conditions (n=6/group). *P<0.05 versus Eng+/+ Su‐Norm; †P<0.05 versus Eng+/− Su‐Norm; ‡P<0.05 Eng+/+ Su‐Hypox versus Eng+/− Su‐Hypox. α‐SMA indicates α‐smooth muscle antigen; RV, right ventricular; TRPC‐6, transient receptor protein channel 6; WT, wild type.
Figure 4.
Figure 4.
Reduced endoglin expression improves survival after right ventricular pressure overload. A and B, Levels of endoglin mRNA and protein expression in WT and Eng+/− mice after PAC (n=6/group). C, Right ventricular systolic pressure in WT and Eng+/− mice after PAC (n=6/group). D, Right ventricular stroke volume in WT and Eng+/− mice after PAC (n=6/group). E, Total body weight in WT and Eng+/− mice after PAC (n=6/group). F, Kaplan‐Meier's survival curves in WT and Eng+/− mice after PAC (n=12/group). *P<0.05 versus sham; †P<0.05 versus WT versus Eng+/− sham; ‡P<0.05 Wt versus Eng+/− PAC. PAC indicates pulmonary artery constriction; RV, right ventricular; WT, wild type.
Figure 5.
Figure 5.
Reduced endoglin expression limits RV fibrosis and hypertrophy after right ventricular pressure overload. A and B, Representative histologic staining for RV collagen abundance in WT and Eng+/− mice after PAC. Quantification of RV fibrosis after PAC is shown (n=6/group). C and D, Representative histological staining for RV cardiomyocyte hypertrophy in WT and Eng+/− mice after PAC. Quantification of cardiomyocyte cross‐sectional area after PAC is shown (n=6/group). *P<0.05 versus sham; †P<0.05 versus WT‐PAC. PAC indicates pulmonary artery constriction; RV, right ventricular; WT, wild type.
Figure 6.
Figure 6.
Reduced endoglin expression limits TGF‐β1 signaling and calcineurin activity in the RV after right ventricular pressure overload. A, Levels of active TGF‐β1 in RV protein lysates from WT and Eng+/− mice (n=6/group). B through D, Quantification of RV type I collagen, pSmad3, and pERK1/2 protein levels in WT and Eng+/− mice after PAC (n=6/group). Representative western blots are shown. E, Levels of RV calcineurin protein in WT and Eng+/− mice after PAC (n=6/group). A representative western blot is shown. F and H, Levels of RV MYH7, TRPC‐6, and α‐SMA mRNA expression in WT and Eng+/− mice after PAC (n=6/group). *P<0.05 versus sham; †P<0.05 versus WT‐PAC. α‐SMA indicates alpha‐smooth muscle antigen; PAC, pulmonary artery constriction; RV, right ventricular; TGF‐β1, transforming growth factor beta 1; TRPC‐6, transient receptor protein channel 6; WT, wild type.
Figure 7.
Figure 7.
Reduced endoglin expression limits calcineurin activity in right ventricular pressure overload. Luciferase activity in right ventricular lysates from Eng+/+‐NFAT‐Luc and Eng+/−‐NFAT‐Luc mice subjected to 7 days of severe RVPO. *P<0.05 versus Eng+/+‐NFAT‐Luc Sham; †P<0.05 versus Eng+/+‐NFAT‐Luc PAC. PAC indicates pulmonary artery constriction; RVPO, RV pressure overload.
Figure 8.
Figure 8.
Neutralizing endoglin activity improves survival and limits the development of RV fibrosis after right ventricular pressure overload. A, Kaplan‐Meier's survival curves in WT mice treated with an IgG control Ab or N‐ Eng Ab after PAC (n=18/group). B and C, Representative histological staining for RV collagen abundance in IgG vs. N‐Eng Ab‐treated mice after PAC. Quantification of RV fibrosis after PAC is shown (n=6/group). D, Quantification of cardiomyocyte cross‐sectional area after PAC is shown (n=6/group). E through H, Quantification of RV type I collagen, pSmad3, pERK1/2, and calcineurin protein levels in IgG versus N‐Eng Ab‐treated mice after PAC (n=6/group). Representative western blots are shown. I through K, Levels of RV MYH7, TRPC‐6, and α‐SMA mRNA expression in IgG versus N‐Eng Ab‐treated mice after PAC (n=6/group). *P<0.05 versus sham; †P<0.05 versus WT+N‐Eng Ab PAC. α‐SMA indicates alpha‐smooth muscle antigen; PAC, pulmonary artery constriction; RV, right ventricular; TRPC‐6, transient receptor protein channel 6; WT, wild type.
Figure 9.
Figure 9.
Neutralizing endoglin activity reverses cardiac fibrosis after chronic right ventricular pressure overload. A, Schematic of randomization to treatment with a N‐Eng Ab or IgG control Ab after 3 weeks of PAC in WT mice. B and C, Quantification of RV fibrosis and representative histologic staining for RV collagen abundance in IgG‐ versus N‐anti‐Eng Ab‐treated mice after moderate RVPO (n=6/group). D through F, Quantification of type I collagen and calcineurin protein levels in WT mice after moderate RVPO for 3 and 6 weeks in the presence and absence of either an IgG control Ab or N‐ Eng Ab. Representative western blots are shown. *P<0.05 versus sham; †P<0.05 versus 3 weeks RVPO; ‡P<0.05 versus 6 weeks RVPO+IgG. PAC indicates pulmonary artery constriction; RV, right ventricular; RVPO, RV pressure overload; WT, wild type.
Figure 10.
Figure 10.
Reduced endoglin activity limits TGF‐β1‐induced calcineurin expression and myofibroblast transformation in right ventricular fibroblasts. Postulated mechanism by which endoglin promotes RV fibrosis by facilitating TGF‐β1 signaling in response to pressure overload through canonical and noncanonical pathways, including calcineurin‐mediated myofibroblast transformation in RVFB. In contrast, reduced endoglin activity attenuates TGF‐β1 signaling through canonical, noncanonical, and calcineurin pathways and limits myofibroblast transformation and fibrosis, thereby improving survival. α‐SMA indicates alpha‐smooth muscle antigen; RV, right ventricular; RVBF, right ventricular fibroblasts; TGF‐β1, transforming growth factor‐beta 1; TRPC‐6, transient receptor protein channel 6.
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