Proapoptotic Rassf1A/Mst1 signaling in cardiac fibroblasts is protective against pressure overload in mice
Dominic P. Del Re, … , Louise Van Der Weyden, Junichi Sadoshima
Dominic P. Del Re, … , Louise Van Der Weyden, Junichi Sadoshima
Published September 20, 2010
Citation Information: J Clin Invest. 2010;120(10):3555-3567. https://doi.org/10.1172/JCI43569.
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Research Article Cardiology

Proapoptotic Rassf1A/Mst1 signaling in cardiac fibroblasts is protective against pressure overload in mice

Abstract

Mammalian sterile 20-like kinase 1 (Mst1) is a mammalian homolog of Drosophila Hippo, the master regulator of cell death, proliferation, and organ size in flies. It is the chief component of the mammalian Hippo pathway and promotes apoptosis and inhibits compensatory cardiac hypertrophy, playing a critical role in mediating heart failure. How Mst1 is regulated, however, remains unclear. Using genetically altered mice in which expression of the tumor suppressor Ras-association domain family 1 isoform A (Rassf1A) was modulated in a cell type–specific manner, we demonstrate here that Rassf1A is an endogenous activator of Mst1 in the heart. Although the Rassf1A/Mst1 pathway promoted apoptosis in cardiomyocytes, thereby playing a detrimental role, the same pathway surprisingly inhibited fibroblast proliferation and cardiac hypertrophy through both cell-autonomous and autocrine/paracrine mechanisms, playing a protective role during pressure overload. In cardiac fibroblasts, the Rassf1A/Mst1 pathway negatively regulated TNF-α, a key mediator of hypertrophy, fibrosis, and resulting cardiac dysfunction. These results suggest that the functional consequence of activating the proapoptotic Rassf1A/Mst1 pathway during pressure overload is cell type dependent in the heart and that suppressing this mechanism in cardiac fibroblasts could be detrimental.

Authors

Dominic P. Del Re, Takahisa Matsuda, Peiyong Zhai, Shumin Gao, Geoffrey J. Clark, Louise Van Der Weyden, Junichi Sadoshima

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Figure 6

rassf1A silencing stimulates TNF-α to promote autocrine-mediated fibroblast proliferation and paracrine-mediated cardiomyocyte hypertrophy.

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rassf1A silencing stimulates TNF-α to promote autocrine-mediated fibrob...
(A) Luciferase reporter assay demonstrated the ability of endogenous Rassf1A to inhibit nf-κb promoter activation. Values are relative to GFP. (B) Representative immunoblot demonstrating activation of Akt and IKK complex in response to Rassf1A depletion. (C) RT-PCR performed with fibroblast mRNA demonstrated selective upregulation of TNF-α mRNA. (D) TNF-α was detected in medium of fibroblasts by ELISA 48 hours after Rassf1A depletion or dnMst1 expression. (E–G) Cardiac fibroblasts were treated with adenovirus expressing scrambled control or Rassf1A shRNA, alone or in combination with LacZ or IκBαSA (SA) mutant, and the resulting conditioned medium was transferred to serum-starved cardiomyocytes. Conditioned medium from control and Rassf1A shRNA–treated cells was also incubated with mouse IgG or TAb (100 ng/ml) for 30 minutes prior to transfer. Values are shown relative to vehicle control. (E) Cells were stained with α-actinin Ab and surface area determined. (F) Total protein content was normalized to DNA content. (G) Cardiomyocytes were stained with ANF Ab and positive cells counted. (H) Cardiac fibroblasts and conditioned media were treated as described above, and the resulting conditioned media were transferred to separate serum-starved fibroblasts. Cells were costained with anti-tubulin Ab and DAPI. Nuclei per ×10 visual field were counted, and the number per square millimeter was expressed as fold of vehicle control. Treatment with TNF-α (10 U/ml), PE (100 μm), and serum (10% FBS) served as controls. Data are mean ± SEM (n = 3). *P < 0.05; **P < 0.01.