RhoA Ambivalently Controls Prominent Myofibroblast Characteritics by Involving Distinct Signaling Routes

Abstract

Introduction: RhoA has been shown to be beneficial in cardiac disease models when overexpressed in cardiomyocytes, whereas its role in cardiac fibroblasts (CF) is still poorly understood. During cardiac remodeling CF undergo a transition towards a myofibroblast phenotype thereby showing an increased proliferation and migration rate. Both processes involve the remodeling of the cytoskeleton. Since RhoA is known to be a major regulator of the cytoskeleton, we analyzed its role in CF and its effect on myofibroblast characteristics in 2 D and 3D models.

Results: Downregulation of RhoA was shown to strongly affect the actin cytoskeleton. It decreased the myofibroblast marker α-sm-actin, but increased certain fibrosis-associated factors like TGF-β and collagens. Also, the detailed analysis of CTGF expression demonstrated that the outcome of RhoA signaling strongly depends on the involved stimulus. Furthermore, we show that proliferation of myofibroblasts rely on RhoA and tubulin acetylation. In assays accessing three different types of migration, we demonstrate that RhoA/ROCK/Dia1 are important for 2D migration and the repression of RhoA and Dia1 signaling accelerates 3D migration. Finally, we show that a downregulation of RhoA in CF impacts the viscoelastic and contractile properties of engineered tissues.

Conclusion: RhoA positively and negatively influences myofibroblast characteristics by differential signaling cascades and depending on environmental conditions. These include gene expression, migration and proliferation. Reduction of RhoA leads to an increased viscoelasticity and a decrease in contractile force in engineered cardiac tissue.

Fig 1. RhoA knockdown influences cytoskeletal protein expression and localization as well as disturbs higher order actin structures.

A) Bar graph summary of real-time PCR data for RhoA in shControl and shRhoA NRCF normalized to PBGD and related to shControl (means ± SEM, n = 6 versus PBGD, *p < 0.05). B) Representative immunoblot of RhoA and β-actin in whole cell lysates from shControl, shScr and shRhoA NRCF (left). Bar graph summary of RhoA protein expression normalized to β-actin (right). The relative change of RhoA in shRhoA NRCF and shScr NRCF to shControl NRCF is given (means ± SEM, n = 22, *p < 0.05). C) Representative immunoblots of investigated cytoskeletal proteins in whole cell lysates from shControl and shRhoA NRCF are shown. D) Bar graph summary of real-time PCR data of α-sm-actin normalized to PBGD and protein expression normalized to β-actin in shControl and shRhoA NRCF relative to shControl (means ± SEM, n = 7, *p < 0.05). E) Bar graph summary of acetylated tubulin normalized to β-actin. Whole cell lysates obtained from shControl and shRhoA NRCF were used. The values are given relative to shControl (means ± SEM, n = 7–12, *p < 0.05). F) Immunofluorescence staining of shControl and shRhoA NRCF for actin isoforms β- (green), γ-actin (red) and DAPI (blue) (320x, upper two rows) or for α-sm-actin (green), actin (red) and DAPI (blue) (lower two rows). G) Analysis of geodesic actin structures (left) and representative immunofluorescence staining of actin structures in shControl and shRhoA NRCF (right). The relative number of geodesic (G), mixed (M) and non-geodesic (NG) NRCF as indicated (arrow) in shControl compared to shRhoA is given (means ± SEM, n = 3, *p < 0.05). H) Immunofluorescence staining of tubulin (green), actin (red) and DAPI (blue) (400x, upper two rows) or acetylated tubulin (red), Golgi apparatus (green) and DAPI (blue) (400x, lower two rows) in shControl and shRhoA NRCF.

Fig 2. Knockdown of RhoA and actin depolymerization by Latrunculin A (LatA) results in changes in the Golgi apparatus structure.

A) Representative immunofluorescence staining of the Golgi apparatus (640x, left) and structural analysis of Golgi apparatus size and density (right) of shControl and shRhoA NRCF (means ± SEM, n = 30, *p < 0.05). B) Immunofluorescence staining of actin (left) and Golgi apparatus (middle) in control and LatA treated NRCF (8.5 μM, 200x) In addition, higher magnifications are shown (640x). C) Bar graph summary of immunoblot analysis of β-actin normalized to total cell lysate protein, α-sm-actin, tubulin normalized to β-actin and acetylated tubulin normalized to total tubulin. Whole cell lysates were obtained from control and LatA treated NRCF. The relative change of expression in LatA treated NRCF to control NRCF is given (8.5 μM, n = 7).

Fig 3. RhoA knockdown changes focal adhesion site size and orientation as well as cell morphology and adhesion velocity.

A) Representative immunoblot of vinculin and β-actin in whole cell lysates from shControl and shRhoA NRCF (upper left). Immunofluorescence stainings of vinculin (green), actin (red) and DAPI (blue) in shControl and shRhoA NRCF is shown (upper right) (320x). Analysis of focal adhesion site length (lower left) and orientation to the cell axis (lower right) in shControl and shRhoA NRCF is depicted (means ± SEM, n = 3, at least 110 focal adhesion sites per condition were analyzed, *p < 0.05). B) Morphometric analysis of cell area (left) and perimeter (right) of shControl and shRhoA NRCF (means ± SEM, n = 3, each 50 NRCF per condition, *p < 0.05). C) Cell volume of detached shControl and shRhoA NRCF measured by resistance in a pulsed low voltage field. Given is the relative cell volume of shRhoA compared to shControl NRCF (n = 3) D) Adhesion assay on cell culture (left graph) and collagen-coated surface (right graph) over a time course of 1 h. The number of adherent shRhoA compared to shControl NRCF in percent of total cell number is given for each time point (means ± SEM, n = 4, *p < 0.05).

Fig 4. Downregulation of RhoA inhibits migration on a focal plane but improves amoeboid migration performance.

A) Bar graph summary of absolute distance (left), average migration velocity (middle) and directness (right) of shControl and shRhoA NRCF migrating on a plane cell culture surface (means ± SEM, n = 3, 15 NRCF per virus type, *p < 0.05). B) Bar graph summary of cell migration of shControl and shRhoA NRCF through a porous membrane (pore size: 8 μm) in the presence of low serum (1%) and high serum (10%) (means ± SEM, n = 5, *p < 0.05). C) Representative images of shControl and shRhoA cells migrating through a collagen membrane in ibidi μ slides^3D. The cells were stained for total actin and with DAPI (left). Bar graph summary of cell migration of shControl and shRhoA NRCF through a collagen matrix (1,5% in DMEM) in ibidi μ-slides^3D. As a bait FCS (10%) was used (means ± SEM, n = 8, *p < 0.05) (right). Immunofluorescence staining of vinculin (green), actin (red) and DAPI (blue) (320x) of shControl (bottom, upper panel) and shRhoA NRCF (bottom, lower panel) inside a collagen matrix (1,5% in DMEM).

Fig 5. The knockdown or RhoA reduces cell doubling time without affecting the cell viability.

A) Proliferation-DAPI assay of shControl and shRhoA NRCF over a time course of 4 days. Doubling time of the involved cell types is shown in the table above (doubling time in days) (means ± SEM, n = 5, measured in 8 replicates each, *p < 0.05). B) AnnexinV-FLUOS and propidium iodide staining of shControl and shRhoA NRCF. Bar graph summary of apototic (left) and necrotic (right) NRCF in percent compared to total cell number (means ± SEM, n = 3).

Fig 6. Expression and secretion of CTGF is impaired with reduced RhoA expression in low serum conditions.

A) Bar graph summary of intracellular (left) and extracellular (right) CTGF under serum reduced conditions (1%) (means ± SEM, n = 6–7, *p < 0.05). Whole cell lysates and culture media were obtained from shControl and shRhoA NRCF and the relative changes of CTGF normalized to β-actin in shRhoA NRCF to shControl are given. B) Luciferase assay depicting the relative serum response factor activation in low serum conditions (1%). The relative change of shRhoA to shControl NRCF is given (means ± SEM, n = 5, *p < 0.05). C) Bar graph summary of intracellular (left) and extracellular (right) CTGF under high serum conditions (10%) (means ± SEM, n = 6–7, *p < 0.05). The relative changes of CTGF normalized to β-actin in shRhoA NRCF to shControl are given. D) Bar graph summary of real-time PCR data for CTGF in shControl and shRhoA NRCF in high serum conditions (10%) normalized to PBGD and related to shControl (means ± SEM, n = 4 versus PBGD, *p < 0.05). E) Luciferase assay depicting the total serum response factor activation in high serum conditions (10%) (left) and the total serum response factor activity in high serum conditions (10%) (right) of shControl and shRhoA NRCF (means ± SEM, n = 5).

Fig 7. Actin disruption, cell morphology, migratory and proliferative performance of RhoA knockdown NRCF can be mimicked using ROCK and HDAC6 inhibitors.

A) Representative actin stainings of control, fasudil (10 μM) and TubA (5 μM) treated NRCF (400x). B) Morphometric analysis of cell area (left) and perimeter (right) of control, fasudil (10 μM) and TubA (5 μM) treated NRCF (means ± SEM, n = 3, 50 NRCF per condition, *p < 0.05). C) Proliferation-DAPI assay of control and fasudil treated NRCF over a time course of 4 days (means ± SEM, n = 5, *p < 0.05). D) Proliferation-DAPI assay of control, TubA and LiCl (50 mM) treated NRCF over a time course of 4 days (means ± SEM, n = 3, *p < 0.05).

Fig 8. Migration through a collagen matrix is impaired after ROCK inhibition but improved after mDia1 knockdown.

A) Bar graph summary of average migration velocity (left), absolute distance (middle) and directness (right) of control, fasudil and TubA treated NRCF migrating on a plane cell culture surface (means ± SEM, n = 3, 15 NRCF per condition, *p < 0.05). B) Bar graph summary of cell migration of control, fasudil and TubA treated NRCF through a porous membrane (pore size: 8 μm) in the presence of low serum (1%) and high serum (10%) (means ± SEM, n = 4, *p < 0.05). C) Representative images of control, fasudil and TubA treated NRCF migrating through a collagen membrane in ibidi μ-slides^3D. The cells were stained for total actin and with DAPI (left). Bar graph summary of cell migration of control, fasudil and TubA treated NRCF through a collagen matrix (1,5% in DMEM) in ibidi μ-slides^3D. As a bait FCS (10%) was used (means ± SEM, n = 8, *p < 0.05) (right). D) Representative immunoblot of mDia1 and β-actin in whole cell lysates from shControl and siDia1 NRCF (left). E) Bar graph summary of average migration velocity (left), absolute distance (middle) and directness (right) of siControl and siDia1 NRCF migrating on a plane cell culture surface (means ± SEM, n = 3, 15 NRCF per condition, *p < 0.05). F) Representative images of siControl and siDia1 NRCF migrating through a collagen membrane in ibidi μ-slides^3D. The cells were stained for total actin and with DAPI (left). Bar graph summary of cell migration of siControl and siDia1 NRCF through a collagen matrix (1,5% in DMEM) in ibidi μ-slides^3D. As a bait FCS (10%) was used (means ± SEM, n = 7, *p < 0.05) (middle). G) Immunofluorescence staining of vinculin (green), actin (red) and DAPI (blue) (320x) of siControl and siDia1 NRCF inside a collagen matrix (1,5% in DMEM) (right).

Fig 9. Impact of the RhoA knockdown on viscoelastic and contractile properties of engineered tissues.

A) Representative stress-strain curves of ECT composed of shRhoA and shControl NRCF. B) Evaluation of the Failing Point (n = 6) and C) the Young´s modulus (n = 4–5) (means ± SEM, *p < 0.05). D) Evaluation of the force of contraction (FOC) in EHM supplemented with either shRhoA or shControl NRCF in the presence of different calcium concentrations (means ± SEM, n = 3, 2–4 EHM per condition and experiment, *p < 0.05) E) Given is the mean resting force of all EHM at 2 mM calcium as determined by the Frank-Starling mechanism (means ± SEM, n = 3, 2–4 EHM per condition and experiment). F) Quantification of an immunoblot analysis of cytoskeletal proteins, calsequestrin, intracellular CTGF and H3 in cell lysates obtained from shControl and shRhoA NRCF complemented EHM. The values are normalized to vimentin and given relative to the expression in shControl NRCF containing EHM (means ± SEM, n = 3, 2–4 EHM per condition were pooled and analyzed, *p < 0.05). G) Quantification of an immunoblot analysis of cytoskeletal proteins, calsequestrin, intracellular CTGF and H3 in cell lysates obtained from shControl and shRhoA NRCF co-cultured with neonatal rat cardiomyocytes for 7 days. The values are normalized to vimentin and given relative to the expression in shControl NRCF co-cultures (means ± SEM, n = 6, *p < 0.05).

Fig 10. Summary scheme of proposed mechanism.