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Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin
Published on: November 18, 2011
Thromboxane-induced actin polymerization in hypoxic pulmonary artery is independent of Rho
Jena Fediuk1, Alexey Gutsol, Nora Nolette
1Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada.
Abstract:
Actin polymerization (APM), regulated by Rho GTPases, promotes myocyte force generation. Hypoxia is known to impede postnatal disassembly of the actin cytoskeleton in pulmonary arterial (PA) myocytes. We compared basal and agonist-induced APM in myocytes from PA and descending aorta (Ao), under hypoxic and normoxic conditions. We also examined effects of thromboxane challenge on force generation and cytoskeletal assembly in resistance PA and renal arteries from neonatal swine with persistent pulmonary hypertension (PPHN) induced by 72-h normobaric hypoxia, compared with age-matched controls. Synthetic and contractile phenotype myocytes from neonatal porcine PA or Ao were grown in hypoxia (10% O(2)) or normoxia (21% O(2)) for 7 days, then challenged with 10(-6) M thromboxane mimetic U46619. F/G actin ratio was quantified by laser-scanning cytometry and by cytoskeletal fractionation. Thromboxane receptor (TP) G protein coupling was measured by immunoprecipitation and probing for Gαq, G12, or G13, RhoA activation by Rhotekin-RBD affinity precipitation, and LIM kinase (LIMK) and cofilin phosphorylation by Western blot. Isometric force to serial concentrations of U46619 was measured in muscular pulmonary and renal arteries from PPHN and control swine; APM was quantified in fixed contracted vessels. Contractile PA myocytes exhibit marked Rho-dependent APM in hypoxia, with increased active RhoA and LIMK phosphorylation. Their additional APM response to U46619 challenge is independent of RhoA, reflecting decreased TP association with G12/13 in favor of Gαq. In contrast, hypoxic contractile Ao myocytes polymerize actin modestly and depolymerize to U46619. Both basal APM and the APM response to U46619 are increased in PPHN PA. APM corresponds with increased force generation to U46619 challenge in PPHN PA but not renal arteries.
Insights
Hypoxia increases actin polymerization in pulmonary artery myocytes, impacting force generation. This effect is amplified in persistent pulmonary hypertension, suggesting a key role in disease progression.
Area of Science:
- Cardiovascular Biology
- Cellular Physiology
- Vascular Medicine
Background:
- Actin polymerization (APM) is crucial for myocyte force generation, regulated by Rho GTPases.
- Hypoxia impairs the normal disassembly of the actin cytoskeleton in pulmonary arterial (PA) myocytes.
- Persistent pulmonary hypertension (PPHN) involves complex vascular remodeling and altered myocyte function.
Purpose of the Study:
- To compare basal and agonist-induced APM in PA and aortic (Ao) myocytes under varying oxygen conditions.
- To investigate the effects of thromboxane challenge on force generation and cytoskeletal assembly in PPHN swine models.
- To elucidate the signaling pathways involved in APM regulation, including Rho GTPases and thromboxane receptor (TP) coupling.
Main Methods:
- Cultured neonatal porcine PA and Ao myocytes were exposed to hypoxia or normoxia.
- Cells and isolated arteries were challenged with thromboxane mimetic U46619.
- Actin polymerization was quantified using laser-scanning cytometry and cytoskeletal fractionation.
- TP G protein coupling, RhoA activation, and LIM kinase (LIMK)/cofilin phosphorylation were assessed.
- Isometric force and APM were measured in arteries from control and PPHN swine.
Main Results:
- Hypoxic PA myocytes showed increased RhoA activation and LIMK phosphorylation, indicating enhanced APM.
- U46619 challenge induced RhoA-independent APM in hypoxic PA myocytes, linked to altered TP-G protein coupling (Gαq favored over G12/13).
- Hypoxic Ao myocytes exhibited modest APM basally and depolymerization upon U46619 challenge.
- PPHN PA myocytes displayed elevated basal APM and increased APM response to U46619, correlating with greater force generation.
Conclusions:
- Contractile PA myocytes exhibit hypoxia-induced, Rho-dependent APM, with further modulation by thromboxane.
- Altered TP signaling contributes to enhanced APM in PA myocytes under hypoxia.
- Increased APM in PPHN PA myocytes is associated with augmented force generation, highlighting its pathological significance.
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