Elucidating the roles of TRPM4 and Cx40 in cardiovascular function
Creators: Gust, Stephen L
Publisher: name: University of Alberta Library
Year: 2024

<p>The modulation of resistance artery diameter is crucial for the regulation of tissue perfusion and systemic blood pressure. This is a multi-factorial process which necessitates the integrated activity of vascular endothelial and smooth muscle cells. A wide variety of endothelial and smooth muscle ion channels are involved in the coordination of the electrical and chemical signals governing the contractile state of vascular smooth muscle cells. Smooth muscle contraction is dependent upon an increase in the intracellular concentration of Ca2+ via Ca2+ release from the sarcoplasmic reticulum and/or Ca2+ influx from the extracellular space. This increase in Ca2+ can be evoked by any of a large range of stimuli, including intravascular pressure. Pressure-evoked vasoconstriction is the basis of the myogenic response, a phenomenon whereby arteries respond to an increase in pressure by contracting to decrease diameter and maintain constant flow. The transient receptor potential melastatin 4 (TRPM4) channel has emerged as a crucial mediator of pressure-evoked vasoconstriction. Most research into its function has involved the pharmacological TRPM4 inhibitor 9-phenanthrol, though recent studies have questioned its selectivity.<br>
Meanwhile, the endothelium regulates the contractile state of smooth muscle cells through two interrelated Ca2+-dependent mechanisms: by the release of diffusible chemical mediators such as nitric oxide (NO), and by electrical coupling with smooth muscle cells via myoendothelial gap junctions (MEGJs). Gap junctions (GJs) are low resistance channels composed of connexins (Cxs) which allow the passage of ions and small molecules from the cytosol of one cell to another. An increase in intracellular Ca2+ causes increased opening of Ca2+-activated K+ (KCa) channels, hyperpolarizing the endothelial cell. This hyperpolarization is conducted along the endothelium by interendothelial GJs and to smooth muscle cells by MEGJs. The hyperpolarization of smooth muscle cells inhibits opening of voltage-operated Ca2+ channels, thereby limiting contraction. Endothelial cells express three Cx subtypes: Cx37, Cx40 and Cx43. Of these, Cx40 has been suggested to be necessary for endothelium-dependent vasodilation. A novel Cx40 knockout (KO) rat has only recently been developed, and the cardiovascular function of this rat has yet to be characterized.<br>
Thus, my over-arching goal is to investigate the functional roles of smooth muscle TRPM4 and endothelial Cx40 by addressing three hypotheses:<br>
1)  TRPM4 plays a role in both myogenic and agonist-induced vasoconstriction in resistance arteries.<br>
2)  The Cx40 KO rat will demonstrate impaired endothelial modulation of arterial diameter versus wild-type (WT).<br>
3)  The introduction of a mild hypercaloric diet will impair endothelial modulation of arterial diameter to a greater extent in the Cx40 KO rat than in WT rats.<br>
    To test these hypotheses, I have used a combination of functional and biochemical techniques to investigate the role of TRPM4 in vasoconstriction and characterize the cardiovascular function of the Cx40 KO rat.<br>
    Firstly, my data show that pharmacological inhibition of TRPM4 with 9-phenanthrol inhibits agonist-, nerve- and pressure-evoked vasoconstriction in rat mesenteric arteries. The additional presence of the intermediate-conductance KCa (IKCa) inhibitors NS6180 and TRAM-34 has no effect on the inhibition of vasoconstriction by 9-phenanthrol, therefore this action of 9-phenanthrol is not mediated by IKCa. Next, my data show that the Cx40 KO rat exhibits pronounced hypertension and heart enlargement. This is accompanied by a large-magnitude increase in nerve-evoked vasoconstriction in the perfused mesenteric vascular bed. A large component of this effect is endothelium-independent, demonstrating that the ablation of Cx40 affects vascular smooth muscle function. Finally, applying the metabolic stress of a mild high fat diet (HFD) causes further increases in nerve-evoked vasoconstriction in the perfused mesenteric vascular bed, with the largest effect in WT rats.<br>
    To conclude, I have shown that 9-phenanthrol does not produce its effect by activating IKCa and demonstrated that TRPM4 is a necessary mediator of agonist-, nerve- and pressure-evoked vasoconstriction. I have also demonstrated that Cx40 has a crucial physiological role in vascular function and the maintenance of normal systemic BP. The onset of endothelial dysfunction as modelled by a mild HFD appears to share an overlapping mechanism with the Cx40 KO-induced increase in vasoconstriction. These results illustrate crucial physiological roles for TRPM4 and Cx40 in the modulation of resistance artery diameter.</p>