Transient Receptor Potential Channel 4 Encodes a Vascular Permeability Defect and High-Frequency Ca2+ Transients in Severe Pulmonary Arterial Hypertension

Abstract

The canonical transient receptor potential channel 4 (TRPC4) comprises an endothelial storeeoperated Ca 2þ entry channel, and TRPC4 inactivation confers a survival benefit in pulmonary arterial hypertension (PAH). Endothelial Ca 2þ signals mediated by TRPC4 enhance vascular permeability in vitro, but the contribution of TRPC4-dependent Ca 2þ signals to the regulation of endothelial permeability in PAH is poorly understood. We tested the hypothesis that TRPC4 increases vascular permeability and alters the frequency of endothelial Ca 2þ transients in PAH. We measured permeability in isolated lungs, and found that TRPC4 exaggerated permeability responses to thapsigargin in Sugen/hypoxia-treated PAH rats. We compared endothelial Ca 2þ activity of wild-type with TRPC4-knockout rats using confocal microscopy, and evaluated how Ca 2þ signals were influenced in response to thapsigargin and sequential treatment with acetylcholine. We found that thapsigargin-stimulated Ca 2þ signals were increased in PAH, and recovered by TRPC4 inactivation. Store depletion revealed bimodal Ca 2þ responses to acetylcholine, with both shortand long-duration populations. Our results show that TRPC4 underlies an exaggerated endothelial permeability response in PAH. Furthermore, TRPC4 increased the frequency of endothelial Ca 2þ transients in severe PAH, suggesting that TRPC4 provides a Ca 2þ source associated with endothelial dysfunction in the pathophysiology of PAH. This phenomenon represents a new facet of the etiology of PAH, and may contribute to PAH vasculopathy by enabling inflammatory mediator flux across the endothelial barrier. The current model of the endothelium extends beyond the concept of a homogeneous cell monolayer in contact with the blood.1e6 Recent advances in high-resolution microscopy have enabled the measurement of individual cellular behavior rather than tissue-wide averaged responses, and we are now beginning to understand the heterogeneous nature of local endothelia and their major impact on physiology and disease.7e10 Endothelial dysfunction underlies the pathogenesis of pulmonary arterial hypertension (PAH), a deadly disease of high pulmonary artery pressure culminating in right-sided heart failure. Endothelial dysfunction in PAH is characterized by the aberrant production of endothelial-dependent vasoconstrictors and vasodilators within pulmonary arterioles, leading to sustained elevation of pulmonary artery pressure, and the initiation of endothelial hyperproliferation, leading to occlusive lesion formation within the microcirculation.

Mind the gap: mechanisms regulating the endothelial barrier

“…However, in the same study, phospholipase Cß (PLCß)-induced Ca 2+ increase was also found to contribute to histamineinduced permeability. Although transient receptor potential canonical 4 (TRPC4) was recently shown to contribute to vascular permeability in a model of pulmonary hypertension in vivo, 199 the finding that stromal interacting molecule 1 (STIM1) decreased barrier function in response to thrombin independent of Ca 2+ entry but rather via direct activation of RhoA 200 suggests that the different signalling pathways may converge on the level of RhoA. In line with this, PKC activation downstream of PLC signalling, which has been shown to reduce VE-cadherin binding and cause intercellular gap formation, 201 is also known to activate RhoA via p115RhoGEF.…”

Treasure troves of pharmacological tools to study transient receptor potential canonical 1/4/5 channels

“…TRPC1/4/5 have been suggested to play multiple roles in various conditions, including epilepsy, fear, pain, severe pulmonary arterial hypertension, vascular inflammation in arthritis, and cardiac remodelling (Figure ). However, the lack of modulators to study the functional roles of these channels have limited progress into their exact role (Alawi et al, ; Camacho Londono et al, ; Francis, Xu, Zhou, & Stevens, ; Minard et al, ; Phelan et al, ; Riccio et al, ; Riccio et al, ; Westlund et al, ; Zheng & Phelan, ). The identification of high‐quality, reliable, selective, and subtype‐specific TRPC1/4/5 pharmacological tools and modulators would perhaps enable a better understanding of TRPC1/4/5 channels in human health and disease.…”

“…There have been numerous studies that have shown the involvement of TRPC channels within the cardiovascular system (Alawi et al, ; Camacho Londono et al, ; Francis et al, ; Lau et al, ; Xiao, Liu, Shen, Cao, & Li, ). Camacho Londono et al () reported that TRPC1 and TRPC4 proteins play a pivotal role in a background Ca 2+ entry pathway, which fine‐tunes Ca 2+ cycling in cardiomyocytes.…”

The role of endothelial leak in pulmonary hypertension (2017 Grover Conference Series)

“…However, by measuring calcium signals at high frequency, we found that cytosolic calcium oscillations are superimposed on the global rise in cytosolic calcium. 44 The number of these calcium oscillations expanded greatly in hypertensive pulmonary arteries and they were significantly reduced in pulmonary artery endothelium from TRPC4 KO animals ( Fig. 4b ).…”

“…To address this issue, conduit pulmonary artery segments were isolated from normotensive and hypertensive animals, the endothelium was loaded with the Fluo-4 calcium dye, and calcium transients were measured. 44 Whereas most studies examine endothelial cell cytosolic calcium with low temporal resolution, in these studies, we measured the calcium signal at 9.8 frames/s. This faster resolution enabled measurement of fast calcium oscillations, or transients, which would be missed when measuring at slower time constants.…”