A paradoxical increase of force development in saphenous and tail arteries from heterozygous ANO1 knockout mice.

Vladimir V Matchkov,Boris V Skryabin,Dmitrii Kamaev,Andreas Hoegh Jensen,Christian Aalkjaer,Henrik Black Joergensen,Hans Christian Beck

doi:10.14814/phy2.14645

Vladimir V Matchkov, Boris V Skryabin + Show 5 more

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https://doi.org/10.14814/phy2.14645

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Abstract

A Ca2+‐activated Cl− channel protein, ANO1, is expressed in vascular smooth muscle cells where Cl− current is thought to potentiate contraction by contributing to membrane depolarization. However, there is an inconsistency between previous knockout and knockdown studies on ANO1’s role in small arteries. In this study, we assessed cardiovascular function of heterozygous mice with global deletion of exon 7 in the ANO1 gene. We found decreased expression of ANO1 in aorta, saphenous and tail arteries from heterozygous ANO1 knockout mice in comparison with wild type. Accordingly, ANO1 knockdown reduced the Ca2+‐activated Cl− current in smooth muscle cells. Consistent with conventional hypothesis, the contractility of aorta from ANO1 heterozygous mice was reduced. Surprisingly, we found an enhanced contractility of tail and saphenous arteries from ANO1 heterozygous mice when stimulated with noradrenaline, vasopressin, and K+‐induced depolarization. This difference was endothelium‐independent. The increased contractility of ANO1 downregulated small arteries was due to increased Ca2+ influx. The expression of L‐type Ca2+ channels was not affected but expression of the plasma membrane Ca2+ ATPase 1 and the Piezo1 channel was increased. Expressional analysis of tail arteries further suggested changes of ANO1 knockdown smooth muscle cells toward a pro‐contractile phenotype. We did not find any difference between genotypes in blood pressure, heart rate, pressor response, and vasorelaxation in vivo. Our findings in tail and saphenous arteries contrast with the conventional hypothesis and suggest additional roles for ANO1 as a multifunctional protein in the vascular wall that regulates Ca2+ homeostasis and smooth muscle cell phenotype.

Highlights

ANO1 protein, known as TMEM16a, is a Ca2+-activated Cl− channel (Caputo et al, 2008; Schroeder et al, 2008; Yang et al, 2008) that is known to be expressed in different tissues (Pedemonte & Galietta, 2014)
We found an increased expression of Rho-related GTP-binding protein RhoE that inhibits on Rho-associated protein kinase (ROCK) and Ca2+-sensitization in vascular smooth muscle cells (Loirand et al, 1999; Shimokawa et al, 2016; Figure 10b)
We found an approximately 50% reduction to in the Ca2+-activated Cl− conductance in tail artery smooth muscle cells from ANO1 knockdown mice, which is in accordance with the importance of ANO1 protein for this conductance

Summary

Introduction

ANO1 protein, known as TMEM16a, is a Ca2+-activated Cl− channel (Caputo et al, 2008; Schroeder et al, 2008; Yang et al, 2008) that is known to be expressed in different tissues (Pedemonte & Galietta, 2014). Influx of Ca2+ through L-type Ca2+ channels leads to an increase in the global intracellular Ca2+ concentration and activation of the contractile machinery of smooth muscle cells This potentially makes ANO1 an important protein involved in the regulation of the tone of small arteries and a regulator of vascular resistance, which defines blood flow and arterial pressure (Mendelsohn, 2005; Mulvany & Aalkjaer, 1990). Consistent with this role, ANO1 has been found to be upregulated in experimental models with circulatory abnormalities, for example, pulmonary and essential hypertension (Askew Page et al, 2019; Forrest et al, 2012; Gui et al, 2015; Papp et al, 2019; Wang et al, 2015). Knockout and knockdown animal models are used to provide information about protein function in vitro and in vivo (Bulley et al, 2012; Dam, et al, 2014; Heinze et al, 2014; Jensen et al, 2018)

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