The TMEM16A-specific inhibitor MONNA is an important regulator of rat coronary artery function

H. Askew Page1,2, T. Dalsgaard2, S. Olesen2, I. Greenwood1,2. 1Vascular Biology, St. George's University of London, London, UNITED KINGDOM, 2Ion Channel Group, University of Copenhagen, Copenhagen, DENMARK.

Introduction: Failure of coronary blood vessels to adequately supply cardiac myocytes with oxygen underlies ischaemic heart disease and ultimately myocardial infarction. Thus it is important to determine the factors that regulate coronary blood flow. Ca2+-activated chloride channels encoded by TMEM16A play a key role in depolarising vascular smooth muscle cell membrane potentials. This can activate voltage-gated Ca2+ channels, causing Ca2+ influx and contraction of the cell. We investigated whether TMEM16A was a viable functional regulator of rat coronary arteries using molecular expression techniques and functional studies with a specific inhibitor, MONNA.

Methods: Male Wistar rats were killed in accordance with Schedule 1 of the United Kingdom Animals Act (1986) or Danish regulations. Quantitative polymerase chain reaction and immunodetection technqiues were used to assess mRNA and protein expression of TMEM16A. Standard perforated patch clamp techniques were used to measure currents from freshly isolated rat coronary artery vascular smooth muscle cells (VSMCs). Wire myography and Langendorff perfused heart setup were used to assess coronary artery segment contractility and coronary blood flow respectively. Microelectrode impalement of rat coronary artery segments were combined with wire myography for membrane potential measurements.

Results: TMEM16A was identified at mRNA and protein levels in rat coronary artery smooth muscle. Application of the TMEM16A-specific blocker MONNA reduced U46619-elicted membrane depolarisation, inhibited caffeine-evoked chloride currents in single smooth muscle cells, attenuated U46619-induced contractions in a wire myograph (maximal contractions were 19.97%±1.56 of the peak contractions pre-incubation while they were 96.09%±1.56 of those in vehicle controls (n=8 p<0.0001) and increased coronary blood flow in Langendorff perfused rat heart preparations. In LAD coronary arteries from spontaneously hypertensive rats (SHRs) TMEM16A transcript was increased compared to those from normotensive rats. Interestingly LAD coronary artery segments from SHRs were more sensitive to contraction with U46619 and 5HT. 30nM U46619 evoked contractions of 0.23±0.06 mN in LAD coronary artery segments from SHRs, compared to 0.06±0.03 mN in arteries from normotensive rats (n=6 p<0.05). Similar effects were seen with 5HT. This increased sensitivity was diminished in the presence of 10µM MONNA.

Conclusion: In conclusion MONNA had pronounced effects on the physiology of rat coronary arteries, implying a significant role for TMEM16A in the regulation of coronary artery function.