TMEM16A is implicated in the regulation of coronary flow and is altered in hypertension

Abstract

Background and purpose: Coronary artery disease leads to ischaemic heart disease and ultimately myocardial infarction. Thus, it is important to determine the factors that regulate coronary blood flow. Ca²⁺ -activated chloride channels contribute to the regulation of arterial tone; however, their role in coronary arteries is unknown. The aim of this study was to investigate the expression and function of the main molecular correlate of Ca²⁺ -activated chloride channels, TMEM16A, in rat coronary arteries.

Experimental approach: We performed mRNA and protein analysis, electrophysiological studies of coronary artery myocytes, and functional studies of coronary artery contractility and coronary perfusion, using novel inhibitors of TMEM16A. Furthermore, we assessed whether any changes in expression and function occurred in coronary arteries from spontaneously hypertensive rats (SHRs).

Key results: TMEM16A was expressed in rat coronary arteries. The TMEM16A-specific inhibitor, MONNA, hyperpolarised the membrane potential in U46619. MONNA, T16A_inh -A01, and Ani9 attenuated 5-HT/U46619-induced contractions. MONNA and T16A_inh -A01 also increased coronary flow in Langendorff perfused rat heart preparations. TMEM16A mRNA was increased in coronary artery smooth muscle cells from SHRs, and U46619 and 5-HT were more potent in arteries from SHRs than in those from normal Wistar rats. MONNA diminished this increased sensitivity to U46619 and 5-HT.

Conclusions and implications: In conclusion, TMEM16A is a key regulator of coronary blood flow and is implicated in the altered contractility of coronary arteries from SHRs.

Figure 1

Tmem16a is expressed in rat coronary arteries. (a) Abundance of Tmem16a and Tmem16b mRNA relative to predetermined house‐keeping genes in left anterior descending (LAD) and septal coronary arteries. n = 3 where n is a pooled sample of arteries from at least three rats. (b) Abundance of Tmem16a mRNA relative to predetermined house‐keeping genes in LAD coronary arteries, mesenteric arteries, and pulmonary arteries. n = 3 where n is a pooled sample of arteries from at least three rats. (c) Western blot with anti‐TMEM16A antibody AB53212 on lysates from rat pulmonary artery, TMEM16A‐GFP‐transfected HEK293 cells, and untransfected HEK293 cells. Each rat sample constitutes three rats worth of tissue. (d) Single isolated vascular smooth muscle cells (VSMCs) from LAD coronary arteries with anti‐TMEM16A antibody AB53212 (i–iv). Cells are co‐stained with anti‐α‐smooth muscle actin (i and v), DAPI (ii and vi), anti‐TMEM16A (iii), and a composite image (iv and viii). Arrows highlight small hotspots of intense staining. Images v–viii show a single VSMC isolated from a LAD coronary artery that was not incubated with an anti‐TMEM16A primary antibody. Images are representative of at least three cells from arteries of three rats. Scale bar is 10 μm. (e) Images of a 16‐μm‐thick slice of a rat heart where a coronary artery can be seen in situ, stained with anti‐α‐smooth muscle actin (i and v), DAPI (ii and vi), +anti‐TMEM16A (iii), −anti‐TMEM16A (vii), and a composite image (iv and viii). Arrows 1 and 3 indicate the arterial wall within the myocardial tissue, and Arrow 2 highlights the lumen of the vessel. Images are representative of slices from two rats and two to three arteries per rat. Scale bar is 30 μm

Figure 2

Inhibition of TMEM16A significantly attenuates coronary artery function. Bar charts showing that contractions of coronary arteries to (a) U46619 and (b) 5‐HT were significantly smaller when the segments were bathed in low chloride physiological saline solution (LCPSS); n = 7 for U46619 and n = 6 for 5‐HT. (c) Pre‐incubation of 10 μmol·L⁻¹ MONNA had no effect on the KPSS‐induced contraction in LADCA segments; n = 6 for both DMSO and MONNA. (d) Pre‐incubation with 1 μmol·L⁻¹ MONNA caused a significant attenuation of the U46619‐induced response in left anterior descending (LAD) coronary arterials segments (n = 8 for both DMSO and 1 μmol·L⁻¹ MONNA), while pre‐incubation with 10 μmol·L⁻¹ MONNA significantly reduced the ability of the vessel to contract to (e) U46619 (n = 8 for both DMSO and 10 μmol·L⁻¹ MONNA). (f) Pre‐incubation with 1 μmol·L⁻¹ MONNA did not cause a significant attenuation of the 5‐HT‐induced response in LAD coronary arterial segments (n = 6 for both DMSO and 1 μmol·L⁻¹ MONNA), while pre‐incubation of 10 μmol·L⁻¹ MONNA significantly reduced the ability of the vessel to contract to (g) 5‐HT (n = 8 for both DMSO and 10 μmol·L⁻¹ MONNA). (h) Concentration effect curves of U46619 in LADCA segments in the presence of K⁺ channel blockers were significantly attenuated after incubation with 10 μmol·L⁻¹ MONNA; n = 8 for both. (i) Scatter graph indicating that the EC₅₀ of U46619 in LAD coronary artery segments in the presence of 10 μmol·L⁻¹ MONNA were similar in both endothelium intact and denuded segments. Data are expressed as mean EC₅₀ of U46619 ± SEM (in 10 μmol·L⁻¹ MONNA), n = 5 for both. (j) The maximal contraction evoked by U46619 in LADCA segments incubated with 10 μmol·L⁻¹ MONNA was not different between endothelium intact and endothelium denuded segments; n = 5 for both. All data are expressed as mean ± SEM. Statistical significance is defined as *P ˂ 0.05

Figure 3

Effect of other TMEM16A inhibitors on coronary artery contractility. (a) Pre‐incubation with 3 μmol·L⁻¹ Ani9 did not cause a significant attenuation of the high K⁺‐induced contraction of coronary arteries (n = 5 for both DMSO and 3 μmol·L⁻¹ Ani9). (b) While pre‐incubation with 3 μmol·L⁻¹ T16A_inh‐A01 did not cause a significant attenuation of high K⁺‐induced contraction of coronary arteries (n = 5 for both DMSO and 3 μmol·L⁻¹ T16A_inh‐A01), pre‐incubation with 10 μmol·L⁻¹ T16A_inh‐A01 reduced the contractility considerably (n = 12 for both DMSO and 10 μmol·L⁻¹ T16A_inh‐A01). (c) U46619 concentration effect curves were significantly attenuated in the presence of 3 μmol·L⁻¹ Ani9 (n = 6 for both DMSO and 3 μmol·L⁻¹ Ani9), (d) 3 μmol·L⁻¹ T16A_inh‐A01 (n = 9 for both DMSO and 3 μmol·L⁻¹ T16A_inh‐A01), and (e) 10 μmol·L⁻¹ T16A_inh‐A01 (n = 10 for both DMSO and 10 μmol·L⁻¹ T16A_inh‐A01). (f) 5‐HT concentration effect curves were also significantly impaired in the presence of 10 μmol·L⁻¹ T16A_inh‐A01 (n = 7 for both DMSO and 10‐μmol·L⁻¹ T16A_inh‐A01). All data are expressed as mean ± SEM. Statistical significance is defined as *P ˂ 0.05

Figure 4

Inhibition of TMEM16A significantly increases coronary flow. (a) Application of MONNA and T16A_inh‐A01 caused a concentration‐dependent increase in coronary flow (n = 6 for MONNA, n = 5 for T16A_inh‐A01, and n = 5 for DMSO). Pre‐incubation with 1 μmol·L⁻¹ MONNA prevented U46619‐induced reductions in coronary flow (b) (n = 5 for both conditions). (c) Shows a representative trace from the experiment described in (b). All data are expressed as mean ± SEM. Statistical significance is defined as *P ˂ 0.05

Figure 5

The scatter plot shows the increase in current evoked from left anterior descending coronary artery vascular smooth muscle cells, at a holding potential of −50 mV, upon application of 10 mmol·L⁻¹ caffeine in the absence and presence of 10 μmol·L⁻¹ MONNA; n = 5. Scatter graph showing the membrane potential (left, mV), and evoked contraction (right, mN) in resting, +U46619, and +U46619 + 10 μmol·L⁻¹ MONNA; n = 4 for resting E_M, n = 6 for E_M in U46619, and n = 7 for E_M in U46619 + 10 μmol·L⁻¹ MONNA, and n = 7 for tension recordings in resting and U46619, and n = 8 in U46619 + 10 μmol·L⁻¹ MONNA. All data are expressed as mean ± SEM. Statistical significance is defined as *P ˂ 0.05

Figure 6

Increased expression of Tmem16a in coronary arteries from spontaneously hypertensive rats (SHRs). (a) Tmem16a mRNA is expressed at levels approximately fourfold greater in coronary arteries smooth muscle cells from SHRs when compared to Wistar rats. There was no increase in Tmem16a expression in the ventricular tissue from SHRs. (b) The expression of the cardiac‐specific marker troponin I3 (Tnni3) was expressed at negligible levels in the coronary artery vascular smooth muscle cell (VSMC) samples of both Wistar rats and SHRs, and expression was significantly greater in the ventricular tissue of the same rats (*for Wistar tissues, ^# for SHR tissues). All data are expressed as mean ± SEM of at least five separate rat samples. Statistical significance is defined as P ˂ 0.05

Figure 7

Altered function of TMEM16A in left anterior descending (LAD) coronary arteries from spontaneously hypertensive rats (SHRs). (a) Increases in contractility of LAD coronary arteries from SHRs in response to lower concentrations of U46619 (n = 8 for Wistar and n = 6 for SHRs) and 5‐HT (b) (n = 9 for Wistars and n = 7 for SHRs) were abolished by pre‐incubation with 10 μmol·L⁻¹ MONNA. LAD coronary arteries from SHRs were more sensitive to constriction with U46619 (n = 27 for Wistar and n = 16 for SHR) and 5‐HT (n = 12 for Wistar and n = 13 in SHRs) (c and d). (e) Pre‐incubation with 1 μmol·L⁻¹ MONNA has no effect on the U46619 concentration effect curve in LAD coronary artery segments of SHRs. (f) Pre‐incubation with 10 μmol·L⁻¹ MONNA significantly attenuates the ability of the vessel to constrict to U46619. (g) Pre‐incubation with 1 μmol·L⁻¹ MONNA has a small but significant effect on the 5‐HT concentration‐effect curve in LAD coronary artery segments of SHRs. (h) Pre‐incubation with 10 μmol·L⁻¹ MONNA significantly attenuates the ability of the vessel to constrict to 5‐HT. (i) Basal tone developed in LAD coronary artery segments of SHRs was significantly suppressed by application of 10 μmol·L⁻¹ MONNA. All data are expressed as mean ± SEM. Statistical significance is defined as *P ˂ 0.05