Spreading vasodilatation in the murine microcirculation: attenuation by oxidative stress-induced change in electromechanical coupling

Abstract

Regulation of blood flow in microcirculatory networks depends on spread of local vasodilatation to encompass upstream arteries; a process mediated by endothelial conduction of hyperpolarization. Given that endothelial coupling is reduced in hypertension, we used hypertensive Cx40ko mice, in which endothelial coupling is attenuated, to investigate the contribution of the renin-angiotensin system and reduced endothelial cell coupling to conducted vasodilatation of cremaster arterioles in vivo. When the endothelium was disrupted by light dye treatment, conducted vasodilatation, following ionophoresis of acetylcholine, was abolished beyond the site of endothelial damage. In the absence of Cx40, sparse immunohistochemical staining was found for Cx37 in the endothelium, and endothelial, myoendothelial and smooth muscle gap junctions were identified by electron microscopy. Hyperpolarization decayed more rapidly in arterioles from Cx40ko than wild-type mice. This was accompanied by a shift in the threshold potential defining the linear relationship between voltage and diameter, increased T-type calcium channel expression and increased contribution of T-type (3 μmol l(-1) NNC 55-0396), relative to L-type (1 μmol l(-1) nifedipine), channels to vascular tone. The change in electromechanical coupling was reversed by inhibition of the renin-angiotensin system (candesartan, 1.0 mg kg(-1) day(-1) for 2 weeks) or by acute treatment with the superoxide scavenger tempol (1 mmol l(-1)). Candesartan and tempol treatments also significantly improved conducted vasodilatation. We conclude that conducted vasodilatation in Cx40ko mice requires the endothelium, and attenuation results from both a reduction in endothelial coupling and an angiotensin II-induced increase in oxidative stress. We suggest that during cardiovascular disease, the ability of microvascular networks to maintain tissue integrity may be compromised due to oxidative stress-induced changes in electromechanical coupling.

Figure 1. Cell layers of cremaster arterioles from Cx40ko mice are coupled by gap junctions

A, cremaster arterioles from Cx40ko mice comprise an inner layer of endothelial cells (ECs) surrounded by a single layer of smooth muscle cells (SMCs) and adventitia (adv). B, endothelial projections penetrate the internal elastic lamina (IEL). Inset shows that myoendothelial gap junctions were identified by pentalaminar membranes, and were also present between adjacent SMCs (C) and endothelial cells (D–G; insets, between arrows). Scale bars are as follows: A, 10 μm; B, 5 μm; C–G, 0.5 μm; and B–G insets, 50 nm. H and I, Cx40 is expressed in the ECs of cremaster arterioles from wild-type (WT) but not Cx40ko mice. J and K, Cx37 is expressed in the ECs of wild-type arterioles and weakly in Cx40ko arterioles. L and M, Cx43 is not expressed in the arterioles of either wild-type or Cx40ko mice.

Figure 2. The endothelium forms the pathway for conducted vasodilatation in cremaster arterioles of Cx40ko mice

A, upper panel, the endothelium was disrupted over 300 μm at a distance of 1 mm from the ACh stimulation site using light dye treatment (LDT). A, lower panel, control experiments were conducted in the same vessels to confirm specificity of endothelial disruption at the LDT treatment site (treat) but not at the control site (con). B, locally initiated dilatation did not spread past the site of LDT (open circles). C, endothelial disruption was confirmed by absence of dilatation in response to the endothelium-dependent dilator, ACh, but only after LDT. D, smooth muscle integrity was confirmed by intact relaxation in response to the nitric oxide donor, SNP, at control and LDT-treated sites, before and after light exposure. n = 5 vessels in 5 animals. *P < 0.05 vs. control. E and F, selectivity of endothelial disruption was confirmed by staining of endothelial cell nuclei with propidium iodide (0.1%), which is excluded from living cells but can penetrate the membrane of dead or dying cells; light microscopy and epi-fluorescence. Only preparations in which endothelial cell nuclei took up the dye, whereas smooth muscle nuclei excluded the dye, were included in the data. Scale bars represent 25 μm.

Figure 3. Hyperpolarization decays with distance in arterioles of Cx40ko mice

A, focal stimulation with ACh (1 mol l⁻¹, 1 s) induced a local dilatation that decayed in Cx40ko mice (7 arterioles in 7 mice) but not in wild-type mice (6 arterioles in 6 mice). B, representative paired recordings of simultaneous membrane potential and diameter from Cx40ko (grey) and wild-type (black) at the local site and 1000 μm upstream, showing a greater decay of hyperpolarization in Cx40ko than in wild-type arterioles. C and D, summary data of paired recordings of membrane potential and diameter in response to local ionophoresis of ACh. Hyperpolarization decayed more steeply in arterioles from Cx40ko (C, open circles; 4 cell pairs in 4 animals) than from wild-type mice (C, filled circles; 7 cell pairs in 7 animals), while vasodilatation decayed only in arterioles from Cx40ko mice (D, open circles). Values are means ± SEM; *P < 0.05. l-NAME and indomethacin were present throughout to block NO and prostaglandins.

Figure 4. Attenuation of conduction of dilatation in Cx40ko arterioles involves change in electromechanical coupling

A, paired recordings of vessel tone and membrane potential at the local site in Cx40ko arterioles, before and during ACh stimulation (1 mol l⁻¹, 1 s; 60 recordings from 30 cells in 7 mice) could be fitted by two-line regression analysis with a threshold of –45 ± 3 mV, beyond which dilatation attenuated linearly with hyperpolarization. B, proposed mechanism underlying attenuated conduction of vasodilatation in Cx40ko arterioles (grey lines) compared with wild-type mice (black lines). Hyperpolarization (lower lines) decays more rapidly in Cx40ko arterioles (grey) than wild-type arterioles (black), due to a reduction in endothelial coupling (change in slope) and a change in the threshold potential (arrows; Cx40ko, −45 mV; and wild-type, −35 mV; Wölfle et al. 2011). C, computational modelling of Cx40ko arterioles, using measured parameters and reduced endothelial coupling resistance (see Supplementary Table 1), shows electrotonic decay of hyperpolarization in endothelial cells (open triangles) and smooth muscle cells (filled triangles). D, attenuation of vasodilatation observed in Cx40ko arterioles can be predicted with application of the −45 mV threshold (open circles), beyond which dilatation attenuates linearly with hyperpolarization, but not using the wild-type threshold of −35 mV (filled circles).

Figure 5. T-type calcium channels play a greater role in vascular tone of arterioles from Cx40ko mice than wild-type mice, but not after normalization of blood pressure with candesartan or inhibition of oxidative stress

A, vascular tone of arterioles did not vary between Cx40ko mice (216 arterioles in 21 mice) and wild-type mice (227 arterioles in 22 mice). B, the contribution of T-type channels (NNC 55-0396 sensitive) was significantly larger in Cx40ko arterioles (122 arterioles in 11 mice) than in wild-type arterioles (124 arterioles in 12 mice), while the contribution of L-type channels (nifedipine sensitive) was not significantly different between the genotypes. The non-L-/non-T-type component was significantly smaller in arterioles from Cx40ko mice compared with wild-type mice. C, vascular tone was not significantly different between wild-type (63 arterioles in 6 mice) and Cx40ko mice (81 arterioles in 7 mice) after chronic candesartan treatment (1.0 mg kg⁻¹ day⁻¹ for 2 weeks). Vascular tone was not significantly different between wild-type (73 arterioles in 7 mice) and Cx40ko mice (46 arterioles in 5 mice) after acute treatment with tempol (1 mmol l⁻¹). D, chronic candesartan treatment did not alter the relative contribution of L- or T-type channels, but did significantly reduce the non-L-/non-T-type component in wild-type mice (63 arterioles in 6 mice). In Cx40ko mice, candesartan treatment significantly reduced the relative contribution of T-type channels and increased the contribution of L-type channels (81 arterioles in 7 mice). In the presence of tempol, the relative contribution of T-type channels to vascular tone was significantly reduced in arterioles from Cx40ko mice (46 arterioles in 5 mice), while the contribution of L-type channels was significantly increased. In wild-type mice, acute tempol treatment significantly increased the contribution of T-type channels (73 arterioles in 7 mice). Values are shown as means + SEM; *P < 0.05 vs. untreated wild-type; #P < 0.05 vs. untreated Cx40ko.

Figure 6. Expression of L- and T-type channels in arterioles of Cx40ko mice

Protein expression (green) of L-type (Ca_v1.2) channels (A and B) and T-type channels (C and D, Ca_v3.1; and E and F, Ca_v3.2) in arterioles from wild-type (A, C and E) and Cx40ko mice (B, D and F). Nuclei of smooth muscle cells (SMCs; arrows), stained with propidium iodide (red), are aligned at right angles to the vessel axis, while endothelial cell (EC) nuclei (arrows) are parallel to the vessel axis. Dashed lines mark arteriolar boundaries.

Figure 7. Expression of T-type channels is increased in arterioles of Cx40ko mice but decreased after treatment with candesartan

Protein expression (green) of L-type (Ca_v1.2) channels (A) and T-type channels (B, Ca_v3.1; and C, Ca_v3.2) in arterioles from Cx40ko mice, after chronic candesartan treatment (1.0 mg kg⁻¹ day⁻¹ for 2 weeks). Nuclei of smooth muscle cells (SMCs; arrows), stained with propidium iodide (red), are aligned at right angles to the vessel axis, while endothelial cell (EC) nuclei (arrows) are parallel to the vessel axis. Dashed lines mark arteriolar boundaries. D, group data show that expression of T-type channels is increased in the smooth muscle of arterioles from Cx40ko mice compared with wild-type mice (WT), but is decreased after chronic candesartan treatment (cand). Values are shown as means + SEM; *P < 0.05 vs. wild-type; 17–20 arterioles in 4–6 mice of each group.

Figure 8. Spreading vasodilatation is improved in arterioles of Cx40ko mice by tempol or chronic candesartan treatment

A, acute treatment with tempol did not alter conducted dilatations of arterioles from wild-type mice (n = 5 vessels in 5 mice). B, in Cx40ko mice, acute tempol treatment significantly improved conducted vasodilatation at 1000 and 1500 μm (*P < 0.05 vs. Cx40ko, n = 6 vessels in 6 mice). C, treatment of Cx40ko mice with candesartan for 14–18 days also significantly improved conducted vasodilatation at 1000 and 1500 μm sites (*P < 0.05 vs. Cx40ko, n = 7 vessels in 7 mice). Acute treatment with tempol had no further effect (n = 6 vessels in 6 mice).