Disruption of Pressure-Induced Ca ²⁺ Spark Vasoregulation of Resistance Arteries, Rather Than Endothelial Dysfunction, Underlies Obesity-Related Hypertension

Figures

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  Figure 1.. Endothelial TRPV4 Ca2+ signal quantification. A and B, Representative en-face images of the mesenteric endothelium from low-fat diet (LFD) and high-fat diet (HFD) fed mice. Scale bars=10 µm. C and D, Representative changes in F/Fo at region of interest positioned on A and B. Dashed lines indicate published levels of TRPV4 fluorescence based on quantal opening of 1 to 4 channels. Traces shown under baseline conditions (CPA and 3 nM GSK101) and subsequently with Carbachol 3 µM. Scale bars=10 µm. E, Number of TRPV4 sparklet sites per field studied indicating activity at MEP rather than non-MEP locations. F, NPo per TRPV4 site studied between LFD (black) and HFD (red) arteries (LFD: n=5 arteries from 4 mice. HFD: n=5 arteries from 3 mice). CPA indicates cyclopiazonic acid; F/Fo, fluorescence intensities relative to baseline; MEP, myoendothelial projection; and TRPV4, vanilloid 4 transient receptor potential ion channel.

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  Figure 2.. Endothelial-mediated vasodilation in obesity. Raw pressure myography traces are presented in this figure. The diameter of the artery (y axis) is shown in relation to time (x axis) and the vasodilatory responses of the arteries from the normal diet fed (black) and high fat–fed (red) mice are shown in response to various endothelial targeted vasodilatory agonists. Arterial diameter traces illustrate the concept of pressure-induced constriction whereby the artery maintains a stable internal diameter in response merely to the presence of 80 mmHg of intraluminal pressure. The artery is then exposed to a cumulative concentration range of endothelial agonists: Carbachol—a muscarinic agonist or GSK101—a TRPV4 (vanilloid 4 transient receptor potential ion channel) agonist. The raw data indicate that the pressure-constricted mesenteric arteries from low fat–fed (LFD) and high fat–fed (HFD) mice vasodilated equivalently to Carbachol (A–C, n=6 arteries from 5 mice for LFD, 12 arteries from 12 mice for HFD), GSK101 (D–F, n=3 arteries from 3 mice for LFD, 10 arteries from 10 mice HFD).

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  Figure 3.. Enhanced pressure-induced constriction due to reduced large conductance Ca2+-activated potassium channel (BK) channel vasodilatory effect in high fat–fed mice. Raw pressure myography traces presented similarly to those in Figure 2. Arterial diameter demonstrates stable pressure-induced constriction before application of the drug. Data are presented showing the effects of BK channel agonist (NS11021) and blocker (IBX). A–C, Representative traces from low fat–fed (LFD) and high fat–fed (HFD) mice are shown in A (LFD) and B (HFD). C, The degree of pressure-induced constriction, calculated as a percentage of passive free diameter, was greater in HFD (n=13 arteries from 10 mice) compared with LFD arteries (LFD: n=13 arteries from 9 mice. LFD: 41±2.6% vs HFD: 49.2±2.4%; P=0.02). D, Mesenteric resistance arteries from LFD mice showed significantly greater constriction to IBX compared with those from the HFD group (LFD: 6 arteries from 4 mice. HFD: 4 arteries from 4 mice. LFD: 13.7±2.6% vs HFD: 5.2.2±1.1%; P=0.005). There was no change in lumen diameter when the arteries from either HFD or LFD mice were incubated with the TRPV4 (vanilloid 4 transient receptor potential ion channel)…

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  Figure 4.. Differential pressure regulation of Ca2+ spark frequency in high fat–fed and low fat–fed arteries. A, Confocal image of an intact pressurized mesenteric artery from a low-fat diet mouse with representative F/Fo traces (B and C) derived from regions of interest (ROI) as shown on the image at 20 mmHg (B) and 80 mmHg (C) of intraluminal pressure. D, Confocal image of artery from high-fat diet mice with changes in F/Fo at ROI at both 20 mmHg (E) and 80 mmHg (F) intraluminal pressure (for quantification of Ca2+ spark characteristics, see Figure S6). F/Fo indicates fluorescence intensities relative to baseline.

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  Figure 5.. Contribution of oxidant-activated PKG to large conductance Ca2+-activated potassium channel (BK) channel dysfunction in high-fat feeding. A, Representative traces from third-order mesenteric arteries from high fat–fed WT and high fat–fed PKG[C42S]KI mice showing basal pressure–induced constriction and incubation with Paxilline. B, There is equivalence in the degree of pressure-induced constriction of the mesenteric arteries between the WT and PKG[C42S]KI mice (WT: n=8 arteries from 4 mice. PKG[C42S]KI: n=5 arteries from 4 mice). C, There is equivalence in the responses to Paxilline between WT (n=5 arteries from 3 mice) and PKG[C42S]KI (n=4 arteries from 3 mice) mouse third order mesenteric arteries. D, Representative Western blot protocols showing the monomer (lower band) and dimer (upper band) formation of PKG following exposure to H2O2 from mesenteric arteries from low fat–fed and high fat–fed arteries from WT mice. There was no difference in the percentage of total PKG dimerization between the low fat–fed (n=5 from 5 mice) and the high fat–fed (n=13 from 13 mice for baseline and 100 µM H2O2, n=7 from 7 mice for 30 µM H2O2) groups. PKG indicates Protein Kinase G; and WT, wild…

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  Figure 6.. Cartoon illustrating proposed pathway by which obesity influences vascular tone of resistance arteries. Intraluminal pressure generates oxidants which activate PKG. Thus, activated PKG targets the sarcoplasmic reticulum to increase Ca2+ spark frequency and these activate BK channels to trigger vasodilation through hyperpolarization. While PKG can still undergo oxidative activation in obesity, the capacity of the SR to generate Ca2+ sparks is abrogated. In tandem, work from Navedo et al has shown that obesity also has a direct effect on the BK channel directly, limiting the Ca2+ sensitivity. In our model, obesity had no effect on the endothelial muscarinic/TRPV4/IKCa vasodilatory pathway. BK indicates large conductance Ca2+-activated potassium channel. EC, endothelial cell; IEL, internal elastic lamina; IKCa, intermediate conductance calcium-activated potassium channel; PKG, Protein Kinase G; RyR, ryanodine receptor; SR, sarcoplasmic reticulum; TRPV4, vanilloid transient receptor potential 4 channel; VDCC, voltage-dependent Ca2+ channel; and VSMC, vascular smooth muscle cell.