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. 2014 May;13(3):251-9.
doi: 10.1016/j.jcf.2013.10.009. Epub 2013 Oct 31.

Genotype-specific Alterations in Vascular Smooth Muscle Cell Function in Cystic Fibrosis Piglets

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Free PMC article

Genotype-specific Alterations in Vascular Smooth Muscle Cell Function in Cystic Fibrosis Piglets

Jinny J Guo et al. J Cyst Fibros. .
Free PMC article

Abstract

Background: The most common CF-causing mutations interfere with CFTR trafficking from the endoplasmic reticulum (CFTR-F508del) or prematurely terminate transcription (CFTR-null). We suspected that genotype-specific patterns of CFTR expression would have differential effects on smooth muscle cell calcium signaling and hence vascular tone. We hypothesized that compared to wild-type or CFTR-null aorta, aorta from CFTR-F508del (dF) piglets will have reduced endoplasmic reticulum calcium mobilization and decreased vasoconstriction.

Methods: Aortic reactivity was assessed by myography, and ratiometric calcium imaging was performed in isolated vascular smooth muscle cells.

Results: Aorta from dF piglets had reduced myogenic tone (P<0.001) and decreased constriction to KCl (P<0.05). Combined inhibition of ryanodine and IP3 receptors decreased wild-type and CFTR-null responses to levels seen in dF aorta. Compared to wild-type cells, dF-expressing smooth muscle cells had reduced calcium transients, while CFTR-null cells had decreased baseline intracellular calcium concentrations.

Conclusions: Expression of CFTR-F508del interferes with smooth muscle cell calcium handling and decreases aortic responsiveness.

Keywords: Cystic fibrosis transmembrane conductance regulator; Endoplasmic reticulum; Inositol triphosphate receptor; Vascular smooth muscle cell.

Figures

Figure 1
Figure 1
Genotype-specific myogenic tone. Aortic segments from 7 piglets (2 WT, 3 CFTR-null, 2 CFTR-F508del) were incubated at 1, 2, 4, 6 or 8g-force resting tension and myogenic tone was assessed (A, symbol size is proportionate to resting tension). Aortic segments from additional WT (open symbols, N=38), null (black symbols, N=29), and dF piglets (gray symbols, N=11) were incubated at 2g resting tension for 3 hours and the ensuing tension was recorded (B). Compared to aorta from WT piglets, aorta from dF piglets had deceased constriction during incubation in PSS buffer, calcium free buffer with EGTA or PSS with ryanodine (C). Combined inhibition of ryanodine and IP3 receptors with both ryanodine (1 μM) and 2-APB (50 μM) reduced the tone of aorta from WT or CFTR-null piglets to approximate the tone of dF aorta. *P<0.05 versus WT, #P<0.05 versus buffer alone.
Figure 2
Figure 2
Aortic response to adrenergic stimulation. Aortic constriction to noradrenaline (NA, 10 μM) was assessed for WT (open bars, N=17), CFTR-null (black bars, N=13), and dF piglets (gray bars, N=6) incubated in either PSS buffer alone or calcium-free buffer with EGTA. Aorta from dF piglets had decreased constriction during the 90 minute incubation, regardless of NA addition 30 minutes prior to study completion. Elimination of extracelluar calcium eliminated the heightened tone of WT and CFTR-null aorta. *P<0.05 versus WT, ‡P<0.01 or †P<0.05 for NA versus buffer, #P<0.05 for NA versus EGTA-NA.
Figure 3
Figure 3
Aortic response to membrane depolarization. Vasoconstriction to cumulative concentrations of KCl was assessed for WT (open symbols, N=21), CFTR-null (black symbols, N=11), and dF piglet aorta (gray symbols, N=13) following one hour incubation in PSS buffer alone (A) or PSS with BaCl2 (B–D). Independent of BaCl2, aorta from dF piglets had decreased KCl-induced constriction (A). Independent of genotype, KIR antagonism with BaCl2 increased aortic constriction to 5, 10 and 15 mM KCl (B), but this did not normalize the KCl responsiveness of dF aorta (C and D). *P<0.05 versus WT, #P<0.05 versus PSS without BaCl2.
Figure 4
Figure 4
Genotype-specific aortic morphology. Following dilation with sodium nitroprusside, aortic segments from WT (left, N=14), Null (middle, N=8) and dF piglets (right, N=10) were fixed and stained for morphometric analysis (A). While the aorta from CFTR-null piglets tended to have increased lumen diameters (B) and decreased wall to lumen ratios (C), these did not approach statistical significance.
Figure 5
Figure 5
Vascular smooth muscle cell CFTR expression and its effect on calcium transients. VSMC were isolated from neonatal piglet aorta and identified by positive immunofluorescence for alpha-smooth muscle actin (A, with DAPI nuclear counterstain). CFTR mRNA was identified in aortic VSMC from WT, dF Het and dF/dF, but not CFTR-null piglets (B, dominant markers in left lane: 200 and 100 bp, expected size: 155 bp). Mean angiotensin II-stimulated calcium transients are provided for WT versus null (C) and WT versus dF genotypes (D). Compared to WT (white bars), VSMC from null piglets (black bars) had decreased baseline [Ca2+]i (E), while VSMC from dF piglets (gray bars) had decreased calcium responses to angiotensin II (F). N= 8 to 17, *P<0.05 versus wild-type.
Figure 6
Figure 6
Simplified model of the potential impact of CFTR mutations on VSMC function. Wild-type CFTR (yellow) localizes within the plasma and ER/SR membranes (A). Increasing extracellular [KCl] leads to membrane depolarization and activation of voltage-dependent calcium channels (red), a process that is attenuated by the calcium chelator EGTA and inward rectifying potassium channels (purple, blocked by barium). An increase in [Ca2+]i induces further calcium release through ryanodine receptors (green, blocked by ryanodine) and IP3 receptors (blue, blocked by 2-APB). Countercurrent chloride efflux from ER/SR CFTR channels may provide charge neutralization (27). Membrane stretch, angiotensin II (AngII) and noradrenaline (NA) mediate vasoconstriction in part by activation of phospholipase C (PLC), leading to the release of IP3 and subsequent IP3 receptor activation. In addition to PLC activation, NA activates adenylate cyclase (AC) leading to the production of cAMP, with subsequent CFTR activation potentially leading to plasma membrane depolarization and enhanced calcium entry. VSMC expressing CFTR-F508del lack plasma membrane CFTR expression (B), potentially contributing to membrane hyperpolarization (28) and a reduction in calcium-induced calcium release. CFTR-null cells also lack ER/SR CFTR expression, and this may contribute to the observed decrease in baseline calcium concentration (C).

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