Fig 1: KCa3.1 inhibition TGFβ1 and bFGF-induced myofibroblast contraction. a) Myofibroblast collagen gel contraction was increased following TGFβ1 stimulation and this was inhibited by TRAM-34 200 nM in both IPF (n=3) and NFC (n=3) donors (data shown is pooled IPF and NFC which did not differ, n=6)(P=0.0014, repeated measures ANOVA, P=0.0023 for TGFβ1 compared to control, P=0.0026 for TRAM-34 compared to TGFβ1 corrected by Bonferroni’s multiple comparisons test). b) TGFβ1-dependent myofibroblast collagen gel contraction was also inhibited by ICA-17043 100 nM (n=6) (All groups; repeated measures ANOVA, P=0.0002, TGFβ1 versus control, P=0.0002 and for ICA-17043 versus TGFβ1, P=0.0006, corrected by Bonferroni’s multiple comparisons test). c) Similarly, myofibroblast collagen gel contraction was increased following bFGF stimulation and was also inhibited with 24h pre-treatment with TRAM-34 200 nM (All groups; repeated measures ANOVA P<0.0001, for bFGF compared to control, P<0.0001, and for TRAM-34 compared to bFGF, P<0.0001 (corrected by Bonferroni’s multiple comparisons test). d) Similarly, 24h pre-treatment with ICA-17043 100 nM significantly reduced bFGF-dependent myofibroblast collagen gel contraction (P=0.0007, repeated measures ANOVA) (P=0.0005 for bFGF versus control and P=0.0053 for ICA-17043 versus bFGF, corrected by Bonferroni’s multiple comparisons test). Data represented as mean±SEM for all the above figures.
Fig 2: KCa3.1 expression within human lung tissue of non-fibrotic and IPF patients. a) Representative KCa3.1 and αSMA immunostaining of healthy lung parenchyma from two NFC tissue donors. All pictures are from sequential sections. Isotype controls are negative. b) Representative immunostaining of lung parenchyma from two IPF tissue donors demonstrating KCa3.1 and αSMA immunostaining in areas of fibrosis. All pictures are from sequential sections. KCa3.1 channel expression is particularly strong in the epithelium and within and surrounding areas positive for αSMA.
Fig 3: KCa3.1 is expressed in microglia and is upregulated by AβO. (A) qPCR was conducted on RNA extracted from cultured primary mouse microglia, astrocytes and neurons from three independent preparations. KCa3.1, Kir2.1, and CD11b were expressed exclusively in microglia, while Nav1.8 and CAMK2‐α expressions were characteristic of neurons and GFAP expression characteristic of astrocytes. n = 3/group; one‐way ANOVA follow by Bonferroni post hoc test. (B–D) Hippocampal slices were treated with 100 nmol/L AβO for 24 h. (B) Slices were immunostained with CD11b (green) or anti‐GFAP (green) and co‐stained with anti‐KCa3.1 (APC064). AβO treatment caused increased staining of KCa3.1, which was largely colocalized with CD11b, but not with the astrocytic marker GFAP. AβO‐induced KCa3.1 upregulation was further corroborated by the increased transcript (qPCR result in C, n = 4) and protein (representative Western blot and quantification in D, n = 4) levels of KCa3.1. Numerical data are presented by mean ± SE from four independent experiments and were analyzed by two‐sample t‐test.
Fig 4: Functional KCa3.1 channels demonstrate greater expression in IPF myofibroblasts compared to NFC myofibroblasts and channel expression is increased by pro-fibrotic growth factors. a) The mean percentage of IPF myofibroblasts per donor developing a KCa3.1 current in response to 1-EBIO was significantly higher than in NFC myofibroblasts (P=0.0285, unpaired t-test). Data presented as mean±SEM. b) The whole-cell current at +40 mV before and after the addition of 1-EBIO in all responding NFC and IPF human lung myofibroblasts. Data presented as median and IQR. c) The subtracted (1-EBIO minus baseline) 1-EBIO-dependent KCa3.1 current at +40 mV was significantly larger in IPF cells than in NFC cells (P=0.0054, Mann Whitney test). Data presented as median and IQR. d) The mean percentage of NFC and IPF myofibroblasts expressing KCa3.1 currents increased after stimulation with TGFβ1 and bFGF (All groups; 1-way ANOVA, P=0.0013). The proportion of IPF cells responding to 1-EBIO after TGFβ1 stimulation was significantly higher (*P=0.0336, corrected by Bonferroni’s multiple comparisons test). Significantly more NFC cells responded to 1-EBIO following bFGF stimulation (**P=0.0035, corrected by Bonferroni’s multiple comparisons test). Data presented as mean±SEM.
Fig 5: KCa3.1 channel protein is present within myofibroblasts and KCa3.1 channels are functional. a) Western blot of human lung myofibroblast lysates using 2 different KCa3.1 channel antibodies, M20 and P4997. All images show a consistent band at the predicted size for the KCa3.1 channel at 48 KDa in human lung myofibroblasts. An additional band at 53 kDa is present as described in other cell types. b) Example of immunofluorescent staining for KCa3.1 in NFC myofibroblasts using M20 and P4997 antibodies. DAPI nuclear staining and negative rabbit isotype control IgG are shown. c) Whole-cell patch-clamp electrophysiology recordings of KCa3.1 in NFC (n=14) and IPF (n=13) human lung myofibroblasts activated with 1-EBIO and blocked with TRAM-34 (200 nM). Upper panels: Mean ± SEM current voltage curves demonstrate a small outwardly rectifying current at baseline, and the IPF donors have a relatively small inwardly rectifying Kir current (confirmed by blocking with 10 µM barium, results not shown). Large currents with a negative reversal potential develop after the addition of the KCa3.1 opener 1-EBIO (100 µM), which are blocked by the selective KCa3.1 blocker TRAM-34 (200 nM). Lower panels: The subtracted (1-EBIO minus TRAM-34) TRAM-34-sensitive KCa3.1 current. d) The voltage protocol and the raw current are demonstrated showing typical electrophysiological features of KCa3.1 in a myofibroblast.
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