Impaired BKCa channel function in native vascular smooth muscle from humans with type 2 diabetes

Figures

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  Figure 1. Adipose arteries from diabetic subjects exhibit blunted response to IBTx. (A) Exemplary image of small resistance adipose arteries used in this study. The image below shows an expanded view of the area in the green square. (B) Representative diameter recordings of pressurized (80 mmHg) adipose arteries from non-diabetic and diabetic patients before and after perfusion of 100 nM iberiotoxin or a Ca2+ free + nifedipine (1 μM) solution. (C) Summary of iberiotoxin-induced constriction in non-diabetic (n = 7 arteries, 4 subjects) and diabetic (n = 5 arteries, 5 subjects) adipose arteries pressurized to 80 mmHg. (D) Scatter/bar plot summarizing vascular tone at 80 mmHg in response to 60 mM extracellular K+ in non-diabetic (n = 8 arteries, 4 subjects) and diabetic (n = 6 arteries, 5 subjects) adipose arteries. *P < 0.05, Mann-Whitney test. Significance was compared between non-diabetic and diabetic datasets.

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  Figure 2. STOCs amplitude and frequency are reduced in vascular smooth muscle from diabetic patients. (A) Representative traces of spontaneous whole-cell BKCa currents (e.g. STOCs) at −40 mV in vascular smooth muscle from non-diabetic and diabetic patients. (B,C) Voltage dependency of STOC amplitude (B) and frequency (C) in cells from non-diabetic (n = 10 cells, 4 subjects) and diabetic (n = 13 cells, 4 subjects) patients. Solid lines represent the best fit of the amplitude data using a linear regression function and of the frequency data using a Boltzmann sigmoidal function. *P < 0.05, unpaired t test. Significance was compared between corresponding non-diabetic and diabetic datasets.

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  Figure 3. The frequency and amplitude of Ca2+ sparks is similar in non-diabetic and diabetic vascular smooth muscle. (A) Representative three-dimensional pseudo-color images of Ca2+ sparks (upper panels) and fractional fluorescence traces (F/F0; lower panels) from those sites in fluo-4 AM-loaded vascular smooth muscle cells from non-diabetic and diabetic patients. Scatter/bar plots summarizing Ca2+ spark amplitude (B) and frequency (C) in cells from non-diabetic (n = 254 events, 14 cells, 3 subjects) and diabetic (n = 333 events, 19 cells, 5 subjects) patients. *P < 0.05, Mann-Whitney test. Significance was compared between non-diabetic and diabetic datasets.

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  Figure 4. Decreased BKCa channel activity in vascular smooth muscle from diabetic patients. (A) Representative single BKCa channel traces at −40 mV from excised membrane patches of isolated human non-diabetic and diabetic vascular smooth muscle cells in the presence of 1 μM and 10 μM free Ca2+ bath solution (C: closed; O: open). (B) Scatter/bar plot summarizing BKCa channel open probability (Po) at the indicated free Ca2+ concentration from human non-diabetic (n = 18 cells, 9 subjects) and diabetic (n = 27 cells, 15 subjects) cells. *P < 0.05, Mann-Whitney test. (C) Open dwell time histograms of BKCa channels in non-diabetic and diabetic vascular smooth muscle. The goodness of the fit was assessed with R2 and the F test was used for comparison between open time fits. Significance was compared between non-diabetic and diabetic datasets.

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  Figure 5. Impaired BKCa β1 subunit function in human diabetic vascular smooth muscle. (A) Representative single BKCa channel records at +40 mV and 1 μM free Ca2+ obtained from excised membrane patches of HEK293 cells expressing only BKCa α subunit or coexpressing BKCa α + BKCa β1 subunits in the absence and presence of tamoxifen (1 μM). (B) Bar plot summarizing the change in open probability (tamoxifen: control) for HEK293 cells expressing BKCa α (n = 11 cells) or BKCa α + BKCa β1 (n = 12 cells) subunits. *P < 0.05, unpaired t test C) Representative single BKCa channel traces recorded from excised membrane patches of vascular smooth muscle from non-diabetic and diabetic patients with 1 μM free Ca2+ in the bath before (−) and after (+) application of 1 μM tamoxifen. (D) Amalgamated data summarizing BKCa open probability in non-diabetic (n = 17 cells, 9 subjects) and diabetic (n = 19 cells, 8 subjects) cells in the presence or absence of tamoxifen. (C: closed; O: open) *P < 0.05, Wilcoxon test. Significance for Fig. 5B was compared between cells co-expressing BKCa α + BKCa β1 vs. BKCa α only. Significance for Fig. 5D was compared to corresponding - tamoxifen datasets.

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  Figure 6. No change in BKCa subunit total protein levels in arterial lysates from non-diabetic and diabetic patients. (A) Representative immunoreactive bands corresponding to BKCa α1 and BKCa β1 subunits in lysates from untransfected HEK293 cells (HEK), HEK293 cells transfected with either BKCa α1 or BKCa β1 subunit and human arteries from non-diabetic patients (n = 3 lysates per condition). Full-length blots are shown in Supplementary Fig. S2A. (B) Representative immunoreactive bands corresponding to BKCa α1 and BKCa β1 subunits, and β-actin as a normalization control. Full-length blots are shown in Supplementary Fig. S2B. (C) Corresponding densitometric summary data for each subunit obtained using arterial lysates from non-diabetic (n = 10 lysates) and diabetic (n = 10 lysates) patients. *P < 0.05, Mann-Whitney test. Significance was compared between non-diabetic and diabetic datasets.

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  Figure 7. Decreased association between BKCa α1 and BKCa β1 subunits in vascular smooth muscle cells from diabetic patients. (A–C) Differential interference contrast (DIC) and confocal fluorescent PLA puncta (red) and DAPI (blue) images of freshly dissociated human vascular smooth muscle labeled with mouse-anti BKCa α1 (A), rabbit-anti BKCa α1 (B), and rabbit-anti BKCa β1 (C) antibodies. (D,E) DIC (left) and fluorescence PLA (red)/DAPI (blue) (right) images of dissociated vascular smooth muscle from non-diabetic (D) and diabetic (E) patients co-labeled with two distinct antibodies for the BKCa α1 subunit. (F,G) DIC (left) and fluorescence PLA (red)/DAPI (blue) (right) images of dissociated human vascular smooth muscle from non-diabetic (F) and diabetic (G) patients co-labeled for BKCa α1 and BKCa β1 subunits. Scale bar = 10 μm. (H) Quantification of PLA fluorescent puncta per μm2 cell area for non-diabetic and diabetic vascular smooth muscle cells labeled for mouse-anti BKCa α1 (n = 19 cells), rabbit-anti BKCa α1 (n = 27 cells), rabbit-anti BKCa β1 (n = 12 cells), mouse-anti BKCa α1 + rabbit-anti BKCa α1 (n = 24 non-diabetic, 14 diabetic cells); mouse-anti BKCa α1 + rabbit-anti BKCa β1 (n…