Myocardial Blood Flow Control by Oxygen Sensing Vascular Kvβ Proteins

Abstract

[Figure: see text].

Keywords: aldo-keto reductases; coronary circulation; ion channels; physiology; potassium channels.

Figure 1:. Loss of Kvβ2 impairs cardiac pump function during stress.

(A) Representative M-mode echocardiographic images obtained from wild type (WT; 129SvEv), and Kvβ2^−/− mice during infusion of 5 μg/kg∙min⁻¹ norepinephrine. (B) Box-and-whisker plot of ejection fraction data for WT and Kvβ2^−/− mice at baseline, after administration of hexamethonium (HX; 5 mg∙kg⁻¹, i.v.), and during norepinephrine infusions (0.5 – 5 μg/kg∙min⁻¹; 2–3 min duration). n = 8 each, **P<0.01, ***P<0.001 (two-way RM ANOVA). (C) Arterial blood pressure recordings obtained via femoral artery catheter in WT and Kvβ2^−/− mice, before and after norepinephrine treatment (NE, 5 μg/kg∙min⁻¹, indicated by arrows).

Figure 2:. Relationship between myocardial blood flow and cardiac workload in Kvβ-null mice.

(A) Long axis MCE images showing signal intensity from myocardial tissue and cavity before destruction frame and during replenishment phase (~10 sec). The left ventricular wall is outlined with a yellow dashed line in the destruction frame. (B) Signal intensity versus time (following destruction frame) in region of interest in the anterior left ventricular myocardial wall of WT (129SvEv), Kvβ1.1^−/−, and Kvβ2^−/− mice. Data were fit with exponential function (see inset). (C,D) Summary of MBF as a function of cardiac workload (double product; heart rate x mean arterial pressure) in Kvβ1.1^−/− (C) and Kvβ2^−/− (D) versus strain-matched wild type (WT) control mice. Data were fit with a simple linear regression model with slopes: WT (0.00192 ± 0.00031), Kvβ1.1^−/− (0.00279 ± 0.00016); n = 6–8 mice; WT (0.00241 ± 0.00014), Kvβ2^−/− (0.00162 ± 0.00022); n = 4–8 mice, *P<0.05, slope of Kvβ2^−/− vs. WT.

Figure 3:. Ablation of Kvβ2 attenuates hypoxia-induced coronary vasodilation.

(A) Summarized bath O₂ (%) measured in normoxic and hypoxic conditions (perfusate aerated with 5% CO₂, balance N₂, + 1 mM Na₂S₂O₄); data are pooled from measurements obtained with wild type (129SvEv) and Kvβ2^−/− coronary arteries. n = 7–9, ***P<0.001 (Mann Whitney U). (B) Representative arterial diameter recordings in isolated preconstricted (100 nM U46619) coronary arteries from wild type (WT; 129SvEv) and Kvβ2^−/− mice in normoxic and hypoxic conditions. Ca²⁺-free perfusate containing nifedipine (nifed; 1 μM) and forskolin (fsk; 0.5 μM) was introduced at the end of the experiment to induce maximum dilation. (C) Scatter-plot and mean ± SEM showing percent decrease in diameter recorded under normoxic (- hypoxia) and hypoxic (+ hypoxia) conditions for arteries from WT and Kvβ2^−/− mice. Normoxic and hypoxic conditions were both applied in continuous presence of U46619, see above (B). n = 5 arteries, 3–4 mice *P<0.05, ns: P≥0.05 (one-way ANOVA, Tukey). (D) Scatter-plot and mean ± SEM showing hypoxia-induced dilation (%) for arteries from WT and Kvβ2^−/− mice. **P<0.01 (Mann-Whitney U test).

Figure 4:. L-lactate enhances I_Kv in coronary arterial myocytes and promotes coronary vasodilation via Kvβ2.

(A, B) Representative outward K⁺ current recordings normalized to cell capacitance (pA/pF) in response to step-wise (10 mV) depolarization to +50 mV from a holding potential of −70 mV in isolated coronary arterial myocytes. Currents were recorded before and after application of 10 mM L-lactate in bath solution lacking (A) or containing (B) 500 nM psora-4. (C, D) Summary current-voltage relationships obtained in coronary arterial myocytes before and after application of 10 mM L-lactate in bath solution lacking (C) or containing (D) 500 nM psora-4. n = 5–7 cells from 4–7 mice. *P <0.05, ns: P≥0.05 (two-way RM ANOVA). (E) Summary of L-lactate-induced currents recorded in the absence and presence of 500 nM psora-4. n = 5–7 cells from 4–7 mice. *P <0.05 (mixed-effects). (F-H) Arterial diameter traces obtained from pressurized (80 mmHg) coronary arteries isolated from wild type (WT; 129SvEv; F,G) and Kvβ2^−/− (H) mice in the absence and presence of L-lactate (5–20 mM, as indicated). Arteries were preconstricted with 100 nM U46619; for WT arteries, L-lactate was applied in the absence (top) and presence (bottom) of psora-4 (500 nM). Maximum passive diameter was recorded at the end of each experiment in Ca²⁺-free saline solution with nifedipine (nifed; 1 μM) and forskolin (fsk; 0.5 μM). (I) Summary plot showing L-lactate-induced dilation, expressed as a percent change from baseline diameter relative to maximum passive diameter, for arteries isolated from WT (129SvEv; ± 500 nM psora-4) and Kvβ2^−/− mice. n = 4 arteries from 4 mice for each. *P<0.001; ns: P≥0.05, lactate vs. baseline (Friedman).

Figure 5:. Kvβ2 controls redox-dependent vasoreactivity in resistance mesenteric arteries.

(A) Representative fluorescence images showing PLA-associated fluorescent punctae (red) in wild type coronary and mesenteric arterial myocytes. Cells were labelled for Kv1.5 alone, or co-labelled for Kv1.5 and Kv1.2, Kv1.5 and Kvβ1.1, Kv1.5 and Kvβ2, or Kvβ1.1 and Kvβ2 proteins. DAPI nuclear stain is shown for each condition (blue). Scale bars represent 5 μm. (B) Summary of PLA-associated punctate sites normalized to total cell footprint area for conditions and groups as in D. P values are shown for coronary versus mesenteric arteries (Mann Whitney U). (C,D) Arterial diameter traces obtained from pressurized (80 mmHg) mesenteric arteries isolated from wild type (C; 129SvEv) and Kvβ2^−/− (D) mice in the absence and presence of L-lactate (5–20 mM, as indicated). Arteries were preconstricted with 100 nM U46619 and L-lactate was applied in the absence (top) and presence (bottom) of the selective Kv1 channel inhibitor psora-4 (500 nM). Maximum passive diameters were recorded at the end of each experiment in Ca²⁺-free saline solution with nifedipine (nifed; 1 μM) and forskolin (fsk; 0.5 μM). (E) Summary plot of L-lactate-induced dilation, expressed as the percent change from baseline diameter relative to maximum passive diameter, for arteries isolated from WT (129SvEv; ± psora-4) and Kvβ2^−/− mice. n = 5 arteries from 4–5 mice for each. *P<0.05; ns: P≥0.05, lactate vs. baseline (Friedman).

Figure 6:. Increasing the ratio of Kvβ1.1:Kvβ2 subunits in smooth muscle inhibits L-lactate-induced vasodilation and suppresses myocardial blood flow.

(A) Schematic diagram describing the SM22α-rtTA:TRE-β1 model. Double transgenic animals (+dox) results in activation of the reverse tetracycline trans-activator (rtTA) in smooth muscle cells, and drives expression of Kvβ1.1. (B) Western blots showing immunoreactive bands for Kvβ1 in whole mesenteric artery and brain lysates from SM22α-rtTA (single transgenic control) and SM22α-rtTA:TRE-β1 (double transgenic) mice after doxycycline treatment. Ponceau-stained membrane (mol. Wt.: ~30–55 kDa) is shown as an internal control for total loaded protein. (C) Summarized relative densities of Kvβ1.1-associated immunoreactive bands in mesenteric arteries and brains of SM22α-rtTA:TRE-β1 relative to SM22α-rtTA. n = 3 each. *P<0.05, ns: P≥0.05 (one sample t test). (D) Representative fluorescence images showing PLA-associated fluorescent punctae (red) in coronary arterial myocytes isolated from SM22α-rtTA and SM22α-rtTA:TRE-β1mice. Cells were labelled for Kv1.5 alone, or co-labelled for Kv1.5 and Kvβ1, or Kv1.5 and Kvβ2 proteins. DAPI nuclear stain is shown for each condition (blue). Scale bars represent 5 μm. (E) Summary of PLA-associated punctate sites normalized to total cell footprint area for conditions and groups as in D. n = 6–19 cells from 2–3 mice for each; *P<0.05, **P<0.001 (Mann Whitney U). (F) Representative arterial diameter recordings from 100 nM U46619-preconstricted mesenteric arteries isolated from SM22α-rtTA and SM22α-rtTA:TRE-β1 mice in the absence and presence of L-lactate (5–20 mM), as in Figure 5C,D. Passive dilation in the presence of Ca²⁺-free solution + nifedipine (1 μM) and forskolin (fsk; 0.5 μM) is shown for each recording. (G) Summary plot of L-lactate-induced dilation for arteries isolated from SM22α-rtTA and SM22α-rtTA:TRE-β1 mice. n = 6–10 arteries from 5–6 mice; *P<0.05; ns: P≥0.05, lactate vs. baseline (Friedman). (H) Summary relationships between myocardial blood flow (MBF) and cardiac workload (double product; heart rate x mean arterial pressure) in SM22α-rtTA:TRE-β1 vs. SM22α-rtTA control mice. n = 5 each; ***P<0.001 (linear regression).