Pyridine nucleotide redox potential in coronary smooth muscle couples myocardial blood flow to cardiac metabolism

Figures

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  Fig. 1. Increased myocardial workload promotes local elevation of lactate:pyruvate ratio in the coronary arterial wall. A Schematic showing protocol for induction of acute cardiac stress prior to collection of tissues for MALDI-MS imaging. To induce high cardiac work, anesthetized mice with stable heart rates of 400–450 bpm were treated with dobutamine (db; 10 mg/kg, i.p.). Heart rate was continuously monitored, and hearts were rapidly cryopreserved in the thoracic cavity and excised after 2–3 min of stabilized heart rate responses to either db or PBS (vehicle). B Symbol plot showing exemplary heart rates recorded every 30 s before (i.e., during induction and stabilization of anesthesia) and after administration of either db (10 mg/kg; i.p.) or PBS. C Images of left ventricular intramyocardial coronary arteries in H & E-stained heart sections and corresponding intensity-coded MALDI-MS images showing lactate (Lac) and pyruvate (Pyr) relative to background signals in hearts from low and high cardiac work mice. x–y resolution: ~20 μm. Insets show magnified region of interest at coronary arterial wall. Scale bars represent 200 μm. Experiment was repeated twice with similar results. D Box and…

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  Fig. 2. Arterial myocyte NADH:NAD+ is sensitive to changes in proximal cardiomyocyte beating frequency. A Schematic illustration depicting the preparation of arterial myocyte/induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) co-cultures. Primary aortic vascular smooth muscle cells (passage 0–2) were treated with adenovirus to express the NADH:NAD+-sensitive fluorescent biosensor peredox-mCherry (i.). Arterial myocytes were then seeded and allowed to integrate (48 h) onto a two-dimensional monolayer of iPSC-CMs (ii.). Imaging was performed on either arterial/cardiac co-cultured myocytes or arterial myocytes alone during electrical stimulation (1–3 Hz; iii.) while monitoring peredox-mCherry green:red fluorescence. B Exemplary brightfield and fluorescence images showing red (mCherry) and green (T-sapphire) fluorescence of peredox-mCherry expressing arterial myocytes in the presence of non-fluorescent cardiac myocytes in an arterial + cardiac myocyte co-culture. Scale bars represent 100 μm. Experiment was repeated six times with similar results. C Exemplary time series of arterial myocyte NADH:NAD+ in either an arterial/cardiac myocyte co-culture (green trace) or arterial…

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  Fig. 3. Modulation of coronary arterial myocyte IKv upon changes in intracellular pyridine nucleotide redox potential. Isolated coronary arterial myocytes were dialyzed with pyridine nucleotides at concentrations as indicated in Supplementary Table 2 for voltage-clamp recordings in the conventional whole-cell configuration. A, B Representative outward K+ currents (A) and IK densities (pA/pF; B) recorded in coronary arterial myocytes from wild type mice (129SvEv) in the presence of either oxidized or reduced pyridine nucleotide redox ratios. Recordings were performed in the absence (i.) and presence (ii.) of the Kv1 channel inhibitor psora-4 (500 nM). Representative psora-4-sensitive currents (i.e., total outward IK – psora-insensitive IK) and summarized densities (mean values ± SEM) are shown in iii. panels. n = 5–6 cells, 4–5 mice for each. *P < 0.001, oxidized vs. reduced (mixed effects analysis). C Summarized I/Imax (mean values ± SEM) from two-pulse activation voltage protocol (i.) and inactivation protocol (ii.) for coronary arterial myocytes from wild type mice in the presence of oxidized or reduced pyridine nucleotide ratios. Curves were fit with a Boltzmann function; V0.5,act and…

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  Fig. 4. Potentiation of native coronary Kv1 activity by NADH requires Kvβ2. A, B Unitary K+ channel currents (-80-80 mV as indicated, left) and summarized I-V relationships (right) from inside-out patch recordings from isolated coronary arterial myocytes (wild type 129SvEv, A) or Cos-7 cells transiently expressing Kv1.5 (B). (A) n = 6 cells; (B) n = 4 cells. C Single K+ channel activity in patches from coronary arterial myocytes at a holding potential of +40 mV in the absence and presence of psora-4 (500 nM). D Summary of K+ channel open probabilities (nPo, mean values ± SEM) recorded in the absence and presence of 500 nM psora-4. n = 6 cells, *p = 0.0312 (two-sided Wilcoxon matched-pairs signed rank test). E Representative single Kv current recordings (holding potential = −40 mV) in the absence (ct) and presence of 1 mM NADH (top) or 1 mM NAD+ (bottom). F Summary of Kv channel nPo (mean values ± SEM) recorded before (ct) and after bath application of 1 mM NADH (n = 27 cells) or NAD+ (n = 15 cells). **p = 0.0076 (paired two-sided t test). G Single Kv channel currents recorded from freshly isolated human coronary arterial myocyte membrane patches before and after application of 1 mM NADH.…

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  Fig. 5. Loss of Kvβ2 catalytic function prevents redox-mediated increases in Kv1 activity and vasodilation, and suppresses MBF. A Schematic illustration depicting abolished NADPH-dependent carbonyl reduction by Kvβ2 resulting from the Y90F point mutation. B Single-channel recordings showing Kv1 activity in membrane patches from Kvβ2Y90F mice before and after application of 1 mM NADH. C Summary of Kv1 open probabilities (nPo; mean values ± SEM) in membrane patches from Kvβ2Y90F mice before and after application of 1 mM NADH. n = 7 cells from 3 mice; ns: P ≥ 0.05 (two-sided Wilcoxon matched-pairs signed rank test). D Representative arterial diameter recordings in arteries from wild type (129SvEv) and Kvβ2Y90F mice in the absence and presence of l-lactate (5–20 mM) in the perfusate (i.); and, summary of percent change in diameter (mean values ± SEM) in response to l-lactate (5–20 mM) relative to baseline (⎯ l-lactate, ii.). wt: n = 5 arteries from 4 mice, Kvβ2Y90F: n = 6 arteries from 5 mice. ***p = 0.00001 (two-way repeated measures ANOVA). E Representative arterial diameter recordings in arteries from wild type (129SvEv) and Kvβ2Y90F mice in the absence and presence of H2O2 (0.1–10 μM) in…

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  Fig. 6. Oxygen demand-sensitive coronary arterial myocyte NADH:NAD+ links blood flow to myocardial metabolism via Kvβ2 catalytic function. Schematic representation showing proposed model of myocardial blood flow regulation via coronary pyridine nucleotide redox potential. Increases in myocardial oxygen consumption and cardiac workload promote the rapid elevation of intracellular NADH:NAD+ in arterial myocytes in the wall of intramyocardial coronary arteries. This change in redox balance modulates the voltage-sensitivity of Kv1 gating via binding and enzymatic oxidation of NADH by Kv-associated Kvβ2 proteins. Resultant enhancement of steady-state K+ efflux leads to membrane potential (VM) hyperpolarization, reduction in intracellular [Ca2+]I, arterial myocyte relaxation, and vasodilation, thus increasing blood flow. Created with BioRender.com.