Acute Glycogen Synthase Kinase-3 Inhibition Modulates Human Cardiac Conduction

Figures

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  Figure 1. Conduction Velocity of Human LV Slices Is Reduced by Short-Term Culture With SB2 (A) Representative activation maps of a control slice (left) compared to an SB2-treated slice (right) from the same heart. Electrical activation from time t0 to t1 is slower in the SB2-treated slice as seen by the greater number of isochrones to reach the same distance. (B) Control optical action potentials (top) at t0(blue) and t1(dashed cyan) shows faster propagation compared to SB2 optical action potentials (bottom) from t0(red) to t1(dashed magenta). (C) Conduction velocities quantified from activation maps show no difference between baseline and 3-hour control but reduced conduction velocity in 3-hour SB2-treated slices (baseline: 62 cm/s; 3-hour control: 63 cm/s; 3-hour SB2: 51 cm/s) at pacing cycle lengths of 1,000 ms, 800 ms, 600 ms, and 400 ms. (D) Conduction velocity restitution of each individual heart shows reduced conduction velocity at all stimulation cycle lengths but no change in restitution slope in SB2-treated slices. Each color represents an individual heart for 3-hour vehicle (solid line) or 3-hour SB2 (dashed line). Statistics were performed using repeated-measures analysis of…

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  Figure 2. Action Potential Upstroke Velocity Is Decreased in SB2-Treated Slices (A) Representative action potentials from control without culture (blue), 3 hours in culture (magenta), and 3-hour SB2-cultured (red) slices paced at a cycle length of 1,000 ms. (B) Averaged action potential of all recorded myocytes from slices for each condition. (C) (Top) Zoom of action potential upstroke from B shows decreased dVm/dtmax in SB2-treated slices compared to control and 3-hour cultured control (baseline: 234.1 V/s; 3-hour control: 249.7 V/s; 3-hour SB2: 195.4 V/s). Action potential upstroke change in membrane potential (ΔVm) (bottom) over the same time period shows a significant decrease in dVm/dtmax for SB2 treatment and not a decrease for the entire duration. (D to G) RMP, action potential amplitude, APD90, and APD restitution are not changed between all conditions for each individual heart, whereas dVm/dtmax is significantly decreased in action potentials from slices cultured with SB2 compared with control and cultured control. Statistics were performed using repeated-measures analysis of variance followed by Tukey’s post hoc test for multiple pairwise comparisons. ∗P < 0.05; ∗∗∗P < 0.001. 3Hr…

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  Figure 3. Computational Simulations Conducted in Virtual Cardiac Organotypic Slices Accurately Reproduce Experimental Electrophysiologic Data (A) Activation sequence in response pacing at a basic cycle length (BCL) of 1,000 ms (top) and membrane voltage over time at points indicated in top graphic (bottom). (B) The same as A except that the excitation in response to a stimulus (S2) was delivered 400 ms later. (C) Longitudinal conduction velocity restitution relationship shows reasonable correspondence between simulations (white squares) and experiments (black circles) at all S1-S2 intervals. (D) Adaptation relationships for upstroke velocity (dVm/dtmax) and (E) 90% action potential duration (APD90) show that the model reproduces values seen in experiments for all BCL values tested.

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  Figure 4. Short-Term SB2 Culture of LV Slices Results in Changes in Nav1.5 Protein But Not Gene Transcription (A) Western blot for NaV1.5 and GAPDH with quantification based on band density relative to GAPDH shows a decrease in NaV1.5 protein (30% average decrease) in the SB2 condition compared to 3-hour control. (B) Reverse transcription quantitative polymerase chain reaction shows no changes between 3 hours in culture and 3 hours in culture with SB2 for ACTC1 (negative control) or electrophysiology-related transcripts (SCN5A) and FGF12, a known regulator of cardiac sodium current. Statistics in A were performed using repeated-measures analysis of variance followed by Tukey’s post hoc test for multiple pairwise comparisons. Statistics in B were performed using Student’s t-test. ∗P < 0.05. NaV1.5 = pore-forming α-subunit protein of the voltage-gated cardiac sodium channel; other abbreviations as in Figure 2.

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  Figure 5. Acute SB2 Treatment Increases β-Catenin Protein in Human LV Slices (A) Western blot of β-catenin and GAPDH levels with quantification based on band density relative to GAPDH shows an increase in β-catenin (31% average increase) in SB2-treated slices. (B) Both chromatin-bound β-catenin and nonphosphorylated β-catenin levels increased with respect to histone H3 in slices cultured with SB2. In contrast to other subcellular fractions, chromatin-bound β-catenin shows multiple bands of lower molecular weights in addition to the primary band at 92 kDa. (C) ABC (green) is present in human myocyte nuclei, marked by PCM1 (red) and DAPI (blue), in baseline, 3-hour control, and 3-hour SB2 conditions. White arrowheads indicate β-catenin–positive cardiomyocyte nuclei, whereas white asterisks mark β-catenin–negative cardiomyocyte nuclei. Scale = 50 μm. Statistics were performed using repeated-measures analysis of variance followed by Tukey’s post hoc test for multiple pairwise comparisons. ∗P < 0.05. ABC = active β-catenin; DAPI = 4′,6-diamidino-2-phenylindole; non-P = nonphosphorylated; other abbreviations as in Figure 2.

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  Figure 6. Myocyte-Specific β-Catenin GOF and Acute GSK-3 Inhibition by SB2 Reduces Conduction Velocity in Murine Hearts (A) Representative activation maps showing slowed electrical activation in β-catenin GOF hearts (right) compared to noninduced controls (left). (B) Activation map showing ventricular tachycardia induced from electrical stimulation of β-catenin GOF ventricles. The white arrows show irregular propagation of 2 different wave fronts, which results in a functional block. Five of 6 β-catenin GOF and 0 of 6 control hearts had spontaneous or stimulation-induced ventricular tachycardia. (C) Conduction velocities of RVs and LVs are significantly reduced in β-catenin GOF hearts compared to controls. (D) Western blot shows expression of stabilized β-catenin (lower-molecule-weight band associated with loss of exon 3) in β-catenin GOF mice and decreased NaV1.5 protein in β-catenin GOF compared to control hearts. (E) Transcription of Wnt/β-catenin target gene Axin2 is increased, and Scn5a is decreased, in β-catenin GOF compared to control hearts. Statistics were performed using Student’s t-test: ∗P < 0.05; ∗∗P < 0.01. β-cat = β-catenin; GOF = gain of function; RV = right ventricle;…