Chronic Sympathetic Hyperactivity Triggers Electrophysiological Remodeling and Disrupts Excitation-Contraction Coupling in Heart

Figures

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  Figure 1. Chronic sympathetic hyperactivity reduces cellular contractility in ventricular cardiomyocytes. Panel (A) shows 5 representative contractions from WT (black) and ARDKO (red) cardiac cells. ARDKO myocytes had a significant reduction in fractional shortening (B), contraction (C) and relaxation (D) velocity. *p < 0.05 when compared to WT at same frequency. All cell contractility data are summarized in Table S5.

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  Figure 2. Reduced LCC currents contribute to impaired cellular contractility in ARDKO VCMs. Panel (A) shows representative LCC currents records from WT (black) and ARDKO (red) VCMs. ARDKO had reduced LCC current (B), with no significant modulation of stationary voltage-dependency for activation (C) and inactivation (D) of the LCC. (WT n = 25 cells/4 hearts; ARDKO n = 29 cells/4 hearts). *p < 0.05 when compared to WT.

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  Figure 3. CSH impairs CICR in heart. (Panel A) shows confocal line-scan images from WT (Top) and ARDKO (Bottom) VCMs loaded with Fluo-4 Ca2+ indicator and paced at 1 Hz. (Panel B) show the initiation phase of [Ca2+]i transient used to analyze CICR delay. We used a custom-made routine to create a mask of Fluo-4 fluorescence signals where the orange pixels in the masked image represent the delay between the stimulus and the initiation of CICR. ARDKO VCMs had reduced [Ca2+]i transient amplitude (C) with slower fluorescence decay (D) (WT n = 90 cells/5 hearts; ARDKO n = 83 cells/5 hearts). Also, ARDKO VCMs had delayed initiation of Ca2+ release (E) (WT n = 20 cells; ARDKO n = 16 cells). *p < 0.05 when compared to WT (Student’s t-test).

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  Figure 4. ARDKO displayed AP prolongation and reduced voltage-gated outward K+ currents. Panel A and B are representative traces of an action potential (A) and outward K+ currents (B) recorded from WT (black) and ARDKO (red) myocytes. ARDKO VCMs displayed delayed AP repolarization, as demonstrated by the increase in time to reach 50% (C) and 90% (D) of repolarization (WT n = 34 cells; ARDKO n = 32 cells). The reduced voltage-gated outward K+ currents (E) contribute to the prolonged AP waveform profile in ARDKO (WT n = 31 cells/4 hearts; ARDKO n = 34 cells/4 hearts). *p < 0.05 when compared to WT.

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  Figure 5. CSH increases NCX expression and current density. ARDKO VCMs have a significant increase in NCX current density recorded in whole-cell patch clamp (A) (WT n = 18 cells/3 hearts; ARDKO n = 9 cells/3 hearts). The inset in panel A shows representative traces NCX currents from WT (black) and ARDKO (red) myocytes. NCX currents reduction associated with an overexpression of the NCX protein (B) (n = 9 samples for each group). *p < 0.05 when compared to WT (Mann-Whitney). Inset of panel B shows bands cropped from the same blot for each respective protein. The full-length blots can be found in Figure S5.

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  Figure 6. Simulations of cardiac CICR suggests impaired ECC in ARDKO mice. (A) Field-stimulated AP for WT (black) and ARDKO (red) simulations. (B) Sarcomere shortening for WT (black) and ARDKO (red) during APs shown in (A). (C) Force generated during the simulated APs.

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  Figure 7. Computational modeling provides mechanistic insights into how CSH alters Ca2+ spark properties. Panel A&B) Simulated Ca2+ sparks (shown as number of open RyR2 channels) during the APs shown in Fig. 6. in WT (A) and ARDKO (B) where each colored line indicates RyR2 cluster activity from a different CRU. For clarity only 10% of events are shown. (C) Average number of Ca2+ sparks triggered during a [Ca2+]i transient. (D) Probability of an LCC opening triggering a Ca2+ spark. E) Average Ca2+ spark duration. Ca2+ spark synchrony measured as average delay time from start of the AP to each Ca2+ spark peak (F) and variance of delay (G). In panels C-G, WT is indicated by black bars and ARDKO by red bars. (n = 10 simulations for each group) *p < 0.05 when compared to WT (Mann-Whitney).

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  Figure 8. Simulations reveal individual contributions of membrane current changes on ARDKO phenotype. Peak systolic [Ca2+]i transient (A), Decay time constant for [Ca2+]i transient (B), time to 90% AP recovery (C), EC coupling gain (D), Ca2+ spark fidelity to LCC openings (E) and contractile force (F) for WT (black bars), ARDKO (red bars), and conditions where each membrane current change associated with ARDKO is implemented in isolation (green, purple, and orange bars for NCX, IK+, and LCC, respectively. Significance (p < 0.05, One-way ANOVA) measured against WT (*) and against ARDKO (‡) and n = 10 simulation runs for each condition.