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. 2018 Jan 23:9:16.
doi: 10.3389/fphys.2018.00016. eCollection 2018.

Sympathetic Nervous Regulation of Calcium and Action Potential Alternans in the Intact Heart

James Winter  1   2 , Martin J Bishop  3 , Catherine D E Wilder  1 , Christopher O'Shea  2 , Davor Pavlovic  2 , Michael J Shattock  1
Affiliations

Sympathetic Nervous Regulation of Calcium and Action Potential Alternans in the Intact Heart

James Winter et al. Front Physiol. .

Abstract

Rationale: Arrhythmogenic cardiac alternans are thought to be an important determinant for the initiation of ventricular fibrillation. There is limited information on the effects of sympathetic nerve stimulation (SNS) on alternans in the intact heart and the conclusions of existing studies, focused on investigating electrical alternans, are conflicted. Meanwhile, several lines of evidence implicate instabilities in Ca handling, not electrical restitution, as the primary mechanism underpinning alternans. Despite this, there have been no studies on Ca alternans and SNS in the intact heart. The present study sought to address this, by application of voltage and Ca optical mapping for the simultaneous study of APD and Ca alternans in the intact guinea pig heart during direct SNS. Objective: To determine the effects of SNS on APD and Ca alternans in the intact guinea pig heart and to examine the mechanism(s) by which the effects of SNS are mediated. Methods and Results: Studies utilized simultaneous voltage and Ca optical mapping in isolated guinea pig hearts with intact innervation. Alternans were induced using a rapid dynamic pacing protocol. SNS was associated with rate-independent shortening of action potential duration (APD) and the suppression of APD and Ca alternans, as indicated by a shift in the alternans threshold to faster pacing rates. Qualitatively similar results were observed with exogenous noradrenaline perfusion. In contrast with previous reports, both SNS and noradrenaline acted to flatten the slope of the electrical restitution curve. Pharmacological block of the slow delayed rectifying potassium current (IKs), sufficient to abolish IKs-mediated APD-adaptation, partially reversed the effects of SNS on pacing-induced alternans. Treatment with cyclopiazonic acid, an inhibitor of the sarco(endo)plasmic reticulum ATPase, had opposite effects to that of SNS, acting to increase susceptibility to alternans, and suggesting that accelerated Ca reuptake into the sarcoplasmic reticulum is a major mechanism by which SNS suppresses alternans in the guinea pig heart. Conclusions: SNS suppresses calcium and action potential alternans in the intact guinea pig heart by an action mediated through accelerated Ca handling and via increased IKs.

Keywords: action potential duration; alternans; calcium transient; intact heart; optical mapping; sarco(endo)plasmic reticulum ATPase; sympathetic nervous system; ventricular fibrillation.

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Figures

Figure 1
Figure 1
The isolated innervated guinea pig heart. (A) Illustrative representation of the isolated innervated guinea pig heart model. See text for details. PP, perfusion pressure; ECG, electrocardiogram. (B) Illustration of analysis parameters used for action potential and Ca transient recordings. Action potential duration (APD) was measured as the period between activation (midpoint of upstroke, TActM) and 80% repolarisation (TRepol80). The rapid decay phase of the Ca transient was fitted to exponential function to derive the time constant (tau). Ca transient decay time was measured from peak of the Ca transient to 50% recovery. (C,D) Data analysis protocols for action potential and Ca transient amplitude alternans. For action potential duration, the maximum-minimum difference between pairs of beats is taken as the amplitude of alternans and the threshold for detection is set to 5 ms. For Ca transient alternans, the relative amplitude of the intracellular Ca transient, measured from the baseline preceding each beat, was used to calculate a Ca alternans ratio (see Equation 1). 0.1 is used as the threshold for Ca recordings.
Figure 2
Figure 2
Transition from concordant to discordant alternans. Illustrative example of the transition from concordant to discordant alternans during dynamic pacing in the guinea pig heart. Representative traces from basal and apical regions are shown, along with representative isochronal maps of activation time, regional action potential duration and total repolarisation time across the guinea pig left ventricle during concordant and discordant alternans. Beats or regions with relatively long or short action potentials are labeled. *denotes a region of tissue with a steep repolarisation gradient, secondary to discordant alternans, that results in regional condition slowing (**) on the next beat.
Figure 3
Figure 3
Effects of sympathetic nerve stimulation on action potential duration and Ca transient alternans. (A,B) Representative traces and data showing the change in action potential duration (APD) with sympathetic nerve stimulation (SNS). CL, cycle length. (C) Representative optical action potential recordings during pacing at a 120 ms cycle length and average cycle length relationships for the magnitude of APD alternans at baseline (black) and during SNS (red). (D) Data showing the change in the APD alternans threshold with SNS. (E,F) Representative trace showing the change in Ca transient kinetics with SNS and data demonstrating the effects of SNS on the time constant (tau) of Ca transient decay. (G) Representative optical Ca transient recordings during pacing at a 120 ms cycle length and average cycle length relationships for the magnitude of the Ca alternans ratio at baseline and during SNS. (H) Data showing the change in the Ca alternans threshold with SNS. Data taken from a 3 x 3 pixel region in the basal left ventricle. Data represent mean ± SEM. Paired Student's t-tests; ***p < 0.001. (n = 11–13 hearts).
Figure 4
Figure 4
Effects of exogenous noradrenaline on action potential duration alternans. (A) Cycle length relationships of action potential duration (APD) alternans in control conditions (black) and with noradrenaline perfusion (blue). (B) Mean data of alternans threshold in both conditions. Data taken from a 3 × 3 pixel region in the basal left ventricle. Data represent mean ± SEM. Paired Student's t-test; ***p < 0.001. (n = 6 hearts).
Figure 5
Figure 5
Modulation of the electrical restitution curve by sympathetic nerve stimulation. (A) Representative action potential duration (APD) restitution curves at baseline and during sympathetic nerve stimulation (SNS). The right panels show the first derivative of the exponential fit of each curve. (B) The same data with normalization to the maximum APD of each curve and the associated first derivative. (C) Data showing the effects of SNS on maximum slope of the APD restitution curve with and without normalization to account for differences in APD between conditions. Data represent mean ± SEM. Two-way ANOVA, with Tukey's post-hoc tests; ***p < 0.001. (n = 13 hearts).
Figure 6
Figure 6
Effects of IKs block on alternans during sympathetic nerve stimulation. (A) Representative recordings of optical action potentials in control conditions and following perfusion of HMR 1556 (2 μmol/L). (B) Data showing ventricular action potential duration (APD) in control conditions and during sympathetic nerve stimulation (SNS) with and without HMR 1556. (C) Relative change in APD from baseline before and after perfusion of HMR 1556. (D) Data showing the change in the alternans threshold with SNS with and without HMR 1556. (E) Relative change in the alternans threshold before and after perfusion of HMR 1556. Data taken from a 3 × 3 pixel region in the basal left ventricle. Data represent mean ± SEM. Two-way ANOVA, with Tukey's post-hoc tests; **p < 0.01, ***p < 0.001. Paired Student's t-test; **p < 0.01, ***p < 0.001. (n = 8 hearts).
Figure 7
Figure 7
Role of altered Ca handling in the action of sympathetic nerve stimulation on cardiac alternans (A) S1S2 (extra-stimulus) Ca transient amplitude restitution relationships in the presence and absence of noradrenaline (200 nmol/L). Inset are representative traces of a single extra-stimulus (S2) at a 130 ms cycle length in the absence and presence of noradrenaline. (B) Amplitude-normalized intracellular Ca transients recorded in control conditions and with cyclopiazonic acid (CPA, 10 μmol/L). (C,D) Data on the effects of CPA on action potential duration (APD) and the time to 50% decay of the intracellular Ca transient. (E,F) Data on the cycle length relationships for APD and Ca amplitude alternans. Data taken from a 3 × 3 pixel region in the basal left ventricle. Paired Student's t-test; **p < 0.01. Data are mean ± SEM. (n = 5–6 hearts).
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