Spatially Discordant Alternans and Arrhythmias in Tachypacing-Induced Cardiac Myopathy in Transgenic LQT1 Rabbits: The Importance of I_Ks and Ca²⁺ Cycling

<div><p>Background</p><p>Remodeling of cardiac repolarizing currents, such as the downregulation of slowly activating K⁺ channels (I_Ks), could underlie ventricular fibrillation (VF) in heart failure (HF). We evaluated the role of <i>I</i>_ks remodeling in VF susceptibility using a tachypacing HF model of transgenic rabbits with Long QT Type 1 (LQT1) syndrome.</p><p>Methods and Results</p><p>LQT1 and littermate control (LMC) rabbits underwent three weeks of tachypacing to induce cardiac myopathy (TICM). <i>In vivo</i> telemetry demonstrated steepening of the QT/RR slope in LQT1 with TICM (LQT1-TICM; pre: 0.26±0.04, post: 0.52±0.01, P<0.05). <i>In vivo</i> electrophysiology showed that LQT1-TICM had higher incidence of VF than LMC-TICM (6 of 11 vs. 3 of 11, respectively). Optical mapping revealed larger APD dispersion (16±4 vs. 38±6 ms, p<0.05) and steep APD restitution in LQT1-TICM compared to LQT1-sham (0.53±0.12 vs. 1.17±0.13, p<0.05). LQT1-TICM developed spatially discordant alternans (DA), which caused conduction block and higher-frequency VF (15±1 Hz in LQT1-TICM vs. 13±1 Hz in LMC-TICM, p<0.05). Ca²⁺ DA was highly dynamic and preceded voltage DA in LQT1-TICM. Ryanodine abolished DA in 5 out of 8 LQT1-TICM rabbits, demonstrating the importance of Ca²⁺ in complex DA formation. Computer simulations suggested that HF remodeling caused Ca²⁺-driven alternans, which was further potentiated in LQT1-TICM due to the lack of I_Ks.</p><p>Conclusions</p><p>Compared with LMC-TICM, LQT1-TICM rabbits exhibit steepened APD restitution and complex DA modulated by Ca²⁺. Our results strongly support the contention that the downregulation of I_Ks in HF increases Ca²⁺ dependent alternans and thereby the risk of VF.</p></div

TICM Protocol.

<p>(<b>A</b>) Three-week pacing protocol. TSM/PPM = transmitter/ pacemaker, PES = programmed electrical stimulation. <b>(B</b>) TICM groups show dilated left ventricle and reduced ejection fraction. The red bars indicate end-systolic and end-diastolic LV internal dimensions (8 and 11 mm for LMC sham pacing and 12 and 15 mm for TICM). <b>(C)</b> Post-pacing protocol LV function presented as left ventricular ejection fraction in LMC-TICM, LQT1-TICM, and their sham controls. TICM rabbits show statistically significant differences in LV ejection fraction compared with sham; *P<0.05. <b>(D)</b> Quantification of I_Ks in LMC rabbits. Current amplitude was normalized to cell capacitance. Compared with LMC-SH-P (n = 12), significant downregulation of I_Ks was seen in LMC-TICM (n = 11).</p

Free-Moving Telemetry: QT-RR Ratios.

<p>(<b>A-D</b>): QT/RR relationship in awake, free-moving rabbits, recorded approximately every 20 minutes for 24 hours in each group. Lines indicate linear regression derived from the mean of all individual regression lines per genotype. Note that LQT1-TICM demonstrates steeper QT/RR slope post-pacing compared to pre-pacing. (<b>E</b>): VERP at BCL = 240 ms in LMC-TICM, LQT1-TICM, and sham animals. No significant difference was found among the four groups (<b>F</b>): VERP (BCL = 240 ms) vs. VF frequency in 5 LMC-TICM and LQT1-TICM animals. Unlike LMC-TICM, LQT1-TICM did not show a correlation between VERP and VF frequency.</p

<i>In vivo</i> VF Inducibility using Pen protocol (S1S2S3S4).

<p>P<0.05 LQT1-TICM vs. LQT1-SH-P; all other comparisons, P = NS.</p><p><i>In vivo</i> VF Inducibility using Pen protocol (S1S2S3S4).</p

Computer simulations of APD and Ca2+ alternans under different conditions.

<p><b>(A</b>) V_m (upper) and whole-cell Ca²⁺ concentration (lower) versus time for the control condition at CL = 280 ms. (<b>B</b>) V_m and whole-cell Ca²⁺ concentration versus time for the HF condition. (<b>C</b>) V_m and whole-cell Ca²⁺ concentration versus time for the same condition as in B but with zero I_Ks. (<b>D</b>) Peak whole-cell Ca²⁺ concentrations of two consecutive beats versus CL for the three conditions. (<b>E</b>) APD of the same two consecutive beats as in D versus CL for the three conditions.</p

Prevalent discordant alternans in LQT1-TICM.

<p>(<b>A</b>) Sample traces of DA from three locations. (<b>B</b>) Space-time plot of DA along the line a-c in panel A. (<b>C</b>) APD (black) and Ca²⁺ duration (red) along the line a-c. Alternation between odd and even beats was larger in Ca²⁺. (<b>D</b>) Maps of activation and nodal lines of DA. Note that activation in odd beats shows markedly slowing conduction toward the apex region. However, the nodal lines were not associated with the alternating activation pattern.</p

APD dispersion and restitution in TICM rabbits.

<p>(<b>A&B</b>): Typical raw data of action potential traces from optical mapping. LQT1-SH-P shows 2:1 block at 240 ms CL, while LQT1-TICM was paced as low as 190 ms CL with marked shortening of APD. (<b>C</b>) Mean APD in each group at basic cycle length of 350 ms. LQT1 rabbits show statistically significant differences in APD compared with LMC; *P<0.05. (<b>D</b>) APD dispersion increased in LQT1-TICM. (<b>E</b>) APD restitution curves from four groups. (<b>F</b>): Maximum slopes of the APD restitution curves. LQT1-TICM demonstrates increase in APD restitution slopes (*P<0.05).</p

VF induction and VF frequency in LQT1-TICM.

<p>(<b>A</b>) Sample traces of V_m and Ca²⁺ from 190 ms during VF induction. DA was prominent before VF. (<b>B</b>) Activation maps of VF induction. Conduction block occurred near the nodal line and formed a rotating wave (grey arrow). (<b>C</b>) VF frequency. VF frequency was higher in LQT1-TICM despite the prolongation of APD at basic CL (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122754#pone.0122754.g003" target="_blank">Fig 3C</a> (<b>D</b>) Correlation between APD and VF frequency. The baseline APD is no longer a predictor of VF frequency as in <i>in vivo</i> EPS in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122754#pone.0122754.g002" target="_blank">Fig 2F</a>.</p