Membrane potential determines calcium alternans through modulation of SR Ca2+ load and L-type Ca2+ current - PubMed (original) (raw)

Membrane potential determines calcium alternans through modulation of SR Ca2+ load and L-type Ca2+ current

Giedrius Kanaporis et al. J Mol Cell Cardiol. 2017 Apr.

Abstract

Alternans is a risk factor for cardiac arrhythmia, including atrial fibrillation. At the cellular level alternans is observed as beat-to-beat alternations in contraction, action potential (AP) morphology and magnitude of the Ca2+ transient (CaT). It is widely accepted that the bi-directional interplay between membrane voltage and Ca2+ is crucial for the development of alternans, however recently the attention has shifted to instabilities in cellular Ca2+ handling, while the role of AP alternation remains poorly understood. This study provides new insights how beat- to-beat alternation in AP morphology affects occurrence of CaT alternans in atrial myocytes. Pacing-induced AP and CaT alternans were studied in rabbit atrial myocytes using combined Ca2+ imaging and electrophysiological measurements. To determine the role of AP morphology for the generation of CaT alternans, trains of two voltage commands in form of APs recorded during large and small alternans CaTs were applied to voltage-clamped cells. APs of longer duration (as observed during small amplitude alternans CaT) and especially beat-to-beat alternations in AP morphology (AP alternans) reduced the pacing frequency threshold and increased the degree of CaT alternans. AP morphology contributes to the development of CaT alternans by two mechanisms. First, the AP waveform observed during small alternans CaTs coincided with higher end-diastolic sarcoplasmic reticulum Ca2+ levels ([Ca2+]SR), and AP alternans resulted in beat-to-beat alternations in end-diastolic [Ca2+]SR. Second, L-type Ca2+ current was significantly affected by AP morphology, where the AP waveform observed during large CaT elicited L-type Ca2+ currents of higher magnitude and faster kinetics, resulting in more efficient triggering of SR Ca2+ release. In conclusion, alternation in AP morphology plays a significant role in the development and stabilization of atrial alternans. The demonstration that CaT alternans can be controlled or even prevented by modulating AP morphology has important ramifications for arrhythmia prevention and therapy strategies.

Keywords: Action potential; Alternans; Arrhythmia; Ca(2+) signaling; Excitation-contraction coupling.

PubMed Disclaimer

Figures

Fig. 1. Simultaneous CaT and AP alternans recording

A, Simultaneous recordings of APs and [Ca2+]i from a single atrial myocyte under current clamp conditions and loaded with Indo-1. B, Typical AP waveforms recorded during large (APCaT_Large) and small (APCaT_Small) alternans CaTs used as voltage commands to pace cells during AP voltage clamp experiments.

Fig. 2. Effect of AP morphology on CaTs properties

A, CaTs (top) elicited with same-shape APCaT_Large, same-shape APCaT_Small and alternans AP-clamp protocols (bottom) recorded from the same atrial myocyte at 1.19 Hz stimulation frequency. B, overlays of CaTs elicited with APCaT_Large and APCaT_Small voltage commands during same-shape and alternans AP clamp protocols. Traces represent average of three consecutive CaTs recorded from the same cell as in panel A. C, Summary data for amplitudes and time to 80% of peak of CaTs recorded during same-shape and alternans AP voltage clamp protocols. Data are normalized to CaTs elicited with same-shape APCaT_Large pacing protocol (n=17). *p<0.01, **p<0.005, ***p<0.0005

Fig. 3. AP waveforms modulate severity of CaT alternans

A, CaTs elicited with same-shape APCaT_Large, same-shape APCaT_Small and alternans AP clamp protocols recorded from the same atrial myocyte at different pacing frequencies. B, Pacing with same-shape APCaT_Small waveforms (open circles, n=30) enhances degree of CaT alternans compared to APCaT_Large stimuli (black squares, n=28). CaT alternans ratio (AR) is further increased during the alternans AP voltage clamp protocol (grey triangles, n=18). Dashed line indicates definition of CaT alternans (AR>0.1).

Fig. 4. In-phase and out-of-phase alternans during AP voltage clamp protocols

Same-shape AP voltage clamp protocols were followed by alternans AP voltage protocol. A, In-phase alternans. Aa, Example of sequence of same shape APCaT_Small voltage commands followed by alternans AP clamp protocol (bottom). CaT alternans are in-phase at the switch to the alternans AP clamp protocol and remain in-phase (top). Ab, average CaT ARs observed during stimulation with same-shape AP voltage commands (left: APCaT_Large; right: APCaT_Small) followed by alternans AP clamp protocol. Data refer only to the cells that are in-phase at the transition to the alternans protocol. B, Out-of-phase alternans. Ba, Examples of CaT alternans that are out-of-phase at the switch to the alternans AP clamp protocol, which leads either to a phase shift to in-phase (top) or remains out-of-phase (middle). Bottom: voltage clamp protocol. Bb, in the majority of cases a phase shift to in-phase occurred when the cell entered the alternans protocol out-of-phase, irrespective whether the preceding same-shape protocol consisted of APCaT_Large (circles) or APCaT_Small (triangles). The ability to change phase was determined by the degree of CaT alternans. Cells exhibiting CaT ARs lower than ~0.6 during the same-shape AP protocols were capable to reverse CaT alternans phase, while cells with higher CaT AR were not likely to change phase. Bc, Change of phase during alternans AP protocol resulted in increased AR. When CaTs alternans remained out-of-phase to AP alternans, no change or a decrease in AR was observed. Data is obtained from a total of 17 myocytes. *p<0.05, **p<0.01, ***p<0.0005.

Fig. 5. AP morphology affects SR Ca2+ load

A, [Ca2+]SR measurements with Fluo-5N from voltage clamped myocytes. The same myocyte was exposed to three different AP clamp protocols. End-diastolic [Ca2+]SR was higher during the same-shape APCaT_Small protocol compared to APCaT_Large and revealed [Ca2+]SR alternans during the alternans AP clamp protocol. B, Simultaneous membrane current (top) and [Ca2+]i (middle) recording from an AP voltage clamped myocyte using the same-shape APCaT_Large protocol (bottom), followed by rapid application of caffeine (10 mM). C, SR Ca2+ load estimated from the integral of the membrane current induced by caffeine and representing extrusion of Ca2+ released from the SR via NCX. Caffeine was applied after pacing with same-shape APCaT_Large (n=7), same-shape APCaT_Small (n=7) and alternans AP voltage clamp (n=6) protocols. D, SR Ca2+ load estimated from the normalized amplitudes of the cytosolic CaTs elicited with caffeine using the same protocols as in panel C. SR Ca2+ load was consistently higher after APCaT_Small. *p<0.05

Fig. 6. APCaT_Large and APCaT_Small waveforms differ in CaT triggering efficiency

Aa, In the absence of CaT alternans, when the same-shape APCaT_Large protocol is followed by the same-shape APCaT_Small protocol the CaT transient elicited by the first APCaT_Small stimulus (indicated by the arrow) exhibits a decreased amplitude. Ab, Summary data of CaT amplitudes during initial stimulation with same-shape APCaT_Large protocol and the first CaTs after the switch of protocol to APCaT_Small (n=17). Data are normalized to the CaT amplitudes observed during the initial stimulation with same-shape APCaT_Large. Ba, When same-shape APCaT_Small protocol is followed by the same-shape APCaT_Large protocol, the first APCaT_Large command elicits CaT of larger amplitude (indicated by the arrow). Bb, Summary data of CaT amplitudes during initial stimulation with same-shape APCaT_Small protocol and the first CaT elicited with APCaT_Large (n=16). Data are normalized to the CaT amplitudes during same-shape APCaT_Small protocol. *p<0.05, **p<0.0001

Fig. 7. AP alternans determines magnitude and kinetics of L-type Ca2+ current

A, Representative traces of ILCC elicited with APCaT_Large and APCaT_Small voltage commands from the same atrial myocyte paced at 0.7 Hz (no CaT alternans). B, Summary data of ILCC kinetics: duration of ILCC measured at 80% inactivation of peak current (Ba; n=6), and (Bb) average peak ILCC and ILCC time integrals elicited with APCaT_Large and APCaT_Small waveforms and normalized to APCaT_Large (n=6). *p<0.05, **p<0.0001

References

1. Comtois P, Nattel S. Atrial repolarization alternans as a path to atrial fibrillation. J Cardiovasc Electrophysiol. 2012;23:1013–5. -PubMed
1. Hiromoto K, Shimizu H, Furukawa Y, Kanemori T, Mine T, Masuyama T, et al. Discordant repolarization alternans-induced atrial fibrillation is suppressed by verapamil. Circ J. 2005;69:1368–73. -PubMed
1. Narayan SM, Bode F, Karasik PL, Franz MR. Alternans of atrial action potentials during atrial flutter as a precursor to atrial fibrillation. Circulation. 2002;106:1968–73. -PubMed
1. Jousset F, Tenkorang J, Vesin JM, Pascale P, Ruchat P, Rollin AG, et al. Kinetics of atrial repolarization alternans in a free-behaving ovine model. J Cardiovasc Electrophysiol. 2012;23:1003–12. -PMC -PubMed
1. Walker ML, Rosenbaum DS. Repolarization alternans: implications for the mechanism and prevention of sudden cardiac death. Cardiovasc Res. 2003;57:599–614. -PubMed

Membrane potential determines calcium alternans through modulation of SR Ca2+ load and L-type Ca2+ current - PubMed (original) (raw)