Calcium‐ and voltage‐driven atrial alternans: Insight from [Ca]i and Vm asynchrony

Figures

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  FIGURE 1. Terminology and experimental protocols. (a) Simultaneous CaT and APD alternans measurement in current clamped rabbit atrial myocyte. APDN: APD recorded during the large amplitude alternans CaT; APDW: APD recorded during the small amplitude alternans CaT. (b) Average APDCaT_Small/APDW and average APDCaT_Large/APDN constructed from experiments as illustrated in (a). To construct the average AP, three consecutive APs recorded from three individual cells paced at 1.3 Hz and exhibiting CaT alternans were averaged. (c) Command voltages for four different AP alternans voltage clamp protocols to deliver extra beats. N: APDCaT_Large or APDN; W: APDCaT_Small or APDW. (d) AP alternans voltage clamp experiments result in either ‘in‐phase’ or ‘out‐of‐phase’ CaT alternans. “In‐phase” refers to the situation typically occurring in current clamped cells (i.e., when the voltage is not clamped and is controlled by the cell; cf. panel a). (e) CaT alternans in cell pairs. Concordant: small and large amplitude CaTs coincide in both cells; Discordant: small amplitude CaT in cell A coincides with large amplitude CaT in cell B and vice versa.

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  FIGURE 2. Synchronized APD and CaT alternans in single rabbit atrial myocyte. Simultaneous recording of APs (current clamp) and CaTs, showing initiation of pacing induced alternans. n = 40 observations of a total of 52 observations. Top: overlays of two consecutive APs recorded at time intervals marked by the gray bars. [Ca]i was measured with Indo‐1. AR, alternans ratio.

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  FIGURE 3. Simultaneous APD (current clamp) and CaT alternans recording in single rabbit atrial myocyte. (a) APD alternans onset precedes CaT alternans. n = 5 observations of a total of 52 observations. (b) CaT alternans onset precedes APD alternans. n = 7 observations of a total of 52 observations. Top in both panels: overlay of two consecutive APs recorded at time intervals marked by the gray bars. [Ca]i was measured with Fluo‐4. AR, alternans ratio.

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  FIGURE 4. Simultaneous APD (current clamp) and CaT alternans recording in single rabbit atrial myocyte. (a) Simultaneous termination of APD and CaT alternans. n = 4 observations of a total of 11 observations. (b) Dyssynchronous termination of APD and CaT alternans. APD alternans continues after cessation of CaT alternans. n = 7 observations of a total of 11 observations. (c) Prolonged episode of APD alternans in the absence of CaT alternans. Top in all panels: overlay of two consecutive APs recorded at time intervals marked by the gray bars. [Ca]i was measured with Indo‐1 (panels a and c) or Fluo‐4 (panel b). AR, alternans ratio.

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  FIGURE 5. CaT alternans in AP alternans voltage‐clamped atrial myocytes elicited with WWW‐ and NNN‐protocols. (a) WWW‐protocol applied to in‐phase CaT alternans failed to induce a phase shift. (b) NNN‐protocol applied to in‐phase CaT alternans failed to induce a phase shift. (c) WWW‐protocol applied to out‐of‐phase CaT alternans failed to induce a phase shift. (d) NNN‐protocol applied to in‐phase CaT alternans caused phase shift from in‐phase to out‐of‐phase alternans. i: in‐phase; o: out‐of‐phase. [Ca]i was measured with Fluo‐4. Number of recordings with WWW‐protocol: 70. Number of recordings with NNN‐protocol: 81.

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  FIGURE 6. CaT alternans in AP alternans voltage‐clamped atrial myocytes elicited with NN‐protocol. (a) NN‐protocol applied to in‐phase CaT alternans caused phase shift from in‐phase to out‐of‐phase alternans. (b) NN‐protocol applied to out‐of‐phase CaT alternans caused phase shift from out‐of‐phase to in‐phase alternans. (c) NN‐protocol applied to in‐phase CaT alternans failed to induce a phase shift. (d) Frequency distribution of effect of NN‐protocol on alternans phase. i: in‐phase; o: out‐of‐phase. [Ca]i was measured with Fluo‐4. Number of recordings with NN‐protocol: 72.

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  FIGURE 7. CaT alternans in AP alternans voltage‐clamped atrial myocytes elicited with WW‐protocol. (a) WW‐protocol applied to in‐phase CaT alternans failed to induce a phase shift. (b) WW‐protocol applied to out‐of‐phase CaT alternans caused phase shift from out‐of‐phase to in‐phase alternans. (c) WW‐protocol applied to in‐phase CaT alternans caused phase shift from in‐phase to out‐of‐phase alternans. (d) Frequency distribution of effect of WW‐protocol on alternans phase. i, in‐phase; o, out‐of‐phase. [Ca]i was measured with Fluo‐4. Number of recordings with WW‐protocol: 80.

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  FIGURE 8. CaT alternans in AP alternans voltage‐clamped atrial myocytes elicited with NN‐protocol. NN‐protocol applied to in‐phase CaT alternans caused a transient phase shift from in‐phase to out‐of‐phase, followed by cessation of CaT alternans. i: in‐phase; o: out‐of‐phase. [Ca]i was measured with Fluo‐4. n = 6 observations.

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  FIGURE 9. Simultaneous measurements of CaT and APD alternans in atrial myocyte pairs. V m and [Ca]i were recorded with fluorescence transverse line scan confocal imaging. V m probe: FluoVolt; Ca probe: Rhod‐2. (a) Confocal line scan (x‐t) [Ca]i (Rhod‐2 fluorescence) images. (b) [Ca]i and V m traces recorded from line scan images in panel (a). Signals were spatially averaged over the width of the cell. Highly synchronized concordant CaT and APD alternans were recorded. Synchronized spontaneous V m depolarization and Ca release during rest after pacing indicate electrical coupling between the two cells. Number of cell pairs with synchronized APD and CaT alternans: 40.

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  FIGURE 10. Simultaneous measurements of CaT and APD alternans in atrial myocyte pairs. V m (FluoVolt) and [Ca]i (Rhod‐2) were recorded with fluorescence transverse line scan confocal imaging. Cell A reveals the development of synchronized CaT and APD alternans. Cell B failed to develop alternans. Two synchronized spontaneous V m depolarizations and Ca release events during rest after pacing indicate electrical coupling between the two cells. Number of cell pairs showing dyssynchrony of APD and CaT alternans: 15.

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  FIGURE 11. Simultaneous measurements of CaT and APD alternans in atrial myocyte pairs. V m (FluoVolt) and [Ca]i (Rhod‐2) were recorded with fluorescence transverse line scan confocal imaging. CaT alternans develop autonomously in the absence of APD alternans at different times in the two cells and are discordant. Synchronized spontaneous V m depolarization and Ca release during rest after pacing indicate electrical coupling of the two cells. Number of cell pairs showing dyssynchrony of APD and CaT alternans: 15.

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  FIGURE 12. Simultaneous measurements of CaT and APD alternans in atrial myocyte pairs. V m (FluoVolt) and [Ca]i (Rhod‐2) were recorded with fluorescence transverse line scan confocal imaging. The two cells show concordant APD alternans; however, only cell B developed CaT alternans. Number of cell pairs showing dyssynchrony of APD and CaT alternans: 15.