Mechanism of carvedilol induced action potential and calcium alternans

Figures

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  Figure 1.. Carvedilol increases CaT alternans susceptibility. A, Representative traces of CaT alternans recorded in control and in presence of carvedilol at 0.5, 0.75 and 1 µM. B, CaT alternans in control and in presence of metoprolol (10 µM) as negative control. C, Mean CaT alternans ratio (AR) from control and carvedilol (0.5 µM: P = 0.31, n = 5; 0.75 µM: * P = 0.039, n = 11; 1 µM: ** P = 0.001, n = 14) or metoprolol (Met; 10 µM; P = 0.41, n = 7) treated atrial myocytes. All experiments are paired measurements of control and drug effect measured in the same cell and statistical analysis (paired t-test) was done against concentration specific controls. For presentation purpose only, all controls were pooled together. D, Representative CaT traces recorded in control and in presence of carvedilol (1 µM) at 1 Hz pacing rate. E, Mean CaT alternans ratio (AR) in control and 1 µM carvedilol (1 Hz; P = 0.008, n = 11). F, Carvedilol effect on SR Ca load was quantified as the amplitude of the CaT elicited by caffeine (10 mM) in control (top) and in the presence of 0.75 µM carvedilol (bottom). G, Average CaT amplitudes in response to caffeine (n = 7).

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  Figure 2.. Carvedilol prolongs the refractoriness of the SR calcium release. A, Double pulse (S1-S2) protocol used to record Ca currents (ICa) and CaT simultaneously in atrial myocytes. Series of S1-S2 stimulations were applied with progressively decreased diastolic intervals (DI) from 1,000 to 2 ms. B, Representative traces of ICa (top) and corresponding [Ca]i profiles (bottom) in control. C, Representative traces of ICa (top) and corresponding [Ca]i profiles (bottom) in presence of carvedilol (0.75 µM). D, ICa recovery from inactivation (RFI) curves in control (gray symbols) and in presence of carvedilol (open symbols). Dashed lines indicate time constant τ of ICa RFI kinetics derived from mono-exponential fit. Inset: average τ of ICa RFI (P = 0.091, n = 6, paired t-test). E, CaT and SR Ca release refractoriness in control (gray symbols) and carvedilol (open symbols). Inset: average time constant τ (mono-exponential fit) of CaT RFI (P = 0.024, n = 6, paired t-test).

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  Figure 3.. Carvedilol induces APD alternans. A, Representative AP traces recorded in control and in presence of carvedilol (0.75 µM) at 1 Hz. B, Overlay of 2 consecutive APs in control and carvedilol treated cells (marked by box in panel A). APs were recorded in current-clamp mode and evoked by 5 ms stimulation pulses of a magnitude ~1.5 times higher than AP activation threshold. C, APD of a pair of consecutive APs were classified as long (l) and short (s) based on APD50 analysis. Comparison of control and carvedilol mean APD50 of long (L: P = 0.19; n = 11) and short (S: P = 0.24) APs. D, ARAPD50 in control and after carvedilol (P = 0.096). E, Representative AP traces recorded in control and in presence of carvedilol (0.75 µM) at 2 Hz. F, Overlay of 2 consecutive APs in control and carvedilol treated cells (marked by box in panel E). G, Comparison of control and carvedilol mean APD50 of long (L: P = 0.12; n = 9) and short (S: P = 0.037) APs. H, Average ARAPD50 in control and after carvedilol treatment (P = 0.034). Statistical analysis with paired t-test.

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  Figure 4.. Carvedilol prolongs AP restitution and steepens APD restitution curve. A, AP restitution was investigated in current-clamp mode using a double pulse protocol (S1-S2). DI ranged from 250 to 1,250 ms. B, Representative AP traces recorded in control conditions at DIs of 1,000 ms, 500 ms and 300 ms. C, Representative AP traces recorded in presence of carvedilol (0.75 µM) at DIs of 1,000 ms, 500 ms and 300 ms. D, AP restitution curves. Mean APD50 S2/S1 ratios at DIs between 250 and 1,250 ms in control (gray symbols) and in presence of carvedilol (open symbols). Dashed lines indicate time constant τ derived from mono-exponential fit of the restitution curves. E, average τ of the restitution curves in control and after carvedilol treatment (P = 0.023, n = 9, paired t-test).

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  Figure 5.. Carvedilol increases APD50 and CaT alternans ratios. Simultaneous AP and [Ca]i recordings in control (A) and in presence of 0.75 µM carvedilol (B) using the fluorescent probes FluoVolt and Rhod-2. Vm and [Ca]i signals were acquired in confocal transverse line scan mode. AP and CaT were elicited by field stimulation at 2 Hz. C, Mean APDs at 50% repolarization (APD50) of long (L) and short (S) APs (panel a) and ARAPD50 (panel b) in control and in presence of carvedilol (n = 19). Statistical analysis with unpaired t-test. D, mean amplitudes of small (S) and large (L) alternans CaTs (panel a) and average CaT ARs (panel b) in control and in presence of carvedilol. E, Correlation between ARAPD50 and CaT AR in control (a) and carvedilol (b) treated cells. r, correlation coefficient. F, distribution of ARAPD50 (panel a) and CaT AR (panel b) in control (gray bars) and in presence of carvedilol (open bars).

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  Figure 6.. Metoprolol does not modify APD or CaT alternans. Vm and [Ca]i traces from atrial myocytes in control (A) and during metoprolol (10 µM) treatment (B). APs and CaTs were elicited by field stimulation at 2 Hz. C, Mean APD50 of long (L; control vs. metoprolol: P = 0.35, n = 11) and short (S; P = 0.21) APs during alternans (panel a) and average ARAPD50 (panel b) in control and metoprolol (Met; P = 0.37, n = 11). D, average CaT amplitudes of large (L: control vs. metoprolol, P = 0.43) and small (S; P = 0.29) amplitude alternans CaTs (panel a) and average CaT AR (panel b) in control and metoprolol (P = 0.41). Statistical analysis with unpaired t-test.