Differential conditions for early after-depolarizations and triggered activity in cardiomyocytes derived from transgenic LQT1 and LQT2 rabbits

Abstract

Early after-depolarization (EAD), or abnormal depolarization during the plateau phase of action potentials, is a hallmark of long-QT syndrome (LQTS). More than 13 genes have been identified as responsible for LQTS, and elevated risks for EADs may depend on genotypes, such as exercise in LQT1 vs. sudden arousal in LQT2 patients. We investigated mechanisms underlying different high-risk conditions that trigger EADs using transgenic rabbit models of LQT1 and LQT2, which lack I(Ks) and I(Kr) (slow and fast components of delayed rectifying K(+) current), respectively. Single-cell patch-clamp studies show that prolongation of action potential duration (APD) can be further enhanced by lowering extracellular potassium concentration ([K(+)](o)) from 5.4 to 3.6 mm. However, only LQT2 myocytes developed spontaneous EADs following perfusion with lower [K(+)](o), while there was no EAD formation in littermate control (LMC) or LQT1 myocytes, although APDs were also prolonged in LMC myocytes and LQT1 myocytes. Isoprenaline (ISO) prolonged APDs and triggered EADs in LQT1 myocytes in the presence of lower [K(+)](o). In contrast, continuous ISO perfusion diminished APD prolongation and reduced the incidence of EADs in LQT2 myocytes. These different effects of ISO on LQT1 and LQT2 were verified by optical mapping of the whole heart, suggesting that ISO-induced EADs are genotype specific. Further voltage-clamp studies revealed that ISO increases L-type calcium current (I(Ca)) faster than I(Ks) (time constant 9.2 s for I(Ca) and 43.6 s for I(Ks)), and computer simulation demonstrated a high-risk window of EADs in LQT2 during ISO perfusion owing to mismatch in the time courses of I(Ca) and I(Ks), which may explain why a sympathetic surge rather than high sympathetic tone can be an effective trigger of EADs in LQT2 perfused hearts. In summary, EAD formation is genotype specific, such that EADs can be elicited in LQT2 myocytes simply by lowering [K(+)](o), while LQT1 myocytes require sympathetic stimulation. Slower activation of I(Ks) than of I(Ca) by ISO may explain why different sympathetic modes, i.e. sympathetic surge vs. high sympathetic tone, are associated with polymorphic ventricular tachycardia in LQTS patients.

Figure 5. Isoprenaline (100 nm) boosted both I_Ca and I_Ks in a time-dependent manner

A, the voltage protocol used to record I_Ca and I_Ks at the same time. Holding potential is −50 mV, voltage first jump to −10 mV for 200 ms to record peak Ca²⁺ current, then further jump to +10 mV for 2 s to record slow activated I_Ks. Tail current of I_Ks could be best observed at −30 mV. The pulse was repeated every 6 s following the ISO perfusion. Recordings were done in the presence of 5 μm E-4031 to eliminate I_Kr. B, the current is shown as a function of time. The I_Ca was measured as peak current at −10 mV. Steady-state I_Ks was measured at the end of the 10 mV testing potential. They could best be fitted with one exponential function. The averaged time constant for I_Ca is 9.2 ± 1.15 s (n = 6) and for I_Kr 43.6 ± 9.82 s (n = 6).

Figure 1. Effect of [K⁺]_o on action potential and EAD formation

Aa, dependence of action potential and resting membrane potential on [K⁺]_o in a single ventricular myocyte from a littermate control animal (LMC). Ab, early after-depolarization (EAD) occurred when [K⁺]_o was switched from 5.4 to 3.6 mm in a single ventricular myocyte from an LQT2 rabbit. B, seven consecutive action potentials in LMC (Ba), LQT1 (Bb) and LQT2 myocytes (Bc, top trace) in 3.6 mm[K⁺]_o; Bc, bottom trace, membrane potential oscillation secondary to EAD in an LQT2 myocyte.

Figure 2. Relative contributions of I_Kr and I_Ks to action potential repolarization

A, L-type Ca²⁺ current (I_Ca) and its peak current I–V curve. Aa, original currents of L-type Ca²⁺ current in an LMC myocyte. Holding is at −50 mV, test potential is from −40 to +40 mV, pulses are 250 ms, and pulse intervals are 2 s. Ab, I–V curve of peak I_Ca in LMC, LQT1 and LQT2 myocytes. B, I_Kr and I_Ks isolated from LMC myocytes using an action potential stimulation protocol. I_Kr or I_Ks was defined as a current sensitive to 5 μm E-4031 or 30 μm chromanol 293B. The stimulation action potential pulse was acquired from an LMC myocyte in 3.6 mm[K⁺]_o; the action potential duration at 90% repolarization was around 500 ms. C, an example of the action potentials (top panels) and derivative of the membrane potential (bottom panels). The action potentials were from the sixth traces of each group in Figure 1B. The derivative of the membrane potential is the rate of membrane change (ΔV/Δt) and was also proportional to transmembrane current.

Figure 3. Effect of isoprenaline (ISO; 50 nm) on action potential duration (APD) and EAD formation

Experiments were done with 3.6 mm[K⁺]_o. A, ISO increased APD in LMC and LQT1 myocytes and shortened APD in LQT2 myocytes. B, ISO prolonged APD in an LMC myocyte. C, ISO prolonged APD and induced EAD in an LQT1 myocyte. D, ISO shortened APD in an LQT2 myocyte. E, spontaneous membrane potential oscillation (Eb) secondary to EAD (Ea) in an LQT1 myocyte.

Figure 4. Response to adrenergic stimulation in Langendorff-perfused hearts

A, optical signal examples of action potentials from LMC, LQT1 and LQT2 hearts at baseline and over the initial 10 s exposure to isoprenaline (100 nm). The atrioventricular node was ablated in all hearts to maintain the slow heart rate during isoprenaline exposure. In LQT1, there was continuous prolongation of the action potential and persistent EADs, while LQT2 shows initial EADs, with subsequent shortening of the action potential at 5 and 10 s. B, change in action potential at 10 s of isoprenaline exposure from baseline. Action potential prolongation is seen in LQT1, while action potential shortening is seen in LQT2.

Figure 6. Computer simulation of transient EAD genesis due to isoprenaline in an LQT2 myocyte

A, the maximal conductances of I_Ca and I_Ksversus time to mimic the effects of isoprenaline. A negative sign was added to the I_Ca conductance to agree with Fig. 5B. B, predicated action potentials at different time points as indicated by the letters in A.