In Vivo and In Silico Investigation Into Mechanisms of Frequency Dependence of Repolarization Alternans in Human Ventricular Cardiomyocytes

Abstract

Rationale: Repolarization alternans (RA) are associated with arrhythmogenesis. Animal studies have revealed potential mechanisms, but human-focused studies are needed. RA generation and frequency dependence may be determined by cell-to-cell variability in protein expression, which is regulated by genetic and external factors.

Objective: To characterize in vivo RA in human and to investigate in silico using human models, the ionic mechanisms underlying the frequency-dependent differences in RA behavior identified in vivo.

Methods and results: In vivo electrograms were acquired at 240 sites covering the epicardium of 41 patients at 6 cycle lengths (600-350 ms). In silico investigations were conducted using a population of biophysically detailed human models incorporating variability in protein expression and calibrated using in vivo recordings. Both in silico and in vivo, 2 types of RA were identified, with Fork- and Eye-type restitution curves, based on RA persistence or disappearance, respectively, at fast pacing rates. In silico simulations show that RA are strongly correlated with fluctuations in sarcoplasmic reticulum calcium, because of strong release and weak reuptake. Large L-type calcium current conductance is responsible for RA disappearance at fast frequencies in Eye-type (30% larger in Eye-type versus Fork-type; P<0.01), because of sarcoplasmic reticulum Ca(2+) ATPase pump potentiation caused by frequency-induced increase in intracellular calcium. Large Na(+)/Ca(2+) exchanger current is the main driver in translating Ca(2+) fluctuations into RA.

Conclusions: In human in vivo and in silico, 2 types of RA are identified, with RA persistence/disappearance as frequency increases. In silico, L-type calcium current and Na(+)/Ca(2+) exchanger current determine RA human cell-to-cell differences through intracellular and sarcoplasmic reticulum calcium regulation.

Keywords: calcium; calibration; electrophysiology; pericardium; sarcoplasmic reticulum.

Figure 1.

In vivo recordings of activation recovery interval (ARI) used for the calibration of the population of human ventricular models. A, ARI as an in vivo surrogate of action potential duration (APD). Red dots represent activation and depolarization times, whereas gray dots represent recovery and repolarization times. Adapted from Potse et al with permission of the publisher. Copyright ©2009, the American Physiological Society. B, Rate dependence and variability in in vivo ARIs aggregated from the different patients as a function of decreasing pacing cycle length (CL). C, Unipolar electrograms and corresponding sequence of ARIs from an alternans-susceptible site. D, Unipolar electrograms and corresponding sequence of ARIs from an alternans-resistant site. Dots and crosses on the electrograms represent activation and recovery times, respectively.

Figure 2.

Population of human ventricular models calibrated with in vivo recordings. A, Action potentials of accepted and rejected models for a cycle length (CL) of 600 ms. B, Partial correlation coefficients (PCC) between action potential biomarkers and current conductance parameters. C, Action potential biomarkers for normal and alternans models for a CL of 350 ms. Biomarker values have been normalized against maximum values in all accepted models. D, Distribution of ionic conductances scaling factors for normal and alternans models with respect to their original value in the ±100% range (0–2). Symbols indicate statistical significance levels (*P<0.05, **P<0.01, ***P<0.001). APA indicates action potential amplitude; APD, action potential duration; CaTD, calcium transient duration; CaT_max, systolic Ca²⁺ level; CaT_min, diastolic Ca²⁺ level; G_CaL, Ca²⁺ channel permeability; G_K1, K1 channel conductance; G_Kr, Kr channel conductance; G_Ks, Ks channel conductance; G_Na, fast Na⁺ channel conductance; G_NaCa, Na⁺/Ca²⁺ exchanger conductance; G_NaK, Na⁺/K⁺ pump activity; G_NaL, late Na⁺ channel conductance; G_to, transient outward potassium channel conductance; ORd, O’Hara–Rudy dynamic model; P_Jrel, Ca²⁺ release permeability via ryanodine receptor to cytoplasm; P_Jup, Ca²⁺ uptake permeability via sarcoplasmic reticulum Ca²⁺ ATPase pump from the cytoplasm; RMP, resting membrane potential; UPD, upstroke duration; and V_max, peak upstroke voltage.

Figure 3.

Types of action potential duration (APD) alternans in vivo and in silico. A, Representative restitution curves of APD (activation recovery interval [ARI]) vs cycle length (CL) exhibiting Eye-type and Fork-type alternans in vivo (top) and in silico (bottom) data. B, Representative restitution curves of APD (ARI) vs diastolic interval (DI) exhibiting normal condition and Eye-type and Fork-type alternans in vivo (top) and in silico (bottom) data.

Figure 4.

SR Ca²⁺ cycling properties and fluctuations in alternans models. A and B, Comparison of ryanodine receptor release (P_Jrel, A) and sarcoplasmic reticulum Ca²⁺ ATPase pump uptake (P_Jup, B) in normal, Eye-type, and Fork-type alternans models. The vertical axis shows parameters scaling. Symbols indicate statistical significance levels (*P<0.05, **P<0.01, ***P<0.001). C, Sarcoplasmic reticulum Ca²⁺ balance (SRCB) magnitudes for Eye-type and Fork-type alternans under all considered cycle lengths (CLs).

Figure 5.

Sarcolemmal Ca²⁺ balance (SCB) in alternans models. A and B, Comparison of L-type Ca²⁺ channel conductance (G_CaL, A) and Na⁺/Ca²⁺ exchanger conductance (G_NaCa, B) in normal, Eye-type, and Fork-type alternans models. C, Sarcolemmal calcium balance (SCB) magnitudes for Eye-type and Fork-type alternans under all considered cycle lengths (CLs).

Figure 6.

Ionic mechanisms resulting in action potential duration (APD) alternans in human in silico ventricular cardiomyocytes. A, From top to bottom, transmembrane potential (Vm), Ca²⁺ concentration in JSR (Ca_JSR), Ca²⁺ release via ryanodine receptor (RyR; J_rel), Ca²⁺ reuptake via sarcoplasmic reticulum Ca²⁺ ATPase pump (SERCA; J_up), intracellular Ca²⁺ transient (CaT), L-type Ca²⁺ current (I_CaL), and Na⁺/Ca²⁺ exchanger current (I_NaCa) in a representative Eye-type model before the generation of alternans (cycle length [CL]=600 ms, left), during alternans (CL=500 ms, middle) and alternans disappearance (CL=350 ms, right). B, Schematic diagram illustrating the network of events explaining APD alternans for long and short beats: In (1), larger/smaller Ca_JSR levels at the start of the beat, results in larger/smaller J_rel and intracellular Ca²⁺ levels (2), leading to (3) increase/decrease in inward current through the I_NaCa forward mode, and a smaller decrease/increase in inward current through I_CaL calcium-induced inactivation. NSR indicates network SR; and PLB, phospholamban.

Figure 7.

Effects of varying L-type calcium current (I_CaL) kinetics on the Eye-type and Fork-type models with biggest alternans magnitudes. Correlation between action potential duration (APD) alternans magnitudes and sarcolemmal calcium balance (SCB) magnitudes in the Eye-type model with biggest alternans (A) and the Fork-type model with biggest alternans (B). Correlation between sarcoplasmic reticulum calcium balance (SRCB) magnitudes and APD alternans magnitudes in the biggest Eye -type model (C) and the biggest Fork-type model (D). Stars, squares, and diamonds represent the variation of I_CaL activation (τd), inactivation (τf), and recovery from Ca²⁺-dependent inactivation (τj) time constants, respectively. Colors represent changes in time constants magnitude. The red circle with a square inside represents the original kinetics.

Figure 8.

Suppression of action potential duration (APD) alternans by Na⁺/Ca²⁺ exchanger current (I_NaCa) inhibition. A, Percentage of different types of alternans models under 20%, 40%, and 60% I_NaCa block. B and C, Effects of I_NaCa suppression on regulating the sarcolemmal calcium balance (SCB) and sarcoplasmic reticulum calcium balance (SRCB), respectively.