Alternans in genetically modified langendorff-perfused murine hearts modeling catecholaminergic polymorphic ventricular tachycardia

Abstract

The relationship between alternans and arrhythmogenicity was studied in genetically modified murine hearts modeling catecholaminergic polymorphic ventricular tachycardia (CPVT) during Langendorff perfusion, before and after treatment with catecholamines and a β-adrenergic antagonist. Heterozygous (RyR2(p/s)) and homozygous (RyR2(s/s)) RyR2-P2328S hearts, and wild-type (WT) controls, were studied before and after treatment with epinephrine (100 nM and 1 μM) and propranolol (100 nM). Monophasic action potential recordings demonstrated significantly greater incidences of arrhythmia in RyR2(p/s) and RyR2(s/s) hearts as compared to WTs. Arrhythmogenicity in RyR2(s/s) hearts was associated with alternans, particularly at short baseline cycle lengths. Both phenomena were significantly accentuated by treatment with epinephrine and significantly diminished by treatment with propranolol, in full agreement with clinical expectations. These changes took place, however, despite an absence of changes in mean action potential durations, ventricular effective refractory periods or restitution curve characteristics. Furthermore pooled data from all hearts in which arrhythmia occurred demonstrated significantly greater alternans magnitudes, but similar restitution curve slopes, to hearts that did not demonstrate arrhythmia. These findings thus further validate the RyR2-P2328S murine heart as a model for human CPVT, confirming an alternans phenotype in common with murine genetic models of the Brugada syndrome and the congenital long-QT syndrome type 3. In contrast to these latter similarities, however, this report demonstrates the dissociation of alternans from changes in the properties of restitution curves for the first time in a murine model of a human arrhythmic syndrome.

Keywords: Brugada syndrome; alternans; arrhythmia; catecholaminergic polymorphic ventricular tachycardia; monophasic action potential; restitution curve; ryanodine receptor; sudden cardiac death.

Figure 1

Incidences of arrhythmia in WT, RyR2^p/s and RyR2^p/s hearts. Sixteen slices were acquired in an interleaved order. Incidences of ventricular arrhythmia occurring in WT (A), RyR2^p/s (B) and RyR2^p/s (C) hearts treated with control solution (a) and with solutions containing 100 nM epinephrine (b) and 1 μM epinephrine (c). The top panel shows traces obtained in the absence of propranolol; the bottom panel shows data obtained in the presence of 100 nM propranolol. Asterisks in the top panel indicate situations in which incidence of arrhythmia was significantly (P < 0.05) higher than in WT controls; brackets indicate significant (P < 0.05) differences in incidence of arrhythmia between other sets of conditions.

Figure 2

Arrhythmia in WT, RyR2^p/s and RyR2^p/s hearts. Monophasic action potential recordings obtained from WT (A), RyR2^p/s (B) and RyR2^p/s (C) hearts treated with control solution (a) and with solutions containing 100 nM epinephrine (b) and 1 μM epinephrine (c). The top panel shows traces obtained in the absence of propranolol; the bottom panel shows traces obtained in the presence of 100 nM propranolol. Traces show arrhythmia where this was observed significantly (P < 0.05) more frequently than in WT controls (see Figure 1). Vertical lines indicate the timing of stimuli.

Figure 3

Relationships between alternans magnitude and baseline cycle length. Relationships between alternans magnitude, defined as the difference between APD (at 90% re-polarization, APD₉₀) for successive odd- and even-numbered action potentials, and BCL in WT (A), RyR2^p/s (B) and RyR2^s/s (C) hearts treated with control solution (diamonds) and with solutions containing 100 nM epinephrine (squares) and 1 μM epinephrine (triangles). The top panel shows traces obtained in the absence of propranolol; the bottom panel shows traces obtained in the presence of 100 nM propranolol.

Figure 4

Cartoon representation of parameters used to construct restitution curves.

Figure 5

Sample restitution curve. Sample curve obtained from WT hearts in the absence of pharmacological manipulations. Data were fitted with a curve of the form y = y₀ + A(1−e^−x/τ) where y₀ = −11.9 ± 3.6, A = 60.0 ± 2.6, τ = 57.8 ± 5.4, χ² = 0.98 (n = 7).

Figure 6

Maximum slopes of restitution curves. Maximum slopes obtained from restitution curves in WT (A), RyR2^p/s (B) and RyR2^s/s(C) hearts treated with control solution (■) and with solution containing 100 nM epinephrine (□), prior to (top panel) and after (bottom panel) addition of 100 nM propranolol. Maximum slopes did not differ significantly between experimental groups.