Catharine A. Goddard

Mouse model of SCN5A-linked hereditary Lenegre's disease - Age-related conduction slowing and myocardial fibrosis

Background—We have previously linked hereditary progressive cardiac conduction defect (hereditary Lenègre’s disease) to a loss-of-function mutation in the gene encoding the main cardiac Na+ channel, SCN5A. In the present study, we investigated heterozygous Scn5a-knockout mice (Scn5a+/− mice) as a model for hereditary Lenègre’s disease. Methods and Results—In Scn5a+/− mice, surface ECG recordings showed age-related lengthening of the P-wave and PR- and QRS-interval duration, coinciding with previous observations in patients with Lenègre’s disease. Old but not young Scn5a+/− mice showed extensive fibrosis of their ventricular myocardium, a feature not seen in wild-type animals. In old Scn5a+/− mice, fibrosis was accompanied by heterogeneous expression of connexin 43 and upregulation of hypertrophic markers, including &bgr;-MHC and skeletal &agr;-actin. Global connexin 43 expression as assessed with Western blots was similar to wild-type mice. Decreased connexin 40 expression was seen in the atria. Using pangenomic microarrays and real-time PCR, we identified in Scn5a+/− mice an age-related upregulation of genes encoding Atf3 and Egr1 transcription factors. Echocardiography and hemodynamic investigations demonstrated conserved cardiac function with aging and lack of ventricular hypertrophy. Conclusions—We conclude that Scn5a+/− mice convincingly recapitulate the Lenègre’s disease phenotype, including progressive impairment with aging of atrial and ventricular conduction associated with myocardial rearrangements and fibrosis. Our work provides the first demonstration that a monogenic ion channel defect can progressively lead to myocardial structural anomalies.

Sinus node dysfunction following targeted disruption of the murine cardiac sodium channel gene Scn5a

We have examined sino‐atrial node (SAN) function in hearts from adult mice with heterozygous targeted disruption of the Scn5a gene to clarify the role of Scn5a‐encoded cardiac Na+ channels in normal SAN function and the mechanism(s) by which reduced Na+ channel function might cause sinus node dysfunction. Scn5a+/− mice showed depressed heart rates and occasional sino‐atrial (SA) block. Their isolated peripheral SAN pacemaker cells showed a reduced Na+ channel expression and slowed intrinsic pacemaker rates. Wild‐type (WT) and Scn5a+/− SAN preparations exhibited similar activation patterns but with significantly slower SA conduction and frequent sino‐atrial conduction block in Scn5a+/− SAN preparations. Furthermore, isolated WT and Scn5a+/− SAN cells demonstrated differing correlations between cycle length, maximum upstroke velocity and action potential amplitude, and cell size. Small myocytes showed similar, but large myocytes reduced pacemaker rates, implicating the larger peripheral SAN cells in the reduced pacemaker rate that was observed in Scn5a+/− myocytes. These findings were successfully reproduced in a model that implicated iNa directly in action potential propagation through the SAN and from SAN to atria, and in modifying heart rate through a coupling of SAN and atrial cells. Functional alterations in the SAN following heterozygous‐targeted disruption of Scn5a thus closely resemble those observed in clinical sinus node dysfunction. The findings accordingly provide a basis for understanding of the role of cardiac‐type Na+ channels in normal SAN function and the pathophysiology of sinus node dysfunction and suggest new potential targets for its clinical management.

Paced electrogram fractionation analysis of arrhythmogenic tendency in DeltaKPQ Scn5a mice.

Introduction: Gain‐of‐function mutations within Scn5a, including the ΔKPQ 1505‐1507 deletion in the inactivation domain compromising myocardial repolarization, are implicated in human long QT 3 syndrome (LQT3), associated with ventricular arrhythmogenesis and sudden death.

Effects of L‐type Ca2+ channel antagonism on ventricular arrhythmogenesis in murine hearts containing a modification in the Scn5a gene modelling human long QT syndrome 3

Ventricular arrhythmogenesis in long QT 3 syndrome (LQT3) involves both triggered activity and re‐entrant excitation arising from delayed ventricular repolarization. Effects of specific L‐type Ca2+ channel antagonism were explored in a gain‐of‐function murine LQT3 model produced by a ΔKPQ 1505–1507 deletion in the SCN5A gene. Monophasic action potentials (MAPs) were recorded from epicardial and endocardial surfaces of intact, Langendorff‐perfused Scn5a+/Δ hearts. In untreated Scn5a+/Δ hearts, epicardial action potential duration at 90% repolarization (APD90) was 60.0 ± 0.9 ms compared with 46.9 ± 1.6 ms in untreated wild‐type (WT) hearts (P < 0.05; n= 5). The corresponding endocardial APD90 values were 52.0 ± 0.7 ms and 53.7 ± 1.6 ms in Scn5a+/Δ and WT hearts, respectively (P > 0.05; n= 5). Epicardial early afterdepolarizations (EADs), often accompanied by spontaneous ventricular tachycardia (VT), occurred in 100% of MAPs from Scn5a+/Δ but not in any WT hearts (n= 10). However, EAD occurrence was reduced to 62 ± 7.1%, 44 ± 9.7%, 10 ± 10% and 0% of MAPs following perfusion with 10 nm, 100 nm, 300 nm and 1 μm nifedipine, respectively (P < 0.05; n= 5), giving an effective IC50 concentration of 79.3 nm. Programmed electrical stimulation (PES) induced VT in all five Scn5a+/Δ hearts (n= 5) but not in any WT hearts (n= 5). However, repeat PES induced VT in 3, 2, 2 and 0 out of 5 Scn5a+/Δ hearts following perfusion with 10 nm, 100 nm, 300 nm and 1 μm nifedipine, respectively. Patch clamp studies in isolated ventricular myocytes from Scn5a+/Δ and WT hearts confirmed that nifedipine (300 nm) completely suppressed the inward Ca2+ current but had no effect on inward Na+ currents. No significant effects were seen on epicardial APD90, endocardial APD90 or ventricular effective refractory period in Scn5a+/Δ and WT hearts following perfusion with nifedipine at 1 nm, 10 nm, 100 nm, 300 nm and 1 μm nifedipine concentrations. We conclude that L‐type Ca2+ channel antagonism thus exerts specific anti‐arrhythmic effects in Scn5a+/Δ hearts through suppression of EADs.

Effects of flecainide and quinidine on arrhythmogenic properties of Scn5a+/− murine hearts modelling the Brugada syndrome

Brugada syndrome (BrS) is associated with a loss of Na+ channel function and an increased incidence of rapid polymorphic ventricular tachycardia (VT) and sudden cardiac death. A programmed electrical stimulation (PES) technique assessed arrhythmic tendency in Langendorff‐perfused wild‐type (WT) and genetically modified (Scn5a+/−) ‘loss‐of‐function’ murine hearts in the presence and absence of flecainide and quinidine, and the extent to which Scn5a+/− hearts model the human BrS. Extra‐stimuli (S2), applied to the right ventricular epicardium, followed trains of pacing stimuli (S1) at progressively reduced S1–S2 intervals. These triggered VT in 16 out of 29 untreated Scn5a+/− and zero out of 31 WT hearts. VT occurred in 11 out of 16 (10 μm) flecainide‐treated WT and nine out of the 13 initially non‐arrhythmogenic Scn5a+/− hearts treated with (1.0 μm) flecainide. Quinidine (10 μm) prevented VT in six out of six flecainide‐treated WT and 13 out of the 16 arrhythmogenic Scn5a+/− hearts in parallel with its clinical effects. Paced electrogram fractionation analysis demonstrated increased electrogram durations, expressed as electrogram duration (EGD) ratios, with shortening S1–S2 intervals in arrhythmogenic Scn5a+/− hearts, and prolonged ventricular effective refractory periods (VERPs) in non‐arrhythmogenic Scn5a+/− hearts. Flecainide increased EGD ratios in WT (at 10 μm) and non‐arrhythmogenic Scn5a+/− hearts (at 1.0 μm), whereas quinidine (10 μm) reduced EGD ratios and prolonged VERPs in WT and arrhythmogenic Scn5a+/− hearts. However, epicardial and endocardial monophasic action potential recordings consistently demonstrated positive gradients of repolarization in WT, arrhythmogenic and non‐arrhythmogenic Scn5a+/− hearts under all pharmacological conditions. Together, these findings demonstrate proarrhythmic effects of flecainide in WT and Scn5a+/− murine hearts that recapitulate its clinical effects. They further attribute the arrhythmogenic phenomena observed here to re‐entrant substrates resulting from delayed epicardial activation despite an absence of transmural heterogeneities of repolarization, in sharp contrast to recent characterizations in ‘gain‐of‐function’Scn5a+/Δ murine hearts modelling the long‐QT(3) syndrome.

Physiological consequences of the P2328S mutation in the ryanodine receptor (RyR2) gene in genetically modified murine hearts

Aim:  To explore the physiological consequences of the ryanodine receptor (RyR2)‐P2328S mutation associated with catecholaminergic polymorphic ventricular tachycardia (CPVT).

Effects of flecainide and quinidine on arrhythmogenic properties of Scn5a+/Δ murine hearts modelling long QT syndrome 3

Long QT(3) (LQT3) syndrome is associated with incomplete Na+ channel inactivation, abnormal repolarization kinetics and prolonged cardiac action potential duration (APD). Electrophysiological effects of flecainide and quinidine were compared in Langendorff‐perfused wild‐type (WT), and genetically modified (Scn5a+/Δ) murine hearts modelling LQT3. Extra stimuli (S2) following trains of pacing stimuli (S1) applied to the right ventricular epicardium triggered ventricular tachycardia (VT) in 16 out of 28 untreated Scn5a+/Δ and zero out of 12 WT hearts. Paced electrogram fractionation analysis then demonstrated increased electrogram durations (EGD), expressed as EGD ratios, in arrhythmogenic Scn5a+/Δ hearts, and prolonged ventricular effective refractory periods in initially non‐arrhythmogenic Scn5a+/Δ hearts. Nevertheless, comparisons of epicardial and endocardial monophasic action potential recordings demonstrated negative transmural repolarization gradients in both groups, giving ΔAPD90 values at 90% repolarization of −20.88 ± 1.93 ms (n= 11) and −16.91 ± 1.43 ms (n= 23), respectively. Flecainide prevented initiation of VT in 13 out of 16 arrhythmogenic Scn5a+/Δ hearts, reducing EGD ratio and restoring ΔAPD90 to + 7.55 ± 2.24 ms (n= 9) (P < 0.05). VT occurred in four out of eight non‐arrhythmogenic Scn5a+/Δ hearts in the presence of quinidine, which increased EGD ratio but left ΔAPD90 unchanged. In contrast (P < 0.05), WT hearts had positive ΔAPD90 values (+ 11.72 ± 2.17 ms) (n= 20). Flecainide then increased arrhythmic tendency and EGD ratio but conserved ΔAPD90; reduced EGD ratios and unaltered ΔAPD90 values accompanied the lower arrhythmogenicity associated with quinidine treatment. In addition to the changes in EGD ratio shown by WT hearts, these findings attribute arrhythmogenesis and its modification by flecainide and quinidine to alterations in ΔAPD90 in Scn5a+/Δ hearts. This is consistent with a hypothesis in which incomplete Na+ channel inactivation in Scn5a+/Δ hearts generates functional substrates dependent on altered refractoriness that cause abnormalities in activation and conduction of subsequent cardiac impulses. Any spatial heterogeneities between the epicardial and endocardial layers would thus cause fragmentation of the activation wavefront and contribute to electrogram spreading.

Mechanisms of ventricular arrhythmogenesis in mice following targeted disruption of KCNE1 modelling long QT syndrome 5

Mutations within KCNE1 encoding a transmembrane protein which coassembles with K+ channels mediating slow K+, IKs, currents are implicated in cardiac action potential prolongation and ventricular arrhythmogenicity in long QT syndrome 5. We demonstrate the following potentially arrhythmogenic features in simultaneously recorded, left ventricular, endocardial and epicardial monophasic action potentials from Langendorff‐perfused murine KCNE1−/− hearts for the first time. (1) Prolonged epicardial (57.1 ± 0.5 ms cf. 36.1 ± 0.07 ms in wild‐type (WT), P < 0.001; n= 5) and endocardial action potential duration at 90% repolarication (APD90) (54.4 ± 2.4 ms cf. 48.5 ± 0.3 ms, P < 0.05; n= 5). (2) Negative transmural repolarization gradients (ΔAPD90: endocardial minus epicardial APD90) (−2.5 ± 2.4 ms, compared with 12.4 ± 1.1 ms in WT, P < 0.001; n= 5). (3) Frequent epicardial early afterdepolarizations (EADs) and spontaneous ventricular tachycardia (VT) in 4 out of 5 KCNE1−/− hearts but not WT (n= 5). EADs were especially frequent following temporary cessations of ventricular pacing. (4) Monomorphic VT lasting 1.36 ± 0.2 s in 5 out of 5 KCNE1−/− hearts, following premature stimuli but not WT (n= 5). (5) Epicardial APD alternans. Perfusion of KCNE1−/− hearts with 1 μm nifedipine induced potentially anti‐arrhythmic changes including: (1) restored epicardial APD90 (from 57.1 ± 0.5 ms to 42.3 ± 0.4 ms, P < 0.001; n= 5); (2) altered ΔAPD90 to values (11.2 ± 2.6) close to WT (P > 0.05; n= 5); (3) EAD suppression during both spontaneous activity and following cessation of ventricular pacing (n= 5) to give similar features to WT controls (n= 5); (4) suppression of programmed electrical stimulation‐induced VT; and (5) suppression of APD alternans. These findings suggest arrhythmic effects of reduced outward currents expected in KCNE1−/− hearts and their abolition by antagonism of inward L‐type Ca2+ current.

Comparison of protein transduction domains in mediating cell delivery of a secreted CRE protein.

Protein transduction domains (PTDs) are versatile peptide sequences that facilitate cell delivery of several cargo molecules including proteins. PTDs usually consist of short stretches of basic amino acids that can cross the plasma membrane and gain entry into cells. Traditionally, to assess PTD mediated protein delivery, PTD-fusion proteins have been used as purified proteins. To overcome the requirement for a protein purification step, we used a secretory signal peptide to allow PTD-CRE fusion proteins to be exported from transfected mammalian cells. PTD induced protein transduction into cells was assessed by a CRE-mediated recombination event that resulted in beta-galactosidase expression. Several PTDs were tested including the prototypic TAT, different TAT variants, Antp, MTS and polyarginine. A negative correlation was observed between the cationic charge on the PTD and the extent of secretion. Poor secretion was found when the PTD charge was greater than +5. One TAT-CRE protein variant had a 14-fold enhancement above CRE alone when added to cells in the presence of chloroquine. This PTD domain also enhanced gene expression after plasmid delivery. These data illustrate that some secreted PTD proteins may be useful reagents to improve protein delivery in mammalian systems and a novel approach to enhancing the response to DNA transfections.

Alternans in Genetically Modified Langendorff-Perfused Murine Hearts Modeling Catecholaminergic Polymorphic Ventricular Tachycardia

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 (RyR2p/s) and homozygous (RyR2s/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 RyR2p/s and RyR2s/s hearts as compared to WTs. Arrhythmogenicity in RyR2s/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.