ATRIAL FIBRILLATION THERAPY

Abstract

Provided is a method of facilitating the determination of treatment of a subject displaying atrial fibrillation, the method comprising determining a level of pitx2c expression in a sample from the subject and selecting a treatment based upon the level of pitx2c expression. Treatment with a sodium channel blocker may be selected if the level of pitx2c expression is determined to be reduced or below a predetermined threshold. Also provided are an assay system and a kit for use in the methods of the invention.

Claims

1. A method of facilitating the determination of treatment of a subject displaying atrial fibrillation, the method comprising determining a level of pitx2c expression in a sample from the subject and selecting a treatment based upon the level of pitx2c expression.

2. The method of claim 1, wherein selecting a treatment comprises selecting treatment with a sodium channel blocker if the level of pitx2c expression is determined to be reduced or below a predetermined threshold.

3. The method of claim 1, wherein the sample comprises at least one of urine, saliva, blood, sputum, semen, faeces, a nasal swab, tears, a vaginal swab, a rectal swab, a cervical smear, a tissue biopsy, and a urethral swab.

4. The method of claim 3, wherein the sample is blood.

5. The method of claim 1, wherein the level of pitx2c expression is determined directly by determining the pitx2c protein level or mRNA level.

6. The method of claim 1, wherein the level of pitx2c expression is determined indirectly by determining the level of a biomarker which is indicative of the level of pitx2c expression.

7. The method of claim 6, wherein the biomarker is ccl21.

8. (canceled)

9. The method of claim 2, wherein the sodium channel blocker type 1 antiarrhythmic drug.

10. The method of claim 2, the sodium channel blocker is flecainide or propafenone.

11. The method of claim 1, wherein determining a level of pitx2c expression in a sample from the subject comprises providing the sample to an assay system comprising: a measurement device that measures pitx2c expression; and a data transformation device that acquires the pitx2c expression data from the measurement device and performs data transformation to calculate whether or not the level determined is higher or lower than a reference or normal value for pitx2c expression.

12. The method of claim 11, wherein the assay system further comprises a user interface output device to output data to a user.

13. The method of claim 11, wherein the assay system further comprises a database of treatment information and, the device identifies the treatment information for the level of pitx2c determined and outputs the treatment information to the user interface output device.

14. A kit comprising at least one antibody, probe and/or primer which is/are capable of specifically binding to pitx2c mRNA or protein, or a biomarker indicative of pitx2c expression, and instructions for use.

15. The kit of claim 14, wherein the at least one antibody, probe and/or primer labeled with a detectable label.

16. A method of treating a subject displaying atrial fibrillation, comprising the steps of: identifying the subject; determining a level of pitx2c expression in the subject; and administering a sodium channel blocking antiarrhythmic drug to the subject in the event the pitx2c expression level is reduced or below a predetermined threshold or administering another class of antiarrhythmic drug to the subject in the event the pitx2c expression level is elevated or approximately equal to or above a predetermined threshold.

17. (canceled)

18. The method of claim 16, wherein the antiarrhythmic drug of another class is a potassium channel blocker.

19. (canceled)

Description

DETAILED DESCRIPTION

[0032] Embodiments of the invention will now be described by way of example and with reference to the accompanying Figures, in which:

[0033] FIG. 1 is an illustrative example of electrophysiological signals recorded from a Pitx2c.sup.+/ mouse heart, namely an atrial monophasic action potential (top tracing, MAP), an intracardiac electrogram recorded from the atrial (middle tracing) and an electrogram recorded from the right ventricle (bottom tracing). Programmed stimulation induced atrial fibrillation at baseline (top tracing), but fails to do so during perfusion with the sodium channel blocker flecainide (bottom tracing);

[0034] FIG. 2 is a graph showing the number of Pitx2c.sup.+/ and wild-type (WT) hearts with inducible atrial arrhythmias elicited with a single atrial premature stimulus, with or without flecainide perfusion. Wild type hearts do not show a difference in inducible arrhythmias. In pitx2c+/ hearts, flecainide completely suppresses atrial arrhythmias;

[0035] FIG. 3 is a plot showing the mean action potential durations (APD) measured by monophasic action potential catheters at different paced cycle lengths in Pitx2c.sup.+/ and WT hearts;

[0036] FIG. 4 is a plot showing the mean action potential durations (APD) in Pitx2c.sup.+/ and WT hearts before and after perfusion with flecainide at three different paced cycle lengths (80-100-120 ms);

[0037] FIG. 5 is a graph showing the change in inter-atrial activation time in Pitx2c.sup.+/ and WT hearts after flecainide perfusion. Flecainide has a pronounced effect on activation times in pitx2c+/ mice, but a much lesser effect in wild-type mice; and

[0038] FIG. 6 is a plot showing the change in effective refractory period (ERP) in Pitx2c.sup.+/ and WT hearts after flecainide perfusion. Flecainide increases effective refractory periods in Pitx2c+/ mice much more than in wild-type mice.

Example 1: Comparison of the Effect of Antiarrhythmic Drugs on Hearts of Wild-Type Mice and Mice with Reduced Pitx2C Expression

Example 1.1: Flecainide

Methods

[0039] The inventors studied the effects of the sodium channel blocker flecainide in the isolated, beating heart of wild type mice and mice with a heterozygous deletion of the Pitx2c gene. This mouse model (provided by Nigel Brown, St George's London) is an established model for reduced Pitx2 mRNA expression that is susceptible to AF. In PITX2c.sup.+/ mice, pitx2c mRNA expression was found to be reduced to about 60% of the WT level in the left atrium (Kirchhof et al., Circ Cardiovasc Genet. 2011; 4:123-133).

Electrophysiological Study in the Isolated Heart

[0040] PITX2c.sup.+/ and WT hearts (3-4 months old), were rapidly excised and Langendorff-perfused to record left atrial (LA) monophasic action potential duration. In this preparation, the beating heart is perfused with a warm, oxygenated modified Krebs-Henseleit solution containing (in mmol/1): NaCl 118; NaHCO.sub.3 24.88; KH.sub.2PO 1.18; Glucose 5.55; Na-Pyruvate 5; MgSO.sub.4 0.83; CaCl.sub.2 1.8; KCl 3.52 (95% O.sub.2-5% CO.sub.2, pH 7.4) at constant perfusion pressure (1005 mmHg) and coronary flow (40.5 ml/min).

[0041] Thereby, detailed electrophysiological measurements can be taken for 1-2 hours in the beating, functioning, intact heart (perfusate temperature 37 C., perfusion flow rate 3-5 mL/min) (Fabritz et al., Basic Res Cardiol 2003; 98:25-32). An octapolar murine electrophysiology catheter (0.5 mm electrode spacing, CIB'ER MOUSE, NuMED, Hopkinton, N.Y., USA) was inserted into the right atrium (RA) and right ventricle for pacing. Atrial and ventricular electrograms and monophasic action potential (MAP) from the left atrium were simultaneously recorded. After instrumentation, the hearts were subjected to a stabilization period of 10 minutes, followed by observation of spontaneous sinus rhythm for 5 minutes to provoke repolarization-related arrhythmias. Then the right atrium was paced at constant physiological heart rates (80-120 ms paced cycle length) for 1.5 minutes of pacing per heart rate to record steady-state action potential duration, and premature right atrial stimulation was performed using trains of 8 beats following by a premature stimulus in 2 ms steps to determine the effective refractory period (ERP) and inducibility of arrhythmias. Arrhythmias>1 second were counted. The pacing protocol was repeated after adding flecainide acetate (1 m) to the perfusate.

[0042] Experiments were accepted for analysis of action potential duration and activation time if they met published quality criteria and if MAPs were stable. Signals>mV were accepted for analysis of action potentials and activation times, and signals>0.5 mV amplitude were accepted for evaluation of arrhythmias. Recordings were manually screened for arrhythmias. Atrial fibrillation was defined as >5 closely couple polymorphic consecutive atrial ectopic beats with a cycle length shorter than sinus-rhythm cycle length.

Transmembrane Potential Recordings in Isolated, Superfused Left Atria

[0043] Transmembrane action potentials were recorded from isolated superfused left atria using borosilicate glass microelectrodes (tip resistance 15-30 M), filled with 3 M KCl. Voltage signals were amplified (Axoclamp 2B; Molecular Devices, USA), digitised and displayed using spike2 software (Cambridge Electronic Design, UK) at 20 kHz sampling frequency. LA were paced at different cycle lengths at 2 ms pulse width at 2 diastolic threshold through bipolar platinum electrodes. APD was analysed off line (spike 2, CED, UK). After 15 min pacing for equilibration, preparations were paced successively at increasing frequencies from 0.5 to 10 Hz, with 200 beats at each frequency to ensure steady state prior to recording.

Modelling of the Electrophysiological Consequences of Increased Resting Membrane Potential.

[0044] The human atrial model of Courtemanche-Ramirez-Nattel for atrial cells was used to assess the effects of 0.5-1 M flecainide. To reproduce the depolarised resting membrane potential and shorter action potential duration observed in the Pitx2c-deficient murine model, the maximal I.sub.K1 conductance was reduced by 25% and I.sub.Kr conductance was doubled. The effect of flecainide on sodium channels was simulated by reducing maximal I.sub.Na.conductance to 50% and 40%. Simulations were run in atrial cell strands of 100 excitable elements (cells; diameter 100 m). To reach steady state, the 5 leftmost cells of the strand were paced (S1) for 2 minutes at 1000 and 500 ms paced cycle length. A premature stimulation (S2) was applied to determine the ERP and CV was measured from cells 25-75 of the model. Resting membrane potential was measured as the minimum potential of the cycle preceding that arising from the S2 stimulus. Values for all other parameters were measured from the 50.sup.th cell. Post-repolarisation refractoriness is reported as a function of the diastolic interval in the 50.sup.th cell.

Statistical Analysis

[0045] Categorical data were compared using Fishers exact test. Numerical data were compared by two sided paired t tests (e.g. measurements before and after perfusion of flecainide or sotalol) and Wilcoxon signed-rank tests. For multiple measurements data were analysed by repeated measures analysis of variance followed by a multiple comparison procedure (Bonferroni t-test) if the overall test was significant. Data were considered significantly different at two-sided p values<0.05. When data are displayed as boxplots, boxes and box limits indicate the data range mean, and standard error. Whiskers indicate the minimum and maximum of the respective data. Individual measurements are shown in the boxplots as points.

Results

[0046] FIGS. 1 and 2 provide an illustrative example of a short episode of atrial fibrillation induced in a pitx2c+/ heart and the suppression of atrial fibrillation in the same heart by perfusion with flecainide. As shown in FIG. 2, flecainide suppressed atrial fibrillation in all Pitx2c+/ hearts that showed atrial fibrillation at baseline (8/21). Flecainide did not suppress atrial fibrillation in mice with normal Pitx2c expression (wild type hearts, 4/16 hearts with arrhythmias at baseline, 4/11 with flecainide). This indicates that flecainide suppresses inducible atrial arrhythmias in Pitx2c.sup.+/ mice.

[0047] Pitx2c+/ mice have shorter action potential durations at high paced cycle lengths than their wild-type littermates (FIG. 3).

[0048] No difference was observed in the effect of flecainide on action potential duration (APD) between wild-type and Pitx2c+/ mice (FIG. 4). This indicates that Flecainide does not alter atrial APD in either WT or PITX2c.sup.+/ hearts. However, flecainide was found to have a marked effect on atrial activation times in Pitx2c+/ mice, while there is only a negligible effect in wild-type mice (FIG. 5).

[0049] As shown in FIG. 6, flecainide induces more post-repolarization refractoriness (i.e. prolongation of the refractory period beyond the end of the action potential) in Pitx2c+/ mice (e.g. 12 ms at 80 ms paced cycle length) compared to WT mice (e.g. 7.5 ms at 80 ms paced cycle length). Post-repolarization refractoriness is a powerful mediator of antiarrhythmic efficacy in large animals (Kirchhof Fabritz et al., Circulation 1998; Kirchhof et al., JPET 2003; Kirchhof P. et al., Heart Rhythm. (2012); Milberg P. et al., Europace. (2013); Milberg P. et al., Journal of Cardiovascular Electrophysiology. 18:658-664 (2007); Kirchhof P, et al., J Pharmacol Exp Ther. 305:257-263 (2003)) and in patients with atrial fibrillation (Kirchhof P, et al. Basic Res Cardiol. 100:112-121 (2005)).

[0050] There are only small differences in action potential duration and refractoriness at baseline between pitx2c+/ and WT atria, but these differences are markedly more pronounced during perfusion with flecainide (lower panels): The refractory period is dramatically prolonged, causing marked post-repolarization refractoriness.

[0051] The analysis described above was initially done blinded. After unblinding, the differential effects discussed above were identified.

[0052] Taken together, these findings demonstrate that the sodium channel blocker flecainide has a profoundly different effect in atria with reduced pitx2c expression compared to atria with normal pitx2c levels. It was not only found that flecainide is much more effective in reducing atrial fibrillation in mice with reduced pitx2c expression, but also identified were several relevant differences in the electrophysiological effects of flecainide in atria with reduced pitx2c expression, e.g. a higher post repolarization refractoriness (FIG. 6).

[0053] Pitx2c.sup.+/ left atria had significantly (p<0.05) more depolarised resting membrane potentials at all cycle lengths tested as measured by sharp electrodes inserted into superfused left atrial preparation, e.g. 680.7 mV (n=31 cells) when compared with wildtype (700.7 mV, n=30 cells) at 100 ms paced CL. Flecainide did not alter resting membrane potential in either genotype. This can potentially explain the altered response to sodium channel blockers, as the binding of sodium channel blockers will be enhanced in cells that are more depolarized during electrical diastole.

[0054] When a more positive resting membrane potential was mimicked in the Courtemanche model of human atrial cardiomyocytes, flecainide did not alter the resting membrane potential, but sodium channels remain in the closed state for longer, and this was more pronounced in the presence of flecainide and during rapid pacing. Flecainide also had an enhanced effect on PRR prolongation when the resting membrane potential was increased compared to the reference model. Thus, the modelling of human atrial tissue replicated our findings in mice.

Example 1.2: Sotalol

[0055] Further experiments suggested that the antiarrhythmic effect of the potassium channel blocker d,l sotalol is lost in Pitx2c+/ hearts: In three pairs of mice studied using the same experimental setup described above, sotalol (10 M) prolonged atrial APD in WT hearts, but did not alter atrial APD in Pitx2c+/ hearts. These initial observations provide an explanation why potassium channel blockers such as sotalol may not be effective antiarrhythmic drugs in patients with reduced atrial pitx2c mRNA expression, and lend further support to the determination of pitx2c mRNA levels to aid the selection of antiarrhythmic drugs in patients with atrial fibrillation.

Conclusions

[0056] The present inventors have found that the effects of the antiarrhythmic drug flecainide are profoundly altered in the left atria of mice with reduced pitx2c expression compared to the wild-type mice. The effective refractory period was prolonged to a much higher value in PITX2c.sup.+/ hearts, and most significantly flecainide was more effective in preventing AF in PITX2c.sup.+/ mouse hearts compared to the wild type hearts. These observations indicate that patients with reduced pitx2c mRNA expression are suitable for therapy with a sodium channel blocking antiarrhythmic drug, such as flecainide, while others may possibly benefit from treatment with another agent, e.g. a potassium channel blocker such as sotalol.

[0057] It was found that PITX2c.sup.+/ left atria have reduced expression of some potassium channel genes, which may contribute to their higher resting membrane potentials and shorter action potential duration. This effect can explain the altered response to sodium channel blockers, as demonstrated in a computer model of human action potentials.

[0058] The invention provides for the first time a test that allows one to determine the effectiveness of a class of antiarrhythmic drug in the prevention of atrial fibrillation at the time point of initiation of such therapy. The method of the invention will enable the identification of subjects who will benefit from treatment with a sodium channel blocker, and thereby aids clinicians in selecting the most appropriate medication.

Example 2: Pitx2 Expression Levels in Human Atria

[0059] RNA was prepared using Qiagen RNeasy fibrous mini-kit and Quiagen QiaShredder Columns. cDNA was generated and RT-PCR reactions were performed using gene specific primers and SYBR Green (Life Technologies) on an ABI 7500 Fast machine.

[0060] We assessed the expression levels of PITX2 mRNA in human left and right atria, and could confirm that PITX2 mRNA is mainly expressed in the left atrium. A clear variation (up to 100-fold) in the expression levels of PITX2 mRNA in human left atria was observed: In some left atria, PITX2 expression was undetectable. More often, PITX2 expression levels are between 1% and 4% of actin levels. In some patients, left atrial PITX2 expression reaches up to 10% of actin expression. These data clearly illustrate that PITX2 expression in the left atrium varies in patients, rendering a selection of therapy based on PITX2 levels useful for the treatment of patients with atrial fibrillation.

Example 3: Use of Pitx2C Level to Select a Treatment for Atrial Fibrillation

[0061] A biological sample (e.g. blood) obtained from a subject displaying atrial fibrillation is analysed and the level of pitx2c mRNA is measured. The pitx2c mRNA level in the sample is compared to a pre-determined reference level. A suitable anti-arrhythmic drug is selected and administered to the subject depending on whether the measured pitx2c mRNA level in the sample is higher, lower or approximately the same as the pre-determined reference level.

[0062] If it is determined that the pitx2c mRNA level is reduced (i.e. the measured pix2c mRNA level is lower than the reference level) such that the subject is identified as having atrial fibrillation which is associated with reduced pitx2c expression, a sodium channel blocking drug (such as flecainide) can be administered. If it is determined that the pitx2c mRNA level is not reduced relative to the reference level, a potassium channel blocker (e.g. sotalol) can be administered to the subject.