Research ArticleRett Syndrome

Pathogenesis of Lethal Cardiac Arrhythmias in Mecp2 Mutant Mice: Implication for Therapy in Rett Syndrome

See allHide authors and affiliations

Science Translational Medicine  14 Dec 2011:
Vol. 3, Issue 113, pp. 113ra125
DOI: 10.1126/scitranslmed.3002982

Abstract

Rett syndrome is a neurodevelopmental disorder typically caused by mutations in methyl-CpG–binding protein 2 (MECP2) in which 26% of deaths are sudden and of unknown cause. To explore the hypothesis that these deaths may be due to cardiac dysfunction, we characterized the electrocardiograms in 379 people with Rett syndrome and found that 18.5% show prolongation of the corrected QT interval (QTc), an indication of a repolarization abnormality that can predispose to the development of an unstable fatal cardiac rhythm. Male mice lacking MeCP2 function, Mecp2Null/Y, also have prolonged QTc and show increased susceptibility to induced ventricular tachycardia. Female heterozygous null mice, Mecp2Null/+, show an age-dependent prolongation of QTc associated with ventricular tachycardia and cardiac-related death. Genetic deletion of MeCP2 function in only the nervous system was sufficient to cause long QTc and ventricular tachycardia, implicating neuronally mediated changes to cardiac electrical conduction as a potential cause of ventricular tachycardia in Rett syndrome. The standard therapy for prolonged QTc in Rett syndrome, β-adrenergic receptor blockers, did not prevent ventricular tachycardia in Mecp2Null/Y mice. To determine whether an alternative therapy would be more appropriate, we characterized cardiomyocytes from Mecp2Null/Y mice and found increased persistent sodium current, which was normalized when cells were treated with the sodium channel–blocking anti-seizure drug phenytoin. Treatment with phenytoin reduced both QTc and sustained ventricular tachycardia in Mecp2Null/Y mice. These results demonstrate that cardiac abnormalities in Rett syndrome are secondary to abnormal nervous system control, which leads to increased persistent sodium current. Our findings suggest that treatment in people with Rett syndrome would be more effective if it targeted the increased persistent sodium current to prevent lethal cardiac arrhythmias.

Introduction

Rett syndrome (RTT) is a severe X-linked dominant neurodevelopmental disorder that typically affects girls and is characterized by regression of spoken language, loss of hand use, problems with ambulation, and development of repetitive hand stereotypies (1). RTT is typically caused by mutations in methyl-CpG–binding protein 2 (MECP2) (2), a gene encoding a protein involved in regulation of gene expression (3). In addition to the cognitive and motor abnormalities present in RTT, affected people also show autonomic dysfunction, with breathing and heart rate irregularities (47). Boys with mutations in MECP2 exhibit more severe autonomic dysfunction, with marked breathing and heart rate abnormalities that result in death within the first year of life (8).

People with RTT have a high incidence of sudden unexpected deaths (26% of all deaths) (9), which are believed to have a cardiac origin. Previous work has indicated that some people with RTT have prolonged QT intervals (LQT) on electrocardiograms (ECGs) (10). In patients with other diseases, LQT is a significant risk factor for sudden arrhythmic cardiac death (11). To date, the cause for LQT in RTT is unknown, and its contribution to the high proportion of sudden death is yet untested.

The most common causes of inherited LQT include mutations in the voltage-gated potassium channels KVLQT1 (LQT1) and HERG (LQT2) and in the voltage-gated sodium channel SCN5A (LQT3), although rare mutations in genes encoding other channel subunits and in other cardiac proteins such as caveolin-3 (12) may also contribute to a few inherited cases (13). In addition to genetic causes, alterations in autonomic nervous system function also alter cardiac repolarization and contribute to the long QT phenotype (14). Patients with potassium channelopathies (LQT1 and LQT2) respond to β-adrenergic receptor blockade, an effective anti-arrhythmic prophylactic therapy; in contrast, LQT patients with sodium channelopathies (LQT3) have a poor response to β-adrenergic receptor blockade for the prevention of arrhythmias, indicating that sodium and potassium channelopathies contribute to LQTs through two different mechanisms (11).

Given that RTT patients have MeCP2 dysfunction, which leads to the LQT phenotype, we sought to define whether (i) MeCP2 dysfunction in mice recapitulates the long QT phenotype and causes predisposition to arrhythmic-induced death after programmed electrical stimulation (PES); (ii) MeCP2 dysfunction in neuronal tissue alone is sufficient to reproduce the LQT phenotype; and (iii) alterations in the sodium current may underlie a significant portion of the LQT phenotype in this mouse model of RTT.

Results

Long QTc is common in people with RTT and reproduced in the animal model of RTT

To define the prevalence of electrophysiological abnormalities in people with RTT, we evaluated ECGs from 379 girls and women with typical RTT enrolled in the Rett Syndrome Natural History Study. Seventy people (18.5%) had QTc of more than 450 ms, the established threshold for LQT (Fig. 1, A and B), consistent with previous small-scale studies (10, 15, 16). Because 26% of deaths in RTT are sudden and unexpected (11), these 18.5% of affected individuals are likely at risk for sudden death.

Fig. 1

Mecp2 deficiency is associated with LQT. (A) A representative ECG from a human patient with RTT with the RR and QT interval identified. (B) A scatter plot of QTc interval as a function of age in years for 379 RTT patients. The red dashed line indicates the cutoff (450 ms) for prolonged QTc. (C) A representative ECG from a wild-type (WT) mouse at baseline. (D) A representative ECG from a Mecp2Null/Y male mouse at 2 months old. (E to H) Quantification of ECGs shows increased QTc (E) and QRS (F) intervals in Mecp2Null/Y mice. Young Mecp2+/− female mice show normal QTc (G) and QRS (H). However, older female Mecp2+/− mice show long QTc (G) and increased QRS duration (H). *P < 0.05; **P < 0.01; ***P < 0.001. n, number of mice as indicated within bars.

To determine the origin of LQT in RTT, we sought to identify electrophysiological abnormalities in mouse models of RTT. Because human boys with mutations in MECP2 are more severely affected than girls, with severe autonomic dysfunction and early death (17), we first characterized male mice lacking MeCP2 at 2 to 3 months of age, the time point at which the animals manifest the most severe motor and behavioral phenotypes. Using surface ECG, we found that male mice hemizygous for Mecp2 (Mecp2Null/Y) exhibited longer QTc intervals than wild-type mice (Fig. 1, C to E). Moreover, QRS intervals in Mecp2Null/Y were longer than those in wild-type mice (Fig. 1F), but none of the other electrical properties that we recorded (RR, PQ, SCL, AV intervals) were different between the two animal groups (Table 1). To determine whether the repolarization abnormality represented by the prolonged QTc interval was associated with deficits in the hearts of Mecp2Null/Y animals, we performed echocardiography and found no evidence of any structural or contractility abnormality in Mecp2Null/Y animals compared with control animals (Table 2).

Table 1

Electrophysiological intervals in wild-type (WT) and Mecp2Null/Y mice at 2 to 3 months old. Data are expressed as means ± SEM. RR, time interval between two consecutive RR waves; PQ, interval from the beginning of the P wave to the peak of the Q wave; QRS, duration of the interval between beginning of Q wave to peak of S wave; QT, interval from beginning of Q wave to the end of the T wave; QTc, QT interval corrected for heart rate; SCL, sinus cycle length time; AV, interval from the beginning of the P wave to the beginning of the QRS complex.

View this table:
Table 2

Echocardiographic parameters of wild-type and Mecp2Null/Y mice at 2 to 3 months of age. Data are expressed as means ± SEM. HR, heart rate; bpm, beats per minute; EF, ejection fraction; FS, left ventricular fractional shortening; ESD, end-systolic diameter; EDD, end-diastolic diameter; IVSs/IVSd, intraventricular septal wall thickness in systole/diastole; LVPWs/LVPWd, left ventricular posterior wall thickness in systole/diastole.

View this table:

We next sought to determine whether the propensity toward LQT found in male hemizygous mice was also present in mice that precisely mimicked RTT, females heterozygous for a null allele of Mecp2. Four- to 5-month-old female heterozygous mice (Mecp2Null/+) did not show a significantly longer QTc interval or changes in QRS duration (Fig. 1, G and H). However, older 9- to 10-month-old female Mecp2Null/+ mice exhibited LQT (Fig. 1G) and prolonged QRS (Fig. 1H), with no change in other ECG parameters (Table 3). As in the male animals, echocardiography did not reveal structural heart abnormalities or contractility deficits in female Mecp2Null/+ mice (Table 4). Thus, hemizygous male Mecp2Null/Y mice have severe early-onset LQT and QRS prolongation, and heterozygous female Mecp2Null/+ show prolongation of both parameters that becomes apparent at older ages. A similar phenomenon is seen in patients with MeCP2 deficiency: Boys lacking MeCP2 function typically die of severe neurological disease within the first years of life (8), whereas females have relatively normal development until about 18 months of age, when they develop progressive onset of neurological regression (1).

Table 3

Electrophysiological intervals recorded in wild-type and Mecp2Null/+ mice. Data are expressed as means ± SEM.

View this table:
Table 4

Echocardiographic parameters of wild-type and Mecp2Null/+ mice. Data are expressed as means ± SEM.

View this table:

RTT mice show increased susceptibility to induced ventricular tachycardia

Given the significant LQT in male Mecp2Null/Y and female Mecp2Null/+ mice and the association between LQT and development of ventricular arrhythmias, we hypothesized that these mice may be more susceptible to developing ventricular arrhythmias. To test this, we inserted a catheter into the right ventricle of anesthetized mice and electrically stimulated the heart using PES to determine susceptibility toward cardiac arrhythmias. Male Mecp2Null/Y mice developed sustained ventricular tachycardia (VT) (defined as VT of more than 1 s) more often than did wild-type mice immediately after ventricular stimulation (Fig. 2, A and B). The duration of any (including nonsustained) arrhythmia episodes was significantly longer in Mecp2Null/Y mice than in wild-type mice (Fig. 2C). We noted that one of the male Mecp2Null/Y died as a result of cardiac arrhythmia induced during PES.

Fig. 2

Mecp2 deficiency is associated with predisposition to induced VT and arrhythmia-induced death in mice. (A) Representative surface ECG tracings of WT (top) and male Mecp2Null/Y mice (middle) after pacing (bottom). (B) Incidence of sustained VT after pacing in Mecp2Null/Y and WT mice. (C) Arrhythmia duration of any type in Mecp2Null/Y and WT mice. (D) Representative surface ECG tracings from WT (top) and Mecp2Null/+ mice (middle) after pacing (bottom). (E) Age-dependent susceptibility to sustained VT. (F) Incidence of sudden cardiac death (SCD) in WT and Mecp2Null/+ mice at 10 months. Two of seven Mecp2Null/+ mice developed VT and ventricular fibrillation after intracardiac pacing, causing death. (G) ECG of a 10-month-old Mecp2+/− mouse showing development of sustained VT leading to asystole at 8 min after pacing. **P < 0.01; ***P < 0.001. n is indicated within the bars of the graphs.

Similar to the age-dependent nature of LQT in female Mecp2Null/+ mice, young 4- to 5-month-old female Mecp2Null/+ mice did not develop a significant number of arrhythmias (Fig. 2E). However, every older female Mecp2Null/+ mouse tested showed PES-induced ventricular arrhythmias, whereas none of the wild-type mice had PES-induced arrhythmias (Fig. 2, D and E). Additionally, 29% (two of seven mice) of female Mecp2Null/+ mice died of VT during ventricular stimulation, suggesting that older female Mecp2Null/+ mice with LQT are at risk for arrhythmia-induced death (Fig. 2, F and G).

Cardiac abnormalities in RTT mice result from loss of MeCP2 function within the nervous system

Removing MeCP2 function from only the nervous system reproduces all the phenotypes of animals lacking MeCP2 in all tissues, including premature death (18). To determine whether loss of MeCP2 function within the nervous system would also cause LQT and increased susceptibility to ventricular arrhythmias, we generated a nervous system–specific conditional knockout (NKO) using the Nestin-Cre/loxP system, which restricts knockout of MeCP2 to the nervous system (19). Mecp2 mRNA expression was eliminated from the brain in these NKO animals, but was unaffected in the heart (Fig. 3A). As previously determined, the conditional allele of Mecp2, FLOX, shows a slight decrease in brain Mecp2 mRNA expression compared with wild type (20); however, Mecp2 mRNA was not significantly reduced in the hearts of FLOX compared with wild-type mice. QTc was longer in male NKO mice than in either FLOX or wild-type controls (Fig. 3B). As expected, the hypomorphic FLOX animals showed an intermediate phenotype, with mildly prolonged QTc duration; however, the QTc duration in the NKO animals was significantly increased over that of the FLOX controls. This LQT was associated with an increased incidence of VT in male NKO when compared to wild-type controls (Fig. 3C) after PES, whereas the VT incidence in the FLOX animals was not different from that in wild type. The incidence of VT was higher in the NKO animals (64%) than in the FLOX animals (25%, P = 0.09). In addition, 2 of the 11 NKO mice that we tested died of VT after pacing, suggesting that neuronal deficiency of Mecp2 is sufficient to cause both LQT and pacing-induced arrhythmias and arrhythmia-induced death (Fig. 3D).

Fig. 3

Male mice with nervous system–specific conditional knockout of MeCP2 (NKO) have LQT and inducible arrhythmias. (A) Mecp2 mRNA expression in brain and heart normalized to WT. *P < 0.05, FLOX compared to WT; ***P < 0.01, NKO compared to WT. (B and C) QTc duration (B) and VT incidence (C) in WT, FLOX, and NKO male mice. *P < 0.05; **P < 0.01. n is shown within the bars of the graphs. NS, nonsignificant. (D) Example of an ECG from one of two NKO mice that died of ventricular arrhythmia, showing the ECG 1 and 10 min after PES-induced VT.

Alteration in sodium current underlies LQT and the susceptibility to ventricular arrhythmia in RTT mice

Because the exact etiology of LQT in RTT is poorly understood, current strategies to prevent sudden arrhythmic events in RTT have been empirical. The standard therapy to prevent arrhythmias in RTT is to treat prophylactically with a β-adrenergic receptor blocker such as propranolol and to reduce exposure to drugs that might lengthen the QTc interval, such as anticholinergic agents. We evaluated the effectiveness of treatment with a β-adrenergic receptor blocker therapy in male Mecp2Null/Y mice using PES. Heart rate decreased in wild-type mice after injection of propranolol (3 mg/kg, intraperitoneally), whereas Mecp2Null/Y mice experienced a less pronounced decrease in heart rate (Fig. 4A). Propranolol did not reduce QTc in either male wild-type or Mecp2Null/Y mice (Fig. 4B). Similarly, propranolol did not protect male Mecp2Null/Y mice from PES-induced arrhythmias. All male Mecp2Null/Y mice treated with propranolol experienced either VT (60%) or atrioventricular block (40%) leading to death (Fig. 4C), whereas none of the treated wild-type mice had arrhythmias. These results suggest that propranolol may not be an effective drug for the treatment of QT prolongation and arrhythmias in people with RTT.

Fig. 4

Block of INa prevents pacing-induced arrhythmias. (A) Effect of treatment with β-adrenergic receptor blocker (BB) propranolol on heart rate in WT and Mecp2Null/Y mice. (B) Effect of propranolol on QTc intervals in WT and Mecp2Null/Y mice. (C) Effect of propranolol on PES-induced arrhythmias in WT and Mecp2Null/Y mice. Two Mecp2Null/Y mice developed severe atrioventricular block (AVB) and the other three developed sustained VT or ventricular fibrillation (VT/VF). (D and E) Effect of PHT on late-phase INa in Mecp2Null/Y cardiomyocytes, WT control cardiomyocytes, and NKO cardiomyocytes. (F) Effect of acute PHT injection on heart rate in WT and Mecp2Null/Y mice. (G) Effect of PHT on QTc interval in WT and Mecp2Null/Y mice. (H) Effect of PHT on arrhythmias in Mecp2Null/Y and WT mice. *P < 0.05; **P < 0.01. n is indicated in bar graphs. NS, nonsignificant. pA/s, the integrated magnitude of the current from 350 to 800 ms after depolarizing pulse.

β-Adrenergic receptor blockers have been described as being efficacious primarily in LQT1 and LQT2 syndromes, which are ascribed to potassium channelopathies; however, β-adrenergic blockers have not been found to be effective anti-arrhythmic agents in primary sodium channelopathies such as LQT3 or Brugada syndrome (21). To determine whether alteration in the voltage-gated sodium channel current caused the LQT phenotype in male Mecp2Null/Y mice, we performed patch clamping in isolated ventricular myocytes to measure the voltage-gated sodium channel current from these animals. Although there was no difference in peak sodium current at −20 mV (wild type: −18.8 ± 1.9 pA/pF; Mecp2Null/Y: −16.6 ± 1.0 pA/pF; P = 0.3) and no difference in the cellular capacitance (wild type: 198 ± 19 pF; Mecp2Null/Y: 176 ± 28 pF), measurements of persistent sodium channel current (INa) showed a larger INa in Mecp2Null/Y mice versus wild type (Fig. 4, D and E). We similarly performed patch clamping in isolated ventricular myocytes from NKO animals and found that they had an increased persistent INa compared with the wild-type littermate animals of the Mecp2Null/Y mice (Fig. 4D). Although this comparison was not made with the exact littermate control animals, the persistent INa is robust within wild-type animals if the genetic strain is identical. In this experiment, the background genetic strain was identical between the Mecp2Null/Y line and the NKO line.

In the genetic forms of LQT that do not respond to β-adrenergic receptor blockers, such as LQT3, drugs that target the sodium channel prevent cardiac arrhythmias (10). Given the observations that (i) the β-adrenergic receptor blocker propranolol could not alter either QTc interval or arrhythmia incidence in Mecp2Null/Y mice, (ii) a persistent late INa current existed in Mecp2Null/Y mice, and (iii) seizures are common in human RTT, we decided to evaluate phenytoin (PHT) as a potential therapeutic agent in RTT. PHT blocks the persistent late INa, which prevents cardiac arrhythmias and neurological epileptic seizures, and thus may be able to prevent ventricular arrhythmias in MeCP2 deficiency. When cardiomyocytes from male Mecp2Null/Y mice were treated with PHT, the late INa decreased compared to that in untreated male Mecp2Null/Y myocytes, demonstrating that the persistent late INa in Mecp2Null/Y mice is reversible by pharmacological blockade of the sodium channel (Fig. 4, D and E). Injection of PHT (50 mg/kg, intraperitoneally) into wild-type and Mecp2Null/Y mice decreased QTc to a value similar to that of PHT-treated wild-type mice (Fig. 4, F to H) and completely abolished ventricular arrhythmias in Mecp2Null/Y mice (Fig. 4H), indicating that INa is a significant component of arrhythmias in RTT and that PHT or drugs with similar pharmacology may reduce arrhythmia risk in people with RTT.

Discussion

Here, we used a large cohort of people with RTT to determine that LQT is found in nearly 20% of people with RTT. This LQT is suspected to underlie the sudden unexpected deaths found in this disease (22). We determined that mouse models of RTT have LQT and are susceptible to VT and sudden cardiac death. The development of these cardiac abnormalities occurs in an age-dependent fashion in female heterozygous animals, indicating that they are likely secondary to the loss of MeCP2 function in this disease. This idea was confirmed by our unexpected finding that the increased persistent INa, LQT, VT, and sudden cardiac death were obtained by removing MeCP2 function within the nervous system.

Although β-adrenergic receptor blockers are the mainstay of therapy for most cases of LQT, and RTT specifically, we found that in the animal model β-adrenergic receptor blockers did not prevent VT and may have increased arrhythmic death, similar to findings in a mouse model of LQT3 syndrome (21). Cardiomyocytes isolated from RTT mouse hearts showed an increased INa, similar to that seen in genetic forms of LQT that affect sodium channels. This increased INa was blocked by treatment with a sodium channel blocker, PHT. Treatment of the mouse model with PHT shortened the QTc interval and prevented VT and cardiac death, suggesting that such treatments should be tested for efficacy in people with RTT.

An unexpected result of this work was the finding that the cardiac arrhythmias present in the animals are the result of changes in MeCP2 function within the nervous system. This was surprising because LQT reflects alteration in the repolarization property of cardiomyocytes themselves, and the genetic causes of idiopathic LQT are the result of mutations in genes that encode proteins within the cardiomyocytes that control the electrical properties of those cells. We found that the electrical properties of cardiomyocytes from both Mecp2Null/Y and NKO animals were indeed changed. Thus, the alteration of the electrical properties in the cardiomyocytes must be an indirect response to alterations of the nervous system control of the heart.

Neurological dysfunction may affect the control of cardiac rate and rhythm. First, repetitive seizures can induce remodeling of the potassium and sodium channels within the heart, leading to QTc prolongation and cardiac arrhythmias (23). Second, autonomic neuropathies prolong QTc interval in patients with primary central nervous system disease (14, 2427), autonomic neuropathy (28, 29), and amyotrophic lateral sclerosis (30). The exact mechanism by which altered nervous system control leads to cardiac arrhythmias in these cases is unknown. It has been suspected that sympathovagal imbalance in people with RTT may contribute to sudden cardiac death (10, 22), even in the presence of normal cardiac function revealed by echocardiography (31). An important question is whether this imbalance indirectly leads to QTc prolongation. Further characterization in mouse models of RTT may be helpful in understanding the mechanism underlying this prolongation, although this characterization may be challenging because heart rate in mice is primarily under sympathetic control (32).

Because sympathetic overactivation appears to be present in people with RTT (10), we were somewhat surprised that inhibition of the sympathetic outflow with β-adrenergic receptor blockers did not appear to be beneficial in the animal model of RTT. The poor response to β-adrenergic receptor blockers in these animals is consistent with the increased late INa, reminiscent of the inability of β-adrenergic receptor blockers to suppress arrhythmias in people with LQT syndromes caused by sodium channel mutations, such as in LQT3. In LQT3, bradycardia caused by unchecked parasympathetic activation can lead to life-threatening cardiac arrhythmias (21). We similarly saw sudden cardiac death after β-adrenergic receptor blockade in female Mecp2Null/+ mice, with some of the animals actually dying in atrioventricular block.

This study has the inherent limitation of using a knockout mouse to model human disease. Mice typically have shorter QT intervals than human patients, and their neurological development may favor parasympathetic control of heart rate, which is greater than that of human patients (33). Another limitation in this work is our inability to determine whether there is an age-dependent change in QTc prolongation in people with RTT. Although the data presented here are based on a very large cohort of affected people, additional information will be gained as the Rett Syndrome Natural History Study progresses and true longitudinal information gathered with repeated ECG recordings. Furthermore, it would have been beneficial to determine from the human data whether exposure to specific drugs or specific MECP2 mutations alter the propensity toward developing QTc prolongation.

Here, mice lacking MeCP2, a genetic mimic of the human condition, showed the same cardiac phenotype as people with RTT, indicating that these mice may be useful for determining the underlying pathophysiological mechanisms as well as for testing therapeutic options for RTT. We have demonstrated this latter point by showing that acute treatment of Mecp2 null mice with PHT may protect against this disorder. This is not presently the standard of care for LQT in RTT: We recommend that alternative therapies should be tested in patients with RTT, focusing on the use of sodium channel–blocking agents. We chose PHT as a representative of a class of sodium channel–blocking agents that are commonly used in neurological diseases for their anti-seizure effects, but these drugs could also be useful for preventing cardiac arrhythmias. Future work using the cellular and animal system presented here will help determine whether PHT, other anti-epileptic drugs that block the sodium channel, or newer antiarrhythmic agents such as ranolazine that inhibit persistent INa are a good therapeutic option in this disease. Such information will then be useful to guide formal clinical trials in people with RTT.

Materials and Methods

Human subjects

This study was approved by the institutional review boards of the participating centers. Written informed consent was obtained from all guardians of patients participating in the study (clinicaltrials.gov, identifier NCT00296764). The study methodology has been described (34). For this work, ECG results obtained by referring physicians were reviewed and QTc interval for the first ECG was obtained, and the age at which this test was performed was recorded.

Animals

Mecp2Tm1.1Bird and Mecp2Tm1Bird mice (35) were obtained as a gift from A. Bird, and experimental heterozygous Mecp2Tm1.1Bird/+ female animals were generated as described (36). To generate a nervous system knockout of MeCP2, we mated a conditional allele of MeCP2, Mecp2Tm1Bird (35), on a pure 129S6 background to Nestin-Cre (Jackson Labs) maintained on a C57BL/6 background to generate F1 129S6.C57BL/6 experimental animals. All animals were housed in the Association for Assessment and Accreditation of Laboratory Animal Care–approved animal facility at Baylor College of Medicine, and experiments were approved by the Baylor Institutional Animal Care and Use Committee.

Transthoracic echocardiography

Mice were anesthetized with 1.5% isoflurane in 95% O2. Body temperature was maintained at 36°C and 37°C on a heated platform, and ECGs and temperature were continuously monitored. Cardiac function was assessed with a VisualSonics VeVo 770 Imaging System (VisualSonics) equipped with a high-frequency 30-MHz probe, as described (37).

Surface ECG

Mice were anesthetized with 1.5% isoflurane in 95% O2 and six-lead ECGs recorded by pad electrodes with bandpass filtering between 0.03 Hz and 1 kHz, according to published methods (38). QT values were calculated as the interval between the onset of the QRS complex and the moment after the T-wave peak [the first derivative (dV/dt) becomes zero]. Corrected QT intervals (QTc) were calculated by the formula QTc = QT + 0.3173 × (170 − RR) as described (39).

Programmed electrical stimulation

Atrial and ventricular intracardiac electrograms were recorded with a 1.1-F octapolar electrode catheter (EPR-800; Millar Instruments) inserted into the right ventricle via the right jugular vein, as described (40, 41). Inducibility of VT was determined with overdrive pacing and extra stimulus protocols, and sustained VT is defined as VT lasting more than 1 s. For acute treatment with antiarrhythmic agents, animals were injected intraperitoneally 40 min before catheterization with a propranolol solution (3 mg/kg) or 120 min before with PHT (50 mg/kg).

RNA isolation and quantitative real-time polymerase chain reaction

Animals were killed by cervical dislocation after Avertin anesthesia, and hearts were rapidly dissected, washed in 1× phosphate-buffered saline to remove blood, and snap-frozen in liquid nitrogen. They were then homogenized in Trizol (Invitrogen) with a Polytron. RNA was extracted according to the manufacturer’s instructions. Complementary DNA (cDNA) was made with qScript cDNA kit (Quanta BioSciences), and quantitative polymerase chain reaction (qPCR) was performed on a Bio-Rad CFX96 qPCR machine with PerfeCTa SYBR Green FastMix (Qantas) as described (20).

Patch clamping of ventricular myocytes

Enzymatic isolation of ventricular myocytes from wild-type and Mecp2Null/Y mice was performed by the Langendorff perfusion system as described (40). Macroscopic INa was recorded by the whole-cell patch clamp technique by Axon 200B amplifier (Axon Instruments). Briefly, isolated myocytes were perfused at room temperature with bath solution: 130 mM NaCl, 10 mM CsCl, 1 mM MgCl2, 10 mM Hepes, 10 mM glucose, 1 mM 4-aminopyridine (pH 7.4), with CsOH. Electrodes were pulled with series resistance around 1.5 megohms and filled with pipette solution: 130 mM CsCl, 10 mM NaCl, 10 mM Hepes, 10 mM EGTA (pH 7.4), with CsOH. Recording protocols were generated with pCLAMP software 9 (Axon Instruments) and digitized at 25 kHz with a Digidata 1332A A/D converter. To activate late INa, we applied a 1-s depolarizing step pulse to −20 mV from a holding potential of −120 mV at a rate of 0.1 Hz. The magnitude of steady late INa during the 350 to 800 ms was calculated by integrating the current over that time period with the integration feature of Clampfit 10.0 software, as described (42). PHT was acutely provided to cardiomyocytes in solution at a final concentration of 6 μg/ml, the cells were incubated for 10 min, and then patch clamping was performed as above.

Statistical analysis

Continuous variables were expressed as mean. Continuous variables were evaluated with an unpaired Student’s t test or analysis of variance (ANOVA). Categorical data were expressed as percentages and compared with the Fisher’s exact test. P < 0.05 was considered statistically significant.

References and Notes

  1. Acknowledgments: We thank T. Ai for his assistance with the electrophysiological experiments, D. Skapura and E. M. Arvide for technical assistance, and S. Sarma for assistance with echocardiography. We also thank J. Fryer, R. Samaco, B. Arenkiel, and H. Zoghbi for critical comments. We thank A. Bird for the gift of the Mecp2 mutant animals. We are indebted to all the families who participate in the Rett Syndrome Natural History Study. Funding: J.L.N. is supported by a Cynthia and Anthony Petrello Scholar Endowment at the Jan and Dan Neurological Research Institute, Texas Children’s Hospital; U.S. NIH grants NS52240, HD062553, and HD24064 (Baylor Intellectual and Developmental Disabilities Research Center); and the International Rett Syndrome Foundation (IRSF). X.H.T.W. is a W. M. Keck Foundation Distinguished Young Scholar in Medical Research and the Juanita P. Quigley Endowed Chair in Cardiology, and is supported by U.S. NIH grants HL089598 and HL091947 and Muscular Dystrophy Association grant 69238. M.D.M. is supported by NIH training grant HL066991. C.S.W. is supported by NS066601. The Rett Syndrome Natural History Study is supported by U54 HD061222, IRSF, and the Blue Bird Circle of Houston. The project described was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (EKS NICHD). The content is solely the responsibility of the authors and does not necessarily represent the official views of the EKS NICHD or the NIH. Author contributions: J.L.N., X.H.T.W., and M.D.M. designed the study, evaluated the data, and wrote the manuscript. M.D.M., T.W., E.M., J.H., D.L.B., T.-W.H., and C.S.W. performed the experiments. A.K.P., D.G.G., S.S., and J.L.N. collected the clinical data and helped edit the manuscript. Competing interests: The authors declare that they have no competing interests.
View Abstract

Navigate This Article