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Keywords:

  • Epilepsy;
  • HCN channels;
  • Cardiac electrophysiology;
  • Gene expression;
  • Genetic Absence Epilepsy Rats from Strasbourg (GAERS);
  • Post–kainic acid–induced status epilepticus

Summary

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Biography
  10. Supporting Information

Objective

Evidence from animal and human studies indicates that epilepsy can affect cardiac function, although the molecular basis of this remains poorly understood. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels generate pacemaker activity and modulate cellular excitability in the brain and heart, with altered expression and function associated with epilepsy and cardiomyopathies. Whether HCN expression is altered in the heart in association with epilepsy has not been investigated previously. We studied cardiac electrophysiologic properties and HCN channel subunit expression in rat models of genetic generalized epilepsy (Genetic Absence Epilepsy Rats from Strasbourg, GAERS) and acquired temporal lobe epilepsy (post–status epilepticus SE). We hypothesized that the development of epilepsy is associated with altered cardiac electrophysiologic function and altered cardiac HCN channel expression.

Methods

Electrocardiography studies were recorded in vivo in rats and in vitro in isolated hearts. Cardiac HCN channel messenger RNA (mRNA) and protein expression were measured using quantitative PCR and Western blotting respectively.

Results

Cardiac electrophysiology was significantly altered in adult GAERS, with slower heart rate, shorter QRS duration, longer QTc interval, and greater standard deviation of RR intervals compared to control rats. In the post-SE model, we observed similar interictal changes in several of these parameters, and we also observed consistent and striking bradycardia associated with the onset of ictal activity. Molecular analysis demonstrated significant reductions in cardiac HCN2 mRNA and protein expression in both models, providing a molecular correlate of these electrophysiologic abnormalities.

Significance

These results demonstrate that ion channelopathies and cardiac dysfunction can develop as a secondary consequence of chronic epilepsy, which may have relevance for the pathophysiology of cardiac dysfunction in patients with epilepsy.

Seizures commonly affect cardiac rate and rhythm via autonomic neuronal dysfunction, which over time with repeated seizures could have major detrimental effects on cardiac function and result in serious clinical consequences. Indeed, epilepsy is associated with an increased risk of sudden unexplained death (sudden unexplained death in epilepsy; SUDEP), possibly due to cardiac arrhythmias.[1] Patients with drug refractory temporal lobe epilepsy (TLE) show greater cardiovascular dysfunction than those with well-controlled TLE,[2] which is in line with one of the risk factors for SUDEP, that being frequent and severe generalized tonic–clonic seizures.[3] It has been reported that approximately 40% of patients with drug-refractory epilepsy have one or more abnormalities in cardiac function.[4] In addition, heart rate variability is an important indicator for abnormalities in autonomic function, and there are many studies of patients with epilepsy showing autonomic alterations related to epilepsy (reviewed in[5]). Partial and generalized seizures can affect autonomic function during (ictal), immediately after (postictal), and between (interictal) seizures,[6] which may contribute to cardiac arrhythmias.

Recently, abnormalities in cardiac repolarization leading to ventricular fibrillation have been reported in patients with epilepsy. The QT interval on the electrocardiography (ECG) recording is a measure of the duration of ventricular depolarization and repolarization, and shortening or prolongation of the QT interval are established risk factors for cardiac arrhythmias and sudden cardiac death.[7, 8] Transient prolonged corrected QT (QTc) interval has been reported to occur periictally in 10–13% of seizures,[9] whereas shortening of the QTc interval has been associated with generalized tonic–clonic seizures.[10]

Little is known about the underlying molecular mechanisms by which epilepsy can affect cardiac function. One prime candidate for such an epilepsy-associated acquired cardiac channelopathy is the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which have been implicated in the dual pathologies of epilepsy and cardiac dysfunction. Pathologic alterations in cerebral HCN expression and function have been described in human epilepsy patients,[11] and in animal models of both acquired and genetic epilepsies.[11-13] The inward depolarizing current associated with HCN channels, termed Ih in the brain and If in the heart, plays an essential role in the generation and modulation of spontaneous electrical activity in the sinoatrial node in heart[14] and thalamocortical relay neurons in brain.[15] Rodent HCN channels are encoded by four genes (Hcn1-4)[16] and they are abundantly expressed throughout the heart, where they play an essential role in the initiation of spontaneous and rhythmic activity. HCN2 and HCN4 are the major isoforms expressed in the heart.[17] They exhibit differential cardiac region expression profiles, with HCN4 being the major isoform expressed in the sinoatrial node,[17] whereas HCN2 is the major isoform expressed in the atrial and ventricular cardiomyocytes.[17] HCN channels play an essential role in the normal functioning of the heart with studies on HCN knockout mice providing evidence in support of this. HCN2 knockout mice display a complex phenotype that includes absence seizures and cardiac sinus dysrhythmias,[18] whereas mice lacking HCN4, either globally or specifically in the heart, die in utero because of the failure to form functional mature pacemaker cells in the sinoatrial node.[19] In addition, conditional deletion of HCN4 in adulthood causes a dramatic reduction (~75%) in sinoatrial If and cardiac arrhythmia characterized by recurrent sinus pauses.[20] In concordance with these findings in mice, four different mutations have been described in the human HCN4 gene, which lead to altered channel expression and/or biophysical properties, and are associated with sinus bradycardia and complex cardiac arrhythmia.[21, 22] A recent study has also identified specific mutations in the HCN2 and HCN4 genes in three patients with epilepsy who died of SUDEP.[23] Furthermore, alterations in HCN expression and function have also been implicated in human patients with failing hearts[24] and hypertrophic cardiomyopathy.[25]

Despite the importance of HCN channels in cardiac function and reported changes in the epileptic brain, investigations into altered channel expression and function as candidate mechanisms for cardiac dysfunction and SUDEP are lacking. Here, we investigated cardiac function and HCN channel expression in two rat models of epilepsy with contrasting underlying causation: a genetic model—Genetic Absence Epilepsy Rats from Strasbourg (GAERS), and an acquired model—the post–kainic acid–induced status epilepticus (post-SE) model of TLE. We have shown here that both altered cardiac function and reduced expression of HCN channel proteins are associated with models of genetic and acquired epilepsy, and that this phenotype appears to be a consequence of epilepsy development. In addition, during ictal activity in the post-SE model, we found pronounced and dramatic effects on heart rate. This suggests that an acquired change in HCN channel expression could contribute to an increased risk of cardiac arrhythmias.

Materials and Methods

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Biography
  10. Supporting Information

Ethics statement

All procedures on rats were approved by The University of Melbourne Animal Ethics committee (ethics numbers 1011823 and 0707408) and followed the Australian Code of Practice for the care and use of animals for scientific purposes. All surgery and procedures were performed under sodium pentobarbital or neurolept anesthesia and all efforts were made to minimize suffering.

Epilepsy models and tissue collection

Genetic absence epilepsy rats from Strasbourg (GAERS)

GAERS are a well-validated rat model of genetic generalized epilepsy with absence seizures that has been used for epilepsy research for more than two decades. The GAERS strain derives from a Wistar rat colony that was selectively inbred for the presence of spontaneous absence-like seizures with behavioral arrest. Seizures in GAERS begin to emerge around 4–8 weeks of age and are fully developed by 4 months. In parallel a counter strain was bred from the same original colony, the non-epileptic controls (NECs), which does not manifest absence seizures even when followed for >12 months. These two closely related strains represent a uniquely powerful tool to identify neurobiologic factors that are associated with the epilepsy phenotype.

For postmortem molecular analyses, male GAERS and NEC rats bred from our colonies at the University of Melbourne were euthanized at 7 days (preepileptic) (NEC n = 4–10; GAERS n = 3–8), 6 weeks (when seizures are just beginning to manifest) (NEC n = 4–8; GAERS n = 7–9), or 16 weeks (fully epileptic) (NEC n = 9–10; GAERS n = 8–10) by an overdose of sodium pentobarbital (1 ml/kg intraperitoneal [i.p.]; Virbac, Australia) followed by rapid extraction of the heart, and microdissection of the four cardiac chambers. Tissue was snap frozen using liquid nitrogen, and stored at −80°C. Another cohort of 16-week-old NEC (n = 9) and GAERS (n = 11) were used for ex vivo Langendorff ECG recordings (see below).

Post–kainic acid–induced status epilepticus (post-SE)

The post-SE model of acquired TLE was generated by inducing a period of continuous seizure activity (status epilepticus [SE]) in previously nonepileptic rats by administering the glutamatergic agonist, kainic acid (KA). This model reflects many features of TLE in humans, including histopathologic changes in limbic structures, a “latent period” following the initiating insult, and the eventual occurrence of spontaneous seizures. Adult (8–10 week old) male NEC rats received repeated i.p. injections of KA (2.5 mg/kg every 45 min) (Enzo Life Sciences, Farmingdale, NY, U.S.A.) (or saline for control animals) until SE was induced. After 4 h, SE was terminated by injection of 4 mg/kg, i.p., diazepam (Mayne Pharma, Adelaide, South Australia, Australia). In our laboratory, 100% of animals that experienced SE develop spontaneous seizures with an average time of onset of 2.8 weeks.[12] Nine weeks after, SE animals were assigned to either a cohort for ex vivo Langendorff ECG recordings (control n = 10, post-SE n = 9) or a cohort for molecular analysis (control n = 7–10, post-SE n = 5–7), for which animals were culled and cardiac tissue collected as described for GAERS and NEC rats. A third cohort of rats (control n = 11, post-SE n = 8) underwent freely moving in vivo EEG-ECG (electrocardiography) recordings, and subsequently cardiac tissue was collected for molecular analysis.

In vivo electroencephalography and electrocardiography surgery, recording and analysis

Simultaneous in vivo EEG and ECG recordings were acquired from male NEC (n = 5) and GAERS (n = 10) rats (15–16 weeks of age) under neurolept analgesia,[26] and from epileptic post-SE rats (9 weeks post-SE) and saline-treated controls in the unanesthetized freely moving state (see Data S1).

Langendorff isolated rat heart perfusion

The Langendorff-perfused isolated heart preparation was performed as previously described[27] using male NEC (n = 9) and GAERS rats (n = 11) (16 weeks of age) and control (n = 10) and post-SE (n = 9) animals 9 weeks after SE (see Data S1). The primary experimental advantage of the Langendorff preparation for this study was that it removed external modulatory neuronal and hormonal influences on cardiac function, thereby allowing an investigation of whether the intrinsic cardiac electrophysiology was altered. The Langendorff preparation has been used in research for over a century and is still widely used today for many different applications.[28, 29]

Ex vivo ECG recording and analysis

See Data S1.

Quantitative polymerase chain reaction (qPCR)

HCN mRNA transcript levels were determined as described previously[12] using catalogued Taqman gene expression assays from Applied Biosystems (Melbourne, Victoria, Australia) (HCN2 Assay ID Rn01408572_mH; HCN4 Assay ID Rn00572232_m1; GAPDH Assay ID Rn99999916_s1 and β-actin Assay ID Rn00667869_m1) (see Data S1).

Western blotting

Whole cell lysate and Western blotting was performed as described previously[30]; see Data S1.

Statistical analysis

For the cardiac ECG data, qPCR, and Western blotting analysis, comparisons between control and experimental groups were evaluated by a two-tailed Mann-Whitney U test with statistical significance set at p < 0.05. Isoprenaline data were analyzed using two-way repeated-measures analysis of variance (ANOVA) and Bonferroni multiple comparisons post hoc test. All data are presented as mean ± standard error of the mean (SEM).

Results

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Biography
  10. Supporting Information

Genetic and acquired models of epilepsy exhibit altered cardiac electrophysiologic function

Cardiac electrophysiologic function was assessed ex vivo (Langendorff-perfused isolated heart preparation) and in vivo under neurolept anesthesia (interictally) in adult GAERS and NEC rats. Examples of in vivo and ex vivo ECG traces for NEC and GAERS are shown in Figure 1A,B, and an example of an interictal in vivo ECG trace from a GAERS highlighting typical irregular heartbeats is shown in Figure 1C. Cardiac electrophysiologic properties were significantly altered ex vivo in GAERS (n = 11) with slower heart rate (p < 0.05), shorter QRS duration (p < 0.001), longer QTc interval (p < 0.05), and greater standard deviation of RR intervals (sdRR; p < 0.05) compared to NEC rats (n = 9; Table 1). These findings were replicated in vivo, with significance obtained for sdRR interval (p < 0.05, NEC n = 5, and GAERS n = 10; Table 1).

Table 1. Interictal in vivo (under neurolept anesthesia) and ex vivo ECG results from NEC (n = 5–9) and GAERS (n = 10–11)
ECG parameterStrainIn vivoEx vivo
  1. Data expressed as mean ± SEM.

  2. *p < 0.05, ***p < 0.001 two tailed Mann-Whitney U test.

Heart rate (bpm)NEC420.9 ± 12.9279.5 ± 6.4
GAERS400.7 ± 12.4255.9 ± 6.9*
sdRR (msec)NEC2.29 ± 0.713.13 ± 7.2
GAERS27.43 ± 9.1*22.27 ± 6.5*
QRS interval (msec)NEC8.03 ± 1.29.66 ± 0.3
GAERS5.34 ± 0.56.24 ± 0.3***
QTc interval (msec)NEC101.82 ± 6.3107.9 ± 5.1
GAERS110.29 ± 7.0126.55 ± 6.6*
ST interval (msec)NEC30.71 ± 2.240.93 ± 3.8
GAERS35.85 ± 0.943.2 ± 3.4
image

Figure 1. Representative ECG traces. In vivo (A) and ex vivo (B) ECG traces from a NEC and GAERS rat and ex vivo (C) ECG traces from a saline and post-SE rat. (D) Example ECG trace from a GAERS (interictal) rat showing irregular heartbeats.

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To assess whether these changes were specific to the GAERS model of genetic generalized epilepsy, or whether they were also seen an acquired epilepsy model, we examined the same cardiac electrophysiology parameters in the post-SE model of acquired TLE in freely moving, unanesthetized rats and ex vivo (Langendorff-perfused isolated heart preparation). An example of ex vivo ECG trace from a saline and post-SE rat is shown in Figure 1D. Over the week of recording, post-SE rats had 57.1 ± 19.5 seizures with an average duration of 37.71 ± 1.99 s. Good quality ECG data during a seizure was obtained from three rats. In the interictal recordings QTc (p < 0.01 in vivo, p < 0.05 ex vivo), ST intervals (p < 0.01 in vivo and ex vivo), and sdRR intervals (p < 0.01 ex vivo) were significantly elevated in post-SE rats (n = 8–9) compared to saline-treated controls (n = 10–11; Table 2). These data indicate that the changes observed in GAERS were not due solely to a genetic predisposition to cardiac abnormalities, and provide the first evidence that genetic and acquired forms of epilepsy lead to cardiac electrophysiologic dysfunction.

Table 2. Interictal in vivo and ex vivo ECG results from saline (n = 10–11) and post-SE rats (n = 8–9)
ECG parameterTreatmentIn vivoEx vivo
  1. Data expressed as mean ± SEM.

  2. *p < 0.05, **p < 0.01 two tailed Mann-Whitney U test.

Heart rate (bpm)Saline314.2 ± 7.07264.6.6 ± 4.8
Post-SE337.0 ± 11.75276.2 ± 4.0
sdRR (msec)Saline0.28 ± 0.033.9 ± 0.2
Post-SE0.41 ± 0.095.2 ± 0.5*
QRS interval (msec)Saline11.49 ± 0.3912.7 ± 0.4
Post-SE13.05 ± 0.7512.4 ± 0.3
QTc interval (msec)Saline128.8 ± 3.18115.6 ± 3.7
Post-SE169.0 ± 13.60**130.6 ± 5.2*
ST interval (msec)Saline44.86 ± 1.2337.2 ± 1.4
Post-SE58.55 ± 5.06**46.7 ± 2.6**

Further similarities between the cardiac electrophysiologic changes seen in these two models were observed following drug treatment to the isolated heart preparations: the increased beat-to-beat variability seen in isolated hearts from GAERS and post-SE rats was no longer observed after exposure to isoprenaline (10 nm) (Fig. 2). A two-way repeated-measures ANOVA revealed a significant effect of isoprenaline treatment for GAERS compared to NEC (p < 0.05) and for post-SE rats compared to saline controls (p < 0.0001). Isoprenaline treatment resulted in a significant reduction in NEC, GAERS, saline, and post-SE rats. However, post hoc analysis showed no significant difference in sdRR after isoprenaline treatment between NEC and GAERS (Fig. 2A, p > 0.05) or between saline and post-SE rats (Fig. 2B, p > 0.05). Heart rate in the two models increased by a similar magnitude after isoprenaline treatment (NEC 18.4 ± 8.6 bpm vs. GAERS 53 ± 24 bpm, p > 0.05; and saline 122.2 ± 9.0 bpm vs post-SE 104.7 ± 11.7 bpm, p > 0.05).

image

Figure 2. Exposure to isoprenaline normalizes the increase in sdRR interval. sdRR interval was increased in GAERS (A, n = 5) and post-SE (B, n = 9) rats prior to isoprenaline treatment (pre-Iso) compared to NEC (n = 5) and saline (n = 9) controls, respectively (see also Tables 1, 2and). Isoprenaline resulted in a significant reduction in the sdRR intervals in NEC and GAERS (p < 0.05) and in saline and post-SE rats (p < 0.0001) (two-way repeated-measures ANOVA). However, after exposure to 10 nm isoprenaline, no significant difference in the sdRR interval was observed between a subset of NEC versus GAERS or saline versus post-SE rats (post-Iso) (p > 0.05, Bonferroni's multiple comparisons test).

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Changes in heart rate during spontaneous seizures post-SE

Three seizures from each of the three post-SE rats with good quality ECG recordings during seizures were analyzed for changes in heart rate during ictal activity (Fig. 3). A representative simultaneous in vivo EEG/ECG recording from a post-SE rat during ictal activity is shown in Figure 3A. Bradycardia occurs almost immediately after seizure onset for all seizures depicted in Figure 3B–D, except for one seizure, which shows a brief tachycardia after seizure onset (Fig. 3D green trace). Consistent with this, there was a significant increase in the in vivo sdRR interval in post-SE rats during ictal activity (n = 3) compared to interictal activity (n = 8; p < 0.05; Table 3). However, there were no other changes in the QRST complex measures between interictal and ictal periods (Table 3).

Table 3. In vivo ECG results from epileptic post-SE rats during seizure activity (ictal) (n = 3–6) compared to between seizure activity (interictal) (n = 8)
ECG parameter 
  1. Data expressed as mean ± SEM.

  2. *p < 0.05, two tailed Mann-Whitney U test.

Heart rate (bpm) 
Interictal337.0 ± 11.75
Ictal297.7 ± 5.52
sdRR (msec) 
Interictal0.41 ± 0.09
Ictal2.89 ± 0.06*
QRS interval (msec) 
Interictal13.05 ± 0.75
Ictal12.47 ± 0.91
QTc interval (msec) 
Interictal169.0 ± 13.60
Ictal161.8 ± 5.71
ST interval (msec) 
Interictal58.55 ± 5.06
Ictal60.85 ± 2.52
image

Figure 3. Changes in heart rate during ictal activity in post-SE rats. (A) Representative EEG/ECG trace from a post-SE rat during ictal activity (also represented by the red trace in B). The point of seizure onset is based on visual identification of motor seizures with changes on EEG occurring later (as indicated with *). Bradycardia occurs at the beginning of the seizure, followed by a tachycardia, which continues into the postictal stage, which is supported by (B), where an elevated heart rate above baseline is evident even after the seizure ends. (B-D) Changes in heart rate during three randomly selected seizures (indicated as different colors) from three post-SE rats. Bradycardia occurs almost immediately after seizure onset (at 0 s) for all seizures depicted except for the green trace in rat 3 (D), which shows a brief tachycardia after seizure onset. ● indicates the end of the seizure.

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Genetic and acquired models of epilepsy exhibit altered cardiac HCN mRNA and protein expression

To investigate a possible molecular mechanism explaining the observed alterations in cardiac function, we analyzed HCN mRNA and protein expression in cardiac tissue. Adult epileptic GAERS (n = 8–10) showed cardiac chamber and HCN channel isoform-specific reductions in HCN expression, compared to NEC (n = 9–10) (Fig. 4). HCN2 mRNA expression was significantly decreased by 20% and 40% in the left (Fig. 4A, p < 0.01) and right (Fig. 4B, p < 0.01) atria, respectively, and by 18% in the left (Fig. 4C, p < 0.05) and right (Fig. 4D, p = 0.065) ventricle in GAERS compared to NEC. HCN4 mRNA expression in GAERS also showed significant reductions of 33% and 53% in the left (Fig. 4C, p < 0.05) and right (Fig. 4D, p < 0.001) ventricles, respectively, but no change in the left or right atria. To determine if reduced mRNA translated to a decrease in protein, Western blotting for HCN2 and HCN4 was performed on left ventricular tissue, and representative blots are shown in Fig. 4E,F. HCN2 protein expression was decreased by 24% (p < 0.01) in GAERS, but HCN4 expression was not significantly changed compared to NEC rats (n = 8–9; Fig. 4G). Changes in HCN mRNA expression were also evident in 6 week old GAERS (an age when seizures begin to be expressed in GAERS), compared to NEC rats: HCN2 mRNA expression was significantly reduced in the right atria (NEC 1.0 ± 0.07 vs. GAERS 0.65 ± 0.07, p < 0.05), left ventricle (NEC 1.0 ± 0.07 vs. GAERS 0.81 ± 0.05, p < 0.05), and right ventricle (NEC 1.0 ± 0.07 vs. GAERS 0.72 ± 0.03, p < 0.01). HCN4 mRNA expression also showed a significant reduction in the right ventricle (NEC 1.3 ± 0.1 vs. GAERS 0.45 ± 0.03 p < 0.001). However, 7-day-old GAERS that are not yet experiencing spontaneous absence seizures do not show a reduction in HCN2 or HCN4 mRNA expression in any of the cardiac chambers compared to NEC rats (data not shown).

image

Figure 4. HCN mRNA and protein expression is decreased in the heart of adult epileptic GAERS. HCN2 and HCN4 mRNA expression was measured using qPCR in the left atria (A), right atria (B), left ventricle (C), and right ventricle (D). HCN2 mRNA expression was significantly reduced in the left and right atria and left ventricle, whereas HCN4 mRNA expression was significantly reduced in the left and right ventricles of adult epileptic GAERS (n = 8–10) compared to NEC rats (n = 9–10). Representative Western blot of HCN2 protein (60 kDa) (E) and HCN4 protein (134kDa) (F) normalized to tubulin (50 kDa). N=NEC; G=GAERS. (G) Western blot analysis showed a significant reduction in HCN2 protein expression in the left ventricle of adult epileptic GAERS (n = 9) compared to NEC rats (n = 8–9). *p < 0.05, **p < 0.01, ***p < 0.001 two tailed Mann-Whitney U test.

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To establish whether similar cardiac abnormalities were associated with the same molecular correlates across the different epilepsy models, we next probed cardiac chambers from post-SE rats for HCN expression alterations. HCN2 mRNA expression was significantly decreased by 20% in the right atria (Fig. 5B) and left ventricle (Fig. 5C) and by 40% in the left atria (Fig. 5A) in post-SE rats (n = 5–7) compared to controls (n = 9–10; p < 0.05 for all chambers), whereas HCN4 mRNA expression was not altered in any chamber. Western blotting for HCN2 and HCN4 was performed on left ventricular tissue, and representative blots are shown in Figure 5E,F. HCN2 protein expression was similarly decreased in the left ventricle of post-SE rats (p < 0.05; Fig. 5G), showing concordance between the extent of reductions in mRNA (18%) and protein (22%). These changes were replicated in the third cohort of post-SE versus saline-treated control rats (data not shown).

image

Figure 5. HCN2 mRNA and protein expression is decreased in the heart of post-SE rats. HCN2 and HNC4 mRNA expression was measured using qPCR in the left atria (A), right atria (B), left ventricle (C), and right ventricle (D). There was a significant decrease in HCN2 mRNA expression in the left and right atria and left ventricle, whereas HCN4 mRNA expression was unaltered in all four cardiac chambers of post-KA induced SE (n = 5–7) rats compared to saline-treated rats (n = 9–10). Representative Western blot of HCN2 protein (60 kDa) (E) and HCN4 protein (134kDa) (F) normalized to tubulin (50 kDa). SE = post-SE; S = saline. (G) Western blot analysis showed a significant reduction in HCN2 protein expression in the left ventricle of post-SE rats (n = 7) compared to saline-treated rats (n = 7). *p < 0.05 two tailed Mann-Whitney U test.

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Discussion

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Biography
  10. Supporting Information

Cardiac dysfunction is a complex and important problem for people with epilepsy, and may have relevance for SUDEP, a major cause of mortality in patients with epilepsy.[1] Alterations in function or expression of ion channels that modulate cellular excitability in both cerebral and cardiac tissue represent strong candidate mechanisms for cardiac dysfunction in patients with epilepsy. The pacemaker function of the sinoatrial node is essential for normal cardiac physiology and the cellular mechanism underlying the generation of automaticity in pacemaker cells has not been completely clarified. Voltage-dependent ionic currents that have been identified in cardiac pacemaker cells include the Na+ current,[31] L- and T-type Ca2+ currents,[32] HCN If current,[14] and various types of delayed rectifier K+ currents.[33] Although there is still debate about the specific contributions of these different ion channels to the pacemaking of the sinoatrial node, there is now general agreement that the If current plays a major role in the generation and control of pacemaker activity.[14] HCN channels and its Ih current also modulate cellular excitability in the brain, and brain alterations in HCN channel expression and function have been reported from patients with epilepsy[11] and from animal models of epilepsy.[11-13] It is important to note that specific mutations in the HCN2 and HCN4 genes have recently been identified in patients who died of SUDEP, potentially implicating these channels in the pathophysiology of fatal cardiac arrhythmias in patients with epilepsy.[23] Here we show for the first time, in two contrasting rat models of epilepsy, the development of pathologic alterations in cardiac electrophysiologic properties coupled with abnormalities in HCN ion channel expression as an acquired consequence of the development of the epilepsy. These data suggest the existence of a common cardiac phenotypic trait broadly applicable across epilepsy types, and provides greater understanding of the molecular causation underlying cardiac dysfunction in epilepsy.

Strikingly consistent molecular and cardioelectrophysiologic results were observed across the epilepsy models studied, despite their different etiology and seizure types, with both exhibiting downregulation of HCN channels (in particular the HCN2 isoform) and both showing similar cardiac phenotypes, consisting of prolonged QT intervals and increased variability of heart rate. Prolonged QTc interval is a clinical characteristic of “long QT syndrome,” an arrhythmogenic disorder commonly caused by ion channel mutations in which delayed repolarization of the heart increases the propensity for episodes of ventricular tachyarrhythmias leading to sudden death.[7] Our finding of longer QTc intervals in epileptic rats raises the possibility that this may also be a mechanism for SUDEP in humans with epilepsy. Patients with chronic drug-resistant epilepsies undergoing simultaneous video-EEG and cardiac monitoring have been reported to not uncommonly have prolongation of the QTc intervals, both during and between seizures,[9] and an increased QTc interval has also been reported in epilepsy patients who later died of SUDEP.[34] Likewise, alterations in heart rate variability are suggestive of faulty regulation of autonomic function, and could also predispose to vulnerability to cardiac dysfunction. Adult epileptic GAERS and post-SE animals have an increased beat-to-beat variability as evident by the increased standard deviation of RR intervals, which was normalized by stimulation of cardiac β-adrenergic receptors by isoprenaline. Isoprenaline activation of cardiac β-adrenergic receptors generates cyclic AMP (cAMP), which binds to HCN channels allowing the channels to open at more depolarized potentials, resulting in a more rapid and complete opening of the HCN channel, thereby normalizing the abnormal rhythm and increasing heart rate. It should be acknowledged that the increased RR interval in the epileptic animals could also be a result of their lower baseline heart rate, rather than being an independent marker of increased arrhythmicity, and the isoprenaline effect to normalize the RR interval being a nonspecific result of its effect to increase the heart rate. It is notable that we have shown that there is increased beat-to-beat variability during seizure activity as compared to between seizure activity, which is evident by the changes that occur in heart rate during a spontaneous seizure (Fig. 3). This is the first report showing changes in heart rate occurring during spontaneous seizures in a rat model of acquired epilepsy. Bradycardia has also been reported to occur during induced status epilepticus in rats.[35] Tachycardia is the most common cardiac change occurring during seizures in patients with epilepsy; however, bradycardia has also been reported to occur in around 2% of patients, and can result in syncopal loss of consciousness.[36] Extreme and frequent changes in cardiac physiology during seizures, whether it is tachycardia or bradycardia, may significantly affect the normal functioning of the heart over time.

Although this study does not prove a causative relationship between the HCN expression changes and the aberrant cardiac electrophysiologic function observed in the epileptic rats, support for this comes from the HCN2 knockout mouse.[18] A complete lack of HCN2 channels results in spontaneous epilepsy and cardiac sinus dysrhythmia, and mirrors the cardiac abnormalities reported in the two epilepsy models here. This includes significantly increased sdRR in the mutants, and normalization of heart rate variability with isoprenaline treatment (QT intervals were not assessed in the HCN2 knockout mouse). The electrophysiologic finding with the most potential clinical significance found in these chronically epileptic rats was the prolonged QTc interval. Although we have demonstrated an association between decreases in cardiac HCN channel expression and prolongation of the QTc interval in vivo and in vitro in hearts of chronically epileptic animals, we have not yet proven a direct causative relationship between them. However, there is rationale for how changes in HCN expression could alter the QT interval. The QT interval represents the time for both ventricular depolarization and repolarization. The QRS complex represents the ventricular depolarization process, whereas the T-wave represents the ventricular repolarization process. Although ventricular repolarization is determined primarily by potassium outward currents, recently HCN channels have been implicated as important players in modulating ventricular repolarization.[37, 38] In addition, MinK-related peptide 1 is a protein that can interact with HCN channels,[39] and mutations in this peptide are associated with long QT syndrome.[40]

Recently there has been considerable interest in the possibility that genetic mutations in ion channels that are expressed in the heart and the brain may cause both the epilepsy and a predisposition to cardiac dysrhythmias, providing a potential mechanistic explanation for SUDEP.[23, 41, 42] However, it is patients with acquired structural/metabolic epilepsies who are more often drug resistant, who are at the greatest risk of SUDEP,[3] and therefore a primarily genetically determined ion channelopathy causing both the epilepsy and a predisposition to cardiac arrhythmias is unlikely to be a common mechanism for SUDEP. The cardiac HCN channel expression changes and electrophysiologic dysfunction demonstrated here appear to be acquired consequences in both models studied. Furthermore, although GAERS are a genetic model of epilepsy, the HCN expression changes in the heart are evident at the onset of the epilepsy (6 weeks old), whereas these changes are not being seen in young animals that have not yet developed seizures (7 days old). The temporal evolution of such changes, the similarities to the post-SE model, and the supporting phenotype of the HCN2 knockout mouse all suggest that the cardiac changes in GAERS are also acquired consequences of the epilepsy. It is interesting to note that decreases in HCN expression have been reported in the brains of both animal models, but only after the development of the epilepsy.[13] This indicates that the HCN changes in both the heart and the brain in these rat models are likely to be a secondary consequence of the recurrent epileptic seizures.

It is interesting to speculate how acquiring a neurologic disorder may influence cardiac ion channel expression and electrophysiologic function. During and following seizures there are profound changes in heart rate and QRS morphology, which are believed to be driven by the major changes in autonomic nervous system output that occur in association with a seizure.[4] In addition, in patients with chronic epilepsy there are interictal autonomic changes that are more common than in those with recent-onset epilepsy.[5] It is therefore possible that repeated exposure of the heart to severe autonomic stresses during seizures, and chronic autonomic dysfunction during seizures, result in secondary cardiac changes,[43] such as plastic changes in HCN expression in the heart, and then ultimately in electrophysiologic changes that predispose to cardiac arrhythmias. Interventional longitudinal experiments will be required to establish the nature of this causative relationship.

Investigating the mechanism by which HCN2 expression is repressed in the heart of animals with acquired and genetic epilepsy will potentially open up avenues for therapeutic interventions. If indeed HCN2 repression is linked to detrimental cardiac function, as our data suggest, then preventing or reversing this protein repression may prove beneficial for restoring normal cardiac function. Neuronal restrictive silencer factor (NRSF) binds to its recognition sequence (neuron restrictive silencer elements [NRSEs]) that are present on hundreds of neuronal genes, including HCN2,[44] to suppress transcription. Blocking the binding of NRSF to NRSEs in the Hcn2 gene, as has been shown for the Hcn1 gene in an epilepsy setting,[45] may prevent the transcriptional downregulation of HCN2 and associated cardiac problems.

In conclusion, we have shown here that both altered cardiac electrophysiology and reduced expression of HCN channel proteins are present in rat models of genetic and acquired epilepsy, and that this phenotype appears to be a consequence of epilepsy development. This suggests that acquired changes in HCN channel expression may also be present in humans with epilepsy, and this could underlie cardiac dysfunction that is an important problem in patients with epilepsy, including potentially being a contributory mechanistic factor to SUDEP.

Acknowledgments

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Biography
  10. Supporting Information

We wish to thank Ashleigh Hicks for technical assistance with RNA extractions and Emma Braine and Gil Rind for performing the EEG and ECG electrode implantation surgeries. This work was supported in part by a NH&MRC Project Grant to Kim Powell (#628723) and also to Christopher Reid and Terence O'Brien (#1009142).

Disclosure

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Biography
  10. Supporting Information

None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

References

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Biography
  10. Supporting Information

Biography

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Biography
  10. Supporting Information
  • Image of creator

    Dr. Kim Powell heads the Molecular Epilepsy Group in The Department of Medicine at The University of Melbourne, Australia.

Supporting Information

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Biography
  10. Supporting Information
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