Edward Glasscock

Arrhythmia in Heart and Brain: KCNQ1 Mutations Link Epilepsy and Sudden Unexplained Death

Mice engineered to carry a human mutation that causes heart problems also have epilepsy, suggesting a cause of SUDEP, sudden unexplained death in epilepsy. Patients with epilepsy face an extra frightening burden. Occasionally, otherwise healthy individuals with this disease die unexpectedly for no apparent cause. The incidence of sudden death is ~10% for epilepsy patients—a risk far greater than that faced by a non-epileptic person. Sudden unexplained death in epilepsy (SUDEP) frequently follows a seizure, and patients who experience many seizures have a greater risk of SUDEP. But the causes of SUDEP remain a mystery. Now, Goldman et al. shed new light on how defective potassium channels contribute to this syndrome. Various causes of SUDEP have been proposed—some that produce irreversible cardiac dysfunction and some that produce respiratory distress. One suggested cause invokes the common dependence of the heart and brain on electrical activity for proper functioning. When ion channels—the membrane proteins that control electrical activity—go awry (by gene mutation or a drug), the brain becomes uncontrollably excited, producing a seizure, during which the regular beating of the heart is disrupted and can cease altogether. Both people and a mouse model display mutations in the KCNQ1 gene—which encodes a potassium channel in the heart—that give rise to heartbeat abnormalities and a higher risk for sometimes fatal arrhythmias. Goldman et al. studied KCNQ1-mutant mice and found that this same potassium channel that causes problems in the heart is also present in neurons in the brain and is in particularly high abundance in regions that are susceptible to epilepsy. A closer look at the brains of these mice disclosed that their electrical discharges display abnormalities characteristic of epilepsy and that these aberrations often occur at the same time as abnormal heartbeats. Continuous video surveillance of these mice revealed that many also experienced overt seizures. In one instance, a mouse with the mutant ion channel suffered increasingly frequent seizures accompanied by irregular abnormal cardiac activity and ultimately went into cardiac arrest, a mouse version of SUDEP. Taken together, these results reinforce hints in the literature that SUDEP may result from common excitability defects in the brain and heart. Cardiac abnormalities that resemble those in the mutant mice can be caused in humans by mutations in ~10 genes. The ability to screen epilepsy patients for these mutations would allow those who are at risk for cardiac-induced sudden death to take preventive measures. Sudden unexplained death is a catastrophic complication of human idiopathic epilepsy, causing up to 18% of patient deaths. A molecular mechanism and an identified therapy have remained elusive. Here, we find that epilepsy occurs in mouse lines bearing dominant human LQT1 mutations for the most common form of cardiac long QT syndrome, which causes syncopy and sudden death. KCNQ1 encodes the cardiac KvLQT1 delayed rectifier channel, which has not been previously found in the brain. We have shown that, in these mice, this channel is found in forebrain neuronal networks and brainstem nuclei, regions in which a defect in the ability of neurons to repolarize after an action potential, as would be caused by this mutation, can produce seizures and dysregulate autonomic control of the heart. That long QT syndrome mutations in KCNQ1 cause epilepsy reveals the dual arrhythmogenic potential of an ion channelopathy coexpressed in heart and brain and motivates a search for genetic diagnostic strategies to improve risk prediction and prevention of early mortality in persons with seizure disorders of unknown origin.

Kv1.1 Potassium Channel Deficiency Reveals Brain-Driven Cardiac Dysfunction as a Candidate Mechanism for Sudden Unexplained Death in Epilepsy

Mice lacking Kv1.1 Shaker-like potassium channels encoded by the Kcna1 gene exhibit severe seizures and die prematurely. The channel is widely expressed in brain but only minimally, if at all, in mouse myocardium. To test whether Kv1.1-potassium deficiency could underlie primary neurogenic cardiac dysfunction, we performed simultaneous video EEG–ECG recordings and found that Kcna1-null mice display potentially malignant interictal cardiac abnormalities, including a fivefold increase in atrioventricular (AV) conduction blocks, as well as bradycardia and premature ventricular contractions. During seizures the occurrence of AV conduction blocks increased, predisposing Kv1.1-deficient mice to sudden unexplained death in epilepsy (SUDEP), which we recorded fortuitously in one animal. To determine whether the interictal AV conduction blocks were of cardiac or neural origin, we examined their response to selective pharmacological blockade of the autonomic nervous system. Simultaneous administration of atropine and propranolol to block parasympathetic and sympathetic branches, respectively, eliminated conduction blocks. When administered separately, only atropine ameliorated AV conduction blocks, indicating that excessive parasympathetic tone contributes to the neurocardiac defect. We found no changes in Kv1.1-deficient cardiac structure, but extensive Kv1.1 expression in juxtaparanodes of the wild-type vagus nerve, the primary source of parasympathetic input to the heart, suggesting a novel site of action leading to Kv1.1-associated cardiac bradyarrhythmias. Together, our data suggest that Kv1.1 deficiency leads to impaired neural control of cardiac rhythmicity due in part to aberrant parasympathetic neurotransmission, making Kcna1 a strong candidate gene for human SUDEP.

Masking epilepsy by combining two epilepsy genes

Inherited errors in ion channel genes comprise the largest subset of monogenic causes of idiopathic epilepsy, and pathogenic variants contribute to genetic risk in the complex inheritance of this common disorder. We generated a digenic mouse model of human idiopathic epilepsy by combining two epilepsy-associated ion channel mutations with mutually opposing excitability defects and overlapping subcellular localization. We found that increasing membrane excitability by removing Shaker-like K+ channels, which are encoded by the Kcna1 gene, masked the absence epilepsy caused by a P/Q-type Ca2+ channelopathy due to a missense mutation in the Cacna1a gene. Conversely, decreasing network excitability by impairing Cacna1a Ca2+-channel function attenuated limbic seizures and sudden death in Kcna1-null mice. We also identified intermediate excitability phenotypes at the network and axonal levels. Protective interactions between pathogenic ion channel variants may markedly alter the clinical expression of epilepsy, highlighting the need for comprehensive profiling of this candidate gene set to improve the accuracy of genetic risk assessment of this complex disease.

Genomic biomarkers of SUDEP in brain and heart

Sudden unexpected death in epilepsy (SUDEP) is the leading cause of epilepsy-related mortality, but how to predict which patients are at risk and how to prevent it remain uncertain. The underlying pathomechanisms of SUDEP are still largely unknown, but the general consensus is that seizures somehow disrupt normal cardiac or respiratory physiology leading to death. However, the proportion of SUDEP cases exhibiting cardiac or respiratory dysfunction as a critical factor in the terminal cascade of events remains unresolved. Although many general risk factors for SUDEP have been identified, the development of reliable patient-specific biomarkers for SUDEP is needed to provide more accurate risk prediction and personalized patient management strategies. Studies in animal models and patient groups have revealed at least nine different brain-heart genes that may contribute to a genetic susceptibility for SUDEP, making them potentially useful as genomic biomarkers. This review summarizes data on the relationship between these neurocardiac genes and SUDEP, discussing their brain-heart expression patterns and genotype-phenotype correlations in mouse models and people with epilepsy. These neurocardiac genes represent good first candidates for evaluation as genomic biomarkers of SUDEP in future studies. The development of validated reliable genomic biomarkers for SUDEP has the potential to transform the clinical treatment of epilepsy by pinpointing patients at risk of SUDEP and allowing optimized, genotype-guided therapeutic and prevention strategies.

Transcompartmental reversal of single fibre hyperexcitability in juxtaparanodal Kv1.1-deficient vagus nerve axons by activation of nodal KCNQ channels

•  Voltage‐gated Kv1.1 potassium channels cluster at juxtaparanodes of myelinated axons in the vagus nerve, which provides parasympathetic innervation to the heart. •  Kcna1 knockout mice lacking Kv1.1 channels exhibit frequent atrioventricular cardiac conduction blocks that are abolished by atropine, suggestive of a vagal mechanism. •  Electrophysiological analysis of single myelinated axons from wild‐type and Kv1.1‐deficient mouse vagus nerves revealed that the absence of Kv1.1 channels rendered large myelinated vagal axons far more susceptible to spontaneous ectopic firing in the presence of 4‐aminopyridine. •  KCNQ2 potassium channels are present within vagal nodes of Ranvier and their activation with flupirtine rescued single axon hyperexcitability mediated by juxtaparanodal Kv1.1‐deficiency. •  These results demonstrate a functional synergy between nodal and extranodal K+ channels and implicate KCNQ channels as potential targets for Kv1‐related peripheral nerve hyperexcitability.

Expression and function of Kv1.1 potassium channels in human atria from patients with atrial fibrillation

Voltage-gated Kv1.1 channels encoded by the Kcna1 gene are traditionally regarded as being neural-specific with no known expression or intrinsic functional role in the heart. However, recent studies in mice reveal low-level Kv1.1 expression in heart and cardiac abnormalities associated with Kv1.1-deficiency suggesting that the channel may have a previously unrecognized cardiac role. Therefore, this study tests the hypothesis that Kv1.1 channels are associated with arrhythmogenesis and contribute to intrinsic cardiac function. In intra-atrial burst pacing experiments, Kcna1-null mice exhibited increased susceptibility to atrial fibrillation (AF). The atria of Kcna1-null mice showed minimal Kv1 family ion channel remodeling and fibrosis as measured by qRT-PCR and Masson’s trichrome histology, respectively. Using RT-PCR, immunocytochemistry, and immunoblotting, KCNA1 mRNA and protein were detected in isolated mouse cardiomyocytes and human atria for the first time. Patients with chronic AF (cAF) showed no changes in KCNA1 mRNA levels relative to controls; however, they exhibited increases in atrial Kv1.1 protein levels, not seen in paroxysmal AF patients. Patch-clamp recordings of isolated human atrial myocytes revealed significant dendrotoxin-K (DTX-K)-sensitive outward current components that were significantly increased in cAF patients, reflecting a contribution by Kv1.1 channels. The concomitant increases in Kv1.1 protein and DTX-K-sensitive currents in atria of cAF patients suggest that the channel contributes to the pathological mechanisms of persistent AF. These findings provide evidence of an intrinsic cardiac role of Kv1.1 channels and indicate that they may contribute to atrial repolarization and AF susceptibility.

Scn2a deletion improves survival and brain–heart dynamics in the Kcna1-null mouse model of sudden unexpected death in epilepsy (SUDEP)

People with epilepsy have greatly increased probability of premature mortality due to sudden unexpected death in epilepsy (SUDEP). Identifying which patients are most at risk of SUDEP is hindered by a complex genetic etiology, incomplete understanding of the underlying pathophysiology and lack of prognostic biomarkers. Here we evaluated heterozygous Scn2a gene deletion (Scn2a+/-) as a protective genetic modifier in the Kcna1 knockout mouse (Kcna1-/-) model of SUDEP, while searching for biomarkers of SUDEP risk embedded in electroencephalography (EEG) and electrocardiography (ECG) recordings. The human epilepsy gene Kcna1 encodes voltage-gated Kv1.1 potassium channels that act to dampen neuronal excitability whereas Scn2a encodes voltage-gated Nav1.2 sodium channels important for action potential initiation and conduction. SUDEP-prone Kcna1-/- mice with partial genetic ablation of Nav1.2 channels (i.e. Scn2a+/-; Kcna1-/-) exhibited a two-fold increase in survival. Classical analysis of EEG and ECG recordings separately showed significantly decreased seizure durations in Scn2a+/-; Kcna1-/- mice compared with Kcna1-/- mice, without substantial modification of cardiac abnormalities. Novel analysis of the EEG and ECG together revealed a significant reduction in EEG-ECG association in Kcna1-/- mice compared with wild types, which was partially restored in Scn2a+/-; Kcna1-/- mice. The degree of EEG-ECG association was also proportional to the survival rate of mice across genotypes. These results show that Scn2a gene deletion acts as protective genetic modifier of SUDEP and suggest measures of brain-heart association as potential indices of SUDEP susceptibility.

Spontaneous seizures in Kcna1-null mice lacking voltage-gated Kv1.1 channels activate Fos expression in select limbic circuits.

Mice lacking voltage‐gated Kv1.1 channels as a result of deletion of the Kcna1 gene are an extensively utilized genetic model of human epilepsy and sudden unexpected death in epilepsy because of their frequent seizures and genotypic–phenotypic similarity to the human condition. Ictal behaviors, electrophysiological recordings, and gene expression studies suggest limbic circuits are critical for epilepsy in Kcna1‐null mice, but the exact brain networks recruited by seizures remain unknown. In this study, Fos protein expression patterns were used to map limbic brain regions with increased neuronal activity at baseline and during spontaneous seizures in Kcna1‐null mice by comparing seizing and non‐seizing knockouts and wild‐type controls. Basal Fos levels were unchanged in non‐seizing knockout mice compared to wild types for all brain regions examined except the dentate gyrus granule cell layer which exhibited a significant decrease in Fos‐positive cells. Following seizures, Kcna1‐null brains exhibited significantly increased Fos labeling in the basolateral amygdala and the dentate hilus region, but not in other principal cell layers of the hippocampal formation. The selective Fos activation in the amygdala following seizures suggests that extra hippocampal limbic circuits may be critically involved with seizure generation or spread in Kcna1‐null mice.

Gradient Index Microlens Implanted in Prefrontal Cortex of Mouse Does Not Affect Behavioral Test Performance over Time.

Implanted gradient index lenses have extended the reach of standard multiphoton microscopy from the upper layers of the mouse cortex to the lower cortical layers and even subcortical regions. These lenses have the clarity to visualize dynamic activities, such as calcium transients, with subcellular and millisecond resolution and the stability to facilitate repeated imaging over weeks and months. In addition, behavioral tests can be used to correlate performance with observed changes in network function and structure that occur over time. Yet, this raises the questions, does an implanted microlens have an effect on behavioral tests, and if so, what is the extent of the effect? To answer these questions, we compared the performance of three groups of mice in three common behavioral tests. A gradient index lens was implanted in the prefrontal cortex of experimental mice. We compared their performance with mice that had either a cranial window or a sham surgery. Three presurgical and five postsurgical sets of behavioral tests were performed over seven weeks. Behavioral tests included rotarod, foot fault, and Morris water maze. No significant differences were found between the three groups, suggesting that microlens implantation did not affect performance. The results for the current study clear the way for combining behavioral studies with gradient index lens imaging in the prefrontal cortex, and potentially other regions of the mouse brain, to study structural, functional, and behavioral relationships in the brain.

Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can potentially increase risk of sudden unexpected death in epilepsy (SUDEP). Electroencephalography (EEG) and electrocardiography (ECG) are widely used clinical diagnostic tools to monitor for abnormal brain and cardiac rhythms in patients. Here, a technique to simultaneously record video, EEG, and ECG in mice to measure behavior, brain, and cardiac activities, respectively, is described. The technique described herein utilizes a tethered (i.e., wired) recording configuration in which the implanted electrode on the head of the mouse is hard-wired to the recording equipment. Compared to wireless telemetry recording systems, the tethered arrangement possesses several technical advantages such as a greater possible number of channels for recording EEG or other biopotentials; lower electrode costs; and greater frequency bandwidth (i.e., sampling rate) of recordings. The basics of this technique can also be easily modified to accommodate recording other biosignals, such as electromyography (EMG) or plethysmography for assessment of muscle and respiratory activity, respectively. In addition to describing how to perform the EEG-ECG recordings, we also detail methods to quantify the resulting data for seizures, EEG spectral power, cardiac function, and heart rate variability, which we demonstrate in an example experiment using a mouse with epilepsy due to Kcna1 gene deletion. Video-EEG-ECG monitoring in mouse models of epilepsy or other neurological disease provides a powerful tool to identify dysfunction at the level of the brain, heart, or brain-heart interactions.