Circulation: Genomics and Precision Medicine, 2018
BACKGROUND: Paroxysmal atrial fibrillation (AF) can be caused by gain-of-
function mutations in g... more BACKGROUND: Paroxysmal atrial fibrillation (AF) can be caused by gain-of- function mutations in genes, encoding the cardiac potassium channel subunits KCNJ2, KCNE1, and KCNH2 that mediate the repolarizing potassium currents Ik1, Iks, and Ikr, respectively. METHODS: Linkage analysis, whole-exome sequencing, and Xenopus oocyte electrophysiology studies were used in this study. RESULTS: Through genetic studies, we showed that autosomal dominant early-onset nocturnal paroxysmal AF is caused by p.S447R mutation in KCND2, encoding the pore-forming (α) subunit of the Kv4.2 cardiac potassium channel. Kv4.2, along with Kv4.3, contributes to the cardiac fast transient outward K+ current, Ito. Ito underlies the early phase of repolarization in the cardiac action potential, thereby setting the initial potential of the plateau phase and governing its duration and amplitude. In Xenopus oocytes, the mutation increased the channel’s inactivation time constant and affected its regulation: p.S447 resides in a protein kinase C (PKC) phosphorylation site, which normally allows attenuation of Kv4.2 membrane expression. The mutant Kv4.2 exhibited impaired response to PKC; hence, Kv4.2 membrane expression was augmented, enhancing potassium currents. Coexpression of mutant and wild-type channels (recapitulating heterozygosity in affected individuals) showed results similar to the mutant channel alone. Finally, in a hybrid channel composed of Kv4.3 and Kv4.2, simulating the mature endogenous heterotetrameric channel underlying Ito, the p.S447R Kv4.2 mutation exerted a gain-of-function effect on Kv4.3. CONCLUSIONS: The mutation alters Kv4.2’s kinetic properties, impairs its inhibitory regulation, and exerts gain-of-function effect on both Kv4.2 homotetramers and Kv4.2-Kv4.3 heterotetramers. These effects presumably increase the repolarizing potassium current Ito, thereby abbreviating action potential duration, creating arrhythmogenic substrate for nocturnal AF. Interestingly, Kv4.2 expression was previously shown to demonstrate circadian variation, with peak expression at daytime in murine hearts (human nighttime), with possible relevance to the nocturnal onset of paroxysmal AF symptoms in our patients. The atrial-specific phenotype suggests that targeting Kv4.2 might be effective in the treatment of nocturnal paroxysmal AF, avoiding adverse ventricular effects.
Organophosphates (OP) are used extensively as pesticides and as chemical weapons. Cardiotoxicity ... more Organophosphates (OP) are used extensively as pesticides and as chemical weapons. Cardiotoxicity is a major concern in survivors of the acute poisoning. To characterize the delayed cardiac effects of OP, rats were poisoned by intraperitoneal administration of dichlorvos. In group I, poisoning (0.25-, 0.75-, 1.4-LD 50) was followed by application of atropine and obidoxime. In group II, poisoning (0.35-, 0.5-LD 50) was done without antidotes. Cardiac evaluation included electrocardiography and echocardiog-raphy 2-and 6-week post-exposure, arrhythmia susceptibility following administration of Isoproterenol (150 mcg/ kg), and histological evaluation. All poisoned animals displayed cholinergic symptoms. In group I, all animals exposed to 1.4-LD 50 (n = 3) had profound convulsions and died despite antidote treatment. However, in the lower doses, all animals survived and no cardiac abnormalities were noted during follow-up. In group II, six animals had convulsions and died. Surviving animals had mild but significant pro-longation of corrected QT at both 2 and 6 weeks, compared to shams. There were no notable echocardiographic, gravimetric , or histological differences between poisoned and sham animals. Our data indicate that dichlorvos poisoning is associated with QT prolongation without anatomical or histopathological abnormalities. This new model can be used to elaborate the molecular mechanism\s of QT prolongation following OP poisoning.
Biventricular pacing is an important modality to improve left ventricular (LV) synchronization an... more Biventricular pacing is an important modality to improve left ventricular (LV) synchronization and long-term function. However, the biological effects of this treatment are far from being elucidated and existing animal models are limited and demanding. Recently, we introduced an implanted device for double-site epicardial pacing in rats and echocardiographically demonstrated favorable effects of LV and biventricular (LV-based) pacing modes typically observed in humans. Here, this new animal model was further characterized. Electrodes were implanted either on the right atria (RA) and right ventricle (RV) or on the RV and LV. Following recovery, rats were either used for invasive hemodynamic measurements (pressure-volume analysis) or exposed to sustained RV vs. biventricular tachypacing for 3 days. RV pacing compromised, while LV-based pacing modes markedly enhanced cardiac performance. Changes in LV performance were associated with prominent compensatory changes in arterial resistance. Sustained RV tachypacing increased the electrocardiogram QTc interval by 7.9 ± 3.1 ms (n = 6, p < 0.05), dispersed refractoriness between the right and left pacing sites and induced important molecular changes mainly in the early-activated septal tissue. These effects were not observed during biventricular tachypacing (n = 6). Our results demonstrate that the rat is an attractive new model to study the biological consequences of LV dyssynchrony and resynchronization. The electromechanical and molecular consequences of various ventricular pacing modes have become a subject of intense interest in recent years due to profound clinical implications 1–3. Compelling evidence indicates that right ventricular (RV) pacing can be detrimental to left ventricular (LV) function. The sequence of electrical activation during RV pacing may largely resemble the activation pattern of left bundle branch block (LBBB) 4 , and can result in dyssynchronous LV contraction 5, 6. In accordance, RV pacing increases the risk for heart failure (HF) even in patients without pre-existing LV dysfunction 7–10. Cardiac resynchronization therapy (CRT) by means of biventricular (BiV) pacing was developed as a strategy to overcome the deleterious effects of electromechanical dyssynchrony in the failing heart and represents a major advance in HF therapy 11. BiV pacing has been demonstrated to improve contractile performance in patients with
Etzion Y, Mor A, Grossman Y. Differential modulation of cerebellar climbing fiber and parallel fi... more Etzion Y, Mor A, Grossman Y. Differential modulation of cerebellar climbing fiber and parallel fiber synaptic responses at high pressure. High pressure, which induces central nervous system (CNS) dysfunction (highpressure neurological syndrome) depresses synaptic transmission at all synapses examined to date. Several lines of evidence indicate an inhibitory effect of pressure on Ca 2ϩ entry into the presynaptic terminal. In the present work we studied for the first time the effect of pressure on the cerebellar climbing fiber (CF) synaptic responses. Pressure modulation of cerebellar synaptic plasticity was tested in both the CF and parallel fiber (PF) pathways using paired-pulse protocols. CF synapses, which normally operate at a high baseline release probability, demonstrate paired-pulse depression (PPD). High pressure reduced CF synaptic responses at 5.1 and 10.1 MPa but did not affect its PPD. High extracellular Ca 2ϩ concentration ([Ca 2ϩ ]o) could not antagonize the effect of pressure on the CF response, whereas low [Ca 2ϩ ]o, in contrast to pressure, decreased both the response amplitude and the observed PPD. PF synapses, which usually operate at low release probability, exhibit paired-pulse facilitation (PPF). Pressure increased PF PPF at all interstimulus intervals (ISIs) tested (20 -200 ms). Several Ca 2ϩ channel blockers as well as low [Ca 2ϩ ]o could mimic the effect of pressure on the PF response but significantly increased the PPF only at the 20-ms ISI. These results, together with previous data, show that the CF synapse is relatively resistant to pressure. The lack of pressure effect on CF PPD is surprising and may suggest that the PPD is not directly linked to synaptic depletion, as generally suggested. The increase in PPF of the PF at pressure, which is mimicked by Ca 2ϩ channel blockers or low [Ca 2ϩ ]o, further supports pressure involvement in synaptic release mechanism(s). These results also indicate that pressure effects may be selective for various types of synapses in the CNS.
European journal of human genetics : EJHG, Jan 30, 2015
Dilated cardiomyopathy (DCM) and malignant ventricular arrhythmias are important causes of conges... more Dilated cardiomyopathy (DCM) and malignant ventricular arrhythmias are important causes of congestive heart failure, heart transplantation, and sudden cardiac death in young patients. Cypher/ZASP is a cytoskeletal protein localized in the sarcomeric Z-line that has a pivotal role in maintaining adult cardiac structure and function. The putative mutation p.(D117N) in Cypher/ZASP has been suggested to cause systolic dysfunction, dilated left ventricle with hypertrabeculated myocardium, and intraventricular conduction disturbance, based on two reported sporadic cases. In two unrelated Bedouin families, one with pediatric DCM and the other with DCM and ventricular arrhythmias at young adulthood searching for the causative mutation by exome sequencing we identified the p.(D117N) variant in Cypher/ZASP. However, p.(D117N) did not segregate as the causative mutation in these families, i.e. it was not present in some patients and was found in several individuals who had no clinical manifest...
Gene mutations, mostly segregating with a dominant mode of inheritance, are important causes of d... more Gene mutations, mostly segregating with a dominant mode of inheritance, are important causes of dilated cardiomyopathy (DCM), a disease characterized by enlarged ventricular dimensions, impaired cardiac function, heart failure and high risk of death. Another myocardial abnormality often linked to gene mutations is left ventricular noncompaction (LVNC) characterized by a typical diffuse spongy appearance of the left ventricle. Here, we describe a large Bedouin family presenting with a severe recessive DCM and LVNC. Homozygosity mapping and exome sequencing identified a single gene variant that segregated as expected and was neither reported in databases nor in Bedouin population controls. The PLEKHM2 cDNA2156_2157delAG variant causes the frameshift p.Lys645AlafsTer12 and/or the skipping of exon 11 that results in deletion of 30 highly conserved amino acids. PLEKHM2 is known to interact with several Rabs and with kinesin-1, affecting endosomal trafficking. Accordingly, patients' p...
Gene mutations, mostly segregating with a dominant mode of inheritance, are important causes of d... more Gene mutations, mostly segregating with a dominant mode of inheritance, are important causes of dilated cardiomyopathy (DCM), a disease characterized by enlarged ventricular dimensions, impaired cardiac function, heart failure and high risk of death. Another myocardial abnormality often linked to gene mutations is left ventricular noncompaction (LVNC) characterized by a typical diffuse spongy appearance of the left ventricle. Here, we describe a large Bedouin family presenting with a severe recessive DCM and LVNC. Homozygosity mapping and exome sequencing identified a single gene variant that segregated as expected and was neither reported in databases nor in Bedouin population controls. The PLEKHM2 cDNA2156_2157delAG variant causes the frameshift p.Lys645AlafsTer12 and/or the skipping of exon 11 that results in deletion of 30 highly conserved amino acids. PLEKHM2 is known to interact with several Rabs and with kinesin-1, affecting endosomal trafficking. Accordingly, patients&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; primary fibroblasts exhibited abnormal subcellular distribution of endosomes marked by Rab5, Rab7 and Rab9, as well as the Golgi apparatus. In addition, lysosomes appeared to be concentrated in the perinuclear region, and autophagy flux was impaired. Transfection of wild-type PLEKHM2 cDNA into patient&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s fibroblasts corrected the subcellular distribution of the lysosomes, supporting the causal effect of PLEKHM2 mutation. PLEKHM2 joins LAMP-2 and BAG3 as a disease gene altering autophagy resulting in an isolated cardiac phenotype. The association of PLEKHM2 mutation with DCM and LVNC supports the importance of autophagy for normal cardiac function.
Dilated cardiomyopathy (DCM) and malignant ventricular arrhythmias are important causes of conges... more Dilated cardiomyopathy (DCM) and malignant ventricular arrhythmias are important causes of congestive heart failure, heart transplantation, and sudden cardiac death in young patients. Cypher/ZASP is a cytoskeletal protein localized in the sarcomeric Z-line that has a pivotal role in maintaining adult cardiac structure and function. The putative mutation p.(D117N) in Cypher/ZASP has been suggested to cause systolic dysfunction, dilated left ventricle with hypertrabeculated myocardium, and intraventricular conduction disturbance, based on two reported sporadic cases. In two unrelated Bedouin families, one with pediatric DCM and the other with DCM and ventricular arrhythmias at young adulthood searching for the causative mutation by exome sequencing we identified the p.(D117N) variant in Cypher/ZASP. However, p.(D117N) did not segregate as the causative mutation in these families, i.e. it was not present in some patients and was found in several individuals who had no clinical manifestations. Furthermore, the carrier frequency in the Bedouin population of origin is estimated to be 5.2%, which is much higher than the incidence of idiopathic DCM in this population. Thus, our data support the notion that the p.(D117N) variant in Cypher/ZASP is not a causative mutation in the families tested by us. The results also indicates that at least in some cases, the p.(D117N) in Cypher/ZASP is not a causative mutation and the role of D117N in Cypher/ZASP in cardiac pathologies should be further clarified and re-evaluated.European Journal of Human Genetics advance online publication, 30 September 2015; doi:10.1038/ejhg.2015.195.
Etzion Y, Mor A, Grossman Y. Differential modulation of cerebellar climbing fiber and parallel fi... more Etzion Y, Mor A, Grossman Y. Differential modulation of cerebellar climbing fiber and parallel fiber synaptic responses at high pressure. High pressure, which induces central nervous system (CNS) dysfunction (highpressure neurological syndrome) depresses synaptic transmission at all synapses examined to date. Several lines of evidence indicate an inhibitory effect of pressure on Ca 2ϩ entry into the presynaptic terminal. In the present work we studied for the first time the effect of pressure on the cerebellar climbing fiber (CF) synaptic responses. Pressure modulation of cerebellar synaptic plasticity was tested in both the CF and parallel fiber (PF) pathways using paired-pulse protocols. CF synapses, which normally operate at a high baseline release probability, demonstrate paired-pulse depression (PPD). High pressure reduced CF synaptic responses at 5.1 and 10.1 MPa but did not affect its PPD. High extracellular Ca 2ϩ concentration ([Ca 2ϩ ]o) could not antagonize the effect of pressure on the CF response, whereas low [Ca 2ϩ ]o, in contrast to pressure, decreased both the response amplitude and the observed PPD. PF synapses, which usually operate at low release probability, exhibit paired-pulse facilitation (PPF). Pressure increased PF PPF at all interstimulus intervals (ISIs) tested (20 -200 ms). Several Ca 2ϩ channel blockers as well as low [Ca 2ϩ ]o could mimic the effect of pressure on the PF response but significantly increased the PPF only at the 20-ms ISI. These results, together with previous data, show that the CF synapse is relatively resistant to pressure. The lack of pressure effect on CF PPD is surprising and may suggest that the PPD is not directly linked to synaptic depletion, as generally suggested. The increase in PPF of the PF at pressure, which is mimicked by Ca 2ϩ channel blockers or low [Ca 2ϩ ]o, further supports pressure involvement in synaptic release mechanism(s). These results also indicate that pressure effects may be selective for various types of synapses in the CNS.
Macrophage-derived foam cells are thought to play a major role in atherosclerotic lesion formatio... more Macrophage-derived foam cells are thought to play a major role in atherosclerotic lesion formation and progression. An automated assay was established to evaluate the uptake of fluorescently labeled oxidized low-density lipoprotein (oxLDL) by a monocyte/macrophage cell line. The assay was used to screen 480 known bioactive compounds. Twenty-two active compounds were identified. Efficacy studies in peritoneal macrophages demonstrated a high rate of concordance with the initial screening results. Inhibitory compounds confirmed important previous findings and identified new drugs of interest including: 3 blockers of nuclear factor b activation, 2 protein kinase C inhibitors, a phospholipase C inhibitor, and 2 antipsychotic drugs. In addition, an opioid receptor agonist was found to increase the oxLDL uptake of macrophages. The involvement of nuclear factor B in oxLDL uptake was validated in peritoneal macrophages in vivo. The results support a model in which oxLDL uptake is dependent on the activation of multiple intracellular signaling pathways that culminate in actin-mediated lipoprotein internalization. (Circ Res. 2009; 105:148-157.)
Chronic wounds of the lower limbs are a cause of severe morbidity in diabetic patients. Low oxyge... more Chronic wounds of the lower limbs are a cause of severe morbidity in diabetic patients. Low oxygen tension around the wound is one of several critical factors, which mutually enhance the progression of a chronic ulcer. Hyperbaric oxygen therapy (HBO) is believed to improve wound healing by enhancing oxygen tension around the wound. While conventional therapies for diabetic foot ulcer are based on scientific evidence, HBO treatment lacks evidence-based support regarding its cost effectiveness and efficacy. Recently, several publications emerged, which improve our knowledge regarding this subject. This paper briefly reviews the pathophysiology of chronic diabetic ulcers and the possible advantage of HBO therapy in this clinical setting. The article also summarizes the results of relevant publications, in which appropriate scientific measures were applied. In conclusion, there is evidence that HBO therapy reduces the need for major amputations among diabetic patients with chronic ulcer...
Recordings from cerebellar Purkinje cell dendrites have revealed that in response to sustained cu... more Recordings from cerebellar Purkinje cell dendrites have revealed that in response to sustained current injection, the cell firing pattern can move from tonic firing of Ca(2+) spikes to doublet firing and even to quadruplet firing or more complex firing. These firing patterns are not modified substantially if Na(+) currents are blocked. We show that the experimental results can be viewed as a slow transition of the neuronal dynamics through a period-doubling bifurcation. To further support this conclusion and to understand the underlying mechanism that leads to doublet firing, we develop and study a simple, one-compartment model of Purkinje cell dendrite. The neuron can also exhibit quadruplet and chaotic firing patterns that are similar to the firing patterns that some of the Purkinje cells exhibit experimentally. The effects of parameters such as temperature, applied current, and potassium reversal potential in the model resemble their effects in experiments. The model dynamics inv...
Abstract Data regarding the atrial electrophysiology of rodents is hard to obtain. We hypothesize... more Abstract Data regarding the atrial electrophysiology of rodents is hard to obtain. We hypothesized that the latency to the first R following programmed S1–S2 stimulation of the atria (defined as RR S2) is affected by atrial capture and may be utilized to determine the ...
ZnT-1 is a Cation Diffusion Facilitator (CDF) family protein, and is present throughout the phylo... more ZnT-1 is a Cation Diffusion Facilitator (CDF) family protein, and is present throughout the phylogenetic tree from bacteria to humans. Since its original cloning in 1995, ZnT-1 has been considered to be the major Zn(2+) extruding transporter, based on its ability to protect cells against zinc toxicity. However, experimental evidence for ZnT-1 induced Zn(2+) extrusion was not convincing. In the present study, based on the 3D crystal structure of the ZnT-1 homologue, YiiP, that predicts a homodimer that utilizes the H(+) electrochemical gradient to facilitate Zn(2+) efflux, we demonstrate ZnT-1 dependent Zn(2+) efflux from HEK 293T cells using FluoZin-3 and Fura 2 by single cell microscope based fluorescent imaging. ZnT-1 facilitates zinc efflux in a sodium-independent, pH-driven and calcium-sensitive manner. Moreover, substitution of two amino acids in the putative zinc binding domain of ZnT-1 led to nullification of Zn(2+) efflux and rendered the mutated protein incapable of protect...
Proceedings of the National Academy of Sciences, 2009
The recent progress made in the bioengineering of cardiac patches offers a new therapeutic modali... more The recent progress made in the bioengineering of cardiac patches offers a new therapeutic modality for regenerating the myocardium after myocardial infarction (MI). We present here a strategy for the engineering of a cardiac patch with mature vasculature by heterotopic transplantation onto the omentum. The patch was constructed by seeding neonatal cardiac cells with a mixture of prosurvival and angiogenic factors into an alginate scaffold capable of factor binding and sustained release. After 48 h in culture, the patch was vascularized for 7 days on the omentum, then explanted and transplanted onto infarcted rat hearts, 7 days after MI induction. When evaluated 28 days later, the vascularized cardiac patch showed structural and electrical integration into host myocardium. Moreover, the vascularized patch induced thicker scars, prevented further dilatation of the chamber and ventricular dysfunction. Thus, our study provides evidence that grafting prevascularized cardiac patch into infarct can improve cardiac function after MI.
Pfl�gers Archiv European Journal of Physiology, 1999
The effects of high pressure (up to 10.1 MPa) on the spontaneous firing of Purkinje neurons in gu... more The effects of high pressure (up to 10.1 MPa) on the spontaneous firing of Purkinje neurons in guineapig cerebellar slices were studied using the macropatch clamp technique. Pressure did not significantly alter the single somatic Na + spike parameters or the frequency of regular Na + spike firing. When Na + currents were blocked by 0.5-1 µM tetrodotoxin (TTX), a pressure of 10.1 MPa slightly reduced the dendritic Ca 2+ spike amplitude to 90.2±3.1% of its control value, and slowed its kinetics. The effects of pressure on the single Ca 2+ spike were even less prominent when K + currents were blocked by 5 mM 4-aminopyridine (4-AP). Pressure prolonged the active period of Ca 2+ spike firing to 152.2±10.4% of the control value. Within the active period pressure increased the inter-spike interval to 164.9±8.7% and suppressed the typical firing of doublets. The latter changes were reversed by a high extracellular potassium concentration ([K + ] o ) and 1 µM 4-AP, whereas in the presence of 5 mM 4-AP the pattern was insensitive to pressure. A high [Ca 2+ ] o reduced the firing frequency and suppressed doublet firing in a manner reminiscent of the pressure effect, but these changes could not be reversed by 4-AP. A low [Ca 2+ ] o slightly increased the firing of doublets. These results show that the single somatic Na + spike is insensitive and the dendritic Ca 2+ spike is only mildly sensitive to pressure. However, alterations in Ca 2+ spike firing pattern suggest that modulation of dendritic K + currents induce depression of dendritic excitability at pressure.
Activation of ERK signaling may promote cardioprotection from ischemia-reperfusion (I/R) injury. ... more Activation of ERK signaling may promote cardioprotection from ischemia-reperfusion (I/R) injury. ZnT-1, a protein that confers resistance from zinc toxicity, was found to interact with Raf-1 kinase through its C-terminal domain, leading to downstream activation of ERK. In the present study, we evaluated the effects of ZnT-1 in cultured murine cardiomyocytes (HL-1 cells) that were exposed to simulated-I/R. Cellular injury was evaluated by lactate dehydrogenase (LDH) release and by staining for pro-apoptotic caspase activation. Overexpression of ZnT-1 markedly reduced LDH release and caspase activation following I/R. Knockdown of endogenous ZnT-1 augmented the I/R-induced release of LDH and increased caspase activation following I/R. Phospho-ERK levels were significantly increased following I/R in cells overexpressing ZnT-1, while knockdown of ZnT-1 reduced phospho-ERK levels. Pretreatment of cells with the MEK inhibitor PD98059 abolished the protective effect of ZnT-1 following I/R. Accordingly, a truncated form of ZnT-1 lacking the C-terminal domain failed to induce ERK activation and did not protect the cells from I/R injury. In contrast, expression of the C-terminal domain by itself was sufficient to induce ERK activation and I/R protection. Interestingly, the C-terminal of the ZnT-1 did not have protective effect against the toxicity of zinc. In the isolated rat heart, global ischemic injury rapidly increased the endogenous levels of ZnT-1. However, following reperfusion ZnT-1 levels were found to be decreased. Our findings indicate that ZnT-1 may have important role in the ischemic myocardium through its ability to interact with Raf-1 kinase.
Circulation: Genomics and Precision Medicine, 2018
BACKGROUND: Paroxysmal atrial fibrillation (AF) can be caused by gain-of-
function mutations in g... more BACKGROUND: Paroxysmal atrial fibrillation (AF) can be caused by gain-of- function mutations in genes, encoding the cardiac potassium channel subunits KCNJ2, KCNE1, and KCNH2 that mediate the repolarizing potassium currents Ik1, Iks, and Ikr, respectively. METHODS: Linkage analysis, whole-exome sequencing, and Xenopus oocyte electrophysiology studies were used in this study. RESULTS: Through genetic studies, we showed that autosomal dominant early-onset nocturnal paroxysmal AF is caused by p.S447R mutation in KCND2, encoding the pore-forming (α) subunit of the Kv4.2 cardiac potassium channel. Kv4.2, along with Kv4.3, contributes to the cardiac fast transient outward K+ current, Ito. Ito underlies the early phase of repolarization in the cardiac action potential, thereby setting the initial potential of the plateau phase and governing its duration and amplitude. In Xenopus oocytes, the mutation increased the channel’s inactivation time constant and affected its regulation: p.S447 resides in a protein kinase C (PKC) phosphorylation site, which normally allows attenuation of Kv4.2 membrane expression. The mutant Kv4.2 exhibited impaired response to PKC; hence, Kv4.2 membrane expression was augmented, enhancing potassium currents. Coexpression of mutant and wild-type channels (recapitulating heterozygosity in affected individuals) showed results similar to the mutant channel alone. Finally, in a hybrid channel composed of Kv4.3 and Kv4.2, simulating the mature endogenous heterotetrameric channel underlying Ito, the p.S447R Kv4.2 mutation exerted a gain-of-function effect on Kv4.3. CONCLUSIONS: The mutation alters Kv4.2’s kinetic properties, impairs its inhibitory regulation, and exerts gain-of-function effect on both Kv4.2 homotetramers and Kv4.2-Kv4.3 heterotetramers. These effects presumably increase the repolarizing potassium current Ito, thereby abbreviating action potential duration, creating arrhythmogenic substrate for nocturnal AF. Interestingly, Kv4.2 expression was previously shown to demonstrate circadian variation, with peak expression at daytime in murine hearts (human nighttime), with possible relevance to the nocturnal onset of paroxysmal AF symptoms in our patients. The atrial-specific phenotype suggests that targeting Kv4.2 might be effective in the treatment of nocturnal paroxysmal AF, avoiding adverse ventricular effects.
Organophosphates (OP) are used extensively as pesticides and as chemical weapons. Cardiotoxicity ... more Organophosphates (OP) are used extensively as pesticides and as chemical weapons. Cardiotoxicity is a major concern in survivors of the acute poisoning. To characterize the delayed cardiac effects of OP, rats were poisoned by intraperitoneal administration of dichlorvos. In group I, poisoning (0.25-, 0.75-, 1.4-LD 50) was followed by application of atropine and obidoxime. In group II, poisoning (0.35-, 0.5-LD 50) was done without antidotes. Cardiac evaluation included electrocardiography and echocardiog-raphy 2-and 6-week post-exposure, arrhythmia susceptibility following administration of Isoproterenol (150 mcg/ kg), and histological evaluation. All poisoned animals displayed cholinergic symptoms. In group I, all animals exposed to 1.4-LD 50 (n = 3) had profound convulsions and died despite antidote treatment. However, in the lower doses, all animals survived and no cardiac abnormalities were noted during follow-up. In group II, six animals had convulsions and died. Surviving animals had mild but significant pro-longation of corrected QT at both 2 and 6 weeks, compared to shams. There were no notable echocardiographic, gravimetric , or histological differences between poisoned and sham animals. Our data indicate that dichlorvos poisoning is associated with QT prolongation without anatomical or histopathological abnormalities. This new model can be used to elaborate the molecular mechanism\s of QT prolongation following OP poisoning.
Biventricular pacing is an important modality to improve left ventricular (LV) synchronization an... more Biventricular pacing is an important modality to improve left ventricular (LV) synchronization and long-term function. However, the biological effects of this treatment are far from being elucidated and existing animal models are limited and demanding. Recently, we introduced an implanted device for double-site epicardial pacing in rats and echocardiographically demonstrated favorable effects of LV and biventricular (LV-based) pacing modes typically observed in humans. Here, this new animal model was further characterized. Electrodes were implanted either on the right atria (RA) and right ventricle (RV) or on the RV and LV. Following recovery, rats were either used for invasive hemodynamic measurements (pressure-volume analysis) or exposed to sustained RV vs. biventricular tachypacing for 3 days. RV pacing compromised, while LV-based pacing modes markedly enhanced cardiac performance. Changes in LV performance were associated with prominent compensatory changes in arterial resistance. Sustained RV tachypacing increased the electrocardiogram QTc interval by 7.9 ± 3.1 ms (n = 6, p < 0.05), dispersed refractoriness between the right and left pacing sites and induced important molecular changes mainly in the early-activated septal tissue. These effects were not observed during biventricular tachypacing (n = 6). Our results demonstrate that the rat is an attractive new model to study the biological consequences of LV dyssynchrony and resynchronization. The electromechanical and molecular consequences of various ventricular pacing modes have become a subject of intense interest in recent years due to profound clinical implications 1–3. Compelling evidence indicates that right ventricular (RV) pacing can be detrimental to left ventricular (LV) function. The sequence of electrical activation during RV pacing may largely resemble the activation pattern of left bundle branch block (LBBB) 4 , and can result in dyssynchronous LV contraction 5, 6. In accordance, RV pacing increases the risk for heart failure (HF) even in patients without pre-existing LV dysfunction 7–10. Cardiac resynchronization therapy (CRT) by means of biventricular (BiV) pacing was developed as a strategy to overcome the deleterious effects of electromechanical dyssynchrony in the failing heart and represents a major advance in HF therapy 11. BiV pacing has been demonstrated to improve contractile performance in patients with
Etzion Y, Mor A, Grossman Y. Differential modulation of cerebellar climbing fiber and parallel fi... more Etzion Y, Mor A, Grossman Y. Differential modulation of cerebellar climbing fiber and parallel fiber synaptic responses at high pressure. High pressure, which induces central nervous system (CNS) dysfunction (highpressure neurological syndrome) depresses synaptic transmission at all synapses examined to date. Several lines of evidence indicate an inhibitory effect of pressure on Ca 2ϩ entry into the presynaptic terminal. In the present work we studied for the first time the effect of pressure on the cerebellar climbing fiber (CF) synaptic responses. Pressure modulation of cerebellar synaptic plasticity was tested in both the CF and parallel fiber (PF) pathways using paired-pulse protocols. CF synapses, which normally operate at a high baseline release probability, demonstrate paired-pulse depression (PPD). High pressure reduced CF synaptic responses at 5.1 and 10.1 MPa but did not affect its PPD. High extracellular Ca 2ϩ concentration ([Ca 2ϩ ]o) could not antagonize the effect of pressure on the CF response, whereas low [Ca 2ϩ ]o, in contrast to pressure, decreased both the response amplitude and the observed PPD. PF synapses, which usually operate at low release probability, exhibit paired-pulse facilitation (PPF). Pressure increased PF PPF at all interstimulus intervals (ISIs) tested (20 -200 ms). Several Ca 2ϩ channel blockers as well as low [Ca 2ϩ ]o could mimic the effect of pressure on the PF response but significantly increased the PPF only at the 20-ms ISI. These results, together with previous data, show that the CF synapse is relatively resistant to pressure. The lack of pressure effect on CF PPD is surprising and may suggest that the PPD is not directly linked to synaptic depletion, as generally suggested. The increase in PPF of the PF at pressure, which is mimicked by Ca 2ϩ channel blockers or low [Ca 2ϩ ]o, further supports pressure involvement in synaptic release mechanism(s). These results also indicate that pressure effects may be selective for various types of synapses in the CNS.
European journal of human genetics : EJHG, Jan 30, 2015
Dilated cardiomyopathy (DCM) and malignant ventricular arrhythmias are important causes of conges... more Dilated cardiomyopathy (DCM) and malignant ventricular arrhythmias are important causes of congestive heart failure, heart transplantation, and sudden cardiac death in young patients. Cypher/ZASP is a cytoskeletal protein localized in the sarcomeric Z-line that has a pivotal role in maintaining adult cardiac structure and function. The putative mutation p.(D117N) in Cypher/ZASP has been suggested to cause systolic dysfunction, dilated left ventricle with hypertrabeculated myocardium, and intraventricular conduction disturbance, based on two reported sporadic cases. In two unrelated Bedouin families, one with pediatric DCM and the other with DCM and ventricular arrhythmias at young adulthood searching for the causative mutation by exome sequencing we identified the p.(D117N) variant in Cypher/ZASP. However, p.(D117N) did not segregate as the causative mutation in these families, i.e. it was not present in some patients and was found in several individuals who had no clinical manifest...
Gene mutations, mostly segregating with a dominant mode of inheritance, are important causes of d... more Gene mutations, mostly segregating with a dominant mode of inheritance, are important causes of dilated cardiomyopathy (DCM), a disease characterized by enlarged ventricular dimensions, impaired cardiac function, heart failure and high risk of death. Another myocardial abnormality often linked to gene mutations is left ventricular noncompaction (LVNC) characterized by a typical diffuse spongy appearance of the left ventricle. Here, we describe a large Bedouin family presenting with a severe recessive DCM and LVNC. Homozygosity mapping and exome sequencing identified a single gene variant that segregated as expected and was neither reported in databases nor in Bedouin population controls. The PLEKHM2 cDNA2156_2157delAG variant causes the frameshift p.Lys645AlafsTer12 and/or the skipping of exon 11 that results in deletion of 30 highly conserved amino acids. PLEKHM2 is known to interact with several Rabs and with kinesin-1, affecting endosomal trafficking. Accordingly, patients' p...
Gene mutations, mostly segregating with a dominant mode of inheritance, are important causes of d... more Gene mutations, mostly segregating with a dominant mode of inheritance, are important causes of dilated cardiomyopathy (DCM), a disease characterized by enlarged ventricular dimensions, impaired cardiac function, heart failure and high risk of death. Another myocardial abnormality often linked to gene mutations is left ventricular noncompaction (LVNC) characterized by a typical diffuse spongy appearance of the left ventricle. Here, we describe a large Bedouin family presenting with a severe recessive DCM and LVNC. Homozygosity mapping and exome sequencing identified a single gene variant that segregated as expected and was neither reported in databases nor in Bedouin population controls. The PLEKHM2 cDNA2156_2157delAG variant causes the frameshift p.Lys645AlafsTer12 and/or the skipping of exon 11 that results in deletion of 30 highly conserved amino acids. PLEKHM2 is known to interact with several Rabs and with kinesin-1, affecting endosomal trafficking. Accordingly, patients&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; primary fibroblasts exhibited abnormal subcellular distribution of endosomes marked by Rab5, Rab7 and Rab9, as well as the Golgi apparatus. In addition, lysosomes appeared to be concentrated in the perinuclear region, and autophagy flux was impaired. Transfection of wild-type PLEKHM2 cDNA into patient&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s fibroblasts corrected the subcellular distribution of the lysosomes, supporting the causal effect of PLEKHM2 mutation. PLEKHM2 joins LAMP-2 and BAG3 as a disease gene altering autophagy resulting in an isolated cardiac phenotype. The association of PLEKHM2 mutation with DCM and LVNC supports the importance of autophagy for normal cardiac function.
Dilated cardiomyopathy (DCM) and malignant ventricular arrhythmias are important causes of conges... more Dilated cardiomyopathy (DCM) and malignant ventricular arrhythmias are important causes of congestive heart failure, heart transplantation, and sudden cardiac death in young patients. Cypher/ZASP is a cytoskeletal protein localized in the sarcomeric Z-line that has a pivotal role in maintaining adult cardiac structure and function. The putative mutation p.(D117N) in Cypher/ZASP has been suggested to cause systolic dysfunction, dilated left ventricle with hypertrabeculated myocardium, and intraventricular conduction disturbance, based on two reported sporadic cases. In two unrelated Bedouin families, one with pediatric DCM and the other with DCM and ventricular arrhythmias at young adulthood searching for the causative mutation by exome sequencing we identified the p.(D117N) variant in Cypher/ZASP. However, p.(D117N) did not segregate as the causative mutation in these families, i.e. it was not present in some patients and was found in several individuals who had no clinical manifestations. Furthermore, the carrier frequency in the Bedouin population of origin is estimated to be 5.2%, which is much higher than the incidence of idiopathic DCM in this population. Thus, our data support the notion that the p.(D117N) variant in Cypher/ZASP is not a causative mutation in the families tested by us. The results also indicates that at least in some cases, the p.(D117N) in Cypher/ZASP is not a causative mutation and the role of D117N in Cypher/ZASP in cardiac pathologies should be further clarified and re-evaluated.European Journal of Human Genetics advance online publication, 30 September 2015; doi:10.1038/ejhg.2015.195.
Etzion Y, Mor A, Grossman Y. Differential modulation of cerebellar climbing fiber and parallel fi... more Etzion Y, Mor A, Grossman Y. Differential modulation of cerebellar climbing fiber and parallel fiber synaptic responses at high pressure. High pressure, which induces central nervous system (CNS) dysfunction (highpressure neurological syndrome) depresses synaptic transmission at all synapses examined to date. Several lines of evidence indicate an inhibitory effect of pressure on Ca 2ϩ entry into the presynaptic terminal. In the present work we studied for the first time the effect of pressure on the cerebellar climbing fiber (CF) synaptic responses. Pressure modulation of cerebellar synaptic plasticity was tested in both the CF and parallel fiber (PF) pathways using paired-pulse protocols. CF synapses, which normally operate at a high baseline release probability, demonstrate paired-pulse depression (PPD). High pressure reduced CF synaptic responses at 5.1 and 10.1 MPa but did not affect its PPD. High extracellular Ca 2ϩ concentration ([Ca 2ϩ ]o) could not antagonize the effect of pressure on the CF response, whereas low [Ca 2ϩ ]o, in contrast to pressure, decreased both the response amplitude and the observed PPD. PF synapses, which usually operate at low release probability, exhibit paired-pulse facilitation (PPF). Pressure increased PF PPF at all interstimulus intervals (ISIs) tested (20 -200 ms). Several Ca 2ϩ channel blockers as well as low [Ca 2ϩ ]o could mimic the effect of pressure on the PF response but significantly increased the PPF only at the 20-ms ISI. These results, together with previous data, show that the CF synapse is relatively resistant to pressure. The lack of pressure effect on CF PPD is surprising and may suggest that the PPD is not directly linked to synaptic depletion, as generally suggested. The increase in PPF of the PF at pressure, which is mimicked by Ca 2ϩ channel blockers or low [Ca 2ϩ ]o, further supports pressure involvement in synaptic release mechanism(s). These results also indicate that pressure effects may be selective for various types of synapses in the CNS.
Macrophage-derived foam cells are thought to play a major role in atherosclerotic lesion formatio... more Macrophage-derived foam cells are thought to play a major role in atherosclerotic lesion formation and progression. An automated assay was established to evaluate the uptake of fluorescently labeled oxidized low-density lipoprotein (oxLDL) by a monocyte/macrophage cell line. The assay was used to screen 480 known bioactive compounds. Twenty-two active compounds were identified. Efficacy studies in peritoneal macrophages demonstrated a high rate of concordance with the initial screening results. Inhibitory compounds confirmed important previous findings and identified new drugs of interest including: 3 blockers of nuclear factor b activation, 2 protein kinase C inhibitors, a phospholipase C inhibitor, and 2 antipsychotic drugs. In addition, an opioid receptor agonist was found to increase the oxLDL uptake of macrophages. The involvement of nuclear factor B in oxLDL uptake was validated in peritoneal macrophages in vivo. The results support a model in which oxLDL uptake is dependent on the activation of multiple intracellular signaling pathways that culminate in actin-mediated lipoprotein internalization. (Circ Res. 2009; 105:148-157.)
Chronic wounds of the lower limbs are a cause of severe morbidity in diabetic patients. Low oxyge... more Chronic wounds of the lower limbs are a cause of severe morbidity in diabetic patients. Low oxygen tension around the wound is one of several critical factors, which mutually enhance the progression of a chronic ulcer. Hyperbaric oxygen therapy (HBO) is believed to improve wound healing by enhancing oxygen tension around the wound. While conventional therapies for diabetic foot ulcer are based on scientific evidence, HBO treatment lacks evidence-based support regarding its cost effectiveness and efficacy. Recently, several publications emerged, which improve our knowledge regarding this subject. This paper briefly reviews the pathophysiology of chronic diabetic ulcers and the possible advantage of HBO therapy in this clinical setting. The article also summarizes the results of relevant publications, in which appropriate scientific measures were applied. In conclusion, there is evidence that HBO therapy reduces the need for major amputations among diabetic patients with chronic ulcer...
Recordings from cerebellar Purkinje cell dendrites have revealed that in response to sustained cu... more Recordings from cerebellar Purkinje cell dendrites have revealed that in response to sustained current injection, the cell firing pattern can move from tonic firing of Ca(2+) spikes to doublet firing and even to quadruplet firing or more complex firing. These firing patterns are not modified substantially if Na(+) currents are blocked. We show that the experimental results can be viewed as a slow transition of the neuronal dynamics through a period-doubling bifurcation. To further support this conclusion and to understand the underlying mechanism that leads to doublet firing, we develop and study a simple, one-compartment model of Purkinje cell dendrite. The neuron can also exhibit quadruplet and chaotic firing patterns that are similar to the firing patterns that some of the Purkinje cells exhibit experimentally. The effects of parameters such as temperature, applied current, and potassium reversal potential in the model resemble their effects in experiments. The model dynamics inv...
Abstract Data regarding the atrial electrophysiology of rodents is hard to obtain. We hypothesize... more Abstract Data regarding the atrial electrophysiology of rodents is hard to obtain. We hypothesized that the latency to the first R following programmed S1–S2 stimulation of the atria (defined as RR S2) is affected by atrial capture and may be utilized to determine the ...
ZnT-1 is a Cation Diffusion Facilitator (CDF) family protein, and is present throughout the phylo... more ZnT-1 is a Cation Diffusion Facilitator (CDF) family protein, and is present throughout the phylogenetic tree from bacteria to humans. Since its original cloning in 1995, ZnT-1 has been considered to be the major Zn(2+) extruding transporter, based on its ability to protect cells against zinc toxicity. However, experimental evidence for ZnT-1 induced Zn(2+) extrusion was not convincing. In the present study, based on the 3D crystal structure of the ZnT-1 homologue, YiiP, that predicts a homodimer that utilizes the H(+) electrochemical gradient to facilitate Zn(2+) efflux, we demonstrate ZnT-1 dependent Zn(2+) efflux from HEK 293T cells using FluoZin-3 and Fura 2 by single cell microscope based fluorescent imaging. ZnT-1 facilitates zinc efflux in a sodium-independent, pH-driven and calcium-sensitive manner. Moreover, substitution of two amino acids in the putative zinc binding domain of ZnT-1 led to nullification of Zn(2+) efflux and rendered the mutated protein incapable of protect...
Proceedings of the National Academy of Sciences, 2009
The recent progress made in the bioengineering of cardiac patches offers a new therapeutic modali... more The recent progress made in the bioengineering of cardiac patches offers a new therapeutic modality for regenerating the myocardium after myocardial infarction (MI). We present here a strategy for the engineering of a cardiac patch with mature vasculature by heterotopic transplantation onto the omentum. The patch was constructed by seeding neonatal cardiac cells with a mixture of prosurvival and angiogenic factors into an alginate scaffold capable of factor binding and sustained release. After 48 h in culture, the patch was vascularized for 7 days on the omentum, then explanted and transplanted onto infarcted rat hearts, 7 days after MI induction. When evaluated 28 days later, the vascularized cardiac patch showed structural and electrical integration into host myocardium. Moreover, the vascularized patch induced thicker scars, prevented further dilatation of the chamber and ventricular dysfunction. Thus, our study provides evidence that grafting prevascularized cardiac patch into infarct can improve cardiac function after MI.
Pfl�gers Archiv European Journal of Physiology, 1999
The effects of high pressure (up to 10.1 MPa) on the spontaneous firing of Purkinje neurons in gu... more The effects of high pressure (up to 10.1 MPa) on the spontaneous firing of Purkinje neurons in guineapig cerebellar slices were studied using the macropatch clamp technique. Pressure did not significantly alter the single somatic Na + spike parameters or the frequency of regular Na + spike firing. When Na + currents were blocked by 0.5-1 µM tetrodotoxin (TTX), a pressure of 10.1 MPa slightly reduced the dendritic Ca 2+ spike amplitude to 90.2±3.1% of its control value, and slowed its kinetics. The effects of pressure on the single Ca 2+ spike were even less prominent when K + currents were blocked by 5 mM 4-aminopyridine (4-AP). Pressure prolonged the active period of Ca 2+ spike firing to 152.2±10.4% of the control value. Within the active period pressure increased the inter-spike interval to 164.9±8.7% and suppressed the typical firing of doublets. The latter changes were reversed by a high extracellular potassium concentration ([K + ] o ) and 1 µM 4-AP, whereas in the presence of 5 mM 4-AP the pattern was insensitive to pressure. A high [Ca 2+ ] o reduced the firing frequency and suppressed doublet firing in a manner reminiscent of the pressure effect, but these changes could not be reversed by 4-AP. A low [Ca 2+ ] o slightly increased the firing of doublets. These results show that the single somatic Na + spike is insensitive and the dendritic Ca 2+ spike is only mildly sensitive to pressure. However, alterations in Ca 2+ spike firing pattern suggest that modulation of dendritic K + currents induce depression of dendritic excitability at pressure.
Activation of ERK signaling may promote cardioprotection from ischemia-reperfusion (I/R) injury. ... more Activation of ERK signaling may promote cardioprotection from ischemia-reperfusion (I/R) injury. ZnT-1, a protein that confers resistance from zinc toxicity, was found to interact with Raf-1 kinase through its C-terminal domain, leading to downstream activation of ERK. In the present study, we evaluated the effects of ZnT-1 in cultured murine cardiomyocytes (HL-1 cells) that were exposed to simulated-I/R. Cellular injury was evaluated by lactate dehydrogenase (LDH) release and by staining for pro-apoptotic caspase activation. Overexpression of ZnT-1 markedly reduced LDH release and caspase activation following I/R. Knockdown of endogenous ZnT-1 augmented the I/R-induced release of LDH and increased caspase activation following I/R. Phospho-ERK levels were significantly increased following I/R in cells overexpressing ZnT-1, while knockdown of ZnT-1 reduced phospho-ERK levels. Pretreatment of cells with the MEK inhibitor PD98059 abolished the protective effect of ZnT-1 following I/R. Accordingly, a truncated form of ZnT-1 lacking the C-terminal domain failed to induce ERK activation and did not protect the cells from I/R injury. In contrast, expression of the C-terminal domain by itself was sufficient to induce ERK activation and I/R protection. Interestingly, the C-terminal of the ZnT-1 did not have protective effect against the toxicity of zinc. In the isolated rat heart, global ischemic injury rapidly increased the endogenous levels of ZnT-1. However, following reperfusion ZnT-1 levels were found to be decreased. Our findings indicate that ZnT-1 may have important role in the ischemic myocardium through its ability to interact with Raf-1 kinase.
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Papers by Yoram Etzion
function mutations in genes, encoding the cardiac potassium channel
subunits KCNJ2, KCNE1, and KCNH2 that mediate the repolarizing
potassium currents Ik1, Iks, and Ikr, respectively.
METHODS: Linkage analysis, whole-exome sequencing, and Xenopus
oocyte electrophysiology studies were used in this study.
RESULTS: Through genetic studies, we showed that autosomal dominant
early-onset nocturnal paroxysmal AF is caused by p.S447R mutation in
KCND2, encoding the pore-forming (α) subunit of the Kv4.2 cardiac
potassium channel. Kv4.2, along with Kv4.3, contributes to the cardiac
fast transient outward K+ current, Ito. Ito underlies the early phase of
repolarization in the cardiac action potential, thereby setting the initial
potential of the plateau phase and governing its duration and amplitude.
In Xenopus oocytes, the mutation increased the channel’s inactivation
time constant and affected its regulation: p.S447 resides in a protein
kinase C (PKC) phosphorylation site, which normally allows attenuation
of Kv4.2 membrane expression. The mutant Kv4.2 exhibited impaired
response to PKC; hence, Kv4.2 membrane expression was augmented,
enhancing potassium currents. Coexpression of mutant and wild-type
channels (recapitulating heterozygosity in affected individuals) showed
results similar to the mutant channel alone. Finally, in a hybrid channel
composed of Kv4.3 and Kv4.2, simulating the mature endogenous
heterotetrameric channel underlying Ito, the p.S447R Kv4.2 mutation
exerted a gain-of-function effect on Kv4.3.
CONCLUSIONS: The mutation alters Kv4.2’s kinetic properties, impairs
its inhibitory regulation, and exerts gain-of-function effect on both
Kv4.2 homotetramers and Kv4.2-Kv4.3 heterotetramers. These effects
presumably increase the repolarizing potassium current Ito, thereby
abbreviating action potential duration, creating arrhythmogenic substrate
for nocturnal AF. Interestingly, Kv4.2 expression was previously shown to
demonstrate circadian variation, with peak expression at daytime in murine
hearts (human nighttime), with possible relevance to the nocturnal onset
of paroxysmal AF symptoms in our patients. The atrial-specific phenotype
suggests that targeting Kv4.2 might be effective in the treatment of
nocturnal paroxysmal AF, avoiding adverse ventricular effects.
function mutations in genes, encoding the cardiac potassium channel
subunits KCNJ2, KCNE1, and KCNH2 that mediate the repolarizing
potassium currents Ik1, Iks, and Ikr, respectively.
METHODS: Linkage analysis, whole-exome sequencing, and Xenopus
oocyte electrophysiology studies were used in this study.
RESULTS: Through genetic studies, we showed that autosomal dominant
early-onset nocturnal paroxysmal AF is caused by p.S447R mutation in
KCND2, encoding the pore-forming (α) subunit of the Kv4.2 cardiac
potassium channel. Kv4.2, along with Kv4.3, contributes to the cardiac
fast transient outward K+ current, Ito. Ito underlies the early phase of
repolarization in the cardiac action potential, thereby setting the initial
potential of the plateau phase and governing its duration and amplitude.
In Xenopus oocytes, the mutation increased the channel’s inactivation
time constant and affected its regulation: p.S447 resides in a protein
kinase C (PKC) phosphorylation site, which normally allows attenuation
of Kv4.2 membrane expression. The mutant Kv4.2 exhibited impaired
response to PKC; hence, Kv4.2 membrane expression was augmented,
enhancing potassium currents. Coexpression of mutant and wild-type
channels (recapitulating heterozygosity in affected individuals) showed
results similar to the mutant channel alone. Finally, in a hybrid channel
composed of Kv4.3 and Kv4.2, simulating the mature endogenous
heterotetrameric channel underlying Ito, the p.S447R Kv4.2 mutation
exerted a gain-of-function effect on Kv4.3.
CONCLUSIONS: The mutation alters Kv4.2’s kinetic properties, impairs
its inhibitory regulation, and exerts gain-of-function effect on both
Kv4.2 homotetramers and Kv4.2-Kv4.3 heterotetramers. These effects
presumably increase the repolarizing potassium current Ito, thereby
abbreviating action potential duration, creating arrhythmogenic substrate
for nocturnal AF. Interestingly, Kv4.2 expression was previously shown to
demonstrate circadian variation, with peak expression at daytime in murine
hearts (human nighttime), with possible relevance to the nocturnal onset
of paroxysmal AF symptoms in our patients. The atrial-specific phenotype
suggests that targeting Kv4.2 might be effective in the treatment of
nocturnal paroxysmal AF, avoiding adverse ventricular effects.