Loss of Intracellular Fibroblast Growth Factor 14 (iFGF14) Increases the Excitability of Mature Hippocampal and Cortical Pyramidal Neurons.

Mutations in FGF14 , which encodes intracellular fibroblast growth factor 14 (iFGF14), have been linked to spinocerebellar ataxia type 27 (SCA27), a multisystem disorder associated with progressive deficits in motor coordination and cognitive function. Mice ( Fgf14 -/- ) lacking iFGF14 display similar phenotypes, and we have previously shown that the deficits in motor coordination reflect reduced excitability of cerebellar Purkinje neurons, owing to the loss of iFGF14-mediated regulation of the voltage-dependence of inactivation of the fast transient component of the voltage-gated Na + (Nav) current, I NaT . Here, we present the results of experiments designed to test the hypothesis that loss of iFGF14 also attenuates the intrinsic excitability of mature hippocampal and cortical pyramidal neurons. Current-clamp recordings from adult mouse hippocampal CA1 pyramidal neurons in acute in vitro slices, however, revealed that repetitive firing rates were higher in Fgf14 -/- , than in wild type (WT), cells. In addition, the waveforms of individual action potentials were altered in Fgf14 -/- hippocampal CA1 pyramidal neurons, and the loss of iFGF14 reduced the time delay between the initiation of axonal and somal action potentials. Voltage-clamp recordings revealed that the loss of iFGF14 altered the voltage-dependence of activation, but not inactivation, of I NaT in CA1 pyramidal neurons. Similar effects of the loss of iFGF14 on firing properties were evident in current-clamp recordings from layer 5 visual cortical pyramidal neurons. Additional experiments demonstrated that the loss of iFGF14 does not alter the distribution of anti-Nav1.6 or anti-ankyrin G immunofluorescence labeling intensity along the axon initial segments (AIS) of mature hippocampal CA1 or layer 5 visual cortical pyramidal neurons in situ . Taken together, the results demonstrate that, in contrast with results reported for neonatal (rat) hippocampal pyramidal neurons in dissociated cell culture, the loss of iFGF14 does not disrupt AIS architecture or Nav1.6 localization/distribution along the AIS of mature hippocampal (or cortical) pyramidal neurons in situ .

Kv12-encoded K+ channels drive the day-night switch in the repetitive firing rates of SCN neurons.

Considerable evidence suggests that day-night rhythms in the functional expression of subthreshold potassium (K+) channels regulate daily oscillations in the spontaneous firing rates of neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in mammals. The K+ conductance(s) driving these daily rhythms in the repetitive firing rates of SCN neurons, however, have not been identified. To test the hypothesis that subthreshold Kv12.1/Kv12.2-encoded K+ channels play a role, we obtained current-clamp recordings from SCN neurons in slices prepared from adult mice harboring targeted disruptions in the Kcnh8 (Kv12.1-/-) or Kcnh3 (Kv12.2-/-) locus. We found that mean nighttime repetitive firing rates were higher in Kv12.1-/- and Kv12.2-/- than in wild type (WT), SCN neurons. In marked contrast, mean daytime repetitive firing rates were similar in Kv12.1-/-, Kv12.2-/-, and WT SCN neurons, and the day-night difference in mean repetitive firing rates, a hallmark feature of WT SCN neurons, was eliminated in Kv12.1-/- and Kv12.2-/- SCN neurons. Similar results were obtained with in vivo shRNA-mediated acute knockdown of Kv12.1 or Kv12.2 in adult SCN neurons. Voltage-clamp experiments revealed that Kv12-encoded current densities in WT SCN neurons are higher at night than during the day. In addition, the pharmacological block of Kv12-encoded currents increased the mean repetitive firing rate of nighttime, but not daytime, in WT SCN neurons. Dynamic clamp-mediated subtraction of modeled Kv12-encoded currents also selectively increased the mean repetitive firing rates of nighttime WT SCN neurons. Despite the elimination of the nighttime decrease in the mean repetitive firing rates of SCN neurons, however, locomotor (wheel-running) activity remained rhythmic in Kv12.1-/-, Kv12.2-/-, and Kv12.1-targeted shRNA-expressing, and Kv12.2-targeted shRNA-expressing animals.

Determinants of iFGF13-mediated regulation of myocardial voltage-gated sodium (NaV) channels in mouse.

Posttranslational regulation of cardiac NaV1.5 channels is critical in modulating channel expression and function, yet their regulation by phosphorylation of accessory proteins has gone largely unexplored. Using phosphoproteomic analysis of NaV channel complexes from adult mouse left ventricles, we identified nine phosphorylation sites on intracellular fibroblast growth factor 13 (iFGF13). To explore the potential roles of these phosphosites in regulating cardiac NaV currents, we abolished expression of iFGF13 in neonatal and adult mouse ventricular myocytes and rescued it with wild-type (WT), phosphosilent, or phosphomimetic iFGF13-VY. While the increased rate of closed-state inactivation of NaV channels induced by Fgf13 knockout in adult cardiomyocytes was completely restored by adenoviral-mediated expression of WT iFGF13-VY, only partial rescue was observed in neonatal cardiomyocytes after knockdown. The knockdown of iFGF13 in neonatal ventricular myocytes also shifted the voltage dependence of channel activation toward hyperpolarized potentials, a shift that was not reversed by WT iFGF13-VY expression. Additionally, we found that iFGF13-VY is the predominant isoform in adult ventricular myocytes, whereas both iFGF13-VY and iFGF13-S are expressed comparably in neonatal ventricular myocytes. Similar to WT iFGF13-VY, each of the iFGF13-VY phosphomutants studied restored NaV channel inactivation properties in both models. Lastly, Fgf13 knockout also increased the late Na+ current in adult cardiomyocytes, and this effect was restored with expression of WT and phosphosilent iFGF13-VY. Together, our results demonstrate that iFGF13 is highly phosphorylated and displays differential isoform expression in neonatal and adult ventricular myocytes. While we found no roles for iFGF13 phosphorylation, our results demonstrate differential effects of iFGF13 on neonatal and adult mouse ventricular NaV channels.

Effects of NALCN-Encoded Na+ Leak Currents on the Repetitive Firing Properties of SCN Neurons Depend on K+-Driven Rhythmic Changes in Input Resistance.

Neurons in the suprachiasmatic nucleus (SCN) generate circadian changes in the rates of spontaneous action potential firing that regulate and synchronize daily rhythms in physiology and behavior. Considerable evidence suggests that daily rhythms in the repetitive firing rates (higher during the day than at night) of SCN neurons are mediated by changes in subthreshold potassium (K+) conductance(s). An alternative "bicycle" model for circadian regulation of membrane excitability in clock neurons, however, suggests that an increase in NALCN-encoded sodium (Na+) leak conductance underlies daytime increases in firing rates. The experiments reported here explored the role of Na+ leak currents in regulating daytime and nighttime repetitive firing rates in identified adult male and female mouse SCN neurons: vasoactive intestinal peptide-expressing (VIP+), neuromedin S-expressing (NMS+) and gastrin-releasing peptide-expressing (GRP+) cells. Whole-cell recordings from VIP+, NMS+, and GRP+ neurons in acute SCN slices revealed that Na+ leak current amplitudes/densities are similar during the day and at night, but have a larger impact on membrane potentials in daytime neurons. Additional experiments, using an in vivo conditional knockout approach, demonstrated that NALCN-encoded Na+ currents selectively regulate daytime repetitive firing rates of adult SCN neurons. Dynamic clamp-mediated manipulation revealed that the effects of NALCN-encoded Na+ currents on the repetitive firing rates of SCN neurons depend on K+ current-driven changes in input resistances. Together, these findings demonstrate that NALCN-encoded Na+ leak channels contribute to regulating daily rhythms in the excitability of SCN neurons by a mechanism that depends on K+ current-mediated rhythmic changes in intrinsic membrane properties.SIGNIFICANCE STATEMENT Elucidating the ionic mechanisms responsible for generating daily rhythms in the rates of spontaneous action potential firing of neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in mammals, is an important step toward understanding how the molecular clock controls circadian rhythms in physiology and behavior. While numerous studies have focused on identifying subthreshold K+ channel(s) that mediate day-night changes in the firing rates of SCN neurons, a role for Na+ leak currents has also been suggested. The results of the experiments presented here demonstrate that NALCN-encoded Na+ leak currents differentially modulate daily rhythms in the daytime/nighttime repetitive firing rates of SCN neurons as a consequence of rhythmic changes in subthreshold K+ currents.

Differential regulation of cardiac sodium channels by intracellular fibroblast growth factors.

Voltage-gated sodium (NaV) channels are responsible for the initiation and propagation of action potentials. In the heart, the predominant NaV1.5 α subunit is composed of four homologous repeats (I-IV) and forms a macromolecular complex with multiple accessory proteins, including intracellular fibroblast growth factors (iFGF). In spite of high homology, each of the iFGFs, iFGF11-iFGF14, as well as the individual iFGF splice variants, differentially regulates NaV channel gating, and the mechanisms underlying these differential effects remain elusive. Much of the work exploring iFGF regulation of NaV1.5 has been performed in mouse and rat ventricular myocytes in which iFGF13VY is the predominant iFGF expressed, whereas investigation into NaV1.5 regulation by the human heart-dominant iFGF12B is lacking. In this study, we used a mouse model with cardiac-specific Fgf13 deletion to study the consequences of iFGF13VY and iFGF12B expression. We observed distinct effects on the voltage-dependences of activation and inactivation of the sodium currents (INa), as well as on the kinetics of peak INa decay. Results in native myocytes were recapitulated with human NaV1.5 heterologously expressed in Xenopus oocytes, and additional experiments using voltage-clamp fluorometry (VCF) revealed iFGF-specific effects on the activation of the NaV1.5 voltage sensor domain in repeat IV (VSD-IV). iFGF chimeras further unveiled roles for all three iFGF domains (i.e., the N-terminus, core, and C-terminus) on the regulation of VSD-IV, and a slower time domain of inactivation. We present here a novel mechanism of iFGF regulation that is specific to individual iFGF isoforms and that leads to distinct functional effects on NaV channel/current kinetics.

FHF2 phosphorylation and regulation of native myocardial Na V 1.5 channels.

Phosphorylation of the cardiac Na V 1.5 channel pore-forming subunit is extensive and critical in modulating channel expression and function, yet the regulation of Na V 1.5 by phosphorylation of its accessory proteins remains elusive. Using a phosphoproteomic analysis of Na V channel complexes purified from mouse left ventricles, we identified nine phosphorylation sites on Fibroblast growth factor Homologous Factor 2 (FHF2). To determine the roles of phosphosites in regulating Na V 1.5, we developed two models from neonatal and adult mouse ventricular cardiomyocytes in which FHF2 expression is knockdown and rescued by WT, phosphosilent or phosphomimetic FHF2-VY. While the increased rates of closed-state and open-state inactivation of Na V channels induced by the FHF2 knockdown are completely restored by the FHF2-VY isoform in adult cardiomyocytes, sole a partial rescue is obtained in neonatal cardiomyocytes. The FHF2 knockdown also shifts the voltage-dependence of activation towards hyperpolarized potentials in neonatal cardiomyocytes, which is not rescued by FHF2-VY. Parallel investigations showed that the FHF2-VY isoform is predominant in adult cardiomyocytes, while expression of FHF2-VY and FHF2-A is comparable in neonatal cardiomyocytes. Similar to WT FHF2-VY, however, each FHF2-VY phosphomutant restores the Na V channel inactivation properties in both models, preventing identification of FHF2 phosphosite roles. FHF2 knockdown also increases the late Na + current in adult cardiomyocytes, which is restored similarly by WT and phosphosilent FHF2-VY. Together, our results demonstrate that ventricular FHF2 is highly phosphorylated, implicate differential roles for FHF2 in regulating neonatal and adult mouse ventricular Na V 1.5, and suggest that the regulation of Na V 1.5 by FHF2 phosphorylation is highly complex. eTOC Summary: Lesage et al . identify the phosphorylation sites of FHF2 from mouse left ventricular Na V 1.5 channel complexes. While no roles for FHF2 phosphosites could be recognized yet, the findings demonstrate differential FHF2-dependent regulation of neonatal and adult mouse ventricular Na V 1.5 channels.

Kv12-Encoded K + Channels Drive the Day-Night Switch in the Repetitive Firing Rates of SCN Neurons.

Considerable evidence suggests that day-night rhythms in the functional expression of subthreshold potassium (K + ) channels regulate daily oscillations in the rates of spontaneous action potential firing of neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in mammals. The K + conductance(s) driving these daily rhythms in repetitive firing rates, however, have not been identified. To test the hypothesis that subthreshold Kv12.1/Kv12.2-encoded K + channels play a role, we obtained current-clamp recordings from SCN neurons in slices prepared from adult mice harboring targeted disruptions in the Kcnh8 (Kv12.1 -/- ) or Kcnh3 (Kv12.2 -/- ) locus. We found that mean nighttime repetitive firing rates were higher in Kv12.1 -/- and Kv12.2 -/- , than in wild type (WT), SCN neurons. In marked contrast, mean daytime repetitive firing rates were similar in Kv12.1 -/- , Kv12.2 -/- and WT SCN neurons, and the day-night difference in mean repetitive firing rates, a hallmark feature of WT SCN neurons, was eliminated in Kv12.1 -/- and Kv12.2 -/- SCN neurons. Similar results were obtained with in vivo shRNA-mediated acute knockdown of Kv12.1 or Kv12.2 in adult SCN neurons. Voltage-clamp experiments revealed that Kv12-encoded current densities in WT SCN neurons are higher at night than during the day. In addition, pharmacological block of Kv12-encoded currents increased the mean repetitive firing rate of nighttime, but not daytime, in WT SCN neurons. Dynamic clamp-mediated subtraction of modeled Kv12-encoded currents also selectively increased the mean repetitive firing rates of nighttime WT SCN neurons. Despite the elimination of nighttime decrease in the mean repetitive firing rates of SCN neurons, however, locomotor (wheel-running) activity remained rhythmic in Kv12.1 -/- , Kv12.2 -/- , Kv12.1-targeted shRNA-expressing, and Kv12.2-targeted shRNA-expressing animals.

Electro-mechanical coupling of KCNQ channels is a target of epilepsy-associated mutations and retigabine.

KCNQ2 and KCNQ3 form the M-channels that are important in regulating neuronal excitability. Inherited mutations that alter voltage-dependent gating of M-channels are associated with neonatal epilepsy. In the homolog KCNQ1 channel, two steps of voltage sensor activation lead to two functionally distinct open states, the intermediate-open (IO) and activated-open (AO), which define the gating, physiological, and pharmacological properties of KCNQ1. However, whether the M-channel shares the same mechanism is unclear. Here, we show that KCNQ2 and KCNQ3 feature only a single conductive AO state but with a conserved mechanism for the electro-mechanical (E-M) coupling between voltage sensor activation and pore opening. We identified some epilepsy-linked mutations in KCNQ2 and KCNQ3 that disrupt E-M coupling. The antiepileptic drug retigabine rescued KCNQ3 currents that were abolished by a mutation disrupting E-M coupling, suggesting that modulating the E-M coupling in KCNQ channels presents a potential strategy for antiepileptic therapy.

Intrinsic mechanisms in the gating of resurgent Na+ currents.

The resurgent component of the voltage-gated sodium current (INaR) is a depolarizing conductance, revealed on membrane hyperpolarizations following brief depolarizing voltage steps, which has been shown to contribute to regulating the firing properties of numerous neuronal cell types throughout the central and peripheral nervous systems. Although mediated by the same voltage-gated sodium (Nav) channels that underlie the transient and persistent Nav current components, the gating mechanisms that contribute to the generation of INaR remain unclear. Here, we characterized Nav currents in mouse cerebellar Purkinje neurons, and used tailored voltage-clamp protocols to define how the voltage and the duration of the initial membrane depolarization affect the amplitudes and kinetics of INaR. Using the acquired voltage-clamp data, we developed a novel Markov kinetic state model with parallel (fast and slow) inactivation pathways and, we show that this model reproduces the properties of the resurgent, as well as the transient and persistent, Nav currents recorded in (mouse) cerebellar Purkinje neurons. Based on the acquired experimental data and the simulations, we propose that resurgent Na+ influx occurs as a result of fast inactivating Nav channels transitioning into an open/conducting state on membrane hyperpolarization, and that the decay of INaR reflects the slow accumulation of recovered/opened Nav channels into a second, alternative and more slowly populated, inactivated state. Additional simulations reveal that extrinsic factors that affect the kinetics of fast or slow Nav channel inactivation and/or impact the relative distribution of Nav channels in the fast- and slow-inactivated states, such as the accessory Navß4 channel subunit, can modulate the amplitude of INaR.

Understanding Circadian Mechanisms of Sudden Cardiac Death: A Report From the National Heart, Lung, and Blood Institute Workshop, Part 1: Basic and Translational Aspects.

Sudden cardiac death (SCD), the unexpected death due to acquired or genetic cardiovascular disease, follows distinct 24-hour patterns in occurrence. These 24-hour patterns likely reflect daily changes in arrhythmogenic triggers and the myocardial substrate caused by day/night rhythms in behavior, the environment, and endogenous circadian mechanisms. To better address fundamental questions regarding the circadian mechanisms, the National Heart, Lung, and Blood Institute convened a workshop, Understanding Circadian Mechanisms of Sudden Cardiac Death. We present a 2-part report of findings from this workshop. Part 1 summarizes the workshop and serves to identify research gaps and opportunities in the areas of basic and translational research. Among the gaps was the lack of standardization in animal studies for reporting environmental conditions (eg, timing of experiments relative to the light dark cycle or animal housing temperatures) that can impair rigor and reproducibility. Workshop participants also pointed to uncertainty regarding the importance of maintaining normal circadian rhythmic synchrony and the potential pathological impact of desynchrony on SCD risk. One related question raised was whether circadian mechanisms can be targeted to reduce SCD risk. Finally, the experts underscored the need for studies aimed at determining the physiological importance of circadian clocks in the many different cell types important to normal heart function and SCD. Addressing these gaps could lead to new therapeutic approaches/molecular targets that can mitigate the risk of SCD not only at certain times but over the entire 24-hour period.

Understanding Circadian Mechanisms of Sudden Cardiac Death: A Report From the National Heart, Lung, and Blood Institute Workshop, Part 2: Population and Clinical Considerations.

Sudden cardiac death (SCD) is the sudden, unexpected death due to abrupt loss of heart function secondary to cardiovascular disease. In certain populations living with cardiovascular disease, SCD follows a distinct 24-hour pattern in occurrence, suggesting day/night rhythms in behavior, the environment, and endogenous circadian rhythms result in daily spans of increased vulnerability. The National Heart, Lung, and Blood Institute convened a workshop, Understanding Circadian Mechanisms of Sudden Cardiac Death to identify fundamental questions regarding the role of the circadian rhythms in SCD. Part 2 summarizes research gaps and opportunities in the areas of population and clinical research identified in the workshop. Established research supports a complex interaction between circadian rhythms and physiological responses that increase the risk for SCD. Moreover, these physiological responses themselves are influenced by several biological variables, including the type of cardiovascular disease, sex, age, and genetics, as well as environmental factors. The emergence of new noninvasive biotechnological tools that continuously measure key cardiovascular variables, as well as the identification of biomarkers to assess circadian rhythms, hold promise for generating large-scale human data sets that will delineate which subsets of individuals are most vulnerable to SCD. Additionally, these data will improve our understanding of how people who suffer from circadian disruptions develop cardiovascular diseases that increase the risk for SCD. Emerging strategies to identify new biomarkers that can quantify circadian health (eg, environmental, behavioral, and internal misalignment) may lead to new interventions and therapeutic targets to prevent the progression of cardiovascular diseases that cause SCD.

Identification of structures for ion channel kinetic models.

Markov models of ion channel dynamics have evolved as experimental advances have improved our understanding of channel function. Past studies have examined limited sets of various topologies for Markov models of channel dynamics. We present a systematic method for identification of all possible Markov model topologies using experimental data for two types of native voltage-gated ion channel currents: mouse atrial sodium currents and human left ventricular fast transient outward potassium currents. Successful models identified with this approach have certain characteristics in common, suggesting that aspects of the model topology are determined by the experimental data. Incorporating these channel models into cell and tissue simulations to assess model performance within protocols that were not used for training provided validation and further narrowing of the number of acceptable models. The success of this approach suggests a channel model creation pipeline may be feasible where the structure of the model is not specified a priori.

Modulation of the effects of class Ib antiarrhythmics on cardiac NaV1.5-encoded channels by accessory NaVß subunits.

Native myocardial voltage-gated sodium (NaV) channels function in macromolecular complexes comprising a pore-forming (α) subunit and multiple accessory proteins. Here, we investigated the impact of accessory NaVß1 and NaVß3 subunits on the functional effects of 2 well-known class Ib antiarrhythmics, lidocaine and ranolazine, on the predominant NaV channel α subunit, NaV1.5, expressed in the mammalian heart. We showed that both drugs stabilized the activated conformation of the voltage sensor of domain-III (DIII-VSD) in NaV1.5. In the presence of NaVß1, the effect of lidocaine on the DIII-VSD was enhanced, whereas the effect of ranolazine was abolished. Mutating the main class Ib drug-binding site, F1760, affected but did not abolish the modulation of drug block by NaVß1/ß3. Recordings from adult mouse ventricular myocytes demonstrated that loss of Scn1b (NaVß1) differentially affected the potencies of lidocaine and ranolazine. In vivo experiments revealed distinct ECG responses to i.p. injection of ranolazine or lidocaine in WT and Scn1b-null animals, suggesting that NaVß1 modulated drug responses at the whole-heart level. In the human heart, we found that SCN1B transcript expression was 3 times higher in the atria than ventricles, differences that could, in combination with inherited or acquired cardiovascular disease, dramatically affect patient response to class Ib antiarrhythmic therapies.

Mechanical dysfunction of the sarcomere induced by a pathogenic mutation in troponin T drives cellular adaptation.

Familial hypertrophic cardiomyopathy (HCM), a leading cause of sudden cardiac death, is primarily caused by mutations in sarcomeric proteins. The pathogenesis of HCM is complex, with functional changes that span scales, from molecules to tissues. This makes it challenging to deconvolve the biophysical molecular defect that drives the disease pathogenesis from downstream changes in cellular function. In this study, we examine an HCM mutation in troponin T, R92Q, for which several models explaining its effects in disease have been put forward. We demonstrate that the primary molecular insult driving disease pathogenesis is mutation-induced alterations in tropomyosin positioning, which causes increased molecular and cellular force generation during calcium-based activation. Computational modeling shows that the increased cellular force is consistent with the molecular mechanism. These changes in cellular contractility cause downstream alterations in gene expression, calcium handling, and electrophysiology. Taken together, our results demonstrate that molecularly driven changes in mechanical tension drive the early disease pathogenesis of familial HCM, leading to activation of adaptive mechanobiological signaling pathways.

Proteomic and functional mapping of cardiac NaV1.5 channel phosphorylation sites.

Phosphorylation of the voltage-gated Na+ (NaV) channel NaV1.5 regulates cardiac excitability, yet the phosphorylation sites regulating its function and the underlying mechanisms remain largely unknown. Using a systematic, quantitative phosphoproteomic approach, we analyzed NaV1.5 channel complexes purified from nonfailing and failing mouse left ventricles, and we identified 42 phosphorylation sites on NaV1.5. Most sites are clustered, and three of these clusters are highly phosphorylated. Analyses of phosphosilent and phosphomimetic NaV1.5 mutants revealed the roles of three phosphosites in regulating NaV1.5 channel expression and gating. The phosphorylated serines S664 and S667 regulate the voltage dependence of channel activation in a cumulative manner, whereas the nearby S671, the phosphorylation of which is increased in failing hearts, regulates cell surface NaV1.5 expression and peak Na+ current. No additional roles could be assigned to the other clusters of phosphosites. Taken together, our results demonstrate that ventricular NaV1.5 is highly phosphorylated and that the phosphorylation-dependent regulation of NaV1.5 channels is highly complex, site specific, and dynamic.

Polycystin 2 is increased in disease to protect against stress-induced cell death.

Polycystin 2 (PC2 or TRPP1, formerly TRPP2) is a calcium-permeant Transient Receptor Potential (TRP) cation channel expressed primarily on the endoplasmic reticulum (ER) membrane and primary cilia of all cell and tissue types. Despite its ubiquitous expression throughout the body, studies of PC2 have focused primarily on its role in the kidney, as mutations in PC2 lead to the development of autosomal dominant polycystic kidney disease (ADPKD), a debilitating condition for which there is no cure. However, the endogenous role that PC2 plays in the regulation of general cellular homeostasis remains unclear. In this study, we measure how PC2 expression changes in different pathological states, determine that its abundance is increased under conditions of cellular stress in multiple tissues including human disease, and conclude that PC2-deficient cells have increased susceptibility to cell death induced by stress. Our results offer new insight into the normal function of PC2 as a ubiquitous stress-sensitive protein whose expression is up-regulated in response to cell stress to protect against pathological cell death in multiple diseases.

The Role of the Voltage-Gated Potassium Channel Proteins Kv8.2 and Kv2.1 in Vision and Retinal Disease: Insights from the Study of Mouse Gene Knock-Out Mutations.

Mutations in the KCNV2 gene, which encodes the voltage-gated K+ channel protein Kv8.2, cause a distinctive form of cone dystrophy with a supernormal rod response (CDSRR). Kv8.2 channel subunits only form functional channels when combined in a heterotetramer with Kv2.1 subunits encoded by the KCNB1 gene. The CDSRR disease phenotype indicates that photoreceptor adaptation is disrupted. The electroretinogram (ERG) response of affected individuals shows depressed rod and cone activity, but what distinguishes this disease is the supernormal rod response to a bright flash of light. Here, we have utilized knock-out mutations of both genes in the mouse to study the pathophysiology of CDSRR. The Kv8.2 knock-out (KO) mice show many similarities to the human disorder, including a depressed a-wave and an elevated b-wave response with bright light stimulation. Optical coherence tomography (OCT) imaging and immunohistochemistry indicate that the changes in six-month-old Kv8.2 KO retinae are largely limited to the outer nuclear layer (ONL), while outer segments appear intact. In addition, there is a significant increase in TUNEL-positive cells throughout the retina. The Kv2.1 KO and double KO mice also show a severely depressed a-wave, but the elevated b-wave response is absent. Interestingly, in all three KO genotypes, the c-wave is totally absent. The differential response shown here of these KO lines, that either possess homomeric channels or lack channels completely, has provided further insights into the role of K+ channels in the generation of the a-, b-, and c-wave components of the ERG.

Regional differences in the expression of tetrodotoxin-sensitive inward Ca2+ and outward Cs+/K+ currents in mouse and human ventricles.

Tetrodotoxin (TTX) sensitive inward Ca2+ currents, ICa(TTX), have been identified in cardiac myocytes from several species, although it is unclear if ICa(TTX) is expressed in all cardiac cell types, and if ICa(TTX) reflects Ca2+ entry through the main, Nav1.5-encoded, cardiac Na+ (Nav) channels. To address these questions, recordings were obtained with 2 mm Ca2+ and 0 mm Na+ in the bath and 120 mm Cs+ in the pipettes from myocytes isolated from adult mouse interventricular septum (IVS), left ventricular (LV) endocardium, apex, and epicardium and from human LV endocardium and epicardium. On membrane depolarizations from a holding potential of -100 mV, ICa(TTX) was identified in mouse IVS and LV endocardial myocytes and in human LV endocardial myocytes, whereas only TTX-sensitive outward Cs+/K+ currents were observed in mouse LV apex and epicardial myocytes and human LV epicardial myocytes. The inward Ca2+, but not the outward Cs+/K+, currents were blocked by mm concentrations of MTSEA, a selective blocker of cardiac Nav1.5-encoded Na+ channels. In addition, in Nav1.5-expressing tsA-201 cells, ICa(TTX) was observed in 3 (of 20) cells, and TTX-sensitive outward Cs+/K+ currents were observed in the other (17) cells. The time- and voltage-dependent properties of the TTX-sensitive inward Ca2+ and outward Cs+/K+ currents recorded in Nav1.5-expressing tsA-201 were indistinguishable from native currents in mouse and human cardiac myocytes. Overall, the results presented here suggest marked regional, cell type-specific, differences in the relative ion selectivity, and likely the molecular architecture, of native SCN5A-/Scn5a- (Nav1.5-) encoded cardiac Na+ channels in mouse and human ventricles.

Circulating long noncoding RNA DKFZP434I0714 predicts adverse cardiovascular outcomes in patients with end-stage renal disease.

BACKGROUND: Cardiovascular (CV) diseases are major causes of mortality in uremic patients. Conventional risk factors fail to identify uremic patients with increased propensity for adverse CV outcomes. We aimed to test the hypothesis that circulating long noncoding RNAs (lncRNAs) could be a prognostic marker to predict adverse CV outcomes in uremic patients. METHODS AND RESULTS: Plasma lncRNAs were profiled in patients with end-stage renal disease (ESRD, n = 28) or chronic kidney disease (CKD, n = 8) and in healthy (n = 12) subjects by RNA sequencing. A total of 179 lncRNAs were significantly dysregulated with ESRD; the expression signature of plasma lncRNAs distinguished ESRD from both CKD and control samples. Analysis on a microarray dataset obtained from renal biopsy samples of patients with advanced kidney disease (GSE66494) revealed that a significant proportion of plasma lncRNAs (30.7%) and mRNAs (49.5%) dysregulated with uremia were similarly dysregulated in diseased kidneys, suggesting that plasma RNA profiles mirror the transcriptomal changes in diseased kidney tissues. Further analyses identified eight plasma lncRNAs as potential predictors of adverse CV outcomes in uremic patients. Validation study in an independent cohort of ESRD patients (n = 111) confirmed that elevated plasma lncRNA DKFZP434I0714 is a significant independent predictor of adverse CV outcomes in uremic patients. Additional experiments demonstrated the functional involvement of DKFZP434I0714 in the pathogenesis of endothelial dysfunction. CONCLUSIONS: In summary, plasma lncRNA expression signature reflects the disease states of uremia. Elevated plasma levels of lncRNA DKFZP434I0714 in uremic patients portend a worse CV outcome and warrant closer monitoring and more aggressive management.