Ofer Beharier

Molecular Basis for Zinc Transporter 1 Action as an Endogenous Inhibitor of L-type Calcium Channels

The L-type calcium channel (LTCC) has a variety of physiological roles that are critical for the proper function of many cell types and organs. Recently, a member of the zinc-regulating family of proteins, ZnT-1, was recognized as an endogenous inhibitor of the LTCC, but its mechanism of action has not been elucidated. In the present study, using two-electrode voltage clamp recordings in Xenopus oocytes, we demonstrate that ZnT-1-mediated inhibition of the LTCC critically depends on the presence of the LTCC regulatory β-subunit. Moreover, the ZnT-1-induced inhibition of the LTCC current is also abolished by excess levels of the β-subunit. An interaction between ZnT-1 and the β-subunit, as demonstrated by co-immunoprecipitation and by fluorescence resonance energy transfer, is consistent with this result. Using surface biotinylation and total internal reflection fluorescence microscopy in HEK293 cells, we show a ZnT-1-dependent decrease in the surface expression of the pore-forming α1-subunit of the LTCC. Similarly, a decrease in the surface expression of the α1-subunit is observed following up-regulation of the expression of endogenous ZnT-1 in rapidly paced cultured cardiomyocytes. We conclude that ZnT-1-mediated inhibition of the LTCC is mediated through a functional interaction of ZnT-1 with the LTCC β-subunit and that it involves a decrease in the trafficking of the LTCC α1-subunit to the surface membrane.

ZnT-1 enhances the activity and surface expression of T-type calcium channels through activation of Ras-ERK signaling

Zinc transporter-1 (ZnT-1) is a putative zinc transporter that confers cellular resistance from zinc toxicity. In addition, ZnT-1 has important regulatory functions, including inhibition of L-type calcium channels and activation of Raf-1 kinase. Here we studied the effects of ZnT-1 on the expression and function of T-type calcium channels. In Xenopus oocytes expressing voltage-gated calcium channel (CaV) 3.1 or CaV3.2, ZnT-1 enhanced the low-threshold calcium currents (I(caT)) to 182 ± 15 and 167.95 ± 9.27% of control, respectively (P < 0.005 for both channels). As expected, ZnT-1 also enhanced ERK phosphorylation. Coexpression of ZnT-1 and nonactive Raf-1 blocked the ZnT-1-mediated ERK phosphorylation and abolished the ZnT-1-induced augmentation of I(caT). In mammalian cells (Chinese hamster ovary), coexpression of CaV3.1 and ZnT-1 increased the I(caT) to 166.37 ± 6.37% compared with cells expressing CaV3.1 alone (P < 0.01). Interestingly, surface expression measurements using biotinylation or total internal reflection fluorescence microscopy indicated marked ZnT-1-induced enhancement of CaV3.1 surface expression. The MEK inhibitor PD-98059 abolished the ZnT-1-induced augmentation of surface expression of CaV3.1. In cultured murine cardiomyocytes (HL-1 cells), transient exposure to zinc, leading to enhanced ZnT-1 expression, also enhanced the surface expression of endogenous CaV3.1 channels. Consistently, in these cells, endothelin-1, a potent activator of Ras-ERK signaling, enhanced the surface expression of CaV3.1 channels in a PD-98059-sensitive manner. Our findings indicate that ZnT-1 enhances the activity of CaV3.1 and CaV3.2 through activation of Ras-ERK signaling. The augmentation of CaV3.1 currents by Ras-ERK activation is associated with enhanced trafficking of the channel to the plasma membrane.

S100B – a potential biomarker for early detection of neonatal brain damage following asphyxia

Birth asphyxia results in a significant percentage of neonatal morbidity and mortality. A key factor in the management of this complication is the early and accurate detection of brain damage following asphyxia. Currently, reliable tools for such diagnosis are absent. Extensive research has focused on biomarkers in an attempt to solve this matter. Recent data marked serum and urine elevation of the S100B protein as an established peripheral biomarker for detection of brain injury including traumatic head injuries and brain damage following cardiac arrest and stroke. In the past decade, a substantial number of studies illustrated the potential use of S100B testing in order to detect brain damage in asphyxiated newborns. This review summarizes the available data regarding the use of S100B as a biomarker of brain damage following birth asphyxia.

ZnT-1 protects HL-1 cells from simulated ischemia–reperfusion through activation of Ras–ERK signaling

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.

Correlation Between Atrial ZnT-1 Expression and Atrial Fibrillation in Humans: A Pilot Study

Background: Until recently, the membrane protein ZnT‐1 was studied mainly in the context of zinc homeostasis. However, new findings indicate that it acts as an inhibitor of L‐type calcium channels. We recently found that acute rapid pacing of the rat atria in vivo augments the expression of ZnT‐1, while knockdown of ZnT‐1 in culture can oppose the inhibition of L‐type calcium channels following rapid pacing. This pilot study, the first to assess cardiac ZnT‐1 in humans, was designed to look for possible correlation between the atrial expression of ZnT‐1 and atrial fibrillation.

Gestational Diabetes Mellitus Is a Significant Risk Factor for Long-Term Maternal Renal Disease

CONTEXTnGestational diabetes mellitus (GDM) was found to be an independent risk factor for recurrent long-term type 2 diabetes mellitus, cardiovascular morbidity, and vascular endothelial dysfunction. However, data on the link between GDM and future risk for long-term maternal renal disease are limited.nnnOBJECTIVEnThe purpose of this study was to investigate whether GDM poses a risk for subsequent long-term maternal renal morbidity.nnnDESIGNnA population-based noninterventional study compared the incidence of future renal morbidity in a cohort of women with and without previous GDM. Deliveries occurred during a 25-year period, with a mean follow-up duration of 11.2 years.nnnSETTINGnThe study was conducted at the Soroka University Medical Center.nnnPARTICIPANTSnThe study population was composed of all singleton pregnancies in women who delivered between January 1988 and December 2013.nnnMAIN OUTCOME MEASUREnThe main outcome was diagnosis of renal morbidities.nnnRESULTSnOf 97,968 women who met the inclusion criteria, 9542 (9.7%) had at least 1 previous pregnancy with GDM. Using a Kaplan-Meier survival curve, we show that women with GDM had higher rates of total renal morbidity (0.1% vs 0.2%, for no GDM and with GDM, respectively; odds ratio, 2.3, 95% confidence interval, 1.4-3.7; P < .001). In addition, we found a significant dose-response association (using the χ(2) test for trends) between the number of pregnancies with GDM and future risk for renal morbidity (0.1%, 0.2%, and 0.4% for no GDM, 1 episode of GDM, and 2 episodes of GDM, respectively; P < .001). In a Cox proportional hazards model, adjusted for confounders, GDM was independently associated with future renal morbidity.nnnCONCLUSIONnGDM is a significant risk factor for future maternal renal morbidity. The risk is more substantial for patients with recurrent episodes of GDM.

New insights into the atrial electrophysiology of rodents using a novel modality: the miniature-bipolar hook electrode

Studies of atrial electrophysiology (EP) in rodents are challenging, and available data are sparse. Herein, we utilized a novel type of bipolar electrode to evaluate the atrial EP of rodents through small lateral thoracotomy. In anesthetized rats and mice, we attached two bipolar electrodes to the right atrium and a third to the right ventricle. This standard setup enabled high-resolution EP studies. Moreover, a permanent implantation procedure enabled EP studies in conscious freely moving rats. Atrial EP was evaluated in anesthetized rats, anesthetized mice (ICR and C57BL6 strains), and conscious rats. Signal resolution enabled atrial effective refractory period (AERP) measurements and first time evaluation of the failed 1:1 atrial capture, which was unexpectedly longer than the AERP recorded at near normal cycle length by 27.2+/-2.3% in rats (P<0.0001; n=35), 31.7+/-8.3% in ICR mice (P=0.0001; n=13), and 57.7+/-13.7% in C57BL6 mice (P=0.015; n=4). While AERP rate adaptation was noted when 10 S1s at near normal basic cycle lengths were followed by S2 at varying basic cycle length and S3 for AERP evaluation, such rate adaptation was absent using conventional S1S2 protocols. Atrial tachypacing in rats shortened the AERP values on a timescale of hours, but a reverse remodeling phase was noted thereafter. Comparison of left vs. right atrial pacing in rats was also feasible with the current technique, resulting in similar AERP values recorded in the low right atrium. In conclusion, our findings indicate that in vivo rate adaptation of the rodent atria is different than expected based on previous ex vivo recordings. In addition, atrial electrical remodeling of rats shows unique remodeling-reverse remodeling characteristics that are described here for the first time. Further understanding of these properties should help to determine the clinical relevance as well as limitations of atrial arrhythmia models in rodents.

Early-term deliveries as an independent risk factor for long-term respiratory morbidity of the offspring

Newborns exhibit the lowest immediate respiratory morbidity rates when born following 39 completed weeks of gestation. We sought to determine whether early‐term delivery (37–38u2009+u20096 weeks’ gestation) impacts on long‐term pediatric respiratory morbidity.

The physiologic anticoagulant and anti-inflammatory role of heparins and their utility in the prevention of pregnancy complications

Accumulating evidence supports the concept of increased thrombin generation, placental vascular lesions, and inflammation as crucial points in the development of the great obstetrical syndromes [preeclampsia, intrauterine growth restriction (IUGR), preterm labor (PTL), preterm prelabor rupture of membranes (PROM), fetal demise and recurrent abortions]. In light of this, the role of heparins for primary or secondary prevention of these syndromes is becoming more and more apparent, mainly due to the antithrombotic and anti-inflammatory effects of heparins. There is agreement regarding the use of heparin in the prevention of gestational complications in patients with antiphospholipid syndrome, while its use for other obstetrical complications is under debate. In the present review we will describe the physiologic role of heparins on coagulation and inflammation and we will discuss current evidence regarding the use of heparins for the prevention/treatment of obstetrical syndromes.

INO-8875, a Highly Selective A1 Adenosine Receptor Agonist: Evaluation of Chronotropic, Dromotropic, and Hemodynamic Effects in Rats

Selective pharmacological activation of the adenosine 1 receptor (A1R) is a promising new approach to achieve a potent block of atrioventricular (A-V)–nodal conduction without significant cardiovascular side effects. The purpose of the present study was to evaluate the cardiovascular profile of INO-8875, a highly selective A1R agonist, and to compare its properties with N-[3(R)-tetrahydrofuranyl]-6-aminopurine riboside (CVT-510), which has already been shown to induce negative dromotropic effects with minimal cardiovascular side effects in animals and in clinical studies. Dose-response experiments in the isolated hearts of rats were used to evaluate the functional selectivity of INO-8875 for the slowing of A-V–nodal conduction. Ventilated adult rats were used to study the effects of INO-8875, in vivo, on arterial blood pressure as well as on supraventricular electrophysiology. Ex vivo, INO-8875 (100 nM to 3 μM) progressively prolonged A-V–nodal conduction without reducing left ventricular function or coronary resistance. In vivo, INO-8875 up to a dose of 50 μg/kg did not reduce the carotid arterial blood pressure (n = 4). INO-8875 (1–50 μg/kg) and CVT-510 (20 and 50 μg/kg) both induced a dose-dependent decrease in heart rate and atrial refractoriness, as well as slowing of A-V–nodal conduction. However, compared with CVT-510, the activity of INO-8875 was more pronounced in A-V–nodal function. INO-8875 exhibited a greater duration of action, lasting up to 2.5 hours post dosing, whereas the effects of CVT-510 dissipated over 1 hour. INO-8875 demonstrates functional properties of a highly selective A1R agonist. INO-8875 exhibits an increased dromotropic effect and greater duration of action compared with CVT-510.