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Background: In depolarized myocardial infarct epicardial border zones, the cardiac sodium channel is largely inactivated, contributing to slow conduction and reentry. We have demonstrated that adenoviral delivery of the skeletal muscle Na(+) channel (SkM1) to epicardial border zones normalizes conduction and reduces induction of ventricular tachycardia/ventricular fibrillation. We now studied the impact of canine mesenchymal stem cells (cMSCs) in delivering SkM1.Methods and Results: cMSCs were isolated and transfected with SkM1. Coculture experiments showed cMSC/SkM1 but not cMSC alone and maintained fast conduction at depolarized potentials. We studied 3 groups in the canine 7d infarct: sham, cMSC, and cMSC/SkM1. In vivo epicardial border zones electrograms were broad and fragmented in sham, narrower in cMSCs, and narrow and unfragmented in cMSC/SkM1 (P<0.05). During programmed electrical stimulation of epicardial border zones, QRS duration in cMSC/SkM1 was shorter than in cMSC and sham (P<0.05). Programmed electrical stimulation-induced ventricular tachycardia/ventricular fibrillation was equivalent in all groups (P>0.05).Conclusion: cMSCs provide efficient delivery of SkM1 current. The interventions performed (cMSCs or cMSC/SkM1) were neither antiarrhythmic nor proarrhythmic. Comparing outcomes with cMSC/SkM1 and viral gene delivery highlights the criticality of the delivery platform to SkM1 antiarrhythmic efficacy. [ABSTRACT FROM AUTHOR]

The prevention and treatment of cardiac arrhythmias conferring major morbidity and mortality is far from optimal, and relies heavily on devices and drugs for the partial successes that have been seen. The greatest success has been in the use of electronic pacemakers to drive the hearts of patients having high degree heart block. Recent years have seen the beginnings of attempts to use novel approaches available through gene and cell therapies to treat both brady- and tachyarrhythmias. By far the most successful approaches to date have been seen in the development of biological pacemakers. However, the far more difficult problems posed by atrial fibrillation and ventricular tachycardia are now being addressed. In the following pages we review the approaches now in progress as well as the specific methodologic demands that must be met if these therapies are to be successful. [ABSTRACT FROM AUTHOR]

Background: A potential concern about biological pacemakers is their possible malfunction, which might create ventricular tachycardias (VTs).Objective: The purpose of this study was to test our hypothesis that should VTs complicate implantation of HCN-channel-based biological pacemakers, they would be suppressed by inhibitors of the pacemaker current, I(f).Methods: We created a chimeric channel (HCN212) containing the N- and C-termini of mouse HCN2 and the transmembrane region of mouse HCN1 and implanted it in HEK293 cells. Forty-eight hours later, in whole-cell patch clamp recordings, mean steady state block induced by 3 microM ivabradine (IVB) showed HCN1 = HCN212 > HCN2 currents. The HCN212 adenoviral construct was then implanted into the canine left bundle branch in 11 dogs. Complete AV block was created via radiofrequency ablation, and a ventricular demand electronic pacemaker was implanted (VVI 45 bpm). Electrocardiogram, 24-hour Holter monitoring, and pacemaker log record check were performed for 11 days.Results: All dogs developed rapid VT (>120 bpm, maximum rate = 285 +/- 37 bpm) at 0.9 +/- 0.3 days after implantation that persisted through 5 +/- 1 days. IVB, 1 mg/kg over 5 minutes, was administered during rapid VT, and three dogs received a second dose 24 hours later. While VT terminated with IBV in all instances within 3.4 +/- 0.6 minutes, no effect of IVB on sinus rate was noted.Conclusion: We conclude that (1) I(f)-associated tachyarrhythmias-if they occur with HCN-based biological pacemakers-can be controlled with I(f)-inhibiting drugs such as IVB; (2) in vitro, IVB appears to have a greater steady state inhibiting effect on HCN1 and HCN212 isoforms than on HCN4; and (3) VT originating from the HCN212 injection site is suppressed more readily than sinus rhythm. This suggests a selectivity of IVB at the concentration attained for ectopic over HCN4-based pacemaker function. This might confer a therapeutic benefit. [ABSTRACT FROM AUTHOR]

This study examined self-esteem in relation to sexual behaviors which often result in teen pregnancy. A sample of 141 male and 172 female adolescents of racial diversity was surveyed to elicit levels of self-esteem, sexual activity, pregnancy and fatherhood status. The Coopersmith Self-Esteem Inventory was used as well to elicit qualitative data about self-esteem, demographics, and sexual activity. Analysis revealed no differences in the self-esteem of males vs. females. Further, sexual activity or virginity was not related to self-esteem in either males or females. Pregnant teens did not have different levels of self-esteem from the nonpregnant. However, males who had fathered a child had lower self-esteem than did nonfathers. The findings support a multifocused approach to sex education for pregnancy prevention and also emphasize a need to include males in both pregnancy prevention efforts as well as in further research on teen pregnancy. [ABSTRACT FROM AUTHOR]

This article reviews work over the past three decades that is related to the contribution of the pacemaker current, I f , to basal and autonomically regulated spontaneous rate in the sinoatrial node. It also addresses how the actions of the pacemaker current relate to those of Ca homeostasis with respect to basal and autonomically regulated rhythm. In this regard, it explores the relative contributions of Ca-sensitive and Ca-insensitive isoforms of adenylyl cyclase to sinoatrial node automaticity. The latter studies include previously unpublished work making use of mice in which both the type 1 and type 8 Ca-sensitive adenylyl cyclase isoforms were knocked out. These studies indicate that the pacemaker current and the L-type Ca current are distinctly influenced by Ca-sensitive and insensitive adenylyl cyclase isoforms., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objective: To determine whether men with unexplained infertility and low total T (TT) have abnormal spermatogenesis and lower fecundity., Design: Secondary analysis of the prospective, randomized, multicenter clinical trial, Assessment of Multiple Intrauterine Gestations from Ovarian Stimulation (AMIGOS)., Setting: Infertility clinics., Patient(s): Nine hundred couples with unexplained infertility enrolled in AMIGOS. Semen analysis with an ejaculate of at least 5 million total motile sperm was required for enrollment. For inclusion in this secondary analysis, a fasting TT was required., Intervention(s): None., Main Outcome Measure(s): Logistic regression, adjusted for age and body mass index, assessed the association between low TT (defined as <264 ng/dL), semen parameters, and pregnancy outcome., Result(s): Seven hundred eighty-one men (mean age, 34.2 ± 5.7 years) with a median (interquartile range) TT of 411 (318-520) ng/dL were included. Men with TT <264 ng/dL were less likely to have normal (≥4% strict Kruger) morphology (unadjusted odds ratio [OR], 0.56; 95% confidence interval [CI], 0.34, 0.92; adjusted OR, 0.59; 95% CI, 0.35, 0.99). There was no association between low TT and semen volume < 1.5 mL, sperm concentration < 15 × 10 6 /mL, or motility < 40%. Among couples whose male partner had low TT, 21 (18.8%) had a live birth, compared with 184 (27.5%) live births in couples with a male partner having TT > 264 ng/dL. The odds of live birth decreased by 40% in couples whose male partner had low TT (unadjusted OR, 0.60; 95% CI, 0.36, 1.00; adjusted OR, 0.65; 95% CI, 0.38, 1.12)., Conclusion(s): In couples with unexplained infertility, low TT in the male partner was associated with abnormal sperm morphology and lower live birth rates., Clinical Trial Registration Number: NCT01044862., (Published by Elsevier Inc.)

In vivo, cardiomyocytes comprise a heterogeneous population of contractile cells defined by unique electrophysiologies, molecular markers and morphologies. The mechanisms directing myocardial cells to specific sub-lineages remain poorly understood. Here we report that overexpression of TGFβ-Activated Kinase (TAK1/Map3k7) in mouse embryonic stem (ES) cells faithfully directs myocardial differentiation of embryoid body (EB)-derived cardiac cells toward the sinoatrial node (SAN) lineage. Most cardiac cells in Map3k7-overexpressing EBs adopt markers, cellular morphologies, and electrophysiological behaviors characteristic of the SAN. These data, in addition to the fact that Map3k7 is upregulated in the sinus venous-the source of cells for the SAN-suggest that Map3k7 may be an endogenous regulator of the SAN fate.

The therapeutic success of human stem cell-derived cardiomyocytes critically depends on their ability to respond to and integrate with the surrounding electromechanical environment. Currently, the immaturity of human cardiomyocytes derived from stem cells limits their utility for regenerative medicine and biological research. We hypothesize that biomimetic electrical signals regulate the intrinsic beating properties of cardiomyocytes. Here we show that electrical conditioning of human stem cell-derived cardiomyocytes in three-dimensional culture promotes cardiomyocyte maturation, alters their automaticity and enhances connexin expression. Cardiomyocytes adapt their autonomous beating rate to the frequency at which they were stimulated, an effect mediated by the emergence of a rapidly depolarizing cell population, and the expression of hERG. This rate-adaptive behaviour is long lasting and transferable to the surrounding cardiomyocytes. Thus, electrical conditioning may be used to promote cardiomyocyte maturation and establish their automaticity, with implications for cell-based reduction of arrhythmia during heart regeneration.

A 31-year-old woman who had successfully undergone bariatric surgery (gastric bypass with Roux-en-Y anastamosis) three years earlier presented with complaints of acute epigastric abdominal pain, nausea, and vomiting. Computed tomography (CT) showed small bowel intussusception, and the patient was taken to the operating room. A mass the size and shape of a football was found; the mass consisted of the proximal limb of the Roux-en-Y intussuscepted in a retrograde manner. The bowel was gently reduced, deemed viable, and the Roux-en-Y anastamosis was revised with resection of the lead point. We urge the surgeon to be highly suspicious of acute bowel obstruction in the post-bariatric surgery population and believe that CT is essential in evaluating these patients. We further recommend resection of the lead point to avoid repeat bouts of intussusception from the same focal etiology.

Few studies investigating the validity of marijuana use have used samples of truant youth. In the current study, self-reports of marijuana use are compared with urine test results for marijuana to identify marijuana underreporting among adolescents participating in a longitudinal Brief Intervention for drug-involved truant youth. It was hypothesized that marijuana underreporting would be associated with alcohol underreporting and engaging in sexual risk behaviors. The results indicated marijuana underreporting was significantly associated with self-denial of alcohol use, but not associated with sexual risk behavior. Also, there was an age effect in marijuana use underreporting such that younger truant youth were more likely to underreport marijuana use, compared to older truant youth. Implications for policy and future research are discussed.

Since its introduction into clinical practice, electronic pacing has saved many lives. Despite continuous improvements, electronic pacemakers have important shortcomings, which stimulated the development of biological alternatives. Biological pacemakers generate the cardiac impulse using genes or cells to treat bradycardias. Over the past decade, significant improvements have been made in biological pacemakers, but issues remain in relation to long-term outcomes and safety. Concurrently, efforts to improve electronic pacemakers have also intensified. Whether new generations of electronic pacemakers will erase lingering concerns with regard to electronic pacing or whether biologicals will ultimately supplement or supplant electronics remains to be seen., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Atrial fibrillation (AF) is a common arrhythmia with significant morbidities and only partially adequate therapeutic options. AF is associated with atrial remodeling processes, including changes in the expression and function of ion channels and signaling pathways. TWIK protein-related acid-sensitive K+ channel (TASK)-1, a two-pore domain K+ channel, has been shown to contribute to action potential repolarization as well as to the maintenance of resting membrane potential in isolated myocytes, and TASK-1 inhibition has been associated with the induction of perioperative AF. However, the role of TASK-1 in chronic AF is unknown. The present study investigated the function, expression, and phosphorylation of TASK-1 in chronic AF in atrial tissue from chronically paced canines and in human subjects. TASK-1 current was present in atrial myocytes isolated from human and canine hearts in normal sinus rhythm but was absent in myocytes from humans with AF and in canines after the induction of AF by chronic tachypacing. The addition of phosphatase to the patch pipette rescued TASK-1 current from myocytes isolated from AF hearts, indicating that the change in current is phosphorylation dependent. Western blot analysis showed that total TASK-1 protein levels either did not change or increased slightly in AF, despite the absence of current. In studies of perioperative AF, we have shown that phosphorylation of TASK-1 at Thr383 inhibits the channel. However, phosphorylation at this site was unchanged in atrial tissue from humans with AF or in canines with chronic pacing-induced AF. We conclude that phosphorylation-dependent inhibition of TASK-1 is associated with AF, but the phosphorylation site responsible for this inhibition remains to be identified., (Copyright © 2015 the American Physiological Society.)

Since the first reports on the isolation and differentiation of stem cells, and in particular since the early success in driving these cells down a cardiac lineage, there has been interest in the potential of such preparations in cardiac regenerative therapy. Much of the focus of such research has been on improving mechanical function after myocardial infarction; however, electrophysiologic studies of these preparations have revealed a heterogeneous mix of action potential characteristics, including some described as "pacemaker" or "nodal-like," which in turn led to interest in the therapeutic potential of these preparations in the treatment of rhythm disorders; several proof-of-concept studies have used these cells to create a biologic alternative to electronic pacemakers. Further, there are additional potential applications of a preparation of pacemaker cells derived from stem cells, for example, in high-throughput screens of new chronotropic agents. All such applications require reasonably efficient methods for selecting or enriching the "nodal-like" cells, however, which in turn depends on first defining what constitutes a nodal-like cell since not all pacemaking cells are necessarily of nodal lineage. This review discusses the current state of the field in terms of characterizing sinoatrial-like cardiomyocytes derived from embryonic and induced pluripotent stem cells, markers that might be appropriate based on the current knowledge of the gene program leading to sinoatrial node development, what functional characteristics might be expected and desired based on studies of the sinoatrial node, and recent efforts at enrichment and selection of nodal-like cells., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Efforts to use gene therapy to create a biological pacemaker as an adjunct or replacement of electronic pacemakers have been ongoing for about 15 years. For the past decade, most of these efforts have focused on the hyperpolarization-activated cyclic nucleotide gated-(HCN) gene family of channels alone or in combination with other genes. The HCN gene family is the molecular correlate of the cardiac pacemaker current, If. It is a suitable basis for a biological pacemaker because it generates a depolarizing inward current primarily during diastole and is directly regulated by cyclic adenosine monophosphate (cAMP), thereby incorporating autonomic responsiveness. However, biological pacemakers based either on native HCN channels or on mutated HCN channels designed to optimize biophysical characteristics have failed to attain the desired basal and maximal physiological heart rates in large animals. More recent work has explored dual gene therapy approaches, combining an HCN variant with another gene to reduce outward current, increase an additional inward current, or enhance cAMP synthesis. Several of these dual gene therapy approaches have demonstrated appropriate basal and maximal heart rates with little or no reliance on a backup electronic pacemaker during the period of study. Future research, besides examining the efficacy of other gene combinations, will need to consider the additional issues of safety and persistence of the viral vectors often used to deliver these genes to a specific cardiac region., (© The Author(s) 2014.)

Aims: Although the right atrium (RA contains subsidiary atrial pacemaker (SAP) tissue that can take over from the sinoatrial node (SAN) in sick sinus syndrome (SSS), SAP tissue is bradycardic. Little is known about SAP tissue and one aim of the study was to characterize ion channel expression to obtain insight into SAP pacemaker mechanisms. A second aim was to determine whether HCN over-expression (a 'biopacemaker'-like strategy) can accelerate the pacemaker rate producing a pacemaker that is similar in nature to the SAN., Methods and Results: SAP tissue was isolated from the rat and the leading pacemaker site was characterized. Cell size at the leading pacemaker site in the SAP was smaller than in the RA and comparable to that in the SAN. mRNA levels showed the SAP to be similar to, but distinct from, the SAN. For example, in the SAN and SAP, expression of Tbx3 and HCN1 was higher and Nav1.5 and Cx43 lower than in the RA. Organ-cultured SAP tissue beat spontaneously, but at a slower rate than the SAN. Adenovirus-mediated gene transfer of HCN2 and the chimeric protein HCN212 significantly increased the pacemaker rate of the SAP close to that of the native SAN, but HCN4 was ineffective., Conclusion: SAP tissue near the inferior vena cava is bradycardic, but shares characteristics with the SAN. Pacing can be accelerated by the over-expression of HCN2 or HCN212. This provides proof of concept for the use of SAP tissue as a substrate for biopacemaking in the treatment of SSS.

Background: Sinus node (SN) dysfunction is observed in some long-QT syndrome (LQTS) patients, but has not been studied as a function of LQTS genotype. LQTS6 involves mutations in the hERG β-subunit MiRP1, which also interacts with hyperpolarization-activated, cyclic nucleotide gated (HCN) channels-the molecular correlate of SN pacemaker current (If ). An LQTS registry search identified a 55-year male with M54T MiRP1 mutation, history of sinus bradycardia (39-56 bpm), and prolonged QTc., Objective: We tested if LQTS6 incorporates sinus bradycardia due to abnormal If ., Methods: We transiently co-transfected neonatal rat ventricular myocytes (to study currents in a myocyte background) with human HCN4 (hHCN4, primary SN isoform) or human HCN2 (hHCN2) and one of the following: empty vector, wild-type hMiRP1 (WT), M54T hMiRP1 (M54T). Current amplitude, voltage dependence, and kinetics were measured by whole cell patch clamp., Results: M54T co-expression decreased HCN4 current density by 80% compared to hHCN4 alone or with WT, and also slowed HCN4 activation at physiologically relevant voltages. Neither WT nor M54T altered HCN4 voltage dependence. A computer simulation predicts that these changes in HCN4 current would decrease rate and be additive with published effects of M54T mutation on hERG kinetics on rate., Conclusions: We conclude that M54T LQTS6 mutation can cause sinus bradycardia through effects on both hERG and HCN currents. Patients with other LQTS6 mutations should be examined for SN dysfunction, and the effect on HCN current determined., (© 2013 Wiley Periodicals, Inc.)

Objectives: This study sought to test the hypothesis that hyperpolarization-activated cyclic nucleotide-gated (HCN)-based biological pacing might be improved significantly by hyperpolarizing the action potential (AP) threshold via coexpression of the skeletal muscle sodium channel 1 (SkM1)., Background: Gene-based biological pacemakers display effective in vivo pacemaker function. However, approaches used to date have failed to manifest optimal pacemaker properties, defined as basal beating rates of 60 to 90 beats/min, a brisk autonomic response achieving maximal rates of 130 to 160 beats/min, and low to absent electronic backup pacing., Methods: We implanted adenoviral SkM1, HCN2, or HCN2/SkM1 constructs into left bundle branches (LBB) or left ventricular (LV) epicardium of atrioventricular-blocked dogs., Results: During stable peak gene expression on days 5 to 7, HCN2/SkM1 LBB-injected dogs showed highly stable in vivo pacemaker activity superior to SkM1 or HCN2 alone and superior to LV-implanted dogs with regard to beating rates (resting approximately 80 beats/min; maximum approximately 130 beats/min), no dependence on electronic backup pacing, and enhanced modulation of pacemaker function during circadian rhythm or epinephrine infusion. In vitro isolated LV of dogs overexpressing SkM1 manifested a significantly more negative AP threshold., Conclusions: LBB-injected HCN2/SkM1 potentially provides a more clinically suitable biological pacemaker strategy than other reported constructs. This superiority is attributable to the more negative AP threshold and injection into the LBB., (Copyright © 2013 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Peri-operative atrial fibrillation (peri-op AF) is a common complication following thoracic surgery. This arrhythmia is thought to be triggered by an inflammatory response and can be reproduced in various animal models. Previous work has shown that the lipid inflammatory mediator, platelet-activating factor (PAF), synthesized by activated neutrophils, can induce atrial and ventricular arrhythmias as well as repolarization abnormalities in isolated ventricular myocytes. We have previously shown that carbamylated PAF-induced repolarization abnormalities result from the protein kinase C (PKC) ε-dependent phosphorylation of the two-pore domain potassium channel TASK-1. We now demonstrate that canine peri-op AF is associated with the phosphorylation-dependent loss of TASK-1 current. Further studies identified threonine 383 in the C terminus of human and canine TASK-1 as the phosphorylation site required for PAF-dependent inhibition of the channel. Using a novel phosphorylation site-specific antibody targeting the phosphorylated channel, we have determined that peri-op AF is associated with the loss of TASK-1 current and increased phosphorylation of TASK-1 at this site.

The voltage-gated Na+ channel is a critical determinant of the action potential (AP) upstroke. Increasing Na+ conductance may speed AP propagation. In this study, we propose use of the skeletal muscle Na+ channel SkM1 as a more favorable gene than the cardiac isoform SCN5A to enhance conduction velocity in depolarized cardiac tissue. We used cells that electrically coupled with cardiac myocytes as a delivery platform to introduce the Na+ channels. Human embryonic kidney 293 cells were stably transfected with SkM1 or SCN5A. SkM1 had a more depolarized (18 mV shift) inactivation curve than SCN5A. We also found that SkM1 recovered faster from inactivation than SCN5A. When coupled with SkM1 expressing cells, cultured myocytes showed an increase in the dV/dtmax of the AP. Expression of SCN5A had no such effect. In an in vitro cardiac syncytium, coculture of neonatal cardiac myocytes with SkM1 expressing but not SCN5A expressing cells significantly increased the conduction velocity under both normal and depolarized conditions. In an in vitro reentry model induced by high-frequency stimulation, expression of SkM1 also enhanced angular velocity of the induced reentry. These results suggest that cells carrying a Na+ channel with a more depolarized inactivation curve can improve cardiac excitability and conduction in depolarized tissues.

Aims: Reentry accounts for most life-threatening arrhythmias, complicating myocardial infarction, and therapies that consistently prevent reentry from occurring are lacking. In this study, we compare antiarrhythmic effects of gene transfer of green fluorescent protein (GFP; sham), the skeletal muscle sodium channel (SkM1), the liver-specific connexin (Cx32), and SkM1/Cx32 in the subacute canine infarct., Methods and Results: Immediately after ligation of the left anterior descending artery, viral constructs were implanted in the epicardial border zone (EBZ). Five to 7 days later, efficient restoration of impulse propagation (narrow QRS and local electrogram duration) occurred in SkM1, Cx32, and SkM1/Cx32 groups (P< 0.05 vs. GFP). Programmed electrical stimulation from the EBZ induced sustained ventricular tachycardia (VT)/ventricular fibrillation (VF) in 15/22 GFP dogs vs. 2/12 SkM1, 6/14 Cx32, and 8/10 SkM1/Cx32 (P< 0.05 SkM1 vs. GFP). GFP, SkM1, and SkM1/Cx32 had predominantly polymorphic VT/VF, whereas in Cx32 dogs, monomorphic VT predominated (P< 0.05 for Cx32 vs. GFP). Tetrazolium red staining showed significantly larger infarcts in Cx32- vs. GFP-treated animals (P< 0.05)., Conclusion: Whereas SkM1 gene transfer reduces the incidence of inducible VT/VF, Cx32 therapy to improve gap junctional conductance results in larger infarct size, a different VT morphology, and no antiarrhythmic efficacy.

Previous observations show that β-adrenergic modulation of pacemaker current (I(f)) in sinoatrial node (SAN) cells is impaired by disruption of normal Ca(2+)-homeostasis with ryanodine or BAPTA. Recently, the presence of Ca(2+)-activated adenylyl cyclase (AC) 1 was reported in SAN, and was proposed as a possible mechanism of Ca(2+)-dependence of β-adrenergic modulation. However, direct evidence that pacemaker (HCN) channels can be regulated by Ca(2+)-activated AC and that such regulation introduces Ca(2+) dependence, is lacking. Here we co-expressed AC1 or AC6 with HCN2 in neonatal rat ventricular myocytes, which lack AC1. Although both isoforms have equivalent expression level and ability to interact with HCN2, only AC1 increases intracellular cAMP content, accelerates spontaneous beating rate and modifies HCN2 biophysics. Measured HCN2 current in the AC1 group activated ~10mV more positive than in GFP or AC6. The β-adrenergic agonist isoproterenol induced a further positive shift under control conditions, but failed to do so after pretreatment with the Ca(2+) chelator BAPTA. In the AC6 group, isoproterenol shifted the HCN2 activation relation to a similar extent in the absence and presence of BAPTA. Thus, AC1 but not AC6 over-expression introduces Ca(2+)-sensitivity to the β-adrenergic response of HCN2. These results demonstrate physical and functional interaction between AC isoforms and the HCN2 pacemaker channel and support a key role of Ca(2+) activated AC1 as a molecular mechanism in Ca(2+)-dependent modulation of β-adrenergic response of heart rate., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Increasing interest has been shown in Brief Interventions for troubled persons, including those with substance abuse problems. Most of the published literature on this topic has focused on adults, and on the efficacy of these interventions. Few of these studies have examined the critical issues of enrollment and engagement in Brief Intervention services. The present paper seeks to address the shortcomings in the current literature by reporting on our experiences implementing NIDA funded, Brief Intervention projects involving truant and diversion program youth.

The field of biological pacing is entering its second decade of active investigation. The inception of this area of study was serendipitous, deriving largely from observations made by several teams of investigators, whose common interest was to understand the mechanisms governing cardiac impulse initiation. Research directions taken have fallen under the broad headings of gene therapy and cell therapy, and biomaterials research has also begun to enter the field. In this Review, we revisit certain milestones achieved through the construction of a 'roadmap' in biological pacing. Whether the end result will be a clinically applicable biological pacemaker is still uncertain. However, promising constructs that achieve physiologically relevant heart rates and good autonomic responsiveness are now available, and proof of principle studies are giving way to translation to large-animal models in long-term studies. Provided that interest in the field continues, the next decade should see either biological pacemakers become a clinical reality or the improvement of electronic pacemakers to a point where the biological approach is no longer a viable alternative.

We demonstrate the hohlraum radiation temperature and symmetry required for ignition-scale inertial confinement fusion capsule implosions. Cryogenic gas-filled hohlraums with 2.2 mm-diameter capsules are heated with unprecedented laser energies of 1.2 MJ delivered by 192 ultraviolet laser beams on the National Ignition Facility. Laser backscatter measurements show that these hohlraums absorb 87% to 91% of the incident laser power resulting in peak radiation temperatures of T(RAD)=300 eV and a symmetric implosion to a 100 μm diameter hot core., (© 2011 American Physical Society)

Increasing connexin43 (Cx43) gap junctional conductance as a means to improve cardiac conduction has been proposed as a novel antiarrhythmic modality. Yet, transmission of molecules via gap junctions may be associated with increased infarct size. To determine whether maintaining open gap junction channels impacts on infarct size and induction of ventricular tachycardia (VT) following coronary occlusion, we expressed the pH- and voltage-independent connexin isoform connexin32 (Cx32) in ventricle and confirmed Cx32 expression. Wild-type (WT) mice injected with adenovirus-Cx32 (Cx32inj) were examined following coronary occlusion to determine infarct size and inducibility of VT. There was an increased infarct size in Cx32inj hearts as compared to WT (WT 22.9 ± 4%; Cx32inj 44.3 ± 5%; p < 0.05). Programmed electrical stimulation showed no difference in VT inducibility in WT and Cx32inj mice (VT was reproducibly inducible in 55% of shams and 50% of Cx32inj mice (p > 0.05). Following coronary occlusion, improving cell-cell communication increased infarct size, and conferred no antiarrhythmic benefit.

Biological pacing has been proposed as a physiologic counterpart to electronic pacing, and the sinoatrial node (SAN) is the general standard for biological pacemakers. We tested the expression of SAN pacemaker cell activity when implanted autologously in the right ventricle (RV). We induced complete heart block and implanted electronic pacemakers in the RV of adult mongrel dogs. Autologous SAN cells isolated enzymatically were studied by patch clamp to confirm SAN identity. SAN cells (400,000) were injected into the RV subepicardial free wall and dogs were monitored for 2 weeks. Pacemaker function was assessed by overdrive pacing and IV epinephrine challenge. SAN cells expressed a time-dependent inward current (I(f)) activating on hyperpolarization: density = 4.3 ± 0.6 pA/pF at -105 mV. Four of the six dogs demonstrated >50% of beats originating from the implant site at 24 h. Biological pacemaker rates on days 7-14 = 45-55 bpm and post-overdrive escape times = 1.5-2.5 s. Brisk catecholamine responsiveness occurred. Dogs implanted with autologous SAN cells manifest biological pacing properties dissimilar from those of the anatomic SAN. This highlights the importance of cell and substrate interaction in generating biological pacemaker function.

Aims: acute myocardial ischaemia induces a decrease in resting membrane potential [which leads to reduction of action potential (AP) V(max)] and intracellular acidification (which closes gap junctions). Both contribute to conduction slowing. We hypothesized that ventricular expression of the skeletal muscle Na(+) channel, Nav1.4 (which activates fully at low membrane potentials), or connexin32 (Cx32, which is less pH-sensitive than connexin43) would support conduction and be antiarrhythmic. We tested this hypothesis in a murine model of ischaemia and reperfusion arrhythmias., Methods and Results: empty adenovirus (Sham) or adenoviral constructs expressing either SkM1 (gene encoding Nav1.4) or Cx32 genes were injected into the left ventricular wall. Four days later, ventricular tachycardia (VT) occurred during reperfusion following a 5 min coronary occlusion. In Nav1.4- and Cx32-expressing mice, VT incidence and duration were lower than in Sham (P < 0.05). In vitro multisite microelectrode mapping was performed in the superfused right ventricular wall. To simulate ischaemic conditions, [K(+)] in solution was increased to 10 mmol/L and/or pH was decreased to 6.0. Western blots revealed Cx32 and Nav1.4 expression in both ventricles. Nav1.4 APs showed higher V(max) and conduction velocity (CV) than Shams at normal and elevated [K(+)]. Exposure of tissue to acid solution reduced intracellular pH to 6.4. There was no difference in CV between Sham and Cx32 groups in control solution. Acid solution slowed CV in Sham (P < 0.05) but not in Cx32., Conclusion: Nav1.4 or Cx32 expression preserved normal conduction in murine hearts and decreased the incidence of reperfusion VT.

Background: Biological pacemakers based on the HCN2 channel isoform respond to beta-adrenergic and muscarinic stimulation, suggesting a capacity to respond to autonomic input., Objective: The purpose of this study was to investigate autonomic response to emotional arousal in canines implanted with murine HCN2-based biological pacemakers using gene therapy., Methods: An electronic pacemaker was implanted with its lead in the right ventricular apical endocardium (VVI 35 bpm). An adenoviral HCN2/GFP construct (Ad-HCN2, n = 7) or saline (control, n = 5) was injected into the left bundle branch on day 2 after radiofrequency ablation of the atrioventricular node to induce complete atrioventricular block. Emotional arousal was achieved by presenting food following an overnight fast. Autonomic control was evaluated with Poincaré plots of R-R(N) against R-R(N+1) intervals to characterize heart rate variability (HRV) and with continuous RR interval assessment via 24-hour ambulatory ECG. The 24-hour ECG and Poincaré plot shape were analyzed., Results: During day 1 after biological pacemaker implantation, Poincaré HRV parameters and RR intervals were unchanged with food presentation. However, on day 7, food presentation was accompanied by an increase in HRV (SD1, p < 0.07, and SD2, p < 0.05) and shortening of RR interval (P < .05) in dogs with Ad-HCN2 but not in controls., Conclusion: This is the first demonstration that biological pacemakers are capable of responding to natural arousal stimuli to elicit appropriate chronotropic responses, a potential advantage over electronic pacemakers., (Copyright © 2010 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Background: Skeletal muscle sodium channel (Nav1.4) expression in border zone myocardium increases action potential upstroke velocity in depolarized isolated tissue. Because resting membrane potential in the 1-week canine infarct is reduced, we hypothesized that conduction velocity (CV) is greater in Nav1.4 dogs compared with in control dogs., Objective: The purpose of this study was to measure CV in the infarct border zone border in dogs with and without Nav1.4 expression., Methods: Adenovirus was injected in the infarct border zone in 34 dogs. The adenovirus incorporated the Nav1.4- and a green fluorescent protein (GFP) gene (Nav1.4 group, n = 16) or only GFP (n = 18). After 1 week, upstroke velocity and CV were measured by sequential microelectrode recordings at 4 and 7 mM [K(+)] in superfused epicardial slabs. High-density in vivo epicardial activation mapping was performed in a subgroup (8 Nav1.4, 6 GFP) at three to four locations in the border zone. Microscopy and antibody staining confirmed GFP or Nav1.4 expression., Results: Infarct sizes were similar between groups (30.6% +/- 3% of left ventricle mass, mean +/- standard error of the mean). Longitudinal CV was greater in Nav1.4 than in GFP sites (58.5 +/- 1.8 vs. 53.3 +/- 1.2 cm/s, 20 and 15 sites, respectively; P <.05). Transverse CV was not different between the groups. In tissue slabs, dV/dt(max) was higher and CV was greater in Nav1.4 than in control at 7 mM [K(+)] (P <.05). Immunohistochemical Nav1.4 staining was seen at the longitudinal ends of the myocytes., Conclusion: Nav1.4 channels in myocardium surviving 1 week infarction increases longitudinal but not transverse CV, consistent with the increased dV/dt(max) and with the cellular localization of Nav1.4., (Copyright 2010 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Current in vivo approaches for the delivery of siRNAs have been hindered by inefficient targeting to cells and by the triggering of immune responses. The cellular delivery of siRNA by immunoprivileged cells avoids the immune response, because the siRNA is delivered from one cell interior to another via gap junctions, thereby avoiding the extracellular compartment. Human mesenchymal stem cells (hMSCs) can be delivered focally or systemically, and have also exhibited the ability to migrate to targeted tissue in vivo. These features suggest the potential use of hMSCs as a cellular delivery system for siRNA. This feature review discusses the role of gap junctions to facilitate the transfer of siRNA directly to target cells, thus avoiding the disadvantages involved with approaches that are dependent on the extracellular space for siRNA delivery.

In rabbit, sodium current (I(Na)) contributes to newborn sinoatrial node (SAN) automaticity but is absent in adult SAN, where heart rate is slower. In contrast, heart rate is high and I(Na) is functional in adult mouse SAN. Given the slower heart rates of large mammals, we asked if I(Na) is functionally active in SAN of newborn or adult canine heart. SAN cells were isolated from newborn (6-10 days), young (40-43 days) and adult mongrels. I(Na) was observed in >80% of cells from each age. However, current density was markedly greater in newborn, decreasing with age. At all ages, I(Na) was sensitive to nanomolar tetrodotoxin (TTX); 100 nmol/L inhibited I(Na) by 46.7%, 59.9% and 90.7% in newborn, young and adult cells, respectively. While high TTX sensitivity suggested the presence of non-cardiac isoforms, steady-state inactivation was relatively negative (midpoints -89.7+/-0.7 mV, -95.1+/-1.2 mV and -93.4+/-1.9 mV from newborn to adult). Consequently, I(Na) should be unavailable at physiological potentials under normal conditions, and 100 nmol/L TTX did not change cycle length or action potential parameters of spontaneous adult SAN cells. However, computer modeling predicts the large newborn I(Na) protects against excess rate slowing from strong vagal stimulation. The results show that canine SAN cells have TTX-sensitive I(Na) which decreases with post-natal age. The current does not contribute to normal automaticity in isolated adult cells but can be recruited to sustain excitability if nodal cells are hyperpolarized. This is particularly relevant in newborn, where I(Na) is large and parasympathetic/sympathetic balance favors vagal tone., (Published by Elsevier Ltd.)

We examined whether coupling of a ventricular myocyte to a non-myocyte cell expressing HCN2 could create a two-cell syncytium capable of generating sustained pacing. Three non-myocyte cell types were transfected with the mHCN2 gene and used as sources of mHCN2-induced currents. They were human mesenchymal stem cells and HEK293 cells, both of which express connexin43 (Cx43), and HeLa cells transfected with Cx43. Cell-cell coupling between heterologous pairs increased with time in co-culture, and hyperpolarization of the myocyte induced HCN2 currents, indicating current transfer from the mHCN2-expressing cell to the myocyte via gap junctions. The magnitude of the HCN2 currents recorded in myocytes increased with increasing junctional conductance. Once a critical level of electrical cell-cell coupling between myocytes and mHCN2 transfected cells was exceeded spontaneous action potentials were generated at frequencies of approximately 0.6 to 1.7 Hz (1.09 +/- 0.05 Hz). Addition of carbenoxolone (200 microM), a gap junction channel blocker, to the media stopped spontaneous activity in heterologous cell pairs. Carbenoxolone washout restored activity. Blockade of HCN2 currents by 100 microM 9-amino-1,2,3,4-tetrahydroacridine (THA) stopped spontaneous activity and subsequent washout restored it. Neither THA nor carbenoxolone affected electrically stimulated action potentials in isolated single myocytes. In summary, the inward current evoked in the genetically engineered (HCN2-expressing) cell was delivered to the cardiac myocyte via gap junctions and generated action potentials such that the cell pair could function as a pacemaker unit. This finding lays the groundwork for understanding cell-based biological pacemakers in vivo once an understanding of delivery and target cell geometry is defined.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels regulate pacemaker activity in some cardiac cells and neurons. In the present study, we have identified the presence of HCN channels in pancreatic beta-cells. We then examined the functional characterization of these channels in beta-cells via modulating HCN channel activity genetically and pharmacologically. Voltage-clamp experiments showed that over-expression of HCN2 in rat beta-cells significantly increased HCN current (I(h)), whereas expression of dominant-negative HCN2 (HCN2-AYA) completely suppressed endogenous I(h). Compared to control beta-cells, over-expression of I(h) increased insulin secretion at 2.8 mmol/l glucose. However, suppression of I(h) did not affect insulin secretion at both 2.8 and 11.1 mmol/l glucose. Current-clamp measurements revealed that HCN2 over-expression significantly reduced beta-cell membrane input resistance (R(in)), and resulted in a less-hyperpolarizing membrane response to the currents injected into the cell. Conversely, dominant negative HCN2-AYA expression led to a substantial increase of R(in), which was associated with a more hyperpolarizing membrane response to the currents injected. Remarkably, under low extracellular potassium conditions (2.5 mmol/l K(+)), suppression of I(h) resulted in increased membrane hyperpolarization and decreased insulin secretion. We conclude that I(h) in beta-cells possess the potential to modulate beta-cell membrane potential and insulin secretion under hypokalemic conditions.

Several hundred thousand electronic pacemakers are implanted in the US each year to treat abnormally slow heart rates. Biological pacemaker research strives to replace this hardware, and the associated monitoring and maintenance, by using gene or cell therapy to create a permanent and autonomically responsive pacemaker. While there are numerous technological hurdles to overcome before this is a therapeutic reality, one critical issue is determining the optimal channel gene to employ in creating a biological pacemaker. This review discusses the pros and cons of various model systems for characterizing and evaluating the function of candidate channel genes. It is argued that a sequential approach that combines in silico, in vitro and in vivo models is required.

Cardiac slow delayed rectifier (IKs) channel is composed of KCNQ1 (pore-forming) and KCNE1 (auxiliary) subunits. Although KCNE1 is an obligate IKs component that confers the uniquely slow gating kinetics, KCNE2 is also expressed in human heart. In vitro experiments suggest that KCNE2 can associate with the KCNQ1-KCNE1 complex to suppress the current amplitude without altering the slow gating kinetics. Our goal here is to test the role of KCNE2 in cardiac IKs channel function. Pulse-chase experiments in COS-7 cells show that there is a KCNE1 turnover in the KCNQ1-KCNE1 complex, supporting the possibility that KCNE1 in the IKs channel complex can be substituted by KCNE2 when the latter is available. Biotinylation experiments in COS-7 cells show that although KCNE1 relies on KCNQ1 coassembly for more efficient cell surface expression, KCNE2 can independently traffic to the cell surface, thus becoming available for substituting KCNE1 in the IKs channel complex. Injecting vesicles carrying KCNE1 or KCNE2 into KCNQ1-expressing oocytes leads to KCNQ1 modulation in the same manner as KCNQ1+KCNEx (where x=1 or 2) cRNA coinjection. Thus, free KCNEx peptides delivered to the cell membrane can associate with existing KCNQ1 channels to modulate their function. Finally, adenovirus-mediated KCNE2 expression in adult guinea pig ventricular myocytes exhibited colocalization with native KCNQ1 protein and reduces the native IKs current density. We propose that in cardiac myocytes the IKs current amplitude is under dynamic control by the availability of KCNE2 subunits in the cell membrane.