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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM PUBLISHER]

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. [ABSTRACT FROM AUTHOR]

An LQTS6 M54T Mutation and Sinus Node Dysfunction 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM PUBLISHER]

Aims Acute myocardial ischaemia induces a decrease in resting membrane potential [which leads to reduction of action potential (AP) Vmax] 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 Vmax 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. [ABSTRACT FROM AUTHOR]

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 &bgr;-cells. We then examined the functional characterization of these channels in &bgr;-cells via modulating HCN channel activity genetically and pharmacologically. Voltage-clamp experiments showed that over-expression of HCN2 in rat &bgr;-cells significantly increased HCN current (Ih), whereas expression of dominant-negative HCN2 (HCN2-AYA) completely suppressed endogenous Ih. Compared to control &bgr;-cells, overexpression of Ih increased insulin secretion at 2.8 mmol/l glucose. However, suppression of Ih did not affect insulin secretion at both 2.8 and 11.1 mmol/l glucose. Current-clamp measurements revealed that HCN2 overexpression significantly reduced &bgr;-cell membrane input resistance (Rin), 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 Rin, which was associated with a more hyperpolarizing membrane response to the currents injected. Remarkably, under low extracellular potassium conditions (2.5 mmol/l KC), suppression of Ih resulted in increased membrane hyperpolarization and decreased insulin secretion. We conclude that Ih in &bgr;-cells possess the potential to modulate &bgr;-cell membrane potential and insulin secretion under hypokalemic conditions. [ABSTRACT FROM AUTHOR]

The pacemaker current, mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, contributes to the initiation and regulation of cardiac rhythm. Previous experiments creating HCN-based biological pacemakers in vivo found that an engineered HCN2/HCN1 chimeric channel (HCN212) resulted in significantly faster rates than HCN2, interrupted by 1–5 s pauses. To elucidate the mechanisms underlying the differences in HCN212 and HCN2 in vivo functionality as biological pacemakers, we studied newborn rat ventricular myocytes over-expressing either HCN2 or HCN212 channels. The HCN2- and HCN212-over-expressing myocytes manifest similar voltage dependence, current density and sensitivity to saturating cAMP concentrations, but HCN212 has faster activation/deactivation kinetics. Compared with HCN2, myocytes expressing HCN212 exhibit a faster spontaneous rate and greater incidence of irregular rhythms (i.e. periods of rapid spontaneous rate followed by pauses). To explore these rhythm differences further, we imposed consecutive pacing and found that activation kinetics of the two channels are slower at faster pacing frequencies. As a result, time-dependent HCN current flowing during diastole decreases for both constructs during a train of stimuli at a rapid frequency, with the effect more pronounced for HCN2. In addition, the slower deactivation kinetics of HCN2 contributes to more pronounced instantaneous current at a slower frequency. As a result of the frequency dependence of both instantaneous and time-dependent current, HCN2 exhibits more robust negative feedback than HCN212, contributing to the maintenance of a stable pacing rhythm. These results illustrate the benefit of screening HCN constructs in spontaneously active myocyte cultures and may provide the basis for future optimization of HCN-based biological pacemakers. [ABSTRACT FROM AUTHOR]

Previously, we have shown that murine HCN2 channels over-expressed in newborn and adult cardiac myocytes produce currents with different biophysical characteristics. To investigate the role of tyrosine kinase modulation in these age-dependent differences, we employed the broad spectrum tyrosine kinase inhibitor erbstatin. Our results demonstrated distinct and separable effects of erbstatin on channel gating and current amplitude and a marked age dependence to these effects. In newborn myocytes, erbstatin decreased current amplitude, shifted the activation relation negative, and slowed activation kinetics. The effect on activation voltage but not that on amplitude was absent when expressing a cAMP-insensitive mutant (HCN2R/E), while a C-terminal truncated form of HCN2 (HCN2ΔCx) exhibited only the voltage dependent but not the amplitude effect of erbstatin. Thus, the action of erbstatin on the activation relation and current amplitude are distinct and separable in newborn myocytes, and the effect on activation voltage depends on the cAMP status of HCN2 channels. In contrast to newborn myocytes, erbstatin had no effect on HCN2 under control conditions in adult myocytes but induced a negative shift with no change in amplitude when saturated cAMP was added to the pipette solution. We conclude that erbstatin’s effects on HCN2 current magnitude and voltage dependence are distinct and separable, and there are fundamental developmental differences in the heart that affect channel function and its modulation by the tyrosine kinase inhibitor erbstatin. [ABSTRACT FROM AUTHOR]

Enhanced recovery of mechanical function in the canine heart by seeding an extracellular matrix patch with mesenchymal stem cells committed to a cardiac lineage. Am J Physiol Heart Circ Physiol 295: H2257-H2263, 2008. First published October 3, 2008; doi: 10.11 52/ajpheart.002 19.2008.-The need to regenerate tissue is par- amount, especially for the heart that lacks the ability to regenerate after injury. The urinary bladder extracellular matrix (ECM), when used to repair a right ventricular defect, successfully regenerated some mechanical function. The objective of the current study was to determine whether the regenerative effect of ECM could be improved by seeding the patch with human mesenchymal stem cells (hMSCs) enhanced to differentiate down a cardiac Image. hMSCs were used to form three-dimensional spheroids. The expression of cardiac proteins was determined in cells exposed to the spheroid formation and compared with nonmanipulated hMSCs. To determine whether functional calcium channels were present, the cells were patch clamped. To evaluate the ability of these cells to regenerate mechanical function, the spheroids were seeded on ECM and then implanted into the canine heart to repair a full-thickness right ventricular defect. As a result, many of the cells spreading from the spheroids expressed cardiac-specific proteins, including sarcomeric x-ac- tinin, cardiotin, and atrial natriuretic peptide, as well as the cell cycle markers cyclin Dl and proliferating cell nuclear antigen. A calcium current similar in amplitude to that of ventricular myocytes was present in 16% of the cells. The cardiogenic cell-seeded scaffolds increased the regional mechanical function in the canine heart compared with the unmanipulated hMSC-seeded scaffolds. In addition, the cells prelabeled with fluorescent markers demonstrated myocyte-specific actinin staining with sarcomere spacing similar to that of normal myocytes. In conclusion, the spheroid-derived cells express cardiac-specific proteins and demonstrate a calcium current similar to adult ventricular myocytes. When these cells are implanted into the canine heart, some of these cells appear striated and mechanical function is improved compared with the unmanipulated hMSCs. Further investigation will be required to deter- mine whether the increased mechanical function is due to a differentiation of the cardiogenic cells to myocytes or to other effects. [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]

The article focuses on the use of human mesenchymal stem cells (hMSCs) in biological pacing. It discusses the origins and the evolution of biological pacing. Types of stem cell approaches to biological pacing include human embryonic stem cell and platform delivery using hMSCs. The article proposes to load hMSCs with HCN genes through electroporation, thus avoiding use of viral vectors.

The sinoatrial node performs its task as a cardiac impulse generator throughout the life of the organism, but this important function is not a constant. Rather, there are significant developmental changes in the expression and function of ion channels and other cellular elements, which lead to a postnatal slowing of heart rate and may be crucial to the reliable functioning of the node during maturation. In this review, we provide an overview of current knowledge regarding these changes, with the main focus placed on maturation of the ion channel expression profile. Studies on Na+ and pacemaker currents have shown that their contribution to automaticity is greater in the newborn than in the adult, but this age-dependent decrease is at least partially opposed by an increased contribution of L-type Ca2+ current. Whereas information regarding age-dependent changes in other transmembrane currents within the sinoatrial node are lacking, there are data on other relevant parameters. These include an increase in the nodal content of fibroblasts and in the area of nonexpression of connexin43, considered a molecular marker of nodal tissue. Although much remains to be done before a comprehensive view of the developmental biology of the node is available, important evidence in support of a molecular interpretation of developmental slowing of the intrinsic sinoatrial rate is beginning to emerge. [ABSTRACT FROM AUTHOR]

Recent results indicate that phosphoinositides, including phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), directly enhance the opening of hyperpolarization-activated, cyclic nucleotide-regulated (HCN) channels by shifting their activation gating to more positive voltages. This contrasts with the action of phosphoinositides to inhibit the opening of the related cyclic nucleotide-gated (CNG) channels involved in sensory signaling. We both review previous studies and present new experiments that investigate whether HCN channels may be regulated by dynamic changes in PI(4,5)P2 levels caused by the receptor-mediated activation of phospholipase C (PLC). We coexpressed HCN1 or HCN2 channels in Xenopus oocytes with the PLC-coupled bradykinin BK2 receptor, the muscarinic M1 receptor, or the TrkA receptor. Activation of all three receptors produced a positive shift in HCN channel voltage gating, the opposite of the effect expected for PI(4,5)P2 depletion. This action was not caused by alterations in cAMP as the effect was preserved in HCN mutant channels that fail to bind cAMP. The receptor effects were mediated by PLC activity, but did not depend on signaling through the downstream products of PI(4,5)P2 hydrolysis: IP3 or diacylglycerol (DAG). Importantly, the modulatory effects on gating were blocked by inhibitors of phosphatidylinositol (PI) kinases, suggesting a role for increased PI(4,5)P2 synthesis. Finally, we found that bradykinin exerted a similar PI kinase-dependent effect on the gating of native HCN channels in cardiac sinoatrial node cells, suggesting that this pathway may represent a novel, physiologically relevant mechanism for enhancing HCN channel function. [ABSTRACT FROM AUTHOR]

Stem cells show promise for repair of damaged cardiac tissue. Little is known with certainty, however, about the distribution of these cells once introduced in vivo. Previous attempts at tracking delivered stem cells have been hampered by the autofluorescence of host tissue and limitations of existing labeling techniques. We have developed a novel loading approach to stably label human mesenchymal stem cells with quantum dot (QD) nanoparticles. We report the optimization and validation of this long-term tracking technique and highlight several important biological applications by delivering labeled cells to the mammalian heart. The bright QD crystals illuminate exogenous stem cells in histologic sections for at least 8 weeks following delivery and permit, for the first time, the complete three-dimensional reconstruction of the locations of all stem cells following injection into the heart. [ABSTRACT FROM AUTHOR]

We investigated effects of the paracrine factors secreted by human mesenchymal stem cells (hMSCs) on endothelial cell migration, extracellular matrix invasion, proliferation, and survival in vitro. Human mesenchymal stem cells were cultured as a monolayer or as three-dimensional aggregates in hanging drops (hMSC spheroids). We performed analysis of paracrine factors in medium conditioned by a monolayer of hMSCs and hMSC spheroids. Concentrations of vascular endothelial growth factor (VEGF), basic fibroblast growth factor, angiogenin, procathepsin B, interleukin (IL)-11, and bone morphogenic protein 2 were increased 5–20 times in medium conditioned by hMSC spheroids, whereas concentrations of IL-6, IL-8, and monocyte hemoattractant protein-1 were not increased. Concentrations of VEGF and angiogenin in medium conditioned by hMSC spheroids showed a weak dependence on the presence of serum, which allows serum-free conditioned medium with elevated concentrations of angiogenic cytokines to be obtained. Medium conditioned by hMSC spheroids was more effective in stimulation of umbilical vein endothelial cell proliferation, migration, and basement membrane invasion than medium conditioned by a monolayer of hMSCs. This medium also promotes endothelial cell survival in vitro. We suggest that culturing of hMSCs as three-dimensional cellular aggregates provides a method to concentrate proangiogenic factors secreted by hMSCs and allows for reduction of serum concentration in conditioned medium. Our data support the hypothesis that hMSCs serve as trophic mediators for endothelial cells. [ABSTRACT FROM AUTHOR]

Biological pacemaking as a replacement for or adjunct to electronic pacemakers has been a subject of interest since the 1990s. In the following pages, we discuss the rational for and progress made using a hyperpolarization activated, cyclic nucleotide gated channel isoform to carry the I(f) pacemaker current in gene and cell therapy approaches. Both strategies have resulted in effective biological pacemaker function over a period of weeks in intact animals. Moreover, the use of adult human mesenchymal stem cells as a platform for carrying pacemaker genes has resulted in the formation of functional gap junctions with cardiac myocytes in situ leading to effective and persistent propagation of pacemaker current. The approaches described are encouraging, suggesting that biological pacemakers based on this strategy can be brought to clinical testing. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Cardiac memory (CM) is characterized by an altered T-wave morphology, which reflects altered repolarization gradients. We hypothesized that the delayed rectifier currents, I Kr and I Ks, might contribute to these repolarization changes. Methods: We studied conscious, chronically instrumented dogs paced from the postero-lateral left ventricular (LV) wall at rates 5–10% faster than sinus rate for 3weeks. ECGs during sinus rhythm were recorded on days 0, 7, 14 and 21 of pacing. Within 3weeks, CM achieved steady state, hearts were excised, and epicardial and endocardial tissues and myocytes were studied. Results: In unpaced controls, action potential duration to 50% and 90% repolarization (APD) in epicardium was shorter than in endocardium (P <0.05); in CM epicardial APD increased at CL≥500ms, while endocardial APD was either unchanged or decreased such that the transmural gradient seen in controls diminished (P <0.05). A transmural I Kr gradient occurred in controls (epicardium>endocardium, P <0.05) and was reversed in CM. No I Ks transmural gradient was found in controls, while in CM endocardial I Ks was greater than epicardial at greater than +50mV. Canine ERG (cERG) mRNA and protein in epicardium>endocardium in controls (P <0.05), and this difference was lost in CM. Expression levels of KCNQ1 and KCNE1 protein were similar in all groups. Conclusions: A transcriptionally induced change in epicardial I Kr contributes to the altered ventricular repolarization that characterizes CM. [Copyright &y& Elsevier]

The advent of gene therapy and cell therapy has led to reconsideration of standard therapies for cardiac disease. One such area of reconsideration is that of the cardiac pacemaker, which has been the mainstay of treatment for high-degree heart block and sinoatrial node dysfunction. Over the past five years, gene therapy has been used to explore the overexpression of β2-adrenergic receptors, the down-regulation of inward rectifier current, and the overexpression of pacemaker current as potential sources of biological pacemakers. Cell therapy approaches have explored the “forcing” of embryonic stem cells to evolve along cardiac (and specifically pacemaker) cell lines and the use of adult mesenchymal stem cells as platforms for delivery of specific gene therapies. This review considers the strengths and weaknesses of each of the approaches used to date and attempts to look to the future of biological alternatives to electronic pacemakers. [Copyright &y& Elsevier]

Human mesenchymal stem cells (hMSCs) are a multipotent cell population with the potential to be a cellular repair or delivery system provided that they communicate with target cells such as cardiac myocytes via gap junctions. Immunostaining revealed typical punctate staining for Cx43 and Cx40 along regions of intimate cell-to-cell contact between hMSCs. The staining patterns for Cx45 rather were typified by granular cytoplasmic staining. hMSCs exhibited cell-to-cell coupling to each other, to HeLa cells transfected with Cx40, Cx43 and Cx45 and to acutely isolated canine ventricular myocytes. The functional currents (Ij) recorded between hMSC pairs exhibited quasi-symmetrical and asymmetrical voltage (Vj) dependence. Ij records from hMSC-HeLaCx43 and hMSC-HeLaCx40 cell pairs also showed symmetrical and asymmetrical Vj dependence, while hMSC-HeLaCx45 pairs always produced asymmetrical Ij with pronounced Vj gating when the Cx45 side was negative. Symmetrical Ij suggests that the dominant functional channel is homotypic, while the asymmetrical Ij suggests the activity of another channel type (heterotypic, heteromeric or both). The hMSCs exhibited a spectrum of single channels with transition conductances (γj) of 30-80 pS. The macroscopic Ij obtained from hMSC-cardiac myocyte cell pairs exhibited asymmetrical Vj dependence, while single channel events revealed γj of the size range 40-100 pS. hMSC coupling via gap junctions to other cell types provides the basis for considering them as a therapeutic repair or cellular delivery system to syncytia such as the myocardium. [ABSTRACT FROM AUTHOR]

Objective: In German shepherd dogs having inherited arrhythmias and sudden death, pause-dependent arrhythmias are triggered by early afterdepolarizations (EADs) originating from left ventricular (LV) Purkinje fibers (PF). Heterogeneity of LV repolarization provides the arrhythmogenic substrate. To elucidate the mechanisms whereby α-adrenergic stimulation exacerbates these arrhythmias we tested the effects of phenylephrine on both arrhythmogenic trigger and substrate. Methods and results: We used microelectrode techniques to record action potentials from LV and right ventricular (RV) PF and from midmyocardial sections of anteroseptal, anterobasal and posterobasal LV wall of unafflicted and afflicted dogs. EADs occurred spontaneously in 8 of 12 LV PF and in no RV PF from afflicted dogs and in no PF from unafflicted dogs. In LV PF from afflicted dogs, phenylephrine (10−9–10−5 M) concentration-dependently decreased membrane potential, induced abnormal automaticity at membrane potentials from −65 to −45 mV in 6 LV PF and potentiated EADs in another 6. To determine the mechanisms of membrane depolarization we studied phenylephrine effects on IK1 in voltage-clamped single LV and RV PF cells from afflicted dogs. In LV PF, phenylephrine (10−5 M) reduced IK1 over the range of −120 to −40 mV and had no effects on RV PF. Regional heterogeneity of LV repolarization was observed in afflicted dogs only. Phenylephrine had no effects on repolarization in either group. Conclusion(s): α-adrenergic stimulation exacerbates arrhythmias in afflicted dogs by increasing the arrhythmogenic trigger while leaving the substrate unchanged. Decrease in IK1 contributes importantly to α-adrenergic effects on LV PF. [Copyright &y& Elsevier]

Strategic partnerships are commonplace in today's global economy. Effective use of partnerships are viewed increasingly as an important issue facing small, growth-oriented firms. A key debate is how small firms should manage supplier partnerships. Contrasting the debate about the extent to which small firms are inertial versus adaptive, the impact of growth on value chain partnerships with suppliers was investigated. Data collected from 91 pharmaceutical-related firms is used to test the proposition that firm growth is related positively to maintaining or to expanding relationships with suppliers, not to terminating partnerships. The paper further posits that this relationship between firm growth and the amount purchased from a supplier is moderated by the perceived effectiveness of the value-chain partnerships such that when effectiveness is higher, the relationship between sales growth and the amount purchased from a value-chain partner is stronger. The propositions, which are tested with regression analysis, are supported partially. [ABSTRACT FROM AUTHOR]

Neuropeptide Y (NPY) is released from sympathetic neurons and exerts short-term (acute) effects on prejunctional nerve terminals and postjunctional cardiac ion channels. However, NPY also exerts long-term (trophic) effects on angiogenesis, cardiac hypertrophy, autonomic signaling, and cardiac ion channels, including effects on L-type Ca2+ and pacemaker channels. [ABSTRACT FROM AUTHOR]

Objective: Clinical and experimental studies suggest that immature hearts are as or more sensitive than adult hearts to adverse effects of IKr blocking drugs. We hypothesized that age-dependent changes in IKr and IKs contribute to the different repolarization reserves and proarrhythmic effects of IKr blockers in the young and adult heart. Methods: Dogs aged 1–150 days and adults were used to study (1) proarrhythmic effects in situ of the IKr blocker dofetilide; (2) dofetilide effects on action potential duration (APD) recorded with microelectrodes from left ventricular (LV) slabs; (3) IKr and IKs in single LV myocytes using whole-cell voltage clamp. Results: In situ, dofetilide-induced proarrhythmia occurred in 40% of adults, 86% of young (20–150 day) dogs and 0% of neonatal (1–19 day) dogs (P<0.05). Isolated tissue experiments showed no transmural gradient for repolarization from neonate through 3 months of age, after which the gradient increased through adulthood. In the presence of dofetilide, the greatest APD prolongation occurred in neonates. Yet, transmural dispersion did not increase in neonates but significantly increased in young and adults. Dofetilide-induced early afterdepolarization (EAD) incidence was 23% in adults, 59% in young and 8% in neonates (P<0.05). IKr but not IKs was expressed at <30 days, whereas both currents were present in adult myocardium. Conclusions: Our data suggest that a lack of IKs results in a greater dependence on IKr for repolarization in neonates and is associated with exaggerated effects of IKr-blockade on APD. However, APD prolongation alone is insufficient for expression of proarrhythmia, which also requires transmural dispersion of repolarization and EADs. The extent to which APD prolongation, transmural dispersion and EADs are manifested at various ages in the absence and presence of IKr blocking drugs appears to be the ultimate determinant of proarrhythmia. [Copyright &y& Elsevier]

Hyperpolarization-activated cation currents, termed If, Ih, or Iq, were initially discovered in heart and nerve cells over 20 years ago. These currents contribute to a wide range of physiological functions, including cardiac and neuronal pacemaker activity, the setting of resting potentials, input conductance and length constants, and dendritic integration. The hyperpolarization-activated, cation nonselective (HCN) gene family encodes the channels that underlie Ih. Here we review the relation between the biophysical properties of recombinant HCN channels and the pattern of HCN mRNA expression with the properties of native Ih in neurons and cardiac muscle. Moreover, we consider selected examples of the expanding physiological functions of Ih with a view toward understanding how the properties of HCN channels contribute to these diverse functional roles. [ABSTRACT FROM AUTHOR]

Pacemaker current (If) encoded by the HCN gene family contributes importantly to cardiac rhythm. That contribution depends on the biophysical characteristics of If, such as voltage dependence, which vary markedly with cardiac region, development and disease. Heterologous expression studies of individual HCN isoforms have failed to account for the diverse functionality of the native current. To investigate the influence of cellular environment on the gating of HCN channels, we compared the functional characteristics of HCN2 and HCN4, the two major ventricular isoforms, when over-expressed in a normal context (neonatal myocytes) and in a heterologous context (HEK 293 cells). Independent of cell type, HCN4 activates substantially slower than HCN2 and with a half-maximum activation voltage ≅10 mV less negative. However, both isoforms activate more positively in myocytes than in HEK 293 cells. The latter result suggests a context dependence (i.e. cell-type specificity) to HCN voltage dependence that exerts a comparable influence on these two isoforms. This is distinct from the inherent difference in the biophysical properties of HCN2 and HCN4. [ABSTRACT FROM AUTHOR]

Presents information on a study which examined the role of beta-adrenergic receptor subtype signaling to cyclic adenosine monophosphate and calcium in the genesis of catecholamine-dependent arrhythmias in German shepherd dogs. Methodology; Results and discussion.

Objectives: Repolarization-prolonging drugs induce torsades de pointes (TdP) in females more than males. The action potential plateau and the early afterdepolarizations that induce TdP are determined, in part, by L-type calcium current (ICa,L). Therefore, we studied gender- and hormone-related differences in ICa,L in age-, and weight-matched normal male, female and hormonally-treated, castrated rabbits. Methods: Oophorectomized (OVX) or orchiectomized (ORCH) 50- to 60-day-old rabbits were subcutaneously implanted with pellets impregnated with placebo (PLA), 5α-dihydroxytestosterone (DHT), or 17β-estradiol (EST). Four to five weeks later, epicardial and endocardial myocytes were isolated from the left ventricle. Patch clamp technique was performed to assess ICa,L. Results: ICa,L density (measured as peak current density [pA/pF] at +15 mV, Vh=−40 mV), was greater in female epicardium (−7.4±0.9) than endocardium (−5.6±0.7, P<0.05), while male epicardial ICa,L density (−6.5±0.7) did not differ from endocardial (−5.9±1.0, P>0.05). OVX-female, DHT and EST-treated groups had epicardial ICa,L density (−5.6±0.6, and −5.9±0.7, respectively) greater than endocardial (−4.3±0.3, and −3.6±0.4, P<0.05). However, OVX-females had hormone levels not significantly different from female controls and EST-treated females had non-physiological levels of estradiol. There were no differences between endocardial and epicardial ICa,L activation and inactivation. In contrast, epicardial–endocardial differences in ICa,L density in EST-treated OVX-females were associated with epicardial–endocardial differences in ICa,L activation and conductance; in DHT-treated OVX-females only epicardial–endocardial activation differed. The other groups, showed no ICa,L transmural gradient, or differences in activation, inactivation or conductance. Conclusions: The greater dispersion in ICa,L density of OVX–DHT and OVX–EST than OVX–PLA suggests both hormones can modulate ICa,L density in females. That gonadal steroids had no effect on ICa,L dispersion in males suggests gender differences in mechanism of action of both hormones. The greater ICa,L dispersion in females may contribute to gender differences in repolarization. [Copyright &y& Elsevier]

Presents a study which investigated the developmental changes in calcium currents in sinus node cells from adult and newborn rabbits. Methods; Results; Discussion.

We have reported previously that the sinoatrial node (SAN) in the newborn rabbit expresses a Na+ current (INa) with properties similar to the neuronal type-I isoform and that this current contributes to the net inward current flowing during diastolic depolarization. To characterize this current further we conducted cell-attached single-channel experiments in isolated newborn SAN myocytes. The Na+ channel was sensitive to divalent cation block and had a single-channel conductance of 25.6 pS in the absence of divalent cations. Kinetic compatibility between single-channel and previous whole-cell data was confirmed by measuring the time constant of current decay. At pacemaker potentials, time constants were of the order of tens of milliseconds. Additional experiments indicated that this slow inactivation arises because the Na+ channels expressed in the neonatal SAN tend to re-open frequently at potentials in the pacemaker range. We suggest that this is the mechanism by which a small tetrodotoxin (TTX)-sensitive current contributes to the total inward current flowing during slow diastolic depolarization in neonatal (but not adult) pacemaker myocytes. [ABSTRACT FROM AUTHOR]

Presents information on a study which examined the direct contribution of the sinoatrial node TTX-sensitive sodium current to maintain diastolic depolarization by testing for the presence of steady-state and time-dependent components. Methodology of the study; Results and discussion on the study.

Introduction: In canine ventricle, a-adrenergic agonists prolong action potential duration (APD) without any effect on the action potential notch, suggesting that, in this species, the effect on repolarization might he independent of inhibition of Ito. The present study investigated the action of the α -adrenergic agonist phenylephrine on the action potential and the repolarizing currents Ito, and Ik, in isolated canine epicardial myocytes. Methods and Results: Isolated cells from canine epicardial tissue, and Purkinje fibers, were studied with the whole cell, voltage clamp method. Phenylephrine 0.1 μM increased APD by 13% ± 4% at 90% repolarization without affecting the notch or amplitude. Under voltage clamp, concentrations of phenylephrine as high as 10 μM had no effect on Ito in canine epicardial myocytes. However, Ito, of isolated canine Purkinje myocytes was reduced to 69% ± 7% of control by 1 μM phenylephrine. Further studies in canine epicardial myocytes revealed an action of phenylephrine to inhibit Ik, and in particular Iks. Using a voltage protocol that included a two-step repolarization to separate Iks and Ikr tail components, the largely Ikr component was not significantly affected by 1 μM phenylephrine, whereas the largely Ikscomponent was reduced to 81% ± 5% of control value. Conclusion: α-.Adrenergic prolongation of repolarization in canine epicardium does not result from inhibition of Ito. Rather, it appears that reduction of Iks contributes to the action of phenylephrine. The unresponsiveness of epicardial Ito is not a general characteristic of the canine heart, because Purkinje myocyte Ito was inhibited, suggesting regional differences in the molecular basis of Ito and/or α-adrenergic signaling in the canine heart. [ABSTRACT FROM AUTHOR]

Deals with a study which tested whether the effect of sympathetic innervation results from a chronic or sustained action of neurally released neuropeptide Y (NPY). Description of the calcium current in control experiments; Effect of innervation on calcium current; Acute and chronic effect of NPY on calcium current.