Coupling an HCN2-expressing cell to a myocyte creates a two-cell pacing unit.

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.

Impulse conduction and gap junctional remodelling by endothelin-1 in cultured neonatal rat ventricular myocytes.

Endothelin-1 (ET-1) is an important contributor to ventricular hypertrophy and failure, which are associated with arrhythmogenesis and sudden death. To elucidate the mechanism(s) underlying the arrhythmogenic effects of ET-1 we tested the hypothesis that long-term (24 hrs) exposure to ET-1 impairs impulse conduction in cultures of neonatal rat ventricular myocytes (NRVM). NRVM were seeded on micro-electrode-arrays (MEAs, Multi Channel Systems, Reutlingen, Germany) and exposed to 50 nM ET-1 for 24 hrs. Hypertrophy was assessed by morphological and molecular methods. Consecutive recordings of paced activation times from the same cultures were conducted at baseline and after 3, 6 and 24 hrs, and activation maps for each time period constructed. Gap junctional Cx43 expression was assessed using Western blot and confocal microscopy of immunofluorescence staining using anti-Cx43 antibodies. ET-1 caused hypertrophy as indicated by a 70% increase in mRNA for atrial natriuretic peptide (P < 0.05), and increased cell areas (P < 0.05) compared to control. ET-1 also caused a time-dependent decrease in conduction velocity that was evident after 3 hrs of exposure to ET-1, and was augmented at 24 hrs, compared to controls (P < 0.01). ET-1 increased total Cx43 protein by approximately 40% (P < 0.05) without affecting non- phosphorylated Cx43 (NP-Cx43) protein expression. Quantitative confocal microscopy showed a approximately 30% decrease in the Cx43 immunofluorescence per field in the ET-1 group (P < 0.05) and a reduced field stain intensity (P < 0.05), compared to controls. ET-1-induced hypertrophy was accompanied by reduction in conduction velocity and gap junctional remodelling. The reduction in conduction velocity may play a role in ET-1 induced susceptibility to arrhythmogenesis.

History of arrhythmias.

A historical overview is given on the techniques to record the electrical activity of the heart, some anatomical aspects relevant for the understanding of arrhythmias, general mechanisms of arrhythmias, mechanisms of some specific arrhythmias and nonpharmacological forms of therapy. The unravelling of arrhythmia mechanisms depends, of course, on the ability to record the electrical activity of the heart. It is therefore no surprise that following the construction of the string galvanometer by Einthoven in 1901, which allowed high-fidelity recording of the body surface electrocardiogram, the study of arrhythmias developed in an explosive way. Still, papers from McWilliam (1887), Garrey (1914) and Mines (1913, 1914) in which neither mechanical nor electrical activity was recorded provided crucial insights into re-entry as a mechanism for atrial and ventricular fibrillation, atrioventricular nodal re-entry and atrioventricular re-entrant tachycardia in hearts with an accessory atrioventricular connection. The components of the electrocardiogram, and of extracellular electrograms directly recorded from the heart, could only be well understood by comparing such registrations with recordings of transmembrane potentials. The first intracellular potentials were recorded with microelectrodes in 1949 by Coraboeuf and Weidmann. It is remarkable that the interpretation of extracellular electrograms was still controversial in the 1950s, and it was not until 1962 that Dower showed that the transmembrane action potential upstroke coincided with the steep negative deflection in the electrogram. For many decades, mapping of the spread of activation during an arrhythmia was performed with a "roving" electrode that was subsequently placed on different sites on the cardiac surface with a simultaneous recording of another signal as time reference. This method could only provide reliable information if the arrhythmia was strictly regular. When multiplexing systems became available in the late 1970s, and optical mapping in the 1980s, simultaneous registrations could be made from many sites. The analysis of atrial and ventricular fibrillation then became much more precise. The old question whether an arrhythmia is due to a focal or a re-entrant mechanism could be answered, and for atrial fibrillation, for instance, the answer is that both mechanisms may be operative. The road from understanding the mechanism of an arrhythmia to its successful therapy has been long: the studies of Mines in 1913 and 1914, microelectrode studies in animal preparations in the 1960s and 1970s, experimental and clinical demonstrations of initiation and termination of tachycardias by premature stimuli in the 1960s and 1970s, successful surgery in the 1980s, the development of external and implantable defibrillators in the 1960s and 1980s, and finally catheter ablation at the end of the previous century, with success rates that approach 99% for supraventricular tachycardias.

Molecular/genetic determinants of repolarization and their modification by environmental stress.

Although a variety of factors, inherited or environmental, can influence expression of ion channel proteins to impact on repolarization, that environment can affect genetic determinants of repolarization for intervals of varying duration is a concept that is not as generally appreciated as it should be. In the following pages we review the molecular/genetic determinants of cardiac repolarization and summarize how pathologic events and environmental intrusions can affect these determinants. Understanding the chains of events involved should yield insights into both the causes and potential avenues of treatment for abnormalities of repolarization.

Connexin-specific cell-to-cell transfer of short interfering RNA by gap junctions.

The purpose of this study was to determine whether oligonucleotides the size of siRNA are permeable to gap junctions and whether a specific siRNA for DNA polymerase beta (pol beta) can move from one cell to another via gap junctions, thus allowing one cell to inhibit gene expression in another cell directly. To test this hypothesis, fluorescently labelled oligonucleotides (morpholinos) 12, 16 and 24 nucleotides in length were synthesized and introduced into one cell of a pair using a patch pipette. These probes moved from cell to cell through gap junctions composed of connexin 43 (Cx43). Moreover, the rate of transfer declined with increasing length of the oligonucleotide. To test whether siRNA for pol beta was permeable to gap junctions we used three cell lines: (1) NRK cells that endogenously express Cx43; (2) Mbeta16tsA cells, which express Cx32 and Cx26 but not Cx43; and (3) connexin-deficient N2A cells. NRK and Mbeta16tsA cells were each divided into two groups, one of which was stably transfected to express a small hairpin RNA (shRNA), which gives rise to siRNA that targets pol beta. These two pol beta knockdown cell lines (NRK-kcdc and Mbeta16tsA-kcdc) were co-cultured with labelled wild type, NRK-wt or Mbeta16tsA-wt cells or N2A cells. The levels of pol beta mRNA and protein were determined by semiquantitative RT-PCR and immunoblotting. Co-culture of Mbeta16tsA-kcdc cells with Mbeta16tsA-wt, N2A or NRK-wt cells had no effect on pol beta levels in these cells. Similarly, co-culture of NRK-kcdc with N2A cells had no effect on pol beta levels in the N2A cells. In contrast, co-culture of NRK-kcdc with NRK-wt cells resulted in a significant reduction in pol beta in the wt cells. The inability of Mbeta16tsA-kcdc cells to transfer siRNA is consistent with the fact that oligonucleotides of the 12 nucleotide length were not permeable to Cx32/Cx26 channels. This suggested that Cx43 but not Cx32/Cx26 channels allowed the cell-to-cell movement of the siRNA. These results support the novel hypothesis that non-hybridized and possible hybridized forms of siRNA can move between mammalian cells through connexin-specific gap junctions.

Mechanisms of cardiac arrhythmias: focus on atrial fibrillation.

Atrial fibrillation (AF) is a major cause of morbidity in the aging population. Initially preponderant in men over age 60, it ultimately becomes a disease of women, in part perhaps because of their greater longevity. The subject of AF is an extremely complex one, as described in this article. The author deals with atrial size and atrial pressure, atrial structure, and electrophysiologic correlates of chronic AF in human subjects. He also discusses pacing-induced models of AF and ionic determinants of pacing-induced AF. He emphasizes the changes that occur in the atrium as a result of rapid rate and/or fibrillation and the interdependence of AF and changes in myocardial structure.

Long-term electrophysiological effects of regional cardiac sympathetic denervation of the neonatal dog.

OBJECTIVE: In many cardiac arrhythmias, both a triggering factor and a favorable myocardial substrate are required. Whereas the sympathetic nervous system may trigger tachyarrhythmias, its function as a long-term modulator of the myocardial substrate is less well understood. Therefore, we tested the hypothesis that regional sympathetic denervation at birth would produce an abnormal myocardial substrate. The comparator was the substrate associated with inherited, lethal tachyarrhythmias at 5 months of age in German shepherd dogs with incomplete sympathetic innervation. METHODS: Mongrel dogs underwent right cardiac stellectomy (RSX) within the first day of life and were terminally studied with control littermates at 5 months of age. RESULTS: On days 1-21 of life, RSX animals manifested significant QT prolongation on ECG and sudden, asystolic death. Beyond this age, QT intervals normalized and deaths did not occur. At 5 months, action potentials (AP) were recorded from Purkinje fibers (PF) and midmyocardial preparations in anteroseptal (AS) and posterobasal (PB) left ventricle. Early afterdepolarizations occurred only in left ventricular PF from RSX dogs. Isoproterenol prolonged AP duration in AS and shortened it in PB of RSX but not control dogs. The incidence of isoproterenol-initiated triggered activity and the amplitude of delayed afterdepolarizations were greater in RSX than control dogs. CONCLUSION: Five months after RSX heterogeneous alterations of LV electrophysiological properties were similar to those previously observed in animals having inherited deficits in sympathetic innervation and sudden death. This implicates the sympathetic nerves as long-term modulators of an arrhythmogenic substrate. That 5-month-old RSX dogs did not experience tachyarrhythmias or sudden death indicates that further anomalies--beyond those explicable by the substrate change--must exist to induce sudden death.

Altering ventricular activation remodels gap junction distribution in canine heart.

INTRODUCTION: Prolonged arrhythmic or paced ventricular activation causes persistent changes in myocardial conduction and repolarization that may result from altered electrotonic current flow, for which gap junctional coupling is the principal determinant. Remodeling of gap junctions and their constituent connexins modifies conduction and has been causally implicated in reentrant arrhythmogenesis. We hypothesized conversely that altering the pattern of ventricular activation causes gap junctional remodeling. METHODS AND RESULTS: Seven dogs were paced from the left ventricular (LV) epicardium (VVO, approximately 120 beats/min) for 21 days before excision of transmural LV samples that were divided into endomyocardial, mid-myocardial, and epimyocardial layers. Another five paced dogs had recording electrodes attached to multiple LV sites. All 12 dogs developed characteristic pacing-induced persistent T wave changes of cardiac memory. After 21 days of pacing, the ventricularly paced QRS duration prolonged by a mean of 4 msec over baseline (P < 0.05), a change that was associated with significant slowing of intraventricular conduction to local sites. These changes in QRS duration and repolarization were associated with a reduction in epimyocardial connexin43 expression on quantitative Western blotting of LV myocardium from close to, but not distant from, the pacing site (61.7+/-18.4 vs 100.9+/-34.0; P < 0.02) and a marked disruption in immunolabeled connexin43 distribution in epimyocardium only. CONCLUSION: Spatially distinct transmural and regional gap junctional remodeling is a consequence of abnormal ventricular activation and is associated with consistent changes in activation that may alter patterns of repolarization and facilitate reentrant arrhythmogenesis.

Impact of sex and gonadal steroids on prolongation of ventricular repolarization and arrhythmias induced by I(K)-blocking drugs.

BACKGROUND: Mechanisms for longer rate-corrected QT intervals and higher incidences of drug-induced torsade de pointes in women than in men are incompletely defined, although gonadal steroids are assumed to be important determinants of these differences. METHODS AND RESULTS: We used microelectrode techniques to study isolated rabbit right ventricular endocardium from control male and female and castrated male (ORCH) and female (OVX) rabbits. Action potential duration to 30% repolarization (APD(30)) was significantly shorter in male than female and in ORCH than OVX at a cycle length of 500 ms. The I(Ks) blocker chromanol 293B had no effect on APD in males or females. The I(Kr) blocker dofetilide prolonged APD in female and ORCH more than in male and OVX. At 10(-)(6) mol/L dofetilide (cycle length=1 second), the incidence of early afterdepolarizations was: female, 67%; ORCH, 56%; male, 40%; and OVX, 28%. Serum 17beta-estradiol levels were unrelated to the effects of dofetilide, but as testosterone levels increased, the dofetilide effect to increase APD diminished, as did early afterdepolarization incidence. CONCLUSIONS: Sex-related differences in basal right ventricular endocardial AP configuration persist in castrated rabbits, suggesting that extragonadal factors contribute to the differences in ventricular repolarization. In this model, drugs that block I(Kr) but not I(Ks) prolong repolarization in a way that suggests that protection from excess prolongation in males is attributable to testosterone, whereas the risk of excess prolongation of repolarization in females is related to sex-determined factors in addition to estrogen.

Interactions between antiarrhythmic drugs and cardiac memory.

OBJECTIVE: Ventricular pacing or arrhythmias can induce cardiac memory (CM). We hypothesized that clinically administered antiarrhythmic drugs alter the expression of CM, and that the repolarization changes characteristic of CM can modulate the effects of antiarrhythmic drugs. METHODS: We studied conscious, chronically-instrumented dogs paced for two 1-h periods to study the effects of drugs on the evolution of memory (protocol 1) or for 21 days (protocol 2) to observe the effects of steady-state memory on drug actions. Dogs were treated in both settings with quinidine, lidocaine or E4031, in random order, and within therapeutic serum concentration ranges. RESULTS: Pacing, alone, for 2 h significantly prolonged ERP only near the left ventricular pacing site, whereas pacing alone for 21 days prolonged ERP at all sites (P<0.05). Quinidine and E4031, but not lidocaine, prolonged repolarization and ERP and suppressed evolution of CM in protocol 1. However, quinidine's effect in prolonging repolarization was diminished in both protocols, while its effect in prolonging ERP was diminished in the 21-day protocol only. In contrast, the effects of E4031 were additive to those of CM, prolonging repolarization and ERP in both protocols, while lidocaine showed no changes in effect at all. CONCLUSIONS: Pacing to induce CM significantly affects ventricular repolarization and refractoriness, and there are interactions between CM, quinidine and E4031. Depending on the specific drug, these interactions have the potential to be anti- or proarrhythmic, and may impact importantly on the clinical efficacy of drugs as well as on electrophysiologic testing of drug actions.

Cardiac memory in canine atrium : identification and implications.

BACKGROUND: Memory is a diverse biological phenomenon whose importance in the ventricle has been demonstrated. We hypothesized its occurrence in the atrium, contributing to the modulation of cardiac rhythm. METHODS AND RESULTS: We analyzed P and Ta waves in conscious chronically instrumented dogs with complete heart block. Animals were atrioventricularly sequentially paced at 5% greater than the sinus rate from the lateral right atrium (RA) during control, followed by 2 periods of 1-hour test pacing at 50% greater than the sinus rate, or by equivalent test pacing from the left atrial appendage (LAA) at 5% or 50% greater than the sinus rate. Recovery RA pacing periods of 20- and 30-minute duration, respectively, succeeded each test pacing period. RA test pacing at either rate did not affect the variables measured, but changing the pacing site from RA to LAA altered the P and Ta waves. Displacement of the spatial atrial gradient vector occurred during recovery from LAA pacing, was more marked at rapid pacing rates, and manifested accumulation and resolution consistent with cardiac memory. Concurrently, the right effective refractory period decreased. CONCLUSIONS: Memory is demonstrable in canine atrium, showing rapid onset, accumulation during successive pacing periods, and resolution on cessation of pacing. Given its association with a reduced effective refractory period, it may contribute to the substrate for atrial arrhythmias.

beta(1) and beta(2)-adrenergic receptor subtype effects in German shepherd dogs with inherited lethal ventricular arrhythmias.

OBJECTIVE: Delayed afterdepolarization-induced triggered activity originating in ventricular myocardium is a mechanism for some age-dependent, inherited ventricular tachycardias in a colony of German shepherd dogs. METHODS: We used standard microelectrode techniques to study beta-adrenergic receptor subtype modulation of the triggered activity in anteroseptal left ventricular myocardium from eleven of these dogs and seven unafflicted, age-matched German shepherd controls. RESULTS: During sustained stimulation at cycle lengths of 300-4000 ms, 10(-9)-10(-7) M isoproterenol concentration-dependently shortened action potential duration (APD) to 90% repolarization more in myocardium from afflicted than from unafflicted dogs. This shortening was prevented by a beta(1)-blocker CGP20712A (10(-7) M) while a beta(2)-blocker ICI118551 (10(-7) M) did not modify the effect of isoproterenol in either group. The beta(2)-agonist zinterol 10(-8)-10(-6) M had no effect on APD. Stimulation at a cycle length of 250 ms in the presence of 10(-7) M isoproterenol induced more triggered AP in myocardium from afflicted than unafflicted dogs. beta(1)-Blockade completely eliminated, while beta(2)-blockade facilitated, and the beta(2)-agonist zinterol did not induce triggered activity in the two groups. CONCLUSION: Isoproterenol effects on APD and triggered activity in the myocardium of dogs with inherited arrhythmias are due primarily to an abnormality of beta(1)-adrenoceptor mediated signaling that is subject to beta(2)-adrenergic modulation.