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The cardiac safety of renzapride, a novel benzamide currently under clinical development for the treatment of irritable bowel syndrome, was investigated in a four-way randomized crossover electrocardiographic clinical study in healthy human subjects and also in an in vitro cardiac conductivity study in sheep isolated Purkinje fibres. The primary endpoint in the clinical study was prolongation of the individually corrected QT interval (QTci). No clinically or statistically significant prolongation of QTci after 4 or 20 mg renzapride compared with placebo was observed. The relative effects of renzapride and cisapride in the in vitro study showed that the cardiac action potential duration was unaltered by 0.2 and 2 microM renzapride, shortened by 20 microM renzapride, and prolonged by 1 microM cisapride. Cisparide was also a 1000-fold more potent inhibitor of human ether-a-go-go related gene (hERG) channels in HEK293 cells than renzapride. These studies indicate that therapeutic doses of renzapride are unlikely to prolong cardiac action potentials and, therefore, are also unlikely to cause cardiac arrhythmias in clinical use.

The various cardiac regions have specific action potential properties appropriate to their electrical specialization, resulting from a specific pattern of ion-channel functional expression. The present study addressed regionally defined differential ion-channel expression in the non-diseased human heart with a genomic approach. High-throughput real-time RT-PCR was used to quantify the expression patterns of 79 ion-channel subunit transcripts and related genes in atria, ventricular epicardium and endocardium, and Purkinje fibres isolated from 15 non-diseased human donor hearts. Two-way non-directed hierarchical clustering separated atria, Purkinje fibre and ventricular compartments, but did not show specific patterns for epicardium versus endocardium, nor left- versus right-sided chambers. Genes that characterized the atria (versus ventricles) included Cx40, Kv1.5 and Kir3.1 as expected, but also Cav1.3, Cav3.1, Cav alpha2 delta2, Nav beta1, TWIK1, TASK1 and HCN4. Only Kir2.1, RyR2, phospholamban and Kv1.4 showed higher expression in the ventricles. The Purkinje fibre expression-portrait (versus ventricle) included stronger expression of Cx40, Kv4.3, Kir3.1, TWIK1, HCN4, ClC6 and CALM1, along with weaker expression of mRNA encoding Cx43, Kir2.1, KChIP2, the pumps/exchangers Na(+),K(+)-ATPase, NCX1, SERCA2, and the Ca(2+)-handling proteins RYR2 and CASQ2. Transcripts that were more strongly expressed in epicardium (versus endocardium) included Cav1.2, KChIP2, SERCA2, CALM3 and calcineurin-alpha. Nav1.5 and Nav beta1 were more strongly expressed in the endocardium. For selected genes, RT-PCR data were confirmed at the protein level. This is the first report of the global portrait of regional ion-channel subunit-gene expression in the non-diseased human heart. Our data point to significant regionally determined ion-channel expression differences, with potentially important implications for understanding regional electrophysiology, arrhythmia mechanisms, and responses to ion-channel blocking drugs. Concordance with previous functional studies suggests that regional regulation of cardiac ion-current expression may be primarily transcriptional.

Objective: To quantitatively detect the expression of connexins (Cx) mRNA in the posterior nodal extension (PNE) of adult rat heart and understand the relationship between Cx expression and atrial ventricular nodal reentrant tachycardia (AVNRT)., Methods: PNE was separated from adult rat heart by means of laser microdissection (LCM), and the cells were also isolated from the atrioventricular node (AVN), sinoatrial node (SAN), Purkinje fiber (PF), right atrium (RA) and right ventricle (RV), to serve as the controls. The Cx mRNA level was detected in these cells with quantitative real-time PCR (QRT-PCR)., Results: The cells were successfully isolated from the PNE and other regions of adult rat heart, where heterogeneous expression of the 3 Cx isoforms (Cx43, Cx45, and Cx40) were observed. Cx45 mRNA showed higher expression in the PNE than in the working myocardium, whereas Cx43 mRNA level was about 25 times higher (P<0.05) in the working myocardium and 18 times higher (P<0.05) in the PF than in the PNE. In the PF, Cx40 mRNA level was proximately 6.8 times (P<0.01) as much as that in the PNE. Cx expression in the PNE was, however, similar to that in the SAN and AVN., Conclusion: Cx mRNAs exhibit heterogeneous expression in the PNE to allow the formation of the slow pathway. In addition, Cx expression in the PNE is very different from that in the adjacent myocardium, resulting in conduction discontinuity at the cellular junction, where, on certain occasion, unidirectional block may occur to cause AVNRT.

The objective of this study was to investigate the effects of nitroglycerin (NG) on the contractile force in order to elucidate regulatory roles of cyclic guanosine monophosphate (cGMP) in the bundle of canine cardiac Purkinje fibers. NG suppressed the developed tension in canine Purkinje fibers immersed in normal Tyrode's solution. The contraction was also elicited in the depolarizing solution containing 30 mM K+ by adding isoproterenol (Iso) or tetraethylammonium (TEA). The suppression caused by NG was more marked in fibers in the depolarizing solution treated with TEA than in the depolarizing solution treated with Iso. 8-Bromo-cGMP and sodium nitroprusside had a slight inhibition on the contraction in the fibers. Methylene blue did not affect the decrease in contractile force induced by NG. The weaker inhibition caused by NG on the contraction in the presence of Iso might result from a possible increase in intracellular cAMP levels in the fibers., ((c) 2007 Prous Science. All rights reserved.)

The action potential of Purkinje fibres is markedly shortened by tetrodotoxin, suggesting the possibility that a slowly inactivating sodium current might flow during the plateau. The aim of the present experiments was to investigate, in canine cardiac Purkinje single cells by means of a whole cell patch clamp technique, whether a sodium current slowly inactivates at less negative potentials and (if so) some of its distinctive characteristics. The results showed that a 500 ms depolarizing step from a holding potential of -90 mV to -50 mV induced the fast inward current I(Na) (labelled here I(Na1)). With steps to -40 mV or less negative values, a slowly decaying component (tentatively labelled here I(Na2)) appeared, which peaked at -30 to -20 mV and decayed slowly and incompletely during the 500 ms steps. The I(Na2) was present also during steps to -10 mV, but then the transient outward current (I(to)) appeared. When the holding potential (V(h)) was decreased to -60 to -50 mV, I(Na2) disappeared even if a small I(Na1) might still be present. Tetrodotoxin (30 mum), lignocaine (100 mum) and cadmium (0.2 mm; but not manganese, 1 mm) blocked I(Na2). During fast depolarizing ramps, the rapid inactivation of I(Na1) was followed by a negative slope region. During repolarizing ramps, a region of positive slope was present, whereas I(Na1) was absent. At less negative values of V(h), the amplitude of the negative and positive slopes became gradually smaller. Gradually faster ramps increased the magnitude of the negative slope, and tetrodotoxin (30 mum) reduced or abolished it. Thus, Purkinje cells have a slowly decaying inward current owing to Na(+) entry (I(Na2)) that is different in several ways from the fast I(Na1) and that appears important for the duration of the plateau.

Alternation in the duration of consecutive cardiac action potentials (electrical alternans) may precipitate conduction block and the onset of arrhythmias. Consequently, suppression of alternans using properly timed premature stimuli may be antiarrhythmic. To determine the extent to which alternans control can be achieved in cardiac tissue, isolated canine Purkinje fibers were paced from one end using a feedback control method. Spatially uniform control of alternans was possible when alternans amplitude was small. However, control became attenuated spatially as alternans amplitude increased. The amplitude variation along the cable was well described by a theoretically expected standing wave profile that corresponds to the first quantized mode of the one-dimensional Helmholtz equation. These results confirm the wavelike nature of alternans and may have important implications for their control using electrical stimuli.

As the beagle dog is a commonly used preclinical species to test the effects of new drugs on cardiac repolarisation and Purkinje fibres have become an established in vitro preparation to assess the effects of these new drugs on action potential duration (APD), the main aim of this study was therefore to evaluate the relative contribution of the inward (I(K1)) and slow delayed (I(Ks)) rectifier cardiac K(+) currents to action potential repolarisation in beagle Purkinje fibres under three different experimental conditions: (i) selective block of I(K1) with BaCl(2), (ii) selective block of I(Ks) with (-) chromanol 293B under basal conditions and (iii) selective block of I(Ks) during beta-adrenoceptor stimulation. Furthermore, the dependence of this contribution on gender and pacing rate was investigated. Microelectrode techniques were employed to measure APD in Purkinje fibres from adult female and male dogs. At stimulation rates of 3.33, 1.0 and 0.2 Hz, the degree of prolongation of APD evoked by BaCl(2) (10 microM) was comparable in fibres from female and male dogs. At the same stimulation rates, 10 microM (-) chromanol 293B did not change the APD in fibres from female and male dogs. During beta-adrenoceptor stimulation with 0.1 microM isoproterenol, an APD prolonging effect of (-) chromanol 293B was detected. In the presence of isoproterenol, action potentials in fibres from male dogs get shorter when changing the stimulation rate from 1.0 to 0.2 Hz, while the opposite is seen in fibres from female dogs. This alteration was completely reversed by (-) chromanol 293B. In conclusion, our findings confirm that beta-adrenoceptor stimulation is one condition where there may be an increased role of I(Ks) in action potential repolarisation. Gender differences in the autonomic modulation of I(Ks) could be a contributing factor to the reported increased susceptibility of female hearts to arrhythmias.

Nkx2-5 gene mutations cause cardiac abnormalities, including deficits of function in the atrioventricular conduction system (AVCS). In the chick, Nkx2-5 is elevated in Purkinje fiber AVCS cells relative to working cardiomyocytes. Here, we show that Nkx2-5 expression rises to a peak as Purkinje fibers progressively differentiate. To disrupt this pattern, we overexpressed Nkx2-5 from embryonic day 10, as Purkinje fibers are recruited within developing chick hearts. Overexpression of Nkx2-5 caused inhibition of slow tonic myosin heavy chain protein (sMHC), a late Purkinje fiber marker but did not affect Cx40 levels. Working cardiomyocytes overexpressing Nkx2-5 in these hearts ectopically up-regulated Cx40 but not sMHC. Isolated embryonic cardiomyocytes overexpressing Nkx2-5 also displayed increased Cx40 and suppressed sMHC. By contrast, overexpression of a human NKX2-5 mutant did not effect these markers in vivo or in vitro, suggesting one possible mechanism for clinical phenotypes. We conclude that a prerequisite for normal Purkinje fiber maturation is precise regulation of Nkx2-5 levels., (2005 Wiley-Liss, Inc.)

Objective: Short QT syndrome (SQTS) is characterized by ventricular arrhythmias and sudden death. One form of SQTS is caused by mutation N588K in human ether-a-go-go-related gene (HERG). In this study we sought to determine the potential role of N588K in arrhythmias., Methods: We measured the characteristics of HERG current generated by wild-type (WT) KCNH2 and the N588K mutant channel expressed in mammalian TSA201 cells., Results: Whole-cell patch-clamp recordings of WT HERG currents showed the usual rapid onset of inactivation (rectification) at potentials more positive than +10 mV. In contrast, N588K currents rectified at potentials over +80 mV. Over the physiological range of potentials, N588K currents do not inactivate. During an action potential clamp, WT currents displayed a "hump" like waveform with slow activation kinetics and a rapid increase during phase 3 repolarization. In contrast, N588K currents were proportional to the amplitude of the action potential and displayed a dome-like configuration and a much larger current during the initial phases in the ventricle. Purkinje cell action potentials display a more negative phase 2 repolarization than the ventricle and elicited much smaller WT and N588K currents of similar amplitudes., Conclusions: Physiologically the N588K mutation abolishes rectification of HERG currents and specifically increases I(Kr) in the ventricle with minimal effects on the Purkinje fiber action potential duration. Such preferential prolongation may explain the separation of the T and U waves observed in the ECG of SQTS patients and lead to re-excitation of the ventricle endocardium.

Purkinje fibers play essential roles in impulse propagation to the ventricles, and their functional impairment can become arrhythmogenic. However, little is known about precise spatiotemporal pattern(s) of interconnection between Purkinje-fiber network and the underlying ventricular myocardium within the heart. To address this issue, we simultaneously visualized intracellular Ca(2+) dynamics at Purkinje fibers and subjacent ventricular myocytes in Langendorff-perfused rat hearts using multi-pinhole type, rapid-scanning confocal microscopy. Under recording of electrocardiogram at room temperature spatiotemporal changes in fluo3-fluorescence intensity were visualized on the subendocardial region of the right-ventricular septum. Staining of the heart with either fluo3, acetylthiocholine iodide (ATCHI), or di-4-ANEPPS revealed characteristic structures of Purkinje fibers. During sinus rhythm (about 60 bpm) or atrial pacing (up to 3 Hz) each Purkinje-fiber exhibited spatiotemporally synchronous Ca(2+) transients nearly simultaneously to ventricular excitation. Ca(2+) transients in individual fibers were still synchronized within the Purkinje-fiber network not only under high-K(+) (8 mM) perfusion-induced Purkinje-to-ventricular (P-V) conduction delay, but also under unidirectional, orthodromic P-V block produced by 10-mM K(+) perfusion. While spontaneous, asynchronous intracellular Ca(2+) waves were identified in injured fibers of Purkinje network locally, surrounding fibers still exhibited Ca(2+) transients synchronously to ventricular excitation. In summary, these results are the first demonstration of intracellular Ca(2+) dynamics in the Purkinje-fiber network in situ. The synchronous Ca(2+) transients, preserved even under P-V conduction disturbances or under emergence of Ca(2+) waves, imply a syncytial role of Purkinje fibers as a specialized conduction system, whereas unidirectional block at P-V junctions indicates a substrate for reentrant arrhythmias.

Ventricular tachycardia is a common heart rhythm disorder and a frequent cause of sudden cardiac death. Aberrant cell-cell coupling through gap junction channels, a process termed gap junction remodeling, is observed in many of the major forms of human heart disease and is associated with increased arrhythmic risk in both humans and in animal models. Genetically engineered mice with cardiac-restricted knockout of Connexin43, the major cardiac gap junctional protein, uniformly develop sudden cardiac death, although a detailed electrophysiological understanding of their profound arrhythmic propensity is unclear. Using voltage-sensitive dyes and high resolution optical mapping techniques, we found that uncoupling of the ventricular myocardium results in ectopic sites of ventricular activation. Our data indicate that this behavior reflects alterations in source-sink relationships and paradoxical conduction across normally quiescent Purkinje-ventricular muscle junctions. The aberrant activation profiles are associated with wavefront collisions, which in the setting of slow conduction may account for the highly arrhythmogenic behavior of Connexin43-deficient hearts. Thus, the extent of gap junction remodeling in diseased myocardium is a critical determinant of cardiac excitation patterns and arrhythmia susceptibility.

Objectives: The aim of this study is to analyze Scn1b mRNA expression levels and protein distribution of Scn1b, a putative modulator of the pore-forming Na(+) channel subunit in the heart, during mouse cardiac development., Methods: Scn1b mRNA levels were determined by real-time RT-PCR using embryonic hearts ranging from E9.5 to E18.5 as well as in postnatal and adult heart. Scn1b protein distribution and subcellular localization during cardiogenesis were analyzed by immunohistochemistry and confocal microscopy., Results: Scn1b mRNA showed a dynamic expression pattern, peaking at stage E12.5 and decreasing at E15.5. Scn1b mRNA increased at later embryonic and neonatal stages, being maximal in the adult heart. Immunohistochemistry experiments revealed comparable distribution of Scn1b protein between the different cardiac chambers at early embryonic stages. With further development, Scn1b protein showed an enhanced expression in the trabeculated myocardium and the bundle branches. At the subcellular level in later embryonic and postnatal mouse cardiomyocytes, Scn1b was present in T-tubules as identified by immunostaining of alpha-actinin, and in the intercalated disks as identified by immunostaining of connexin 43., Conclusion: These results demonstrate that Scn1b is expressed during mouse heart development, suggesting it can play an important role in the action potential configuration of the cardiomyocytes during heart morphogenesis.

Objective: To determine the presence and the potential contribution of neuronal sodium channels to dog cardiac function., Methods: We used a combination of electrophysiological (patch clamp), RT-PCR, biochemical and immunohistochemical techniques to identify and localize neuronal Na(+) channels in dog heart and determine their potential contribution to the fast sodium current., Results: In all cardiac tissues investigated, Na(v)1.1, Na(v)1.2 and Na(v)1.3 transcripts were detected. In immunoblots, we found Na(v)1.1 and Na(v)1.2 proteins in the ventricle (V) and in Purkinje fibers (PF). Na(v)1.3 immunoblots suggested strong proteolytic activity against this isoform in the heart. Na(v)1.6 was not found in any of the tissues tested. Confocal immunofluorescence on cardiac myocytes showed that Na(v)1.1 was predominantly localized at the intercalated disks in V and PF and around the nucleus (V). Na(v)1.2 was only present at the Z lines (V). Consistent with the immunoblot data, an intense but diffuse intracellular staining was observed for Na(v)1.3. Na(v)1.6 fluorescence staining was faint and diffuse. Surprisingly, immunoblots indicated the presence of two Na(v)beta 2 variants: a 42-kDa protein that co-localized with Na(v)1.2 at the Z lines in V and a 34-kDa protein that co-localized with Na(v)1.1 at the intercalated disks in PF. In agreement with the biochemical data, electrophysiological results suggest that neuronal sodium channels generate 10+/-5% and 22+/-5% of the peak sodium current in dog ventricle and Purkinje fibers, respectively., Conclusions: Our results suggest that neuronal NaChs are more abundant in Purkinje fibers than in ventricles, and this suggests a role for them in cardiac conduction.

Objective: The aim of this work was to target a vital reporter gene in the mouse cardiac conduction system (CS) to distinguish this tissue from the surrounding myocardium in the adult heart., Methods: A transgenic mouse line has been created in which EGFP is expressed under the control of the Cx40 gene. Correlative investigations associating EGFP imaging and electrophysiological techniques were carried out on the adult heart and isolated cardiomyocytes., Results: In the heart of the Cx40(EGFP/+) mice, EGFP signal was seen in the coronary arteries, the atria, the atrioventricular (AV) node and the His-Purkinje system. The latter was found to be structurally and functionally asymmetrical. The anatomical asymmetry was apparent in both the number of strands or fasciculi making up the His bundle branches (BBs) (1 strand on the right, 20 or so on the left), and the density (low on the right, high on the left) of the network of Purkinje fibers (PFs) that extends over the ventricular wall surfaces. The profiles of the electrical activation patterns recorded on the right and left flanks of the septum were also asymmetrical, mirroring the architecture of the branches. EGFP made it easy to identify the Purkinje cells in populations of dissociated cardiomyocytes and they were investigated using the patch-clamp technique. The hyperpolarization-activated current (If) was recorded in all spontaneously active Purkinje cells., Conclusions: This investigation provides positive evidence of the asymmetry of the His-Purkinje system of the adult mouse, and the first patch-clamp recording data on murine cardiac Purkinje cells. This mouse model opens up new perspectives for investigating the contribution of specific genes to the morphology and function of the His-Purkinje system.

Brain natriuretic peptide (BNP) might be of importance during heart development and is described to be increasingly expressed in congestive heart failure and to affect the progress of this condition. However, details in the normal expression of BNP are still unclear in various parts of the adult and growing heart, including the conduction system. In this study, we investigated the expression of BNP in relation to that of atrial natriuretic peptide (ANP) in the growing as well as in the adult rat heart. The effects of chemical sympathectomy in adult rats were also examined. Contrary to previous BNP immunohistochemical studies, the BNP antiserum was preabsorbed with an excess of ANP before staining to abolish the crossreactivity with ANP. There was a pronounced BNP immunoreaction in the auricles, the trabeculated ventricular walls, and the peripheral parts of the conduction system at 0-1 days postnatally. The degree of immunoreaction gradually decreased with increasing age. A similar developmental pattern was seen concerning ANP expression, but the magnitude of the latter clearly exceeded that for BNP. Immunoreaction for BNP was never detected in the atrioventricular (AV) node and AV bundle at any stage. In contrast to the situation for ANP previously observed, no obvious changes in BNP immunoreaction patterns were observed in response to sympathectomy. This is the first study to thoroughly demonstrate the expression of BNP in the various regions of the rat heart during growth and in the normal and sympathectomized adult stage. The observations are related to possible functions of natriuretic peptides in the growing and adult heart., (Copyright 2004 Wiley-Liss, Inc.)

The presence and functional role of the swelling-activated Cl(-) current (I(Cl(swell))) in rabbit cardiac Purkinje cells was examined using patch-clamp methodology. Extracellular hypotonicity (210 or 135 mOsm) activated an outwardly rectifying, time-independent current with a reversal potential close to the calculated Cl(-) equilibrium potential (E(Cl)). The magnitude of this current was related to tonicity of the superfusate. The current was blocked by 0.5 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). These features are comparable to those of I(Cl(swell)) found in sinoatrial nodal, atrial, and ventricular myocytes. I(Cl(swell)) activation at 210 and 135 mOsm depolarized the resting membrane potential with 6 and 10 mV and shortened the action potential by approximately 18 and approximately 33%, respectively. DIDS partially reversed I(Cl(swell))-induced action potential changes. We conclude that I(Cl(swell)) is present in Purkinje cells and its activation leads to action potential shortening and resting membrane potential depolarization, both of which can promote the development of reentrant arrhythmias.

Impulse-conducting Purkinje fibers differentiate from myocytes during embryogenesis. The conversion of contractile myocytes into conduction cells is induced by the stretch/pressure-induced factor, endothelin (ET). Active ET is produced via proteolytic processing from its precursor by ET-converting enzyme 1 (ECE1) and triggers signaling by binding to its receptors. In the embryonic chick heart, ET receptors are expressed by all myocytes, but ECE1 is predominantly expressed in endothelial cells of coronary arteries and endocardium along which Purkinje fiber recruitment from myocytes takes place. Furthermore, co-expression of exogenous ECE1 and ET-precursor in the embryonic heart is sufficient to ectopically convert cardiomyocytes into Purkinje fibers. Thus, localized expression of ECE1 defines the site of Purkinje fiber recruitment in embryonic myocardium. However, it is not known how ECE1 expression is regulated in the embryonic heart. The unique expression pattern of ECE1 in the embryonic heart suggests that blood flow-induced stress/stretch may play a role in patterning ECE1 expression and subsequent induction of Purkinje fiber differentiation. We show that gadolinium, an antagonist for stretch-activated cation channels, downregulates the expression of ECE1 and a conduction cell marker, Cx40, in ventricular chambers, concurrently with delayed maturation of a ventricular conduction pathway. Conversely, pressure-overload in the ventricle by conotruncal banding results in a significant expansion of endocardial ECE1 expression and Cx40-positive putative Purkinje fibers. Coincident with this, an excitation pattern typical of the mature heart is precociously established. These in vivo data suggest that biomechanical forces acting on, and created by, the cardiovascular system during embryogenesis play a crucial role in Purkinje fiber induction and patterning.

Four dopamine receptor agonists used for the treatment of Parkinson's disease (apomorphine, pergolide, ropinirole and sumanirole) were evaluated for the ability to block human ether-a-go-go related gene (hERG) K(+) channels and to modify the duration of canine Purkinje fiber action potentials. Apomorphine, pergolide and ropinirole blocked the hERG-mediated currents with IC(50) values of 2.4, 0.12 and 1.2 microM, respectively. When evaluated in an action potential duration assay, pergolide significantly shortened action potential duration at 90% repolarization (APD(90)) whereas apomorphine and ropinirole significantly prolonged repolarization. Sumanirole only partially blocked hERG K(+) channels at the highest tested concentration (10 microM) and did not modify action potential duration over the tested concentration range (0.65-65 microM). Taken together, these data provide evidence that dopamine receptor agonists developed for the treatment of Parkinson's disease differentially influence hERG K(+) channel function and cardiac action potential duration.

Mutations in minK-related peptide 1 (MiRP1), the product of the KCNE2 gene, have been associated with malignant ventricular arrhythmia syndromes related to impaired repolarization. MiRP1 interacts with a variety of ion-channel alpha-subunits, dysfunction of which could account for arrhythmia syndromes; however, the observation of very low-level expression of MiRP1 in ventricular tissue has led to doubts about its relevance. The specialized His-Purkinje system plays a key role in cardiac electrophysiology and is an important contributor to ventricular arrhythmias related to abnormal repolarization. We examined the relative abundance of MiRP1 in canine Purkinje versus ventricular tissue and found much greater expression at both mRNA and protein levels in Purkinje tissue. Thus, the cardiac Purkinje system is a strong candidate to play a role in arrhythmic syndromes due to MiRP1 abnormalities.

Cardiac Purkinje fibers play an important role in cardiac arrhythmias, but no information is available about ionic currents in human cardiac Purkinje cells (PCs). PCs and midmyocardial ventricular myocytes (VMs) were isolated from explanted human hearts. K(+) currents were evaluated at 37 degrees C with whole cell patch clamp. PCs had clear inward rectifier K(+) current (I(K1)), with a density not significantly different from VMs between -110 and -20 mV. A Cs(+)-sensitive, time-dependent hyperpolarization-activated current was measurable negative to -60 mV. Transient outward current (I(to)) density was smaller, but end pulse sustained current (I(sus)) was larger, in PCs vs. VMs. I(to) recovery was substantially slower in PCs, leading to strong frequency dependence. Unlike VM I(to), which was unaffected by 10 mM tetraethylammonium, Purkinje I(to) was strongly inhibited by tetraethylammonium, and Purkinje I(to) was 10-fold more sensitive to 4-aminopyridine than VM. PC I(sus) was also reduced strongly by 10 mM tetraethylammonium. In conclusion, human PCs demonstrate a prominent I(K1), a time-dependent hyperpolarization-activated current, and an I(to) with pharmacological sensitivity and recovery kinetics different from those in the atrium or ventricle and compatible with a different molecular basis.

Although Purkinje fibers (PFs) play an important role in cardiac electrophysiology, almost nothing is known about the expression of ion-channel subunits in PFs. We applied competitive reverse transcription-polymerase chain reaction, Western blotting, and immunocytochemistry to compare the expression of ion-channel subunit mRNA and protein in canine PFs versus ventricular muscle (VM). For transient outward current-related subunits, Kv4.2 was not detected, and Kv1.4 expression was extremely low. Kv4.3 expression was of the same order for VM and PFs. The tetraethylammonium chloride-sensitive subunit Kv3.4 was expressed much more strongly in PFs than in VM, and Kv channel-interacting protein transcript expression was 25-fold stronger in VM than in PFs. For delayed rectifiers, ERG and KvLQT1 expression was lower in PFs at both mRNA and protein levels. Although minK transcripts were more numerous in PFs, minK protein was significantly more strongly expressed in VM. L-type Ca2+ current alpha-subunit (Ca(V)1.2) and Na+-Ca2+ exchanger proteins were more strongly expressed in VM than in PFs. For T-type Ca2+ current, Ca(V)3.1, Ca(V)3.2, and Ca(V)3.3 transcripts were all more strongly expressed in PFs. For the nonselective cation current, hyperpolarization-activated cation channel 1 (HCN1) expression was subquantifiable, HCN2 transcript expression was comparable in PFs and VM, and HCN4 mRNA expression was strong in PFs but below the detection threshold in VM. HCN2 and HCN4 protein expression was much stronger in PFs than in VM. We conclude that ion-channel subunit expression in PFs differs from that in VM in ways that are consistent with, and shed light on the molecular basis of, well-recognized fundamental PF ionic properties.

In avian, there are three slow skeletal myosin heavy chain (MHC) isoforms, slow skeletal MHC 1, 2, and 3. While slow skeletal MHC 3 has been characterized, slow skeletal MHC 1 and 2 are not yet fully studied. To determine the complete sequence of slow skeletal MHC 2, we isolated six overlapping cDNA clones, each encoding a portion of chick slow skeletal MHC 2, using the reverse transcription polymerase chain reaction (RT-PCR). The entire slow skeletal MHC 2 cDNA consisted of 5927 nucleotides including a 104 bp 3'-untranslated region and encoded 1941 amino acids. Using one of the cDNA clones, we made a probe for in situ hybridization. We also used immunohistochemistry to localize slow skeletal MHC 2 in skeletal and cardiac tissues. These studies showed that in addition to its expected expression in the adult chicken slow skeletal muscle, slow skeletal MHC 2 was expressed in the subendocardial cluster of cells and around the blood vessels within the ventricle of late embryos and adults. This isoform was not expressed in the myocardium throughout the life of the chicken. Based on morphological criteria as well as rich desmin expression, we concluded that the subendocardial cluster of cells were Purkinje cells. Although the physiological significance of the slow skeletal MHC expression remains elusive at this time, this MHC isoform may be used as a specific marker for Purkinje cells.

Background: Cardiac Purkinje cells (PCs) are important for the generation of triggered arrhythmias, particularly in association with abnormal repolarization. The effects of congestive heart failure (CHF) on the ionic properties of PCs are unknown., Methods and Results: PCs were isolated from false tendons of control dogs and dogs with ventricular tachypacing-induced CHF. CHF PCs were hypertrophied (capacitance, mean+/-SEM, 149+/-4 pF, n=130; versus 128+/-3 pF, n=150, control; P<0.001). Transient outward current density was reduced in CHF PCs without change in voltage dependence or kinetics. CHF also reduced inward-rectifier current density, with no change in form of the current-voltage relationship. Densities of L- and T-type calcium, rapid and slow delayed rectifier, and Na(+)-Ca(2+) exchange currents were unaltered by CHF, but L-type calcium current inactivation was slowed at positive potentials. Purkinje fiber action potentials from CHF dogs showed decreased phase 1 amplitudes and elevated plateau voltages and demonstrated twice as much prolongation on exposure to the rapid delayed rectifier blocker E-4031 as control Purkinje fibers., Conclusions: CHF causes remodeling of important K(+) and Ca(2+) currents in cardiac PCs, decreasing repolarization reserve and causing an exaggerated repolarization delay in response to a class III drug. These results have important potential implications regarding ventricular arrhythmogenesis, particularly related to triggered activity in PCs, in patients with CHF.

Cells with high and fluctuating energy demands such as cardiomyocytes need efficient systems to link energy production to energy utilization. This is achieved in part by compartmentalized energy transfer enzymes such as creatine kinase (CK). However, hearts from CK-deficient mice develop normal cardiac function under conditions of moderate workload. We have therefore investigated whether a direct functional interplay exists between mitochondria and sarcoplasmic reticulum or between mitochondria and myofilaments in cardiac cells that catalyzes direct energy and signal channeling between organelles. We used the selective permeabilization of sarcolemmal membranes with saponin to study the functional interactions between organelles within the cellular architecture. We measured contractile kinetics, oxygen consumption, and caffeine-induced tension transients. The results show that in hearts of normal mice, ATP produced by mitochondria (supplied with substrates, oxygen, and adenine nucleotides) was able to sustain calcium uptake and contractile speed. Moreover, direct mitochondrially supplied ATP was nearly as effective as CK-supplied ATP and much more effective than externally supplied ATP, suggesting that a direct ATP/ADP channeling exists between the sites of energy production (mitochondria) and energy utilization (sarcoplasmic reticulum and myofilaments). On the other hand, in cardiac cells of mice deficient in mitochondrial and cytosolic CK, marked cytoarchitectural modifications were observed, and direct adenine nucleotide channeling between mitochondria and organelles was still effective for sarcoplasmic reticulum and myofilaments. Such direct crosstalk between organelles may explain the preserved cardiac function of CK-deficient mice under moderate workloads.

A number of different phenotypes emerge from the mesoderm-derived cardiomyogenic cells of the embryonic tubular heart, including those comprising the cardiac conduction system. The transcriptional regulation of this phenotypic divergence within the cardiomyogenic lineage remains poorly characterized. A relationship between expression of the transcription factor Nkx-2.5 and patterning to form cardiogenic mesoderm subsequent to gastrulation is well established. Nkx-2.5 mRNA continues to be expressed in myocardium beyond the looped, tubular heart stage. To investigate the role of Nkx-2.5 in later development, we have determined the expression pattern of Nkx-2.5 mRNA by in situ hybridization in embryonic chick, fetal mouse, and human hearts, and of Nkx-2.5 protein by immunolocalization in the embryonic chick heart. As development progresses, significant nonuniformities emerge in Nkx-2.5 expression levels. Relative to surrounding force-generating ("working") myocardium, elevated Nkx-2.5 mRNA signal becomes apparent in the specialized cells of the conduction system. Similar differences are found in developing chick, human, and mouse fetal hearts, and nuclear-localized Nkx-2.5 protein is prominently expressed in differentiating chick conduction cells relative to adjacent working myocytes. This tissue-restricted expression of Nkx-2.5 is transient and correlates with the timing of spatio-temporal recruitment of cells to the central and the peripheral conduction system. Our data represent the first report of a transcription factor showing a stage-dependent restriction to different parts of the developing conduction system, and suggest some commonality in this development between birds and mammals. This dynamic pattern of expression is consistent with the hypothesis that Nkx-2.5, and its level of expression, have a role in regulation and/or maintenance of specialized fate selection by embryonic myocardial cells., (Copyright 2001 Wiley-Liss, Inc.)

The intramural dynamics of ventricular fibrillation (VF) remain poorly understood. Recent investigations have suggested that stable intramural reentry may underlie the mechanisms of VF. We performed optical mapping studies of VF in isolated swine right ventricles (RVs) and left ventricles (LVs). Nine RV walls were cut obliquely in their distal edge exposing the transmural surface. Six LV wedge preparations were also studied. Results showed that intramural reentry was present. In RV, 28 of 44 VF episodes showed reentry; 15% of the activation pathways were reentrant. Except for 4 episodes, reentry was transmural, involving subendocardial structures as the papillary muscle (PM) or trabeculae. In LV, reentry was observed in 27 of 27 VF episodes; 23% of the activations were part of reentrant pathways (P<0.05 compared with RV). All LV reentrant pathways were truly intramural (confined to the wall) and were frequently located at the PM insertion. In both ventricles, reentry was spatially and temporally unstable. Histological studies showed abrupt changes in fiber orientation at sites of reentry and wave splitting. Connexin 40 immunostaining demonstrated intramyocardial Purkinje fibers at sites of reentry in the PM root and around endocardial trabeculae. Our results confirm that reentry is frequent-but unstable-in the myocardial wall during VF. In RV, reentry is mostly transmural and requires participation of subendocardial structures. The LV has a greater incidence of reentry and is intramural. Anisotropic anatomic structures played key roles in the generation of wave splitting and in the maintenance of reentry.

Recent studies have shown the presence of postjunctional alpha(2)-adrenergic receptors on canine Purkinje fibers but not muscle cells. Stimulation of these receptors results in prolongation of the action potential duration and the Purkinje relative refractory period. We studied the effect of alpha(2)-adrenergic agonists on inducible ischemic ventricular tachycardia (VT) of both Purkinje fiber and myocardial origin. Open-chest dogs in whom VT was induced with extrastimuli after occlusion of the anterior descending coronary artery were studied. A mapping system, incorporating Purkinje signals, characterized the mechanisms of VT. The alpha(2)-adrenergic agonists clonidine (0.5-4.0 microg/kg) or UK 14,304 (4-5 microg/kg) versus saline were given intravenously after reproducibility of inducible sustained monomorphic VT had been demonstrated. Eighteen dogs were given clonidine, eleven of which had focal Purkinje VT. Of these 11 dogs, clonidine blocked VT induction in 9 (81.9%) and rendered VT nonsustained in 1 (9.1%), and VT remained inducible in 1 dog (9.1%), although this was focal midmyocardial VT only. In the seven dogs with VT of myocardial origin, six (85.6%) remained inducible with clonidine, whereas one dog (14.4%) had only nonsustained VT after clonidine. Of the six dogs, UK 14,304 blocked VT induction in four (66.6%) and rendered VT nonsustained in one (16.7%), and VT remained inducible in one dog (16.7%). In four dogs with VT of myocardial origin, VT remained inducible. In the eight control dogs that were given saline, focal Purkinje VT was repeatedly inducible. Pharmacological stimulation of postjunctional alpha(2)-adrenoceptors on Purkinje fibers may selectively prevent induction of VT of Purkinje fiber origin in the ischemic canine ventricle.

Objectives: To study the blocking effects of H 345/52 on ionic currents of rabbit ventricular myocytes and how these features translate into a proarrhythmic potential., Methods: The single electrode voltage clamp technique was used to study the effects of H 345/52 on the rapid component of the delayed rectifying potassium current, I(Kr), and the L-type calcium current (I(Ca)). Differential effects of H 345/52 and almokalant on APD prolongation were studied in a rabbit Purkinje fibre/ventricular muscle preparation. The temporal variability of the action potential duration (APD) and its relation to proarrhythmias was examined in Langendorff-perfused rabbit hearts administered H 345/52 or almokalant. Anaesthetised, methoxamine-sensitised rabbits were used to assess the propensity of intravenous H 345/52 and ibutilide to induce torsades de pointes (TdP)., Results: H 345/52 potently blocked I(Kr) (IC(50)=40 nM) without consequential use-dependency. The I(Ca) was also blocked, but at higher concentrations (IC(50)=1.3 microM). Block of I(Ca) was markedly frequency-dependent (positive) and influenced by membrane potential, such that H 345/52 was more effective following clamp steps from plateau potentials than from -80 mV. In the Purkinje fibre-ventricular muscle preparation, almokalant prolonged the Purkinje fibre APD preferentially, whereas H 345/52 homogeneously prolonged APD in both tissue types. In the perfused rabbit heart, H 345/52 (1 microM) and almokalant (0.3 microM) prolonged APD to a similar degree but increased the temporal variability of APD differently, from 3+/-0.4 ms in control hearts to 8+/-1.2 ms and to 38+/-7.5 ms (P<0.001 vs. H 345/52), respectively. Unequivocal early after-depolarisations were seen in 5/6 almokalant-perfused hearts but in no heart administered H 345/52 (P<0.05). In anaesthetised rabbits, H 345/52 (17.4 micromol/kg) or ibutilide (2.6 micromol/kg maximum), maximally lengthened the QT interval from 133+/-4.5 to 177+/-8.0 ms and from 125+/-5.1 to 166+/-9.3 ms (P<0.001, n=8). However, whereas ibutilide induced TdP in all animals at 0.06+/-0.009 micromol/kg, H 345/52 did not induce TdP (P=0.0002) at up to 17.4 micromol/kg., Conclusions: H 345/52 blocks I(Kr) with high potency and I(Ca) with somewhat lower potency and was found to delay ventricular repolarisation without substantially increasing temporal or spatial dispersion and without inducing early after-depolarisations or TdP.

Connexin40 (Cx40) is a major gap junction protein that is expressed in the His-Purkinje system and thought to be a critical determinant of cell-to-cell communication and conduction of electrical impulses. Video maps of the ventricular epicardium and the proximal segment of the right bundle branch (RBB) were obtained using a high-speed CCD camera while simultaneously recording volume-conducted ECGs. In Cx40(-/-) mice, the PR interval was prolonged (47.4+/-1.4 in wild-type [WT] [n=6] and 57.5+/-2.8 in Cx40(-/-) [n=6]; P<0.01). WT ventricular epicardial activation was characterized by focused breakthroughs that originated first on the right ventricle (RV) and then the left ventricle (LV). In Cx40(-/-) hearts, the RV breakthrough occurred after the LV breakthrough. Additionally, Cx40(-/-) mice showed RV breakthrough times that were significantly delayed with respect to QRS complex onset (3.7+/-0.7 ms in WT [n=6] and 6.5+/-0.7 ms in Cx40(-/-) [n=6]; P<0.01), whereas LV breakthrough times did not change. Conduction velocity measurements from optical mapping of the RBB revealed slow conduction in Cx40(-/-) mice (74.5+/-3 cm/s in WT [n=7] and 43.7+/-6 cm/s in Cx40(-/-) [n=7]; P<0.01). In addition, simultaneous ECG records demonstrated significant delays in Cx40(-/-) RBB activation time with respect to P time (P-RBB time; 41.6+/-1.9 ms in WT [n=7] and 55.1+/-1.3 ms in [n=7]; P<0.01). These data represent the first direct demonstration of conduction defects in the specialized conduction system of Cx40(-/-) mice and provide new insight into the role of gap junctions in cardiac impulse propagation.

HF-1 b, an SP1 -related transcription factor, is preferentially expressed in the cardiac conduction system and ventricular myocytes in the heart. Mice deficient for HF-1 b survive to term and exhibit normal cardiac structure and function but display sudden cardiac death and a complete penetrance of conduction system defects, including spontaneous ventricular tachycardia and a high incidence of AV block. Continuous electrocardiographic recordings clearly documented cardiac arrhythmogenesis as the cause of death. Single-cell analysis revealed an anatomic substrate for arrhythmogenesis, including a decrease and mislocalization of connexins and a marked increase in action potential heterogeneity. Two independent markers reveal defects in the formation of ventricular Purkinje fibers. These studies identify a novel genetic pathway for sudden cardiac death via defects in the transition between ventricular and conduction system cell lineages.

It is becoming clear that mutations in the KVLQT1, human "ether-a-go-go" related gene, cardiac voltage-dependent sodium channel gene, minK and MiRP1 genes, respectively, are responsible for the LQT1, LQT2, LQT3, LQT5 and LQT6 variants of the Romano-Ward syndrome, characterized by autosomal dominant transmission and no deafness. The much rarer Jervell-Lange-Nielsen syndrome (with marked QT prolongation and sensorineural deafness) arises when a child inherits mutant KVLQT1 or minK alleles from both parents. In addition, some families are not linked to the known genetic loci. Cardiac voltage-dependent sodium channel gene encodes the cardiac sodium channel, and long QT syndrome (LQTS) mutations prolong action potentials by increasing inward plateau sodium current. The other mutations cause a decrease in net repolarizing current by reducing potassium currents through "dominant negative" or "loss of function" mechanisms. Polymorphic ventricular tachycardia (torsade de pointes) is thought to be initiated by early after-depolarizations in the Purkinje system and maintained by reentry in the myocardium. Clinical presentations vary with the specific gene affected and the specific mutation. Nevertheless, patients with identical mutations can also present differently, and some patients with LQTS mutations may have no manifest baseline phenotype. The question of whether the latter situation is one of high risk for administration of QT prolonging drugs or during myocardial ischemia is under active investigation. More generally, the identification of LQTS genes has provided tremendous new insights for our understanding of normal cardiac electrophysiology and its perturbation in a wide range of conditions associated with sudden death. It seems likely that the approach of applying information from the genetics of uncommon congenital syndromes to the study of common acquired diseases will be an increasingly important one in the next millennium.

Background: The ionic mechanism underlying the transient inward current (I(ti)), the current responsible for delayed afterdepolarizations (DADs), appears to be different in ventricular myocytes and Purkinje fibers. In ventricular myocytes, I(ti) was ascribed to a Na(+)-Ca(2+) exchange current, whereas in Purkinje fibers, it was additionally ascribed to a Cl(-) current and a nonselective cation current. If Cl(-) current contributes to I(ti) and thus to DADs, Cl(-) current blockade may be potentially antiarrhythmogenic. In this study, we investigated the ionic nature of I(ti) in single sheep Purkinje and ventricular myocytes and the effects of Cl(-) current blockade on DADs., Methods and Results: In whole-cell patch-clamp experiments, I(ti) was induced by repetitive depolarizations from -93 to +37 mV in the presence of 1 micromol/L norepinephrine. In both Purkinje and ventricular myocytes, I(ti) was inward at negative potentials and outward at positive potentials. The anion blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) blocked outward I(ti) completely but inward I(ti) only slightly. The DIDS-sensitive component of I(ti) was outwardly rectifying, with a reversal close to the reversal potential of Cl(-) currents. Blockade of Na(+)-Ca(2+) exchange by substitution of extracellular Na(+) by equimolar Li(+) abolished the DIDS-insensitive component of I(ti). DIDS reduced both DAD amplitude and triggered activity based on DADs. Conclusions-In both Purkinje and ventricular myocytes, I(ti) consists of 2 ionic mechanisms: a Cl(-) current and a Na(+)-Ca(2+) exchange current. Blockade of the Cl(-) current may be potentially antiarrhythmogenic by lowering DAD amplitude and triggered activity based on DADs.

Purkinje cells contain sarcoplasmic reticulum (SR) directly under the surface membrane, are devoid of t-tubuli, and are packed with myofibrils surrounded by central SR. Several studies have reported that electrical excitation induces a biphasic Ca(2+) transient in Purkinje fiber bundles. We determined the nature of the biphasic Ca(2+) transient in aggregates of Purkinje cells. Aggregates (n=12) were dispersed from the subendocardial Purkinje fiber network of normal canine left ventricle, loaded with Fluo-3/AM, and studied in normal Tyrode's solution (24 degrees C). Membrane action potentials were recorded with fine-tipped microelectrodes, and spatial and temporal changes in [Ca(2+)](i) were obtained from fluorescent images with an epifluorescent microscope (x20; Nikon). Electrical stimulation elicited an action potential as well as a sudden increase in fluorescence (L(0)) compared with resting levels. This was followed by a further increase in fluorescence (L(1)) along the edges of the cells. Fluorescence then progressed toward the Purkinje cell core (velocity of propagation 180 to 313 microm/s). In 62% of the aggregates, initial fluorescent changes of L(0) were followed by focally arising Ca(2+) waves (L(2)), which propagated at 158+/-14 microm/s (n=13). Spontaneous Ca(2+) waves (L(2)*) propagated like L(2) (164+/-10 microm/s) occurred between stimuli and caused slow membrane depolarization; 28% of L(2)* elicited action potentials. Both spontaneous Ca(2+) wave propagation and resulting membrane depolarization were thapsigargin sensitive. Early afterdepolarizations were not accompanied by Ca(2+) waves. Action potentials in Purkinje aggregates induced a rapid rise of Ca(2+) through I(CaL) and release from a subsarcolemmal compartment (L(0)). Ca(2+) release during L(0) either induced further Ca(2+) release, which propagated toward the cell core (L(1)), or initiated Ca(2+) release from small regions and caused L(2) Ca(2+) waves, which propagated throughout the aggregate. Spontaneous Ca(2+) waves (L(2)*) induce action potentials.

Objective: Our goal was to identify the ATP-sensitive potassium (KATP) channels in cardiac Purkinje cells and to document the functional properties that might distinguish them from KATP channels in other parts of the heart., Methods: Single Purkinje cells and ventricular myocytes were isolated from rabbit heart. Standard patch-clamp techniques were used to record action potential waveforms. and whole-cell and single-channel currents., Results: The KATP channel opener levcromakalim (10 microM) caused marked shortening of the Purkinje cell action potential. Under whole-cell voltage-clamp, levcromakalim induced an outward current, which was blocked by glibenclamide (5 microM), in both Purkinje cells and ventricular myocytes. Metabolic poisoning of Purkinje cells with NaCN and 2-deoxyglucose caused a significant shortening of the action potential (control 376 +/- 51 ms; 6 min NaCN/2-deoxyglucose 153 +/- 21 ms). This effect was reversed with the application of glibenclamide. Inside-out membrane patches from Purkinje cells showed unitary current fluctuations which were inhibited by cytoplasmic ATP with an IC50 of 119 microM and a Hill coefficient of 2.1. This reflects approximately five-fold lower sensitivity to ATP inhibition than for KATP channels from ventricular myocytes under the same conditions. The slope conductance of Purkinje cell KATP channels, with symmetric, 140 mM K+, was 60.1 +/- 2.0 pS (mean +/- SEM). Single-channel fluctuations showed mean open and closed times of 3.6 +/- 1.5 ms and 0.41 +/- 0.2 ms, respectively, at -60 mV and approximately 21 degrees C. At positive potentials. KATP channels exhibited weak inward rectification that was dependent on the concentration of internal Mg2+. Computer simulations, based on the above results, predict significant shortening of the Purkinje cell action potential via activation of KATP channels in the range 1-5 mM cytoplasmic ATP., Conclusions: Purkinje cell KATP channels may represent a molecular isoform distinct from that present in ventricular myocytes. The presence of KATP channels in the Purkinje network suggests that they may have an important influence on cardiac rhythm and conduction during periods of ischemia.

Abnormal dystrophin expression is directly responsible for Duchenne and Becker muscular dystrophies. In skeletal muscle, dystrophin provides a link between the actin network and the extracellular matrix via the dystrophin-associated protein complex. In mature skeletal muscle, utrophin is a dystrophin-related protein localized mainly at the neuromuscular junction, with the same properties as dystrophin in terms of linking the protein complex. Utrophin could potentially overcome the absence of dystrophin in dystrophic skeletal muscles. In cardiac muscle, dystrophin and utrophin were both found to be present with a distinct subcellular distribution in Purkinje fibres, i.e. utrophin was limited to the cytoplasm, while dystrophin was located in the cytoplasmic membrane. In this study, we used this particular characteristic of cardiac Purkinje fibres and demonstrated that associated proteins of dystrophin and utrophin are different in this structure. We conclude, contrary to skeletal muscle, dystrophin-associated proteins do not form a complex in Purkinje fibres. In addition, we have indirect evidence of the presence of two different 400 kDa dystrophins in Purkinje fibres.

In this study, the effects of carbachol (CCh) on twitch tension, intracellular Na+ activity (aiNa), and action potential were simultaneously measured in canine cardiac Purkinje fibers in order to examine the regulation of inotropy through muscarinic receptors and its relation to aiNa. In fibers driven at 1 Hz, CCh (10 microM) initially and transiently decreased and then increased the twitch tension by 36 +/- 8%. The action potential showed a significant elevation of the plateau and a significant shortening of the duration at 90% repolarization (APD90), from 403 +/- 7 to 389 +/- 7 ms. The aiNa decreased from 7.4 +/- 0.4 to 6.7 +/- 0.3 mM (n = 23, p < 0.05). Atropine (1 microM) decreased the twitch tension by 21 +/- 6% (n = 7, p < 0.05) without significant effects on the action potential and aiNa, and inhibited the effects of CCh. Cs+ (20 mM) increased the plateau height and APD90, enhanced the twitch tension by 66 +/- 24%, but decreased aiNa from 7.3 +/- 0.3 to 6.3 +/- 0.4 mM (n = 6, p < 0.05). In the presence of 20 mM Cs+, some fibers generated slow responses. The addition of 10 microM CCh further increased the twitch tension and APD90, and decreased aiNa from 6.3 +/- 0.4 to 5.3 +/- 0.3 mM. Ouabain (0.3 microM) increased the twitch tension and aiNa, and inhibited the CCh-induced decrease of aiNa. In the presence of ouabain, 20 mM Cs+ depolarized the fiber and generated slow responses with a decreased aiNa. The addition of 10 microM CCh enhanced the slow action potential, and increased aiNa although there was a transient decrease during early exposure. These results suggest that activation of muscarinic receptors in canine Purkinje fibers results in an enhancement of the Na+-K+ pump activity and a biphasic inotropic response, probably via different receptor subtypes. The inhibitory effect, most likely through M2 receptors, is associated with the activation of K+ channels. The stimulatory effect, on the other hand, is probably due to the action on the M1 receptors, resulting in increases in Ca2+ currents.

1. Capsinolol (1.0-30.0 microM) in a cumulating manner decreased the maximum upstroke velocity (Vmax), the action potential amplitude and twitch tension in isolated guinea-pig atria and papillary muscle, rabbit papillary muscle, dog Purkinje fibers and human ventricle tissues. 2. In the isolated guinea-pig atrium, perfusing with capsinolol at 3 microM for 3 min temporarily increased the twitch force and decreased spontaneous cycle length; however, the results were reversed after longer exposure of the tissue. 3. Capsinolol prolonged the duration of action potential in the guinea-pig atrium and rabbit papillary muscles. The maximum diastolic potential was shifted to a less-negative level in dog Purkinje fibers and human ventricular muscles.

Atrial natriuretic peptide has frequently been detected in the cardiac conduction system and has been shown to regulate some intracellular effects in Purkinje fibers. To determine if atrial natriuretic peptide works as an autocrine and/or paracrine hormone on cardiac Purkinje fibers, we examined the different parts of the conduction system in the bovine heart by use of in vitro receptor autoradiography. In no parts of the bovine conduction system were specific binding sites for [125I] atrial natriuretic peptide observed, whereas the ventricular myocardium exhibited a large number of [125I] atrial natriuretic peptide binding sites. This is the first morphologic study showing the presence of [125I] atrial natriuretic peptide binding sites in the ventricular myocardium and their absence in the conduction system. The present observations together with results obtained in studies using other methods strongly suggest that natriuretic peptide receptors are localized on ventricular myocytes.

Heart contraction is coordinated by conduction of electrical excitation through specialized tissues of the cardiac conduction system. By retroviral single-cell tagging and lineage analyses in the embryonic chicken heart, we have recently demonstrated that a subset of cardiac muscle cells terminally differentiates as cells of the peripheral conduction system (Purkinje fibers) and that this occurs invariably in perivascular regions of developing coronary arteries. Cis regulatory elements that function in transcriptional regulation of cells in the conducting system have been distinguished from those in contractile cardiac muscle cells; eg, 5' regulatory sequences of the desmin gene act as enhancer elements in skeletal muscle and in the conduction system but not in cardiac muscle. We hypothesize that Purkinje fiber differentiation involves a switch of the gene expression program from that characteristic of cardiac muscle to one typical of skeletal muscle. To test this hypothesis, we examined the expression of myosin binding protein-H (MyBP-H) in Purkinje fibers of chicken hearts. This unique myosin binding protein is present in skeletal but not cardiac myocytes. A site-directed polyclonal antibody (AB105) was generated against MyBP-H. Immunohistological analysis of the myocardium mapped the AB105 antigen predominantly to A bands of myofibrils within Purkinje fibers. Western blot analysis of whole extracts from the ventricular wall of adult chicken hearts revealed that the AB105 epitope was restricted to a single protein of approximately 86 kD, the same size as MyBP-H in skeletal muscle. Biochemical properties of the Purkinje fiber 86-kD protein and RNase protection analyses of its mRNA indicate that Purkinje fiber 86-kD protein is indistinguishable from skeletal muscle MyBP-H. The results provide evidence that skeletal muscle MyBP-H is expressed in a subset of cardiac muscle cells that differentiate into Purkinje fibers of the heart.

Substances of abuse exert adrenergic and/or depressant actions on the cellular processes responsible for cytosolic calcium overload. This investigation attempted to determine whether substances of abuse, through catechol-mediated effects or cellular actions, elicit or inhibit the production of arrhythmias caused by delayed afterdepolarizations (DADs) and triggered activity (TA). The papillary muscles of rats and Purkinje fibers of dogs were superfused in vitro with Tyrode's solution at 37 degrees C. Intracellular microelectrodes were used to record membrane potentials. Overdrives failed to induce DADs and TA in the canine Purkinje fibers exposed to either Tyrode's solution alone, or containing ethanol or harmine. Instead, ethanol and harmine inhibited DADs and TA induced by overdrives in the presence of strophanthidin. On the contrary, in the presence of acetaldehyde and amphetamine, overdrives did produce TA, which was inhibited by propranolol. In conclusion, substances of abuse may either elicit or inhibit the production of DADs and TA, depending on the balance between adrenergic and depressant actions on the cellular mechanisms responsible for the calcium overload of the cytosol.

The interaction between Na+ and H+ ions upon Na-H exchange (NHE) was examined in sheep cardiac Purkinje fibers. Acid equivalent fluxes through NHE were examined using recordings of intracellular pH and Na+ in isolated preparations measured with ion selective microelectrodes. The extent of acid-extrusion by NHE was estimated from pH(i) recovery-rate, multiplied by beta(i) (intracellular buffering power) in response to an internal acid load induced by 20 mm NH4Cl removal (nominally HCO3- free media). A mixed inhibitory effect was found of extracellular H+ on external Na+-activation of NHE (i.e. an increase, at low pH(o), in the apparent Michaelis constant for external Na+ ions [K(Nao)(0.5)] and a decrease in the maximum transport rate [V(Nao)(max)]). In addition, we confirmed that the stoichiometry of Na(o) binding is unaffected by the pH(o) (between 7.5 and 6.5), showing a Hill coefficient close to one. The interaction between Na+ and H+ ions at the internal face of the cardiac NHE was also studied. Our evidence suggests that an increase in the intracellular Na+ ion concentration ([Na+]i) inhibits acid efflux and that this inhibition can be approximated by the decrease in thermodynamic driving force caused by reducing the transmembrane Na+ gradient. It appears, however, that small variations in [Na+]i from the normal resting level (intracellular sodium activity, a(i)Na = 7 to 13 mm) have little or no effect on acid efflux, suggesting that variation of a(i)Na is not a physiologically important controller of NHE activity in heart.

We used standard microelectrode techniques to study the developmental changes and beta-adrenergic modulation of membrane potential and of Na-K pump activity in adult (> 1 yr of age) and neonatal (2-10 days) canine Purkinje fibers. Isoproterenol (10(-7) M) increased the rate of development and magnitude of pacing-induced hyperpolarization of adult fibers driven at a 1-s cycle length. This effect of isoproterenol was attenuated by treating dogs with pertussis toxin (PTX), (30 micrograms/kg). Other adult and neonatal fibers were superfused with a Tyrode solution containing Ba2+ 0.2 mM, Cs+ 2 mM, and 10(-6) M verapamil, thus leading to depolarization and cessation of spontaneous activity. The Na-K pump was studied by alternating solutions containing [K] at 0 mM (inhibiting the pump) and 4 mM (reactivating the pump). Although the kinetics of the Na-K pump appeared faster in neonatal fibers than in adult fibers, measurement of cell surface-to-volume ratio compensated for the difference. We therefore conclude that 1) the apparent age-related changes in Na-K pump activity in canine Purkinje fibers in fact reflect cell surface-to-volume ratio and, 2) the beta-adrenergic agonist-induced hyperpolarization in adults requires the presence of a PTX-sensitive G protein for its occurrence.

Aim: To explain the effect of daurisoline (DS) on delayed afterdepolarization (DAD)., Methods: Ca(2+)-sensitive microelectrode technic was used to record intracellular Ca2+ activity (alpha Cai) and triggered activity (TA) arising from DAD in myocardium., Results: Strophantin G 3 mumol.L-1 yielded an increase in resting myocardial alpha Cai by 0.19 +/- 0.11 mumol.L-1 and transient elevations of alpha Cai by 1.48 +/- 0.55 and 4.96 +/- 1.81 mumol.L-1, respectively during the development of DAD and TA. By pretreatment with DS or verapamil, strophantin G-caused elevations of the alpha Cai in resting and provoked myocardia were eliminated and TA disappeared. DS 50 mumol.L-1 reduced Na(+)-free medium-induced elevation of dog Purkinje fibrous alpha Cai and abolished caffeine-induced increase of dog myocardial alpha Cai., Conclusions: DS inhibited DAD and TA by preventing an increase of alpha Cai via transmembrane Ca2+ entry and Ca2+ release from the reticulum.

Autoradiograms were developed from free running strands of Purkinje fibers and from left ventricular myocardium of dog hearts exposed to [7-methoxy-3H]prazosin or [O-methyl-3H]yohimbine in the presence or absence of excessive non-radioactive ligands. Both Purkinje fiber and ventricular myocardium show high density, tissue-specific binding to [3H]prazosin. In contrast, high density, tissue-specific binding to [3H]yohimbine was present in Purkinje fibers but not in ventricular myocardium. Membrane fractions showed high affinity, saturable, and displaceable binding with [3H]yohimbine in preparations from canine cardiac Purkinje fibers but not those from canine cardiac ventricular myocardium. Scatchard analysis of the canine Purkinje membrane alpha 2-adrenergic receptor binding showed a Bmax of 54.9 fmol/mg protein with a Kd of 6.25 nM. These results confirm the electrophysiological findings that post-junctional alpha 2-adrenergic receptors are present in Purkinje fibers.

The many routes by which Li can cross the cardiac sarcolemma were examined using Li-selective microelectrodes. In the presence of 70 mmol/l extracellular Li there was a rapid increase in intracellular Li activity (aiLi) equivalent to an influx of 0.45 mmol/l/min (or 2.1 pmol/cm2.s). Depolarisation (voltage-clamp or high [K]O) had no significant effect on the rate of aiLi increase, or recovery on removal of LiO. Following addition of 20 mmol/l [Li]O to the normal Tyrode, the rate of aiLi increase was stimulated by 49.7% by removal of extracellular Mg. However Li entry was inhibited by SITS (100 mumol/l) by 20.6%; tetrodotoxin (10(15)g/ml) 20.0%; caesium (2 mmol/l) 22.5%; verapamil (20 mumol/l) 14.3%; and manganese (1 mmol/l) 37.6%. With 5 mmol/l [Li]O, aiLi stabilized at 2.3 mmol/l, i.e. much lower than expected assuming passive distribution of Li (predicted level 61 mmol/l), implying active extrusion of Li from the cells. This stable level of Li was not significantly affected by short exposures to strophanthidin, bumetanide, or ethyl isopropylamiloride, but was decreased by increasing the [Ca]O or adding manganese. The aiLi was increased on removal of [Na]O or addition of phloretin (100 mumol/l) suggesting that NaO-Lii exchange helps to maintain a low aiLi. The removal of KO produced an increase in aiLi. This effect was inhibited by strophanthidin, suggesting Li can enter via the Na/K pump in K-free conditions.

The gating charge and voltage dependence of the open state to the inactivated state (O-->I) transition was measured for the voltage-dependent mammalian cardiac Na channel. Using the site 3 toxin, Anthopleurin-A (Ap-A), which selectively modifies the O-->I transition (see Hanck, D. A., and M. F. Sheets. 1995. Journal of General Physiology. 106:601-616), we studied Na channel gating currents (Ig) in voltage-clamped single canine cardiac Purkinje cells at approximately 12 degrees C. Comparison of Ig recorded in response to step depolarizations before and after modification by Ap-A toxin showed that toxin-modified gating currents decayed faster and had decreased initial amplitudes. The predominate change in the charge-voltage (Q-V) relationship was a reduction in gating charge at positive potentials such that Qmax was reduced by 33%, and the difference between charge measured in Ap-A toxin and in control represented the gating charge associated with Na channels undergoing inactivation by O-->I. By comparing the time course of channel activation (represented by the gating charge measured in Ap-A toxin) and gating charge associated with the O-->I transition (difference between control and Ap-A charge), the influence of activation on the time course of inactivation could be accounted for and the inherent voltage dependence of the O-->I transition determined. The O-->I transition for cardiac Na channels had a valence of 0.75 e-. The total charge of the cardiac voltage-gated Na channel was estimated to be 5 e-. Because charge is concentrated near the opening transition for this isoform of the channel, the time constant of the O-->I transition at 0 mV could also be estimated (0.53 ms, approximately 12 degrees C). Prediction of the mean channel open time-voltage relationship based upon the magnitude and valence of the O-->C and O-->I rate constants from INa and Ig data matched data previously reported from single Na channel studies in heart at the same temperature.