Protease XIV abolishes NHE inhibition by empagliflozin in cardiac cells.

Background: SGLT2i directly inhibit the cardiac sodium-hydrogen exchanger-1 (NHE1) in isolated ventricular cardiomyocytes (CMs). However, other studies with SGLT2i have yielded conflicting results. This may be explained by methodological factors including cell isolation techniques, cell types and ambient pH. In this study, we tested whether the use of protease XIV (PXIV) may abrogate inhibition of SGLT2i on cardiac NHE1 activity in isolated rabbit CMs or rat cardiomyoblast cells (H9c2), in a pH dependent manner. Methods: Rabbit ventricular CMs were enzymatically isolated from Langendorff-perfused hearts during a 30-min perfusion period followed by a 25-min after-dissociation period, using a collagenase mixture without or with a low dose PXIV (0.009 mg/mL) present for different periods. Empagliflozin (EMPA) inhibition on NHE activity was then assessed at pH of 7.0, 7.2 and 7.4. In addition, effects of 10 min PXIV treatment were also evaluated in H9c2 cells for EMPA and cariporide NHE inhibition. Results: EMPA reduced NHE activity in rabbit CMs that were not exposed to PXIV treatment or undergoing a 35-min PXIV treatment, independent of pH levels. However, when exposure time to PXIV was extended to 55 min, NHE inhibition by Empa was completely abolished at all three pH levels. In H9c2 cells, NHE inhibition by EMPA was evident in non-treated cells but lost after 10-min incubation with PXIV. NHE inhibition by cariporide was unaffected by PXIV. Conclusion: The use of protease XIV in cardiac cell isolation procedures obliterates the inhibitory effects of SGLT2i on NHE1 activity in isolated cardiac cells, independent of pH.

MicroRNAs in extracellular vesicles released from epicardial adipose tissue promote arrhythmogenic conduction slowing.

Background: Patients with excess epicardial adipose tissue (EAT) are at increased risk of developing cardiac arrhythmias. EAT promotes arrhythmias by depolarizing the resting membrane of cardiomyocytes, which slows down conduction and facilitates re-entrant arrhythmias. We hypothesized that EAT slows conduction by secreting extracellular vesicles (EVs) and their microRNA (miRNA) cargo. Objective: We aimed to determine the role of EAT-derived EVs and their miRNA cargo in conduction slowing. Methods: EAT and subcutaneous adipose tissue (SAT) were collected from patients with atrial fibrillation. Adipose tissue explants were incubated in culture medium and secretome was collected. The numbers of EVs in the EAT and SAT secretome were measured by calibrated flow cytometry. EVs in the EAT secretome were isolated by size exclusion chromatography and miRNAs were sequenced. Pathway analysis was performed to predict candidates involved in cardiac electrophysiology. The candidates were validated in the EAT and SAT by quantitative real-time polymerase chain reaction. Finally, miRNA candidates were overexpressed in neonatal rat ventricular myocytes. Results: The EV concentration was higher in the EAT secretome than in the SAT and control secretomes. miRNA sequencing of EAT-derived EVs detected a total of 824 miRNAs. Pathway analysis led to the identification of 7 miRNAs potentially involved in regulation of cardiac resting membrane potential. Validation of those miRNA candidates showed that they were all expressed in EAT, and that miR-1-3p and miR-133a-3p were upregulated in EAT in comparison with SAT. Overexpression of miR-1-3p and miR-133a-3p in neonatal rat ventricular myocytes led to conduction slowing and reduced Kcnj2 and Kcnj12 expression. Conclusion: miR-1-3p and miR-133a-3p are potential mediators of EAT arrhythmogenicity.

Secretome of atrial epicardial adipose tissue facilitates reentrant arrhythmias by myocardial remodeling.

BACKGROUND: Epicardial adipose tissue (EAT) accumulation is associated with cardiac arrhythmias. The effect of EAT secretome (EATs) on cardiac electrophysiology remains largely unknown. OBJECTIVE: The purpose of this study was to investigate the arrhythmogenicity of EATs and its underlying molecular and electrophysiological mechanisms. METHODS: We collected atrial EAT and subcutaneous adipose tissue (SAT) from 30 patients with atrial fibrillation (AF), and EAT from 3 donors without AF. The secretome was collected after a 24-hour incubation of the adipose tissue explants. We cultured neonatal rat ventricular myocytes (NRVMs) with EATs, subcutaneous adipose tissue secretome (SATs), and cardiomyocytes conditioned medium (CCM) for 72 hours. We implemented the electrophysiological changes observed after EATs incubation into a model of human left atrium and tested arrhythmia inducibility. RESULTS: Incubation of NRVMs with EATs decreased expression of the potassium channel subunit Kcnj2 by 26% and correspondingly reduced the inward rectifier K+ current IK1 by 35% compared to incubation with CCM, resulting in a depolarized resting membrane of cardiomyocytes. EATs decreased expression of connexin43 (29% mRNA, 46% protein) in comparison to CCM. Cells incubated with SATs showed no significant differences in Kcnj2 or Gja1 expression in comparison to CCM, and their resting potential was not depolarized. Cardiomyocytes incubated with EATs showed reduced conduction velocity and increased conduction heterogeneity compared to SATs and CCM. Computer modeling of human left atrium revealed that the electrophysiological changes induced by EATs promote sustained reentrant arrhythmias if EAT partially covers the myocardium. CONCLUSION: EAT slows conduction, depolarizes the resting potential, alters electrical cell-cell coupling, and facilitates reentrant arrhythmias.

Low human dystrophin levels prevent cardiac electrophysiological and structural remodelling in a Duchenne mouse model.

Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disorder caused by loss of dystrophin. This lack also affects cardiac structure and function, and cardiovascular complications are a major cause of death in DMD. Newly developed therapies partially restore dystrophin expression. It is unclear whether this will be sufficient to prevent or ameliorate cardiac involvement in DMD. We here establish the cardiac electrophysiological and structural phenotype in young (2-3 months) and aged (6-13 months) dystrophin-deficient mdx mice expressing 100% human dystrophin (hDMD), 0% human dystrophin (hDMDdel52-null) or low levels (~ 5%) of human dystrophin (hDMDdel52-low). Compared to hDMD, young and aged hDMDdel52-null mice displayed conduction slowing and repolarisation abnormalities, while only aged hDMDdel52-null mice displayed increased myocardial fibrosis. Moreover, ventricular cardiomyocytes from young hDMDdel52-null animals displayed decreased sodium current and action potential (AP) upstroke velocity, and prolonged AP duration at 20% and 50% of repolarisation. Hence, cardiac electrical remodelling in hDMDdel52-null mice preceded development of structural alterations. In contrast to hDMDdel52-null, hDMDdel52-low mice showed similar electrophysiological and structural characteristics as hDMD, indicating prevention of the cardiac DMD phenotype by low levels of human dystrophin. Our findings are potentially relevant for the development of therapeutic strategies aimed at restoring dystrophin expression in DMD.

Genetic variation in GNB5 causes bradycardia by augmenting the cholinergic response via increased acetylcholine-activated potassium current (IK,ACh).

Mutations in GNB5, encoding the G-protein ß5 subunit (Gß5), have recently been linked to a multisystem disorder that includes severe bradycardia. Here, we investigated the mechanism underlying bradycardia caused by the recessive p.S81L Gß5 variant. Using CRISPR/Cas9-based targeting, we generated an isogenic series of human induced pluripotent stem cell (hiPSC) lines that were either wild type, heterozygous or homozygous for the GNB5 p.S81L variant. These were differentiated into cardiomyocytes (hiPSC-CMs) that robustly expressed the acetylcholine-activated potassium channel [I(KACh); also known as IK,ACh]. Baseline electrophysiological properties of the lines did not differ. Upon application of carbachol (CCh), homozygous p.S81L hiPSC-CMs displayed an increased acetylcholine-activated potassium current (IK,ACh) density and a more pronounced decrease of spontaneous activity as compared to wild-type and heterozygous p.S81L hiPSC-CMs, explaining the bradycardia in homozygous carriers. Application of the specific I(KACh) blocker XEN-R0703 resulted in near-complete reversal of the phenotype. Our results provide mechanistic insights and proof of principle for potential therapy in patients carrying GNB5 mutations.This article has an associated First Person interview with the first author of the paper.

Neurokinin-3 receptor activation selectively prolongs atrial refractoriness by inhibition of a background K+ channel.

The cardiac autonomic nervous system (ANS) controls normal atrial electrical function. The cardiac ANS produces various neuropeptides, among which the neurokinins, whose actions on atrial electrophysiology are largely unknown. We here demonstrate that the neurokinin substance-P (Sub-P) activates a neurokinin-3 receptor (NK-3R) in rabbit, prolonging action potential (AP) duration through inhibition of a background potassium current. In contrast, ventricular AP duration was unaffected by NK-3R activation. NK-3R stimulation lengthened atrial repolarization in intact rabbit hearts and consequently suppressed arrhythmia duration and occurrence in a rabbit isolated heart model of atrial fibrillation (AF). In human atrial appendages, the phenomenon of NK-3R mediated lengthening of atrial repolarization was also observed. Our findings thus uncover a pathway to selectively modulate atrial AP duration by activation of a hitherto unidentified neurokinin-3 receptor in the membrane of atrial myocytes. NK-3R stimulation may therefore represent an anti-arrhythmic concept to suppress re-entry-based atrial tachyarrhythmias, including AF.

AAV9-mediated Rbm24 overexpression induces fibrosis in the mouse heart.

The RNA-binding protein Rbm24 has recently been identified as a pivotal splicing factor in the developing heart. Loss of Rbm24 in mice disrupts cardiac development by governing a large number of muscle-specific splicing events. Since Rbm24 knockout mice are embryonically lethal, the role of Rbm24 in the adult heart remained unexplored. Here, we used adeno-associated viruses (AAV9) to investigate the effect of increased Rbm24 levels in adult mouse heart. Using high-resolution microarrays, we found 893 differentially expressed genes and 1102 differential splicing events in 714 genes in hearts overexpressing Rbm24. We found splicing differences in cardiac genes, such as PDZ and Lim domain 5, Phospholamban, and Titin, but did not find splicing differences in previously identified embryonic splicing targets of Rbm24, such as skNAC, αNAC, and Coro6. Gene ontology enrichment analysis demonstrated increased expression of extracellular matrix (ECM)-related and immune response genes. Moreover, we found increased expression of Tgfß-signaling genes, suggesting enhanced Tgfß-signaling in these hearts. Ultimately, this increased activation of cardiac fibroblasts, as evidenced by robust expression of Periostin in the heart, and induced extensive cardiac fibrosis. These results indicate that Rbm24 may function as a regulator of cardiac fibrosis, potentially through the regulation of TgfßR1 and TgfßR2 expression.

Recombinant human collagen-based microspheres mitigate cardiac conduction slowing induced by adipose tissue-derived stromal cells.

BACKGROUND: Stem cell therapy to improve cardiac function after myocardial infarction is hampered by poor cell retention, while it may also increase the risk of arrhythmias by providing an arrhythmogenic substrate. We previously showed that porcine adipose tissue-derived-stromal cells (pASC) induce conduction slowing through paracrine actions, whereas rat ASC (rASC) and human ASC (hASC) induce conduction slowing by direct coupling. We postulate that biomaterial microspheres mitigate the conduction slowing influence of pASC by interacting with paracrine signaling. AIM: To investigate the modulation of ASC-loaded recombinant human collagen-based microspheres, on the electrophysiological behavior of neonatal rat ventricular myocytes (NRVM). METHOD: Unipolar extracellular electrograms, derived from microelectrode arrays (8x8 electrodes) containing NRVM, co-cultured with ASC or ASC loaded microspheres, were used to determine conduction velocity (CV) and conduction heterogeneity. Conditioned medium (Cme) of (co)cultures was used to assess paracrine mechanisms. RESULTS: Microspheres did not affect CV in control (NRVM) monolayers. In co-cultures of NRVM and rASC, hASC or pASC, CV was lower than in controls (14.4±1.0, 13.0±0.6 and 9.0± 1.0 vs. 19.5±0.5 cm/s respectively, p<0.001). Microspheres loaded with either rASC or hASC still induced conduction slowing compared to controls (13.5±0.4 and 12.6±0.5 cm/s respectively, p<0.001). However, pASC loaded microspheres increased CV of NRVM compared to pASC and NRMV co-cultures (16.3±1.3 cm/s, p< 0.001) and did not differ from controls (p = NS). Cme of pASC reduced CV in control monolayers of NRVM (10.3±1.1 cm/s, p<0.001), similar to Cme derived from pASC-loaded microspheres (11.1±1.7 cm/s, p = 1.0). The presence of microspheres in monolayers of NRVM abolished the CV slowing influence of Cme pASC (15.9±1.0 cm/s, p = NS vs. control). CONCLUSION: The application of recombinant human collagen-based microspheres mitigates indirect paracrine conduction slowing through interference with a secondary autocrine myocardial factor.

Differential Mechanisms of Myocardial Conduction Slowing by Adipose Tissue-Derived Stromal Cells Derived from Different Species.

Stem cell therapy is a promising therapeutic option to treat patients after myocardial infarction. However, the intramyocardial administration of large amounts of stem cells might generate a proarrhythmic substrate. Proarrhythmic effects can be explained by electrotonic and/or paracrine mechanisms. The narrow therapeutic time window for cell therapy and the presence of comorbidities limit the application of autologous cell therapy. The use of allogeneic or xenogeneic stem cells is a potential alternative to autologous cells, but differences in the proarrhythmic effects of adipose-derived stromal cells (ADSCs) across species are unknown. Using microelectrode arrays and microelectrode recordings, we obtained local unipolar electrograms and action potentials from monolayers of neonatal rat ventricular myocytes (NRVMs) that were cocultured with rat, human, or pig ADSCs (rADSCs, hADSCs, pADSCs, respectively). Monolayers of NRVMs were cultured in the respective conditioned medium to investigate paracrine effects. We observed significant conduction slowing in all cardiomyocyte cultures containing ADSCs, independent of species used (p < .01). All cocultures were depolarized compared with controls (p < .01). Only conditioned medium taken from cocultures with pADSCs and applied to NRVM monolayers demonstrated similar electrophysiological changes as the corresponding cocultures. We have shown that independent of species used, ADSCs cause conduction slowing in monolayers of NRVMs. In addition, pADSCs exert conduction slowing mainly by a paracrine effect, whereas the influence on conduction by hADSCs and rADSCs is preferentially by electrotonic interaction. Stem Cells Translational Medicine 2017;6:22-30.

Spatial Heterogeneity of Cx43 is an Arrhythmogenic Substrate of Polymorphic Ventricular Tachycardias during Compensated Cardiac Hypertrophy in Rats.

BACKGROUND: Ventricular remodeling increases the propensity of ventricular tachyarrhythmias and sudden death in patients. We studied the mechanism underlying these fatal arrhythmias, electrical and structural cardiac remodeling, as well as arrhythmogeneity during early, compensated hypertrophy in a rat model of chronic pressure overload. METHODS: Twenty-six Wistar rats were subjected to transverse aortic constriction (TAC) (n = 13) or sham operation (n = 13). Four weeks postoperative, echo- and electrocardiography was performed. Epicardial (208 or 455 sites) and transmural (30 sites) ventricular activation mapping was performed on Langendorff perfused hearts. Subsequently, hearts were processed for (immuno)histological and molecular analyses. RESULTS: TAC rats showed significant hypertrophy with preserved left ventricular (LV) function. Epicardial conduction velocity (CV) was similar, but more dispersed in TAC. Transmural CV was slowed in TAC (37.6 ± 2.9 cm s(-1)) compared to sham (58.5 ± 3.9 cm s(-1); P < 0.01). Sustained polymorphic ventricular tachycardias were induced from LV in 8/13 TAC and in 0/13 sham rats. During VT, electrical activation patterns showed variable sites of earliest epicardial activation and altering sites of functional conduction block. Wandering epicardial reentrant activation was sporadically observed. Collagen deposition was significantly higher in TAC compared to sham, but not different between arrhythmogenic and non-arrhythmogenic TAC animals. Connexin43 (Cx43) expression was heterogeneous with a higher prevalence of non-phosphorylated Cx43 in arrhythmogenic TAC animals. CONCLUSION: In TAC rats with compensated cardiac hypertrophy, dispersion of conduction correlated to arrhythmogenesis, an increased heterogeneity of Cx43, and a partial substitution with non-phosphorylated Cx43. These alterations may result in the increased vulnerability to polymorphic VTs.

Atrial fibrosis and conduction slowing in the left atrial appendage of patients undergoing thoracoscopic surgical pulmonary vein isolation for atrial fibrillation.

BACKGROUND: Atrial fibrosis is an important component of the arrhythmogenic substrate in patients with atrial fibrillation (AF). We studied the effect of interstitial fibrosis on conduction velocity (CV) in the left atrial appendage of patients with AF. METHODS AND RESULTS: Thirty-five left atrial appendages were obtained during AF surgery. Preparations were superfused and stimulated at 100 beats per minute. Activation was recorded with optical mapping. Longitudinal CV (CVL), transverse CV (CVT), and activation times (> 2 mm distance) were measured. Interstitial collagen was quantified and graded qualitatively. The presence of fibroblasts and myofibroblasts was assessed immunohistochemically. Mean CVL was 0.55 ± 0.22 m/s, mean CVT was 0.25 ± 0.15 m/s, and the mean activation time was 9.31 ± 5.45 ms. The amount of fibrosis was unrelated to CV or patient characteristics. CVL was higher in left atrial appendages with thick compared with thin interstitial collagen strands (0.77 ± 0.22 versus 0.48 ± 0.19 m/s; P = 0.012), which were more frequently present in persistent patients with AF. CVT was not significantly different (P = 0.47), but activation time was 14.93 ± 4.12 versus 7.95 ± 4.12 ms in patients with thick versus thin interstitial collagen strands, respectively (P = 0.004). Fibroblasts were abundantly present and were associated with the presence of thick interstitial collagen strands (P = 0.008). Myofibroblasts were not detected in the left atrial appendage. CONCLUSIONS: In patients with AF, thick interstitial collagen strands are associated with higher CVL and increased activation time. Our observations demonstrate that the severity and structure of local interstitial fibrosis is associated with atrial conduction abnormalities, presenting an arrhythmogenic substrate for atrial re-entry.

Increased amount of atrial fibrosis in patients with atrial fibrillation secondary to mitral valve disease.

OBJECTIVE: Atrial fibrosis is related to atrial fibrillation but may differ in patients with mitral valve disease or lone atrial fibrillation. Therefore, we studied atrial fibrosis in patients with atrial fibrillation+mitral valve disease or with lone atrial fibrillation and compared it with controls. METHODS: Left and right atrial appendages amputated during Maze III surgery for lone atrial fibrillation (n=85) or atrial fibrillation+mitral valve disease (n=26) were embedded in paraffin, sectioned, and stained with picrosirius red. Atria from 10 deceased patients without a cardiovascular history served as controls. A total of 1048 images (4-µm sections, 10-fold magnification, 4 images per appendage) were obtained and digitized. The percentage of fibrous tissue was calculated by quantitative morphometry. RESULTS: Irrespective of the presence or absence of atrial fibrillation or mitral valve disease, more fibrous tissue was present in right atrial appendages than in left atrial appendages (12.7%±5.7% vs 8.2%±3.9%; P<.0001). The mean amount of fibrous tissue in the atria was significantly larger in patients with atrial fibrillation+mitral valve disease than in patients with lone AF and controls (13.6%±5.8%, 9.7%±3.2%, and 8.8%±2.4%, respectively; P<.01). No significant differences existed between patients with lone atrial fibrillation and patients without a cardiovascular history (controls). CONCLUSIONS: Atria of patients with atrial fibrillation and mitral valve disease have more fibrosis than atria of patients with lone atrial fibrillation. However, patients with lone atrial fibrillation have an equal amount of atrial fibrosis compared with controls. These findings support the notion that fibrosis plays a more important role in the pathogenesis of atrial fibrillation secondary to mitral valve disease than in lone atrial fibrillation and potentially explains the relatively poor success of antiarrhythmic surgery in patients with mitral valve disease.

Load-reducing therapy prevents development of arrhythmogenic right ventricular cardiomyopathy in plakoglobin-deficient mice.

OBJECTIVES: We used a murine model of arrhythmogenic right ventricular cardiomyopathy (ARVC) to test whether reducing ventricular load prevents or slows development of this cardiomyopathy. BACKGROUND: At present, no therapy exists to slow progression of ARVC. Genetically conferred dysfunction of the mechanical cell-cell connections, often associated with reduced expression of plakoglobin, is thought to cause ARVC. METHODS: Littermate pairs of heterozygous plakoglobin-deficient mice (plako(+/-)) and wild-type (WT) littermates underwent 7 weeks of endurance training (daily swimming). Mice were randomized to blinded load-reducing therapy (furosemide and nitrates) or placebo. RESULTS: Therapy prevented training-induced right ventricular (RV) enlargement in plako(+/-) mice (RV volume: untreated plako(+/-) 136 ± 5 µl; treated plako(+/-) 78 ± 5 µl; WT 81 ± 5 µl; p < 0.01 for untreated vs. WT and untreated vs. treated; mean ± SEM). In isolated, Langendorff-perfused hearts, ventricular tachycardias (VTs) were more often induced in untreated plako(+/-) hearts (15 of 25), than in treated plako(+/-) hearts (5 of 19) or in WT hearts (6 of 21, both p < 0.05). Epicardial mapping of the RV identified macro-re-entry as the mechanism of ventricular tachycardia. The RV longitudinal conduction velocity was reduced in untreated but not in treated plako(+/-) mice (p < 0.01 for untreated vs. WT and untreated vs. treated). Myocardial concentration of phosphorylated connexin43 was lower in plako(+/-) hearts with VTs compared with hearts without VTs and was reduced in untreated plako(+/-) compared with WT (both p < 0.05). Plako(+/-) hearts showed reduced myocardial plakoglobin concentration, whereas ß-catenin and N-cadherin concentration was not changed. CONCLUSIONS: Load-reducing therapy prevents training-induced development of ARVC in plako(+/-) mice.

Mechanism of right precordial ST-segment elevation in structural heart disease: excitation failure by current-to-load mismatch.

BACKGROUND: The Brugada sign has been associated with mutations in SCN5A and with right ventricular structural abnormalities. Their role in the Brugada sign and the associated ventricular arrhythmias is unknown. OBJECTIVE: The purpose of this study was to delineate the role of structural abnormalities and sodium channel dysfunction in the Brugada sign. METHODS: Activation and repolarization characteristics of the explanted heart of a patient with a loss-of-function mutation in SCN5A (G752R) and dilated cardiomyopathy were determined after induction of right-sided ST-segment elevation by ajmaline. In addition, right ventricular structural discontinuities and sodium channel dysfunction were simulated in a computer model encompassing the heart and thorax. RESULTS: In the explanted heart, disappearance of local activation in unipolar electrograms at the basal right ventricular epicardium was followed by monophasic ST-segment elevation. The local origin of this phenomenon was confirmed by coaxial electrograms. Neither early repolarization nor late activation correlated with ST-segment elevation. At sites of local ST-segment elevation, the subepicardium was interspersed with adipose tissue and contained more fibrous tissue than either the left ventricle or control hearts. In computer simulations entailing right ventricular structural discontinuities, reduction of sodium channel conductance or size of the gaps between introduced barriers resulted in subepicardial excitation failure or delayed activation by current-to-load mismatch and in the Brugada sign on the ECG. CONCLUSION: Right ventricular excitation failure and activation delay by current-to-load mismatch in the subepicardium can cause the Brugada sign. Therefore, current-to-load mismatch may underlie the ventricular arrhythmias in patients with the Brugada sign.

Engineering physiologically controlled pacemaker cells with lentiviral HCN4 gene transfer.

BACKGROUND: Research on biological pacemakers for the heart has so far mainly focused on short-term gene and cell therapies. To develop a clinically relevant biological pacemaker, long-term function and incorporation of autonomic modulation are crucial. Lentiviral vectors can mediate long-term gene expression, while isoform 4 of the Hyperpolarization-activated Cyclic Nucleotide-gated channel (encoded by HCN4) contributes to pacemaker function and responds maximally to cAMP, the second messenger in autonomic modulation. MATERIAL AND METHODS: Action potential (AP) properties and pacemaker current (I(f)) were studied in single neonatal rat ventricular myocytes that overexpressed HCN4 after lentiviral gene transduction. Autonomic responsiveness and cycle length stability were studied using extracellular electrograms of confluent cultured monolayers. RESULTS: Perforated patch-clamp experiments demonstrated that HCN4-transduced single cardiac myocytes exhibited a 10-fold higher I(f) than non-transduced single myocytes, along with slow diastolic depolarization, comparable to pacemaker cells of the sinoatrial node, the dominant native pacemaker. HCN4-transduced monolayers exhibited a 47% increase in beating rate, compared to controls. Upon addition of DBcAMP, HCN4-transduced monolayers had beating rates which were 54% faster than baseline and significantly more regular than controls. CONCLUSIONS: Lentiviral vectors efficiently transduce cardiac myocytes and mediate functional gene expression. Because HCN4-transduced myocytes demonstrate an increase in spontaneous beating rate and responsiveness to autonomic modulation, this approach may be useful to create a biological pacemaker.

Inhibition of cardiomyocyte automaticity by electrotonic application of inward rectifier current from Kir2.1 expressing cells.

A biological pacemaker might be created by generation of a cellular construct consisting of cardiac cells that display spontaneous membrane depolarization, and that are electrotonically coupled to surrounding myocardial cells by means of gap junctions. Depending on the frequency of the spontaneously beating cells, frequency regulation might be required. We hypothesized that application of Kir2.1 expressing non-cardiac cells, which provide I (K1) to spontaneously active neonatal cardiomyocytes (NCMs) by electrotonic coupling in such a cellular construct, would generate an opportunity for pacemaker frequency control. Non-cardiac Kir2.1 expressing cells were co-cultured with spontaneously active rat NCMs. Electrotonic coupling between the two cell types resulted in hyperpolarization of the cardiomyocyte membrane potential and silencing of spontaneous activity. Either blocking of gap-junctional communication by halothane or inhibition of I (K1) by BaCl(2) restored the original membrane potential and spontaneous activity of the NCMs. Our results demonstrate the power of electrotonic coupling for the application of specific ion currents into an engineered cellular construct such as a biological pacemaker.

Pulsed-field gel electrophoresis analysis of Bordetella pertussis populations in various European countries with different vaccine policies.

The increasing incidence of pertussis in a number of countries, despite good vaccination coverage, is a cause for concern. We used pulsed-field gel electrophoresis (PFGE) typing to examine the genetic diversity of 101 clinical isolates of Bordetella pertussis, recovered during 1999-2001, and circulating in five different European countries to evaluate temporal and geographical distribution. This DNA fingerprinting approach seems to be a more discriminative epidemiological tool than sequencing of individual genes. Despite differences in vaccination policies in the five countries, these European isolates were found to be very similar and fell into the same major PFGE profile groups, with a predominance of one profile group. There was no evidence of geographic clustering, except that one new profile subgroup was predominantly found in one country. This study provides a baseline for continued surveillance of the B. pertussis population in Europe.

Analysis of the rat connexin 43 proximal promoter in neonatal cardiomyocytes.

Altered transcriptional control is likely to contribute to the down-regulation of connexin 43 (Cx43) expression observed in many forms of heart disease. However, little is known about the factors regulating Cx43 transcription in the heart under (patho)physiological conditions. Therefore, a systematic study of rat Cx43 (rCx43) proximal promoter regulation in rat primary neonatal ventricular cardiomyocytes (NCM) and, for comparison, different cell types was initiated. Luciferase assays revealed that, in NCM, the proximal promoter is preserved in a conserved region extending from 148 nucleotides upstream towards 281 nucleotides downstream relative to the transcription initiation site (TIS). Further deletional analysis suggested the involvement of four putative Sp- and two AP1-binding sites. The binding of both Sp1 and Sp3 to the Sp-binding elements and AP1 to the AP1-binding elements was demonstrated by electrophoretic mobility shift assays (EMSA). Promoter-luciferase assays using the natural rCx43 proximal promoter and mutated derivatives in NCM, HL-1 and A7r5 cells revealed that all sites contribute to basal promoter activity. Trans-activation of the Cx43 proximal promoter with Sp1 and Sp3 in Drosophila Schneider line 2 (SL2) cells demonstrated that Sp1 and, to a lesser extent, Sp3 determine rCx43 promoter activation. Thus Sp1, Sp3 and AP1 determine basal Cx43 expression. In addition, we studied the effect of the cardiac transcription factor Nkx2.5 on Cx43 regulation. NCM were infected with adenovirus encoding either beta-galactosidase (control) or Nkx2.5. Cx43 protein and mRNA were significantly decreased after Nkx2.5 infection as shown by Western and Northern blot analyses. Promoter-reporter assays demonstrated that the rCx43 promoter was down-regulated approximately twofold upon Nkx2.5 overexpression. Therefore, in NCM, Nkx2.5 appears to play a role in the regulation of Cx43 expression.

Sp1 and Sp3 activate the rat connexin40 proximal promoter.

The rat gap junction protein connexin40 (rCx40) has a characteristic developmental and regional expression pattern, for which the exact regulatory mechanisms are not known. To identify the molecular factors controlling Cx40 expression, its proximal promoter was characterized. The proximal rCx40 promoter is the most conserved noncoding region within the Cx40-gene known thus far and contains five potential binding sites for Sp-family transcription factors. The binding of both Sp1 and Sp3 to each of these DNA elements was demonstrated by EMSA. Luciferase assays of the natural rCx40 proximal promoter or mutated derivatives in Cx40-expressing (NCM, primary rat neonatal cardiomyocytes and A7r5, rat smooth muscle embryonic thoracic aorta cells) and -nonexpressing cells (N2A, mouse neuroblastoma cells) revealed that all sites are contributing to basal promoter activity. Trans-activation assays in Drosophila Schneider line 2 cells demonstrated that Sp1 and Sp3 activate the rCx40 proximal promoter in a dose-dependent and additive manner.