Barriers in participant recruitment of diverse ethnicities in the state of Kuwait.

BACKGROUND: High rejection rates of subject recruitments for research studies have been reported in immigrants in many countries. However, the barriers in recruiting members of the expatriate population in Kuwait have not yet been investigated. This study was therefore designed to identify barriers in recruiting expatriates for research studies in the state of Kuwait. METHODS: A population-based cross-sectional study was conducted on expatriate subject's aged 18 years and older living in Kuwait. Difference between groups of continuous independent variables was analyzed using the t-test. Different categories such as ethnicity and gender were compared using the chi-square test. RESULTS: 3460 (85.1%) participants were recruited and 617 (14.2%) refused to participate in the study while 2530 (38%) calls were unreachable from the total of 6607 calls placed. Younger subjects (mean age 41.1 years) were more hesitant to be part of the study compared to older participants. The rejections among South Asians was (41.8%), Arabs (32.6%), Southeast Asians (18.9%) while the others (6.6%) category was least to refuse among all the nationalities. Gender was not significantly associated with refusal. CONCLUSION: There is an acute lack of appropriate recording of the problems faced while recruiting the participants. The findings suggest important messages for the decision makers in the area of expatriate recruitments, to understand the challenge and design new strategies to overcome the problem of recruitment in the state of Kuwait for research studies.

Global array-based transcriptomics from minimal input RNA utilising an optimal RNA isolation process combined with SPIA cDNA probes.

BACKGROUND: Technical advances in the collection of clinical material, such as laser capture microdissection and cell sorting, provide the advantage of yielding more refined and homogenous populations of cells. However, these attractive advantages are counter balanced by the significant difficulty in obtaining adequate nucleic acid yields to allow transcriptomic analyses. Established technologies are available to carry out global transcriptomics using nanograms of input RNA, however, many clinical samples of low cell content would be expected to yield RNA within the picogram range. To fully exploit these clinical samples the challenge of isolating adequate RNA yield directly and generating sufficient microarray probes for global transcriptional profiling from this low level RNA input has been addressed in the current report. We have established an optimised RNA isolation workflow specifically designed to yield maximal RNA from minimal cell numbers. This procedure obtained RNA yield sufficient for carrying out global transcriptional profiling from vascular endothelial cell biopsies, clinical material not previously amenable to global transcriptomic approaches. In addition, by assessing the performance of two linear isothermal probe generation methods at decreasing input levels of good quality RNA we demonstrated robust detection of a class of low abundance transcripts (GPCRs) at input levels within the picogram range, a lower level of RNA input (50 pg) than previously reported for global transcriptional profiling and report the ability to interrogate the transcriptome from only 10 pg of input RNA. By exploiting an optimal RNA isolation workflow specifically for samples of low cell content, and linear isothermal RNA amplification methods for low level RNA input we were able to perform global transcriptomics on valuable and potentially informative clinically derived vascular endothelial biopsies here for the first time. These workflows provide the ability to robustly exploit ever more common clinical samples yielding extremely low cell numbers and RNA yields for global transcriptomics.

Peroxisome proliferator-activated receptor-delta genotype influences metabolic phenotype and may influence lipid response to statin therapy in humans: a genetics of diabetes audit and research Tayside study.

CONTEXT: Previous studies have identified a single-nucleotide polymorphism in the gene encoding peroxisome proliferator-activated receptor-delta (PPARD), rs2016520, that is associated with changes in metabolic disease in some but not all studies, which suggests that PPARD agonists may have therapeutic benefits for the treatment of metabolic disorders, including dyslipidemia, type 2 diabetes, and obesity. OBJECTIVE: The objective of the study was to determine whether rs2016520 or other single-nucleotide polymorphism in the PPARD locus influenced the risk of developing various characteristics of metabolic disease. DESIGN: Haplotype tagging analysis across PPARD was performed in 11,074 individuals from the Welcome Trust U.K. Type 2 Diabetes Case Control Collection. RESULTS: In subjects with and without type 2 diabetes, rs2016520 was associated with body mass index, high-density lipoprotein cholesterol, leptin, and TNFalpha and was dependent on gender. CONCLUSION: The current results suggest differential effects of PPARdelta in males and females.

Alternans in genetically modified langendorff-perfused murine hearts modeling catecholaminergic polymorphic ventricular tachycardia.

The relationship between alternans and arrhythmogenicity was studied in genetically modified murine hearts modeling catecholaminergic polymorphic ventricular tachycardia (CPVT) during Langendorff perfusion, before and after treatment with catecholamines and a ß-adrenergic antagonist. Heterozygous (RyR2(p/s)) and homozygous (RyR2(s/s)) RyR2-P2328S hearts, and wild-type (WT) controls, were studied before and after treatment with epinephrine (100 nM and 1 µM) and propranolol (100 nM). Monophasic action potential recordings demonstrated significantly greater incidences of arrhythmia in RyR2(p/s) and RyR2(s/s) hearts as compared to WTs. Arrhythmogenicity in RyR2(s/s) hearts was associated with alternans, particularly at short baseline cycle lengths. Both phenomena were significantly accentuated by treatment with epinephrine and significantly diminished by treatment with propranolol, in full agreement with clinical expectations. These changes took place, however, despite an absence of changes in mean action potential durations, ventricular effective refractory periods or restitution curve characteristics. Furthermore pooled data from all hearts in which arrhythmia occurred demonstrated significantly greater alternans magnitudes, but similar restitution curve slopes, to hearts that did not demonstrate arrhythmia. These findings thus further validate the RyR2-P2328S murine heart as a model for human CPVT, confirming an alternans phenotype in common with murine genetic models of the Brugada syndrome and the congenital long-QT syndrome type 3. In contrast to these latter similarities, however, this report demonstrates the dissociation of alternans from changes in the properties of restitution curves for the first time in a murine model of a human arrhythmic syndrome.

Epithelial IgG and its relationship to the loss of F508 in the common mutant form of the cystic fibrosis transmembrane conductance regulator.

The most debilitating feature of cystic fibrosis (CF) disease is uncontrolled inflammation of respiratory epithelium. The relationship between the commonest mutated form of CFTR (F508del or DeltaF508) and inflammation has not yet been elucidated. Here, we present a new paradigm suggesting that CFTR can interact with intra-epithelial IgG, establishing a direct link between normal CFTR and the immune system. Further, our data show that the amino-acid sequence local to F508 can bind IgG with high affinity, dependent on F508, such that loss of F508 abolishes this link both in vitro and in the intact cell.

A single nucleotide polymorphism on exon-4 of the gene encoding PPARdelta is associated with reduced height in adults and children.

CONTEXT: Peroxisome proliferator-activated receptor (PPAR)-delta is a nuclear transcription factor that plays a key role in many metabolic processes, including energy metabolism, and lipid and glucose metabolism. Candidate gene studies have identified a putative functional variant, rs2016520, in the gene encoding PPARdelta (PPARD), which is associated in some studies with metabolic traits. In addition, this single-nucleotide polymorphism was associated with adult height in several whole-genome scans, but this association did not achieve whole genome significance. OBJECTIVE: This study sought to determine whether PPARD variation influenced height. DESIGN: Haplotype tagging analysis across PPARD was performed in about 11,000 individuals from the Wellcome Trust U.K. Type 2 Diabetes Case Control Collection (Go-DARTS2). RESULTS: There was an association between rs2016520 and height in both patients with type 2 diabetes and controls without diabetes (combined P = 5 x 10(-5)). In a metaanalysis using published data from Caucasian cohorts totaling more than 38,000 participants, compelling evidence was found for this locus and its association with height (P = 10(-8)) with an overall effect size of about 0.5 cm per allele. A similar analysis in a group of 2700 prepubescent children also displayed a similar effect size to that seen in the adults. CONCLUSION: PPARD variation is clearly associated with a phenotype of reduced stature in both adults and children. Because height is an important indicator of metabolic and nutritional status, this provides additional support for a key role for PPARdelta in critical metabolic functions. PPARdelta may affect height through a variety of mechanisms including altered metabolic efficiency or effects on osteoclast function.

Defective formation of PKA/CnA-dependent annexin 2-S100A10/CFTR complex in DeltaF508 cystic fibrosis cells.

Cystic fibrosis (CF) is characterised by impaired epithelial ion transport and is caused by mutations in the cystic fibrosis conductance regulator protein (CFTR), a cAMP/PKA and ATP-regulated chloride channel. We recently demonstrated a cAMP/PKA/calcineurin (CnA)-driven association between annexin 2 (anx 2), its cognate partner -S100A10 and cell surface CFTR. The complex is required for CFTR and outwardly rectifying chloride channel function in epithelia. Since the cAMP/PKA-induced Cl(-) current is absent in CF epithelia, we hypothesized that the anx 2-S100A10/CFTR complex may be defective in CFBE41o cells expressing the commonest F508del-CFTR (DeltaF-CFTR) mutation. Here, we demonstrate that, despite the presence of cell surface DeltaF-CFTR, cAMP/PKA fails to induce anx 2-S100A10/CFTR complex formation in CFBE41o- cells homozygous for F508del-CFTR. Mechanistically, PKA-dependent serine phosphorylation of CnA, CnA-anx 2 complex formation and CnA-dependent dephosphorylation of anx 2 are all defective in CFBE41o- cells. Immunohistochemical analysis confirms an abnormal cellular distribution of anx 2 in human and CF mouse epithelia. Thus, we demonstrate that cAMP/PKA/CnA signaling pathway is defective in CF cells and suggest that loss of anx 2-S100A10/CFTR complex formation may contribute to defective cAMP/PKA-dependent CFTR channel function.

Comparison of protein transduction domains in mediating cell delivery of a secreted CRE protein.

Protein transduction domains (PTDs) are versatile peptide sequences that facilitate cell delivery of several cargo molecules including proteins. PTDs usually consist of short stretches of basic amino acids that can cross the plasma membrane and gain entry into cells. Traditionally, to assess PTD mediated protein delivery, PTD-fusion proteins have been used as purified proteins. To overcome the requirement for a protein purification step, we used a secretory signal peptide to allow PTD-CRE fusion proteins to be exported from transfected mammalian cells. PTD induced protein transduction into cells was assessed by a CRE-mediated recombination event that resulted in beta-galactosidase expression. Several PTDs were tested including the prototypic TAT, different TAT variants, Antp, MTS and polyarginine. A negative correlation was observed between the cationic charge on the PTD and the extent of secretion. Poor secretion was found when the PTD charge was greater than +5. One TAT-CRE protein variant had a 14-fold enhancement above CRE alone when added to cells in the presence of chloroquine. This PTD domain also enhanced gene expression after plasmid delivery. These data illustrate that some secreted PTD proteins may be useful reagents to improve protein delivery in mammalian systems and a novel approach to enhancing the response to DNA transfections.

Criteria for arrhythmogenicity in genetically-modified Langendorff-perfused murine hearts modelling the congenital long QT syndrome type 3 and the Brugada syndrome.

The experiments investigated the applicability of two established criteria for arrhythmogenicity in Scn5a+/Delta and Scn5a+/- murine hearts modelling the congenital long QT syndrome type 3 (LQT3) and the Brugada syndrome (BrS). Monophasic action potentials (APs) recorded during extrasystolic stimulation procedures from Langendorff-perfused control hearts and hearts treated with flecainide (1 microM) or quinidine (1 or 10 microM) demonstrated that both agents were pro-arrhythmic in wild-type (WT) hearts, quinidine was pro-arrhythmic in Scn5a+/Delta hearts, and that flecainide was pro-arrhythmic whereas quinidine was anti-arrhythmic in Scn5a+/- hearts, confirming clinical findings. Statistical analysis confirmed a quadratic relationship between epicardial and endocardial AP durations (APDs) in WT control hearts. However, comparisons between plots of epicardial against endocardial APDs and this reference curve failed to correlate with arrhythmogenicity. Restitution curves, relating APD to diastolic interval (DI), were then constructed for the first time in a murine system and mono-exponential growth functions fitted to these curves. Significant (P<0.05) alterations in the DI at which slopes equalled unity, an established indicator of arrhythmogenicity, now successfully predicted the presence or absence of arrhythmogenicity in all cases. We thus associate changes in the slopes of restitution curves with arrhythmogenicity in models of LQT3 and BrS.

Transient alterations in transmural repolarization gradients and arrhythmogenicity in hypokalaemic Langendorff-perfused murine hearts.

Clinical hypokalaemia is associated with acquired electrocardiographic QT prolongation and arrhythmic activity initiated by premature ventricular depolarizations and suppressed by lidocaine (lignocaine). Nevertheless, regular (S1) pacing at a 125 ms interstimulus interval resulted in stable waveforms and rhythm studied using epicardial and endocardial monophasic action potential (MAP) electrodes in Langendorff-perfused murine hearts whether under normokalaemic (5.2 mM K+) or hypokalaemic (3.0 mM K+) conditions, in both the presence and absence of lidocaine (10 microM). Furthermore, the transmural gradient in repolarization time, known to be altered in the congenital long-QT syndromes, and reflected in the difference between endocardial and epicardial MAP duration at 90% repolarization (DeltaAPD(90)), did not differ significantly (P > 0.05) between normokalaemic (5.5 +/- 4.5 ms, n = 8, five hearts), hypokalaemic (n = 8, five hearts), or lidocaine-treated normokalaemic (n = 8, five hearts) or hypokalaemic (n = 8, five hearts) hearts. However, premature ventricular depolarizations occurring in response to extrasystolic (S2) stimulation delivered at S1S2 intervals between 0 and 22 +/- 6 ms following recovery from refractoriness initiated arrhythmic activity specifically in hypokalaemic (n = 8, five hearts) as opposed to normokalaemic (n = 25, 14 hearts), or lidocaine-treated hypokalaemic (n = 8, five hearts) or normokalaemic hearts (n = 8, five hearts). This was associated with sharp but transient reversals in DeltaAPD(90) in MAPs initiated within the 250 ms interval directly succeeding premature ventricular depolarizations, from 3.3 +/- 5.6 ms to -31.8 +/- 11.8 ms (P < 0.05) when they were initiated immediately after recovery from refractoriness. In contrast the corresponding latency differences consistently remained close to the normokalaemic value (-1.6 +/- 1.4 ms, P > 0.05). These findings empirically associate arrhythmogenesis in hypokalaemic hearts with transient alterations in transmural repolarization gradients resulting from premature ventricular depolarizations. This is in contrast to sustained alterations in transmural repolarization gradients present on regular stimulation in long-QT syndrome models.

Effects of flecainide and quinidine on arrhythmogenic properties of Scn5a+/- murine hearts modelling the Brugada syndrome.

Brugada syndrome (BrS) is associated with a loss of Na+ channel function and an increased incidence of rapid polymorphic ventricular tachycardia (VT) and sudden cardiac death. A programmed electrical stimulation (PES) technique assessed arrhythmic tendency in Langendorff-perfused wild-type (WT) and genetically modified (Scn5a+/-) 'loss-of-function' murine hearts in the presence and absence of flecainide and quinidine, and the extent to which Scn5a+/- hearts model the human BrS. Extra-stimuli (S2), applied to the right ventricular epicardium, followed trains of pacing stimuli (S1) at progressively reduced S1-S2 intervals. These triggered VT in 16 out of 29 untreated Scn5a+/- and zero out of 31 WT hearts. VT occurred in 11 out of 16 (10 microM) flecainide-treated WT and nine out of the 13 initially non-arrhythmogenic Scn5a+/- hearts treated with (1.0 microM) flecainide. Quinidine (10 microM) prevented VT in six out of six flecainide-treated WT and 13 out of the 16 arrhythmogenic Scn5a+/- hearts in parallel with its clinical effects. Paced electrogram fractionation analysis demonstrated increased electrogram durations, expressed as electrogram duration (EGD) ratios, with shortening S1-S2 intervals in arrhythmogenic Scn5a+/- hearts, and prolonged ventricular effective refractory periods (VERPs) in non-arrhythmogenic Scn5a+/- hearts. Flecainide increased EGD ratios in WT (at 10 microM) and non-arrhythmogenic Scn5a+/- hearts (at 1.0 microM), whereas quinidine (10 microM) reduced EGD ratios and prolonged VERPs in WT and arrhythmogenic Scn5a+/- hearts. However, epicardial and endocardial monophasic action potential recordings consistently demonstrated positive gradients of repolarization in WT, arrhythmogenic and non-arrhythmogenic Scn5a+/- hearts under all pharmacological conditions. Together, these findings demonstrate proarrhythmic effects of flecainide in WT and Scn5a+/- murine hearts that recapitulate its clinical effects. They further attribute the arrhythmogenic phenomena observed here to re-entrant substrates resulting from delayed epicardial activation despite an absence of transmural heterogeneities of repolarization, in sharp contrast to recent characterizations in 'gain-of-function' Scn5a+/Delta murine hearts modelling the long-QT(3) syndrome.

Mechanisms of ventricular arrhythmogenesis in mice following targeted disruption of KCNE1 modelling long QT syndrome 5.

Mutations within KCNE1 encoding a transmembrane protein which coassembles with K+ channels mediating slow K+, I(Ks), currents are implicated in cardiac action potential prolongation and ventricular arrhythmogenicity in long QT syndrome 5. We demonstrate the following potentially arrhythmogenic features in simultaneously recorded, left ventricular, endocardial and epicardial monophasic action potentials from Langendorff-perfused murine KCNE1-/- hearts for the first time. (1) Prolonged epicardial (57.1 +/- 0.5 ms cf. 36.1 +/- 0.07 ms in wild-type (WT), P < 0.001; n = 5) and endocardial action potential duration at 90% repolarication (APD90) (54.4 +/- 2.4 ms cf. 48.5 +/- 0.3 ms, P < 0.05; n = 5). (2) Negative transmural repolarization gradients (DeltaAPD90: endocardial minus epicardial APD90) (-2.5 +/- 2.4 ms, compared with 12.4 +/- 1.1 ms in WT, P < 0.001; n = 5). (3) Frequent epicardial early afterdepolarizations (EADs) and spontaneous ventricular tachycardia (VT) in 4 out of 5 KCNE1-/- hearts but not WT (n = 5). EADs were especially frequent following temporary cessations of ventricular pacing. (4) Monomorphic VT lasting 1.36 +/- 0.2 s in 5 out of 5 KCNE1-/- hearts, following premature stimuli but not WT (n = 5). (5) Epicardial APD alternans. Perfusion of KCNE1-/- hearts with 1 mum nifedipine induced potentially anti-arrhythmic changes including: (1) restored epicardial APD90 (from 57.1 +/- 0.5 ms to 42.3 +/- 0.4 ms, P < 0.001; n = 5); (2) altered DeltaAPD90 to values (11.2 +/- 2.6) close to WT (P > 0.05; n = 5); (3) EAD suppression during both spontaneous activity and following cessation of ventricular pacing (n = 5) to give similar features to WT controls (n = 5); (4) suppression of programmed electrical stimulation-induced VT; and (5) suppression of APD alternans. These findings suggest arrhythmic effects of reduced outward currents expected in KCNE1-/- hearts and their abolition by antagonism of inward L-type Ca2+ current.

Effects of flecainide and quinidine on arrhythmogenic properties of Scn5a+/Delta murine hearts modelling long QT syndrome 3.

Long QT3 (LQT3) syndrome is associated with incomplete Na+ channel inactivation, abnormal repolarization kinetics and prolonged cardiac action potential duration (APD). Electrophysiological effects of flecainide and quinidine were compared in Langendorff-perfused wild-type (WT), and genetically modified (Scn5a+/Delta) murine hearts modelling LQT3. Extra stimuli (S2) following trains of pacing stimuli (S1) applied to the right ventricular epicardium triggered ventricular tachycardia (VT) in 16 out of 28 untreated Scn5a+/Delta and zero out of 12 WT hearts. Paced electrogram fractionation analysis then demonstrated increased electrogram durations (EGD), expressed as EGD ratios, in arrhythmogenic Scn5a+/Delta hearts, and prolonged ventricular effective refractory periods in initially non-arrhythmogenic Scn5a+/Delta hearts. Nevertheless, comparisons of epicardial and endocardial monophasic action potential recordings demonstrated negative transmural repolarization gradients in both groups, giving DeltaAPD(90) values at 90% repolarization of -20.88 +/- 1.93 ms (n = 11) and -16.91 +/- 1.43 ms (n = 23), respectively. Flecainide prevented initiation of VT in 13 out of 16 arrhythmogenic Scn5a+/Delta hearts, reducing EGD ratio and restoring DeltaAPD90 to + 7.55 +/- 2.24 ms (n = 9) (P < 0.05). VT occurred in four out of eight non-arrhythmogenic Scn5a+/Delta hearts in the presence of quinidine, which increased EGD ratio but left DeltaAPD90 unchanged. In contrast (P < 0.05), WT hearts had positive DeltaAPD90 values (+ 11.72 +/- 2.17 ms) (n = 20). Flecainide then increased arrhythmic tendency and EGD ratio but conserved DeltaAPD90; reduced EGD ratios and unaltered DeltaAPD90 values accompanied the lower arrhythmogenicity associated with quinidine treatment. In addition to the changes in EGD ratio shown by WT hearts, these findings attribute arrhythmogenesis and its modification by flecainide and quinidine to alterations in DeltaAPD90 in Scn5a+/Delta hearts. This is consistent with a hypothesis in which incomplete Na+ channel inactivation in Scn5a+/Delta hearts generates functional substrates dependent on altered refractoriness that cause abnormalities in activation and conduction of subsequent cardiac impulses. Any spatial heterogeneities between the epicardial and endocardial layers would thus cause fragmentation of the activation wavefront and contribute to electrogram spreading.

Effects of L-type Ca2+ channel antagonism on ventricular arrhythmogenesis in murine hearts containing a modification in the Scn5a gene modelling human long QT syndrome 3.

Ventricular arrhythmogenesis in long QT 3 syndrome (LQT3) involves both triggered activity and re-entrant excitation arising from delayed ventricular repolarization. Effects of specific L-type Ca2+ channel antagonism were explored in a gain-of-function murine LQT3 model produced by a DeltaKPQ 1505-1507 deletion in the SCN5A gene. Monophasic action potentials (MAPs) were recorded from epicardial and endocardial surfaces of intact, Langendorff-perfused Scn5a+/Delta hearts. In untreated Scn5a+/Delta hearts, epicardial action potential duration at 90% repolarization (APD90) was 60.0 +/- 0.9 ms compared with 46.9 +/- 1.6 ms in untreated wild-type (WT) hearts (P < 0.05; n = 5). The corresponding endocardial APD(90) values were 52.0 +/- 0.7 ms and 53.7 +/- 1.6 ms in Scn5a+/Delta and WT hearts, respectively (P > 0.05; n = 5). Epicardial early afterdepolarizations (EADs), often accompanied by spontaneous ventricular tachycardia (VT), occurred in 100% of MAPs from Scn5a+/Delta but not in any WT hearts (n = 10). However, EAD occurrence was reduced to 62 +/- 7.1%, 44 +/- 9.7%, 10 +/- 10% and 0% of MAPs following perfusion with 10 nm, 100 nm, 300 nm and 1 mum nifedipine, respectively (P < 0.05; n = 5), giving an effective IC50 concentration of 79.3 nm. Programmed electrical stimulation (PES) induced VT in all five Scn5a+/Delta hearts (n = 5) but not in any WT hearts (n = 5). However, repeat PES induced VT in 3, 2, 2 and 0 out of 5 Scn5a+/Delta hearts following perfusion with 10 nm, 100 nm, 300 nm and 1 mum nifedipine, respectively. Patch clamp studies in isolated ventricular myocytes from Scn5a+/Delta and WT hearts confirmed that nifedipine (300 nm) completely suppressed the inward Ca2+ current but had no effect on inward Na+ currents. No significant effects were seen on epicardial APD90, endocardial APD90 or ventricular effective refractory period in Scn5a+/Delta and WT hearts following perfusion with nifedipine at 1 nm, 10 nm, 100 nm, 300 nm and 1 microm nifedipine concentrations. We conclude that L-type Ca2+ channel antagonism thus exerts specific anti-arrhythmic effects in Scn5a+/Delta hearts through suppression of EADs.

Sinus node dysfunction following targeted disruption of the murine cardiac sodium channel gene Scn5a.

We have examined sino-atrial node (SAN) function in hearts from adult mice with heterozygous targeted disruption of the Scn5a gene to clarify the role of Scn5a-encoded cardiac Na+ channels in normal SAN function and the mechanism(s) by which reduced Na+ channel function might cause sinus node dysfunction. Scn5a+/- mice showed depressed heart rates and occasional sino-atrial (SA) block. Their isolated peripheral SAN pacemaker cells showed a reduced Na+ channel expression and slowed intrinsic pacemaker rates. Wild-type (WT) and Scn5a+/- SAN preparations exhibited similar activation patterns but with significantly slower SA conduction and frequent sino-atrial conduction block in Scn5a+/- SAN preparations. Furthermore, isolated WT and Scn5a+/- SAN cells demonstrated differing correlations between cycle length, maximum upstroke velocity and action potential amplitude, and cell size. Small myocytes showed similar, but large myocytes reduced pacemaker rates, implicating the larger peripheral SAN cells in the reduced pacemaker rate that was observed in Scn5a+/- myocytes. These findings were successfully reproduced in a model that implicated i(Na) directly in action potential propagation through the SAN and from SAN to atria, and in modifying heart rate through a coupling of SAN and atrial cells. Functional alterations in the SAN following heterozygous-targeted disruption of Scn5a thus closely resemble those observed in clinical sinus node dysfunction. The findings accordingly provide a basis for understanding of the role of cardiac-type Na+ channels in normal SAN function and the pathophysiology of sinus node dysfunction and suggest new potential targets for its clinical management.

Mouse model of SCN5A-linked hereditary Lenègre's disease: age-related conduction slowing and myocardial fibrosis.

BACKGROUND: We have previously linked hereditary progressive cardiac conduction defect (hereditary Lenègre's disease) to a loss-of-function mutation in the gene encoding the main cardiac Na+ channel, SCN5A. In the present study, we investigated heterozygous Scn5a-knockout mice (Scn5a+/- mice) as a model for hereditary Lenègre's disease. METHODS AND RESULTS: In Scn5a+/- mice, surface ECG recordings showed age-related lengthening of the P-wave and PR- and QRS-interval duration, coinciding with previous observations in patients with Lenègre's disease. Old but not young Scn5a+/- mice showed extensive fibrosis of their ventricular myocardium, a feature not seen in wild-type animals. In old Scn5a+/- mice, fibrosis was accompanied by heterogeneous expression of connexin 43 and upregulation of hypertrophic markers, including beta-MHC and skeletal alpha-actin. Global connexin 43 expression as assessed with Western blots was similar to wild-type mice. Decreased connexin 40 expression was seen in the atria. Using pangenomic microarrays and real-time PCR, we identified in Scn5a+/- mice an age-related upregulation of genes encoding Atf3 and Egr1 transcription factors. Echocardiography and hemodynamic investigations demonstrated conserved cardiac function with aging and lack of ventricular hypertrophy. CONCLUSIONS: We conclude that Scn5a+/- mice convincingly recapitulate the Lenègre's disease phenotype, including progressive impairment with aging of atrial and ventricular conduction associated with myocardial rearrangements and fibrosis. Our work provides the first demonstration that a monogenic ion channel defect can progressively lead to myocardial structural anomalies.

A functional analysis of mouse models of cardiac disease through metabolic profiling.

Since the completion of the human and mouse genomes, the focus in mammalian biology has been on assessing gene function. Tools are needed for assessing the phenotypes of the many mouse models that are now being generated, where genes have been "knocked out," "knocked in," or mutated, so that gene expression can be understood in its biological context. Metabolic profiling of cardiac tissue through high resolution NMR spectroscopy in conjunction with multivariate statistics has been used to classify mouse models of cardiac disease. The data sets included metabolic profiles from mouse models of Duchenne muscular dystrophy, two models of cardiac arrhythmia, and one of cardiac hypertrophy. The metabolic profiles demonstrate that the strain background is an important component of the global metabolic phenotype of a mouse, providing insight into how a given gene deletion may result in very different responses in diverse populations. Despite these differences associated with strain, multivariate statistics were capable of separating each mouse model from its control strain, demonstrating that metabolic profiles could be generated for each disease. Thus, this approach is a rapid method of phenotyping mouse models of disease.

Paced electrogram fractionation analysis of arrhythmogenic tendency in DeltaKPQ Scn5a mice.

INTRODUCTION: Gain-of-function mutations within Scn5a, including the DeltaKPQ 1505-1507 deletion in the inactivation domain compromising myocardial repolarization, are implicated in human long QT 3 syndrome (LQT3), associated with ventricular arrhythmogenesis and sudden death. METHODS AND RESULTS: Patch clamp studies on isolated ventricular Scn5a+/Delta myocytes from DeltaKPQ mice produced by homologous recombination in embryonic stem (ES) cells confirmed such altered electrophysiological properties of the mutant channel. Programmed electrical stimulation (PES) with decremental pacing from the basal right ventricular epicardial surface and paced electrogram fractionation analysis (PEFA) of electrograms recorded from the basal left ventricular epicardial surface of Langendorff-perfused whole heart preparations demonstrated ventricular tachycardia (VT) in 8 of 9 Scn5a+/Delta mutant (but no Scn5a+/+ (wild-type (WT)) controls; n = 17), with increased electrogram durations (EGD) and more dispersed conduction curves. Isoproterenol (100 nM) was without effect on tachycardic Scn5a+/Delta hearts (n = 9) yet propranolol (1 microM) prevented VT in all isoproterenol-infused WT control (n = 4) but no Scn5a+/Delta hearts (n = 4). Furthermore propranolol itself increased EGD and dispersion in Scn5a+/Delta hearts. In contrast, mexiletine (10 microM) suppressed VTs in 4 of 5 Scn5a+/Delta hearts without altering EGD or dispersion. CONCLUSION: Beta-adrenoreceptor blockade does not confer an antiarrhythmic effect and may even enhance arrhythmogenesis by increasing reentrant substrate in Scn5a+/Delta hearts while mexiletine protects against VT without modifying conduction characteristics. Together these findings permit a scheme where VT in LQT3 is initiated by triggered mechanisms but propagated by reentry.