Pharmacogenomics of anesthetic drugs in transgenic LQT1 and LQT2 rabbits reveal genotype-specific differential effects on cardiac repolarization

Anesthetic agents prolong cardiac repolarization by blocking ion currents. However, the clinical relevance of this blockade in subjects with reduced repolarization reserve is unknown. We have generated transgenic long QT syndromes type 1 (LQT1) and type 2 (LQT2) rabbits that lack slow delayed rectifier K+ currents (IKs) or rapidly activating K+ currents (IKr) and used them as a model system to detect the channel-blocking properties of anesthetic agents. Therefore, LQT1, LQT2, and littermate control (LMC) rabbits were administered isoflurane, thiopental, midazolam, propofol, or ketamine, and surface ECGs were analyzed. Genotype-specific heart rate correction formulas were used to determine the expected QT interval at a given heart rate. The QT index (QTi) was calculated as percentage of the observed QT/expected QT. Isoflurane, a drug that blocks IKs) prolonged the QTi only in LQT2 and LMC but not in LQT1 rabbits. Midazolam, which blocks inward rectifier K+ current (IK1), prolonged the QTi in both LQT1 and LQT2 but not in LMC. Thiopental, which blocks both IKs and IK1, increased the QTi in LQT2 and LMC more than in LQT1. By contrast, ketamine, which does not block IKr, IKs, or IK1, did not alter the QTi in any group. Finally, anesthesia with isoflurane or propofol resulted in lethal polymorphic ventricular tachycardia (pVT) in three out of nine LQT2 rabbits. Transgenic LQT1 and LQT2 rabbits could serve as an in vivo model in which to examine the pharmacogenomics of drug-induced QT prolongation of anesthetic agents and their proarrhythmic potential. Transgenic LQT2 rabbits developed pVT under isoflurane and propofol, underlining the proarrhythmic risk of IKs blockers in subjects with reduced IKr.     

Overlapping LQT1 and LQT2 phenotype in a patient with long QT syndrome associated with loss-of-function variations in KCNQ1 and KCNH2

Long QT syndrome (LQTS) is an inherited disorder characterized by prolonged QT intervals and potentially life-threatening arrhythmias. Mutations in 12 different genes have been associated with LQTS. Here we describe a patient with LQTS who has a mutation in KCNQ1 as well as a polymorphism in KCNH2. The proband (MMRL0362), a 32-year-old female, exhibited multiple ventricular extrasystoles and one syncope. Her ECG (QT interval corrected for heart rate (QTc) = 518ms) showed an LQT2 morphology in leads V4-V6 and LQT1 morphology in leads V1-V2. Genomic DNA was isolated from lymphocytes. All exons and intron borders of 7 LQTS susceptibility genes were amplified and sequenced. Variations were detected predicting a novel missense mutation (V110I) in KCNQ1, as well as a common polymorphism in KCNH2 (K897T). We expressed wild-type (WT) or V110I Kv7.1 channels in CHO-K1 cells cotransfected with KCNE1 and performed patch-clamp analysis. In addition, WT or K897T Kv11.1 were also studied by patch clamp. Current-voltage (I-V) relations for V110I showed a significant reduction in both developing and tail current densities compared with WT at potentials >+20 mV (p < 0.05; n = 8 cells, each group), suggesting a reduction in IKs currents. K897T- Kv11.1 channels displayed a significantly reduced tail current density compared with WT-Kv11.1 at potentials >+10 mV. Interestingly, channel availability assessed using a triple-pulse protocol was slightly greater for K897T compared with WT (V0.5 = -53.1 ± 1.13 mV and -60.7 ± 1.15 mV for K897T and WT, respectively; p < 0.05). Comparison of the fully activated I-V revealed no difference in the rectification properties between WT and K897T channels. We report a patient with a loss-of-function mutation in KCNQ1 and a loss-of-function polymorphism in KCNH2. Our results suggest that a reduction of both IKr and IKs underlies the combined LQT1 and LQT2 phenotype observed in this patient.     

兔LQT2模型心室肌复极化的性别差异

本研究旨在探讨长QT综合征（long QT syndromes,LQTS）室性心律失常发生的性别差异及其电生理机制,初步观察了不同性别兔LQT2模型左心事原已存在的电生理异质性和心事复极动力学的特征。实验分为3组,上下常组以标准台氏液灌流;LQT2模型组给予含100gmol／L dl-sotalol的台式液灌流;LQT2模型＋低钾组给了含3．0mmol／LKCl、100μmol／L dl-sotalol的台式液灌流。采用冠状动脉旋支灌注兔左室心肌楔形组织块标本,应用浮置玻璃微电极记录技术进行记录。给予基础刺激周长（basic cycle length,BCL）为500、l000和2000ms的S1刺激,同步记录心事肌内膜侧、外膜侧细胞动作电位,并记录跨壁心电图：在BCL为500和1000ms时加用S2程序刺激以记录动作电位时程（action potential duration,APD）恢复曲线。研究发现：在不同刺激频率时,3组实验雌兔心肌细胞的跨壁复极化离散（transmural dispersion of repolarization,TDR）、APD恢复曲线斜率均大于雄兔,有显著性差异（P〈0．05）,并呈频率依赖性;LQT2模型组及LQT2模型＋低钾组雌雄兔TDR、APD恢复曲线斜率较正常组明显增人（P〈0．01）。BCL为1000ms时,LQT2模型组雌兔7例中1例发生尖端扭转性窀性心动过速（torsade de pointes,TdP）;LQT2模型＋低钾组雌兔7例中5例诱发TdP,雄兔7例中2例诱发TdP,有显著性差异（P〈0．05）。结果提示：LQT2模型心肌原已存在的电生理异质性和动态异质性均有明显的性别差异,并≯频率依赖性。存LQT2模型中,TDR以及APD恢复曲线斜率的增大可能是雌性动物较雄性更易发生尖端扭转性心律失常的原因。     

Potential biomarkers for ischemic heart damage identified in mitochondrial proteins by comparative proteomics

We used proteomics to detect regional differences in protein expression levels from mitochondrial fractions of control, ischemia-reperfusion (IR), and ischemic preconditioned (IPC) rabbit hearts. Using 2-DE, we identified 25 mitochondrial proteins that were differentially expressed in the IR heart compared with the control and IPC hearts. For three of the spots, the expression patterns were confirmed by Western blotting analysis. These proteins included 3-hydroxybutyrate dehydrogenase, prohibitin, 2-oxoglutarate dehydrogenase, adenosine triphosphate synthases, the reduced form of nicotinamide adenine dinucleotide (NADH) oxidoreductase, translation elongation factor, actin alpha, malate dehydrogenase, NADH dehydrogenase, pyruvate dehydrogenase and the voltage-dependent anion channel. Interestingly, most of these proteins are associated with the mitochondrial respiratory chain and energy metabolism. The successful use of multiple techniques, including 2-DE, MALDI-TOF-MS and Western blotting analysis demonstrates that proteomic analysis provides appropriate means for identifying cardiac markers for detection of ischemia-induced cardiac injury.     

Spatially Discordant Alternans and Arrhythmias in Tachypacing-Induced Cardiac Myopathy in Transgenic LQT1 Rabbits: The Importance of IKs and Ca2+ Cycling

BackgroundRemodeling of cardiac repolarizing currents, such as the downregulation of slowly activating K⁺ channels (I_Ks), could underlie ventricular fibrillation (VF) in heart failure (HF). We evaluated the role of I_ks remodeling in VF susceptibility using a tachypacing HF model of transgenic rabbits with Long QT Type 1 (LQT1) syndrome.ConclusionsCompared with LMC-TICM, LQT1-TICM rabbits exhibit steepened APD restitution and complex DA modulated by Ca²⁺. Our results strongly support the contention that the downregulation of I_Ks in HF increases Ca²⁺ dependent alternans and thereby the risk of VF.     

Thapsigargin selectively rescues the trafficking defective LQT2 channels G601S and F805C

Several mutations in the human ether-a-go-go-related K+ channel gene (HERG or KCNH2) cause long QT syndrome (LQT2) by reducing the intracellular transport (trafficking) of the channel protein to the cell surface. Drugs that bind to and block HERG channels (i.e. E4031) rescue the surface expression of some trafficking defective LQT2 mutations. Because these drugs potently block HERG current, their ability to correct congenital LQT is confounded by their risk of causing acquired LQT. We tested the hypothesis that pharmacological rescue can occur without HERG channel block. Thapsigargin (1 microM), a sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor, rescued the surface expression of G601S, and it did so without blocking current. Thapsigargin-induced rescue and E4031-induced rescue caused complex glycosylation that was evident within 3 h of drug exposure. Disruption of the Golgi apparatus with brefeldin A prevented thapsigargin- and E4031-induced rescue of IG01S. Confocal imaging showed that G601S protein is predominantly "trapped" intracellularly and that both thapsigargin and E4031 promote its relocation to the surface membrane. We also studied two other trafficking defective LQT2 mutations. Thapsigargin rescued the C terminus mutation F805C but not N470D, whereas E4031 rescued N470D but not F805C. Other sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitors did not rescue G601S or F805C. This study 1) supports the hypothesis that the LQT2 trafficking defective phenotype can be reversed without blocking the channel; 2) demonstrates pharmacological rescue of a C terminus LQT2 mutation; and 3) shows that thapsigargin can correct trafficking defective phenotypes in more than one channel type and disease (i.e. LQT2 and cystic fibrosis).     

Lipogenesis in rabbit isolated fat-cells

1. Fat-cells isolated from rabbit perirenal adipose tissue were incubated with the following U-(14)C-labelled substrates: 5mm-glucose (+insulin), 5mm-pyruvate, 5mm-lactate, 5mm-glucose+5mm-acetate (+insulin), and the relative rates of incorporation of these substrates into glyceride fatty acids determined. In general total rates of fatty acid synthesis were similar whatever substrate was supplied to the cells. 2. Rabbit fat-cells incorporated [U-(14)C]acetate into fatty acids and CO(2) as well in the absence of glucose as in the presence of this substrate. 3. The disposition of the utilization of glucose-derived carbon through various metabolic pathways was determined. 4. Extramitochondrial and mitochondrial activities were determined for 11 enzymes. The cells contained a very low activity of pyruvate carboxylase, undetectable NADP-malate dehydrogenase activity and a high mitochondrial phosphoenolpyruvate carboxylase activity. 5. Various rabbit fat-cell metabolic parameters based on the measurement of (14)C incorporation and enzyme activity were compared with the same parameters previously measured in rat and guinea-pig fat-cells. In general guinea pig occupied a position between rat and rabbit with respect to these parameters. 6. The profiles of substrate incorporation into fatty acids and of relative enzyme activities in rabbit fat-cells indicated that the operation of a ;citrate-cleavage' pathway may not be obligatory for the supply of lipogenic acetyl units.     

Cardiac Metabolic Pathways Affected in the Mouse Model of Barth Syndrome

Cardiolipin (CL) is a mitochondrial phospholipid essential for electron transport chain (ETC) integrity. CL-deficiency in humans is caused by mutations in the tafazzin (Taz) gene and results in a multisystem pediatric disorder, Barth syndrome (BTHS). It has been reported that tafazzin deficiency destabilizes mitochondrial respiratory chain complexes and affects supercomplex assembly. The aim of this study was to investigate the impact of Taz-knockdown on the mitochondrial proteomic landscape and metabolic processes, such as stability of respiratory chain supercomplexes and their interactions with fatty acid oxidation enzymes in cardiac muscle. Proteomic analysis demonstrated reduction of several polypeptides of the mitochondrial respiratory chain, including Rieske and cytochrome c1 subunits of complex III, NADH dehydrogenase alpha subunit 5 of complex I and the catalytic core-forming subunit of F₀F₁-ATP synthase. Taz gene knockdown resulted in upregulation of enzymes of folate and amino acid metabolic pathways in heart mitochondria, demonstrating that Taz-deficiency causes substantive metabolic remodeling in cardiac muscle. Mitochondrial respiratory chain supercomplexes are destabilized in CL-depleted mitochondria from Taz knockdown hearts resulting in disruption of the interactions between ETC and the fatty acid oxidation enzymes, very long-chain acyl-CoA dehydrogenase and long-chain 3-hydroxyacyl-CoA dehydrogenase, potentially affecting the metabolic channeling of reducing equivalents between these two metabolic pathways. Mitochondria-bound myoglobin was significantly reduced in Taz-knockdown hearts, potentially disrupting intracellular oxygen delivery to the oxidative phosphorylation system. Our results identify the critical pathways affected by the Taz-deficiency in mitochondria and establish a future framework for development of therapeutic options for BTHS.     

Pronounced Effects of HERG-Blockers E-4031 and Erythromycin on APD,Spatial APD Dispersion and Triangulation in Transgenic Long-QT Type 1 Rabbits

Background

Prolongation of action potential duration (APD), increased spatial APD dispersion, and triangulation are major factors promoting drug-induced ventricular arrhythmia. Preclinical identification of HERG/I_Kr-blocking drugs and their pro-arrhythmic potential, however, remains a challenge. We hypothesize that transgenic long-QT type 1 (LQT1) rabbits lacking repolarizing I_Ks current may help to sensitively detect HERG/I_Kr-blocking properties of drugs.

Methods

Hearts of adult female transgenic LQT1 and wild type littermate control (LMC) rabbits were Langendorff-perfused with increasing concentrations of HERG/I_Kr-blockers E-4031 (0.001–0.1 µM, n = 9/7) or erythromycin (1–300 µM, n = 9/7) and APD, APD dispersion, and triangulation were analyzed.

Results

At baseline, APD was longer in LQT1 than in LMC rabbits in LV apex and RV mid. Erythromycin and E-4031 prolonged APD in LQT1 and LMC rabbits in all positions. However, erythromycin-induced percentaged APD prolongation related to baseline (%APD) was more pronounced in LQT1 at LV base-lateral and RV mid positions (100 µM, LQT1, +40.6±9.7% vs. LMC, +24.1±10.0%, p<0.05) and E-4031-induced %APD prolongation was more pronounced in LQT1 at LV base-lateral (0.01 µM, LQT1, +29.6±10.6% vs. LMC, +19.1±3.8%, p<0.05) and LV base-septal positions. Moreover, erythromycin significantly increased spatial APD dispersion only in LQT1 and increased triangulation only in LQT1 in LV base-septal and RV mid positions. Similarly, E-4031 increased triangulation only in LQT1 in LV apex and base-septal positions.

Conclusions

E-4031 and erythromycin prolonged APD and increased triangulation more pronouncedly in LQT1 than in LMC rabbits. Moreover, erythromycin increased APD dispersion only in LQT1, indicating that transgenic LQT1 rabbits could serve as sensitive model to detect HERG/I_Kr-blocking properties of drugs.     

Phenotype guided characterization and molecular analysis of Indian patients with long QT syndromes

BackgroundLong QT syndromes (LQTS) are characterized by prolonged QTc interval on electrocardiogram (ECG) and manifest with syncope, seizures or sudden cardiac death. Long QT 1–3 constitute about 75% of all inherited LQTS. We classified a cohort of Indian patients for the common LQTS based on T wave morphology and triggering factors to prioritize the gene to be tested. We sought to identify the causative mutations and mutation spectrum, perform genotype-phenotype correlation and screen family members.MethodsThirty patients who fulfilled the criteria were enrolled. The most probable candidate gene among KCNQ1, KCNH2 and SCN5A were sequenced.ResultsOf the 30 patients, 22 were classified at LQT1, two as LQT2 and six as LQT3. Mutations in KCNQ1 were identified in 17 (77%) of 22 LQT1 patients, KCNH2 mutation in one of two LQT2 and SCN5A mutations in two of six LQT3 patients. We correlated the presence of the specific ECG morphology in all mutation positive cases. Eight mutations in KCNQ1 and one in SCN5A were novel and predicted to be pathogenic by in-silico analysis. Of all parents with heterozygous mutations, 24 (92%) of 26 were asymptomatic. Ten available siblings of nine probands were screened and three were homozygous and symptomatic, five heterozygous and asymptomatic.ConclusionsThis study in a cohort of Asian Indian patients highlights the mutation spectrum of common Long QT syndromes. The clinical utility for prevention of unexplained sudden cardiac deaths is an important sequel to identification of the mutation in at-risk family members.     

The dominant negative LQT2 mutation A561V reduces wild-type HERG expression

HERG(1) K(+) channel mutations are responsible for one form of dominantly inherited long QT syndrome (LQT). Some LQT mutations exert a dominant negative effect on wild-type current expression. To investigate mechanisms of dominant-negative behavior, we co-expressed wild-type HERG with the A561V mutant in mammalian cells. Transfection with various cDNA ratios produced HERG K(+) current densities that approached a predicted binomial distribution where mutant and wild-type subunits co-assemble in a tetramer with nearly complete dominance. Using C terminus myc-tagged wild-type HERG we specifically followed the mutant's effect on full-length wild-type HERG protein expression. Co-expression with A561V reduced the abundance of full-length wild-type HERG protein comparable to the current reduction. Reduction of wild-type protein was due to decreased synthesis and increased turnover. Conditions facilitating protein folding (growth at 30 degrees C, or in 10% glycerol) resulted in partial rescue from the dominant effect, as did the 26 S proteosome inhibitor ALLN. Thus, for A561V, dominant negative effects result from assembly of wild-type subunits with mutant very early in production leading to rapid recognition of mutant channels and targeting for proteolysis. These results establish protein misfolding, cellular proofreading, and bystander involvement as contributing mechanisms for dominant effects in LQT2.     

An LQT mutant minK alters KvLQT1 trafficking

Cardiac I_Ks, the slowly activated delayed-rectifier K⁺ current, is produced by the protein complex composed of - and -subunits: KvLQT1 and minK. Mutations of genes encoding KvLQT1 and minK are responsible for the hereditary long QT syndrome (loci LQT1 and LQT5, respectively). MinK-L51H fails to traffic to the cell surface, thereby failing to produce effective I_Ks. We examined the effects that minK-L51H and an endoplasmic reticulum (ER)-targeted minK (minK-ER) exerted over the electrophysiology and biosynthesis of coexpressed KvLQT1. Both minK-L51H and minK-ER were sequestered primarily in the ER as confirmed by lack of plasma membrane expression. Glycosylation and immunofluorescence patterns of minK-L51H were qualitatively different for minK-ER, suggesting differences in trafficking. Cotransfection with the minK mutants resulted in reduced surface expression of KvLQT1 as assayed by whole cell voltage clamp and immunofluorescence. MinK-L51H reduced current amplitude by 91% compared with wild-type (WT) minK/KvLQT1, and the residual current was identical to KvLQT1 without minK. The phenotype of minK-L51H on I_Ks was not dominant because coexpressed WT minK rescued the current and surface expression. Collectively, our data suggest that ER quality control prevents minK-L51H/KvLQT1 complexes from trafficking to the plasma membrane, resulting in decreased I_Ks. This is the first demonstration that a minK LQT mutation is capable of conferring trafficking defects onto its associated -subunit. potassium channel; hereditary arrhythmia; electrophysiology; protein interaction     

The congenital long QT syndrome Type 3: An update

Congenital long QT syndrome type 3 (LQT3) is the third in frequency compared to the 15 forms known currently of congenital long QT syndrome (LQTS). Cardiac events are less frequent in LQT3 when compared with LQT1 and LQT2, but more likely to be lethal; the likelihood of dying during a cardiac event is 20% in families with an LQT3 mutation and 4% with either an LQT1 or an LQT2 mutation. LQT3 is consequence of mutation of gene SCN5A which codes for the Nav1.5 Na⁺ channel α-subunit and electrocardiographically characterized by a tendency to bradycardia related to age, prolonged QT/QTc interval (mean QTc value 478 ± 52 ms), accentuated QT dispersion consequence of prolonged ST segment, late onset of T wave and frequent prominent U wave because of longer repolarization of the M cell across left ventricular wall.     

Mechanistic basis for LQT1 caused by S3 mutations in the KCNQ1 subunit of IKs

Long QT interval syndrome (LQTS) type 1 (LQT1) has been reported to arise from mutations in the S3 domain of KCNQ1, but none of the seven S3 mutations in the literature have been characterized with respect to trafficking or biophysical deficiencies. Surface channel expression was studied using a proteinase K assay for KCNQ1 D202H/N, I204F/M, V205M, S209F, and V215M coexpressed with KCNE1 in mammalian cells. In each case, the majority of synthesized channel was found at the surface, but mutant I_Ks current density at +100 mV was reduced significantly for S209F, which showed ∼75% reduction over wild type (WT). All mutants except S209F showed positively shifted V_1/2’s of activation and slowed channel activation compared with WT (V_1/2 = +17.7 ± 2.4 mV and τ_activation of 729 ms at +20 mV; n = 18). Deactivation was also accelerated in all mutants versus WT (126 ± 8 ms at −50 mV; n = 27), and these changes led to marked loss of repolarizing currents during action potential clamps at 2 and 4 Hz, except again S209F. KCNQ1 models localize these naturally occurring S3 mutants to the surface of the helices facing the other voltage sensor transmembrane domains and highlight inter-residue interactions involved in activation gating. V207M, currently classified as a polymorphism and facing lipid in the model, was indistinguishable from WT I_Ks. We conclude that S3 mutants of KCNQ1 cause LQTS predominantly through biophysical effects on the gating of I_Ks, but some mutants also show protein stability/trafficking defects, which explains why the kinetic gain-of-function mutation S209F causes LQT1.     

Analysis of the cyclic nucleotide binding domain of the HERG potassium channel and interactions with KCNE2

Mutations in the cyclic nucleotide binding domain (CNBD) of the human ether-a-go-go-related gene (HERG) K+ channel are associated with LQT2, a form of hereditary Long QT syndrome (LQTS). Elevation of cAMP can modulate HERG K+ channels both by direct binding and indirect regulation through protein kinase A. To assess the physiological significance of cAMP binding to HERG, we introduced mutations to disrupt the cyclic nucleotide binding domain. Eight mutants including two naturally occurring LQT2 mutants V822M and R823W were constructed. Relative cAMP binding capacity was reduced or absent in CNBD mutants. Mutant homotetramers carry little or no K+ current despite normal protein abundance and surface expression. Co-expression of mutant and wild-type HERG resulted in currents with altered voltage dependence but without dominant current suppression. The data from co-expression of V822M and wild-type HERG best fit a model where one normal subunit within a tetramer allows nearly normal current expression. The presence of KCNE2, an accessory protein that associates with HERG, however, conferred a partially dominant current suppression by CNBD mutants. Thus KCNE2 plays a pivotal role in determining the phenotypic severity of some forms of LQT2, which suggests that the CNBD of HERG may be involved in its interaction with KCNE2.     

Proteomic analysis of mitochondria reveals a metabolic switch from fatty acid oxidation to glycolysis in the failing heart

This work characterizes the mitochondrial proteomic profile in the failing heart and elucidates the molecular basis of mitochondria in heart failure. Heart failure was induced in rats by myocardial infarction, and mitochondria were isolated from hearts by differential centrifugation. Using two-dimen- sional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry, a system biology approach was employed to investigate differences in mitochondrial proteins between normal and failing hearts. Mass spectrometry identified 27 proteins differentially expressed that involved in energy metabolism. Among those, the up-regulated proteins included tricarboxylic acid cycle enzymes and pyruvate dehydrogenase complex subunits while the down-regulated proteins were involved in fatty acid oxidation and the OXPHOS complex. These results suggest a substantial metabolic switch from free fatty acid oxidation to glycolysis in heart failure and provide molecular evidence for alterations in the structural and functional parameters of mitochondria that may contribute to cardiac dysfunction during ischemic injury.     

Ischemic preconditioning exaggerates cardiac damage in PKC-delta null mice

Ischemic preconditioning confers cardiac protection during subsequent ischemia-reperfusion, in which protein kinase C (PKC) is believed to play an essential role, but controversial data exist concerning the PKC-delta isoform. In an accompanying study (26), we described metabolic changes in PKC-delta knockout mice. We now wanted to explore their effect on early preconditioning. Both PKC-delta(-/-) and PKC-delta(+/+) mice underwent three cycles of 5-min left descending artery occlusion/5-min reperfusion, followed by 30-min occlusion and 2-h reperfusion. Unexpectedly, preconditioning exaggerated ischemia-reperfusion injury in PKC-delta(-/-) mice. Whereas ischemic preconditioning increased superoxide anion production in PKC-delta(+/+) hearts, no increase in reactive oxygen species was observed in PKC-delta(-/-) hearts. Proteomic analysis of preconditioned PKC-delta(+/+) hearts revealed profound changes in enzymes related to energy metabolism, e.g., NADH dehydrogenase and ATP synthase, with partial fragmentation of these mitochondrial enzymes and of the E(2) component of the pyruvate dehydrogenase complex. Interestingly, fragmentation of mitochondrial enzymes was not observed in PKC-delta(-/-) hearts. High-resolution NMR analysis of cardiac metabolites demonstrated a similar rise of phosphocreatine in PKC-delta(+/+) and PKC-delta(-/-) hearts, but the preconditioning-induced increase in phosphocholine, alanine, carnitine, and glycine was restricted to PKC-delta(+/+) hearts, whereas lactate concentrations were higher in PKC-delta(-/-) hearts. Taken together, our results suggest that reactive oxygen species generated during ischemic preconditioning might alter mitochondrial metabolism by oxidizing key mitochondrial enzymes and that metabolic adaptation to preconditioning is impaired in PKC-delta(-/-) hearts.     

Mutational screening of SCN5A linked disorders in Polish patients and their family members

Mutations in SCN5A lead to a broad spectrum of phenotypes, including the Long QT syndrome, Brugada syndrome, Idiopathic ventricular fibrillation (IVF), Sudden infant death syndrome (SIDS) (probably regarded as a form of LQT3), Sudden unexplained nocturnal death syndrome (SUNDS) and isolated progressive cardiac conduction defect (PCCD) (Lev-Lenegre disease). Brugada Syndrome (BS) is a form of idiopathic ventricular fibrillation characterized by the right bundle-branch block pattern and ST elevation (STE) in the right precordial leads of the ECG. Mutations of the cardiac sodium channel SCN5A cause the disorder, and an implantable cardioverter-defibrillator is often recommended for affected individuals. In this study sequences of the coding region of the SCN5A gene were analysed in patients with the LQT3, Brugada Syndrome and other arrythmogenic disorders. Different mSSCP patterns are described with no disease-related SSCP conformers in any sample. Direct sequencing of the SCN5A gene confirmed the absence of mutations. This suggests that the analysed region of the SCN5A gene is not commonly involved in the pathogenesis of the Brugada Syndrome and associated disorders.