Patrick Friederich

Sudden infant death syndrome and long QT syndrome: an epidemiological and genetic study

Sudden infant death syndrome (SIDS) is a frequent cause of death among infants. The etiology of SIDS is unknown and several theories, including fatal ventricular arrhythmias, have been suggested. We performed an epidemiological and genetic investigation of SIDS victims to estimate the presence of inherited long QT syndrome (LQTS) as a contributor for SIDS. Forty-one consecutively collected and unrelated SIDS cases were characterized by clinical and epidemiological criteria. We performed a comprehensive gene mutation screening with single-strand conformation polymorphism analysis and sequencing techniques of the most relevant LQTS genes to assess mutation frequencies. In vitro characterization of identified mutants was subsequently performed by heterologous expression experiments in Chinese hamster ovary cells and in Xenopus laevis oocytes. A positive family history for LQTS was suspected by mild prolonged Q-T interval in family members in 2 of the 41 SIDS cases (5%). In neither case, a family history of sudden cardiac death was present nor a mutation could be identified after thorough investigation. In another SIDS case, a heterozygous missense mutation (H105L) was identified in the N-terminal region of the KCNQ1 (LQTS 1) gene. Despite absence of this mutation in the general population and a high conservational degree of the residue H105 during evolution, electrophysiological investigations failed to show a significant difference between wild-type and KCNQ1H105L/minK-mediated IKs currents. Our data suggest that a molecular diagnosis of SIDS related to LQTS genes is rare and that, even when an ion channel mutation is identified, this should be regarded with caution unless a pathophysiological relationship between SIDS and the electrophysiological characterization of the mutated ion channel has been demonstrated.

Local anesthetic interaction with human ether-a-go-go-related gene (HERG) channels: role of aromatic amino acids Y652 and F656.

Background:Human ether-a-go-go–related gene (HERG) potassium channels constitute a potential target involved in cardiotoxic side effects of amino-amide local anesthetics. The molecular interaction site of these low-affinity blockers with HERG channels is currently unknown. The aim of this study was to determine the effect of the mutations Y652A and F656A in the putative drug binding region of HERG on the inhibition by bupivacaine, ropivacaine, and mepivacaine. Methods:The authors examined the inhibition of wild-type and mutant HERG channels, transiently expressed in Chinese hamster ovary cells by bupivacaine, ropivacaine, and mepivacaine. Whole cell patch clamp recordings were performed at room temperature. Results:Inhibition of HERG wild-type and mutant channels by the different local anesthetics was concentration dependent, stereoselective, and reversible. The sensitivity decreased in the order bupivacaine > ropivacaine > mepivacaine for wild-type and mutant channels. The mutant channels were approximately 4–30 times less sensitive to the inhibitory action of the different local anesthetics than the wild-type channel. The concentration–response data were described by Hill functions (bupivacaine: wild-type IC50 = 22 ± 2 &mgr;m, n = 38; Y652A IC50 = 95 ± 5 &mgr;m, n = 31). The mutations resulted in a change of the stereoselectivity of HERG channel block by ropivacaine. The potency of the local anesthetics to inhibit wild-type and mutant channels correlated with the lipophilicity of the drug (r > 0.9). Conclusions:These results indicate that local anesthetics specifically but not exclusively interact with the aromatic residues Y652 and F656 in S6 of HERG channels.

Inhibition of human TREK-1 channels by bupivacaine.

Human TWIK-related K+ channels (TREK-1) stabilize the membrane potential (mp) of neurons and have a major role in the regulation of membrane excitability. In view of their physiological significance, interaction of bupivacaine with TREK-1 channels may be clinically important. Our aim was to characterize with the patch-clamp technique the properties of human TREK-1 channels and the effects of bupivacaine on these channels expressed in Chinese hamster ovary (CHO) cells. Transfection of CHO cells with TREK-1 channels (CHOTREK-1 cells) hyperpolarized the mp from −33 ± 13 to −78 ± 4 mV. The channels were stimulated by intracellular acidosis. Inhibition of TREK-1 channels by bupivacaine was reversible, concentration-dependent, voltage-independent, and increased with intracellular acidosis. Bupivacaine depolarized the mp of CHOTREK-1 cells in a reversible and concentration-dependent manner. Concentrations for channel inhibition and membrane depolarization were not linearly related (50% inhibitory concentration value for channel inhibition 370 ± 20 &mgr;M, Hill coefficient 1.8 ± 0.1, n = 51; 50% inhibitory concentration value for membrane depolarization 856 ± 14 &mgr;M, Hill coefficient 2.4 ± 0.1, mean ± SEM, n = 27). The results suggest that protonated bupivacaine elicits the observed effects via a site of interaction accessible from the intracellular space. Inhibition of TREK-1 channels and consecutive depolarization of the cell membrane by bupivacaine may contribute to blockade of neuronal signal conduction during regional anesthesia. IMPLICATIONS: The interaction of bupivacaine with human TREK-1 channels was studied with the patch-clamp technique. Bupivacaine inhibited TREK-1 channels and depolarized the membrane potential of cells expressing TREK-1 channels in a concentration-dependent and reversible manner. Both effects may contribute to conductance block caused by bupivacaine.

Long QT 1 mutation KCNQ1A344V increases local anesthetic sensitivity of the slowly activating delayed rectifier potassium current.

Background:Anesthesia in patients with long QT syndrome (LQTS) is a matter of concern. Congenital LQTS is most frequently caused by mutations in KCNQ1 (Kv7.1), whereas drug-induced LQTS is a consequence of HERG (human ether-a-go-go–related gene) channel inhibition. The aim of this study was to investigate whether the LQT1 mutation A344V in the S6 region of KCNQ1, at a position corresponding to the local anesthetic binding site in HERG, may render drug insensitive KCNQ1 channels into a toxicologically relevant target of these pharmacologic agents. This may suggest that LQTS constitutes not only a nonspecific but also a specific pharmacogenetic risk factor for anesthesia. Methods:The authors examined electrophysiologic and pharmacologic properties of wild-type and mutant KCNQ1 channels. The effects of bupivacaine, ropivacaine, and mepivacaine were investigated using two-electrode voltage clamp and whole cell patch clamp recordings. Results:The mutation A344V induced voltage-dependent inactivation in homomeric KCNQ1 channels and shifted the voltage dependence of KCNQ1/KCNE1 channel activation by +30 mV. The mutation furthermore increased the sensitivity of KCNQ1/KCNE1 channels for bupivacaine 22-fold (KCNQ1wt/KCNE1: IC50 = 2,431 ± 582 &mgr;m, n = 20; KCNQ1A344V/KCNE1: IC50 = 110 ± 9 &mgr;m, n = 24). Pharmacologic effects of the mutant channels were dominant when mutant and wild-type channels were coexpressed. Simulation of cardiac action potentials with the Luo-Rudy model yielded a prolongation of the cardiac action potential duration and induction of early afterdepolarizations by the mutation A344V that were aggravated by local anesthetic intoxication. Conclusions:The results indicate that certain forms of the LQTS may constitute a specific pharmacogenetic risk factor for regional anesthesia.

Molecular interaction of droperidol with human ether-a-go-go-related gene channels: prolongation of action potential duration without inducing early afterdepolarization.

Background:The cardiac safety of droperidol given at antiemetic doses is a matter of debate. Although droperidol potently inhibits human ether-a-go-go-related gene (HERG) channels, the molecular mode of this interaction is unknown. The role of amino acid residues typically mediating high-affinity block of HERG channels is unclear. It is furthermore unresolved whether droperidol at antiemetic concentrations induces action potential prolongation and arrhythmogenic early afterdepolarizations in cardiac myocytes. Methods:Molecular mechanisms of HERG current inhibition by droperidol were established using two-electrode voltage clamp recordings of Xenopus laevis oocytes expressing wild-type and mutant channels. The mutants T623A, S624A, V625A, Y652A, and F656A were generated by site-directed mutagenesis. The effect of droperidol on action potentials was investigated in cardiac myocytes isolated from guinea pig hearts using the patch clamp technique. Results:Droperidol inhibited currents through HERG wild-type channels with a concentration of half-maximal inhibition of 0.6–0.9 &mgr;m. Droperidol shifted the channel activation and the steady state inactivation toward negative potentials while channel deactivation was not affected. Current inhibition increased with membrane potential and with increasing duration of current activation. Inhibition of HERG channels was similarly reduced by all mutations. Droperidol at concentrations between 5 and 100 nm prolonged whereas concentrations greater than 300 nm shortened action potentials. Early afterdepolarizations were not observed. Conclusions:Droperidol is a high-affinity blocker of HERG channels. Amino acid residues typically involved in high-affinity block mediate droperidol effects. Patch clamp results and computational modeling allow the hypothesis that interaction with calcium currents may explain why droperidol at antiemetic concentrations prolongs the action potential without inducing early afterdepolarizations.

Amitriptyline is a potent blocker of human Kv1.1 and Kv7.2/7.3 channels.

BACKGROUND: Kv1.1 and Kv7.2/7.3 channels control excitability of neuronal cells. As hyperexcitability is a sign of neuropathic pain, epilepsy, and anxiety disorders, these channels may be important molecular targets of amitriptyline that cause pharmacological as well as toxicological effects by altering neuronal excitability. Since the molecular mechanisms underlying these effects of amitriptyline have not been fully elucidated, we aimed to characterize the interaction of amitriptyline with human Kv1.1 and Kv7.2/7.3 channels. We also intended to establish the interaction of amitriptyline with the Kv7.2/7.3 channel opener, retigabine. METHODS: Kv1.1 and Kv7.2/7.3 channels were expressed in human embryonic kidney cells and in Chinese hamster ovary cells. The effects of amitriptyline and retigabine were studied with the patch-clamp technique. RESULTS: Amitriptyline inhibited Kv1.1 and Kv7.2/7.3 channels in a concentration-dependent and reversible manner. The IC50-value was 22 ± 3 &mgr;M (n = 33) and 10 ± 1 &mgr;M (n = 40), respectively. Deactivating inward currents of Kv7.2/7.3 channels were inhibited with an IC50-value of 4.2 ± 0.6 &mgr;M (n = 32). Inhibition of Kv7.2/7.3 channels by amitriptyline reversibly depolarized the resting membrane potential. Retigabine reversed both the inhibitory action of amitriptyline on Kv7.2/7.3 channels as well as the depolarization of the membrane potential. CONCLUSIONS: Since amitriptyline inhibited Kv1.1 and Kv7.2/7.3 channels only at toxicologically relevant plasma concentrations, our results suggest a role for these channels in the neuroexcitatory side effects of amitriptyline. As the inhibitory effects of amitriptyline were reversed by retigabine, a combination of amitriptyline and retigabine could be of additional benefit in the therapy of neuropathic pain.

Structural Requirements of Human Ether-a-go-go– related Gene Channels for Block by Bupivacaine

Background: Local anesthetics interact with human ether-a-go-go–related gene (HERG) channels via the aromatic amino acids Y652 and F656 in the S6 region. This study aimed to establish whether the residues T623, S624, and V625 residing deeper within the pore are also involved in HERG channel block by bupivacaine. In addition, the study aimed to further define the role of the aromatic residues Y652 and F656 in bupivacaine inhibition by mutating these residues to threonine. Methods: Alanine and threonine mutants were generated by site-directed mutagenesis. Electrophysiologic and pharmacologic properties of wild-type and mutant HERG channels were established using two-electrode voltage-clamp recordings of Xenopus laevis oocytes expressing HERG channels. Results: Tail currents at −120 mV through HERG wild-type channels were inhibited with an IC50 value of 132 ± 22 &mgr;m (n = 33). Bupivacaine (300 &mgr;m) inhibited wild-type tail currents by 62 ± 12% (n = 7). Inhibition of HERG tail currents by bupivacaine (300 &mgr;m) was reduced by all mutations (P < 0.001). The effect was largest for F656A (inhibition 5 ± 2%, n = 6) in the lower S6 region and for T623A (inhibition 13 ± 4%, n = 9) near the selectivity filter. Introducing threonine at positions 656 and 652 significantly reduced inhibition by bupivacaine compared with HERG wild type (P < 0.001). Conclusions: The authors’ results indicate that not only the aromatic residues Y652 and F656 but also residues residing deeper within the pore and close to the selectivity filter of HERG channels are involved in inhibition of HERG channels by the low-affinity blocker bupivacaine.

Inhibition of Kv4.3/KChIP2.2 channels by bupivacaine and its modulation by the pore mutation Kv4.3V401I.

Background: The transient outward current Ito is an important repolarizing K current in human ventricular myocardium mediated by Kv4.3 and KChIP2.2 subunits. Inhibition of Ito by amino-amide local anesthetics may be involved in severe cardiotoxic side effects. This study elucidates the molecular mechanisms of bupivacaine interaction with complexes formed by Kv4.3 and KChIP2.2 as well as the modulatory effect of KChIP2.2. For this purpose, the pharmacologic effects of bupivacaine on Kv4.3wt/KChIP2.2 channels and on the pore mutant Kv4.3V401I were investigated. Methods: Kv4.3/KChIP2.2 cDNA was transiently expressed in Chinese hamster ovary cells. Site-directed mutagenesis and patch clamp experiments were performed to analyze the effects of bupivacaine on wild-type and mutant channels. Results: Inhibition of Kv4.3wt/KChIP2.2 channels by bupivacaine was concentration-dependent and reversible. The IC50s for inhibition of the charge conducted by Kv4.3wt/KChIP2.2 channels by bupivacaine and levobupivacaine were 55 ± 8 and 50 ± 5 &mgr;m, respectively. The local anesthetic accelerated macroscopic current decline of Kv4.3wt/KChIP2.2 and slowed recovery from inactivation without altering steady state inactivation. KChIP2.2 altered the response of Kv4.3wt channels to bupivacaine and bupivacaine modulated KChIP2.2 effects on Kv4.3wt channels. The pore mutation V401I slowed macroscopic current decline of Kv4.3 channels and recovery from inactivation, and it diminished modulation of gating by KChIP2.2. Bupivacaine inhibition of Kv4.3V401I resembled Kv4.3wt and was not changed by coexpression of KChIP2.2. Conclusions: These results indicate that bupivacaine blocks Kv4.3/KChIP2.2 channels from the open state. They furthermore give structural evidence that amino-amide local anesthetics interfere with the effects of KChIP2.2 on Kv4.3 by an indirect mechanism.

Interaction of Ropivacaine with Cloned Cardiac Kv4.3/KChIP2.2 Complexes

Background:Inhibition of cardiac K channels by local anesthetic may contribute to QTc interval prolongation of the electrocardiogram and induction of ventricular arrhythmia. The transient outward current Ito has been identified as a toxicologically relevant target of bupivacaine. S(−)-ropivacaine has been developed as a safer alternative to bupivacaine. The effects of S(−)-ropivacaine on Ito have not been investigated. In human ventricular myocardium, Ito is formed by Kv4.3 and KChIP2.2 subunits. Therefore, the aim of this study was to establish the effects of S(−)-ropivacaine on human Kv4.3/KChIP2.2 channels. Methods:Kv4.3/KChIP2.2 complementary DNA cloned from human heart was transiently transfected in Chinese hamster ovary cells. The pharmacologic effects of S(−)-ropivacaine were investigated with the patch clamp method. Results:Ropivacaine inhibited Kv4.3/KChIP2.2 channels in a concentration-dependent, stereospecific, and reversible manner. The IC50 value of S(−)-ropivacaine for inhibition of the charge conducted by Kv4.3/KChIP2.2 channel was 117 ± 21 &mgr;m (n = 30). The local anesthetic accelerated macroscopic current decline with an IC50 value of 77 ± 11 &mgr;m (n = 30). It shifted the midpoint of channel activation into the depolarizing direction, and it slowed recovery from inactivation without altering steady state inactivation. Kv4.3 channels are more sensitive to the inhibitory effect than Kv4.3/KChIP2.2 channels. Conclusions:The results are consistent with the idea that ropivacaine, by blocking Kv4.3/KChIP2.2 from the open state, interferes with the gating modifying effects of KChIP2.2 on Kv4.3 channels. Inhibition of Kv4.3/KChIP2.2 channels by the local anesthetic may contribute to the deterioration of cardiac function during events of intoxication.

Superficial cervical plexus neuropathy after single-injection interscalene brachial plexus block.

BACKGROUND: Interscalene brachial plexus block (ISB) using the modified lateral approach provides a well-established method of anesthesia and analgesia for patients undergoing shoulder surgery. Considering the neural anatomy at the site of injection, the superficial cervical plexus may be at risk of injury. We evaluated the incidence and characteristics of superficial cervical plexus neuropathy. METHODS: During a 1-yr period, 273 consecutive patients requiring single-injection ISB for shoulder or proximal arm surgery were studied. Patients were examined for symptoms compatible with superficial cervical plexus injury before surgery, 24 h postoperatively, and contacted by telephone 31 days after surgery. Symptomatic patients received an additional phone call 6 mo after surgery. RESULTS: Twenty-four hours after shoulder surgery, 21 patients (7.7%) showed symptoms consistent with superficial cervical plexus neuropathy. Symptoms consisted of hypesthesia in 1–4 cutaneous branches of the cervical plexus. Five patients (1.8%) reported symptoms that lasted for >31 days. All symptoms had entirely resolved after 6 mo. CONCLUSIONS: Superficial cervical plexus neuropathy is not uncommon after ISB using the modified lateral approach and the possibility should be discussed with patients preprocedurally.