Jin O-Uchi

Mutations in Cytoplasmic Loops of the KCNQ1 Channel and the Risk of Life-Threatening Events: Implications for Mutation-Specific Response to Beta-Blocker Therapy in Type-1 Long QT Syndrome

Background— &bgr;-Adrenergic stimulation is the main trigger for cardiac events in type 1 long-QT syndrome (LQT1). We evaluated a possible association between ion channel response to &bgr;-adrenergic stimulation and clinical response to &bgr;-blocker therapy according to mutation location. Methods and Results— The study sample comprised 860 patients with genetically confirmed mutations in the KCNQ1 channel. Patients were categorized into carriers of missense mutations located in the cytoplasmic loops (C loops), membrane-spanning domain, C/N terminus, and nonmissense mutations. There were 27 aborted cardiac arrest and 78 sudden cardiac death events from birth through 40 years of age. After multivariable adjustment for clinical factors, the presence of C-loop mutations was associated with the highest risk for aborted cardiac arrest or sudden cardiac death (hazard ratio versus nonmissense mutations=2.75; 95% confidence interval, 1.29–5.86; P=0.009). &bgr;-Blocker therapy was associated with a significantly greater reduction in the risk of aborted cardiac arrest or sudden cardiac death among patients with C-loop mutations than among all other patients (hazard ratio=0.12; 95% confidence interval, 0.02–0.73; P=0.02; and hazard ratio=0.82; 95% confidence interval, 0.31–2.13; P=0.68, respectively; P for interaction=0.04). Cellular expression studies showed that membrane spanning and C-loop mutations produced a similar decrease in current, but only C-loop mutations showed a pronounced reduction in channel activation in response to &bgr;-adrenergic stimulation. Conclusions— Patients with C-loop missense mutations in the KCNQ1 channel exhibit a high risk for life-threatening events and derive a pronounced benefit from treatment with &bgr;-blockers. Reduced channel activation after sympathetic activation can explain the increased clinical risk and response to therapy in patients with C-loop mutations.Background— β-Adrenergic stimulation is the main trigger for cardiac events in type 1 long-QT syndrome (LQT1). We evaluated a possible association between ion channel response to β-adrenergic stimulation and clinical response to β-blocker therapy according to mutation location. Methods and Results— The study sample comprised 860 patients with genetically confirmed mutations in the KCNQ1 channel. Patients were categorized into carriers of missense mutations located in the cytoplasmic loops (C loops), membrane-spanning domain, C/N terminus, and nonmissense mutations. There were 27 aborted cardiac arrest and 78 sudden cardiac death events from birth through 40 years of age. After multivariable adjustment for clinical factors, the presence of C-loop mutations was associated with the highest risk for aborted cardiac arrest or sudden cardiac death (hazard ratio versus nonmissense mutations=2.75; 95% confidence interval, 1.29–5.86; P =0.009). β-Blocker therapy was associated with a significantly greater reduction in the risk of aborted cardiac arrest or sudden cardiac death among patients with C-loop mutations than among all other patients (hazard ratio=0.12; 95% confidence interval, 0.02–0.73; P =0.02; and hazard ratio=0.82; 95% confidence interval, 0.31–2.13; P =0.68, respectively; P for interaction=0.04). Cellular expression studies showed that membrane spanning and C-loop mutations produced a similar decrease in current, but only C-loop mutations showed a pronounced reduction in channel activation in response to β-adrenergic stimulation. Conclusions— Patients with C-loop missense mutations in the KCNQ1 channel exhibit a high risk for life-threatening events and derive a pronounced benefit from treatment with β-blockers. Reduced channel activation after sympathetic activation can explain the increased clinical risk and response to therapy in patients with C-loop mutations. # Clinical Perspective {#article-title-29}

Prognostic significance of epidermal growth factor receptor phosphorylation and mutation in head and neck squamous cell carcinoma.

The molecular status of the epidermal growth factor receptor (EGFR) has not been as well studied in head and neck squamous cell carcinoma (HNSCC) as in lung cancer. We examined the frequencies of EGFR mutations as well as the expression/phosphorylation status of the EGFR protein in HNSCC patients. Moreover, we tried to elucidate associations between EGFR molecular status and patient characteristics and disease-free survival. In this prospective cohort study, clinical data and samples were obtained from 82 consecutive patients who had not been treated with EGFR molecular targeting therapy. Full-length EGFR was sequenced, and expression and phosphorylation of the EGFR protein were measured by Western blotting. Four novel mutations (E709K, V765G, Ins770G, and G1022S) and one mutation well-known in lung cancer (L858R) were identified in six HNSCC samples (7%), but we could not find any mutations in the extracellular domain of EGFR, such as EGFRvIII, in this study. E709K and Ins770G as well as L858R appear to be functional mutations based on the use of Ba/F3 cells. In terms of patient characteristics, the number of metastatic lymph nodes and node stage were associated with phosphorylation of EGFR. No patients with EGFR mutations relapsed during the study period. Excluding mutated cases, patients whose tumor samples showed phosphorylated EGFR relapsed significantly earlier than those without phosphorylated EGFR. This finding was still significant after adjusting for mutation and overexpression of EGFR protein using the Cox proportional hazard model. In conclusion, phosphorylated EGFR without mutations may be a marker of poor prognosis in patients with HNSCC.

Interaction of α1-Adrenoceptor Subtypes With Different G Proteins Induces Opposite Effects on Cardiac L-type Ca2+ Channel

We examined the effect of &agr;1-adrenoceptor subtype-specific stimulation on L-type Ca2+ current (ICa) and elucidated the subtype-specific intracellular mechanisms for the regulation of L-type Ca2+ channels in isolated rat ventricular myocytes. We confirmed the protein expression of &agr;1A- and &agr;1B-adrenoceptor subtypes at the transverse tubules (T-tubules) and found that simultaneous stimulation of these 2 receptor subtypes by nonsubtype selective agonist, phenylephrine, showed 2 opposite effects on ICa (transient decrease followed by sustained increase). However, selective &agr;1A-adrenoceptor stimulation (≥0.1 &mgr;mol/L A61603) only potentiated ICa, and selective &agr;1B-adrenoceptor stimulation (10 &mgr;mol/L phenylephrine with 2 &mgr; mol/L WB4101) only decreased ICa. The positive effect by &agr;1A-adrenoceptor stimulation was blocked by the inhibition of phospholipase C (PLC), protein kinase C (PKC), or Ca2+/calmodulin-dependent protein kinase II (CaMKII). The negative effect by &agr;1B-adrenoceptor stimulation disappeared after the treatment of pertussis toxin or by the prepulse depolarization, but was not attriburable to the inhibition of cAMP-dependent pathway. The translocation of PKC&dgr; and ϵ to the T-tubules was observed only after &agr;1A-adrenoceptor stimulation, but not after &agr;1B-adrenoceptor stimulation. Immunoprecipitaion analysis revealed that &agr;1A-adrenoceptor was associated with Gq/11, but &agr;1B-adrenoceptor interacted with one of the pertussis toxin-sensitive G proteins, Go. These findings demonstrated that the interactions of &agr;1-adrenoceptor subtypes with different G proteins elicit the formation of separate signaling cascades, which produce the opposite effects on ICa. The coupling of &agr;1A-adrenoceptor with Gq/11-PLC-PKC-CaMKII pathway potentiates ICa. In contrast, &agr;1B-adrenoceptor interacts with Go, of which the &bgr;&ggr;-complex might directly inhibit the channel activity at T-tubules.

Molecular Basis of Decreased Kir4.1 Function in SeSAME/EAST Syndrome

SeSAME/EAST syndrome is a channelopathy consisting of a hypokalemic, hypomagnesemic, metabolic alkalosis associated with seizures, sensorineural deafness, ataxia, and developmental abnormalities. This disease links to autosomal recessive mutations in KCNJ10, which encodes the Kir4.1 potassium channel, but the functional consequences of these mutations are not well understood. In Xenopus oocytes, all of the disease-associated mutant channels (R65P, R65P/R199X, G77R, C140R, T164I, and A167V/R297C) had decreased K(+) current (0 to 23% of wild-type levels). Immunofluorescence demonstrated decreased surface expression of G77R, C140R, and A167V expressed in HEK293 cells. When we coexpressed mutant and wild-type subunits to mimic the heterozygous state, R199X, C140R, and G77R currents decreased to 55, 40, and 20% of wild-type levels, respectively, suggesting that carriers of these mutations may present with an abnormal phenotype. Because Kir4.1 subunits can form heteromeric channels with Kir5.1, we coexpressed the aforementioned mutants with Kir5.1 and found that currents were reduced at least as much as observed when we expressed mutants alone. Reduction of pH(i) from approximately 7.4 to 6.8 significantly decreased currents of all mutants except R199X but did not affect wild-type channels. In conclusion, perturbed pH gating may underlie the loss of channel function for the disease-associated mutant Kir4.1 channels and may have important physiologic consequences.

Use of Mutant-Specific Ion Channel Characteristics for Risk Stratification of Long QT Syndrome Patients

Mutations that slow the opening of potassium channels in the heart can predict risk for long QT syndrome, a heart arrhythmia that can cause sudden death. Keeping the Heart in Tune The decades-long, 24/7 beating of the human heart is sustained by a symphony of ion channels rhythmically opening and closing on cue. Hearts of those born with mutations in these channels can occasionally hit a bad note, which can cause heart palpitations or—sometimes—sudden death. This so-called long QT syndrome can be diagnosed from an electrocardiogram (ECG) and other clinical parameters, but the distance between the Q and the T waves of the ECG predicts disease well only when the gap is very long—more than 500 ms. Now, Jons et al. have found an electrical characteristic of the IKs channel—the mutation-specific rate at which it opens—that allows the accurate diagnosis of individuals with a QT interval less than 500 ms. Seventeen different mutations in the IKs channel were identified in a group of 387 patients with long QT syndrome. To scrutinize the details of these mutation-carrying channels, the authors expressed the mutated subunits in frog oocytes and then analyzed their function with electrophysiological electrodes and stimulation. The mutated channels carried about 30% less current and tended to activate (open) more slowly than the wild-type ones, but in contrast, the aberrant channels deactivated (closed) at the same rate as their normal counterparts. By using multivariate regression, the authors showed that the diminished amount of current contributed directly to the longer QT interval seen in these patients (and so did not add to the information provided by an ECG), but the slow activation was an independent parameter relative to the QT gap. Further, when the authors analyzed the clinical history of the patients carrying these mutations, they found that the extent of the slowing of channel activation correlated positively with episodes of cardiac dysfunction—syncope (loss of consciousness), cardiac arrest requiring defibrillation, and sudden death—before age 30. But would the slowing of activation of IKs channels really disturb the beating heart enough to cause these cardiac problems? The authors used a computer model to find out. This analysis revealed that beating heart cells that carry the slowly activating mutant channels exhibit prolonged action potentials, which would compromise the cell’s ability to recover from any early beats experienced by the heart. This impairment could trigger arrhythmias such as those seen in the patients. These results could ensure better care for some patients with long QT syndrome, particularly those with only modest increases in their QT intervals. Right now, such patients sometimes remain untreated. Screening patients for channel mutations that cause slower activation could help to identify those at greatest risk, allowing proper intervention to prevent the electrical dissonance that can lead to lethal cardiac arrhythmias. Inherited long QT syndrome (LQTS) is caused by mutations in ion channels that delay cardiac repolarization, increasing the risk of sudden death from ventricular arrhythmias. Currently, the risk of sudden death in individuals with LQTS is estimated from clinical parameters such as age, gender, and the QT interval, measured from the electrocardiogram. Even though a number of different mutations can cause LQTS, mutation-specific information is rarely used clinically. LQTS type 1 (LQT1), one of the most common forms of LQTS, is caused by mutations in the slow potassium current (IKs) channel α subunit KCNQ1. We investigated whether mutation-specific changes in IKs function can predict cardiac risk in LQT1. By correlating the clinical phenotype of 387 LQT1 patients with the cellular electrophysiological characteristics caused by an array of mutations in KCNQ1, we found that channels with a decreased rate of current activation are associated with increased risk of cardiac events (hazard ratio = 2.02), independent of the clinical parameters usually used for risk stratification. In patients with moderate QT prolongation (a QT interval less than 500 ms), slower activation was an independent predictor for cardiac events (syncope, aborted cardiac arrest, and sudden death) (hazard ratio = 2.10), whereas the length of the QT interval itself was not. Our results indicate that genotype and biophysical phenotype analysis may be useful for risk stratification of LQT1 patients and suggest that slow channel activation is associated with an increased risk of cardiac events.

Trigger-specific ion-channel mechanisms, risk factors, and response to therapy in type 1 long QT syndrome

BACKGROUND Arrhythmic events in long-QT syndrome type 1 (LQT1) may be associated with exercise, acute arousal, or rest/sleep. OBJECTIVES We aimed to identify trigger-specific risk factors for cardiac events in patients with LQT1. METHODS The study population comprised 721 genetically confirmed patients with LQT1 from the US portion of the International LQTS Registry. Multivariate analysis was used to assess the independent contribution of prespecified clinical and mutation-specific factors to the development of a first reported triggered event, associated with exercise, arousal, or sleep/rest. RESULTS Cardiac events occurred in 221 study patients, of whom 121 (55%) were associated with exercise, 30 (14%) with arousal, 47 (21%) with sleep/rest, and 23 (10%) with other triggers. Multivariate analysis showed that males <13 years had a 2.8-fold (P < .001) increase in the risk for exercise-triggered events whereas females ≥13 years showed a 3.5-fold (P = .002) increase in the risk for sleep/rest nonarousal events. Cytoplasmic-loop mutations within the transmembrane region, involved in adrenergic channel regulation, were associated with the increased risk for both exercise- and arousal-triggered events (hazard ratio = 6.19 [P < .001] and 4.99 [P < .001], respectively) but were not associated with events during sleep/rest (hazard ratio = 0.72; P = .46). Beta-blocker therapy was associated with a pronounced 78% (P < .001) reduction in the risk for exercise-triggered events but did not have a significant effect on events associated with arousal or sleep/rest. CONCLUSIONS In patients with LQT1, cardiac events triggered by exercise, arousal, or rest/sleep are associated with distinctive risk factors and response to medical therapy. These findings can be used for improved recommendations for lifestyle modifications and therapeutic management in this population.

Acidosis or inorganic phosphate enhances the length dependence of tension in rat skinned cardiac muscle

1 We investigated the effect of acidosis on the sarcomere length (SL) dependence of tension generation, in comparison with the effect of inorganic phosphate (Pi), in rat skinned ventricular trabeculae. The shift of the mid‐point of the pCa‐tension relationship associated with an increase in SL from 1.9 to 2.3 μm (ΔpCa50) was studied. 2 Decreasing pH from 7.0 to 6.2 lowered maximal and submaximal Ca2+‐activated tension and increased ΔpCa50 in a pH‐dependent manner (from 0.21 ± 0.01 to 0.30 ± 0.01 pCa units). The addition of Pi (20 mm) decreased maximal tension and enhanced the SL dependence, both to a similar degree as observed when decreasing pH to 6.2 (ΔpCa50 increased from 0.20 ± 0.01 to 0.29 ± 0.01 pCa units). 3 Further experiments were performed using 6 % (w/v) Dextran T‐500 (molecular weight ∼500 000) to osmotically reduce interfilament lattice spacing (SL, 1.9 μm). Compared with that at pH 7.0, in the absence of Pi the increase in the Ca2+ sensitivity of tension induced by osmotic compression was enhanced at pH 6.2 (0.18 ± 0.01 vs. 0.25 ± 0.01 pCa units) or in the presence of 20 mm Pi (0.17 ± 0.01 vs. 0.24 ± 0.01 pCa units). 4 H+, as well as Pi, has been reported to decrease the number of strongly binding cross‐bridges, which reduces the co‐operative activation of the thin filament and increases the pool of detached cross‐bridges available for interaction with actin. It is therefore considered that during acidosis, the degree of increase in the number of force‐generating cross‐bridges upon reduction of interfilament lattice spacing is enhanced, resulting in greater SL dependence of tension generation. 5 Our results suggest that the Frank‐Starling mechanism may be enhanced when tension development is suppressed due to increased H+ and/or Pi under conditions of myocardial ischaemia or hypoxia.

Role of Ca2+/calmodulin-dependent protein kinase II in the regulation of the cardiac L-type Ca2+ current during endothelin-1 stimulation

Endothelin-1 (ET-1) shows a positive inotropic effect on cardiac muscle. Although the L-type Ca(2+) current (I(Ca)) is one of the important determinants of cardiac excitation-contraction coupling, the effect of ET-1 on the I(Ca) is not always clear. The controversial results appear to be due to different patch-clamp methods. The present study measured the effect of ET-1 on the I(Ca) of rat ventricular myocytes using the perforated patch-clamp technique. The holding potential was set to -40 mV, and depolarization was applied every 10 s. ET-1 (10 nM) increased the I(Ca) in a monophasic manner. The current reached a steady state 15 min after the application of ET-1, when the measurement was done. Endothelin receptor subtype expression was also investigated using Western immunoblotting. ET(A)-receptor protein was expressed, but ET(B)-receptor protein was not expressed, in the cell membranes of rat ventricular myocytes. The effect of ET-1 on the I(Ca) was inhibited by a selective ET(A)-receptor antagonist, BQ-123, but not by a selective ET(B)-receptor antagonist, BQ-788. The effect was inhibited by protein kinase C (PKC) inhibitor chelerythrine and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93, but not by its inactive analog KN-92. The effect of ET-1 was also blocked by another CaMKII inhibitor, autocamtide-2-related inhibitory peptide. These results suggest that ET-1 increases the I(Ca) via the ET(A)-receptor-PKC-CaMKII pathway.

Adrenergic signaling regulates mitochondrial Ca2+ uptake through Pyk2-dependent tyrosine phosphorylation of the mitochondrial Ca2+ uniporter.

AIMS Mitochondrial Ca2+ homeostasis is crucial for balancing cell survival and death. The recent discovery of the molecular identity of the mitochondrial Ca2+ uniporter pore (MCU) opens new possibilities for applying genetic approaches to study mitochondrial Ca2+ regulation in various cell types, including cardiac myocytes. Basal tyrosine phosphorylation of MCU was reported from mass spectroscopy of human and mouse tissues, but the signaling pathways that regulate mitochondrial Ca2+ entry through posttranslational modifications of MCU are completely unknown. Therefore, we investigated α1-adrenergic-mediated signal transduction of MCU posttranslational modification and function in cardiac cells. RESULTS α1-adrenoceptor (α1-AR) signaling translocated activated proline-rich tyrosine kinase 2 (Pyk2) from the cytosol to mitochondrial matrix and accelerates mitochondrial Ca2+ uptake via Pyk2-dependent MCU phosphorylation and tetrametric MCU channel pore formation. Moreover, we found that α1-AR stimulation increases reactive oxygen species production at mitochondria, mitochondrial permeability transition pore activity, and initiates apoptotic signaling via Pyk2-dependent MCU activation and mitochondrial Ca2+ overload. INNOVATION Our data indicate that inhibition of α1-AR-Pyk2-MCU signaling represents a potential novel therapeutic target to limit or prevent mitochondrial Ca2+ overload, oxidative stress, mitochondrial injury, and myocardial death during pathophysiological conditions, where chronic adrenergic stimulation is present. CONCLUSION The α1-AR-Pyk2-dependent tyrosine phosphorylation of the MCU regulates mitochondrial Ca2+ entry and apoptosis in cardiac cells.

Mitochondrial Ion Channels/Transporters as Sensors and Regulators of Cellular Redox Signaling

SIGNIFICANCE Mitochondrial ion channels/transporters and the electron transport chain (ETC) serve as key sensors and regulators for cellular redox signaling, the production of reactive oxygen species (ROS) and nitrogen species (RNS) in mitochondria, and balancing cell survival and death. Although the functional and pharmacological characteristics of mitochondrial ion transport mechanisms have been extensively studied for several decades, the majority of the molecular identities that are responsible for these channels/transporters have remained a mystery until very recently. RECENT ADVANCES Recent breakthrough studies uncovered the molecular identities of the diverse array of major mitochondrial ion channels/transporters, including the mitochondrial Ca2+ uniporter pore, mitochondrial permeability transition pore, and mitochondrial ATP-sensitive K+ channel. This new information enables us to form detailed molecular and functional characterizations of mitochondrial ion channels/transporters and their roles in mitochondrial redox signaling. CRITICAL ISSUES Redox-mediated post-translational modifications of mitochondrial ion channels/transporters and ETC serve as key mechanisms for the spatiotemporal control of mitochondrial ROS/RNS generation. FUTURE DIRECTIONS Identification of detailed molecular mechanisms for redox-mediated regulation of mitochondrial ion channels will enable us to find novel therapeutic targets for many diseases that are associated with cellular redox signaling and mitochondrial ion channels/transporters.