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The 'mitochondrial permeability transition', characterized by a sudden induced change of the inner mitochondrial membrane permeability for water as well as for small substances (/=1.5 kDa), has been known for three decades. Research interest in the entity responsible for this phenomenon, the 'mitochondrial permeability transition pore' (mPTP), has dramatically increased after demonstration that it plays a key role in the life and death decision in cells. Therefore, a better understanding of this phenomenon and its regulation by environmental stresses, kinase signalling, and pharmacological intervention is vital. The characterization of the molecular identity of the mPTP will allow identification of possible pharmacological targets and assist in drug design for its precise regulation. However, despite extensive research efforts, at this point the pore-forming core component(s) of the mPTP remain unidentified. Pivotal new genetic evidence has shown that components once believed to be core elements of the mPTP (namely mitochondrial adenine nucleotide translocator and cyclophilin D) are instead only mPTP regulators (or in the case of voltage-dependent anion channels, probably entirely dispensable). This review provides an update on the current state of knowledge regarding the regulation of the mPTP.

The mitochondrial permeability transition (MPT) pore complex is a key participant in the machinery that controls mitochondrial fate and, consequently, cell fate. The quest for the pore identity has been ongoing for several decades and yet the main structure remains unknown. Established "dogma" proposes that the core of the MPT pore is composed of an association of voltage-dependent anion channel (VDAC) and adenine nucleotide translocase (ANT). Recent genetic knockout experiments contradict this commonly accepted interpretation and provide a basis for substantial revision of the MPT pore identity. There is now sufficient evidence to exclude VDAC and ANT as the main pore structural components. Regarding MPT pore regulation, the role of cyclophilin D is confirmed and ANT may still serve some regulatory function, although the involvement of hexokinase II and creatine kinase remains unresolved. When cell protection signaling pathways are activated, we have found that the Bcl-2 family members relay the signal from glycogen synthase kinase-3 beta onto a target at or in close proximity to the pore. Our experimental findings in intact cardiac myocytes and neurons indicate that the current "dogma" related to the role of Ca2+ in MPT induction requires reevaluation. Emerging evidence suggests that after injury-producing stresses, reactive oxygen species (but not Ca2+) are largely responsible for the pore induction. In this article we discuss the current state of knowledge and provide new data related to the MPT pore structure and regulation.

The inward rectifier current I(K1) is tightly regulated regionally within the heart, downregulated in heart failure, and genetically suppressed in Andersen syndrome. We used in vivo viral gene transfer to dissect the role of I(K1) in cardiac repolarization and maintenance of the resting membrane potential (RMP) in guinea pig ventricular myocytes. Kir2.1 overexpression boosted Ba(2+)-sensitive I(K1) by more than 100% (at -50mV), significantly shortened action potential durations (APDs), accelerated phase 3 repolarization, and hyperpolarized RMP compared with control cells (nongreen cells from the same hearts and green cells from GFP-transduced hearts). The dominant-negative Kir2.1AAA reduced I(K1) by 50-90%; those cells with less than 80% reduction of I(K1) exhibited prolonged APDs, decelerated phase 3 repolarization, and depolarization of the RMP. Further reduction of I(K1) resulted in a pacemaker phenotype, as previously described. ECGs revealed a 7.7% +/- 0.9% shortening of the heart rate-corrected QT interval (QTc interval) in Kir2.1-transduced animals (n = 4) and a 16.7% +/- 1.8% prolongation of the QTc interval (n = 3) in Kir2.1AAA-transduced animals 72 hours after gene delivery compared with immediate postoperative recordings. Thus, I(K1) is essential for establishing the distinctive electrical phenotype of the ventricular myocyte: rapid terminal repolarization to a stable and polarized resting potential. Additionally, the long-QT phenotype seen in Andersen syndrome is a direct consequence of dominant-negative suppression of Kir2 channel function.

Abstract —The cardiac delayed rectifier potassium current mediates repolarization of the action potential and underlies the QT interval of the ECG. Mutations in either of the two molecular components of the rapid delayed rectifier ( I K,r ), HERG and KCNE2, have been linked to heritable or acquired long-QT syndrome. Mechanisms whereby mutations of KCNE2 produce fatal cardiac arrhythmias characteristic of long-QT syndrome remain unclear. In this study, we characterize functional interactions between HERG and KCNE2 with a view to defining underlying mechanisms for action potential prolongation and long-QT syndrome. Whereas coexpression of hKCNE2 with HERG alters both kinetics and density of ionic current, incorporation of these effects into a quantitative model of the action potential predicts that only changes in current density significantly affect repolarization. Thus, the primary functional consequence of hKCNE2 on action potential morphology is through modulation of I K,r density, as predicted by the model. Mutations associated with long-QT syndrome that result only in modest changes of gating kinetics may be epiphenomena or may modulate action potential repolarization via interaction with alternative pore-forming potassium channel α subunits.

When depolarized from typical resting membrane potentials (V(rest) approximately -90 mV), cardiac sodium (Na) currents are more sensitive to local anesthetics than brain or skeletal muscle Na currents. When expressed in Xenopus oocytes, lidocaine block of hH1 (human cardiac) Na current greatly exceeded that of mu1 (rat skeletal muscle) at membrane potentials near V(rest), whereas hyperpolarization to -140 mV equalized block of the two isoforms. Because the isoform-specific tonic block roughly parallels the drug-free voltage dependence of channel availability, isoform differences in the voltage dependence of fast inactivation could underlie the differences in block. However, after a brief (50 ms) depolarizing pulse, recovery from lidocaine block is similar for the two isoforms despite marked kinetic differences in drug-free recovery, suggesting that differences in fast inactivation cannot entirely explain the isoform difference in lidocaine action. Given the strong coupling between fast inactivation and other gating processes linked to depolarization (activation, slow inactivation), we considered the possibility that isoform differences in lidocaine block are explained by differences in these other gating processes. In whole-cell recordings from HEK-293 cells, the voltage dependence of hH1 current activation was approximately 20 mV more negative than that of mu1. Because activation and closed-state inactivation are positively coupled, these differences in activation were sufficient to shift hH1 availability to more negative membrane potentials. A mutant channel with enhanced closed-state inactivation gating (mu1-R1441C) exhibited increased lidocaine sensitivity, emphasizing the importance of closed-state inactivation in lidocaine action. Moreover, when the depolarization was prolonged to 1 s, recovery from a "slow" inactivated state with intermediate kinetics (I(M)) was fourfold longer in hH1 than in mu1, and recovery from lidocaine block in hH1 was similarly delayed relative to mu1. We propose that gating processes coupled to fast inactivation (activation and slow inactivation) are the key determinants of isoform-specific local anesthetic action.

Abstract Membrane current abnormalities have been described in human heart failure. To determine whether similar current changes are observed in a large animal model of heart failure, we studied dogs with pacing-induced cardiomyopathy. Myocytes isolated from the midmyocardium of 13 dogs with heart failure induced by 3 to 4 weeks of rapid ventricular pacing and from 16 nonpaced control dogs did not differ in cell surface area or resting membrane potential. Nevertheless, action potential duration (APD) was significantly prolonged in myocytes isolated from failing ventricles (APD at 90% repolarization, 1097±73 milliseconds [failing hearts, n=30] versus 842±56 milliseconds [control hearts, n=25]; P 2+ current and whole-cell Na + current did not differ in cells from failing and from control hearts, but significant differences in K + currents were observed. The density of the inward rectifier K + current (I K1 ) was reduced in cells from failing hearts at test potentials below −90 mV (at −150 mV, −19.1±2.2 pA/pF [failing hearts, n=18] versus −32.2±5.1 pA/pF [control hearts, n=15]; P K1 was also reduced in cells from failing hearts (at −60 mV, 1.7±0.2 pA/pF [failing hearts] versus 2.5±0.2 pA/pF [control hearts]; P 2+ -independent transient outward current (I to ) was dramatically reduced in myocytes isolated from failing hearts compared with nonfailing control hearts (at +80 mV, 7.0±0.9 pA/pF [failing hearts, n=20] versus 20.4±3.2 pA/pF [control hearts, n=15]; P to kinetics or single-channel conductance. A reduction in the number of functional I to channels was demonstrated by nonstationary fluctuation analysis (0.4±0.03 channels per square micrometer [failing hearts, n=5] versus 1.2±0.1 channels per square micrometer [control hearts, n=3]; P to by 4-aminopyridine in control myocytes decreased the notch amplitude and prolonged the APD. Current clamp–release experiments in which current was injected for 8 milliseconds to reproduce the notch sufficed to shorten the APD significantly in cells from failing hearts. These data support the hypothesis that downregulation of I to in pacing-induced heart failure is at least partially responsible for the action potential prolongation. Because the repolarization abnormalities mimic those in cells isolated from failing human ventricular myocardium, canine pacing-induced cardiomyopathy may provide insights into the development of repolarization abnormalities and the mechanisms of sudden death in patients with heart failure.

Limitation of infarct size by ischemic/pharmacological pre- and postconditioning involves activation of a complex set of cell-signaling pathways. Multiple lines of evidence implicate the mitochondrial permeability transition pore (mPTP) as a key end effector of ischemic/pharmacological pre- and postconditioning. Increasing the ROS threshold for mPTP induction enhances the resistance of cardiomyocytes to oxidant stress and results in infarct size reduction. Here, we survey and synthesize the present knowledge about the role of glycogen synthase kinase (GSK)-3β in cardioprotection, including pre- and postconditioning. Activation of a wide spectrum of cardioprotective signaling pathways is associated with phosphorylation and inhibition of a discrete pool of GSK-3β relevant to mitochondrial signaling. Therefore, GSK-3β has emerged as the integration point of many of these pathways and plays a central role in transferring protective signals downstream to target(s) that act at or in proximity to the mPTP. Bcl-2 family proteins and mPTP-regulatory elements, such as adenine nucleotide translocator and cyclophilin D (possibly voltage-dependent anion channel), may be the functional downstream target(s) of GSK-3β. Gaining a better understanding of these interactions to control and prevent mPTP induction when appropriate will enable us to decrease the negative impact of the reperfusion-induced ROS burst on the fate of mitochondria and perhaps allow us to limit propagation of damage throughout and between cells and consequently, to better limit infarct size.

Cardiac arrhythmias continue to pose a major medical challenge and significant public health burden. Atrial fibrillation, the most prevalent arrhythmia, affects more than two million Americans annually and is associated with a twofold increase in mortality. In addition, more than 250,000 Americans each year suffer ventricular arrhythmias, often resulting in sudden cardiac death. Despite the high incidence and societal impact of cardiac arrhythmias, presently there are insufficient insights into the molecular mechanisms involved in arrhythmia generation, propagation, and/or maintenance or into the molecular determinants of disease risk, prognosis, and progression. In addition, present therapeutic strategies for arrhythmia abatement often are ineffective or require palliative device therapy after persistent changes in the electrical and conduction characteristics of the heart have occurred, changes that appear to increase the risk for arrhythmia progression. This article reviews our present understanding of the complexity of mechanisms that regulate cardiac membrane excitability and cardiac function and explores the role of derangements in these mechanisms that interact to induce arrhythmogenic substrates. Approaches are recommended for future investigations focused on providing new mechanistic insights and therapeutic interventions.

Regulatory subunit KCNE3 (E3) interacts with KCNQ1 (Q1) in epithelia, regulating its activation kinetics and augmenting current density. Since E3 is expressed weakly in the heart, we hypothesized that ectopic expression of E3 in cardiac myocytes might abbreviate action potential duration (APD) by interacting with Q1 and augmenting the delayed rectifier current (I(K)). Thus, we transiently coexpressed E3 with Q1 and KCNE1 (E1) in Chinese hamster ovary cells and found that E3 coexpression increased outward current at potentials byor = -80 mV and accelerated activation. We then examined the changes in cardiac electrophysiology following injection of adenovirus-expressed E3 into the left ventricular cavity of guinea pigs. After 72 hours, the corrected QT interval of the electrocardiogram was reduced by approximately 10%. APD was reduced by3-fold in E3-transduced cells relative to controls, while E-4031-insensitive I(K) and activation kinetics were significantly augmented. Based on quantitative modeling of a transmural cardiac segment, we demonstrate that the degree of QT interval abbreviation observed results from electrotonic interactions in the face of limited transduction efficiency and that heterogeneous transduction of E3 may actually potentiate arrhythmias. Provided that fairly homogeneous ectopic ventricular expression of regulatory subunits can be achieved, this approach may be useful in enhancing repolarization and in treating long QT syndrome.

1. IKs, the slow component of the delayed rectifier potassium current, figures prominently in the repolarization of heart cells. The K+ channel gene KvLQT1 is mutated in the heritable long QT (LQT) syndrome. Heterologous coexpression of KvLQT1 and the accessory protein minK yields an IKs-like current. Nevertheless, the links between KvLQT1 and cardiac IKs are largely inferential. 2. Since the LQT syndrome mutant KvLQT1-G306R suppresses channel activity when coexpressed with wild-type KvLQT1 in a heterologous system, overexpression of this mutant in cardiomyocytes should reduce or eliminate native IKs if KvLQT1 is indeed the major molecular component of this current. To test this idea, we created the adenovirus AdRMGI-KvLQT1-G306R, which overexpress KvLQT1-G306R channels. 3. In > 60% of neonatal mouse myocytes, a sizable IKs could be measured using perforated-patch recordings (8.0 ± 1.6 pA pF-1, n = 13). IKs was increased by forskolin and blocked by clofilium or indapamide but not by E-4031. While cells infected with a reporter virus expressing only green fluorescent protein (GFP) displayed IKs similar to that in uninfected cells, AdRMGI-KvLQT1-G306R-infected cells showed a significantly reduced IKs (2.4 ± 1.1 pA pF-1, n = 10, P < 0.01) when measured 60-72 h after infection. Similar results were observed in adult guinea-pig myocytes (5.9 ± 1.2 pA pF-1, n = 9, for control vs. 0.1 ± 0.1 pA pF-1, n = 5, for AdRMGI-KvLQT1-G306R-infected cells). 4. We conclude that KvLQT1 is the major molecular component of IKs. Our results further establish a dominant-negative mechanism for the G306R LQT syndrome mutation., link_to_subscribed_fulltext

The high incidence of sudden death in heart failure may reflect abnormalities of repolarization and heightened susceptibility to arrhythmogenic early afterdepolarizations (EADs). We hypothesized that overexpression of the human K+ channel HERG (human ether-a-go-go-related gene) could enhance repolarization and suppress EADs. Adult rabbit ventricular myocytes were maintained in primary culture, which suffices to prolong action potentials and predisposes to EADs. To achieve efficient gene transfer, we created AdHERG, a recombinant adenovirus containing the HERG gene driven by a Rous sarcoma virus (RSV) promoter. The virally expressed HERG current exhibited pharmacologic and kinetic properties like those of native IKr. Transient outward currents in AdHERG-infected myocytes were similar in magnitude to those in control cells, while stimulated action potentials (0.2 Hz, 37 degrees C) were abbreviated compared with controls. The occurrence of EADs during a train of action potentials was reduced by more than fourfold, and the relative refractory period was increased in AdHERG-infected myocytes compared with control cells. Gene transfer of delayed rectifier potassium channels represents a novel and effective strategy to suppress arrhythmias caused by unstable repolarization.

Background —A heritable form of the long-QT syndrome (LQT3) has been linked to mutations in the cardiac sodium channel gene (SCN5A). Recently, a sporadic SCN5A mutation was identified in a Japanese girl afflicted with the long-QT syndrome. In contrast to the heritable mutations, this externally positioned domain IV, S4 mutation (R1623Q) neutralized a charged residue that is critically involved in activation-inactivation coupling. Methods and Results —We have characterized the R1623Q mutation in the human cardiac sodium channel (hH1) using both whole-cell and single-channel recordings. In contrast to the autosomal dominant LQT3 mutations, R1623Q increased the probability of long openings and caused early reopenings, producing a threefold prolongation of sodium current decay. Lidocaine restored rapid decay of the R1623Q macroscopic current. Conclusions —The R1623Q mutation produces inactivation gating defects that differ mechanistically from those caused by LQT3 mutations. These findings provide a biophysical explanation for this severe long-QT phenotype and extend our understanding of the mechanistic role of the S4 segment in cardiac sodium channel inactivation gating and class I antiarrhythmic drug action.