Mutations in DNAJC19 cause altered mitochondrial structure and increased mitochondrial respiration in human iPSC-derived cardiomyocytes.

BACKGROUND: Dilated cardiomyopathy with ataxia (DCMA) is an autosomal recessive disorder arising from truncating mutations in DNAJC19, which encodes an inner mitochondrial membrane protein. Clinical features include an early onset, often life-threatening, cardiomyopathy associated with other metabolic features. Here, we aim to understand the metabolic and pathophysiological mechanisms of mutant DNAJC19 for the development of cardiomyopathy. METHODS: We generated induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) of two affected siblings with DCMA and a gene-edited truncation variant (tv) of DNAJC19 which all lack the conserved DnaJ interaction domain. The mutant iPSC-CMs and their respective control cells were subjected to various analyses, including assessments of morphology, metabolic function, and physiological consequences such as Ca2+ kinetics, contractility, and arrhythmic potential. Validation of respiration analysis was done in a gene-edited HeLa cell line (DNAJC19tvHeLa). RESULTS: Structural analyses revealed mitochondrial fragmentation and abnormal cristae formation associated with an overall reduced mitochondrial protein expression in mutant iPSC-CMs. Morphological alterations were associated with higher oxygen consumption rates (OCRs) in all three mutant iPSC-CMs, indicating higher electron transport chain activity to meet cellular ATP demands. Additionally, increased extracellular acidification rates suggested an increase in overall metabolic flux, while radioactive tracer uptake studies revealed decreased fatty acid uptake and utilization of glucose. Mutant iPSC-CMs also showed increased reactive oxygen species (ROS) and an elevated mitochondrial membrane potential. Increased mitochondrial respiration with pyruvate and malate as substrates was observed in mutant DNAJC19tv HeLa cells in addition to an upregulation of respiratory chain complexes, while cellular ATP-levels remain the same. Moreover, mitochondrial alterations were associated with increased beating frequencies, elevated diastolic Ca2+ concentrations, reduced sarcomere shortening and an increased beat-to-beat rate variability in mutant cell lines in response to ß-adrenergic stimulation. CONCLUSIONS: Loss of the DnaJ domain disturbs cardiac mitochondrial structure with abnormal cristae formation and leads to mitochondrial dysfunction, suggesting that DNAJC19 plays an essential role in mitochondrial morphogenesis and biogenesis. Moreover, increased mitochondrial respiration, altered substrate utilization, increased ROS production and abnormal Ca2+ kinetics provide insights into the pathogenesis of DCMA-related cardiomyopathy.

Lipid regulation of hERG1 channel function.

The lipid regulation of mammalian ion channel function has emerged as a fundamental mechanism in the control of electrical signalling and transport specificity in various cell types. In this work, we combine molecular dynamics simulations, mutagenesis, and electrophysiology to provide mechanistic insights into how lipophilic molecules (ceramide-sphingolipid probe) alter gating kinetics and K+ currents of hERG1. We show that the sphingolipid probe induced a significant left shift of activation voltage, faster deactivation rates, and current blockade comparable to traditional hERG1 blockers. Microseconds-long MD simulations followed by experimental mutagenesis elucidated ceramide specific binding locations at the interface between the pore and voltage sensing domains. This region constitutes a unique crevice present in mammalian channels with a non-swapped topology. The combined experimental and simulation data provide evidence for ceramide-induced allosteric modulation of the channel by a conformational selection mechanism.

Refinement of a cryo-EM structure of hERG: Bridging structure and function.

The human-ether-a-go-go-related gene (hERG) encodes the voltage-gated potassium channel (KCNH2 or Kv11.1, commonly known as hERG). This channel plays a pivotal role in the stability of phase 3 repolarization of the cardiac action potential. Although a high-resolution cryo-EM structure is available for its depolarized (open) state, the structure surprisingly did not feature many functionally important interactions established by previous biochemical and electrophysiology experiments. Using molecular dynamics flexible fitting (MDFF), we refined the structure and recovered the missing functionally relevant salt bridges in hERG in its depolarized state. We also performed electrophysiology experiments to confirm the functional relevance of a novel salt bridge predicted by our refinement protocol. Our work shows how refinement of a high-resolution cryo-EM structure helps to bridge the existing gap between the structure and function in the voltage-sensing domain (VSD) of hERG.

Direct Effects of Empagliflozin on Extracellular Matrix Remodelling in Human Cardiac Myofibroblasts: Novel Translational Clues to Explain EMPA-REG OUTCOME Results.

BACKGROUND: Empagliflozin, an SGLT2 inhibitor, has shown remarkable reductions in cardiovascular mortality and heart failure admissions (EMPA-REG OUTCOME). However, the mechanism underlying the heart failure protective effects of empagliflozin remains largely unknown. Cardiac fibroblasts play an integral role in the progression of structural cardiac remodelling and heart failure, in part, by regulating extracellular matrix (ECM) homeostasis. The objective of this study was to determine if empagliflozin has a direct effect on human cardiac myofibroblast-mediated ECM remodelling. METHODS: Cardiac fibroblasts were isolated via explant culture from human atrial tissue obtained at open heart surgery. Collagen gel contraction assay was used to assess myofibroblast activity. Cell morphology and cell-mediated ECM remodelling was examined with the use of confocal microscopy. Gene expression of profibrotic markers was assessed with the use of reverse-transcription quantitative polymerase chain reaction. RESULTS: Empagliflozin significantly attenuated transforming growth factor ß1-induced fibroblast activation via collagen gel contraction after 72-hour exposure, with escalating concentrations (0.5 µmol/L, 1 µmol/L, and 5 µmol/L) resulting in greater attenuation. Morphologic assessment showed that myofibroblasts exposed to empagliflozin were smaller in size with shorter and fewer number of extensions, indicative of a more quiescent phenotype. Moreover, empagliflozin significantly attenuated cell-mediated ECM remodelling as measured by collagen fibre alignment index. Gene expression profiling revealed significant suppression of critical profibrotic markers by empagliflozin, including COL1A1, ACTA2, CTGF, FN1, and MMP-2. CONCLUSIONS: We provide novel data showing a direct effect of empagliflozin on human cardiac myofibroblast phenotype and function by attenuation of myofibroblast activity and cell-mediated collagen remodelling. These data provide critical insights into the profound effects of empagliflozin as noted in the EMPA-REG OUTCOME study.

The Pore-Lipid Interface: Role of Amino-Acid Determinants of Lipophilic Access by Ivabradine to the hERG1 Pore Domain.

Abnormal cardiac electrical activity is a common side effect caused by unintended block of the promiscuous drug target human ether-à-go-go-related gene (hERG1), the pore-forming domain of the delayed rectifier K+ channel in the heart. hERG1 block leads to a prolongation of the QT interval, a phase of the cardiac cycle that underlies myocyte repolarization detectable on the electrocardiogram. Even newly released drugs such as heart-rate lowering agent ivabradine block the rapid delayed rectifier current IKr, prolong action potential duration, and induce potentially lethal arrhythmia known as torsades de pointes. In this study, we describe a critical drug-binding pocket located at the lateral pore surface facing the cellular membrane. Mutations of the conserved M651 residue alter ivabradine-induced block but not by the common hERG1 blocker dofetilide. As revealed by molecular dynamics simulations, binding of ivabradine to a lipophilic pore access site is coupled to a state-dependent reorientation of aromatic residues F557 and F656 in the S5 and S6 helices. We show that the M651 mutation impedes state-dependent dynamics of F557 and F656 aromatic cassettes at the protein-lipid interface, which has a potential to disrupt drug-induced block of the channel. This fundamentally new mechanism coupling the channel dynamics and small-molecule access from the membrane into the hERG1 intracavitary site provides a simple rationale for the well established state-dependence of drug blockade. SIGNIFICANCE STATEMENT: The drug interference with the function of the cardiac hERG channels represents one of the major sources of drug-induced heart disturbances. We found a novel and a critical drug-binding pocket adjacent to a lipid-facing surface of the hERG1 channel, which furthers our molecular understanding of drug-induced QT syndrome.

Macrophage Uptake of Necrotic Cell DNA Activates the AIM2 Inflammasome to Regulate a Proinflammatory Phenotype in CKD.

Nonmicrobial inflammation contributes to CKD progression and fibrosis. Absent in melanoma 2 (AIM2) is an inflammasome-forming receptor for double-stranded DNA. AIM2 is expressed in the kidney and activated mainly by macrophages. We investigated the potential pathogenic role of the AIM2 inflammasome in kidney disease. In kidneys from patients with diabetic or nondiabetic CKD, immunofluorescence showed AIM2 expression in glomeruli, tubules, and infiltrating leukocytes. In a mouse model of unilateral ureteral obstruction (UUO), Aim2 deficiency attenuated the renal injury, fibrosis, and inflammation observed in wild-type (WT) littermates. In bone marrow chimera studies, UUO induced substantially more tubular injury and IL-1ß cleavage in Aim2-/- or WT mice that received WT bone marrow than in WT mice that received Aim2-/- bone marrow. Intravital microscopy of the kidney in LysM(gfp/gfp) mice 5-6 days after UUO demonstrated the significant recruitment of GFP+ proinflammatory macrophages that crawled along injured tubules, engulfed DNA from necrotic cells, and expressed active caspase-1. DNA uptake occurred in large vacuolar structures within recruited macrophages but not resident CX3CR1+ renal phagocytes. In vitro, macrophages that engulfed necrotic debris showed AIM2-dependent activation of caspase-1 and IL-1ß, as well as the formation of AIM2+ ASC specks. ASC specks are a hallmark of inflammasome activation. Cotreatment with DNaseI attenuated the increase in IL-1ß levels, confirming that DNA was the principal damage-associated molecular pattern in this process. Therefore, the activation of the AIM2 inflammasome by DNA from necrotic cells drives a proinflammatory phenotype that contributes to chronic injury in the kidney.

Role of the pH in state-dependent blockade of hERG currents.

Mutations that reduce inactivation of the voltage-gated Kv11.1 potassium channel (hERG) reduce binding for a number of blockers. State specific block of the inactivated state of hERG block may increase risks of drug-induced Torsade de pointes. In this study, molecular simulations of dofetilide binding to the previously developed and experimentally validated models of the hERG channel in open and open-inactivated states were combined with voltage-clamp experiments to unravel the mechanism(s) of state-dependent blockade. The computations of the free energy profiles associated with the drug block to its binding pocket in the intra-cavitary site display startling differences in the open and open-inactivated states of the channel. It was also found that drug ionization may play a crucial role in preferential targeting to the open-inactivated state of the pore domain. pH-dependent hERG blockade by dofetilie was studied with patch-clamp recordings. The results show that low pH increases the extent and speed of drug-induced block. Both experimental and computational findings indicate that binding to the open-inactivated state is of key importance to our understanding of the dofetilide's mode of action.

Ivabradine prolongs phase 3 of cardiac repolarization and blocks the hERG1 (KCNH2) current over a concentration-range overlapping with that required to block HCN4.

In Europe, ivabradine has recently been approved to treat patients with angina who have intolerance to beta blockers and/or heart failure. Ivabradine is considered to act specifically on the sinoatrial node by inhibiting the If current (the funny current) to slow automaticity. However, in vitro studies show that ivabradine prolongs phase 3 repolarization in ventricular tissue. No episodes of Torsades de Pointes have been reported in randomized clinical studies. The objective of this study is to assess whether ivabradine blocked the hERG1 current. In the present study we discovered that ivabradine prolongs action potential and blocks the hERG current over a range of concentrations overlapping with those required to block HCN4. Ivabradine produced tonic, rather than use-dependent block. The mutation Y652A significantly suppressed pharmacologic block of hERG by ivabradine. Disruption of C-type inactivation also suppressed block of hERG1 by ivabradine. Molecular docking and molecular dynamics simulations indicate that ivabradine may access the inner cavity of the hERG1 via a lipophilic route and has a well-defined binding site in the closed state of the channel. Structural organization of the binding pockets for ivabradine is discussed. Ivabradine blocks hERG and prolongs action potential duration. Our study is potentially important because it indicates the need for active post marketing surveillance of ivabradine. Importantly, proarrhythmia of a number of other drugs has only been discovered during post marketing surveillance.

Ketones prevent oxidative impairment of hippocampal synaptic integrity through KATP channels.

Dietary and metabolic therapies are increasingly being considered for a variety of neurological disorders, based in part on growing evidence for the neuroprotective properties of the ketogenic diet (KD) and ketones. Earlier, we demonstrated that ketones afford hippocampal synaptic protection against exogenous oxidative stress, but the mechanisms underlying these actions remain unclear. Recent studies have shown that ketones may modulate neuronal firing through interactions with ATP-sensitive potassium (KATP) channels. Here, we used a combination of electrophysiological, pharmacological, and biochemical assays to determine whether hippocampal synaptic protection by ketones is a consequence of KATP channel activation. Ketones dose-dependently reversed oxidative impairment of hippocampal synaptic integrity, neuronal viability, and bioenergetic capacity, and this action was mirrored by the KATP channel activator diazoxide. Inhibition of KATP channels reversed ketone-evoked hippocampal protection, and genetic ablation of the inwardly rectifying K+ channel subunit Kir6.2, a critical component of KATP channels, partially negated the synaptic protection afforded by ketones. This partial protection was completely reversed by co-application of the KATP blocker, 5-hydoxydecanoate (5HD). We conclude that, under conditions of oxidative injury, ketones induce synaptic protection in part through activation of KATP channels.

NS1643 interacts around L529 of hERG to alter voltage sensor movement on the path to activation.

Activators of hERG1 such as NS1643 are being developed for congenital/acquired long QT syndrome. Previous studies identify the neighborhood of L529 around the voltage-sensor as a putative interacting site for NS1643. With NS1643, the V1/2 of activation of L529I (-34 ± 4 mV) is similar to wild-type (WT) (-37 ± 3 mV; P > 0.05). WT and L529I showed no difference in the slope factor in the absence of NS1643 (8 ± 0 vs. 9 ± 0) but showed a difference in the presence of NS1643 (9 ± 0.3 vs. 22 ± 1; P < 0.01). Voltage-clamp-fluorimetry studies also indicated that in L529I, NS1643 reduces the voltage-sensitivity of S4 movement. To further assess mechanism of NS1643 action, mutations were made in this neighborhood. NS1643 shifts the V1/2 of activation of both K525C and K525C/L529I to hyperpolarized potentials (-131 ± 4 mV for K525C and -120 ± 21 mV for K525C/L529I). Both K525C and K525C/K529I had similar slope factors in the absence of NS1643 (18 ± 2 vs. 34 ± 5, respectively) but with NS1643, the slope factor of K525C/L529I increased from 34 ± 5 to 71 ± 10 (P < 0.01) whereas for K525C the slope factor did not change (18 ± 2 at baseline and 16 ± 2 for NS1643). At baseline, K525R had a slope factor similar to WT (9 vs. 8) but in the presence of NS1643, the slope factor of K525R was increased to 24 ± 4 vs. 9 ± 0 mV for WT (P < 0.01). Molecular modeling indicates that L529I induces a kink in the S4 voltage-sensor helix, altering a salt-bridge involving K525. Moreover, docking studies indicate that NS1643 binds to the kinked structure induced by the mutation with a higher affinity. Combining biophysical, computational, and electrophysiological evidence, a mechanistic principle governing the action of some activators of hERG1 channels is proposed.

Kinetic model for NS1643 drug activation of WT and L529I variants of Kv11.1 (hERG1) potassium channel.

Congenital and acquired (drug-induced) forms of the human long-QT syndrome are associated with alterations in Kv11.1 (hERG) channel-controlled repolarizing IKr currents of cardiac action potentials. A mandatory drug screen implemented by many countries led to a discovery of a large group of small molecules that can activate hERG currents and thus may act as potent antiarrhythmic agents. Despite significant progress in identification of channel activators, little is known about their mechanism of action. A combination of electrophysiological studies with molecular and kinetic modeling was used to examine the mechanism of a model activator (NS1643) action on the hERG channel and its L529I mutant. The L529I mutant has gating dynamics similar to that of wild-type while its response to application of NS1643 is markedly different. We propose a mechanism compatible with experiments in which the model activator binds to the closed (C3) and open states (O). We suggest that NS1643 is affecting early gating transitions, probably during movements of the voltage sensor that precede the opening of the activation gate.

Role of mutation and pharmacologic block of human KCNH2 in vasculogenesis and fetal mortality: partial rescue by transforming growth factor-ß.

BACKGROUND: N629D KCNH2 is a human missense long-QT2 mutation. Previously, we reported that the N629D/N629D mutation embryos disrupted cardiac looping, right ventricle development, and ablated IKr activity at E9.5. The present study evaluates the role of KCNH2 in vasculogenesis. METHODS AND RESULTS: N629D/N629D yolk sac vessels and aorta consist of sinusoids without normal arborization. Isolated E9.5 +/+ first branchial arches showed normal outgrowth of mouse ERG-positive/α-smooth muscle actin coimmunolocalized cells; however, outgrowth was grossly reduced in N629D/N629D. N629D/N629D aortas showed fewer α-smooth muscle actin positive cells that were not coimmunolocalized with mouse ERG cells. Transforming growth factor-ß treatment of isolated N629D/N629D embryoid bodies partially rescued this phenotype. Cultured N629D/N629D embryos recapitulate the same cardiovascular phenotypes as seen in vivo. Transforming growth factor-ß treatment significantly rescued these embryonic phenotypes. Both in vivo and in vitro, dofetilide treatment, over a narrow window of time, entirely recapitulated the N629D/N629D fetal phenotypes. Exogenous transforming growth factor-ß treatment also rescued the dofetilide-induced phenotype toward normal. CONCLUSIONS: Loss of function of KCNH2 mutations results in defects in cardiogenesis and vasculogenesis. Because many medications inadvertently block the KCNH2 potassium current, these novel findings seem to have clinical relevance.

Phenotypic analysis of arrhythmogenic cardiomyopathy in the Hutterite population: role of electrocardiogram in identifying high-risk desmocollin-2 carriers.

BACKGROUND: The p.Gln554X mutation in desmocollin-2 (DSC2) is prevalent in ≈10% of the Hutterite population. While the homozygous mutation causes severe biventricular arrhythmogenic right ventricular cardiomyopathy, the phenotypic features and prognosis of heterozygotes remain incompletely understood. METHODS AND RESULTS: Eleven homozygotes (mean age 32±8 years, 45% female), 28 heterozygotes (mean age 40±15 years, 50% female), and 22 mutation-negatives (mean age 43±17 years, 41% female) were examined. Diagnostic testing was performed as per the arrhythmogenic right ventricular cardiomyopathy modified Task Force Criteria. Inverted T waves in the right precordial leads on ECG were seen in all homozygotes but not in their counterparts (P<0.001). Homozygotes had higher median daily premature ventricular complex burden than did heterozygotes or mutation-negatives (1407 [IQR 1080 to 2936] versus 2 [IQR 0 to 6] versus 6 [IQR 0 to 214], P=0.0002). Ventricular tachycardia was observed in 60% of homozygotes but in none of the remaining individuals (P<0.001). On cardiac magnetic resonance imaging, homozygotes had significantly larger indexed end-diastolic volumes (right ventricular: 122±24 versus 83±17 versus 83±12 mL/m(2), P<0.0001; left ventricular: 93±18 versus 76±13 versus 80±11 mL/m(2), P=0.0124) and lower ejection fraction values compared with heterozygotes and mutation-negatives (right ventricular ejection fraction: 41±9% versus 59±9% versus 61±6%, P<0.0001; left ventricular ejection fraction: 53±8% versus 65±5% versus 64±5%, P<0.0001). Most affected individuals lacked right ventricular wall motion abnormalities. Thus, few met cardiac magnetic resonance imaging task force criteria. CONCLUSIONS: The ECG reliably identifies homozygous p.Gln554X carriers and may be useful as an initial step in the screening of high-risk Hutterites. The cardiac phenotype of heterozygotes appears benign, but further prospective follow-up of their arrhythmic risk is needed.

Structure driven design of novel human ether-a-go-go-related-gene channel (hERG1) activators.

One of the main culprits in modern drug discovery is apparent cardiotoxicity of many lead-candidates via inadvertent pharmacologic blockade of K+, Ca2+ and Na+ currents. Many drugs inadvertently block hERG1 leading to an acquired form of the Long QT syndrome and potentially lethal polymorphic ventricular tachycardia. An emerging strategy is to rely on interventions with a drug that may proactively activate hERG1 channels reducing cardiovascular risks. Small molecules-activators have a great potential for co-therapies where the risk of hERG-related QT prolongation is significant and rehabilitation of the drug is impractical. Although a number of hERG1 activators have been identified in the last decade, their binding sites, functional moieties responsible for channel activation and thus mechanism of action, have yet to be established. Here, we present a proof-of-principle study that combines de-novo drug design, molecular modeling, chemical synthesis with whole cell electrophysiology and Action Potential (AP) recordings in fetal mouse ventricular myocytes to establish basic chemical principles required for efficient activator of hERG1 channel. In order to minimize the likelihood that these molecules would also block the hERG1 channel they were computationally engineered to minimize interactions with known intra-cavitary drug binding sites. The combination of experimental and theoretical studies led to identification of functional elements (functional groups, flexibility) underlying efficiency of hERG1 activators targeting binding pocket located in the S4-S5 linker, as well as identified potential side-effects in this promising line of drugs, which was associated with multi-channel targeting of the developed drugs.

Rehabilitating drug-induced long-QT promoters: in-silico design of hERG-neutral cisapride analogues with retained pharmacological activity.

BACKGROUND: The human ether-a-go-go related gene 1 (hERG1), which codes for a potassium ion channel, is a key element in the cardiac delayed rectified potassium current, IKr, and plays an important role in the normal repolarization of the heart's action potential. Many approved drugs have been withdrawn from the market due to their prolongation of the QT interval. Most of these drugs have high potencies for their principal targets and are often irreplaceable, thus "rehabilitation" studies for decreasing their high hERG1 blocking affinities, while keeping them active at the binding sites of their targets, have been proposed to enable these drugs to re-enter the market. METHODS: In this proof-of-principle study, we focus on cisapride, a gastroprokinetic agent withdrawn from the market due to its high hERG1 blocking affinity. Here we tested an a priori strategy to predict a compound's cardiotoxicity using de novo drug design with molecular docking and Molecular Dynamics (MD) simulations to generate a strategy for the rehabilitation of cisapride. RESULTS: We focused on two key receptors, a target interaction with the (adenosine) receptor and an off-target interaction with hERG1 channels. An analysis of the fragment interactions of cisapride at human A2A adenosine receptors and hERG1 central cavities helped us to identify the key chemical groups responsible for the drug activity and hERG1 blockade. A set of cisapride derivatives with reduced cardiotoxicity was then proposed using an in-silico two-tier approach. This set was compared against a large dataset of commercially available cisapride analogs and derivatives. CONCLUSIONS: An interaction decomposition of cisapride and cisapride derivatives allowed for the identification of key active scaffolds and functional groups that may be responsible for the unwanted blockade of hERG1.

Phospholamban knockout breaks arrhythmogenic Ca²âº waves and suppresses catecholaminergic polymorphic ventricular tachycardia in mice.

RATIONALE: Phospholamban (PLN) is an inhibitor of cardiac sarco(endo)plasmic reticulum Ca²âº ATPase. PLN knockout (PLN-KO) enhances sarcoplasmic reticulum Ca²âº load and Ca²âº leak. Conversely, PLN-KO accelerates Ca²âº sequestration and aborts arrhythmogenic spontaneous Ca²âº waves (SCWs). An important question is whether these seemingly paradoxical effects of PLN-KO exacerbate or protect against Ca²âº-triggered arrhythmias. OBJECTIVE: We investigate the impact of PLN-KO on SCWs, triggered activities, and stress-induced ventricular tachyarrhythmias (VTs) in a mouse model of cardiac ryanodine-receptor (RyR2)-linked catecholaminergic polymorphic VT. METHODS AND RESULTS: We generated a PLN-deficient, RyR2-mutant mouse model (PLN-/-/RyR2-R4496C+/-) by crossbreeding PLN-KO mice with catecholaminergic polymorphic VT-associated RyR2-R4496C mutant mice. Ca²âº imaging and patch-clamp recording revealed cell-wide propagating SCWs and triggered activities in RyR2-R4496C+/- ventricular myocytes during sarcoplasmic reticulum Ca²âº overload. PLN-KO fragmented these cell-wide SCWs into mini-waves and Ca²âº sparks and suppressed the triggered activities evoked by sarcoplasmic reticulum Ca²âº overload. Importantly, these effects of PLN-KO were reverted by partially inhibiting sarco(endo)plasmic reticulum Ca²âº ATPase with 2,5-di-tert-butylhydroquinone. However, Bay K, caffeine, or Li⁺ failed to convert mini-waves to cell-wide SCWs in PLN-/-/RyR2-R4496C+/- ventricular myocytes. Furthermore, ECG analysis showed that PLN-KO mice are not susceptible to stress-induced VTs. On the contrary, PLN-KO protected RyR2-R4496C mutant mice from stress-induced VTs. CONCLUSIONS: Our results demonstrate that despite severe sarcoplasmic reticulum Ca²âº leak, PLN-KO suppresses triggered activities and stress-induced VTs in a mouse model of catecholaminergic polymorphic VT. These data suggest that breaking up cell-wide propagating SCWs by enhancing Ca²âº sequestration represents an effective approach for suppressing Ca²âº-triggered arrhythmias.

Homozygous founder mutation in desmocollin-2 (DSC2) causes arrhythmogenic cardiomyopathy in the Hutterite population.

BACKGROUND: Dominant mutations in cellular junction proteins are the major cause of arrhythmogenic cardiomyopathy, whereas recessive mutations in those proteins cause cardiocutaneous syndromes such as Naxos and Carvajal syndrome. The Hutterites are distinct genetic isolates who settled in North America in 1874. Descended from <100 founders, they trace their origins to 16th-century Europe. METHODS AND RESULTS: We clinically and genetically evaluated 2 large families of the Alberta Hutterite population with a history of sudden death and found several individuals with severe forms of biventricular cardiomyopathy characterized by mainly left-sided localized aneurysms, regions of wall thinning with segmental akinesis, in addition to typical electric and histological features known for arrhythmogenic right ventricular cardiomyopathy. We identified a homozygous truncation mutation, c.1660C>T (p.Q554X) in desmocollin-2 (DSC2), in affected individuals and determined a carrier frequency of this mutation of 9.4% (1 in 10.6) among 1535 Schmiedeleut Hutterites, suggesting a common founder in that subgroup. Immunohistochemistry of endomyocardial biopsy samples revealed altered expression of the truncated DSC2 protein at the intercalated discs but only minor changes in immunoreactivity of other desmosomal proteins. Recombinant expressed mutant DSC2 protein in cells confirmed a stable, partially processed truncated protein with cytoplasmic and membrane localization. CONCLUSIONS: A homozygous truncation mutation in DSC2 leads to a cardiac-restricted phenotype of an early onset biventricular arrhythmogenic cardiomyopathy. The truncated protein remains partially stable and localized at the intercalated discs. These data suggest that the processed DSC2 protein plays a role in maintaining desmosome integrity and function.

Inflammasome-independent NLRP3 augments TGF-ß signaling in kidney epithelium.

Tubulointerstitial inflammation and fibrosis are strongly associated with the outcome of chronic kidney disease. We recently demonstrated that the NOD-like receptor, pyrin domain containing-3 (NLRP3) contributes to renal inflammation, injury, and fibrosis following unilateral ureteric obstruction in mice. NLRP3 expression in renal tubular epithelial cells (TECs) was found to be an important component of experimental disease pathogenesis, although the biology of NLRP3 in epithelial cells is unknown. In human and mouse primary renal TECs, NLRP3 expression was increased in response to TGF-ß1 stimulation and associated with epithelial-mesenchymal transition (EMT) and the expression of α-smooth muscle actin (αSMA) and matrix metalloproteinase (MMP) 9. TGF-ß1-induced EMT and the induction of MMP-9 and αSMA were significantly decreased in mouse Nlrp3(-/-) renal TECs, suggesting a role for Nlrp3 in TGF-ß-dependent signaling. Although apoptosis-associated speck-like protein containing a CARD domain(-/-) TECs demonstrated a phenotype similar to that of Nlrp3(-/-) cells in response to TGF-ß1, the effect of Nlrp3 on MMP-9 and αSMA expression was inflammasome independent, as IL-1ß, IL-18, MyD88, and caspase-1 were dispensable. Smad2 and Smad3 phosphorylation in response to TGF-ß1 was attenuated in Nlrp3(-/-) and apoptosis-associated speck-like protein containing a CARD domain(-/-) cells, accounting for the dampened EMT and TGF-ß1 responsiveness in these cells. Consistent with these findings, overexpression of NLRP3 in 293T cells resulted in increased Smad3 phosphorylation and activity. Taken together, these data support a novel and direct role for NLRP3 in promoting TGF-ß signaling and R-Smad activation in epithelial cells independent of the inflammasome.

Modeling of open, closed, and open-inactivated states of the hERG1 channel: structural mechanisms of the state-dependent drug binding.

The human ether-a-go-go related gene 1 (hERG1) K ion channel is a key element for the rapid component of the delayed rectified potassium current in cardiac myocytes. Since there are no crystal structures for hERG channels, creation and validation of its reliable atomistic models have been key targets in molecular cardiology for the past decade. In this study, we developed and vigorously validated models for open, closed, and open-inactivated states of hERG1 using a multistep protocol. The conserved elements were derived using multiple-template homology modeling utilizing available structures for Kv1.2, Kv1.2/2.1 chimera, and KcsA channels. Then missing elements were modeled with the ROSETTA De Novo protein-designing suite and further refined with all-atom molecular dynamics simulations. The final ensemble of models was evaluated for consistency to the reported experimental data from biochemical, biophysical, and electrophysiological studies. The closed state models were cross-validated against available experimental data on toxin footprinting with protein-protein docking using hERG state-selective toxin BeKm-1. Poisson-Boltzmann calculations were performed to determine gating charge and compare it to electrophysiological measurements. The validated structures offered us a unique chance to assess molecular mechanisms of state-dependent drug binding in three different states of the channel.