Hyperphosphorylation of RyRs underlies triggered activity in transgenic rabbit model of LQT2 syndrome.

RATIONALE: Loss-of-function mutations in human ether go-go (HERG) potassium channels underlie long QT syndrome type 2 (LQT2) and are associated with fatal ventricular tachyarrhythmia. Previously, most studies focused on plasma membrane-related pathways involved in arrhythmogenesis in long QT syndrome, whereas proarrhythmic changes in intracellular Ca(2+) handling remained unexplored. OBJECTIVE: We investigated the remodeling of Ca(2+) homeostasis in ventricular cardiomyocytes derived from transgenic rabbit model of LQT2 to determine whether these changes contribute to triggered activity in the form of early after depolarizations (EADs). METHODS AND RESULTS: Confocal Ca(2+) imaging revealed decrease in amplitude of Ca(2+) transients and sarcoplasmic reticulum Ca(2+) content in LQT2 myocytes. Experiments using sarcoplasmic reticulum-entrapped Ca(2+) indicator demonstrated enhanced ryanodine receptor (RyR)-mediated sarcoplasmic reticulum Ca(2+) leak in LQT2 cells. Western blot analyses showed increased phosphorylation of RyR in LQT2 myocytes versus controls. Coimmunoprecipitation experiments demonstrated loss of protein phosphatases type 1 and type 2 from the RyR complex. Stimulation of LQT2 cells with ß-adrenergic agonist isoproterenol resulted in prolongation of the plateau of action potentials accompanied by aberrant Ca(2+) releases and EADs, which were abolished by inhibition of Ca(2+)/calmodulin-dependent protein kinase type 2. Computer simulations showed that late aberrant Ca(2+) releases caused by RyR hyperactivity promote EADs and underlie the enhanced triggered activity through increased forward mode of Na(+)/Ca(2+) exchanger type 1. CONCLUSIONS: Hyperactive, hyperphosphorylated RyRs because of reduced local phosphatase activity enhance triggered activity in LQT2 syndrome. EADs are promoted by aberrant RyR-mediated Ca(2+) releases that are present despite a reduction of sarcoplasmic reticulum content. Those releases increase forward mode Na(+)/Ca(2+) exchanger type 1, thereby slowing repolarization and enabling L-type Ca(2+) current reactivation.

Proteomic analyses of transgenic LQT1 and LQT2 rabbit hearts elucidate an increase in expression and activity of energy producing enzymes.

Various biochemical and genomic mechanisms are considered to be a hallmark of metabolic remodeling in the stressed heart, including the hypertrophied and failing heart. In this study, we used quantitative proteomic 2-D Fluorescence Difference In-Gel Electrophoresis (2-D DIGE) in conjunction with mass spectrometry to demonstrate differential protein expression in the hearts of transgenic rabbit models of Long QT Syndrome 1 (LQT1) and Long QT Syndrome 2 (LQT2) as compared to littermate controls (LMC). The results of our proteomic analysis revealed upregulation of key metabolic enzymes involved in all pathways associated with ATP generation, including creatine kinase in both LQT1 and LQT2 rabbit hearts. Additionally, the expression of lamin-A protein was increased in both LQT1 and LQT2 rabbit hearts as was the expression of mitochondrial aldehyde dehydrogenase and desmoplakin in LQT1 and LQT 2 rabbit hearts, respectively. Results of the proteomic analysis also demonstrated down regulation in the expression of protein disulfide-isomerase A3 precuorsor and dynamin-like 120 kDa protein (mitochondrial) in LQT1, and of alpha-actinin 2 in LQT2 rabbit hearts. Up regulation of the expression of the enzymes associated with ATP generation was substantiated by the results of selective enzyme assays in LQT1 and LQT2 hearts, as compared to LMC, which revealed increases in the activities of glycogen phosphorylase (+50%, +65%, respectively), lactate dehydrogenase (+25%, +25%) pyruvate dehydrogenase (+31%, +22%), and succinate dehydrogenase (+32%, +60%). The activity of cytochrome c-oxidase, a marker for the mitochondrial function was also found to be significantly elevated (+80%) in LQT1 rabbit hearts as compared with LMC. Western blot analysis in LQT1 and LQT2 hearts compared to LMC revealed an increase in the expression of very-long chain-specific acyl-CoA dehydrogenase (+35%, +33%), a rate-limiting enzymes in ß-oxidation of fatty acids. Collectively, our results demonstrate similar increases in the expression and activities of key ATP-generating enzymes in LQT1 and LQT2 rabbit hearts, suggesting an increased demand, and in turn, increased energy supply across the entire metabolic pathway by virtue of the upregulation of enzymes involved in energy generation.

MicroRNA-1 and -133 increase arrhythmogenesis in heart failure by dissociating phosphatase activity from RyR2 complex.

In heart failure (HF), arrhythmogenic spontaneous sarcoplasmic reticulum (SR) Ca(2+) release and afterdepolarizations in cardiac myocytes have been linked to abnormally high activity of ryanodine receptors (RyR2s) associated with enhanced phosphorylation of the channel. However, the specific molecular mechanisms underlying RyR2 hyperphosphorylation in HF remain poorly understood. The objective of the current study was to test the hypothesis that the enhanced expression of muscle-specific microRNAs (miRNAs) underlies the HF-related alterations in RyR2 phosphorylation in ventricular myocytes by targeting phosphatase activity localized to the RyR2. We studied hearts isolated from canines with chronic HF exhibiting increased left ventricular (LV) dimensions and decreased LV contractility. qRT-PCR revealed that the levels of miR-1 and miR-133, the most abundant muscle-specific miRNAs, were significantly increased in HF myocytes compared with controls (2- and 1.6-fold, respectively). Western blot analyses demonstrated that expression levels of the protein phosphatase 2A (PP2A) catalytic and regulatory subunits, which are putative targets of miR-133 and miR-1, were decreased in HF cells. PP2A catalytic subunit mRNAs were validated as targets of miR-133 by using luciferase reporter assays. Pharmacological inhibition of phosphatase activity increased the frequency of diastolic Ca(2+) waves and afterdepolarizations in control myocytes. The decreased PP2A activity observed in HF was accompanied by enhanced Ca(2+)/calmodulin-dependent protein kinase (CaMKII)-mediated phosphorylation of RyR2 at sites Ser-2814 and Ser-2030 and increased frequency of diastolic Ca(2+) waves and afterdepolarizations in HF myocytes compared with controls. In HF myocytes, CaMKII inhibitory peptide normalized the frequency of pro-arrhythmic spontaneous diastolic Ca(2+) waves. These findings suggest that altered levels of major muscle-specific miRNAs contribute to abnormal RyR2 function in HF by depressing phosphatase activity localized to the channel, which in turn, leads to the excessive phosphorylation of RyR2s, abnormal Ca(2+) cycling, and increased propensity to arrhythmogenesis.

Pore mutants of HERG and KvLQT1 downregulate the reciprocal currents in stable cell lines.

We previously reported a transgenic rabbit model of long QT syndrome based on overexpression of pore mutants of repolarizing K(+) channels KvLQT1 (LQT1) and HERG (LQT2).The transgenes in these rabbits eliminated the slow and fast components of the delayed rectifier K(+) current (I(Ks) and I(Kr), respectively), as expected. Interestingly, the expressed pore mutants of HERG and KvLQT1 downregulated the remaining reciprocal repolarizing currents, I(Ks) and I(Kr), without affecting the steady-state levels of the native polypeptides. Here, we sought to further explore the functional interactions between HERG and KvLQT1 in heterologous expression systems. Stable Chinese hamster ovary (CHO) cell lines expressing KvLQT1-minK or HERG were transiently transfected with expression vectors coding for mutant or wild-type HERG or KvLQT1. Transiently expressed pore mutant or wild-type KvLQT1 downregulated I(Kr) in HERG stable CHO cell lines by 70% and 44%, respectively. Immunostaining revealed a severalfold lower surface expression of HERG, which could account for the reduction in I(Kr) upon KvLQT1 expression. Deletion of the KvLQT1 NH(2)-terminus did not abolish the downregulation, suggesting that the interactions between the two channels are mediated through their COOH-termini. Similarly, transiently expressed HERG reduced I(Ks) in KvLQT1-minK stable cells. Coimmunoprecipitations indicated a direct interaction between HERG and KvLQT1, and surface plasmon resonance analysis demonstrated a specific, physical association between the COOH-termini of KvLQT1 and HERG. Here, we present an in vitro model system consistent with the in vivo reciprocal downregulation of repolarizing currents seen in transgenic rabbit models, illustrating the importance of the transfection method when studying heterologous ion channel expression and trafficking. Moreover, our data suggest that interactions between KvLQT1 and HERG are mediated through COOH-termini.

Posttranslational modification of voltage-dependent potassium channel Kv1.5: COOH-terminal palmitoylation modulates its biological properties.

The physiological function of ion channels is affected by protein-protein and protein-membrane interactions that modulate their activity and/or localization. Palmitoylation modulates protein function by facilitating targeted membrane association, interaction with other proteins, and determining subcellular localization. In this study, we demonstrate that the voltage-dependent potassium (Kv) channel Kv1.5 is palmitoylated and that the mutation of COOH-terminal cysteines is sufficient to abolish the palmitoylation of the Kv1.5 polypeptide in Chinese hamster ovary (CHO) cells. The labeling represented the thioester linkage of the labeled palmitic acid to cysteine rather than amide and oxygen ester linkages as judged by the release of the palmitic acid upon the treatment of the gel with hydroxylamine at a neutral pH. Site-directed mutagenesis and radiolabeling studies revealed that C593 was the sole site of palmitoylation. The elucidation of the biological function of palmitoylation revealed that the expression of the FLAG-Kv1.5 palmitoylation-deficient mutant (FL-Kv1.5(Palm-)) in stable CHO cells increased membrane expression as determined by the biotinylation of surface proteins and quantitative immunofluorescence analyses of these cells, in turn enhancing the outward potassium current. This enhanced surface expression and the currents were consequential to the slower rate of internalization, causing an increased localization of FL-Kv1.5(Palm-) in the plasma membrane compared with the wild-type FL-Kv1.5 channels. We conclude that the Kv1.5 channel is palmitoylated and that its palmitoylation modulates its biological functions and, therefore, might provide a physiological link between the metabolic state and the expression of Kv1.5 on the plasma membrane.

Purification of the NF2 tumor suppressor protein from human erythrocytes.

BACKGROUND: Neurofibromatosis type 2 (NF2) is an autosomal dominant disease predisposing individuals to the risk of developing tumors of cranial and spinal nerves. The NF2 tumor suppressor protein, known as Merlin/Schwanomin, is a member of the protein 4.1 superfamily that function as links between the cytoskeleton and the plasma membrane. METHODS: Upon selective extraction of membrane-associated proteins from erythrocyte plasma membrane (ghosts) using low ionic strength solution, the bulk of NF2 protein remains associated with the spectrin-actin depleted inside-out-vesicles. Western blot analysis showed a approximately 70 kDa polypeptide in the erythrocyte plasma membrane. Furthermore, quantitative removal of NF2 protein from the inside-out-vesicles was achieved using 1.0 M potassium iodide, a treatment known to remove tightly-bound peripheral membrane proteins. RESULTS: These results suggest a novel mode of NF2 protein association with the erythrocyte membrane that is distinct from the known membrane interactions of protein 4.1. Based on these biochemical properties, several purification strategies were devised to isolate native NF2 protein from human erythrocyte ghosts. Using purified and recombinant NF2 protein as internal standards, we quantified approximately 41-65,000 molecules of NF2 protein per erythrocyte. CONCLUSION: We provide evidence for the presence of NF2 protein in the human erythrocyte membrane. The identification of NF2 protein in the human erythrocyte membrane will make it feasible to discover novel interactions of NF2 protein utilizing powerful techniques of erythrocyte biochemistry and genetics in mammalian cells.