Human Cardiac cDNA Library Research Articles

Sumoylation-independent activation of Calcineurin-NFAT-signaling via SUMO2 mediates cardiomyocyte hypertrophy.

The objective of this study was to identify unknown modulators of Calcineurin (Cn)-NFAT signaling. Measurement of NFAT reporter driven luciferase activity was therefore utilized to screen a human cardiac cDNA-library (~107 primary clones) in C2C12 cells through serial dilutions until single clones could be identified. This extensive screening strategy culminated in the identification of SUMO2 as a most efficient Cn-NFAT activator. SUMO2-mediated activation of Cn-NFAT signaling in cardiomyocytes translated into a hypertrophic phenotype. Prohypertrophic effects were also observed in mice expressing SUMO2 in the heart using AAV9 (Adeno-associated virus), complementing the in vitro findings. In addition, increased SUMO2-mediated sumoylation in human cardiomyopathy patients and in mouse models of cardiomyopathy were observed. To decipher the underlying mechanism, we generated a sumoylation-deficient SUMO2 mutant (ΔGG). Surprisingly, ΔGG replicated Cn-NFAT-activation and the prohypertrophic effects of native SUMO2, both in vitro and in vivo, suggesting a sumoylation-independent mechanism. Finally, we discerned a direct interaction between SUMO2 and CnA, which promotes CnA nuclear localization. In conclusion, we identified SUMO2 as a novel activator of Cn-NFAT signaling in cardiomyocytes. In broader terms, these findings reveal an unexpected role for SUMO2 in cardiac hypertrophy and cardiomyopathy, which may open the possibility for therapeutic manipulation of this pathway.

Filamin C: a novel component of the KCNE2 interactome during hypoxia

SummaryAimKCNE2 encodes for the potassium voltage-gated channel, KCNE2. Mutations in KCNE2 have been associated with long-QT syndrome (LQTS). While KCNE2 has been extensively studied, the functions of its C-terminal domain remain inadequately described. Here, we aimed to elucidate the functions of this domain by identifying its protein interactors using yeast two-hybrid analysis.MethodsThe C-terminal domain of KCNE2 was used as bait to screen a human cardiac cDNA library for putative interacting proteins. Co-localisation and co-immunoprecipitation analyses were used for verification.ResultsFilamin C (FLNC) was identified as a putative interactor with KCNE2. FLNC and KCNE2 co-localised within the cell, however, a physical interaction was only observed under hypoxic conditions.ConclusionThe identification of FLNC as a novel KCNE2 ligand not only enhances current understanding of ion channel function and regulation, but also provides valuable information about possible pathways likely to be involved in LQTS pathogenesis.

A Novel Role for PP5 in Regulating Titin Phosphorylation and Function in the Heart

Within the N2B/N2BA isoforms of the giant protein titin, a cardiac-specific unique sequence (N2Bus) can be phosphorylated by various protein kinases, including PKA, PKG, ERK2, and CaMKII, which reduces titin-based stiffness. Failing human hearts are hypo-phosphorylated at the N2Bus. Specific phosphatases acting on N2Bus are not known. We carried out a yeast-2-hybrid screen using human N2Bus (“bait”) and a human cardiac cDNA library (“prey”) and found the catalytic domain of protein-phosphatase-5 (PP5c) as a binding partner of N2Bus. The interaction was verified by GST-pulldown assays. PP5 is unique within the family of serine/threonine protein phosphatases because of the presence of tetratricopeptide repeats (TPR) at the molecule's N-terminus. PP5 is well expressed in human and mouse hearts and elevated in failing hearts. PP5 is autoinhibited due to interaction of the TPR domain with the catalytic C-terminus. However, PP5 can be activated by proteolytic cleavage of the TPR region, through binding of the TPR domain by arachidonic acid, or via binding of heat-shock-protein-90 (HSP90). In PP5-overexpressing transgenic (TG) mouse hearts, we found PP5 to co-localize with the titin-N2Bus in the sarcomeric I-band, as shown by immunofluorescence and immunoelectron microscopy. Autoradiography/back-phosphorylation assays and the detection of N2Bus-phosphosites by phospho-specific anti-titin antibodies showed that recombinant PP5 dephosphorylates recombinant human N2Bus previously phosphorylated by ERK2, PKA, or PKG. PP5 also dephosphorylates the cardiac titin isoforms, N2B and N2BA, in human heart tissue. PP5 TG mouse hearts showed reduced N2Bus phosphorylation compared to wildtype (WT) hearts. Force measurements on permeabilized single cardiomyocytes revealed increased passive stiffness in PP5 TG vs. WT hearts. To conclude, PP5 binds to titin N2Bus and acts as an antagonist to the protein-kinase-mediated effects on titin stiffness. Inhibition of the PP5 effect on titin might benefit stiff hearts.

Ankyrin-B Regulates Kir6.2 Membrane Expression and Function in Heart

Ankyrin polypeptides are critical for normal membrane protein expression in diverse cell types, including neurons, myocytes, epithelia, and erythrocytes. Ankyrin dysfunction results in defects in membrane expression of ankyrin-binding partners (including ion channels, transporters, and cell adhesion molecules), resulting in aberrant cellular function and disease. Here, we identify a new role for ankyrin-B in cardiac cell biology. We demonstrate that cardiac sarcolemmal K(ATP) channels directly associate with ankyrin-B in heart via the K(ATP) channel alpha-subunit Kir6.2. We demonstrate that primary myocytes lacking ankyrin-B display defects in Kir6.2 protein expression, membrane expression, and function. Moreover, we demonstrate a secondary role for ankyrin-B in regulating K(ATP) channel gating. Finally, we demonstrate that ankyrin-B forms a membrane complex with K(ATP) channels and the cardiac Na/K-ATPase, a second key membrane transporter involved in the cardiac ischemia response. Collectively, our new findings define a new role for cardiac ankyrin polypeptides in regulation of ion channel membrane expression in heart.

Phospholamban Interacts with HAX-1, a Mitochondrial Protein with Anti-apoptotic Function

Phospholamban (PLN) is a key regulator of Ca 2+ homeostasis and contractility in the heart. Its regulatory effects are mediated through its interaction with the sarcoplasmic reticulum Ca 2+-ATPase, (SERCA2a), resulting in alterations of its Ca 2+-affinity. To identify additional proteins that may interact with PLN, we used the yeast-two-hybrid system to screen an adult human cardiac cDNA library. HS-1 associated protein X-1 (HAX-1) was identified as a PLN-binding partner. The minimal binding regions were mapped to amino acid residues 203–245 for HAX-1 and residues 16–22 for PLN. The interaction between the two proteins was confirmed using GST-HAX-1, bound to the glutathione-matrix, which specifically adsorbed native PLN from human or mouse cardiac homogenates, while in reciprocal binding studies, recombinant His-HAX-1 bound GST-PLN. Kinetic studies using surface plasmon resonance yielded a K D of ∼ 1 μM as the binding affinity for the PLN/HAX-1 complex. Phosphorylation of PLN by cAMP-dependent protein kinase reduced binding to HAX-1, while increasing concentrations of Ca 2+ diminished the PLN/HAX-1 interaction in a dose-dependent manner. HAX-1 concentrated to mitochondria, but upon transient co-transfection of HEK 293 cells with PLN, HAX-1 redistributed and co-localized with PLN at the endoplasmic reticulum. Analysis of the anti-apoptotic function of HAX-1 revealed that the presence of PLN enhanced the HAX-1 protective effects from hypoxia/reoxygenation-induced cell death. These findings suggest a possible link between the Ca 2+ handling by the sarcoplasmic reticulum and cell survival mediated by the PLN/HAX-1 interaction.

Cardiac sodium channel Na v1.5 interacts with and is regulated by the protein tyrosine phosphatase PTPH1

In order to identify proteins interacting with the cardiac voltage-gated sodium channel Na v1.5, we used the last 66 amino acids of the C-terminus of the channel as bait to screen a human cardiac cDNA library. We identified the protein tyrosine phosphatase PTPH1 as an interacting protein. Pull-down experiments confirmed the interaction, and indicated that it depends on the PDZ-domain binding motif of Na v1.5. Co-expression experiments in HEK293 cells showed that PTPH1 shifts the Na v1.5 availability relationship toward hyperpolarized potentials, whereas an inactive PTPH1 or the tyrosine kinase Fyn does the opposite. The results of this study suggest that tyrosine phosphorylation destabilizes the inactivated state of Na v1.5.

A common human SCN5A polymorphism modifies expression of an arrhythmia causing mutation.

SCN5A encodes the alpha-subunit of the ion channel that carries Na current in human heart. From a human cardiac cDNA library we recloned SCN5A. The new clone hH1b differed from existing clones hH1 in four and from hH1a in three positions. The common polymorphism H558R was uniquely present in hH1b. Voltage clamp study showed minor but potentially important kinetic differences between hH1b and the other clones. More dramatically, when the LQT3 mutation M1766L was introduced into the different clones, Na current was markedly reduced in the hH1 and hH1a backgrounds, whereas in hH1b the Na current was not reduced. Immunocytochemistry experiments showed a trafficking defect for M1766L Na channels in hH1 and hH1a but not in hH1b. The double-mutation M1766L/H558R in the hH1a background restored normal trafficking and current including persistent late current, suggesting the disease phenotype was the result of a "double hit" that included the common polymorphism, H558R. These results show that the choice of background clone must be carefully considered in mutagenesis studies. This also represents an example of intragenic complementation, the first for such a large protein.

Identification of novel interactions between domains of Myosin binding protein-C that are modulated by hypertrophic cardiomyopathy missense mutations.

Cardiac myosin binding protein-C (cMyBPC) is a modular protein consisting of 11 domains whose precise function and sarcomeric arrangement are incompletely understood. Identification of hypertrophic cardiomyopathy (HCM)--causing missense mutations in cMyBPC has highlighted the significance of certain domains. Of particular interest is domain C5, an immunoglobulin-like domain with a cardiac-specific insert, which is of unknown function yet is the site of two HCM-causing missense mutations. To identify interactors with this region, a human cardiac cDNA library was screened in a yeast two-hybrid (Y2H) assay using the C5 sequence as bait. Screening >7x10(6) clones surprisingly revealed that domain C5 preferentially bound to clones encoding C-terminal fragments of cMyBPC; the interacting region was narrowed to domain C8 by deletion mapping. A surface plasmon resonance assay using purified recombinant cMyBPC domains was used to measure the affinity of C5 and C8 in vitro (K(a)=1x10(5) mol/L(-1)). This affinity was decreased about 2-fold by the HCM mutation R654H, and by at least 10-fold by the mutation N755K. Further Y2H assays also demonstrated specific binding between domains C7 and C10 of cMyBPC. Based on these novel interactions, and previous biochemical and structural data, we propose that cMyBPC molecules trimerize into a collar around the thick filament, with overlaps of domains C5-C7 of one cMyBPC with C8-C10 of another. We speculate that this interaction may be dynamically formed and released, thereby restricting or favoring cross-bridge formation, respectively. We suggest that the HCM mutations act by altering the cMyBPC collar, indicating its importance in thick filament structure and regulation.

Regulation of the voltage-gated K+ channel KCNA10 by KCNA4B, a novel β-subunit

Voltage-gated K+ (Kv) channels are heteromultimeric complexes consisting of pore-forming alpha-subunits and accessory beta-subunits. Several beta-subunits have been identified and shown to interact with specific alpha-subunits to modify their levels of expression or some of their kinetic properties. The aim of the present study was to isolate accessory proteins for KCNA10, a novel Kv channel alpha-subunit functionally related to Kv and cyclic nucleotide-gated cation channels. Because one distinguishing feature of KCNA10 is a putative cyclic nucleotide-binding domain located at the COOH terminus, the entire COOH-terminal region was used to probe a human cardiac cDNA library using the yeast two-hybrid system. Interacting clones were then rescreened in a functional assay by coinjection with KCNA10 in Xenopus oocytes. One of these clones (KCNA4B), when injected alone in oocytes, produced no detectable current. However, when coinjected with KCNA10, it increased KCNA10 current expression by nearly threefold. In addition, the current became more sensitive to activation by cAMP. KCNA4B can be coimmunoprecipitated with the COOH terminus of KCNA10 and full-length KCNA10. It encodes a soluble protein (141 aa) with no amino acid homology to known beta-subunits but with limited structural similarity to the NAD(P)H-dependent oxidoreductase superfamily. KCNA4B is located on chromosome 13 and spans approximately16 kb, and its coding region is made up of five exons. In conclusion, KCNA4B represents the first member of a new class of accessory proteins that modify the properties of Kv channels.

Co-regulation of Tissue-specific Alternative Human Carnitine Palmitoyltransferase Iβ Gene Promoters by Fatty Acid Enzyme Substrate

Carnitine palmitoyltransferase I (CPT-I) catalyzes the rate-determining step in mitochondrial fatty acid beta-oxidation. CPT-I has two structural genes (alpha and beta) that are differentially expressed among tissues. Our CPT-Ibeta isolates from a human cardiac cDNA library contained two different extreme 5'-sequences derived from short alternative first untranslated exons that utilize a common splice acceptor site in exon 2. Primer extension identified single dominant start sites for each transcript, and ribonuclease protection assays showed the presence of one 5'-exon in liver, muscle, and heart mRNAs, indicating that the cognate promoter U (upstream/ubiquitous) is active in each of these tissues. By contrast, mRNAs containing the alternative 5'-exon were present only in muscle and heart, indicating a muscle-specific promoter M (muscle). CPT-Ibeta mRNA levels increased markedly in tissues of fasted rats, when circulating free fatty acid concentrations are elevated. Using CPT-Ibeta promoter/reporter transient transfection of murine C2C12 myotubes and HepG2 hepatocytes, fatty acids were found to increase promoter activity in a peroxisome proliferator-activated receptor alpha (PPARalpha)-dependent fashion. A promoter fatty acid response element (FARE) was mapped, mutation of which ablated fatty acid-mediated production of both transcripts. PPARalpha/retinoid X receptor alpha formed specific complexes with oligonucleotides containing the FARE, and anti-PPARalpha antibody shifted nuclear protein-DNA complexes, confirming the role of this factor in regulating the expression of this critical metabolic enzyme gene. The constitutive repressor chicken ovalbumin upstream promoter transcription factor competitively binds at the FARE and modulates fatty acid induction of the promoters.

Expression of novel isoforms of carnitine palmitoyltransferase I (CPT-1) generated by alternative splicing of the CPT-ibeta gene.

Carnitine palmitoyltransferase I (CPT-I) catalyses the rate-determining step in mitochondrial fatty acid beta-oxidation. The enzyme has two cognate structural genes that are preferentially expressed in liver (alpha) or fat and muscle (beta). We hypothesized the existence of additional isoforms in heart to account for unique kinetic characteristics of enzyme activity in this tissue. Hybridization and PCR screening of a human cardiac cDNA library revealed the expression of two novel CPT-I isoforms generated by alternative splicing of the CPT-Ibeta transcript, in addition to the beta and alpha cDNA species previously described. Ribonuclease protection and reverse transcriptase-mediated PCR assays confirmed the presence of mRNA species of each splicing variant in heart, skeletal muscle and liver, with differing relative concentrations in the tissues. The novel splicing variants omit exons or utilize a cryptic splice donor site within an exon. Deduced polypeptide sequences of the novel enzymes include omissions in the region of putative membrane-spanning and malonyl-CoA regulatory domains compared with the previously described CPT-Is, implying that the encoded enzymes will exhibit unique features with respect to outer mitochondrial membrane topology and response to physiological and pharmacological inhibitors.

Molecular cloning, characterization, and genomic localization of a human potassium channel gene

Potassium (K+) channels are critical for a variety of cell functions, including modulation of action potentials, determination of resting membrane potential, and development of memory and learning. In addition to their role in regulating myocyte excitability, cardiac K+ channels control heart rate and coronary vascular tone and are implicated in the development of arrhythmias. We report here the cloning and sequencing of a K+ channel gene, KCNA1, derived from a human cardiac cDNA library and the chromosomal localization of the corresponding genomic clone. Oligonucleotides based on a delayed rectifier K+ channel gene were used in PCR reactions with human genomic DNA to amplify the S4–S6 regions of several different K+ channel genes. These sequences were used to isolate clones from a human cardiac cDNA library. We sequenced one of these clones, HCK1. HCK1 contains putative S2–S6 domains and shares ∼70% sequence homology with previously isolated Shaker homologues. HCK1 was used to screen human cosmid libraries and a genomic clone was isolated. By sequencing the genomic clones, a putative S1 domain and translation initiation sequences were identified. Genomic mapping using human-rodent somatic cell panels and in situ hybridization with human metaphase chromosomes have localized KCNA1 to the distal short arm of human chromosome 12. This work is an important step in the study of human cardiac K+ channel structure and function and will be of use in the study of human inherited disease.