Human Heart cDNA Research Articles

Abstract 105: Novel Functions For Synaptosomal-associated Protein 29 (snap29) In Cardiac Arrhythmias

Human genetic studies and mouse models have classically linked mutations/deficiencies in components of the desmosomal cell-cell junction to arrhythmogenic right ventricular cardiomyopathy (ARVC). However, growing evidence points to the importance of the desmosome in cardiac diseases beyond ARVC, which include electrical diseases that have no impact on cardiac structure/morphology. The mechanisms of how defects in cardiac desmosomal protein homeostasis drive these distinct forms of cardiac disease remain elusive to the field. To uncover mechanisms that underlie the distinct pathophysiology (structural versus non-structural) encompassed by desmosomal mutations/loss, we performed an unbiased yeast-two-hybrid screen using an adult human heart cDNA library and the desmosomal protein, desmoplakin (DSP) to uncover new regulators of cardiac desmosomal protein homeostasis. We identified synaptosomal-associated protein 29 (SNAP29), as a novel DSP-interacting protein in the adult human heart. Traditional functions of SNAP29 are to regulate membrane fusion and play a role in autophagy; however, its role at the desmosome and in the heart is undefined. We show that SNAP29 is a subcomponent of the cardiac desmosome, as it co-localizes with DSP in the adult heart and DSP-deficient hearts harbor loss of SNAP29. Cardiomyocyte-specific SNAP29 knockout (SNAP29-cKO) mice displayed baseline and pacing-induced ventricular arrhythmias in an age-dependent manner in the absence of cardiac structural and functional deficits. We show that a loss of a subset of desmosomal proteins and connexin43 as well as upregulation of selective autophagy-mediated degradation underlie SNAP29 deficient cardiomyocyte arrhythmias. In line with this, acute blockade of autophagy was sufficient to rescue desmosomal and connexin43 protein levels as well as arrhythmias in SNAP29 deficient cardiomyocytes. In conclusion, SNAP29 insulates a subset of desmosomal and gap junction proteins from selective autophagy-mediated degradation to restrict cardiac arrhythmias. Thus, loss of SNAP29-desmosome-gap junction interactome may predispose the heart to desmosomal based diseases of an electrical nature.

Discovery of Protein Degradation Machinery at the Desmosome Reveals Novel Triggers of the Desmosomal Disease, Arrhythmogenic Right Ventricular Cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic‐based cardiac disease which is characterized by right/bi‐ventricular dysfunction, fibro‐fatty replacement of the myocardium and ventricular arrhythmias, leading to sudden death in young people and athletes. ARVC is termed a “disease of the desmosome” (mechanical cell‐cell junction) as 40% of ARVC patients harbor mutations and/or loss of desmosomal components. However, limited information exists on how cardiac desmosomal protein loss/degradation triggers ARVC. Through a yeast‐two‐hybrid screen in an adult human heart cDNA library, we uncovered a new interaction between the desmosomal protein, desmoplakin (DSP, N terminus) and a protein degradation machinery component, CSN6 (COP9 signalosome subunit 6). Traditionally, CSN proteins are thought to prevent proteins from being degraded; however, a role for CSN6 in the heart is undefined. We show that CSN6 localizes to desmosomal junctions in cardiac muscle and structurally complex with desmoplakin, suggesting that the novel hypothesis that CSN6 may protect the cardiac desmosome from degradation and loss/destablization this pathway may lead to ARVC. Using two novel cardiac‐specific CSN6 knockout (CSN6‐KO) mice, we further show that CSN6 loss accelerates desmosome destruction and triggers ARVC, as CSN6‐KO hearts exhibit selective loss of desmosomal proteins and recapitulate the key cardiomyopathy features and sudden death associated with ARVC, similar to classic mouse model of ARVC (DSP‐cKO). CSN6‐KO and DSP‐cKO hearts also harbor underlying hyper‐accumulation of protein degradation machinery specifically at cell‐cell junctions. Restoration of CSN function by the protein degradation inhibitor (MLN4924) could rescue the loss of desmosomal protein expression associated with CSN6 loss in vitro, further highlighting that the cardiac desmosome is regulated by protein degradation. CSN6 pathways are also relevant to human ARVC as a N‐terminus desmoplakin mutation (R315C) identified in a human ARVC patient was sufficient to abrogate DSP‐CSN6 binding and result in loss of CSN6 localization at the cell‐cell junction in cardiac biopsies. DSP R315C knock‐in mice also exhibited cardiac CSN6 reduction and early electrical defects found in ARVC. We altogether identify CSN6 as a novel “brake” to prevent desmosomal protein degradation, and reveal that disruption of this complex is an important “accelerator” of sudden death and ARVC.Support or Funding InformationAmerican Heart Association Postdoctoral Fellowship 17POST33670205This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Policing Cell‐Cell Connections: A Novel Role for the COP9 Signalosome in Mechanisms Underlying Arrhythmogenic Heart Disease and Sudden Death

Arrhythmogenic right ventricular cardiomyopathy is a genetic based cardiomyopathy, characterized by ventricular dysfunction, fibrofatty replacement of the ventricle and ventricular arrhythmias leading to sudden cardiac death in young people. ARVC is termed a “disease of the desmosome”; however, limited information exists on how desmosomal protein dysregulation/loss triggers ARVC. To identify novel protein targets that impact desmosomal biology and the desmosomal disease, ARVC, we performed a yeast two hybrid screen using the desmosomal gene, desmoplakin (DSP) as bait to screen an adult human heart cDNA library. We identified CSN6, subunit 6 of the COP9 signalosome, as a novel cardiac desmosomal interacting protein (via desmoplakin, DSP). Traditional functions of the CSN complex (composed of subunits 1–8) are to inhibit protein degradation by “turning off” ubiquitinatin‐mediated protein degradation via deneddlylation; however, the role of CSN6 in the heart is undefined. We show that CSN6 uniquely protects the cardiac desmosome and its loss accelerates desmosome destruction as (i) CSN6 co‐localizes to desmosomal junctions, (ii) CSN6 co‐immunoprecipitates with desmosomal proteins and (iii) hearts from novel cardiac‐specific CSN6 knockout (CSN6‐cKO) mice display selective loss of desmosomal protein levels (and its primary target, connexin43). CSN6 loss is a trigger for ARVC as CSN6‐cKO mice exhibit sudden death and cardiac disease features associated with a biventricular form of ARVC, similar to our cardiac‐specific DSP knockout mice (DSP‐cKO) that is a classic biventricular model of ARVC. CSN6 and DSP‐cKO hearts both selectively display underlying hyper‐accumulation of protein degradation machinery at the cell junction, specifically linking CSN6 pathways to the desmosome. CSN6 pathways are relevant to human ARVC as (i) CSN6 levels are down‐regulated in ARVC hiPSC‐derived cardiac cells that exhibit striking desmosomal defects and arrhythmogenic behavior and (ii) CSN6 localization is lost from cell junctions in a cardiac biopsy from an ARVC patient harboring desmosomal (DSP (R315C) and plakophilin‐2 (PKP2 IVS10‐1 G>C) mutations. Yeast and in silico modeling assays reveal that this DSP R315C mutation is sufficient to abrogate DSP binding to CSN6. We highlight the importance of CSN6 at the cardiac desmosome in maintaining desmosomal protein turnover (“desmophagy”), and suggest that loss of CSN6 and desmosomal protein turnover as new underlying mechanisms driving ARVC and sudden death.Support or Funding InformationF.S. is supported by grants from National Institutes of Health (NHLBI), American Heart Association and Tobacco Related Disease Research Program.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Production and functional characterization of human insulin-like growth factor 1

Insulin-like growth factor 1 (IGF-1) is a polypeptide hormone produced mainly by the liver in response to the endocrine growth hormone (GH) stimulus. This protein is involved in a wide range of cellular functions, including cellular differentiation, transformation, apoptosis suppression, migration and cell-cycle progression and other metabolic processes. In the current study, human heart cDNA was employed to isolate IGF-1 encoding fragment using reverse transcriptase (RT) PCR. The isolated fragment was cloned into pET32a expression vector and then transformed into the competent Escherichia coli Origami 2. After selecting the correct colony with the highest expression level, the colony was cultured and induced with IPTG. Recombinant IGF-1 expression was detected by SDS-PAGE and His-tagged protein purification was performed with the affinity chromatography. In order to confirm the activity of the resultant protein, biological activity of the recombinant IGF-1 was assayed through inducing proliferation of MCF-7 cells. Molecular techniques, including PCR, restriction digestion, mass spectrometry analyses, SDS-PAGE and biological activity analyses of this protein confirmed the correct cloning, expression, and function of IGF-1 in this study. Overall, we provided a rapid and cost effective production and purification method for IGF-1 protein, which is biologically active and functional.

Metallothionein 2A an interactive protein linking phosphorylated FADD to NF-κB pathway leads to colorectal cancer formation.

The rapid increase in the incidence rate of colorectal cancer has led to the search and identification of biomarkers that can predict risk for and future behavior of this malignancy and management. To study the biological role of the phosphorylated Fas associated death domain (pFADD) gene in colorectal cancer, we performed a GAL4-based yeast two-hybrid screening of a human heart cDNA library. A series of two yeast hybrid method was used to identification of protein-protein interaction. It was confirmed by glutathione S-transferase (GST) pull down assay and co-immunoprecipitation (co-IP). Three channeled fluorescence microscopy further confirmed the interaction in cellular level. Xenograft in vivo model was developed and knockdown relevant genes by RNAi techniques and confirmed the relationship which leads to colorectal cancer. Using the FADD cDNA as bait, we identified six putative clones as associated proteins. The interaction of pFADD and metallothionein 2A (MT2A) was confirmed by GST pull-down assays in vitro and co-IP experiments in vivo. FADD co-localized with MT2A mostly to nuclei and slightly to cytoplasm, as shown by three channel fluorescence microscopy. Co-transfection of pFADD with MT2A gene inhibited cell apoptosis and induced cell proliferation in colorectal cancer cells compared with control groups. When we used antisense MT2A and pFADD which is serine 194 in the C terminal of FADD gene that has been reported to be phosphorylated to interdict the effect of respective genes the inhibition of cell proliferation and induction of apoptosis were significantly enhanced in animal model. Further in this study we identify non-canonical nuclear factor-κB (NF-κB) signaling up regulated and it was directly linked with the tumor necrosis with MT2A and pFADD genes. pFADD with MT2A can inhibit the apoptosis and promote proliferation, of colorectal cancer cells, and antisense sequence of MT2A and pFADD approaches which might swell the combination of deregulated proliferation and suppressed apoptosis.

17β-Estradiol-induced interaction of estrogen receptor α and human atrial essential myosin light chain modulates cardiac contractile function.

Chronicincreased workload of the human heart causes ventricular hypertrophy, re-expression of the atrial essential myosin light chain (hALC-1), and improved contractile function. Although hALC-1 is an important positive inotropic regulator of the human heart, little is known about its regulation. Therefore, we investigated the role of the sex hormone 17β-estradiol (E2) on hALC-1 gene expression, the underlying molecular mechanisms, and the impact of this regulatory process on cardiac contractile function. We showed that E2 attenuated hALC-1 expression in human atrial tissues of both sexes and in human ventricular AC16 cells. E2 induced the nuclear translocation of estrogen receptor alpha (ERα) and hALC-1 in AC16 cells, where they cooperatively regulate the transcriptional activity of hALC-1 gene promoter. E2-activated ERα required the estrogen response element (ERE) motif within the hALC-1 gene promoter to reduce its transcriptional activity (vehicle: 15.55±4.80 vs. E2: 6.51±3.69; ~2 fold). This inhibitory effect was potentiated in the presence of hALC-1 (vehicle: 11.13±3.66 vs. E2: 2.18±1.10; ~5 fold), and thus, hALC-1 acts as a co-repressor of ERα-mediated transcription. Yeast two-hybrid screening of a human heart cDNA library revealed that ERα interacts physically with hALC-1 in the presence of E2. This interaction was confirmed by Co-Immunoprecipitation and immunofluorescence in human atrium. As a further novel effect, we showed that chronic E2-treatment of adult mouse cardiomyocytes overexpressing hALC-1 resulted in reduced cell-shortening amplitude and twitching kinetics of these cells independent of Ca2+ activation levels. Together, our data showed that the expression of hALC-1 gene is, at least partly, regulated by E2/ERα, while hALC-1 acts as a co-repressor. The inotropic effect of hALC-1 overexpression in cardiomyocytes can be significantly repressed by E2.

Human heart cell proteins interacting with a C-terminally truncated 2A protein of coxsackie B3 virus: identification by the yeast two-hybrid system.

Protein 2A is a non-structural protein of coxsackievirus B3 (CVB3), an important human pathogen that can cause a variety of human diseases. Protein 2A not only participates in viral life cycle, but also regulates host cell functions; however, the underlying mechanisms remain poorly understood. In order to better understand the molecular mechanisms of CVB3 2A's function, the yeast two-hybrid (Y2H) system was adopted to screen for CVB3 2A interactive proteins in the human heart cDNA library. Full-length 2A shows strong transcriptional activity in yeast cells, which interferes with the application of Y2H system; therefore, a series of 2A deletion mutants were constructed. Analysis of transcriptional self-activation revealed that 2A lost its transcriptional activity after truncation of 60 amino acids (aa) at the N-terminus or deletion of 17 aa at the C-terminus. Choosing the 2A mutant with 17 aa deletion at the C-terminus as the bait protein, four interactive cellular proteins were identified, including TIMP4, MYL2, COX7C, and ENO1. These proteins are mostly related to protein degradation and metabolism. Although the interactions detected by the Y2H system should be considered as preliminary results, the finding of proteins translated from a human heart cDNA library that interacts with the CVB3 2A will stimulate experiments testing the reactivity of a translational mixture derived from that library with full-length 2A protein, followed by co-immunoprecipitation studies.

In vitro interaction between coxsackievirus B3 VP1 protein and human pleckstrin homology domain retinal protein (PHR1).

Coxsackievirus B3 (CVB3) infection causes central nervous system diseases including aseptic meningitis and encephalitis. To understand the mechanism of this virus, a yeast two-hybrid system was used to screen cellular proteins from a human heart cDNA library. The results revealed that the human Pleckstrin Homology Domain Retinal protein (PHR1), a PH domain-containing protein with low expression in the heart and high expression in the brain, interacts with CVB3 VP1, a major structural protein of CVB3. Yeast mating assays and in vitro coimmunoprecipitation verified the interaction between CVB3 VP1 and PHR1. An α-galactosidase assay indicated that of α-galactosidase activity was higher in positive clones than in controls suggesting a strong interaction. Furthermore, assay of deletion mutants defined the minimal region of PHR1 required for its interaction with VP1 as amino acids 95-172 and two regions of VP1 required for its interaction with PHR1 as amino acids 729-767 and 811-859. The results revealed multiple binding sites between PHR1 and CVB3 VP1 and suggested that the strong interaction between these two proteins might play an important role in central nervous system disease in the human brain.

172 Junctophilin-2 interacts with the cardiac L-Type voltage-gated calcium channel

Background Excitation contraction (E-C) coupling in cardiac muscle is regulated by the L-type voltage-gated calcium channels (LTCC) within the t-tubules, and surface membrane, and the ryanodine receptors (RyR2) localised to the junctional sarcoplasmic reticulum (jSR). The juxtaposition of the t-tubules and jSR forms a microdomain termed a dyad. It is now widely accepted that junctophilin-2 (JP2) acts as a protein bridge to maintain the dyad geometry. JP2 is anchored in the SR, via its C-terminal transmembrane domain, and is thought to span the cytosol and interact with the plasma membrane thereby constraining the dyad architecture to provide the optimal proximal distance between the LTCCs and RyR2s to facilitate effective calcium induced calcium release for cardiac contraction. More recently, data has emerged to suggest that JP2 has an additional role, influencing E-C coupling through an interaction with RyR2. Furthermore, there is evidence for an association between JP2 and the LTCC in skeletal muscle. However, the region of the LTCC involved in binding to JP2 was not established nor was whether there is an interaction between JP2 and the LTCC in the heart. Methods We employed yeast-2-hybrid (Y2H) (Hybrigenics) experiments to investigate JP2 binding partners using human heart cDNA library. Domains of the LTCC were expressed as GST-fusion proteins and JP2 full-length and domains were purified from E.coli . Pull-down experiments were employed to investigate the interaction between the JP2 and the LTCC protein domains. Results Data from Y2H experiments identified the C-terminal region within the ion channel pore of LTCCs as a JP2 binding partner. Therefore, we designed a series of constructs encompassing the C-terminal domain of the Cav1.2 subunit and expressed them as GST-fusion proteins. Employing purified JP2 we showed a direct interaction with the C-terminal tail of Ca v 1.2. Furthermore, we determined that the N-terminal portion of JP2 mediates this interaction. We also investigated whether this interaction is Ca 2+ dependent since we have previously reported that JP2 has a cation binding domain. Our experiments revealed that the JP2-Ca v 1.2 interaction is independent of [Ca 2+ ]. Conclusion This study has provided novel data revealing that not only is there a direct interaction between the cardiac LTCC and JP2 but that the binding domains involve the C-terminal region of Ca v 1.2 and the N-terminal domain of JP2. Significantly, the Ca v 1.2 C-terminal domain binds calmodulin (CaM), both apoCaM and Ca 2+ CaM, regulating both calcium dependent inactivation (CDI) and facilitation (CDF) of the channel. Therefore, our future studies are directed at understanding the significance of JP2 binding, for example whether JP2 influences channel function (CDI/CDF) and/or whether a JP2-Ca v 1.2 is important for LTCC localisation to the dyad.

Screening for cardiac HERG potassium channel interacting proteins using the yeast two‐hybrid technique

The human ERG protein (HERG or Kv 11.1) encoded by the human ether-a-go-go-related gene (herg) is the pore-forming subunit of the cardiac delayed rectifier potassium current (IKr) responsible for action potential (AP) repolarization. Mutations in HERG lead to long-QT syndrome, a major cause of arrhythmias. Protein-protein interactions are fundamental for ion channel trafficking, membrane localization, and functional modulation. To identify proteins involved in the regulation of the HERG channel, we conducted a yeast two-hybrid screen of a human heart cDNA library using the C-terminus or N-terminus of HERG as bait. Fifteen proteins were identified as HERG amino terminal (HERG-NT)-interacting proteins, including Caveolin-1 (a membrane scaffold protein with multiple interacting partners, including G-proteins, kinases and NOS), the zinc finger protein, FHL2 and PTPN12 (a non-receptor tyrosine phosphatase). Eight HERG carboxylic terminal (HERG-CT)-interacting proteins were also identified, including the NF-κB-interacting protein myotrophin, We have identified multiple potential interacting proteins that may regulate cardiac IKr through cytoskeletal interactions, G-protein modulation, phosphorylation and downstream second messenger and transcription cascades. These findings provide further insight into dynamic modulation of HERG under physiological conditions and arrhythmogenesis.

Abstract 314: The Interaction Of The Estrogen Receptor Alpha With The Cardiac Myosin Essential Light Chain Isoform 4 As A New Regulatory Mechanism In The Heart

Estrogen (17β-estradiol, E2) is one of the key regulators of growth, differentiation and physiological function in different tissues, including the heart. The effects of E2 are mainly mediated by estrogen receptor (ER) alpha and beta, which act in concert with many cofactors to mediate estrogenic effects. So far, only a few cofactors of ER have been described in the human heart. To gain a better understanding of E2-mediated ERα action in the human heart, we identified and characterized the novel interaction partners of ERα Yeast two hybrid screening of a human heart cDNA library revealed that ERα interacts with the cardiac myosin essential light chain isoform 4 (ALC1) in presence of E2. ALC1, as a member of contractile proteins, is expressed in the fetal heart and becomes restricted to the atria of the adult heart under physiological conditions, and is reexpressed in ventricle of adult hypertrophic hearts. This switch is accompanied by alteration of contractile performance, thus improving the heart function. Retransformation experiments showed that ALC1 interacts with full-length ERα and ERα-EF domain in presence of E2. The interaction of ERα with ALC1 was also confirmed by Co-IP in human atrium. Double irnmunofluorescence (IF) analysis of paraffin-embedded sections from human atrium tissues showed co-localization of ERα and ALC1 proteins in a striated sarcomeric pattern. Co-localization corresponds most likely to the H-zone of sarcomere. IF analysis of AC16 cells (a human cardiomyocytes cell line) showed that ERα and ALC1 were mainly localized in the cytoplasm in the absence of E2. However, E2 treatment of AC16 cells led to a translocation of ALC1 and ERα into the nuclei of AC16 cells, where they also co-localized. Expression analysis in AC16 cells showed that E2 increases the expression of ALC1 gene (MYL4). Using ERE-based luciferase reporter assays we showed that E2-induced interaction of ERα with the ALC1 represses the transcriptional activity of ERα. We characterize for the first time an E2-regulated interaction of ALC1 with ERα in cardiomyocytes that may be crucial in physiological and/or pathological processes by regulating of transcriptional activity of ERa in the heart and/or by modulating contractile properties of cardiomyocytes.

Protein-L-isoaspartate (D-aspartate) O-methyltransferase protects cardiomyocytes against hypoxia induced apoptosis through inhibiting proapoptotic kinase Mst1

Mammalian sterile 20-like kinase 1 (Mst1) is a mammalian homolog of Hippo kinase from Drosophila and it is a critical component of the Hippo signaling pathway, which regulates a variety of biological processes ranging from cell contact inhibition, organ size control, apoptosis and tumor suppression in mammals. Mst1 plays essential roles in heart disease since its activation causes cardiomyocyte apoptosis and dilated cardiomyopathy. However, the mechanism underlying Mst1 activation in the heart is not known. To identify novel cardiac proteins that may regulate Mst1 activity in the heart under pathophysiological conditions, a yeast two-hybrid screening of a human heart cDNA library with a dominant-negative Mst1 (K59R) mutant used as bait was performed. As a result, protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) was identified as an Mst1-interacting protein. The interaction of PCMT1 with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK293 cells and native cardiomyocytes, in which PCMT1 interacted with the kinase domain of Mst1, but not with its C-terminal regulatory domain. Overexpression of PCMT1 did not affect the Mst1 expression, but significantly attenuated the Mst1 activation and its apoptotic effects in response to the hypoxia/reoxygenation induced injury in cardiomyocytes. Indeed, upregulation of PCMT1 by CGP3466B, a compound related to the anti-Parkinson's drug R-(-)-deprenyl with potent antiapoptotic effects, inhibited the hypoxia/reoxygenation induced Mst1 activation and cardiomyocyte apoptosis. These findings implicate PCMT1 as a novel inhibitor of Mst1 activation in cardiomyocytes and suggest that targeting PCMT1 may prevent myocardial apoptosis through inhibition of Mst1.

Glyceraldehyde-3-Phosphate Dehydrogenase Interacts with Proapoptotic Kinase Mst1 to Promote Cardiomyocyte Apoptosis

Mammalian sterile 20-like kinase 1 (Mst1) is a critical component of the Hippo signaling pathway, which regulates a variety of biological processes ranging from cell contact inhibition, organ size control, apoptosis and tumor suppression in mammals. Mst1 plays essential roles in the heart disease since its activation causes cardiomyocyte apoptosis and dilated cardiomyopathy. However, the mechanism underlying Mst1 activation in the heart remains unknown. In a yeast two-hybrid screen of a human heart cDNA library with Mst1 as bait, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as an Mst1-interacting protein. The interaction of GAPDH with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK293 cells and mouse heart homogenates, in which GAPDH interacted with the kinase domain of Mst1, whereas the C-terminal catalytic domain of GAPDH mediated its interaction with Mst1. Moreover, interaction of Mst1 with GAPDH caused a robust phosphorylation of GAPDH and markedly increased the Mst1 activity in cells. Chelerythrine, a potent inducer of apoptosis, substantially increased the nuclear translocation and interaction of GAPDH and Mst1 in cardiomyocytes. Overexpression of GAPDH significantly augmented the Mst1 mediated apoptosis, whereas knockdown of GAPDH markedly attenuated the Mst1 activation and cardiomyocyte apoptosis in response to either chelerythrine or hypoxia/reoxygenation. These findings reveal a novel function of GAPDH in Mst1 activation and cardiomyocyte apoptosis and suggest that disruption of GAPDH interaction with Mst1 may prevent apoptosis related heart diseases such as heart failure and ischemic heart disease.

Abstract 29: Inhibition of Mst1 Kinase by Protein-l-Isoaspartate (d-aspartate) O-Methyltransferase Protects Cardiomyocytes from Hypoxia-Induced Apoptosis

Background: Mammalian sterile 20-like kinase 1 (Mst1) is a mammalian homolog of Hippo kinase from Drosophila and it is a critical component of the Hippo signaling pathway, which regulates a variety of biological processes ranging from cell contact inhibition, organ size control, apoptosis and tumor suppression in mammals. Activation of Mst1 causes cardiomyocyte apoptosis and dilated cardiomyopathy. However, the mechanism underlying Mst1 activation in the heart is not known. Method and result: In a yeast two-hybrid screen of a human heart cDNA library with a dominant-negative Mst1 (K59R) mutant used as bait, protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) was identified as an Mst1-interacting protein. The interaction of PCMT1 with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK-293 cells and native cardiomyocytes, in which PCMT1 interacted with the kinase domain of Mst1, but not with its C-terminal regulatory domain. Knockdown of PCMT1 augments hypoxia/reoxygenation induced Mst1 activation and apoptosis. Overexpression of PCMT1 did not affect the Mst1 expression, but significantly attenuated the Mst1 activation and its apoptotic effects in response to the hypoxia/reoxygenation induced injury in cardiomyocytes. Moreover, upregulation of PCMT1 by CGP3466B, a compound related to the anti-Parkinson’s drug R-(-)-deprenyl with potent antiapoptotic effects, inhibited the hypoxia/reoxygenation induced Mst1 activation and cardiomyocyte apoptosis. Conclusion: PCMT1 is a novel inhibitor of Mst1 activation in cardiomyocytes and suggest that targeting PCMT1 may prevent myocardial apoptosis through inhibition of Mst1.

Exon 2 of the TFPI Gene Is a Translational Repressor of TFPIβ Protein Expression

AbstractAbstract 3358 Background:Tissue factor pathway inhibitor (TFPI) is an essential anticoagulant protein located on endothelium and within platelets. Two TFPI isoforms, termed TFPIα and TFPIβ, are generated through alternative splicing toward the 3’ end of the TFPI gene. Alternative splicing also occurs within the 5’ untranslated region (UTR) of TFPI mRNA, resulting in the removal of exon 2 (X2). Previous studies have demonstrated that TFPIα and TFPIβ mRNA are produced, on average, in a ratio of 10:1 (TFPIα:TFPIβ) in human tissues. However, this does not appear to correlate with protein expression, suggesting that TFPI protein isoform production is regulated during translation. Biochemical studies were undertaken to determine how alternative splicing within the 5’ or 3’ UTRs of TFPI mRNA may function in the translational regulation of TFPI protein isoform production. Methods:Northern analysis of human lung RNA using probes directed toward Exon 1 (X1) or Exon 2 (both 5’ UTR), Exon 6 (common to both TFPIα and TFPIβ), Exon 8 (TFPIβ specific) or Exons 9 and 10 (TFPIα specific), and RT-PCR analysis of multiple human tissue cDNAs using an exon 1 specific forward primer and TFPIα or TFPIβ specific reverse primers, were used to investigate isoform specific alternative splicing of exon 2. Polysome profiling of HUVEC and EA.hy926 cellular RNA was performed to investigate translational control of TFPI isoform expression. Luciferase (Luc) reporter assays were established to quantify the effect of exon 2 on TFPI isoform protein expression. Constructs were produced where both splice variants of the 5’ UTR, and the TFPIα or TFPIβ specific 3’ UTRs, were inserted at their respective ends of a Gaussia Luc expression cassette. Luc activity was assessed in stably transfected CHO or EA.hy926 cells and normalized to GFP production or Luc mRNA. Results:Northern analysis revealed the presence of two TFPIα specific bands at 4.1 and 1.4 kb, produced via alternative polyadenylation, and one TFPIβ specific band at 1.1 kb. Exon 1 and 2 probes bind both TFPIα species, demonstrating that 5’ UTR alternative splicing occurs in TFPIα mRNA. RT-PCR analysis of human placenta, lung, and heart cDNA confirmed this, and demonstrated that exon 2 is also alternatively spliced in TFPIβ message. Polysome analysis identified TFPIβ-specific translational repression by exon 2. Luciferase reporter assays quantified this repression at 93% and 86% in CHO and EA.hy926 cells, respectively (see Table 1 for normalized data). In contrast, exon 2 had minimal effect on luciferase expression when the TFPIα 3’ UTR was present. RT-PCR analysis of 15 human tissues revealed that testis, thymus, brain, and spleen had the highest relative amount of exon 2 containing TFPIβ message, although all tissues had more exon 2-absent TFPIβ message compared to that containing exon 2. Conclusion:Exon 2 is a strong translational repressor of TFPIβ production, and a much weaker repressor of TFPIα. To our knowledge, this is the first time that alternative splicing within a 5’ UTR has been demonstrated to affect a specific protein isoform produced via alternative splicing at the 3’ end of the same gene. While the mechanism of repression has yet to be elucidated, the repression of TFPIβ, and not TFPIα, by exon 2 suggests that regions within both exon 2 and the 3’ UTR are required for this effect and is consistent with the hypothesis of mRNA circularization via interactions between the 5’ and 3’ UTRs. We hypothesize that the physiological relevance of the demonstrated repression will include tissue-specific regulation of TFPIβ expression and cellular regulation of TFPIβ expression in inflammatory disease.Table 1Normalised Luciferase activities determined via luciferase reporter assayCell LineMean Normalised Luc Activity [CPS/(RFU/μg protein)]Mean Normalised Luc Activity (CPS/Relative Luc mRNA)X1-Luc-TFPIβX1+X2-Luc-TFPIβX1-Luc-TFPIαX1+X2-Luc-TFPIαCHO94.96.3**426.0331.5#EA.hy926194.826.3*39316.536450.5#**- p < 0.0001 vs X1-Luc-TFPIβ;*- p < 0.01 vs X1-Luc-TFPIβ;#- p < 0.01 vs X1-Luc-TFPIα. Disclosures:Mast:Novo Nordisk A/S: Research Funding.

17β-Estradiol-induced interaction of ERα with NPPA regulates gene expression in cardiomyocytes

17β-Oestradiol (E2) and its receptors (ERα and ERβ) are important regulators of physiological and pathological processes in the cardiovascular system. ER act in concert with other regulatory factors mediating oestrogenic effects. However, the underlying mechanisms modulating ER transcriptional activity are not fully elucidated. To gain better understanding of E2-induced ERα action in the human heart, we aimed to identify and functionally analyse interaction partners of ERα. Using yeast two-hybrid assays with a human heart cDNA library, we identified atrial natriuretic peptide precursor A (NPPA), a well-known cardiac hypertrophy marker, as a novel ERα interaction partner interacting in an E2-dependent manner. Mutation analyses and immunofluorescence data indicated that the LXXLL motif within NPPA is necessary for its E2-induced interaction with ERα, its action as a co-repressor of ERα, and its translocation into the nucleus of human and rat cardiomyocytes. Expression analysis and chromatin immunoprecipitation assays in a human left ventricular cardiomyocyte cell line, AC16, showed that NPPA interacts with E2/ERα, suppressing the transcriptional activity of ERα on E2-target genes, such as NPPA, connexin43, αactinin-2, nuclear factor of activated T-cells, and collagens I and III. We characterize for the first time an E2-regulated interaction of NPPA with ERα in cardiomyocytes, that may be crucial in physiological and/or pathological cardiac processes, thereby representing a potential therapeutic target.

AMP-activated protein kinase phosphorylates cardiac troponin I and alters contractility of murine ventricular myocytes.

AMP-activated protein kinase (AMPK) is an important regulator of energy balance and signaling in the heart. Mutations affecting the regulatory γ2 subunit have been shown to cause an essentially cardiac-restricted phenotype of hypertrophy and conduction disease, suggesting a specific role for this subunit in the heart. The γ isoforms are highly conserved at their C-termini but have unique N-terminal sequences, and we hypothesized that the N-terminus of γ2 may be involved in conferring substrate specificity or in determining intracellular localization. A yeast 2-hybrid screen of a human heart cDNA library using the N-terminal 273 residues of γ2 as bait identified cardiac troponin I (cTnI) as a putative interactor. In vitro studies showed that cTnI is a good AMPK substrate and that Ser150 is the principal residue phosphorylated. Furthermore, on AMPK activation during ischemia, Ser150 is phosphorylated in whole hearts. Using phosphomimics, measurements of actomyosin ATPase in vitro and force generation in demembraneated trabeculae showed that modification at Ser150 resulted in increased Ca(2+) sensitivity of contractile regulation. Treatment of cardiomyocytes with the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) resulted in increased myocyte contractility without changing the amplitude of Ca(2+) transient and prolonged relaxation despite shortening the time constant of Ca(2+) transient decay (tau). Compound C prevented the effect of AICAR on myocyte function. These results suggest that AMPK activation increases myocyte contraction and prolongs relaxation by increasing myofilament Ca(2+) sensitivity. We conclude that cTnI phosphorylation by AMPK may represent a novel mechanism of regulation of cardiac function.

Untangling the Roles of Anti-Apoptosis in Regulating Programmed Cell Death using Humanized Yeast Cells

Genetically programmed cell death (PCD) mechanisms, including apoptosis, are important for the survival of metazoans since it allows, among things, the removal of damaged cells that interfere with normal function. Cell death due to PCD is observed in normal processes such as aging and in a number of pathophysiologies including hypoxia (common causes of heart attacks and strokes) and subsequent tissue reperfusion. Conversely, the loss of normal apoptotic responses is associated with the development of tumors. So far, limited success in preventing unwanted PCD has been reported with current therapeutic approaches despite the fact that inhibitors of key apoptotic inducers such as caspases have been developed. Alternative approaches have focused on mimicking anti-apoptotic processes observed in cells displaying increased resistance to apoptotic stimuli. Hormesis and pre-conditioning are commonly observed cellular strategies where sub-lethal levels of pro-apoptotic stimuli lead to increased resistance to higher or lethal levels of stress. Increased expression of anti-apoptotic sequences is a common mechanism mediating these protective effects. The relevance of the latter observation is exemplified by the observation that transgenic mice overexpressing anti-apoptotic genes show significant reductions in tissue damage following ischemia. Thus strategies aimed at increasing the levels of anti-apoptotic proteins, using gene therapy or cell penetrating recombinant proteins are being evaluated as novel therapeutics to decrease cell death following acute periods of cell death inducing stress. In spite of its functional and therapeutic importance, more is known regarding the processes involved in apoptosis than anti-apoptosis. The genetically tractable yeast Saccharomyces cerevisiae has emerged as an exceptional model to study multiple aspects of PCD including the mitochondrial mediated apoptosis observed in metazoans. To increase our knowledge of the process of anti-apoptosis, we screened a human heart cDNA expression library in yeast cells undergoing PCD due to the conditional expression of a mammalian pro-apoptotic Bax cDNA. Analysis of the multiple Bax suppressors identified revealed several previously known as well as a large number of clones representing potential novel anti-apoptotic sequences. The focus of this review is to report on recent achievements in the use of humanized yeast in genetic screens to identify novel stress-induced PCD suppressors, supporting the use of yeast as a unicellular model organism to elucidate anti-apoptotic and cell survival mechanisms.

Protein Phosphatase-5 is a Novel Titin Ligand Which Dephosphorylates Cardiac Titin and Reduces Passive Myocyte Stiffness

Titin's elastic I-band region is a hotspot for protein-protein interactions and important for myocyte extensibility and passive stiffness. The titin springs can be phosphorylated at the N2B-unique sequence (N2Bus) by protein kinases (PK)A or PKG and at the PEVK-domain by PKCalpha, which affects passive stiffness. We searched for protein phosphatase(s) acting on the N2Bus. A yeast-2-hybrid (Y2H) screen with the human N2Bus (bait) and a human heart cDNA library (prey) detected the catalytic domain of the serine/threonine protein phosphatase-5 (PP5) as a binding partner of the cardiac N2Bus. The interaction was confirmed in forced Y2H screens with the N2Bus and full-length PP5 or PP5 catalytic subunit (PP5c), and also in GST-pulldown assays. In cardiomyocytes, PP5 was mainly in the cytosol but also in the nucleus and at the sarcomeric I-bands. Recombinant PP5 was found by autoradiography to dephosphorylate recombinant, PKG-phosphorylated, N2Bus, and PP5 bound more strongly to (PKA-/PKG-)phosphorylated N2Bus than to non-phosphorylated N2Bus. Phosphorylation of titin could be reduced in human heart tissue treated ex vivo with recombinant PP5c, an effect detected with phospho-N2Bus (S469) specific antibodies. A transgenic mouse model with PP5 overexpression revealed reduced cardiac titin phosphorylation levels compared to wildtype mouse hearts. PP5 expression was elevated in human heart failure, while titin phosphorylation was depressed. PP5c treatment of enzymatically skinned single human cardiomyocytes significantly reduced passive stiffness, which can potentially be explained by dephosphorylation of the PEVK-domain adjacent to the N2Bus. In conclusion, PP5 is a novel binding partner of cardiac titin at the N2Bus and acts to reduce passive myocyte stiffness by dephosphorylating the titin springs. PP5 may participate in mechanical signaling pathways converging on the titin springs.

Cardiac Troponin T: A Sarcomeric AKAP, Tethers Protein Kinase a at the Myofilaments

Efficient and specific phosphorylation of PKA substrates, elicited in response to beta-adrenergic stimulation, require spatially confined pools of PKA anchored in proximity of its substrates. PKA-dependent phosphorylation of cardiac sarcomeric proteins has been the subject of intense investigations. Yet, the identity, composition, and function of PKA complexes at the sarcomeres have remained elusive. Here we report the identification and characterization of a novel sarcomeric AKAP (A kinase anchoring protein), cardiac troponin T (cTnT). Using the yeast two-hybrid technology in screening two adult human heart cDNA libraries, we identified the regulatory subunit of PKA as interacting with human cTnT bait. Immunoprecipitation studies show that cTnT is a dual specificity AKAP, interacting with both PKA-regulatory subunits type I and II. The disruptor peptide Ht31, but not Ht31P (control), abolished cTnT/PKA-R association. Truncations and point mutations identified an amphipathic helix domain in cTnT as the PKA binding site. This was confirmed by a peptide SPOT assay in the presence of Ht31 or Ht31P (control). Gelsolin-dependent removal of thin filament proteins also reduced myofilament-bound PKA-type II. Using a cardiac troponin (cTn) exchange procedure that substitutes the endogenous cTn complex with a recombinant cTn complex we show PKA-type II is troponin-bound in the myofilament lattice. Displacement of PKA-cTnT complexes correlates with a significant decrease in myofibrillar PKA activity. Taken together, our data propose a novel role for cTnT as a dual-specificity sarcomeric AKAP.