Ventricular TLR4 Levels Abrogate TLR2-Mediated Adverse Cardiac Remodeling upon Pressure Overload in Mice.

Involvement of the Toll-like receptor 4 (TLR4) in maladaptive cardiac remodeling and heart failure (HF) upon pressure overload has been studied extensively, but less is known about the role of TLR2. Interplay and redundancy of TLR4 with TLR2 have been reported in other organs but were not investigated during cardiac dysfunction. We explored whether TLR2 deficiency leads to less adverse cardiac remodeling upon chronic pressure overload and whether TLR2 and TLR4 additively contribute to this. We subjected 35 male C57BL/6J mice (wildtype (WT) or TLR2 knockout (KO)) to sham or transverse aortic constriction (TAC) surgery. After 12 weeks, echocardiography and electrocardiography were performed, and hearts were extracted for molecular and histological analysis. TLR2 deficiency (n = 14) was confirmed in all KO mice by PCR and resulted in less hypertrophy (heart weight to tibia length ratio (HW/TL), smaller cross-sectional cardiomyocyte area and decreased brain natriuretic peptide (BNP) mRNA expression, p < 0.05), increased contractility (QRS and QTc, p < 0.05), and less inflammation (e.g., interleukins 6 and 1ß, p < 0.05) after TAC compared to WT animals (n = 11). Even though TLR2 KO TAC animals presented with lower levels of ventricular TLR4 mRNA than WT TAC animals (13.2 ± 0.8 vs. 16.6 ± 0.7 mg/mm, p < 0.01), TLR4 mRNA expression was increased in animals with the largest ventricular mass, highest hypertrophy, and lowest ejection fraction, leading to two distinct groups of TLR2 KO TAC animals with variations in cardiac remodeling. This variation, however, was not seen in WT TAC animals even though heart weight/tibia length correlated with expression of TLR4 in these animals (r = 0.078, p = 0.005). Our data suggest that TLR2 deficiency ameliorates adverse cardiac remodeling and that ventricular TLR2 and TLR4 additively contribute to adverse cardiac remodeling during chronic pressure overload. Therefore, both TLRs may be therapeutic targets to prevent or interfere in the underlying molecular processes.

Microdissected pancreatic cancer proteomes reveal tumor heterogeneity and therapeutic targets.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a relative paucity of cancer cells that are surrounded by an abundance of nontumor cells and extracellular matrix, known as stroma. The interaction between stroma and cancer cells contributes to poor outcome, but how proteins from these individual compartments drive aggressive tumor behavior is not known. Here, we report the proteomic analysis of laser-capture microdissected (LCM) PDAC samples. We isolated stroma, tumor, and bulk samples from a cohort with long- and short-term survivors. Compartment-specific proteins were measured by mass spectrometry, yielding what we believe to be the largest PDAC proteome landscape to date. These analyses revealed that, in bulk analysis, tumor-derived proteins were typically masked and that LCM was required to reveal biology and prognostic markers. We validated tumor CALB2 and stromal COL11A1 expression as compartment-specific prognostic markers. We identified and functionally addressed the contributions of the tumor cell receptor EPHA2 to tumor cell viability and motility, underscoring the value of compartment-specific protein analysis in PDAC.

Combined Expression of Plasma Thrombospondin-2 and CA19-9 for Diagnosis of Pancreatic Cancer and Distal Cholangiocarcinoma: A Proteome Approach.

BACKGROUND: Minimally invasive diagnostic biomarkers for patients with pancreatic ductal adenocarcinoma (PDAC) and distal cholangiocarcinoma (dCCA) are warranted to facilitate accurate diagnosis. This study identified diagnostic plasma proteins based on proteomics of tumor secretome. MATERIALS AND METHODS: Secretome of tumor and normal tissue was collected after resection of PDAC and dCCA. Differentially expressed proteins were measured by mass spectrometry. Selected candidate biomarkers and carbohydrate antigen 19-9 (CA19-9) were validated by enzyme-linked immunosorbent assay in plasma from patients with PDAC (n = 82), dCCA (n = 29), benign disease (BD; n = 30), and healthy donors (HDs; n = 50). Areas under the curve (AUCs) of receiver operator characteristic curves were calculated to determine the discriminative power. RESULTS: In tumor secretome, 696 discriminatory proteins were identified, including 21 candidate biomarkers. Thrombospondin-2 (THBS2) emerged as promising biomarker. Abundance of THBS2 in plasma from patients with cancer was significantly higher compared to HDs (p < .001, AUC = 0.844). Combined expression of THBS2 and CA19-9 yielded the optimal discriminatory capacity (AUC = 0.952), similarly for early- and late-stage disease (AUC = 0.971 and AUC = 0.911). Remarkably, this combination demonstrated a power similar to CA19-9 to discriminate cancer from BD (AUC = 0.764), and THBS2 provided an additive value in patients with high expression levels of bilirubin. CONCLUSION: Our proteome approach identified a promising set of candidate biomarkers. The combined plasma expression of THBS2/CA19-9 is able to accurately distinguish patients with PDAC or dCCA from HD and BD. IMPLICATIONS FOR PRACTICE: The combined plasma expression of thrombospondin-2 and carbohydrate antigen 19-9 is able to accurately diagnose patients with pancreatic cancer and distal cholangiocarcinoma. This will facilitate minimally invasive diagnosis for these patients by distinguishing them from healthy individuals and benign diseases.

Identification of a PEST Sequence in Vertebrate KIR2.1 That Modifies Rectification.

KIR2.1 potassium channels, producing inward rectifier potassium current (I K1 ), are important for final action potential repolarization and a stable resting membrane potential in excitable cells like cardiomyocytes. Abnormal KIR2.1 function, either decreased or increased, associates with diseases such as Andersen-Tawil syndrome, long and short QT syndromes. KIR2.1 ion channel protein trafficking and subcellular anchoring depends on intrinsic specific short amino acid sequences. We hypothesized that combining an evolutionary based sequence comparison and bioinformatics will identify new functional domains within the C-terminus of the KIR2.1 protein, which function could be determined by mutation analysis. We determined PEST domain signatures, rich in proline (P), glutamic acid (E), serine (S), and threonine (T), within KIR2.1 sequences using the "epestfind" webtool. WT and ΔPEST KIR2.1 channels were expressed in HEK293T and COS-7 cells. Patch-clamp electrophysiology measurements were performed in the inside-out mode on excised membrane patches and the whole cell mode using AxonPatch 200B amplifiers. KIR2.1 protein expression levels were determined by western blot analysis. Immunofluorescence microscopy was used to determine KIR2.1 subcellular localization. An evolutionary conserved PEST domain was identified in the C-terminus of the KIR2.1 channel protein displaying positive PEST scores in vertebrates ranging from fish to human. No similar PEST domain was detected in KIR2.2, KIR2.3, and KIR2.6 proteins. Deletion of the PEST domain in California kingsnake and human KIR2.1 proteins (ΔPEST), did not affect plasma membrane localization. Co-expression of WT and ΔPEST KIR2.1 proteins resulted in heterotetrameric channel formation. Deletion of the PEST domain did not increase protein stability in cycloheximide assays [T½ from 2.64 h (WT) to 1.67 h (ΔPEST), n.s.]. WT and ΔPEST channels, either from human or snake, produced typical I K1 , however, human ΔPEST channels displayed stronger intrinsic rectification. The current observations suggest that the PEST sequence of KIR2.1 is not associated with rapid protein degradation, and has a role in the rectification behavior of I K1 channels.

Proteomic analysis of gemcitabine-resistant pancreatic cancer cells reveals that microtubule-associated protein 2 upregulation associates with taxane treatment.

BACKGROUND: Chemoresistance hampers the treatment of patients suffering from pancreatic ductal adenocarcinoma (PDAC). Here we aimed to evaluate the (phospho)proteome of gemcitabine-sensitive and gemcitabine-resistant PDAC cells to identify novel therapeutic targets and predictive biomarkers. METHODS: The oncogenic capabilities of gemcitabine-sensitive and resistant PDAC cells were evaluated in vitro and in vivo. Cultured cells were analyzed by label-free proteomics. Differential proteins and phosphopeptides were evaluated by gene ontology and for their predictive or prognostic biomarker potential with immunohistochemistry of tissue microarrays. RESULTS: Gemcitabine-resistant cells had increased potential to induce xenograft tumours (p value < 0.001). Differential analyses showed that proteins associated with gemcitabine resistance are correlated with microtubule regulation. Indeed, gemcitabine-resistant cells displayed an increased sensitivity for paclitaxel in vitro (p < 0.001) and nab-paclitaxel had a strong anti-tumour efficacy in vivo. Microtubule-associated protein 2 (MAP2) was found to be highly upregulated (p = 0.002, fold change = 10) and phosphorylated in these resistant cells. Expression of MAP2 was correlated with a poorer overall survival in patients treated with gemcitabine in the palliative (p = 0.037) and adjuvant setting (p = 0.014). CONCLUSIONS: These data show an explanation as to why the combination of gemcitabine with nab-paclitaxel is effective in PDAC patients. The identified gemcitabine-resistance marker, MAP2, emerged as a novel prognostic marker in PDAC patients treated with gemcitabine and warrants further clinical investigation.

Optogenetic sensors in the zebrafish heart: a novel in vivo electrophysiological tool to study cardiac arrhythmogenesis.

Cardiac arrhythmias are among the most challenging human disorders to diagnose and treat due to their complex underlying pathophysiology. Suitable experimental animal models are needed to study the mechanisms causative for cardiac arrhythmogenesis. To enable in vivo analysis of cardiac cellular electrophysiology with a high spatial and temporal resolution, we generated and carefully validated two zebrafish models, one expressing an optogenetic voltage indicator (chimeric VSFP-butterfly CY) and the other a genetically encoded calcium indicator (GCaMP6f) in the heart. Methods: High-speed epifluorescence microscopy was used to image chimeric VSFP-butterfly CY and GCaMP6f in the embryonic zebrafish heart, providing information about the spatiotemporal patterning of electrical activation, action potential configuration and intracellular Ca2+ dynamics. Plotting VSFP or GCaMP6f signals on a line along the myocardial wall over time facilitated the visualization and analysis of electrical impulse propagation throughout the heart. Administration of drugs targeting the sympathetic nervous system or cardiac ion channels was used to validate sensitivity and kinetics of both zebrafish sensor lines. Using the same microscope setup, we imaged transparent juvenile casper fish expressing GCaMP6f, demonstrating the feasibility of imaging cardiac optogenetic sensors at later stages of development. Results: Isoproterenol slightly increased heart rate, diastolic Ca2+ levels and Ca2+ transient amplitudes, whereas propranolol caused a profound decrease in heart rate and Ca2+ transient parameters in VSFP-Butterfly and GCaMP6f embryonic fish. Ikr blocker E-4031 decreased heart rate and increased action potential duration in VSFP-Butterfly fish. ICa,L blocker nifedipine caused total blockade of Ca2+ transients in GCaMP6f fish and a reduced heart rate, altered ventricular action potential duration and disrupted atrial-ventricular electrical conduction in VSFP-Butterfly fish. Imaging of juvenile animals demonstrated the possibility of employing an older zebrafish model for in vivo cardiac electrophysiology studies. We observed differences in atrial and ventricular Ca2+ recovery dynamics between 3 dpf and 14 dpf casper fish, but not in Ca2+ upstroke dynamics. Conclusion: By introducing the optogenetic sensors chimeric VSFP-butterfly CY and GCaMP6f into the zebrafish we successfully generated an in vivo cellular electrophysiological readout tool for the zebrafish heart. Complementary use of both sensor lines demonstrated the ability to study heart rate, cardiac action potential configuration, spatiotemporal patterning of electrical activation and intracellular Ca2+ homeostasis in embryonic zebrafish. In addition, we demonstrated the first successful use of an optogenetic sensor to study cardiac function in older zebrafish. These models present a promising new research tool to study the underlying mechanisms of cardiac arrhythmogenesis.

SCA1+ Cells from the Heart Possess a Molecular Circadian Clock and Display Circadian Oscillations in Cellular Functions.

Stem cell antigen 1-positive (SCA1+) cells (SPCs) have been investigated in cell-based cardiac repair and pharmacological research, although improved cardiac function after injection has been variable and the mode of action remains unclear. Circadian (24-hr) rhythms are biorhythms regulated by molecular clocks that play an important role in (patho)physiology. Here, we describe (1) the presence of a molecular circadian clock in SPCs and (2) circadian rhythmicity in SPC function. We isolated SPCs from human fetal heart and found that these cells possess a molecular clock based on typical oscillations in core clock components BMAL1 and CRY1. Functional analyses revealed that circadian rhythmicity also governs SPC proliferation, stress tolerance, and growth factor release, with large differences between peaks and troughs. We conclude that SPCs contain a circadian molecular clock that controls crucial cellular functions. Taking circadian rhythms into account may improve reproducibility and outcome of research and therapies using SPCs.

Calmodulin/CaMKII inhibition improves intercellular communication and impulse propagation in the heart and is antiarrhythmic under conditions when fibrosis is absent.

AIM: In healthy hearts, ventricular gap junctions are mainly composed by connexin43 (Cx43) and localize in the intercalated disc, enabling appropriate electrical coupling. In diseased hearts, Cx43 is heterogeneously down-regulated, whereas activity of calmodulin/calcium-calmodulin protein kinase II (CaM/CaMKII) signalling increases. It is unclear if CaM/CaMKII affects Cx43 expression/localization or impulse propagation. We analysed different models to assess this. METHODS AND RESULTS: AC3-I mice with CaMKII genetically inhibited were subjected to pressure overload (16 weeks, TAC vs. sham). Optical and epicardial mapping was performed on Langendorff-perfused rabbit and AC3-I hearts, respectively. Cx43 subcellular distribution from rabbit/mouse ventricles was evaluated by immunoblot after Triton X-100-based fractionation. In mice with constitutively reduced CaMKII activity (AC3-I), conduction velocity (CV) was augmented (n = 11, P < 0.01 vs. WT); in AC3-I, CV was preserved after TAC, in contrast to a reduction seen in TAC-WT mice (-20%). Cx43 expression was preserved after TAC in AC3-I mice, though arrhythmias and fibrosis were still present. In rabbits, W7 (CaM inhibitor, 10 µM) increased CV (6-13%, n= 6, P< 0.05), while susceptibility to arrhythmias decreased. Immunoconfocal microscopy revealed enlarged Cx43 cluster sizes at intercalated discs of those hearts. Total Cx43 did not change by W7 (n= 4), whereas Triton X-100 insoluble Cx43 increased (+21%, n= 4, P< 0.01). Similar findings were obtained in AC3-I mouse hearts when compared with control, and in cultured dog cardiomyocytes. Functional implication was shown through increased intercellular coupling in cultured neonatal rat cardiomyocytes. CONCLUSION: Both acute and chronic CaM/CaMKII inhibition improves conduction characteristics and enhances localization of Cx43 in the intercalated disc. In the absence of fibrosis, this reduced the susceptibility for arrhythmias.

Spatial Heterogeneity of Cx43 is an Arrhythmogenic Substrate of Polymorphic Ventricular Tachycardias during Compensated Cardiac Hypertrophy in Rats.

BACKGROUND: Ventricular remodeling increases the propensity of ventricular tachyarrhythmias and sudden death in patients. We studied the mechanism underlying these fatal arrhythmias, electrical and structural cardiac remodeling, as well as arrhythmogeneity during early, compensated hypertrophy in a rat model of chronic pressure overload. METHODS: Twenty-six Wistar rats were subjected to transverse aortic constriction (TAC) (n = 13) or sham operation (n = 13). Four weeks postoperative, echo- and electrocardiography was performed. Epicardial (208 or 455 sites) and transmural (30 sites) ventricular activation mapping was performed on Langendorff perfused hearts. Subsequently, hearts were processed for (immuno)histological and molecular analyses. RESULTS: TAC rats showed significant hypertrophy with preserved left ventricular (LV) function. Epicardial conduction velocity (CV) was similar, but more dispersed in TAC. Transmural CV was slowed in TAC (37.6 ± 2.9 cm s(-1)) compared to sham (58.5 ± 3.9 cm s(-1); P < 0.01). Sustained polymorphic ventricular tachycardias were induced from LV in 8/13 TAC and in 0/13 sham rats. During VT, electrical activation patterns showed variable sites of earliest epicardial activation and altering sites of functional conduction block. Wandering epicardial reentrant activation was sporadically observed. Collagen deposition was significantly higher in TAC compared to sham, but not different between arrhythmogenic and non-arrhythmogenic TAC animals. Connexin43 (Cx43) expression was heterogeneous with a higher prevalence of non-phosphorylated Cx43 in arrhythmogenic TAC animals. CONCLUSION: In TAC rats with compensated cardiac hypertrophy, dispersion of conduction correlated to arrhythmogenesis, an increased heterogeneity of Cx43, and a partial substitution with non-phosphorylated Cx43. These alterations may result in the increased vulnerability to polymorphic VTs.

CTGF knockout does not affect cardiac hypertrophy and fibrosis formation upon chronic pressure overload.

BACKGROUND: One of the main contributors to maladaptive cardiac remodeling is fibrosis. Connective tissue growth factor (CTGF), a matricellular protein that is secreted into the cardiac extracellular matrix by both cardiomyocytes and fibroblasts, is often associated with development of fibrosis. However, recent studies have questioned the role of CTGF as a pro-fibrotic factor. Therefore, we aimed to investigate the effect of CTGF on cardiac fibrosis, and on functional, structural, and electrophysiological parameters in a mouse model of CTGF knockout (KO) and chronic pressure overload. METHODS AND RESULTS: A new mouse model of global conditional CTGF KO induced by tamoxifen-driven deletion of CTGF, was subjected to 16weeks of chronic pressure overload via transverse aortic constriction (TAC, control was sham surgery). CTGF KO TAC mice presented with hypertrophic hearts, and echocardiography revealed a decrease in contractility on a similar level as control TAC mice. Ex vivo epicardial mapping showed a low incidence of pacing-induced ventricular arrhythmias (2/12 in control TAC vs. 0/10 in CTGF KO TAC, n.s.) and a tendency towards recovery of the longitudinal conduction velocity of CTGF KO TAC hearts. Picrosirius Red staining on these hearts unveiled increased fibrosis at a similar level as control TAC hearts. Furthermore, genes related to fibrogenesis were also similarly upregulated in both TAC groups. Histological analysis revealed an increase in fibronectin and vimentin protein expression, a significant reduction in connexin43 (Cx43) protein expression, and no difference in NaV1.5 expression of CTGF KO ventricles as compared with sham treated animals. CONCLUSION: Conditional CTGF inhibition failed to prevent TAC-induced cardiac fibrosis and hypertrophy. Additionally, no large differences were found in other parameters between CTGF KO and control TAC mice. With no profound effect of CTGF on fibrosis formation, other factors or pathways are likely responsible for fibrosis development.

A systematic evaluation of protein kinase A-A-kinase anchoring protein interaction motifs.

Protein kinase A (PKA) in vertebrates is localized to specific locations in the cell via A-kinase anchoring proteins (AKAPs). The regulatory subunits of the four PKA isoforms (RIα, RIß, RIIα, and RIIß) each form a homodimer, and their dimerization domain interacts with a small helical region present in each of the more than 40 AKAPs reported so far. This allows for tight anchoring of PKA and efficient communication with other signaling proteins that interact with the AKAP scaffold in a spatial and temporal manner. The hydrophobic interaction surfaces of the PKA-R dimer and several AKAP helices have been investigated in great detail. Despite this knowledge, not every suggested AKAP has had its binding motif specified. Here we created an efficient bioinformatic tool, termed THAHIT, to accurately map the PKA binding motif and/or additional motifs of all previously reported AKAPs. Moreover, THAHIT predicts its specificity toward PKA-RIα and/or PKA-RIIα binding. To verify the validity of these newly predicted anchoring sites and their putative specificities, we used computational modeling approaches (HADDOCK), biochemical affinity studies (fluorescence anisotropy), and cellular colocalization studies. We further demonstrate the potential of THAHIT to identify novel AKAPs in cAMP-based chemical proteomics discovery data sets, and the human proteome. We retrieved numerous novel AKAP candidates, including a never reported 330 kDa AKAP observed in heart tissue, which we further characterized biochemically as a PKA-RIIα binder. Altogether, THAHIT provides a comprehensive overview of known and novel PKA-AKAP interaction domains and their PKA-R specificities.

Insights in KIR2.1 channel structure and function by an evolutionary approach; cloning and functional characterization of the first reptilian inward rectifier channel KIR2.1, derived from the California kingsnake (Lampropeltis getula californiae).

Potassium inward rectifier KIR2.1 channels contribute to the stable resting membrane potential in a variety of muscle and neuronal cell-types. Mutations in the KIR2.1 gene KCNJ2 have been associated with human disease, such as cardiac arrhythmias and periodic paralysis. Crystal structure and homology modelling of KIR2.1 channels combined with functional current measurements provided valuable insights in mechanisms underlying channel function. KIR2.1 channels have been cloned and analyzed from all main vertebrate phyla, except reptilians. To address this lacuna, we set out to clone reptilian KIR2.1 channels. Using a degenerated primer set we cloned the KCNJ2 coding regions from muscle tissue of turtle, snake, bear, quail and bream, and compared their deduced amino acid sequences with those of KIR2.1 sequences from 26 different animal species obtained from Genbank. Furthermore, expression constructs were prepared for functional electrophysiological studies of ectopically expressed KIR2.1 ion channels. In general, KCNJ2 gene evolution followed normal phylogenetic patterns, however turtle KIR2.1 ion channel sequence is more homologues to avians than to snake. Alignment of all 31 KIR2.1 sequences showed that all disease causing KIR2.1 mutations, except V93I, V123G and N318S, are fully conserved. Homology models were built to provide structural insights into species specific amino acid substitutions. Snake KIR2.1 channels became expressed at the plasmamembrane and produced typical barium sensitive (IC50 ∼6µM) inward rectifier currents.

Changes in Cx43 and NaV1.5 expression precede the occurrence of substantial fibrosis in calcineurin-induced murine cardiac hypertrophy.

In mice, the calcium-dependent phosphatase calcineurin A (CnA) induces a transcriptional pathway leading to pathological cardiac hypertrophy. Interestingly, induction of CnA has been frequently noticed in human hypertrophic and failing hearts. Independently, the arrhythmia vulnerability of such hearts has been regularly associated with remodeling of parameters determining electrical conduction (expression level of connexin43 (Cx43) and NaV1.5, connective tissue architecture), for which the precise molecular basis and sequence of events is still unknown. Recently, we observed reduced Cx43 and NaV1.5 expression in 4-week old mouse hearts, overexpressing a constitutively active form of CnA (MHC-CnA model), but the order of events is still unknown. Therefore, three key parameters of conduction (Cx43, NaV1.5 and connective tissue expression) were characterized in MHC-CnA ventricles versus wild-type (WT) during postnatal development on a weekly basis. At postnatal week 1, CnA overexpression induced cardiac hypertrophy in MHC-CnA. Moreover, protein and RNA levels of both Cx43 and NaV1.5 were reduced by at least 50% as compared to WT. Cx43 immunoreactive signal was reduced at week 2 in MHC-CnA. At postnatal week 3, Cx43 was less phosphorylated and RNA level of Cx43 normalized to WT values, although the protein level was still reduced. Additionally, MHC-CnA hearts displayed substantial fibrosis relative to WT, which was accompanied by increased RNA levels for genes previously associated with fibrosis such as Col1a1, Col1a2, Col3a1, Tgfb1, Ctgf, Timp1 and microRNA miR-21. In MHC-CnA, reduction in Cx43 and NaV1.5 expression thus coincided with overexpression of CnA and hypertrophy development and preceded significant presence of fibrosis. At postnatal week 4 the alterations in conductional parameters observed in the MHC-CnA model lead to abnormal conduction and arrhythmias, similar to those observed in cardiac remodeling in heart failure patients. The MHC-CnA model, therefore, provides for a unique model to resolve the molecular origin of conductional remodeling in detail.

Experimental Mapping of the Canine KCNJ2 and KCNJ12 Gene Structures and Functional Analysis of the Canine K(IR)2.2 ion Channel.

For many model organisms traditionally in use for cardiac electrophysiological studies, characterization of ion channel genes is lacking. We focused here on two genes encoding the inward rectifier current, KCNJ2 and KCNJ12, in the dog heart. A combination of RT-PCR, 5'-RACE, and 3'-RACE demonstrated the status of KCNJ2 as a two exon gene. The complete open reading frame (ORF) was located on the second exon. One transcription initiation site was mapped. Four differential transcription termination sites were found downstream of two consensus polyadenylation signals. The canine KCNJ12 gene was found to consist of three exons, with its ORF located on the third exon. One transcription initiation and one termination site were found. No alternative splicing was observed in right ventricle or brain cortex. The gene structure of canine KCNJ2 and KCNJ12 was conserved amongst other vertebrates, while current GenBank gene annotation was determined as incomplete. In silico translation of KCN12 revealed a non-conserved glycine rich stretch located near the carboxy-terminus of the K(IR)2.2 protein. However, no differences were observed when comparing dog with human K(IR)2.2 protein upon ectopic expression in COS-7 or HEK293 cells with respect to subcellular localization or electrophysiological properties.

Alternative promoter usage and splicing of the human SCN5A gene contribute to transcript heterogeneity.

The sodium channel isoform Na(v)1.5 mediates sodium current, excitability, and electrical conduction in the human heart. Recent studies have indicated alternative splicing within the protein-coding portion of its gene, SCN5A, as a mechanism to generate diversity in Na(v)1.5 protein structure and function. In the present study we identified several novel SCN5A transcripts in human heart, displaying distinct 5'-untranslated regions but identical protein-coding sequences. These transcripts originated from the splicing of alternative exons 1 (designated 1A, 1B, 1C, and 1D) to the translational start codon-containing exon 2, and were preferentially expressed in the heart as compared to other tissues. Comparison of their expression level between adult and fetal heart demonstrated that exon 1C- and 1D-derived sequences were more prominent in adult than in fetal heart. Two new promoters (designated P2 and P3) for the SCN5A gene were identified and functionally characterized in myocardial- and nonmyocardial-derived cell lines. Translation of the transcript containing exon 1D-derived sequences proved to be significantly impaired in these cell lines, which could be restored by mutation of an upstream translational start codon. These results implicate the usage of alternative promoters and 5'-untranslated regions as new mechanisms in the regulation of human Na(v)1.5 expression.

Human cardiomyocyte progenitor cell-derived cardiomyocytes display a maturated electrical phenotype.

Cardiomyocyte progenitor cells (CMPCs) can be isolated from the human heart and differentiated into cardiomyocytes in vitro. A comprehensive assessment of their electrical phenotype upon differentiation is essential to predict potential future applications of this cell source. CMPCs isolated from human fetal heart were differentiated in vitro and examined using immunohistochemistry, Western blotting, RT-PCR, voltage clamp and current clamp techniques. Differentiated cultures presented up to 95% alpha-actinin positive cardiomyocytes. Adherens junction and desmosomal proteins beta-catenin, N-cadherin, desmin and plakophilin2 were upregulated. Expression levels of cardiac connexins were not affected by differentiation, however Cx43 phosphorylation was increased upon differentiation, accompanied by translocation of connexins to the cell border. RT-PCR analysis demonstrated upregulation of all major cardiac ion channel constituents during differentiation. Patch clamp experiments showed that cardiomyocytes had a stable resting membrane potential of -73.4+/-1.8 mV. Infusion of 1 mM BaCl(2) resulted in depolarization to -59.9+/-2.8 mV, indicating I(K1) channel activity. Subsequent voltage clamp experiments confirmed presence of near mature I(Na), I(Ca,L) and I(K1) current densities. Infusion of the I(Kr) blocker Almokalant caused prolongation of the action potential by 40%. Differentiated monolayers were not spontaneously contracting in the absence of serum, but responded to field stimulation, displaying adult ventricular-like action potentials. Human fetal CMPC-derived cardiomyocytes have a homogenous and rather mature electrical phenotype that benefits to in vitro physiology and pharmacology. In the context of cardiac repair, their properties may translate into a reduced pro-arrhythmic risk and enhanced electrical integration upon transplantation.

Cloning, sequence analysis and phylogeny of connexin43 isolated from American black bear heart.

Conduction in the heart requires gap junctions. In mammalian ventricular myocytes these consist of connexin43 (Cx43). Hearts of non-hibernating species display conduction disturbances at reduced temperatures. These may exacerbate into lethal arrhythmias. Hibernating species are protected against these arrhythmias by a non-resolved mechanism. To analyze whether the amino acid composition of Cx43 from the hibernating American black bear displays specific features, we cloned the full coding sequence of Ursus americanus Cx43 and compared with that of other (non)hibernating species. UaCx43 displays 99.7% identity to rabbit Cx43 at the amino acid level. No specific features were observed in UaCx43 when compared to previously cloned Cx43 from hibernating and non-hibernating mammals. Phylogenetic tree reconstruction of this and other published full-length Cx43 sequences reveals a very high level of conservation from fish to men. Finally, one of the previously identified six mammalian characteristic amino acids, is not conserved in the black bear.

Beta-, not alpha-adrenergic stimulation enhances conduction velocity in cultures of neonatal cardiomyocytes.

BACKGROUND: During both cardiac maturation and myopathy, elevated levels of circulating catecholamines coincide with alterations in impulse propagation. An in vitro model of cultured cardiomyocytes was used to study the effects of adrenergic stimulation on the conduction characteristics of immature heart cells. METHODS AND RESULTS: Neonatal rat cardiomyocytes were cultured on preparations designed to measure conduction velocity (CV). CV was measured on the same preparation twice at t=0 and at t=24 h. Under control conditions (n=7), CV at t=0 (30.9+/-1.9 cm/s) and t=24 (32.4+/-4.4 cm/s) was similar (p=0.70). Immunohistochemistry revealed expression of the gap junction proteins connexin (Cx) 40, Cx43 and Cx45, with Cx43 being highly predominant. Stimulation for 24 h with the beta-adrenergic agonist isoproterenol (ISO) significantly increased CV from 28.0 +/-2.0 cm/s at t=0 to 34.8+/-2.2 cm/s at t=24 (p=0.002, n=5). Microelectrode recordings showed a faster upstroke of the action potential (AP) of ISO-treated cells. Reverse transcribed-polymerase chain reactions (RT-PCR) showed that ISO increased expression of SCN5A and alpha(1c) (alpha-subunit of the cardiac sodium and L-type calcium channel, respectively). Stimulation of cells with ISO did not induce alterations in distribution or expression of Cx40, Cx43 and Cx45 (both mRNA and protein), but slightly increased the phosphorylation of Cx43. Stimulation for 24 h with the alpha-adrenergic agonist phenylephrine did neither affect CV nor the expression of the connexin isoforms, SCN5A and alpha(1c). CONCLUSIONS: Alpha- and beta-adrenergic stimulation differently affect propagation of the electric impulse, which is primarily not caused by a differential effect on intercellular coupling. RT-PCR analysis and an enhanced AP upstroke velocity indicate a higher functional expression level of alpha(1c) and SCN5A in beta-adrenergic stimulated cells, which may explain the observed increase in CV.

Connexin43 repression following epithelium-to-mesenchyme transition in embryonal carcinoma cells requires Snail1 transcription factor.

Embryonic stem (ES) cells and embryonal carcinoma (EC) cells express high amounts of functional connexin43 (Cx43). During mesoderm formation and subsequent cardiac differentiation, Cx43 is initially down-regulated but is up-regulated again as the emerging cardiomyocytes mature. In this study, we investigated the regulation of Cx43 expression during early phases of differentiation in F9 and P19 EC cells. We found a striking inverse correlation between the expression of Cx43 and that of the transcriptional repressor Snail1. No clear relationship was found with Smad-interacting-protein1 (SIP1), another transcription factor inducing epithelium-to-mesenchyme transition (EMT). Promoter-reporter assays indicated Cx43 repression at the promoter level by ectopically expressed Snail1. To establish whether the Cx43 down-regulation depends on endogenous Snail1, MES-1 cells, differentiated derivatives of P19 EC, were stably transfected by an siRNA construct silencing Snail1 expression. This resulted in a mesenchyme-to-epithelium transition, which was accompanied by increased levels of Cx43 mRNA and protein and enhanced metabolic and electrical coupling. We conclude that Snail1-mediated EMT results in a Cx43 repression.

The human Cx40 promoter polymorphism -44G-->A differentially affects transcriptional regulation by Sp1 and GATA4.

Expression of the tissue-specific gap junction protein connexin(Cx)40 is regulated by the interaction of ubiquitous and tissue-specific factors such as Sp1 and GATA4. Cardiac Cx40 expression is altered under pathological conditions such as atrial fibrillation. A human promoter polymorphism, a G-->A change at position -44 that has been associated with atrial-specific arrhythmias, is located between the TBE-NKE-Sp and GATA consensus transcription factor binding sites important for the regulation of the mouse Cx40 gene. The presence of the A-allele at position -44 in promoter-reporter constructs significantly reduces promoter activity. Using electrophoretic mobility shift assays and luciferase reporter assays in various cell types, we show that Sp1 and GATA4 are important regulators of human Cx40 gene transcription and that the -44 G-->A polymorphism negatively affects the promoter regulation by the transcription factors Sp1 and GATA4.