Endoplasmic Reticulum Protein TXNDC5 Augments Myocardial Fibrosis by Facilitating Extracellular Matrix Protein Folding and Redox-Sensitive Cardiac Fibroblast Activation.

Rationale: Cardiac fibrosis plays a critical role in the pathogenesis of heart failure. Excessive accumulation of extracellular matrix (ECM) resulting from cardiac fibrosis impairs cardiac contractile function and increases arrhythmogenicity. Current treatment options for cardiac fibrosis, however, are limited, and there is a clear need to identify novel mediators of cardiac fibrosis to facilitate the development of better therapeutics.

Differential Expression and Remodeling of Transient Outward Potassium Currents in Human Left Ventricles.

Background: Myocardial, transient, outward currents, , have been shown to play pivotal roles in action potential (AP) repolarization and remodeling in animal models. The properties and contribution of to left ventricular (LV) repolarization in the human heart, however, are poorly defined.

Methods And Results: Whole-cell, voltage-clamp recordings, acquired at physiological (35°C to 37°C) temperatures, from myocytes isolated from the LV of nonfailing human hearts identified 2 distinct transient currents, () and (), with significantly (<0.

Arrhythmogenic remodeling of β2 versus β1 adrenergic signaling in the human failing heart.

Background: Arrhythmia is the major cause of death in patients with heart failure, for which β-adrenergic receptor blockers are a mainstay therapy. But the role of β-adrenergic signaling in electrophysiology and arrhythmias has never been studied in human ventricles.

Methods And Results: We used optical imaging of action potentials and [Ca(2+)]i transients to compare the β1- and β2-adrenergic responses in left ventricular wedge preparations of human donor and failing hearts.

Deep RNA sequencing reveals dynamic regulation of myocardial noncoding RNAs in failing human heart and remodeling with mechanical circulatory support.

Background: Microarrays have been used extensively to profile transcriptome remodeling in failing human heart, although the genomic coverage provided is limited and fails to provide a detailed picture of the myocardial transcriptome landscape. Here, we describe sequencing-based transcriptome profiling, providing comprehensive analysis of myocardial mRNA, microRNA (miRNA), and long noncoding RNA (lncRNA) expression in failing human heart before and after mechanical support with a left ventricular (LV) assist device (LVAD).

Methods And Results: Deep sequencing of RNA isolated from paired nonischemic (NICM; n=8) and ischemic (ICM; n=8) human failing LV samples collected before and after LVAD and from nonfailing human LV (n=8) was conducted.

Heterogeneity and function of K(ATP) channels in canine hearts.

Background: The concept that pore-forming Kir6.2 and regulatory SUR2A subunits form cardiac ATP-sensitive potassium (K(ATP)) channels is challenged by recent reports that SUR1 is predominant in mouse atrial K(ATP) channels.

Objective: To assess SUR subunit composition of K(ATP) channels and consequence of K(ATP) activation for action potential duration (APD) in dog hearts.

How to build an integrated biobank: the Washington University Translational Cardiovascular Biobank & Repository experience.

Translational studies that assess and extend observations made in animal models of human pathology to elucidate relevant and important determinants of human diseases require the availability of viable human tissue samples. However, there are a number of technical and practical obstacles that must be overcome in order to perform cellular and electrophysiological studies of the human heart. In addition, changing paradigms of how diseases are diagnosed, studied and treated require increasingly complex integration of rigorous disease phenotyping, tissue characterization and detailed delineation of a multitude of "_omics".

Gender differences in electrophysiological gene expression in failing and non-failing human hearts.

The increasing availability of human cardiac tissues for study are critically important in increasing our understanding of the impact of gender, age, and other parameters, such as medications and cardiac disease, on arrhythmia susceptibility. In this study, we aimed to compare the mRNA expression of 89 ion channel subunits, calcium handling proteins, and transcription factors important in cardiac conduction and arrhythmogenesis in the left atria (LA) and ventricles (LV) of failing and nonfailing human hearts of both genders. Total RNA samples, prepared from failing male (n = 9) and female (n = 7), and from nonfailing male (n = 9) and female (n = 9) hearts, were probed using custom-designed Taqman gene arrays.

CD36 protein influences myocardial Ca2+ homeostasis and phospholipid metabolism: conduction anomalies in CD36-deficient mice during fasting.

Sarcolemmal CD36 facilitates myocardial fatty acid (FA) uptake, which is markedly reduced in CD36-deficient rodents and humans. CD36 also mediates signal transduction events involving a number of cellular pathways. In taste cells and macrophages, CD36 signaling was recently shown to regulate store-responsive Ca(2+) flux and activation of Ca(2+)-dependent phospholipases A(2) that cycle polyunsaturated FA into phospholipids.

Differential calcium handling in two canine models of right ventricular pressure overload.

Background: The purpose of this investigation was to characterize differential right atrial (RA) and ventricular (RV) molecular changes in Ca(2+)-handling proteins consequent to RV pressure overload and hypertrophy in two common, yet distinct models of pulmonary hypertension: dehydromonocrotaline (DMCT) toxicity and pulmonary artery (PA) banding.

Methods: A total of 18 dogs underwent sternotomy in four groups: (1) DMCT toxicity (n = 5), (2) mild PA banding over 10 wk to match the RV pressure rise with DMCT (n = 5); (3) progressive PA banding to generate severe RV overload (n = 4); and (4) sternotomy only (n = 4).

Results: In the right ventricle, with DMCT, there was no change in sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) or phospholamban (PLB), but we saw a trend toward down-regulation of phosphorylated PLB at serine-16 (p[Ser-16]PLB) (P = 0.

Residual Cx45 and its relationship to Cx43 in murine ventricular myocardium.

Gap junction channels in ventricular myocardium are required for electrical and metabolic coupling between cardiac myocytes and for normal cardiac pump function. Although much is known about expression patterns and remodeling of cardiac connexin(Cx)43, little is known about the less abundant Cx45, which is required for embryonic development and viability, is downregulated in adult hearts, and is pathophysiologically upregulated in human end-stage heart failure. We applied quantitative immunoblotting and immunoprecipitation to native myocardial extracts, immunogold electron microscopy to cardiac tissue and membrane sections, electrophysiological recordings to whole hearts, and high-resolution tandem mass spectrometry to Cx45 fusion protein, and developed two new tools, anti-Cx45 antisera and Cre(+);Cx45 floxed mice, to facilitate characterization of Cx45 in adult mammalian hearts.

Identification of CaMKII phosphorylation sites in Connexin43 by high-resolution mass spectrometry.

Connexin43 (Cx43) is a major cardiac gap junction channel protein required for normal electrical and contractile activity. Gap junction channel assembly, function, and turnover are regulated by phosphorylation under both normal and disease conditions. The carboxyl terminus (CT) of Cx43 contains numerous amino acid residues that are phosphorylated by protein kinases.

Remodeling of cardiac fibroblasts following myocardial infarction results in increased gap junction intercellular communication.

Background: We have recently shown that native murine ventricular fibroblasts express both connexin43 (Cx43) and Cx45, and that the level of Cx43 expression influences intercellular coupling and cell proliferation. Relatively little is known, however, about how myocardial infarction (MI) influences expression of Cx43, or how altered Cx43 expression may affect fibroblast function post-MI. Fibroblasts are critical for infarct healing and post-infarct ventricular remodeling.

Reduced expression of Cx43 attenuates ventricular remodeling after myocardial infarction via impaired TGF-beta signaling.

In addition to mediating cell-to-cell electrical coupling, gap junctions are important in tissue repair, wound healing, and scar formation. The expression and distribution of connexin43 (Cx43), the major gap junction protein expressed in the heart, are altered substantially after myocardial infarction (MI); however, the effects of Cx43 remodeling on wound healing and the attendant ventricular dysfunction are incompletely understood. Cx43-deficient and wild-type mice were subjected to proximal ligation of the anterior descending coronary artery and followed for 6 days or 4 wk to test the hypothesis that reduced expression of Cx43 influences wound healing, fibrosis, and ventricular remodeling after MI.

Connexin43 expression levels influence intercellular coupling and cell proliferation of native murine cardiac fibroblasts.

Little is known about connexin expression and function in murine cardiac fibroblasts. The authors isolated native ventricular fibroblasts from adult mice and determined that although they expressed both connexin43 (Cx43) and connexin45 (Cx45), the relative abundance of Cx45 was greater than that of Cx43 in fibroblasts compared to myocytes, and the electrophoretic mobility of both Cx43 and Cx45 differed in fibroblasts and in myocytes. Increasing Cx43 expression by adenoviral infection increased intercellular coupling, whereas decreasing Cx43 expression by genetic ablation decreased coupling.

Role of activated CaMKII in abnormal calcium homeostasis and I(Na) remodeling after myocardial infarction: insights from mathematical modeling.

Ca(2+)/calmodulin-dependent protein kinase II is a multifunctional serine/threonine kinase with diverse cardiac roles including regulation of excitation contraction, transcription, and apoptosis. Dynamic regulation of CaMKII activity occurs in cardiac disease and is linked to specific disease phenotypes through its effects on ion channels, transporters, transcription and cell death pathways. Recent mathematical models of the cardiomyocyte have incorporated limited elements of CaMKII signaling to advance our understanding of how CaMKII regulates cardiac contractility and excitability.

Protein kinase Cepsilon mediates salutary effects on electrical coupling induced by ischemic preconditioning.

Background: Ischemic preconditioning delays the onset of electrical uncoupling and prevents loss of the primary ventricular gap junction protein connexin 43 (Cx43) from gap junctions during subsequent ischemia.

Objective: To test the hypothesis that these effects are mediated by protein kinase C epsilon (PKCepsilon), we studied isolated Langendorff-perfused hearts from mice with homozygous germline deletion of PKCepsilon (PKCepsilon-KO).

Methods: Cx43 phosphorylation and distribution were measured by quantitative immunoblotting and confocal microscopy.

Diminished zonula occludens-1 expression in the failing human heart.

Background: Reduced expression of the major gap junction protein connexin 43 (Cx43) in the failing human heart may lead to arrhythmias and sudden cardiac death. Cx43 interacts with the actin binding protein, zonula occludens-1 (ZO-1), and it has recently been demonstrated that ZO-1 regulates the formation and function of Cx43 gap junctions. We hypothesize that normal expression of ZO-1 and its interaction with Cx43 are required for appropriate assembly and function of Cx43 gap junctions in the heart.

Arrhythmia susceptibility and premature death in transgenic mice overexpressing both SUR1 and Kir6.2[DeltaN30,K185Q] in the heart.

Sarcolemmal ATP-sensitive potassium (K(ATP)) channels are activated after pathological depletion of intracellular ATP, unlike their pancreatic beta-cell counterparts, which dynamically regulate membrane excitability in response to changes in blood glucose. We recently engineered a series of transgenic (TG) mice overexpressing an ATP-insensitive inward rectifying K(+) channel protein (Kir)6.2 mutant (Kir6.

Relative contributions of connexins 40 and 43 to atrial impulse propagation in synthetic strands of neonatal and fetal murine cardiomyocytes.

Atrial tissue expresses both connexin 40 (Cx40) and 43 (Cx43) proteins. To assess the relative roles of Cx40 and Cx43 in atrial electrical propagation, we synthesized cultured strands of atrial myocytes derived from mice with genetic deficiency in Cx40 or Cx43 expression and measured propagation velocity (PV) by high-resolution optical mapping of voltage-sensitive dye fluorescence. The amount of Cx40 and/or Cx43 in gap junctions was measured by immunohistochemistry and total or sarcolemmal Cx43 or Cx40 protein by immunoblotting.

Targeted deletion of Kv4.2 eliminates I(to,f) and results in electrical and molecular remodeling, with no evidence of ventricular hypertrophy or myocardial dysfunction.

Previous studies have demonstrated a role for voltage-gated K+ (Kv) channel alpha subunits of the Kv4 subfamily in the generation of rapidly inactivating/recovering cardiac transient outward K+ current, I(to,f), channels. Biochemical studies suggest that mouse ventricular I(to,f) channels reflect the heteromeric assembly of Kv4.2 and Kv4.

Fibroblast growth factor 14 is an intracellular modulator of voltage-gated sodium channels.

Genetic ablation of the fibroblast growth factor (Fgf) 14 gene in mice or a missense mutation in Fgf14 in humans causes ataxia and cognitive deficits. These phenotypes suggest that the neuronally expressed Fgf14 gene is essential for regulating normal neuronal activity. Here, we demonstrate that FGF14 interacts directly with multiple voltage-gated Na(+) (Nav) channel alpha subunits heterologously expressed in non-neuronal cells or natively expressed in a murine neuroblastoma cell line.

Overexpression of cardiac connexin45 increases susceptibility to ventricular tachyarrhythmias in vivo.

Electrophysiological remodeling involving gap junctions has been demonstrated in failing hearts and may contribute to intercellular uncoupling, delayed conduction, enhanced arrhythmias, and vulnerability to sudden death in patients with heart failure. Recently, we showed that failing human hearts exhibit marked increases in connexin45 (Cx45) expression in addition to previously documented decreases in connexin43 (Cx43) expression. Each of these changes results in reduced gap junction coupling.