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. Given the importance of CaMKII in cardiac disease, it is imperative that computer models evolve to capture the dynamic range of CaMKII activity. In this study, using mathematical modeling combined with biochemical and imaging techniques, we test the hypothesis that CaMKII signaling in the canine infarct border zone (BZ) contributes to impaired calcium homeostasis and electrical remodeling. We report that the level of CaMKII autophosphorylation is significantly increased in the BZ region. Computer simulations using an updated mathematical model of CaMKII signaling reproduce abnormal Ca 2+ transients and action potentials characteristic of the BZ. Our simulations show that CaMKII hyperactivity contributes to abnormal Ca 2+ homeostasis and reduced action potential upstroke velocity due to effects on I Na gating kinetics. In conclusion, we present a new mathematical tool for studying effects of CaMKII signaling on cardiac excitability and Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access

Abstract-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. Progressive genetic reduction in Cx43 expression decreased PV from 34Ϯ6 cm/sec in Cx43 ϩ/ϩ to 30Ϯ8 cm/sec in Cx43 ϩ/Ϫ and 19Ϯ11 cm/sec in Cx43 Ϫ/Ϫ cultures. Concomitantly, the cell area occupied by Cx40 immunosignal in gap junctions decreased from 2.0Ϯ1.6% in Cx43 ϩ/ϩ to 1.7Ϯ0.5% in Cx43 ϩ/Ϫ and 1.0Ϯ0.2% in Cx43 Ϫ/Ϫ strands. In contrast, progressive genetic reduction in Cx40 expression increased PV from 30Ϯ2 cm/sec in Cx40 ϩ/ϩ to 40Ϯ7 cm/sec in Cx40 ϩ/Ϫ and 45Ϯ10 cm/sec in Cx40 Ϫ/Ϫ cultures. Concomitantly, the cell area occupied by Cx43 immunosignal in gap junctions increased from 1.2Ϯ0.9% in Cx40 ϩ/ϩ to 2.8Ϯ1.4% in Cx40 ϩ/Ϫ and 3.1Ϯ0.6% in Cx40 Ϫ/Ϫ cultures. In accordance with the immunostaining results, immunoblots of the Triton X-100 -insoluble fraction revealed an increase of Cx43 in gap junctions in extracts from Cx40-ablated atria, whereas total cellular Cx43 remained unchanged. Our results suggest that the relative abundance of Cx43 and Cx40 is an important determinant of atrial impulse propagation in neonatal hearts, whereby dominance of Cx40 decreases and dominance of Cx43 increases local propagation velocity. (Circ Res. 2006;99:1216-1224.)Key Words: atrial myocyte Ⅲ basic science Ⅲ cardiac gap junction connexins Ⅲ cardiovascular genomics Ⅲ cell culture Ⅲ conduction velocity Ⅲ connexin 40 Ⅲ connexin 43 Ⅲ mapping Ⅲ neonatal mouse cardiomyocytes Ⅲ optical mapping C onnexin (Cx) proteins enable the intercellular exchange of ions and small regulatory molecules and are important determinants of cardiac electrical propagation. 1 Three major connexins, Cx43, Cx40, and Cx45, are expressed in heart. 2,3 Cx43 is abundant in ventricular and atrial myocardium; Cx40 is expressed in atrial tissue and in the Purkinje system; Cx45 is present in the sinoatrial (SA) and atrioventricular (AV) nodes and colocalizes with Cx43 in ventricular myocardium. 4,5 Cx43 and Cx40 each form channels with relatively large pores, whereas Cx45 forms narrow channels (unitary channel conductances of 75, 130, and 30 pS, respectively). 6 Ventricular myocytes express abundant Cx43 and small amounts of Cx45. In contrast, atrial myocytes express large amounts of Cx43 and Cx40. In ventricular myocytes and transfected cells, Cx43 and Cx45 colocalize in gap junctions and may form heteromeric/heterotypic channels. 5,7-10 Heterotypic Cx43/Cx40 gap junction channels have been described in HeLa cell pairs, whereas the role of heteromeric Cx43/ Cx40 channels has not been fully clarified. 7,11 Changes in cell-to-cell...

The endoplasmic reticulum protein TXNDC5 promotes cardiac fibrosis by facilitating ECM protein folding and CF activation via redox-sensitive c-Jun N-terminal kinase signaling. Loss of TXNDC5 protects against β agonist-induced cardiac fibrosis and contractile dysfunction. Targeting TXNDC5, therefore, could be a powerful new therapeutic approach to mitigate excessive cardiac fibrosis, thereby improving cardiac function and outcomes in patients with heart failure.

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. However, our knowledge of the specific residues and kinases involved is incomplete. The objective of this study was to identify amino acid residues in the Cx43-CT that are targets of the multi-functional protein kinase, Ca2+/calmodulin protein kinase II (CaMKII), an enzyme known to play critical roles in Ca2+ homeostasis, transcription, apoptosis and ischemic heart disease. We subjected fusion protein containing the Cx43-CT to phosphorylation by CaMKII in vitro, digestion with Lys-C and trypsin followed by enrichment for phosphorylated peptides using TiO2, and analysis in an LTQ XL Orbitrap with collision-induced dissociation and electron transfer dissociation. We deduced the sites of modification by interpreting tandem spectra from these “orthogonal” methods of gas phase peptide fragmentation. We have identified 15 serine residues, including one novel site, in the Cx43-CT that are phosphorylated by CaMKII, the activity of which may be important in regulating Cx43 in normal and diseased hearts.

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. Interestingly, increasing Cx43 expression reduced fibroblast proliferation, whereas decreasing Cx43 expression increased proliferation. These data demonstrate that native fibroblasts isolated from the mouse heart exhibit intercellular coupling via gap junctions containing both Cx43 and Cx45. Fibroblast proliferation is inversely related to the expression level of Cx43. Thus, connexin expression and remodeling is likely to alter fibroblast function, maintenance of the extracellular matrix, and ventricular remodeling in both normal and diseased hearts.

Long-chain acylcarnitines increase intracellular Ca2+ (Ca2+i) and induce electrophysiologic alterations that likely contribute to the genesis of malignant ventricular arrhythmias induced during myocardial ischemia. The mechanisms by which long-chain acylcarnitines increase Ca2+i are not known, although it occurs in the presence of Ca2+ channel blockade and inhibition of Na+/Ca2+ exchange. Long-chain acylcarnitines activate Ca2+ release channels from skeletal muscle sarcoplasmic reticulum (SR), but their effect on cardiac SR is unclear. To test the hypothesis that long-chain acylcarnitines increase Ca2+i from the SR, SR-enriched membrane fractions were prepared from rabbit left ventricular myocardium using sucrose density-gradient centrifugation and characterized by marker enzyme analysis. 45Ca2+ efflux was assessed in the presence or absence of long-chain acylcarnitines. Palmitoylcarnitine and stearoylcarnitine produced concentration-dependent efflux of 45Ca2+, whereas shorter chain acylcarnitines, palmitate, and palmitoyl-coenzyme A did not. Pretreatment of cardiac SR vesicles with ryanodine did not prevent palmitoylcarnitine-induced Ca2+ release. In addition, palmitoylcarnitine did not influence specific [3H]ryanodine binding, suggesting a mechanism independent of alterations in ryanodine receptor/Ca2+ release channel binding. In summary, long-chain acylcarnitines enhance Ca2+ release from cardiac SR vesicles and may thereby mobilize Ca2+i to induce electrophysiologic derangements under conditions, such as ischemia, in which these amphiphiles accumulate.

Background-We have recently shown that native murine ventricular fibroblasts express both 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. They can couple electrically with cardiac myocytes and influence myocardial activation patterns. Thus, Cx43 remodeling and the level of intercellular communication in fibroblasts expressed in the infarcted heart were the subject of the present investigation.

Although the total content of Cx45 is not upregulated in Cx43-deficient hearts, the localization of Cx45 to cardiac gap junctions depends on the expression level of Cx43 and is dramatically altered in mice that express no Cx43.