Calreticulin Induces Dilated Cardiomyopathy

Background

Calreticulin, a Ca²⁺-buffering chaperone of the endoplasmic reticulum, is highly expressed in the embryonic heart and is essential for cardiac development. After birth, the calreticulin gene is sharply down regulated in the heart, and thus, adult hearts have negligible levels of calreticulin. In this study we tested the role of calreticulin in the adult heart.

Methodology/Principal Findings

We generated an inducible transgenic mouse in which calreticulin is targeted to the cardiac tissue using a Cre/loxP system and can be up-regulated in adult hearts. Echocardiography analysis of hearts from transgenic mice expressing calreticulin revealed impaired left ventricular systolic and diastolic function and impaired mitral valve function. There was altered expression of Ca²⁺ signaling molecules and the gap junction proteins, Connexin 43 and 45. Sarcoplasmic reticulum associated Ca²⁺-handling proteins (including the cardiac ryanodine receptor, sarco/endoplasmic reticulum Ca²⁺-ATPase, and cardiac calsequestrin) were down-regulated in the transgenic hearts with increased expression of calreticulin.

Conclusions/Significance

We show that in adult heart, up-regulated expression of calreticulin induces cardiomyopathy in vivo leading to heart failure. This is due to an alternation in changes in a subset of Ca²⁺ handling genes, gap junction components and left ventricle remodeling.     

Chromatin compaction and cell death by high molecular weight FGF-2 depend on its nuclear localization, intracrine ERK activation, and engagement of mitochondria

Fibroblast growth factor 2 (FGF-2) is produced as CUG-initiated, 22-34 kDa or AUG-initiated 18 kDa isoforms (hi- and lo-FGF-2, respectively), with potentially distinct functions. We report that expression of hi-FGF-2 in HEK293 cells elicited chromatin compaction preceding cell death with apoptotic features. Nuclear localization of the intact protein was required as expression of a non-nuclear hi-FGF-2 mutant failed to elicit chromatin compaction. Equally ineffective, despite nuclear localization, was the over-expression of the 18 kDa core sequence (lo-FGF-2). Chromatin compaction by hi-FGF-2 was accompanied by increased cytosolic cytochrome C, and was attenuated either by over-expression of Bcl-2 or by a peptide inhibitor of the pro-apoptotic protein Bax. In addition hi-FGF-2 elicited sustained activation of total and nuclear extracellular signal regulated kinase (ERK1/2) by an intracrine route, as it was not prevented by neutralizing anti-FGF-2 antibodies. Inhibition of the ERK1/2 activating pathway by dominant negative upstream activating kinase, or by PD 98059, prevented chromatin compaction by hi-FGF-2. ERK1/2 activation was not affected by the Bax-inhibiting peptide suggesting that it occurred upstream of mitochondrial involvement. We conclude that the hi-FGF-2-induced chromatin compaction and cell death requires its nuclear localization, intracrine ERK1/2 activation and mitochondrial engagement.     

Connexin43 phosphorylation and cytoprotection in the heart

The fundamental role played by connexins including connexin43 (Cx43) in forming intercellular communication channels (gap junctions), ensuring electrical and metabolic coupling between cells, has long been recognized and extensively investigated. There is also increasing recognition that Cx43, and other connexins, have additional roles, such as the ability to regulate cell proliferation, migration, and cytoprotection. Multiple phosphorylation sites, targets of different signaling pathways, are present at the regulatory, C-terminal domain of Cx43, and contribute to constitutive as well as transient phosphorylation Cx43 patterns, responding to ever-changing environmental stimuli and corresponding cellular needs. The present paper will focus on Cx43 in the heart, and provide an overview of the emerging recognition of a relationship between Cx43, its phosphorylation pattern, and development of resistance to injury. We will also review our recent work regarding the role of an enhanced phosphorylation state of Cx43 in cardioprotection. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Biochemical and ultrastructural evidence for the association of basic fibroblast growth factor with cardiac gap junctions

Basic fibroblast growth factor (bFGF) is a ubiquitous and multifunctional polypeptide that is believed to have a role in tissue repair and to act as a morphogen in embryonic development. Here, we have used immunohistochemical and biochemical methods with antibodies directed against the amino-terminal domain of bFGF, designated IS2, which recognize native and denatured bFGF, to demonstrate that in addition to its known intracellular and extracellular localization in heart, bFGF is also associated with cardiomyocyte gap junctions. In tissue sections, IS2 labeled regions of intercalated discs, producing an immunofluorescence pattern virtually indistinguishable from that obtained with antibodies against the heart gap junction protein connexin-43. By electron microscopy, gap junctions but not other regions of plasma membrane were heavily immunolabeled with this antibody. By solid phase immunoassay, bFGF was found to be more concentrated in a fraction enriched in cardiac gap junctions than in whole sarcolemmal preparations. Finally, an 18-kDa protein was recognized by several different antibodies specific for bFGF on Western blots of heart subcellular fractions enriched in gap junctions. We suggest that bFGF-like peptides are either an integral part of, or exist in close association with, cardiac gap junctions and thus may play a role in modulating gap junctional intercellular communication.     

Stimulation and inhibition of cardiac myocyte proliferation in vitro

Summary We have examined the effect of crude cardiac tissue extracts as well as that of several growth factors and triiodothyronin (T3) on DNA synthesis of cardiac myocytes in culture. Extracts from embryonic and adult cardiac tissue stimulated DNA synthesis of myocytes. Atrial myocytes exhibited overall higher degree of stimulation than their ventricular counterparts and extracts from adult atrial tissue had the highest apparent mitogenic activity for atrial myocytes. We have shown that adult heart contains basic fibroblast growth factor (bFGF), especially in the atria [1]. Transforming growth factor (TGF) and insulin-like growth factors (IGFs) are also accumulated in cardiac tissues [2, 3]. We found that bFGF and the IGFs stimulate myocyte cell proliferation and DNA synthesis. These factors also stimulate cardiac non-muscle proliferation, especially in the presence of serum. TGF inhibited proliferation and DNA synthesis and cancelled the effect of bFGF or IGFs on the myocytes. T3 also diminished the bFGF-induced mitogenic stimulation of cardiomyocytes. Our data suggest that these factors may be involved in the regulation of cardiomyocyte proliferation in vivo.Abbreviations bFGF basic Fibroblast Growth Factor - BSA Bovine Serum Albumin - DM Defined Medium - Fes Fetal calf serum - FITC Fluorescein - IGF Insulin-like Growth Factor - IgG Immunoglobulin - LI Labeling Index - PBS Phosphate Buffered Saline - T3 Triiodothyronine - TGF Transforming Growth Factor      

Basic fibroblast growth factor is cardioprotective in ischemia-reperfusion injury

To examine whether basic fibroblast growth factor (bFGF) administered to the heart by perfusion can improve cardiac resistance to injury we employed an isolated rat heart model of ischemia-reperfusion injury and determined the extent of functional recovery in bFGF-treated and control hearts. Global ischemia was simulated by interruption of flow for 60 min. Recovery of developed force of contraction (DF), recorded after reestablishment of flow for 30 min, reached 63.8±1.5% and 96.5±3.5% of preischemic levels in control and bFGF-treated hearts (10 g/heart), respectively, indicating that bFGF induced significantly improved recovery of mechanical function. Recoveries of the rates of contraction or relaxation were also significantly improved in bFGF-treated hearts. Extent of myocardial injury, assessed by determination of phosphocreatine kinase in the effluent, was reduced as a result of bFGF treatment. As a first step towards understanding the mechanism and direct cellular target(s) of bFGF-induced cardioprotection, we investigated its fate after perfusion. Perfusion of 10 g bFGF/heart resulted in a 4-fold increase in bFGF associated with the heart compared to control levels, as estimated by biochemical fractionation and immunoblotting. Immunofluorescent staining of the bFGF-perfused hearts revealed intense anti-bFGF staining in association with blood vessels as well as the periphery of cardiomyocytes, suggesting that the latter may be a target for direct bFGF action. In conclusion, our findings of bFGF-induced increases in cardiac resistance to, and improved functional recovery from, ischemia-reperfusion injury indicate that bFGF may have clinical applications in the treatment of ischemic heart disease.     

Elevation in phosphatidylethanolamine is an early but not essential event for cardiac cell differentiation

The biosynthesis of phosphatidylethanolamine was examined during differentiation of P19 teratocarcinoma cells into cardiac myocytes. P19 cells were induced to undergo differentiation into cardiac myocytes by the addition of dimethyl sulfoxide to the medium. Immunofluorescence labeling confirmed the expression of striated myosin 10 days postinduction of differentiation. The content of phosphatidylethanolamine increased significantly within the first 2 days of differentiation. [1,3-(3)H]Glycerol incorporation into phosphatidylethanolamine was increased 7.2-fold during differentiation, indicating an elevation in de novo synthesis from 1, 2-diacyl-sn-glycerol. The mechanism for the increase in phosphatidylethanolamine levels during cardiac cell differentiation was a 2.8-fold increase in the activity of ethanolaminephosphotransferase, the 1,2-diacyl-sn-glycerol utilizing reaction of the cytidine 5'-diphosphate-ethanolamine pathway of phosphatidylethanolamine biosynthesis. Incubation of P19 cells with the phosphatidylethanolamine biosynthesis inhibitor 8-(4-chlorophenylthio)-cAMP inhibited the differentiation-induced elevation in phosphatidylethanolamine levels but did not affect the expression of striated myosin. The results suggest that elevation in phosphatidylethanolamine is an early event of P19 cell differentiation into cardiac myocytes, but is not essential for differentiation to proceed.     

Lysophosphatidylethanolamine acyltransferase activity is elevated during cardiac cell differentiation

This work attempts to explain several aspects of the response of plasminogen to 6-aminohexanoate (6-AH). These responses include the overall fluorescent changes that occur when plasminogen binds the ligand, the changes shown by the individual domains when they bind the ligand, and the changes in structure shown by the holoprotein when it binds 6-AH. The results have implications for understanding the physicochemical behavior of all kringle based proteins.     

Expression of fibroblast growth factor receptor-1 in rat heart H9c2 myoblasts increases cell proliferation

Aspergillus fumigatus is a highly pathogenic fungus causing a wide spectrum of diseases in immunocompromised as well as immunocompetent hosts. The present work was undertaken to evaluate the cytotoxic nature of fractionated antigens of A. fumigatus against the mammalian cell lines (J774, RAW, CHO and L929). An enriched protein antigenic fraction of A. fumigatus was subjected to con A Sepharose and phenyl Sepharose chromatography. Antigenic fractions, ConAub (conA unbound) and PSC III (fraction III of phenyl Sepharose column) containing low mw antigens showed higher cytotoxicity as compared to other antigenic fractions. PSC III was further purified on HPLC resulting in an 18 kDa homogeneous protein. The purified protein showed high ELISA absorbance values for specific IgG and IgE antibodies in sera of ABPA patients. Monoclonal antibody raised against Asp fl, a major allergen/antigen of A. fumigatus recognised the purified 18 kDa by ELISA and western blot. The 18 kDa allergen/antigen or Asp fl showed similar toxicity towards all the four cell lines (macrophage and fibroblast) with an IC50 of 75 ng/ml or 4.16 nM. Reduction in toxicity of 18 kDa at low temperatures and potentiation in presence of ammonium chloride and monensin indicates mechanism of internalisation of 18 kDa in eukaryotic cells is similar to -sarcin. The present work shows that the 18 kDa allergen/antigen (Asp fl) is a major cytotoxin secreted by A. fumigatus which may play multiple roles in the pathogenesis of Aspergillosis through allergenicity, antigenicity and cytotoxicity. (Mol Cell Biochem 167: 89-97, 1997)     

Administration of FGF-2 to the Heart Stimulates Connexin-43 Phosphorylation at Protein Kinase C Target Sites

Fibroblast growth factor-2 (FGF-2) confers acute, preconditioning-like cardiac resistance to ischemic injury in a protein kinase C (PKC)-dependent fashion. One of the downstream targets of PKC is the gap junction protein connexin-43 (Cx43). We thus examined the effects of FGF-2 on Cx43 phosphorylation at specific PKC sites in the adult heart. Rat hearts perfused ex vivo for 20 min with an FGF-2-containing solution displayed increased levels of phosphorylated 44-45 kDa Cx43, assessed by western blotting. In addition, FGF-2 significantly upregulated phosphorylation of the PKC target serines 262 and 368 on Cx43 at intercalated disks, assessed using phosphospecific antibodies in immunolocalization and western blotting assays. Our data show that FGF-2, administered by perfusion, can alter the phosphorylation status of Cx43 at cardiomyocyte intercalated disks, and suggest a link between phosphorylation of Cx43 at specific PKC sites and FGF-2 cardioprotection.