Ian M.C. Dixon

Role of extracellular matrix proteins in heart function

The cardiac interstitium is populated by nonmyocyte cell types including transcriptionally active cardiac fibroblasts and endothelial cells. Since these cells are the source of many components of the cardiac extracellular matrix, and because changes in cardiac extracellular matrix are suspected of contributing to the genesis of cardiovascular complications in disease states such as diabetes, hypertension, cardiac hypertrophy and congestive heart failure, interest in the mechanisms of activation of fibroblasts and endothelial cells has led to progress in understanding these processes. Recent work provides evidence for the role of the renin-angiotensin-aldosterone system in the pathogenesis of abnormal deposition of extracellular matrix in the cardiac interstitium during the development of inappropriate cardiac hypertrophy and failure. The cardiac extracellular matrix is also known to change in response to altered cardiac performance associated with post-natal aging, and in response to environmental stimuli including intermittent hypoxia and abnormal nutrition. It is becoming clear that the extracellular matrix mainly consists of molecules of collagen types I and III; they form fibrils and provide most of the connective material for tying together myocytes and other structures in the myocardium and thus is involved in the transmission of developed mechanical force. The data available in the literature support the view that the extracellular matrix is a dynamic entity and alterations in this structure result in the development of heart dysfunction.

Modification of the extracellular matrix following myocardial infarction monitored by FTIR spectroscopy

Comparison of mid- and near-infrared spectra of control and infarcted rat ventricular tissue reveals the presence of absorptions in infarcted tissue which are highly characteristics of collagen, indicating large scale deposition of type I collagen in the myocardium following infarction. These results demonstrate that IR spectroscopy may be used to rapidly monitor the modifications of the extracellular matrix associated with myocardial infarction.

The basic helix―loop―helix transcription factor scleraxis regulates fibroblast collagen synthesis

The transcription factor scleraxis has been implicated in regulating the development of collagen-rich tissues such as tendons and cardiac valves, but its role in general collagen synthesis in the heart is unknown. Scleraxis expression in cardiac fibroblasts was examined, and its ability to regulate gene expression of collagen I alpha 2, the predominant cardiac collagen isoform, was assayed. Using real-time PCR, we demonstrate here that scleraxis mRNA is up-regulated by the profibrotic agonist TGF-beta(1) in rat cardiac myofibroblasts, and that phenoconversion of fibroblasts to myofibroblasts similarly increases scleraxis expression. Over-expression of scleraxis in NIH-3T3 or primary rat cardiac fibroblasts by adenoviral gene delivery is sufficient to significantly increase collagen I alpha 2 gene expression. Using luciferase reporter assays, we demonstrate that scleraxis transactivates the human collagen I alpha 2 promoter in a DNA- and protein-binding dependent manner. Intriguingly, examination of infarcted rat hearts reveals a nearly four-fold increase in scleraxis expression in the infarct scar, but not in non-infarcted tissue. These data support a novel and previously unknown role for scleraxis in the regulation of collagen gene expression in the heart, including in post-infarct scar formation.

ALTERATIONS IN CARDIAC MEMBRANE CA2+ TRANSPORT DURING OXIDATIVE STRESS

Although cardiac dysfunction due to ischemia-reperfusion injury is considered to involve oxygen free radicals, the exact manner by which this oxidative stress affects the myocardium is not clear. As the occurrence of intracellular Ca2+ overload has been shown to play a critical role in the genesis of cellular damage due to ischemia-reperfusion, this study was undertaken to examine whether oxygen free radicals are involved in altering the sarcolemmal Ca2+-transport activities due to reperfusion injury. When isolated rat hearts were made globally ischemic for 30 min and then reperfused for 5 min, the Ca2+ -pump and Na+-Ca2+ exchange activities were depressed in the purified sarcolemmal fraction; these alterations were prevented when a free radical scavenger enzymes (superoxide dismutase plus catalase) were added to the reperfusion medium. Both the Ca2+- pump and Na+- Ca2+ exchange activities in control heart sarcolemmal preparations were depressed by activated oxygen-generating systems containing xanthine plus xanthine oxidase and H2O2; these changes were prevented by the inclusion of superoxide dismutase and catalase in the incubation medium. These results support the view that oxidative stress during ischemia-reperfusion may contribute towards the occurrence of intracellular Ca2+ overload and subsequent cell damage by depressing the sarcolemmal mechanisms governing the efflux of Ca2+ from the cardiac cell.

Apoptosis, autophagy and ER stress in mevalonate cascade inhibition-induced cell death of human atrial fibroblasts

3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors (statins) are cholesterol-lowering drugs that exert other cellular effects and underlie their beneficial health effects, including those associated with myocardial remodeling. We recently demonstrated that statins induces apoptosis and autophagy in human lung mesenchymal cells. Here, we extend our knowledge showing that statins simultaneously induces activation of the apoptosis, autophagy and the unfolded protein response (UPR) in primary human atrial fibroblasts (hATF). Thus we tested the degree to which coordination exists between signaling from mitochondria, endoplasmic reticulum and lysosomes during response to simvastatin exposure. Pharmacologic blockade of the activation of ER-dependent cysteine-dependent aspartate-directed protease (caspase)-4 and lysosomal cathepsin-B and -L significantly decreased simvastatin-induced cell death. Simvastatin altered total abundance and the mitochondrial fraction of proapoptotic and antiapoptotic proteins, while c-Jun N-terminal kinase/stress-activated protein kinase mediated effects on B-cell lymphoma 2 expression. Chemical inhibition of autophagy flux with bafilomycin-A1 augmented simvastatin-induced caspase activation, UPR and cell death. In mouse embryonic fibroblasts that are deficient in autophagy protein 5 and refractory to autophagy induction, caspase-7 and UPR were hyper-induced upon treatment with simvastatin. These data demonstrate that mevalonate cascade inhibition-induced death of hATF manifests from a complex mechanism involving co-regulation of apoptosis, autophagy and UPR. Furthermore, autophagy has a crucial role in determining the extent of ER stress, UPR and permissiveness of hATF to cell death induced by statins.

Fourier transform infrared evaluation of microscopic scarring in the cardiomyopathic heart: effect of chronic AT1 suppression.

Our primary aim was to investigate the use of Fourier transform infrared (FTIR) spectromicroscopy as an accurate assay of cardiac extracellular matrix remodeling. Abnormal rearrangement or remodeling of the cardiac extracellular matrix is known to contribute to cardiac dysfunction. The microscopic multifocal necrosis and scarring are modulated by chronic AT(1) receptor blockade in experimental cardiomyopathy; thus, we also wished to rationalize the spectromicroscopic differences among control, untreated cardiomyopathic (CMP), and losartan-treated cardiomyopathic (LOS) hearts according to the pathogenesis of experimental cardiomyopathy. Male UM-X7.1 cardiomyopathic Syrian hamsters at early and late (65 and 200 days) stages of cardiomyopathy were subjected to 4-week losartan (15 mg/kg/day continuous infusion) treatment. Focal collagen microdomain distribution was confirmed spectroscopically by observation of the collagen IR fingerprint in the 1000-1800 cm(-1) region. Synchrotron FTIR spectromicroscopic map data were obtained from control (F1-beta strain) hamsters, nontreated cardiomyopathic, and losartan-treated CMP animals and imaged with mapping software, according to intensity of collagen fingerprint. Compared to controls, untreated late-stage CMP myocardium was characterized by elevated levels of fibrillar collagens and this was partially normalized with a 4-week losartan treatment. FTIR spectromicroscopy revealed that elevated collagen expression in focal microdomains is present in late-stage cardiomyopathy, and 4-week AT(1) blockade is associated with attenuation of collagen absorption in these lesions.

Autophagy is a regulator of TGF-β1-induced fibrogenesis in primary human atrial myofibroblasts.

Transforming growth factor-β1 (TGF-β1) is an important regulator of fibrogenesis in heart disease. In many other cellular systems, TGF-β1 may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown. Thus we tested whether or not TGF-β1-induced autophagy has a regulatory function on fibrosis in human atrial myofibroblasts (hATMyofbs). Primary hATMyofbs were treated with TGF-β1 to assess for fibrogenic and autophagic responses. Using immunoblotting, immunofluorescence and transmission electron microscopic analyses, we found that TGF-β1 promoted collagen type Iα2 and fibronectin synthesis in hATMyofbs and that this was paralleled by an increase in autophagic activation in these cells. Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells. ATG7 knockdown in hATMyofbs and ATG5 knockout (mouse embryonic fibroblast) fibroblasts decreased the fibrotic effect of TGF-β1 in experimental versus control cells. Furthermore, using a coronary artery ligation model of myocardial infarction in rats, we observed increases in the levels of protein markers of fibrosis, autophagy and Smad2 phosphorylation in whole scar tissue lysates. Immunohistochemistry for LC3β indicated the localization of punctate LC3β with vimentin (a mesenchymal-derived cell marker), ED-A fibronectin and phosphorylated Smad2. These results support the hypothesis that TGF-β1-induced autophagy is required for the fibrogenic response in hATMyofbs.

Cardiac sarcolemmal Na+-Ca2+ exchange and Na+-K+ ATPase activities and gene expression in alloxan-induced diabetes in rats

To determine the sequence of alterations in cardiac sarcolemmal (SL) Na+-Ca2+ exchange, Na+-K+ ATPase and Ca2+-transport activities during the development of diabetes, rats were made diabetic by an intravenous injection of 65 mg/kg alloxan. SL membranes were prepared from control and experimental hearts 1-12 weeks after induction of diabetes. A separate group of 4 week diabetic animals were injected with insulin (3 U/day) for an additional 4 weeks. Both Na+-K+ ATPase and Ca2+-stimulated ATPase activities were depressed as early as 10 days after alloxan administration; Mg2+ ATPase activity was not depressed throughout the experimental periods. Both Na+-Ca2+ exchange and ATP-dependent Ca2+-uptake activities were depressed in diabetic hearts 2 weeks after diabetes induction. These defects in SL Na+-K+ ATPase and Ca-transport activities were normalized upon treatment of diabetic animals with insulin. Northern blot analysis was employed to compare the relative mRNA abundances of α--subunit of Na+-K+ ATPase and Na+-Ca2+ exchanger in diabetic ventricular tissue vs. control samples. At 6 weeks after alloxan administration, a significant depression of the Na+-K+ ATPase α-- subunit mRNA was noted in diabetic heart. A significant increase in the Na+-Ca2+ exchanger mRNA abundance was observed at 3 weeks which returned to control by 5 weeks. The results from the alloxan-rat model of diabetes support the view that SL membrane abnormalities in Na+-K+ ATPase, Na+Ca2+ exchange and Ca2+-pump activities may lead to the occurrence of intracellular Ca2+ overload during the development of diabetic cardiomyopathy but these defects may not be the consequence of depressed expression of genes specific for those SL proteins.

Distribution of Collagen Deposition in Cardiomyopathic Hamster Hearts Determined by Infrared Microscopy

Fibrillar collagens are major proteins of the cardiac extracellular matrix and play a significant role in the structural organization of the healthy heart. The aim of this study was (i) to investigate and compare the patterns of cardiac collagen deposition in different layers taken from both cardiomyopathic and normal myocardium using infrared (IR) microspectroscopy and (ii) to evaluate IR microspectroscopy as an alternative means for in vitro detection of collagen deposition in heart. Frozen sections from UM-X7.1 strain hamsters expressing the cardiomyopathic phenotype associated with ventricular remodeling and age-matched control (F1-beta) strain hamsters were examined using IR microspectroscopy. The presence of collagen was identified by the appearance of a typical collagen band at 1204 cm(-1), and the results were compared with identical tissue sections stained with trichrome, a routine discriminator for interstitial matrix proteins in cardiac myocytes. Spatial information addressing collagen deposition was obtained and viewed using contour mapping and three-dimensional band intensity maps at 1204 cm(-1). Perivascular and interstitial collagen deposition was detected in control samples taken from both ventricles as indicated with relative low intensities of the band of 1204 cm(-1). When compared with these control levels, the concentration of collagen was increased in cardiomyopathic left-ventricular samples with some focal depositions, and these results were confirmed with the trichrome references. Our study suggests that collagen deposition from normal and diseased hearts may be successfully analyzed directly in the absence of any chemihistological or immunological staining, by infrared microscopy.

The participation of the Na+–Ca2+ exchanger in primary cardiac myofibroblast migration, contraction, and proliferation

Cardiac ventricular myofibroblast motility, proliferation, and contraction contribute to post‐myocardial infarct wound healing, infarct scar formation, and remodeling of the ventricle remote to the site of infarction. The Na+–Ca2+ exchanger (NCX1) is involved in altered calcium handling in cardiac myocytes during cardiac remodeling associated with heart failure, however, its role in cardiac myofibroblast cell function is unexplored. In this study we investigated the involvement of NCX1 as well as the role of non‐selective‐cation channels (NSCC) in cardiac myofibroblast cell function in vitro. Immunofluorescence and Western blots revealed that P1 cells upregulate α‐smooth muscle actin (αSMA) and embryonic smooth muscle myosin heavy chain (SMemb) expression. NCX1 mRNA and proteins as well as Cav1.2a protein are also expressed in P1 myofibroblasts. Myofibroblast motility in the presence of 50 ng/ml PDGF‐BB was blocked with AG1296. Myofibroblast motility, contraction, and proliferation were sensitive to KB‐R7943, a specific NCX1 reverse‐mode inhibitor. In contrast, only proliferation and contraction, but not motility were sensitive to nifedipine, while gadolinium (NSCC blocker) was only associated with decreased motility. ML‐7 treatment was associated with inhibition of the chemotactic response and contraction. Thus cardiac myofibroblast chemotaxis, contraction, and proliferation were sensitive to different pharmacologic treatments suggesting that regulation of transplasmalemmal calcium movements may be important in growth factor receptor‐mediated processes. NCX1 may represent an important moiety in suppression of myofibroblast functions. J. Cell. Physiol. 213: 540–551, 2007.