Yuanwen Chen

Microarray identifies extensive downregulation of noncollagen extracellular matrix and profibrotic growth factor genes in chronic isolated mitral regurgitation in the dog.

Background— The volume overload of isolated mitral regurgitation (MR) in the dog results in left ventricular (LV) dilatation and interstitial collagen loss. To better understand the mechanism of collagen loss, we performed a gene array and overlaid regulated genes into ingenuity pathway analysis. Methods and Results— Gene arrays from LV tissue were compared in 4 dogs before and 4 months after MR. Cine-magnetic resonance–derived LV end-diastolic volume increased 2-fold (P=0.005), and LV ejection fraction increased from 41% to 53% (P<0.007). LV interstitial collagen decreased 40% (P<0.05) compared with controls, and replacement collagen was in short strands and in disarray. Ingenuity pathway analysis identified Marfan syndrome, aneurysm formation, LV dilatation, and myocardial infarction, all of which have extracellular matrix protein defects and/or degradation. Matrix metalloproteinase-1 and -9 mRNA increased 5- (P=0.01) and 10-fold (P=0.003), whereas collagen I did not change and collagen III mRNA increased 1.5-fold (P=0.02). However, noncollagen genes important in extracellular matrix structure were significantly downregulated, including decorin, fibulin 1, and fibrillin 1. In addition, connective tissue growth factor and plasminogen activator inhibitor were downregulated, along with multiple genes in the transforming growth factor-β signaling pathway, resulting in decreased LV transforming growth factor-β1 activity (P=0.03). Conclusions— LV collagen loss in isolated, compensated MR is chiefly due to posttranslational processing and degradation. The downregulation of multiple noncollagen genes important in global extracellular matrix structure, coupled with decreased expression of multiple profibrotic factors, explains the failure to replace interstitial collagen in the MR heart.

Dynamic molecular and histopathological changes in the extracellular matrix and inflammation in the transition to heart failure in isolated volume overload

Left ventricular (LV) volume overload (VO) causes eccentric remodeling with inflammatory cell infiltration and extracellular matrix (ECM) degradation, for which there is currently no proven therapy. To uncover new pathways that connect inflammation and ECM homeostasis with cellular dysfunction, we determined the cardiac transciptome in subacute, compensated, and decompensated stages based on in vivo hemodynamics and echocardiography in the rat with aortocaval fistula (ACF). LV dilatation at 5 wk was associated with a normal LV end-diastolic dimension-to-posterior wall thickness ratio (LVEDD/PWT; compensated), whereas the early 2-wk (subacute) and late 15-wk (decompensated) ACF groups had significant increases in LVEDD/PWT. Subacute and decompensated stages had a significant upregulation of genes related to inflammation, the ECM, the cell cycle, and apoptosis. These changes were accompanied by neutrophil and macrophage infiltration, nonmyocyte apoptosis, and interstitial collagen loss. At 15 wk, there was a 40-fold increase in the matricellular protein periostin, which inhibits connections between collagen and cells, thereby potentially mediating a side-to-side slippage of cardiomyocytes and LV dilatation. The majority of downregulated genes was composed of mitochondrial enzymes whose suppression progressed from 5 to 15 wk concomitant with LV dilatation and systolic heart failure. The profound decrease in gene expression related to fatty acid, amino acid, and glucose metabolism was associated with the downregulation of peroxisome proliferator associated receptor (PPAR)-α-related and bioenergetic-related genes at 15 wk. In VO, an early phase of inflammation subsides at 5 wk but reappears at 15 wk with marked periostin production along with the suppression of genes related to PPAR-α and energy metabolism.

Chymase Inhibition Prevents Fibronectin and Myofibrillar Loss and Improves Cardiomyocyte Function and LV Torsion Angle in Dogs With Isolated Mitral Regurgitation

Background— The left ventricular (LV) dilatation of isolated mitral regurgitation (MR) is associated with an increase in chymase and a decrease in interstitial collagen and extracellular matrix. In addition to profibrotic effects, chymase has significant antifibrotic actions because it activates matrix metalloproteinases and kallikrein and degrades fibronectin. Thus, we hypothesize that chymase inhibitor (CI) will attenuate extracellular matrix loss and LV remodeling in MR. Methods and Results— We studied dogs with 4 months of untreated MR (MR; n=9) or MR treated with CI (MR+CI; n=8). Cine MRI demonstrated a >40% increase in LV end-diastolic volume in both groups, consistent with a failure of CI to improve a 25% decrease in interstitial collagen in MR. However, LV cardiomyocyte fractional shortening was decreased in MR versus normal dogs (3.71±0.24% versus 4.81±0.31%; P<0.05) and normalized in MR+CI dogs (4.85±0.44%). MRI with tissue tagging demonstrated an increase in LV torsion angle in MR+CI versus MR dogs. CI normalized the significant decrease in fibronectin and FAK phosphorylation and prevented cardiomyocyte myofibrillar degeneration in MR dogs. In addition, total titin and its stiffer isoform were increased in the LV epicardium and paralleled the changes in fibronectin and FAK phosphorylation in MR+CI dogs. Conclusions— These results suggest that chymase disrupts cell surface–fibronectin connections and FAK phosphorylation that can adversely affect cardiomyocyte myofibrillar structure and function. The greater effect of CI on epicardial versus endocardial titin and noncollagen cell surface proteins may be responsible for the increase in torsion angle in chronic MR.

Tumor Necrosis Factor-α Produced in Cardiomyocytes Mediates a Predominant Myocardial Inflammatory Response to Stretch in Early Volume Overload

Acute stretch caused by volume overload (VO) of aorto-caval fistula (ACF) induces a variety of myocardial responses including mast cell accumulation, matrix metalloproteinase (MMP) activation, and collagen degradation, all of which are critical in dictating long-term left ventricle (LV) outcome to VO. Meanwhile, these responses can be part of myocardial inflammation dictated by tumor necrosis factor-alpha (TNF-alpha), which is elevated after acute ACF. However, it is unknown whether TNF-alpha mediates a major myocardial inflammatory response to stretch in early VO. In 24-h ACF and sham rats, microarray gene expression profiling and subsequent Ingenuity Pathway Analysis identified a predominant inflammatory response and a gene network of biologically interactive genes strongly linked to TNF-alpha. Western blot demonstrated increased local production of TNF-alpha in the LV (1.71- and 1.66-fold in pro- and active-TNF-alpha over control, respectively, P<0.05) and cardiomyocytes (2- and 4-fold in pro- and active-TNF-alpha over control, respectively, P<0.05). TNF-alpha neutralization with infliximab (5.5 mg/kg) attenuated the myocardial inflammatory response to acute VO, as indicated by inhibition of inflammatory gene upregulation, myocardial infiltration (total CD45+ cells, mast cells, and neutrophils), MMP-2 activation, collagen degradation, and cardiac cell apoptosis, without improving LV remodeling and function. These results indicate that TNF-alpha produced by cardiomyocytes mediates a predominant inflammatory response to stretch in the early VO in the ACF rat, suggesting an important role of TNF-alpha in initiating pathophysiological response of myocardium to VO.

Loss of interstitial collagen causes structural and functional alterations of cardiomyocyte subsarcolemmal mitochondria in acute volume overload

Volume overload (VO) caused by aortocaval fistula (ACF) is associated with oxidative/inflammatory stress. The resulting inflammation, matrix metalloproteinase (MMP) activation, and collagen degradation is thought to play a pivotal role in left ventricular (LV) dilatation and failure. Since mitochondria are also targets for inflammation and oxidative stress, we hypothesized that there would be bioenergetic dysfunction with acute VO. In Sprague-Dawley rats subjected to 24 hrs of ACF, there was a two-fold increase in LV pressure-volume area in vivo, consistent with increased LV myocardial oxygen usage and increased bioenergetic demand in cardiomyocytes. Isolated cardiomyocytes from ACF LVs demonstrated increased hydrogen peroxide and superoxide formation and increased MMP activity. Subsarcolemmal mitochondria (SSM) showed a 40% decrease in state 3 respiration and proteomic analysis of SSM demonstrated decreased levels of complexes I-V in ACF. Immunohistochemical analysis revealed disruption of the subsarcolemmal location of the SSM network in ACF. To test for a potential link between SSM dysfunction and loss of interstitial collagen, rats were treated with the MMP-inhibitor PD166793 prior to ACF. MMP-inhibitor preserved interstitial collagen, integrin-α5 and the SSM structural arrangement. In addition, the decrease in state 3 mitochondrial respiration with ACF was prevented by PD166793. These studies established an important interaction between degradation of interstitial collagen in acute VO and the disruption of SSM structure and function which could contribute to progression to heart failure.

Novel insights into interactions between mitochondria and xanthine oxidase in acute cardiac volume overload.

Xanthine oxidoreductase (XOR) is increased in the left ventricle (LV) of humans with volume overload (VO), and mitochondrial inhibition of the respiratory chain occurs in animal models of VO. Because mitochondria are both a source and a target of reactive oxygen and nitrogen species, we hypothesized that activation of XOR and mitochondrial dysfunction are interdependent. To test this we used the aortocaval fistula (ACF) rat model of VO and a simulation of the stretch response in isolated adult cardiomyocytes with and without the inhibitor of XOR, allopurinol, or the mitochondrially targeted antioxidant MitoQ. Xanthine oxidase (XO) activity was increased in cardiomyocytes from ACF vs sham rats (24h) without an increase in XO protein. A twofold increase in LV end-diastolic pressure/wall stress and a decrease in LV systolic elastance with ACF were improved when allopurinol treatment (100mg/kg) was started at ACF induction. Subsarcolemmal State 3 mitochondrial respiration was significantly decreased in ACF and normalized by allopurinol. Cardiomyocytes subjected to 3h cyclical stretch resulted in an increase in XO activity and mitochondrial swelling, which was prevented by allopurinol or MitoQ pretreatment. These studies establish an early interplay between cardiomyocyte XO activation and bioenergetic dysfunction that may provide a new target that prevents progression to heart failure in VO.

Preservation of basal AcSDKP attenuates carbon tetrachloride-induced fibrosis in the rat liver

BACKGROUND & AIMS N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is an endogenous tetrapeptide which has antifibrogenic effects at physiological concentrations in various tissues. AcSDKP is produced locally in the liver, however, little is known about its biological effect in this organ. We hypothesize that basal levels of endogenous AcSDKP decrease during the development of liver fibrosis and preservation of basal AcSDKP attenuates liver fibrosis. METHODS Endogenous levels of AcSDKP in the liver were measured by enzyme immunoassay after 2, 6, and 10 weeks of carbon tetrachloride (CCl(4))-induced liver fibrosis in rats. Subcutaneous osmotic pump infusion of vehicle or AcSDKP (800 microg/kg/day) was administered to CCl(4)-treated rats for 8 weeks to study the effect of exogenous AcSDKP on liver fibrosis. The effect of AcSDKP on profibrogenic properties of hepatic stellate cells was studied in vitro. RESULTS Endogenous AcSDKP was significantly decreased in the liver of CCl(4)-treated rats. Chronic AcSDKP infusion preserved basal levels of AcSDKP and reduced liver injury, inflammation, fibrosis, and profibrogenic transforming growth factor-beta signaling. This was demonstrated by decreased aminotransferase serum levels, CD45 positive cells, collagen accumulation, alpha-smooth muscle actin positivity, transforming growth factor-beta1, phosphorylated Smad2/3 protein, increased bone morphogenetic protein-7, and phosphorylated Smad1/5/8. Further, AcSDKP exerts antifibrogenic effects on hepatic stellate cells (HSCs) by downregulation of HSC activation in vitro. CONCLUSIONS Maintaining physiological levels of AcSDKP is critical in negatively regulating the development of fibrosis in chronic liver injury. Preservation of AcSDKP may be a useful therapeutic approach in the management of liver fibrosis.

Mast cell stabilization decreases cardiomyocyte and LV function in dogs with isolated mitral regurgitation.

BACKGROUND Mast cells are increased in isolated mitral regurgitation (MR) in the dog and may mediate extracellular matrix loss and left ventricular (LV) dilatation. We tested the hypothesis that mast cell stabilization would attenuate LV remodeling and improve function in the MR dog. METHODS AND RESULTS MR was induced in adult dogs randomized to no treatment (MR, n = 5) or to the mast cell stabilizer, ketotifen (MR + MCS, n = 4) for 4 months. LV hemodynamics were obtained at baseline and after 4 months of MR and magnetic resonance imaging (MRI) was performed at sacrifice. MRI-derived, serial, short-axis LV end-diastolic (ED) and end-systolic (ES) volumes, LVED volume/mass ratio, and LV 3-dimensional radius/wall thickness were increased in MR and MR + MCS dogs compared with normal dogs (n = 6) (P < .05). Interstitial collagen was decreased by 30% in both MR and MR + MCS versus normal dogs (P < .05). LV contractility by LV maximum time-varying elastance was significantly depressed in MR and MR + MCS dogs. Furthermore, cardiomyocyte fractional shortening was decreased in MR versus normal dogs and further depressed in MR + MCS dogs (P < .05). In vitro administration of ketotifen to normal cardiomyocytes also significantly decreased fractional shortening and calcium transients. CONCLUSIONS Chronic mast cell stabilization did not attenuate eccentric LV remodeling or collagen loss in MR. However, MCS therapy had a detrimental effect on LV function because of a direct negative inotropic effect on cardiomyocyte function.

Whole-genome profiling highlights the molecular complexity underlying eccentric cardiac hypertrophy

Objectives: Heart failure is typically preceded by myocardial hypertrophy and remodeling, which can be concentric due to pressure overload (PO), or eccentric because of volume overload (VO). The molecular mechanisms that underlie these differing patterns of hypertrophy are distinct and have yet to be fully elucidated. Thus, the goal of this work is to identify novel therapeutic targets for cardiovascular conditions marked by hypertrophy that have previously been resistant to medical treatment, such as a pure VO. Methods: Concentric or eccentric hypertrophy was induced in rats for 2 weeks with transverse aortic constriction (TAC) or aortocaval fistula (ACF), respectively. Hemodynamic and echocardiographic analysis were used to assess the development of left ventricular (LV) hypertrophy and functional differences between groups. Changes in gene expression were determined by microarray and further characterized with Ingenuity Pathway Analysis. Results: Both models of hypertrophy increased LV mass. Rats with TAC demonstrated concentric LV remodeling while rats with ACF exhibited eccentric LV remodeling. Microarray analysis associated eccentric remodeling with a more extensive alteration of gene expression compared with concentric remodeling. Rats with VO had a marked activation of extracellular matrix genes, promotion of cell cycle genes, downregulation of genes associated with oxidative metabolism, and dysregulation of genes critical to cardiac contractile function. Rats with PO demonstrated similar categorical changes, but with the involvement of fewer individual genes. Conclusions: Our results indicate that eccentric remodeling is a far more complex process than concentric remodeling. This study highlights the importance of several key biological functions early in the course of VO, including regulation of matrix, metabolism, cell proliferation, and contractile function. Thus, the results of this analysis will inform the ongoing search for new treatments to prevent the progression to heart failure in VO.