Xianyao Xu

Regulation of cellular oxidative stress and apoptosis by G protein-coupled receptor kinase-2; The role of NADPH oxidase 4

Cardiac myocyte oxidative stress and apoptosis are considered important mechanisms for the development of heart failure (HF). Chronic HF is characterized by increased circulating catecholamines to augment cardiac output. Long-term stimulation of myocardial β-adrenergic receptors (β-ARs) is deleterious in cardiac myocytes, however, the potential mechanisms underlying increased cell death are unclear. We hypothesize that GRK2, a critical regulator of myocardial β-AR signaling, plays an important role in mediating cellular oxidative stress and apoptotic cell death in response to β-agonist stimulation. Stimulation of H9c2 cells with a non-selective β-agonist, isoproterenol (Iso) caused increased oxidative stress and apoptosis. There was also increased Nox4 expression, but no change in Nox2, the primary NADPH isoforms and major sources of ROS generation in cardiac myocytes. Adenoviral-mediated overexpression of GRK2 led to similar increases in ROS production and apoptosis as seen with Iso stimulation. These increases in oxidative stress were abolished by pre-treatment with the non-specific Nox inhibitor, apocynin, or siRNA knockdown of Nox4. Adenoviral-mediated expression of a GRK2 inhibitor prevented ROS production and apoptosis in response to Iso stimulation. β-Arrestins are signaling proteins that function downstream of GRK2 in β-AR uncoupling. Adenoviral-mediated overexpression of β-arrestins increased ROS production and Nox4 expression. Chronic β-agonist stimulation in mice increased Nox4 expression and apoptosis compared to PBS or AngII treatment. These data demonstrate that GRK2 may play an important role in regulating oxidative stress and apoptosis in cardiac myocytes and provides an additional novel mechanism for the beneficial effects of cardiac-targeted GRK2 inhibition to prevent the development of HF.

β-Arrestins regulate human cardiac fibroblast transformation and collagen synthesis in adverse ventricular remodeling

Cardiac fibroblasts (CFs) produce and degrade the myocardial extracellular matrix and are critical in maladaptive ventricular remodeling that can result in heart failure (HF). β-Arrestins are important signaling molecules involved in β-adrenergic receptor (β-AR) desensitization and can also mediate signaling in a G protein-independent fashion. We hypothesize that β-arrestins play an important role in the regulation of adult human CF biology with regard to myofibroblast transformation, increased collagen synthesis, and myocardial fibrosis which are important in the development of HF. β-Arrestin1 & 2 expression is significantly upregulated in adult human CF isolated from failing left ventricles and β-AR signaling is uncoupled with loss of β-agonist-mediated inhibition of collagen synthesis versus normal control CF. Knockdown of either β-arrestin1 or 2 restored β-AR signaling and β-agonist mediated inhibition of collagen synthesis. Overexpression of β-arrestins in normal CF led to a failing phenotype with increased baseline collagen synthesis, impaired β-AR signaling, and loss of β-agonist-mediated inhibition of collagen synthesis. β-Arrestin knockdown in failing CF diminished TGF-β stimulated collagen synthesis and also inhibited ERK phosphorylation. Overexpression of β-arrestins in normal CF increased basal ERK1/2 and Smad2/3 phosphorylation and enhanced TGF-β-stimulated collagen synthesis. This was prevented by pre-treatment with a MEK1/2 inhibitor. Enhanced β-arrestin signaling appears to be deleterious in CF by promoting a pro-fibrotic phenotype via uncoupling of β-AR signaling as well as potentiating ERK and Smad signaling. Targeted inhibition of β-arrestins in CF may represent a therapeutic strategy to prevent maladaptive myocardial fibrosis.

Regulation of mitochondrial oxidative stress by β-arrestins in cultured human cardiac fibroblasts.

ABSTRACT Oxidative stress in cardiac fibroblasts (CFs) promotes transformation to myofibroblasts and collagen synthesis leading to myocardial fibrosis, a precursor to heart failure (HF). NADPH oxidase 4 (Nox4) is a major source of cardiac reactive oxygen species (ROS); however, mechanisms of Nox4 regulation are unclear. β-arrestins are scaffold proteins that signal in G-protein-dependent and -independent pathways; for example, in ERK activation. We hypothesize that β-arrestins regulate oxidative stress in a Nox4-dependent manner and increase fibrosis in HF. CFs were isolated from normal and failing adult human left ventricles. Mitochondrial ROS/superoxide production was quantitated using MitoSox. β-arrestin and Nox4 expressions were manipulated using adenoviral overexpression or short interfering RNA (siRNA)-mediated knockdown. Mitochondrial oxidative stress and Nox4 expression in CFs were significantly increased in HF. Nox4 knockdown resulted in inhibition of mitochondrial superoxide production and decreased basal and TGF-β-stimulated collagen and α-SMA expression. CF β-arrestin expression was upregulated fourfold in HF. β-arrestin knockdown in failing CFs decreased ROS and Nox4 expression by 50%. β-arrestin overexpression in normal CFs increased mitochondrial superoxide production twofold. These effects were prevented by inhibition of either Nox or ERK. Upregulation of Nox4 seemed to be a primary mechanism for increased ROS production in failing CFs, which stimulates collagen deposition. β-arrestin expression was upregulated in HF and plays an important and newly identified role in regulating mitochondrial superoxide production via Nox4. The mechanism for this effect seems to be ERK-mediated. Targeted inhibition of β-arrestins in CFs might decrease oxidative stress as well as pathological cardiac fibrosis. Summary: β-arrestins regulate oxidative stress in a Nox4-dependent manner leading to increased extracellular-matrix protein synthesis by cardiac fibroblasts (CFs). Targeted inhibition of β-arrestins in CFs might decrease pathological fibrosis.

High Molecular Weight Polyethylene Glycol Inhibits Myocardial Ischemia-Reperfusion Injury In Vivo

OBJECTIVES Cardiac ischemia-reperfusion (I-R) injury remains a significant problem as there are no therapies available to minimize the cell death that can lead to impaired function and heart failure. We have shown that high-molecular-weight polyethylene glycol (PEG) (15-20 kD) can protect cardiac myocytes in vitro from hypoxia-reoxygenation injury. In this study, we investigated the potential protective effects of PEG in vivo. METHODS Adult rats underwent left anterior descending artery occlusion for 60 minutes followed by 48 hours or 4 weeks of reperfusion. One milliliter of 10% PEG solution or phosphate-buffered saline (PBS) control (n = 10 per group) was administered intravenously (IV) immediately before reperfusion. RESULTS Fluorescein-labeled PEG was robustly visualized in the myocardium 1 hour after IV delivery. The PEG group had significant recovery of left ventricular ejection fraction at 4 weeks versus a 25% decline in the PBS group (P < .01). There was 50% less LV fibrosis in the PEG group versus PBS with smaller peri-infarct and remote territory fibrosis (P < .01). Cell survival signaling was upregulated in the PEG group with increased Akt (3-fold, P < .01) and ERK (4-fold, P < .05) phosphorylation compared to PBS controls at 48 hours. PEG also inhibited apoptosis as measured by TUNEL-positive nuclei (56% decrease, P < .02) and caspase 3 activity (55% decrease, P < .05). CONCLUSIONS High-molecular-weight PEG appears to have a significant protective effect from I-R injury in the heart when administered IV immediately before reperfusion. This may have important clinical translation in the setting of acute coronary revascularization and myocardial protection in cardiac surgery.

ID: 137: GRK2 INHIBITION REDUCES POST-MYOCARDIAL INFARCTION CARDIAC FIBROBLAST MEDIATED ADVERSE REMODELING

Objectives Remote (non-infarct) territory fibrosis is a significant cause of post-infarction heart failure (HF). We have previously shown that increased G protein-coupled receptor kinase-2 (GRK2) activity in adult human cardiac fibroblasts (CF) isolated from failing hearts is an important mechanism of cardiac fibrosis through uncoupling β-adrenergic receptor (β-AR) signaling. This study investigates the potential therapeutic role of GRK2 inhibition on CF biology in vivo. Methods Adult male rats underwent LAD ligation to induce post-MI HF. Left ventricular (LV) function was assessed by echocardiography. Myocardial fibrosis was quantitated by histologic staining. LV CF were isolated and cultured. GRK2 was inhibited by intra-coronary adenoviral-mediated delivery of a GRK2 inhibitor (Ad-GRK2ct) immediately following LAD ligation (n=11). Control rats received a null adenovirus (n=10). Animals were studied prior to and 12 weeks post-MI and adenoviral delivery. Results There was a significant decline in LV function at 12 weeks post-MI which [Fractional shortening: 0.35±0.01 vs. 0.52±0.01, p<0.01]. There was significant increase in remote territory (non-infarct area) fibrosis at 12 weeks post-MI compared to control [12±1% vs. 2±1% fibrosis, p<0.05], consistent with adverse remodeling. Additionally, collagen synthesis was significantly upregulated in isolated CF 12 weeks post-MI compared to control CF [3559±760 vs. 1029±45 cmp/mg protein, p<0.02]. At 12 weeks post-MI, GRK2 activity was increased 1.4-fold [p<0.01]. There was a 42% decrease in intracellular cAMP [p<0.05] and loss of b-agonist (isoproterenol)-stimulated inhibition of collagen synthesis characteristic of normal CF, indicating uncoupling of β-AR signaling post-MI. Adenoviral mediated overexpression of GRK2ct, GRK2 inhibitor, in vitro in the cultured CF post-MI led to a 50% decrease in aSMA expression (p<0.01) as well as a significant decreased collagen expression and synthesis compared to null adenovirus (Ad-Null) control [1928±126 vs. 2611±213 cmp/mg protein, p<0.05], restoring the control CF phenotype. Intra-coronary delivery of Ad-GRK2ct following MI significantly reduced post-MI LV dysfunction vs. Ad-Null as measured by improved fractional shortening [0.42±0.01 vs. 0.30±0.02, p<0.01] and ejection fraction [72±1% vs. 57±2%, p<0.03]. Ad-GRK2ct also decreased peri-infarct and remote territory fibrosis by 60% [p<0.03]. Consistent with these findings, Ad-GRK2ct resulted in an over 25% decreased in α-SMA, collagen I, and collagen III expression in CF isolated 12 weeks post-MI vs. Ad-Null [p<0.04] providing evidence of decreased post-MI CF activation and myofibroblast transformation with Ad-GRK2ct. Conclusions Uncoupling of β-adrenergic signaling in CF via increased GRK2 appears to be a key mechanism of post-MI fibrosis. Targeted inhibition of GRK2 and restoration of b-adrenergic signaling/cAMP production in CF may represent a novel therapeutic approach to prevent pathological fibrosis and maladaptive remodeling.