Livia C. Hool

Transient Exposure to Hydrogen Peroxide Causes an Increase in Mitochondria-Derived Superoxide As a Result of Sustained Alteration in L-Type Ca2+ Channel Function in the Absence of Apoptosis in Ventricular Myocytes

We sought to understand the effect of a transient exposure of cardiac myocytes to H2O2 at a concentration that did not induce apoptosis. Myocytes were exposed to 30 &mgr;mol/L H2O2 for 5 minutes followed by 10 U/mL catalase for 5 minutes to degrade the H2O2. Cellular superoxide was measured using dihydroethidium. Transient exposure to H2O2 caused a 66.4% increase in dihydroethidium signal compared with controls exposed to only catalase, without activation of caspase 3 or evidence of necrosis. The increase in dihydroethidium signal was attenuated by the mitochondrial inhibitors myxothiazol or carbonyl cyanide p-(trifluoromethoxy)phenyl-hydrazone and when calcium uptake by the mitochondria was inhibited with Ru360. We investigated the L-type Ca2+ channel (ICa-L) as a source of calcium influx. Nisoldipine, an inhibitor of ICa-L, attenuated the increase in superoxide. Basal channel activity increased from 5.4 to 8.9 pA/pF. Diastolic calcium was significantly increased in quiescent and contracting myocytes after H2O2. The response of ICa-L to &bgr;-adrenergic receptor stimulation was used as a functional reporter because decreasing intracellular H2O2 alters the sensitivity of ICa-L to isoproterenol. H2O2 increased the K0.5 required for activation of ICa-L by isoproterenol from 5.8 to 27.8 nmol/L. This effect and the increase in basal current density persisted for several hours after H2O2. We propose that extracellular H2O2 is associated with an increase in superoxide from the mitochondria caused by an increase in Ca2+ influx from ICa-L. The effect persists because a positive feedback exists among increased basal channel activity, elevated intracellular calcium, and superoxide production by the mitochondria.

Hypoxia Increases the Sensitivity of the L-Type Ca2+ Current to β-Adrenergic Receptor Stimulation via a C2 Region–Containing Protein Kinase C Isoform

Abstract— The effects of hypoxia on the L-type Ca2+ current (ICa-L) in the absence and presence of the &bgr;-adrenergic receptor agonist isoproterenol (Iso) were examined. Exposing guinea pig ventricular myocytes to hypoxia alone resulted in a reversible inhibition of basal ICa-L. When cells were exposed to Iso in the presence of hypoxia, the K0.5 for activation of ICa-L by Iso was significantly decreased from 5.3±0.7 to 1.6±0.1 nmol/L. The membrane-impermeant thiol-specific oxidizing compound 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) attenuated the inhibition of basal ICa-L by hypoxia 81.3±9.4% but had no effect on the increase in sensitivity of ICa-L to Iso. In addition, DTT mimicked the effects of hypoxia on basal ICa-L and the increase in sensitivity to Iso. Neither the inhibitors of guanylate cyclase LY-83583 or methylene blue nor the NO synthase inhibitor NG-monomethyl-l-arginine monoacetate had any effect on the basal inhibition of ICa-L or the decrease in K0.5 for activation of ICa-L by Iso during hypoxia. However, the protein kinase C (PKC) inhibitors bisindolylmaleimide I and Gö 7874 significantly attenuated the increase in sensitivity of ICa-L to Iso. More specifically, the response was attenuated when cells were dialyzed with a peptide inhibitor of the C2 region–containing classical PKC isoforms. The same effect was not observed with the PKC&egr; peptide inhibitor. These results suggest that hypoxia regulates ICa-L through the following 2 distinct mechanisms: direct inhibition of basal ICa-L and an indirect effect on the sensitivity of the channel to &bgr;-adrenergic receptor stimulation that is mediated through a classical PKC isoform.

REACTIVE OXYGEN SPECIES IN CARDIAC SIGNALLING: FROM MITOCHONDRIA TO PLASMA MEMBRANE ION CHANNELS

1 Reactive oxygen species (ROS) have been considered deleterious to cell function and there is good evidence to suggest that they play a role in the pathophysiology of a number of cardiac disease states. However, ROS are also now being recognized as important regulators of cell function by altering the redox state of proteins. 2 Possible sources of production of ROS in cardiac myocytes are the mitochondria and nicotinamide adenine dinucleotide phosphate‐oxidase. The generation of ROS and anti‐oxidant defence mechanisms in the heart are discussed. 3 The evidence for a role for ROS in the development of disease states, such as atherosclerosis, ischaemia, cardiac hypertrophy and hypertension, is presented. It is now recognized that cardiac ion channel function is regulated by ROS. Implications with respect to cardiac arrhythmia are discussed.

Decreasing Cellular Hydrogen Peroxide With Catalase Mimics the Effects of Hypoxia on the Sensitivity of the L-Type Ca2+ Channel to β-Adrenergic Receptor Stimulation in Cardiac Myocytes

Abstract— In cardiac myocytes, hypoxia inhibits the basal L-type Ca2+ current (ICa-L) and increases the sensitivity of ICa-L to &bgr;-adrenergic receptor stimulation. We investigated whether hydrogen peroxide (H2O2) is involved in the hypoxic response. Guinea pig ventricular myocytes were dialyzed with catalase, which specifically catalyzes the conversion of H2O2 to H2O and oxygen, and then ICa-L was recorded during exposure to isoproterenol (Iso). Catalase decreased the K0.5 for activation of ICa-L by Iso from 2.7±0.3 nmol/L (in cells dialyzed with heat-inactivated catalase) to 0.4±0.1 nmol/L. The increase in sensitivity to Iso by catalase may be attenuated when cells are preexposed to H2O2. A significant increase in sensitivity of ICa-L to Iso was recorded when mitochondrial function was inhibited with myxothiazol or FCCP, suggesting that the source of H2O2 was from the mitochondria. Prior exposure of cells to H2O2 attenuated the inhibition of basal ICa-L during hypoxia and the increase in sensitivity of ICa-L to Iso during hypoxia. Additionally, extracellularly applied catalase mimicked the effect of hypoxia on basal ICa-L. Measurement of the rate of production of hydrogen peroxide using 5- (and 6-)chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate acetyl ester indicated that hypoxia was associated with a significant decrease in the production of hydrogen peroxide in the cells. These data suggest that hypoxia mediates changes in channel activity through a lowering in H2O2 levels and that H2O2 is a key intermediate in modifying basal channel activity and the &bgr;-adrenergic responsiveness of the channel during hypoxia.

Secreted Frizzled-Related Protein 4: An Angiogenesis Inhibitor

Wnt signaling is involved in developmental processes, cell proliferation, and cell migration. Secreted frizzled-related protein 4 (sFRP4) has been demonstrated to be a Wnt antagonist; however, its effects on endothelial cell migration and angiogenesis have not yet been reported. Using various in vitro assays, we show that sFRP4 inhibits endothelial cell migration and the development of sprouts and pseudopodia as well as disrupts the stability of endothelial rings in addition to inhibiting proliferation. sFRP4 interfered with endothelial cell functions by antagonizing the canonical Wnt/beta-catenin signaling pathway and the Wnt/planar cell polarity pathway. Furthermore, sFRP4 blocked the effect of vascular endothelial growth factor on endothelial cells. sFRP4 also selectively induced apoptotic events in endothelial cells by increasing cellular levels of reactive oxygen species. In vivo assays demonstrated a reduction in vascularity after sFRP4 treatment. Most importantly, sFRP4 restricted tumor growth in mice by interfering with endothelial cell function. The data demonstrate sFRP4 to be a potent angiogenesis inhibitor that warrants further investigation as a therapeutic agent in the control of angiogenesis-associated pathology.

Evidence for regulation of mitochondrial function by the L-type Ca2+ channel in ventricular myocytes.

The L-type Ca(2+) channel is responsible for initiating contraction in the heart. Mitochondria are responsible for meeting the cellular energy demands and calcium is required for the activity of metabolic intermediates. We examined whether activation of the L-type Ca(2+) channel alone is sufficient to alter mitochondrial function. The channel was activated directly with the dihydropyridine agonist BayK(-) or voltage-clamp of the plasma membrane and indirectly by depolarization of the membrane with high KCl. Activation of the channel increased superoxide production (assessed as changes in dihydroethidium fluorescence), NADH production and metabolic activity (assessed as formation of formazan from tetrazolium) in a calcium-dependent manner. Activation of the channel also increased mitochondrial membrane potential assessed as changes in JC-1 fluorescence. The response was reversible upon inactivation of the channel during voltage-clamp of the plasma membrane and did not appear to require calcium. We examined whether the response may be mediated through movement of cytoskeletal proteins. Depolymerization of actin or exposing cells to a peptide directed against the alpha-interacting domain of the alpha(1C)-subunit of the channel (thereby preventing movement of the beta-subunit) attenuated the increase in mitochondrial membrane potential. We conclude that activation of the L-type Ca(2+) channel can regulate mitochondrial function and the response appears to be modulated by movement through the cytoskeleton.

Evidence For The Regulation Of L-Type Ca2+ Channels In The Heart by Reactive Oxygen Species: Mechanism For Mediating Pathology

1 It is well recognized that reactive oxygen species (ROS) can activate transduction pathways to mediate pathophysiology. An increase in ROS has been implicated in a number of cardiovascular disorders. Reactive oxygen species regulate cell function through redox modification of target proteins. One of these target proteins is the L‐type Ca2+ channel. 2 There is good evidence that thiol reducing and oxidizing compounds, including hydrogen peroxide, can influence calcium channel function. The evidence for regulation of the channel protein and regulatory proteins by thiol‐specific modifying agents and relevance to hypoxia and oxidative stress is presented. 3 Clinical studies suggest that calcium channel antagonists may be beneficial in reducing myocardial injury associated with oxidative stress. The identification of cysteines as possible targets for intervention during hypoxic trigger of arrhythmia or chronic pathological remodelling is discussed.

Regulator of G-Protein Signaling 5 Controls Blood Pressure Homeostasis and Vessel Wall Remodeling

Rationale: Regulator of G-protein signaling 5 (RGS5) modulates G-protein-coupled receptor signaling and is prominently expressed in arterial smooth muscle cells. Our group first reported that RGS5 is important in vascular remodeling during tumor angiogenesis. We hypothesized that RGS5 may play an important role in vessel wall remodeling and blood pressure regulation. Objective: To demonstrate that RGS5 has a unique and nonredundant role in the pathogenesis of hypertension and to identify crucial RGS5-regulated signaling pathways. Methods and Results: We observed that arterial RGS5 expression is downregulated with chronically elevated blood pressure after angiotensin II infusion. Using a knockout mouse model, radiotelemetry, and pharmacological inhibition, we subsequently showed that loss of RGS5 results in profound hypertension. RGS5 signaling is linked to the renin–angiotensin system and directly controls vascular resistance, vessel contractility, and remodeling. RGS5 deficiency aggravates pathophysiological features of hypertension, such as medial hypertrophy and fibrosis. Moreover, we demonstrate that protein kinase C, mitogen-activated protein kinase/extracellular signal–regulated kinase, and Rho kinase signaling pathways are major effectors of RGS5-mediated hypertension. Conclusions: Loss of RGS5 results in hypertension. Loss of RGS5 signaling also correlates with hyper-responsiveness to vasoconstrictors and vascular stiffening. This establishes a significant, distinct, and causal role of RGS5 in vascular homeostasis. RGS5 modulates signaling through the angiotensin II receptor 1 and major G&agr;q-coupled downstream pathways, including Rho kinase. So far, activation of RhoA/Rho kinase has not been associated with RGS molecules. Thus, RGS5 is a crucial regulator of blood pressure homeostasis with significant clinical implications for vascular pathologies, such as hypertension.

Cav1.2 calcium channel is glutathionylated during oxidative stress in guinea pig and ischemic human heart

Glutathionylation as a posttranslational modification of proteins is becoming increasingly recognized, but its role in many diseases has not been demonstrated. Oxidative stress and alterations in calcium homeostasis are associated with the development of cardiac hypertrophy. Because the cardiac L-type Ca(2+) channel can be persistently activated after exposure to H(2)O(2), the aim of this study was to determine whether alterations in channel function were associated with glutathionylation of the α(1C) subunit (Ca(v)1.2) channel protein. Immunoblot analysis indicated that Ca(v)1.2 protein is significantly glutathionylated after exposure to H(2)O(2) and glutathione in vitro and after ischemia-reperfusion injury. L-type Ca(2+) channel macroscopic current and intracellular calcium were significantly increased in myocytes after exposure to oxidized glutathione and reversed by glutaredoxin. The increase in current correlated with an increase in open probability of the channel assessed as changes in single-channel activity after exposing the human long N-terminal Ca(v)1.2 to H(2)O(2) or oxidized glutathione. We also demonstrate that the Ca(v)1.2 channel is significantly glutathionylated in ischemic human heart. We conclude that oxidative stress is associated with an increase in glutathionylation of the Ca(v)1.2 channel protein. We suggest that the associated constitutive activity contributes to the development of pathology in ischemic heart disease.

Contributions of Ion Channel Currents to Ventricular Action Potential Changes and Induction of Early Afterdepolarizations During Acute Hypoxia

Rationale: Variability in delivery of oxygen can lead to electric instability in the myocardium and the generation of arrhythmias. In addition ischemic heart disease and angina are associated with an increase in circulating catecholamines that further increases the risk of developing ventricular tachyarrhythmias. Objective: We investigated the net effects of acute hypoxia and catecholamines on the cardiac action potential. Methods and Results: We incorporated all published data on the effects of hypoxia on the late Na+ current (INa-L), the fast Na+ current (INa), the basal L-type Ca2+ channel current (ICa-L), and the slow (IKs) and rapid components of the delayed rectifier K+-current (IKr) in the absence and presence of &bgr;-adrenergic receptor (&bgr;-AR) stimulation into the Luo–Rudy model of the action potential. Hypoxia alone had little effect on the action potential configuration or action potential duration. However in the presence of &bgr;-AR stimulation, hypoxia caused a prolongation of the action potential and early afterdepolarizations (EADs) and spontaneous tachycardia were induced. Experiments performed in guinea pig ventricular myocytes confirmed the modeling results. Conclusions: EADs occur predominantly because of the increased sensitivity of ICa-L to &bgr;-AR stimulation during hypoxia. &bgr;-AR stimulation is necessary to induce EADs as EADs are never observed during hypoxia in the absence of &bgr;-AR stimulation.