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1. It is well recognised 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. ROS 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 oxidising 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 will be 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.
The production of reactive oxygen species (ROS) is usually considered to be directly detrimental to the health of organisms because ROS can damage key macromolecules such as DNA, proteins and lipids.1 However, ROS can also act as signalling molecules able to stimulate and modulate a variety of biochemical and genetic systems including the regulation of signal transduction pathways, gene expression, proliferation and cell death by apoptosis.2 The activity of some second messengers appears to be under the tonic regulation of ROS and this is necessary for normal function.3,4
Reactive oxygen species are generally speaking oxygen molecules in different states of reduction as well as compounds of oxygen with hydrogen and nitrogen. Superoxide is formed from the reduction of oxygen but since it cannot easily cross membranes its reactivity is limited to the organelle in which it is produced.2 At physiological pH it is rapidly dismutated to hydrogen peroxide by superoxide dismutase. The biologically active reactive species are hydrogen peroxide, hydroxyl radicals, hypochlorite and peroxynitrites. Since hydrogen peroxide can readily cross plasma membranes it is considered a good candidate as a signal molecule.5,6
The cell normally experiences a predominantly reduced intracellular environment due to the large pool of reduced thioredoxin and glutathione.5 An alteration in the cell’s redox state such as increased ROS production is associated with pathology. The regulation of signalling pathways by hydrogen peroxide (H2O2) and superoxide has been linked to the development of various cardiovascular diseases. These include ischemic heart disease, hypertension, cardiomyopathies, cardiac hypertrophy and congestive heart failure7-9. It would appear therefore that consistent with homeostatic mechanisms necessary to maintain normal cellular function, an alteration in production of ROS may be capable of inducing pathology because the redox state of the cell or target protein has been altered.
The cellular transport of ions is vital to life. In addition to participating in a number of physiological responses, ion channels underlie the electrical activity of cells. Ion channels have a unique functional role, because not only do they participate in electrical activity, they form the means by which electrical signals are converted to responses within the cell. Calcium is a good example. Plasma membrane calcium channels such as the L-type Ca2+ channel activate quickly and can rapidly change the cytoplasmic environment. Once inside the cell, calcium acts as a ‘second messenger’ prompting responses by binding to a variety of calcium sensitive proteins. Calcium channels are known to play an important role in stimulating muscle contraction, in neurotransmitter secretion, gene regulation, activating other ion channels, and controlling the shape and duration of action potentials. Since excessive quantities can be toxic, its movement is tightly regulated and controlled. Calcium is also a mediator of pathology in that it is necessary for activation of second messengers such as MAPK, calcineurin and nuclear factor for activated T cell (NFAT) transcription signalling that are implicated in the development of cardiac hypertrophy.10,11
There is good evidence that ion channel function can be modified by ROS. The ion channel may be the direct target of ROS. Alternatively an intermediate regulatory protein may be the target of ROS that then modifies the function of the channel. The thiol redox state of the channel protein may be an important determinant of channel function and the cell’s fate. In this article the evidence for regulation of the cardiac L-type Ca2+ channel by reactive oxygen species will be presented and the role for the channel as possible mediator of pathology during alterations in cellular redox state will be discussed. Specifically the evidence for altered channel function during hypoxia and during increased production of ROS (or oxidative stress) will be presented.
The L-type Ca2+ channel is a member of a gene superfamily of transmembrane ion channel proteins that includes voltage-gated K+ and Na+ channels. Ca2+ channels share structural similarities with K+ and Na+ channels in that they possess a pore-forming α1 subunit comprising four repeats of a domain with six transmembrane-spanning segments that include the voltage sensing S4 segment and the pore-forming region (Figure 1). The α1 subunit is large (190-250 kDa) and incorporates the majority of the known sites regulated by second messengers, toxins and drugs. This subunit is usually complexed with at least three auxiliary subunits, α2, δ, β and γ, with the α2 and δ subunits always linked by a disulfide bond.12
Figure 1. The four homologous domains of the α1 subunit of the L-type Ca2+ channel indicating the pore-forming region (Pore) between S5 and S6, voltage sensor S4 and region in the C terminal domain that is proposed to be oxygen sensitive with cysteines shown in Italics (see text). Predicted α helices are depicted as cylinders.
Voltage gated calcium channels contain many cysteines. The α1 subunit from L-type channels, for example, has 48. Cysteine residues are generally thought to be the most likely target of redox or nitrosylation modification in proteins, as free thiols can easily react with oxygen or reactive nitrogen species and can be assisted in forming intramolecular disulfide bonds13-15. Not all of these will be susceptible to oxidation or reactions, however, as many will already be involved in disulfide bonds.
In vitro studies examining the effect of thiol-specific reducing or oxidising agents on L-type Ca2+ channel function have demonstrated that free thiols in the protein are likely to be sensitive to the oxidation state of the cell16-22. In guinea pig ventricular myocytes, the thiol-reducing agent dithiothreitol decreases basal native L-type Ca2+ current while 5,5′-dithio-bis[2-nitrobenzoic acid] (DTNB), a thiol-specific oxidising agent increases basal current.17 Similarly in frog ventricular myocytes, thiol oxidising agents increase L-type Ca2+ current.23 The effect can be prevented by dithiothreitol but is unaffected by an inhibitor of cAMP or application of GDPβS implying that a regulatory protein is not involved in the response. In contrast the oxidising mercury compound p-hydroxy-mercuric-phenylsulphonic acid (PHMPS) has been reported to decrease basal L-type Ca2+ current.20 Dithiothreitol has no effect on human cardiac L-type Ca2+ channels expressed in HEK 293 cells but reverses the inhibitory effects of another oxidising agent thimerosal.19 Therefore thiol oxidising or reducing agents can affect channel function but the responses appear to vary. This may be due to a contribution by the auxiliary subunits of the native channel that is absent in the expressed (cloned) channel in which only the α subunit is present. For example the auxiliary β3 subunit of Ca2+-dependent K+ channels participates in movement of ions through the α subunit by forming gates that block pore access.24 This gating mechanism is abolished by reduction of extracellular disulfide linkages. Therefore auxiliary subunits may play important regulatory roles where reactive cysteines alter subunit function.
In cardiac myocytes rapid changes in cellular oxygen and generation of ROS can contribute to electrophysiological instability in the myocardium and the development of arrhythmias.25-30 Understanding how ion channels respond to changes in oxygen tension may help with developing strategies to prevent the trigger of arrhythmias. Acute hypoxia (rapidly decreasing O2 pressure to approx 15 mmHg without depletion of cellular ATP) causes a decrease in basal L-type Ca2+ current in cardiac myocytes.17,18,31-33 This has also been demonstrated in the recombinant α1C subunit of the L-type Ca2+ channel.19,34,35 In addition the region of the α1C subunit that is responsive to hypoxia has been proposed (Figure 1). Only one of three splice variants expressed in the heart (hHT variant) appears to confer sensitivity to hypoxia.35 In the case of the native L-type Ca2+ channel, dithiothreitol mimics the effects of acute hypoxia (PO2 17mmHg) and DTNB attenuates the hypoxic inhibition of basal channel activity.17 In addition exposure of myocytes to catalase (that specifically converts hydrogen peroxide to water and oxygen) mimics the effect of hypoxia and the effect of dithiothreitol.32
Although controversial, acute hypoxia (that does not necessarily deplete cellular ATP) has been reported to be associated with a decrease in ROS.32,36-38 This is controversial because some studies have reported that ROS increase during hypoxia.39-41 However the duration and degree of hypoxia appears to be an important determinant of ROS production. In the mitochondria, the reduced state of the electron transfer proteins, the mitochondrial membrane potential and the level and duration of hypoxia/anoxia are critical in determining production of reactive oxygen species. On the other hand production of superoxide by NAD(P)H-oxidase is dependent upon oxygen as a substrate. If available oxygen decreases superoxide production also decreases. Therefore, it is important to stress that not all hypoxic conditions are alike and cells appear to respond differentially to oxygen deprivation depending on the site of ROS generation in the cell, the cells’ intrinsic requirements for oxygen and the duration of hypoxia. In adult ventricular myocytes a decrease in O2 pressure from 150 mmHg (room air) to 15 mmHg results in a 41% decrease in superoxide (assessed with the fluorescent indicator dihydroethidium)33 and a significant decrease in cellular hydrogen peroxide (assessed with 5-(and -6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester).32 The alteration in production of reactive oxygen species in cardiac myocytes in response to acute hypoxia would appear to be due to altered mitochondrial function and not an alteration in activity of NAD(P)H-oxidase.32,33 Therefore there is good evidence that cellular ROS production decreases during hypoxia and that a reduction in cellular redox state that occurs with hypoxia can significantly alter channel function.
A decrease in calcium influx during the plateau phase of the action potential may be necessary to prevent arrhythmias associated with prolongation of the QT interval during hypoxia that occurs without activation of ATP-sensitive K+ channels.42 Therefore a decrease in L-type Ca2+ channel basal current may be adaptive during hypoxia. However, hypoxia also increases the sensitivity of the L-type Ca2+ channel to the β-adrenergic receptor agonist isoproterenol and this may not be beneficial to the cell.17,32,33 Typically β-adrenergic receptor stimulation increases channel activity through activation of cAMP and subsequent protein kinase A-dependent phosphorylation of the channel.43 This increases the mode 2 state of channel openings (long openings). During cardiac ischemia there is an increase in sympathetic drive and circulating catecholamines. An increase in the sensitivity of the channel to β-adrenergic receptor stimulation would be expected to cause a prolongation in action potential duration that is usually associated with life-threatening arrhythmias. This may explain the increased incidence of early afterdepolarisations (that typically precede life-threatening ventricular tachyarrhythmias) associated with hypoxia and ischemia.17,30 The increase in sensitivity of the channel to isoproterenol can be mimicked by dithiothreitol17 and perfusing cells intracellularly with catalase.32 Consistent with this, exposing myocytes to the sulfhydryl oxidant phenylarsine oxide, is associated with a decrease in sensitivity of the channel to isoproterenol.44 Therefore increased sympathetic stimulation to the heart during ischemia and hypoxia may trigger arrhythmia as a result of altered sensitivity of the L-type Ca2+ channel to β-adrenergic receptor stimulation. The increase in sensitivity of the channel to β-adrenergic receptor stimulation does not involve nitric oxide and occurs downstream from the β-adrenergic receptor at the level of protein kinase A or the channel protein.17 Since the activity of protein kinase A can be altered by oxidation of thiols on the protein, protein kinase A may be an important regulator of cell function during changes in redox state.45-47 Understanding how the channel and β-adrenergic receptor pathway are altered during hypoxia would assist in identifying a target site for designing antiarrhythmic drugs to prevent trigger of arrhythmia during ischemic events.
Hydrogen peroxide can also directly alter channel function. Oxidation of the channel by hydrogen peroxide enhances L-type calcium channel activity.32,48 The enhanced calcium influx suggests oxidation of the channels may contribute to alterations in calcium homeostasis during oxidative stress. The generation of ROS by the mitochondria is dependent on intracellular Ca2+ and enhanced production of ROS occurs with enhanced mitochondrial matrix Ca2+ uptake.49,50 As mentioned above the regulation of signalling pathways by ROS has been linked to the development of various cardiovascular diseases. In addition Ca2+ is an important mediator of cell growth and a persistent increase in intracellular Ca2+ is associated with disease states such as heart failure.51 Therefore Ca2+ and ROS participate in pathological remodelling. However the mechanisms by which they cooperate in mediating pathology are poorly understood.
Figure 2. Application of hydrogen peroxide externally causes a further increase in cellular superoxide assessed with the fluorescent indicator dihydroethidium (DHE). A: DHE recorded from a guinea pig ventricular myocyte prior to and following exposure to increasing concentrations of H2O2 as indicated. Slope values are indicated below each H2O2 concentration. B: Mean±SE of ratio of fluorescence expressed as % of slope recorded at 5-25 min for guinea pig ventricular myocytes exposed to concentrations of H2O2 as indicated. Number of cells tested is shown in parentheses above each bar. 25-30 μM H2O2 consistently increased cellular superoxide without apoptosis or necrosis (for details see text).
Exposing cardiac myocytes to ROS further increases mitochondrial ROS production28,50 (Figure 2). It has been proposed that ROS production increases due to a direct effect of ROS on mitochondrial function. Alternatively ROS production may increase due to increased calcium uptake into the mitochondria as a result of enhanced calcium influx through the L-type Ca2+ channel. We exposed guinea pig ventricular myocytes to a transient oxidative stress (that mimics the burst of ROS that occurs in vivo with ischemia-reperfusion) and examined the effect on cellular ROS and calcium. Exposure to 30 μM hydrogen peroxide for 5 min followed by 10U/ml catalase for 5 min to degrade the hydrogen peroxide caused a 65% increase in dihydroethidium (DHE) signal without induction of apoptosis (assessed by caspase 3 assay) or necrosis (assessed by propidium iodide uptake) in the cells (data published as an abstract Circ. Res. 2006, 99:E19-E50). Exposure of the myocytes to nisoldipine (L-type Ca2+ channel antagonist) or Ru360 (an inhibitor of the mitochondria calcium uniporter) before or after the 5 min exposure to 30 μM hydrogen peroxide significantly attenuated the increase in DHE signal. However dantrolene, an inhibitor of sarcoplasmic reticulum calcium release did not attenuate the increase in DHE signal. This suggested that an increase in calcium influx through the channel and increased calcium uptake by the mitochondria were necessary for the increase in cellular superoxide. We tested this directly by activating the channel with the pharmacological agonist (-)-Bay K8644. An increase in influx of calcium through the channel alone was sufficient to increase cellular superoxide by 79%. In addition L-type Ca2+ channel basal current density was significantly increased from 5.4 to 8.9 pA/pF after transient exposure to hydrogen peroxide. This effect persisted for up to 9 hours after the transient exposure to hydrogen peroxide. Therefore it would appear that transient oxidation of the channel by hydrogen peroxide is sufficient to increase superoxide from the mitochondria as a result of increased calcium influx through the L-type Ca2+ channel. The effect persists because a positive feedback exists between increased basal channel activity, elevated intracellular calcium and superoxide production by the mitochondria (Figure 3). This may be sufficient to activate hypertrophic signalling pathways and induce pathological remodelling.
Figure 3. Model explaining persistent increase in cellular superoxide and increase in L-type Ca2+ channel basal current density as a result of a transient oxidative stress. Transient exposure to extracellular hydrogen peroxide oxidises the channel causing an increase in channel basal current density. An increase in calcium uptake into the mitochondria follows (as a result of an increase in calcium influx through the L-type Ca2+ channel) that further increases superoxide production by the mitochondria. The increase in cellular superoxide causes further oxidation of the channel (for further details see text).
Results from clinical studies have suggested that the channel may be an attractive target for treatment of myocardial injury during oxidative stress. A combination of calcium channel blockers and antioxidants are effective in reducing the progression of atherosclerosis that involves increased ROS production and lipid peroxidation in the vasculature.52,53 Administration of the L-type Ca2+ channel antagonist verapamil before coronary perfusion with thrombolytic therapy improves recovery of the region around the infarct54 and is associated with lower restenosis after percutaneous coronary intervention.55 In animal studies calcium channel antagonists have been effective in reducing myocardial oxidative stress in stroke prone hypertensive rats,56 protecting against myocardial reperfusion injury57 and stunned myocardium.58 Therefore the channel represents an attractive candidate for targeting under conditions of oxidative stress.
There is now good evidence that ROS can act as signalling molecules to mediate pathology. ROS can also regulate L-type Ca2+ channel function most likely as a result of direct redox modification of cysteines on the channel protein, although redox modification of regulatory proteins may also influence channel function. Cellular responses to changes in oxygen tension such as hypoxia are generally thought to be adaptive to prevent the deleterious effects of systemic hypoxemia. However acute changes in cellular ROS production and redox state can also represent a trigger for arrhythmia as a result of acute alterations in channel function. Pathological remodelling may involve persistent oxidation of the channel, increased ROS and increased calcium influx during oxidative stress. In order to treat disorders that involve modulation of channel function during alterations in redox state a greater understanding of how the channel protein is affected is required. Greater knowledge of the structural details of calcium channel regulation by ROS is an important step in designing drugs that could target specific sites on the protein. If a cysteine or cysteines are altered and identified this would provide a potential site to target for modification of channel function.
This study was supported by the National Health and Medical Research Council of Australia. Dr Hool is a National Health and Medical Research Council Career Development Award Fellow.
1. Halliwell B. The role of oxygen radicals in human disease, with particular reference to the vascular system. Haemostasis 1993; 23: 118-26.
2. Thannickal VJ, Fanburg BL. Reactive oxygen species in cell signaling. Am. J. Physiol. 2000; 279: L1005-28.
3. Salmeen A, Andersen JN, Myers MP, Meng TC, Hinks JA, Tonks NK, Barford D. Redox regulation of protein tyrosine phosphatase 1B involves a sulphenyl-amide intermediate. Nature 2003; 423: 769-73.
4. van Montfort RL, Congreve M, Tisi D, Carr R, Jhoti H. Oxidation state of the active-site cysteine in protein tyrosine phosphatase 1B. Nature. 2003; 423: 773-7.
5. Stone JR, Yang S. Hydrogen peroxide: a signaling messenger. Antioxid. Redox. Signal. 2006; 8: 243-70.
6. Wolin MS. Interactions of oxidants with vascular signaling systems. Arterioscler. Throm. Vasc. Biol. 2000; 20: 1430-42.
7. Sabri A, Hughie HH, Lucchesi PA. Regulation of hypertrophic and apoptotic signaling pathways by reactive oxygen species in cardiac myocytes. Antioxid Redox. Signal. 2003; 5: 731-40.
8. Nakagami H, Takemoto M, Liao JK. NADPH oxidase-derived superoxide anion mediates angiotensin II-induced cardiac hypertrophy. J. Mol. Cell. Cardiol. 2003; 35: 851-9.
9. Bolli R. Causative role of oxyradicals in myocardial stunning: a proven hypothesis. A brief review of the evidence demonstrating a major role of reactive oxygen species in several forms of postischemic dysfunction. Basic Res. Cardiol. 1998; 93: 156-62.
10. Molkentin JD, Lu JR, Antos CL, Markham B, Richardson J, Robbins J, Grant SR, Olson EN. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell. 1998; 93: 215-28.
11. Ruwhof C, van der Laarse A. Mechanical stress-induced cardiac hypertrophy: mechanisms and signal transduction pathways. Cardiovasc. Res. 2000; 47: 23-37.
12. Abernethy DR, Soldatov NM. Structure-functional diversity of human L-type Ca2+ channel: perspectives for new pharmacological targets. J. Pharmacol. Exp. Ther. 2002; 300: 724-8.
13. Campbell DL, Stamler JS, Strauss HC. Redox modulation of L-type calcium channels in ferret ventricular myocytes. Dual mechanism regulation by nitric oxide and S-nitrosothiols. J. Gen. Physiol. 1996; 108: 277-93.
14. Lipton SA, Choi YB, Pan ZH, Lei SZ, Chen HS, Sucher NJ, Loscalzo J, Singel DJ, Stamler JS. A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds. Nature. 1993; 364: 626-32.
15. Gilbert HF. Molecular and cellular aspects of thiol-disulfide exchange. Adv. Enzymol. Relat. Areas. Mol. Biol. 1990; 63: 69-172.
16. Chiamvimonvat N, O'Rourke B, Kamp TJ, Kallen RG, Hofmann F, Flockerzi V, Marban E. Functional consequences of sulfhydryl modification in the pore-forming subunits of cardiovascular Ca2+ and Na+ channels. Circ. Res. 1995; 76: 325-34.
17. Hool LC. Hypoxia increases the sensitivity of the L-type Ca2+ current to β-adrenergic receptor stimulation via a C2 region-containing protein kinase C isoform. Circ. Res. 2000; 87: 1164-71.
18. Hool LC. Hypoxia alters the sensitivity of the L-type Ca2+ channel to a-adrenergic receptor stimulation in the presence of β-adrenergic receptor stimulation. Circ. Res. 2001; 88: 1036-43.
19. Fearon IM, Palmer AC, Balmforth AJ, Ball SG, Varadi G, Peers C. Modulation of recombinant human cardiac L-type Ca2+ channel α1C subunits by redox agents and hypoxia. J. Physiol. 1999; 514: 629-37.
20. Lacampagne A, Duittoz A, Bolanos P, Peineau N, Argibay JA. Effect of sulfhydryl oxidation on ionic and gating currents associated with L-type calcium channels in isolated guinea-pig ventricular myocytes. Cardiovasc. Res. 1995; 30: 799-806.
21. Nelson MT, Joksovic PM, Perez-Reyes E, Todorovic SM. The endogenous redox agent L-cysteine induces T-type Ca2+ channel-dependent sensitization of a novel subpopulation of rat peripheral nociceptors. J. Neurosci. 2005; 25: 8766-75.
22. Todorovic SM, Jevtovic-Todorovic V, Meyenburg A, Mennerick S, Perez-Reyes E, Romano C, Olney JW, Zorumski CF. Redox modulation of T-type calcium channels in rat peripheral nociceptors. Neuron. 2001; 31: 75-85.
23. Yamaoka K, Yakehiro M, Yuki T, Fujii H, Seyama I. Effect of sulfhydryl reagents on the regulatory system of the L-type Ca2+ channel in frog ventricular myocytes. Pflügers Arch. 2000; 440: 207-215.
24. Zeng XH, Xia XM, Lingle CJ. Redox-sensitive extracellular gates formed by auxiliary b subunits of calcium-activated potassium channels. Nat. Struct. Biol. 2003; 10: 448-54.
25. Jeroudi MO, Hartley CJ, Bolli R. Myocardial reperfusion injury: role of oxygen radicals and potential therapy with antioxidants. Am. J. Cardiol. 1994; 73: 2B-7B.
26. Sanguinetti MC. When the KChIPs are down. Nature Med. 2002; 8: 18-19.
27. Keating MT, Sanguinetti MC. Molecular and cellular mechanisms of cardiac arrhythmias. Cell. 2001; 104: 569-80.
28. Aon MA, Cortassa S, Marban E, O'Rourke B. Synchronized whole cell oscillations in mitochondrial metabolism triggered by a local release of reactive oxygen species in cardiac myocytes. J. Biol. Chem. 2003; 278: 44735-44.
29. Akar FG, Aon MA, Tomaselli GF, O'Rourke B. The mitochondrial origin of postischemic arrhythmias. J. Clin. Invest. 2005; 115: 3527-35.
30. Tanskanen AJ, Greenstein JL, O'Rourke B, Winslow RL. The role of stochastic and modal gating of cardiac L-type Ca2+ channels on early after-depolarizations. Biophys. J. 2005; 88: 85-95.
31. Smani T, Hernandez A, Urena J, Castellano AG, Franco-Obregon A, Ordonez A, Lopez-Barneo J. Reduction of Ca2+ channel activity by hypoxia in human and porcine coronary myocytes. Cardiovasc. Res. 2002; 53: 97-104.
32. Hool LC, Arthur PG. Decreasing cellular hydrogen peroxide with catalase mimics the effects of hypoxia on the sensitivity of the L-type Ca2+ channel to beta-adrenergic receptor stimulation in cardiac myocytes. Circ. Res. 2002; 91: 601-9.
33. Hool LC, Di Maria CA, Viola HM, Arthur PG. Role of NAD(P)H oxidase in the regulation of cardiac L-type Ca2+ channel function during acute hypoxia. Cardiovasc Res 2005; 67: 624-35.
34. Fearon IM, Palmer AC, Balmforth AJ, Ball SG, Mikala G, Schwartz A, Peers C. Hypoxia inhibits the recombinant α1C subunit of the human cardiac L-type Ca2+ channel. J. Physiol. 1997; 500: 551-6.
35. Fearon IM, Varadi G, Koch SE, Isaacsohn I, Ball SG, Peers C. Splice variants reveal the region involved in oxygen sensing by recombinant human L-type Ca2+ channels. Circ. Res. 2000; 87: 537-9.
36. Michelakis ED, Rebeyka I, Wu X, Nsair A, Thebaud B, Hashimoto K, Dyck JR, Haromy A, Harry G, Barr A, et al. O2 sensing in the human ductus arteriosus: regulation of voltage-gated K+ channels in smooth muscle cells by a mitochondrial redox sensor. Circ. Res. 2002; 91: 478-86.
37. Weir EK, Archer SL. The mechanism of acute hypoxic pulmonary vasoconstriction: the tale of two channels. FASEB Journal. 1995; 9: 183-9.
38. Weir EK, Hong Z, Porter VA, Reeve HL. Redox signaling in oxygen sensing by vessels. Resp. Physiol. Neurobiol. 2002; 132: 121-30.
39. Budinger GR, Duranteau J, Chandel NS, Schumacker PT. Hibernation during hypoxia in cardiomyocytes. Role of mitochondria as the O2 sensor. J Biol. Chem. 1998; 273: 3320-6.
40. Waypa GB, Guzy R, Mungai PT, Mack MM, Marks JD, Roe MW, Schumacker PT. Increases in mitochondrial reactive oxygen species trigger hypoxia-induced calcium responses in pulmonary artery smooth muscle cells. Circ. Res. 2006; 99: 970-8.
41. Ganitkevich V, Reil S, Schwethelm B, Schroeter T, Benndorf K. Dynamic responses of single cardiomyocytes to graded ischemia studied by oxygen clamp in on-chip picochambers. Circ. Res. 2006; 99: 165-71.
42. Carmeliet E. Cardiac ionic currents and acute ischemia: from channels to arrhythmias. Physiol. Rev. 1999, 79: 917-1017.
43. McDonald TF, Pelzer S, Trautwein W, Pelzer DJ. Regulation and modulation of calcium channels in cardiac, skeletal, and smooth muscle cells. Physiol. Rev. 1994; 74: 365-507.
44. Sims C, Harvey RD. Redox modulation of basal and β-adrenergically stimulated cardiac L-type Ca2+ channel activity by phenylarsine oxide. Br. J. Pharmacol. 2004; 142: 797-807.
45. Humphries KM, Juliano C, Taylor SS. Regulation of cAMP-dependent protein kinase activity by glutathionylation. J. Biol. Chem. 2002; 277: 43505-11.
46. Humphries KM, Deal MS, Taylor SS. Enhanced dephosphorylation of cAMP-dependent protein kinase by oxidation and thiol modification. J. Biol. Chem. 2005; 280: 2750-8.
47. Brennan JP, Bardswell SC, Burgoyne JR, Fuller W, Schroder E, Wait R, Begum S, Kentish JC, Eaton P. Oxidant-induced activation of type I protein kinase A is mediated by RI subunit interprotein disulfide bond formation. J. Biol. Chem. 2006; 281: 21827-36.
48. Hudasek K, Brown ST, Fearon IM. H2O2 regulates recombinant Ca2+ channel α1C subunits but does not mediate their sensitivity to acute hypoxia. Biochem. Biophys. Res. Commun. 2004; 318: 135-41.
49. Brookes PS, Yoon Y, Robotham JL, Anders MW, Sheu SS. Calcium, ATP, and ROS: a mitochondrial love-hate triangle. Am. J. Physiol. Cell Physiol. 2004; 287: C817-33.
50. Zorov DB, Filburn CR, Klotz LO, Zweier JL, Sollott SJ. Reactive oxygen species (ROS)-induced ROS release: a new phenomenon accompanying induction of the mitochondrial permeability transition in cardiac myocytes. J. Exper. Med. 2000; 192: 1001-14.
51. McIntosh MA, Cobbe SM, Smith GL. Heterogeneous changes in action potential and intracellular Ca2+ in left ventricular myocyte sub-types from rabbits with heart failure. Cardiovasc. Res. 2000; 45: 397-409.
52. Napoli C, Sica V, Pignalosa O, de Nigris F. New trends in anti-atherosclerotic agents. Curr. Med. Chem. 2005; 12: 1755-72.
53. Schwartz CJ, Valente AJ, Sprague EA, Kelley JL, Cayatte AJ, Mowery J. Atherosclerosis. Potential targets for stabilization and regression. Circulation 1992; 86: III117-23.
54. Pizzetti G, Mailhac A, Li Volsi L, Di Marco F, Lu C, Margonato A, Chierchia SL. Beneficial effects of diltiazem during myocardial reperfusion: a randomized trial in acute myocardial infarction. Ital. Heart J. 2001; 2: 757-65.
55. Bestehorn HP, Neumann FJ, Buttner HJ, Betz P, Sturzenhofecker P, von Hodenberg E, Verdun A, Levai L, Monassier JP, Roskamm H. Evaluation of the effect of oral verapamil on clinical outcome and angiographic restenosis after percutaneous coronary intervention: the randomized, double-blind, placebo-controlled, multicenter Verapamil Slow-Release for Prevention of Cardiovascular Events After Angioplasty (VESPA) Trial. J. Am. Coll. Cardiol. 2004; 43: 2160-5.
56. Umemoto S, Tanaka M, Kawahara S, Kubo M, Umeji K, Hashimoto R, Matsuzaki M. Calcium antagonist reduces oxidative stress by upregulating Cu/Zn superoxide dismutase in stroke-prone spontaneously hypertensive rats. Hypertens. Res. 2004; 27: 877-85.
57. Wang QD, Pernow J, Sjoquist PO, Ryden L. Pharmacological possibilities for protection against myocardial reperfusion injury. Cardiovasc. Res. 2002; 55: 25-37.
58. Kloner RA, Jennings RB. Consequences of brief ischemia: stunning, preconditioning, and their clinical implications: part 1. Circulation 2001; 104: 2981-9.