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Cardiovascular Research 2005 67(4):578-580; doi:10.1016/j.cardiores.2005.06.010
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Copyright © 2005, European Society of Cardiology

Hypoxic modulation of cardiac L-type Ca2+ current: Interaction of reactive oxygen species and β-adrenergic signaling

Robert T. Mallet*

Department of Integrative Physiology, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, Texas 76107-2699, USA

* Tel.: +1 817 735 2260; fax: +1 817 735 5084. Email address: malletr{at}hsc.unt.edu

Received 16 June 2005; accepted 17 June 2005

KEYWORDS calcium channels; hypoxia; isoproterenol; mitochondria; superoxide

See article by Hool et al. [5] (pages 624–635) in this issue.


   1. Introduction
Top
 1. Introduction
2. Redox modulation of...
 3. Sources of {middle...
 References
 
Sarcolemmal L-type Ca2+ channels are integral components of the excitation–contraction coupling mechanism in cardiomyocytes. Membrane depolarization to –30 mV opens these channels [1], allowing rapid influx of Ca2+ which peaks within 2–3 ms [2]. This inward Ca2+ current (ICa) produces the plateau phase of the cardiomyocyte action potential and triggers a massive release of Ca2+ from the sarcoplasmic reticulum via ryanodine-sensitive Ca2+ channels, arrayed in close proximity to the L-type channels. The resultant cytosolic Ca2+ transient activates crossbridge cycling and mechanical force production by the contractile machinery and inactivates the L-type Ca2+ current [2]. Cardiac L-type channels contain four subunits ({alpha}1C, {alpha}2, β2, {delta}) [1,2]. Each of the four homologous domains of the large {alpha}1C subunit harbor six membrane-spanning {alpha} helices; helices 5 and 6 of the four {alpha}1C domains combine to form a Ca2+ pore [2].

L-type Ca2+ current is physiologically regulated by protein kinases that covalently modify the {alpha}1C subunit to modulate intrinsic channel gating properties. β-Adrenergic activity increases ICa via protein kinase A phosphorylation of Ser-1928 of {alpha}1C [2] (Fig. 1), which shifts gating behavior of the channels from mode 0 (rarely or never open) to modes 1 (low open probability; channels open only briefly) and 2 (high open probability; prolonged opening of channels) [1,3]. This mechanism contributes importantly to β-adrenergic enhancement of myocardial contractile performance. Protein kinase C may phosphorylate Thr-27 and/or Thr-31 near the amino terminus of {alpha}1C, producing either a monophasic decrease or a transient increase followed by a decrease in ICa [1].


Figure 1
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  		Fig. 1 Modulation of sarcolemmal Ca2+ current by β-adrenergic stimulation and reactive oxygen intermediates. The β-adrenergic signaling cascade activates sarcolemmal Ca2+ current (ICa) by phosphorylating ser-1928 of the L-type Ca2+ channel's pore-forming {alpha}1C subunit. Reactive oxygen intermediates modulate channel activity by altering the reduction state of cysteinyl sulfhydryls (SH) on the cytosolic and extracellular (EC) sides of {alpha}1C. Oxidation of extracellular SH increases basal ICa; oxidation of intracellular SH blunts β-adrenergic activation of ICa. Hypoxia attenuates formation of these intermediates by decreasing intracellular O2 concentration. Work by Hool et al. [5,6,9] supports the pathways indicated by dashed lines. Proposed pathways involving S-nitrosoglutathione (GSNO) are indicated by dotted lines. AC: adenylate cyclase; β-AR: sarcolemmal β-adrenergic receptor; cAMP: cyclic AMP; eNOS: nitric oxide synthase (endothelial isoform); GSH: glutathione; ISO: isoproterenol; NO: nitric oxide; ONOO–: peroxynitrite; PKA, PKB: protein kinases A and B; Q: ubiquinone; .Q: ubisemiquinone; SL: sarcolemma. I, III and IV indicate mitochondrial respiratory complexes.

 
Protein kinase G, activated by the nitric oxide/guanylate cyclase/cyclic GMP cascade, exerts complex effects on ICa: direct phosphorylation of the L-type channel by this kinase activates ICa, but the kinase also suppresses β-adrenergic activation of ICa [2,4] by activating protein phosphatase, which dephosphorylates ser-1928 of {alpha}1C, and phosphodiesterase 2, which degrades cyclic AMP [1].


   2. Redox modulation of ICa and hypoxia–β-adrenergic interaction
Top
 1. Introduction
 2. Redox modulation of...
3. Sources of {middle...
 References
 
In this issue of Cardiovascular Research, Hool et al. report an investigation of redox modulation of Ca2+ currents in guinea-pig ventricular cardiomyocytes subjected to hypoxia and β-adrenergic stimulation [5]. These investigators [6] and Fearon et al. [7] previously demonstrated that hypoxia directly suppressed basal ICa. On the other hand, hypoxia enhanced the β-adrenergic sensitivity of the L-type channels; thus, hypoxia augmented ICa activation by submaximal isoproterenol [6]. The membrane-impermeable, sulfhydryl-selective oxidant 5,5'-dithio-bis(2-nitrobenzoic acid) prevented hypoxic attenuation of basal ICa, but did not interfere with hypoxic enhancement of ICa activation by isoproterenol [6]. The membrane-permeable sulfhydryl reductant 1,4-dithiothreitol mimicked both hypoxic attenuation of basal ICa and its sensitization of the channel to isoproterenol [6]. Fearon et al. [7] reported that the extracellular oxidants thimerosal and PCMBS prevented hypoxic attenuation of ICa in cultured cells that stably express {alpha}1C subunits of human L-type channels. The sulfhydryl oxidants 2,2'-dithiodipyridine and thimerosal dampened ICa in cultured cells that stably express {alpha}1 subunits from rabbit lung [3], and the organomercurial sulfhydryl oxidant p-hydroxy-mercuric-phenylsulfonic acid similarly blunted ICa response in guinea-pig ventricular cardiomyocytes [8]. These results implicated the {alpha}1C subunit as the target of hypoxia and, taken with Hool's report [6], indicate that hypoxia dampens basal ICa by reducing extracellular sulfhydryls, while sensitizing the channels to isoproterenol by reducing intracellular sulfhydryls on {alpha}1C or its accessory proteins.

Extracellular application of the H2O2-scavenging enzyme catalase dampened basal ICa, but introduction of the enzyme to the intracellular milieu by dialysis sensitized the Ca2+ channels to isoproterenol [9]. Glutathione peroxidase, the principal intracellular H2O2-scavenging enzyme in cardiomyocytes, also enhanced ICa activation by isoproterenol. These results supported a scenario in which endogenous H2O2 maintains basal ICa while restraining the β-adrenergic sensitivity of the Ca2+ current by oxidizing extracellular and intracellular sulfhydryls, respectively, and hypoxia modulates channel activity by suppressing H2O2 formation.


   3. Sources of ·O2– and H2O2 that dampen β-adrenergic activation of ICa
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 1. Introduction
 2. Redox modulation of...
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Hydrogen peroxide is generated in cardiomyocytes by univalent reduction of its precursor, the free radical superoxide anion (·O2–), by superoxide dismutase. Several cellular processes, including mitochondrial respiration, NAD(P)H oxidase, xanthine oxidase and cyclooxygenase can generate ·O2–. Hool et al. [5] sought to determine which of these ·O2– sources was blunted by hypoxia (PO2 15–17 mm Hg) to enhance ICa activation by isoproterenol. The impact of hypoxia on basal and isoproterenol-stimulated Ca2+ currents, measured by the whole-cell patch-clamp technique, was studied in the absence and presence of inhibitors of NAD(P)H oxidase and mitochondrial respiration, the two principal sources of ·O2– in oxygenated cardiomyocytes. Apocynin, a selective NAD(P)H oxidase inhibitor [10], only tended to increase β-adrenergic sensitivity of ICa, but diphenyleneiodonium (DPI), a general inhibitor of flavin-containing oxidases including NAD(P)H oxidase [11] and mitochondrial respiratory complex I [12], proved as effective as hypoxia at augmenting isoproterenol activation of ICa. Hypoxia and DPI similarly attenuated ·O2– formation in mitochondria isolated from the cardiomyocytes. The role of the mitochondria in generating hypoxia-sensitive ·O2– was interrogated further with myxothiazol, an inhibitor of respiratory complex III [13,14]. Myxothiazol dampened ·O2– formation and, like hypoxia, enhanced ICa activation by submaximal isoproterenol.

This report has several implications. The mitochondrial respiratory chain, particularly its ubisemiquinone site [13,15], is likely the principal source of reactive oxygen species that modulate β-adrenergic activation of the L-type Ca2+ channels. Indeed, NAD(P)H oxidase and other sources of ·O2– appear to be at most only minor contributors to redox modulation of ICa. In light of DPI's lack of specificity for NAD(P)H oxidase, results of experimentation with this inhibitor should be interpreted cautiously. The present results suggest a mechanism for the enhancement of β-adrenergic inotropism by pharmacological and metabolic antioxidants in ischemically stunned guinea-pig myocardium [16], if the antioxidants increased the reduction state of intracellular {alpha}1C sulfhydryls.

Although Hool et al.'s findings support the proposed actions of H2O2 on the L-type channels [5], mechanisms mediated by S-nitroso-glutathione (GSNO) could impact the channels, too. Peroxynitrite (ONOO–), produced by the irreversible condensation of ·O2– and nitric oxide, reacts with glutathione to generate GSNO [17]. The latter compound inactivates ICa by nitrosating sulfhydryls on the intracellular side of {alpha}1C [18], which decreases channel open probability. Moreover, H2O2 activates protein kinase B/Akt [11], which in turn phosphorylates and activates the constitutive endothelial nitric oxide synthase [19], the principal constitutive source of NO in myocardium. Accordingly, decreased ·O2– and H2O2 during hypoxia could decrease GSNO formation (Fig. 1). Decreased extracellular GSNO content could contribute to hypoxic dampening of basal ICa if the latter process involves extracellular sulfhydryls; indeed, extracellular GSNO activated ICa [20]. Moreover, hypoxia could enhance β-adrenergic activation of ICa by decreasing intracellular GSNO, thereby ameliorating S-nitrosylation of intracellular sulfhydryls.


   Acknowledgements
 
This work was supported by a grant from the U.S. National Heart, Lung and Blood Institute (HL-071684).


   References
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 1. Introduction
 2. Redox modulation of...
 3. Sources of {middle...
 References
 

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