Glutathionylation of the L-type Ca2+ channel in oxidative stress-induced pathology of the heart

Abstract

There is mounting evidence to suggest that protein glutathionylation is a key process contributing to the development of pathology. Glutathionylation occurs as a result of posttranslational modification of a protein and involves the addition of a glutathione moiety at cysteine residues. Such modification can occur on a number of proteins, and exerts a variety of functional consequences. The L-type Ca2+ channel has been identified as a glutathionylation target that participates in the development of cardiac pathology. Ca2+ influx via the L-type Ca2+ channel increases production of mitochondrial reactive oxygen species (ROS) in cardiomyocytes during periods of oxidative stress. This induces a persistent increase in channel open probability, and the resulting constitutive increase in Ca2+ influx amplifies the cross-talk between the mitochondria and the channel. Novel strategies utilising targeted peptide delivery to uncouple mitochondrial ROS and Ca2+ flux via the L-type Ca2+ channel following ischemia-reperfusion have delivered promising results, and have proven capable of restoring appropriate mitochondrial function in myocytes and in vivo.

Figure 1

Transient exposure to H₂O₂ leads to L-type Ca²⁺ channel-dependent increase in mitochondrial superoxide, triggering ROS-induced-ROS release. (a) dihydroethidium (DHE) fluorescence recorded from individual guinea pig cardiomyocytes before and after exposure to 30 µM H₂O₂ for 5 min followed by 10 U/mL catalase for 5 min then 2 nM FCCP (an uncoupler of oxidative phosphorylation) as indicated (left panel) or 7 nM myxothiazol (to block electron transport at mitochondrial complex III) (right panel); (b) DHE fluorescence recorded from individual guinea pig cardiomyocytes before and after exposure to 30 µM H₂O₂ for 5 min followed by 10 U/mL catalase for 5 min and the L-type Ca²⁺ channel antagonist nisoldipine (nisol; 2 µM) as indicated (left panel) or ryanodine receptor antagonist dantrolene (20 µM; right panel); and (c) Schematic illustrating the persistent increase in intracellular ROS and intracellular Ca²⁺ following a transient H₂O₂ exposure or ROS-induced ROS-release. Reproduced with permission from [40].

Figure 2

Transient exposure of myocytes to H₂O₂ alters mitochondrial protein synthesis. Exposure of guinea-pig myocytes to 30 µM H₂O₂ for 5 min followed by 10 U/mL catalase for 5 min is sufficient to induce alterations in cellular protein synthesis consistent with the development of myocyte hypertrophy. iTRAQ-facilitated proteomics analysis indicated that the majority of changes in protein synthesis are mitochondrial in origin. For further details see text. Reproduced with permission from [50].

Figure 3

Oxidative stress causes persistent enhancement of the L-type Ca²⁺ channel via ROS-mediated glutathionylation. (a) Time course of changes in membrane current recorded from guinea pig cardiac myocytes during extracellular exposure to 1 mM GSH followed by 2 mM GSSG as indicated; (b) Mean (±SEM) of L-type Ca²⁺ channel current density under control conditions (no drugs) and after exposure to GSH applied either extracellularly or in the patch pipette (GSH pip). NS, not significant; (c) Mean (±SEM) of L-type Ca²⁺ channel current density under control conditions (no drugs) and after exposure to GSSG applied either extracellularly or in the patch pipette (GSSG pip). #, p < 0.05 vs. control; (d) Current-voltage relationship for representative myocytes during voltage steps from −60 to +80 mV and exposure to GSH or GSSG as indicated; (e) Representative single channel currents recorded at −100 mV in the absence and presence of 200 µM DTNB followed by 1 mM DTT and then 2 µM nisoldipine. The open probability (P₀) for each treatment is indicated; (f) Immunoblot demonstrating glutathionylation of protein after probing with anti-GSH antibody (left) and anti-channel antibody (right) in immunoprecipitated Ca_v1.2 channel protein samples from control nonischemic human heart (Con) and ischemic human heart (IHD); (g) Densitometry analysis of immunoblots for glutathionylated protein band normalised to the channel protein in the same lane for Con samples and IHD samples (mean ± SEM); and (h) Concentration of protein-glutathione mixed-disulfides in channel protein from Con and IHD heart samples (mean ± SEM). Reproduced with permission from [74].

Figure 4

Targeted peptide delivery disrupts cross-talk between the L-type Ca²⁺ channel and the mitochondria and reduces ischemia-reperfusion injury. (A) Exposure to AID–TAT attenuates the increase in mitochondrial membrane potential (Ψ_m) associated with activation of the channel. Ψ_m assessed as changes in JC-1 fluorescence recorded from two representative guinea pig myocytes before and after exposure to either 1 µM scrambled AID–TAT (AID–TAT(S)) or 1 µM AID–TAT followed by 45 mM KCl to activate the L-type Ca²⁺ channel. Arrow indicates where KCl was added. Mean ± SEM of changes in JC-1 fluorescence for myocytes exposed to AID–TAT(S) and AID–TAT shown on the right; (B) L-type Ca²⁺ channel current traces activated by voltage step to +10 mV from a holding potential of −30 mV from representative myocytes that were pre-incubated with either AID–TAT(S) or AID–TAT; and (C) GSH/GSSG ratio of guinea pig hearts treated with 1 or 10 µM scrambled AID–TAT (AID(S)–TAT) or active AID–TAT within 5 min after commencement of reperfusion following 30 min of no-flow ischemia. Panels A and B reproduced with permission from [104] and panel C reproduced with permission from [114].