Hypoxic regulation of cardiac Ca2+ channel: possible role of haem oxygenase

Abstract

Acute and chronic hypoxias are common cardiac diseases that lead often to arrhythmia and impaired contractility. At the cellular level it is unclear whether the suppression of cardiac Ca(2+) channels (Ca(V)1.2) results directly from oxygen deprivation on the channel protein or is mediated by intermediary proteins affecting the channel. To address this question we measured the early effects of hypoxia (5-60 s, P(O(2)) < 5 mmHg) on Ca(2+) current (I(Ca)) and tested the involvement of protein kinase A (PKA) phosphorylation, Ca(2+)/calmodulin-mediated signalling and the haem oxygenase (HO) pathway in the hypoxic regulation of Ca(V)1.2 in rat and cat ventricular myocytes and HEK-293 cells. Hypoxic suppression of ICa) and Ca(2+) transients was significant within 5 s and intensified in the following 50 s, and was reversible. Phosphorylation by cAMP or the phosphatase inhibitor okadaic acid desensitized I(Ca) to hypoxia, while PKA inhibition by H-89 restored the sensitivity of I(Ca) to hypoxia. This phosphorylation effect was specific to Ca(2+), but not Ba(2+) or Na(+), permeating through the channel. CaMKII inhibitory peptide and Bay K8644 reversed the phosphorylation-induced desensitization to hypoxia. Mutation of CAM/CaMKII-binding motifs of the α(1c) subunit of Ca(V)1.2 fully desensitized the Ca(2+) channel to hypoxia. Rapid application of HO inhibitors (zinc protoporphyrin (ZnPP) and tin protoporphyrin (SnPP)) suppressed the channel in a manner similar to acute hypoxia such that: (1) I(Ca) and I(Ba) were suppressed within 5 s of ZnPP application; (2) PKA activation and CaMKII inhibitors desensitized I(Ca), but not I(Ba), to ZnPP; and (3) hypoxia failed to further suppress I(Ca) and I(Ba) in ZnPP-treated myocytes. We propose that the binding of HO to the CaM/CaMKII-specific motifs on Ca(2+) channel may mediate the rapid response of the channel to hypoxia.

Figure 2. The suppression of I_Ca by hypoxia is accompanied by a similar suppression of the I_Ca-triggered intracellular Ca²⁺ transients

A, simultaneous measurements of I_Ca and Ca²⁺ transients measured with 0.4 mm K₄Fura-2 in the patch pipette in non-cAMP dialysed rat cardiomyocytes before (O₂) and after 40 s of hypoxia (N₂). B, time course of suppression I_Ca and Ca²⁺ transients during exposure to hypoxic solution.

Figure 1. Suppression of current through the L-type Ca²⁺ channel during brief periods of hypoxia

A, time course of hypoxia-induced changes in the peak inward current in rat cardiomyocytes where the permeating ion was Ca²⁺ (I_Ca), Ba²⁺ (I_Ba) or Na⁺ (I_Na) and in HEK cells where the recombinant channel conducted Ca²⁺ (I_Ca). B, current–voltage relations for I_Ca in rat ventricular cardiomyocytes measured under normoxic control conditions (O₂) and after 1–2 min exposure to hypoxia (N₂). C, average suppression of peak inward current measured with different permeating ions (Ca²⁺ vs. Ba²⁺ vs. Na⁺) and preparations (rat vs. cat cardiomyocytes vs. HEK-cell expression system) after 45–60 s superfusion with solutions equilibrated with N₂, He or O₂. The inset panels show that currents activated by depolarization from −60 to +10 mV displayed different rates of inactivation, but similar degrees of suppression during hypoxia (I_Ca vs. I_Ba in rat cardiomyocytes vs. I_Ca in HEK cells). All illustrated experiments were conducted in the absence of cAMP or other interventions that might evoke cAMP-dependent phosphorylation. The experiments with cat cardiomyocytes were performed at 30–32°C, the rest at room temperature, 23–25°C.

Figure 3. Phosphorylation of the Ca²⁺ channel makes it insensitive to hypoxia when the permeating ion is Ca²⁺ (I_Ca), but not when it is Ba²⁺ (I_Ba) or Na⁺ (I_Na)

A, time course of I_Ca and I_Ba during 40 s hypoxia (N₂) in rat ventricular myocytes dialysed with 0.2 mm cAMP. B, the I–V relation is insensitive to hypoxia even when SR Ca²⁺ stores are depleted by pre-incubation with 1 μm thapsigargin (Thap). The inset panel shows sample currents recorded with depolarization to +10 mV. C, bar graph showing the reduction in the currents through the Ca²⁺ channel during 45–60 s of hypoxia in cells dialysed with 0.2 mm cAMP or incubated with 1 μm of the phosphatase inhibitor okadaic acid (OA). Results obtained with the PKA inhibitor H-89 and thapsigargin (Thap) are included. Results obtained under non-phosphorylating conditions are reproduced for comparison from Fig. 1C as hatched bars. All illustrated results were obtained using rat ventricular cells and hypoxic solutions equilibrated with 100% N₂.

Figure 4. Inhibition of Ca²⁺-dependent inactivation of I_Ca by calmodulin domain peptide_290−309 and Bay K is associated with enhanced suppression of I_Ca by anoxia

A, putative CaM-binding (IQ and hydrophobic 1–5–10 motifs) and phosphorylation sites (grey highlight) in the partial AA sequences of CaMKII, peptide_290−309 (PEP), wild-type and mutant α_1c subunits of the Ca²⁺ channel (α_1c77 and α_1c86) and haem oxygenase (HO-2) (Colbran, 1993; Rhoads & Friedberg, 1997; Soldatov et al. 1997; Boehning et al. 2004). B, time course of suppression of I_Ca upon removal of extracellular O₂ in cAMP-dialysed rat cardiomyocytes that in addition were dialysed with 100 μm PEP, treated with 1 μm Bay K8644 (BayK) or both. For comparison purposes, I_Ca in cardiac control cells dialysed with 0.2 mm of cAMP is reproduced from Fig. 3A (dashed line). C, comparison of the anoxic suppressions of I_Ca in cardiomyocytes measured after 60–90 s of hypoxia (during recording of I–V relations). A, amino acid sequences of α_1c77 and α_1c86 subunits.

Figure 5. The cytoplasmic CaM/CaMKII binding sites of the α_1C subunit of the Ca²⁺ channel is required for its hypoxia-induced suppression

The measured Ca²⁺ and Ba²⁺ currents (I_Ca and I_Ba) show that Ca²⁺-dependent inactivation (A) and anoxic suppression (C) are present in HEK-293 cell expressing the α_1c77 of the L-type Ca²⁺ channel, but not in cells expressing the mutant α_1c86 subunit lacking CaM/CAMKII binding sites (B and D; cf. Fig. 4B). The currents in panels A and B were normalized to emphasize differences and similarities in kinetics. Extracellular Ba²⁺ at 20 mm was used to enhance the currents shown in panels C and D. All measurements were done under non-phosphorylating conditions.

Figure 6. Suppression the Ca²⁺ channel by inhibition of haem oxygenase

A, brief exposures to the haem oxygenase inhibitor ZnPP in concentrations of 10 and 100 nm cause rapid, reversible, dose-dependent suppression of I_Ca. B, effect of 10 nm ZnCl₂ or 100 nm ZnPP on I_Ca. Insets in panels A and B show traces of I_Ca measured at the indicated times (a, b and c). C and D, normalized I–V relations of I_Ba measured in control cells (Control, open circles) and in cells exposed to 100 nm ZnPP (C,filled triangles) that was applied either in the external solution (C) or via the patch pipette by 9 min of dialysis (D). All experiments were performed on rat ventricular cardiomyocytes under normoxic and non-phosphorylating conditions.

Figure 7. Inhibition of the Ca²⁺ channel in rat cardiomyocytes by haem oxygenase inhibitors with respect to phosphorylation, permeating ion and hypoxia

A and B, effect of 100 nm ZnPP on I_Ca (A) and I_Ba (B) measured in the absence of phosphorylating interventions (no cAMP, filled circles) and with dialysis of either 0.2 mm cAMP (cAMP, open circles) or 60 nm okadaic acid (OA, open triangles). C, pretreatment with an inhibitor of CaMKII (10 μm CK59) restored the suppressive effect (double arrow) of 100 nm ZnPP on I_Ca in cardiomyocytes dialysed with 0.2 mm cAMP. Notice that in this, as in all our experiments, the suppression of I_Ca was quantified based on a normalization that yielded 100% immediately before the exposure to hypoxia or ZnPP. D, hypoxia (N₂) had no additional suppressive effects after I_Ba had been suppressed by 1 μm ZnPP. E, summary of the quantitative effects of 100 nm ZnPP on I_Ca and I_Ba, as measured in panels A–C. This bar graph has the same layout as used for hypoxic effects in Fig. 3C, but shows data obtained with a different protein kinase inhibitor (CK59 vs. H-89).

Figure 8. Proposed model for modulation of L-type Ca²⁺ channel by oxygen

The carboxy-tail of the α_1C subunit includes LA and IQ motifs for calmodulin- (CaM) mediated interactions and a binding site for protein phosphatase 2a (PP2a). CaM is shown as also interacting with CaMKII, haem oxygenase-2 (HO-2) and inhibitory peptide_290−309 (PEP). The primary inputs to the system are thought to be: cAMP leading to PKA-mediated phosphorylation (left), permeation of Ca²⁺ through the channel to a restricted space (middle), and molecular oxygen (O₂) that binds to haem/HO. PKA may act directly or via CaMKII and may have multiple phoshporylation targets (including HO-2). Similarly okadaic acid (OA) may promote phosphorylation inhibiting different phosphatases. ROS generation by mitochondria or membrane bound NOX proteins, CO-mediated signalling and I_Ca-triggered release of Ca²⁺ from the SR are thought to be of little or no importance for the rapid suppression of the Ca²⁺ channel at the onset of hypoxia.