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Plasma membrane K_ATP channel-mediated cardioprotection involves posthypoxic reductions in calcium overload and contractile dysfunction: mechanistic insights into cardioplegia

István Baczkó^*, Lynn Jones^*, Claire F. McGuigan^*, Jocelyn E. Manning Fox^*, Manoj Gandhi^*, Wayne R. Giles^,, Alexander S. Clanachan^* and Peter E. Light^*,1

^* Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada;
Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada; and
Department of Bioengineering, University of California San Diego, La Jolla, California, USA

¹ Correspondence: Department of Pharmacology, University of Alberta, 9-58 Medical Sciences, Edmonton, Alberta T6G 2H7, Canada. E-mail: peter.light{at}ualberta.ca

SPECIFIC AIMS

Activation of plasma membrane (pm) K_ATP channels in cardiac myocytes has been shown to be cardioprotective under hypoxic or ischemic conditions.

A major clinical correlate of ischemia/reperfusion injury is contractile dysfunction in which Ca²⁺ overload via increased Na⁺/Ca²⁺ exchange (NCX) is a major contributor. Our recent data suggest that a major function of pmK_ATP channels is to favor a polarized membrane potential and thereby reduce reverse mode NCX-mediated Ca²⁺ overload during metabolic stress such as ischemia/reperfusion or intense cardiac work, thus ameliorating cellular injury.

We investigated the relationship between pmK_ATP activation, reverse mode NCX activity, intracellular Ca²⁺ transients, and contractile dysfunction during recovery after metabolic inhibition (MI) in field-stimulated rat ventricular myocytes. We then investigated the implications of our initial findings in experimental cardioplegia using hyperkalemic-depolarizing and hypokalemic-polarizing solutions in single-cell and whole heart models.

PRINCIPAL FINDINGS

1. Activation of pmK_ATP and inhibition of the NCX exchanger reduce the duration of post-MI contractile dysfunction in rat ventricular myocytes
Reoxygenation of ventricular myocytes subjected to 6 min MI resulted in a transient decrease in contractile amplitude during early reoxygenation. Myocytes reoxygenated with the pmK_ATP opener P-1075 (5 µM), the NCX inhibitor KB-R7943 (5 µM), or both exhibited a similar transient decrease in contractile amplitude (Fig. 1 ). However, the duration of the decrease in contractile amplitude was significantly less in the P-1075 and KB-R7943 groups than with control (Fig. 1E ). The pmK_ATP inhibitor HMR 1098 (20 µM) blocked the effect of 5 µM P-1075 on dysfunction (Fig. 1E ). HMR 1098 alone did not increase the duration of dysfunction (Fig. 1E ), but caused a significant reduction in contractile recovery at the end of the reoxygenation period (Fig. 1F ). The myocyte cell length, fractional cell shortening, and ± dL/dt values were not significantly different among groups at basal conditions. Fractional cell shortening and its first derivative were reduced (±dL/dt) in all groups at 2 min reoxygenation. However, fractional cell shortening, the maximum rate of shortening (+dL/dt), and maximum rate of relaxation (–dL/dt) were significantly higher in groups superfused with P-1075 (5 µM), KB-R7943 (5 µM), or their combination, indicating improved contractility and relaxation at early stages of reoxygenation. In the HMR 1098 group, fractional cell shortening and ± dL/dt values were similar to those of the control group but significantly lower than those in the P-1075 group at 2 min reoxygenation.

View larger version (38K): [in this window] [in a new window]   Figure 1. Representative cell shortening recordings from field-stimulated (1 Hz) rat ventricular myocytes subjected to 2 min of superfusion with control solution (Basal), 6 min metabolic inhibition (MI), and 10 min reoxygenation with control solution containing: A) vehicle, B) KB-R7943 (5 µM), C) P-1075 (5 µM), D) P-1075 (5 µM) + KB-R7943 (5 µM). Grouped data show that inhibition of the NCX (KB-R7943) and activation of pmKATP channels (P-1075) decrease the duration of post-MI contractile dysfunction (E). Significant impairment of cell shortening was observed in cardiac myocytes superfused with the pmKATP channel blocker HMR 1098 at the end of recovery (F). L (%): % change in cell length. Arrows: duration of post-MI contractile dysfunction. Horizontal bar: 1 min interval. The duration of dysfunction was defined as the period when the cell exhibited <50% of basal fractional cell-shortening value in reoxygenation. I = 7–10 cells/group, **P < 0.01, ***P < 0.001 vs. control group.

2. Functional pmK_ATP channels are needed for improved recovery during early reoxygenation
To confirm the pharmacological evidence for pmK_ATP channel-mediated protection, we used a dominant negative adenoviral construct of the pmK_ATP channel subunit Kir6.2, AdVKir6.2(AAA), to functionally silence endogenous pmK_ATP channel activity [coexpressing green fluorescent protein, (GFP)]. Single-cell contractility experiments were performed on myocytes infected in vivo with AdVKir6.2(AAA) 96 h prior to enzymatic dissociation. GFP-tagged ventricular myocytes were then subjected to the MI/reoxygenation protocol. There was no difference in basal cell length and fractional cell shortening between AdVKir6.2(AAA) and control groups. However, after reoxygenation-induced contractile dysfunction, myocytes expressing the Kir6.2(AAA) construct failed to recover. In the presence of nonfunctional pmK_ATP channels, P-1075 (5 µM) failed to alleviate contractile dysfunction during early reoxygenation, confirming the critical role pmK_ATP channels play in posthypoxic functional recovery.

3. Reduction of post-MI diastolic [Ca²⁺]_i after pmK_ATP activation and inhibition of the NCX: possible mechanism for amelioration of contractile dysfunction
Reverse mode NCX activity plays an important role in the development of diastolic Ca²⁺ overload. Therefore, we determined the effects of pmK_ATP channel activation and NCX inhibition on dynamic changes in intracellular calcium. Field-stimulated ventricular myocytes loaded with calcium green-1AM were subjected to the MI/reoxygenation protocol; Ca²⁺ transients were continuously recorded. Reoxygenation resulted in an increase in diastolic [Ca²⁺]_i, followed by a recovery to basal values. Application of KB-R7943 (5 µM), P-1075 (5 µM), and their combination in the reoxygenation solution significantly reduced elevations in [Ca²⁺]_i. HMR 1098 prevented the beneficial effects of P-1075 on diastolic [Ca²⁺]_i but did not cause a further increase when applied alone. The time course of changes in diastolic [Ca²⁺]_i and development of contractile dysfunction were similar, suggesting a close relationship between diastolic Ca²⁺ levels and myocardial function in reoxygenation after metabolic inhibition. There were no significant differences in the duration of the calcium transient at 90% recovery level, time to peak, or amplitude of the Ca²⁺ transient in any of the experimental groups in basal conditions and during reoxygenation. These results suggest increases in diastolic [Ca²⁺]_i and not alterations in calcium handling are responsible for the development of posthypoxic contractile dysfunction.

4. Diastolic resting membrane potential of ventricular myocytes is critical in recovery of function and calcium homeostasis during simulated cardioplegia
Our data suggest that maneuvers designed to hyperpolarize the resting membrane potential of cardiomyocytes may afford protection from ischemia/reperfusion injury in the setting of cardioplegia. We compared the contractility of cardiac myocytes subjected to cardioplegia with modified depolarizing St. Thomas’ solution (STC, containing 16 mM K⁺) and a hyperpolarizing cardioplegia solution (HPC, containing 3.2 mM K⁺ + P-1075). During superfusion with STC, large contractions were observed but not in the HPC group (Fig. 2 A, B). The improved recovery of function (L and dL/dt) in the HPC group compared with the STC group is illustrated on Fig. 2A, B . Fractional cell shortening was significantly better at 6 and 12 min of recovery after cardioplegia in the HPC group compared with STC (Fig. 2C ). Diastolic cell length was reduced in the STC group and increased in the HPC group compared with basal values. Diastolic cell length was shorter in the STC group compared with HPC during cardioplegia + MI and, unlike the HPC group, did not return to basal cell length (Fig. 2D ). Calcium transients were recorded from another two groups of ventricular myocytes subjected to the cardioplegia protocol with STC or HPC. Depolarizing cardioplegia and subsequent MI resulted in a significantly elevated diastolic [Ca²⁺]_i during recovery; recovery after HPC cardioplegia did not alter diastolic [Ca²⁺]_i. These data suggest a crucial role for resting membrane potential in the modulation of diastolic [Ca²⁺]_i and cardiomyocyte function during recovery after cardioplegia.

View larger version (30K): [in this window] [in a new window]   Figure 2. Representative cell shortening recordings from field-stimulated rat ventricular myocytes superfused with A) depolarizing St. Thomas’ cardioplegia (STC), MI, and reoxygenation solutions; and (B) hyperpolarizing cardioplegia (HPC), MI, and reoxygenation solutions. Myocytes in both groups were superfused with control solution during recovery. Cardiomyocytes in the STC group exhibited occasional large contractions during superfusion with the cardioplegia solution, not observed in the HPC group. Note the improved post-MI contractile recovery in the HPC group (B) vs. the reduced contractility and rate of relaxation in the STC group (A) as demonstrated by individual cell shortening, rate of cell shortening traces (averaged for 10 steady-state beats), and C) grouped data. D) Diastolic cell length was significantly shorter in the STC group during cardioplegia + MI and at the end of the 12 min recovery periods. In the HPC group, diastolic cell length significantly increased during cardioplegia + MI and returned to control by the end of recovery. Arrows (A, B) indicate times when cell shortening traces were used to construct individual traces. n = 7 in all groups; *P < 0.05 vs. STC group; # #P < 0.01, # # #P < 0.001 vs. control period in the same group.

5. Hyperpolarizing cardioplegia improves cardiac function in isolated working rat hearts
To determine whether the beneficial effects of the hyperpolarizing cardioplegic solution on Ca²⁺ homeostasis of ventricular myocytes directly translate to improved cardiac recovery, experiments were carried out on isolated rat hearts. Hearts were subjected to 1) 6 h of cold storage in STC cardioplegia solution; 2) 6 h of cold storage in HPC cardioplegia solution; and 3) a group of hearts not subjected to cold cardioplegic storage with a time-matched recovery perfusion protocol (control). Cardiac function of rat hearts subjected to 6 h of STC cardioplegia was impaired compared with the HPC group and the control group.

CONCLUSIONS AND SIGNIFICANCE

We confirm that activation of pmK_ATP channels is cardioprotective in a cellular model of contractility. Our results show that activation of pmK_ATP channels and inhibition of the NCX reduce the duration of contractile dysfunction upon reoxygenation after MI and that this is the result of improved calcium homeostasis in early reoxygenation. Activation of pmK_ATP reduces the increase in diastolic [Ca²⁺]_i during reoxygenation. The mechanism likely involves hyperpolarization of the resting membrane potential and reduction in NCX-mediated elevation of diastolic [Ca²⁺]_i. We propose it is a novel and plausible mechanism by which activation of pmK_ATP offers cardioprotection against reperfusion injury and has direct clinical relevance in the setting of cardioplegia.

View larger version (48K): [in this window] [in a new window]   Figure 3. Proposed model for the cardioprotective effects of pmKATP channel activation via membrane potential-mediated reductions in reverse mode NCX. A) During ischemia/reperfusion, membrane depolarization and elevations in intracellular sodium lead to detrimental calcium overload via increased reverse mode NCX. B) Activation of pmKATP channels and subsequent membrane hyperpolarization during reperfusion leads to a reduction in reverse mode NCX and less calcium overload, resulting in improved calcium homeostasis and cardiac function. KB-R7943: NCX inhibitor; P-1075 and HMR 1098: pmKATP opener and blocker, respectively.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/1096/fj.04-3008fje; doi: 10.1096/fj.04-3008fje

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