Cardioprotective effect of mitochondrial ATP-dependent potassium channel: the role of metabolic activators

 

Kachaeva E. V.

Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow region, Russia

 

Brief episodes of hypoxia protect myocardium against subsequent ischemia. Opening of mitochondrial ATP-dependent potassium channels causes the same effect. Many synthetic mitoK_ATP openers are studied. We study natural channel activators, which are potent cardioprotectors. Uridine-5’-diphosphate (UDP) activates mitoK_ATP, and 5-hydroxydecanoate (5-HD) eliminates this effect. In experimental animals, UDP precursors, uridine and UMP decreased myocardium ischemic alteration index and T-wave amplitude. Glibenclamide (Glib) and 5-HD inhibited both effects. UMP and uridine significantly decreased number of premature ventricular beats and duration of ventricular tachycardia. Glib and 5-HD inhibited the antiarrhythmic parameters, 5-HD being less effective. Uridine and UMP decreased duration of fibrillation, Glib and 5-HD did not abolish this effect. MitoK_ATP prevents both ischemic injuries and rhythm disorders, and partly fibrillation. Activity of mitochondrial ATP-dependent potassium channel (mitoK_ATP) of rat heart and liver mitochondria was shown to decrease during aging. These facts partially explain increase of ischemia risk at mature age.

 

 

Cardiovascular diseases are a common cause of death in technically developed countries, so the search for principally new ways of their prevention and treatment is a problem of great concern in modern medicine. A development of new approaches based on the data of basic studies obtained lately is important in this perspective. During the last years the role of the activators of ATP-dependent potassium channels of sarcoplasmic and mitochondrial membranes in myocardium protection against ischemic injury has been widely discussed [1]. At present it is generally accepted that it is the mitochondrial channel that plays a special role in the system of anti-ischemic protection of the cell [2]. As was shown firstly in Prof. Garlid lab. the pharmacological activators of the mitochondrial ATP-dependent K⁺ channel (mitoK_ATP) have a clear cardioprotecting properties and prevented ATP disintegration in experimental myocardial ischemia . During the last 3 years in our lab we are trying to find the natural (metabolic) activators of mitoK_ATP .We examined the capacity of diphosphonucleotides to reactivate mitoK_ATP after its inhibition by ATP and found that ADP and GDP at a concentration of 0.2 – 0.5 mM reactivated this channel. It was also found that uridine-5¢-diphosphate (UDP) produced more significant activating effect on mitoK_ATP (Fig.1).

 

Fig. 1. Effect of UDP on the channel-forming subunit of mitoK_ATP reconstituted into BLM (the left figure) and on the whole channel in intact mitochondria (the right figure). The incubation medium for BLM contained: 20 mМ Tris-HCl, 100 mМ KCl, рН 7.2; for intact mitochondria: 50 mМ KCl, 5 mM Hepes, 5 mМ NaH₂PO₄, 5 mM succinic acid, 0.5 mM MgCl₂, 0.1 nM EGTА, 5 μM cytochrome C, 2 μM rotenone, and 2 μM cyclosporin A, pH 7.2. Swelling was initiated by addition of 0.1 mg/ml mitochondria. Additions: ATP –50 μM, UDP – 30 μM, uridine and UMP – 30 μM, 5-HD – 100 μM.

 

The studies were performed not only on mitoK_ATP reconstituted into artificial membranes (BLM and liposomes) but in intact mitochondria. As it is seen from the Fig, 50 mM ATP leads to the inhibition of the energy-dependent influx of K⁺ into mitochondria, and 30 mM UDP in the presence of ATP reactivates this transport. The effect of UDP is specific since 5-HD (a selective inhibitor of mitoK_ATP) prevented the activating effect of UDP. It should be noted that precursors od UDP, uridine and UMP, did not affect the channel in intact mitochondria

Data obtained assumes that UDP influence on the channel subunit of mitoK_ATP Earlier we suggested the working model of structural organization of mitochondrial ATP-dependent potassium channel. According to this model the channel consists of 4 protein subunits, forming cation pore, and 4 subunits, forming channel regulatory domain. Action of different channel inhibitors and activators is shown on the figure. Classical channel modulators act through regulatory subunit. But UDP realizes its effect through the channel-forming subunit.

The main goal of this study is to investigate a possible role of metabolic activators of mitoK_ATP in cardioprotection. We studied the cardioprotective properties of the precursors of UDP, uridine and uridine-5¢-monophosphate (UMP). These precursors opposed to UDP possess the capacity to penetrate into the cell.

On the model of acute ischemia of the rat heart left ventricle ( 60 min. coronary artert occlusion), the size of the ischemia alteration zone was histochemically revealed. Uridine and UMP were injected 5 min before occlusion. It was shown that the myocardium infarct zone decreased in 1.9 and 3.5 times for uridine and UMP, respectively (Fig.2). The administration of Glib and 5-HD prior to UMP injection eliminated the decrease in IAI and thus the anti-ischemic effect of the nucleotide.

Fig.2. Effect of uridine and UMP (both 30 mg/kg, i.v.) injected 5 min prior to occlusion of the left coronary artery on ischemic alteration index 60 min after occlusion. 5-HD (5 mg/kg) or glibenclamide (1 mg/kg) were injected intravenously 10 min and 35 min before occlusion, respectively. *- р<0.05 as compared with control.

 

Glib or 5-HD alone, without subsequent introduction of uridine or UMP, did not affect the size of the ischemic alteration zone.

It should be noted that UDP produced practically no cardioprotective action (data not shown).

Another important indicator of progression of ischemic damage is the T-wave amplitude that can be registered on early stage of disease by electrocardiography. The administration of uridine and UMP diminished the occlusion-induced increase in the T-wave amplitude (Fig.3). The cardioprotective anti-ischemic effect of the preparations was evident even at the initial stage of ischemia, at 3 min after the occlusion, and persisted throughout the observation period.

Since in studying of both parameters the inhibitors of mitoK_ATP glibenclamide and 5-hydroxydecanoic acid (5-HD) prevented the cardioprotective effect of uridine and UMP we can conclude that mitoK_ATP is involved in the realization of the anti-ischemic effect of uridine and its phosphonucleotide.

Since the antiarrhythmic action of 5-HD was less manifested, than the effect of glibenclamide we suppose that their antiarrhythmic action  is associated mainly with the activation of the cell membrane ATP-dependent potassium channel (cellK_ATP). Thus both preparations can be considered as potential cardiotropic agents, but the mechanism of their action can be different.

 

Fig.3. Uridine and UMP effect on T-wave amplitude on ECG of rats 60 min after left coronary artery occlusion (experimental conditions as at Fig. 2).

*- р<0.05 as compared with control.

 

Besides anti-ischemic action, uridine and UMP demonstrated also antiarrhythmic effect. The following markers of early post-occlusion disturbances of heart rhythm were registered in the experiments: the time of arrhythmia onset after LCA occlusion, the total arrhythmia duration, the number of PVB, the duration of VT and VF.

On the the figure 4 the uridine and UMP effect on arrhythmic indices is shown. Uridine and particularly UMP affected favorably the development of serious myocardial rhythm disturbances such as PVB and VT. The administration of UMP decreased the number of PVB 5.6 times and reduced the duration of VT 9.4 times compared with control.

Fig. 4. Uridine and UMP effct on early postocclusion rhythm disturbances of rat heart (experimental conditions as at Fig. 2).

*- р<0.05 as compared with control.

Uridine also had the beneficial effect on the recovery of cardiac rhythm: the number of PVB decreased 1.9 times, and the duration of VT was reduced 4.1 times compared with control. Glibenclamide prevented the manifestation of the antiarrhythmic activity of both preparations. At the same time, positive effects of uridine and UMP on VT and PVB practically were not abolished by 5-HD. Thus it leads us to a conclusion that antiarrhythmic action of uridine and UMP is determined by cellK_ATP rather than mitochondrial channel. It should be noted that Glib and 5-HD themselves did not affect the development of early occlusion arrhythmias except that Glib reduced to some extent the duration of VF (Fig.4).

Moreover, both uridine and UMP demonstrated a significant antifibrillatory action (Fig.5), decreasing the duration of VF 3.6 and 10.8 times, respectively. However, their effects were not related to the activation of either cellK_ATP or mitoK_ATP. Glib and 5-HD did not eliminate the positive effect of the nucleotide and nucleoside.

Fig. 5. Antifibrillatory effect of uridine and UMP (experimental conditions as at Fig. 2).

*- р<0.05 as compared with control.

Consequently, the results of the experiments showed that mitochondrial channel activation leads to decrease of infarct size during ischemia. At the same time, antiarrhythmic action is determined mainly by cellK_ATP channel.