In heart, glycolysis may be a preferential source of adenosine triphosphate (ATP) for membrane functions. In this study the patch-clamp technique was used to study potassium channels sensitive to intracellular ATP levels in permeabilized ventricular myocytes. Activation of these K+ channels has been implicated in marked cellular K+ loss leading to electrophysiological abnormalities and arrhythmias during myocardial ischemia. The results showed that glycolysis was more effective than oxidative phosphorylation in preventing ATP-sensitive K+ channels from opening. Experiments in excised inside-out patches suggested that key glycolytic enzymes located in the membrane or adjacent cytoskeleton near the channels may account for their preference for glycolytic ATP.

SUMMARY1. The contribution of ATP-sensitive K+ (KATP) channels to the rapid increase in cellular K+ efflux and shortening of action potential duration (APD) during early myocardial ischaemia and hypoxia remains controversial, because for the first 10 min of ischaemia or hypoxia in intact hearts cytosolic [ATP] remains about two orders of magnitude greater than the [ATP] causing half-maximal blockade of KATP channels in excised membrane patches. The purpose of this study was to investigate this apparent discrepancy.2. During substrate-free hypoxia, total, diastolic and systolic unidirectional K+ efflux rates increased by 43, 26 and 103 % respectively after 8-3 min in isolated arterially perfused rabbit interventricular septa loaded with 42K+. APD shortened by 39 %. From the Goldman-Hodgkin-Katz equation, the relative increases in systolic and diastolic K+ efflux rates were consistent with activation of a voltage-independent K+ conductance.3. During total global ischaemia, [K+]. measured with intramyocardial valinomycin K+-sensitive electrodes increased at a maximal rate of 0-68 mm min-', which could be explained by a < 26 % increase in unidirectional K+ efflux rate (assuming no change in K+ influx), less than the increase during hypoxia. APD shortened by 23 % over 10 min.

Sulfonylurea derivatives glibenclamide and tolbutamide are selective blockers of ATP-sensitive K+ (KATP) channels. However, their ability to prevent cellular K+ loss and shortening of action potential duration during ischemia or hypoxia in the intact heart is modest compared with their efficacy at blocking KATP channels in excised membrane patches. In the isolated arterially perfused rabbit interventricular septum, the increase in unidirectional K+ efflux and shortening of action potential duration during substrate-free hypoxia were effectively blocked by glibenclamide, but only by very high concentrations (100 microM); during hypoxia with glucose present, glibenclamide was only partially effective at reducing K+ loss. During total global ischemia (10 minutes), up to 100 microM glibenclamide or 1 mM tolbutamide attenuated shortening of action potential duration but only reduced [K+]0 accumulation by a maximum of 32 +/- 6%. In isolated patch-clamped guinea pig ventricular myocytes in which the whole-cell ATP-sensitive K+ current was activated by exposure to the metabolic inhibitors, glibenclamide (up to 100 microM) and tolbutamide (10 mM) were only partially effective at blocking the whole-cell ATP-sensitive K+ current (maximum block, 51 +/- 10% and 50 +/- 9%, respectively), especially when ADP was included in the patch electrode solution. In inside-out membrane patches excised from these myocytes, glibenclamide blocked unitary currents through KATP channels with a Kd of 0.5 microM and a Hill coefficient of 0.5 in the absence of ADP at the cytosolic membrane surface, but block was incomplete when 100 microM ADP (+2 mM free Mg2+) was present. ADP had a similar effect on block of KATP channels by tolbutamide. These findings suggest that free cytosolic [ADP], which rises rapidly to the 100 microM range during early myocardial ischemia and hypoxia, may account for the limited efficacy of sulfonylureas at blocking ischemic and hypoxic cellular K+ loss under these conditions.

In sinoatrial node (SAN) cells, electrogenic sodium-calcium exchange (NCX) is the dominant calcium (Ca) efflux mechanism. However, the role of NCX in the generation of SAN automaticity is controversial. To investigate the contribution of NCX to pacemaking in the SAN, we performed optical voltage mapping and high-speed 2D laser scanning confocal microscopy (LSCM) of Ca dynamics in an ex vivo intact SAN/atrial tissue preparation from atrial-specific NCX knockout (KO) mice. These mice lack P waves on electrocardiograms, and isolated NCX KO SAN cells are quiescent. Voltage mapping revealed disorganized and arrhythmic depolarizations within the NCX KO SAN that failed to propagate into the atria. LSCM revealed intermittent bursts of Ca transients. Bursts were accompanied by rising diastolic Ca, culminating in long pauses dominated by Ca waves. The L-type Ca channel agonist BayK8644 reduced the rate of Ca transients and inhibited burst generation in the NCX KO SAN whereas the Ca buffer 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (acetoxymethyl ester) (BAPTA AM) did the opposite. These results suggest that cellular Ca accumulation hinders spontaneous depolarization in the NCX KO SAN, possibly by inhibiting L-type Ca currents. The funny current (I f ) blocker ivabradine also suppressed NCX KO SAN automaticity. We conclude that pacemaker activity is present in the NCX KO SAN, generated by a mechanism that depends upon I f . However, the absence of NCX-mediated depolarization in combination with impaired Ca efflux results in intermittent bursts of pacemaker activity, reminiscent of human sinus node dysfunction and "tachy-brady" syndrome.sinoatrial node | sodium-calcium exchange | pacemaker activity | arrhythmia | intracellular calcium P hysiological heart rhythm originates in the sinoatrial node (SAN), a cluster of specialized pacemaker cells located on the endocardial surface of the right atrium (RA). SAN dysfunction (SND) leads to serious arrhythmias characterized by pathological pauses, often alternating with rapid heart rates or atrial fibrillation (1). Each year in the United States, close to 200,000 patients affected with SAN disease require surgical implantation of an electronic pacemaker (2). Therefore, advances in our understanding of SAN pacemaker activity are essential for developing new therapies to avoid this costly procedure and its related morbidity.In SAN pacemaker cells, action potentials (APs) are thought to be triggered by spontaneous diastolic depolarization (SDD) produced by a coupled system of cellular "clocks" (3). The first clock, known as the "membrane clock," initiates SDD in response to inward funny current (I f ) carried mostly by HCN4 channels (4) although other ion channels, like voltage-dependent Ca channels, have also been implicated (5). The second (and more controversial) clock is referred to as the "Ca clock." This clock produces a depolarizing current in late diastole when local Ca released by ryanodine receptors (RyRs) on the sarcoplasmic reticulum (SR) is extruded by the e...

The ability of glycolysis, oxidative phosphorylation, the creatine kinase system, and exogenous ATP to suppress ATP-sensitive K § channels and prevent cell shortening were compared in patch-clamped single guinea pig ventricular myocytes. In cell-attached patches on myocytes permeabilized at one end with saponin, ATP-sensitive K § channels were activated by removing ATP from the bath, and could be closed equally well by exogenous ATP or substrates for endogenous ATP production by glycolysis (with the mitochondrial inhibitor FCCP present), mitochondrial oxidative phosphorylation, or the creatine kinase system. In the presence of an exogenous ATP-consuming system, however, glycolytic substrates (with FCCP present) were superior to substrates for either oxidative phosphorylation or the creatine kinase system at suppressing ATP-sensitive K + channels. All three groups of substrates were equally effective at preventing cell shortening. In 6 of 38 excised inside-out membrane patches, ATP-sensitive K + channels activated by removing ATP from the bath were suppressed by a complete set of substrates for the ATP-producing steps of glycolysis but not by individual glycolytic substrates, which is consistent with the presence of key glycolytic enzymes located near the channels in these patches. Under whole-cell voltage-clamp conditions, inclusion of 15 mM ATP in the patch electrode solution dialyzing the interior of the cell did not prevent activation of the ATP-sensitive K § current under control conditions or during exposure to complete metabolic inhibition. In isolated arterially perfused rabbit interventricular septa, selective inhibition of glycolysis caused an immediate increase in 42K+ efflux rate, which was prevented by 100 ~M glyburide, a known blocker of ATP-sensitive K § channels. These observations suggest that key glycolytic enzymes are associated with cardiac ATP-sensitive K + channels and under conditions in which intracellular competition for ATP is high (e.g., in beating heart) that act as a preferential source of ATP for these channels.