The microelectrode array (MEA) was used to evaluate the cardioprotective effects of adenosine triphosphate sensitive potassium (K(ATP)) channel activation using potassium channel openers (KCOs) on HL-1 cardiomyocytes subjected to acute chemically induced metabolic inhibition. Beat frequency and extracellular action potential (exAP) amplitude were measured in the presence of metabolic inhibitors (sodium azide (NaN(3)) or 2-deoxyglucose (2-DG)) or KCOs (pinacidil (PIN, a cyanoguanidine derivative, activates sarcolemmal K(ATP) channels) or SDZ PCO400 (SDZ, a benzopyran derivative, activates mitochondrial K(ATP) channels)). The protective effects of these KCOs on metabolically inhibited HL-1 cells were subsequently investigated. Signal shapes indicated that NaN(3) and 2-DG reduced the rate of the sodium (Na(+)) influx signal as reflected by a reduction in beat frequency. PIN and SDZ appeared to reduce both rate of depolarization and extent of the Na(+) influx signals. Pre-treating cardiomyocytes with PIN (0.1 mM), but not SDZ, prevented the reduction of beat frequency associated with NaN(3)- or 2-DG-induced metabolic inhibition. The exAP amplitude was not affected by either KCO. The cardioprotective effect of PIN relative to SDZ may be due to the opening of different K(ATP) channels. This metabolic inhibition model on the MEA may provide a stable platform for the study of cardiac pathophysiology in the future.