Jovanović, Aleksandar

Discovered in the cardiac sarcolemma, ATP-sensitive K+(KATP) channels have more recently also been identified within the inner mitochondrial membrane. Yet the consequences of mitochondrial KATPchannel activation on mitochondrial function remain partially documented. Therefore, we isolated mitochondria from rat hearts and used K+channel openers to examine the effect of mitochondrial KATPchannel opening on mitochondrial membrane potential, respiration, ATP generation, Ca2+transport, and matrix volume. From a mitochondrial membrane potential of −180 ± 15 mV, K+channel openers, pinacidil (100 μM), cromakalim (25 μM), and levcromakalim (20 μM), induced membrane depolarization by 10 ± 7, 25 ± 9, and 24 ± 10 mV, respectively. This effect was abolished by removal of extramitochondrial K+or application of a KATPchannel blocker. K+channel opener-induced membrane depolarization was associated with an increase in the rate of mitochondrial respiration and a decrease in the rate of mitochondrial ATP synthesis. Furthermore, treatment with a K+channel opener released Ca2+from mitochondria preloaded with Ca2+, an effect also dependent on extramitochondrial K+concentration and sensitive to KATPchannel blockade. In addition, K+channel openers, cromakalim and pinacidil, increased matrix volume and released mitochondrial proteins, cytochrome cand adenylate kinase. Thus, in isolated cardiac mitochondria, KATPchannel openers depolarized the membrane, accelerated respiration, slowed ATP production, released accumulated Ca2+, produced swelling, and stimulated efflux of intermembrane proteins. These observations provide direct evidence for a role of mitochondrial KATPchannels in regulating functions vital for the cardiac mitochondria.

Background —Mechanical load and humoral stimuli such as endothelin (ET) and angiotensin II (Ang II) are potent modulators of cardiac structure and endocrine function, specifically gene expression and production and release of atrial natriuretic peptide (ANP). We define the contribution of mechanical load compared with neurohumoral stimulation in vivo with specific focus on myocardial and circulating ANP during chronic myocardial unloading produced by thoracic inferior vena caval constriction (TIVCC). Methods and Results —TIVCC was produced by banding the IVC for 10 days in 7 dogs, whereas in the 6 control dogs, the band was not constricted. TIVCC was characterized by a decrease in cardiac output, right atrial pressure, and left ventricular (LV) end-diastolic diameter and marked activation of ET and Ang II in plasma and atrial and ventricular myocardium. Despite neurohumoral stimulation, LV mass index and myocyte diameters in unloaded hearts decreased, reflecting myocyte atrophy. The total number of myocytes in the LV remained unchanged. Atrial stores of ANP increased, but plasma ANP did not change, in association with a trend toward ANP gene expression to decrease in unloaded hearts. Conclusions —Chronic mechanical unloading of the heart results in myocardial atrophy and lack of activation of ANP synthesis despite marked neurohumoral stimulation by the growth promoters ET and Ang II.

Background—Opening of cardiac ATP-sensitive K+(KATP) channels has emerged as a promising but still controversial cardioprotective mechanism. Defining KATPchannel function at the level of recombinant channel proteins is a necessary step toward further evaluation of the cardioprotective significance of this ion conductance.Methods and Results—KATPchannel–deficient COS-7 cells were found to be vulnerable to chemical hypoxia-reoxygenation injury that induced significant cytosolic Ca2+loading (from 97±3 to 236±11 nmol/L). In these cells, the potassium channel opener pinacidil (10 μmol/L) did not prevent Ca2+loading (from 96±3 nmol/L before to 233±12 nmol/L after reoxygenation) or evoked membrane current. Cotransfection withKir6.2/SUR2Agenes, which encode cardiac KATPchannel subunits, resulted in a cellular phenotype that, in the presence of pinacidil (10 μmol/L), expressed K+current and gained resistance to hypoxia-reoxygenation (Ca2+concentration from 99±7 to 127±11 nmol/L;P>0.05). Both properties were abolished by the KATPchannel blocker glyburide (1 μmol/L). In COS-7 cells transfected with individual channel subunitsKir6.2orSUR2A, which alone do not form functional cardiac KATPchannels, pinacidil did not protect against hypoxia-reoxygenation.Conclusions—The fact that transfer of cardiac KATPchannel subunits protected natively KATPchannel–deficient cells provides direct evidence that the cardiac KATPchannel protein complex harbors intrinsic cytoprotective properties. These findings validate the concept that targeting cardiac KATPchannels should be considered a valuable approach to protect the myocardium against injury.

Although tight regulation of intracellular pH (pHi) is critical for the survival under stress, paradoxically a slowed recovery of pHi under hypoxic injury may be cardioprotective. In this study, we investigated the recovery of pHi after hypoxia-induced intracellular acidosis in cardiomyocytes loaded with the H+-sensitive dye SNARF-1. Exposure of single cardiomyocytes to 2,4-dinitrophenol (DNP), an inhibitor of mitochondrial oxidative phosphorylation, induced significant intracellular acidification. However, within 10–12 min upon removal of DNP, cardiomyocytes restituted their intracellular H+ concentration. The presence either of 5-N-ethyl-N-isopropylamiloride (EIPA) an inhibitor of Na/H antiporter, or 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), an inhibitor of bicarbonate-dependent exchange, did not modify the cellular response to DNP. But, combined use of EIPA and DIDS prevented the restitution of intracellular pH following removal of DNP. This study, thus, demonstrated, for the first time, that blockade of both Na/H and bicarbonate-dependent exchange is necessary and sufficient to maintain the hypoxia-induced intracellular acidification. Therefore, concomitant blockade of both pH-regulating mechanisms deserves to be further considered as a novel strategy against hypoxia-reoxygenation injury in the heart.

Diadenosine tetraphosphate (Ap4A) has been recently discovered in the pancreatic γ cells where targets ATP-sensitive K+ (KATP) channels, depolarizes the cell membrane and induces insulin secretion. However, whether Ap4A inhibit pancreatic KATP channels by targeting protein channel complex itself was unknown. Therefore, we coexpressed pancreatic KATP channel subunits, Kir6.2 and SUR1, in COS-7 cells and examined the effect of Ap4A on the single channel behavior using the inside-out configuration of the patch-clamp technique. Ap4A inhibited channel opening in a concentration-dependent manner. Analysis of single channels demonstrated that Ap4A did not change intraburst kinetic behavior of KATP channels, but rather decreased burst duration and increased between-burst duration. It is concluded that Ap4A antagonizes KATP channel opening by targeting channel subunits themselves and by keeping channels longer in closed interburst states.