Brian O’Rourke

Evidence for Mitochondrial K+ Channels and Their Role in Cardioprotection

Abstract— Twenty years after the discovery of sarcolemmal ATP-sensitive K+ channels and 12 years after the discovery of mitochondrial KATP (mitoKATP) channels, progress has been remarkable, but many questions remain. In the case of the former, detailed structural information is available, and it is well accepted that the channel couples bioenergetics to cellular electrical excitability; however, in the heart, a clear physiological or pathophysiological role has yet to be defined. For mitoKATP, structural information is lacking, but there is abundant evidence linking the opening of the channel to protection against ischemia-reperfusion injury or apoptosis. This review updates recent progress in understanding the physiological role of mitoKATP and highlights outstanding questions and controversies, with the intent of stimulating additional investigation on this topic.

Selective pharmacological agents implicate mitochondrial but not sarcolemmal K(ATP) channels in ischemic cardioprotection

BACKGROUND Pharmacological evidence has implicated ATP-sensitive K(+) (K(ATP)) channels as the effectors of cardioprotection, but the relative roles of mitochondrial (mitoK(ATP)) and sarcolemmal (surfaceK(ATP)) channels remain controversial. METHODS AND RESULTS We examined the effects of the K(ATP) channel blocker HMR1098 and the K(ATP) channel opener P-1075 on surfaceK(ATP) and mitoK(ATP) channels in rabbit ventricular myocytes. HMR1098 (30 micromol/L) inhibited the surfaceK(ATP) current activated by metabolic inhibition, whereas the drug did not blunt diazoxide (100 micromol/L)-induced flavoprotein oxidation, an index of mitoK(ATP) channel activity. P-1075 (30 micromol/L) did not increase flavoprotein oxidation but did elicit a robust surfaceK(ATP) current that was completely inhibited by HMR1098. These results indicate that HMR1098 selectively inhibits surfaceK(ATP) channels, whereas P-1075 selectively activates surface K(ATP) channels. In a cellular model of simulated ischemia, the mitoK(ATP) channel opener diazoxide (100 micromol/L), but not P-1075, blunted cellular injury. The cardioprotection afforded by diazoxide or by preconditioning was prevented by the mitoK(ATP) channel blocker 5-hydroxydecanoate (500 micromol/L) but not by the surfaceK(ATP) channel blocker HMR1098 (30 micromol/L). CONCLUSIONS The cellular effects of mitochondria- or surface-selective agents provide further support for the emerging consensus that mitoK(ATP) channels rather than surfaceK(ATP) channels are the likely effectors of cardioprotection.

Antiarrhythmic Engineering of Skeletal Myoblasts for Cardiac Transplantation

Skeletal myoblasts are an attractive cell type for transplantation because they are autologous and resistant to ischemia. However, clinical trials of myoblast transplantation in heart failure have been plagued by ventricular tachyarrhythmias and sudden cardiac death. The pathogenesis of these arrhythmias is poorly understood, but may be related to the fact that skeletal muscle cells, unlike heart cells, are electrically isolated by the absence of gap junctions. Using a novel in vitro model of myoblast transplantation in cardiomyocyte monolayers, we investigated the mechanisms of transplant-associated arrhythmias. Cocultures of human skeletal myoblasts and rat cardiomyocytes resulted in reentrant arrhythmias (spiral waves) that reproduce the features of ventricular tachycardia seen in patients receiving myoblast transplants. These arrhythmias could be terminated by nitrendipine, an l-type calcium channel blocker, but not by the Na channel blocker lidocaine. Genetic modification of myoblasts to express the gap junction protein connexin43 decreased arrhythmogenicity in cocultures, suggesting a specific means for increasing the safety (and perhaps the efficacy) of myoblast transplantation in patients.

Elevated Cytosolic Na+ Decreases Mitochondrial Ca2+ Uptake During Excitation-Contraction Coupling and Impairs Energetic Adaptation in Cardiac Myocytes

Mitochondrial Ca2+ ([Ca2+]m) regulates oxidative phosphorylation and thus contributes to energy supply and demand matching in cardiac myocytes. Mitochondria take up Ca2+ via the Ca2+ uniporter (MCU) and extrude it through the mitochondrial Na+/Ca2+ exchanger (mNCE). It is controversial whether mitochondria take up Ca2+ rapidly, on a beat-to-beat basis, or slowly, by temporally integrating cytosolic Ca2+ ([Ca2+]c) transients. Furthermore, although mitochondrial Ca2+ efflux is governed by mNCE, it is unknown whether elevated intracellular Na+ ([Na+]i) affects mitochondrial Ca2+ uptake and bioenergetics. To monitor [Ca2+]m, mitochondria of guinea pig cardiac myocytes were loaded with rhod-2–acetoxymethyl ester (rhod-2 AM), and [Ca2+]c was monitored with indo-1 after dialyzing rhod-2 out of the cytoplasm. [Ca2+]c transients, elicited by voltage-clamp depolarizations, were accompanied by fast [Ca2+]m transients, whose amplitude (Δ) correlated linearly with Δ[Ca2+]c. Under β-adrenergic stimulation, [Ca2+]m decay was ≈2.5-fold slower than that of [Ca2+]c, leading to diastolic accumulation of [Ca2+]m when amplitude or frequency of Δ[Ca2+]c increased. The MCU blocker Ru360 reduced Δ[Ca2+]m and increased Δ[Ca2+]c, whereas the mNCE inhibitor CGP-37157 potentiated diastolic [Ca2+]m accumulation. Elevating [Na+]i from 5 to 15 mmol/L accelerated mitochondrial Ca2+ decay, thus decreasing systolic and diastolic [Ca2+]m. In response to gradual or abrupt changes of workload, reduced nicotinamide-adenine dinucleotide (NADH) levels were maintained at 5 mmol/L [Na+]i, but at 15 mmol/L, the NADH pool was partially oxidized. The results indicate that (1) mitochondria take up Ca2+ rapidly and contribute to fast buffering during a [Ca2+]c transient; and (2) elevated [Na+]i impairs mitochondrial Ca2+ uptake, with consequent effects on energy supply and demand matching. The latter effect may have implications for cardiac diseases with elevated [Na+]i.

Decreased Sarcoplasmic Reticulum Calcium Content Is Responsible for Defective Excitation-Contraction Coupling in Canine Heart Failure

BackgroundAltered excitation-contraction (E-C) coupling in canine pacing-induced heart failure involves decreased sarcoplasmic reticulum (SR) Ca uptake and enhanced Na/Ca exchange, which could be expected to decrease SR Ca content (CaSR) and may explain the reduced intracellular Ca (Cai) transient. Studies in other failure models have suggested that the intrinsic coupling between L-type Ca current (ICa,L) and SR Ca release is reduced without a change in SR Ca load. The present study investigates whether CaSR and/or coupling is altered in midmyocardial myocytes from failing canine hearts (F). Methods and ResultsMyocytes were indo-1-loaded via patch pipette (37°C), and Cai transients were elicited with voltage-clamp steps applied at various frequencies. ICa,L density was not significantly decreased in F, but steady-state Cai transients were reduced to 20% to 40% of normal myocytes (N). CaSR, measured by integrating Na/Ca exchange currents during caffeine-induced release, was profoundly decreased in F, to 15% to 25% of N. When CaSR was normalized in F by preloading in 5 mmol/L external Ca before a test pulse at 2 mmol/L Ca, a normal-amplitude Cai transient was elicited. E-C coupling gain was dependent on CaSR but was affected similarly in both groups, indicating that intrinsic coupling is unaltered in F. ConclusionsA decrease in CaSR is sufficient to explain the diminished Cai transients in F, without a change in the effectiveness of coupling. Therefore, therapeutic approaches that increase CaSR may be able to fully correct the Ca handling deficit in heart failure.

Nicorandil, a potent cardioprotective agent, acts by opening mitochondrial ATP-dependent potassium channels☆

OBJECTIVES To determine the mechanism of cardioprotection afforded by nicorandil, an orally efficacious antianginal drug, we examined its effects on ATP-dependent potassium (K(ATP)) channels. BACKGROUND Nicorandil can mimic ischemic preconditioning, while mitochondrial K(ATP) (mitoK(ATP)) channels rather than sarcolemmal K(ATP) (surfaceK(ATP)) channels have emerged as the likely effectors. METHODS Flavoprotein fluorescence and membrane current in intact rabbit ventricular myocytes were measured simultaneously to assay mitoK(ATP) channel and surface K(ATP) channel activities, respectively. In a cell-pelleting model of ischemia, cells permeable to trypan blue were counted as killed by 60 and 120 min of ischemia. RESULTS Nicorandil (100 micromol/liter) increased flavoprotein oxidation but not membrane current; a 10-fold higher concentration recruits both mitoK(ATP) and surfaceK(ATP) channels. Pooled dose-response data confirm that nicorandil concentrations as low as 10 micromol/liter turn on mitoK(ATP) channels, while surfaceK(ATP) current requires exposure to millimolar concentrations. Nicorandil blunted the rate of cell death in a pelleting model of ischemia; this cardioprotective effect was prevented by the mitoK(ATP) channel blocker 5-hydroxydecanoate but was unaffected by the surfaceK(ATP) channel blocker HMR1098. CONCLUSIONS Nicorandil exerts a direct cardioprotective effect on heart muscle cells, an effect mediated by selective activation of mitoK(ATP) channels.

Synergistic Modulation of ATP-Sensitive K+ Currents by Protein Kinase C and Adenosine Implications for Ischemic Preconditioning

Ischemic preconditioning has been shown to involve the activation of adenosine receptors, protein kinase C (PKC), and ATP-sensitive K+ (K ATP) channels. We investigated the effects of PKC activation and adenosine on K(ATP) current (I KATP) and action potentials in isolated rabbit ventricular myocytes. Responses to pinacidil (100 to 400 micromol/L), an opener of K(ATP) channels, were markedly increased by preexposure to the PKC activator phorbol 12-myristate 13-acetate (PMA, 100 nmol/L). I(KATP) measured at 0 mV was increased by PMA pretreatment from 0.55 +/- 0.32 to 3.25 +/- 0.47 nA (n=6, P < .01). We next determined whether PKC activation abbreviates the time required to turn on I(KATP) developed after an average of 15.1 +/- 2.4 minutes (n=8). Ten-minute pretreatment with PMA alone (PMA+MI) did not significantly alter this latency (11.9 +/- 2.0 minutes, n=8). Since adenosine receptor activation has been shown to play an important role in the preconditioning response, two groups of myocytes were studied with adenosine (10 micromol/L) included during MI. Without PMA, adenosine alone (MI+Ado) did not affect the latency to develop I(KATP) (12.3 +/- 1.5 minutes, n=8). However, if cells were pretreated with PMA and then subjected to MI in the presence of adenosine (PMA+MI+Ado), the latency was greatly shortened to 5.5 +/- 1.6 minutes (n=8;P < .02 versus MI, PMA+MI, and MI+Ado groups). This effect could not be reproduced by an inactive phorbol but was completely abolished by the adenosine receptor antagonist 8-(p-sulfophenyl)-theophylline. The opening of K(ATP) channels may be cardioprotective because of the abbreviation of action potential duration (APD) during ischemia. Therefore, we tested whether PKC activation could modify the time course of APD shortening during MI. Consistent with the ionic current measurements, PMA pretreatment significantly accelerated APD shortening, but only when adenosine (10 micromol/L) was included during MI. The effects were not attributable to accelerated ATP consumption: PMA pretreatment did not alter the time required to induce rigor during MI, whether or not adenosine was included. Our results indicate that PKC activation increases the I(KATP) Induced by pinacidil or by MI. The latter effect requires concomitant adenosine receptor activation. The synergistic modulation of I(KATP) by PKC and adenosine provides an explicit basis for current paradigms of ischemic preconditioning.

Role of Sodium-Calcium Exchanger in Modulating the Action Potential of Ventricular Myocytes From Normal and Failing Hearts

Abstract— Increased Na+-Ca2+ exchange (NCX) activity in heart failure and hypertrophy may compensate for depressed sarcoplasmic reticular Ca2+ uptake, provide inotropic support through reverse-mode Ca2+ entry, and/or deplete intracellular Ca2+ stores. NCX is electrogenic and depends on Na+ and Ca2+ transmembrane gradients, making it difficult to predict its effect on the action potential (AP). Here, we examine the effect of [Na+]i on the AP in myocytes from normal and pacing-induced failing canine hearts and estimate the direction of the NCX driving force using simultaneously recorded APs and Ca2+ transients. AP duration shortened with increasing [Na+]i and was correlated with a shift in the reversal point of the NCX driving force. At [Na+]i ≥10 mmol/L, outward NCX current during the plateau facilitated repolarization, whereas at 5 mmol/L [Na+]i, NCX had a depolarizing effect, confirmed by partially inhibiting NCX with exchange inhibitory peptide. Exchange inhibitory peptide shortened the AP duration at 5 mmol/L [Na+]i and prolonged it at [Na+]i ≥10 mmol/L. With K+ currents blocked, total membrane current was outward during the late plateau of an AP clamp at 10 mmol/L [Na+]i and became inward close to the predicted reversal point for the NCX driving force. The results were reproduced using a computer model. These results indicate that NCX plays an important role in shaping the AP of the canine myocyte, helping it to repolarize at high [Na+]i, especially in the failing heart, but contributing a depolarizing, potentially arrhythmogenic, influence at low [Na+]i.

Adenosine Primes the Opening of Mitochondrial ATP-Sensitive Potassium Channels A Key Step in Ischemic Preconditioning?

BACKGROUND Adenosine can initiate ischemic preconditioning, and mitochondrial ATP-sensitive potassium (K(ATP)) channels have emerged as the likely effectors. We sought to determine the mechanistic interactions between these 2 observations. METHODS AND RESULTS The mitochondrial flavoprotein oxidation induced by diazoxide (100 micromol/L) was used to quantify mitochondrial K(ATP) channel activity in intact rabbit ventricular myocytes. Adenosine (100 micromol/L) increased mitochondrial K(ATP) channel activity and abbreviated the latency to mitochondrial K(ATP) channel opening. These potentiating effects were entirely prevented by the adenosine receptor antagonist 8-(p-sulfophenyl)-theophylline (100 micromol/L) or by the protein kinase C inhibitor polymyxin B (50 micromol/L). The effects of adenosine and diazoxide reflected mitochondrial K(ATP) channel activation, because they could be blocked by the mitochondrial K(ATP) channel blocker 5-hydroxydecanoate (500 micromol/L). In a cellular model of simulated ischemia, adenosine mitigated cell injury; this cardioprotective effect was blocked by 5-hydroxydecanoate but not by the surface-selective K(ATP) channel blocker HMR1098. Moreover, adenosine augmented the cardioprotective effect of diazoxide. A quantitative model of mitochondrial K(ATP) channel gating reproduced the major experimental findings. CONCLUSIONS Our results support the hypothesis that adenosine receptor activation primes the opening of mitochondrial K(ATP) channels in a protein kinase C-dependent manner. The findings provide tangible links among various key elements in the preconditioning cascade.

Mitochondrial Oscillations in Physiology and Pathophysiology

Oscillations in chemical reactions and metabolic pathways have historically served as prototypes for understanding the dynamics of complex nonlinear systems. This chapter reviews the oscillatory behavior of mitochondria, with a focus on the mitochondrial oscillator dependent on reactive oxygen species (ROS), as first described in heart cells. Experimental and theoretical evidence now indicates that mitochondrial energetic variables oscillate autonomously as part of a network of coupled oscillators under both physiological and pathological conditions. The physiological domain is characterized by small-amplitude oscillations in mitochondrial membrane potential (delta psi(m)) showing correlated behavior over a wide range of frequencies, as determined using Power Spectral Analysis and Relative Dispersion Analysis of long term recordings of delta psi(m). Under metabolic stress, when the balance between ROS generation and ROS scavenging is perturbed, the mitochondrial network throughout the cell locks to one main low-frequency, high-amplitude oscillatory mode. This behavior has major pathological implications because the energy dissipation and cellular redox changes that occur during delta psi(m) depolarization result in suppression of electrical excitability and Ca2+ handling, the two main functions of the cardiac cell. In an ischemia/reperfusion scenario these alterations scale up to the level of the whole organ, giving rise to fatal arrhythmias.