Precise matching of energy supply with demand requires delicately balanced control of the enzymes involved in substrate metabolism. In response to a change in substrate supply, the nonlinear properties of metabolic control may induce complex dynamic behavior. Using confocal imaging of f lavoprotein redox potential and mitochondrial membrane potential, we show that substrate deprivation leads to subcellular heterogeneity of mitochondrial energization in intact cells. The complex spatiotemporal patterns of redox and matrix potential included local metabolic transients, cell-wide coordinated redox transitions, and propagated metabolic waves both within and between coupled cells. Loss of metabolic synchrony during mild metabolic stress reveals that intra-and intercellular control of mitochondrial function involves diffusible cytoplasmic messengers.Nonlinear dynamic control in metabolic pathways plays a crucial role in conferring sensitivity and rapid responses to changes in cellular workload or environmental conditions. Through a combination of allosteric and stoichiometric effects on multiple control points, the enzymatic pathways involved in energy metabolism can undergo large changes in activity in response to small perturbations of key effector molecules (1). Experimental and theoretical studies have demonstrated that, under some conditions, such finely tuned systems may become unstable and display self-organizing oscillations, bistability, or chaotic behavior (reviewed in ref.2). Early observations of metabolic oscillations in yeast (3-5) and our own investigation of oscillations of NADH and sarcolemmal K,ATP current in substrate-deprived heart cells (6) provide evidence that such phenomena can be observed in intact cells. In our previous work, modulation of the amplitude and͞or frequency of the oscillations by external glucose led to the hypothesis that they were driven by a glycolytic oscillator, but the predominantly mitochondrial origin of the NADH f luorescence in cardiomyocytes (7) suggested that metabolic transients in oxidative phosphorylation were also present.To investigate the mitochondrial component of the oscillations, the present study uses the endogenous fluorescence of flavoproteins to image redox oscillations in the mitochondrial matrix. Subcellular imaging reveals a remarkable degree of spatial and temporal heterogeneity in mitochondrial redox and electrical potential, including local transients and propagated metabolic waves. The last observation suggests that communication between mitochondria in the same or neighboring cells is likely to involve diffusible cytosolic messengers acting to synchronize the energy state of the entire population of mitochondria.

Cardiac tissue can display unusual responses to certain stimulation protocols. In the wake of a conditioning wave of excitation, spiral waves can be initiated by applying stimuli timed to occur during a period of vulnerability (VP). Although vulnerability is well known in cardiac and chemical media, the determinants of the VP and its boundaries have received little theoretical and analytical study. From numerical and analytical studies of reaction-diffusion equations, we have found that 1) vulnerability is an inherent property of Beeler-Reuter and FitzHugh-Nagumo models of excitable media; 2) the duration of the vulnerable window (VW) the one-dimensional analog of the VP, is sensitive to the medium properties and the size of the stimulus field; and 3) the amplitudes of the excitatory and recovery processes modulate the duration of the VW. The analytical results reveal macroscopic behavior (vulnerability) derived from the diffusion of excitation that is not observable at the level of isolated cells or single reaction units.

Na channels open upon depolarization but then enter inactivated states from which they cannot readily reopen. After brief depolarizations, native channels enter a fastinactivated state from which recovery at hyperpolarized potentials is rapid (<20 ms). Prolonged depolarization induces a slow-inactivated state that requires much longer periods for recovery (> 1 s). The slow-inactivated state therefore assumes particular importance in pathological conditions, such as ischemia, in which tissues are depolarized for prolonged periods. While use-dependent block of Na channels by local anesthetics has been explained on the basis of delayed recovery of fast-inactivated Na channels, the potential contribution of slow-inactivated channels has been ignored. The principal (tx) subunits from skeletal muscle or brain Na channels display anomalous gating behavior when expressed in Xen0pus oocytes, with a high percentage entering slow-inactivated states after brief depolarizations. This enhanced slow inactivation is eliminated by coexpressing the r subunit with the subsidiary [31 subunit. We compared the lidocaine sensitivity ofct subunits expressed in the presence and absence of the [~1 subunit to determine the relative contributions of fast-inactivated and slow-inactivated channel block. Coexpression of ~31 inhibited the use-dependent accumulation of lidocaine block during repetitive (1-Hz) depolarizations from -100 to -20 mV. Therefore, the time required for recovery from inactivated channel block was measured at -100 mV. Fast-inactivated (ct + [31) channels were mostly unblocked within 1 s of repolarization; however, slowinactivated (et alone) channels remained blocked for much longer repriming intervals (>5 s). The affinity of the slow-inactivated state for lidocaine was estimated to be 15-25 izM, versus 24 izM for the fast-inactivated state. We conclude that slow-inactivated Na channels are blocked by lidocaine with an affinity comparable to that of fast-inactivated channels. A prominent functional consequence is potentiation of use-dependent block through a delay in repriming of lidocaine-bound slow-inactivated channels. Key words: sodium channel 9 slow inactivation 9 lidocaine 9 Xenopus oocytes 9 [31 subunit INTRODUCTION

Torsadelike (polymorphic) ECGs can be derived from spiral wave reentry in a medium of identical cells. Under normal conditions, the spiral core around which a reentrant wave front rotates is stationary. As the balance of repolarizing currents becomes less outward (eg, secondary to potassium channel blockade), the APD is prolonged. When the wavelength (APD.velocity) exceeds the perimeter of the stationary unexcited core, the core will become unstable, causing spiral core drift. Large repolarizing currents shorten the APD and result in a monomorphic reentrant process (stationary core), whereas smaller currents prolong the APD and amplify spiral core instability, resulting in a polymorphic process. We conclude that, similar to sodium channel blockade, the proarrhythmic potential of potassium channel blockade in the setting of propagation may be directly linked to its cellular antiarrhythmic potential, ie, arrhythmia suppression resulting from a prolonged APD may, on initiation of a reentrant wave front, destabilize the core of a rotating spiral, resulting in complex motion (precession) of the spiral tip around a nonstationary region of unexcited cells. In tissue with inhomogeneities, core instability alters the activation sequence from one reentry cycle to the next and can lead to spiral wave fractination as the wave front collides with inhomogeneous regions. Depending on the nature of the inhomogeneities, wave front fragments may annihilate one another, producing a nonsustained arrhythmia, or may spawn new spirals (multiple wavelets), producing fibrillation and sudden cardiac death.