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Mitochondrial networks in cardiac myocytes under oxidative stress show collective (cluster) behavior through synchronization of their inner membrane potentials (ΔΨ m ). However, it is unclear whether the oscillation frequency and coupling strength between individual mitochondria affect the size of the cluster and vice versa. We used the wavelet transform and developed advanced signal processing tools that allowed us to capture individual mitochondrial ΔΨ m oscillations in cardiac myocytes and examine their dynamic spatio-temporal properties. Heterogeneous frequency behavior prompted us to sort mitochondria according to their frequencies. Signal analysis of the mitochondrial network showed an inverse relationship between cluster size and cluster frequency as well as between cluster amplitude and cluster size. High cross-correlation coefficients between neighboring mitochondria clustered longitudinally along the myocyte striations, indicated anisotropic communication between mitochondria. Isochronal mapping of the onset of myocyte-wide ΔΨ m depolarization further exemplified heterogeneous ΔΨ m among mitochondria. Taken together, the results suggest that frequency and amplitude modulation of clusters of synchronized mitochondria arises by means of strong changes in local coupling between neighboring mitochondria.wavelets | frequency | amplitude | mitochondrial oscillator | mitochondrial coupling M itochondria in cardiac myocytes under the influence of substrate deprivation or oxidative stress have a fundamental role as determinants of cell life or death, with implications that scale to affect the function of the whole heart (1, 2). Cardiac mitochondria are organized in a highly ordered network consisting of intermyofibrillar, subcarcolemmal, and perinuclear (3) mitochondria whose coordinated function controls the energy metabolism of the myocyte (4).In cardiac myocytes, the localized perturbation of a few mitochondria within the overall mitochondrial network can trigger the transition from the physiological to the pathophysiological state by producing synchronized whole-myocyte oscillations of ΔΨ m that can be monitored with the fluorescent dye tetramethylrhodamine ethyl ester (TMRE) (5). The imbalance between reactive oxygen species (ROS) generation and ROS scavenging capacity in a significant proportion of the network (∼60%) (5) is thought to destabilize ΔΨ m beyond a critical point into a state of ROS-induced ROS release. Increased ROS overflow exceeding a threshold level results in the appearance of a spanning cluster of mitochondria oscillating in apparent synchrony throughout the cell as the mitochondrial network locks into a low-frequency, high-amplitude oscillatory mode (6, 7).In many disparate examples of physically and chemically coupled oscillators, synchronization of the system generally arises from an initial nucleus of (spontaneously) synchronized oscillators that integrate neighboring oscillators, thereby increasing the size and signal amplitude of the initial oscillatory nucleus (8-11).When the clus...

Action potential (AP) prolongation is a hallmark of failing myocardium. Functional downregulation of K currents is a prominent feature of cells isolated from failing ventricles. The detailed changes in K current expression differ depending on the species, the region of the heart, and the mechanism of induction of heart failure. We used complementary approaches to study K current downregulation in pacing tachycardia-induced heart failure in the rabbit. The AP duration (APD) at 90% repolarization was significantly longer in cells isolated from failing hearts compared with controls (539 +/- 162 failing vs. 394 +/- 114 control, P < 0.05). The major K currents in the rabbit heart, inward rectifier potassium current (I(K1)), transient outward (I(to)), and delayed rectifier current (I(K)) were functionally downregulated in cells isolated from failing ventricles. The mRNA levels of Kv4.2, Kv1.4, KChIP2, and Kir2.1 were significantly downregulated, whereas the Kv4.3, Erg, KvLQT1, and minK were unaltered in the failing ventricles compared with the control left ventricles. Significant downregulation in the long splice variant of Kv4.3, but not in the total Kv4.3, Kv4.2, and KChIP2 immunoreactive protein, was observed in cells isolated from the failing ventricle with no change in Kv1.4, KvLQT1, and in Kir2.1 immunoreactive protein levels. Multiple cellular and molecular mechanisms underlie the downregulation of K currents in the failing rabbit ventricle.

Abstract-Increased Na ϩ -Ca 2ϩ exchange (NCX) activity in heart failure and hypertrophy may compensate for depressed sarcoplasmic reticular Ca 2ϩ uptake, provide inotropic support through reverse-mode Ca 2ϩ entry, and/or deplete intracellular Ca 2ϩ stores. NCX is electrogenic and depends on Na ϩ and Ca 2ϩ transmembrane gradients, making it difficult to predict its effect on the action potential (AP). Here, we examine the effect of [Na ϩ ] 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 Ca 2ϩ transients. AP duration shortened with increasing [Na ϩ ] i and was correlated with a shift in the reversal point of the NCX driving force. At [Na ϩ ] i Ն10 mmol/L, outward NCX current during the plateau facilitated repolarization, whereas at 5 mmol/L [Na ϩ ] 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 ϩ ] i and prolonged it at [Na ϩ ] i Ն10 mmol/L. With K ϩ currents blocked, total membrane current was outward during the late plateau of an AP clamp at 10 mmol/L [Na ϩ ] 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 ϩ ] i , especially in the failing heart, but contributing a depolarizing, potentially arrhythmogenic, influence at low [Na Key Words: heart failure Ⅲ Na ϩ -Ca 2ϩ exchanger Ⅲ reversal potential Ⅲ Ca 2ϩ transients T he Na ϩ -Ca 2ϩ exchanger (NCX) catalyzes the electrogenic exchange of Na ϩ for Ca 2ϩ across the cardiac sarcolemma and is reversible, operating in either forward (Ca 2ϩ -efflux) or reverse (Ca 2ϩ -influx) modes, depending on the prevailing electrochemical driving forces for Ca 2ϩ and Na ϩ . This complex dependence on transmembrane ion and voltage gradients, which are both rapidly changing during the cellular action potential (AP), makes predictions about the overall influence of NCX current (I NCX ) on excitation-contraction coupling challenging. NCX is the primary Ca 2ϩ extrusion mechanism in the heart 1,2 and is required to remove the increment of Ca 2ϩ entering the myocyte via Ca 2ϩ channels on each beat, 3 but the timing of the transition from reverse-mode exchange (outward I NCX ) to forward-mode exchange (inward I NCX ) has been difficult to determine. Because the membrane impedance during the AP plateau of large mammals (including humans) is high, the net direction of current flow through NCX is likely to be a fundamental determinant of AP duration (APD).Early investigations into the net effect of I NCX on the AP demonstrated that it contributed net inward current and prolonged the AP in the rat. 4 than that reported by intracellular Ca 2ϩ dyes. In the failing heart, where SR function is impaired, a greater dependence on NCX for...