SUMMARY The mitochondrion is the primary source of reactive oxygen species (ROS) in eukaryotic cells. With the aid of a novel mitochondrial matrix-targeted superoxide indicator, here we show that individual mitochondria undergo spontaneous bursts of superoxide generation, termed “superoxide flashes”. Superoxide flashes occur randomly in space and time, exhibit all-or-none properties, and reflect elementary events of superoxide production within single mitochondria across a wide diversity of cells. Individual flashes are triggered by transient openings of the mitochondrial permeability transition pore (mPTP) and are fueled by electron transfer complexes-dependent superoxide production. While decreased during cardiac hypoxia/anoxia, a flurry of superoxide flash activity contributes to the destructive rebound ROS burst observed during early reoxygenation after anoxia. The discovery of superoxide flashes reveals a novel mechanism for quantal ROS production by individual mitochondria and substantiates the central role of mPTP in oxidative stress related pathology in addition to its well-known role in apoptosis.

The calcium ion (Ca(2+)) is the simplest and most versatile intracellular messenger known. The discovery of Ca(2+) sparks and a related family of elementary Ca(2+) signaling events has revealed fundamental principles of the Ca(2+) signaling system. A newly appreciated "digital" subsystem consisting of brief, high Ca(2+) concentration over short distances (nanometers to microns) comingles with an "analog" global Ca(2+) signaling subsystem. Over the past 15 years, much has been learned about the theoretical and practical aspects of spark formation and detection. The quest for the spark mechanisms [the activation, coordination, and termination of Ca(2+) release units (CRUs)] has met unexpected challenges, however, and raised vexing questions about CRU operation in situ. Ample evidence shows that Ca(2+) sparks catalyze many high-threshold Ca(2+) processes involved in cardiac and skeletal muscle excitation-contraction coupling, vascular tone regulation, membrane excitability, and neuronal secretion. Investigation of Ca(2+) sparks in diseases has also begun to provide novel insights into hypertension, cardiac arrhythmias, heart failure, and muscular dystrophy. An emerging view is that spatially and temporally patterned activation of the digital subsystem confers on intracellular Ca(2+) signaling an exquisite architecture in space, time, and intensity, which underpins signaling efficiency, stability, specificity, and diversity. These recent advances in "sparkology" thus promise to unify the simplicity and complexity of Ca(2+) signaling in biology.

Directional movement is a property common to all cell types during development and is critical to tissue remodelling and regeneration after damage1–3. In migrating cells, calcium plays a multifunctional role in directional sensing, cytoskeleton redistribution, traction force generation, and relocation of focal adhesions1, 4–7. Here we visualise, for the first time, high-calcium microdomains (“calcium flickers”), and their patterned activation in migrating fibroblasts. Calcium flicker activity is dually coupled to membrane tension (via TRPM7, a stretch-activated Ca2+-permeant channel of the transient receptor potential superfamily8) and chemoattractant signal transduction (via type 2 inositol 1,4,5-trisphosphate receptors). Interestingly, calcium flickers are most active at the leading lamella of migrating cells, displaying a 4:1 front-to-rear polarisation opposite to the global calcium gradient6. When exposed to a PDGF gradient perpendicular to cell movement, asymmetric calcium flicker activity develops across the lamella and promotes the turning of migrating fibroblasts. These findings illustrate how the exquisite spatiotemporal organisation of calcium microdomains can orchestrate complex cellular processes such as cell migration.

Abstract-Local, rhythmic, subsarcolemmal Ca 2ϩ releases (LCRs) from the sarcoplasmic reticulum (SR) during diastolic depolarization in sinoatrial nodal cells (SANC) occur even in the basal state and activate an inward Na ϩ -Ca 2ϩ exchanger current that affects spontaneous beating. Why SANC can generate spontaneous LCRs under basal conditions, whereas ventricular cells cannot, has not previously been explained. Here we show that a high basal cAMP level of isolated rabbit SANC and its attendant increase in protein kinase A (PKA)-dependent phosphorylation are obligatory for the occurrence of spontaneous, basal LCRs and for spontaneous beating. Gradations in basal PKA activity, indexed by gradations in phospholamban phosphorylation effected by a specific PKA inhibitory peptide were highly correlated with concomitant gradations in LCR spatiotemporal synchronization and phase, as well as beating rate. Higher levels of basal PKA inhibition abolish LCRs and spontaneous beating ceases. Stimulation of ␤-adrenergic receptors extends the range of PKA-dependent control of LCRs and beating rate beyond that in the basal state. The link between SR Ca 2ϩ cycling and beating rate is also present in vivo, as the regulation of beating rate by local ␤-adrenergic receptor stimulation of the sinoatrial node in intact dogs is markedly blunted when SR Ca 2ϩ cycling is disrupted by ryanodine. Thus, PKA-dependent phosphorylation of proteins that regulate cell Ca 2ϩ balance and spontaneous SR Ca 2ϩ cycling, ie, phospholamban and L-type Ca 2ϩ channels (and likely others not measured in this study), controls the phase and size of LCRs and the resultant Na ϩ -Ca 2ϩ exchanger current and is crucial for both basal and reserve cardiac pacemaker function. R ecent studies have demonstrated that in sinoatrial (SA) nodal cells (SANC) generate local, rhythmic, subsarcolemmal Ca 2ϩ releases (LCRs) under basal conditions, ie, even in the absence of experimental Ca 2ϩ loading or stimulation of ␤-adrenergic receptors (␤-ARs). [1][2][3] In rabbit SANC, spontaneous, rhythmic LCRs occur during the late diastolic depolarization and activate Na ϩ -Ca 2ϩ exchanger (NCX) to generate an inward current that accelerates the depolarization rate, and, thus, LCRs are involved in control of spontaneous beating rate of SANC. 1 The mechanisms that permit SANC, but not ventricular myocytes, to generate rhythmic LCRs under basal conditions, however, have not been delineated.Spontaneous SR Ca 2ϩ release is facilitated by factors that increase the rate at which the SR can pump Ca 2ϩ , foremost among which are elevated cell Ca 2ϩ or elevated cAMP and its attendant protein kinase A (PKA)-dependent protein phosphorylation that results from intense ␤-AR stimulation. Whereas the cytosolic Ca 2ϩ concentration does not appreciably differ in rabbit ventricular cells and SANC, 2,4 the cAMP level of the intact SA node is high, 5 and it has been suspected that the basal cAMP level within SANC is elevated. 6,7 The SA node, however, is highly innervated, and neither the basal cAMP le...

Developments in miniaturized microscopes have enabled visualization of brain activities and structural dynamics in animals engaging in self-determined behaviors. However, it remains a challenge to resolve activity at single dendritic spines in freely behaving animals. Here, we report the design and application of a fast high-resolution, miniaturized two-photon microscope (FHIRM-TPM) that accomplishes this goal. With a headpiece weighing 2.15 g and a hollow-core photonic crystal fiber delivering 920-nm femtosecond laser pulses, the FHIRM-TPM is capable of imaging commonly used biosensors (GFP and GCaMP6) at high spatiotemporal resolution (0.64 μm laterally and 3.35 μm axially, 40 Hz at 256 × 256 pixels for raster scanning and 10,000 Hz for free-line scanning). We demonstrate the microscope's robustness with hour-long recordings of neuronal activities at the level of spines in mice experiencing vigorous body movements.

Luminal Ca 2؉ in the endoplasmic and sarcoplasmic reticulum (ER͞ SR) plays an important role in regulating vital biological processes, including store-operated capacitative Ca 2؉ entry, Ca 2؉ -induced Ca 2؉ release, and ER͞SR stress-mediated cell death. We report rapid and substantial decreases in luminal [Ca 2؉ ], called ''Ca 2؉ blinks,'' within nanometer-sized stores (the junctional cisternae of the SR) during elementary Ca 2؉ release events in heart cells. Blinks mirror small local increases in cytoplasmic Ca 2؉ , or Ca 2؉ sparks, but changes of [Ca 2؉ ] in the connected free SR network were below detection. Store microanatomy suggests that diffusional strictures may account for this paradox. Surprisingly, the nadir of the store depletion trails the peak of the spark by about 10 ms, and the refilling of local store occurs with a rate constant of 35 s ؊1 , which is Ϸ6-fold faster than the recovery of local Ca 2؉ release after a spark. These data suggest that both local store depletion and some time-dependent inhibitory mechanism contribute to spark termination and refractoriness. Visualization of local store Ca 2؉ signaling thus broadens our understanding of cardiac store Ca 2؉ regulation and function and opens the possibility for local regulation of diverse store-dependent functions.calcium-induced calcium release ͉ calcium spark ͉ cardiac myocytes ͉ endoplasmic reticulum ͉ sarcoplasmic reticulum L ocal Ca 2ϩ releases from the endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) in muscle have been shown to underlie neurosecretion, memory encoding, neurite growth, muscle contraction, and apoptosis (1-4). Whereas the ER͞SR serves primarily as the intracellular Ca 2ϩ store, luminal Ca 2ϩ plays an active role in many regulatory systems, including store-operated capacitative Ca 2ϩ entry (5, 6), Ca 2ϩ -induced Ca 2ϩ release (7-9), and ER͞SR stress-mediated cell death (10, 11). Over the last decade, the elementary Ca 2ϩ release events have been directly visualized as Ca 2ϩ ''sparks'' (12-15), ''puffs'' (16), ''syntillas'' (17), or the equivalent (18) in the cytoplasm of both excitable and nonexcitable cells. However, the reciprocal store depletion signals, which were speculated in various models of spark termination (refs. 7 and 19; see ref. 20 for a review), have not been seen experimentally. In theory, a rapid refilling of local store Ca 2ϩ from the bulk of ER͞SR might occur and prevent significant local Ca 2ϩ depletion (21,22). Alternatively, this failing could be due to lack of a means to probe Ca 2ϩ inside this delicate membrane-bound intracellular structure with the required sensitivity, resolution, and speed, given the extremely small release flux involved (Ϸ2⅐10 Ϫ19 mol of Ca 2ϩ ) (12, 17). Using confocal imaging, electron microscopy, and electrophysiological approaches, we investigated dynamic Ca 2ϩ regulation inside nanometer-sized SR structures during elementary Ca 2ϩ release events in intact heart muscle cells. Our results afforded insights into mechanisms underlying spark termination and refract...

Rapid development of transgenic and gene-targeted mice and acute genetic manipulation via gene transfer vector systems have provided powerful tools for cardiovascular research. To facilitate the phenotyping of genetically engineered murine models at the cellular and subcellular levels and to implement acute gene transfer techniques in single mouse cardiomyocytes, we have modified and improved current enzymatic methods to isolate a high yield of high-quality adult mouse myocytes (5.3 +/- 0.5 x 10(5) cells/left ventricle, 83.8 +/- 2.5% rod shaped). We have also developed a technique to culture these isolated myocytes while maintaining their morphological integrity for 2-3 days. The high percentage of viable myocytes after 1 day in culture (72.5 +/- 2.3%) permitted both physiological and biochemical characterization. The major functional aspects of these cells, including excitation-contraction coupling and receptor-mediated signaling, remained intact, but the contraction kinetics were significantly slowed. Furthermore, gene delivery via recombinant adenoviral infection was highly efficient and reproducible. In adult beta(1)/beta(2)-adrenergic receptor (AR) double-knockout mouse myocytes, adenovirus-directed expression of either beta(1)- or beta(2)-AR, which occurred in 100% of cells, rescued the functional response to beta-AR agonist stimulation. These techniques will permit novel experimental settings for cellular genetic physiology.

Most excitable cells maintain tight control of intracellular Ca(2+) through coordinated interaction between plasma membrane and endoplasmic or sarcoplasmic reticulum. Quiescent sarcoplasmic reticulum Ca(2+) release machinery is essential for the survival and normal function of skeletal muscle. Here we show that subtle membrane deformations induce Ca(2+) sparks in intact mammalian skeletal muscle. Spontaneous Ca(2+) sparks can be reversibly induced by osmotic shock, and participate in a normal physiological response to exercise. In dystrophic muscle with fragile membrane integrity, stress-induced Ca(2+) sparks are essentially irreversible. Moreover, moderate exercise in mdx muscle alters the Ca(2+) spark response. Thus, membrane-deformation-induced Ca(2+) sparks have an important role in physiological and pathophysiological regulation of Ca(2+) signalling, and uncontrolled Ca(2+) spark activity in connection with chronic activation of store-operated Ca(2+) entry may function as a dystrophic signal in mammalian skeletal muscle.