Trajectory mapping of human embryonic stem cell cardiogenesis reveals lineage branch points and an ISL1 progenitor-derived cardiac fibroblast lineage.

A family of multipotent heart progenitors plays a central role in the generation of diverse myogenic and nonmyogenic lineages in the heart. Cardiac progenitors in particular play a significant role in lineages involved in disease, and have also emerged to be a strong therapeutic candidate. Based on this premise, we aimed to deeply characterize the progenitor stage of cardiac differentiation at a single-cell resolution. Integrated comparison with an embryonic 5-week human heart transcriptomic dataset validated lineage identities with their late stage in vitro counterparts, highlighting the relevance of an in vitro differentiation for progenitors that are developmentally too early to be accessed in vivo. We utilized trajectory mapping to elucidate progenitor lineage branching points, which are supported by RNA velocity. Nonmyogenic populations, including cardiac fibroblast-like cells and endoderm, were found, and we identified TGFBI as a candidate marker for human cardiac fibroblasts in vivo and in vitro. Both myogenic and nonmyogenic populations express ISL1, and its loss redirected myogenic progenitors into a neural-like fate. Our study provides important insights into processes during early heart development.

Genome-wide CRISPR screen identifies ZIC2 as an essential gene that controls the cell fate of early mesodermal precursors to human heart progenitors.

Cardiac progenitor formation is one of the earliest committed steps of human cardiogenesis and requires the cooperation of multiple gene sets governed by developmental signaling cascades. To determine the key regulators for cardiac progenitor formation, we have developed a two-stage genome-wide CRISPR-knockout screen. We mimicked the progenitor formation process by differentiating human pluripotent stem cells (hPSCs) into cardiomyocytes, monitored by two distinct stage markers of early cardiac mesodermal formation and commitment to a multipotent heart progenitor cell fate: MESP1 and ISL1, respectively. From the screen output, we compiled a list of 15 candidate genes. After validating seven of them, we identified ZIC2 as an essential gene for cardiac progenitor formation. ZIC2 is known as a master regulator of neurogenesis. hPSCs with ZIC2 mutated still express pluripotency markers. However, their ability to differentiate into cardiomyocytes was greatly attenuated. RNA-Seq profiling of the ZIC2-mutant cells revealed that the mutants switched their cell fate alternatively to the noncardiac cell lineage. Further, single cell RNA-seq analysis showed the ZIC2 mutants affected the apelin receptor-related signaling pathway during mesoderm formation. Our results provide a new link between ZIC2 and human cardiogenesis and document the potential power of a genome-wide unbiased CRISPR-knockout screen to identify the key steps in human mesoderm precursor cell- and heart progenitor cell-fate determination during in vitro hPSC cardiogenesis.

SMAD4 Is Essential for Human Cardiac Mesodermal Precursor Cell Formation.

Understanding stage-specific molecular mechanisms of human cardiomyocyte (CM) progenitor formation and subsequent differentiation are critical to identify pathways that might lead to congenital cardiovascular defects and malformations. In particular, gene mutations in the transforming growth factor (TGF)ß superfamily signaling pathways can cause human congenital heart defects, and murine loss of function studies of a central component in this pathway, Smad4, leads to early embryonic lethality. To define the role of SMAD4 at the earliest stages of human cardiogenesis, we generated SMAD4 mutant human embryonic stem cells (hESCs). Herein, we show that the loss of SMAD4 has no effect on hESC self-renewal, or neuroectoderm formation, but is essential for the formation of cardiac mesoderm, with a subsequent complete loss of CM formation during human ES cell cardiogenesis. Via transcriptional profiling, we show that SMAD4 mutant cell lines fail to generate cardiac mesodermal precursors, clarifying a role of NODAL/SMAD4 signaling in cardiac mesodermal precursor formation via enhancing the expression of primitive streak genes. Since SMAD4 relative pathways have been linked to congenital malformations, it will become of interest to determine whether these may due, in part, to defective cell fate decision during cardiac mesodermal precursor formation. Stem Cells 2018 Stem Cells 2019;37:216-225.

Human ISL1+ Ventricular Progenitors Self-Assemble into an In Vivo Functional Heart Patch and Preserve Cardiac Function Post Infarction.

The generation of human pluripotent stem cell (hPSC)-derived ventricular progenitors and their assembly into a 3-dimensional in vivo functional ventricular heart patch has remained an elusive goal. Herein, we report the generation of an enriched pool of hPSC-derived ventricular progenitors (HVPs), which can expand, differentiate, self-assemble, and mature into a functional ventricular patch in vivo without the aid of any gel or matrix. We documented a specific temporal window, in which the HVPs will engraft in vivo. On day 6 of differentiation, HVPs were enriched by depleting cells positive for pluripotency marker TRA-1-60 with magnetic-activated cell sorting (MACS), and 3 million sorted cells were sub-capsularly transplanted onto kidneys of NSG mice where, after 2 months, they formed a 7 mm × 3 mm × 4 mm myocardial patch resembling the ventricular wall. The graft acquired several features of maturation: expression of ventricular marker (MLC2v), desmosomes, appearance of T-tubule-like structures, and electrophysiological action potential signature consistent with maturation, all this in a non-cardiac environment. We further demonstrated that HVPs transplanted into un-injured hearts of NSG mice remain viable for up to 8 months. Moreover, transplantation of 2 million HVPs largely preserved myocardial contractile function following myocardial infarction. Taken together, our study reaffirms the promising idea of using progenitor cells for regenerative therapy.

Absence of physiological Ca2+ transients is an initial trigger for mitochondrial dysfunction in skeletal muscle following denervation.

BACKGROUND: Motor neurons control muscle contraction by initiating action potentials in muscle. Denervation of muscle from motor neurons leads to muscle atrophy, which is linked to mitochondrial dysfunction. It is known that denervation promotes mitochondrial reactive oxygen species (ROS) production in muscle, whereas the initial cause of mitochondrial ROS production in denervated muscle remains elusive. Since denervation isolates muscle from motor neurons and deprives it from any electric stimulation, no action potentials are initiated, and therefore, no physiological Ca2+ transients are generated inside denervated muscle fibers. We tested whether loss of physiological Ca2+ transients is an initial cause leading to mitochondrial dysfunction in denervated skeletal muscle. METHODS: A transgenic mouse model expressing a mitochondrial targeted biosensor (mt-cpYFP) allowed a real-time measurement of the ROS-related mitochondrial metabolic function following denervation, termed "mitoflash." Using live cell imaging, electrophysiological, pharmacological, and biochemical studies, we examined a potential molecular mechanism that initiates ROS-related mitochondrial dysfunction following denervation. RESULTS: We found that muscle fibers showed a fourfold increase in mitoflash activity 24 h after denervation. The denervation-induced mitoflash activity was likely associated with an increased activity of mitochondrial permeability transition pore (mPTP), as the mitoflash activity was attenuated by application of cyclosporine A. Electrical stimulation rapidly reduced mitoflash activity in both sham and denervated muscle fibers. We further demonstrated that the Ca2+ level inside mitochondria follows the time course of the cytosolic Ca2+ transient and that inhibition of mitochondrial Ca2+ uptake by Ru360 blocks the effect of electric stimulation on mitoflash activity. CONCLUSIONS: The loss of cytosolic Ca2+ transients due to denervation results in the downstream absence of mitochondrial Ca2+ uptake. Our studies suggest that this could be an initial trigger for enhanced mPTP-related mitochondrial ROS generation in skeletal muscle.

Protons Trigger Mitochondrial Flashes.

Emerging evidence indicates that mitochondrial flashes (mitoflashes) are highly conserved elemental mitochondrial signaling events. However, which signal controls their ignition and how they are integrated with other mitochondrial signals and functions remain elusive. In this study, we aimed to further delineate the signal components of the mitoflash and determine the mitoflash trigger mechanism. Using multiple biosensors and chemical probes as well as label-free autofluorescence, we found that the mitoflash reflects chemical and electrical excitation at the single-organelle level, comprising bursting superoxide production, oxidative redox shift, and matrix alkalinization as well as transient membrane depolarization. Both electroneutral H(+)/K(+) or H(+)/Na(+) antiport and matrix proton uncaging elicited immediate and robust mitoflash responses over a broad dynamic range in cardiomyocytes and HeLa cells. However, charge-uncompensated proton transport, which depolarizes mitochondria, caused the opposite effect, and steady matrix acidification mildly inhibited mitoflashes. Based on a numerical simulation, we estimated a mean proton lifetime of 1.42 ns and diffusion distance of 2.06 nm in the matrix. We conclude that nanodomain protons act as a novel, to our knowledge, trigger of mitoflashes in energized mitochondria. This finding suggests that mitoflash genesis is functionally and mechanistically integrated with mitochondrial energy metabolism.

Identification of EFHD1 as a novel Ca(2+) sensor for mitoflash activation.

Mitochondrial flashes (mitoflashes) represent stochastic and discrete mitochondrial events that each comprises a burst of superoxide production accompanied by transient depolarization and matrix alkalinization in a respiratory mitochondrion. While mitochondrial Ca(2+) is shown to be an important regulator of mitoflash activity, little is known about its specific mechanism of action. Here we sought to determine possible molecular players that mediate the Ca(2+) regulation of mitoflashes by screening mitochondrial proteins containing the Ca(2+)-binding motifs. In silico analysis and targeted siRNA screening identified four mitoflash activators (MICU1, EFHD1, SLC25A23, SLC25A25) and one mitoflash inhibitor (LETM1) in terms of their ability to modulate mitoflash response to hyperosmotic stress. In particular, overexpression or down-regulation of EFHD1 enhanced or depressed mitoflash activation, respectively, under various conditions of mitochondrial Ca(2+) elevations. Yet, it did not alter mitochondrial Ca(2+) handling, mitochondrial respiration, or ROS-induced mitoflash production. Further, disruption of the two EF-hand motifs of EFHD1 abolished its potentiating effect on the mitoflash responses. These results indicate that EFHD1 functions as a novel mitochondrial Ca(2+) sensor underlying Ca(2+)-dependent activation of mitoflashes.

Endothelin-1 supports clonal derivation and expansion of cardiovascular progenitors derived from human embryonic stem cells.

Coronary arteriogenesis is a central step in cardiogenesis, requiring coordinated generation and integration of endothelial cell and vascular smooth muscle cells. At present, it is unclear whether the cell fate programme of cardiac progenitors to generate complex muscular or vascular structures is entirely cell autonomous. Here we demonstrate the intrinsic ability of vascular progenitors to develop and self-organize into cardiac tissues by clonally isolating and expanding second heart field cardiovascular progenitors using WNT3A and endothelin-1 (EDN1) human recombinant proteins. Progenitor clones undergo long-term expansion and differentiate primarily into endothelial and smooth muscle cell lineages in vitro, and contribute extensively to coronary-like vessels in vivo, forming a functional human-mouse chimeric circulatory system. Our study identifies EDN1 as a key factor towards the generation and clonal derivation of ISL1(+) vascular intermediates, and demonstrates the intrinsic cell-autonomous nature of these progenitors to differentiate and self-organize into functional vasculatures in vivo.

Cyclophilin D regulates mitochondrial flashes and metabolism in cardiac myocytes.

Cyclophilin D (CyP-D) is the mitochondrial-specific member of the evolutionally conserved cyclophilin family, and plays an important role in the regulation of mitochondrial permeability transition (MPT) under stress. Recently we have demonstrated that respiratory mitochondria undergo mitochondrial flash ("mitoflash") activity which is coupled with transient MPT under physiological conditions. However, whether and how CyP-D regulates mitoflashes remain incompletely understood. By using both loss- and gain-of-function approaches in isolated cardiomyocytes, beating hearts, and skeletal muscles in living mice, we revisited the role of CyP-D in the regulation of mitoflashes. Overexpression of CyP-D increased, and knockout of it halved, cardiac mitoflash frequency, while mitoflash amplitude and kinetics remained unaffected. However, CyP-D ablation did not alter mitoflash frequency, with mitoflash amplitude increased, in gastrocnemius muscles. This disparity was accompanied by 4-fold higher CyP-D expression in mouse cardiac than skeletal muscles. The mitochondrial maximal respiration rate and reserved capacity were reduced in CyP-D-null cardiomyocytes. These data indicate that CyP-D is a significant regulator of mitoflash ignition and mitochondrial metabolism in heart. In addition, tissue-specific CyP-D expression may partly explain the differential regulation of mitoflashes in the two types of striated muscles.

Remodeling of Mitochondrial Flashes in Muscular Development and Dystrophy in Zebrafish.

Mitochondrial flash (mitoflash) is a highly-conserved, universal, and physiological mitochondrial activity in isolated mitochondria, intact cells, and live organisms. Here we investigated developmental and disease-related remodeling of mitoflash activity in zebrafish skeletal muscles. In transgenic zebrafish expressing the mitoflash reporter cpYFP, in vivo imaging revealed that mitoflash frequency and unitary properties underwent multiphasic and muscle type-specific changes, accompanying mitochondrial morphogenesis from 2 to 14 dpf. In particular, short (S)-type mitoflashes predominated in early muscle formation, then S-, transitory (T)- and regular (R)-type mitoflashes coexisted during muscle maturation, followed by a switch to R-type mitoflashes in mature skeletal muscles. In early development of muscular dystrophy, we found accelerated S- to R-type mitoflash transition and reduced mitochondrial NAD(P)H amidst a remarkable cell-to-cell heterogeneity. This study not only unravels a profound functional and morphological remodeling of mitochondria in developing and diseased skeletal muscles, but also underscores mitoflashes as a useful reporter of mitochondrial function in milieu of live animals under physiological and pathophysiological conditions.

Next-generation models of human cardiogenesis via genome editing.

Cardiogenesis is one of the earliest and most important steps during human development and is orchestrated by discrete families of heart progenitors, which build distinct regions of the fetal heart. For the past decade, a lineage map for the distinct subsets of progenitors that generate the embryonic mammalian heart has begun to lay a foundation for the development of new strategies for rebuilding the adult heart after injury, an unmet clinical need for the vast majority of patients with end-stage heart failure who are not heart transplant recipients. The studies also have implications for the root causes of congenital heart disease, which affects 1 in 50 live births, the most prevalent malformations in children. Although much of this insight has been generated in murine models, it is becoming increasingly clear that there can be important divergence with principles and pathways for human cardiogenesis, as well as for regenerative pathways. The development of human stem cell models, coupled with recent advances in genome editing with RNA-guided endonucleases, offers a new approach for the primary study of human cardiogenesis. In addition, application of the technology to the in vivo setting in large animal models, including nonhuman primates, has opened the door to genome-edited large animal models of adult and congenital heart disease, as well as a detailed mechanistic dissection of the more diverse and complex set of progenitor families and pathways, which guide human cardiogenesis. Implications of this new technology for a new generation of human-based, genetically tractable systems are discussed, along with potential therapeutic applications.

Mitoflash frequency in early adulthood predicts lifespan in Caenorhabditis elegans.

It has been theorized for decades that mitochondria act as the biological clock of ageing, but the evidence is incomplete. Here we show a strong coupling between mitochondrial function and ageing by in vivo visualization of the mitochondrial flash (mitoflash), a frequency-coded optical readout reflecting free-radical production and energy metabolism at the single-mitochondrion level. Mitoflash activity in Caenorhabditis elegans pharyngeal muscles peaked on adult day 3 during active reproduction and on day 9 when animals started to die off. A plethora of genetic mutations and environmental factors inversely modified the lifespan and the day-3 mitoflash frequency. Even within an isogenic population, the day-3 mitoflash frequency was negatively correlated with the lifespan of individual animals. Furthermore, enhanced activity of the glyoxylate cycle contributed to the decreased day-3 mitoflash frequency and the longevity of daf-2 mutant animals. These results demonstrate that the day-3 mitoflash frequency is a powerful predictor of C. elegans lifespan across genetic, environmental and stochastic factors. They also support the notion that the rate of ageing, although adjustable in later life, has been set to a considerable degree before reproduction ceases.

Imaging Ca2+ nanosparks in heart with a new targeted biosensor.

RATIONALE: In cardiac dyads, junctional Ca2+ directly controls the gating of the ryanodine receptors (RyRs), and is itself dominated by RyR-mediated Ca2+ release from the sarcoplasmic reticulum. Existing probes do not report such local Ca2+ signals because of probe diffusion, so a junction-targeted Ca2+ sensor should reveal new information on cardiac excitation-contraction coupling and its modification in disease states. OBJECTIVE: To investigate Ca2+ signaling in the nanoscopic space of cardiac dyads by targeting a new sensitive Ca2+ biosensor (GCaMP6f) to the junctional space. METHODS AND RESULTS: By fusing GCaMP6f to the N terminus of triadin 1 or junctin, GCaMP6f-triadin 1/junctin was targeted to dyadic junctions, where it colocalized with t-tubules and RyRs after adenovirus-mediated gene transfer. This membrane protein-tagged biosensor displayed ≈4× faster kinetics than native GCaMP6f. Confocal imaging revealed junctional Ca2+ transients (Ca2+ nanosparks) that were ≈50× smaller in volume than conventional Ca2+ sparks (measured with diffusible indicators). The presence of the biosensor did not disrupt normal Ca2+ signaling. Because no indicator diffusion occurred, the amplitude and timing of release measurements were improved, despite the small recording volume. We could also visualize coactivation of subclusters of RyRs within a single junctional region, as well as quarky Ca2+ release events. CONCLUSIONS: This new, targeted biosensor allows selective visualization and measurement of nanodomain Ca2+ dynamics in intact cells and can be used to give mechanistic insights into dyad RyR operation in health and in disease states such as when RyRs become orphaned.

Proinflammatory Cytokines Stimulate Mitochondrial Superoxide Flashes in Articular Chondrocytes In Vitro and In Situ.

OBJECTIVE: Mitochondria play important roles in many types of cells. However, little is known about mitochondrial function in chondrocytes. This study was undertaken to explore possible role of mitochondrial oxidative stress in inflammatory response in articular chondrocytes. METHODS: Chondrocytes and cartilage explants were isolated from wild type or transgenic mice expressing the mitochondrial superoxide biosensor - circularly permuted yellow fluorescent protein (cpYFP). Cultured chondrocytes or cartilage explants were incubated in media containing interleukin-1ß (10 ng/ml) or tumor necrosis factor-α (10 ng/ml) to stimulate an inflammatory response. Mitochondrial imaging was carried out by confocal and two-photon microscopy. Mitochondrial oxidative status was evaluated by "superoxide flash" activity recorded with time lapse scanning. RESULTS: Cultured chondrocytes contain abundant mitochondria that show active motility and dynamic morphological changes. In intact cartilage, mitochondrial abundance as well as chondrocyte density declines with distance from the surface. Importantly, sudden, bursting superoxide-producing events or "superoxide flashes" occur at single-mitochondrion level, accompanied by transient mitochondrial swelling and membrane depolarization. The superoxide flash incidence in quiescent chondrocytes was ∼4.5 and ∼0.5 events/1000 µm(2)*100 s in vitro and in situ, respectively. Interleukin-1ß or tumor necrosis factor-α stimulated mitochondrial superoxide flash activity by 2-fold in vitro and 5-fold in situ, without altering individual flash properties except for reduction in spatial size due to mitochondrial fragmentation. CONCLUSIONS: The superoxide flash response to proinflammatory cytokine stimulation in vitro and in situ suggests that chondrocyte mitochondria are a significant source of cellular oxidants and are an important previously under-appreciated mediator in inflammatory cartilage diseases.

Superoxide constitutes a major signal of mitochondrial superoxide flash.

AIMS: Mitochondrial flashes detected with an N- and C-terminal circularly-permuted yellow fluorescent protein (cpYFP) have been thought to represent transient and quantal bursts of superoxide production under physiological, stressful and pathophysiological conditions. However, the superoxide nature of the cpYFP-flash has been challenged, considering the pH-sensitivity of cpYFP and the distinctive regulation of the flash versus the basal production of mitochondrial reactive oxygen species (ROS). Thus, the aim of the study is to further determine the origin of mitochondrial flashes. MAIN METHODS: We investigated the origin of the flashes using the widely-used pH-insensitive ROS indicators, mitoSOX, an indicator for superoxide, and 2, 7-dichlorodihydrofluorescein diacetate (DCF), an indicator for H2O2 and other oxidants. KEY FINDINGS: Robust, quantal, and stochastic mitochondrial flashes were detected with either mitoSOX or DCF in several cell-types and in mitochondria isolated from the heart. Both mitoSOX-flashes and DCF-flashes showed similar incidence and kinetics to those of cpYFP-flashes, and were equally sensitive to mitochondria-targeted antioxidants. Furthermore, they were markedly decreased by inhibitors or an uncoupler of the mitochondrial electron transport chain, as is the case with cpYFP-flashes. The involvement of the mitochondrial permeability transition pore in DCF-flashes was evidenced by the coincidental loss of mitochondrial membrane potential and matrix-enriched rhod-2, as well as by their sensitivity to cyclosporine A. SIGNIFICANCE: These data indicate that all the three types of mitochondrial flashes stem from the common physiological process of bursting superoxide and ensuing H2O2 production in the matrix of single mitochondrion.

Kissing and nanotunneling mediate intermitochondrial communication in the heart.

Mitochondria in many types of cells are dynamically interconnected through constant fusion and fission, allowing for exchange of mitochondrial contents and repair of damaged mitochondria. However, constrained by the myofibril lattice, the ∼6,000 mitochondria in the adult mammalian cardiomyocyte display little motility, and it is unclear how, if at all, they communicate with each other. By means of target-expressing photoactivatable green fluorescent protein (PAGFP) in the mitochondrial matrix or on the outer mitochondrial membrane, we demonstrated that the local PAGFP signal propagated over the entire population of mitochondria in cardiomyocytes on a time scale of ∼10 h. Two elemental steps of intermitochondrial communications were manifested as either a sudden PAGFP transfer between a pair of adjacent mitochondria (i.e., "kissing") or a dynamic nanotubular tunnel (i.e., "nanotunneling") between nonadjacent mitochondria. The average content transfer index (fractional exchange) was around 0.5; the rate of kissing was 1‰ s(-1) per mitochondrial pair, and that of nanotunneling was about 14 times smaller. Electron microscopy revealed extensive intimate contacts between adjacent mitochondria and elongated nanotubular protrusions, providing a structural basis for the kissing and nanotunneling, respectively. We propose that, through kissing and nanotunneling, the otherwise static mitochondria in a cardiomyocyte form one dynamically continuous network to share content and transfer signals.

Synergistic triggering of superoxide flashes by mitochondrial Ca2+ uniport and basal reactive oxygen species elevation.

Mitochondrial superoxide flashes reflect a quantal, bursting mode of reactive oxygen species (ROS) production that arises from stochastic, transient opening of the mitochondrial permeability transition pore (mPTP) in many types of cells and in living animals. However, the regulatory mechanisms and the exact nature of the flash-coupled mPTP remain poorly understood. Here we demonstrate a profound synergistic effect between mitochondrial Ca(2+) uniport and elevated basal ROS production in triggering superoxide flashes in intact cells. Hyperosmotic stress potently augmented the flash activity while simultaneously elevating mitochondrial Ca(2+) and ROS. Blocking mitochondrial Ca(2+) transport by knockdown of MICU1 or MCU, newly identified components of the mitochondrial Ca(2+) uniporter, or scavenging mitochondrial basal ROS markedly diminished the flash response. More importantly, whereas elevating Ca(2+) or ROS production alone was inefficacious in triggering the flashes, concurrent physiological Ca(2+) and ROS elevation served as the most powerful flash activator, increasing the flash incidence by an order of magnitude. Functionally, superoxide flashes in response to hyperosmotic stress participated in the activation of JNK and p38. Thus, physiological levels of mitochondrial Ca(2+) and ROS synergistically regulate stochastic mPTP opening and quantal ROS production in intact cells, marking the flash as a coincidence detector of mitochondrial Ca(2+) and ROS signals.

Superoxide flashes: elemental events of mitochondrial ROS signaling in the heart.

The role of mitochondrial reactive oxygen species (mitoROS) in cellular function remains obscure. By synthesizing recent data, we propose here that local dynamic mitoROS in the form of "superoxide flashes" serve as "signaling ROS" rather than "homeostatic ROS", distinguishable from basal mitoROS due to constitutive leakage of the electron transfer chain (ETC). Individual superoxide flashes are 10-s mitoROS bursts that are compartmentalized to a single mitochondrion or local mitochondrial networks. As a highly-conserved universal mitochondrial activity, it occurs in intact cells, in ex vivo beating hearts, and even in living animals. Unlike basal mitoROS, superoxide flashes are ignited by transient openings of a type of mitochondrial permeability transition pore (mPTP), and their incidence is richly regulated by an array of factors that converge on either the mPTP or ETC. Emerging evidence has shown that superoxide flashes decode dietary and metabolic status or exercise, gauge oxidative stress (e.g., during reoxygenation after hypoxia or anoxia), and constitute early mitochondrial signals that initiate oxidative stress-related apoptosis in a context-dependent manner. That they make only a miniscule contribution to global ROS attests to the high efficiency of local ROS signaling. However, the exact mechanisms underlying superoxide flash formation, regulation and function remain uncertain. Future investigation is warranted to uncover the cellular logic and molecular pathways of local dynamic mitoROS signaling in heart muscle cells and many other cell types.