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.

Despite current treatment regimens, heart failure remains the leading cause of morbidity and mortality in the developed world due to the limited capacity of adult mammalian ventricular cardiomyocytes to divide and replace ventricular myocardium lost from ischemia-induced infarct1,2. As a result, there is great interest to identify potential cellular sources and strategies to generate new ventricular myocardium3. Past studies have shown that lower vertebrate and early postnatal mammalian ventricular cardiomyocytes can proliferate to help regenerate injured ventricles4–6; however, recent studies have suggested that additional endogenous cellular sources may contribute to this overall ventricular regeneration3. Here, we have developed in the zebrafish a combination of fluorescent reporter transgenes, genetic fate-mapping strategies, and a ventricle-specific genetic ablation system to discover that differentiated atrial cardiomyocytes can transdifferentiate into ventricular cardiomyocytes to contribute to zebrafish cardiac ventricular regeneration. Using in vivo time-lapse and confocal imaging, we monitored the dynamic cellular events during atrial-to-ventricular cardiomyocyte transdifferentiation to define intermediate cardiac reprogramming stages. Importantly, we observed that Notch signaling becomes activated in the atrial endocardium following ventricular ablation, and discovered that inhibiting Notch signaling blocked the atrial-to-ventricular transdifferentiation and cardiac regeneration. Overall, these studies not only provide evidence for the plasticity of cardiac lineages during myocardial injury, but more importantly reveal an abundant new potential cardiac resident cellular source for cardiac ventricular regeneration.

Rationale: Unrepaired cardiomyocyte membrane injury causes irreplaceable cell loss, leading to myocardial fibrosis and eventually heart failure. However, the cellular and molecular mechanisms of cardiac membrane repair are largely unknown. MG53, a newly identified striated muscle-specific protein, is involved in skeletal muscle membrane repair. But the role of MG53 in the heart has not been determined. Objective: We sought to investigate whether MG53 mediates membrane repair in cardiomyocytes and, if so, the cellular and molecular mechanism underlying MG53-mediated membrane repair in cardiomyocytes. Moreover, we determined possible cardioprotective effect of MG53-mediated membrane repair. Methods and Results: We demonstrated that MG53 is crucial to the emergency membrane repair response in cardiomyocytes and protects the heart from stress-induced loss of cardiomyocytes. Disruption of the sarcolemmal membrane by mechanical, electric, chemical, or metabolic insults caused rapid and robust translocation of MG53 toward the injury sites. Ablation of MG53 prevented sarcolemmal resealing after infrared laser-induced membrane damage in intact heart, and exacerbated mitochondrial dysfunction and loss of cardiomyocytes during ischemia/reperfusion injury. Unexpectedly, the MG53-mediated cardiac membrane repair was mediated by a cholesterol-dependent mechanism: depletion of membrane cholesterol abolished, and its recovery restored injury-induced membrane translocation of MG53. The redox status of MG53 did not affect initiation of MG53 translocation, whereas MG53 oxidation conferred stability to the membrane repair patch. Conclusions: Thus, cholesterol-dependent MG53-mediated membrane repair is a vital, heretofore unappreciated cardioprotective mechanism against a multitude of insults and may bear important therapeutic implications. (Circ Res. 2010;107:76-83.)Key Words: membrane repair Ⅲ MG53 Ⅲ cholesterol Ⅲ ischemia/reperfusion injury Ⅲ heart I n eukaryotic cells, the plasma membrane partitions a Ϸ10 000-fold Ca 2ϩ gradient and prevents loss of vital intracellular constituents, thus representing the last line of defense for cell integrity, homeostasis, and function. Physical, chemical or metabolic disruption of the plasma membrane leads to a repairor-die emergency of the cell. Although the natural tendency to reseal the lipid biomembrane acts constitutively, recent studies indicate that plasma membrane disruption requires active emergency response mechanisms to mend the broken membrane. 1 In the heart, plasma membrane repair is of particular importance because cardiomyocytes are terminally differentiated cells, displaying only very limited self-renewal capability. 2 Cardiomyocytes undergo transient membrane injuries that occur as accidents under physiological conditions and can be exacerbated by various pathophysiological stresses. 3 Progressive necrotic and apoptotic cell death causes onset of myocardial fibrosis and undermines cardiac contractile and electrophysiological performance, ultimately leading to heart failure....

While the adult human heart has very limited regenerative potential, the adult zebrafish heart can fully regenerate after 20% ventricular resection. Although previous reports suggest that developmental signaling pathways such as FGF and PDGF are reused in adult heart regeneration, the underlying intracellular mechanisms remain largely unknown. Here we show that H 2 O 2 acts as a novel epicardial and myocardial signal to prime the heart for regeneration in adult zebrafish. Live imaging of intact hearts revealed highly localized H 2 O 2 (~30 µM) production in the epicardium and adjacent compact myocardium at the resection site. Decreasing H 2 O 2 formation with the Duox inhibitors diphenyleneiodonium (DPI) or apocynin, or scavenging H 2 O 2 by catalase overexpression markedly impaired cardiac regeneration while exogenous H 2 O 2 rescued the inhibitory effects of DPI on cardiac regeneration, indicating that H 2 O 2 is an essential and sufficient signal in this process. Mechanistically, elevated H 2 O 2 destabilized the redox-sensitive phosphatase Dusp6 and hence increased the phosphorylation of Erk1/2. The Dusp6 inhibitor BCI achieved similar pro-regenerative effects while transgenic overexpression of dusp6 impaired cardiac regeneration. H 2 O 2 plays a dual role in recruiting immune cells and promoting heart regeneration through two relatively independent pathways. We conclude that H 2 O 2 potentially generated from Duox/Nox2 promotes heart regeneration in zebrafish by unleashing MAP kinase signaling through a derepression mechanism involving Dusp6.

Pacemaker cardiomyocytes that create the sinoatrial node are essential for the initiation and maintenance of proper heart rhythm. However, illuminating developmental cues that direct their differentiation has remained particularly challenging due to the unclear cellular origins of these specialized cardiomyocytes. By discovering the origins of pacemaker cardiomyocytes, we reveal an evolutionarily conserved Wnt signaling mechanism that coordinates gene regulatory changes directing mesoderm cell fate decisions, which lead to the differentiation of pacemaker cardiomyocytes. We show that in zebrafish, pacemaker cardiomyocytes derive from a subset of Nkx2.5+ mesoderm that responds to canonical Wnt5b signaling to initiate the cardiac pacemaker program, including activation of pacemaker cell differentiation transcription factors Isl1 and Tbx18 and silencing of Nkx2.5. Moreover, applying these developmental findings to human pluripotent stem cells (hPSCs) notably results in the creation of hPSC-pacemaker cardiomyocytes, which successfully pace threedimensional bioprinted hPSC-cardiomyocytes, thus providing potential strategies for biological cardiac pacemaker therapy.

Many organs are composed of complex tissue walls that are structurally organized to optimize organ function. In particular, the ventricular myocardial wall of the heart is comprised of an outer compact layer that concentrically encircles the ridge-like inner trabecular layer. Although disruption in the morphogenesis of this myocardial wall can lead to various forms of congenital heart disease (CHD)1 and non-compaction cardiomyopathies2, it remains unclear how embryonic cardiomyocytes assemble to form ventricular wall layers of appropriate spatial dimensions and myocardial mass. Here, we utilize advanced genetic and imaging tools in zebrafish to reveal an interplay between myocardial Notch and Erbb2 signaling that directs the spatial allocation of myocardial cells to their proper morphologic positions in the ventricular wall. Although previous studies have shown that endocardial Notch signaling non-cell-autonomously promotes myocardial trabeculation through Erbb2 and BMP signaling3, we discover that distinct ventricular cardiomyocyte clusters exhibit myocardial Notch activity that cell-autonomously inhibits Erbb2 signaling and prevents cardiomyocyte sprouting and trabeculation. Myocardial-specific Notch inactivation leads to ventricles of reduced size and increased wall thickness due to excessive trabeculae, whereas widespread myocardial Notch activity results in ventricles of increased size with a single-cell thick wall but no trabeculae. Notably, this myocardial Notch signaling is activated non-cell-autonomously by neighboring Erbb2-activated cardiomyocytes that sprout and form nascent trabeculae. Thus, these findings support an interactive cellular feedback process that guides the assembly of cardiomyocytes to morphologically create the ventricular myocardial wall and more broadly provides insight into the cellular dynamics of how diverse cell lineages organize to create form.

During inflammation, the proper inflammatory infiltration of neutrophils is crucial for the host to fight against infections and remove damaged cells and detrimental substances. IL-1β and NADPH oxidase–mediated reactive oxygen species (ROS) have been implicated to play important roles in this process. However, the cellular and molecular basis underlying the actions of IL-1β and ROS and their relationship during inflammatory response remains undefined. In this study, we use the zebrafish model to investigate these issues. We find that, similar to that of NADPH oxidase–mediated ROS signaling, the Il-1β–Myd88 pathway is required for the recruitment of neutrophils, but not macrophages, to the injury-induced inflammatory site, whereas it is dispensable for bacterial-induced inflammation. Interestingly, the Il-1β–Myd88 pathway is independent of NADPH oxidase–mediated ROS signaling and critical for the directional migration, but not the basal random movement, of neutrophils. In contrast, the NADPH oxidase–mediated ROS signaling is required for both basal random movement and directional migration of neutrophils. We further document that ectopic expression of Il-1β in zebrafish induces an inflammatory disorder, which can be suppressed by anti-inflammatory treatment. Our findings reveal that the Il-1β–Myd88 axis and NADPH oxidase–mediated ROS signaling are two independent pathways that differentially regulate neutrophil migration during sterile inflammation. In addition, Il-1β overexpressing Tg(hsp70:mil-1β_eGFP;lyz:DsRed2)hkz10t;nz50 transgenic zebrafish provides a useful animal model for the study of chronic inflammatory disorder and for anti-inflammatory drug discovery.

Rationale: Rad (Ras associated with diabetes) GTPase, a monomeric small G protein, binds to Ca v ␤ subunit of the L-type Ca 2؉ channel (LCC) and thereby regulates LCC trafficking and activity. Emerging evidence suggests that Rad is an important player in cardiac arrhythmogenesis and hypertrophic remodeling. However, whether and how Rad involves in the regulation of excitation-contraction (EC) coupling is unknown. Objective: This study aimed to investigate possible role of Rad in cardiac EC coupling and ␤-adrenergic receptor (␤AR) inotropic mechanism. Methods and Results: Adenoviral overexpression of Rad by 3-fold in rat cardiomyocytes suppressed LCC current (I Ca ), [Ca 2؉ ] i transients, and contractility by 60%, 42%, and 38%, respectively, whereas the "gain" function of EC coupling was significantly increased, due perhaps to reduced "redundancy" of LCC in triggering sarcoplasmic reticulum release. Conversely, Ϸ70% Rad knockdown by RNA interference increased I Ca (50%), [Ca 2؉ ] i transients (52%) and contractility (58%) without altering EC coupling efficiency; and the dominant negative mutant RadS105N exerted a similar effect on I Ca . Rad upregulation caused depolarizing shift of LCC activation and hastened time-dependent LCC inactivation; Rad downregulation, however, failed to alter these attributes. Key Words: Rad Ⅲ GTPase Ⅲ Ca 2ϩ signaling Ⅲ excitation-contraction coupling Ⅲ ␤-adrenergic signaling C ardiac excitation-contraction (EC) coupling is mainly mediated by intermolecular signaling between two types of Ca 2ϩ channels, the voltage-gated L-type Ca 2ϩ channel (LCC), and the ryanodine receptor (RyR) Ca 2ϩ release channel that reside in the plasma membrane and the sarcoplasmic reticulum (SR), respectively. During EC coupling, LCC Ca 2ϩ influx activates a large number of "Ca 2ϩ sparks" 1 from clusters of RyRs, via the Ca 2ϩ -induced Ca 2ϩ release mechanism. 2 Summation of Ca 2ϩ sparks across the cell gives rise to an intracellular Ca 2ϩ transient that signals contractile myofilaments to generate force and movement. Return to the diastolic Ca 2ϩ level, to relax the muscle, is controlled by Ca 2ϩ cycling via the SR Ca 2ϩ -ATPase (SERCA) and, to a lesser extent, the sarcolemmal Na ϩ /Ca 2ϩ exchanger (NCX). Albeit controversial, recurrent evidence also suggests the involvement of trigger mechanism other than LCC. In particular, it has been suggested that reverse NCX allosterically activated by LCC current augments the trigger Ca 2ϩ at high membrane voltage. 3 Rad (Ras associated with diabetes), a monomeric small G protein that was initially identified by subtractive cloning as genes overexpressed in the skeletal muscle of a subset of patients with type 2 diabetes, is expressed most abundantly in the heart, 4 along with its cousin Rem, but not Gem/Kir 5 in the RGK family. At the molecular level, Rad comprises multiple functional domains including calmodulin binding and 14-3-3 protein-binding domains, as well as regulatory phosphorylation sites. 6,7 A common feature of Rad and other RGK proteins is to bin...