Major features of the transcellular signaling mechanism responsible for endothelium-dependent regulation of vascular smooth muscle tone are unresolved. We identified local calcium (Ca2+) signals (“sparklets”) in the vascular endothelium of resistance arteries that represent Ca2+ influx through single TRPV4 cation channels. Gating of individual TRPV4 channels within a four-channel cluster was cooperative, with activation of as few as three channels per cell causing maximal dilation through activation of endothelial cell intermediate (IK)- and small (SK)-conductance, Ca2+-sensitive potassium (K+) channels. Endothelial-dependent muscarinic receptor signaling also acted largely through TRPV4 sparklet-mediated stimulation of IK and SK channels to promote vasodilation. These results support the concept that Ca2+ influx through single TRPV4 channels is leveraged by the amplifier effect of cooperative channel gating and the high Ca2+ sensitivity of IK and SK channels to cause vasodilation.

Transplantation studies in mice and rats have shown that human embryonic stem cell-derived cardiomyocytes (hESC-CMs) can improve the function of infarcted hearts1–3, but two critical issues related to their electrophysiological behavior in vivo remain unresolved. First, the risk of arrhythmias following hESC-CM transplantation in injured hearts has not been determined. Second, the electromechanical integration of hESC-CMs in injured hearts has not been demonstrated, so it is unclear if these cells improve contractile function directly through addition of new force-generating units. Here we use a guinea pig model to show hESC-CM grafts in injured hearts protect against arrhythmias and can contract synchronously with host muscle. Injured hearts with hESC-CM grafts show improved mechanical function and a significantly reduced incidence of both spontaneous and induced ventricular tachycardia (VT). To assess the activity of hESC-CM grafts in vivo, we transplanted hESC-CMs expressing the genetically-encoded calcium sensor, GCaMP34, 5. By correlating the GCaMP3 fluorescent signal with the host ECG, we found that grafts in uninjured hearts have consistent 1:1 host-graft coupling. Grafts in injured hearts are more heterogeneous and typically include both coupled and uncoupled regions. Thus, human myocardial grafts meet physiological criteria for true heart regeneration, providing support for the continued development of hESC-based cardiac therapies for both mechanical and electrical repair.

Calcium (Ca 2؉ ) release through inositol 1,4,5-trisphosphate receptors (IP 3Rs) regulates the function of virtually every mammalian cell. Unlike ryanodine receptors, which generate local Ca 2؉ events (''sparks'') that transmit signals to the juxtaposed cell membrane, a similar functional architecture has not been reported for IP 3Rs. Here, we have identified spatially fixed, local Ca 2؉ release events (''pulsars'') in vascular endothelial membrane domains that project through the internal elastic lamina to adjacent smooth muscle membranes. Ca 2؉ pulsars are mediated by IP3Rs in the endothelial endoplasmic reticulum of these membrane projections. Elevation of IP 3 by the endothelium-dependent vasodilator, acetylcholine, increased the frequency of Ca 2؉ pulsars, whereas blunting IP3 production, blocking IP3Rs, or depleting endoplasmic reticulum Ca 2؉ inhibited these events. The elementary properties of Ca 2؉ pulsars were distinct from ryanodine-receptor-mediated Ca 2؉ sparks in smooth muscle and from IP3-mediated Ca 2؉ puffs in Xenopus oocytes. The intermediate conductance, Ca 2؉ -sensitive potassium (K Ca3.1) channel also colocalized to the endothelial projections, and blockage of this channel caused an 8-mV depolarization. Inhibition of Ca 2؉ pulsars also depolarized to a similar extent, and blocking K Ca3.1 channels was without effect in the absence of pulsars. Our results support a mechanism of IP 3 signaling in which Ca 2؉ release is spatially restricted to transmit intercellular signals.calcium ͉ endothelium ͉ calcium biosensor ͉ intermediate conductance Ca 2ϩ -sensitive potassium channel ͉ calcium pulsar

We have investigated the mechanism of mitochondrialnuclear crosstalk during cellular stress in mouse C2C12 myocytes. For this purpose, we used cells with reduced mitochondrial DNA (mtDNA) contents by ethidium bromide treatment or myocytes treated with known mitochondrial metabolic inhibitors, including carbonyl cyanide m-chlorophenylhydrazone (CCCP), antimycin, valinomycin and azide. Both genetic and metabolic stresses similarly affected mitochondrial membrane potential (Δψ m ) and electron transport-coupled ATP synthesis, which was also accompanied by an elevated steady-state cytosolic Ca 2⍣ level ([Ca 2⍣ ] i ). The mitochondrial stress resulted in: (i) an enhanced expression of the sarcoplasmic reticular ryanodine receptor-1 (RyR-1), hence potentiating the Ca 2⍣ release in response to its modulator, caffeine; (ii) enhanced levels of Ca 2⍣ -responsive factors calineurin, calcineurindependent NFATc (cytosolic counterpart of activated T-cell-specific nuclear factor) and c-Jun N-terminal kinase (JNK)-dependent ATF2 (activated transcription factor 2); (iii) reduced levels of transcription factor, NF-κB; and (iv) enhanced transcription of cytochrome oxidase Vb (COX Vb) subunit gene. These cellular changes, including the steady-state [Ca 2⍣ ] i were normalized in genetically reverted cells which contain near-normal mtDNA levels. We propose that the mitochondria-to-nucleus stress signaling occurs through cytosolic [Ca 2⍣ ] i changes, which are likely to be due to reduced ATP and Ca 2⍣ efflux. Our results indicate that the mitochondrial stress signal affects a variety of cellular processes, in addition to mitochondrial membrane biogenesis.

Genetically encoded sensor proteins provide unique opportunities to advance the understanding of complex cellular interactions in physiologically relevant contexts; however, previously described sensors have proved to be of limited use to report cell signaling in vivo in mammals. Here, we describe an improved Ca 2؉ sensor, GCaMP2, its inducible expression in the mouse heart, and its use to examine signaling in heart cells in vivo. The high brightness and stability of GCaMP2 enable the measurement of myocyte Ca 2؉ transients in all regions of the beating mouse heart and prolonged pacing and mapping studies in isolated, perfused hearts. Transgene expression is efficiently temporally regulated in cardiomyocyte GCaMP2 mice, allowing recording of in vivo signals 4 weeks after transgene induction. High-resolution imaging of Ca 2؉ waves in GCaMP2-expressing embryos revealed key aspects of electrical conduction in the preseptated heart. At embryonic day (e.d.) 10.5, atrial and ventricular conduction occur rapidly, consistent with the early formation of specialized conduction pathways. However, conduction is markedly slowed through the atrioventricular canal in the e.d. 10.5 heart, forming the basis for an effective atrioventricular delay before development of the AV node, as rapid ventricular activation occurs after activation of the distal AV canal tissue. Consistent with the elimination of the inner AV canal muscle layer at e.d. 13.5, atrioventricular conduction through the canal was abolished at this stage. These studies demonstrate that GCaMP2 will have broad utility in the dissection of numerous complex cellular interactions in mammals, in vivo. atrioventricular node ͉ Ca 2ϩ imaging ͉ genetic sensor ͉ heart development ͉ fluorescent Ca 2ϩ sensor T ransient, highly regulated elevations in cytosolic free Ca 2ϩ underlie numerous cellular processes that enable organ function (1-5). In the mammalian heart, for example, efficient function depends upon the coordinated release and reuptake of Ca 2ϩ ions from intracellular organelles in millions of cells, at rates between 0.5 and 15 Hz throughout life, and even subtle dysfunctions of this process can result in cardiac arrythmias and sudden death. Whereas fluorescent imaging using purpose-designed small Ca 2ϩ -binding indicator molecules has enabled important advances in the understanding of the regulatory processes underlying Ca 2ϩ signaling in single cells (6, 7), these approaches have significant limitations in the context of a complex, multicellular organ such as the beating heart. Thus, difficulties in obtaining an adequate and stable concentration of indicator molecules within cells deep in complex tissues, the incompatibility of loading procedures in the in vivo setting, and the inability to selectively load specific cell lineages constitute substantial experimental constraints on the study of multicellular, processive Ca 2ϩ signaling in complex organ function. Genetically encoded sensors of Ca 2ϩ signaling (7-13) hold great promise in this regard and have been used t...

The domestic dog is becoming an increasingly valuable model species in medical genetics, showing particular promise to advance our understanding of cancer and orthopaedic disease. Here we undertake the largest canine genome-wide association study to date, with a panel of over 4,200 dogs genotyped at 180,000 markers, to accelerate mapping efforts. For complex diseases, we identify loci significantly associated with hip dysplasia, elbow dysplasia, idiopathic epilepsy, lymphoma, mast cell tumour and granulomatous colitis; for morphological traits, we report three novel quantitative trait loci that influence body size and one that influences fur length and shedding. Using simulation studies, we show that modestly larger sample sizes and denser marker sets will be sufficient to identify most moderate- to large-effect complex disease loci. This proposed design will enable efficient mapping of canine complex diseases, most of which have human homologues, using far fewer samples than required in human studies.

Ventricular tachyarrhythmias are the main cause of sudden death in patients after myocardial infarction. Here we show that transplantation of embryonic cardiomyocytes (eCMs) in myocardial infarcts protects against the induction of ventricular tachycardia (VT) in mice. Engraftment of eCMs, but not skeletal myoblasts (SMs), bone marrow cells or cardiac myofibroblasts, markedly decreased the incidence of VT induced by in vivo pacing. eCM engraftment results in improved electrical coupling between the surrounding myocardium and the infarct region, and Ca2+ signals from engrafted eCMs expressing a genetically encoded Ca2+ indicator could be entrained during sinoatrial cardiac activation in vivo. eCM grafts also increased conduction velocity and decreased the incidence of conduction block within the infarct. VT protection is critically dependent on expression of the gap-junction protein connexin 43 (Cx43; also known as Gja1): SMs genetically engineered to express Cx43 conferred a similar protection to that of eCMs against induced VT. Thus, engraftment of Cx43-expressing myocytes has the potential to reduce life-threatening post-infarct arrhythmias through the augmentation of intercellular coupling, suggesting autologous strategies for cardiac cell-based therapy.

In the epithelium of the lower airways, a cell type of unknown function has been termed "brush cell" because of a distinctive ultrastructural feature, an apical tuft of microvilli. Morphologically similar cells in the nose have been identified as solitary chemosensory cells responding to taste stimuli and triggering trigeminal reflexes. Here we show that brush cells of the mouse trachea express the receptors (Tas2R105, Tas2R108), the downstream signaling molecules (α-gustducin, phospholipase C β2 ) of bitter taste transduction, the synthesis and packaging machinery for acetylcholine, and are addressed by vagal sensory nerve fibers carrying nicotinic acetylcholine receptors. Tracheal application of an nAChR agonist caused a reduction in breathing frequency. Similarly, cycloheximide, a Tas2R108 agonist, evoked a drop in respiratory rate, being sensitive to nicotinic receptor blockade and epithelium removal. This identifies brush cells as cholinergic sensors of the chemical composition of the lower airway luminal microenvironment that are directly linked to the regulation of respiration.airway sensory innervation | respiratory epithelium | jugular-nodose ganglion | bitter-tasting substances