Search

Aversive responses to bright light (photoaversion) require signaling from the eye to the brain. Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) encode absolute light intensity and are thought to provide the light signals for photoaversion. Consistent with this, neonatal mice exhibit photoaversion before the developmental onset of image vision, and melanopsin deletion abolishes photoaversion in neonates. It is not well understood how the population of ipRGCs, which constitutes multiple physiologically distinct types (denoted M1-M6 in mouse), encodes light stimuli to produce an aversive response. Here, we provide several lines of evidence that M1 ipRGCs that lack the Brn3b transcription factor drive photoaversion in neonatal mice. First, neonatal mice lacking TRPC6 and TRPC7 ion channels failed to turn away from bright light, while two photon Ca2+ imaging of their acutely isolated retinas revealed reduced photosensitivity in M1 ipRGCs, but not other ipRGC types. Second, mice in which all ipRGC types except for Brn3b-negative M1 ipRGCs are ablated exhibited normal photoaversion. Third, pharmacological blockade or genetic knockout of gap junction channels expressed by ipRGCs, which reduces the light sensitivity of M2-M6 ipRGCs in the neonatal retina, had small effects on photoaversion only at the brightest light intensities. Finally, M1s were not strongly depolarized by spontaneous retinal waves, a robust source of activity in the developing retina that depolarizes all other ipRGC types. M1s therefore constitute a separate information channel between the neonatal retina and brain that could ensure behavioral responses to light but not spontaneous retinal waves.SIGNIFICANCE STATEMENT At an early stage of development, before the maturation of photoreceptor input to the retina, neonatal mice exhibit photoaversion. On exposure to bright light, they turn away and emit ultrasonic vocalizations, a cue to their parents to return them to the ne

Podocyte, the gatekeeper of the glomerular filtration barrier, is a primary target for growth factor and Ca2+ signaling whose perturbation leads to proteinuria. However, the effects of insulin action on store-operated Ca2+ entry (SOCE) in podocytes remain unknown. Here, we demonstrated that insulin stimulates SOCE by VAMP2-dependent Orai1 trafficking to the plasma membrane. Insulin-activated SOCE triggers actin remodeling and transepithelial albumin leakage via the Ca2+-calcineurin pathway in podocytes. Transgenic Orai1 overexpression in mice causes podocyte fusion and impaired glomerular filtration barrier. Conversely, podocyte-specific Orai1 deletion prevents insulin-stimulated SOCE, synaptopodin depletion, and proteinuria. Podocyte injury and albuminuria coincide with Orai1 upregulation at the hyperinsulinemic stage in diabetic (db/db) mice, which can be ameliorated by the suppression of Orai1-calcineurin signaling. Our results suggest that tightly balanced insulin action targeting podocyte Orai1 is critical for maintaining filter integrity, which provides novel perspectives on therapeutic strategies for proteinuric diseases, including diabetic nephropathy.

BACKGROUND Cation channels play an essential role in red blood cells (RBCs) ion homeostasis. One set of ion channels are the transient receptor potential channels of canonical type (TRPC channels). The abundance of these channels in primary erythroblasts, erythroid cell lines and RBCs was associated with an increase in intracellular Ca2+ upon stimulation with Erythropoietin (Epo). In contrast two independent studies on Epo-treated patients revealed diminished basal Ca2+ concentration or reduced phosphatidylserine exposure to the outer membrane leaflet. METHODS To resolve the seemingly conflicting reports we challenged mature human and mouse RBCs of several genotypes with Epo and Prostaglandin E2 (PGE2) and recorded the intracellular Ca2+ content. Next Generation Sequencing was utilised to approach a molecular analysis of reticulocytes. RESULTS/CONCLUSIONS Our results allow concluding that Epo and PGE2 regulation of the Ca2+ homeostasis is distinctly different between murine and human RBCs and that changes in intracellular Ca2+ upon Epo treatment is a primary rather than a compensatory effect. In human RBCs, Epo itself has no effect on Ca2+ fluxes but inhibits the PGE2-induced Ca2+ entry. In murine mature RBCs functional evidence indicates TRPC4/C5 mediated Ca2+ entry activated by Epo whereas PGE2 leads to a TRPC independent Ca2+ entry.

Inhibitory G (G(i)) proteins have been proposed to be cardioprotective. We investigated effects of G alpha(i2) knockout on cardiac function and survival in a murine heart failure model of cardiac beta(1)-adrenoceptor overexpression. beta(1)-transgenic mice lacking G alpha(i2) (beta(1)-tg/G alpha(-/-)(i2)) were compared with wild-type mice and littermates either overexpressing cardiac beta(1)-adrenoceptors (beta(1)-tg) or lacking G alpha(i2) (G alpha(-/-)(i2)). At 300 days, mortality of mice only lacking G alpha(i2) was already higher compared with wild-type or beta(1)-tg, but similar to beta(1)-tg/G alpha(-/-)(i2), mice. Beyond 300 days, mortality of beta(1)-tg/G alpha(-/-)(i2) mice was enhanced compared with all other genotypes (mean survival time: 363 +/- 21 days). At 300 days of age, echocardiography revealed similar cardiac function of wild-type, beta(1)-tg, and G alpha(-/-)(i2) mice, but significant impairment for beta(1)-tg/G alpha(-/-)(i2) mice (e.g. ejection fraction 14 +/- 2 vs. 40 +/- 4% in wild-type mice). Significantly increased ventricle-to-body weight ratio (0.71 +/- 0.06 vs. 0.48 +/- 0.02% in wild-type mice), left ventricular size (length 0.82 +/- 0.04 vs. 0.66 +/- 0.03 cm in wild types), and atrial natriuretic peptide and brain natriuretic peptide expression (mRNA: 2819 and 495% of wild-type mice, respectively) indicated hypertrophy. G alpha(i3) was significantly up-regulated in G alpha(i2) knockout mice (protein compared with wild type: 340 +/- 90% in G alpha(-/-)(i2) and 394 +/- 80% in beta(1)-tg/G alpha(-/-)(i2), respectively). G alpha(i2) deficiency combined with cardiac beta(1)-adrenoceptor overexpression strongly impaired survival and cardiac function. At 300 days of age, beta(1)-adrenoceptor overexpression alone had not induced cardiac hypertrophy or dysfunction while there was overt cardiomyopathy in mice additionally lacking G alpha(i2). We propose an enhanced effect of increased beta(1)-adrenergic drive by the lack of protection via G alpha

Inhibitory G (G(i)) proteins have been proposed to be cardioprotective. We investigated effects of G alpha(i2) knockout on cardiac function and survival in a murine heart failure model of cardiac beta(1)-adrenoceptor overexpression. beta(1)-transgenic mice lacking G alpha(i2) (beta(1)-tg/G alpha(-/-)(i2)) were compared with wild-type mice and littermates either overexpressing cardiac beta(1)-adrenoceptors (beta(1)-tg) or lacking G alpha(i2) (G alpha(-/-)(i2)). At 300 days, mortality of mice only lacking G alpha(i2) was already higher compared with wild-type or beta(1)-tg, but similar to beta(1)-tg/G alpha(-/-)(i2), mice. Beyond 300 days, mortality of beta(1)-tg/G alpha(-/-)(i2) mice was enhanced compared with all other genotypes (mean survival time: 363 +/- 21 days). At 300 days of age, echocardiography revealed similar cardiac function of wild-type, beta(1)-tg, and G alpha(-/-)(i2) mice, but significant impairment for beta(1)-tg/G alpha(-/-)(i2) mice (e.g. ejection fraction 14 +/- 2 vs. 40 +/- 4% in wild-type mice). Significantly increased ventricle-to-body weight ratio (0.71 +/- 0.06 vs. 0.48 +/- 0.02% in wild-type mice), left ventricular size (length 0.82 +/- 0.04 vs. 0.66 +/- 0.03 cm in wild types), and atrial natriuretic peptide and brain natriuretic peptide expression (mRNA: 2819 and 495% of wild-type mice, respectively) indicated hypertrophy. G alpha(i3) was significantly up-regulated in G alpha(i2) knockout mice (protein compared with wild type: 340 +/- 90% in G alpha(-/-)(i2) and 394 +/- 80% in beta(1)-tg/G alpha(-/-)(i2), respectively). G alpha(i2) deficiency combined with cardiac beta(1)-adrenoceptor overexpression strongly impaired survival and cardiac function. At 300 days of age, beta(1)-adrenoceptor overexpression alone had not induced cardiac hypertrophy or dysfunction while there was overt cardiomyopathy in mice additionally lacking G alpha(i2). We propose an enhanced effect of increased beta(1)-adrenergic drive by the lack of protection via G alpha

We previously showed in vitro that calcium entry through Trpc1 ion channels regulates myoblast migration and differentiation. In the present work, we used primary cell cultures and isolated muscles from Trpc1(-/-) and Trpc1(+/+) murine model to investigate the role of Trpc1 in myoblast differentiation and in muscle regeneration. In these models, we studied regeneration consecutive to cardiotoxin-induced muscle injury and observed a significant hypotrophy and a delayed regeneration in Trpc1(-/-) muscles consisting in smaller fiber size and increased proportion of centrally nucleated fibers. This was accompanied by a decreased expression of myogenic factors such as MyoD, Myf5, and myogenin and of one of their targets, the developmental MHC (MHCd). Consequently, muscle tension was systematically lower in muscles from Trpc1(-/-) mice. Importantly, the PI3K/Akt/mTOR/p70S6K pathway, which plays a crucial role in muscle growth and regeneration, was down-regulated in regenerating Trpc1(-/-) muscles. Indeed, phosphorylation of both Akt and p70S6K proteins was decreased as well as the activation of PI3K, the main upstream regulator of the Akt. This effect was independent of insulin-like growth factor expression. Akt phosphorylation also was reduced in Trpc1(-/-) primary myoblasts and in control myoblasts differentiated in the absence of extracellular Ca(2+) or pretreated with EGTA-AM or wortmannin, suggesting that the entry of Ca(2+) through Trpc1 channels enhanced the activity of PI3K. Our results emphasize the involvement of Trpc1 channels in skeletal muscle development in vitro and in vivo, and identify a Ca(2+)-dependent activation of the PI3K/Akt/mTOR/p70S6K pathway during myoblast differentiation and muscle regeneration.

Skeletal muscle contraction is reputed not to depend on extracellular Ca2+. Indeed, stricto sensu, excitation-contraction coupling does not necessitate entry of Ca2+. However, we previously observed that, during sustained activity (repeated contractions), entry of Ca2+ is needed to maintain force production. In the present study, we evaluated the possible involvement of the canonical transient receptor potential (TRPC)1 ion channel in this entry of Ca2+ and investigated its possible role in muscle function. The influx of Ca2+ through TRPC1 represents a minor part of the entry of Ca2+ into muscle fibers at rest, and the activity of the channel is not store dependent. The lack of TRPC1 does not affect intracellular Ca2+ concentration ([Ca2+](i)) transients reached during a single isometric contraction. However, the involvement of TRPC1-related Ca2+ entry is clearly emphasized in muscle fatigue. Indeed, muscles from TRPC1-/- mice stimulated repeatedly progressively display lower [Ca2+](i) transients than those observed in TRPC1+/+ fibers, and they also present an accentuated progressive loss of force. Interestingly, muscles from TRPC1-/- mice display a smaller fiber cross-sectional area (CSA), generate less force per CSA, and contain less myofibrillar proteins than their controls. To study the role of TRPC1 in skeletal muscle functions, we investigated in vivo the ability of the TRPC1-/- mice to perform voluntary (running wheel and escape test) or endurance exercises (treadmill and wire test). If TRPC1-/- mice do not exhibit any difficulty to perform voluntary exercise, they show a predisposition to muscle fatigue. Indeed, the performances of the TRPC1-/- mice in the treadmill and wire test were lower than those observed in a control mice. We also compared basal activity (home cage activity) and coordination (rotarod, balance-beam test). We conclude that TRPC1 ion channels modulate the entry of Ca2+ during repeated contractions and help muscles to maintain their force

We have previously showed in vitro that Ca2+ entry through TRPC1 ion channels regulates myoblasts migration and differentiation by activating calpain, a Ca2+-dependent protease which cleaves myristoylated alanine-rich C-kinase substrate (MARCKS) protein to allow myoblasts migration. To explore, in vivo, whether the absence of TRPC1 channel impairs skeletal muscle regeneration, we used cardiotoxin injections to induce muscle injury in adult TRPC1 +/+ and TRPC1-/- mice. Interestingly, we observed that regenerated TRPC1-/- muscles had a smaller fibre size and a decreased specific force respectively after 10 and 14 days of regeneration. We also observed an increase of central nuclei at day 14 of regeneration in TRPC1-/- whereas, at this stage, in TRPC1+/+ muscles, nuclei were essentially situated in the periphery of the fibres. These observations indicate a delay in muscle regeneration in TRPC1-/- mice in comparison with their controls. To understand the molecular mechanisms which sustain this delayed regeneration in TRPC1-/- mice, we investigated myogenic transcription factors implicated in the control of myogenesis. In comparison with TRPC1-/-, TRPC1+/+ muscles showed an earlier increase of the mRNA level and of the protein expression of MyoD , Myf5 and myogenin. Interestingly, developmental Myosin Heavy Chain (MHCd), a well known downstream target of MyoD during muscle regeneration, was also expressed earlier in TRPC1+/+ than in TRPC1 -/- muscles. Finally, we also observed a more important and earlier phosphorylation of both Akt and P70S6k in TRPC1+/+ muscles than in TRPC1-/- muscles, suggesting, as previously reported, an involvement of Akt / mTOR/ P70S6k pathway in the control of protein synthesis, muscles fibres size and muscle regeneration in vivo. Altogether, our results demonstrate the importance of TRPC1 channels in skeletal muscles development both in vitro and in vivo and identify Akt / mTOR / P70S6K as the main pathway affected in TRPC1 -/- during muscle re

The trafficking of leukocytes from the blood to sites of inflammation is the cumulative result of receptor-ligand-mediated signaling events associated with the leukocytes themselves as well as with the underlying vascular endothelium. Our data show that G{alpha}i signaling pathways in the vascular endothelium regulate a critical step required for leukocyte diapedesis. In vivo studies using knockout mice demonstrated that a signaling event in a non-lymphohematopoietic compartment of the lung prevented the recruitment of proinflammatory leukocytes. Intravital microscopy showed that blockade was at the capillary endothelial surface and ex vivo studies of leukocyte trafficking demonstrated that a G{alpha}i-signaling event in endothelial cells was required for transmigration. Collectively, these data suggest that specific G{alpha}i2-mediated signaling between endothelial cells and leukocytes is required for the extravasation of leukocytes and for tissue-specific accumulation.

The trafficking of leukocytes from the blood to sites of inflammation is the cumulative result of receptor-ligand-mediated signaling events associated with the leukocytes themselves as well as with the underlying vascular endothelium. Our data show that G{alpha}i signaling pathways in the vascular endothelium regulate a critical step required for leukocyte diapedesis. In vivo studies using knockout mice demonstrated that a signaling event in a non-lymphohematopoietic compartment of the lung prevented the recruitment of proinflammatory leukocytes. Intravital microscopy showed that blockade was at the capillary endothelial surface and ex vivo studies of leukocyte trafficking demonstrated that a G{alpha}i-signaling event in endothelial cells was required for transmigration. Collectively, these data suggest that specific G{alpha}i2-mediated signaling between endothelial cells and leukocytes is required for the extravasation of leukocytes and for tissue-specific accumulation.

The heterotrimeric guanine nucleotide-binding protein Galphai2 is involved in regulation of immune responses against microbial and nonmicrobial stimuli. Galphai2-/- mice have a selectively impaired IgM response consistent with a disorder in B cell development yet have augmented T cell effector function associated with increased production of IFN-gamma and IL-4. The goal of the present study was to determine if a deficiency in the Galphai2 protein in mice would affect the protective immune response against Strongyloides stercoralis, which is IL-4-, IL-5-, and IgM-dependent. Galphai2-/- and wild-type mice were immunized and challenged with S. stercoralis larvae and analyzed for protective immune responses against infection. Galphai2-/- mice failed to kill the larvae in the challenge infection as compared with wild-type mice despite developing an antigen-specific Th2 response characterized by increased IL-4, IL-5, IgM, and IgG. Transfer of serum collected from immunized Galphai2-/- mice to naïve wild-type mice conferred passive protective immunity against S. stercoralis infection thus confirming the development of a protective antibody response in Galphai2-/- mice. Differential cell analyses and myeloperoxidase assays for quantification of neutrophils showed a significantly reduced recruitment of neutrophils into the microenvironment of the parasites in immunized Galphai2-/- mice. However, cell transfer studies demonstrated that neutrophils from Galphai2-/- mice are competent in killing larvae. These data demonstrate that Galphai2 signaling events are not required for the development of the protective immune responses against S. stercoralis; however, Galphai2 is essential for the recruitment of neutrophils required for host-dependent killing of larvae.