Neutrophils incite and macrophages avert electrical storm after myocardial infarction

Sudden cardiac death, arising from abnormal electrical conduction, occurs frequently in patients with coronary heart disease. Myocardial ischemia simultaneously induces arrhythmia and massive myocardial leukocyte changes. In this study, we optimized a mouse model in which hypokalemia combined with myocardial infarction triggered spontaneous ventricular tachycardia in ambulatory mice, and we showed that major leukocyte subsets have opposing effects on cardiac conduction. Neutrophils increased ventricular tachycardia via lipocalin-2 in mice, whereas neutrophilia associated with ventricular tachycardia in patients. In contrast, macrophages protected against arrhythmia. Depleting recruited macrophages in Ccr2−/− mice or all macrophage subsets with Csf1 receptor inhibition increased both ventricular tachycardia and fibrillation. Higher arrhythmia burden and mortality in Cd36−/− and Mertk−/− mice, viewed together with reduced mitochondrial integrity and accelerated cardiomyocyte death in the absence of macrophages, indicated that receptor-mediated phagocytosis protects against lethal electrical storm. Thus, modulation of leukocyte function provides a potential therapeutic pathway for reducing the risk of sudden cardiac death

Adhesion-GPCR Gpr116 (ADGRF5) is a Regulator of Urine Acidification and Surface Expression of the Vacuolar-type H+-ATPase in Renal α-Intercalated Cells

The G protein-coupled receptor (GPCR) superfamily is among the largest in the human genome. Their diversity and nearly universal expression underlie their significance in many physiologic processes. GPCRs are a common target of pharmaceutical drug development, and uncovering the function of understudied GPCRs in the kidney represents a wealth of untapped therapeutic potential. We previously identified Gpr116, an adhesion-class GPCR, as one of the most highly expressed GPCRs in the kidney. In the present study, we confirm the localization of Gpr116 to the luminal membrane of acid-secreting α-intercalated cells (αICs) in the nephron using both imaging and functional studies, where we demonstrate in situ receptor activation using an agonist peptide unique to Gpr116. Additionally, kidney-specific knockout (KO) of Gpr116 caused a significant reduction to urine pH. Notably, the loss of acid in the urine is accompanied by a small, but significant, increase in blood pH, and a small, but significant, decrease in pCO2 compared to wild-type littermates. Results from transmission electron micrographs show greater accumulation of V-ATPase proton pumps at the surface of αICs in KO mice, suggesting a possible role for Gpr116 in the regulation of V-ATPase trafficking. We conclude that loss of Gpr116 from the nephron causes a primary loss of acid in the urine which results in a mild metabolic alkalosis (“renal tubular alkalosis”) due to reabsorption of HCO3- by αICs. This study establishes a significant physiologic role of the previously understudied Gpr116 in the murine kidney and demonstrates the scientific potential of future investigations into novel GPCRs

Ultrastructural Characterization of the Glomerulopathy in Alport Mice by Helium Ion Scanning Microscopy (HIM)

Abstract The glomerulus exercises its filtration barrier function by establishing a complex filtration apparatus consisting of podocyte foot processes, glomerular basement membrane and endothelial cells. Disruption of any component of the glomerular filtration barrier leads to glomerular dysfunction, frequently manifested as proteinuria. Ultrastructural studies of the glomerulus by transmission electron microscopy (TEM) and conventional scanning electron microscopy (SEM) have been routinely used to identify and classify various glomerular diseases. Here we report the application of newly developed helium ion scanning microscopy (HIM) to examine the glomerulopathy in a Col4a3 mutant/Alport syndrome mouse model. Our study revealed unprecedented details of glomerular abnormalities in Col4a3 mutants including distorted podocyte cell bodies and disorganized primary processes. Strikingly, we observed abundant filamentous microprojections arising from podocyte cell bodies and processes, and presence of unique bridging processes that connect the primary processes and foot processes in Alport mice. Furthermore, we detected an altered glomerular endothelium with disrupted sub-endothelial integrity. More importantly, we were able to clearly visualize the complex, three-dimensional podocyte and endothelial interface by HIM. Our study demonstrates that HIM provides nanometer resolution to uncover and rediscover critical ultrastructural characteristics of the glomerulopathy in Col4a3 mutant mice

Meiotic gatekeeper STRA8 suppresses autophagy by repressing Nr1d1 expression during spermatogenesis in mice.

The transition from mitotic to meiotic cell cycles is essential for haploid gamete formation and fertility. Stimulated by retinoic acid gene 8 (Stra8) is an essential gatekeeper of meiotic initiation in vertebrates; yet, the molecular role of STRA8 remains principally unknown. Here we demonstrate that STRA8 functions as a suppressor of autophagy during spermatogenesis in mice. Stra8-deficient germ cells fail to enter meiosis and present aberrant upregulation of autophagy-lysosome genes, commensurate with autophagy activation. Biochemical assays show that ectopic expression of STRA8 alone is sufficient to inhibit both autophagy induction and maturation. Studies also revealed that, Nr1d1, a nuclear hormone receptor gene, is upregulated in Stra8-deficient testes and that STRA8 binds to the Nr1d1 promoter, indicating that Nr1d1 is a direct target of STRA8 transcriptional repression. In addition, it was found that NR1D1 binds to the promoter of Ulk1, a gene essential for autophagy initiation, and that Nr1d1 is required for the upregulated Ulk1 expression in Stra8-deficient testes. Furthermore, both genetic deletion of Nr1d1 and pharmacologic inhibition of NR1D1 by its synthetic antagonist SR8278 exhibit rescuing effects on the meiotic initiation defects observed in Stra8-deficient male germ cells. Together, the data suggest a novel link between STRA8-mediated autophagy suppression and meiotic initiation

A Circadian Clock Gene, <i>Cry</i>, Affects Heart Morphogenesis and Function in <i>Drosophila</i> as Revealed by Optical Coherence Microscopy

<div><p>Circadian rhythms are endogenous, entrainable oscillations of physical, mental and behavioural processes in response to local environmental cues such as daylight, which are present in the living beings, including humans. Circadian rhythms have been related to cardiovascular function and pathology. However, the role that circadian clock genes play in heart development and function in a whole animal <i>in vivo</i> are poorly understood. The <i>Drosophila</i> cryptochrome <i>(dCry)</i> is a circadian clock gene that encodes a major component of the circadian clock negative feedback loop. Compared to the embryonic stage, the relative expression levels of <i>dCry</i> showed a significant increase (>100-fold) in <i>Drosophila</i> during the pupa and adult stages. In this study, we utilized an ultrahigh resolution optical coherence microscopy (OCM) system to perform non-invasive and longitudinal analysis of functional and morphological changes in the <i>Drosophila</i> heart throughout its post-embryonic lifecycle for the first time. The <i>Drosophila</i> heart exhibited major morphological and functional alterations during its development. Notably, heart rate (HR) and cardiac activity period (CAP) of <i>Drosophila</i> showed significant variations during the pupa stage, when heart remodeling took place. From the M-mode (2D + time) OCM images, cardiac structural and functional parameters of <i>Drosophila</i> at different developmental stages were quantitatively determined. In order to study the functional role of <i>dCry</i> on <i>Drosophila</i> heart development, we silenced <i>dCry</i> by RNAi in the <i>Drosophila</i> heart and mesoderm, and quantitatively measured heart morphology and function in those flies throughout its development. Silencing of <i>dCry</i> resulted in slower HR, reduced CAP, smaller heart chamber size, pupal lethality and disrupted posterior segmentation that was related to increased expression of a posterior compartment protein, wingless. Collectively, our studies provided novel evidence that the circadian clock gene, <i>dCry</i>, plays an essential role in heart morphogenesis and function.</p></div

Silencing of <i>dCry</i> resulted in segment polarity phenotypes.

<p>(<b>a, c and d</b>) Control larva (24B-GAL4/+) showed regular denticle belts in posterior A6 and A7 segments. (<b>b, e and f</b>) Silencing of <i>dCry</i> (UAS-dCry-RNAi; 24B-GAL4) results in disorganized cuticular morphologies in A6 denticle belt and significantly increased, enlarged and disorganized A7 denticle belt (denoted by arrows). (<b>g, h</b>) Control flies showed normal and organized notum bristles and A6 and A7 denticle belts. (<b>i, j</b>) The few emerged UAS-dCry-RNAi; 24B-GAL4 adult flies showed a smaller notum with disoriented and up-pointing bristles in notum (arrow in <b>i</b>), and disorganized and partially absent A6 and A7 denticle belts (arrow in <b>j</b>).</p

Renal Atp6ap2/(Pro)renin Receptor Is Required for Normal Vacuolar H + -ATPase Function but Not for the Renin-Angiotensin System

International audienceATPase H+-transporting lysosomal accessory protein 2 (Atp6ap2), also known as the (pro)renin receptor, is a type 1 transmembrane protein and an accessory subunit of the vacuolar H+-ATPase (V-ATPase) that may also function within the renin-angiotensin system. However, the contribution of Atp6ap2 to renin-angiotensin-dependent functions remains unconfirmed. Using mice with an inducible conditional deletion of Atp6ap2 in mouse renal epithelial cells, we found that decreased V-ATPase expression and activity in the intercalated cells of the collecting duct impaired acid-base regulation by the kidney. In addition, these mice suffered from marked polyuria resistant to desmopressin administration. Immunoblotting revealed downregulation of the medullary Na+-K+-2Cl- cotransporter NKCC2 in these mice compared with wild-type mice, an effect accompanied by a hypotonic medullary interstitium and impaired countercurrent multiplication. This phenotype correlated with strong autophagic defects in epithelial cells of medullary tubules. Notably, cells with high accumulation of the autophagosomal substrate p62 displayed the strongest reduction of NKCC2 expression. Finally, nephron-specific Atp6ap2 depletion did not affect angiotensin II production, angiotensin II-dependent BP regulation, or sodium handling in the kidney. Taken together, our results show that nephron-specific deletion of Atp6ap2 does not affect the renin-angiotensin system but causes a combination of renal concentration defects and distal renal tubular acidosis as a result of impaired V-ATPase activity

Silencing of <i>dCry</i> resulted in segment polarity phenotypes.

<p>(<b>a, c and d</b>) Control larva (24B-GAL4/+) showed regular denticle belts in posterior A6 and A7 segments. (<b>b, e and f</b>) Silencing of <i>dCry</i> (UAS-dCry-RNAi; 24B-GAL4) results in disorganized cuticular morphologies in A6 denticle belt and significantly increased, enlarged and disorganized A7 denticle belt (denoted by arrows). (<b>g, h</b>) Control flies showed normal and organized notum bristles and A6 and A7 denticle belts. (<b>i, j</b>) The few emerged UAS-dCry-RNAi; 24B-GAL4 adult flies showed a smaller notum with disoriented and up-pointing bristles in notum (arrow in <b>i</b>), and disorganized and partially absent A6 and A7 denticle belts (arrow in <b>j</b>).</p

3D and M-mode OCM imaging of post-embryonic <i>Drosophila</i> lifecycle.

<p>(<b>a</b>) 3D OCM renderings of a 24B-GAL4/+ <i>Drosophila</i> flies at larva, pupa and adult stages. (<b>b</b>) Schematic representation of heart metamorphosis. Red arrows on larva and adult schematic denote the OCM M-mode imaging locations until PD1 24h and for subsequent time points, respectively. (<b>c</b>) Enface OCM projections showing heart metamorphosis. (<b>d</b>) Axial OCM sections showing heart remodelling during Drosophila lifecycle. * denotes the air bubble location during early hours of pupa development. (<b>e</b>) M-mode images at different developmental stages showing HR changes across lifecycle. (<b>f</b>) Examples demonstrating cardiac activity period (CAP) calculation. Scale bars in (c) and (d) represent 500 μm.</p

Silencing of <i>dCry</i> led to abnormal wing vein distribution and Wg expression.

<p>(<b>a, b and c</b>) Control fly with heterozygous <i>En-GAL4; UAS-GFP</i> alone (En-GAL4; UAS-GFP /+) exhibited normal wing. (<b>d, e and f</b>) Silencing of <i>dCry</i> in the wing (UAS-dCry-RNAi/UAS-GFP; En-GAL4) resulted in a marked increase in the acv, pcv (arrows in <b>e</b>), M, L3 and L4 wing veins. L4 vein was disorganized with extra veins in the distal part (arrow in <b>f</b>). (<b>g)</b> Control flies showed normal Wg expression pattern (a broad strip in the notum, a thinner strip in the prospective wing margin-dorsal/ventral (D/V) boundary, and a strip encircling the prospective wing blade). (<b>h)</b> Merged images of the expression of <i>Wg</i> and co-overexpression of GFP in the pattern of <i>En</i> in control flies. (<b>i)</b> In the dCry-RNAi wing discs, Wg expression level was markedly increased and Wg expression pattern was disorganized. (<b>j)</b> Merged images of the expression of Wg and co-overexpression of GFP in the pattern of <i>En</i> in UAS-dCry-RNAi/UAS-GFP; En-GAL4 flies.</p