A century of exercise physiology: key concepts in neural control of the circulation.

Early in the twentieth century, Walter B. Cannon (1871-1945) introduced his overarching hypothesis of "homeostasis" (Cannon 1932)-the ability to sustain physiological values within a narrow range necessary for life during periods of stress. Physical exercise represents a stress in which motor, respiratory and cardiovascular systems must be integrated across a range of metabolic stress to match oxygen delivery to oxygen need at the cellular level, together with appropriate thermoregulatory control, blood pressure adjustments and energy provision. Of these, blood pressure regulation is a complex but controlled variable, being the function of cardiac output and vascular resistance (or conductance). Key in understanding blood pressure control during exercise is the coordinating role of the autonomic nervous system. A long history outlines the development of these concepts and how they are integrated within the exercise context. This review focuses on the renaissance observations and thinking generated in the first three decades of the twentieth century that opened the doorway to new concepts of inquiry in cardiovascular regulation during exercise. The concepts addressed here include the following: (1) exercise and blood pressure, (2) central command, (3) neurovascular transduction with emphasis on the sympathetic nerve activity and the vascular end organ response, and (4) tonic neurovascular integration.

Sex-dependent cholinergic effects on amyloid pathology: A translational study.

INTRODUCTION: About two-thirds of Alzheimer's Disease (AD) patients are women, who exhibit more severe pathology and cognitive decline than men. Whether biological sex causally modulates the relationship between cholinergic signaling and amyloid pathology remains unknown. METHODS: We quantified amyloid beta (Aß) in male and female App-mutant mice with either decreased or increased cholinergic tone and examined the impact of ovariectomy and estradiol replacement in this relationship. We also investigated longitudinal changes in basal forebrain (cholinergic function) and Aß in elderly individuals. RESULTS: We show a causal relationship between cholinergic tone and amyloid pathology in males and ovariectomized female mice, which is decoupled in ovary-intact and ovariectomized females receiving estradiol. In elderly humans, cholinergic loss exacerbates Aß. DISCUSSION: Our findings emphasize the importance of reflecting human menopause in mouse models. They also support a role for therapies targeting estradiol and cholinergic signaling to reduce Aß. HIGHLIGHTS: Cholinergic tone regulates amyloid beta (Aß) pathology in males and ovariectomized female mice. Estradiol uncouples the relationship between cholinergic tone and Aß. In elderly humans, cholinergic loss correlates with increased Aß in both sexes.

CaV3.1 channels facilitate calcium wave generation and myogenic tone development in mouse mesenteric arteries.

The arterial myogenic response to intraluminal pressure elicits constriction to maintain tissue perfusion. Smooth muscle [Ca2+] is a key determinant of constriction, tied to L-type (CaV1.2) Ca2+ channels. While important, other Ca2+ channels, particularly T-type could contribute to pressure regulation within defined voltage ranges. This study examined the role of one T-type Ca2+ channel (CaV3.1) using C57BL/6 wild type and CaV3.1-/- mice. Patch-clamp electrophysiology, pressure myography, blood pressure and Ca2+ imaging defined the CaV3.1-/- phenotype relative to C57BL/6. CaV3.1-/- mice had absent CaV3.1 expression and whole-cell current, coinciding with lower blood pressure and reduced mesenteric artery myogenic tone, particularly at lower pressures (20-60 mmHg) where membrane potential is hyperpolarized. This reduction coincided with diminished Ca2+ wave generation, asynchronous events of Ca2+ release from the sarcoplasmic reticulum, insensitive to L-type Ca2+ channel blockade (Nifedipine, 0.3 µM). Proximity ligation assay (PLA) confirmed IP3R1/CaV3.1 close physical association. IP3R blockade (2-APB, 50 µM or xestospongin C, 3 µM) in nifedipine-treated C57BL/6 arteries rendered a CaV3.1-/- contractile phenotype. Findings indicate that Ca2+ influx through CaV3.1 contributes to myogenic tone at hyperpolarized voltages through Ca2+-induced Ca2+ release tied to the sarcoplasmic reticulum. This study helps establish CaV3.1 as a potential therapeutic target to control blood pressure.

High levels of lipoprotein(a) in transgenic mice exacerbate atherosclerosis and promote vulnerable plaque features in a sex-specific manner.

BACKGROUND AND AIMS: Despite increased clinical interest in lipoprotein(a) (Lp(a)), many questions remain about the molecular mechanisms by which it contributes to atherosclerotic cardiovascular disease. Existing murine transgenic (Tg) Lp(a) models are limited by low plasma levels of Lp(a) and have not consistently shown a pro-atherosclerotic effect of Lp(a). METHODS: We generated Tg mice expressing both human apolipoprotein(a) (apo(a)) and human apoB-100, with pathogenic levels of plasma Lp(a) (range 87-250 mg/dL). Female and male Lp(a) Tg mice (Tg(LPA+/0;APOB+/0)) and human apoB-100-only controls (Tg(APOB+/0)) (n = 10-13/group) were fed a high-fat, high-cholesterol diet for 12 weeks, with Ldlr knocked down using an antisense oligonucleotide. FPLC was used to characterize plasma lipoprotein profiles. Plaque area and necrotic core size were quantified and immunohistochemical assessment of lesions using a variety of cellular and protein markers was performed. RESULTS: Male and female Tg(LPA+/0;APOB+/0) and Tg(APOB+/0) mice exhibited proatherogenic lipoprotein profiles with increased cholesterol-rich VLDL and LDL-sized particles and no difference in plasma total cholesterol between genotypes. Complex lesions developed in the aortic sinus of all mice. Plaque area (+22%), necrotic core size (+25%), and calcified area (+65%) were all significantly increased in female Tg(LPA+/0;APOB+/0) mice compared to female Tg(APOB+/0) mice. Immunohistochemistry of lesions demonstrated that apo(a) deposited in a similar pattern as apoB-100 in Tg(LPA+/0;APOB+/0) mice. Furthermore, female Tg(LPA+/0;APOB+/0) mice exhibited less organized collagen deposition as well as 42% higher staining for oxidized phospholipids (OxPL) compared to female Tg(APOB+/0) mice. Tg(LPA+/0;APOB+/0) mice had dramatically higher levels of plasma OxPL-apo(a) and OxPL-apoB compared to Tg(APOB+/0) mice, and female Tg(LPA+/0;APOB+/0) mice had higher plasma levels of the proinflammatory cytokine MCP-1 (+3.1-fold) compared to female Tg(APOB+/0) mice. CONCLUSIONS: These data suggest a pro-inflammatory phenotype exhibited by female Tg mice expressing Lp(a) that appears to contribute to the development of more severe lesions with greater vulnerable features.

Minimal Kidney Disease Phenotype in Shroom3 Heterozygous Null Mice.

Background: Shroom family member 3 (SHROOM3) encodes an actin-associated protein that regulates epithelial morphology during development. Several genome-wide association studies (GWAS) have identified genetic variances primarily in the 5' region of SHROOM3, associated with chronic kidney disease (CKD) and poor transplant outcomes. These genetic variants are associated with alterations in Shroom3 expression. Objective: Characterize the phenotypic abnormalities associated with reduced Shroom3 expression in postnatal day 3-, 1-month and 3-month-old mice. Methods: The Shroom3 protein expression pattern was determined by immunofluorescence. We generated Shroom3 heterozygous null mice (Shroom3Gt/+) and performed comparative analyses with wild type littermates based on somatic and kidney growth, gross renal anatomy, renal histology, renal function at postnatal day 3, 1 month, and 3 months. Results: The Shroom3 protein expression localized to the apical regions of medullary and cortical tubular epithelium in postnatal wild type kidneys. Co-immunofluorescence studies confirmed protein expression localized to the apical side of the tubular epithelium in proximal convoluted tubules, distal convoluted tubules, and collecting ducts. While Shroom3 heterozygous null mice exhibited reduced Shroom3 protein expression, no differences in somatic and kidney growth were observed when compared to wild type mice. Although, rare cases of unilateral hypoplasia of the right kidney were observed at postnatal 1 month in Shroom3 heterozygotes. Yet renal histological analysis did not reveal any overt abnormalities in overall kidney structure or in glomerular and tubular organization in Shroom3 heterozygous null mice when compared to wild type mice. Analysis of the apical-basolateral orientation of the tubule epithelium demonstrated alterations in the proximal convoluted tubules and modest disorganization in the distal convoluted tubules at 3 months in Shroom3 heterozygotes. Additionally, these modest abnormalities were not accompanied by tubular injury or physiological defects in renal and cardiovascular function. Conclusion: Taken together, our results describe a mild kidney disease phenotype in adult Shroom3 heterozygous null mice, suggesting that Shroom3 expression and function may be required for proper structure and maintenance of the various tubular epithelial parenchyma of the kidney.

Contexte: Le gène SHROOM3 (membre 3 de la famille Shroom) code pour une protéine associée à l'actine qui régule la morphologie épithéliale pendant le développement. Plusieurs études d'association à l'échelle du génome (GWAS ­ Genome-wide association studies) ont identifié des variations génétiques, principalement dans la région 5' du gène SHROOM3, associées à l'insuffisance rénale chronique (IRC) et à de mauvais résultats de transplantation. Ces variations génétiques sont associées à des altérations dans l'expression de (Shroom3). Objectif: Caractériser les anomalies phénotypiques associées à une diminution de l'expression de Shroom3 chez des souris à l'âge postnatal de 3 jours, 1 mois et 3 mois. Méthodologie: Le profil d'expression des protéines Shroom3 a été déterminé par immunofluorescence. Nous avons généré des souris hétérozygotes Shroom3 (Shroom3Gt/+) et procédé à des analyses comparatives avec des congénères de type sauvage en ce qui concerne la croissance somatique et rénale, l'anatomie rénale, l'histologie rénale et la fonction rénale à l'âge postnatal de 3 jours, 1 mois et 3 mois. Résultats: L'expression de la protéine Shroom3 est localisée dans les régions apicales de l'épithélium tubulaire médullaire et cortical des reins des souris de type sauvage après la naissance. Des études de co-immunofluorescence ont confirmé l'expression des protéines localisée sur le côté apical de l'épithélium tubulaire dans les tubules contournés proximaux, les tubules contournés distaux et les tubes collecteurs. Les souris hétérozygotes Shroom3 ont présenté une expression réduite de la protéine Shroom3, mais aucune différence dans la croissance somatique et rénale n'a été observée par rapport aux souris de type sauvage. Cependant, de rares cas d'hypoplasie unilatérale du rein droit ont été observés à l'âge postnatal de 1 mois chez les souris hétérozygotes Shroom3. L'analyze histologique rénale n'a révélé aucune anomalie manifeste dans la structure globale des reins ou dans l'organization des glomérules et des tubules chez les souris hétérozygotes Shroom3 par rapport aux souris de type sauvage. L'analyze de l'orientation apicale-basolatérale de l'épithélium tubulaire a montré des altérations dans les tubules contournés proximaux et une légère désorganisation dans les tubules contournés distaux à l'âge de 3 mois chez les souris hétérozygotes Shroom3. En outre, ces légères anomalies n'étaient pas accompagnées d'une lésion tubulaire ou d'anomalies physiologiques dans la fonction rénale et cardiovasculaire. Conclusion: Pris dans leur ensemble, nos résultats décrivent un phénotype d'insuffisance rénale légère chez les souris hétérozygotes Shroom3 adultes, ce qui suggère que l'expression et la fonction de la protéine Shroom3 peuvent être nécessaires pour la structure et le maintien appropriés des différents parenchymes épithéliaux tubulaires du rein.

Correlation of functional and radioligand binding characteristics of GPER ligands confirming aldosterone as a GPER agonist.

Aldosterone exerts some of its effects not by binding to mineralocorticoid receptors, but rather by acting via G protein-coupled estrogen receptors (GPER). To determine if aldosterone binds directly to GPER, we studied the ability of aldosterone to compete for the binding of [3 H] 2-methoxyestradiol ([3 H] 2-ME), a high potency GPER-selective agonist. We used GPER gene transfer to engineer Sf9-cultured insect cells to express GPER. We chose insect cells to avoid interactions with any intrinsic mammalian receptors for aldosterone. [3 H] 2-ME binding was saturable and reversible to a high-affinity population of receptors with Kd  = 3.7 nM and Bmax  = 2.2 pmol/mg. Consistent with agonist binding to G Protein-coupled receptors, [3 H] 2-ME high-affinity state binding was reduced in the presence of the hydrolysis-resistant GTP analog, GppNHp. [3 H] 2-ME binding was competed for by the GPER agonist G1, the GPER antagonist G15, estradiol (E2), as well as aldosterone (Aldo). The order of potency for competing for [3 H] 2-ME binding, namely 2ME > Aldo > E2 ≥ G1, paralleled the orders of potency for inhibition of cell proliferation and inhibition of ERK phosphorylation by ligands acting at GPER. These data confirm the ability of aldosterone to interact with the GPER, consistent with the interpretation that aldosterone likely mediates its GPER-dependent effects by direct binding to the GPER. SIGNIFICANCE STATEMENT: Despite the growing evidence for aldosterone's actions via G protein-coupled estrogen receptors (GPER), there remains significant skepticism that aldosterone can directly interact with GPER. The current studies are the first to demonstrate directly that aldosterone indeed is capable of binding to the GPER and thus likely mediates its GPER-dependent effects by direct binding to the receptor.

Pannexin 3 deletion reduces fat accumulation and inflammation in a sex-specific manner.

BACKGROUND: Pannexin 3 (PANX3) is a channel-forming glycoprotein that enables nutrient-induced inflammation in vitro, and genetic linkage data suggest that it regulates body mass index. Here, we characterized inflammatory and metabolic parameters in global Panx3 knockout (KO) mice in the context of forced treadmill running (FEX) and high-fat diet (HFD). METHODS: C57BL/6N (WT) and KO mice were randomized to either a FEX running protocol or no running (SED) from 24 until 30 weeks of age. Body weight was measured biweekly, and body composition was measured at 24 and 30 weeks of age. Male WT and KO mice were fed a HFD from 12 to 28 weeks of age. Metabolic organs were analyzed for a panel of inflammatory markers and PANX3 expression. RESULTS: In females there were no significant differences in body composition between genotypes, which could be due to the lack of PANX3 expression in female white adipose tissue, while male KOs fed a chow diet had lower body weight and lower fat mass at 24 and 30 weeks of age, which was reduced to the same extent as 6 weeks of FEX in WT mice. In addition, male KO mice exhibited significantly lower expression of multiple pro-inflammatory genes in white adipose tissue compared to WT mice. While on a HFD body weight differences were insignificant, multiple inflammatory genes were significantly different in quadriceps muscle and white adipose tissue resulting in a more anti-inflammatory phenotype in KO mice compared to WT. The lower fat mass in male KO mice may be due to significantly fewer adipocytes in their subcutaneous fat compared to WT mice. Mechanistically, adipose stromal cells (ASCs) cultured from KO mice grow significantly slower than WT ASCs. CONCLUSION: PANX3 is expressed in male adult mouse adipose tissue and may regulate adipocyte numbers, influencing fat accumulation and inflammation.

Striatal Acetylcholine Helps to Preserve Functional Outcomes in a Mouse Model of Stroke.

Acetylcholine (ACh) has been suggested to facilitate plasticity and improve functional recovery after different types of brain lesions. Interestingly, numerous studies have shown that striatal cholinergic interneurons are relatively resistant to acute ischemic insults, but whether ACh released by these neurons enhances functional recovery after stroke is unknown. We investigated the role of endogenous striatal ACh in stroke lesion volume and functional outcomes following middle cerebral artery occlusion to induce focal ischemia in striatum-selective vesicular acetylcholine transporter-deficient mice (stVAChT-KO). As transporter expression is almost completely eliminated in the striatum of stVAChT-KO mice, ACh release is nearly abolished in this area. Conversely, in other brain areas, VAChT expression and ACh release are preserved. Our results demonstrate a larger infarct size after ischemic insult in stVAChT-KO mice, with more pronounced functional impairments and increased mortality than in littermate controls. These changes are associated with increased activation of GSK-3, decreased levels of ß-catenin, and a higher permeability of the blood-brain barrier in mice with loss of VAChT in striatum neurons. These results support a framework in which endogenous ACh secretion originating from cholinergic interneurons in the striatum helps to protect brain tissue against ischemia-induced damage and facilitates brain recovery by supporting blood-brain barrier function.

Proteinase-Activated Receptor 4 Activation Triggers Cell Membrane Blebbing through RhoA and ß-Arrestin.

Proteinase-activated receptors (PARs) are a four-member family of G-protein-coupled receptors that are activated via proteolysis. PAR4 is a member of this family that is cleaved and activated by serine proteinases such as thrombin, trypsin, and cathepsin-G. PAR4 is expressed in a variety of tissues and cell types, including platelets, vascular smooth muscle cells, and neuronal cells. In studying PAR4 signaling and trafficking, we observed dynamic changes in the cell membrane, with spherical membrane protrusions that resemble plasma membrane blebbing. Since nonapoptotic membrane blebbing is now recognized as an important regulator of cell migration, cancer cell invasion, and vesicular content release, we sought to elucidate the signaling pathway downstream of PAR4 activation that leads to such events. Using a combination of pharmacological inhibition and CRISPR/CRISPR-associated protein 9 (Cas9)-mediated gene editing approaches, we establish that PAR4-dependent membrane blebbing occurs independently of the Gα q/11- and Gα i-signaling pathways and is dependent on signaling via the ß-arrestin-1/2 and Ras homolog family member A (RhoA) signaling pathways. Together these studies provide further mechanistic insight into PAR4 regulation of cellular function. SIGNIFICANCE STATEMENT: We find that the thrombin receptor PAR4 triggers cell membrane blebbing in a RhoA-and ß-arrestin-dependent manner. In addition to identifying novel cellular responses mediated by PAR4, these data provide further evidence for biased signaling in PAR4 since membrane blebbing was dependent on some, but not all, signaling pathways activated by PAR4.

Modulation of hippocampal neuronal resilience during aging by the Hsp70/Hsp90 co-chaperone STI1.

Chaperone networks are dysregulated with aging, but whether compromised Hsp70/Hsp90 chaperone function disturbs neuronal resilience is unknown. Stress-inducible phosphoprotein 1 (STI1; STIP1; HOP) is a co-chaperone that simultaneously interacts with Hsp70 and Hsp90, but whose function in vivo remains poorly understood. We combined in-depth analysis of chaperone genes in human datasets, analysis of a neuronal cell line lacking STI1 and of a mouse line with a hypomorphic Stip1 allele to investigate the requirement for STI1 in aging. Our experiments revealed that dysfunctional STI1 activity compromised Hsp70/Hsp90 chaperone network and neuronal resilience. The levels of a set of Hsp90 co-chaperones and client proteins were selectively affected by reduced levels of STI1, suggesting that their stability depends on functional Hsp70/Hsp90 machinery. Analysis of human databases revealed a subset of co-chaperones, including STI1, whose loss of function is incompatible with life in mammals, albeit they are not essential in yeast. Importantly, mice expressing a hypomorphic STI1 allele presented spontaneous age-dependent hippocampal neurodegeneration and reduced hippocampal volume, with consequent spatial memory deficit. We suggest that impaired STI1 function compromises Hsp70/Hsp90 chaperone activity in mammals and can by itself cause age-dependent hippocampal neurodegeneration in mice. Cover Image for this issue: doi: 10.1111/jnc.14749.

Forebrain Cholinergic Signaling Regulates Innate Immune Responses and Inflammation.

The brain regulates physiological functions integral to survival. However, the insight into brain neuronal regulation of peripheral immune function and the neuromediator systems and pathways involved remains limited. Here, utilizing selective genetic and pharmacological approaches, we studied the role of forebrain cholinergic signaling in the regulation of peripheral immune function and inflammation. Forebrain-selective genetic ablation of acetylcholine release and vagotomy abolished the suppression of serum TNF by the centrally-acting cholinergic drug galantamine in murine endotoxemia. Selective stimulation of acetylcholine action on the M1 muscarinic acetylcholine receptor (M1 mAChR) by central administration of the positive allosteric modulator benzyl quinolone carboxylic acid (BQCA) suppressed serum TNF (TNFα) levels in murine endotoxemia. This effect was recapitulated by peripheral administration of the compound. BQCA also improved survival in murine endotoxemia and these effects were abolished in M1 mAChR knockout (KO) mice. Selective optogenetic stimulation of basal forebrain cholinergic neurons innervating brain regions with abundant M1 mAChR localization reduced serum TNF in endotoxemic mice. These findings reveal that forebrain cholinergic neurons regulate innate immune responses and inflammation, suggesting the possibility that in diseases associated with cholinergic dysfunction, including Alzheimer's disease this anti-inflammatory regulation can be impaired. These results also suggest novel anti-inflammatory approaches based on targeting forebrain cholinergic signaling in sepsis and other disorders characterized by immune dysregulation.

Selective decrease of cholinergic signaling from pedunculopontine and laterodorsal tegmental nuclei has little impact on cognition but markedly increases susceptibility to stress.

The pedunculopontine tegmental nucleus (PPT) and laterodorsal tegmental nucleus (LDT) are heterogeneous brainstem structures that contain cholinergic, glutamatergic, and GABAergic neurons. PPT/LDT neurons are suggested to modulate both cognitive and noncognitive functions, yet the extent to which acetylcholine (ACh) signaling from the PPT/LDT is necessary for normal behavior remains uncertain. We addressed this issue by using a mouse model in which PPT/LDT cholinergic signaling is highly decreased by selective deletion of the vesicular ACh transporter (VAChT) gene. This approach interferes exclusively with ACh signaling, leaving signaling by other neurotransmitters from PPT/LDT cholinergic neurons intact and sparing other cells. VAChT mutants were examined on different PPT/LDT-associated cognitive domains. Interestingly, VAChT mutants showed no attentional deficits and only minor cognitive flexibility impairments while presenting large deficiencies in both spatial and cued Morris water maze (MWM) tasks. Conversely, working spatial memory determined with the Y-maze and spatial memory measured with the Barnes maze were not affected, suggesting that deficits in MWM were unrelated to spatial memory abnormalities. Supporting this interpretation, VAChT mutants exhibited alterations in anxiety-like behavior and increased corticosterone levels after exposure to the MWM, suggesting altered stress response. Thus, PPT/LDT VAChT-mutant mice present little cognitive impairment per se, yet they exhibit increased susceptibility to stress, which may lead to performance deficits in more stressful conditions.-Janickova, H., Kljakic, O., Rosborough, K., Raulic, S., Matovic, S., Gros, R., Saksida, L. M., Bussey, T. J., Inoue, W., Prado, V. F., Prado, M. A. M. Selective decrease of cholinergic signaling from pedunculopontine and laterodorsal tegmental nuclei has little impact on cognition but markedly increases susceptibility to stress.

Ablation of both Cx40 and Panx1 results in similar cardiovascular phenotypes exhibited in Cx40 knockout mice.

Connexins (Cxs) and pannexins (Panxs) are highly regulated large-pore channel-forming proteins that participate in cellular communication via small molecular exchange with the extracellular microenvironment, or in the case of connexins, directly between cells. Given the putative functional overlap between single membrane-spanning connexin hemichannels and Panx channels, and cardiovascular system prevalence, we generated the first Cx40-/-Panx1-/- mouse with the anticipation that this genetic modification would lead to a severe cardiovascular phenotype. Mice null for both Cx40 and Panx1 produced litter sizes and adult growth progression similar to wild-type (WT), Cx40-/- and Panx1-/- mice. Akin to Cx40-/- mice, Cx40-/-Panx1-/- mice exhibited cardiac hypertrophy and elevated systolic, diastolic, and mean arterial blood pressure compared with WT and Panx1-/- mice; however assessment of left ventricular ejection fraction and fractional shortening revealed no evidence of cardiac dysfunction between groups. Furthermore, Cx40-/-, Panx1-/-, and Cx40-/-Panx1-/- mice demonstrated impaired endothelial-mediated vasodilation of aortic segments to increasing concentrations of methacholine (MCh) compared with WT, highlighting roles for both Cx40 and Panx1 in vascular endothelial cell (EC) function. Surprisingly, elevated kidney renin mRNA expression, plasma renin activity, and extraglomerular renin-producing cell populations found in Cx40-/- mice was further exaggerated in double knockout mice. Thus, while gestation and gross development were conserved in Cx40-/-Panx1-/- mice, they exhibit cardiac hypertrophy, hypertension, and impaired endothelial-mediated vasodilation that phenocopies Cx40-/- mice. Nevertheless, the augmented renin homeostasis observed in the double knockout mice suggests that both Cx40 and Panx1 may play an integrative role.

Pannexin 1 regulates adipose stromal cell differentiation and fat accumulation.

Pannexin 1 (Panx1) is a channel-forming glycoprotein important in paracrine signaling and cellular development. In this study, we discovered that mice globally lacking Panx1 (KO) have significantly greater total fat mass and reduced lean mass compared to wild type (WT) mice under a normal diet. Despite having higher fat content, Panx1 KO mice on a high fat diet exhibited no differences in weight gain and blood markers of obesity as compared to WT controls, except for an increase in glucose and insulin levels. However, metabolic cage data revealed that these Panx1 KO mice display significantly increased activity levels, higher ambulatory activity, and reduced sleep duration relative to their WT littermates on a high-fat diet. To uncover the cellular mechanism responsible for the increased fat content in the KO, we isolated primary cultures of adipose-derived stromal cells (ASCs) from WT and KO fat pads. In WT ASCs we observed that Panx1 protein levels increase upon induction into an adipogenic lineage. ASCs isolated from Panx1 KO mice proliferate less but demonstrate enhanced adipogenic differentiation with increased intracellular lipid accumulation, glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, and adipokine secretion, as compared to WT ASCs. This was consistent with the increased adipocyte size and decreased adipocyte numbers observed in subcutaneous fat of the Panx1 KO mice compared to WT. We concluded that Panx1 plays a key role in adipose stromal cells during the early stages of adipogenic proliferation and differentiation, regulating fat accumulation in vivo.

Changes in aortic reactivity associated with the loss of equilibrative nucleoside transporter 1 (ENT1) in mice.

Slc29a1 encodes for equilibrative nucleoside transporter subtype 1 (ENT1), the primary mechanism of adenosine transfer across cell membranes. Previous studies showed that tissues isolated from Slc29a1-null mice are relatively resistant to injury caused by vascular ischemia-reperfusion. To determine if there are similar changes in the microvasculature, and investigate underlying mechanism, we examined aortas isolated from wildtype and Slc29a1-null mice. Aorta macrostructure and gene expression were examined histologically and by qPCR, respectively. Wire myography was used to assess the contractile properties of isolated thoracic aortic rings and their response to adenosine under both normoxic and hypoxic conditions. In vivo haemodynamic parameters were assessed using the tail-cuff method. Slc29a1-null mice had significantly (P<0.05) increased plasma adenosine (2.75-fold) and lower blood pressure (~15% ↓) than wild-type mice. Aortas from Slc29a1-null mice were stiffer with a smaller circumference (11% ↓), and had an enhanced contractile response to KCl and receptor-mediated stimuli. Blockade of ENT1 with nitrobenzylthioinosine significantly enhanced (by ~3.5-fold) the response of aorta from wild-type mice to phenylephrine, but had minimal effect on aortas from Slc29a1-null mice. Adenosine enhanced phenylephrine-mediated constriction in the wild-type tissue under both normoxic (11.7-fold) and hypoxic (3.6-fold) conditions, but had no effect on the Slc29a1-null aortic aorta. In conclusion, aortas from Slc29a1-null mice respond to hypoxic insult in a manner comparable to wild-type tissues that have been pharmacologically preconditioned with adenosine. These data also support a role for ENT1 in the regulation of the protective effects of adenosine on contractile function in elastic conduit arteries such as thoracic aorta.

GRK2 knockdown in mice exacerbates kidney injury and alters renal mechanisms of blood pressure regulation.

The renin-angiotensin system regulates blood pressure and fluid balance in the body primarily via angiotensin receptor 1 (AT1R). Renal AT1R was found to be primarily responsible for Ang II-mediated hypertension. G protein-coupled receptor kinase 2 (GRK2) modulates AT1R desensitization and increased GRK2 protein expression is reported in hypertensive patients. However, the consequences of GRK2 inhibition on kidney functions remain unknown. We employed shGRK2 knockdown mice (shGRK2 mice) to test the role of GRK2 in kidney development and function that can be ultimately linked to the hypertensive phenotype detected in shGRK2 mice. GRK2 knockdown reduced kidney size, nephrogenesis and glomerular count, and impaired glomerular filtration. Glomerular damage in adult shGRK2 mice was associated with increased renin- and AT1R-mediated production of reactive oxygen species. The AT1R blocker, Losartan, normalized elevated blood pressure and markedly improved glomerular filtration in the shGRK2 knockdown mice. Our findings provide evidence for the crucial role of GRK2 in renal regulation of blood pressure. It also suggests that the detrimental outcomes of GRK2 inhibitors on the kidney should be carefully examined when used as antihypertensive.

Cardiac-specific inducible overexpression of human plasma membrane Ca2+ ATPase 4b is cardioprotective and improves survival in mice following ischemic injury.

Background: Heart failure (HF) is associated with reduced expression of plasma membrane Ca2+-ATPase 4 (PMCA4). Cardiac-specific overexpression of human PMCA4b in mice inhibited nNOS activity and reduced cardiac hypertrophy by inhibiting calcineurin. Here we examine temporally regulated cardiac-specific overexpression of hPMCA4b in mouse models of myocardial ischemia reperfusion injury (IRI) ex vivo, and HF following experimental myocardial infarction (MI) in vivoMethods and results: Doxycycline-regulated cardiomyocyte-specific overexpression and activity of hPMCA4b produced adaptive changes in expression levels of Ca2+-regulatory genes, and induced hypertrophy without significant differences in Ca2+ transients or diastolic Ca2+ concentrations. Total cardiac NOS and nNOS-specific activities were reduced in mice with cardiac overexpression of hPMCA4b while nNOS, eNOS and iNOS protein levels did not differ. hMPCA4b-overexpressing mice also exhibited elevated systolic blood pressure vs. controls, with increased contractility and lusitropy in vivo In isolated hearts undergoing IRI, hPMCA4b overexpression was cardioprotective. NO donor-treated hearts overexpressing hPMCA4b showed reduced LVDP and larger infarct size versus vehicle-treated hearts undergoing IRI, demonstrating that the cardioprotective benefits of hPMCA4b-repressed nNOS are lost by restoring NO availability. Finally, both pre-existing and post-MI induction of hPMCA4b overexpression reduced infarct expansion and improved survival from HF.Conclusions: Cardiac PMCA4b regulates nNOS activity, cardiac mass and contractility, such that PMCA4b overexpression preserves cardiac function following IRI, heightens cardiac performance and limits infarct progression, cardiac hypertrophy and HF, even when induced late post-MI. These data identify PMCA4b as a novel therapeutic target for IRI and HF.

Nicotinamide Phosphoribosyltransferase in Smooth Muscle Cells Maintains Genome Integrity, Resists Aortic Medial Degeneration, and Is Suppressed in Human Thoracic Aortic Aneurysm Disease.

RATIONALE: The thoracic aortic wall can degenerate over time with catastrophic consequences. Vascular smooth muscle cells (SMCs) can resist and repair artery damage, but their capacities decline with age and stress. Recently, cellular production of nicotinamide adenine dinucleotide (NAD+) via nicotinamide phosphoribosyltransferase (Nampt) has emerged as a mediator of cell vitality. However, a role for Nampt in aortic SMCs in vivo is unknown. OBJECTIVES: To determine whether a Nampt-NAD+ control system exists within the aortic media and is required for aortic health. METHODS AND RESULTS: Ascending aortas from patients with dilated aortopathy were immunostained for NAMPT, revealing an inverse relationship between SMC NAMPT content and aortic diameter. To determine whether a Nampt-NAD+ control system in SMCs impacts aortic integrity, mice with Nampt-deficient SMCs were generated. SMC-Nampt knockout mice were viable but with mildly dilated aortas that had a 43% reduction in NAD+ in the media. Infusion of angiotensin II led to aortic medial hemorrhage and dissection. SMCs were not apoptotic but displayed senescence associated-ß-galactosidase activity and upregulated p16, indicating premature senescence. Furthermore, there was evidence for oxidized DNA lesions, double-strand DNA strand breaks, and pronounced susceptibility to single-strand breakage. This was linked to suppressed poly(ADP-ribose) polymerase-1 activity and was reversible on resupplying NAD+ with nicotinamide riboside. Remarkably, we discovered unrepaired DNA strand breaks in SMCs within the human ascending aorta, which were specifically enriched in SMCs with low NAMPT. NAMPT promoter analysis revealed CpG hypermethylation within the dilated human thoracic aorta and in SMCs cultured from these tissues, which inversely correlated with NAMPT expression. CONCLUSIONS: The aortic media depends on an intrinsic NAD+ fueling system to protect against DNA damage and premature SMC senescence, with relevance to human thoracic aortopathy.

Deletion of the vesicular acetylcholine transporter from pedunculopontine/laterodorsal tegmental neurons modifies gait.

Postural instability and gait disturbances, common disabilities in the elderly and frequently present in Parkinson's disease (PD), have been suggested to be related to dysfunctional cholinergic signaling in the brainstem. We investigated how long-term loss of cholinergic signaling from mesopontine nuclei influence motor behaviors. We selectively eliminated the vesicular acetylcholine transporter (VAChT) in pedunculopontine and laterodorsal tegmental nuclei cholinergic neurons to generate mice with selective mesopontine cholinergic deficiency (VAChTEn1-Cre-flox/flox ). VAChTEn1-Cre-flox/flox mice did not show any gross health or neuromuscular abnormality on metabolic cages, wire-hang and grip-force tests. Young VAChTEn1-Cre-flox/flox mice (2-5 months-old) presented motor learning/coordination deficits on the rotarod; moved slower, and had smaller steps on the catwalk, but showed no difference in locomotor activity on the open field. Old VAChTEn1-Creflox/flox mice (13-16 months-old) showed more pronounced motor learning/balance deficits on the rotarod, and more pronounced balance deficits on the catwalk. Furthermore, old mutants moved faster than controls, but with similar step length. Additionally, old VAChT-deficient mice were hyperactive. These results suggest that dysfunction of cholinergic neurons from mesopontine nuclei, which is commonly seen in PD, has causal roles in motor functions. Prevention of mesopontine cholinergic failure may help to prevent/improve postural instability and falls in PD patients. Read the Editorial Highlight for this article on page 688.