Single nuclei profiling identifies cell specific markers of skeletal muscle aging, frailty, and senescence.

Aging is accompanied by a loss of muscle mass and function, termed sarcopenia, which causes numerous morbidities and economic burdens in human populations. Mechanisms implicated in age-related sarcopenia or frailty include inflammation, muscle stem cell depletion, mitochondrial dysfunction, and loss of motor neurons, but whether there are key drivers of sarcopenia are not yet known. To gain deeper insights into age-related muscle loss, we performed transcriptome profiling on lower limb muscle biopsies from 72 young, elderly, and frail human subjects using bulk RNA-seq (N = 72) and single-nuclei RNA-seq (N = 17). This combined approach revealed changes in gene expression that occur with age and frailty in multiple cell types comprising mature skeletal muscle. Notably, we found increased expression of the genes MYH8 and PDK4, and decreased expression of the gene IGFN1, in aged muscle. We validated several key genes changes in fixed human muscle tissue using digital spatial profiling. We also identified a small population of nuclei that express CDKN1A, present only in aged samples, consistent with p21cip1-driven senescence in this subpopulation. Overall, our findings identify unique cellular subpopulations in aged and sarcopenic skeletal muscle, which will facilitate the development of new therapeutic strategies to combat age-related frailty.

Senolysis induced by 25-hydroxycholesterol targets CRYAB in multiple cell types.

Cellular senescence is a driver of many age-related pathologies. There is an active search for pharmaceuticals termed senolytics that can mitigate or remove senescent cells in vivo by targeting genes that promote the survival of senescent cells. We utilized single-cell RNA sequencing to identify CRYAB as a robust senescence-induced gene and potential target for senolysis. Using chemical inhibitor screening for CRYAB disruption, we identified 25-hydroxycholesterol (25HC), an endogenous metabolite of cholesterol biosynthesis, as a potent senolytic. We then validated 25HC as a senolytic in mouse and human cells in culture and in vivo in mouse skeletal muscle. Thus, 25HC represents a potential class of senolytics, which may be useful in combating diseases or physiologies in which cellular senescence is a key driver.

The Ror1 receptor tyrosine kinase plays a critical role in regulating satellite cell proliferation during regeneration of injured muscle.

The Ror family receptor tyrosine kinases, Ror1 and Ror2, play important roles in regulating developmental morphogenesis and tissue- and organogenesis, but their roles in tissue regeneration in adult animals remain largely unknown. In this study, we examined the expression and function of Ror1 and Ror2 during skeletal muscle regeneration. Using an in vivo skeletal muscle injury model, we show that expression of Ror1 and Ror2 in skeletal muscles is induced transiently by the inflammatory cytokines, TNF-α and IL-1ß, after injury and that inhibition of TNF-α and IL-1ß by neutralizing antibodies suppresses expression of Ror1 and Ror2 in injured muscles. Importantly, expression of Ror1, but not Ror2, was induced primarily in Pax7-positive satellite cells (SCs) after muscle injury, and administration of neutralizing antibodies decreased the proportion of Pax7-positive proliferative SCs after muscle injury. We also found that stimulation of a mouse myogenic cell line, C2C12 cells, with TNF-α or IL-1ß induced expression of Ror1 via NF-κB activation and that suppressed expression of Ror1 inhibited their proliferative responses in SCs. Intriguingly, SC-specific depletion of Ror1 decreased the number of Pax7-positive SCs after muscle injury. Collectively, these findings indicate for the first time that Ror1 has a critical role in regulating SC proliferation during skeletal muscle regeneration. We conclude that Ror1 might be a suitable target in the development of diagnostic and therapeutic approaches to manage muscular disorders.

Changes in the nitric oxide-soluble guanylate cyclase system and natriuretic peptide receptor system in placentas of pregnant Dahl salt-sensitive rats.

AIM: Diminished vasodilator activity during pregnancy, which augments vascular responses to vasoconstrictors, is one reason for the onset of pre-eclampsia and superimposed pre-eclampsia. It is known that Dahl salt-sensitive (Dahl-S) rats develop salt-sensitive hypertension like African-Americans. The present study attempted to assess the changes and the interactions of the NOS-NO-sGC-cGMP and NP-NPR-cGMP systems in the hypertensive placenta using Dahl-S rats as an animal model of superimposed pre-eclampsia. MATERIAL AND METHODS: Pregnant Dahl-S rats were fed a high-salt diet to induce the development of hypertension and fetal growth restriction. Using these rats, we investigated the regulation of these two vasodilatation systems, including the kinetics of cyclic guanosine monophosphate (cGMP), soluble guanylate cyclase (sGC), endothelial nitric oxide synthase (NOS), cytokine-inducible NOS, natriuretic peptides (NP) (atrial NP, brain NP and C-type NP), and NP receptors (NPR) (NPR-A, NPR-B, NPR-C). RESULTS: Dahl-S rats fed a high-salt diet exhibited hypertension, fetal growth restriction and thickening of the walls in decidual vessels. The placental cGMP level in the rats fed the high-salt diet was significantly decreased compared with that in controls. The expression levels of endothelial NOS and cytokine-inducible NOS mRNA increased significantly, while that of sGCα2-sunbnit declined significantly. Messenger RNA levels of NPR-C, a clearance-type receptor of NP, declined significantly, whereas those of NP and their functional receptors NPR-A and NPR-B were unchanged. CONCLUSIONS: As Dahl-S rats with excess salt-loading during pregnancy exhibited pathological changes similar to those observed in female humans with pre-eclampsia/superimposed pre-eclampsia, this rat could be useful as an animal model of superimposed pre-eclampsia. In the placentas of hypertensive Dahl-S rats, vasodilatation seemed to be disturbed by the deregulation of both the NO-sGC-cGMP and NP-NPR-cGMP systems.

Harmine lengthens circadian period of the mammalian molecular clock in the suprachiasmatic nucleus.

The circadian clock is a cell-autonomous endogenous system that generates circadian rhythms in the behavior and physiology of most organisms. We previously reported that the harmala alkaloid, harmine, lengthens the circadian period of Bmal1 transcription in NIH 3T3 fibroblasts. Clock protein dynamics were examined using real-time reporter assays of PER2::LUC to determine the effects of harmine on the central clock in the suprachiasmatic nucleus (SCN). Harmine significantly lengthened the period of PER2::LUC expression in embryonic fibroblasts, in neuronal cells differentiated from neuronal progenitor cells and in SCN slices obtained from PER2::LUC mice. Although harmine did not induce the transient mRNA expression of clock genes such as Per1, Per2 and Bmal1 in embryonic fibroblasts, it significantly extended the half-life of PER2::LUC protein in neuronal cells and SCN slices. Harmine might lengthen the circadian period of the molecular clock by increasing PER2 protein stability in the SCN.

Critical role of Frizzled1 in age-related alterations of Wnt/ß-catenin signal in myogenic cells during differentiation.

Activation of Wnt/ß-catenin signal in muscle satellite cells (mSCs) of aged mice during myogenic differentiation has been appreciated as an important age-related feature of the skeletal muscles, resulting in impairment of their regenerative ability following muscle injury. However, it remains elusive about molecules involved in this age-related alteration of Wnt/ß-catenin signal in myogenic cells. To clarify this issue, we carried out expression analyses of Wnt receptor genes using real-time RT-PCR in mSCs isolated from the skeletal muscles of young and aged mice. Here, we show that expression of Frizzled1 (Fzd1) was detected at high levels in mSCs of aged mice. Higher expression levels of Fzd1 were also detected in mSC-derived myogenic cells from aged mice and associated with activation of Wnt/ß-catenin signal during their myogenic differentiation in vitro. We also provide evidence that suppressed expression of Fzd1 in myogenic cells from aged mice results in a significant increase in myogenic differentiation, and its forced expression in those from young mice results in its drastic inhibition. These findings indicate the critical role of Fzd1 in altered myogenic differentiation associated with aging.

Ketogenic diet and fasting induce the expression of cold-inducible RNA-binding protein with time-dependent hypothermia in the mouse liver.

Cold-inducible RNA-binding protein (CIRBP) induced by cold stress modulates the molecular circadian clock in vitro. The present study examines the effect of a ketogenic diet (KD) and fasting on Cirbp expression in the mouse liver. Chronic KD administration induced time-dependent Cirbp expression with hypothermia in mice. The circadian expression of clock genes such as Bmal1 and Clock was phase-advanced and augmented in the liver of mice fed with a KD. Transient food deprivation also induced time-dependent Cirbp expression with hypothermia in mice. These findings suggest that hypothermia is involved in the increased expression of Cirbp under ketogenic or fasting conditions.

The circadian binding of CLOCK protein to the promoter of C/ebpα gene in mouse cells.

C/EBPα plays important roles in metabolism as well as in the maintenance of energy homeostasis. Here we describe loss of the circadian oscillation of C/ebpα expression in liver of Clock mutant mice. Reporter assays indicate Clock and Bmal significantly induced C/ebpα gene expression whereas Cry suppressed. Real time reporter assays showed that two mutated E-boxes disrupted C/ebpα promoter dependent-oscillation. Chromatin immunoprecipitation suggests Clock can bind to two E-boxes in the C/ebpα promoter with a circadian manner in vivo. Thus, C/ebpα gene transcription is under circadian control of a core clock component, Clock. The data suggests that circadian disturbances may affect metabolic abnormalities through the C/ebpα pathway in liver.

Repeated application of glucocorticoids exacerbate pruritus via inhibition of prostaglandin D2 production of mast cells in a murine model of allergic contact dermatitis.

Rebound is known to occur most typically when topical glucocorticoids are abruptly discontinued; however, its frequency and severity are poorly characterized. We previously created a novel murine model of topical glucocorticoid-induced pruritus; however, the mechanism underlying pruritus in this model has not been elucidated. Using this murine model, we aimed to determine the cause of augmentation of pruritus with a focus on the production of prostaglandin (PG) D(2). BALB/c mice with chronic allergic contact dermatitis induced by 5 weeks of repeated application of 2,4,6-trinitro-1-chlorobenzene (TNCB) were treated topically with dexamethasone for 5 weeks immediately after the elicitation of dermatitis and after ear-swelling and scratching behavior were measured. RBL-2H3 mast cells were used to investigate the effect of dexamethasone on degranulation or PGD(2) production in IgE/antigen-stimulated mast cells. The scratching behavior induced by TNCB was augmented by topical application of dexamethasone, but dexamethasone did not have any effect on scratching bouts in mice that had not been treated with TNCB. Topical dexamethasone reduced the PGD(2) level, which increase in TNCB-treated mice, to the baseline level. Moreover, dexamethasone significantly decreased the PGD(2) production in IgE/antigen-stimulated RBL-2H3 mast cells; however, the same concentration of dexamethasone did not have any effect on the degranulation of stimulated mast cells. Topical glucocorticoids may exacerbate pruritus in a mouse model of allergic contact dermatitis via inhibition of PGD(2) production in antigen-mediated activated mast cells in the skin.

Separation of oil-in-water emulsions by microbubble treatment and the effect of adding coagulant or cationic surfactant on removal efficiency.

This study examined the efficiencies of microbubble (MB) treatment, MB treatment with polyaluminium chloride (PAC) as a coagulant, and MB treatment with cetyltrimethylammonium chloride (CTAC) as a cationic surfactant in the separation of emulsified oil (EO) by modified column flotation. Batch mode experiments were conducted by synthesizing emulsified palm oil (d<20 µm), and the chemical oxygen demand (COD) of the influent and effluent was measured to evaluate the treatment performance. MB treatment with PAC and MB treatment with CTAC were found to be more efficient in EO removal than the MB treatment alone. At an EO concentration of ∼1,000 mg L(-1) (pH 7) and under identical treatment conditions (MB generation time: 2.5 min, flotation time: 30 min), MB treatment with PAC (50 mg L(-1)) and that with CTAC (0.5 mg L(-1)) showed equally high EO removal efficiencies of 92 and 89%, respectively. This result is of significant relevance to studies focusing on the development of economical and high-efficiency flotation systems. Furthermore, the effect of pH was investigated by varying the sample pH from 3 to 8, which showed that the EO separation efficiency of MB alone increased drastically from slightly alkaline to acidic condition.

Ror family receptor tyrosine kinases regulate the maintenance of neural progenitor cells in the developing neocortex.

The Ror family receptor tyrosine kinases (RTKs), Ror1 and Ror2, have been shown to play crucial roles in developmental morphogenesis by acting as receptors or co-receptors to mediate Wnt5a-induced signaling. Although Ror1, Ror2 and Wnt5a are expressed in the developing brain, little is known about their roles in the neural development. Here we show that Ror1, Ror2 and their ligand Wnt5a are highly expressed in neocortical neural progenitor cells (NPCs). Small interfering RNA (siRNA)-mediated suppression of Ror1, Ror2 or Wnt5a in cultured NPCs isolated from embryonic neocortex results in the reduction of ßIII-tubulin-positive neurons that are produced from NPCs possibly through the generation of T-box brain 2 (Tbr2)-positive intermediate progenitors. BrdU-labeling experiments further reveal that the proportion of proliferative and neurogenic NPCs, which are positive for neural progenitor cell marker (Pax6) but negative for glial cell marker (glial fibrillary acidic protein; GFAP), is reduced within a few days in culture following knockdown of these molecules, suggesting that Ror1, Ror2 and Wnt5a regulate neurogenesis through the maintenance of NPCs. Moreover, we show that Dishevelled 2 (Dvl2) is involved in Wnt5a-Ror1 and Wnt5a-Ror2 signaling in NPCs, and that suppressed expression of Dvl2 indeed reduces the proportion of proliferative and neurogenic NPCs. Interestingly, suppressed expression of either Ror1 or Ror2 in NPCs in the developing neocortex results in the precocious differentiation of NPCs into neurons, and their forced expression results in delayed differentiation. Collectively, these results indicate that Wnt5a-Ror1 and Wnt5a-Ror2 signaling pathways play roles in maintaining proliferative and neurogenic NPCs during neurogenesis of the developing neocortex.

A novel animal model of pruritus induced by successive application of glucocorticoid to mouse skin.

Topical glucocorticoids are commonly applied for treatment of atopic dermatitis, and are often administered over a long period. However, itching often occurs as a rebound phenomenon after cessation of long-term glucocorticoid application. The present study was an initial trial designed to establish an animal model of glucocorticoid-induced pruritus by topical application of dexamethasone over a long period in mice with contact dermatitis. BALB/c mice with chronic allergic contact dermatitis induced by 5 weeks of repeated application of 2,4,6-trinitro-1-chlorobenzene (TNCB) were treated topically with dexamethasone for 3 weeks from 2 weeks after the elicitation of dermatitis. The effects of dexamethasone on inflammation and pruritus were evaluated by measurement of ear-swelling and scratching behavior, respectively. Significant enhancement of pruritus was confirmed after chronic application of dexamethasone. The increased frequency of scratching behavior was reduced by withdrawal of dexamethasone. On the other hand, ear-swelling was markedly ameliorated by dexamethasone treatment, but rapidly relapsed after dexamethasone withdrawal. The level of interleukin (IL)-4 mRNA in ear skin and that of IgE in serum were increased in the mice with dermatitis and reduced by dexamethasone treatment. On the other hand, the level of nerve growth factor (NGF) mRNA was slightly increased by dexamethasone treatment and remained high even after its discontinuation. It is anticipated that this novel animal model of glucocorticoid-induced pruritus will be useful for clarifying the mechanisms of the rebound phenomenon induced by chronic treatment with topical glucocorticoids, and for developing a new form of therapy.

Plasma levels of n-decanoyl ghrelin, another acyl- and active-form of ghrelin, in human subjects and the effect of glucose- or meal-ingestion on its dynamics.

Besides n-octanoyl ghrelin (O-ghrelin), there is another acyl-form of ghrelin; n-decanoyl ghrelin (D-ghrelin), which has a decanoic acid modification. In this study, we examined the kinetics of D-ghrelin immunoreactivity in human plasma in comparison to O-ghrelin or total ghrelin by using a D-ghrelin-specific radioimmunoassay. The dynamics of plasma D-ghrelin was assessed following glucose- or meal-ingestion in healthy, non-obese subjects (5 males and 5 females). Correlations were also analyzed between the levels of plasma D-ghrelin and anthropometric or metabolic indicators in healthy human subjects (n=111, BMI 17.4-34.3). The plasma levels of D-ghrelin, like O- or T-ghrelin, significantly declined (p<0.05 for male and p<0.01 for female) 60 min after the ingestion of glucose in non-obese subjects. However, in the same subjects, no significant decline was noted in the levels of D-ghrelin, unlike O- or T-ghrelin, upon the meal ingestion. A significant increase was observed in the proportion of plasma D-ghrelin levels to that of T-ghrelin (p<0.05) in the healthy human subjects as BMI increased, unlike the proportion of O-ghrelin to T-ghrelin, which did not change. Since D-ghrelin possesses almost the same potential as that of O-ghrelin with regard to the feeding-stimulation, these differences between the dynamics of D- and O-ghrelin in human plasma might influence appetite-control, especially in those with increased BMI.

CLOCK regulates circadian rhythms of hepatic glycogen synthesis through transcriptional activation of Gys2.

Hepatic glycogen content is important for glucose homeostasis and exhibits robust circadian rhythms that peak at the end of the active phase in mammals. The activities of the rate-limiting enzymes for glycogenesis and glycogenolysis also show circadian rhythms, and the balance between them forms the circadian rhythm of the hepatic glycogen content. However, no direct evidence has yet implicated the circadian clock in the regulation of glycogen metabolism at the molecular level. We show here that a Clock gene mutation damps the circadian rhythm of the hepatic glycogen content, as well as the circadian mRNA and protein expression of Gys2 (glycogen synthase 2), which is the rate-limiting enzyme of glycogenesis in the liver. Transient reporter assays revealed that CLOCK drives the transcriptional activation of Gys2 via two tandemly located E-boxes. Chromatin immunoprecipitation assays of liver tissues revealed that CLOCK binds to these E-box elements in vivo, and real time reporter assays showed that these elements are sufficient for circadian Gys2 expression in vitro. Thus, CLOCK regulates the circadian rhythms of hepatic glycogen synthesis through transcriptional activation of Gys2.

Ketogenic diet disrupts the circadian clock and increases hypofibrinolytic risk by inducing expression of plasminogen activator inhibitor-1.

OBJECTIVE: Metabolic disorders such as diabetes and obesity are considered risk factors for cardiovascular diseases by increasing levels of blood plasminogen activator inhibitor-1 (PAI-1). Ketogenic diets (KDs) have been used as an approach to weight loss in both obese and nonobese individuals. We examined circadian changes in plasma PAI-1 and its mRNA expression levels in tissues from mice fed with a KD (KD mice), to evaluate its effects on fibrinolytic functions. METHODS AND RESULTS: Two weeks on the kDa increased plasma levels of free fatty acids and ketones accompanied by hypoglycemia in mice. Plasma PAI-1 concentrations were extremely elevated in accordance with mRNA expression levels in the heart and liver, but not in the kidneys of KD mice. Circadian expression of PAI-1 mRNA was phase-advanced for 4.7, 7.9, and 7.8 hours in the heart, kidney, and adipose tissues, respectively, as well as that of circadian genes mPer2 and DBP in KD mice, suggesting that peripheral clocks were phase-advanced by ketosis despite feeding ad libitum under a periodic light-dark cycle. The circadian clock that regulates behavioral activity rhythms was also phase-advanced, and its free-running period was significantly shortened in KD mice. CONCLUSIONS: Our findings suggest that ketogenic status increases hypofibrinolytic risk by inducing abnormal circadian expression of PAI-1.

PERIOD2 is a circadian negative regulator of PAI-1 gene expression in mice.

An increased level of obesity-induced plasma plasminogen activator inhibitor-1 (PAI-1) is considered a risk factor for cardiovascular disease. To determine whether the circadian clock component PERIOD2 (PER2) is involved in the regulation of PAI-1 gene expression, we performed transient transfection assays in vitro, and generated transgenic (Tg) mice overexpressing PER2. We then compared PAI-1 expression in Tg and wild-type (WT) mice with or without obesity induced by a high-fat/high-sucrose diet. PER2 suppressed CLOCK:BMAL1- and CLOCK:BMAL2-dependent transactivation of the PAI-1 promoter in vitro. Furthermore, nuclear translocation is dispensable for PER2 to suppress CLOCK:BMAL1-dependent transactivation of the PAI-1 promoter, because functional loss of the nuclear localization domain did not affect either the interaction with BMAL1 or the suppressive role of PER2. The diurnal expression of clock and clock-controlled genes was disrupted in a gene-specific manner, whereas that of PAI-1 mRNA was significantly damped in the hearts of PER2 Tg mice fed with a normal diet. Obesity-induced plasma PAI-1 increase was significantly suppressed in Tg mice in accordance with cardiac PAI-1 mRNA levels, whereas body weight gain and changes in metabolic parameters were identical between WT and Tg mice. Endogenous PAI-1 gene expression induced by transforming growth factor-beta1 was significantly attenuated in embryonic fibroblasts derived from Tg mice compared with those from WT mice. Our results demonstrated that PER2 represses PAI-1 gene transcription in a BMAL1/2-dependent manner. The present findings also suggest that PER2 attenuates obesity-induced hypofibrinolysis by downregulating PAI-1 expression independently of metabolic disorders.