Lower female survival from an opportunistic infection reveals progesterone-driven sex bias in trained immunity.

Immune responses differ between females and males, although such sex-based variance is incompletely understood. Observing that bacteremia of the opportunistic pathogen Burkholderia gladioli caused many more deaths of female than male mice bearing genetic deficiencies in adaptive immunity, we determined that this was associated with sex bias in the innate immune memory response called trained immunity. Female attenuation of trained immunity varies with estrous cycle stage and correlates with serum progesterone, a hormone that decreases glycolytic capacity and recall cytokine secretion induced by antigen non-specific stimuli. Progesterone receptor antagonism rescues female trained immune responses and survival from controlled B. gladioli infection to magnitudes similar to those of males. These data demonstrate progesterone-dependent sex bias in trained immunity where attenuation of female responses is associated with survival outcomes from opportunistic infection.

TRPV4 increases cardiomyocyte calcium cycling and contractility yet contributes to damage in the aged heart following hypoosmotic stress.

Aims: Cardiomyocyte Ca2+ homeostasis is altered with aging via poorly-understood mechanisms. The Transient Receptor Potential Vanilloid 4 (TRPV4) ion channel is an osmotically-activated Ca2+ channel, and there is limited information on the role of TRPV4 in cardiomyocytes. Our data show that TRPV4 protein expression increases in cardiomyocytes of the aged heart. The objective of this study was to examine the role of TRPV4 in cardiomyocyte Ca2+ homeostasis following hypoosmotic stress and to assess the contribution of TRPV4 to cardiac contractility and tissue damage following ischaemia-reperfusion (I/R), a pathological condition associated with cardiomyocyte osmotic stress. Methods and results: TRPV4 protein expression increased in cardiomyocytes of Aged (24-27 months) mice compared with Young (3-6 months) mice. Immunohistochemistry revealed TRPV4 localization to microtubules and the t-tubule network of cardiomyocytes of Aged mice, as well as in left ventricular myocardium of elderly patients undergoing surgical aortic valve replacement for aortic stenosis. Following hypoosmotic stress, cardiomyocytes of Aged, but not Young exhibited an increase in action-potential induced Ca2+ transients. This effect was mediated via increased sarcoplasmic reticulum Ca2+ content and facilitation of Ryanodine Receptor Ca2+ release and was prevented by TRPV4 antagonism (1 µmol/L HC067047). A similar hypoosmotic stress-induced facilitation of Ca2+ transients was observed in Young transgenic mice with inducible TRPV4 expression in cardiomyocytes. Following I/R, isolated hearts of Young mice with transgenic TRPV4 expression exhibited enhanced contractility vs. hearts of Young control mice. Similarly, hearts of Aged mice exhibited enhanced contractility vs. hearts of Aged TRPV4 knock-out (TRPV4-/-) mice. In Aged, pharmacological inhibition of TRPV4 (1 µmol/L, HC067047) prevented hypoosmotic stress-induced cardiomyocyte death and I/R-induced cardiac damage. Conclusions: Our findings provide a new mechanism for hypoosmotic stress-induced cardiomyocyte Ca2+ entry and cell damage in the aged heart. These finding have potential implications in treatment of elderly populations at increased risk of myocardial infarction and I/R injury.

Increased susceptibility to OVX-associated metabolic dysfunction in UCP1-null mice.

Premenopausal females are protected against adipose tissue inflammation and insulin resistance, until loss of ovarian hormone production (e.g., menopause). There is some evidence that females have greater brown adipose tissue (BAT) thermogenic capacity. Because BAT mass correlates inversely with insulin resistance, we hypothesized that increased uncoupling protein 1 (UCP1) expression contributes to the superior metabolic health of females. Given that UCP1 transiently increases in BAT following ovariectomy (OVX), we hypothesized that UCP1 may 'buffer' OVX-mediated metabolic dysfunction. Accordingly, female UCP1 knock-out (KO) and wild-type (Digby, et al.) mice received OVX or sham (SHM) surgeries at 12 weeks of age creating four groups (n=10/group), which were followed for 14 weeks and compared for: body weight and adiposity, food intake, energy expenditure and spontaneous physical activity (metabolic chambers), insulin resistance (HOMA-IR, ADIPO-IR, and glucose tolerance testing), and adipose tissue phenotype (histology, gene, and protein expression). Two-way ANOVA was used to assess main effects of genotype (G), OVX treatment (O), and genotype by treatment (GxO) interactions, which were considered significant when P<0.05. UCP1KO mice experienced a more adverse metabolic response to OVX than WT. Whereas OVX-induced weight gain was not synergistically greater for KO compared to WT (GxO, NS), OVX-induced insulin resistance was significantly exacerbated in KO compared to WT (GxO for HOMA-IR, P<0.05). These results suggest UCP1 is protective against metabolic dysfunction associated with loss of ovarian hormones and support the need for more research into therapeutics to selectively target UCP1 for prevention and treatment of metabolic dysfunction following ovarian hormone loss.

Deletion of UCP1 enhances ex vivo aortic vasomotor function in female but not male mice despite similar susceptibility to metabolic dysfunction.

Females are typically more insulin sensitive than males, which may be partly attributed to greater brown adipose tissue (BAT) activity and uncoupling protein 1 (UCP1) content. Accordingly, we tested the hypothesis that UCP1 deletion would abolish sex differences in insulin sensitivity and that whitening of thoracic periaortic BAT caused by UCP1 loss would be accompanied with impaired thoracic aortic function. Furthermore, because UCP1 exerts antioxidant effects, we examined whether UCP1 deficiency-induced metabolic dysfunction was mediated by oxidative stress. Compared with males, female mice had lower HOMA- and AT-insulin resistance (IR) despite no significant differences in BAT UCP1 content. UCP1 ablation increased HOMA-IR, AT-IR, and whitening of BAT in both sexes. Expression of UCP1 in thoracic aorta was greater in wild-type females compared with males. Importantly, deletion of UCP1 enhanced aortic vasomotor function in females only. UCP1 ablation did not promote oxidative stress in interscapular BAT. Furthermore, daily administration of the free radical scavenger tempol for 8 wk did not abrogate UCP1 deficiency-induced increases in adiposity, hyperinsulinemia, or liver steatosis. Collectively, we report that 1) in normal chow-fed mice housed at 25°C, aortic UCP1 content was greater in females than males and its deletion improved ex vivo aortic vasomotor function in females only; 2) constitutive UCP1 content in BAT was similar between females and males and loss of UCP1 did not abolish sex differences in insulin sensitivity; and 3) the metabolic disruptions caused by UCP1 ablation did not appear to be contingent upon increased oxidative stress in mice under normal dietary conditions.

Loss of UCP1 exacerbates Western diet-induced glycemic dysregulation independent of changes in body weight in female mice.

We tested the hypothesis that female mice null for uncoupling protein 1 (UCP1) would have increased susceptibility to Western diet-induced "whitening" of brown adipose tissue (AT) and glucose intolerance. Six-week-old C57BL/6J wild-type (WT) and UCP1 knockout (UCP1-/-) mice, housed at 25°C, were randomized to either a control diet (10% kcal from fat) or Western diet (45% kcal from fat and 1% cholesterol) for 28 wk. Loss of UCP1 had no effect on energy intake, energy expenditure, spontaneous physical activity, weight gain, or visceral white AT mass. Despite similar susceptibility to weight gain compared with WT, UCP1-/- exhibited whitening of brown AT evidenced by a striking ~500% increase in mass and appearance of large unilocular adipocytes, increased expression of genes related to inflammation, immune cell infiltration, and endoplasmic reticulum/oxidative stress (P < 0.05), and decreased mitochondrial subunit protein (COX I, II, III, and IV, P < 0.05), all of which were exacerbated by Western diet (P < 0.05). UCP1-/- mice also developed liver steatosis and glucose intolerance, which was worsened by Western diet. Collectively, these findings demonstrate that loss of UCP1 exacerbates Western diet-induced whitening of brown AT, glucose intolerance, and induces liver steatosis. Notably, the adverse metabolic manifestations of UCP1-/- were independent of changes in body weight, visceral adiposity, and energy expenditure. These novel findings uncover a previously unrecognized metabolic protective role of UCP1 that is independent of its already established role in energy homeostasis.

Decreasing maternal myostatin programs adult offspring bone strength in a mouse model of osteogenesis imperfecta.

During fetal development, the uterine environment can have effects on offspring bone architecture and integrity that persist into adulthood; however, the biochemical and molecular mechanisms remain unknown. Myostatin is a negative regulator of muscle mass. Parental myostatin deficiency (Mstntm1Sjl/+) increases muscle mass in wild-type offspring, suggesting an intrauterine programming effect. Here, we hypothesized that Mstntm1Sjl/+ dams would also confer increased bone strength. In wild-type offspring, maternal myostatin deficiency altered fetal growth and calvarial collagen content of newborn mice and conferred a lasting impact on bone geometry and biomechanical integrity of offspring at 4 mo of age, the age of peak bone mass. Second, we sought to apply maternal myostatin deficiency to a mouse model with osteogenesis imperfecta (Col1a2oim), a heritable connective tissue disorder caused by abnormalities in the structure and/or synthesis of type I collagen. Femora of male Col1a2oim/+ offspring from natural mating of Mstntm1Sjl/+ dams to Col1a2oim/+sires had a 15% increase in torsional ultimate strength, a 29% increase in tensile strength, and a 24% increase in energy to failure compared with age, sex, and genotype-matched offspring from natural mating of Col1a2oim/+ dams to Col1a2oim/+ sires. Finally, increased bone biomechanical strength of Col1a2oim/+ offspring that had been transferred into Mstntm1Sjl/+ dams as blastocysts demonstrated that the effects of maternal myostatin deficiency were conferred by the postimplantation environment. Thus, targeting the gestational environment, and specifically prenatal myostatin pathways, provides a potential therapeutic window and an approach for treating osteogenesis imperfecta.

Transcription enhancer factor 1 binds multiple muscle MEF2 and A/T-rich elements during fast-to-slow skeletal muscle fiber type transitions.

In adult mouse skeletal muscle, beta-myosin heavy chain (betaMyHC) gene expression is primarily restricted to slow type I fibers; however, its expression can be induced in fast type II fibers in response to a sustained increase in load-bearing work (mechanical overload [MOV]). Our previous betaMyHC transgenic and protein-DNA interaction studies have identified an A/T-rich element (betaA/T-rich -269/-258) that is required for slow muscle expression and which potentiates MOV responsiveness of a 293-bp betaMyHC promoter (beta293wt). Despite the GATA/MEF2-like homology of this element, we found binding of two unknown proteins that were antigenically distinct from GATA and MEF2 isoforms. By using the betaA/T-rich element as bait in a yeast one-hybrid screen of an MOV-plantaris cDNA library, we identified nominal transcription enhancer factor 1 (NTEF-1) as the specific betaA/T-rich binding factor. Electrophoretic mobility shift assay analysis confirmed that NTEF-1 represents the enriched binding activity obtained only when the betaA/T-rich element is reacted with MOV-plantaris nuclear extract. Moreover, we show that TEF proteins bind MEF2 elements located in the control region of a select set of muscle genes. In transient-coexpression assays using mouse C2C12 myotubes, TEF proteins transcriptionally activated a 293-bp betaMyHC promoter devoid of any muscle CAT (MCAT) sites, as well as a minimal thymidine kinase promoter-luciferase reporter gene driven by three tandem copies of the desmin MEF2 or palindromic Mt elements or four tandem betaA/T-rich elements. These novel findings suggest that in addition to exerting a regulatory effect by binding MCAT elements, TEF proteins likely contribute to regulation of skeletal, cardiac, and smooth muscle gene networks by binding select A/T-rich and MEF2 elements under basal and hypertrophic conditions.

Combinatorial evolution of high-affinity peptides that bind to the Thomsen-Friedenreich carcinoma antigen.

Thomsen-Friedenreich (TF) antigen occurs on approximately 90% of human carcinomas, is likely involved in carcinoma cell homotypic aggregation, and has clinical value as a prognostic indicator and marker of metastasized cells. Previously, we isolated anti-TF antigen peptides from bacteriophage display libraries. These bound to TF antigen on carcinoma cells but were of low affinity and solubility. We hypothesized that peptide amino acid sequence changes would result in increased affinity and solubility, which would translate into improved carcinoma cell binding and increased inhibition of aggregation. The new peptides were more soluble and exhibited up to fivefold increase in affinity (Kd approximately equal to 60 nM). They bound cultured human breast and prostate carcinoma cells at low concentrations, whereas the earlier peptides did not. Moreover, the new peptides were potent inhibitors of homotypic aggregation. The maturated peptides will have expanded applications in basic studies of the TF antigen and particular utility as clinical carcinoma-targeting agents.

Divergence in species and regulatory role of beta -myosin heavy chain proximal promoter muscle-CAT elements.

We examined the functional role of distinct muscle-CAT (MCAT) elements during non-weight-bearing (NWB) regulation of a wild-type 293-base pair beta-myosin heavy chain (beta MyHC) transgene. Electrophoretic mobility shift assays (EMSA) revealed decreased NTEF-1, poly(ADP-ribose) polymerase, and Max binding at the human distal MCAT element when using NWB soleus vs. control soleus nuclear extract. Compared with the wild-type transgene, expression assays revealed that distal MCAT element mutation decreased basal transgene expression, which was decreased further in response to NWB. EMSA analysis of the human proximal MCAT (pMCAT) element revealed low levels of NTEF-1 binding that did not differ between control and NWB extract, whereas the rat pMCAT element displayed robust NTEF-1 binding that decreased when using NWB soleus extracts. Differences in binding between human and rat pMCAT elements were consistent whether using rat or mouse nuclear extract or in vitro synthesized human TEF-1 proteins. Our results provide the first evidence that 1) different binding properties and likely regulatory functions are served by the human and rat pMCAT elements, and 2) previously unrecognized beta MyHC proximal promoter elements contribute to NWB regulation.

Identification and characterization of peptides that bind human ErbB-2 selected from a bacteriophage display library.

The ErbB-2 receptor, a member of the tyrosine kinase type 1 family of receptors, has been implicated in many human malignancies. The overexpression of ErbB-2 in cancer cells as well as its extracellular accessibility makes it an attractive target for the development of tumor-specific agents. In this study, random peptide bacteriophage display technology was employed to identify peptides that bound the extracellular domain of human ErbB-2. The peptide KCCYSL, most frequently occurring in the affinity-selected phage population, was chemically synthesized and characterized for its binding activities to ErbB-2. The synthetic peptide exhibited high specificity for ErbB-2 and an equilibrium dissociation constant of 30 microM. Peptide binding to ErbB-2 positive human breast and prostate carcinoma cells was visualized in direct cell binding assays. In conclusion, the peptide KCCYSL has the potential to be developed into a cancer imaging or therapeutic agent targeting malignant cells overexpressing the ErbB-2 receptor.