Nicotine and Microvascular Responses in Skeletal Muscle from Acute Exposure to Cigarettes and Vaping.

Despite claims of safety or harm reduction for electronic cigarettes (E-cig) use (also known as vaping), emerging evidence indicates that E-cigs are not likely safe, or necessarily safer than traditional cigarettes, when considering the user's risk of developing vascular dysfunction/disease. E-cigs are different from regular cigarettes in that E-cig devices are highly customizable, and users can change the e-liquid composition (such as the base solution, flavors, and nicotine level). Since the effects of E-cigs on the microvascular responses in skeletal muscle are poorly understood, we used intravital microscopy with an acute (one-time 10 puff) exposure paradigm to evaluate the individual components of e-liquid on vascular tone and endothelial function in the arterioles of the gluteus maximus muscle of anesthetized C57Bl/6 mice. Consistent with the molecular responses seen with endothelial cells, we found that the peripheral vasoconstriction response was similar between mice exposed to E-cig aerosol or cigarette smoke (i.e., 3R4F reference cigarette); this response was not nicotine dependent, and endothelial cell-mediated vasodilation was not altered within this acute exposure paradigm. We also report that, regardless of the base solution component [i.e., vegetable glycerin (VG)-only or propylene glycol (PG)-only], the vasoconstriction responses were the same in mice with inhalation exposure to 3R4F cigarette smoke or E-cig aerosol. Key findings from this work reveal that some component other than nicotine, in inhaled smoke or aerosol, is responsible for triggering peripheral vasoconstriction in skeletal muscle, and that regardless of one's preference for an E-cig base solution composition (i.e., ratio of VG-to-PG), the acute physiological response to blood vessels appears to be the same. The data suggest that vaping is not likely to be 'safer' than smoking towards blood vessels and can be expected to produce and/or result in the same adverse vascular health outcomes associated with smoking cigarettes.

Metabolic physiology and skeletal muscle phenotypes in male and female myoglobin knockout mice.

Myoglobin (Mb) is a regulator of O2 bioavailability in type I muscle and heart, at least when tissue O2 levels drop. Mb also plays a role in regulating cellular nitric oxide (NO) pools. Robust binding of long-chain fatty acids and long-chain acylcarnitines to Mb, and enhanced glucose metabolism in hearts of Mb knockout (KO) mice, suggest additional roles in muscle intermediary metabolism and fuel selection. To evaluate this hypothesis, we measured energy expenditure (EE), respiratory exchange ratio (RER), body weight gain and adiposity, glucose tolerance, and insulin sensitivity in Mb knockout (Mb-/-) and wild-type (WT) mice challenged with a high-fat diet (HFD, 45% of calories). In males (n = 10/genotype) and females (n = 9/genotype) tested at 5-6, 11-12, and 17-18 wk, there were no genotype effects on RER, EE, or food intake. RER and EE during cold (10°C, 72 h), and glucose and insulin tolerance, were not different compared with within-sex WT controls. At ∼18 and ∼19 wk of age, female Mb-/- adiposity was ∼42%-48% higher versus WT females (P = 0.1). Transcriptomics analyses (whole gastrocnemius, soleus) revealed few consistent changes, with the notable exception of a 20% drop in soleus transferrin receptor (Tfrc) mRNA. Capillarity indices were significantly increased in Mb-/-, specifically in Mb-rich soleus and deep gastrocnemius. The results indicate that Mb loss does not have a major impact on whole body glucose homeostasis, EE, RER, or response to a cold challenge in mice. However, the greater adiposity in female Mb-/- mice indicates a sex-specific effect of Mb KO on fat storage and feed efficiency.NEW & NOTEWORTHY The roles of myoglobin remain to be elaborated. We address sexual dimorphism in terms of outcomes in response to the loss of myoglobin in knockout mice and perform, for the first time, a series of comprehensive metabolic studies under conditions in which fat is mobilized (high-fat diet, cold). The results highlight that myoglobin is not necessary and sufficient for maintaining oxidative metabolism and point to alternative roles for this protein in muscle and heart.

Sex differences in skeletal muscle revealed through fiber type, capillarity, and transcriptomics profiling in mice.

Skeletal muscle anatomy and physiology are sexually dimorphic but molecular underpinnings and muscle-specificity are not well-established. Variances in metabolic health, fitness level, sedentary behavior, genetics, and age make it difficult to discern inherent sex effects in humans. Therefore, mice under well-controlled conditions were used to determine female and male (n = 19/sex) skeletal muscle fiber type/size and capillarity in superficial and deep gastrocnemius (GA-s, GA-d), soleus (SOL), extensor digitorum longus (EDL), and plantaris (PLT), and transcriptome patterns were also determined (GA, SOL). Summed muscle weight strongly correlated with lean body mass (r2  = 0.67, p < 0.0001, both sexes). Other phenotypes were muscle-specific: e.g., capillarity (higher density, male GA-s), myofiber size (higher, male EDL), and fiber type (higher, lower type I and type II prevalences, respectively, in female SOL). There were broad differences in transcriptomics, with >6000 (GA) and >4000 (SOL) mRNAs differentially-expressed by sex; only a minority of these were shared across GA and SOL. Pathway analyses revealed differences in ribosome biology, transcription, and RNA processing. Curation of sexually dimorphic muscle transcripts shared in GA and SOL, and literature datasets from mice and humans, identified 11 genes that we propose are canonical to innate sex differences in muscle: Xist, Kdm6a, Grb10, Oas2, Rps4x (higher, females) and Ddx3y, Kdm5d, Irx3, Wwp1, Aldh1a1, Cd24a (higher, males). These genes and those with the highest "sex-biased" expression in our study do not contain estrogen-response elements (exception, Greb1), but a subset are proposed to be regulated through androgen response elements. We hypothesize that innate muscle sexual dimorphism in mice and humans is triggered and then maintained by classic X inactivation (Xist, females) and Y activation (Ddx3y, males), with coincident engagement of X encoded (Kdm6a) and Y encoded (Kdm5d) demethylase epigenetic regulators that are complemented by modulation at some regions of the genome that respond to androgen.