Loss of endogenous estrogen alters mitochondrial metabolism and muscle clock-related protein Rbm20 in female mdx mice.

Female carriers of a Duchenne muscular dystrophy (DMD) gene mutation manifest exercise intolerance and metabolic anomalies that may be exacerbated following menopause due to the loss of estrogen, a known regulator of skeletal muscle function and metabolism. Here, we studied the impact of estrogen depletion (via ovariectomy) on exercise tolerance and muscle mitochondrial metabolism in female mdx mice and the potential of estrogen replacement therapy (using estradiol) to protect against functional and metabolic perturbations. We also investigated the effect of estrogen depletion, and replacement, on the skeletal muscle proteome through an untargeted proteomic approach with TMT-labelling. Our study confirms that loss of estrogen in female mdx mice reduces exercise capacity, tricarboxylic acid cycle intermediates, and citrate synthase activity but that these deficits are offset through estrogen replacement therapy. Furthermore, ovariectomy downregulated protein expression of RNA-binding motif factor 20 (Rbm20), a critical regulator of sarcomeric and muscle homeostasis gene splicing, which impacted pathways involving ribosomal and mitochondrial translation. Estrogen replacement modulated Rbm20 protein expression and promoted metabolic processes and the upregulation of proteins involved in mitochondrial dynamics and metabolism. Our data suggest that estrogen mitigates dystrophinopathic features in female mdx mice and that estrogen replacement may be a potential therapy for post-menopausal DMD carriers.

The chronic leukocyte and inflammatory cytokine responses of older adults to resistance training in normobaric hypoxia; a randomized controlled trial.

TRIAL DESIGN: Older adults experience chronic dysregulation of leukocytes and inflammatory cytokines, both at rest and in response to resistance training. Systemic hypoxia modulates leukocytes and cytokines, therefore this study characterized the effects of normobaric hypoxia on the leukocyte and cytokine responses of older adults to resistance training. METHODS: 20 adults aged 60-70 years performed eight weeks of moderate-intensity resistance training in either normoxia or normobaric hypoxia (14.4% O2), consisting of two lower body and two upper body exercises. Venous blood was drawn before and after the training intervention and flow cytometry was used to quantify resting neutrophils, lymphocytes, monocytes, eosinophils and basophils, in addition to the subsets of lymphocytes (T, B and natural killer (NK) cells). Inflammatory cytokines were also quantified; interleukin 1 beta (IL-1ß), IL-4, IL-6, IL-8, IL-10 and tumor necrosis factor alpha (TNF-α). Acute changes in leukocytes and cytokines were also measured in the 24 h following the last training session. RESULTS: After the intervention there was a greater concentration of resting white blood cells (p = 0.03; 20.3% higher) T cells (p = 0.008; 25.4% higher), B cells (p = 0.004; 32.6% higher), NK cells (p = 0.012; 43.9% higher) and eosinophils (p = 0.025; 30.8% higher) in hypoxia compared to normoxia, though the cytokines were unchanged. No acute effect of hypoxia was detected in the 24 h following the last training session for any leukocyte population or inflammatory cytokine (p < 0.05). CONCLUSIONS: Hypoxic training caused higher concentrations of resting lymphocytes and eosinophils, when compared to normoxic training. Hypoxia may have an additional beneficial effect on the immunological status of older adults. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (ANZCTR). TRIAL NUMBER: ACTRN12623001046695. Registered 27/9/2023. Retrospectively registered. All protocols adhere to the COSORT guidelines.

Pure and Mixed Variants of Desmoplastic Melanoma: A Single-Center, Retrospective, Clinicopathologic Review.

BACKGROUND: Desmoplastic melanoma (DM) is a rare melanoma variant. Prognostic indicators and survival vary widely and are further confounded by the histopathologic distinction between pure DM (pDM) and mixed DM (mDM) subtypes. The utility of current treatment guidelines is limited by the lack of evidence-based recommendations. OBJECTIVE: To compare the clinicopathologic characteristics of pure and mixed subtypes of DMs. METHODS: All cases of DM were identified from the Washington University in St Louis institutional pathology database between January 2000 and September 2022. Fifty-two cases were identified and subsequently categorized as pure ( n = 26) or mixed ( n = 26). Clinical and histopathologic data were collected and compared. RESULTS: There were no differences in demographics or tumor location between pure and mixed subtypes. Patients with mDM were more likely to have mitoses present ( p = .03). There were no differences in Breslow depth, tumor diameter, level of invasion, ulceration, and lymphovascular or perineural invasion. The utilization of sentinel lymph node biopsy ( p = .17) and sentinel lymph node positivity ( p = .67) were also similar. CONCLUSION: Despite histopathologic distinction between pDM and mDM, these subtypes were found to have similar clinicopathologic characteristics, including similar rates of sentinel lymph node metastasis.

CD30+ lymphoproliferative disorder masquerading as an atypical melanocytic proliferation.

Heuristics are cognitive strategies used to facilitate decision-making. They can be helpful tools for expediting pathologic diagnoses, however, they can also affect judgment and lead to biases that guide the pathologist astray. We report the case of a 52-year-old female who presented with two unusual pigmented lesions on the wrist and thigh that clinically and histopathologically resembled an atypical melanocytic proliferation. A biopsy of the thigh revealed a broad proliferation of large, atypical cells forming nests within a heavily pigmented epidermis. The lesion was initially misdiagnosed as melanoma in situ, despite equivocal staining for melanocytic markers, likely due to anchoring and adjustment as well as availability biases, which restricted the differential diagnosis and limited the selection of immunohistochemical stains. It was later discovered through chart review that the patient had a prior history of a cutaneous CD30+ lymphoproliferative disorder, which eventually led to the appropriate diagnosis in this case. Herein, we highlight a rare and unusual presentation of a pigmented epidermotropic CD30+ lymphoproliferative disorder, along with the biases leading to its misdiagnosis and the steps leading to the revelation of the actual diagnosis.

Primary Cutaneous Mucinous Carcinoma: A Review of the Literature.

BACKGROUND: Primary cutaneous mucinous carcinoma (PCMC) is an exceedingly rare, low-grade tumor that histologically resembles mucinous carcinoma from other primary sites, such as the breast, gastrointestinal tract, and lungs. OBJECTIVE: The purpose of this article was to review the current literature on PCMC as it relates to epidemiology, clinical presentation, histopathology, immunohistochemistry, treatment, and prognosis. MATERIALS AND METHODS: An extensive literature review was conducted using PubMed and Ovid MEDLINE to identify articles related to PCMC. RESULTS: Several hundred cases have been reported in the medical literature, and surgical resection, whenever feasible, is the standard of care. CONCLUSION: The diagnosis of primary cutaneous mucinous carcinoma is one of exclusion, requiring a metastatic work-up to rule out distant primary. Mohs micrographic surgery is a tissue sparing technique that allows complete margin control of these rare neoplasia.

The acute leukocyte and cytokine response of older adults to resistance exercise in normobaric hypoxia.

Ageing causes a decline in leukocyte function and blunted leukocyte responses to resistance exercise. Systemic hypoxia exposure augments the leukocyte response to resistance exercise in young adults, yet this response remains uncharacterised in older adults. This study characterised the effects of normobaric hypoxia on the acute leukocyte and inflammatory cytokine responses to resistance exercise in older adults. We recruited 20 adults aged 60-70 years to perform an acute bout of resistance exercise in normobaric hypoxia (FiO2 14.4%; n = 10) or normoxia (FiO2 20.93%; n = 10). Participants completed 4 × 10 repetitions of lower and upper body exercises at 70% of their predicted 1-repetition maximum. Venous blood was sampled before and up to 24 hours post-exercise to quantify neutrophils, lymphocytes, monocytes, eosinophils, basophils and cytokines (IL-1ß, IL-4, IL-6, IL-8, IL-10, TNFα). Flow cytometry was used to classify lymphocytes as T (CD4+ helper and CD8+ cytotoxic), B and NK cells, in addition to the expression of the senescence marker CD45RA on T cells. The hypoxic group showed a larger lymphocyte response over the 24 hours post-exercise compared to the normoxic group (p = 0.035). Specifically, there were greater concentrations of CD4+ T helper cells following hypoxic exercise compared to normoxia (p = 0.046). There was also a greater proportion of CD45RA+ CD4+ T helper cells, suggesting that the cells were more senescent (p = 0.044). Hypoxia did not impact any other leukocyte population or cytokine following exercise. Normobaric hypoxia increases the lymphocyte response to an acute bout of resistance exercise in older adults.

The unconditioned fear response in dystrophin-deficient mice is associated with adrenal and vascular function.

Loss of function mutations in the gene encoding dystrophin elicits a hypersensitive fear response in mice and humans. In the dystrophin-deficient mdx mouse, this behaviour is partially protected by oestrogen, but the mechanistic basis for this protection is unknown. Here, we show that female mdx mice remain normotensive during restraint stress compared to a hypotensive and hypertensive response in male mdx and male/female wildtype mice, respectively. Partial dystrophin expression in female mdx mice (heterozygous) also elicited a hypertensive response. Ovariectomized (OVX) female mdx mice were used to explain the normotensive response to stress. OVX lowered skeletal muscle mass and lowered the adrenal mass and zona glomerulosa area (aldosterone synthesis) in female mdx mice. During a restraint stress, OVX dampened aldosterone synthesis and lowered the corticosterone:11-dehydrocorticosterone. All OVX-induced changes were restored with replacement of oestradiol, except that oestradiol lowered the zona fasciculata area of the adrenal gland, dampened corticosterone synthesis but increased cortisol synthesis. These data suggest that oestrogen partially attenuates the unconditioned fear response in mdx mice via adrenal and vascular function. It also suggests that partial dystrophin restoration in a dystrophin-deficient vertebrate is an effective approach to develop an appropriate hypertensive response to stress.

IFN-γ blockade after genetic inhibition of PD-1 aggravates skeletal muscle damage and impairs skeletal muscle regeneration.

BACKGROUND: Innate immune responses play essential roles in skeletal muscle recovery after injury. Programmed cell death protein 1 (PD-1) contributes to skeletal muscle regeneration by promoting macrophage proinflammatory to anti-inflammatory phenotype transition. Interferon (IFN)-γ induces proinflammatory macrophages that appear to hinder myogenesis in vitro. Therefore, we tested the hypothesis that blocking IFN-γ in PD-1 knockout mice may dampen inflammation and promote skeletal muscle regeneration via regulating the macrophage phenotype and neutrophils. METHODS: Anti-IFN-γ antibody was administered in PD-1 knockout mice, and cardiotoxin (CTX) injection was performed to induce acute skeletal muscle injury. Hematoxylin and eosin (HE) staining was used to view morphological changes of injured and regenerated skeletal muscle. Masson's trichrome staining was used to assess the degree of fibrosis. Gene expressions of proinflammatory and anti-inflammatory factors, fibrosis-related factors, and myogenic regulator factors were determined by real-time polymerase chain reaction (PCR). Changes in macrophage phenotype were examined by western blot and real-time PCR. Immunofluorescence was used to detect the accumulation of proinflammatory macrophages, anti-inflammatory macrophages, and neutrophils. RESULTS: IFN-γ blockade in PD-1 knockout mice did not alleviate skeletal muscle damage or improve regeneration following acute cardiotoxin-induced injury. Instead, it exacerbated skeletal muscle inflammation and fibrosis, and impaired regeneration via inhibiting macrophage accumulation, blocking macrophage proinflammatory to anti-inflammatory transition, and enhancing infiltration of neutrophils. CONCLUSION: IFN-γ is crucial for efficient skeletal muscle regeneration in the absence of PD-1.

Loss of skeletal muscle estrogen-related receptors leads to severe exercise intolerance.

OBJECTIVE: Skeletal muscle oxidative capacity is central to physical activity, exercise capacity and whole-body metabolism. The three estrogen-related receptors (ERRs) are regulators of oxidative metabolism in many cell types, yet their roles in skeletal muscle remain unclear. The main aim of this study was to compare the relative contributions of ERRs to oxidative capacity in glycolytic and oxidative muscle, and to determine defects associated with loss of skeletal muscle ERR function. METHODS: We assessed ERR expression, generated mice lacking one or two ERRs specifically in skeletal muscle and compared the effects of ERR loss on the transcriptomes of EDL (predominantly glycolytic) and soleus (oxidative) muscles. We also determined the consequences of the loss of ERRs for exercise capacity and energy metabolism in mice with the most severe loss of ERR activity. RESULTS: ERRs were induced in human skeletal muscle in response to an exercise bout. Mice lacking both ERRα and ERRγ (ERRα/γ dmKO) had the broadest and most dramatic disruption in skeletal muscle gene expression. The most affected pathway was "mitochondrial function", in particular Oxphos and TCA cycle genes, and transcriptional defects were more pronounced in the glycolytic EDL than the oxidative soleus. Mice lacking ERRß and ERRγ, the two isoforms expressed highly in oxidative muscles, also exhibited defects in lipid and branch chain amino acid metabolism genes, specifically in the soleus. The pronounced disruption of oxidative metabolism in ERRα/γ dmKO mice led to pale muscles, decreased oxidative capacity, histochemical patterns reminiscent of minicore myopathies, and severe exercise intolerance, with the dmKO mice unable to switch to lipid utilization upon running. ERRα/γ dmKO mice showed no defects in whole-body glucose and energy homeostasis. CONCLUSIONS: Our findings define gene expression programs in skeletal muscle that depend on different combinations of ERRs, and establish a central role for ERRs in skeletal muscle oxidative metabolism and exercise capacity. Our data reveal a high degree of functional redundancy among muscle ERR isoforms for the protection of oxidative capacity, and show that ERR isoform-specific phenotypes are driven in part, but not exclusively, by their relative levels in different muscles.

Muscle Adaptations to Heavy-Load and Blood Flow Restriction Resistance Training Methods.

Resistance-based blood flow restriction training (BFRT) improves skeletal muscle strength and size. Unlike heavy-load resistance training (HLRT), there is debate as to whether strength adaptations following BFRT interventions can be primarily attributed to concurrent muscle hypertrophy, as the magnitude of hypertrophy is often minor. The present study aimed to investigate the effect of 7 weeks of BFRT and HLRT on muscle strength and hypertrophy. The expression of protein growth markers from muscle biopsy samples was also measured. Male participants were allocated to moderately heavy-load training (HL; n = 9), low-load BFRT (LL + BFR; n = 8), or a control (CON; n = 9) group to control for the effect of time. HL and LL + BFR completed 21 training sessions (3 d.week-1) comprising bilateral knee extension and knee flexion exercises (HL = 70% one-repetition maximum (1-RM), LL + BFR = 20% 1-RM + blood flow restriction). Bilateral knee extension and flexion 1-RM strength were assessed, and leg muscle CSA was measured via peripheral quantitative computed tomography. Protein growth markers were measured in vastus lateralis biopsy samples taken pre- and post the first and last training sessions. Biopsy samples were also taken from CON at the same time intervals as HL and LL + BFR. Knee extension 1-RM strength increased in HL (19%) and LL + BFR (19%) but not CON (2%; p < 0.05). Knee flexion 1-RM strength increased similarly between all groups, as did muscle CSA (50% femur length; HL = 2.2%, LL + BFR = 3.0%, CON = 2.1%; TIME main effects). 4E-BP1 (Thr37/46) phosphorylation was lower in HL and LL + BFR immediately post-exercise compared with CON in both sessions (p < 0.05). Expression of other growth markers was similar between groups (p > 0.05). Overall, BFRT and HLRT improved muscle strength and size similarly, with comparable changes in intramuscular protein growth marker expression, both acutely and chronically, suggesting the activation of similar anabolic pathways. However, the low magnitude of muscle hypertrophy was not significantly different to the non-training control suggesting that strength adaptation following 7 weeks of BFRT is not driven by hypertrophy, but rather neurological adaptation.

The Effect of Normobaric Hypoxia on Resistance Training Adaptations in Older Adults.

ABSTRACT: Allsopp, GL, Hoffmann, SM, Feros, SA, Pasco, JA, Russell, AP, and Wright, CR. The effect of normobaric hypoxia on resistance training adaptations in older adults. J Strength Cond Res 36(8): 2306-2312, 2022-The effect of normobaric hypoxia on strength, body composition, and cardiovascular fitness was investigated after a resistance training intervention in older adults. A single-blinded, randomized control trial recruited 20 healthy adults aged 60-75 years for an 8-week resistance training intervention in normoxia ( n = 10) or normobaric hypoxia (14.4% O 2 ; n = 10). Subjects performed 2 sessions per week of upper-body and lower-body exercises at 70% of 1 repetition maximum (1RM). Pretraining and post-training, maximal oxygen uptake (V̇O 2 max), muscular endurance (30 maximal knee flexions/extensions), and 5RM were assessed, with 5RM used to calculate 1RM. Subjects underwent whole-body dual-energy x-ray absorptiometry (DXA) at pretraining and post-training for fat and lean mass quantification. Significance was set at p < 0.05. Subjects in both groups substantially improved their calculated 1RM strength for leg extension, pectoral fly, row, and squat (normoxia; 30, 38, 27, and 29%, hypoxia; 43, 50, 28, and 64%, respectively); however, hypoxia did not augment this response. Hypoxia did not enhance V̇O 2 max or muscular endurance responses after the training intervention, with no improvements seen in either group. Fat mass and lean mass remained unchanged in both groups after the intervention. In summary, 8 weeks of resistance training in hypoxia was well tolerated in healthy older adults and increased upper-body and lower-body strength. However, the magnitude of strength and lean muscle improvements in hypoxia was no greater than normoxia; therefore, there is currently no evidence to support the use of hypoxic resistance training in older adults.

miR-23a suppression accelerates functional decline in the rNLS8 mouse model of TDP-43 proteinopathy.

Skeletal muscle dysfunction may contribute to the progression and severity of amyotrophic lateral sclerosis (ALS). In the present study, we characterized the skeletal muscle pathophysiology in an inducible transgenic mouse model (rNLS8) that develops a TAR-DNA binding protein (TDP-43) proteinopathy and ALS-like neuropathology and disease progression; representative of >90% of all familial and sporadic ALS cases. As we previously observed elevated levels of miR-23a in skeletal muscle of patients with familial and sporadic ALS, we also investigated the effect of miR-23a suppression on skeletal muscle pathophysiology and disease severity in rNLS8 mice. Five weeks after disease onset TDP-43 protein accumulation was observed in tibialis anterior (TA), quadriceps (QUAD) and diaphragm muscle lysates and associated with skeletal muscle atrophy. In the TA muscle TDP-43 was detected in muscle fibres that appeared atrophied and angular in appearance and that also contained ß-amyloid aggregates. These fibres were also positive for neural cell adhesion molecule (NCAM), but not embryonic myosin heavy chain (eMHC), indicating TDP-43/ ß-amyloid localization in denervated muscle fibres. There was an upregulation of genes associated with myogenesis and NMJ degeneration and a decrease in the MURF1 atrophy-related protein in skeletal muscle. Suppression of miR-23a impaired rotarod performance and grip strength and accelerated body weight loss during early stages of disease progression. This was associated with increased AchRα mRNA expression and decreased protein levels of PGC-1α. The TDP-43 proteinopathy-induced impairment of whole body and skeletal muscle functional performance is associated with muscle wasting and elevated myogenic and NMJ stress markers. Suppressing miR-23a in the rNLS8 mouse model of ALS contributes to an early acceleration of disease progression as measured by decline in motor function.

Sensitivity to behavioral stress impacts disease pathogenesis in dystrophin-deficient mice.

Mutation to the gene encoding dystrophin can cause Duchenne muscular dystrophy (DMD) and increase the sensitivity to stress in vertebrate species, including the mdx mouse model of DMD. Behavioral stressors can exacerbate some dystrophinopathy phenotypes of mdx skeletal muscle and cause hypotension-induced death. However, we have discovered that a subpopulation of mdx mice present with a wildtype-like response to mild (forced downhill treadmill exercise) and moderate (scruff restraint) behavioral stressors. These "stress-resistant" mdx mice are more physically active, capable of super-activating the hypothalamic-pituitary-adrenal and renin-angiotensin-aldosterone pathways following behavioral stress and they express greater levels of mineralocorticoid and glucocorticoid receptors in striated muscle relative to "stress-sensitive" mdx mice. Stress-resistant mdx mice also presented with a less severe striated muscle histopathology and greater exercise and skeletal muscle oxidative capacity at rest. Most interestingly, female mdx mice were more physically active following behavioral stressors compared to male mdx mice; a response abolished after ovariectomy and rescued with estradiol. We demonstrate that the response to behavioral stress greatly impacts disease severity in mdx mice suggesting the management of stress in patients with DMD be considered as a therapeutic approach to ameliorate disease progression.

Histopathologic and immunophenotypic features of cutaneous solid organ transplant-associated graft-vs-host disease: Comparison with acute hematopoietic cell transplant-associated graft-vs-host disease and cutaneous drug eruption.

BACKGROUND: Although it is relatively common after hematopoietic cell transplant (HCT), graft-vs-host disease (GVHD) is a rare complication following solid organ transplantation (SOT). METHODS: This study evaluated skin biopsy specimens from five cases of SOT GVHD, 15 cases of HCT GVHD, and 15 cases of cutaneous drug eruption. Immunohistochemical staining for CD3, CD4, CD8, T-bet, and GATA-3 was performed to examine the density and immune phenotype of skin-infiltrating lymphocytes. RESULTS: Similar to HCT GVHD, the predominant histopathologic findings in skin biopsy specimens of SOT GVHD were widespread vacuolar interface dermatitis with scattered necrotic keratinocytes. However, the density of dermal inflammation was considerably higher in SOT GVHD. Features that were more predictive of a cutaneous drug eruption over GVHD included spongiosis, confluent parakeratosis, and many eosinophils. Involvement of the hair follicle epithelium was seen in all three disorders. Both forms of cutaneous GVHD showed a predominance of Th1 (CD3+/T-bet+) lymphocytes within the inflammatory infiltrates. This shift was more pronounced in SOT GVHD, particularly among intraepidermal T-cells. CONCLUSIONS: SOT GVHD shares many histopathologic features with HCT GVHD. However, SOT GVHD has a greater tendency to develop brisk lichenoid inflammation.

Striated muscle activator of Rho signalling (STARS) overexpression in the mdx mouse enhances muscle functional capacity and regulates the actin cytoskeleton and oxidative phosphorylation pathways.

NEW FINDINGS: What is the central question of this study? Striated muscle activator of rho signalling (STARS) is an actin-binding protein that regulates transcriptional pathways controlling muscle function, growth and myogenesis, processes that are impaired in dystrophic muscle: what is the regulation of the STARS pathway in Duchenne muscular dystrophy (DMD)? What is the main finding and its importance? Members of the STARS signalling pathway are reduced in the quadriceps of patients with DMD and in mouse models of muscular dystrophy. Overexpression of STARS in the dystrophic deficient mdx mouse model increased maximal isometric specific force and upregulated members of the actin cytoskeleton and oxidative phosphorylation pathways. Regulating STARS may be a therapeutic approach to enhance muscle health. ABSTRACT: Duchenne muscular dystrophy (DMD) is characterised by impaired cytoskeleton organisation, cytosolic calcium handling, oxidative stress and mitochondrial dysfunction. This results in progressive muscle damage, wasting and weakness and premature death. The striated muscle activator of rho signalling (STARS) is an actin-binding protein that activates the myocardin-related transcription factor-A (MRTFA)/serum response factor (SRF) transcriptional pathway, a pathway regulating cytoskeletal structure and muscle function, growth and repair. We investigated the regulation of the STARS pathway in the quadriceps muscle from patients with DMD and in the tibialis anterior (TA) muscle from the dystrophin-deficient mdx and dko (utrophin and dystrophin null) mice. Protein levels of STARS, SRF and RHOA were reduced in patients with DMD. STARS, SRF and MRTFA mRNA levels were also decreased in DMD muscle, while Stars mRNA levels were decreased in the mdx mice and Srf and Mrtfa mRNAs decreased in the dko mice. Overexpressing human STARS (hSTARS) in the TA muscles of mdx mice increased maximal isometric specific force by 13% (P < 0.05). This was not associated with changes in muscle mass, fibre cross-sectional area, fibre type, centralised nuclei or collagen deposition. Proteomics screening followed by pathway enrichment analysis identified that hSTARS overexpression resulted in 31 upregulated and 22 downregulated proteins belonging to the actin cytoskeleton and oxidative phosphorylation pathways. These pathways are impaired in dystrophic muscle and regulate processes that are vital for muscle function. Increasing the STARS protein in dystrophic muscle improves muscle force production, potentially via synergistic regulation of cytoskeletal structure and energy production.