Serum protein and imaging biomarkers after intermittent steroid treatment in muscular dystrophy.

Background: Weekly Steroids in Muscular Dystrophy (WSiMD) was a pilot study to evaluate once weekly prednisone in patients with Limb Girdle and Becker muscular dystrophy (LGMD and BMD, respectively). At study endpoint, there were trends towards increased lean mass, reduced fat mass, reduced creatine kinase and improved motor function. The investigation was motivated by studies in mouse muscular dystrophy models in which once weekly glucocorticoid exposure enhanced muscle strength and reduced fibrosis. Methods: WSiMD participants provided blood samples for aptamer serum profiling at baseline and after 6 months of weekly steroids. A subset completed magnetic resonance (MR) evaluation of muscle at study onset and endpoint. Results/Conclusions: At baseline compared to age and sex-matched healthy controls, the aggregate serum protein profile in the WSiMD cohort was dominated by muscle proteins, reflecting leak of muscle proteins into serum. Disease status produced more proteins differentially present in serum compared to steroid-treatment effect. Nonetheless, a response to prednisone was discernable in the WSiMD cohort, even at this low dose. Glucocorticoids downregulated muscle proteins and upregulated certain immune process- and matrix-associated proteins. Muscle MR fat fraction showed trends with functional status. The prednisone-responsive markers could be used in larger trial of prednisone efficacy.

An Open Label Exploratory Clinical Trial Evaluating Safety and Tolerability of Once-Weekly Prednisone in Becker and Limb-Girdle Muscular Dystrophy.

BACKGROUND: Glucocorticoid steroids are standard of care in Duchenne Muscular Dystrophy (DMD) to slow disease course. Use of glucocorticoids in other muscular dystrophies, including Becker (BMD) and Limb Girdle (LGMD), has been less explored. Recently, preclinical studies conducted in DMD and LGMD mouse models showed once-weekly prednisone was associated with improved muscle performance without activation of muscle atrophy genes. OBJECTIVE: To determine safety and tolerability of once-weekly prednisone in patients with LGMD and BMD. METHODS: We conducted an open label, exploratory single center study of of once-weekly prednisone at 0.75-1 mg/Kg in LGMD (n = 19) and BMD (n = 1) (mean age 35, range 18-60). The LGMD participants represented multiple different LGMD subtypes, and the study included ambulatory and non-ambulatory participants. Participants were assessed at baseline and 24 weeks for vital signs, blood biomarkers, and for patient-reported side effects. As secondary endpoints, functional muscle testing and body composition were measured. RESULTS: Over the 24-week study, there were no significant changes in blood pressure, HgbA1C, or lipid profiles. We observed a reduction in serum creatine kinase over the study interval. Whole body DEXA scanning suggested a possible increase in lean mass and a reduction in adiposity. Functional measures suggested trends in improved muscle performance. CONCLUSIONS: In this single center, open label pilot study, once-weekly prednisone was safe and well tolerated. Additional investigation of once-weekly prednisone in a larger cohort and for a longer period of time is warranted.

Mechanisms and Clinical Applications of Glucocorticoid Steroids in Muscular Dystrophy.

Glucocorticoid steroids are widely used as immunomodulatory agents in acute and chronic conditions. Glucocorticoid steroids such as prednisone and deflazacort are recommended for treating Duchenne Muscular Dystrophy where their use prolongs ambulation and life expectancy. Despite this benefit, glucocorticoid use in Duchenne Muscular Dystrophy is also associated with significant adverse consequences including adrenal suppression, growth impairment, poor bone health and metabolic syndrome. For other forms of muscular dystrophy like the limb girdle dystrophies, glucocorticoids are not typically used. Here we review the experimental evidence supporting multiple mechanisms of glucocorticoid action in dystrophic muscle including their role in dampening inflammation and myofiber injury. We also discuss alternative dosing strategies as well as novel steroid agents that are in development and testing, with the goal to reduce adverse consequences of prolonged glucocorticoid exposure while maximizing beneficial outcomes.

High seroprevalence for SARS-CoV-2 among household members of essential workers detected using a dried blood spot assay.

OBJECTIVE: Serological testing is needed to investigate the extent of transmission of SARS-CoV-2 from front-line essential workers to their household members. However, the requirement for serum/plasma limits serological testing to clinical settings where it is feasible to collect and process venous blood. To address this problem we developed a serological test for SARS-CoV-2 IgG antibodies that requires only a single drop of finger stick capillary whole blood, collected in the home and dried on filter paper (dried blood spot, DBS). We describe assay performance and demonstrate its utility for remote sampling with results from a community-based study. METHODS: An ELISA to the receptor binding domain of the SARS-CoV-2 spike protein was optimized to quantify IgG antibodies in DBS. Samples were self-collected from a community sample of 232 participants enriched with health care workers, including 30 known COVID-19 cases and their household members. RESULTS: Among 30 individuals sharing a household with a virus-confirmed case of COVID-19, 80% were seropositive. Of 202 community individuals without prior confirmed acute COVID-19 diagnoses, 36% were seropositive. Of documented convalescent COVID-19 cases from the community, 29 of 30 (97%) were seropositive for IgG antibodies to the receptor binding domain. CONCLUSION: DBS ELISA provides a minimally-invasive alternative to venous blood collection. Early analysis suggests a high rate of transmission among household members. High rates of seroconversion were also noted following recovery from infection. Serological testing for SARS-CoV-2 IgG antibodies in DBS samples can facilitate seroprevalence assessment in community settings to address epidemiological questions, monitor duration of antibody responses, and assess if antibodies against the spike protein correlate with protection from reinfection.

Pulsed glucocorticoids enhance dystrophic muscle performance through epigenetic-metabolic reprogramming.

In humans, chronic glucocorticoid use is associated with side effects like muscle wasting, obesity, and metabolic syndrome. Intermittent steroid dosing has been proposed in Duchenne Muscular Dystrophy patients to mitigate the side effects seen with daily steroid intake. We evaluated biomarkers from Duchenne Muscular Dystrophy patients, finding that, compared with chronic daily steroid use, weekend steroid use was associated with reduced serum insulin, free fatty acids, and branched chain amino acids, as well as reduction in fat mass despite having similar BMIs. We reasoned that intermittent prednisone administration in dystrophic mice would alter muscle epigenomic signatures, and we identified the coordinated action of the glucocorticoid receptor, KLF15 and MEF2C as mediators of a gene expression program driving metabolic reprogramming and enhanced nutrient utilization. Muscle lacking Klf15 failed to respond to intermittent steroids. Furthermore, coadministration of the histone acetyltransferase inhibitor anacardic acid with steroids in mdx mice eliminated steroid-specific epigenetic marks and abrogated the steroid response. Together, these findings indicate that intermittent, repeated exposure to glucocorticoids promotes performance in dystrophic muscle through an epigenetic program that enhances nutrient utilization.

Moderate exercise improves function and increases adiponectin in the mdx mouse model of muscular dystrophy.

The loss of dystrophin produces a mechanically fragile sarcolemma, causing muscle membrane disruption and muscle loss. The degree to which exercise alters muscular dystrophy has been evaluated in humans with Duchenne Muscular Dystrophy (DMD) and in mouse models including the mdx mouse but with inconsistent findings. We now examined two different levels of exercise, moderate and low intensity, in the mdx mouse model in the DBA2J background. mdx mice at 4-5 months of age were subjected to two different doses of exercise. We found a dose-dependent benefit for low and moderate exercise, defined as 4 m/min or 8 m/min, for 30 minutes three times a week. After six months, exercised mdx mice showed improved tetanic and specific force compared to the sedentary group. We also observed increased respiratory capacity manifesting as greater minute volume, as well as enhanced cardiac function mitigating the decline of fractional shortening that is normally seen. Exercised mdx mice also showed a dose-dependent increase in serum adiponectin with a concomitant reduced adipocyte cross sectional area. These findings identify moderate intensity exercise as a means to improve muscle performance in the mdx DBA2J mice and suggest serum adiponectin as a biomarker for beneficial exercise effect in DMD.

Motor neuron mitochondrial dysfunction in spinal muscular atrophy.

Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, predominantly affects high metabolic tissues including motor neurons, skeletal muscles and the heart. Although the genetic cause of SMA has been identified, mechanisms underlying tissue-specific vulnerability are not well understood. To study these mechanisms, we carried out a deep sequencing analysis of the transcriptome of spinal motor neurons in an SMA mouse model, in which we unexpectedly found changes in many genes associated with mitochondrial bioenergetics. Importantly, functional measurement of mitochondrial activities showed decreased basal and maximal mitochondrial respiration in motor neurons from SMA mice. Using a reduction-oxidation sensitive GFP and fluorescence sensors specifically targeted to mitochondria, we found increased oxidative stress level and impaired mitochondrial membrane potential in motor neurons affected by SMA. In addition, mitochondrial mobility was impaired in SMA disease conditions, with decreased retrograde transport but no effect on anterograde transport. We also found significantly increased fragmentation of the mitochondrial network in primary motor neurons from SMA mice, with no change in mitochondria density. Electron microscopy study of SMA mouse spinal cord revealed mitochondria fragmentation, edema and concentric lamellar inclusions in motor neurons affected by the disease. Intriguingly, these functional and structural deficiencies in the SMA mouse model occur during the presymptomatic stage of disease, suggesting a role in initiating SMA. Altogether, our findings reveal a critical role for mitochondrial defects in SMA pathogenesis and suggest a novel target for improving tissue health in the disease.