Revised ISHAM-ABPA working group clinical practice guidelines for diagnosing, classifying and treating allergic bronchopulmonary aspergillosis/mycoses.

BACKGROUND: The International Society for Human and Animal Mycology (ISHAM) working group proposed recommendations for managing allergic bronchopulmonary aspergillosis (ABPA) a decade ago. There is a need to update these recommendations due to advances in diagnostics and therapeutics. METHODS: An international expert group was convened to develop guidelines for managing ABPA (caused by Aspergillus spp.) and allergic bronchopulmonary mycosis (ABPM; caused by fungi other than Aspergillus spp.) in adults and children using a modified Delphi method (two online rounds and one in-person meeting). We defined consensus as ≥70% agreement or disagreement. The terms "recommend" and "suggest" are used when the consensus was ≥70% and <70%, respectively. RESULTS: We recommend screening for A. fumigatus sensitisation using fungus-specific IgE in all newly diagnosed asthmatic adults at tertiary care but only difficult-to-treat asthmatic children. We recommend diagnosing ABPA in those with predisposing conditions or compatible clinico-radiological presentation, with a mandatory demonstration of fungal sensitisation and serum total IgE ≥500 IU·mL-1 and two of the following: fungal-specific IgG, peripheral blood eosinophilia or suggestive imaging. ABPM is considered in those with an ABPA-like presentation but normal A. fumigatus-IgE. Additionally, diagnosing ABPM requires repeated growth of the causative fungus from sputum. We do not routinely recommend treating asymptomatic ABPA patients. We recommend oral prednisolone or itraconazole monotherapy for treating acute ABPA (newly diagnosed or exacerbation), with prednisolone and itraconazole combination only for treating recurrent ABPA exacerbations. We have devised an objective multidimensional criterion to assess treatment response. CONCLUSION: We have framed consensus guidelines for diagnosing, classifying and treating ABPA/M for patient care and research.

Evaluation of Acebilustat, a Selective Inhibitor of Leukotriene B4 Biosynthesis, for Treatment of Outpatients With Mild-Moderate Coronavirus Disease 2019: A Randomized, Double-Blind, Placebo-Controlled Phase 2 Trial.

BACKGROUND: The vast majority of coronavirus disease 2019 (COVID-19) disease occurs in outpatients where treatment is limited to antivirals for high-risk subgroups. Acebilustat, a leukotriene B4 inhibitor, has potential to reduce inflammation and symptom duration. METHODS: In a single-center trial spanning Delta and Omicron variants, outpatients were randomized to 100 mg/d of oral acebilustat or placebo for 28 days. Patients reported daily symptoms via electronic query through day 28 with phone follow-up on day 120 and collected nasal swab samples on days 1-10. The primary outcome was sustained symptom resolution to day 28. Secondary 28-day outcomes included time to first symptom resolution, area under the curve (AUC) for longitudinal daily symptom scores, duration of viral shedding through day 10, and symptoms on day 120. RESULTS: Sixty participants were randomized to each study arm. At enrollment, the median duration was 4 days (interquartile range, 3-5 days), and the median number of symptoms was 9 (7-11). Most patients (90%) were vaccinated, with 73% having neutralizing antibodies. A minority of participants (44%; 35% in the acebilustat arm and 53% in placebo) had sustained symptom resolution at day 28 (hazard ratio, 0.6 [95% confidence interval, .34-1.04]; P = .07 favoring placebo). There was no difference in the mean AUC for symptom scores over 28 days (difference in mean AUC, 9.4 [95% confidence interval, -42.1 to 60.9]; P = .72). Acebilustat did not affect viral shedding or symptoms at day 120. CONCLUSIONS: Sustained symptoms through day 28 were common in this low-risk population. Despite this, leukotriene B4 antagonism with acebilustat did not shorten symptom duration in outpatients with COVID-19. Clinical Trials Registration. NCT04662060.

FDA Public Workshop Summary-Addressing Challenges in Inhaled Antifungal Drug Development.

Allergic bronchopulmonary aspergillosis and invasive fungal diseases represent distinct infectious entities that cause significant morbidity and mortality. Currently, administered inhaled antifungal therapies are unapproved, have suboptimal efficacy, and are associated with considerable adverse reactions. The emergence of resistant pathogens is also a growing concern. Inhaled antifungal development programs are challenged by inadequate nonclinical infection models, highly heterogenous patient populations, low prevalence rates of fungal diseases, difficulties defining clinical trial enrollment criteria, and lack of robust clinical trial endpoints. On September 25, 2020, the US Food and Drug Administration (FDA) convened a workshop with experts in pulmonary medicine and infectious diseases from academia, industry, and other governmental agencies. Key discussion topics included regulatory incentives to facilitate development of inhaled antifungal drugs and combination inhalational devices, limitations of existing nonclinical models and clinical trial designs, patient perspectives, and industry insights.

Distinct hypertrophic cardiomyopathy genotypes result in convergent sarcomeric proteoform profiles revealed by top-down proteomics.

Hypertrophic cardiomyopathy (HCM) is the most common heritable heart disease. Although the genetic cause of HCM has been linked to mutations in genes encoding sarcomeric proteins, the ability to predict clinical outcomes based on specific mutations in HCM patients is limited. Moreover, how mutations in different sarcomeric proteins can result in highly similar clinical phenotypes remains unknown. Posttranslational modifications (PTMs) and alternative splicing regulate the function of sarcomeric proteins; hence, it is critical to study HCM at the level of proteoforms to gain insights into the mechanisms underlying HCM. Herein, we employed high-resolution mass spectrometry-based top-down proteomics to comprehensively characterize sarcomeric proteoforms in septal myectomy tissues from HCM patients exhibiting severe outflow track obstruction (n = 16) compared to nonfailing donor hearts (n = 16). We observed a complex landscape of sarcomeric proteoforms arising from combinatorial PTMs, alternative splicing, and genetic variation in HCM. A coordinated decrease of phosphorylation in important myofilament and Z-disk proteins with a linear correlation suggests PTM cross-talk in the sarcomere and dysregulation of protein kinase A pathways in HCM. Strikingly, we discovered that the sarcomeric proteoform alterations in the myocardium of HCM patients undergoing septal myectomy were remarkably consistent, regardless of the underlying HCM-causing mutations. This study suggests that the manifestation of severe HCM coalesces at the proteoform level despite distinct genotype, which underscores the importance of molecular characterization of HCM phenotype and presents an opportunity to identify broad-spectrum treatments to mitigate the most severe manifestations of this genetically heterogenous disease.

The Acute Effects of Theragun™ Percussive Therapy on Viscoelastic Tissue Dynamics and Hamstring Group Range of Motion.

Handheld percussive therapy (PT) massage guns have seen a rapid rise in use and with-it increased attention within injury prevention and sport performance settings. Early studies have proposed beneficial effects upon range of motion (ROM), however the mechanism behind these increases remains unreported. This study aimed to determine the influence of a minimal frequency PT dose upon ROM and myotonometry outcomes. Twenty participants (N = 20; 13 males and 7 females, height 1.78cm ± 9.62; weight 77.35kg ± 8.46) participants were allocated to either a PT group receiving 2 x 60-seconds (plus 30-seconds rest) via a Theragun™ Pro4 to the hamstrings covering a standardised 20 lengths from proximal to distal via the standard ball attachment at 1 bar of pressure or a control group (CON) of 2-minutes 30-seconds passive supine rest. Pre and post intervention outcomes were measured for ROM via passive straight leg raise (PSLR) and tissue dynamics via MyotonPro (Tone, Stiffness, Elasticity, Relaxation Time). Results showed significant within-group increases (p < 0.0001, ηp2 0.656, +11.4%) in ROM following PT and between group difference against CON (P < 0.026). Significant within-group differences in stiffness (p < 0.016, ηp2 0.144, -6%), tone (p < 0.003, ηp2 0.213, +2%) and relaxation time (p < 0.002, ηp2 0.232, +6.3%) were also reported following PT. No significant difference was reported in elasticity (P > 0.05) or any other between group outcomes. PT therapy can provide an acute increase in hamstring group ROM following a resultant decrease in tissue stiffness.

Manifestations of pulmonary aspergillosis in pediatrics.

PURPOSE OF REVIEW: Aspergillus spp. cause a clinical spectrum of disease with severity of disease dependent on degree of immune compromise, nature and intensity of inflammatory host response, and/or underlying lung disease. Chronic pulmonary aspergillosis encompasses a spectrum of diseases including aspergilloma, Aspergillus nodules, chronic cavitary pulmonary aspergillosis, chronic fibrosing pulmonary aspergillosis, and subacute invasive pulmonary aspergillosis. Allergic bronchopulmonary aspergillosis (ABPA) paradoxically is an immune hypersensitivity manifestation in the lungs that almost always occurs in the setting of underlying asthma or cystic fibrosis. These chronic Aspergillus conditions are now becoming more prevalent than invasive Aspergillus, thus it is important to be aware of the current literature of these conditions. RECENT FINDINGS: High-level research assessing the clinical significance and treatment options of these chronic diseases are lacking. Recent literature suggests colonization is antecedent for local airway infection (Aspergillus bronchitis), chronic or allergic bronchopulmonary disease, or invasive and potentially disseminated disease. There have been few advances in assessment of treatment of ABPA. SUMMARY: Research assessing the clinical significance and treatment options is currently needed.

Novel Sarcopenia-related Alterations in Sarcomeric Protein Post-translational Modifications (PTMs) in Skeletal Muscles Identified by Top-down Proteomics.

Sarcopenia, the age-related loss of skeletal muscle mass and strength, is a significant cause of morbidity in the elderly and is a major burden on health care systems. Unfortunately, the underlying molecular mechanisms in sarcopenia remain poorly understood. Herein, we utilized top-down proteomics to elucidate sarcopenia-related changes in the fast- and slow-twitch skeletal muscles of aging rats with a focus on the sarcomeric proteome, which includes both myofilament and Z-disc proteins-the proteins that constitute the contractile apparatuses. Top-down quantitative proteomics identified significant changes in the post-translational modifications (PTMs) of critical myofilament proteins in the fast-twitch skeletal muscles of aging rats, in accordance with the vulnerability of fast-twitch muscles to sarcopenia. Surprisingly, age-related alterations in the phosphorylation of Cypher isoforms, proteins that localize to the Z-discs in striated muscles, were also noted in the fast-twitch skeletal muscle of aging rats. This represents the first report of changes in the phosphorylation of Z-disc proteins in skeletal muscle during aging. In addition, increased glutathionylation of slow skeletal troponin I, a novel modification that may help protect against oxidative damage, was observed in slow-twitch skeletal muscles. Furthermore, we have identified and characterized novel muscle type-specific proteoforms of myofilament proteins and Z-disc proteins, including a novel isoform of the Z-disc protein Enigma. The finding that the phosphorylation of Z-disc proteins is altered in response to aging in the fast-twitch skeletal muscles of aging rats opens new avenues for the investigation of the role of Z-discs in age-related muscle dysfunction.

Phosphorylation of cardiac myosin binding protein C releases myosin heads from the surface of cardiac thick filaments.

Cardiac myosin binding protein C (cMyBP-C) has a key regulatory role in cardiac contraction, but the mechanism by which changes in phosphorylation of cMyBP-C accelerate cross-bridge kinetics remains unknown. In this study, we isolated thick filaments from the hearts of mice in which the three serine residues (Ser273, Ser282, and Ser302) that are phosphorylated by protein kinase A in the m-domain of cMyBP-C were replaced by either alanine or aspartic acid, mimicking the fully nonphosphorylated and the fully phosphorylated state of cMyBP-C, respectively. We found that thick filaments from the cMyBP-C phospho-deficient hearts had highly ordered cross-bridge arrays, whereas the filaments from the cMyBP-C phospho-mimetic hearts showed a strong tendency toward disorder. Our results support the hypothesis that dephosphorylation of cMyBP-C promotes or stabilizes the relaxed/superrelaxed quasi-helical ordering of the myosin heads on the filament surface, whereas phosphorylation weakens this stabilization and binding of the heads to the backbone. Such structural changes would modulate the probability of myosin binding to actin and could help explain the acceleration of cross-bridge interactions with actin when cMyBP-C is phosphorylated because of, for example, activation of ß1-adrenergic receptors in myocardium.

Cystic fibrosis transmembrane conductance regulator modulators: precision medicine in cystic fibrosis.

PURPOSE OF REVIEW: The aim of this study was to describe the newest development in cystic fibrosis (CF) care, CF transmembrane conductance regulator (CFTR) modulator therapies. RECENT FINDINGS: Phase II results showing CFTR modulator triple therapies are more effective than current CFTR modulators. SUMMARY: CFTR modulator therapy targets the protein defective in CF and boosts its function, but the drug must match mutation pathobiology. Ivacaftor, a CFTR potentiator, was the first modulator approved in 2012, with impressive improvement in lung function and other measures of disease in patients with gating and other residual function mutations (∼10% of CF patients). In 2015, the combination of lumacaftor, a CFTR corrector, and ivacaftor was approved for patients homozygous for the F508del mutation (∼40-50% of the CF population) with positive but less impressive clinical response and 10-20% incidence of intolerance. A next-generation CFTR corrector, tezacaftor, with ivacaftor equally effective and better tolerated than lumacaftor, has also received US Food and Drug Administration approval. Novel CFTR correctors, entering Phase 3 trials in triple modulator combination with tezacaftor-ivacaftor, appear substantially more effective for patients who are homozygous for the F508del mutation and can provide benefit for patients with a single F508del mutation. This offers promise of effective CFTR modulator therapy for nearly 90% of CF patients.

Arrhythmogenesis in a catecholaminergic polymorphic ventricular tachycardia mutation that depresses ryanodine receptor function.

Current mechanisms of arrhythmogenesis in catecholaminergic polymorphic ventricular tachycardia (CPVT) require spontaneous Ca(2+) release via cardiac ryanodine receptor (RyR2) channels affected by gain-of-function mutations. Hence, hyperactive RyR2 channels eager to release Ca(2+) on their own appear as essential components of this arrhythmogenic scheme. This mechanism, therefore, appears inadequate to explain lethal arrhythmias in patients harboring RyR2 channels destabilized by loss-of-function mutations. We aimed to elucidate arrhythmia mechanisms in a RyR2-linked CPVT mutation (RyR2-A4860G) that depresses channel activity. Recombinant RyR2-A4860G protein was expressed equally as wild type (WT) RyR2, but channel activity was dramatically inhibited, as inferred by [(3)H]ryanodine binding and single channel recordings. Mice heterozygous for the RyR2-A4860G mutation (RyR2-A4860G(+/-)) exhibited basal bradycardia but no cardiac structural alterations; in contrast, no homozygotes were detected at birth, suggesting a lethal phenotype. Sympathetic stimulation elicited malignant arrhythmias in RyR2-A4860G(+/-) hearts, recapitulating the phenotype originally described in a human patient with the same mutation. In isoproterenol-stimulated ventricular myocytes, the RyR2-A4860G mutation decreased the peak of Ca(2+) release during systole, gradually overloading the sarcoplasmic reticulum with Ca(2+). The resultant Ca(2+) overload then randomly caused bursts of prolonged Ca(2+) release, activating electrogenic Na(+)-Ca(2+) exchanger activity and triggering early afterdepolarizations. The RyR2-A4860G mutation reveals novel pathways by which RyR2 channels engage sarcolemmal currents to produce life-threatening arrhythmias.

Tiered Approach to Resilience Assessment.

Regulatory agencies have long adopted a three-tier framework for risk assessment. We build on this structure to propose a tiered approach for resilience assessment that can be integrated into the existing regulatory processes. Comprehensive approaches to assessing resilience at appropriate and operational scales, reconciling analytical complexity as needed with stakeholder needs and resources available, and ultimately creating actionable recommendations to enhance resilience are still lacking. Our proposed framework consists of tiers by which analysts can select resilience assessment and decision support tools to inform associated management actions relative to the scope and urgency of the risk and the capacity of resource managers to improve system resilience. The resilience management framework proposed is not intended to supplant either risk management or the many existing efforts of resilience quantification method development, but instead provide a guide to selecting tools that are appropriate for the given analytic need. The goal of this tiered approach is to intentionally parallel the tiered approach used in regulatory contexts so that resilience assessment might be more easily and quickly integrated into existing structures and with existing policies.

Small Colony Variants of Pseudomonas aeruginosa Display Heterogeneity in Inhibiting Aspergillus fumigatus Biofilm.

Pseudomonas aeruginosa and Aspergillus fumigatus are major microbes in cystic fibrosis (CF). We reported non-mucoid P. aeruginosa isolates more inhibitory to A. fumigatus than mucoid ones. Another CF P. aeruginosa phenotype, small colony variants (SCVs), is an unknown factor in intermicrobial competition with A. fumigatus. Clinical SCV isolates and reference CF non-mucoid isolate (Pa10, producing normal-sized colonies) were compared. Live cells of P. aeruginosa or filtrates from P. aeruginosa planktonic or biofilm cultures were co-incubated with A. fumigatus growing under conditions allowing biofilm formation or with preformed biofilm. Metabolic activity of A. fumigatus biofilm was then measured. When necessary, assays were done after adjustment for growth differences by adding fresh medium to the planktonic culture filtrate. Pyoverdine determinations were performed spectrophotometrically on the planktonic culture filtrates. In all experimental conditions (live cells and planktonic or biofilm culture filtrates of P. aeruginosa versus A. fumigatus biofilm formation or preformed biofilm), three SCV isolates were less inhibitory than Pa10, two equal or more inhibitory. Adjusting planktonic culture filtrates for growth differences showed SCV inhibition differences variably related to growth or deficient inhibitor production. Studies suggested the principal P. aeruginosa inhibitor to be pyoverdine. SCV isolates appear heterogeneous in their capacity to inhibit A. fumigatus biofilm. SCV isolates can be important in the CF microbiome, because they are capable of intermicrobial inhibition.

Complete Characterization of Cardiac Myosin Heavy Chain (223 kDa) Enabled by Size-Exclusion Chromatography and Middle-Down Mass Spectrometry.

Myosin heavy chain (MHC), the major component of the myosin motor molecule, plays an essential role in force production during muscle contraction. However, a comprehensive analysis of MHC proteoforms arising from sequence variations and post-translational modifications (PTMs) remains challenging due to the difficulties in purifying MHC (∼223 kDa) and achieving complete sequence coverage. Herein, we have established a strategy to effectively purify and comprehensively characterize MHC from heart tissue by combining size-exclusion chromatography (SEC) and middle-down mass spectrometry (MS). First, we have developed a MS-compatible SEC method for purifying MHC from heart tissue with high efficiency. Next, we have optimized the Glu-C, Asp-N, and trypsin limited digestion conditions for middle-down MS. Subsequently, we have applied this strategy with optimized conditions to comprehensively characterize human MHC and identified ß-MHC as the predominant isoform in human left ventricular tissue. Full sequence coverage based on highly accurate mass measurements has been achieved using middle-down MS combining 1 Glu-C, 1 Asp-N, and 1 trypsin digestion. Three different PTMs: acetylation, methylation, and trimethylation were identified in human ß-MHC and the corresponding sites were localized to the N-terminal Gly, Lys34, and Lys129, respectively, by electron capture dissociation (ECD). Taken together, we have demonstrated this strategy is highly efficient for purification and characterization of MHC, which can be further applied to studies of the role of MHC proteoforms in muscle-related diseases. We also envision that this integrated SEC/middle-down MS strategy can be extended for the characterization of other large proteins over 200 kDa.

Cardiac MyBP-C regulates the rate and force of contraction in mammalian myocardium.

Cardiac myosin-binding protein-C (cMyBP-C) is a thick filament-associated protein that seems to contribute to the regulation of cardiac contraction through interactions with either myosin or actin or both. Several studies over the past several years have suggested that the interactions of cardiac myosin-binding protein-C with its binding partners vary with its phosphorylation state, binding predominantly to myosin when dephosphorylated and to actin when it is phosphorylated by protein kinase A or other kinases. Here, we summarize evidence suggesting that phosphorylation of cardiac myosin binding protein-C is a key regulator of the kinetics and amplitude of cardiac contraction during ß-adrenergic stimulation and increased stimulus frequency. We propose a model for these effects via a phosphorylation-dependent regulation of the kinetics and extent of cooperative recruitment of cross bridges to the thin filament: phosphorylation of cardiac myosin binding protein-C accelerates cross bridge binding to actin, thereby accelerating recruitment and increasing the amplitude of the cardiac twitch. In contrast, enhanced lusitropy as a result of phosphorylation seems to be caused by a direct effect of phosphorylation to accelerate cross-bridge detachment rate. Depression or elimination of one or both of these processes in a disease, such as end-stage heart failure, seems to contribute to the systolic and diastolic dysfunction that characterizes the disease.

Susceptibility of Candida albicans from Cystic Fibrosis Patients.

Candida albicans is a common microbe, colonizer and potential pathogen found in respiratory cultures of cystic fibrosis (CF) patients. Because of possible development of resistance in patient isolates resulting from residence in the abnormal milieu of CF patient airways, or from exposure to antifungals, and considering the possibility of patient-to-patient spread of microbes and reports of elevated resistance to other fungal pathogens, it was important to assay the susceptibility of isolates of Candida and compare that profile to isolates from the community. In our center, and unlike another fungal pathogen, no increase in resistance of Candida isolates of the CF cohort was found.

Are Cystic Fibrosis Aspergillus fumigatus Isolates Different? Intermicrobial Interactions with Pseudomonas.

Pseudomonas aeruginosa and Aspergillus fumigatus are the leading bacterial and fungal pathogens in cystic fibrosis (CF). We have shown that Af biofilms are susceptible to Pseudomonas, particularly CF phenotypes. Those studies were performed with a reference virulent non-CF Aspergillus. Pseudomonas resident in CF airways undergo profound genetic and phenotypic adaptations to the abnormal environment. Studies have also indicated Aspergillus from CF patients have unexpected profiles of antifungal susceptibility. This would suggest that Aspergillus isolates from CF patients may be different or altered from other clinical isolates. It is important to know whether Aspergillus may also be altered, as a result of that CF environment, in susceptibility to Pseudomonas. CF Aspergillus proved not different in that susceptibility.

The basophil surface marker CD203c identifies Aspergillus species sensitization in patients with cystic fibrosis.

BACKGROUND: Colonization by Aspergillus fumigatus in patients with cystic fibrosis (CF) can cause A fumigatus sensitization and/or allergic bronchopulmonary aspergillosis (ABPA), which affects pulmonary function and clinical outcomes. Recent studies show that specific allergens upregulate the surface-expressed basophil marker CD203c in sensitized subjects, a response that can be readily measured by using flow cytometry. OBJECTIVE: We sought to identify A fumigatus sensitization in patients with CF by using the basophil activation test (BAT). METHODS: Patients with CF attending Beaumont Hospital were screened for study inclusion. BAT was used to identify A fumigatus sensitization. Serologic (total and A fumigatus-specific IgE), pulmonary function, and body mass index measurements were performed. RESULTS: The BAT discriminates A fumigatus-sensitized from nonsensitized patients with CF. Persistent isolation of A fumigatus in sputum is a significant risk factor for A fumigatus sensitization. Levels of the A fumigatus-stimulated basophil activation marker CD203c inversely correlated with pulmonary function and body mass index in A fumigatus-sensitized but not nonsensitized patients with CF. Total and A fumigatus-specific IgE, but not IgG, levels are increased in A fumigatus-sensitized patients with CF and ABPA when compared with those in A fumigatus-sensitized and nonsensitized patients with CF without ABPA. Itraconazole treatment did not affect A fumigatus sensitization. CONCLUSION: Combining the BAT with routine serologic testing allows classification of patients with CF into 3 groups: nonsensitized, A fumigatus-sensitized, and ABPA. Accurate and prompt identification of A fumigatus-associated clinical status might allow early and targeted therapeutic intervention, potentially improving clinical outcomes.

Top-Down Targeted Proteomics Reveals Decrease in Myosin Regulatory Light-Chain Phosphorylation That Contributes to Sarcopenic Muscle Dysfunction.

Sarcopenia, the loss of skeletal muscle mass and function with advancing age, is a significant cause of disability and loss of independence in the elderly and thus, represents a formidable challenge for the aging population. Nevertheless, the molecular mechanism(s) underlying sarcopenia-associated muscle dysfunction remain poorly understood. In this study, we employed an integrated approach combining top-down targeted proteomics with mechanical measurements to dissect the molecular mechanism(s) in age-related muscle dysfunction. Top-down targeted proteomic analysis uncovered a progressive age-related decline in the phosphorylation of myosin regulatory light chain (RLC), a critical protein involved in the modulation of muscle contractility, in the skeletal muscle of aging rats. Top-down tandem mass spectrometry analysis identified a previously unreported bis-phosphorylated proteoform of fast skeletal RLC and localized the sites of decreasing phosphorylation to Ser14/15. Of these sites, Ser14 phosphorylation represents a previously unidentified site of phosphorylation in RLC from fast-twitch skeletal muscle. Subsequent mechanical analysis of single fast-twitch fibers isolated from the muscles of rats of different ages revealed that the observed decline in RLC phosphorylation can account for age-related decreases in the contractile properties of sarcopenic fast-twitch muscles. These results strongly support a role for decreasing RLC phosphorylation in sarcopenia-associated muscle dysfunction and suggest that therapeutic modulation of RLC phosphorylation may represent a new avenue for the treatment of sarcopenia.