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Background: Contractures following neonatal brachial plexus injury (NBPI) are associated with growth deficits in denervated muscles. This impairment is mediated by an increase in muscle protein degradation, as contractures can be prevented in an NBPI mouse model with bortezomib (BTZ), a proteasome inhibitor (PI). However, BTZ treatment causes substantial toxicity (0% to 80% mortality). The current study tested the hypothesis that newer-generation PIs can prevent contractures with less severe toxicity than BTZ., Methods: Unilateral brachial plexus injuries were surgically created in postnatal (5-day-old) mice. Following NBPI, mice were treated with either saline solution or various doses of 1 of 3 different PIs: ixazomib (IXZ), carfilzomib (CFZ), or marizomib (MRZ). Four weeks post-NBPI, mice were assessed for bilateral passive range of motion at the shoulder and elbow joints, with blinding to the treatment group, through an established digital photography technique to determine contracture severity. Drug toxicity was assessed with survival curves., Results: All PIs prevented contractures at both the elbow and shoulder (p < 0.05 versus saline solution controls), with the exception of IXZ, which did not prevent shoulder contractures. However, their efficacies and toxicity profiles differed. At lower doses, CFZ was limited by toxicity (30% to 40% mortality), whereas MRZ was limited by efficacy. At higher doses, CFZ was limited by loss of efficacy, MRZ was limited by toxicity (50% to 60% mortality), and IXZ was limited by toxicity (80% to 100% mortality) and loss of efficacy. Comparisons of the data on these drugs as well as data on BTZ generated in prior studies revealed BTZ to be optimal for preventing contractures, although it, too, was limited by toxicity., Conclusions: All of the tested second-generation PIs were able to reduce NBPI-induced contractures, offering further proof of concept for a regulatory role of the proteasome in contracture formation. However, the narrow dose ranges of efficacy for all PIs highlight the necessity of precise proteasome regulation for preventing contractures. Finally, the substantial toxicity stemming from proteasome inhibition underscores the importance of identifying muscle-targeted strategies to suppress protein degradation and prevent contractures safely., Clinical Relevance: Although PIs offer unique opportunities to establish critical mechanistic insights into contracture pathophysiology, their clinical use is contraindicated in patients with NPBI at this time., Competing Interests: Disclosure: This study received funding from National Institutes of Health grant R01HD098280-01 and the Cincinnati Children’s Hospital Junior Cooperative Society. The funding sources were not involved at any stage in the research investigation and manuscript preparation. The Disclosure of Potential Conflicts of Interest forms are provided with the online version of the article ( http://links.lww.com/JBJS/H837 )., (Copyright © 2024 by The Journal of Bone and Joint Surgery, Incorporated.)

Neonatal brachial plexus injury (NBPI), a leading cause of pediatric upper limb paralysis, results in disabling and incurable muscle contractures that are driven by impaired longitudinal growth of denervated muscles. A rare form of NBPI, which maintains both afferent and sympathetic muscle innervation despite motor denervation, protects against contractures. We have previously ruled out a role for NRG/ErbB signaling, the predominant pathway governing antegrade afferent neuromuscular transmission, in modulating the formation of contractures. Our current study therefore investigated the contributions of sympathetic innervation of skeletal muscle in modulating NBPI-induced contractures. Through chemical sympathectomy and pharmacologic modification with a β 2 -adrenergic agonist, we discovered that sympathetic innervation alone is neither required nor sufficient to modulate contracture formation in neonatal mice. Despite this, sympathetic innervation plays an intriguing sex-specific role in mediating neonatal muscle growth, as the cross-sectional area (CSA) and volume of normally innervated male muscles were diminished by ablation of sympathetic neurons and increased by β-adrenergic stimulation. Intriguingly, the robust alterations in CSA occurred with minimal changes to normal longitudinal muscle growth as determined by sarcomere length. Instead, β-adrenergic stimulation exacerbated sarcomere overstretch in denervated male muscles, indicating potentially discrete regulation of muscle width and length. Future investigations into the mechanistic underpinnings of these distinct aspects of muscle growth are thus essential for improving clinical outcomes in patients affected by muscle disorders in which both length and width are affected., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Neonatal brachial plexus injury (NBPI) causes disabling and incurable muscle contractures that result from impaired longitudinal growth of denervated muscles. This deficit in muscle growth is driven by increased proteasome-mediated protein degradation, suggesting a dysregulation of muscle proteostasis. The myostatin (MSTN) pathway, a prominent muscle-specific regulator of proteostasis, is a putative signaling mechanism by which neonatal denervation could impair longitudinal muscle growth, and thus a potential target to prevent NBPI-induced contractures. Through a mouse model of NBPI, our present study revealed that pharmacologic inhibition of MSTN signaling induces hypertrophy, restores longitudinal growth, and prevents contractures in denervated muscles of female but not male mice, despite inducing hypertrophy of normally innervated muscles in both sexes. Additionally, the MSTN-dependent impairment of longitudinal muscle growth after NBPI in female mice is associated with perturbation of 20S proteasome activity, but not through alterations in canonical MSTN signaling pathways. These findings reveal a sex dimorphism in the regulation of neonatal longitudinal muscle growth and contractures, thereby providing insights into contracture pathophysiology, identifying a potential muscle-specific therapeutic target for contracture prevention, and underscoring the importance of sex as a biological variable in the pathophysiology of neuromuscular disorders., Competing Interests: ME, PA, KS, QG, RC No competing interests declared, SL consulting fees from Biohaven Pharmaceuticals, Inc, and Alnylam Pharmaceuticals, Inc; payment/honoraria: Regeneron Pharmaceuticals, Inc; patent: "Therapeutics targeting transforming growth factor beta family signaling", (© 2022, Emmert et al.)

Neonatal brachial plexus injury (NBPI) causes disabling and incurable muscle contractures that are driven by impaired growth of denervated muscles. A rare form of NBPI, which maintains afferent muscle innervation despite motor denervation, does not cause contractures. As afferent innervation regulates various aspects of skeletal muscle homeostasis through NRG/ErbB signaling, our current study investigated the role of this pathway in modulating contracture development. Through pharmacologic modification with an ErbB antagonist and NRG1 isoforms, we discovered that NRG/ErbB signaling does not modulate the development of contractures in neonatal mice. Instead, ErbB inhibition impeded growth in nondenervated skeletal muscles, whereas increased ErbB activation exacerbated denervation-induced skeletal muscle atrophy. This potential regulatory effect of NRG/ErbB signaling on neonatal muscle growth warrants deeper investigation., (© 2021 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Neonatal brachial plexus injury (NBPI) causes disabling and incurable contractures, or limb stiffness, which result from proteasome-mediated protein degradation impairing the longitudinal growth of neonatally denervated muscles. We recently showed in a mouse model that the 20S proteasome inhibitor, bortezomib, prevents contractures after NBPI. Given that contractures uniquely follow neonatal denervation, the current study tests the hypothesis that proteasome inhibition during a finite window of neonatal development can prevent long-term contracture development. Following neonatal forelimb denervation in P5 mice, we first outlined the minimum period for proteasome inhibition to prevent contractures 4 weeks post-NBPI by treating mice with saline or bortezomib for varying durations between P8 and P32. We then compared the ability of varying durations of longer-term proteasome inhibition to prevent contractures at 8 and 12 weeks post-NBPI. Our findings revealed that proteasome inhibition can be delayed 3-4 days after denervation but is required throughout skeletal growth to prevent contractures long term. Furthermore, proteasome inhibition becomes less effective in preventing contractures beyond the neonatal period. These therapeutic effects are primarily associated with bortezomib-induced attenuation of 20S proteasome β1 subunit activity. Our collective results, therefore, demonstrate that temporary neonatal proteasome inhibition is not a viable strategy for preventing contractures long term. Instead, neonatal denervation causes a permanent longitudinal growth deficiency that must be continuously ameliorated during skeletal growth. Additional mechanisms must be explored to minimize the necessary period of proteasome inhibition and reduce the risk of toxicity from long-term treatment., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Rationale: Compromised protein quality control can result in proteotoxic intracellular protein aggregates in the heart, leading to cardiac disease and heart failure. Defining the participants and understanding the underlying mechanisms of cardiac protein aggregation is critical for seeking therapeutic targets. We identified Ube2v1 (ubiquitin-conjugating enzyme E2 variant 1) in a genome-wide screen designed to identify novel effectors of the aggregation process. However, its role in the cardiomyocyte is undefined., Objective: To assess whether Ube2v1 regulates the protein aggregation caused by cardiomyocyte expression of a mutant αB crystallin (CryAB R120G ) and identify how Ube2v1 exerts its effect., Methods and Results: Neonatal rat ventricular cardiomyocytes were infected with adenoviruses expressing either wild-type CryAB (CryAB WT ) or CryAB R120G . Subsequently, loss- and gain-of-function experiments were performed. Ube2v1 knockdown decreased aggregate accumulation caused by CryAB R120G expression. Overexpressing Ube2v1 promoted aggregate formation in CryAB WT and CryAB R120G -expressing neonatal rat ventricular cardiomyocytes. Ubiquitin proteasome system performance was analyzed using a ubiquitin proteasome system reporter protein. Ube2v1 knockdown improved ubiquitin proteasome system performance and promoted the degradation of insoluble ubiquitinated proteins in CryAB R120G cardiomyocytes but did not alter autophagic flux. Lys (K) 63-linked ubiquitination modulated by Ube2v1 expression enhanced protein aggregation and contributed to Ube2v1's function in regulating protein aggregate formation. Knocking out Ube2v1 exclusively in cardiomyocytes by using AAV9 (adeno-associated virus 9) to deliver multiplexed single guide RNAs against Ube2v1 in cardiac-specific Cas9 mice alleviated CryAB R120G -induced protein aggregation, improved cardiac function, and prolonged lifespan in vivo., Conclusions: Ube2v1 plays an important role in protein aggregate formation, partially by enhancing K63 ubiquitination during a proteotoxic stimulus. Inhibition of Ube2v1 decreases CryAB R120G -induced aggregate formation through enhanced ubiquitin proteasome system performance rather than autophagy and may provide a novel therapeutic target to treat cardiac proteinopathies.

Rationale: Hypertrophic cardiomyopathy occurs with a frequency of about 1 in 500 people. Approximately 30% of those affected carry mutations within the gene encoding cMyBP-C (cardiac myosin binding protein C). Cardiac stress, as well as cMyBP-C mutations, can trigger production of a 40kDa truncated fragment derived from the amino terminus of cMyBP-C (Mybpc3 40kDa ). Expression of the 40kDa fragment in mouse cardiomyocytes leads to hypertrophy, fibrosis, and heart failure. Here we use genetic approaches to establish a causal role for excessive myofibroblast activation in a slow, progressive genetic cardiomyopathy-one that is driven by a cardiomyocyte-intrinsic genetic perturbation that models an important human disease., Objective: TGFβ (transforming growth factor-β) signaling is implicated in a variety of fibrotic processes, and the goal of this study was to define the role of myofibroblast TGFβ signaling during chronic Mybpc3 40kDa expression., Methods and Results: To specifically block TGFβ signaling only in the activated myofibroblasts in Mybpc3 40kDa transgenic mice and quadruple compound mutant mice were generated, in which the TGFβ receptor II (TβRII) alleles ( Tgfbr2) were ablated using the periostin ( Postn) allele, myofibroblast-specific, tamoxifen-inducible Cre ( Postnmcm) gene-targeted line. Tgfbr2 was ablated either early or late during pathological fibrosis. Early myofibroblast-specific Tgfbr2 ablation during the fibrotic response reduced cardiac fibrosis, alleviated cardiac hypertrophy, preserved cardiac function, and increased lifespan of the Mybpc3 40kDa transgenic mice. Tgfbr2 ablation late in the pathological process reduced cardiac fibrosis, preserved cardiac function, and prolonged Mybpc3 40kDa mouse survival but failed to reverse cardiac hypertrophy., Conclusions: Fibrosis and cardiac dysfunction induced by cardiomyocyte-specific expression of Mybpc3 40kDa were significantly decreased by Tgfbr2 ablation in the myofibroblast. Surprisingly, preexisting fibrosis was partially reversed if the gene was ablated subsequent to fibrotic deposition, suggesting that continued TGFβ signaling through the myofibroblasts was needed to maintain the heart fibrotic response to a chronic, disease-causing cardiomyocyte-only stimulus.

Background: Cardiac stress can trigger production of a 40-kDa peptide fragment derived from the amino terminus of the cardiac myosin-binding protein C. Cardiac stress, as well as cMyBP-C mutations, can trigger production of 1 such truncated protein fragment, a 40-kDa peptide fragment derived from the amino terminus of cMyBP-C. Genetic expression of this 40-kDa fragment in mouse cardiomyocytes (cMyBP-C 40k ) leads to cardiac disease, fibrosis, and death within the first year. Fibrosis can occur in many cardiovascular diseases, and mitogen-activated protein kinase--activated protein kinase-2 signaling has been implicated in a variety of fibrotic processes. Recent studies demonstrated that mitogen-activated protein kinase--activated protein kinase-2 inhibition using the cell-permeant peptide inhibitor MMI-0100 is protective in the setting of acute myocardial infarction. We hypothesized that MMI-0100 might also be protective in a chronic model of fibrosis, produced as a result of cMyBP-C 40k cardiomyocyte expression., Methods and Results: Nontransgenic and cMyBP-C 40k inducible transgenic mice were given MMI-0100 or PBS daily for 30 weeks. In control groups, long-term MMI-0100 was benign, with no measurable effects on cardiac anatomy, function, cell viability, hypertrophy, or probability of survival. In the inducible transgenic group, MMI-0100 treatment reduced cardiac fibrosis, decreased cardiac hypertrophy, and prolonged survival., Conclusions: Pharmaceutical inhibition of mitogen-activated protein kinase--activated protein kinase-2 signaling via MMI-0100 treatment is beneficial in the context of fibrotic cMyBPC 40k disease., (© 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.)

The early events that signal renal dysfunction in presymptomatic heart failure are unclear. We tested the hypothesis that functional and mechanistic changes occur in the kidney that precede the development of symptomatic heart failure. We employed a transgenic mouse model with cardiomyocyte-specific overexpression of mutant α-B-crystallin that develops slowly progressive cardiomyopathy. Presymptomatic transgenic mice displayed an increase in serum creatinine (1.17 ± 0.34 vs. wild type 0.65 ± 0.16 mg/dl, P < 0.05) and in urinary neutrophil gelatinase-associated lipocalin (NGAL; 278.92 ± 176.24 vs. wild type 49.11 ± 22.79 ng/ml, P < 0.05) but no renal fibrosis. Presymptomatic transgenic mouse kidneys exhibited a twofold upregulation of the Ren1 gene, marked overexpression of renin protein in the tubules, and a worsened response to ischemia-reperfusion injury based on serum creatinine (2.77 ± 0.66 in transgenic mice vs. 2.01 ± 0.58 mg/dl in wild type, P < 0.05), urine NGAL (9,198.79 ± 3,799.52 in transgenic mice vs. 3,252.94 ± 2,420.36 ng/ml in wild type, P < 0.05), tubule dilation score (3.4 ± 0.5 in transgenic mice vs. 2.6 ± 0.5 in wild type, P < 0.05), tubule cast score (3.2 ± 0.4 in transgenic mice vs. 2.5 ± 0.5 in wild type, P < 0.05), and TdT-mediated dUTP nick-end labeling (TUNEL)-positive nuclei (10.1 ± 2.1 in the transgenic group vs. 5.7 ± 1.6 per 100 cells counted in wild type, P < 0.01). Our findings indicate functional renal impairment, urinary biomarker elevations, and induction of renin gene and protein expression in the kidney that occur in early presymptomatic heart failure, which increase the susceptibility to subsequent acute kidney injury., (Copyright © 2017 the American Physiological Society.)