The Relative Efficacy of Available Proteasome Inhibitors in Preventing Muscle Contractures Following Neonatal Brachial Plexus Injury.

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.

NRG/ErbB signaling regulates neonatal muscle growth but not neuromuscular contractures in neonatal brachial plexus injury.

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.

Ube2v1 Positively Regulates Protein Aggregation by Modulating Ubiquitin Proteasome System Performance Partially Through K63 Ubiquitination.

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 (CryABR120G) and identify how Ube2v1 exerts its effect. METHODS AND RESULTS: Neonatal rat ventricular cardiomyocytes were infected with adenoviruses expressing either wild-type CryAB (CryABWT) or CryABR120G. Subsequently, loss- and gain-of-function experiments were performed. Ube2v1 knockdown decreased aggregate accumulation caused by CryABR120G expression. Overexpressing Ube2v1 promoted aggregate formation in CryABWT and CryABR120G-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 CryABR120G 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 CryABR120G-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 CryABR120G-induced aggregate formation through enhanced ubiquitin proteasome system performance rather than autophagy and may provide a novel therapeutic target to treat cardiac proteinopathies.

MMI-0100 Inhibits Cardiac Fibrosis in a Mouse Model Overexpressing Cardiac Myosin Binding Protein C.

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-C40k) 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-C40k cardiomyocyte expression. METHODS AND RESULTS: Nontransgenic and cMyBP-C40k 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 cMyBPC40k disease.

Increased susceptibility to structural acute kidney injury in a mouse model of presymptomatic cardiomyopathy.

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.