MICB Genomic Variant Is Associated with NKG2D-mediated Acute Lung Injury and Death.

Rationale: Acute lung injury (ALI) carries a high risk of mortality but has no established pharmacologic therapy. We previously found that experimental ALI occurs through natural killer (NK) cell NKG2D receptor activation and that the cognate human ligand, MICB, was associated with ALI after transplantation. Objectives: To investigate the association of a common missense variant, MICBG406A, with ALI. Methods: We assessed MICBG406A genotypes within two multicenter observational study cohorts at risk for ALI: primary graft dysfunction (N = 619) and acute respiratory distress syndrome (N = 1,376). Variant protein functional effects were determined in cultured and ex vivo human samples. Measurements and Main Results: Recipients of MICBG406A-homozygous allografts had an 11.1% absolute risk reduction (95% confidence interval [CI], 3.2-19.4%) for severe primary graft dysfunction after lung transplantation and reduced risk for allograft failure (hazard ratio, 0.36; 95% CI, 0.13-0.98). In participants with sepsis, we observed 39% reduced odds of moderately or severely impaired oxygenation among MICBG406A-homozygous individuals (95% CI, 0.43-0.86). BAL NK cells were less frequent and less mature in participants with MICBG406A. Expression of missense variant protein MICBD136N in cultured cells resulted in reduced surface MICB and reduced NKG2D ligation relative to wild-type MICB. Coculture of variant MICBD136N cells with NK cells resulted in less NKG2D activation and less susceptibility to NK cell killing relative to the wild-type cells. Conclusions: These data support a role for MICB signaling through the NKG2D receptor in mediating ALI, suggesting a novel therapeutic approach.

Mitochondria-cytokine crosstalk following skeletal muscle injury and disuse: a mini-review.

Skeletal muscle mitochondria are highly adaptable, highly dynamic organelles that maintain the functional integrity of the muscle fiber by providing ATP for contraction and cellular homeostasis (e.g., Na+/K+ ATPase). Emerging as early modulators of inflammation, mitochondria sense and respond to cellular stress. Mitochondria communicate with the environment, in part, by release of physical signals called mitochondrial-derived damage-associated molecular patterns (mito-DAMPs) and deviation from routine function (e.g., reduced ATP production, Ca2+ overload). When skeletal muscle is compromised, mitochondria contribute to an acute inflammatory response necessary for myofibril regeneration; however, exhaustive signaling associated with altered or reduced mitochondrial function can be detrimental to muscle outcomes. Here, we describe changes in mitochondrial content, structure, and function following skeletal muscle injury and disuse and highlight the influence of mitochondria-cytokine crosstalk on muscle regeneration and recovery. Although the appropriate therapeutic modulation following muscle stressors remains unknown, retrospective gene expression analysis reveals that interleukin-6 (IL-6), interleukin-1ß (IL-1ß), chemokine C-X-C motif ligand 1 (CXCL1), and monocyte chemoattractant protein 1 (MCP-1) are significantly upregulated following three unique muscle injuries. These cytokines modulate mitochondrial function and execute bona fide pleiotropic roles that can aid functional recovery of muscle, however, when aberrant, chronically disrupt healing partly by exacerbating mitochondrial dysfunction. Multidisciplinary efforts to delineate the opposing regulatory roles of inflammatory cytokines in the muscle mitochondrial environment are required to modulate regenerative behavior following skeletal muscle injury or disuse. Future therapeutic directions to consider include quenching or limited release of mito-DAMPs and cytokines present in cytosol or circulation.

Lifelong Ulk1-Mediated Autophagy Deficiency in Muscle Induces Mitochondrial Dysfunction and Contractile Weakness.

The accumulation of damaged mitochondria due to insufficient autophagy has been implicated in the pathophysiology of skeletal muscle aging. Ulk1 is an autophagy-related kinase that initiates autophagosome assembly and may also play a role in autophagosome degradation (i.e., autophagy flux), but the contribution of Ulk1 to healthy muscle aging is unclear. Therefore, the purpose of this study was to investigate the role of Ulk1-mediated autophagy in skeletal muscle aging. At age 22 months (80% survival rate), muscle contractile and metabolic function were assessed using electrophysiology in muscle-specific Ulk1 knockout mice (MKO) and their littermate controls (LM). Specific peak-isometric torque of the ankle dorsiflexors (normalized by tibialis anterior muscle cross-sectional area) and specific force of the fast-twitch extensor digitorum longus muscles was reduced in MKO mice compared to LM mice (p < 0.03). Permeabilized muscle fibers from MKO mice had greater mitochondrial content, yet lower mitochondrial oxygen consumption and greater reactive oxygen species production compared to fibers from LM mice (p ≤ 0.04). Alterations in neuromuscular junction innervation patterns as well as changes to autophagosome assembly and flux were explored as possible contributors to the pathological features in Ulk1 deficiency. Of primary interest, we found that Ulk1 phosphorylation (activation) to total Ulk1 protein content was reduced in older muscles compared to young muscles from both human and mouse, which may contribute to decreased autophagy flux and an accumulation of dysfunctional mitochondria. Results from this study support the role of Ulk1-mediated autophagy in aging skeletal muscle, reflecting Ulk1's dual role in maintaining mitochondrial integrity through autophagosome assembly and degradation.

Mitochondrial-specific autophagy linked to mitochondrial dysfunction following traumatic freeze injury in mice.

The objective of this study was to interrogate the link between mitochondrial dysfunction and mitochondrial-specific autophagy in skeletal muscle. C57BL/6J mice were used to establish a time course of mitochondrial function and autophagy induction after fatigue (n = 12), eccentric contraction-induced injury (n = 20), or traumatic freeze injury (FI, n = 28); only FI resulted in a combination of mitochondrial dysfunction, i.e., decreased mitochondrial respiration, and autophagy induction. Moving forward, we tested the hypothesis that mitochondrial-specific autophagy is important for the timely recovery of mitochondrial function after FI. Following FI, there is a significant increase in several mitochondrial-specific autophagy-related protein contents including dynamin-related protein 1 (Drp1), BCL1 interacting protein (BNIP3), Pink1, and Parkin (~2-fold, P < 0.02). Also, mitochondrial-enriched fractions from FI muscles showed microtubule-associated protein light chain B1 (LC3)II colocalization suggesting autophagosome assembly around the damaged mitochondrial. Unc-51 like autophagy activating kinase (Ulk1) is considered necessary for mitochondrial-specific autophagy and herein we utilized a mouse model with Ulk1 deficiency in adult skeletal muscle (myogenin-Cre). While Ulk1 knockouts had contractile weakness compared with littermate controls (-27%, P < 0.02), the recovery of mitochondrial function was not different, and this may be due in part to a partial rescue of Ulk1 protein content within the regenerating muscle tissue of knockouts from differentiated satellite cells in which Ulk1 was not genetically altered via myogenin-Cre. Lastly, autophagy flux was significantly less in injured versus uninjured muscles (-26%, P < 0.02) despite the increase in autophagy-related protein content. This suggests autophagy flux is not upregulated to match increases in autophagy machinery after injury and represents a potential bottleneck in the clearance of damaged mitochondria by autophagy.

Mitochondrial dysfunction in skeletal muscle of fukutin-deficient mice is resistant to exercise- and 5-aminoimidazole-4-carboxamide ribonucleotide-induced rescue.

NEW FINDINGS: What is the central question of this study? Does fukutin deficiency in skeletal muscle cause mitochondrial dysfunction, and if so, can AMP-activated protein kinase (AMPK) stimulation via 5-aminoimidazole-4-carboxamide ribonucleotide attenuate this through regulation of mitochondrial biogenesis and autophagy? What is the main finding and its importance? Mitochondrial dysfunction is associated with fukutin deficiency and AMPK stimulation may benefit muscle contractility to a greater extent than mitochondrial function. ABSTRACT: Disruptions in the dystrophin-glycoprotein complex (DGC) are clearly the primary basis underlying various forms of muscular dystrophies and dystroglycanopathies, but the cellular consequences of DGC disruption are still being investigated. Mitochondrial abnormalities are becoming an apparent consequence and contributor to dystrophy disease pathology. Herein, we demonstrate that muscle-specific deletion of the fukutin gene (Myf5/fktn-KO mice (Fktn KO)), a model of secondary dystroglycanopathy, results in ∼30% lower muscle strength (P < 0.001) and 16% lower mitochondrial respiratory function (P = 0.002) compared to healthy littermate controls (LM). We also observed ∼80% lower expression of the gene for peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) (P = 0.004), a primary transcription factor for mitochondrial biogenesis, in Fktn KO mice that likely contributes to the mitochondrial defects. PGC-1α is post-translationally regulated via phosphorylation by AMP-activated protein kinase (AMPK). Treatment with the AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) failed to rescue mitochondrial deficits in Fktn KO mice (P = 0.458) but did have beneficial (∼30% greater) effects on recovery of muscle contractility following injury in both LM and Fktn KO mice compared to saline treatment (P = 0.006). The beneficial effects of AMPK stimulation via AICAR on muscle contractile function may be partially explained by AMPK's other role of regulating skeletal muscle autophagy, a cellular process critical for clearance of damaged and/or dysfunctional organelles. Two primary conclusions can be drawn from this data: (1) fukutin deletion produces intrinsic muscular metabolic defects that likely contribute to dystroglycanopathy disease pathology, and (2) AICAR treatment accelerates recovery of muscle contractile function following injury suggesting AMPK signalling as a possible target for therapeutic strategies.

Novel SPEG mutations in congenital myopathies: Genotype-phenotype correlations.

INTRODUCTION: Centronuclear myopathies (CNMs) are a subtype of congenital myopathies (CMs) characterized by muscle weakness, predominant type 1 fibers, and increased central nuclei. SPEG (striated preferentially expressed protein kinase) mutations have recently been identified in 7 CM patients (6 with CNMs). We report 2 additional patients with SPEG mutations expanding the phenotype and evaluate genotype-phenotype correlations associated with SPEG mutations. METHODS: Using whole exome/genome sequencing in CM families, we identified novel recessive SPEG mutations in 2 patients. RESULTS: Patient 1, with severe muscle weakness requiring respiratory support, dilated cardiomyopathy, ophthalmoplegia, and findings of nonspecific CM on muscle biopsy carried a homozygous SPEG mutation (p.Val3062del). Patient 2, with milder muscle weakness, ophthalmoplegia, and CNM carried compound heterozygous mutations (p.Leu728Argfs*82) and (p.Val2997Glyfs*52). CONCLUSIONS: The 2 patients add insight into genotype-phenotype correlations of SPEG-associated CMs. Clinicians should consider evaluating a CM patient for SPEG mutations even in the absence of CNM features. Muscle Nerve 59:357-362, 2019.

Novel founder intronic variant in SLC39A14 in two families causing Manganism and potential treatment strategies.

Congenital disorders of manganese metabolism are rare occurrences in children, and medical management of these disorders is complex and challenging. Homozygous exonic mutations in the manganese transporter SLC39A14 have recently been associated with a pediatric-onset neurodegenerative disorder characterized by brain manganese accumulation and clinical signs of manganese neurotoxicity, including parkinsonism-dystonia. We performed whole exome sequencing on DNA samples from two unrelated female children from the United Arab Emirates with progressive movement disorder and brain mineralization, identified a novel homozygous intronic mutation in SLC39A14 in both children, and demonstrated that the mutation leads to aberrant splicing. Both children had consistently elevated serum manganese levels and were diagnosed with SLC39A14-associated manganism. Over a four-year period, we utilized a multidisciplinary management approach for Patient 1 combining decreased manganese dietary intake and chelation with symptomatic management of dystonia. Our treatment strategy appeared to slow disease progression, but did not lead to a cure or reversal of already established deficits. Clinicians should consider testing for noncoding mutations in the diagnosis of congenital disorders of manganese metabolism and utilizing multidisciplinary approaches in the management of these disorders.

Atypical presentations associated with non-polyalanine repeat PHOX2B mutations.

Congenital central hypoventilation syndrome (CCHS) is a disorder of ventilatory control and autonomic dysregulation that can be caused by mutations in the paired-like homeobox 2B (PHOX2B) gene. The majority of CCHS cases are caused by polyalanine repeat mutations (PARMs) in PHOX2B; however, in rare cases, non-polyalanine repeat mutations (NPARMs) have been identified. Here, we report two patients with NPARMs in PHOX2B. Patient 1 has a mild CCHS phenotype seen only on polysomnogram, which was performed for desaturations and stridor following a bronchiolitis episode, and characterized by night-time hypoventilation and a history of ganglioneuroblastoma. She carried a novel de novo missense variant, p.R102S (c.304C > A), in exon 2. Patient 2 has an atypical CCHS phenotype including micrognathia, gastroesophageal reflux, stridor, hypopnea, and intermittent desaturations. Sleep study demonstrated that Patient 2 had daytime and night-time hypercarbia with obstructive sleep apnea, requiring tracheostomy. On PHOX2B sequencing, she carried a recently identified nonsense variant, p.Y78* (c.234C > G), in exon 1. In summary, we present two patients with CCHS and identified NPARMs in PHOX2B who have distinct differences in phenotype severity, further elucidating the range of clinical outcomes in CCHS and illustrating the necessity of considering PHOX2B mutations when encountering atypical CCHS presentations.

Early rehabilitation for volumetric muscle loss injury augments endogenous regenerative aspects of muscle strength and oxidative capacity.

BACKGROUND: Volumetric muscle loss (VML) injuries occur due to orthopaedic trauma or the surgical removal of skeletal muscle and result in debilitating long-term functional deficits. Current treatment strategies do not promote significant restoration of function; additionally appropriate evidenced-based practice physical therapy paradigms have yet to be established. The objective of this study was to develop and evaluate early rehabilitation paradigms of passive range of motion and electrical stimulation in isolation or combination to understand the genetic and functional response in the tissue remaining after a multi-muscle VML injury. METHODS: Adult male mice underwent an ~ 20% multi-muscle VML injury to the posterior compartment (gastrocnemius, soleus, and plantaris muscle) unilaterally and were randomized to rehabilitation paradigm twice per week beginning 2 days post-injury or no treatment. RESULTS: The most salient findings of this work are: 1) that the remaining muscle tissue after VML injury was adaptable in terms of improved muscle strength and mitigation of stiffness; but 2) not adaptable to improvements in metabolic capacity. Furthermore, biochemical (i.e., collagen content) and gene (i.e., gene arrays) assays suggest that functional adaptations may reflect changes in the biomechanical properties of the remaining tissue due to the cellular deposition of non-contractile tissue in the void left by the VML injury and/or differentiation of gene expression with early rehabilitation. CONCLUSIONS: Collectively this work provides evidence of genetic and functional plasticity in the remaining skeletal muscle with early rehabilitation approaches, which may facilitate future evidenced-based practice of early rehabilitation at the clinical level.

PGC-1α overexpression partially rescues impaired oxidative and contractile pathophysiology following volumetric muscle loss injury.

Volumetric muscle loss (VML) injury is characterized by a non-recoverable loss of muscle fibers due to ablative surgery or severe orthopaedic trauma, that results in chronic functional impairments of the soft tissue. Currently, the effects of VML on the oxidative capacity and adaptability of the remaining injured muscle are unclear. A better understanding of this pathophysiology could significantly shape how VML-injured patients and clinicians approach regenerative medicine and rehabilitation following injury. Herein, the data indicated that VML-injured muscle has diminished mitochondrial content and function (i.e., oxidative capacity), loss of mitochondrial network organization, and attenuated oxidative adaptations to exercise. However, forced PGC-1α over-expression rescued the deficits in oxidative capacity and muscle strength. This implicates physiological activation of PGC1-α as a limiting factor in VML-injured muscle's adaptive capacity to exercise and provides a mechanistic target for regenerative rehabilitation approaches to address the skeletal muscle dysfunction.