Extracting high-quality RNA from formaldehyde-fixed naturally aged neuromusculoskeletal tissues.

Modern approaches to discovering molecular mechanisms and validating treatments for age-related neuromusculoskeletal dysfunction typically rely on high-throughput transcriptome analysis. Previously harvested and fixed tissues offer an incredible reservoir of untapped molecular information. However, obtaining RNA from such formaldehyde-fixed neuromusculoskeletal tissues, especially fibrotic aged tissues, is technically challenging and often results in RNA degradation, chemical modification and yield reduction, prohibiting further analysis. Therefore, we developed a protocol to extract high-quality RNA from formaldehyde-fixed brain, cartilage, muscle and peripheral nerve isolated from naturally aged mice. Isolated RNA produced reliable gene expression data comparable to fresh and flash-frozen tissues and was sensitive enough to detect age-related changes, making our protocol valuable to researchers in the field of aging.

Wrist extensor pathomechanics: implications for tendon and nerve transfer.

Central and peripheral nervous system lesions may disrupt the intricate balance of the prime movers of the wrist. In spasticity, hyperactive wrist flexors create a flexion moment and, if untreated, can lead to flexion contractures. In patients with C6 spinal cord injury and tetraplegia, the posterior interosseus nerve is typically affected by a complex pattern of upper and/or lower motoneuron lesions causing radial deviation of the wrist due to loss of ulnar deviation actuators. In this report, we illustrate severe pathomechanics that may occur even with relatively modest changes in wrist balance. These results illustrate how thorough understanding of muscle-tendon-joint interaction aids in designing tendon and nerve reconstructive surgeries to normalize wrist positions and balance in neuromuscular conditions.

Gait speed is a biomarker of cancer-associated cachexia decline and recovery.

Background: Progressive functional decline is a key element of cancer-associated cachexia. No therapies have successfully translated to the clinic due to an inability to measure and improve physical function in cachectic patients. Major barriers to translating pre-clinical therapies to the clinic include lack of cancer models that accurately mimic functional decline and use of non-specific outcome measures of function, like grip strength. New approaches are needed to investigate cachexia-related function at both the basic and clinical science levels. Methods: Survival extension studies were performed by testing multiple cell lines, dilutions, and vehicle-types in orthotopic implantation of K-ras LSL.G12D/+ ; Trp53 R172H/+ ; Pdx-1-Cre (KPC) derived cells. 128 animals in this new model were then assessed for muscle wasting, inflammation, and functional decline using a battery of biochemical, physiologic, and behavioral techniques. In parallel, we analyzed a 156-subject cohort of cancer patients with a range of cachexia severity, and who required rehabilitation, to determine the relationship between gait speed via six-minute walk test (6MWT), grip strength (hGS), and functional independence measures (FIM). Cachectic patients were identified using the Weight Loss Grading Scale (WLGS), Fearon consensus criteria, and the Prognostic Nutritional Index (PNI). Results: Using a 100-cell dose of DT10022 KPC cells, we extended the survival of the KPC orthotopic model to 8-9 weeks post-implantation compared to higher doses used (p<0.001). In this Low-dose Orthotopic (LO) model, both progressive skeletal and cardiac muscle wasting were detected in parallel to systemic inflammation; skeletal muscle atrophy at the fiber level was detected as early as 3 weeks post-implantation compared to controls (p<0.001). Gait speed in LO animals declined as early 2 week post-implantation whereas grip strength change was a late event and related to end of life. Principle component analysis (PCA) revealed distinct cachectic and non-cachectic animal populations, which we leveraged to show that gait speed decline was specific to cachexia (p<0.01) while grip strength decline was not (p=0.19). These data paralleled our observations in cancer patients with cachexia who required rehabilitation. In cachectic patients (identified by WLGS, Fearon criteria, or PNI, change in 6MWT correlated with motor FIM score changes while hGS did not (r 2 =0.18, p<0.001). This relationship between 6MWT and FIM in cachectic patients was further confirmed through multivariate regression (r 2 =0.30, p<0.001) controlling for age and cancer burden. Conclusion: Outcome measures linked to gait are better associated with cachexia related function and preferred for future pre-clinical and clinical cachexia studies.

The effect of fatty infiltration, revision surgery, and sex on lumbar multifidus passive mechanical properties.

Purpose: Previous research has demonstrated increased stiffness in the multifidus muscle compared to other paraspinal muscles at the fiber bundle level. We aimed to compare single fiber and fiber bundle passive mechanical properties of multifidus muscle: (1) in 40 patients undergoing primary versus revision surgery and (2) in muscle with mild versus severe fatty infiltration. Methods: The degree of muscle fatty infiltration was graded using the patients' spine magnetic resonance images. Average single fiber and fiber bundle passive mechanical properties across three tests were compared between primary (N = 30) and revision (N = 10) surgery status, between mild and severe fatty infiltration levels, between sexes, and with age from passive stress-strain tests of excised multifidus muscle intraoperative biopsies. Results: At the single fiber level, elastic modulus was unaffected by degree of fatty infiltration or surgery status. Female sex (p = 0.001) and younger age (p = 0.04) were associated with lower multifidus fiber elastic modulus. At the fiber bundle level, which includes connective tissue around fibers, severe fatty infiltration (p = 0.01) and younger age (p = 0.06) were associated with lower elastic modulus. Primary surgery also demonstrated a moderate, but non-significant effect for lower elastic modulus (p = 0.10). Conclusions: Our results demonstrate that female sex is the primary driver for reduced single fiber elastic modulus of the multifidus, while severity of fatty infiltration is the primary driver for reduced elastic modulus at the level of the fiber bundle in individuals with lumbar spine pathology.

Optimal Distal Tendon Insertion Point for Elbow Flexion in Free-Functioning Gracilis Muscle Transfer for Panbrachial Plexus Injuries: A Cadaveric Study.

PURPOSE: Following pan-brachial plexus injuries, restoration of elbow flexion is widely accepted as the reconstructive priority. A gracilis free functioning muscle transfer (FFMT) can be used to restore elbow flexion alone with insertion into the biceps brachii (BIC) or brachioradialis (BRD) tendons or restore combined elbow and finger flexion with a more distal insertion into the flexor digitorum profundus (FDP) tendons. Using cadaveric experiments, we determined the peak instantaneous moment arm for each insertion option. METHODS: Six simulated gracilis transfer surgeries were performed using both arms of three fresh-frozen full body cadaveric specimens (age: 79 + 10 years. 2 female). The gracilis muscles from both legs were harvested and transferred to the contralateral upper extremity. The elbow was manually moved through three flexion-extension cycles while the instantaneous moment arm was calculated from measurements of gracilis excursion and elbow joint angle for the three distal insertion sites. RESULTS: Peak instantaneous moment arm for all three insertions occurred at an elbow angle between 83° to 92° with a magnitude ranging from 33 mm to 54 mm. The more distal (FDP/BRD) insertions produced a significantly greater (∼1.5 times) peak elbow flexion instantaneous moment arm compared to the BIC insertion. CONCLUSIONS: Based on the instantaneous moment arm, the gracilis FFMT distal insertion locations could result in greater reconstructed elbow flexion strength. In addition, direct measurement of the shape and magnitude of the moment arm curve for differing insertion sites allows high resolution surgical planning and model testing. CLINICAL RELEVANCE: This study presents the first direct experimental quantification of the gracilis FFMT instantaneous moment arm. The experimental evidence supports the use of FDP/BRD insertion locations by providing a quantitative explanation for the increased elbow flexion torque observed clinically in patients with a gracilis FFMT and distal FDP insertion.

Direct intraoperative measurement of isometric contractile properties in living human muscle.

Skeletal muscle's isometric contractile properties are one of the classic structure-function relationships in all of biology allowing for extrapolation of single fibre mechanical properties to whole muscle properties based on the muscle's optimal fibre length and physiological cross-sectional area (PCSA). However, this relationship has only been validated in small animals and then extrapolated to human muscles, which are much larger in terms of length and PCSA. The present study aimed to measure directly the in situ properties and function of the human gracilis muscle to validate this relationship. We leveraged a unique surgical technique in which a human gracilis muscle is transferred from the thigh to the arm, restoring elbow flexion after brachial plexus injury. During this surgery, we directly measured subject specific gracilis muscle force-length relationship in situ and properties ex vivo. Each subject's optimal fibre length was calculated from their muscle's length-tension properties. Each subject's PCSA was calculated from their muscle volume and optimal fibre length. From these experimental data, we established a human muscle fibre-specific tension of 171 kPa. We also determined that average gracilis optimal fibre length is 12.9 cm. Using this subject-specific fibre length, we observed an excellent fit between experimental and theorical active length-tension curves. However, these fibre lengths were about half of the previously reported optimal fascicle lengths of 23 cm. Thus, the long gracilis muscle appears to be composed of relatively short fibres acting in parallel that may not have been appreciated based on traditional anatomical methods. KEY POINTS: Skeletal muscle's isometric contractile properties represent one of the classic structure-function relationships in all of biology and allow scaling single fibre mechanical properties to whole muscle properties based on the muscle's architecture. This physiological relationship has only been validated in small animals but is often extrapolated to human muscles, which are orders of magnitude larger. We leverage a unique surgical technique in which a human gracilis muscle is transplanted from the thigh to the arm to restore elbow flexion after brachial plexus injury, aiming to directly measure muscles properties in situ and test directly the architectural scaling predictions. Using these direct measurements, we establish human muscle fibre-specific tension of ∼170 kPa. Furthermore, we show that the gracilis muscle actually functions as a muscle with relatively short fibres acting in parallel vs. long fibres as previously assumed based on traditional anatomical models.

There Must Be a Better Way.

ABSTRACT: The National Institutes of Health (NIH) R01 grant represents the gold standard for research independence across the United States. The barrier to entry for this grant mechanism is extremely high, with <10% of grants being funded. Interestingly, after this tremendously high barrier, there is very little accountability for the grantee to accomplish the aims that were originally proposed. While there is certainly value to requiring investigators to provide a logical and compelling research plan, excessive grant writing creates a large inefficiency. Thus, a "fail quickly" model, which is not without cost, should be considered. Such a model involves a much lower barrier to entry and much higher short-term accountability so that more aims can be tested in less time at lower cost.

Biomechanical Modeling of Brachialis-to-Wrist Extensor Muscle Transfer Function for Daily Activities in Tetraplegia.

We recently reported a novel case demonstrating the feasibility of a brachialis (BRA)-to-extensor carpi radialis brevis (ECRB) tendon transfer, but it is not yet known whether this transfer provides robust functional results across activities. The purpose of this study was to use biomechanical modeling to define the functional capacity of the BRA-to-ECRB tendon transfer in terms of enabling the performance of several activities of daily living. Methods: A model of the transferred BRA-ECRB muscle-tendon unit was developed to calculate isometric elbow and wrist joint torque as a function of elbow and wrist angles resulting from different BRA reattachment locations from 50 to 80 mm proximal to the wrist joint crease. Using this model, mathematical optimization predicted the optimal location for BRA reattachment in order to perform each of a number of important upper extremity tasks as well as to calculate a global optimum for performing all of the tasks. Results: Analysis of active joint torque showed that the entire elbow torque-angle curve surface shifted "diagonally" toward elbow flexion and wrist extension as the attachment location approached the wrist joint; peak wrist torque was produced at extended wrist angles. Our model predicted that the optimal attachment location for each different task ranged from 54.3 to 74.6 mm proximal to the wrist joint, which is feasible given the anatomy of the muscle-tendon unit. The attachment location to optimize performing all tasks was calculated as 63.5 mm proximal to the wrist joint. Conclusions: This study clearly demonstrates that the BRA, which is underused as a donor in tetraplegia surgery, is an excellent donor muscle to provide wrist extension. Biomechanical simulation further highlighted the need to consider not only donor-muscle appropriateness but the patient's desired function when planning surgical tendon transfers. Clinical Relevance: Quantitative evaluation of the way that surgery affects daily tasks rather than simply matching muscle properties may be a more appropriate approach for surgeons to use when choosing and tensioning donor muscles.

An estrogen-sensitive fibroblast population drives abdominal muscle fibrosis in an inguinal hernia mouse model.

Greater than 25% of all men develop an inguinal hernia in their lifetime, and more than 20 million inguinal hernia repair surgeries are performed worldwide each year. The mechanisms causing abdominal muscle weakness, the formation of inguinal hernias, or their recurrence are largely unknown. We previously reported that excessively produced estrogen in the lower abdominal muscles (LAMs) triggers extensive LAM fibrosis, leading to hernia formation in a transgenic male mouse model expressing the human aromatase gene (Aromhum). To understand the cellular basis of estrogen-driven muscle fibrosis, we performed single-cell RNA sequencing on LAM tissue from Aromhum and wild-type littermates. We found a fibroblast-like cell group composed of 6 clusters, 2 of which were validated for their enrichment in Aromhum LAM tissue. One of the potentially novel hernia-associated fibroblast clusters in Aromhum was enriched for the estrogen receptor-α gene (Esr1hi). Esr1hi fibroblasts maximally expressed estrogen target genes and seemed to serve as the progenitors of another cluster expressing ECM-altering enzymes (Mmp3hi) and to upregulate expression of proinflammatory, profibrotic genes. The discovery of these 2 potentially novel and unique hernia-associated fibroblasts may lead to the development of novel treatments that can nonsurgically prevent or reverse inguinal hernias.

Procedures for obtaining muscle physiology parameters during a gracilis free-functioning muscle transfer in adult patients with brachial plexus injury.

A complete understanding of muscle mechanics allows for the creation of models that closely mimic human muscle function so they can be used to study human locomotion and evaluate surgical intervention. This includes knowledge of muscle-tendon parameters required for accurate prediction of muscle forces. However, few studies report experimental data obtained directly from whole human muscle due to the invasive nature of these experiments. This article presents an intraoperative, in vivo measurement protocol for whole muscle-tendon parameters that include muscle-tendon unit length, sarcomere length, passive tension, and active tension in response to external stimulation. The advantage of this protocol is the ability to obtain these rare experimental data in situ in addition to muscle volume and weight since the gracilis is also completely removed from the leg. The entire protocol including the surgical steps for gracilis harvest takes ~ 3 h. Actual testing of the gracilis where experimental data is measured takes place within a 30-min window during surgery.

Teamwork Pays! Ten Tips for a Great Surgeon-Scientist Collaboration.

This review represents our summary of what makes a great collaboration between a surgeon and a scientist. At first, with no perspective, such a collaboration seems easy and natural. But as time goes on, with more perspective, you realize how special it is. Now, in our 60s, with approximately 35 years of collaboration and 75 coauthored papers (most of them in The Journal of Hand Surgery), we are thankful and humbled for this tremendously fruitful and, importantly, enjoyable collaboration. We are not so foolish to think that we made this great collaboration-it was a gift. However, we now recognize many characteristics that make it great and have developed the following 10 tips.

In vivo human gracilis whole-muscle passive stress-sarcomere strain relationship.

We measured the passive mechanical properties of intact, living human gracilis muscles (n=11 individuals, 10 male and 1 female, age: 33±12 years, mass: 89±23 kg, height: 177±8 cm). Measurements were performed in patients undergoing surgery for free-functioning myocutaneous tissue transfer of the gracilis muscle to restore elbow flexion after brachial plexus injury. Whole-muscle force of the gracilis tendon was measured in four joint configurations (JC1-JC4) with a buckle force transducer placed at the distal tendon. Sarcomere length was also measured by biopsy from the proximal gracilis muscle. After the muscle was removed, a three-dimensional volumetric reconstruction of the muscle was created via photogrammetry. Muscle length from JC1 to JC4 increased by 3.3±1.0, 7.7±1.2, 10.5±1.3 and 13.4±1.2 cm, respectively, corresponding to 15%, 34%, 46% and 59% muscle fiber strain, respectively. Muscle volume and an average optimal fiber length of 23.1±0.7 cm yielded an average muscle physiological cross-sectional area of 6.8±0.7 cm2 which is approximately 3 times that measured previously from cadaveric specimens. Absolute passive tension increased from 0.90±0.21 N in JC1 to 16.50±2.64 N in JC4. As expected, sarcomere length also increased from 3.24±0.08 µm at JC1 to 3.63±0.07 µm at JC4, which are on the descending limb of the human sarcomere length-tension curve. Peak passive muscle stress was 27.8±5.5 kPa in JC4 and muscle modulus ranged from 44.8 MPa in JC1 to 125.7 MPa in JC4. Comparison with other mammalian species indicates that human muscle passive mechanical properties are more similar to rodent muscle than to rabbit muscle. These data provide direct measurements of whole-human muscle passive mechanical properties that can be used in modeling studies and for understanding comparative passive mechanical properties among mammalian muscles.

Outcome from a brachialis donor for wrist extension in tetraplegia-time to reconsider the International Classification for Surgery of the Hand in Tetraplegia (ICSHT).

INTRODUCTION: Surgical reconstruction after quadriplegia represents a powerful solution to restore lost function by injury. A case is presented in which surgical reconstruction of a patient with a C4 level spinal cord injury is performed using the brachialis (BRA) muscle as the donor. CASE PRESENTATION: The patient previously had no hand function. This transfer, in combination with fusion of the thumb CMC joint and transfer of the flexor pollicis longus (FPL) tendon to the radius, gives the patient full thumb key pinch powered by BRA transferred to the wrist extensors. Theoretical analysis of muscle architectural properties demonstrates that the BRA has sufficient force and excursion to substitute for both the long and short radial wrist extensors. Furthermore, based on the fact that the BRA has almost twice the excursion compared to the extensor carpi radialis longus (ECRL), wrist extension can occur throughout the entire wrist and elbow ranges of motion. Finally, peak tension is lower than the rupture tension previously measured by us using this type of tendon-to-tendon attachment technique, suggesting that the transfer itself is safe and, importantly, can be immediately mobilized for neuromuscular rehabilitation. DISCUSSION: This procedure can thus restore tremendous functional capacity in patients who were previously categorized as group 0 by the International Classification of Hand Surgery in Tetraplegia (ICSHT). We suggest that, based on the BRA being an excellent donor for surgical reconstruction, that the ICHST system be reconsidered.

Stretch-induced satellite cell deformation incontracturedmuscles in children with cerebral palsy.

Satellite cells (SCs) are quiescent, adult skeletal muscle stem cells responsible for postnatal muscle growth and repair. Children with cerebral palsy (CP) have muscle contractures with reduced SC abundance, extracellular matrix abnormalities and reduced serial sarcomere number resulting in greatly increased in vivo sarcomere length, perhaps due to impaired sarcomere addition, compared to children with typical development (TD). Stretch is a strong activator of SCs that leads to addition of sarcomeres during bone-muscle growth. Mechanical loading and subsequent deformation of intracellular structures can lead to activation and proliferation, perhaps by cytoskeletal transmissions of extracellular mechanical signals to the nuclei. The primary aim of this study was to determine the effect of ex vivo stretch-induced sarcomere length change on SC deformation in children with CP and TD. Muscle biopsies were obtained from twelve children (7 CP, 5 TD) during surgery. Fiber bundles were labeled with fluorescent antibodies for Pax7 (SC), DRAQ5 (nuclei), and alpha-actinin (sarcomere protein). Fibers were stretched using a custom jig and imaged using confocal microscopy. SC nuclear length, height and aspect ratio underwent increased deformation with increasing sarcomere length (p < 0.05) in both groups. Slopes of association for SC nuclear length, aspect ratio and sarcomere lengths were similar between CP and TD. Our results indicate that SC in children with CP undergo similar deformation as TD across sarcomere lengths.

Muscle-tendon unit in children with cerebral palsy.

Muscle-tendon unit surgery for correction of deformities and movement dysfunction in children with cerebral palsy (CP) is fairly complicated. An understanding of basic muscle-tendon unit properties and their adaptation to both CP and surgery are important to develop advances in this field. In this review, we provide information to therapists, surgeons, and scientists regarding the short- and long-term adaptations of the muscle-tendon unit. Surgical releases, lengthening, and transpositions are discussed, as are some of the tissue, cellular, and molecular adaptations. What this paper adds Muscle strength, tone, and control must be considered in surgical interventions for cerebral palsy (CP). Muscle-tendon unit lengthening causes significant and lasting weakness requiring prolonged rehabilitation. Sarcomere length increases in CP muscle may be one of the underlying causes of muscle weakness. Muscle satellite cells are decreased and epigenetically modified in a way that may limit muscle growth in CP.

Skeletal muscle maximal mitochondrial activity in ambulatory children with cerebral palsy.

AIM: To compare skeletal muscle mitochondrial enzyme activity and mitochondrial content between independently ambulatory children with cerebral palsy (CP) and typically developing children. METHOD: Gracilis biopsies were obtained from 12 children during surgery (n=6/group, children with CP: one female, five males, mean age 13y 4mo, SD 5y 1mo, 4y 1mo-17y 10mo; typically developing children: three females, three males, mean age 16y 5mo, SD 1y 4mo, 14y 6mo-18y 2mo). Spectrophotometric enzymatic assays were used to evaluate the activity of mitochondrial electron transport chain complexes. Mitochondrial content was evaluated using citrate synthase assay, mitochondrial DNA copy number, and immunoblots for specific respiratory chain proteins. RESULTS: Maximal enzyme activity was significantly (50-80%) lower in children with CP versus typically developing children, for complex I (11nmol/min/mg protein, standard error of the mean [SEM] 1.7 vs 20.7nmol/min/mg protein, SEM 4), complex II (6.9nmol/min/mg protein, SEM 1.2 vs 21nmol/min/mg protein, SEM 2.7), complex III (31.9nmol/min/mg protein, SEM 7.4 vs 72.7nmol/min/mg protein, SEM 7.2), and complex I+III (7.4nmol/min/mg protein, SEM 2.5 vs 31.8nmol/min/mg protein, SEM 9.3). Decreased electron transport chain activity was not the result of lower mitochondrial content. INTERPRETATION: Skeletal muscle mitochondrial electron transport chain enzymatic activity but not mitochondrial content is reduced in independently ambulatory children with CP. Decreased mitochondrial oxidative capacity might explain reported increased energetics of movement and fatigue in ambulatory children with CP. What this paper adds Skeletal muscle mitochondrial electron transport chain enzymatic activity is reduced in independently ambulatory children with cerebral palsy (CP). Mitochondrial content appears to be similar between children with CP and typically developing children.

The Lumbricals Are Not the Workhorse of Digital Extension and Do Not Relax Their Own Antagonist.

That the lumbrical muscles are the workhorse of digital extension and that they can relax their own antagonist have been time-honored principles. However, we believe this dogma is incorrect and an oversimplification. We base our assertion on anatomy, innervation, and the notion that muscle architecture is the most important determinant of muscle function. Wang and colleagues proposed the lumbrical to be a sophisticated tension monitoring device. We elaborate on their well-supported thesis, further proposing that the lumbricals also function as a constant tension spring within the closed loop composed of the digital flexors and the extensor mechanism.

Surgical Mobilization of Skeletal Muscles Changes Functional Properties-Implications for Tendon Transfers.

PURPOSE: Tendon transfer surgery restores function by rerouting working muscle-tendon units to replace the function of injured or paralyzed muscles. This procedure requires mobilizing a donor muscle relative to its surrounding myofascial connections, which improves the muscle's new line of action and increases excursion. However, the biomechanical effect of mobilization on a donor muscle's force-generating function has not been previously studied under in vivo conditions. The purpose of this study was to quantify the effect of surgical mobilization on active and passive biomechanical properties of 3 large rabbit hind limb muscles. METHODS: Myofascial connections were mobilized stepwise from the distal end to the proximal end of muscles (0%, 25%, 50%, and 75% of muscle length) and their active and passive length-tension curves were measured after each degree of mobilization. RESULTS: Second toe extensor, a short-fibered muscle, exhibited a 30% decline in peak stress and 70% decline in passive stress, whereas extensor digitorum longus, a short-fibered muscle, and tibialis anterior, a long-fibered muscle, both exhibited similar smaller declines in active (about 18%) and passive stress (about 65%). CONCLUSIONS: The results highlight 3 important points: (1) a trade-off exists between increasing muscle mobility and decreasing force-generating capacity; (2) intermuscular force transmission is important, especially in second toe extensor, because it was able to generate 70% of its premobilization active force although most fibers were freed from their native origin; and (3) muscle architecture is not the major influence on mobilization-induced force impairment. CLINICAL RELEVANCE: These data demonstrate that surgical mobilization itself alters the passive and active force-generating capacity of skeletal muscles. Thus, surgical mobilization should not be viewed simply as a method to redirect the line of action of a donor muscle because this procedure has an impact on the functional properties of the donor muscle itself.

Vitamin D ameliorates adipose browning in chronic kidney disease cachexia.

Patients with chronic kidney disease (CKD) are often 25(OH)D3 and 1,25(OH)2D3 insufficient. We studied whether vitamin D repletion could correct aberrant adipose tissue and muscle metabolism in a mouse model of CKD-associated cachexia. Intraperitoneal administration of 25(OH)D3 and 1,25(OH)2D3 (75 µg/kg/day and 60 ng/kg/day respectively for 6 weeks) normalized serum concentrations of 25(OH)D3 and 1,25(OH)2D3 in CKD mice. Vitamin D repletion stimulated appetite, normalized weight gain, and improved fat and lean mass content in CKD mice. Vitamin D supplementation attenuated expression of key molecules involved in adipose tissue browning and ameliorated expression of thermogenic genes in adipose tissue and skeletal muscle in CKD mice. Furthermore, repletion of vitamin D improved skeletal muscle fiber size and in vivo muscle function, normalized muscle collagen content and attenuated muscle fat infiltration as well as pathogenetic molecular pathways related to muscle mass regulation in CKD mice. RNAseq analysis was performed on the gastrocnemius muscle. Ingenuity Pathway Analysis revealed that the top 12 differentially expressed genes in CKD were correlated with impaired muscle and neuron regeneration, enhanced muscle thermogenesis and fibrosis. Importantly, vitamin D repletion normalized the expression of those 12 genes in CKD mice. Vitamin D repletion may be an effective therapeutic strategy for adipose tissue browning and muscle wasting in CKD patients.

Vitamin D repletion ameliorates adipose tissue browning and muscle wasting in infantile nephropathic cystinosis-associated cachexia.

BACKGROUND: Ctns-/- mice, a mouse model of infantile nephropathic cystinosis, exhibit hypermetabolism with adipose tissue browning and profound muscle wasting. Ctns-/- mice are 25(OH)D3 and 1,25(OH)2 D3 insufficient. We investigated whether vitamin D repletion could ameliorate adipose tissue browning and muscle wasting in Ctns-/- mice. METHODS: Twelve-month-old Ctns-/- mice and wild-type controls were treated with 25(OH)D3 and 1,25(OH)2 D3 (75 µg/kg/day and 60 ng/kg/day, respectively) or an ethylene glycol vehicle for 6 weeks. Serum chemistry and parameters of energy homeostasis were measured. We quantitated total fat mass and studied expression of molecules regulating adipose tissue browning, energy metabolism, and inflammation. We measured lean mass content, skeletal muscle fibre size, in vivo muscle function (grip strength and rotarod activity), and expression of molecules regulating muscle metabolism. We also analysed the transcriptome of skeletal muscle in Ctns-/- mice using RNAseq. RESULTS: Supplementation of 25(OH)D3 and 1,25(OH)2 D3 normalized serum concentration of 25(OH)D3 and 1,25(OH)2 D3 in Ctns-/- mice, respectively. Repletion of vitamin D partially or fully normalized food intake, weight gain, gain of fat, and lean mass, improved energy homeostasis, and attenuated perturbations of uncoupling proteins and adenosine triphosphate content in adipose tissue and muscle in Ctns-/- mice. Vitamin D repletion attenuated elevated expression of beige adipose cell biomarkers (UCP-1, CD137, Tmem26, and Tbx1) as well as aberrant expression of molecules implicated in adipose tissue browning (Cox2, Pgf2α, and NF-κB pathway) in inguinal white adipose tissue in Ctns-/- mice. Vitamin D repletion normalized skeletal muscle fibre size and improved in vivo muscle function in Ctns-/- mice. This was accompanied by correcting the increased muscle catabolic signalling (increased protein contents of IL-1ß, IL-6, and TNF-α as well as an increased gene expression of Murf-2, atrogin-1, and myostatin) and promoting the decreased muscle regeneration and myogenesis process (decreased gene expression of Igf1, Pax7, and MyoD) in skeletal muscles of Ctns-/- mice. Muscle RNAseq analysis revealed aberrant gene expression profiles associated with reduced muscle and neuron regeneration, increased energy metabolism, and fibrosis in Ctns-/- mice. Importantly, repletion of 25(OH)D3 and 1,25(OH)2 D3 normalized the top 20 differentially expressed genes in Ctns-/- mice. CONCLUSIONS: We report the novel findings that correction of 25(OH)D3 and 1,25(OH)2 D3 insufficiency reverses cachexia and may improve quality of life by restoring muscle function in an animal model of infantile nephropathic cystinosis. Mechanistically, vitamin D repletion attenuates adipose tissue browning and muscle wasting in Ctns-/- mice via multiple cellular and molecular mechanisms.