Combining electromyographic and electrical impedance data sets through machine learning: A study in D2-mdx and wild-type mice.

INTRODUCTION/AIMS: Needle impedance-electromyography (iEMG) assesses the active and passive electrical properties of muscles concurrently by using a novel needle with six electrodes, two for EMG and four for electrical impedance myography (EIM). Here, we assessed an approach for combining multifrequency EMG and EIM data via machine learning (ML) to discriminate D2-mdx muscular dystrophy and wild-type (WT) mouse skeletal muscle. METHODS: iEMG data were obtained from quadriceps of D2-mdx mice, a muscular dystrophy model, and WT animals. EIM data were collected with the animals under deep anesthesia and EMG data collected under light anesthesia, allowing for limited spontaneous movement. Fourier transformation was performed on the EMG data to provide power spectra that were sampled across the frequency range using three different approaches. Random forest-based, nested ML was applied to the EIM and EMG data sets separately and then together to assess healthy versus disease category classification using a nested cross-validation procedure. RESULTS: Data from 20 D2-mdx and 20 WT limbs were analyzed. EIM data fared better than EMG data in differentiating healthy from disease mice with 93.1% versus 75.6% accuracy, respectively. Combining EIM and EMG data sets yielded similar performance as EIM data alone with 92.2% accuracy. DISCUSSION: We have demonstrated an ML-based approach for combining EIM and EMG data obtained with an iEMG needle. While EIM-EMG in combination fared no better than EIM alone with this data set, the approach used here demonstrates a novel method of combining the two techniques to characterize the full electrical properties of skeletal muscle.

Design and pilot testing of a 26-gauge impedance-electromyography needle in wild-type and ALS mice.

INTRODUCTION/AIMS: Needle impedance-electromyography (iEMG) is a diagnostic modality currently under development that combines intramuscular electrical impedance with concentric electromyography (EMG) in a single needle. We designed, manufactured, and tested a prototype iEMG needle in a cohort of wild-type (WT) and SOD1G93A amyotrophic lateral sclerosis (ALS) mice to assess its ability to record impedance and EMG data. METHODS: A new six-electrode, 26-gauge, iEMG needle was designed, manufactured and tested. Quantitative impedance and qualitative "gestalt" EMG were performed sequentially on bilateral quadriceps of 16-wk-old SOD1G93A ALS (N = 6) and WT (N = 6) mice by connecting the needle first to an impedance analyzer (with the animal at rest) and then to a standard EMG system (with the animal fully under anesthesia to measure spontaneous activity and briefly during awakening to measure voluntary activity). The needle remained in the muscle throughout the measurement period. RESULTS: EMG data were qualitatively similar to that observed with a commercially available concentric EMG needle; fibrillation potentials were observed in 84% of the ALS mice and none of the WT mice; motor unit potentials were also readily identified. Impedance data revealed significant differences in resistance, reactance, and phase values between the two groups, with ALS animals having reduced reactance and resistance values. DISCUSSION: This work demonstrates the feasibility of a single iEMG needle conforming to standard dimensions of size and function. Further progress of iEMG technology for enhanced neuromuscular diagnosis and quantification of disease status is currently in development.

Using machine learning algorithms to enhance the diagnostic performance of electrical impedance myography.

INTRODUCTION/AIMS: We assessed the classification performance of machine learning (ML) using multifrequency electrical impedance myography (EIM) values to improve upon diagnostic outcomes as compared to those based on a single EIM value. METHODS: EIM data was obtained from unilateral excised gastrocnemius in eighty diseased mice (26 D2-mdx, Duchenne muscular dystrophy model, 39 SOD1G93A ALS model, and 15 db/db, a model of obesity-induced muscle atrophy) and 33 wild-type (WT) animals. We assessed the classification performance of a ML random forest algorithm incorporating all the data (multifrequency resistance, reactance and phase values) comparing it to the 50 kHz phase value alone. RESULTS: ML outperformed the 50 kHz analysis as based on receiver-operating characteristic curves and measurement of the area under the curve (AUC). For example, comparing all diseases together versus WT from the test set outputs, the AUC was 0.52 for 50 kHz phase, but was 0.94 for the ML model. Similarly, when comparing ALS versus WT, the AUCs were 0.79 for 50 kHz phase and 0.99 for ML. DISCUSSION: Multifrequency EIM using ML improves upon classification compared to that achieved with a single-frequency value. ML approaches should be considered in all future basic and clinical diagnostic applications of EIM.

Estimating myofiber cross-sectional area and connective tissue deposition with electrical impedance myography: A study in D2-mdx mice.

INTRODUCTION: Surface electrical impedance myography (sEIM) has the potential for providing information on muscle composition and structure noninvasively. We sought to evaluate its use to predict myofiber size and connective tissue deposition in the D2-mdx model of Duchenne muscular dystrophy (DMD). METHODS: We applied a prediction algorithm, the least absolute shrinkage and selection operator, to select specific EIM measurements obtained with surface and ex vivo EIM data from D2-mdx and wild-type (WT) mice (analyzed together or separately). We assessed myofiber cross-sectional area histologically and hydroxyproline (HP), a surrogate measure for connective tissue content, biochemically. RESULTS: Using WT and D2-mdx impedance values together in the algorithm, sEIM gave average root-mean-square errors (RMSEs) of 26.6% for CSA and 45.8% for HP, which translate into mean errors of ±363 µm2 for a mean CSA of 1365 µm2 and of ±1.44 µg HP/mg muscle for a mean HP content of 3.15 µg HP/mg muscle. Stronger predictions were obtained by analyzing sEIM data from D2-mdx animals alone (RMSEs of 15.3% for CSA and 34.1% for HP content). Predictions made using ex vivo EIM data from D2-mdx animals alone were nearly equivalent to those obtained with sEIM data (RMSE of 16.59% for CSA), and slightly more accurate for HP (RMSE of 26.7%). DISCUSSION: Surface EIM combined with a predictive algorithm can provide estimates of muscle pathology comparable to values obtained using ex vivo EIM, and can be used as a surrogate measure of disease severity and progression and response to therapy.

Predicting myofiber cross-sectional area and triglyceride content with electrical impedance myography: A study in db/db mice.

BACKGROUND: Electrical impedance myography (EIM) provides insight into muscle composition and structure. We sought to evaluate its use in a mouse obesity model characterized by myofiber atrophy. METHODS: We applied a prediction algorithm, ie, the least absolute shrinkage and selection operator (LASSO), to surface, needle array, and ex vivo EIM data from db/db and wild-type mice and assessed myofiber cross-sectional area (CSA) histologically and triglyceride (TG) content biochemically. RESULTS: EIM data from all three modalities provided acceptable predictions of myofiber CSA with average root mean square error (RMSE) of 15% in CSA (ie, ±209 µm2 for a mean CSA of 1439 µm2 ) and TG content with RMSE of 30% in TG content (ie, ±7.3 nmol TG/mg muscle for a mean TG content of 25.4 nmol TG/mg muscle). CONCLUSIONS: EIM combined with a predictive algorithm provides reasonable estimates of myofiber CSA and TG content without the need for biopsy.

Altered muscle electrical tissue properties in a mouse model of premature aging.

INTRODUCTION: Improved methods are needed to detect and quantify age-related muscle change. In this study we assessed the electrical properties of muscle impacted by acquired mitochondrial DNA mutations via the PolG mouse, which exhibits typical age-associated features, and the impact of a potential therapy, nicotinamide mononucleotide (NMN). METHODS: The gastrocnemii of 24 PolG and 30 wild-type (WT) mice (8 PolG and 17 WT treated with NMN) were studied in an electrical impedance-measuring cell. Conductivity and relative permittivity were determined from the impedance data. Myofiber cross-sectional area (CSA) was quantified histologically. RESULTS: Untreated PolG mice demonstrated alterations in several impedance features, including 50-kHz relative permittivity and center frequency. A potential effect of NMN was also observed in these parameters in PolG but not WT animals. Impedance values correlated with myofiber CSA. DISCUSSION: Electrical impedance is sensitive to myofiber features considered characteristic of aging and to the impact of a potential therapy.

Early Actions of Anti-Vascular Endothelial Growth Factor/Vascular Endothelial Growth Factor Receptor Drugs on Angiogenic Blood Vessels.

Tumors induce their heterogeneous vasculature by secreting vascular endothelial growth factor (VEGF)-A. Anti-VEGF/VEGF receptor (VEGFR) drugs treat cancer, but the underlying mechanisms remain unclear. An adenovirus expressing VEGF-A (Ad-VEGF-A164) replicates the tumor vasculature in mice without tumor cells. Mother vessels (MV) are the first angiogenic vessel type to form in tumors and after Ad-VEGF-A164. Multiday treatments with a VEGF trap reverted MV back to normal microvessels. We now show that, within hours, a single dose of several anti-VEGF drugs collapsed MV to form glomeruloid microvascular proliferations (GMP), accompanied by only modest endothelial cell death. GMP, common in many human cancers but of uncertain origin, served as an intermediary step in MV reversion to normal microvessels. The vasodisruptive drug combretastatin CA4 also targeted MV selectively but acted differently, extensively killing MV endothelium. Antivascular changes were quantified with a novel Evans blue dye assay that measured vascular volumes. As in tumors, Ad-VEGF-A164 strikingly increased endothelial nitric oxide synthase (eNOS) expression. The eNOS inhibitor N(G)-Nitro-l-arginine methyl ester mimicked anti-VEGF/VEGFR drugs, rapidly collapsing MV to GMP. Inhibition of eNOS reduces synthesis of its vasodilatory product, nitric oxide, leading to arterial contraction. Patients and mice receiving anti-VEGF/VEGFR drugs develop hypertension, reflecting systemic arterial contraction. Together, anti-VEGF/VEGFR drugs act in part by inhibiting eNOS, causing vasocontraction, MV collapse to GMP, and subsequent reversion of GMP to normal microvessels, all without extensive vascular killing.

Estimating Myofiber Size With Electrical Impedance Myography: a Study In Amyotrophic Lateral Sclerosis MICE.

INTRODUCTION: A method for quantifying myofiber size noninvasively would find wide use, including primary diagnosis and evaluating response to therapy. METHODS: Using prediction algorithms, including the least absolute shrinkage and selection operator, we applied multifrequency electrical impedance myography (EIM) to amyotrophic lateral sclerosis superoxide dismutase 1 G93A mice of different ages and assessed myofiber size histologically. RESULTS: Multifrequency EIM data provided highly accurate predictions of myofiber size, with a root mean squared error (RMSE) of only 14% in mean myofiber area (corresponding to ± 207 µm2 for a mean area of 1,488 µm2 ) and an RMSE of only 8.8% in predicting the coefficient of variation in fiber size distribution. DISCUSSION: This impedance-based approach provides predictive variables to assess myofiber size and distribution with good accuracy, particularly in diseases in which myofiber atrophy is the predominant histological feature, without the requirement for biopsy or burdensome quantification. Muscle Nerve 58: 713-717, 2018.

Predicting myofiber size with electrical impedance myography: A study in immature mice.

INTRODUCTION: Electrical impedance can be used to estimate cellular characteristics. We sought to determine whether it could be used to approximate myofiber size using standard prediction modeling approaches. METHODS: Forty-four C57BL/6J wild-type immature mice of varying ages underwent electrical impedance myography (EIM) with a needle electrode array placed in the gastrocnemius. Animals were then humanely killed and muscle fixed, stained, and myofiber size quantified. Two different statistical prediction models were then applied. RESULTS: Impedance parameters showed major variation with increasing myofiber size. The prediction models based on EIM data alone were able to predict fiber size, with errors in the range of ±69.05-78.44 µm2 (16.19%-18.40% with respect to the average myofiber size). DISCUSSION: By using well-established statistical models, EIM data alone can provide a satisfactory estimate of myofiber size. Additional study of this approach for approximating myofiber size without the requirement of removing tissue for histological analysis is warranted. Muscle Nerve, 2018.

PGC-1α induces SPP1 to activate macrophages and orchestrate functional angiogenesis in skeletal muscle.

RATIONALE: Mechanisms of angiogenesis in skeletal muscle remain poorly understood. Efforts to induce physiological angiogenesis hold promise for the treatment of diabetic microvascular disease and peripheral artery disease but are hindered by the complexity of physiological angiogenesis and by the poor angiogenic response of aged and patients with diabetes mellitus. To date, the best therapy for diabetic vascular disease remains exercise, often a challenging option for patients with leg pain. Peroxisome proliferation activator receptor-γ coactivator-1α (PGC-1α), a powerful regulator of metabolism, mediates exercise-induced angiogenesis in skeletal muscle. OBJECTIVE: To test whether, and how, PGC-1α can induce functional angiogenesis in adult skeletal muscle. METHODS AND RESULTS: Here, we show that muscle PGC-1α robustly induces functional angiogenesis in adult, aged, and diabetic mice. The process involves the orchestration of numerous cell types and leads to patent, nonleaky, properly organized, and functional nascent vessels. These findings contrast sharply with the disorganized vasculature elicited by induction of vascular endothelial growth factor alone. Bioinformatic analyses revealed that PGC-1α induces the secretion of secreted phosphoprotein 1 and the recruitment of macrophages. Secreted phosphoprotein 1 stimulates macrophages to secrete monocyte chemoattractant protein-1, which then activates adjacent endothelial cells, pericytes, and smooth muscle cells. In contrast, induction of PGC-1α in secreted phosphoprotein 1(-/-) mice leads to immature capillarization and blunted arteriolarization. Finally, adenoviral delivery of PGC-1α into skeletal muscle of either young or old and diabetic mice improved the recovery of blood flow in the murine hindlimb ischemia model of peripheral artery disease. CONCLUSIONS: PGC-1α drives functional angiogenesis in skeletal muscle and likely recapitulates the complex physiological angiogenesis elicited by exercise.

Pathological angiogenesis is induced by sustained Akt signaling and inhibited by rapamycin.

Endothelial cells in growing tumors express activated Akt, which when modeled by transgenic endothelial expression of myrAkt1 was sufficient to recapitulate the abnormal structural and functional features of tumor blood vessels in nontumor tissues. Sustained endothelial Akt activation caused increased blood vessel size and generalized edema from chronic vascular permeability, while acute permeability in response to VEGF-A was unaffected. These changes were reversible, demonstrating an ongoing requirement for Akt signaling for the maintenance of these phenotypes. Furthermore, rapamycin inhibited endothelial Akt signaling, vascular changes from myrAkt1, tumor growth, and tumor vascular permeability. Akt signaling in the tumor vascular stroma was sensitive to rapamycin, suggesting that rapamycin may affect tumor growth in part by acting as a vascular Akt inhibitor.

Distinct vascular endothelial growth factor signals for lymphatic vessel enlargement and sprouting.

Lymphatic vessel growth, or lymphangiogenesis, is regulated by vascular endothelial growth factor-C (VEGF-C) and -D via VEGF receptor 3 (VEGFR-3). Recent studies suggest that VEGF, which does not bind to VEGFR-3, can also induce lymphangiogenesis through unknown mechanisms. To dissect the receptor pathway that triggers VEGFR-3-independent lymphangiogenesis, we used both transgenic and adenoviral overexpression of placenta growth factor (PlGF) and VEGF-E, which are specific activators of VEGFR-1 and -2, respectively. Unlike PlGF, VEGF-E induced circumferential lymphatic vessel hyperplasia, but essentially no new vessel sprouting, when transduced into mouse skin via adenoviral vectors. This effect was not inhibited by blocking VEGF-C and -D. Postnatal lymphatic hyperplasia, without increased density of lymphatic vessels, was also detected in transgenic mice expressing VEGF-E in the skin, but not in mice expressing PlGF. Surprisingly, VEGF-E induced lymphatic hyperplasia postnatally, and it did not rescue the loss of lymphatic vessels in transgenic embryos where VEGF-C and VEGF-D were blocked. Our data suggests that VEGFR-2 signals promote lymphatic vessel enlargement, but unlike in the blood vessels, are not involved in vessel sprouting to generate new lymphatic vessels in vivo.

Active Rac1 improves pathologic VEGF neovessel architecture and reduces vascular leak: mechanistic similarities with angiopoietin-1.

Architecturally defective, leaky blood vessels typify pathologic angiogenesis induced by vascular endothelial growth factor-A (VEGF-A). Such neovascular defects aggravate disease pathology and seriously compromise the therapeutic utility of VEGF. Endothelial cell (EC) transduction with active L61Rac1 strongly improved VEGF-driven angiogenesis in vivo as measured by increased neovascular density, enhanced lumen formation, and reduced vessel leakiness. Conversely, transduction with dominant-negative N17Rac1 strongly inhibited neovascularization. In vitro, active L61Rac1 promoted organization of cortical actin filaments and vascular cords and improved EC-EC junctions, indicating that improved cytoskeletal dynamics are important to the mechanism by which active L61Rac1 rectifies VEGF-driven angiogenesis. SEW2871, a sphingosine 1-phosphate receptor-1 agonist that activates Rac1 in ECs, improved cord formation and EC-EC junctions in vitro similarly to active L61Rac. Moreover, SEW2871 administration in vivo markedly improved VEGF neovessel architecture and reduced neovascular leak. Angiopoietin-1, a cytokine that "normalizes" VEGF neovessels in vivo, activated Rac1 and improved cord formation and EC-EC junctions in vitro comparably to active L61Rac1, and a specific Rac1 inhibitor blocked these effects. These studies distinguish augmentation of Rac1 activity as a means to rectify the pathologic angioarchitecture and dysfunctionality of VEGF neovessels, and they identify a rational pharmacologic strategy for improving VEGF angiogenesis.

RhoJ is an endothelial cell-restricted Rho GTPase that mediates vascular morphogenesis and is regulated by the transcription factor ERG.

ERG is a member of the ETS transcription factor family that is highly enriched in endothelial cells (ECs). To further define the role of ERG in regulating EC function, we evaluated the effect of ERG knock-down on EC lumen formation in 3D collagen matrices. Blockade of ERG using siRNA completely interferes with EC lumen formation. Quantitative PCR (QPCR) was used to identify potential downstream gene targets of ERG. In particular, we identified RhoJ as the Rho GTPase family member that is closely related to Cdc42 as a target of ERG. Knockdown of ERG expression in ECs led to a 75% reduction in the expression of RhoJ. Chromatin immunoprecipitation and transactivation studies demonstrated that ERG could bind to functional sites in the proximal promoter of the RhoJ gene. Knock-down of RhoJ similarly resulted in a marked reduction in the ability of ECs to form lumens. Suppression of either ERG or RhoJ during EC lumen formation was associated with a marked increase in RhoA activation and a decrease in Rac1 and Cdc42 activation and their downstream effectors. Finally, in contrast to other Rho GTPases, RhoJ exhibits a highly EC-restricted expression pattern in several different tissues, including the brain, heart, lung, and liver.

Inhibition of vessel permeability by TNP-470 and its polymer conjugate, caplostatin.

Angiogenesis inhibitors, such as TNP-470 and the nontoxic HPMA copolymer-TNP-470 (caplostatin), are emerging as a class of anticancer drugs. We report that TNP-470 and caplostatin inhibit vascular hyperpermeability of tumor blood vessels as well as that induced in mouse skin by different mediators. Treatment with TNP-470 or angiostatin for 3 days was sufficient to reduce permeability of tumor blood vessels, delayed-type hypersensitivity, and pulmonary edema induced by IL-2. TNP-470 also inhibited VPF/VEGF-induced phosphorylation of VEGFR-2, calcium influx, and RhoA activation in endothelial cells. These results identify an activity of TNP-470, that of inhibiting vessel hyperpermeability. This activity likely contributes to TNP-470's antiangiogenic effect and suggests that caplostatin can be used in the treatment of cancer and inflammation.

Electrical Impedance Myography Correlates with Functional Measures of Disease Progression in D2-mdx Mice and Boys with Duchenne Muscular Dystrophy.

BACKGROUND: Sensitive, objective, and longitudinal outcome measures applicable to both pre-clinical and clinical interventions are needed to assess muscle health in Duchenne muscular dystrophy (DMD). Electrical impedance myography (EIM) has the potential to non-invasively measure disease progression in mice and boys with DMD. OBJECTIVE: We sought to evaluate how electrical impedance values (i.e., phase, reactance, and resistance) correlate to established measures of disease in both D2-mdx and wild type (WT) mice and boys with and without DMD. METHODS: Histological, functional, and EIM data collected from previous studies of WT and D2-mdx mice at 6, 13, 21 and 43 weeks of age were reanalyzed. In parallel, previously collected functional outcome measures and EIM values were reanalyzed from boys with and without DMD at four different age groups from 2 to 14 years old. RESULTS: In mice, disease progression as detected by histological, functional, and EIM measures, was appreciable over this time period and grip strength best correlated to longitudinal phase and reactance impedance values. In boys, disease progression quantified through commonly utilized functional outcome measures was significant and longitudinal phase demonstrated the strongest correlation with functional outcome measures. CONCLUSION: Similar changes in EIM values, specifically in longitudinal reactance and phase, were found to show significant correlations to functional measures in both mice and boys. Thus, EIM demonstrates applicability in both pre-clinical and clinical settings and can be used as a safe, non-invasive, and longitudinal proxy biomarker to assess muscle health in DMD.

Novel phenotype characterization utilizing electrical impedance myography signatures in murine spinal cord injury neurogenic bladder models.

Neurogenic bladder (NB) affects people of all ages. Electric impedance myography (EIM) assesses localized muscle abnormalities. Here, we sought to investigate whether unique detrusor EIM signatures are present in NB due to spinal cord injury (SCI). Twenty-eight, 8-10 weeks old, C57BL/6J female mice were studied. Twenty underwent spinal cord transection; 8 served as controls. Cohorts were euthanized at 4 and 6 weeks after spinal cord transection. Each bladder was measured in-situ with EIM with applied frequencies of 1 kHz to 10 MHz, and then processed for molecular and histologic study. SCI mice had greater bladder-to-body weight ratio (p < 0.0001), greater collagen deposition (p = 0.009), and greater smooth-muscle-myosin-heavy-chain isoform A/B ratio (p < 0.0001). Compared with the control group, the SCI group was associated with lower phase, reactance, and resistance values (p < 0.01). Significant correlations (p < 0.001) between bladder-to-body weight ratios and EIM measurements were observed across the entire frequency spectrum. A severely hypertrophied phenotype was characterized by even greater bladder-to-body weight ratios and more depressed EIM values. Our study demonstrated distinct EIM alterations in the detrusor muscle of mice with NB due to SCI. With further refinement, EIM may offer a potential point-of-care tool for the assessment of NB and its response to treatment.

Electrical impedance myography for the early detection of muscle ischemia secondary to compartment syndrome: a study in a rat model.

Acute Compartment Syndrome (ACS) is one of the most devastating orthopedic conditions, affecting any of the body's many compartments, which, if sufficiently severe, may result in disability and amputation. Currently, intra-compartmental pressure measurements serve as the gold standard for diagnosing ACS. Diagnosing limbs at risk for ACS before irreversible damage to muscle and nerve is critical. Standard approaches for diagnosing impending compartment syndrome include clinical evaluation of the limb, such as assessment for "tightness" of the overlying skin, reduced pulses distally, and degree of pain, none of which are specific or sensitive. We have proposed a novel method to detect ACS via electrical impedance myography (EIM), where a weak, high-frequency alternating current is passed between one pair of electrodes through a region of tissue, and the resulting surface voltages are measured via a second pair. We evaluated the ability of EIM to detect early muscle ischemia in an established murine model of compression-induced muscle injury, where we collected resistance, reactance, and their dimensionless product, defined as Relative Injury Index (RII) during the study. Our model generated reproducible hypoxia, confirmed by Hypoxyprobe™ staining of endothelial regions within the muscle. Under conditions of ischemia, we demonstrated a reproducible, stable, and significant escalation in resistance, reactance, and RII values, compared to uninjured control limbs. These data make a reasonable argument for additional investigations into using EIM for the early recognition of muscle hypoperfusion and ischemia. However, these findings must be considered preliminary steps, requiring further pre-clinical and clinical validation.

Surface Electrical Impedance Myography Detects Skeletal Muscle Atrophy in Aged Wildtype Zebrafish and Aged gpr27 Knockout Zebrafish.

Throughout a vertebrate organism's lifespan, skeletal muscle mass and function progressively decline. This age-related condition is termed sarcopenia. In humans, sarcopenia is associated with risk of falling, cardiovascular disease, and all-cause mortality. As the world population ages, projected to reach 2 billion older adults worldwide in 2050, the economic burden on the healthcare system is also projected to increase considerably. Currently, there are no pharmacological treatments for sarcopenia, and given the long-term nature of aging studies, high-throughput chemical screens are impractical in mammalian models. Zebrafish is a promising, up-and-coming vertebrate model in the field of sarcopenia that could fill this gap. Here, we developed a surface electrical impedance myography (sEIM) platform to assess skeletal muscle health, quantitatively and noninvasively, in adult zebrafish (young, aged, and genetic mutant animals). In aged zebrafish (~85% lifespan) as compared to young zebrafish (~20% lifespan), sEIM parameters (2 kHz phase angle, 2 kHz reactance, and 2 kHz resistance) robustly detected muscle atrophy (p < 0.000001, q = 0.000002; p = 0.000004, q = 0.000006; p = 0.000867, q = 0.000683, respectively). Moreover, these same measurements exhibited strong correlations with an established morphometric parameter of muscle atrophy (myofiber cross-sectional area), as determined by histological-based morphometric analysis (r = 0.831, p = 2 × 10-12; r = 0.6959, p = 2 × 10-8; and r = 0.7220; p = 4 × 10-9, respectively). Finally, the genetic deletion of gpr27, an orphan G-protein coupled receptor (GPCR), exacerbated the atrophy of skeletal muscle in aged animals, as evidenced by both sEIM and histology. In conclusion, the data here show that surface EIM techniques can effectively discriminate between healthy young and sarcopenic aged muscle as well as the advanced atrophied muscle in the gpr27 KO animals. Moreover, these studies show how EIM values correlate with cell size across the animals, making it potentially possible to utilize sEIM as a "virtual biopsy" in zebrafish to noninvasively assess myofiber atrophy, a valuable measure for muscle and gerontology research.

Electrical impedance myography detects age-related skeletal muscle atrophy in adult zebrafish.

Age-related deficits in skeletal muscle function, termed sarcopenia, are due to loss of muscle mass and changes in the intrinsic mechanisms underlying contraction. Sarcopenia is associated with falls, functional decline, and mortality. Electrical impedance myography (EIM)-a minimally invasive, rapid electrophysiological tool-can be applied to animals and humans to monitor muscle health, thereby serving as a biomarker in both preclinical and clinical studies. EIM has been successfully employed in several species; however, the application of EIM to the assessment of zebrafish-a model organism amenable to high-throughput experimentation-has not been reported. Here, we demonstrated differences in EIM measures between the skeletal muscles of young (6 months of age) and aged (33 months of age) zebrafish. For example, EIM phase angle and reactance at 2 kHz showed significantly decreased phase angle (5.3 ± 2.1 versus 10.7 ± 1.5°; p = 0.001) and reactance (89.0 ± 3.9 versus 172.2 ± 54.8 ohms; p = 0.007) in aged versus young animals. Total muscle area, in addition to other morphometric features, was also strongly correlated to EIM 2 kHz phase angle across both groups (r = 0.7133, p = 0.01). Moreover, there was a strong correlation between 2 kHz phase angle and established metrics of zebrafish swimming performance, including turn angle, angular velocity, and lateral motion (r = 0.7253, r = 0.7308, r = 0.7857, respectively, p < 0.01 for all). In addition, the technique was shown to have high reproducibility between repeated measurements with a mean percentage difference of 5.34 ± 1.17% for phase angle. These relationships were also confirmed in a separate replication cohort. Together, these findings establish EIM as a fast, sensitive method for quantifying zebrafish muscle function and quality. Moreover, identifying the abnormalities in the bioelectrical properties of sarcopenic zebrafish provides new opportunities to evaluate potential therapeutics for age-related neuromuscular disorders and to interrogate the disease mechanisms of muscle degeneration.