Deletion of exons 2 and 3 from Actb and cell immortalization lead to widespread, ß-actin independent alterations in gene expression associated with cell cycle control.

The cytoplasmic actin proteins, ß- and γ-actin, are 99% identical but thought to perform non-redundant functions. The nucleotide coding regions of cytoplasmic actin genes, Actb and Actg1, are 89% identical. Knockout (KO) of Actb by Cre-mediated deletion of first coding exons 2 and 3 in mice is embryonic lethal and fibroblasts derived from KO embryos (MEFs) fail to proliferate. In contrast, Actg1 KO MEFs display with a much milder defect in cell proliferation and Actg1 KO mice are viable, but present with increased perinatal lethality. Recent studies have identified important protein-independent functions for both Actb and Actg1 and demonstrate that deletions within the Actb nucleotide sequence, and not loss of the ß-actin protein, cause the most severe phenotypes in KO mice and cells. Here, we use a multi-omics approach to better understand what drives the phenotypes of Actb KO MEFs. RNA-sequencing and mass spectrometry reveal largescale changes to the transcriptome, proteome, and phosphoproteome in cells lacking Actb but not those only lacking ß-actin protein. Pathway analysis of genes and proteins differentially expressed upon Actb KO suggest widespread dysregulation of genes involved in the cell cycle that may explain the severe defect in proliferation.

Nucleotide- and Protein-Dependent Functions of Actg1.

Cytoplasmic ß- and γ-actin proteins are 99% identical but support unique organismal functions. The cytoplasmic actin nucleotide sequences Actb and Actg1, respectively, are more divergent but still 89% similar. Actb-/- mice are embryonic lethal and Actb-/- cells fail to proliferate, but editing the Actb gene to express γ-actin (Actbc-g) resulted in none of the overt phenotypes of the knockout revealing protein-independent functions for Actb. To determine if Actg1 has a protein-independent function, we crossed Actbc-g and Actg1-/- mice to generate the bG/0 line, where the only cytoplasmic actin expressed is γ-actin from Actbc-g. The bG/0 mice were viable but showed a survival defect despite expressing γ-actin protein at levels no different from bG/gG with normal survival. A unique myopathy phenotype was also observed in bG/0 mice. We conclude that impaired survival and myopathy in bG/0 mice are due to loss of Actg1 nucleotide-dependent function(s). On the other hand, the bG/0 genotype rescued functions impaired by Actg1-/-, including cell proliferation and auditory function, suggesting a role for γ-actin protein in both fibroblasts and hearing. Together, these results identify nucleotide-dependent functions for Actg1 while implicating γ-actin protein in more cell-/tissue-specific functions.

Social stress is lethal in the mdx model of Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is caused by the loss of dystrophin. Severe and ultimately lethal, DMD progresses relatively slowly in that patients become wheelchair bound only around age twelve with a survival expectancy reaching the third decade of life. METHODS: The mildly-affected mdx mouse model of DMD, and transgenic DysΔMTB-mdx and Fiona-mdx mice expressing dystrophin or utrophin, respectively, were exposed to either mild (scruffing) or severe (subordination stress) stress paradigms and profiled for their behavioral and physiological responses. A subgroup of mdx mice exposed to subordination stress were pretreated with the beta-blocker metoprolol. FINDINGS: Subordination stress caused lethality in ∼30% of mdx mice within 24 h and ∼70% lethality within 48 h, which was not rescued by metoprolol. Lethality was associated with heart damage, waddling gait and hypo-locomotion, as well as marked up-regulation of the hypothalamus-pituitary-adrenocortical axis. A novel cardiovascular phenotype emerged in mdx mice, in that scruffing caused a transient drop in arterial pressure, while subordination stress caused severe and sustained hypotension with concurrent tachycardia. Transgenic expression of dystrophin or utrophin in skeletal muscle protected mdx mice from scruffing and social stress-induced responses including mortality. INTERPRETATION: We have identified a robust new stress phenotype in the otherwise mildly affected mdx mouse that suggests relatively benign handling may impact the outcome of behavioural experiments, but which should also expedite the knowledge-based therapy development for DMD. FUNDING: Greg Marzolf Jr. Foundation, Summer's Wish Fund, NIAMS, Muscular Dystrophy Association, University of Minnesota and John and Cheri Gunvalson Trust.

Mechanical factors tune the sensitivity of mdx muscle to eccentric strength loss and its protection by antioxidant and calcium modulators.

BACKGROUND: Dystrophin deficiency sensitizes skeletal muscle of mice to eccentric contraction (ECC)-induced strength loss. ECC protocols distinguish dystrophin-deficient from healthy, wild type muscle, and test the efficacy of therapeutics for Duchenne muscular dystrophy (DMD). However, given the large lab-to-lab variability in ECC-induced strength loss of dystrophin-deficient mouse skeletal muscle (10-95%), mechanical factors of the contraction likely impact the degree of loss. Therefore, the purpose of this study was to evaluate the extent to which mechanical variables impact sensitivity of dystrophin-deficient mouse skeletal muscle to ECC. METHODS: We completed ex vivo and in vivo muscle preparations of the dystrophin-deficient mdx mouse and designed ECC protocols within physiological ranges of contractile parameters (length change, velocity, contraction duration, and stimulation frequencies). To determine whether these contractile parameters affected known factors associated with ECC-induced strength loss, we measured sarcolemmal damage after ECC as well as strength loss in the presence of the antioxidant N-acetylcysteine (NAC) and small molecule calcium modulators that increase SERCA activity (DS-11966966 and CDN1163) or lower calcium leak from the ryanodine receptor (Chloroxine and Myricetin). RESULTS: The magnitude of length change, work, and stimulation duration ex vivo and in vivo of an ECC were the most important determinants of strength loss in mdx muscle. Passive lengthening and submaximal stimulations did not induce strength loss. We further showed that sarcolemmal permeability was associated with muscle length change, but it only accounted for a minimal fraction (21%) of the total strength loss (70%). The magnitude of length change also significantly influenced the degree to which NAC and small molecule calcium modulators protected against ECC-induced strength loss. CONCLUSIONS: These results indicate that ECC-induced strength loss of mdx skeletal muscle is dependent on the mechanical properties of the contraction and that mdx muscle is insensitive to ECC at submaximal stimulation frequencies. Rigorous design of ECC protocols is critical for effective use of strength loss as a readout in evaluating potential therapeutics for muscular dystrophy.

Variable cytoplasmic actin expression impacts the sensitivity of different dystrophin-deficient mdx skeletal muscles to eccentric contraction.

Eccentric contractions (ECCs) induce force loss in several skeletal muscles of dystrophin-deficient mice (mdx), with the exception of the soleus (Sol). The eccentric force : isometric force (ECC : ISO), expression level of utrophin, fiber type distribution, and sarcoendoplasmic reticulum calcium ATPase expression are factors that differ between muscles and may contribute to the sensitivity of mdx skeletal muscle to ECC. Here, we confirm that the Sol of mdx mice loses only 13% force compared to 87% in the extensor digitorum longus (EDL) following 10 ECC of isolated muscles. The Sol has a greater proportion of fibers expressing Type I myosin heavy chain (MHC) and expresses 2.3-fold more utrophin compared to the EDL. To examine the effect of ECC : ISO, we show that the mdx Sol is insensitive to ECC at ECC : ISO up to 230 ± 15%. We show that the peroneus longus (PL) muscle presents with similar ECC : ISO compared to the EDL, intermediate force loss (68%) following 10 ECC, and intermediate fiber type distribution and utrophin expression relative to EDL and Sol. The combined absence of utrophin and dystrophin in mdx/utrophin-/- mice rendered the Sol only partially susceptible to ECC and exacerbated force loss in the EDL and PL. Most interestingly, the expression levels of cytoplasmic ß- and γ-actins correlate inversely with a given muscle's sensitivity to ECC; EDL < PL < Sol. Our data indicate that fiber type, utrophin, and cytoplasmic actin expression all contribute to the differential sensitivities of mdxEDL, PL, and Sol muscles to ECC.

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.

Two-photon deep-tissue spatially resolved mitochondrial imaging using membrane potential fluorescence fluctuations.

Cell metabolism and viability are directly reflected in their mitochondria. Imaging-based analysis of mitochondrial morphological structure, size and dynamic characteristics can therefore provide critical insight into cell function. However, mitochondria are often very abundant, and due to their close to diffraction-limit size, it is often non-trivial to distinguish a tubular or large mitochondrion from an ensemble of punctate mitochondria. In this paper, we use membrane potential dependent fluorescence fluctuations of individual mitochondria to resolve them using an approach similar to single molecule localization microscopy. We use 2-photon microscopy to image mitochondrial intensity fluctuations at 200 µm deep inside an intact in-vivo mouse soleus muscle. By analyzing the acquired images, we can reconstruct images with an extra layer of information about individual mitochondria, separated from their ensemble. Our analysis shows a factor of 14 improvement in detection of mitochondria.

Skeletal muscle metabolic adaptations to endurance exercise training are attainable in mice with simvastatin treatment.

We tested the hypothesis that a 6-week regimen of simvastatin would attenuate skeletal muscle adaptation to low-intensity exercise. Male C57BL/6J wildtype mice were subjected to 6-weeks of voluntary wheel running or normal cage activities with or without simvastatin treatment (20 mg/kg/d, n = 7-8 per group). Adaptations in in vivo fatigue resistance were determined by a treadmill running test, and by ankle plantarflexor contractile assessment. The tibialis anterior, gastrocnemius, and plantaris muscles were evaluated for exercised-induced mitochondrial adaptations (i.e., biogenesis, function, autophagy). There was no difference in weekly wheel running distance between control and simvastatin-treated mice (P = 0.51). Trained mice had greater treadmill running distance (296%, P<0.001), and ankle plantarflexor contractile fatigue resistance (9%, P<0.05) compared to sedentary mice, independent of simvastatin treatment. At the cellular level, trained mice had greater mitochondrial biogenesis (e.g., ~2-fold greater PGC1α expression, P<0.05) and mitochondrial content (e.g., 25% greater citrate synthase activity, P<0.05), independent of simvastatin treatment. Mitochondrial autophagy-related protein contents were greater in trained mice (e.g., 40% greater Bnip3, P<0.05), independent of simvastatin treatment. However, Drp1, a marker of mitochondrial fission, was less in simvastatin treated mice, independent of exercise training, and there was a significant interaction between training and statin treatment (P<0.022) for LC3-II protein content, a marker of autophagy flux. These data indicate that whole body and skeletal muscle adaptations to endurance exercise training are attainable with simvastatin treatment, but simvastatin may have side effects on muscle mitochondrial maintenance via autophagy, which could have long-term implications on muscle health.

Measuring reactive hyperemia in the lower limb using near-infrared spectroscopy.

Near-infrared spectroscopy (NIRS) has been used to measure reactive hyperemia following a vascular occlusion. However, the procedures and methods of analysis used have varied. The purpose of the present study is to identify reproducible methods for measuring reactive hyperemia using HbO2 NIRS signals in the calf and foot. Healthy participants (10 male, 10 female) aged 19 to 28 years performed one of two tests: reproducibility trials or elevation protocol (30 and 60 cm limb elevation above the heart). The time to 50% reperfusion (T1/2) and the second (R2q) quartile rates of reperfusion were found to be the most reproducible parameters (coefficient of variation= 7.12 to 14.1%). The time to 95% reperfusion (T95) was 12.7% more reproducible on average than the previously reported parameter of time to peak hyperemia. Measures of reperfusion time and rate slowed with increasing limb elevation. Correlations were identified between the calf and foot in the measurements of R2q (R2 = 0.713, p = 0.021), T1/2 (R2 = 0.673, p = 0.033), and T95 (R2 = 0.792, p = 0.006). Half and 95% recovery times and second and third quartile rates expressed good reproducibility and sensitivity to change with reduced perfusion pressure. NIRS measures of reactive hyperemia have the potential to evaluate microvascular perfusion in clinical populations.

Comparisons of ultrasound-estimated intramuscular fat with fitness and health indicators.

INTRODUCTION: We examined ultrasound-estimated intramuscular fat of 4 muscles: rectus femoris (RF), biceps femoris (BF), tibialis anterior (TA), and medial gastrocnemius (MG), and compared the results with other health measures. METHODS: Forty-two participants were tested. Muscle echo intensity was quantified into percent intramuscular fat using previously published equations. RESULTS: Strong correlations were found in percent intramuscular fat in the 4 muscles (r ≥ 0.8). Weak to moderate correlations were found between intramuscular fat and body mass index (r ≥ 0.2), waist/hip ratio (r ≥ 0.2), muscle thickness (r = -0.5 in RF, r = -0.4 in TA, r = -0.7 in MG), and muscle strength (leg extension: r = 0.4; leg flexion: r = -0.5). A relationship between intramuscular fat in RF and MG and physical activity was also observed (P < 0.05). CONCLUSION: Ultrasound-estimated intramuscular fat was associated with other health measures and may provide physiological insight into the health consequences of obesity. Muscle Nerve 54: 743-749, 2016.

Reduced skeletal muscle oxidative capacity and impaired training adaptations in heart failure.

Systolic heart failure (HF) is associated with exercise intolerance that has been attributed, in part, to skeletal muscle dysfunction. The purpose of this study was to compare skeletal muscle oxidative capacity and training-induced changes in oxidative capacity in participants with and without HF. Participants with HF (n = 16, 65 ± 6.6 years) were compared with control participants without HF (n = 23, 61 ± 5.0 years). A subset of participants (HF: n = 7, controls: n = 5) performed 4 weeks of wrist-flexor exercise training. Skeletal muscle oxidative capacity was determined from the recovery kinetics of muscle oxygen consumption measured by near-infrared spectroscopy (NIRS) following a brief bout of wrist-flexor exercise. Oxidative capacity, prior to exercise training, was significantly lower in the HF participants in both the dominant (1.31 ± 0.30 min(-1) vs. 1.59 ± 0.25 min(-1), P = 0.002; HF and control groups, respectively) and nondominant arms (1.29 ± 0.24 min(-1) vs. 1.46 ± 0.23 min(-1), P = 0.04; HF and control groups, respectively). Following 4 weeks of endurance training, there was a significant difference in the training response between HF and controls, as the difference in oxidative training adaptations was 0.69 ± 0.12 min(-1) (P < 0.001, 95% CI 0.43, 0.96). The wrist-flexor training induced a ~50% improvement in oxidative capacity in participants without HF (mean difference from baseline = 0.66 ± 0.09 min(-1), P < 0.001, 95% CI 0.33, 0.98), whereas participants with HF showed no improvement in oxidative capacity (mean difference from baseline = -0.04 ± 0.08 min(-1), P = 0.66, 95% CI -0.24, 0.31), suggesting impairments in mitochondrial biogenesis. In conclusion, participants with HF had reduced oxidative capacity and impaired oxidative adaptations to endurance exercise compared to controls.

Reproducibility of near-infrared spectroscopy measurements of oxidative function and postexercise recovery kinetics in the medial gastrocnemius muscle.

The purpose of this study was to assess the reproducibility of resting blood flow, resting oxygen consumption, and mitochondrial capacity in skeletal muscle using near-infrared spectroscopy (NIRS). We also determined the influence of 2 exercise modalities (ergometer and rubber exercise bands) on the NIRS measurements. Fifteen young, healthy participants (5 female, 10 male) were tested on 2 nonconsecutive occasions within an 8-day period. The NIRS device was placed on the medial gastrocnemius. Venous and arterial occlusions were performed to obtain blood flow and oxygen consumption. A series of repeated arterial occlusions was used to measure the recovery kinetics of muscle oxygen consumption after ∼7-10 s of voluntary plantar flexion exercise. Resting blood flow had mean coefficients of variation (CV) of 42% and 38% for bands and ergometer, respectively, and resting metabolism had mean CVs of 17% and 12% for bands and ergometer, respectively. The recovery time constant of oxygen consumption (day 1 bands and ergometer: 23.2 ± 3.7 s, 27.6 ± 6.5 s, respectively; day 2 bands and ergometer: 25.5 ± 5.4 s, 25.0 ± 4.9 s, respectively) had mean CVs of 10% and 11% for bands and ergometer, respectively. We conclude that measurements of oxygen consumption and mitochondrial capacity using NIRS can be obtained with good reproducibility.