Prolonged Unloading Research Articles

Limited impact of blood flow restriction on muscle quality of young individuals during prolonged unloading: Implications for therapeutics approaches.

Limited impact of blood flow restriction on muscle quality of young individuals during prolonged unloading: Implications for therapeutics approaches.

Prolonged unloading of the cardiovascular system during bedrest and spaceflight weakens neural coupling between blood pressure and heart rate

Impaired baroreflex control following bedrest and spaceflight severely affects rate of recovery and return to ambulatory tasks. Post-bedrest/spaceflight orthostatic intolerance remains a major concern for bedrest confined older patients and for astronauts on long-duration missions. Here, we investigated how baroreflex changes following cardiovascular unloading were associated with autonomic neural coupling between blood pressure and heart rate in returning astronauts and in healthy participants who had undergone prolonged bedrest. Cardiovascular data collected from 27 shuttle astronauts (8–16 days) were compared with 19 head-down tilt bedrest (HDBR, 60 days) volunteers. In each group, beat-by-beat systolic blood pressure (SBP) and heart period (R-to-R intervals, RR) were collected simultaneously during a supine-to-stand test (5–10 min) conducted around 10 days before launch or HDBR and within 120 min of landing or exiting HDBR. Indices characterizing interaction time (fraction time active), response gain (gain), and control directionality (causality) between SBP and RR (SBP→RR: neural reflex, and RR→SBP: mechanical coupling) during standing were derived from the beat-by-beat data. Following spaceflight, cardiac baroreflex fraction time active was reduced along with neural reflex causality, with much larger reductions seen with HDBR. No change in mechanical causality pre-post flight or HDBR was observed; however, comparison of male and female astronauts indicated that neural and mechanical causality were both lower in females. Altogether, these findings suggest that changes in blood pressure regulation after bedrest and spaceflight are primarily reflex/neurally mediated and provides a target for therapeutic approaches to cardiovascular deconditioning.

Muscle unloading: A comparison between spaceflight and ground-based models.

Prolonged unloading of skeletal muscle, a common outcome of events such as spaceflight, bed rest and hindlimb unloading, can result in extensive metabolic, structural and functional changes in muscle fibres. With advancement in investigations of cellular and molecular mechanisms, understanding of disuse muscle atrophy has significantly increased. However, substantial gaps exist in our understanding of the processes dictating muscle plasticity during unloading, which prevent us from developing effective interventions to combat muscle loss. This review aims to update the status of knowledge and underlying mechanisms leading to cellular and molecular changes in skeletal muscle during unloading. We have also discussed advances in the understanding of contractile dysfunction during spaceflights and in ground-based models of muscle unloading. Additionally, we have elaborated on potential therapeutic interventions that show promising results in boosting muscle mass and strength during mechanical unloading. Finally, we have identified key gaps in our knowledge as well as possible research direction for the future.

PO-064 A potential role of stretch-activated channels in anabolic mechanotransduction in the atrophied rat soleus muscle

Objective Prolonged immobilization or unloading of skeletal muscle causes muscle disuse atrophy, which is characterized by a reduction in muscle cross-sectional area and compromised contractile function. To date, the mechanisms of anabolic mechanotransduction in the atrophied mammalian skeletal muscle remain poorly understood. The aim of the present study was to assess a possible role of stretch-activated ion channels (SAC) in the propagation of a mechanical signal to anabolic signaling and protein synthesis (PS) in an isolated rat soleus muscle following mechanical unloading.
 Methods The mechanical unloading was performed via hindlimb suspension (HS). Twenty-eight male Wistar rats weighing were randomly assigned to the following 4 groups (n=7/group): 1) vivarium control (C), 2) control + SAC inhibitor (gadolinium) (C+Gd3+), 3) 7-day HS (HS), 4) 7-day HS + SAC inhibitor (HS+Gd3+). Following unloading, an isolated rat soleus was placed in an organ culture medium and subjected to a bout of eccentric contractions (EC). Upon completion of the EC, muscles were collected for Western blot analyses to determine the content of the key anabolic markers. The rate of PS was measured by SUnSET technique.
 Results EC-induced increase in PS was significantly less in the HS and HS+ Gd3+ groups vs. the C group. There was no statistically significant difference between the HS and HS+ Gd3+ groups in terms of EC-induced increase in muscle PS. A decrease in EC-induced phosphorylation of p70S6K, 4E-BP1, RPS6 and GSK-3beta in the 7-day unloaded soleus treated with SAC inhibitor did not differ from that of the 7-day unloaded soleus without SAC blockade. Thus, the inhibition of SAC with gadolinium did not lead to further decline in EC-induced phosphorylation of the key anabolic markers and muscle PS.
 Conclusions The results of the study suggest that attenuation of mTORC1-signaling and PS in response to EC in unloaded soleus muscle may be associated with inactivation of SAC. The study was supported by the RFBR grant # 16-34-60055.

Spinal Health during Unloading and Reloading Associated with Spaceflight.

Spinal elongation and back pain are recognized effects of exposure to microgravity, however, spinal health has received relatively little attention. This changed with the report of an increased risk of post-flight intervertebral disc (IVD) herniation and subsequent identification of spinal pathophysiology in some astronauts post-flight. Ground-based analogs, particularly bed rest, suggest that a loss of spinal curvature and IVD swelling may be factors contributing to unloading-induced spinal elongation. In flight, trunk muscle atrophy, in particular multifidus, may precipitate lumbar curvature loss and reduced spinal stability, but in-flight (ultrasound) and pre- and post-flight (MRI) imaging have yet to detect significant IVD changes. Current International Space Station missions involve short periods of moderate-to-high spinal (axial) loading during running and resistance exercise, superimposed upon a background of prolonged unloading (microgravity). Axial loading acting on a dysfunctional spine, weakened by anatomical changes and local muscle atrophy, might increase the risk of damage/injury. Alternatively, regular loading may be beneficial. Spinal pathology has been identified in-flight, but there are few contemporary reports of in-flight back injury and no recent studies of post-flight back injury incidence. Accurate routine in-flight stature measurements, in- and post-flight imaging, and tracking of pain and injury (herniation) for at least 2 years post-flight is thus warranted. These should be complemented by ground-based studies, in particular hyper buoyancy floatation (HBF) a novel analog of spinal unloading, in order to elucidate the mechanisms and risk of spinal injury, and to evaluate countermeasures for exploration where injury could be mission critical.

Myocellular Responses to Concurrent Flywheel Training during 70 Days of Bed Rest.

This investigation evaluated myocellular responses to an integrated resistance and aerobic training program during 70 d of bed rest. Training was 6 d·wk on a small-footprint gravity-independent flywheel resistance and aerobic device; 3 d of maximal flywheel supine quadriceps and calf exercises with continuous rowing separated by 4 to 6 h, and 3 d of interval rowing. Vastus lateralis (VL) and soleus (SOL) muscle biopsies were obtained from eight healthy males (age, 28 ± 4 yr; BMI, 25 ± 3 kg·m; V˙O2max, 42 ± 6 mL·kg·min) before and after 6° head-down tilt bed rest. Vastus lateralis and SOL myosin heavy chain (MHC) I and IIa single muscle fiber size and functional characteristics, as well as overall fiber type distribution, capillarization, and metabolic enzyme activities were evaluated. In the VL, MHC I size and power (absolute and normalized) were preserved. The MHC IIa fibers hypertrophied (+6%, P < 0.05) without a change in absolute power, so normalized power declined (-7%, P < 0.05). In the SOL, MHC I fibers atrophied (-9%) and absolute power declined (-17%) (P < 0.05), whereas normalized power was maintained. Size, absolute power, and normalized power were protected in the less-abundant MHC IIa fibers. Reduced MHC coexpressing hybrid fibers, generally indicative of an exercise training effect, was apparent in the VL, whereas fiber type was maintained in the SOL. Capillarization and metabolic enzymes were generally preserved or increased in VL and SOL. The integrated resistance and aerobic training protocol on a device maintains several key myocellular characteristics during prolonged unloading, but further refinement of the exercise approach to fully protect the SOL is warranted.

From the international space station to the clinic: how prolonged unloading may disrupt lumbar spine stability

Background ContextProlonged microgravity exposure is associated with localized low back pain and an elevated risk of post-flight disc herniation. Although the mechanisms by which microgravity impairs the spine are unclear, they should be foundational for developing in-flight countermeasures for maintaining astronaut spine health. Because human spine anatomy has adapted to upright posture on Earth, observations of how spaceflight affects the spine should also provide new and potentially important information on spine biomechanics that benefit the general population. PurposeThis study compares quantitative measures of lumbar spine anatomy, health, and biomechanics in astronauts before and after 6 months of microgravity exposure on board the International Space Station (ISS). Study DesignThis is a prospective longitudinal study. SampleSix astronaut crewmember volunteers from the National Aeronautics and Space Administration (NASA) with 6-month missions aboard the ISS comprised our study sample. Outcome MeasuresFor multifidus and erector spinae at L3–L4, measures include cross-sectional area (CSA), functional cross-sectional area (FCSA), and FCSA/CSA. Other measures include supine lumbar lordosis (L1–S1), active (standing) and passive (lying) flexion-extension range of motion (FE ROM) for each lumbar disc segment, disc water content from T2-weighted intensity, Pfirrmann grade, vertebral end plate pathology, and subject-reported incidence of chronic low back pain or disc injuries at 1-year follow-up. Methods3T magnetic resonance imaging and dynamic fluoroscopy of the lumbar spine were collected for each subject at two time points: approximately 30 days before launch (pre-flight) and 1 day following 6 months spaceflight on the ISS (post-flight). Outcome measures were compared between time points using paired t tests and regression analyses. ResultsSupine lumbar lordosis decreased (flattened) by an average of 11% (p=.019). Active FE ROM decreased for the middle three lumbar discs (L2–L3: −22.1%, p=.049; L3–L4: −17.3%, p=.016; L4–L5: −30.3%, p=.004). By contrast, no significant passive FE ROM changes in these discs were observed (p>.05). Disc water content did not differ systematically from pre- to post-flight. Multifidus and erector spinae changed variably between subjects, with five of six subjects experiencing an average decrease 20% for FCSA and 8%–9% for CSA in both muscles. For all subjects, changes in multifidus FCSA strongly correlated with changes in lordosis (r2=0.86, p=.008) and active FE ROM at L4–L5 (r2=0.94, p=.007). Additionally, changes in multifidus FCSA/CSA correlated with changes in lordosis (r2=0.69, p=.03). Although multifidus-associated changes in lordosis and ROM were present among all subjects, only those with severe, pre-flight end plate irregularities (two of six subjects) had post-flight lumbar symptoms (including chronic low back pain or disc herniation). ConclusionsWe observed that multifidus atrophy, rather than intervertebral disc swelling, associated strongly with lumbar flattening and increased stiffness. Because these changes have been previously linked with detrimental spine biomechanics and pain in terrestrial populations, when combined with evidence of pre-flight vertebral end plate insufficiency, they may elevate injury risk for astronauts upon return to gravity loading. Our results also have implications for deconditioned spines on Earth. We anticipate that our results will inform new astronaut countermeasures that target the multifidus muscles, and research on the role of muscular stability in relation to chronic low back pain and disc injury.

P5840Structural and functional plasticity of myocardial slices in response to prolonged mechanical loading and unloading in vitro

P5840Structural and functional plasticity of myocardial slices in response to prolonged mechanical loading and unloading in vitro

Response of Rat Tibia to Prolonged Unloading Under the Influence of Electrical Stimulation at the Dorsal Root Ganglion

Immobilization of weight bearing skeletons or microgravity results in disuse osteoporosis in both human and animals. Our previous study demonstrated that electrical stimulation at the dorsal root ganglion (DRG) with an implantable micro-electrical stimulation system (IMESS) could trigger secretion of bone anabolic calcitonin gene-related peptide (CGRP) and prevent bone loss in a short-term hindlimb unloading rat model. This study was designed to further investigate whether electrical stimulation to the DRG could prevent bone loss due to prolonged unloading. Eighteen adult rats were randomly assigned into three groups: cage control (CC), hindlimb unloading (HU), and hindlimb unloading with electrical stimulation (HUES). Electrical stimulation was applied via IMESS to the right DRGs at vertebral levels L4-L6 in HUES group for 6 weeks. Following unloading for 6 weeks, proximal tibia metaphysis was shown 64.0% decrease in bone mineral content (BMC) and 47.0% decrease in bone mineral density (BMD) in HU group while significant reduced bone lose with 2.7% increase in total BMC and only 9.2% decrease in total BMD in HUES group. Diaphyseal BMD decreased significantly in both HU and HUES group as compared with CC group. There was enhancement of CGRP expression in the DRGs in HUES group. This experimental study proved the proposed concept using electrical stimulation at the DRG for prevention of disuse-induced bone loss in a rat hindlimb suspension model.

Simulated Microgravity and Low-Dose/Low-Dose-Rate Radiation Induces Oxidative Damage in the Mouse Brain.

Microgravity and radiation are stressors unique to the spaceflight environment that can have an impact on the central nervous system (CNS). These stressors could potentially lead to significant health risks to astronauts, both acutely during the course of a mission or chronically, leading to long-term, post-mission decrements in quality of life. The CNS is sensitive to oxidative injury due to high concentrations of oxidizable, unsaturated lipids and low levels of antioxidant defenses. The purpose of this study was to evaluate oxidative damage in the brain cortex and hippocampus in a ground-based model for spaceflight, which includes prolonged unloading and low-dose radiation. Whole-body low-dose/low-dose-rate (LDR) gamma radiation using (57)Co plates (0.04 Gy at 0.01 cGy/h) was delivered to 6 months old, mature, female C57BL/6 mice (n = 4-6/group) to simulate the radiation component. Anti-orthostatic tail suspension was used to model the unloading, fluid shift and physiological stress aspects of the microgravity component. Mice were hindlimb suspended and/or irradiated for 21 days. Brains were isolated 7 days or 9 months after irradiation and hindlimb unloading (HLU) for characterization of oxidative stress markers and microvessel changes. The level of 4-hydroxynonenal (4-HNE) protein, an oxidative specific marker for lipid peroxidation, was significantly elevated in the cortex and hippocampus after LDR + HLU compared to controls (P < 0.05). The combination group also had the highest level of nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) expression compared to controls (P < 0.05). There was a significant decrease in superoxide dismutase (SOD) expression in the animals that received HLU only or combined LDR + HLU compared to control (P < 0.05). In addition, 9 months after LDR and HLU exposure, microvessel densities were the lowest in the combination group, compared to age-matched controls in the cortex (P < 0.05). Our data provide the first evidence that prolonged exposure to simulated microgravity and LDR radiation is associated with increased oxidative stress biomarkers that may increase the likelihood of brain injury and reduced antioxidant defense. NOX2-containing nicotinamide adenosine dinucleotide phosphate (NADPH oxidase) may contribute to spaceflight environment-induced oxidative stress.

The Effect of OSM on MC3T3-E1 Osteoblastic Cells in Simulated Microgravity with Radiation.

Bone deterioration is a challenge in long-term spaceflight with significant connections to patients experiencing disuse bone loss. Prolonged unloading and radiation exposure, defining characteristics of space travel, have both been associated with changes in inflammatory signaling via IL-6 class cytokines in bone. While there is also evidence for perturbed IL-6 class signaling in spaceflight, there has been scant examination of the connections between microgravity, radiation, and inflammatory stimuli in bone. Our lab and others have shown that the IL-6 class cytokine oncostatin M (OSM) is an important regulator of bone remodeling. We hypothesize that simulated microgravity alters osteoblast OSM signaling, contributing to the decoupling of osteolysis and osteogenesis in bone homeostasis. To test this hypothesis, we induced OSM signaling in murine MC3T3-E1 pre-osteoblast cells cultured in modeled microgravity using a rotating wall vessel bioreactor with and without exposure to radiation typical of a solar particle event. We measured effects on inflammatory signaling, osteoblast activity, and mineralization. Results indicated time dependent interactions among all conditions in the regulation of IL-6 production. Furthermore, OSM induced the transcription of OSM receptor ß, IL 6 receptor α subunits, collagen α1(I), osteocalcin, sclerostin, RANKL, and osteoprotegerin. Measurements of osteoid mineralization suggest that the spatial organization of the osteoblast environment is an important consideration in understanding bone formation. Taken together, these results support a role for altered OSM signaling in the mechanism of microgravity-induced bone loss.

Rebuttal from Michael B. Reid, Andrew R. Judge and Sue C. Bodine.

The arguments put forward by Phillips and McGlory do not take into consideration advances in the field over the past 5 years. These new discoveries have provided critical information on disuse atrophy in both human and rodent muscle. They also call into question key elements of our opponents’ reasoning. The assertion that disuse atrophy largely reflects a decline in protein synthesis is based solely on data from human vastus lateralis muscle. Recent studies demonstrate the limitations of this approach. For example, during cast immobilization, disuse atrophy occurs in a variety of leg muscles at least 2 weeks before significant atrophy is detectable in vastus lateralis (Psatha et al. 2012), suggesting the response of vastus lateralis is delayed relative to other muscles. New data also show that atrophy of vastus lateralis is non-uniform, affecting some regions more than others (Miokovic et al. 2012). Such confounding variables preclude the use of localized measurements in vastus lateralis, indeed any single muscle, as the sole source of information on disuse atrophy. Our opponents argue for limiting research to human vastus lateralis because ‘inherent species-specific differences’ exist between rodent and human muscles. Again, a plethora of recent research has debunked this misconception. As in rodent limb muscle, new data show that human type I fibres undergo greater atrophy than human type II fibres in soleus, gastrocnemius and vastus lateralis muscles after prolonged unloading (Fitts et al. 2010; Brocca et al. 2012). As in rodents, disuse atrophy of human muscle can be detected in as few as 4–5 days (Suetta et al. 2012; Wall et al. 2014), causes losses of up to 2.5–6% per day (Suetta et al. 2012; Grosu et al. 2012; Wall et al. 2014), and occurs at rates that differ widely among human muscles (Belavý et al. 2009; Miokovic et al. 2012; Psatha et al. 2012). Recent data also confirm that human muscle mimics rodent muscle by upregulating proteolytic gene products (Suetta et al. 2012). This consistency among species is fundamental, broad based, and compelling. It provides a solid scientific foundation for translational research in the field. Preclinical studies in rodents have been instrumental in defining the mechanisms of disuse atrophy, identifying atrophy-related pathways, and discovering genes that regulate the process. Rodent research has also demonstrated that proteolytic blockade can inhibit disuse atrophy (Jamart et al. 2011; Talbert et al. 2013), establishing a causal role for proteolysis. Modern evidence leads to the inexorable conclusion that proteolysis is not simply important for disuse atrophy. It is essential.

Abstract 12636: Pro-Atrial Natriuretic Peptide: A Novel Guanylyl Cyclase-A Receptor Activator Which Goes Beyond Carperitide and Nesiritide

Introduction: ProANP is a 108 amino acid (AA) prohormone which is produced in the atria and processed to the mature 28 AA ANP which activates the guanylyl cyclase receptor-A (GC-A) and its second messenger cGMP. ProANP is found in the human circulation but its bioavailability is undefined. Hypothesis: ProANP represents a novel cardiac peptide with more sustained natriuretic actions than either carperitide (ANP) or nesiritide (BNP). ProANP itself can activate GC-A in vitro and the proANP can be processed in human serum from normals and in patients with heart failure (HF). Methods: We examined the in vivo cardiorenal actions of proANP compared to ANP, BNP, or placebo in normal canines (667 pmol/kg, n=5/group). We determined cGMP activation of proANP, ANP and BNP in GC-A expressing HEK293 cells. ProANP processing and degradation was also defined in serum from normal subjects (n=13) and patients with HF (n=14) ex vivo. Results: ProANP had significantly greater diuretic and natriuretic properties with increased renal blood flow and sustained renal tubular actions compared to ANP or BNP in canines. ProANP also resulted in greater and more prolonged cardiac unloading than ANP, and much less hypotensive effects than BNP. The plasma half-life of proANP was 4.5 times longer than ANP and 3.8 times longer than BNP. ProANP itself stimulated cGMP generation in GC-A expressing HEK cells which was equal to ANP. ProANP was processed to ANP in fresh serum from normals as well as HF patients ex vivo and the processed peptide activated cGMP in GC-A expressing HEK cells. Conclusions: ProANP represents a novel GC-A activator with a longer half-life and greater natriuresis and diuresis than ANP and BNP and less hypotensive effects than BNP. The bioactivity of exogenous proANP may be through direct stimulation of GC-A, as well as through processing into ANP, as proANP is processed in the circulation of both normal and HF. As proANP circulates in the human circulation, proANP may represent a key circulating hormone in cardiorenal and blood pressure homeostasis as well as a potential innovative therapeutic beyond ANP (carperitide) or BNP (nesiritide) for cardiorenal diseases including HF.

Changes in Left Ventricular Ejection Fraction Following Implantation of Left Ventricular Assist Device as Destination Therapy

Purpose Recovery of left ventricular (LV) function has been reported following prolonged myocardial unloading with left ventricular assist device (LVAD) therapy. However, recovery appears to be limited to younger patients presenting acutely with non-ischemic heart failure (HF), and it is unclear how often LV recovery occurs after destination LVAD. Methods and Materials Patients undergoing destination LVAD at the Mayo Clinic in Minnesota from April 2007 to February 2012 with at least 6 months of follow-up were included. Ejection fraction (EF) was obtained from transthoracic echocardiographic studies pre-LVAD and at 6 and 12 months post-LVAD. Use of beta blockers, ACE inhibitors, angiotensin-receptor blockers, and aldosterone antagonists at hospital dismissal were recorded. Patients were censored at death or last follow-up. Results Among 69 patients (mean age 64 years, 16% female, 59% ischemic HF, 97% continuous-flow LVAD), the mean EF was 18% pre-LVAD, 23% 6 months post-LVAD, and 22% 12 months post-LVAD. At 6 and 12 months post-LVAD, the EF increased by a mean of 5% and 4% compared with baseline, respectively (paired t-test p=0.001). By one year post-LVAD, 13 (22%) patients had an increase in EF of ≥10%, with greater improvements in those with lower pre-LVAD EF. The change in EF was similar in patients with ischemic (mean EF increase 3%) vs. nonischemic HF (mean EF increase 7%, p=0.13). Use of HF medications had no impact on the change in EF. There were only 2 patients who had an improvement in EF to ≥50% by 1 year. There was no association between change in EF at 6 months and subsequent mortality. Conclusions In our experience, EF only improved slightly after destination LVAD, and rarely reached a normal range. This suggests that mechanical unloading alone is not the main mechanism for LV recovery in end-stage HF. It is possible that use of LVADs may allow myocardial recovery in the setting of novel pharmacologic or cell based interventions.

Prolonged unloading in growing rats reduces cortical osteocyte lacunar density and volume in the distal tibia

Bone dynamically adapts its structure to the environmental demands placed upon it. Load-related stimuli play an important role in this adaptation. It has been postulated that osteocytes sense changes in these stimuli and initiate adaptive responses, across a number of scales, through a process known as mechanotransduction. While much research has focused on gross and tissue-level adaptation, relatively little is known regarding the relation between cellular-level features (e.g. osteocyte lacunar density, volume and shape) and loading. The increasing availability of high resolution 3D imaging modalities, including synchrotron-based techniques, has made studying 3D cellular-level features feasible on a scale not previously possible. The primary objective of this study was to test the hypothesis that unloading (sciatic neurectomy) during growth results in altered osteocyte lacunar density in the tibial diaphysis of the rat. Secondarily, we explored a potential effect of unloading on mean lacunar volume. Lacunar density was significantly (p<0.05) lower in immobilized bones (49,642±11,955 lacunae per mm3; n=6) than in control bones (63,138±1956 lacunae per mm3; n=6). Mean lacunar volume for immobilized bones (209±72μm3; n=6) was significantly smaller (p<0.05) than that for the control bones (284±28μm3; n=6). Our results demonstrate that extreme differences in loading conditions, such as those created by paralysis, do indeed result in changes in osteocyte lacunar density and volume. Further investigation is warranted to examine relations between these measures and more subtle variation in loading as well as pathological states, which have been linked to alterations in mechanotransduction.

Biphasic Stress Response in the Soleus during Reloading after Hind Limb Unloading

Extreme disuse and spaceflight elicit rapid skeletal muscle atrophy, accompanied by elevated proinflammatory signaling and impaired stress response proteins (e.g., heat shock proteins (HSP), insulin-like growth factor 1 (IGF-1)). Recovery of muscle mass is delayed during the early stage of reloading after prolonged unloading, with a concomitant impairment of HSP70 and IGF-1. We postulated that proinflammatory signaling and stress response alterations would characterize early and late phases of signaling during reloading. Twenty-four adult SD rats were divided into the following groups: controls, 28 d of hind limb unloading (HU), HU + early (7 d) reloading (HU-R7), and HU + late (28 d) reloading (HU-R28). Soleus mass decreased (-55%) with HU and remained depressed (-41%) at HU-R7. Nuclear factor κB activation and oxidative stress were elevated with HU and remained high during reloading. HU elevated inducible nitric oxide synthase and returned to baseline during reloading, whereas 3-nitrotyrosine did not increase with HU and peaked at HU-R7. HU depressed levels of HSP25 phosphorylation at Ser82 and IGF-1. Although p-HSP25 and Akt phosphorylation (Ser473) recovered during early reloading, HSP70, heat shock factor 1, and IGF-1 remained depressed. HSP70, heat shock factor 1, and IGF-1 recovered, whereas p-Akt and 3-nitrotyrosine decreased to control levels at HU-R28. Reloading elicited an early phase characterized by elevated nuclear factor κB activation, 3-nitrotyrosine, p-HSP25, and p-Akt levels and a delayed phase with recovery of HSP70, IGF-1, and muscle mass. We conclude that the reloading phenotype in skeletal muscle is expressed in two distinct phases related to (a) pro-inflammatory signaling and (b) muscle mass recovery.

The paradox of left ventricular assist device unloading and myocardial recovery in end-stage dilated cardiomyopathy: implications for heart failure in the elderly

Dilated cardiomyopathy (DCM) is a common debilitating condition with limited therapeutic options besides heart transplantation or palliation. It is characterized by maladaptive remodeling of cardiomyocytes, extracellular collagen matrix (ECCM) and left ventricular (LV) geometry which contributes to further dysfunction. LV assist devices (LVADs) can reverse adverse remodeling in end-stage DCM. However, there is a disconnect between the benefits of prolonged unloading with LVAD at molecular and cellular levels and the low rate of bridge to recovery (BTR). Potential explanations for this paradox include insufficient reverse ECCM remodeling and/or excessive reverse cardiomyocyte remodeling with atrophy. LVAD therapy is associated with decreased collagen turnover and cross-linking and increased tissue angiotensin II (AngII), whereas LVAD combined with angiotensin-converting enzyme inhibition results in decreased tissue AngII and collagen cross-linking, normalizes LV end-diastolic pressure volume relationships and is associated with modestly higher rates of BTR. Much remains to be learned about ventricular reverse remodeling after LVAD. This can be facilitated through systematic collection and comparison of recovered and unrecovered myocardium. Importantly, vigilant monitoring for ventricular recovery among LVAD patients is needed, particularly in older patients receiving LVAD for destination therapy. In addition, prospective multicenter trials are needed to clarify the potential benefit of concomitant heart failure therapy with selective β2 agonism on ventricular recovery.

Regulation of Contractile Proteins and Protein Translational Signaling in Disused Muscle

Background/Aims: Muscle disuse can lead to muscle atrophy and impaired skeletal muscle function. How skeletal muscle modulates protein translational signaling in response to prolonged muscle disuse is not well understood. Using the hindlimb unloading (HU) model of muscle atrophy we examined how hindlimb unweighting affects protein translational signaling, including the activation of Akt/mTOR/p70S6K/S6 signaling and the inhibitory association of 4EBP1 with translation initiation factor eIF4E. Methods: Male F344BN rats were randomized into baseline control, or subjected to HU for 3, 7 or 14 days. Body weight, gastrocnemius muscle, and individual myofiber cross-sectional area were measured to evaluate the degree of muscle atrophy. The amounts of myosin and related muscle contractile proteins were assessed using SDS-PAGE and immunoblotting. Microarray analysis was used to evaluate changes in the mRNA expression of muscle contractile proteins. Total and phosphorylated proteins of Akt/mTOR/p70S6K/S6 pathway were determined via immunoblotting, while the association of 4EBP1 with eIF4E was detected via co-immunoprecipitation. Results: Unloading for 3 days significantly reduced cytosolic myosin content and was associated with increased binding of 4EBP1 to eIF4E, while prolonged unloading (14 days) was associated with the activation of Akt/mTOR/p70S6K/S6 signaling, decreased binding of 4EBP1 to eIF4E, increased cytosolic myosin and elevations in myofibrillar mRNA levels. Conclusion: Taken together, these data suggest that prolonged muscle disuse induces a biphasic translational signaling response that is associated with diminished and then increased muscle contractile protein expression.

T-Tubule Morphology and Local Ca2+-Induced Ca2+ Release in Rat Ventricular Cardiomyocytes are Sensitive to Chronic Changes in Mechanical Load of the Heart

Transverse (t) tubules optimise cardiomyocyte excitation-contraction coupling by promoting cross-talk between L-type Ca2+ channels and ryanodine receptors, and are disrupted in several cardiac diseases, leading to heart failure. A unifying hypothesis is that t-tubule structure is altered by chronically varying the mechanical load of the heart, consequently disrupting local Ca2+-induced Ca2+ release (CICR).To test this hypothesis, rat hearts were unloaded for 4 or 8 weeks using heterotopic abdominal heart-lung transplantation, or overloaded for 6 or 10 weeks using thoracic aortic constriction. Left ventricular cardiomyocytes were isolated enzymatically and studied using confocal microscopy.Chronic overloading increased cardiomyocyte volume at 10 but not 6 weeks (Control 45,348±2373μm3, n=47 vs. OV 6 weeks 48,785±2237μm3, n=60 vs. OV 10 weeks 56,066±3091μm3, n=42; p<0.05) and disrupted t-tubule regularity, which was measured as the power of the dominant frequency of the Fourier transform of Di-8-ANEPPS images (Control 1.69±0.1x107, n=44 vs. OV 10 weeks 9.45±1.0x106, n=33; p<0.001), despite preserved Ca2+ transients. Unloading decreased cardiomyocyte size and induced time-dependent Ca2+ transient changes. Ca2+ release synchronicity (measured by the variance of the time-to-peak of the Ca2+ transient) was disrupted at 8 but not 4 weeks (Control 269.6±17.13ms2, n=106 vs. UN 4 weeks 331.6±23.67ms2, n=46 vs. UN 8 weeks 403.5±35.4ms2, n=50; p<0.01), as was the t-tubule regularity (Control 1.59±0.01x107, n=107 vs. UN 4 weeks 1.75±0.1x107, n=53 vs. UN 8 weeks 1.1±0.0007x107, n=82; p<0.01).We demonstrate that an increase or decrease in load results in time-dependent disrupted t-tubule morphology. This can provide a mechanism for inefficient CICR observed in chronic heart failure and the functional deterioration after prolonged unloading of the heart using left ventricular assist devices.

Nutrition and Resistance Exercise During Reconditioning From Unloading

The recovery of muscle size and function following musculoskeletal unloading has received little attention in the scientific literature. Nutritional factors such as total energy, protein intake, and the pre- and/or post-exercise consumption of amino acid-carbohydrate (AACHO) have been shown to be important for enhancing training adaptations in recreational exercisers. A preliminary study was conducted to explore the interaction between nutrition and resistance exercise during reconditioning from unloading. Muscle CSA, strength, and endurance were measured during a control period following 30 d of unilateral lower limb suspension (Post-ULLS) and after 18 d of reconditioning (ReCon). Six participants consumed either AACHO (979 kJ, 36 g carbohydrate, 22.5 g protein) or placebo (PLAC) prior to resistance exercise (3 d x wk(-1)) during reconditioning. Total daily energy and macronutrient intake were evaluated from dietary journals. From Post-ULLS to ReCon, muscle endurance increased 1.1 +/- 0.6 min in AACHO and decreased 1.3 +/- 0.7 min in PLAC. Muscle CSA (6 +/- 2 vs. 5 +/- 3 cm2) and strength (105 +/- 53 vs. 81 +/- 37 N) increased similarly in AACHO and PLAC, respectively. When groups were pooled there was a significant correlation between daily protein intake and the recovery of muscle CSA (r = 0.81). Although our findings are preliminary, timing AACHO intake during reconditioning was beneficial for muscular endurance, while overall protein intake was associated with increased muscle size. A systematic evaluation into the synergistic relationship between nutrition and exercise during muscular recovery from prolonged unloading is warranted.