IU Indianapolis ScholarWorks :: Browsing by Author "Rupert, Joseph E."

Browsing by Author "Rupert, Joseph E."

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Age- and sex-dependent role of osteocytic pannexin1 on bone and muscle mass and strength
(Nature Research, 2019-09-25) Aguilar-Perez, Alexandra; Pacheco-Costa, Rafael; Atkinson, Emily G.; Deosthale, Padmini; Davis, Hannah M.; Essex, Alyson L.; Dilley, Julian E.; Gomez, Leland; Rupert, Joseph E.; Zimmers, Teresa A.; Thompson, Roger J.; Allen, Matthew R.; Plotkin, Lilian I.; Anatomy and Cell Biology, School of Medicine
Pannexins (Panxs), glycoproteins that oligomerize to form hemichannels on the cell membrane, are topologically similar to connexins, but do not form cell-to-cell gap junction channels. There are 3 members of the family, 1-3, with Panx1 being the most abundant. All Panxs are expressed in bone, but their role in bone cell biology is not completely understood. We now report that osteocytic Panx1 deletion (Panx1Δot) alters bone mass and strength in female mice. Bone mineral density after reaching skeletal maturity is higher in female Panx1Δot mice than in control Panx1fl/fl mice. Further, osteocytic Panx1 deletion partially prevented aging effects on cortical bone structure and mechanical properties. Young 4-month-old female Panx1Δot mice exhibited increased lean body mass, even though pannexin levels in skeletal muscle were not affected; whereas no difference in lean body mass was detected in male mice. Furthermore, female Panx1-deficient mice exhibited increased muscle mass without changes in strength, whereas Panx1Δot males showed unchanged muscle mass and decreased in vivo maximum plantarflexion torque, indicating reduced muscle strength. Our results suggest that osteocytic Panx1 deletion increases bone mass in young and old female mice and muscle mass in young female mice, but has deleterious effects on muscle strength only in males.
The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia
(JoVE, 2016-11-30) Bonetto, Andrea; Rupert, Joseph E.; Barreto, Rafael; Zimmers, Teresa A.; Surgery, School of Medicine
Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.
Exogenous GDF11 Induces Cardiac and Skeletal Muscle Dysfunction and Wasting
(Springer, 2017-07) Zimmers, Teresa A.; Jiang, Yanling; Wang, Meijing; Liang, Tiffany W.; Rupert, Joseph E.; Au, Ernie D.; Marino, Francesco E.; Couch, Marion E.; Koniaris, Leonidas G.; Surgery, School of Medicine
Growth differentiation factor 11 (GDF11), a TGF-beta superfamily member, is highly homologous to myostatin and essential for embryonic patterning and organogenesis. Reports of GDF11 effects on adult tissues are conflicting, with some describing anti-aging and pro-regenerative activities on the heart and skeletal muscle while others opposite or no effects. Herein, we sought to determine the in vivo cardiac and skeletal muscle effects of excess GDF11. Mice were injected with GDF11 secreting cells, an identical model to that used to initially identify the in vivo effects of myostatin. GDF11 exposure in mice induced whole body wasting and profound loss of function in cardiac and skeletal muscle over a 14-day period. Loss of cardiac mass preceded skeletal muscle loss. Cardiac histologic and echocardiographic evaluation demonstrated loss of ventricular muscle wall thickness, decreased cardiomyocyte size, and decreased cardiac function 10 days following initiation of GDF11 exposure. Changes in skeletal muscle after GDF11 exposure were manifest at day 13 and were associated with wasting, decreased fiber size, and reduced strength. Changes in cardiomyocytes and skeletal muscle fibers were associated with activation of SMAD2, the ubiquitin–proteasome pathway and autophagy. Thus, GDF11 over administration in vivo results in cardiac and skeletal muscle loss, dysfunction, and death. Here, serum levels of GDF11 by Western blotting were 1.5-fold increased over controls. Although GDF11 effects in vivo are likely dose, route, and duration dependent, its physiologic changes are similar to myostatin and other Activin receptors ligands. These data support that GDF11, like its other closely related TGF-beta family members, induces loss of cardiac and skeletal muscle mass and function.
Exogenous Oncostatin M Induces Cardiac Dysfunction, Musculoskeletal Atrophy, and Fibrosis
(Elsevier, 2022) Jengelley, Daenique H. A.; Wang, Meijing; Narasimhan, Ashok; Rupert, Joseph E.; Young, Andrew R.; Zhong, Xiaoling; Horan, Daniel J.; Robling, Alexander G.; Koniaris, Leonidas G.; Zimmers, Teresa A.; Biochemistry and Molecular Biology, School of Medicine
Musculoskeletal diseases such as muscular dystrophy, cachexia, osteoarthritis, and rheumatoid arthritis impair overall physical health and reduce survival. Patients suffer from pain, dysfunction, and dysmobility due to inflammation and fibrosis in bones, muscles, and joints, both locally and systemically. The Interleukin-6 (IL-6) family of cytokines, most notably IL-6, is implicated in musculoskeletal disorders and cachexia. Here we show elevated circulating levels of OSM in murine pancreatic cancer cachexia and evaluate the effects of the IL-6 family member, Oncostatin M (OSM), on muscle and bone using adeno-associated virus (AAV) mediated over-expression of murine OSM in wildtype and IL-6 deficient mice. Initial studies with high titer AAV-OSM injection yielded high circulating OSM and IL-6, thrombocytosis, inflammation, and 60% mortality without muscle loss within 4 days. Subsequently, to mimic OSM levels in cachexia, a lower titer of AAV-OSM was used in wildtype and Il6 null mice, observing effects out to 4 weeks and 12 weeks. AAV-OSM caused muscle atrophy and fibrosis in the gastrocnemius, tibialis anterior, and quadriceps of the injected limb, but these effects were not observed on the non-injected side. In contrast, OSM induced both local and distant trabecular bone loss as shown by reduced bone volume, trabecular number, and thickness, and increased trabecular separation. OSM caused cardiac dysfunction including reduced ejection fraction and reduced fractional shortening. RNA-sequencing of cardiac muscle revealed upregulation of genes related to inflammation and fibrosis. None of these effects were different in IL-6 knockout mice. Thus, OSM induces local muscle atrophy, systemic bone loss, tissue fibrosis, and cardiac dysfunction independently of IL-6, suggesting a role for OSM in musculoskeletal conditions with these characteristics, including cancer cachexia.
Forelimb muscle architecture and myosin isoform composition in the groundhog (Marmota monax)
(2015) Rupert, Joseph E.; Rose, Jacob A.; Organ, Jason M.; Butcher, Michael T.; Department of Anatomy & Cell Biology, IU School of Medicine
Scratch-digging mammals are commonly described as having large, powerful forelimb muscles for applying high force to excavate earth, yet studies quantifying the architectural properties of the musculature are largely unavailable. To further test hypotheses about traits that represent specializations for scratch-digging, we quantified muscle architectural properties and myosin expression in the forelimb of the groundhog (Marmota monax), a digger that constructs semi-complex burrows. Architectural properties measured were muscle moment arm, muscle mass (MM), belly length (ML), fascicle length (lF), pennation angle and physiological cross-sectional area (PCSA), and these metrics were used to estimate maximum isometric force, joint torque and power. Myosin heavy chain (MHC) isoform composition was determined in selected forelimb muscles by SDS-PAGE and densitometry analysis. Groundhogs have large limb retractors and elbow extensors that are capable of applying moderately high torque at the shoulder and elbow joints, respectively. Most of these muscles (e.g. latissimus dorsi and pectoralis superficialis) have high lF/ML ratios, indicating substantial shortening ability and moderate power. The unipennate triceps brachii long head has the largest PCSA and is capable of the highest joint torque at both the shoulder and elbow joints. The carpal and digital flexors show greater pennation and shorter fascicle lengths than the limb retractors and elbow extensors, resulting in higher PCSA/MM ratios and force production capacity. Moreover, the digital flexors have the capacity for both appreciable fascicle shortening and force production, indicating high muscle work potential. Overall, the forelimb musculature of the groundhog is capable of relatively low sustained force and power, and these properties are consistent with the findings of a predominant expression of the MHC-2A isoform. Aside from the apparent modifications to the digital flexors, the collective muscle properties observed are consistent with its behavioral classification as a less-specialized burrower and these may be more representative of traits common to numerous rodents with burrowing habits or mammals with some fossorial ability.
Gemcitabine plus nab-paclitaxel preserves skeletal and cardiac mass and function in a murine model of pancreatic cancer cachexia
(bioRxiv, 2023-04-18) Narasimhan, Ashok; Jengelley, Daenique H. A.; Huot, Joshua R.; Umberger, Tara S.; Doud, Emma H.; Mosley, Amber L.; Wang, Meijing; Zhong, Xiaoling; Counts, Brittany R.; Rupert, Joseph E.; Young, Andrew R.; Bonetto, Andrea; Horan, Daniel J.; Robling, Alexander G.; Fishel, Melissa L.; Kelley, Mark R.; Koniaris, Leonidas G.; Zimmers, Teresa A.; Surgery, School of Medicine
More than 85% of patients with pancreatic ductal adenocarcinoma (PDAC) suffer from cachexia, a debilitating syndrome characterized by the loss of muscle and fat and remains an unmet medical need. While chemotherapy remains an effective treatment option, it can also induce weight and muscle loss in patients with cancer. Gemcitabine combined with nab paclitaxel (GnP) is a first line treatment option for patients with PDAC but GnP’s effect on cachexia has not been comprehensively investigated. We interrogated the effects of GnP in a murine model of pancreatic cancer cachexia. Mice were orthotopically implanted with the cachexia inducing pancreatic cell line (KPC) and were administered GnP or vehicle. The controls underwent sham surgery. We defined GnP effects on cachexia and tumor burden by evaluating muscle and cardiac mass and function, fat mass, bone morphometry, and hematology measurements. We completed RNA sequencing and deep proteome profiling in skeletal and cardiac muscle. KPC+GnP reduced tumor burden over 50% and increased survival compared to KPC. KPC vehicle group had more than 15% muscle mass loss and decreased left ventricular mass, this was not present in KPC+GnP when compared to controls. RNA Seq and deep proteomics analyses suggested that muscle and cardiac dysfunction pathways activated in KPC group were either reversed or decreased in KPC+GnP. In all, our data suggests that GnP protects against muscle and cardiac wasting in an experimental model of PDAC cachexia.
IL-6 Trans-Signaling and Crosstalk Among Tumor, Muscle and Fat Mediate Pancreatic Cancer Cachexia
(BioRxiv, 2020-09-17) Rupert, Joseph E.; Bonetto, Andrea; Narasimhan, Ashok; Liu, Yunlong; O’Connell, Thomas M.; Koniaris, Leonidas G.; Zimmers, Teresa A.; Biochemistry and Molecular Biology, School of Medicine
Most patients with pancreatic adenocarcinoma (PDAC) suffer unintentional weight loss, or cachexia. Interleukin-6 causes cachexia in mice and associates with mortality in PDAC. Here we show that tumor cell-derived IL-6 mediates crosstalk between tumor and peripheral tissues to promote cachexia. Tumor-cell IL-6 elicits expression of IL-6 in fat and IL-6 and IL-6 receptor (IL6R) in muscle, concomitantly raising both in blood. Inflammation-induced adipose lipolysis elevates circulating fatty acids, which cooperate with IL-6 to induce skeletal muscle dysmetabolism and wasting. Thus, PDAC induces crosstalk among tumor, fat and muscle via a feed-forward, IL-6 signaling loop. Tumor talks to muscle and fat through IL-6, and muscle to fat via IL6R trans-signaling, and fat to muscle through lipids and fatty acids. Disruption of this crosstalk by depletion of tumor-derived IL-6 halved fat wasting and abolished muscle loss, supporting IL-6, IL-6R and lipids as causal nodes for tissue crosstalk in PDAC cachexia. Significance PDAC-associated cachexia significantly increases patient morbidity and mortality. This study identifies muscle and fat crosstalk via IL6R trans-signaling in concert with muscle steatosis as a main driver of PDAC-associated cachexia.
In Vitro, In Vivo, and In Silico Methods for Assessment of Muscle Size and Muscle Growth Regulation
(Wolters Kluwer, 2020-04-14) Rupert, Joseph E.; Jengelley, Daenique H. A.; Zimmers, Teresa A.; Biochemistry and Molecular Biology, School of Medicine
Trauma, burn injury, sepsis, and ischemia lead to acute and chronic loss of skeletal muscle mass and function. Healthy muscle is essential for eating, posture, respiration, reproduction, and mobility, as well as for appropriate function of the senses including taste, vision, and hearing. Beyond providing support and contraction, skeletal muscle also exerts essential roles in temperature regulation, metabolism, and overall health. As the primary reservoir for amino acids, skeletal muscle regulates whole-body protein and glucose metabolism by providing substrate for protein synthesis and supporting hepatic gluconeogenesis during illness and starvation. Overall, greater muscle mass is linked to greater insulin sensitivity and glucose disposal, strength, power, and longevity. In contrast, low muscle mass correlates with dysmetabolism, dysmobility, and poor survival. Muscle mass is highly plastic, appropriate to its role as reservoir, and subject to striking genetic control. Defining mechanisms of muscle growth regulation holds significant promise to find interventions that promote health and diminish morbidity and mortality after trauma, sepsis, inflammation, and other systemic insults. In this invited review, we summarize techniques and methods to assess and manipulate muscle size and muscle mass in experimental systems, including cell culture and rodent models. These approaches have utility for studies of myopenia, sarcopenia, cachexia, and acute muscle growth or atrophy in the setting of health or injury.
Mechanical Effects of Fine-Wire Climbing on the Hindlimb Skeleton of Mice
(Office of the Vice Chancellor for Research, 2015-04-17) Joll, Jeffery E.; Vickery, Ben; Rupert, Joseph E.; Biro, Kelly C.; Wallace, Joseph M.; Byron, Craig D.; Organ, Jason M.
High-impact exercise (running/jumping) can stimulate multiple anabolic responses (increased trabecular bone volume, BV/TV) in the skeleton, but is also linked to an increased incidence of skeletal fracture. Thus, it is not an appropriate treatment for patients with elevated fracture risks. However, multi-directional offaxis mechanical loading can also elicit anabolic responses, even when magnitudes are relatively low. This represents a potential alternative to high-impact exercise for improving skeletal mechanical properties. To test this hypothesis, we raised twelve weanling female C57BL/6 mice to 4 months of age in custom enclosures that prevent (control) or require (experimental) manual and pedal grasping while balancing and climbing above narrow wire substrates. At sacrifice, we measured whole mouse bone density (DEXA) and performed architectural (μCT) and mechanical (4-pt bending) analyses of the femur and tibia. Body mass was similar between groups, although exercised mice were leaner (-35% fat mass). Bone mineral density was also similar, while bone mineral content was increased (+7%) in the exercised mice. Femoral midshaft polar moment of inertia was similar between groups, but exercised mice had lower BV/TV (-46%) of the distal femur and greater trabecular spacing (+21%). Exercised femora showed more total displacement (+58%) and post yield displacement (+115%) in bending than controls, and increased material toughness (+40%). Patterns were similar for the tibia. Mechanical data are consistent with high-impact exercise studies, but architectural data are not. Together they suggest that our exercise model may improve bone mechanical properties by redistributing mineral within the skeleton, and not by increasing net bone formation.
Mouse Hind Limb Skeletal Muscle Functional Adaptation in a Simulated Fine Branch Arboreal Habitat
(Wiley, 2018-03) Rupert, Joseph E.; Joll, J. Ethan; Elkhatib, Wiaam Y.; Organ, Jason M.; Anatomy and Cell Biology, School of Medicine
The musculoskeletal system is remarkably plastic during growth. The purpose of this study was to examine the muscular plasticity in functional and structural properties in a model known to result in significant developmental plasticity of the postcranial skeleton. Fifteen weanling C57BL/6 mice were raised to 16 weeks of age in one of two enclosures: a climbing enclosure that simulates a fine branch arboreal habitat and is traversed by steel wires crossing at 45° relative to horizontal at multiple intersections, and a control enclosure that resembles a parking deck with no wires but the same volume of habitable space. At killing, ex vivo contractility properties of the soleus (SOL) and extensor digitorum longus (EDL) muscles were examined. Our results demonstrate that EDL muscles of climbing mice contracted with higher specific forces and were comprised of muscle fibers with slower myosin heavy chain isoforms. EDL muscles also fatigued at a higher rate in climbing mice compared to controls. SOL muscle function is not affected by the climbing environment. Likewise, mass and architecture of both EDL and SOL muscles were not different between climbing and control mice. Our data demonstrate that functional adaptation does not require concomitant architectural adaptation in order to increase contractile force.

Browsing by Author "Rupert, Joseph E."

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