Diabetes mellitus is associated with various disorders of the locomotor system including the decline in mass and function of skeletal muscle. The mechanism underlying this association has remained ambiguous, however. We now show that the abundance of the transcription factor KLF15 as well as the expression of genes related to muscle atrophy are increased in skeletal muscle of diabetic model mice, and that mice with muscle-specific KLF15 deficiency are protected from the diabetes-induced decline of skeletal muscle mass. Hyperglycemia was found to upregulate the KLF15 protein in skeletal muscle of diabetic animals, which is achieved via downregulation of the E3 ubiquitin ligase WWP1 and consequent suppression of the ubiquitin-dependent degradation of KLF15. Our results revealed that hyperglycemia, a central disorder in diabetes, promotes muscle atrophy via a WWP1/KLF15 pathway. This pathway may serve as a therapeutic target for decline in skeletal muscle mass accompanied by diabetes mellitus.
Yu Hirata, Kazuhiro Nomura, Yoko Senga, Yuko Okada, Kenta Kobayashi, Shiki Okamoto, Yasuhiko Minokoshi, Michihiro Imamura, Shin’ichi Takeda, Tetsuya Hosooka, Wataru Ogawa
Mono-ADP-ribosylation of an (arginine) protein catalyzed by ADP-ribosyltransferase 1 (ART1) — i.e., transfer of ADP-ribose from NAD to arginine — is reversed by ADP-ribosylarginine hydrolase 1 (ARH1) cleavage of the ADP-ribose–arginine bond. ARH1-deficient mice developed cardiomyopathy with myocardial fibrosis, decreased myocardial function under dobutamine stress, and increased susceptibility to ischemia/reperfusion injury. The membrane repair protein TRIM72 was identified as a substrate for ART1 and ARH1; ADP-ribosylated TRIM72 levels were greater in ARH1-deficient mice following ischemia/reperfusion injury. To understand better the role of TRIM72 and ADP-ribosylation, we used C2C12 myocytes. ARH1 knockdown in C2C12 myocytes increased ADP-ribosylation of TRIM72 and delayed wound healing in a scratch assay. Mutant TRIM72 (R207K, R260K) that is not ADP-ribosylated interfered with assembly of TRIM72 repair complexes at a site of laser-induced injury. The regulatory enzymes ART1 and ARH1 and their substrate TRIM72 were found in multiple complexes, which were coimmunoprecipitated from mouse heart lysates. In addition, the mono-ADP-ribosylation inhibitors vitamin K1 and novobiocin inhibited oligomerization of TRIM72, the mechanism by which TRIM72 is recruited to the site of injury. We propose that a mono-ADP-ribosylation cycle involving recruitment of TRIM72 and other regulatory factors to sites of membrane damage is critical for membrane repair and wound healing following myocardial injury.
Hiroko Ishiwata-Endo, Jiro Kato, Akihiko Tonouchi, Youn Wook Chung, Junhui Sun, Linda A. Stevens, Jianfeng Zhu, Angel M. Aponte, Danielle A. Springer, Hong San, Kazuyo Takeda, Zu-Xi Yu, Victoria Hoffmann, Elizabeth Murphy, Joel Moss
The mitochondrial Ca2+ uniporter (MCU) complex mediates acute mitochondrial Ca2+ influx. In skeletal muscle, MCU links Ca2+ signaling to energy production by directly enhancing the activity of key metabolic enzymes in the mitochondria. Here, we examined the role of MCU in skeletal muscle development and metabolic function by generating mouse models for the targeted deletion of Mcu in embryonic, postnatal, and adult skeletal muscle. Loss of Mcu did not affect muscle growth and maturation or otherwise cause pathology. Skeletal muscle–specific deletion of Mcu in mice also did not affect myofiber intracellular Ca2+ handling, but it did inhibit acute mitochondrial Ca2+ influx and mitochondrial respiration stimulated by Ca2+, resulting in reduced acute exercise performance in mice. However, loss of Mcu also resulted in enhanced muscle performance under conditions of fatigue, with a preferential shift toward fatty acid metabolism, resulting in reduced body fat with aging. Together, these results demonstrate that MCU-mediated mitochondrial Ca2+ regulation underlies skeletal muscle fuel selection at baseline and under enhanced physiological demands, which affects total homeostatic metabolism.
Jennifer Q. Kwong, Jiuzhou Huo, Michael J. Bround, Justin G. Boyer, Jennifer A. Schwanekamp, Nasab Ghazal, Joshua T. Maxwell, Young C. Jang, Zaza Khuchua, Kevin Shi, Donald M. Bers, Jennifer Davis, Jeffery D. Molkentin
Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant or digenic disorder linked to derepression of the toxic DUX4 gene in muscle. There is currently no pharmacological treatment. The emergence of DUX4 enabled development of cell and animal models that could be used for basic and translational research. Since DUX4 is toxic, animal model development has been challenging, but progress has been made, revealing that tight regulation of DUX4 expression is critical for creating viable animals that develop myopathy. Here, we report such a model — the tamoxifen-inducible FSHD mouse model called TIC-DUX4. Uninduced animals are viable, born in Mendelian ratios, and overtly indistinguishable from WT animals. Induced animals display significant DUX4-dependent myopathic phenotypes at the molecular, histological, and functional levels. To demonstrate the utility of TIC-DUX4 mice for therapeutic development, we tested a gene therapy approach aimed at improving muscle strength in DUX4-expressing muscles using adeno-associated virus serotype 1.Follistatin (AAV1.Follistatin), a natural myostatin antagonist. This strategy was not designed to modulate DUX4 but could offer a mechanism to improve muscle weakness caused by DUX4-induced damage. AAV1.Follistatin significantly increased TIC-DUX4 muscle mass and strength even in the presence of DUX4 expression, suggesting that myostatin inhibition may be a promising approach to treat FSHD-associated weakness. We conclude that TIC-DUX4 mice are a relevant model to study DUX4 toxicity and, importantly, are useful in therapeutic development studies for FSHD.
Carlee R. Giesige, Lindsay M. Wallace, Kristin N. Heller, Jocelyn O. Eidahl, Nizar Y. Saad, Allison M. Fowler, Nettie K. Pyne, Mustafa Al-Kharsan, Afrooz Rashnonejad, Gholamhossein Amini Chermahini, Jacqueline S. Domire, Diana Mukweyi, Sara E. Garwick-Coppens, Susan M. Guckes, K. John McLaughlin, Kathrin Meyer, Louise R. Rodino-Klapac, Scott Q. Harper
Patients with severe, treatment-refractory asthma are at risk for death from acute exacerbations. The cytokine IL17A has been associated with airway inflammation in severe asthma, and novel therapeutic targets within this pathway are urgently needed. We recently showed that IL17A increases airway contractility by activating the procontractile GTPase RhoA. Here, we explore the therapeutic potential of targeting the RhoA pathway activated by IL17A by inhibiting RhoA guanine nucleotide exchange factors (RhoGEFs), intracellular activators of RhoA. We first used a ribosomal pulldown approach to profile mouse airway smooth muscle by qPCR and identified Arhgef12 as highly expressed among a panel of RhoGEFs. ARHGEF12 was also the most highly expressed RhoGEF in patients with asthma, as found by RNA sequencing. Tracheal rings from Arhgef12-KO mice and WT rings treated with a RhoGEF inhibitor had evidence of decreased contractility and RhoA activation in response to IL17A treatment. In a house dust mite model of allergic sensitization, Arhgef12-KO mice had decreased airway hyperresponsiveness without effects on airway inflammation. Taken together, our results show that Arhgef12 is necessary for IL17A-induced airway contractility and identify a therapeutic target for severe asthma.
Valerie Fong, Austin Hsu, Esther Wu, Agnieszka P. Looney, Previn Ganesan, Xin Ren, Dean Sheppard, Sarah A. Wicher, Michael A. Thompson, Rodney D. Britt Jr., Y.S. Prakash, Mallar Bhattacharya
Connexin 43 (Cx43), a product of the GJA1 gene, is a gap junction protein facilitating intercellular communication between cardiomyocytes. Cx43 protects the heart from ischemic injury by mechanisms that are not well understood. GJA1 mRNA can undergo alternative translation, generating smaller isoforms in the heart, with GJA1-20k being the most abundant. Here, we report that ischemic and ischemia/reperfusion (I/R) injuries upregulate endogenous GJA1-20k protein in the heart, which targets to cardiac mitochondria and associates with the outer mitochondrial membrane. Exploring the functional consequence of increased GJA1-20k, we found that AAV9-mediated gene transfer of GJA1-20k in mouse hearts increases mitochondrial biogenesis while reducing mitochondrial membrane potential, respiration, and ROS production. By doing so, GJA1-20k promotes a protective mitochondrial phenotype, as seen with ischemic preconditioning (IPC), which also increases endogenous GJA1-20k in heart lysates and mitochondrial fractions. As a result, AAV9-GJA1-20k pretreatment reduces myocardial infarct size in mouse hearts subjected to in vivo ischemic injury or ex vivo I/R injury, similar to an IPC-induced cardioprotective effect. In conclusion, GJA1-20k is an endogenous stress response protein that induces mitochondrial biogenesis and metabolic hibernation, preconditioning the heart against I/R insults. Introduction of exogenous GJA1-20k is a putative therapeutic strategy for patients undergoing anticipated ischemic injury.
Wassim A. Basheer, Ying Fu, Daisuke Shimura, Shaohua Xiao, Sosse Agvanian, Diana M. Hernandez, Tara C. Hitzeman, TingTing Hong, Robin M. Shaw
Physiological and premature aging are frequently associated with an accumulation of prelamin A, a precursor of lamin A, in the nuclear envelope of various cell types. Here, we aimed to underpin the hitherto unknown mechanisms by which prelamin A alters myonuclear organization and muscle fiber function. By experimentally studying membrane-permeabilized myofibers from various transgenic mouse lines, our results indicate that, in the presence of prelamin A, the abundance of nuclei and myosin content is markedly reduced within muscle fibers. This leads to a concept by which the remaining myonuclei are very distant from each other and are pushed to function beyond their maximum cytoplasmic capacity, ultimately inducing muscle fiber weakness.
Yotam Levy, Jacob A. Ross, Marili Niglas, Vladimir A. Snetkov, Steven Lynham, Chen-Yu Liao, Megan J. Puckelwartz, Yueh-Mei Hsu, Elizabeth M. McNally, Manfred Alsheimer, Stephen D.R. Harridge, Stephen G. Young, Loren G. Fong, Yaiza Español, Carlos Lopez-Otin, Brian K. Kennedy, Dawn A. Lowe, Julien Ochala
Zebrafish are a powerful tool for studying muscle function owing to their high numbers of offspring, low maintenance costs, evolutionarily conserved muscle functions, and the ability to rapidly take up small molecular compounds during early larval stages. Fukutin-related protein (FKRP) is a putative protein glycosyltransferase that functions in the Golgi apparatus to modify sugar chain molecules of newly translated proteins. Patients with mutations in the FKRP gene can have a wide spectrum of clinical symptoms with varying muscle, eye, and brain pathologies depending on the location of the mutation in the FKRP protein. Patients with a common L276I FKRP mutation have mild adult-onset muscle degeneration known as limb-girdle muscular dystrophy 2I (LGMD2I), whereas patients with more C-terminal pathogenic mutations develop the severe Walker-Warburg syndrome (WWS)/muscle-eye-brain (MEB) disease. We generated fkrp-mutant zebrafish that phenocopy WWS/MEB pathologies including severe muscle breakdowns, head malformations, and early lethality. We have also generated a milder LGMD2I-model zebrafish via overexpression of a heat shock–inducible human FKRP (L276I) transgene that shows milder muscle pathology. Screening of an FDA-approved drug compound library in the LGMD2I zebrafish revealed a strong propensity towards steroids, antibacterials, and calcium regulators in ameliorating FKRP-dependent pathologies. Together, these studies demonstrate the utility of the zebrafish to both study human-specific FKRP mutations and perform compound library screenings for corrective drug compounds to treat muscular dystrophies.
Peter R. Serafini, Michael J. Feyder, Rylie M. Hightower, Daniela Garcia-Perez, Natássia M. Vieira, Angela Lek, Devin E. Gibbs, Omar Moukha-Chafiq, Corinne E. Augelli-Szafran, Genri Kawahara, Jeffrey J. Widrick, Louis M. Kunkel, Matthew S. Alexander
Generation of homogeneous populations of subtype-specific cardiomyocytes (CMs) derived from human induced pluripotent stem cells (iPSCs) and their comprehensive phenotyping is crucial for a better understanding of the subtype-related disease mechanisms and as tools for the development of chamber-specific drugs. The goals of this study were to apply a simple and efficient method for differentiation of iPSCs into defined functional CM subtypes in feeder-free conditions and to obtain a comprehensive understanding of the molecular, cell biological, and functional properties of atrial and ventricular iPSC-CMs on both the single-cell and engineered heart muscle (EHM) level. By a stage-specific activation of retinoic acid signaling in monolayer-based and well-defined culture, we showed that cardiac progenitors can be directed towards a highly homogeneous population of atrial CMs. By combining the transcriptome and proteome profiling of the iPSC-CM subtypes with functional characterizations via optical action potential and calcium imaging, and with contractile analyses in EHM, we demonstrated that atrial and ventricular iPSC-CMs and -EHM highly correspond to the atrial and ventricular heart muscle, respectively. This study provides a comprehensive understanding of the molecular and functional identities characteristic of atrial and ventricular iPSC-CMs and -EHM and supports their suitability in disease modeling and chamber-specific drug screening.
Lukas Cyganek, Malte Tiburcy, Karolina Sekeres, Kathleen Gerstenberg, Hanibal Bohnenberger, Christof Lenz, Sarah Henze, Michael Stauske, Gabriela Salinas, Wolfram-Hubertus Zimmermann, Gerd Hasenfuss, Kaomei Guan
Cardiac myosin binding protein C (MYBPC3) is the most commonly mutated gene associated with hypertrophic cardiomyopathy (HCM). Haploinsufficiency of full-length MYBPC3 and disruption of proteostasis have both been proposed as central to HCM disease pathogenesis. Discriminating the relative contributions of these 2 mechanisms requires fundamental knowledge of how turnover of WT and mutant MYBPC3 proteins is regulated. We expressed several disease-causing mutations in MYBPC3 in primary neonatal rat ventricular cardiomyocytes. In contrast to WT MYBPC3, mutant proteins showed reduced expression and failed to localize to the sarcomere. In an unbiased coimmunoprecipitation/mass spectrometry screen, we identified HSP70-family chaperones as interactors of both WT and mutant MYBPC3. Heat shock cognate 70 kDa (HSC70) was the most abundant chaperone interactor. Knockdown of HSC70 significantly slowed degradation of both WT and mutant MYBPC3, while pharmacologic activation of HSC70 and HSP70 accelerated degradation. HSC70 was expressed in discrete striations in the sarcomere. Expression of mutant MYBPC3 did not affect HSC70 localization, nor did it induce a protein folding stress response or ubiquitin proteasome dysfunction. Together these data suggest that WT and mutant MYBPC3 proteins are clients for HSC70, and that the HSC70 chaperone system plays a major role in regulating MYBPC3 protein turnover.
Amelia A. Glazier, Neha Hafeez, Dattatreya Mellacheruvu, Venkatesha Basrur, Alexey I. Nesvizhskii, Lap Man Lee, Hao Shao, Vi Tang, Jaime M. Yob, Jason E. Gestwicki, Adam S. Helms, Sharlene M. Day
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