Profiling age-related muscle weakness and wasting: neuromuscular junction transmission as a driver of age-related physical decline
Physiological changes that lead to skeletal muscle dysfunction and atrophy during aging are poorly understood and thus therapeutic interventions which
Efficient precise in vivo base editing in adultdystrophic mice
Duchenne muscular dystrophy (DMD) is a muscular disease caused by alterations in dystrophin expression, which has a functional role in muscle force transmission and sarcolemma stability. As such, a loss of dystrophin can lead to muscle weakening and wasting, cardiomyopathy, and several other health conditions. This study focuses on the efficacy of using NG-targeting base editors to restore dystrophin levels in mdx4cv mice. Five weeks following base editor AAv9-iNG injection, mdx4cv mouse hearts exhibited increased levels of dystrophin. Furthermore, approximately 42% of cardiomyocytes became dystrophin-positive following systemic AAV9-iNG treatment, whereas dystrophin expression remained almost completely absent in the control group. A long-term analysis conducted at the 10-month mark showed that dystrophin was almost completely rescued in mdx4cv mouse hearts. In addition to this, dystrophin rescue was also observed in skeletal muscles, including the gastrocnemius and diaphragm, of mdx4cv mice treated with AAV9-iNG. Long-term analysis following intravenous administration of AAV9-iNG also showed almost complete dystrophin restoration (95% of wild type level) in mdx4cv hearts. Through trichome staining analysis, the increased percentage of fibrotic areas seen in the muscle of mdx4cv mice was significantly reduced in those treated with the base editor. Lastly, to determine if AAV9-iNG could improve muscle function, the authors used Aurora’s 1300A 3-in-1 Whole Animal System to measure the maximum plantarflexion tetanic torque during supramaximal electric stimulation of the tibial nerve. The results showed that mdx4cv mice produced significantly less torque compared to wild type mice. Moreover, a significant increase in tetanic torque was observed in AAV9-iNG treated mdx4cv mice. These findings underscore the importance of optimized base editing techniques and their potential use in treating monogenic diseases such as DMD.
Dose-Escalation Study of Systemically Delivered rAAVrh74.MHCK7.micro-dystrophin in the mdx Mouse Model of Duchenne Muscular Dystrophy
Duchenne muscular dystrophy (DMD) is a rare neuromuscular disease caused by mutations in the DMD gene. These mutations disrupt the expression of dystrophin, a protein important for muscle fiber stability. DMD is characterized by a progressive loss of skeletal and cardiac muscle strength. This study focuses on characterizing the effects of rAAVrh74.MHCK7.micro-dystrophin, an adeno-associated virus vector containing a codon-optimized human micro-dystrophin transgene, on DMD mice. The authors administered systemic injections of the dystrophin-restoring vector to mdx mice at low, intermediate, and high doses. Three months post-treatment, they analyzed micro-dystrophin positive fibers via immunofluorescent staining. They observed mean expression percentages of 46.7%, 66.8%, and 78.3% for low, intermediate, and high doses, respectively, across all muscles assessed (TA, GAS, QD, PSO, TRI, GLUT and DIA). Fiber diameter and fibrosis were then assessed, where low and intermediate doses reduced fibrosis and normalized fiber size in the diaphragm, similar to that of wild type. Force production was analyzed using Aurora’s 1200A isolated muscle system. In vitro analysis of the dystrophic diaphragm revealed improvement in specific force output following high dose treatment similar to that of wild type. To determine if restoring dystrophin would have a functional effect on hindlimb muscle, the authors further assessed the tibialis anterior (TA) in situ using the 1300A Whole Animal System. rAAVrh74.MHCK7.micro-dystrophin not only improved force output in the TA but rescued the muscle from eccentric contraction-induced damage. Lastly, western blot analysis of tissues from injected mice showed micro-dystrophin protein expression across all skeletal muscles. No micro-dystrophin was detected in off-target organs at low and intermediate doses, with high dose exhibiting faint expression in only the liver. These findings demonstrate safety and efficacy of systemic delivery of rAAVrh74.MHCK7.micro-dystrophin, supporting the use of this vector in Phase I/II safety study in boys with DMD.
Influence of external forces on actin-dependent T cell protrusions during immune synapse formation
T-cell activation is necessary for producing an adaptive immune response. For T-cell activation to occur, an antigen-presenting cell (APC) must make direct contact with it. This contact region has been shown to control many T-cell functions, and it is from this contact point that protrusions have been observed. To further characterize these protrusions, this study investigated the effect of various APC-mimicking forces on protrusion dynamics. To mimic APC-induced T-cell activation, a micropipette force probe attached to an activating microbead was used. To evaluate micropipette flexibility, the bending stiffness was measured against a microindenter calibrated with Aurora’s 406A force probe. T-cell activation was induced once the microbead would make contact with the cell at a desired compressive or pulling force. To determine if protrusion was affected by external forces produced by APCs during activation, protrusion lengths were monitored following the application of various forces. It was found that the higher the compressive force, the shorter the protrusion was. Conversely, the larger the absolute pulling force was, the longer the protrusion grew. It was also observed longer protrusions were smaller in diameter, at all forces. To determine whether protrusion dynamics were affected by Arp2/3, a protein complex regulating actin polymerization, T-cells were treated with an Arp2/3-specific inhibitor. Not only were protrusion maximum lengths reduced following treatment, but they began growth later in time and at slower speeds. Finally, confocal imaging was used to assess F actin localization within protrusions. The results showed that the walls were rich with F actin whereas the center was depleted. A similar analysis of T cells and model APCs showed the same F actin localization. This study suggests that protrusion growth is set by an intracellular constant time and that protrusion dynamics are influenced by external forces. In addition to this, actin assembly within protrusions is facilitated by the Arp2/3 complex.
Disparate bone anabolic cues activate bone formation by regulating the rapid lysosomal degradation of sclerostin protein
Sclerostin is an osteocyte-derived glycoprotein with an inhibitory role in bone formation. Deletion of the Sost gene, which controls sclerostin protein expression, has been shown to increase bone mass in mice. As such, sclerostin shows therapeutic potential in treating bone mass conditions such as osteoporosis. This study focuses on elucidating the molecular control of this protein in both in vitro and in vivo settings. Cell lines Ocy454 and UMR106 were first subjected to fluid shear stress-mediated bone mechanical loading. This resulted in a loss of sclerostin protein abundance. An in vivo assessment was then conducted where mice were subjected to ulnar loads using Aurora’s 305C Dual-Mode lever system. Ulnae were harvested post-load and osteocytes were assessed, showing reduced sclerostin abundance compared to limbs without loading. A previous study showed that NOX2-mediated ROS is an essential part of the mechano-transduction pathway involving sclerostin protein loss. To confirm these findings in a whole animal analysis, the authors analyzed the effect of ulnar loading on mice pre-treated with apocynin, a NOX2 inhibitor. The findings showed that apocynin prevented bone formation that was otherwise load-induced. Therefore, NOX2 ROS was found to be necessary for sclerostin degradation as inhibition of NOX2 lessened the degradation of sclerostin. Furthermore, it was found that inhibition of lysosomal degradation reduced sclerostin protein degradation. These results suggest that inhibiting NOX2 or lysosomal function prevents load-induced sclerostin degradation and subsequent bone formation. The authors then analyzed induced pluripotent stem cell (iPSC)-derived osteoblasts from patients with Gaucher disease, a disease categorized as a lysosomal storage disorder. This analysis showed a significant increase in levels of sclerostin compared to iPSC-derived osteoblasts from patients without Gaucher disease. It was also found that treating Gaucher iPSC-derived osteoblasts with an enzyme restoring lysosomal function resulted in a decrease in sclerostin. These data provide insights into the molecular regulation of osteocyte sclerostin protein and shed light on potential therapeutic targets in the treatment of bone-related conditions.
Impact of syndecan-2-selected mesenchymal stromal cells on the early onset of diabetic cardiomyopathy in diabetic db/db mice
Diabetic cardiomyopathy is characterized by structural and functional alterations to the heart including inflammation, fibrosis, and muscle stiffness. These alterations can ultimately lead to dysfunction in the left ventricle (LV). Bone marrow-derived mesenchymal stromal cells (MSCs) have many potential cardiac-aiding properties with demonstrated pro-angiogenic, anti-fibrotic, and general immunomodulatory effects. This study focuses on characterizing the ability of MSCs to reduce cardiomyopathic alterations in a mouse model for human type 2 diabetes mellitus. Twenty-week old db/db and control mice were administered CD362+, CD362−, and wild type MSCs intravenously. After four weeks, cardiomyocyte passive force (Fpassive) was assessed as a measure of stiffness. Using Aurora’s Permeabilized Myocyte Test System (1600A), it was found that all three MSCs had restorative effect on previously elevated Fpassive in db/db cardiomyocytes, with the effect of CD362+ being the least pronounced. Similarly, all three MSCs reestablished titin phosphorylation-regulating nitric oxide (NO) and cGMP levels to those seen in control mice, with CD362+ having the smallest effect. Arteriole density was then assessed to evaluate the pro-angiogenic properties of MSCs. CD362- and wild type were most effective in increasing arteriole density in db/db mice. Lastly, due to their marked influence on the progression of heart failure, a splenic cell analysis was conducted. Diabetic mice showed higher percentages of splenic apoptotic Tregs, lower amounts anti-inflammatory cells, and increased splenic pro-inflammatory cells. Application of all MSCs resulted in lower percentages of apoptotic Tregs compared to control mice. Treatment with CD362+ and wild type MSCs also resulted in increased anti-inflammatory splenic cells, whereas treatment with CD362- MSCs was most effective at decreasing pro-inflammatory cells. Overall, this study shows that the application of MSCs can reduce cardiomyocyte stiffness, restore NO and cGMP levels, and increase arteriole densities in diabetic mice, although CD362+ MSC were less effective.
Long-Term Evaluation of Functional Outcomes Following Rat Volumetric Muscle Loss Injury and Repair
Tissue-engineered muscle repair (TEMR) technology is used to facilitate the regeneration of muscle by providing a favorable microenvironment for regenerative growth. This is done by seeding muscle progenitor cells (MPCs) onto a porcine bladder acellular matrix (BAM), which is then implanted into the animal. Although tissue-engineered constructs are a good restorative option for volumetric muscle loss (VML) injuries, the variability between different muscles poses a challenge in creating fully compatible constructs. This study focuses on improving the matching geometry of TEMR to the TA muscle, specifically, in the rat animal model. Using Aurora’s 1305A 3-in-1 Whole Animal System, an in vivo analysis of peak isometric torque was conducted. At 6 months post VML injury, 67% of TEMR-implanted rats showed significantly greater peak isometric torque compared to other treatment groups. Moreover, 38% of TEMR responders reached approximately 90% of the maximum force production, thus demonstrating near full recovery. In addition to functional assessment, the authors conducted histological and immunofluorescence analyses. Fiber cross sectional area (FCSA) was quantified in both the experimental and control TA muscles of maximum responder rats. It was found that the median FCSA was lower in the experimental TA muscles than in the TA muscles of the control leg. Vascularization and macrophage counts were also assessed, although no significant differences were found. This study highlights the importance of adapting and improving existing tissue engineering technology to allow for optimal treatment of VML injuries in various muscles.