American Journal of Physiology-Cell Physiology

BackgroundSkeletal muscle in wasting conditions often exhibits a common set of phenotypes that include atrophy, mitochondrial respiratory dysfunction, and fragmentation of the acetylcholine receptor (AChR) cluster at the endplate. Mitochondria are frequently implicated in driving muscle pathology in these conditions, although which aspects of mitochondrial function are most relevant is poorly understood. MethodsTo address this gap, we focused on mitochondrial permeability transition (mPT), a well-established pathological mechanism in ischemia-reperfusion injury and neurodegeneration but poorly studied in skeletal muscle. We performed a broad assessment of the consequences of mPT in skeletal muscle, focusing on features that are common in wasting conditions. We then tested whether tumor-host factors could promote mPT and compared differentially expressed genes (DEGs) with mPT and a mouse model of pancreatic cancer cachexia. ResultsInducing mPT in mouse skeletal muscle bundles in a Ca2+ retention capacity assay progressively altered mitochondrial morphology, beginning with cristae swirling and condensation, progressing to mitochondrial cristae displacement, and culminating in breach of the outer mitochondrial membrane; features that are common in wasting conditions. Inducing mPT with Bz423 in single mouse muscle fibers increased mROS and Caspase 3 (Casp3) activity and was prevented by inhibitors of mPT, mROS or Casp3. Incubating single muscle fibers with Bz423 for 24 h reduced fiber diameter by [~]20% which was prevented by inhibiting mPT, mROS, or Casp3. Inducing mPT caused a complex I-specific mitochondrial respiratory impairment and increased co-localization of lysosomes with mitochondria. Inducing mPT also fragmented the AChR cluster at the muscle endplate and was prevented by inhibiting mPT or Casp3. The Ca2+ threshold for mPT and mitochondrial calcein colocalization were reduced by pancreatic tumor-conditioned media in skeletal muscle or C2C12 myoblasts, respectively, and these effects were counteracted by mPT inhibition or cyclophilin D knockout. Finally, there was significant overlap between the transcriptome of mPT and that seen in diaphragm muscle in a mouse model of pancreatic cancer cachexia, particularly during the muscle wasting phase. ConclusionsWe conclude that inducing mPT in skeletal muscle recapitulates muscle phenotypes common with muscle wasting conditions like cachexia. Furthermore, mPT is engaged by tumor-host factors and had significant overlap with DEGs seen during the muscle wasting phase in a mouse model of pancreatic cancer cachexia, warranting further investigation of mPT as a therapeutic target.

BackgroundPeripheral nerve trauma denervates skeletal muscle resulting in paralysis and atrophy that is reversible if timely reinnervation occurs, due to its regenerative capacity. If reinnervation is delayed muscles regenerative ability is exhausted and resident fibroadipogenic progenitors (FAPs) differentiate into adipocytes and fibroblasts that replace muscle with non-contractile fibrotic tissue and fat, resulting in physical disability. Prostaglandin E2 (PGE2) inhibits adipogenesis and fibrosis in other tissues. We determined whether PGE2 could inhibit fibro-fatty degradation of long-term denervated muscle. MethodsWe utilized the rat tibial nerve transection model, denervating the gastrocnemius and selected a 5 week post-denervation time point to represent short-term muscle denervation injury (reversible with reinnervation), and 12 weeks to represent sustained, irreversible injury. Gastrocnemius FAPs were isolated via FACS and grown in culture to assess endogenous PGE2 production and the proliferative and differentiation response to exogenous PGE2. We evaluated transcript and protein expression of PGE2 synthesizing enzyme PTGS2, PGE2 degrading enzyme 15-PGDH and markers of proliferation, adipogenesis and fibrogenesis using RT-qPCR, immunofluorescence and SDS-PAGE/Western blotting. Paracrine impact of FAPs produced PGE2 was assessed by treating C2C12 myoblasts with FAPs conditioned media. ResultsTranscript expression of PTGS2 was increased and 15-PGDH decreased (4.37{+/-}2.63 and -3.06{+/-}0.85 fold change respectively, p<0.05) in 5 week, but not 12 week denervated gastrocnemius, consistent with increased PGE2 production in 5 week denervated muscle. Similarly, PTGS2 transcript levels were significantly increased (2.58{+/-}0.33 fold change, p<0.05) and 15-PGDH decreased (-5.24{+/-}3.19 fold change, p<0.05) in FAPs isolated from 5 week, but not 12 week denervated muscle, demonstrating that FAPs are a source of PGE2 in short-term denervated muscle. 16,16-dimethyl PGE2 did not impact naive FAPs in vitro proliferation, but significantly inhibited their differentiation as demonstrated by 88.9%, 82.3% and 94.2% decreases in FAPs expression of adipogenic marker perilipin-1, fibrogenic marker -smooth muscle actin (-SMA) and lipid content respectively, mediated via PGE2 binding to the FAPs EP4 receptor. FAPs isolated from 12 week denervated muscle demonstrated increased adipogenesis and fibrogenesis vs. naive FAPs (perilipin-1 and -SMA 7.93{+/-}2.96 and 2.00{+/-}0.33 fold increase respectively, p<0.05) and remained fully susceptible to PGE2 inhibition of fibro-adipogenic differentiation. Conditioned media from FAPs derived from 5 week, but not 12 week, denervated gastrocnemius stimulated C2C12 myoblast proliferation which was prevented by EP4 blockade. ConclusionsPGE2 is identified as a novel negative regulator of FAPs differentiation in traumatically denervated muscle, suggesting the therapeutic potential of PGE2 to prevent fibro-fatty degradation of long-term denervated muscle awaiting reinnervation.

Duchenne muscular dystrophy (DMD) is a fatal genetic disorder characterized by skeletal muscle degeneration and cardiomyopathy without a cure. This study examined the therapeutic potential of the sodium-glucose cotransporter 2 (SGLT2) inhibitor empagliflozin (EMPA) on cardiac function in the dystrophin-deficient mdx mouse model of DMD. Male mice were fed control chow or EMPA-containing chow ([~]25 mg/kg/day), and cardiac function was evaluated longitudinally by four-dimensional ultrasound imaging. EMPA did not alter left ventricular mass or chamber volume but preserved ejection fraction (EF) for 12 weeks, maintained significantly higher EF through 24 weeks, and attenuated global impairment of systolic and diastolic myocardial deformation. These functional improvements were accompanied by reduced cardiomyocyte hypertrophy and decreased expression of cardiac stress genes. EMPA reduced mitochondrial DNA damage, increased mitochondrial DNA copy number, and induced transcriptional signatures consistent with enhanced fatty acid and ketone metabolism, contributing to increased myocardial ATP content. Systemically, EMPA improved body mass trajectory, preserved relative lean mass, enhanced skeletal muscle torque, and did not adversely affect renal function. Together, these findings demonstrate that EMPA improves cardiac performance and mitochondrial integrity while enhancing myocardial energy availability in mdx mice, supporting SGLT2 inhibitors as a promising therapeutic strategy for individuals with DMD.

Progressive cardiomyopathy is the leading cause of death in Duchenne muscular dystrophy (DMD). Dysregulation of calcium handling has been implicated in cardiomyopathy progression in DMD. Here we describe a therapeutic approach to improve calcium homeostasis in a mouse model of DMD using the novel therapeutic NDC-1171, which is a positive allosteric modulator of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pump. We synthesized NDC-1171 and treated 4-week-old D2.mdx mice (n=9) via oral gavage. A group of D2.mdx mice (n=9) and a group of DBA/2J mice (n=9; background strain) received a vehicle on the same schedule. We used ultrasound to assess left ventricular function, followed by a treadmill exhaustion test and a 4-paw grip strength test to assess skeletal muscle function. NDC-1171 attenuated cardiac functional decline in D2.mdx mice. At 16 weeks of age, left ventricular ejection fraction (LVEF) was significantly preserved in mice treated with NDC-1171 (57.7{square}{+/-}{square}0.5%) compared to mice treated with a vehicle (50.7{square}{+/-}{square}0.9%, p{square}<{square}0.05), though remained lower than background strain controls (62.4{square}{+/-}{square}0.6%). In contrast, functional behavior testing revealed no significant improvement in skeletal muscle function with treatment. These data suggest that treatment with the SERCA pump modulator NDC-1171 helps preserve cardiac function in a murine model of DMD, even as skeletal muscle function was impaired. Future work will be needed to determine if the benefits of this novel SERCA activator translate to large animal and clinical studies, but these initial results are promising and could help guide development of future treatments for pediatric patients with muscular dystrophy.

Myocyte Enhancer Factor 2D (Mef2D) is a member of the Mef2 family. As a transcription factor, Mef2D regulates the expression of genes that impinge on cellular viability, tissue development, and fuel metabolism in a tissue dependent manner. Mef2D is expressed in the beta-cell, and overexpression and knockdown have been shown to modulate glucose stimulated insulin secretion. We sought to understand the role of Mef2D on beta-cell function and survival. To determine the function of Mef2D in the beta-cell, we built overexpression and knockdown INS-1 832/13 cell lines. We determined the effect of Mef2D overexpression or knockdown on mitochondrial respiration, insulin secretion, cell survival, and gene expression. Our data demonstrates that Mef2D knockdown enhances mitochondrial respiration, insulin secretion, and cell survival. Conversely, Mef2D overexpression inhibits mitochondrial respiration, insulin secretion, and cell survival. We demonstrate that some of this effect is due to modulated expression of the mitochondrial gene mtND6. These findings demonstrate that Mef2D overexpression is detrimental to beta-cell function and that Mef2D knockdown is beneficial. These data suggest that Mef2D may be a viable target to enhance functional beta-cell mass as a treatment for Type 1 and Type 2 Diabetes.

BackgroundQuadriceps weakness may reduce sagittal plane shock absorption during landing, shifting load toward the frontal plane and increasing knee abduction moment (KAM), a biomechanical risk factor for anterior cruciate ligament (ACL) injuries. PurposeThe purpose of this study was to evaluate the association between isokinetic quadriceps strength and peak KAM during drop vertical jump landing in adolescent athletes. Study DesignSecondary analysis of previously collected data. MethodsHealthy adolescent athletes completed quadriceps strength testing using an isokinetic dynamometer and a biomechanical assessment during a drop vertical jump task. Quadriceps strength was quantified as peak concentric torque and the peak external KAM was calculated during the landing phase on the dominant limb. Both strength and KAM were normalized to body mass. Linear regression was used to examine the association between normalized quadriceps strength and peak external KAM on the dominant limb. ResultsThe association between quadriceps strength and peak normalized KAM on the dominant limb was not statistically significant ({beta} = -0.053 (95% CI [-0.137 to 0.030]), F(1,119) = 1.62, R2 = 0.013, p = 0.206). Quadriceps strength explained only 1.3% of the variance in peak KAM, indicating a negligible association between these variables in this cohort. DiscussionQuadriceps strength was not associated with peak normalized KAM during landing, suggesting that frontal-plane knee loading during a drop vertical jump is not meaningfully explained by maximal concentric quadriceps strength alone. KAM appears to be driven more by multi-joint movement strategy and neuromuscular coordination than by the capacity of a single muscle group.

BackgroundPsychological readiness to return to sport and subjective knee function are critical outcomes following ACL reconstruction (ACLR), yet they do not always progress in parallel. An athlete may demonstrate high subjective knee function but low psychological readiness, suggesting a mental barrier to return, or conversely, report high readiness despite persistent functional limitations, raising concerns of overconfidence and reinjury risk. Understanding how these domains change together during recovery is essential for identifying mismatches that may require targeted intervention. PurposeThe purpose of this study is to examine the relationship between changes in psychological readiness (ACL-RSI) and subjective knee function (IKDC) from early to late recovery following ACLR. Study DesignSecondary analysis of prospectively collected data. MethodsAthletes (N = 48, Age at ACLR = 17.7 {+/-} 1.8 y) aged 15-25 years who underwent ACLR with an ipsilateral autograft, had a pre-injury MARX score > 8, and completed the ACL-RSI and IKDC questionnaires at 3.5 {+/-} 1 and 7 {+/-} 1 months post-ACLR were included. Percent changes in ACL-RSI and IKDC scores between early and late recovery were calculated. Spearmans rank correlation was used to examine the association between changes in psychological readiness and subjective knee function. Significance was set to p < .05. ResultsThe mean percent change in ACL-RSI was 40.7 {+/-} 57.1% and the mean percent change in IKDC was 24.8 {+/-} 18.1% from 3.5 {+/-} 1 months to 7 {+/-} 1 months post-ACLR. The percent changes in ACL-RSI and IKDC scores from 3.5 {+/-} 1 months to 7 {+/-} 1 months post-ACLR were moderately correlated ({rho} = 0.350 (95% CI [0.089, 0.584]), p = 0.012). DiscussionThe main finding of this study was that subjective knee function and psychological readiness to return to sport changed in parallel from 3.5 to 7 months following ACLR. Clinicians can use this information regarding the concordant progression of psychological readiness to return to sport and subjective knee function to personalize ACL rehabilitation for future patients. Overall, clinicians can understand that if psychological readiness improves, subjective knee function will likely improve over the 3.5- to 7-month post-ACLR time frame, and vice versa. Therefore, focusing on both of these components at multiple time points during the recovery process may be influential to ensure the greatest likelihood of returning to sport in athletes following ACLR.

BackgroundDiabetes mellitus leads to skeletal muscle dysfunction associated with loss of strength, impaired blood perfusion, lipid accumulation, and inflammation. The opening of large-pore channels has been linked to increased membrane permeability and inflammatory signaling in several pathologies. Boldine, an alkaloid from Peumus boldus, blocks large-pore channel activity and exhibits antioxidant and anti-inflammatory properties. This study evaluated whether boldine prevents skeletal muscle alterations induced by diabetes and explored potential underlying mechanisms. MethodsDiabetes was induced in male C57BL/6J mice using streptozotocin (STZ, 40 mg/kg/day for 5 days). Diabetic mice were treated with boldine (50 mg/kg/day) for four weeks. Muscle strength and resting membrane potential were analyzed in vivo. Also, right gastrocnemius muscle blood perfusion at basal and after acetylcholine (10 M) stimulation were analyzed in vivo. Lipid accumulation was assessed using Oil Red O staining, and CD31 immunodetection was used to evaluate capillary density. mRNA levels of NLRP3 were evaluated in muscle by qPCR. In human myoblasts (AB1167) cultured under low (8 mM) or high glucose (25 mM) conditions, with or without boldine, membrane permeability (ethidium uptake), intracellular Ca{superscript 2} (Fura-2), nitric oxide (DAF-FM), and levels of NLRP3 and Casp1 (qPCR) and reactivity PPAR{gamma} (Immunofluorescence) were determined. ResultsSTZ mice showed reduced muscle strength and depolarized resting membrane potential, both prevented by boldine. Basal muscle perfusion was [~]20% lower in diabetic mice (160.1 {+/-} 17.2 vs. 199.1 {+/-} 13.8 units), whereas boldine preserved perfusion (184.6 {+/-} 14.3 units). Oil Red O-positive fibers increased to 52.4 {+/-} 3.6% in diabetic mice and decreased to 15.2 {+/-} 4.1% with boldine (control: 3.1 {+/-} 1.3%; p<0.05). NLRP3 mRNA increased 17.7 {+/-} 2.8-fold in diabetic muscle and was reduced by [~]50% with boldine. In myoblasts, high glucose increased ethidium uptake, nitric oxide production, NLRP3 and caspase-1 expression, and nuclear PPAR{gamma} ([~]45% positive nuclei); all effects were prevented by boldine. ConclusionsBoldine preserves skeletal muscle function and vascular reactivity in diabetes and prevents lipid accumulation and inflammasome activation both in vivo and in vitro. These effects are associated with inhibition of large-pore channel activity and attenuation of downstream calcium-dependent, inflammatory, and adipogenic pathways, supporting boldine as a promising therapeutic candidate for diabetes-associated skeletal muscle dysfunction. Graphical abstractIn myoblasts, high glucose activates large-pore channels, elevating cytoplasmic Ca{superscript 2} concentration and nitric oxide generation, which increases the activity of Cx-formed hemichannels, raises the levels of inflammasome components, and promotes lipid accumulation. In STZ-diabetic mice, de novo expression of large-pore channels in skeletal muscles contributes to reduced blood perfusion, accumulation of intramuscular fat, muscle weakness, and reduced resting membrane potential of myofibers. Boldine inhibits large-pore channel activity, preventing these alterations and preserving muscle physiology in vivo. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/707704v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@19179b4org.highwire.dtl.DTLVardef@1cd3d21org.highwire.dtl.DTLVardef@16851d6org.highwire.dtl.DTLVardef@1d4e77c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Skeletal muscle hypertrophy results from the integrated regulation of anabolic and proteolytic processes in response to mechanical loading. Although increases in resistance training (RT) volume are used to increase mechanical stress, it remains uncertain whether large and abrupt volume progressions could exceed muscle adaptive capacity by disrupting the balance between anabolic and catabolic signaling. The present study investigated whether a large increase in weekly RT volume (+120%) leads to impaired hypertrophic outcomes and intracellular regulatory responses compared with a modest increase (+20%). Twenty-five resistance-trained men and women (18-35 years old) completed an 8-week randomized, single-blind, within-subject unilateral intervention. Each participant trained both legs twice weekly, with one leg assigned to the large (VOL120) and the contralateral leg to the modest (VOL20) weekly volume progressions relative to habitual training volume. Vastus lateralis muscle cross-sectional area (mCSA) was assessed by ultrasonography before and after training. Muscle biopsies were obtained at baseline, post-intervention, and 24 h after the last session to quantify muscle fiber cross-sectional area (fCSA), satellite cell myonuclear content, and anabolic/catabolic signaling markers. Both protocols induced increases in mCSA over time (p<0.001), with no protocol vs. time interaction. No significant effects were observed for fCSA nor satellite cell number or myonuclear content. Additionally, molecular responses related to translational regulation and protein degradation were largely similar between protocols. Collectively, these data indicate that a large, abrupt increase in weekly set volume does not impair hypertrophic adaptations or meaningfully alter the anabolic-catabolic signaling profile in resistance-trained individuals.

ContextACL injury prevention in young athletes has traditionally relied on a dichotomous classification of contact versus noncontact mechanisms; however, this framework may not adequately capture the movement processes underlying many injuries. ObjectiveTo classify ACL injury mechanisms in athletes aged [&le;]22 years with a specific focus on landing-related movements and to examine their associations with sport contact characteristics and age. DesignRetrospective observational study. SettingSingle sports injury clinic. Patients or Other ParticipantsA total of 151 athletes aged [&le;]22 years (mean age, 17.7 {+/-} 2.1 years) diagnosed with ACL injury between January 2017 and November 2025. Main Outcome Measure(s)ACL injury mechanisms were classified as landing-related without contact (L), landing-related with contact (Lc), or direct contact injury (C). Landing-related injuries (L + Lc) were compared with direct contact injuries. Multivariable logistic regression was used to identify factors associated with landing-related injury. ResultsLanding-related injuries accounted for 123 cases (81.5%), including 88 noncontact and 35 contact-related landing injuries, whereas direct contact injuries occurred in 24 cases (15.9%). Athletes with direct contact injuries were significantly older than those with landing-related injuries (19.2 {+/-} 1.7 vs 17.5 {+/-} 2.5 years, p = 0.03). In multivariable analysis, participation in noncollision sports was strongly associated with landing-related injury (odds ratio [OR] = 9.80; 95% confidence interval [CI] = 3.03-31.5; p < 0.001), whereas increasing age was inversely associated with landing-related injury (OR per year = 0.71; 95% CI = 0.56-0.90). Sex was not independently associated with injury mechanism. ConclusionsMost ACL injuries in athletes aged [&le;]22 years occurred through landing-related mechanisms, regardless of contact. These findings highlight insufficient control of landing and foot contact as a fundamental mechanism of ACL injury in young athletes and support prevention strategies focused on movement quality during sport-specific tasks. Key Points{blacksquare} Most ACL injuries in athletes aged [&le;]22 years occurred during landing or foot contact, regardless of whether external contact was present. {blacksquare}Noncollision sports and younger age were strongly associated with landing-related ACL injury mechanisms. {blacksquare}ACL injury prevention in young athletes should prioritize improving landing and foot contact control rather than focusing solely on contact characteristics.

Maintenance of skeletal muscle function is essential for functional independence, quality of life and healthspan. Muscle RING-finger protein-1 (MuRF1) negatively regulates muscle function and mass through ubiquitination and degradation of muscle proteins. Accordingly, genetic and pharmacological inhibition of MuRF1 attenuates muscle wasting and weakness under catabolic stress. To explore the potential of MuRF1 inhibitors (e.g., MyoMed-205) to improve muscle health, we investigated here the long-term effects of MyoMed-205 on functional capacity and muscle physiology in rats under basal conditions. Wistar rats were randomized to control or MyoMed-205 groups and were followed for 4 or 8 weeks. Body weight, food and water intake, and exercise capacity were monitored weekly. At each endpoint, the soleus muscle was collected for histological analyses. MyoMed-205-treated rats showed normal basic survival-related behaviors and body growth. After 8 weeks, MyoMed-205-treated animals exhibited enhanced exercise capacity (speed (m/min): +45%, p = 0.01; endurance (min): +47%, p = 0.03; and distance covered (m): +87%, p = 0.04) compared with baseline performance. Conversely, no differences were found in soleus fiber type distribution, cross-sectional area, or lipid and collagen content. Our findings indicate that MyoMed-205 enhances functional exercise capacity independently of changes in soleus muscle structure in rats under basal conditions.

BackgroundLongitudinal bone growth occurs via the process of endochondral ossification, involving a complex interplay of chondrocyte proliferation, differentiation, and matrix remodelling. As with all mammalian cells, chondrocytes require dynamin for mitochondrial fission, to shuttle vesicles from the Golgi apparatus, and for both clathrin- and caveolin-mediated endocytosis. Here, we aimed to test the functions of dynamin on bone growth. To do so, we applied dynasore - a small molecule that is a reversible dynamin inhibitor - to mouse metatarsal bones cultured ex vivo. We assessed gross changes using bone length measurements and histomorphometry, and combined this with EdU detection, immunostaining, super-resolution microscopy and transmission electron microscopy. ResultsDynasore induced a dose-dependent hormetic effect on bone elongation: while high concentrations (220 {micro}M) impaired growth and abolished chondrocyte proliferation, low-dose treatment (40 {micro}M) significantly increased longitudinal bone growth. Histological analysis demonstrated that low dose dynasore augmented epiphyseal cartilage expansion and matrix accumulation, particularly within the resting and proliferative zones, while reducing chondrocyte proliferation. Immunostaining indicated that 40 {micro}M dynasore preserved collagen type X synthesis, activated mTORC1 signalling, and blocked autophagy, based on SQSTM1 accumulation. Low dose dynasore treatment expanded the thickness of the filamentous actin layer at the plasma membrane and deepened collagen fiber-containing endocytic pits, indicating that impaired cartilage remodelling was associated with growth-associated matrix accumulation. ConclusionsThis study reveals that dynasore exerts hormetic effects on growth plate chondrocytes, wherein low doses stimulate bone elongation, and high doses impair chondrocyte function.

ObjectivesCompare Anterior Cruciate Ligament (ACL) Return to Sport after Injury (ACL-RSI) scores over time following ACL reconstruction (ACLR) between male and female patients aged 15 to 25 years with primary ACL injuries and ACL reinjuries. DesignRetrospective cohort design. SettingSports physical therapy clinics. Participants332 patients aged 15-25 years who underwent ACLR following either primary ACL injury or ACL reinjury, either contralateral or ipsilateral graft reinjury, and had at least one observation of the ACL-RSI. Main Outcome MeasuresACL-RSI score. ResultsACL-RSI scores significantly increased over time post- ACLR (p < .001), males reported significantly higher scores compared to females (p < .001), and patients with contralateral ACL reinjury demonstrated higher scores than those with ipsilateral ACL graft reinjury (p = .006), though there was no difference in scores between patients with primary ACL injury and ACL reinjury. A significant interaction effect of sex and injury status was also observed (p = .009), generally demonstrating that females had lower psychological readiness compared to males across injury statuses. ConclusionsACL-RSI following ACLR varies based on biological sex and time post-ACLR, though ACL reinjury, independent of the reinjured leg, does not appear to effect scores compared to primary ACL injury.

Intervertebral disc degeneration is associated with loss of nucleus pulposus (NP) cell phenotype and extracellular matrix, both processes linked to changes in cytoskeletal contractility and cell shape. Here, we tested whether microenvironment-specific modulation of RhoA signaling can restore NP-like morphology and gene expression in NP cells cultured in 2D and in 3D alginate. In 2D monolayer culture, where cells are spread and mechanically activated, pharmacologic inhibition of RhoA with CT04 reduced RhoA activity, decreased actomyosin contractility gene expression, and shifted morphology toward a smaller, more circular phenotype. Bulk RNA sequencing showed that CT04 treatment increased expression of NP phenotypic and matrix-related genes including ACAN, GDF5, CHST3, and MUSTN1 while decreasing expression of catabolic and fibroblast-associated genes including ADAMTS1/9 and COL1, consistent with enrichment of extracellular matrix pathways. In contrast, RhoA activation with CN03 in 2D culture increased actin and phosphorylated myosin light chain intensity but produced limited phenotypic improvement. In 3D alginate, which minimizes integrin-mediated adhesion, baseline actomyosin markers were reduced relative to 2D culture. In alginate, RhoA activation with CN03 increased the amount of actin, phosphorylated myosin light chain, and actomyosin gene expression, yet also promoted a more compact, circular morphology and increased NP markers, including ACAN and KRT19 with repeated dosing. Across culture conditions, increased cell roundness was consistently associated with increased ACAN expression, indicating strong coupling between cytoskeletal state, morphology, and NP matrix programs. Together, these findings demonstrate that RhoA pathway perturbation can promote NP phenotypic gene expression in both 2D and 3D culture, but the direction of optimal modulation depends on the microenvironment, supporting RhoA signaling as a context-dependent therapeutic target for disc regeneration.

Right ventricular failure (RVF) is a serious disease with a high mortality but no effective pharmacologic treatments. We reported RVF was reversed by chronic treatment with an 1A-adrenergic receptor (1A-AR) agonist. Recent studies suggest mitochondrial dysfunction contributes to RVF. Therefore, we investigated if reversal of RVF by chronic 1A-AR agonist treatment involved improved mitochondrial function. A mouse model of RVF caused by pulmonary artery constriction (PAC) for 2 wk was chronically treated for a further 2 wk. with a low dose of the 1A-AR agonist A61603 (10 ng/kg/day) or vehicle (no drug control). RV dysfunction was assessed from the fractional shortening of the RV outflow tract (RVOT FS). RVOT FS for sham controls (46.5 {+/-} 1.3 %, n = 9) was reduced 4 wk after PAC (27.6 {+/-} 1.5 %, n = 13, P < 0.0001), but was higher after PAC plus 2 wk A61603 treatment (34.5 {+/-} 0.6 %, n = 14, P < 0.001). RV myocardial respiration rate (O2 consumption) for sham controls (776 {+/-} 51 pM/s/mg, n = 9) was reduced 4 wk after PAC (493 {+/-} 28 pM/s/mg, n = 15, P <0.0001), but was higher after PAC plus 2 wk A61603 treatment (634 {+/-} 30 pM/s/mg, n = 11, P <0.05). RV myocardial ATP level for sham controls (3.3 {+/-} 0.1 mM, n = 10) was reduced 4 wk after PAC (1.9 {+/-} 0.1 mM, n = 6, P < 0.0001), but was higher after PAC plus 2 wk A61603 treatment (2.6 {+/-} 0.13 mM, n = 7, P < 0.01). In conclusion, reversal of RVF after chronic A61603 treatment involved reversal of mitochondrial dysfunction. Consistent with our previous studies, this study suggests that the 1A-AR is a therapeutic target to treat RVF. HighlightsRV failure is reported to involve mitochondrial dysfunction which might impair RV contraction by decreasing cardiomyocyte ATP level. Using the pulmonary artery constriction model of RV failure, we found that chronic treatment with an 1A-adrenergic receptor agonist increased RV myocardial respiration rate, increased RV myocardial ATP level, and increased RV function. These findings suggest that the 1A-adrenergic receptor is a therapeutic target for treating RV failure, and that the mechanism involves improved RV cardiomyocyte bioenergetic status.

BackgroundDexamethasone (DEX) is used in vitro to promote osteogenic differentiation of human bone marrow mesenchymal stromal cells (hBMSCs). In clinical use, however, glucocorticoids induce osteoblast and osteocyte apoptosis while increasing osteoclast survival, leading overall to osteoporosis and high fracture risk. The overall impact of DEX on the differentiation of human progenitor cells remains contradictory and not fully understood, highlighting the need for further investigation using sequencing approaches as in vitro results will naturally influence further translational research. MethodshBMSCs were induced to osteogenic differentiation for 7 days using different concentrations of either DEX or the nonsteroidal glucocorticoid receptor agonist (+)-ZK216348. cDNA library preparation and RNA sequencing (RNAseq) were performed using Oxford Nanopore Technologies. Differentially expressed genes and pathways associated to the transactivation or transrepression activity of DEX were identified. Sequencing results were validated by qPCR, protein analysis, and with a functional assay on peripheral blood mononuclear cells to determine the overall effect of the BMSC supernatant. ResultsHierarchical clustering of RNAseq data identified eight subclusters with shared regulatory patterns. Enrichment analysis revealed that both upregulated and downregulated genes are involved in ossification and extracellular matrix organization pathways. Several pro- and anti-inflammatory genes were differentially regulated. qPCR analysis validated the upregulation of CXCL1, CXCL8, IL18, and COL8A1, while MMP1 and CXCL12 expression decreased in response to DEX. Comparing DEX results with those obtained using (+)-ZK216348 helped distinguish the potential mechanisms regulating the expression of specific genes. Notably, CXCL8 upregulation occurred through transactivation, whereas COL8A1 upregulation is downstream of a transrepressed gene. Further in vitro experiments confirmed that DEX significantly increased CXCL8 expression and IL-8 secretion. However, hPBMC responses indicated no significant pro- or anti-inflammatory effects from hBMSC conditioned medium. ConclusionsIn conclusion, the effects of DEX on the transcriptome of hBMSCs in a pro-osteogenic environment do not fully replicate the acquisition of an osteogenic phenotype. Several genes associated with ossification, extracellular matrix organization, and inflammation were dysregulated. The unique expression patterns of pro-inflammatory cytokines and collagen types warrant further investigation to elucidate their roles in osteogenic differentiation and bone homeostasis.

BackgroundAnterior knee pain (AKP) is common in adolescent athletes and encompasses heterogeneous osseous and soft tissue pathologies, yet its developmental mechanisms remain poorly integrated. HypothesisPain-generating tissues within the knee extensor mechanism are redistributed from osseous to soft tissue structures with skeletal maturation. Study DesignRetrospective observational cohort study. Level of EvidenceLevel 3. MethodsA total of 1,595 patients with sports-related knee injuries (2017-2025) were included. Skeletal maturity was determined by proximal tibial physeal status on radiographs, classifying participants into open-physes (n = 707) and closed-physes (n = 888) groups. AKP was classified into bony and non-bony subtypes based on maximal tenderness. Prevalence was compared using odds ratios (ORs) with 95% confidence intervals (CIs). ResultsOverall, 575 patients (36.1%) had AKP. AKP was more prevalent in the open-physes group than in the closed-physes group (60.1% vs 16.9%; OR, 7.4; 95% CI, 5.9-9.3; p < 0.001). Bony AKP showed a marked difference (42.4% vs 3.7%; OR, 19.1; 95% CI, 12.8-28.6; p < 0.001), whereas non-bony AKP showed only a modest difference (17.7% vs 13.2%; OR, 1.4; 95% CI, 1.1-1.9; p = 0.013). ConclusionThe association between AKP and skeletal maturity was primarily driven by bony AKP, supporting structural redistribution of pain-generating tissues during growth. Clinical RelevanceTenderness-based classification may aid identification of tissue-specific vulnerability and inform growth-stage-specific load management.

Pathological cardiac remodeling is driven by the proliferation and differentiation of resident fibroblasts into active myofibroblasts and results in excessive extracellular matrix (ECM) deposition and tissue stiffening. Expression of the matricellular protein WISP1 has previously been shown to be increased with cardiac fibrosis and promote myofibroblast activity, but the mechanisms by which this occurs remain unknown. Primary cardiac fibroblasts were isolated from adult mouse hearts and treated with recombinant WISP1 or TGF{beta}1 both alone and in combination to determine the functional role of the matricellular protein WISP1 in driving cardiac myofibroblast activity. WISP1 significantly increased alpha-smooth muscle actin and collagen type I expression, total collagen secretion, collagen gel contractility, and wound healing equally in fibroblasts from both male and female mice. However, WISP1 alone failed to induce expression of periostin, a hallmark myofibroblast marker, suggesting the resulting WISP1-dependent cell phenotype is unique and/or acting through non-canonical pathways. Indeed, inhibition of P38 MAPK completely ablated the WISP1-dependent increase in SMA and collagen expression, while having little to no impact on TGF{beta}1-dependent expression of myofibroblast marker genes. We next employed a multi-omics approach to define the functional impact of WISP1 on fibroblast cell-state within the transcriptome, cytosolic, and secreted ECM proteome. RNA-seq results show that WISP1 broadly promotes the expression of proliferative and immune modulatory genes at the transcriptomic level, while having very little impact on traditional myofibroblast and ECM modifying gene expression programs. At the proteome level, WISP1 was again a much weaker mediator of traditional myofibroblast and ECM proteins. However, in agreement with RNA-seq data, we observed a strong WISP1-dependent enrichment for proliferation-associated proteins in the cytosolic proteome and inflammation-associated proteins in the ECM proteome. Interestingly, WISP1 also showed a context-dependent response with TGF{beta}1, suggesting a more complex and yet to be elucidated signaling interaction between these independent mediators of myofibroblast activity. In conclusion, our data suggests that WISP1 promotes a unique proliferative and immune-modulatory myofibroblast phenotype. HighlightsO_LIWISP1 is sufficient to drive myofibroblast SMA and collagen expression and ECM deposition C_LIO_LIWISP1 promotes canonical myofibroblast contractility and wound healing activity C_LIO_LIWISP1 mediates myofibroblast activity via a non-canonical, P38 MAPK-dependent signaling pathway C_LIO_LIMulti-omics analysis of WISP1-dependent RNA and protein expression show that WISP promotes a proliferative and immune modulatory myofibroblast phenotype C_LI

BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease characterized by aberrantly activated, apoptosis-resistant profibrotic lung (myo)fibroblasts. Prior research has demonstrated that lung fibroblasts from patients with IPF exhibit resistance to DNA damage, suggesting that this behavior contributes to their persistent survival and continuous proliferation. We propose that elevated levels of the DNA damage repair protein RAD51 regulate myofibroblast activation and apoptosis and provide a potential therapeutic target to impede fibrosis progression. MethodsHuman lung fibroblasts were transfected with siRNA against RAD51 or treated with RAD51-specific inhibitor B02 and markers of fibrosis, DNA damage, apoptosis, metabolic reprogramming, and mitochondrial dynamics were assessed. The preclinical efficacy of B02 was evaluated in human precision cut lung slices (PCLS) and in a mouse model of pulmonary fibrosis. FindingsRAD51 expression was significantly upregulated in the lungs and lung fibroblasts of IPF patients. Knockdown or inhibition of RAD51 in fibroblasts reduced profibrotic marker expression, suppressed mTORC1 signaling and mitochondrial function, and increased apoptosis susceptibility. Pharmacological inhibition of RAD51 shifted the profibrotic phenotype towards a fibrosis-resolving state in human and mouse PCLS, and in a bleomycin-induced mouse model of lung fibrosis. InterpretationThe inhibition of RAD51 exerts therapeutic benefits in lung fibrosis by promoting apoptosis. Our findings identify that inhibiting RAD51 with B02 in fibroblasts impairs DNA repair and induces metabolic reprogramming, making it a potential therapeutic target. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSPulmonary fibrosis (PF) is characterized by excessive fibroblast activation and subsequent deposition of extracellular matrix (ECM) proteins, which ultimately disrupt normal lung architecture. A significant contributing factor to the pathogenesis of pulmonary fibrosis is the presence of fibroblasts that are resistant to apoptosis, preventing normal wound healing. Recent studies highlight the DNA repair protein RAD51 as effective in protecting fibroblasts from death induced by chemotherapy and ionizing radiation. These finding suggested that RAD51 could have a role in fibroblast activation and apoptosis resistance in pulmonary fibrosis. Added value of this studyWe demonstrated that RAD51 is important for maintaining apoptosis-resistant fibrotic fibroblasts and their metabolic abnormalities. Our findings indicated that TGF{beta}-mediated upregulation of RAD51 reduces DNA damage, activates multiple pathways related to fibroblast activation and proliferation, and induces metabolic reprogramming, ultimately regulating apoptosis. Mechanistically, RAD51 inhibition enhanced p53 acetylation at lysine 120 and upregulated the expression proapoptotic proteins PUMA/BAK in mitochondria, promoting apoptosis. Pharmacological inhibition of RAD51 using the specific inhibitor B02 during the fibrotic phase of experimental lung disease effectively ameliorated pulmonary fibrosis. Implications of all the available evidenceOur findings establish that RAD51 plays an important role in the survival of apoptosis-resistant fibrotic fibroblasts. We propose that reducing RAD51 expression leads to the metabolic reprogramming of activated fibroblasts, resulting in decreased mitochondrial respiration, reduced ATP levels, and diminished glycolysis or glutaminolysis. These observations suggest that targeting energy metabolism through RAD51 inhibition could be a viable strategy to enhance apoptosis, thereby creating a therapeutically targetable pathway in fibrotic cells. These findings highlight the potential of RAD51 as a therapeutic target for the treatment of IPF.

ObjectivesFree fatty acid receptors 2 and 3 (FFA2 and FFA3) are activated by nutrient-derived metabolites such as short-chain fatty acids (SCFAs) and ketone bodies, produced by the gut microbiota and host, respectively. This study aimed to investigate the intracellular signaling pathways recruited in glucagon-like peptide-1 (GLP-1) releasing enteroendocrine cells following activation of FFA2 and FFA3 to resolve the impact of nutrient status on enteroendocrine cell function. MethodsExperiments were performed using primary mouse colonic cultures and the mouse enteroendocrine cell line, GLUTag cells. Expression analysis by bulk RNA sequencing was used to determine expression of FFA2 and FFA3 in GLP-1 releasing cells. Measurement of GLP-1 secretion by sandwich ELISA was used to assess enteroendocrine cell function. Live-cell measurements of intracellular calcium and cAMP levels were performed to assess canonical second messenger signaling pathways. ResultsA SCFA mixture stimulated GLP-1 secretion from both primary mouse colonic cultures and GLUTag cells. In GLUTag cells, the FFA2 ligand 4-CMTB inhibited GLP-1 release independent of Gaq- and Gai-signaling as neither YM-254890 (Gaq inhibitor) nor pertussis toxin (Gai- uncoupler) altered its effect. However, 4-CMTB did elevate cAMP levels, suggesting an indirect mechanism for the increase in cAMP production. Stimulation of FFA2 with the Gai-biased ligand AZ1729 or the ketone body acetoacetate inhibited GLP-1 release and cAMP accumulation. AZ1729 was insensitive to pertussis toxin and OZITX, supporting atypical FFA2 signaling. Stimulation of FFA3 with AR420626 or the ketone body {beta}-hydroxybutyrate increased GLP-1 secretion from GLUTag cells, an effect that was not mediated by cAMP production. AR420626, but not {beta}-hydroxybutyrate increased intracellular calcium levels. ConclusionsOverall, activation of FFA2 inhibited secretory function in GLP-1-releasing enteroendocrine cells, whereas activation of FFA3 stimulated GLP-1 secretion via distinct intracellular signaling mechanisms. O_FIG O_LINKSMALLFIG WIDTH=184 HEIGHT=200 SRC="FIGDIR/small/708924v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@175dbaforg.highwire.dtl.DTLVardef@a9cc3eorg.highwire.dtl.DTLVardef@1a026e5org.highwire.dtl.DTLVardef@15997af_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG HighlightsO_LIExposure to physiological levels of SCFAs stimulates GLP-1 secretion from colonic EECs C_LIO_LIFFA2 and FFA3 regulate GLP-1 release via non-canonical signaling pathways C_LIO_LIKetone bodies activate SCFA receptors to differentially modulate GLP-1 levels C_LIO_LILigand bias enables nutrient-dependent tuning of EEC gut hormone secretion C_LI