The research team, led by Zeliha Celik of Amasya University together with colleagues from Nigde Omer Halisdemir University, Gazi University, and Gazi University’s pulmonary medicine department, recruited 26 adolescent female volleyball players for the trial, which was registered at ClinicalTrials.gov under identifier NCT06759779. The participants were randomly assigned to one of two groups. One group performed inspiratory muscle training, known as IMT, which uses a handheld resistance device that makes it harder to draw air into the lungs, forcing the diaphragm and other inspiratory muscles to work against a load much as a bicep curls a dumbbell. The other group performed expiratory muscle training, or EMST, which instead adds resistance to breathing out, targeting the abdominal muscles and internal intercostals that power forced exhalation.

Both interventions followed an identical schedule: four weeks of training, five days per week, twice daily. That brevity is one of the study’s most striking features. Many training adaptations in skeletal muscle take eight to twelve weeks to become detectable, yet the researchers were able to document changes in respiratory muscle structure and performance after only a month of added work, layered on top of the athletes’ regular volleyball training. This compressed timeline matters for practical coaching, because competitive seasons are short and any supplementary program must deliver results without disrupting the existing schedule.

What sets this trial apart from much of the earlier respiratory training literature is its measurement strategy. Rather than relying solely on performance numbers, the team used ultrasonography to peer directly at the diaphragm, measuring its thickness during both inspiration and expiration as well as its stiffness. Ultrasound has become an increasingly valuable window into respiratory muscle physiology because the diaphragm, sitting deep inside the torso, is otherwise difficult to examine in living people. When the diaphragm contracts, it thickens, and the degree of thickening correlates with the force it generates. Stiffness, assessed with shear wave or elastographic techniques, offers clues about the muscle’s passive and active mechanical properties. Combining these imaging measures with classic pulmonary function tests gave the researchers a two-dimensional picture: what the respiratory system can do and what the muscle driving it actually looks like.

The results were clear on several fronts. The researchers observed significant time effects for diaphragm thickness during both inspiration and expiration, meaning that across the whole study population, the diaphragm became measurably thicker over the four weeks regardless of which training style the athletes had followed. The same pattern held for respiratory muscle strength: maximal inspiratory pressure, or MIP, and maximal expiratory pressure, MEP, both improved significantly over time, with p values below 0.001 indicating a very low probability that these changes arose by chance. MIP and MEP are the standard clinical gauges of respiratory muscle strength, essentially the maximum suction a person can generate breathing in and the maximum pressure they can push out, and gains in both suggest that the training load, whichever direction it came from, strengthened the entire respiratory pump.

Perhaps the most scientifically interesting finding, however, is what did not differ. When the researchers tested for group-by-time interactions, the statistical signature of whether one training modality outperformed the other, they found none for any of the primary outcomes. The inspiratory group and the expiratory group improved to a statistically similar degree on diaphragm thickness, on MIP, and on MEP. Diaphragm stiffness, by contrast, did not change significantly over time in either group, and neither did Tmax, the measure of respiratory muscle endurance used in the study. Pulmonary function parameters likewise showed a significant time effect only for percent-predicted peak expiratory flow, with no group-by-time interaction detected.

This absence of a differential effect invites a deeper physiological interpretation. One possibility is that the diaphragm, as the dominant inspiratory muscle, is recruited even during expiratory training, because forceful exhalation requires precise coordination of the entire respiratory cycle and the diaphragm must eccentrically control the recoil of the system. Another possibility is that four weeks is sufficient to produce general adaptations, such as improved neural drive and coordination of the respiratory muscles, that precede the more modality-specific structural changes that longer programs might reveal. The unchanged stiffness measurement hints that the adaptations observed were primarily hypertrophic and neural rather than involving alterations in the muscle’s elastic properties, though the authors note that respiratory muscle training added to regular sports training may be associated with structural and strength adaptations in adolescent athletes.

The choice of population is also significant. Adolescent female volleyball players occupy a distinctive physiological niche: they are still growing, their rib cages and lungs are developing, and volleyball demands repeated explosive efforts interspersed with short recoveries, a pattern in which respiratory muscle fatigue can subtly degrade performance by triggering the metaboreflex, a protective mechanism that shunts blood away from limb muscles when the breathing muscles struggle. Prior research in adult athletes has suggested that respiratory muscle training can attenuate this reflex and delay fatigue, but evidence in adolescents has been sparse, and studying young athletes raises particular ethical and methodological questions that this team addressed through ethics approval from the Gazi University Ethics Committee, written informed consent from both participants and their parents, and conduct in accordance with the Declaration of Helsinki.

For coaches and sports scientists, the practical takeaway is refreshingly simple. If the goal is to strengthen the respiratory pump of a young athlete, the evidence from this trial suggests that either inspiratory or expiratory resistance training, performed for a modest four weeks at five days per week in two daily sessions, can thicken the diaphragm and boost maximal inspiratory and expiratory pressures on top of a normal sport training load. The devices required are inexpensive handheld resistive trainers, and the time commitment is small compared with the hours devoted to technical and tactical work. What remains open is the question of endurance: neither training modality moved the needle on respiratory muscle endurance in this study, which may require longer programs, higher loads, or different testing protocols to detect.

The study also carries caveats worth weighing. The sample of 26 athletes is small, which limits the statistical power available to detect subtle differences between the two training modalities, and the trial was retrospectively registered, a detail the authors disclose transparently. All participants were adolescent female volleyball players, so generalizing to male athletes, older competitors, or athletes in endurance sports requires further study. Still, as one of the first randomized controlled trials to pair diaphragm ultrasound with comparative respiratory muscle training in adolescents, the work opens a promising line of inquiry: if a month of targeted breathing exercise can visibly remodel the body’s most tireless muscle, the respiratory system may deserve a far more prominent place in the training plans of young athletes than it has traditionally received.

Subject of Research: Comparative effects of inspiratory and expiratory muscle training on diaphragm structure and respiratory function in adolescent athletes

Article Title: Comparative effects of inspiratory and expiratory muscle training on diaphragm adaptations and respiratory function in adolescent athletes: a randomized-controlled study

Article References: Celik, Z., Demirtas, G., Guzel, N. A., Sendur, H. N., Keles, M. N., Karatas, N., & Kokturk, N. (2026). Comparative effects of inspiratory and expiratory muscle training on diaphragm adaptations and respiratory function in adolescent athletes: a randomized-controlled study. BMC Pediatrics. https://doi.org/10.1186/s12887-026-07817-z

Image Credits: AI Generated

DOI: 10.1186/s12887-026-07817-z

Keywords: respiratory muscle training, inspiratory muscle training, expiratory muscle training, diaphragm, ultrasonography, adolescent athletes, volleyball, respiratory function, maximal inspiratory pressure, maximal expiratory pressure, randomized controlled trial, sports physiology

The research, led by José Castro and Mamede de Carvalho at the Hospital de Santa Maria and the Universidade de Lisboa, drew on one of the largest single-centre ALS cohorts ever assembled for this purpose: 1,486 patients contributing 2,968 serial phrenic nerve conduction studies. In this test, a small electrical stimulus is applied over the nerve in the neck, and electrodes record the compound muscle action potential, the summed electrical response of the diaphragm. Because the diaphragm cannot voluntarily influence the evoked signal, the amplitude is, by design, effort-independent. The investigators expressed each amplitude as a percentage of an age- and sex-predicted normative value and asked a deceptively simple question: does this number forecast when a patient will need breathing support, above and beyond the established clinical measures?

The answer, delivered through a pre-registered analysis plan lodged publicly on the Open Science Framework, was emphatic. Using cause-specific Cox proportional hazards models with death treated as a competing risk, the team found that each 10 percent decrease in predicted phrenic amplitude was associated with a 22 percent higher hazard of starting non-invasive ventilation, with an adjusted hazard ratio of 1.22 and a 95 percent confidence interval of 1.16 to 1.27. The statistical weight of the association was extraordinary: a likelihood-ratio chi-square of 77.6, corresponding to a p-value of roughly ten to the minus eighteenth. In a field where biomarker candidates routinely collapse on replication, a signal of this magnitude in nearly three thousand serial measurements commands attention.

Perhaps the most clinically useful finding is the temporal structure of the prediction. The association was concentrated in the near term: within the first year of follow-up, the hazard ratio climbed to 1.31 per 10 percent decrement, before attenuating in later intervals. This makes physiological sense. The phrenic compound muscle action potential reflects the number and integrity of functioning motor units in the diaphragm, so a low or rapidly falling amplitude signals that the respiratory motor pool is approaching a functional threshold. Once that threshold is crossed, ventilatory decline accelerates. For clinicians, the message is that the nerve test is not a distant oracle but a near-term alarm, precisely the window in which the timing of ventilation matters most for survival and quality of life.

The quantitative gains over existing practice were substantial. Adding phrenic amplitude to a clinical model built on forced vital capacity and the revised ALS Functional Rating Scale, the standard severity instrument that incorporates respiratory items, improved discrimination by an optimism-corrected increment of 0.055, with a bootstrap-validated confidence interval of 0.034 to 0.073. That may sound modest, but in prognostic modelling, where incremental improvements of this size are considered meaningful, it represents a genuine advance. More striking is the absolute risk translation: patients whose phrenic amplitude fell below the age- and sex-adjusted normal threshold had approximately double the twelve-month risk of requiring ventilation, 62 percent versus 29 percent. A single bedside-adjacent measurement effectively stratifies patients into two very different futures.

To capture the dynamic nature of the disease, the team also deployed a Bayesian joint longitudinal-survival model, a sophisticated statistical architecture that links repeated measurements over time to the hazard of the clinical event. This analysis separated two distinct prognostic signals: the current amplitude itself, carrying a hazard ratio of 1.57 per 10 percent decrement, and the rate of decline of that amplitude, carrying an independent hazard ratio of 1.30. In other words, both where the diaphragm’s electrical output stands today and how fast it is eroding contribute independent information. This dual signal mirrors the logic clinicians apply intuitively to forced vital capacity, but here it operates on a measurement that no amount of weakness elsewhere in the body can distort.

The problem the study solves is well documented in the ALS literature. Forced vital capacity depends on bulbar competence to seal the mouthpiece, on respiratory muscle strength to generate flow, and on cognitive capacity to follow instructions. Slow vital capacity has been proposed as a gentler alternative, and supine measurements add sensitivity to diaphragmatic weakness, but all remain effort-dependent to some degree. Recent work from the same Lisbon group showed that cognitive dysfunction is associated with an underestimation of respiratory function on these tests, meaning the very patients whose breathing is deteriorating may appear deceptively stable. Guidelines from the European Academy of Neurology and its predecessors have long acknowledged the difficulty of timing ventilation, and the randomised trial by Bourke and colleagues that established the survival benefit of non-invasive ventilation relied on thresholds that cooperative patients can demonstrate but severely affected ones cannot.

The phrenic nerve conduction study itself is not new. It has been studied as a prognostic marker in ALS since at least 2009, when Pinto, Turkman and colleagues showed its value in predicting respiratory insufficiency, and the Lisbon group has since published normative data establishing age- and sex-referenced amplitude thresholds. What distinguishes the new work is scale, rigour and statistical ambition. The pre-registered plan, the competing-risk framework, the bootstrap validation, the time-dependent discrimination analyses and the joint modelling collectively meet the standards set by the TRIPOD reporting guidelines for clinical prediction models. The authors also positioned the measurement as a candidate outcome for clinical trials, where an objective, effort-independent readout of respiratory motor unit loss could serve as a sensitive endpoint for drugs targeting motor neuron degeneration.

Important caveats remain, and the authors state them plainly. The cohort is single-centre, and although it is large, the amplitude values were expressed against normative data derived from the same investigative tradition, raising the possibility of centre-specific calibration. External validation in independent cohorts, ideally spanning different health systems and ethnicities, is required before the marker can be embedded in guidelines, and the models will need recalibration to local populations. The association also attenuates beyond the first year, so the marker complements rather than replaces longitudinal clinical assessment. And while the test involves a mild electrical stimulus that most patients tolerate well, it is still an invasive neurophysiological procedure compared with a spirometry manoeuvre, which shapes how it might be deployed in routine monitoring.

Even with those qualifications, the implications are considerable. Respiratory failure remains the dominant cause of death in ALS, and the window for initiating ventilation comfortably, with time for adjustment, education and psychological preparation, is narrow and easy to miss when the usual yardsticks falter. An effort-independent electrical signature of the diaphragm’s remaining motor units, measured in a clinic room in minutes, that roughly doubles the predicted twelve-month risk when abnormal, offers a way to keep that window open for the patients who need it most, including those who can no longer perform the breathing manoeuvres their own care depends on. As disease-modifying therapies multiply and trials search for responsive endpoints, the humble phrenic nerve, stimulated and recorded for decades in neurophysiology labs, may be poised to become one of the most consequential numbers on the ALS chart.

Subject of Research: Phrenic nerve conduction studies as prognostic biomarkers of respiratory failure in amyotrophic lateral sclerosis

Article Title: Phrenic nerve amplitude as an effort-independent prognostic biomarker of respiratory failure in amyotrophic lateral sclerosis

Article References: Castro, J., Sá, V., Lopes, D., Alves, I., Oliveira Santos, M., & de Carvalho, M. (2026). Phrenic nerve amplitude as an effort-independent prognostic biomarker of respiratory failure in amyotrophic lateral sclerosis. Journal of Neurology, 273(10), Article 574. https://doi.org/10.1007/s00415-026-14125-0

Image Credits: AI Generated

DOI: 10.1007/s00415-026-14125-0

Keywords: amyotrophic lateral sclerosis, phrenic nerve, nerve conduction studies, non-invasive ventilation, respiratory failure, prognosis, biomarkers, forced vital capacity, diaphragm, neurophysiology, motor neuron disease, ALSFRS-R

The diaphragm is the primary engine of breathing, contracting rhythmically from birth to death, and like all skeletal muscles it is vulnerable to the slow erosion of age. Yet respiratory muscles often resist sarcopenia, the age-related loss of muscle mass and strength, better than limb muscles do. Researchers have long suspected that compensatory mechanisms operating at the neuromuscular junction might explain this resilience, but the precise presynaptic adjustments involved have remained poorly defined. To probe the question, the team compared neuromuscular transmission in the diaphragms of 3-month-old adult mice and 24-month-old animals, an age that in mouse terms corresponds to a late stage of the lifespan.

The first surprise came from the postsynaptic side of the synapse. In aged diaphragms, the amplitude of evoked postsynaptic responses was reduced, and miniature end-plate potentials, the tiny electrical blips caused by the spontaneous release of single acetylcholine quanta, were diminished far more dramatically. This indicates that each individual packet of neurotransmitter was producing a weaker effect on the muscle membrane, most likely because the density or sensitivity of nicotinic acetylcholine receptors on the muscle had declined. In many neural systems, such a postsynaptic deficit would spell trouble for reliable signaling, particularly during the sustained high-frequency firing that breathing demands.

But the presynaptic terminal had other plans. The researchers recorded a striking increase in spontaneous exocytosis intensity, meaning the aged nerve endings were leaking or spontaneously releasing more acetylcholine quanta than their young counterparts. They also measured a faster conduction velocity for presynaptic action potentials, a larger quantal content of end-plate potentials evoked by single stimuli, and a bigger asynchronous component of evoked release, where vesicles discharge in a delayed, staggered fashion after the main synchronous burst. Taken together, these changes paint a picture of a nerve terminal that has turned up its overall output, pushing more neurotransmitter into the synaptic cleft to compensate for the fact that each packet now carries less punch.

The compensation became even more evident under stress. When the researchers drove the phrenic nerve with short trains of stimuli at frequencies ranging from 10 to 70 hertz, mimicking the varying demands of breathing, the aged junctions released more acetylcholine than young ones. The effect was most pronounced during prolonged intermittent stimulation at 20 hertz, a pattern that resembles sustained respiratory effort. Rather than fatiguing, the old synapses kept pouring out transmitter, suggesting that the aging diaphragm’s nerve supply has been tuned specifically to withstand prolonged activity rather than just single pulses.

Digging into the mechanism, the team found two key presynaptic adaptations. First, the estimated size of the readily releasable pool, the small cadre of synaptic vesicles parked at the membrane and ready to fuse at a moment’s notice, was larger in aged terminals. Second, the facilitation of neurotransmitter release between bursts, a classic electrophysiological signature of synaptic vesicle mobilization, was enhanced, indicating that reserve vesicles were being recruited to the release sites more efficiently. In effect, the aged nerve terminal had expanded both its standing inventory of ready vesicles and its supply chain for replenishing them.

Perhaps the most intriguing molecular clue involves synapsin 1, a protein that tethers synaptic vesicles into clusters within the reserve pool, holding them back from immediate release. The aged diaphragm junctions showed reduced vesicle clustering and diminished immunolabeling for synapsin 1. This decrease makes functional sense: with less synapsin restraint, more vesicles would be free to join the readily releasable pool and participate in active transmission. The finding suggests that the aging terminal partially dismantles its own vesicle storage architecture to keep the release pipeline full, a trade-off that boosts short-term output even if it may reduce the terminal’s long-term reserves.

The researchers also tracked vesicle recycling directly using FM1-43, a fluorescent dye that loads into synaptic vesicles as they undergo exocytosis and endocytosis. Monitoring the destaining of the dye during 20-hertz activity revealed that vesicle recycling was faster in the aged junctions. This means the old terminals were not just releasing more vesicles; they were also retrieving membrane, refilling the vesicles with neurotransmitter, and returning them to service at an accelerated pace. An accelerated vesicle cycle, combined with a larger releasable pool and enhanced mobilization, provides a coherent mechanistic account of how the aged synapse sustains transmitter output through prolonged, breath-like patterns of stimulation.

The broader significance of the work lies in what it says about homeostatic plasticity in the aging nervous system. Synapses are not passive victims of time; they actively adjust their properties to maintain stable function in the face of degradation elsewhere in the circuit. In the aged diaphragm, the postsynaptic muscle membrane has become less responsive to each quantum of acetylcholine, and the presynaptic terminal has responded by boosting release on nearly every measurable axis: spontaneous release, evoked quantal content, asynchronous release, pool size, mobilization, and recycling speed. The result is that end-plate potentials, while individually somewhat smaller, remain sufficient to trigger muscle contraction, preserving neuromuscular transmission at a stage of life when many other synapses are failing.

For human health, the implications are tantalizing, even if the study was conducted in mice. Respiratory failure is a major contributor to morbidity in the elderly, and understanding how the diaphragm’s neuromuscular junctions defend themselves against aging could point toward ways to reinforce that defense when it falters. The synapsin pathway, the vesicle recycling machinery, and the mechanisms governing the readily releasable pool all represent potential targets for interventions aimed at preserving respiratory muscle function in old age. The Kazan team’s work also adds an important caveat to the conventional narrative of synaptic aging: decline is not the whole story. Deep inside the aging diaphragm, the nerve terminal is fighting back, and for now, at least in these mice, it is winning.

Subject of Research: Presynaptic compensatory mechanisms sustaining neuromuscular transmission in the aging mouse diaphragm

Article Title: Presynaptic compensation sustains neuromuscular transmission in the mouse diaphragm at a late stage of aging

Article References: Presynaptic compensation sustains neuromuscular transmission in the mouse diaphragm at a late stage of aging. (n.d.). https://doi.org/10.1007/s10522-026-10516-y

Image Credits: AI Generated

DOI: 10.1007/s10522-026-10516-y

Keywords: neuromuscular junction, diaphragm, aging, acetylcholine, synaptic vesicles, readily releasable pool, synapsin 1, vesicle recycling, neurotransmitter release, sarcopenia, Biogerontology, mouse study

Every mammalian cell contains a molecular clock built from interlocking transcription-translation feedback loops. Core activators such as BMAL1 and CLOCK drive the rhythmic expression of thousands of genes, which in turn coordinate time-of-day-dependent processes including glucose handling, lipid oxidation, protein quality control and immune surveillance. When these clocks falter, the consequences are well documented: inflammation, impaired glucose homeostasis and distorted lipid metabolism, all of which are hallmark features of cancer cachexia. Previous work has shown that tumours can remotely rewire circadian programs in the liver, and the Florida group had already demonstrated that pancreatic cancer disrupts circadian gene expression in a limb muscle, the tibialis anterior. What remained unknown was whether the cardiorespiratory muscles, whose wasting is thought to be a key driver of cachexia-related morbidity and mortality, suffer the same fate.

To find out, the researchers used an orthotopic model of pancreatic ductal adenocarcinoma in which aggressive KPC tumour cells are injected directly into the pancreas of mice. Twelve days after inoculation, a time point corresponding precisely to the onset of cachexia, they collected diaphragm and heart tissue every four hours across a full twenty-four-hour cycle in constant darkness, ensuring that any rhythms they detected reflected endogenous clocks rather than external light cues. They then performed bulk RNA sequencing on each sample and applied sophisticated statistical models that treat gene expression as a continuous sinusoidal function over the day, allowing them to identify rhythmically expressed genes and to detect changes in amplitude, phase and basal expression. The design was powered at over ninety-seven percent to detect circadian rhythmicity, lending confidence to the results.

The findings were striking. In healthy mice, the diaphragm showed robust rhythmicity in 308 genes, but in tumour-bearing mice sixty-four percent of those genes lost their daily oscillations entirely. At the same time, 718 genes that were never rhythmic in healthy diaphragm gained rhythmicity in the cancer state, a wholesale remodelling of the tissue’s temporal architecture. Among the genes losing their rhythm were Nrf1, which guards mitochondrial homeostasis; Pik3c3, a key mediator of autophagy; and Fgf1, a positive modulator of glucose disposal. Genes that gained rhythmicity included inflammatory players such as Tlr7, Il18 and Il18r1, alongside troubling time-of-day-dependent losses in Glut4, the major insulin-regulated glucose transporter, and in Tfeb and Psme4, master regulators of the autophagy-lysosome system and proteasomal quality control.

The heart told a partially different story. Of 1274 genes rhythmic in healthy hearts, fifty-six percent lost their oscillations in tumour-bearing animals, while 246 genes gained rhythmicity. Crucially, core clock gene rhythmicity was largely preserved in the heart, with only Per1 showing elevated basal expression, whereas the diaphragm exhibited significant repression of core clock components including Bmal1 itself, the gene whose deletion in muscle is known to cause metabolic reprogramming, weakness and atrophy. Only about ten percent of the rhythmic genes disrupted in the cancerous hearts overlapped with those altered by cardiomyocyte-specific Bmal1 deletion, suggesting that factors extrinsic to the cardiac clock, such as systemic inflammatory or autonomic signals, are driving much of the disruption.

The temporal coordination of biological processes also collapsed in a tissue-specific manner. In healthy diaphragms, peak expression times of rhythmic genes are spread evenly across the day, reflecting a smooth distribution of daily functions. In cachectic diaphragms, that even distribution vanished, with peak expression re-clustering around the transitions between rest and activity. Genes governing lipid metabolism and amino acid sensing, which normally peak during rest and activity respectively, no longer fired at their proper times. Instead, inflammatory genes and components of ubiquitin-dependent protein breakdown dominated those windows, implying that the muscle’s daily schedule had been hijacked to prioritize immune activation and protein catabolism over metabolic maintenance.

In the heart, the overall distribution of peak expression remained even, but the content of the schedule changed. Genes that regulate heart rate, including Hcn4 and Pde4d, normally peak during the active phase to support the diurnal rise in heart rate and cardiac output driven by sympathetic tone. In tumour-bearing mice, this temporal upregulation was lost, and inflammatory genes such as Il33 and Edn1 peaked during the active phase instead. Phase-stratified analysis revealed that genes involved in adrenergic signalling and contractility, including the beta-1 adrenergic receptor Adrb1, the SERCA2 calcium pump encoded by Atp2a2, and the fatty acid oxidation regulator Ppara, were downregulated in a time-of-day-dependent fashion, while platelet activation genes rose specifically during the active phase, echoing processes implicated in cancer-associated thromboinflammation.

Perhaps the most clinically provocative discovery came from a time-course analysis spanning the entire cachexia trajectory. Drawing on a published RNA-sequencing dataset from diaphragms collected before cachexia, during mild-to-moderate disease and at severe endpoint, the team found that Bmal1 and Clock were significantly repressed by day eight after tumour inoculation, days before any measurable loss of body or muscle mass, and remained suppressed thereafter. The secondary-loop genes Rora and Nr1d1, which encode RORα and REV-ERBα, were also repressed early. Both factors have clock-independent roles in muscle: REV-ERBα promotes mitochondrial biogenesis, represses atrophy genes and is essential for muscle mass maintenance, while RORα regulates lipid homeostasis and protects against pathological fat accumulation. Their early suppression, combined with the fact that both normally restrain NF-κB inflammatory signalling, suggests that clock disruption may precede and actively contribute to diaphragm wasting rather than merely accompanying it.

Why the diaphragm is hit hardest remains an open question, but the authors point to its anatomical proximity to the pancreas, which may expose it to a concentrated bath of tumour-derived factors and a heightened inflammatory microenvironment. Previous work from the same group documented pronounced immune cell infiltration in the diaphragm before cachexia onset, and NF-κB signalling is known to suppress core clock gene expression and interfere with CLOCK:BMAL1 activity. The researchers caution that their study has limitations, including the absence of phenotypic measurements on the same animals used for circadian sequencing and uncertainty about whether the milder cardiac disruption reflects a distinct pathology or simply an earlier stage of a shared trajectory.

The therapeutic implications are considerable. Because the transcriptional damage in these muscles is time-of-day dependent, the findings support testing chronotherapy-based interventions timed to protect muscle mass and function, and they reinforce growing evidence that restoring circadian machinery can halt wasting. Recent work has shown that re-establishing hepatic circadian function in tumour-bearing mice through REV-ERBα overexpression is sufficient to spare both limb muscles and the heart from atrophy, pointing to circadian programs as master regulators of tissue cross-talk during cancer. If similar strategies can be developed for the diaphragm and heart, they could offer a fundamentally new way to defend the muscles that pancreatic cancer patients can least afford to lose.

Subject of Research: Circadian clock disruption in cardiorespiratory muscles during pancreatic cancer cachexia

Article Title: Pancreatic Cancer Disrupts Circadian Patterns of Gene Expression in Cardiorespiratory Muscles

Article References: Ducharme, J. B., Schonk, M. M., Gutierrez‐Monreal, M. A., Ferreira, L. F., Esser, K. A., Judge, A. R., & Judge, S. M. (2026). Pancreatic Cancer Disrupts Circadian Patterns of Gene Expression in Cardiorespiratory Muscles. Journal of Cachexia, Sarcopenia and Muscle, 17(5), Article e70383. https://doi.org/10.1002/jcsm.70383

Image Credits: AI Generated

DOI: 10.1002/jcsm.70383

Keywords: pancreatic cancer, cachexia, circadian clock, diaphragm, heart, gene expression, Bmal1, inflammation, muscle wasting, RNA sequencing, metabolism, chronotherapy

The rationale for targeting PKR was compelling. PKR, encoded by the EIF2AK2 gene, is canonically activated by double-stranded RNA during viral infection, but it can also be switched on by inflammatory signaling, oxidative stress, and ER stress, all of which are hallmarks of dystrophic muscle. Once active, PKR phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF2α) at serine 51, shutting down global protein synthesis, and it simultaneously amplifies inflammatory pathways through NFκB, MAPK, and the NLRP3 inflammasome. Prior work from the same research group had shown that PKR expression and activation are elevated in dystrophic skeletal muscle, and separate studies demonstrated that imoxin could alleviate ER stress in cultured muscle cells and suppress lipopolysaccharide-induced inflammation and atrophy gene expression in rodent muscle. The hypothesis was straightforward: if PKR is a driver of pathology, blocking it chronically should preserve muscle function and dampen disease progression.

To test this, researchers at Iowa State University and the University of Florida treated male C57BL/10 control mice and mdx mice beginning at eight weeks of age, injecting imoxin subcutaneously at 0.5 mg/kg five days per week for approximately 24 weeks, with vehicle-treated animals serving as controls. The dose and delivery route mirrored protocols that had successfully suppressed PKR activity in models of obesity, neuroinflammation, hypertension, and acute lung injury. At roughly 28 weeks of age, mice underwent in vitro functional testing of the costal diaphragm, the respiratory muscle that most faithfully reproduces the degenerative course of human DMD. Investigators measured isometric tetanic specific force, half-relaxation time, time to peak tension, and both dynamic and elastic passive forces across a range of stretches, providing a comprehensive mechanical portrait of each muscle before tissue was apportioned for histology and biochemical analysis.

The first surprise came at the molecular level. Western blot analysis revealed that dystrophin deficiency drove dramatic increases in total PKR and phosphorylated PKR at Thr446, along with a constellation of PKR regulatory proteins including PACT, phosphorylated PACT, PP1α, TRBP, and both total and phosphorylated eIF2α. Compared with vehicle-treated controls, mdx mice showed 180 to 300 percent higher levels of PKR and pPKR and 112 to 409 percent higher levels of eIF2α and peIF2α. Yet imoxin treatment changed none of these measures. Phosphorylated PKR remained just as elevated in drug-treated mdx mice as in their vehicle-treated counterparts, meaning the dosing strategy failed to achieve its primary biochemical objective of inhibiting PKR activation in the dystrophic diaphragm.

Functional testing told a similarly disappointing story. Specific force production was reduced by roughly 44 to 46 percent in mdx diaphragms regardless of treatment, half-relaxation time was prolonged by disease, and both dynamic and elastic passive forces, indicators of muscle stiffness, were significantly elevated in dystrophic muscle. Imoxin did not improve specific tension, relaxation kinetics, or time to peak tension in either healthy or dystrophic animals. The only flicker of benefit appeared in the histological data: trichrome staining showed that fibrosis occupied a dramatically larger fraction of mdx diaphragm than control tissue, a 477 to 564 percent increase, but imoxin reduced that fibrotic area by 18 percent in mdx mice. Intriguingly, this histological improvement was not accompanied by any reduction in the protein abundance of fibrosis drivers such as fibronectin, TGF-β1, or collagens I and III, all of which remained strongly elevated in drug-treated dystrophic muscle.

The broader signaling landscape was equally resistant to the drug. Endoplasmic reticulum stress markers, including BiP, the phosphorylated-to-total PERK ratio, ATF4, and spliced XBP1, were elevated 79 to 438 percent in mdx muscle, and imoxin left them untouched. Inflammatory signaling proteins told the same story: TLR4, MyD88, IKKα, total and phosphorylated NFκB, TNFα, pro- and cleaved IL-1β, and NLRP3 were all substantially increased by dystrophin deficiency, with TNFα rising nearly fourfold, and none of these measures differed between vehicle- and imoxin-treated mdx groups. The drug that had quieted inflammatory cascades in cell culture and in other disease models appeared powerless against the chronic, self-sustaining inflammatory milieu of the dystrophin-deficient diaphragm.

Why did imoxin fail where it had previously succeeded? The authors offer several technical and biological explanations. Pharmacologically, imoxin blocks the autophosphorylation of inactive PKR but may not strip phosphate groups from already-activated enzyme, and prior successes often involved acute inflammatory insults rather than lifelong, genetically driven stress. In cancer cachexia models, a fivefold higher dose of 5 mg/kg prevented muscle loss while 1 mg/kg did not, suggesting a therapeutic threshold that the 0.5 mg/kg regimen used here may not have reached. More fundamentally, the dystrophic intracellular environment appears poised to sustain PKR activation through multiple redundant inputs: chronic inflammation activates PACT, an upstream PKR kinase, while ER stress and oxidative stress provide additional activation routes that persist even without double-stranded RNA. The researchers observed that even endogenous inhibitors of PKR phosphorylation, TRBP and PP1α, were elevated alongside the activating signals, indicating that dystrophic muscle is differentially regulating the kinase but that the net balance favors activation, a situation the authors liken to the difficulty of pharmacologically inhibiting calpain in dystrophic muscle.

The study’s implications cut both ways. On one hand, it is a negative result: a widely used PKR inhibitor, delivered chronically at a dose validated in other contexts, does not attenuate disease severity in the dystrophic diaphragm of male mice. On the other hand, the consistent, coordinated upregulation of PKR alongside ER stress and inflammatory markers reinforces the kinase’s status as a potential central regulatory node in DMD pathology. The authors suggest that future strategies might bypass autophosphorylation altogether, instead preventing activators from engaging PKR or enhancing its endogenous inhibitors, approaches that could prove more effective against a kinase embedded in such a densely interconnected stress-signaling web. Whether higher imoxin doses, alternative inhibitors, or combination therapies could succeed remains an open question, as does whether female mdx mice, which experience a milder phenotype, would respond differently.

For the DMD research community, the work is a reminder that respiratory muscle, the ultimate arbiter of patient survival, presents a uniquely hostile pharmacological environment. The diaphragm of the mdx mouse endures decades of contraction against a fibrotic, inflamed, calcium-dysregulated substrate, and interventions that succeed in limb muscle or in acute injury models may simply be overwhelmed there. As clinical trials of gene therapies, exon skipping, and anti-inflammatory agents continue to reshape the DMD treatment landscape, this study tempers expectations for stress-kinase inhibitors while sharpening the field’s understanding of why dystrophic muscle is so difficult to quiet. PKR remains a suspect worth pursuing, but catching it, the findings suggest, will require more than blocking a single phosphorylation site.

Subject of Research: PKR inhibition with imoxin and disease progression in the dystrophic diaphragm of mdx mice, a model of Duchenne muscular dystrophy

Article Title: Long‐term imoxin treatment does not attenuate disease severity in dystrophic diaphragm of male mice

Article References: Lee, J. H., Vorwald, M. E., Roths, M., Selsby, J. T., & Valentine, R. J. (2026). Long‐term imoxin treatment does not attenuate disease severity in dystrophic diaphragm of male mice. Physiological Reports, 14(18), Article e71115. https://doi.org/10.14814/phy2.71115

Image Credits: AI Generated

DOI: 10.14814/phy2.71115

Keywords: Duchenne muscular dystrophy, mdx mice, protein kinase R, imoxin, diaphragm, ER stress, inflammation, fibrosis, muscle function, eIF2alpha, NFkB, integrated stress response