Electric field stimulation in Caenorhabditis elegans as a novel approach to investigate mitochondrial Ca2+ homeostasis during in vivo muscle aging.

Muscle tissues are core components of biohybrid robots. However, the lack of in-depth contraction dynamics analysis of muscle tissues affects their control and actuation methods, which in turn limits their performance. We aim to develop a new approach to explore muscle contraction properties to provide a reference and foundation for the control and actuation of muscle tissues. We proposed a method using continuous vector sets (a sequence of vectors connected end-to-end) to characterize the flexible contraction properties of muscles on both of the overall and regional contraction. Based on this, we carried out electrical stimulation experiments on muscle tissues. (1) We found a nonlinear three-stage fatigue response of the muscle tissues under prolonged electric field stimulation with a significant performance degradation point (192s at 1Hz stimulation). (2) We investigated the effects of different parameters such as duty cycle, baseline voltage, and waveform on the contraction dynamics of the muscle tissues. We found that the muscle is highly sensitive to changes in electrical signals and could produce two different contraction behaviors within a single stimulation cycle under the appropriate duty cycle (14.5-85.5% at 1Hz frequency). (3) We found significant regional response capability of muscle tissues under unsaturated external electric fields. The difference in contractile strain of the same region within the muscle was up to about 10% under different stimulus electric fields. We believe that this study provides a reference for the optimization of control strategies for biohybrid robots and is expected to offer the possibility of programmable muscle contraction behavior for further engineering applications.

Loss of PINCH2 leads to myogenic urinary retention in mice.

: Spinal cord injury (SCI) frequently impairs defecation, severely affecting the quality of life. This study examines compensatory neural remodeling after SCI, focusing on basal colonic contractility, neural responses to electrical field stimulation, and alterations in excitatory cholinergic and inhibitory nitrergic pathways. : Female Sprague-Dawley rats underwent either sham surgery or T10 spinal cord transection and were categorized into 3 groups: sham, 1-week post-SCI (acute), and 4-week post-SCI (chronic). Colonic contractility was assessed in an organ bath using electrical field stimulation in the presence of a nitric oxide synthase inhibitor. Neural protein expression was analyzed by immunofluorescence and Western blotting. : SCI produced region- and time-dependent impairments in colonic contractility, with distinct alterations in the proximal circular and longitudinal muscles across acute and chronic phases. Neural excitability shifted dynamically, showing enhanced excitatory activity in the proximal longitudinal muscle at 1-week and the distal circular muscle at 4-week post-SCI. Protein analysis revealed increased neuronal nitric oxide synthase in the proximal colon, decreased soluble guanylyl cyclase in the distal colon, upregulated muscarinic M3 receptor in the proximal colon, and reduced vasoactive intestinal peptide receptor 1 in both proximal and distal regions. : SCI induces spatiotemporal remodeling of excitatory and inhibitory neural pathways, contributing to colonic dysmotility and revealing potential targets for therapeutic intervention.

Electric field (EF) stimulation is an emerging neuromodulatory strategy for promoting the repair and functional recovery of degenerated neural networks in neurodegenerative conditions such as glaucoma. EF stimulation therapeutic potential is thought to arise, in part, from modulation of calcium-dependent signaling pathways that regulate neuronal survival and plasticity. However, despite extensive use of EF stimulation in retinal research and clinical studies, it remains unclear how EF waveform frequency and shape govern population-level intracellular calcium dynamics in retinal ganglion cells (RGCs), limiting the rational design of stimulation protocols. Here we address this gap by combining large-scale ex-vivo calcium imaging of Thy1-GCaMP6f mouse retinas with controlled EF stimulation spanning a wide frequency range and a uniform, non-contact stimulation geometry. This approach enables direct measurement of intracellular calcium responses across thousands of individual RGCs under stimulation conditions relevant to non-invasive and translational paradigms. We further develop a morphologically detailed RGC model in NEURON incorporating reaction-diffusion calcium dynamics and admittance-based extracellular stimulation to mechanistically interpret the frequency-dependent responses observed under sinusoidal EF stimulation. Using this integrated experimental–computational framework, we reveal how electric field stimulation modulates population-level calcium signaling in retinal ganglion cells, enabling simultaneous characterization of spatial response patterns and ensemble-averaged activity across thousands of cells. At this scale, RGC calcium responses are constrained to a distinct frequency regime: low frequencies (below 5 Hz) evoke oscillatory transients, intermediate frequencies (10–100 Hz) produce sustained calcium elevation across the population, and high-frequency stimulation (>3 kHz) leads to a sharp attenuation of calcium responses. Among all tested waveforms, a 1:4 asymmetric charge-balanced stimulus at 50 Hz most effectively and consistently elevated intracellular calcium across the RGC population. The computational model reproduces the experimentally observed frequency dependence for sinusoidal stimulation and reveals that the behavior of these different frequency regimes emerges from the interplay between calcium influx, calcium-activated potassium feedback, calcium extrusion kinetics, and soma geometry. Beyond these findings, this work delivers, to our knowledge, the first large-scale dataset of single-cell calcium responses from RGC populations exposed to diverse EF waveforms and frequencies. This dataset enables future data-driven and hybrid modeling approaches that require rich mappings between extracellular stimulation parameters and intracellular calcium dynamics, and establishes a foundation for systematic, physiology-informed optimization of EF stimulation strategies targeting retinal neurodegenerative disease.

Gastric slow waves fail to propagate through the pyloric sphincter (PS), thus isolating the specialized motility patterns of the stomach and small intestine. We investigated the role of interstitial cells of Cajal (ICC) in PS of mice. Ca2+ waves in ICC, events responsible for electrical slow waves, propagated along the gastric wall but failed to propagate into the PS. ICC in PS fired localized Ca2+ transients and displayed low expression of voltage-dependent Ca2+ conductances. These are properties of intramuscular ICC (ICC-IM) that cannot regenerate and propagate slow waves. A T-type Ca2+ channel antagonist had no effect on Ca2+ transients, but these events were blocked by thapsigargin and CPA, suggesting they result from Ca2+ release. PS ICC expressed ANO1, a Ca2+-activated Cl- conductance. Ca2+ released from stores actives ANO1 channels, thus exerting a depolarizing influence on PS. Ani9, selective antagonist of ANO1 channels, hyperpolarized cells and reduced contractile tone. Electrical field stimulation (EFS) of intrinsic neurons yielded inhibitory junction potentials (IJPs), and cessation of EFS resulted in post-stimulus depolarization and contraction. L-NNA abolished relaxation responses to EFS and switched responses to contractions. Application of atropine or Ani9 (in the presence of L-NNA) abolished contraction during EFS. Our results describe new and fundamental functions of ICC-IM in the PS. The inability of these cells to propagate slow waves provides the insulator function of PS muscles and localized Ca2+ transients and activation of ANO1 regulates PS tone and mediates inputs from enteric neurons.

Pain in children and adolescents remains an underestimated and undertreated condition, with long-term physical and psychosocial consequences. Non-invasive neuromodulation has emerged as a promising, low-risk approach for managing acute and chronic pain by modulating central and peripheral neural pathways. This systematic review followed PRISMA 2020 guidelines to evaluate the efficacy, safety, and clinical applicability of non-invasive neuromodulation techniques in pediatric pain. Searches were conducted in PubMed, Embase, Scopus, Web of Science, Cochrane CENTRAL, and ScienceDirect for randomized controlled trials (RCTs) published between 2015 and 2025. Six RCTs met the inclusion criteria, encompassing percutaneous electrical nerve field stimulation (PENFS), transcutaneous auricular vagus nerve stimulation (taVNS), transcutaneous electrical acupoint stimulation (TEAS), and transcutaneous electrical nerve stimulation (TENS). Four trials reported significant reductions in pain intensity alongside improvements in functional outcomes and quality of life, particularly in functional abdominal pain and postoperative contexts. Most studies showed low or moderate risk across domains, with appropriate randomization and blinded assessment. No serious adverse events were reported, confirming an excellent safety profile. These findings support non-invasive neuromodulation as a feasible and well-tolerated adjunct to conventional pediatric pain management. Further high-quality trials are warranted to standardize protocols and explore mechanisms of neuroplasticity in the developing nervous system. PROSPERO (CRD420251170866).

The continuous extension of human life expectancy and the global trend of population aging have contributed to a marked increase in the incidence of musculoskeletal diseases, with fractures and osteoporosis being prominent examples. Consequently, promoting bone regeneration is a crucial medical challenge that demands immediate attention. As early as the mid-20th century, researchers revealed that electrical stimulation could effectively promote the healing and regeneration of bone tissue. This is achieved by mimicking the endogenous electric field within bone tissue, which influences cellular behavior and molecular mechanisms. In recent years, electroactive hydrogels responsive to electric field stimulation have been developed and applied to regulate cell functions at different stages of bone regeneration. This paper elaborates on the regulatory effects of electrical stimulation on MSCs, macrophages, and vascular endothelial cells during the process of bone regeneration. It also involves the activation of relevant ion channels and signaling pathways. Subsequently, it comprehensively reviews various electric-field-responsive hydrogels developed in recent years, covering aspects such as material selection, preparation methods, characteristics, and their applications in bone regeneration. Ultimately, it provides an objective summary of the existing deficiencies in hydrogel materials and research, and looks ahead to future development directions.

Basal release of 6-cyanodopamine in human vas deferens, a new endogenous catecholamine that enhances smooth muscle contractility.

Direct current electric field suppresses NPFFR2 expression in macrophages and alters its predicted conformational dynamics.

Subthreshold electric fields bidirectionally modulate neurotransmitter release through axon polarization.

ABSTRACTGas therapy has been limited in its application as a robust standalone antitumor strategy due to the restricted gas production and cytotoxicity. To address this challenge, we employed electrotoxic PtRu composite metal nano‐berries (PR) loaded with various therapeutic gas donors to construct a groundbreaking electric field‐induced cascade gas therapy (EGT) platform, which generated a great electro‐stress storm at tumor sites, exerting electrotoxicity and immunity functions against solid tumors, including those of large volume, through three pathways. Initially, electric field stimulation effectively boosted the release rate and yield of therapeutic gases from the EGT platform. Further, gas molecules reacted with reactive oxygen species (ROS) to either form oxidation coordination (CO and ROS) or generate more potent therapeutic components (RNS produced from ROS and NO), contributing to an electro‐stress storm that augmented the cytotoxic potential of the gas components. Subsequently, this electro‐stress storm further activated the tumor immune response, identifying and capturing escaped tumor cells, which held significant implications for treating tumors, including non‐solid tumors with indistinct boundaries. In summary, the EGT platform leveraged an electro‐stress storm to achieve ablation of large volume solid tumors and suppressed metastatic tumors, paving new pathways for gas‐based therapeutic strategies.

Cell migration is a cornerstone of biological systems, enabling organisms to adapt to environmental stimuli and maintain homeostasis. Disruptions in this process can lead to functional impairment or system failure. In many cases, cells do not move randomly; instead, they migrate directionally in response to external cues, allowing them to perform essential biological functions. This directed movement is especially important in processes such as morphogenesis, cancer invasion, and wound healing. To unravel the complexities of directional cell migration, investigating natural guiding stimuli is crucial. Among these, electrical fields stand out as precise and physiologically relevant stimulus. Using a platform designed to apply programmable electric fields, the SCHEEPDOG device, we applied controlled electric field of varying intensities to keratocytes and quantitatively analyzed their migratory behavior. Our findings reveal that electric field stimulation not only induces robust directional migration but also enhances migration speed in an intensity-dependent manner. Additionally, cells initially moving in random directions gradually align with the field vector, with higher intensities accelerating the alignment. Intriguingly, while both speed and alignment time can be modulated through stimulation, the overall shape of migration trajectories remains unchanged. In other terms, for cells initially moving to the opposite direction of the field, the alignment is accompanied by making a turn and the size and shape of this turn are not affected by the magnitude of the electrical stimulation. Together, these results demonstrate that electrical stimulation can tune the speed and directional alignment of keratocyte migration without altering turning dynamics. These findings contribute to a deeper understanding of electrotaxis and offers new insights into how biophysical cues regulate cell migration in both physiological and pathological contexts.

Electric field (EF) stimulation is an emerging biophysical approach that enhances stem cell function by mimicking endogenous wound currents. However, its effects on tonsil-derived mesenchymal stem cells (TMSCs) remain poorly understood. A low-intensity EF stimulation system (0-12mV; potential difference between parallel electrodes, 5mm apart; 5min on/5s off for 38h) was established to examine the effects of EF on TMSC viability, proliferation, stemness, and chondrogenic differentiation. Young and senescent TMSCs were evaluated for metabolic activity, cell cycle distribution, and expression of stemness- and chondrogenesis-related markers. For differentiation assays, cells were preconditioned with EF stimulation before chondrogenic induction. Moderate EF intensities (4-8mV) enhanced the viability, metabolic activity, and proliferation of both young and senescent TMSCs, whereas excessive stimulation (12mV) reduced these functions without causing cell death. In senescent TMSCs, EF stimulation promoted S-phase entry and upregulated Cyclin A2 and Cyclin B1 expression, suggesting partial restoration of proliferative potential. In young TMSCs, EF stimulation increased NANOG, OCT4, and SOX2 expression, thereby supporting stemness maintenance. EF stimulation enhanced glycosaminoglycan deposition and chondrogenic marker expression (Aggrecan, COL2A1, and SOX9) when applied before chondrogenic induction but exerted an inhibitory effect when applied during the differentiation phase. Low-intensity EF stimulation serves as a tunable bioelectric cue that enhances the proliferation, stemness, and early chondrogenic potential of TMSCs in an intensity- and state-dependent manner, providing a non-invasive strategy to improve mesenchymal stem cell function for regenerative applications.

Fluorescence-based optical techniques have significantly advanced our comprehension of living systems, providing invaluable insights into cellular intricacies and facilitating fundamental research and practical diagnostic applications. To explore intracellular environments and the communication among intracellular organelles, we utilized fluorescence lifetime imaging microscopy with exogenous fluorophores of Oregon Green 488 BAPTA-1 AM and Fluovolt and endogenous fluorophores of NAD(P)H and FAD in breast carcinoma MCF7 and MDA-MB-231 and normal MCF10A cells. Fluorescence lifetime images have uncovered a characteristic triangular interrelationship among intracellular calcium ion concentration (Ca2+), mitochondrial function, and membrane electric potential in cancerous and normal cells. To delve deeper into the effects of electric field stimulation on intracellular conditions, the nanosecond pulsed electric field (nsPEF) was applied to the breast cancerous and normal cells. Our findings revealed a unique electric field modulation pattern across breast cells; MCF7 cells exhibited initial modulation in cytosolic Ca2+ levels, followed by changes in mitochondrial function, whereas MDA-MB-231 and MCF10A cells mostly remained unaffected, demonstrating that intracellular conditions and the effect of nsPEF are different not only between cancerous and normal cells but also between subtypes of cancerous cells.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-32020-y.

Abstract Introduction Ehlers-Danlos syndrome (EDS) is an inherited connective tissue disorder characterized by collagen abnormalities, leading to tissue fragility. Individuals with EDS often experience urinary incontinence, pelvic pain, and sexual dysfunction, with a higher prevalence in female (73–89%). Classic EDS (type 1) arises from mutations in the collagen type V (Col5a1) gene. Collagen V plays a pivotal role in the regulation of fibrillogenesis. However, the role of collagen type V on the pelvic organs has not been assessed. The EDS murine model has a mutation in the collagen V (Col5a1) gene and displays a similar phenotype to the clinical condition. Objective The goal of our study is to determine the role of collagen type V in smooth muscle function in the penis and vagina in the EDS mouse model. Methods We assessed male and female 16-week old wild-type (WT; Col5a1 +/+) and heterozygous (EDS; Col5a1 +/-) mice (n=13–14/sex). Tissue bath experiments were conducted on isolated cavernosal and vaginal strips. Contractions were induced by high potassium to assess tissue viability, followed by concentration-response curves to phenylephrine, nitric oxide donor DEA NONOate, acetylcholine and relaxation to electrical field stimulation (EFS) in the penis. Additionally, the vaginal tissue underwent concentration-response curves to norepinephrine, DEA NONOate, and EFS relaxation. The iliococcygeal muscles from the female pelvic floor were also isolated for isometric force production. Results Penile contractions from the EDS mice were greater to phenylephrine, contracting approximately 50% and 66% more at 3x10–7 and 10–7 M, respectfully (p<0.05). This was also reflected in a lower EC50 concentration for EDS penises. However, we saw no difference in penile relaxation to DEA NONOate, acetylcholine and EFS. Similarly, the female EDS mice had greater vaginal contractions to norepinephrine and a lower EC50 concentration (p<0.05). There was no difference in vaginal relaxation to DEA NONOate or EFS. When the iliococcygeal muscle was stimulated at increasing frequencies, there was no difference in muscle contractions between the two genotypes. Conclusions Our findings suggest that the Col5a1 heterozygous mouse model holds potential as an animal model for replicating the genitourinary issues observed in clinical EDS. We are currently looking at the penile and vaginal morphology and measuring collagen protein and gene expression. Our study has established a foundational framework for future research endeavors to further understand the pathophysiological mechanisms that may occur with age or birth trauma in EDS. The ultimate is to have a clinically relevant model to assess potential therapies to improve EDS urogenital symptoms. Disclosure No

Abstract Introduction Radical prostatectomy (RP) is one of the most common therapies for prostate cancer treatment. Unfortunately, most of the RP operations end up with erectile dysfunction (ED) due to cavernous nerve damage. Recently it is discovered that mitochondrial transplantation has a therapeutic potential for various conditions including neurodegenerative diseases, ischemia and nerve injuries. Objective The objective of this study was to evaluate the efficacy of mitochondrial transplantation for RP related ED. Methods Total of 32 Sprague Dawley rats were divided as sham (n=7), bilateral cavernous nerve injury (BCNI, n=7), BCNI + mitochondria (n=7), BCNI + vehicle (n=7), mitochondria analysis (n=3) and mitochondria donor (n=1) groups. BCNI procedure was selected for modeling RP related ED. Isolated mitochondria were transplanted to cavernous tissue of subjects right after BCNI or sham operations. Following the treatment, animals were sacrificed for in vivo intracavernous pressure (ICP) measurements with cavernous nerve stimulation and in vitro isolated organ bath studies. Isometric tension of cavernosal smooth muscle strips was recorded for potassium chloride, acetylcholine, electric field stimulation (EFS), sodium nitroprusside (SNP), and phenylephrine. Tissue samples were collected for Western blotting analysis and histological examinations. Results The results show that mitochondrial transplantation increased ICP values. Acetylcholine induced and EFS relaxation responses were enhanced with mitochondrial transplantation according to vehicle and BCNI groups. There are no significant difference between groups for SNP relaxation responses. eNOS expressions in the cavernous tissue were increased in mitochondria group. Conclusions Improved erectile function, increased eNOS expression and isometric relaxation responses suggest that mitochondrial transplantation is involved in endothelial and neuronal mechanisms of erection. Disclosure No

Abstract Introduction Dapagliflozin (DAPA), a sodium glucose co-transporter 2 inhibitor, is an oral antidiabetic medication. Although effects of long-term DAPA treatment is well known for diabetes related ED, no research has conducted for influence of DAPA on relaxation responses in cavernous tissue. Objective In this study we aim to investigate effects of DAPA on human cavernous tissue. Methods Human corpus cavernosum tissues were acquired from adult males undergoing penile prosthesis surgeries due to diabetes or radical prostatectomy related ED. Tissue strips were evaluated for DAPA in organ bath. Results DAPA incubations were found to significantly reduce relaxation responses for acetylcholine (ACH), and electric field stimulation (EFS). Conclusions Reduced relaxation responses for EFS and ACH indicate that DAPA is impairing neuronal and endothelial mechanisms. Disclosure No

Abstract Introduction Hydroxymethylglutaryl-CoA reductase inhibitors, known as statins, are commonly used for hypercholesterolemia treatment among with diet. Although it is established that statins aid in the recovery of erectile dysfunction by enhancing endothelial function, there are no studies examining the impact of statins on the functioning of erectile tissue. Objective In this study we aimed to investigate effects of statins on human corpus cavernosum tissue. Methods Cavernosal strips were incubated with atorvastatin (ATR) and rosuvastatin (RSV) in organ bath. Relaxation responses were recorded. Results ATR and RSV exhibited considerably diminished relaxation responses to acetylcholine and electric field stimulation, with no discernible difference between ATR and RSV. Conclusions Our data show that ATR and RSV reduce relaxation responses possibly by interfering with neuronal and endothelial mechanisms. Further studies are required for in vivo erectile function evaluation. Disclosure No

Abstract Introduction While obesity is a recognized contributor to erectile dysfunction (ED), the impact of early-life obesity on the long-term function of the corpus cavernosum (CC) and pudendal artery (PA) is not well defined. We hypothesized that nutritional overload in early development triggers cellular senescence and structural changes, impairing neurovascular components of erectile function in adulthood. Objective We hypothesized that nutritional overload in early development triggers cellular senescence and structural changes, impairing neurovascular components of erectile function in adulthood. To determine whether early-life overnutrition induces long-term structural and functional alterations in the corpus cavernosum and pudendal artery. We aimed to assess cavernous and vascular reactivity, along with molecular markers of senescence and tissue remodeling, using a rat model of postnatal nutritional overload. Methods Wistar rats were nursed in small litter (SL; 3 pups/dam) to induce postnatal overnutrition or in normal litter (NL; 10 pups/dam) as controls. After weaning on postnatal day (PND) 21, animals were maintained on standard chow until PND160. Body weight, fat pad mass, and systolic blood pressure (SBP) were recorded. Functional assays in CC and PA evaluated responses to phenylephrine (PE), acetylcholine (ACh), and electrical field stimulation (EFS). Transcriptomic profiling by RNA sequencing and gene validation via RT-qPCR were conducted to investigate markers of senescence and tissue remodeling. Results Final body mass did not differ between groups, but SL rats exhibited higher retroperitoneal and perigonadal fat mass, along with elevated SBP (SL: 120.5 ± 1.2 mmHg vs NL: 114.9 ± 0.58 mmHg). In the CC, EFS-induced relaxation was reduced in SL animals (16 Hz: SL 2.39 ± 0.53 mN vs NL 3.06 ± 0.28 mN), while PE- and EFS-induced contractions were increased and partially reversed by indomethacin, suggesting prostanoid involvement. In the PA, ACh-mediated relaxation was significantly impaired (Emax: SL 28 ± 6% vs NL 68 ± 5.5%), without changes in PE-induced tone. Transcriptomic analysis revealed 124 differentially expressed genes (DEGs) in the CC, 99 in male PA, and 252 in female PA. Enrichment mapping in male tissues highlighted pathways involved in extracellular matrix remodeling, metabolic control, and secretin-related GPCR signaling (e.g., Col1a1, Fstl3, Adamts6, Thbs6, PI3K-Akt-mTOR). In contrast, DEGs in female PA were enriched for innate immune and metabolic/hormonal response pathways. RT-qPCR in CC confirmed increased Bgn and Col1a2, along with altered expression of Calcrl, Vipr2, and Glp1/2r, consistent with senescence-associated remodeling. Cross-tissue comparison identified 11 shared genes, possibly involved in neurovascular tone, metabolic stress responses, and architectural remodeling. Conclusions Early-life obesity induces persistent structural and functional alterations in erectile and pudendal tissues, likely contributing to erectile dysfunction in adulthood. These changes appear to be mediated by pathways involving senescence, fibrosis, and secretin GPCR signaling. Additionally, the identification of shared molecular signatures across tissues highlights a potential neurovascular-metabolic axis disrupted by early metabolic stress. Ongoing studies aim to elucidate the specific roles of these candidate genes and pathways in the pathophysiology of obesity-induced sexual dysfunction. Disclosure No