Journal of Molecular Histology & Medical Physiology

The Enteric Nervous System (ENS) plays a pivotal role in governing the motor, secretory, and defensive functions of the gastrointestinal tract. These enteric neurons intricately process both mechanical and chemical cues from the gut lumen, translating them into intricate motor responses. However, the precise manner in which intact enteric neural networks react to shifts within the gut environment remains a puzzle. To unravel this enigma, we conducted live-cell confocal recordings, capturing intracellular calcium activity in neurons extracted from intact portions of mouse intestine. Our aim was to investigate how neurons respond to various luminal mechanical and chemical stimuli. Utilizing specialized Wnt1, ChAT, and Calb1-GCaMP6 mice, we focused on neurons residing in the jejunum and colon. Our experimental design encompassed an examination of neuronal calcium responses triggered by diverse stimuli, including KCl (75 mM), veratridine (10 μM), 1,1-dimethyl-4-phenylpiperazinium (DMPP; 100 μM), and luminal nutrients (Ensure®), all under conditions of either intraluminal distension or its absence. The outcomes were particularly intriguing: in both the jejunum and colon, the presence of luminal content (chyme in the jejunum and faecal pellets in the colon) induced distension, rendering the underlying enteric circuit unresponsive to depolarizing stimuli. Notably, in the distal colon, heightened levels of distension displayed an inhibitory effect on neuronal reactions to KCl. Moreover, intermediary distension levels orchestrated a reconfiguration of Ca²⁺ responses, particularly influencing the circumferential propagation of slow waves. Our experimentation also revealed the key role of mechanosensitive channels; the inhibition of these channels effectively suppressed distension-induced Ca²⁺ elevations. Furthermore, we uncovered that inhibiting calciumactivated potassium channels restored neuronal responses to KCl in the distended colon, but not to DMPP. In a novel discovery, distension in the jejunum halted a tetrodotoxin-resistant neuronal response to luminal nutrient stimulation. In summation, our findings demonstrate that intestinal distension operates as a regulator of ENS circuit excitability, with mechanosensitive channels acting as key mediators. The dynamic interplay between physiological levels of distension and neural synchronicity or suppression showcases the ENS's ability to fine-tune its responses based on the gut's luminal content.

A growing body of evidence points towards the involvement of myocardial steatosis in the development of left ventricular diastolic dysfunction, although conclusive proof in humans is hampered by complicating coexisting conditions. To address this, we employed a 48-hour food restriction protocol to induce an acute elevation in Myocardial Triglyceride (mTG) content quantified using 1H magnetic resonance spectroscopy in a cohort of 27 young and healthy volunteers (comprising 13 men and 14 women). The results of the fasting regimen exhibited a remarkable over threefold rise in mTG content (P<0.001). Interestingly, the early diastolic circumferential strain rate (CSRd), a marker of diastolic function, remained unaffected following the 48-hour fasting intervention. However, a noteworthy elevation in systolic circumferential strain rate was observed (P<0.001), indicating a decoupling between systolic and diastolic phases. This phenomenon of uncoupling was further substantiated by an additional experiment involving 10 individuals, where the administration of low-dose dobutamine (2 μg/kg/min) resulted in a similar alteration in systolic circumferential strain rate as the one observed during the 48-hour food restriction. Moreover, this change was accompanied by a proportionate increase in CSRd, thereby preserving the synchronization between the two metrics. The collective findings of this study underscore the role of myocardial steatosis in instigating diastolic dysfunction by disrupting the coupling between diastole and systole in the context of healthy adult subjects. Furthermore, these findings postulate a potential contributory role of steatosis in the progression of cardiovascular ailments.

In the cardiac context, glucose and glycolysis play pivotal roles in supporting anaplerosis and potentially influencing the oxidation of d-β-hydroxybutyrate (βHB). The presence of glycogen, serving as a reservoir for glucose, could also contribute to anaplerosis. To delve into the intricate connections between glycogen content, βHB oxidation, glycolytic rates, and their consequential impact on energy dynamics, an isolated rat heart model was employed. Hearts with high glycogen (HG) and low glycogen (LG) levels were perfused with 11 mM [5-3H] glucose and/or 4 mM [14C] βHB to assess glycolysis and βHB oxidation, respectively. Subsequently, freeze-clamping was carried out for glycogen and metabolomic analyses. The ratio of free cytosolic [NAD+]/[NADH] and mitochondrial [Q+]/[QH2] was estimated using the lactate dehydrogenase and succinate dehydrogenase reactions. 31P-nuclear magnetic resonance spectroscopy was utilized to measure phosphocreatine (PCr) and inorganic phosphate (Pi) concentrations. Notably, βHB oxidation rates in LG hearts were found to be half of those in HG hearts, exhibiting a direct correlation with glycogen content. Remarkably, βHB oxidation led to a reduction in glycolysis across all heart conditions. In glycogen-rich hearts perfused solely with βHB, glycogenolysis was twofold compared to hearts perfused with both βHB and glucose. This latter group demonstrated elevated levels of glycolytic intermediates, specifically fructose 1,6-bisphosphate and 3-phosphoglycerate, alongside a higher free cytosolic [NAD+]/[NADH] ratio. The influence of βHB oxidation was further evident through heightened levels of Krebs cycle intermediates, such as citrate, 2-oxoglutarate, and succinate. Additionally, the total NADP/H pool increased, mitochondrial [Q⁺]/[QH₂] decreased, and the calculated free energy of ATP hydrolysis (ΔGATP) was elevated. Intriguingly, while βHB oxidation exerted an inhibitory effect on glycolysis, the reserves of glycolytic intermediates remained intact, and cytosolic free NAD sustained its oxidized state. Furthermore, βHB oxidation in isolation not only amplified Krebs cycle intermediates but also resulted in reduced mitochondrial Q levels and an enhanced ΔGATP. In summation, our findings underscore the facilitating role of glycogen in promoting cardiac βHB oxidation through anaplerosis.

During exercise, the culmination of the O₂ cascade is contingent upon the interaction between microvascular to intramyocyte differences and muscle O₂ diffusion capacity. Presently, there is a lack of non-invasive techniques for determining in human subjects. Utilizing near-infrared spectroscopy (NIRS) and intermittent arterial occlusions to measure the recovery rate constant (k) of muscle oxygen uptake (m), we have established its correlation with in vivo muscle oxidative capacity. We postulated that k would be constrained by under conditions of low muscle oxygenation (kLOW). We proposed two hypotheses: (i) k in optimally oxygenated muscle (kHIGH) is linked to the maximum O₂ flux within fiber bundles; and (ii) the difference (Δk) between kHIGH and kLOW is associated with capillary density (CD). In a study involving 12 participants, we employed NIRS to assess the vastus lateralis k following moderate exercise. The timing and duration of arterial occlusions were manipulated to maintain the tissue saturation index within a 10% range either below (LOW) or above (HIGH) half-maximal desaturation, which was determined during sustained arterial occlusion. The maximal O₂ flux in the phosphorylating state was determined to be 37.7 ± 10.6 pmol s⁻¹ mg⁻¹ (∼5.8 ml min⁻¹ 100 g⁻¹), and CD ranged from 348 to 586 mm^–2. kHIGH surpassed kLOW (3.15 ± 0.45 vs. 1.56 ± 0.79 min^–1, P<0.001). While maximal O₂ flux correlated positively with kHIGH (r=0.80, P=0.002), there was no correlation with kLOW (r=–0.10, P=0.755). The range of Δk extended from –0.26 to –2.55 min^–1, and it exhibited a negative correlation with CD (r=–0.68, P=0.015). It is evident that solely reflects muscle oxidative capacity under conditions of optimal oxygenation. Moreover, the disparity (Δk) between well-oxygenated and poorly oxygenated muscle was found to be linked to CD, a key factor in. The assessment of muscle k and Δk through NIRS offers a non-invasive avenue for gaining insights into muscle oxidative and O2 diffusion capacities.

Duchenne Muscular Dystrophy (DMD) stands as a debilitating condition triggered by mutations in the dystrophin gene. These mutations culminate in compromised sarcolemmal integrity, initiating a cascade of events marked by progressive myofibre necrosis and deteriorated muscle function. Earlier investigations from our lab underscored the significance of lipin1 in bolstering skeletal muscle regeneration and upholding myofibre integrity. Moreover, our studies unveiled a substantial reduction in lipin1 mRNA expression within the skeletal muscle of both DMD patients and the mdx mouse model, a classic model for DMD. Seeking a deeper comprehension of lipin1's role in dystrophic muscle, we embarked on generating dystrophin/lipin1 Double Knockout (DKO) mice. Through a comparative analysis encompassing wild-type B10 mice, muscle-specific lipin1 deficient (lipin1Myf5cKO) mice, mdx mice, and DKO mice, we uncovered a more severe phenotype in the DKO cohort, characterized by intensified necroptosis, fibrosis, and aggravated membrane damage relative to mdx mice. Intriguingly, barium chloride-induced muscle injury spotlighted prolonged regeneration at day 14 post-injection in both lipin1Myf5cKO and DKO mice, underscoring the critical role of lipin1 in muscle regeneration. In situ contractile function assays disclosed diminished specific force production in dystrophic muscles lacking lipin1. Cellular experimentation further solidified these findings, as lipin1-deficient cells exhibited elevated levels of necroptotic markers and medium creatine kinase, potentially stemming from sarcolemmal damage. Significantly, the restoration of lipin1 curbed the elevation of necroptotic markers in differentiated primary lipin1-deficient myoblasts. Collectively, our findings paint a picture of lipin1's dual contribution to myofibre stability and muscle function within the realm of dystrophic muscles. The tantalizing prospect of leveraging lipin1 overexpression as a therapeutic strategy for dystrophic muscles beckons as a beacon of hope.

Serotonergic neuromodulation plays a role in enhancing voluntary muscle activation. However, the influence of the potential motoneuron receptor candidate (5-HT2) on the firing rate and activation threshold of motor units (MUs) in humans remains uncertain. This study aimed to investigate the impact of 5-HT2 receptor activity on human MU behavior during gradually increasing contractions of varying intensity. The tibialis anterior muscle's high-density surface electromyography (HDsEMG) was recorded while participants performed ramped isometric dorsiflexions at 10%, 30%, 50%, and 70% of their maximum voluntary contraction (MVC). MU characteristics were extracted from HDsEMG data collected from 11 young adults (including four females) before and after taking either an 8 mg cyproheptadine dose or a placebo. Blocking 5-HT2 receptors led to a decrease in MU discharge rate during steady-state muscle activation, regardless of the contraction intensity (P<0.001; estimated mean difference (Δ)=1.06 pulses/s). Additionally, there was an elevated MU derecruitment threshold (P<0.013, Δ=1.23% MVC), while maximal voluntary contraction force remained unchanged (P=0.652). At 10% MVC (P<0.001, Δ=0.99 Hz) and 30% MVC (P=0.003, Δ=0.75 Hz), there was a reduction in estimates of persistent inward current amplitude, aligning with changes in MU firing behavior attributed to 5-HT2 receptor antagonism. Overall, these findings underscore the role of 5-HT2 receptor activity in regulating discharge rate among groups of spinal motoneurons during voluntary contractions. This study offers evidence of a direct connection between MU discharge properties, persistent inward current activity, and 5-HT2 receptor activity in humans.

Residing at high altitudes (>2500 m or 8200 ft) leads to a decline in blood flow through the uterine artery during pregnancy, contributing to a higher occurrence of preeclampsia and intrauterine growth restriction. Nevertheless, not all pregnancies experience the effects of prolonged hypoxia associated with high-altitude living. Potassium (K⁺) channels play a pivotal role in the uterine blood vessel adjustments during pregnancy, promoting the relaxation of muscle tone and an augmentation in blood circulation. We postulated that in pregnancies with normal fetal growth at high altitudes, there is an augmented K⁺ channel-mediated vasodilation in myometrial arteries compared to those in healthy pregnant women residing at lower altitudes (approximately 1700 m). Through the manipulation of two K⁺ channels the ATP-sensitive (KATP) and large-conductance Ca²⁺-activated (BKCa) K⁺ channels we evaluated the vasodilation response in myometrial arteries derived from pregnancies with appropriate gestational age (AGA) in women living at varying altitudes. Additionally, we investigated the spatial distribution of these channels within myometrial arteries using immunofluorescence techniques. Our findings revealed an increase in endothelium-dependent KATP-mediated vasodilation in myometrial arteries from high-altitude residents compared to those from lower altitudes, whereas vasodilation triggered by activation of BKCa channels was diminished in these arteries. Furthermore, the co-localization of KATP channels with endothelial markers was reduced in myometrial arteries from high-altitude residents, suggesting that the heightened KATP activity might be governed by mechanisms unrelated to channel localization regulation. These observations underscore the significant contribution of K⁺ channels to the adaptive response of human uterine blood vessels during pregnancy at high altitudes, crucial for maintaining normal fetal growth in the face of chronic hypoxia conditions.

Maintaining the equilibrium of bodily water involves a delicate interplay between water intake and excretion through processes such as urination, defecation, perspiration, and respiration. Notably, elevated levels of the antidiuretic hormone vasopressin have been observed to reduce urine volume, thereby preventing excessive loss of water from the system. The canonical pathway for this regulation occurs within the renal collecting ducts, where vasopressin triggers a sequence involving cAMP and protein kinase A (PKA) signalling. This cascade culminates in the phosphorylation of Aquaporin-2 (AQP2) water channels, facilitating the reabsorption of water from urine. While recent omics data have shed light on downstream targets influenced by PKA, the pivotal regulators that oversee PKA-induced AQP2 phosphorylation remain elusive. A primary challenge arises from the widespread application of vasopressin as a positive control to activate PKA, an approach that can induce non-specific phosphorylation across various PKA substrates due to vasopressin's potent and indiscriminate effects. The intracellular positioning of PKA is under meticulous control, largely orchestrated by specialized scaffold proteins termed A-Kinase Anchoring Proteins (AKAPs). These AKAPs possess distinct target domains dictating their precise cellular localization, thereby creating discrete PKA signalling networks. Though vasopressin typically triggers PKA activation regardless of intracellular localization, certain chemical agents selectively target PKAs situated on vesicles containing AQP2. These agents concurrently induce phosphorylation of AQP2 and its associated PKA substrates. Through immunoprecipitation coupled with mass spectrometry analysis, it was discovered that lipopolysaccharide-responsive and beige-like anchor stood proximal to AQP2 as a PKA substrate. Subsequent investigations utilizing Lrba knockout models underscored the essential role of LRBA in vasopressin-induced AQP2 phosphorylation.

The Carotid Body (CB) stands as a prototypical organ specialized in acute oxygen (O₂) sensing, orchestrating reflexive hyperventilation and heightened cardiac output during instances of hypoxemia. The recurrence of intermittent hypoxia generates a repetitive stimulus that potentially triggers CB overactivation, contributing to the sympathetic hyperactivity observed in sleep apnea sufferers. Despite evidence showcasing the malleability of CB function due to chronic intermittent hypoxia (CIH), the intricate mechanisms underpinning this phenomenon remain partially understood. This study unveils that CIH elicits a modest expansion in CB dimensions and prompts a reconfiguration of cell types within the CB structure. Notably, this involves the mobilization of latent, immature neuroblasts, which embark upon a journey of differentiation, ultimately transforming into mature, O₂-sensing, neuron-like chemoreceptor glomus cells. Through the prospective isolation of distinct cell classes, we demonstrate that as CB neuroblasts mature, there is a concurrent surge in the expression of specific genes associated with acute O₂-sensing in glomus cells. Furthermore, CIH enhances the hypoxia-responsive capacity of mitochondria both in the progressing neuroblasts and the established glomus cells. This novel insight into the mechanisms governing CB-mediated sympathetic overflow offers a fresh perspective and the potential for the development of innovative pharmacological interventions with implications for sleep apnea patients.

Comprehensive insights into muscle physiology necessitate in-depth analyses of fiber types, yet the laborious nature of dissecting and typing individual fibers has posed significant challenges. This often limits investigations to a scant number of fibers from a handful of participants, casting doubts on the representativeness of both the fiber and participant populations. To surmount these obstacles and enable large-scale, fiber-specific studies, an innovative and rapid technique for high-throughput fiber typing of individually dissected fibers has been developed and named Thrifty (High-Throughput Immunofluorescence Fiber Typing). In the Thrifty approach, 400 fiber segments are affixed to microscope slides with a pre-printed grid system, subjected to antibody probing against myosin heavy chain (MyHC)-I and MyHC-II, and classified under a fluorescence microscope. The effectiveness and expediency of Thrifty are benchmarked against a previously established protocol (dot blot) on a fiber-by-fiber basis, while the purity of fiber pools is verified using the gold standard SDS-PAGE and silver staining. Additionally, a modified Thrifty protocol utilizing fluorescence western blot equipment has been successfully validated. Thrifty exhibits exceptional concurrence with the dot blot protocol, yielding a K value of 0.955 (95% CI: 0.928, 0.982), P<0.001. Both the original and modified Thrifty methods consistently yield type I and type II fiber pools of absolute purity. Impressively, the Thrifty procedure accomplishes the typing of 400 fibers in just under 11 hours, nearly three times faster than the dot blot method. Notably, Thrifty emerges as an inventive and dependable approach, boasting remarkable versatility for swift fiber typing of individual fibers. Consequently, Thrifty stands poised to streamline the generation of extensive fiber pools, thereby paving the way for more comprehensive explorations into the intricate mechanisms governing skeletal muscle physiology.