Muscle mitochondrial partial pressure of oxygen (PmitoO2) is a key determinant of skeletal muscle oxygen consumption (V̇O2), metabolism, gene regulation, and adaptation in health and disease. Yet PmitoO2 remains difficult to measure directly in vivo in humans during exercise. Myoglobin-associated PO2 (PMbO2), measured non-invasively using proton magnetic resonance spectroscopy, may provide a measure of PmitoO2 during maximal exercise in humans because Mb is coupled to mitochondrial oxygen availability and PMbO2 falls in the low PO2 range expected for PmitoO2 in vivo. However, it remains unknown whether PMbO2 during maximal exercise varies systematically with muscle V̇O2max, as would be expected if PMbO2 reflects PmitoO2 in vivo. We analyzed data from five initially sedentary males before and after 8 weeks of single-leg knee-extensor exercise (KE) training. During maximal KE under 0.12, 0.21, and 1.00 fractions of inspired oxygen (FIO2), we measured PMbO2 using myoglobin desaturation measured by proton magnetic resonance spectroscopy and muscle V̇O2max from leg blood flow and the arterial-femoral venous O2 content difference. Across FIO2 conditions, muscle V̇O2max varied systematically with PMbO2 both before and after training, consistent with O2-dependent mitochondrial respiration. When interpreted within a canonical hyperbolic framework, the V̇O2-PMbO2 relationship yielded low apparent mitochondrial P50 values that were broadly consistent with values reported for human skeletal muscle isolated mitochondria studied in vitro. A complementary linear P50 analysis that did not assume a hyperbolic relationship supported the same interpretation. Together, these findings provide data-supported, proof-of-concept evidence that PMbO2 reflects in vivo PmitoO2 during maximal exercise in humans.
The pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) mediates airway responses to acute inflammation. Previously we demonstrated that, in human airway smooth muscle (hASM) cells, TNFα increases reactive oxygen species (ROS) formation. TNFα also selectively activates the inositol-requiring enzyme 1α (pIRE1αS724 autophosphorylation) endoplasmic reticulum (ER) stress pathway involving splicing of X-box binding protein 1 (XBP1s) and transcriptionally activates cyclin-dependent kinases 1 and 5 (CDK1 and CDK5), promoting dynamin-related protein 1 (DRP1) phosphorylation at serine 616 (pDRP1S616) and mitochondrial fragmentation. In the present study, we hypothesized that in hASM, TNFα-induced ROS triggers pIRE1αS724/XBP1s ER stress pathway. To test this hypothesis, we examined the impact of the ROS scavenger Tempol on TNFα-induced pIRE1αS724/XBP1s ER stress pathway and downstream signaling mediating mitochondrial fragmentation. Bronchiolar tissue samples were obtained from 6 patients with no history of smoking or chronic pulmonary disease. The smooth muscle layer was dissected, and hASM cells were dissociated and randomly assigned to four treatment groups: 1) Vehicle, 2) Vehicle + TNFα (20 ng/mL, 6 h), 3) Tempol (500 μM) only, and 4) Tempol (500 μM) + TNFα (20 ng/mL, 6 h). ROS formation was determined by confocal imaging using MitoSOX™ Red. Mitochondria were labeled with MitoTracker Red and imaged using confocal microscopy. Using Western blot, we demonstrated that Tempol reduced cellular ROS formation and mitigated the TNFα-induced increase in pIRE1αS724, XBP1s, CDK1/5, pDRP1S616 protein levels and reduced mitochondrial fragmentation. These findings support our hypothesis and indicate a role of ROS in mediating TNFα-induced ER stress and mitochondrial fragmentation.
Circadian rhythms are endogenous ∼24-h cycles that regulate cardiovascular physiology. Although circadian regulation of the left ventricle (LV) is well established, whether the right ventricle (RV) exhibits intrinsic rhythmicity is not known. Here, we provide the first evidence that the healthy RV exhibits robust rhythms in function and molecular gene expression. Cardiovascular disease is the leading cause of mortality in type 1 diabetes (T1D). T1D disrupts circadian rhythms, yet how T1D alters chamber-specific circadian control remains unclear. We investigated RV and LV function and gene expression across the 24-h light-dark cycle in male streptozotocin-induced T1D and control mice. Echocardiographic assessment of diabetes-induced remodeling at ZT0-4 and ZT12-16 [zeitgeber time (ZT)] revealed time-dependent functional impairment, including reversal of the normal diurnal heart rate pattern. T1D impaired day-night RV and LV systolic function, with loss of day-night difference in LV ejection fraction and impaired active period RV stroke volume. Cosinor analysis of RV and LV gene expression demonstrated preserved 24-h rhythmicity of core clock genes Arntl1 and Per2 in both ventricles with T1D, whereas the clock output genes Nr1d1 and Dbp had dampened amplitude in the RV but amplified expression in the T1D LV. These findings demonstrate that T1D differentially disrupts circadian regulation of the RV and LV, with selective vulnerability of the clock-controlled output genes despite preservation of the core oscillator. Chamber-specific circadian remodeling may contribute to the elevated cardiovascular risk in T1D and has implications for the timing of diagnostic and therapeutic interventions.NEW & NOTEWORTHY This study provides the first evidence that the healthy right ventricle (RV), like the left ventricle (LV), possesses an intrinsic circadian clock with robust 24-h rhythmicity. Using a mouse model of type 1 diabetes, we show that diabetes does not uniformly suppress cardiac clocks but instead differentially rewires RV and LV rhythms, dampening clock-output gene amplitude in the RV while amplifying it in the LV. Together, we reveal chamber-specific circadian vulnerability with implications for diabetic cardiomyopathy and chronotherapy.
Brief hypoxic episodes drive neuroplasticity in animal models and humans. Pretreatment with an allosteric α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (R) modulator (“ampakine”) enables a single hypoxic exposure to induce sustained increases in phrenic motor activity [“phrenic motor facilitation” (pMF)]. Phrenic nerve activity was recorded in anesthetized rats to determine whether the ampakine-hypoxia (A-H) combination is unique in its ability to evoke pMF and to determine its underlying mechanisms. Pairing ampakine CX717 with brief moderate or severe hypercapnia failed to produce pMF. Pairing doxapram, a respiratory stimulant, with hypoxia did not produce pMF. We then sequentially tested the hypotheses that A-H-induced pMF requires spinal serotonin, adenosine, or NMDA receptor activation. Cervical intrathecal delivery of serotonin (methysergide) or adenosine 2 A receptor (MSX-3) antagonists before A-H failed to prevent pMF. In contrast, the NMDA-R blocker MK-801 prevented pMF when administered before but not after A-H. Finally, as a step in the translational pathway, we tested the safety and efficacy of acute A-H exposure in unanesthetized rats with indwelling diaphragm electromyogram (EMG) wires after cervical spinal cord injury (SCI). A-H was well tolerated, and at 3 mo after SCI, increased diaphragm EMG output. We conclude that the mechanism driving sustained increases in phrenic motor output after A-H is independent of spinal adenosine or serotonin receptor activation, but requires spinal NMDA-R activation for the induction, but not maintenance, of A-H pMF. Ampakine pretreatment may be useful to increase the efficacy of hypoxia-based rehabilitation paradigms after SCI, particularly since clinical trials report a substantial number of “low responders.”NEW & NOTEWORTHY Rehabilitation paradigms using brief hypoxia exposure can improve recovery after spinal cord injury. A low dose of ampakines, which enhance AMPA neurotransmission, coupled with brief hypoxia (A-H), uniquely evokes respiratory neuroplasticity (“phrenic motor facilitation” or pMF) via a mechanism that requires spinal NMDA, but not serotonin or adenosine receptor activation. A-H also increased diaphragm activation in rats with chronic SCI, suggesting that the A-H pairing may be useful in neurorehabilitation.
Aberrant mechanistic target of rapamycin complex 1 (mTORC1) signaling in skeletal muscle has been implicated in aging and insulin resistance; however, it is not known whether chronic mTORC1 activation directly causes glucose intolerance. We tested the hypothesis that constitutive mTORC1 activation in mouse skeletal muscle impairs glucose homeostasis. Six-month-old female and male mice with tamoxifen-inducible, muscle-specific knockout of the DEP domain containing 5, GATOR1 subcomplex subunit gene (Depdc5), a key component of the GAP activity toward rag 1 (GATOR1) complex and negative regulator of mTORC1, were fed normal chow or Western diet (WD; 45% fat, 17% sucrose) for 12 wk. Depdc5 knockout (KO) increased mTORC1 signaling and altered autophagy markers. WD increased body and fat mass and impaired glucose tolerance independent of genotype. KO had minimal effects on fasting glucose, insulin, Homeostatic Model Assessment of Insulin Resistance (HOMA-IR), glycosylated hemoglobin (HbA1c), or oral glucose tolerance, although female KO mice showed a modest increase in WD-induced weight gain and fasting glucose. Mitochondrial respiration and content were unchanged by KO or WD. KO increased mitochondrial H2O2 production capacity but did not drive clear signs of oxidative stress. Transcriptomic analysis revealed robust KO-driven upregulation of genes related to cell division and immune pathways. Consistent with this, KO increased TNF-α and IL-6 protein expression and shifted macrophage polarization toward an M2-like phenotype without altering total macrophage content. Collectively, these findings indicate that chronic activation of mTORC1 in skeletal muscle promotes inflammatory remodeling but is insufficient to impair systemic glucose homeostasis, even under dietary stress.NEW & NOTEWORTHY Chronic skeletal muscle mTORC1 hyperactivation is widely assumed to drive insulin resistance and metabolic decline, yet direct causal evidence remains limited. Using adult-onset Depdc5 deletion to constitutively activate mTORC1 via GATOR1 disruption, we show that this is insufficient to impair glucose homeostasis even under a Western diet challenge. Strikingly, glucose tolerance and mitochondrial respiration remained preserved despite inflammation and immune transcriptional reprogramming-challenging the prevailing model of mTORC1-driven metabolic dysfunction in muscle.
Alcohol misuse and calorie-dense diets, especially those rich in sugar and fat, independently increase comorbidity risk among people with HIV. The objective of this study was to define the combined effects of chronic binge alcohol administration and consumption of a high-fat high-sucrose diet (HFSD) on systemic metabolic, immunologic, and body composition outcomes under controlled conditions in an established nonhuman primate (NHP) model of HIV infection. Male NHPs fed HFSD diet were administered alcohol or vehicle. A subset of animals in the vehicle (VEH) and alcohol groups was infected with simian immunodeficiency virus (SIV) and treated with antiretroviral therapy (ART). ART treatment decreased viral loads, which did not differ between the vehicle- and alcohol-administered NHPs. There was a main effect of alcohol to significantly reduce peripheral CD4+ T cell counts and the CD4+/CD8+ T-cell ratio. Overall body composition was not significantly different between the treatment groups. However, bone mineral density was significantly lower in the alcohol/SIV+ group. Alcohol decreased the acute insulin response to glucose. ART effectively suppressed viral loads in vehicle and alcohol/SIV NHPs. Alcohol produced significant immunological alterations, impaired glucose-insulin dynamics, and decreased bone mineral density. These findings highlight alcohol misuse as a modifiable driver of metabolic and immunologic dysfunction. Our ongoing studies will dissect the tissue-specific effects of alcohol and calorie-dense diets that contribute to the systemic changes.NEW & NOTEWORTHY This study examined the interactions of a calorie-dense diet and alcohol in SIV-infected and SIV-seronegative nonhuman primates on systemic viral loads and immunological and metabolic measures. ART effectively suppressed viral loads, irrespective of alcohol administration. Alcohol produced significant immunological alterations and decreased bone mineral density in SIV infection. Alcohol impaired glucose-insulin dynamics irrespective of SIV infection, highlighting a significant metabolic maladaptation. The results highlight the interaction of diet and alcohol on increasing the risk for comorbidities in HIV.
The hibernating 13-lined ground squirrel kidney is a unique natural model of resistance to damage caused by cold storage and warm reperfusion. Over months, the kidney is exposed to cycles between multiday periods of torpor with low perfusion at ice-cold temperature and rapid warm reperfusion during arousals. Serum creatinine accumulates during torpor but normalizes during arousal, and animals emerge each spring with functioning kidneys. After confirming a lack of kidney histopathology in sections from 11 animals that had completed 11-22 torpor-arousal cycles, we collected RNA sequencing (RNA-Seq) data from 32 ground squirrel kidneys representing 6 key transitional time points based on seasonal and torpor-arousal cycle physiology. Hibernation state-specific gene expression changes were identified after removing three informative outliers. Both seasonal and torpor-arousal cycle-specific gene expression changes were found. These differentially expressed genes illuminated molecular mechanisms that mitigate damage while supporting full recovery during each ∼12 h rewarming. As with the response to renal ischemia-reperfusion injury in other species, the arousing hibernator induced immediate early genes during warm reperfusion. But, in the hibernator, this response did not precipitate the gene expression program of maladaptive repair that is characterized by cell death, immune system activation, and fibrosis. Rather, it appears that induction of immediate early genes activated a universal 12-h, "circatidal" rhythm. The efficient unfolding of this rhythm across each arousal from torpor was facilitated by the seasonally changed background primed for rapid cell division and minimal energy consumption. Adaptive repair was achieved, with proteostasis and cell type-specific function restored, assuring that minor damage did not accumulate.NEW & NOTEWORTHY The hibernator kidney exhibits no damage after 10-22 cycles between prolonged cold exposure and rapid warm reperfusion occurring over several months. As found with kidney damage in other species, each rewarming activates an immediate early gene response. Unique to the hibernator, adaptive repair is completed within 12 h, facilitated by rapid activation of the universal "circatidal" rhythm unfolding on a seasonally primed background that assures minimal cell death, rapid cell division, and repair.
Traumatic knee injury leads to posttraumatic osteoarthritis (PTOA) and significant skeletal muscle weakness, resulting in chronic disability. The current standard of care frequently fails to prevent musculoskeletal dysfunction, underscoring the need to identify therapeutic mechanisms of PTOA. Using an established preclinical anterior cruciate ligament (ACL) transection model of PTOA and leveraging an innovative SPiDER-senescence-associated β-galactosidase stain to discern senescent cells, we investigated cellular senescence at single-cell resolution and identified anti-inflammatory macrophages as a predominant contributor to the senescent cell burden in both muscle and knee joint after injury. Clearance of senescent cells using the senolytic dasatinib and quercetin (D + Q) mitigated injury-induced muscle atrophy and cartilage degradation, with greater senescent cell clearance within muscle compared with cartilage. We also provide clinical evidence of elevated senescent cell burden in the muscle of patients following ACL injury and with PTOA, which is obstinate to standard of care, highlighting cellular senescence as a strong therapeutic target to improve functional recovery after traumatic joint injury.NEW & NOTEWORTHY Posttraumatic osteoarthritis (PTOA) and disability are consequences of ACL injury, but the cellular effectors that facilitate this process are unknown. Our findings demonstrate increased senescent macrophage burden in the muscle and knee joint in a preclinical ACL injury model, as well as in patients who predict poor muscle quality and weakness. We provide evidence that treatment with senolytics is an effective strategy to improve tissue quality and function and mitigate PTOA severity.
Acute intermittent hypoxia (AIH) elicits respiratory motor plasticity in the phrenic, intercostal, and hypoglossal motor pools and has emerged as a promising therapeutic strategy to improve respiratory function in people with neuromuscular disorders that compromise breathing. Although we recently reported that time-of-day regulates moderate ([Formula: see text] ∼40-50 mmHg) AIH-induced respiratory motor plasticity, it is unknown whether diurnal effects on AIH-induced phrenic (pLTF) or ventilatory (vLTF) long-term facilitation are mediated via the endogenous circadian clock versus other factors. Since many biological rhythms are driven by the endogenous clock and clock genes (including the essential clock gene Bmal1) are rhythmically expressed in the phrenic motor system, we hypothesized that the molecular clock within respiratory motor neurons exerts time-of-day effects on pLTF and vLTF in Sprague Dawley rats (3-6 mo old males). Intrapleural injections of small-interfering RNAs (siRNAs) were used to selectively knock down Bmal1 within respiratory motor neurons by ∼30%. AIH consisting of 15, 1-min hypoxic episodes ([Formula: see text] = 0.09) was delivered in the midrest (i.e., light) or midactive (i.e., dark) phases, and pLTF (Δintegrated phrenic burst amplitude) and vLTF (ΔV̇e/V̇co2) were assessed in rats given siRNAs targeting Bmal1 versus nontargeting controls. In midrest phase, pLTF was reduced, and vLTF was abolished in rats given siBmal1 versus nontargeting siRNA. However, siBmal1 had no significant effect on either pLTF or vLTF in the midactive phase. Thus, the phrenic motor neuron circadian clock regulates AIH-induced respiratory motor plasticity in a time-of-day-dependent manner. It is important to consider circadian biology in future studies of AIH-induced respiratory motor plasticity.NEW & NOTEWORTHY Although diurnal cycle influences respiratory motor plasticity elicited by acute intermittent hypoxia (AIH), it is unknown how endogenous circadian clock mechanisms contribute to time-of-day effects on plasticity. We report that knockdown of the circadian clock protein Bmal1 within respiratory motor neurons attenuates phrenic and ventilatory long-term facilitation in a manner dependent on diurnal phase. Thus, circadian biology is an important consideration for studies of respiratory motor plasticity.
A healthy cardiovascular system requires a healthy endothelium. The endothelium is a central regulator of hemostasis, vascular permeability, inflammation, blood pressure, and, perhaps most fundamentally, blood flow1. It is therefore of no surprise that endothelial dysfunction underlies many of the vascular complications that occur in metabolic disease (e.g. peripheral artery disease, vascular retinopathy, and coronary artery disease)2. Endothelial function is often assessed using flow mediated dilation (FMD)3, and in their recent study, Power and colleagues4 provide novel insight into the mechanisms by which FMD is impaired in type 2 diabetes (T2D). In T2D, loss of FMD is driven, in part, by the shedding of the glycocalyx (Figure 1). This hair-like structure on the surface of the endothelium maintains an erythrocyte-free zone, preserves redox homeostasis, mitigates inflammation, and senses shear stress5. The glycocalyx is a complex matrix made up of proteoglycans, glycoproteins, glycolipids, and glycosaminoglycans, and each of these components plays an essential role in its functions5. Power and colleagues found patients with T2D had impaired FMD and elevated plasma hyaluronan, suggesting glycocalyx shedding. Hyaluronan is a glycosaminoglycan in the glycocalyx that works in concert with CD44 to propagate flow-induced signaling6. Notably, they found endothelial CD44 expression was decreased in the db/db model of T2D. Using isolated mouse mesenteric arteries, they demonstrated cleavage of hyaluronan with hyaluronidase or blocking the binding site of hyaluronan on CD44 blunted FMD. Similarly, knockdown of CD44 in cultured endothelial cells blunted shear stress induced increases in intracellular calcium and endothelial nitric oxide synthase. Together these data demonstrate the importance of hyaluronan-CD44 signaling for endothelial mechanotransduction. Building on their previous work which reported upregulation of a disintegrin and metalloprotease 17 (ADAM17) in arteries from patients with T2D, they found elevated plasma ADAM17 activity in their patient population and increased endothelial ADAM17 expression in db/db mice. They therefore investigated whether cleavage of CD44 by ADAM17 contributes to impaired FMD in T2D. They found both activation and overexpression of ADAM17 blunted the response to shear stress in cultured endothelial cells. Furthermore, ADAM17 overexpression cleaved CD44 as demonstrated by reduced cell-surface CD44 and increased CD44 in the supernatant. Similar results were achieved when endothelial cells were treated with recombinant ADAM17, and recombinant ADAM17 was sufficient to impair CD44-hyaluronan binding in vitro. Perhaps most importantly, recombinant ADAM17 was sufficient to blunt FMD in isolated mouse mesenteric arteries. Altogether these data suggest ADAM17 upregulation contributes to the endothelial dysfunction observed in patients with T2D. These studies build on previous work implicating ADAM17 as a driver of endothelial dysfunction in metabolic disease and provide a novel mechanism by which ADAM17 impairs endothelial-dependent vasodilation. Previous work has demonstrated ADAM17 cleaves glypican-1, a proteoglycan component of the glycocalyx, thereby blunting FMD7, and ADAM17 cleaves the insulin receptor, blunting insulin-dependent vasodilation8,9. Still questions remain regarding how ADAM17 activity impacts other functions of the glycocalyx and how other flow-sensing mechanisms such as piezo channels may compensate in these conditions. The use of endothelial-specific ADAM17 knockout mice offers an exciting avenue to pursue these questions in vivo7. Altogether, ADAM17 may be a promising target to preserve or restore the endothelial glycocalyx and endothelial function in T2D. Vascular dysfunction gives rise to major T2D comorbidities such as nephropathy, neuropathy, and cardiovascular disease10. Continued efforts to investigate the mechanisms that drive endothelial dysfunction may offer new therapeutic targets to treat or prevent these comorbidities, thereby decreasing mortality and improving quality of life for patients with T2D.
Ascent to high-altitude induces systemic and ocular changes that alter vision. The functional responses of the choroidal vasculature at different depths to hypoxia remain incompletely understood. In this study, we used functional optical coherence tomography (f-OCT) to quantify changes in choroidal vascular perfusion density (VPD) across superficial, deep, and total choroidal layers in 16 healthy participants. Imaging was performed at baseline (1,100 m) and during acute (day 2) and prolonged exposure (day 9) to high altitude (3,800 m). Our depth-resolved analysis showed a modest but statistically significant decline in superficial choroidal VPD from baseline to day 2 (P = 0.031; Cohen's d = -0.73). In contrast, both deep and total choroidal VPD increased significantly from baseline to day 2 (P = 0.002 and P = 0.003, respectively; Cohen's d > 0.9). Changes in superficial, deep, and total choroidal VPD persisted from day 2 through day 9 at 3,800 m. [Formula: see text] showed a strong negative correlation with deep choroidal (r = -0.655; P = 0.0003) and total choroid VPD (r = -0.566; P = 0.0014), but no significant correlation was observed with the VPD of the superficial layer (r = 0.018; P = 0.93). The strong correlation between VPD and the stimulus index ([Formula: see text]/[Formula: see text]) in the total choroid suggests that hypoxia-induced vasodilation outweighs hypocapnic vasoconstriction, leading to increased perfusion (r = 0.629; P = 0.0003). These findings suggest that deep choroidal hyperperfusion on ascent to high altitude helps sustain outer retinal oxygenation. Depth-resolved choroid imaging may offer new insights into ocular resilience to environmental challenges and provide a valuable tool to monitor spaceflight-related ocular changes associated with neuro-ocular syndrome.NEW & NOTEWORTHY This study reveals that the human choroid mounts a depth-specific vascular response to high-altitude hypoxia. Rather than showing uniform vasodilation, superficial choroidal perfusion decreases, whereas deep choroidal perfusion increases and remains elevated after prolonged altitude exposure. The strong relationship between deep choroidal perfusion and arterial blood gases suggests that hypoxia-driven vasodilation outweighs hypocapnic vasoconstriction. These data highlight functional OCT as a powerful tool for measuring how physiological stress reshapes ocular vascular function and suggest that combining high-altitude exposure with depth-resolved functional OCT may provide a useful terrestrial model for vascular changes relevant to spaceflight-associated neuro-ocular syndrome.
Disruption of PKD2 function causes polycystic kidney disease (PKD) and leads to the formation of cysts in the kidney in both mice and humans. PKD is progressive, initiating with the formation of tubule dilations that expand into large cysts, causing the destruction of renal parenchyma and loss of kidney function. The mechanisms initiating tubule dilation and expansion are uncertain. Tubule obstruction has been proposed as a factor contributing to cystogenesis. Here, we utilize a proximal tubule fluorescent dextran uptake assay to evaluate changes in tubule flow that occur during cyst initiation or expansion and functional decline. We did not observe overt signs of tubule obstruction during cyst initiation or expansion, even in larger cysts; however, the rate of tubule flow within individual nephrons was greatly diminished in cystic kidneys. Surprisingly, along with reduced tubule flow rate, we observe an increase in newly labeled dextran cells associated with disease in the Pkd2 mutants. These cells are spatially distinct from those absorbing the initial dextran and are detected in both cortical and medullary regions of mutant kidneys. Marker analysis indicated that the dextran+ medullary cells were in the loop of Henle and interstitial macrophages. These cells are not present in wild-type controls. This may be due to tubule or vasculature leakage and utilization of renal reserve capacity in mutants that become exhausted, associated with declining glomerular filtration rate (GFR) as cysts expand.
Exposure to mechanical stimuli can modulate skeletal muscle structure and metabolism, yet the extent to which repeated, isolated mechanical stress promotes adaptive remodeling in humans has not been defined. We investigated whether repeated percussive massage (PM)—a widely used but poorly validated therapeutic modality—induces beneficial skeletal muscle adaptations under ambulatory conditions and whether such adaptations confer resilience during limb disuse in humans. In a 6-wk randomized trial, PM did not alter myofiber cross-sectional area, satellite cell abundance, or capillary density, but RNA-Seq pathway analysis revealed enrichment of extracellular matrix (ECM) remodeling networks, which was supported by increases in the expression of basement membrane and focal adhesion components. PM also reduced subcutaneous fat thickness and increased fatty acid-supported mitochondrial respiration while lowering mitochondrial H2O2 emission. In a separate 10-day immobilization study, PM failed to attenuate unloading-induced reductions in muscle size or strength. However, PM partially preserved fatty acid-supported respiratory capacity relative to a control group, indicating a selective metabolic resilience. Finally, in an acute mechanistic experiment, unilateral PM did not increase subcutaneous adipose tissue lipolysis, as interstitial glycerol concentrations rose similarly in treated and untreated limbs, suggesting that chronic reductions in subcutaneous fat thickness were not driven by lipolytic activation. Collectively, these findings demonstrate that repeated PM promotes targeted skeletal muscle metabolic adaptations, yet is insufficient to induce overt structural remodeling or prevent disuse-induced functional decline.
IL-1β is typically associated with the innate response, often produced following the detection of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). It is also identified as a cytokine involved in bridging the innate and adaptive immune responses, in which innate cells, like dendritic cells, use IL-1β to activate T cells for the adaptive immune response. The role of IL-1 signaling has been established in the context of T cell differentiation and function, particularly in its regulation of T follicular helper (TFH) cells. Recent work has demonstrated that with TFH function primarily acting within the germinal center (GC), a local source of IL-1β must be present, identifying GC B cells as a critical source of IL-1β. Here we discuss the roles of cells within the GC milieu, the cytokines they produce, and their impact on the GC. In addition, we also discuss the findings from studies examining the molecular mechanisms underlying IL-1β production in B cells and its impact on B cell function. This review is especially relevant as it draws together findings from various disease pathologies to consolidate our current understanding of B cell subsets producing IL-1β and IL-1β signaling within the GC, in both T cells and B cells.
The body's circadian rhythm is coordinated by core clock proteins [period (PER), cryptochrome circadian regulator (CRY), circadian locomotor output cycles kaput (CLOCK), and basic helix-loop-helix ARNT-like protein 1 (BMAL1)] that function in both the central hypothalamic and peripheral tissue molecular clocks. Our recent study demonstrated that deletion of Per1 in Dahl salt-sensitive (SS) rats (SSPer1-/-) exacerbated SS hypertension (HTN), kidney injury, and disrupted blood pressure rhythms. To define time-of-day-, genotype-, and diet-dependent alterations in the renal transcriptome and proteome associated with SS HTN, kidney cortex samples were collected from SS and SSPer1-/- rats fed either a normal-salt (NS, 0.4% NaCl) or high-salt (HS, 4% NaCl) diet, during both the active (night) and inactive (day) periods. Dietary challenges were conducted for 3 wk in male rats. Bulk RNA-sequencing was performed on both NS- and HS-fed groups, and proteomic analyses were performed in HS-fed groups. In SS rats, HS intake blunted time-of-day-dependent transcriptional changes. Pathway analyses predicted significant stress and immune responses, as well as metabolic adaptations, induced by the HS diet. Specifically, the remodeling of the pyruvate dehydrogenase complex was identified as a key prediction in both transcriptomic and phosphoproteomic datasets. As expected, Per1 deletion further exacerbated disruptions in immune regulation and metabolic adaptation. Collectively, these findings demonstrate that numerous renal genes exhibit diurnal oscillations under physiological conditions and are profoundly disrupted in SS HTN, likely contributing to impaired kidney function and circadian misalignment of blood pressure regulation.
Sickle cell nephropathy (SCN) significantly shortens the life expectancy of patients with sickle cell disease (SCD). We previously reported that endothelin-1 (ET-1) and endothelin A receptor (ETA) are upregulated in SCN, and ETA antagonism mitigates SCN early in the disease progression in a humanized mouse model of SCD. We hypothesized that endothelium-derived ET-1 mediates the progression of SCN and T cell inflammation in the kidney of SCD mice. To test this hypothesis, we first used allogenic bone marrow transplantation from humanized sickle cell mice (HbSS) into endothelial-derived ET-1 knockout (VEET KO) mice, revealing that endothelial-derived ET-1 mitigates SCN and regulates the renal inflammatory response and T cell infiltration. Second, using young (4-5 mo old) and middle-aged (10-15 mo old) HbSS mice lacking endothelial-specific ET-1 (HbSS-VEET KO), we found a temporal maintenance of glomerular filtration rate, reduced infiltration of T cells to the kidney, and reduced progression of SCN. Furthermore, 2-wk ETA antagonism in middle-aged HbSS mice reduced infiltration of T cells. Finally, flow cytometric analyses revealed blunting of kidney T helper 17 (TH17) cells without a change in kidney T regulatory cells in HbSS-VEET KO mice, suggesting T cell subset-specific regulation by endothelial-derived ET-1 signaling. In vitro studies showed that ETA antagonism directly inhibits TH17 polarization and IL-17A production, suggesting that in established sickle cell disease, the ETA receptor-TH17 cell axis may play a key role in maintenance of fibrosis in SCN. Taken together, these data indicate that endothelial-derived ET-1 mediates the progression of SCN and strengthens the rationale for targeting ET-1 signaling as a new therapeutic approach.
In individuals with type 2 diabetes (T2D), blood flow-mediated increases in endothelial shear stress fail to elicit a robust vasodilatory response. This defective flow-mediated dilation (FMD) is associated with loss of the endothelial glycocalyx, a mechanosensitive extracellular structure that lines the luminal side of blood vessels. Hyaluronan (HA), a polysaccharide constituent of the glycocalyx, is anchored to the plasma membrane by its primary cell-surface receptor, CD44, which is implicated in mechanotransduction of shear stress. Critically, CD44 is also a substrate of a disintegrin and metalloproteinase 17 (ADAM17), a sheddase that is elevated in T2D. However, it is currently unknown whether elevated ADAM17 activity enhances CD44 cleavage from the endothelium and whether this contributes to impaired mechanotransduction and reduced FMD in T2D. Herein, we report elevated plasma HA and ADAM17 activity in a cohort of women and men with T2D and impaired FMD. Moreover, reduced endothelial CD44 is coupled with impaired FMD in arteries isolated from diabetic (db/db) mice. We also provide support for CD44 as a mechanotransducer of HA-associated shear stress mechanosensation and ADAM17-mediated cleavage of CD44 attenuating shear stress mechanotransduction. Finally, using an in-vitro assay and surface plasmon resonance, we show that active recombinant human ADAM17 (ADAM17-r) cleaves recombinant human CD44 and that intraluminal incubation of isolated arteries with ADAM17-r reduces FMD. Collectively, this work supports the role of ADAM17-mediated cleavage of CD44 in impairing endothelial shear stress mechanotransduction.NEW & NOTEWORTHY Impaired flow-mediated dilation (FMD) is an indicator of endothelial dysfunction in type 2 diabetes (T2D). However, the exact cellular and molecular mechanisms contributing to impaired mechanotransduction of shear stress in T2D are incompletely understood. This work supports the novel concept that increased endothelial activity of a disintegrin and metalloproteinase 17 (ADAM17) causes cleavage of cell-surface CD44, leading to loss of hyaluronan (HA)-associated shear stress mechanotransduction.
Neurodevelopmental impairments after preterm birth remain prevalent. Preterm brain injury, such as intraventricular hemorrhage (IVH) and periventricular leukomalacia (PVL), occurs due to immature vascularization and impaired cerebral blood flow. We explored epigenetic changes in the vascular endothelial growth factor A (VEGFA) and cerebrovascular autoregulation as biomarkers for preterm brain injury. Preterm neonates <30 wk gestational age and/or <1,000 g were prospectively enrolled in a single-center observational cohort study. We compared cerebral hemodynamics and cerebrovascular autoregulation during the first 3 postnatal days and VEGFA methylation from buccal samples during the first 2 wk between neonates without brain injury and those with IVH, PVL, or with IVH and/or PVL. Of 73 neonates with a means ± SD gestational age and birth weight of 27.7 ± 1.6 wk and 1,017 ± 235 g, 33 (45.2%) had IVH and/or PVL. Upon multivariable regression, IVH was associated with sedation, cerebral hypoxia, and impaired cerebrovascular autoregulation on day 3. Neonates with PVL had more methylated VEGFA in both weeks (medianweek1 3.12% vs. 2.50%, P = 0.022). Upon multivariable regression, PVL was associated with lower Apgar scores and VEGFA hypermethylation in week 2. Neonates with IVH and/or PVL had lower cerebral tissue oxygenation (Pday1 = 0.005, Pday3 = 0.001) and more impaired cerebrovascular autoregulation on day 1 (P < 0.001) than those without cerebral injury. Our findings indicate associations between both hemodynamic and epigenetic biomarkers and preterm brain injury. IVH and PVL showed associations with impaired cerebrovascular autoregulation, whereas IVH was associated with cerebral hypoxia and PVL with epigenetic downregulation of VEGFA. Hemodynamic changes appeared early postnatally, underscoring their clinical relevance.
