In vivo CRISPR screens reveal SCAF1 and USP15 as drivers of pancreatic cancer.

Functionally characterizing the genetic alterations that drive pancreatic cancer is a prerequisite for precision medicine. Here, we perform somatic CRISPR/Cas9 mutagenesis screens to assess the transforming potential of 125 recurrently mutated pancreatic cancer genes, which revealed USP15 and SCAF1 as pancreatic tumor suppressors. Mechanistically, we find that USP15 functions in a haploinsufficient manner and that loss of USP15 or SCAF1 leads to reduced inflammatory TNFα, TGF-ß and IL6 responses and increased sensitivity to PARP inhibition and Gemcitabine. Furthermore, we find that loss of SCAF1 leads to the formation of a truncated, inactive USP15 isoform at the expense of full-length USP15, functionally coupling SCAF1 and USP15. Notably, USP15 and SCAF1 alterations are observed in 31% of pancreatic cancer patients. Our results highlight the utility of in vivo CRISPR screens to integrate human cancer genomics and mouse modeling for the discovery of cancer driver genes with potential prognostic and therapeutic implications.

Pulmonary hypertension impairs vasomotor function in rat diaphragm arterioles.

Pulmonary hypertension (PH) is a chronic, progressive condition in which respiratory muscle dysfunction is a primary contributor to exercise intolerance and dyspnea in patients. Contractile function, blood flow distribution, and the hyperemic response are altered in the diaphragm with PH, and we sought to determine whether this may be attributed, in part, to impaired vasoreactivity of the resistance vasculature. We hypothesized that there would be blunted endothelium-dependent vasodilation and impaired myogenic responsiveness in arterioles from the diaphragm of PH rats. Female Sprague-Dawley rats were randomized into healthy control (HC, n = 9) and monocrotaline-induced PH rats (MCT, n = 9). Endothelium-dependent and -independent vasodilation and myogenic responses were assessed in first-order arterioles (1As) from the medial costal diaphragm in vitro. There was a significant reduction in endothelium-dependent (via acetylcholine; HC, 78 ± 15% vs. MCT, 47 ± 17%; P < 0.05) and -independent (via sodium nitroprusside; HC, 89 ± 10% vs. MCT, 66 ± 10%; P < 0.05) vasodilation in 1As from MCT rats. MCT-induced PH also diminished myogenic constriction (P < 0.05) but did not alter passive pressure responses. The diaphragmatic weakness, impaired hyperemia, and blood flow redistribution associated with PH may be due, in part, to diaphragm vascular dysfunction and thus compromised oxygen delivery which occurs through both endothelium-dependent and -independent mechanisms.

Monitoring the 5'UTR landscape reveals isoform switches to drive translational efficiencies in cancer.

Transcriptional and translational control are key determinants of gene expression, however, to what extent these two processes can be collectively coordinated is still poorly understood. Here, we use Nanopore long-read sequencing and cap analysis of gene expression (CAGE-seq) to document the landscape of 5' and 3' untranslated region (UTR) isoforms and transcription start sites of epidermal stem cells, wild-type keratinocytes and squamous cell carcinomas. Focusing on squamous cell carcinomas, we show that a small cohort of genes with alternative 5'UTR isoforms exhibit overall increased translational efficiencies and are enriched in ribosomal proteins and splicing factors. By combining polysome fractionations and CAGE-seq, we further characterize two of these UTR isoform genes with identical coding sequences and demonstrate that the underlying transcription start site heterogeneity frequently results in 5' terminal oligopyrimidine (TOP) and pyrimidine-rich translational element (PRTE) motif switches to drive mTORC1-dependent translation of the mRNA. Genome-wide, we show that highly translated squamous cell carcinoma transcripts switch towards increased use of 5'TOP and PRTE motifs, have generally shorter 5'UTRs and expose decreased RNA secondary structures. Notably, we found that the two 5'TOP motif-containing, but not the TOP-less, RPL21 transcript isoforms strongly correlated with overall survival in human head and neck squamous cell carcinoma patients. Our findings warrant isoform-specific analyses in human cancer datasets and suggest that switching between 5'UTR isoforms is an elegant and simple way to alter protein synthesis rates, set their sensitivity to the mTORC1-dependent nutrient-sensing pathway and direct the translational potential of an mRNA by the precise 5'UTR sequence.

Pulmonary hypertension alters blood flow distribution and impairs the hyperemic response in the rat diaphragm.

Pulmonary hypertension (PH) is characterized by pulmonary vascular remodeling, respiratory muscle and cardiac impairments, and exercise intolerance. Specifically, impaired gas exchange increases work of the diaphragm; however, compromised contractile function precludes the diaphragm from meeting the increased metabolic demand of chronic hyperventilation in PH. Given that muscle contractile function is in part, dependent upon adequate blood flow (Q˙), diaphragmatic dysfunction may be predicated by an inability to match oxygen delivery with oxygen demand. We hypothesized that PH rats would demonstrate a decreased hyperemic response to contractions compared to healthy controls. Methods: Sprague-Dawley rats were randomized into healthy (HC, n = 7) or PH (n = 7) groups. PH rats were administered monocrotaline (MCT) while HC rats received vehicle. Disease progression was monitored via echocardiography. Regional and total diaphragm blood flow and vascular conductance at baseline and during 3 min of electrically-stimulated contractions were determined using fluorescent microspheres. Results: PH rats displayed morphometric and echocardiographic criteria for disease (i.e., acceleration time/ejection time, right ventricular hypertrophy). In all rats, total costal diaphragm Q˙ increased during contractions and did not differ between groups. In HC rats, there was a greater increase in medial costal Q˙ compared to PH rats (55% ± 3% vs. 44% ± 4%, p < 0.05), who demonstrated a redistribution of Q˙ to the ventral costal region. Conclusion: These findings support a redistribution of regional diaphragm perfusion and an impaired medial costal hyperemic response in PH, suggesting that PH alters diaphragm vascular function and oxygen delivery, providing a potential mechanism for PH-induced diaphragm contractile dysfunction.

DAP5 enables main ORF translation on mRNAs with structured and uORF-containing 5' leaders.

Half of mammalian transcripts contain short upstream open reading frames (uORFs) that potentially regulate translation of the downstream coding sequence (CDS). The molecular mechanisms governing these events remain poorly understood. Here, we find that the non-canonical initiation factor Death-associated protein 5 (DAP5 or eIF4G2) is required for translation initiation on select transcripts. Using ribosome profiling and luciferase-based reporters coupled with mutational analysis we show that DAP5-mediated translation occurs on messenger RNAs (mRNAs) with long, structure-prone 5' leader sequences and persistent uORF translation. These mRNAs preferentially code for signalling factors such as kinases and phosphatases. We also report that cap/eIF4F- and eIF4A-dependent recruitment of DAP5 to the mRNA facilitates main CDS, but not uORF, translation suggesting a role for DAP5 in translation re-initiation. Our study reveals important mechanistic insights into how a non-canonical translation initiation factor involved in stem cell fate shapes the synthesis of specific signalling factors.

Supplemental oxygen administration during mechanical ventilation reduces diaphragm blood flow and oxygen delivery.

During mechanical ventilation (MV), supplemental oxygen (O2) is commonly administered to critically ill patients to combat hypoxemia. Previous studies demonstrate that hyperoxia exacerbates MV-induced diaphragm oxidative stress and contractile dysfunction. Whereas normoxic MV (i.e., 21% O2) diminishes diaphragm perfusion and O2 delivery in the quiescent diaphragm, the effect of MV with 100% O2 is unknown. We tested the hypothesis that MV supplemented with hyperoxic gas (100% O2) would increase diaphragm vascular resistance and reduce diaphragmatic blood flow and O2 delivery to a greater extent than MV alone. Female Sprague-Dawley rats (4-6 mo) were randomly divided into two groups: 1) MV + 100% O2 followed by MV + 21% O2 (n = 9) or 2) MV + 21% O2 followed by MV + 100% O2 (n = 10). Diaphragmatic blood flow (mL/min/100 g) and vascular resistance were determined, via fluorescent microspheres, during spontaneous breathing (SB), MV + 100% O2, and MV + 21% O2. Compared with SB, total diaphragm vascular resistance was increased, and blood flow was decreased with both MV + 100% O2 and MV + 21% O2 (all P < 0.05). Medial costal diaphragmatic blood flow was lower with MV + 100% O2 (26 ± 6 mL/min/100 g) versus MV + 21% O2 (51 ± 15 mL/min/100 g; P < 0.05). Second, the addition of 100% O2 during normoxic MV exacerbated the MV-induced reductions in medial costal diaphragm perfusion (23 ± 7 vs. 51 ± 15 mL/min/100 g; P < 0.05) and O2 delivery (3.4 ± 0.2 vs. 6.4 ± 0.3 mL O2/min/100 g; P < 0.05). These data demonstrate that administration of supplemental 100% O2 during MV increases diaphragm vascular resistance and diminishes perfusion and O2 delivery to a significantly greater degree than normoxic MV. This suggests that prolonged bouts of MV (i.e., 6 h) with hyperoxia may accelerate MV-induced vascular dysfunction in the quiescent diaphragm and potentially exacerbate downstream contractile dysfunction.NEW & NOTEWORTHY This is the first study, to our knowledge, demonstrating that supplemental oxygen (i.e., 100% O2) during mechanical ventilation (MV) augments the MV-induced reductions in diaphragmatic blood flow and O2 delivery. The accelerated reduction in diaphragmatic blood flow with hyperoxic MV would be expected to potentiate MV-induced diaphragm vascular dysfunction and consequently, downstream contractile dysfunction. The data presented herein provide a putative mechanism for the exacerbated oxidative stress and diaphragm dysfunction reported with prolonged hyperoxic MV.

Effects of pulmonary hypertension on microcirculatory hemodynamics in rat skeletal muscle.

Pulmonary hypertension (PH) has previously been characterized as a disease of the pulmonary vasculature that subsequently results in myocardial dysfunction. Heart failure compromises skeletal muscle microvascular function, which contributes to exercise intolerance. Therefore, we tested the hypothesis that such changes might be present in PH. Thus, we investigated skeletal muscle oxygen (O2) transport in the rat model of PH to determine if O2 delivery (Q̇O2) is impaired at the level of the microcirculation as evidenced via reduced red blood cell (RBC) flux, velocity, hematocrit, and percentage of capillaries flowing in quiescent muscle. Adult male Sprague-Dawley rats were randomized into healthy (n = 9) and PH groups (n = 9). Progressive PH was induced via a one-time intraperitoneal injection of monocrotaline (MCT; 50 mg/kg) and rats were monitored weekly via echocardiography. Intravital microscopy in the spinotrapezius muscle was performed when echocardiograms confirmed moderate PH (preceding right ventricular (RV) failure). At 25 ± 9 days post-MCT, PH rats displayed RV hypertrophy (RV/(Left ventricle + Septum): 0.28 ± 0.05 vs. 0.44 ± 0.11), pulmonary congestion, and increased right ventricular systolic pressure (21 ± 8 vs. 55 ± 14 mm Hg) compared to healthy rats (all P < 0.05). Reduced capillary RBC velocity (403 ± 140 vs. 227 ± 84 µm/s; P = 0.01), RBC flux (33 ± 12 vs. 23 ± 5 RBCs/s; P = 0.04) and % of capillaries supporting continuous RBC flux at rest (79 ± 8 vs. 56 ± 13%; P = 0.01) were evident in PH rats compared to healthy rats. When Q̇O2 within a given field of view was quantified (RBC flux x % of capillaries supporting continuous RBC flux), PH rats demonstrated lower overall Q̇O2 (↓ 50%; P = 0.002). These data support that microcirculatory hemodynamic impairments (↓ Q̇O2 and therefore altered Q̇O2-to-V̇O2 matching) may compromise blood-myocyte O2 transport in PH. The mechanistic bases for decreased capillary RBC flux, velocity, and percentage of capillaries supporting RBC flow remains an important topic.

What Do We Know About the Genetic Architecture of Psychopathology?

In the second half of the twentieth century, twin and family studies established beyond a reasonable doubt that all forms of psychopathology are substantially heritable and highly polygenic. These conclusions were simultaneously an important theoretical advance and a difficult methodological obstacle, as it became clear that heritability is universal and undifferentiated across forms of psychopathology, and the radical polygenicity of genetic effects limits the biological insight provided by genetically informed studies at the phenotypic level. The paradigm-shifting revolution brought on by the Human Genome Project has recapitulated the great methodological promise and the profound theoretical difficulties of the twin study era. We review these issues using the rubric of genetic architecture, which we define as a search for specific genetic insight that adds to the general conclusion that psychopathology is heritable and polygenic. Although significant problems remain, we see many promising avenues for progress.

Capillary hemodynamics and contracting skeletal muscle oxygen pressures in male rats with heart failure: Impact of soluble guanylyl cyclase activator.

In heart failure with reduced ejection fraction (HFrEF), nitric oxide-soluble guanylyl cyclase (sGC) pathway dysfunction impairs skeletal muscle arteriolar vasodilation and thus capillary hemodynamics, contributing to impaired oxygen uptake (V̇O2) kinetics. Targeting this pathway with sGC activators offers a new treatment approach to HFrEF. We tested the hypotheses that sGC activator administration would increase the O2 delivery (Q̇O2)-to-V̇O2 ratio in the skeletal muscle interstitial space (PO2is) of HFrEF rats during twitch contractions due, in part, to increases in red blood cell (RBC) flux (fRBC), velocity (VRBC), and capillary hematocrit (Hctcap). HFrEF was induced in male Sprague-Dawley rats via myocardial infarction. After 3 weeks, rats were treated with 0.3 mg/kg of the sGC activator BAY 60-2770 (HFrEF + BAY; n = 11) or solvent (HFrEF; n = 9) via gavage b.i.d for 5 days prior to phosphorescence quenching (PO2is, in contracting muscle) and intravital microscopy (resting) measurements in the spinotrapezius muscle. Intravital microscopy revealed higher fRBC (70 ± 9 vs 25 ± 8 RBC/s), VRBC (490 ± 43 vs 226 ± 35 µm/s), Hctcap (16 ± 1 vs 10 ± 1%) and a greater number of capillaries supporting flow (91 ± 3 vs 82 ± 3%) in HFrEF + BAY vs HFrEF (all P < 0.05). Additionally, PO2is was especially higher during 12-34s of contractions in HFrEF + BAY vs HFrEF (P < 0.05). Our findings suggest that sGC activators improved resting Q̇O2 via increased fRBC, VRBC, and Hctcap allowing for better Q̇O2-to-V̇O2 matching during the rest-contraction transient, supporting sGC activators as a potential therapeutic to target skeletal muscle vasomotor dysfunction in HFrEF.

Post-occlusive reactive hyperemia and skeletal muscle capillary hemodynamics.

Post-occlusive reactive hyperemia (PORH) is an accepted diagnostic tool for assessing peripheral macrovascular function. While conduit artery hemodynamics have been well defined, the impact of PORH on capillary hemodynamics remains unknown, despite the microvasculature being the dominant site of vascular control. Therefore, the purpose of this investigation was to determine the effects of 5 min of feed artery occlusion on capillary hemodynamics in skeletal muscle. We tested the hypothesis that, upon release of arterial occlusion, there would be: 1) an increased red blood cell flux (fRBC) and red blood cell velocity (VRBC), and 2) a decreased proportion of capillaries supporting RBC flow compared to the pre-occlusion condition. METHODS: In female Sprague-Dawley rats (n = 6), the spinotrapezius muscle was exteriorized for evaluation of capillary hemodynamics pre-occlusion, 5 min of feed artery occlusion (Occ), and 5 min of reperfusion (Post-Occ). RESULTS: There were no differences in mean arterial pressure (MAP) or capillary diameter (Dc) between pre-occlusion and post-occlusion (P > 0.05). During 30 s of PORH, capillary fRBC was increased (pre: 59 ± 4 vs. 30 s-post: 77 ± 2 cells/s; P < 0.05) and VRBC was not changed (pre: 300 ± 24 vs. 30 s post: 322 ± 25 µm/s; P > 0.05). Capillary hematocrit (Hctcap) was unchanged across the pre- to post-occlusion conditions (P > 0.05). Following occlusion, there was a 20-30% decrease in the number of capillaries supporting RBC flow at 30 s and 300 s-post occlusion (pre: 92 ± 2%; 30 s-post: 66 ± 3%; 300 s-post: 72 ± 6%; both P < 0.05). CONCLUSION: Short-term feed artery occlusion (i.e. 5 min) resulted in a more heterogeneous capillary flow profile with the presence of capillary no-reflow, decreasing the percentage of capillaries supporting RBC flow. A complex interaction between myogenic and metabolic mechanisms at the arteriolar level may play a role in the capillary no-reflow with PORH. Measurements at the level of the conduit artery mask significant alterations in blood flow distribution in the microcirculation.

The effects of pulmonary hypertension on skeletal muscle oxygen pressures in contracting rat spinotrapezius muscle.

NEW FINDINGS: What is the central question of this study? Does impairment in the dynamics of O2 transport in skeletal muscle during a series of contractions constitute a potential mechanism underlying reduced exercise capacity in pulmonary hypertension? What is the main finding and its importance? Pulmonary hypertension compromises the dynamic matching of skeletal muscle O2 delivery-to-utilization following contraction onset in the rat spinotrapezius muscle. These results implicate a role for vascular dysfunction in the slow V̇O2 kinetics and exercise intolerance present in pulmonary hypertension. ABSTRACT: Pulmonary hypertension (PH) is characterized by pulmonary vascular dysfunction and exercise intolerance due, in part, to compromised pulmonary and cardiac function. We tested the hypothesis that there are peripheral (i.e., skeletal muscle) aberrations in O2 delivery ( Q̇O2 )-to-O2 utilization ( V̇O2 ) matching and vascular control that might help to explain poor exercise tolerance in PH. Furthermore, we investigated the peripheral effects of nitric oxide (NO) in attenuating these decrements. Male Sprague-Dawley rats (n = 21) were administered monocrotaline (MCT; 50 mg/kg, i.p.) to induce PH. Disease progression was monitored via echocardiography. Phosphorescence quenching determined the O2 partial pressure in the interstitial space ( PO2is ) in the spinotrapezius muscle at rest and during contractions under control (SNP-) and NO-donor (sodium nitroprusside, SNP+) conditions. MCT rats displayed right ventricular (RV) hypertrophy (right ventricle/(left ventricle + septum): 0.44 (0.13) vs. 0.28 (0.05)), pulmonary congestion, increased RV systolic pressure (48 (18) vs. 20 (8) mmHg) and arterial hypoxaemia ( PaO2 : 64 (9) vs. 82 (9) mmHg) compared to healthy controls (HC) (P < 0.05). PO2is was significantly lower in MCT rats during the first 30 s of SNP- contractions. SNP superfusion elevated PO2is in both groups; however, MCT rats demonstrated a lower PO2is throughout SNP+ contractions versus HC (P < 0.05). Thus, for small muscle mass exercise in MCT rats, muscle oxygenation is impaired across the rest-to-contractions transition and exogenous NO does not raise the Q̇O2 -to- V̇O2 ratio in contracting muscle to the same levels as HC. These data support muscle Q̇O2 -to- V̇O2 mismatch as a potential contributor to slow V̇O2 kinetics and therefore exercise intolerance in PH and suggest peripheral vascular dysfunction or remodelling as a possible mechanism.

Does wearing a facemask decrease arterial blood oxygenation and impair exercise tolerance?

INTRODUCTION: Concerns have been raised that COVID-19 face coverings compromise lung function and pulmonary gas exchange to the extent that they produce arterial hypoxemia and hypercapnia during high intensity exercise resulting in exercise intolerance in recreational exercisers. This study therefore aimed to investigate the effects of a surgical, flannel or vertical-fold N95 masks on cardiorespiratory responses to incremental exercise. METHODS: This investigation studied 11 adult males and females at rest and while performing progressive cycle exercise to exhaustion. We tested the hypotheses that wearing a surgical (S), flannel (F) or horizontal-fold N95 mask compared to no mask (control) would not promote arterial deoxygenation or exercise intolerance nor alter primary cardiovascular variables during submaximal or maximal exercise. RESULTS: Despite the masks significantly increasing end-expired peri-oral %CO2 and reducing %O2, each ∼0.8-2% during exercise (P < 0.05), our results supported the hypotheses. Specifically, none of these masks reduced sub-maximal or maximal exercise arterial O2 saturation (P = 0.744), but ratings of dyspnea were significantly increased (P = 0.007). Moreover, maximal exercise capacity was not compromised nor were there any significant alterations of primary cardiovascular responses (mean arterial pressure, stroke volume, cardiac output) found during sub-maximal exercise. CONCLUSION: Whereas these results are for young healthy recreational male and female exercisers and cannot be applied directly to elite athletes, older or patient populations, they do support that arterial hypoxemia and exercise intolerance are not the obligatory consequences of COVID-19-indicated mask-wearing at least for cycling exercise.

Sexual dimorphism in vascular ATP-sensitive K+ channel function supporting interstitial PO2 via convective and/or diffusive O2 transport.

KEY POINTS: Inhibition of pancreatic ATP-sensitive K+ (KATP ) channels is the intended effect of oral sulphonylureas to increase insulin release in diabetes. However, pertinent to off-target effects of sulphonylurea medication, sex differences in cardiac KATP channel function exist, whereas potential sex differences in vascular KATP channel function remain unknown. In the present study, we assessed vascular KATP channel function (topical glibenclamide superfused onto fast-twitch oxidative skeletal muscle) supporting blood flow and interstitial O2 delivery-utilization matching ( PO2 is) during twitch contractions in male, female during pro-oestrus and ovariectomized female (F+OVX) rats. Glibenclamide decreased blood flow (convective O2 transport) and interstitial PO2 in male and female, but not F+OVX, rats. Compared to males, females also demonstrated impaired diffusive O2 transport and a faster fall in interstitial PO2 . Our demonstration, in rats, that sex differences in vascular KATP channel function exist support the tentative hypothesis that oral sulphonylureas may exacerbate exercise intolerance and morbidity, especially in premenopausal females. ABSTRACT: Vascular ATP-sensitive K+ (KATP ) channels support skeletal muscle blood flow ( Q̇m ), interstitial O2 delivery ( Q̇O2 )-utilization ( V̇O2 ) matching (i.e. interstitial-myocyte O2 flux driving pressure; PO2 is) and exercise tolerance. Potential sex differences in skeletal muscle vascular KATP channel function remain largely unexplored. We hypothesized that local skeletal muscle KATP channel inhibition via glibenclamide superfusion (5 mg kg-1 GLI; sulphonylurea diabetes medication) in anaesthetized female Sprague-Dawley rats, compared to males, would demonstrate greater reductions in contracting (1 Hz, 7 V, 180 s) fast-twitch oxidative mixed gastrocnemius (97% type IIA+IID/X+IIB) Q̇m (15 µm microspheres) and PO2 is (phosphorescence quenching), resulting from more compromised convective ( Q̇O2 ) and diffusive ( DO2  ) O2 conductances. Furthermore, these GLI-induced reductions in ovary-intact females measured during pro-oestrus would be diminished following ovariectomy (F+OVX). GLI similarly impaired mixed gastrocnemius V̇O2 in both males (↓28%) and females (↓33%, both P < 0.032) via reduced Q̇m (male: ↓31%, female: ↓35%, both P < 0.020), Q̇O2 (male: 5.6 ± 0.5 vs. 4.0 ± 0.5, female: 6.4 ± 1.1 vs. 4.2 ± 0.6 mL O2  min-1 100 g tissue-1 , P < 0.022) and the resulting PO2 is, with females also demonstrating a reduced DO2  (0.40 ± 0.07 vs. 0.30 ± 0.04 mL O2  min-1 100 g tissue-1 , P < 0.042) and a greater GLI-induced speeding of PO2 is fall (mean response time: Sex × Drug interaction, P = 0.026). Conversely, GLI did not impair the mixed gastrocnemius of F+OVX rats. Therefore, in patients taking sulphonylureas, these results support the potential for impaired vascular KATP channel function to compromise muscle Q̇m and therefore exercise tolerance. Such an effect, if present, would likely contribute to adverse cardiovascular events in premenopausal females more than males.

4EHP and GIGYF1/2 Mediate Translation-Coupled Messenger RNA Decay.

Current models of mRNA turnover indicate that cytoplasmic degradation is coupled with translation. However, our understanding of the molecular events that coordinate ribosome transit with the mRNA decay machinery is still limited. Here, we show that 4EHP-GIGYF1/2 complexes trigger co-translational mRNA decay. Human cells lacking these proteins accumulate mRNAs with prominent ribosome pausing. They include, among others, transcripts encoding secretory and membrane-bound proteins or tubulin subunits. In addition, 4EHP-GIGYF1/2 complexes fail to reduce mRNA levels in the absence of ribosome stalling or upon disruption of their interaction with the cap structure, DDX6, and ZNF598. We further find that co-translational binding of GIGYF1/2 to the mRNA marks transcripts with perturbed elongation to decay. Our studies reveal how a repressor complex linked to neurological disorders minimizes the protein output of a subset of mRNAs.

Vascular ATP-sensitive K+ channels support maximal aerobic capacity and critical speed via convective and diffusive O2 transport.

KEY POINTS: Oral sulphonylureas, widely prescribed for diabetes, inhibit pancreatic ATP-sensitive K+ (KATP ) channels to increase insulin release. However, KATP channels are also located within vascular (endothelium and smooth muscle) and muscle (cardiac and skeletal) tissue. We evaluated left ventricular function at rest, maximal aerobic capacity ( V̇ O2 max) and submaximal exercise tolerance (i.e. speed-duration relationship) during treadmill running in rats, before and after systemic KATP channel inhibition via glibenclamide. Glibenclamide impaired critical speed proportionally more than V̇ O2 max but did not alter resting cardiac output. Vascular KATP channel function (topical glibenclamide superfused onto hindlimb skeletal muscle) resolved a decreased blood flow and interstitial PO2 during twitch contractions reflecting impaired O2 delivery-to-utilization matching. Our findings demonstrate that systemic KATP channel inhibition reduces V̇ O2 max and critical speed during treadmill running in rats due, in part, to impaired convective and diffusive O2 delivery, and thus V̇ O2 , especially within fast-twitch oxidative skeletal muscle. ABSTRACT: Vascular ATP-sensitive K+ (KATP ) channels support skeletal muscle blood flow and microvascular oxygen delivery-to-utilization matching during exercise. However, oral sulphonylurea treatment for diabetes inhibits pancreatic KATP channels to enhance insulin release. Herein we tested the hypotheses that: i) systemic KATP channel inhibition via glibenclamide (GLI; 10 mg kg-1 i.p.) would decrease cardiac output at rest (echocardiography), maximal aerobic capacity ( V̇ O2 max) and the speed-duration relationship (i.e. lower critical speed (CS)) during treadmill running; and ii) local KATP channel inhibition (5 mg kg-1 GLI superfusion) would decrease blood flow (15 µm microspheres), interstitial space oxygen pressures (PO2 is; phosphorescence quenching) and convective and diffusive O2 transport ( Q̇ O2 and DO2 , respectively; Fick Principle and Law of Diffusion) in contracting fast-twitch oxidative mixed gastrocnemius muscle (MG: 9% type I+IIa fibres). At rest, GLI slowed left ventricular relaxation (2.11 ± 0.59 vs. 1.70 ± 0.23 cm s-1 ) and decreased heart rate (321 ± 23 vs. 304 ± 22 bpm, both P < 0.05) while cardiac output remained unaltered (219 ± 64 vs. 197 ± 39 ml min-1 , P > 0.05). During exercise, GLI reduced V̇ O2 max (71.5 ± 3.1 vs. 67.9 ± 4.8 ml kg-1 min-1 ) and CS (35.9 ± 2.4 vs. 31.9 ± 3.1 m min-1 , both P < 0.05). Local KATP channel inhibition decreased MG blood flow (52 ± 25 vs. 34 ± 13 ml min-1 100 g tissue-1 ) and PO2 isnadir (5.9 ± 0.9 vs. 4.7 ± 1.1 mmHg) during twitch contractions. Furthermore, MG V̇ O2 was reduced via impaired Q̇ O2 and DO2 (P < 0.05 for each). Collectively, these data support that vascular KATP channels help sustain submaximal exercise tolerance in healthy rats. For patients taking sulfonylureas, KATP channel inhibition may exacerbate exercise intolerance.

Effects of elevated positive end-expiratory pressure on diaphragmatic blood flow and vascular resistance during mechanical ventilation.

Although mechanical ventilation (MV) is a life-saving intervention, prolonged MV can lead to deleterious effects on diaphragm function, including vascular incompetence and weaning failure. During MV, positive end-expiratory pressure (PEEP) is used to maintain small airway patency and mitigate alveolar damage. We tested the hypothesis that increased intrathoracic pressure with high levels of PEEP would increase diaphragm vascular resistance and decrease perfusion. Female Sprague-Dawley rats (~6 mo) were randomly divided into two groups receiving low PEEP (1 cmH2O; n = 10) or high PEEP (9 cmH2O; n = 9) during MV. Blood flow, via fluorescent microspheres, was determined during spontaneous breathing (SB), low-PEEP MV, high-PEEP MV, low-PEEP MV + surgical laparotomy (LAP), and high-PEEP MV + pneumothorax (PTX). Compared with SB, both low-PEEP MV and high-PEEP MV increased total diaphragm and medial costal vascular resistance (P ≤ 0.05) and reduced total and medial costal diaphragm blood flow (P ≤ 0.05). Also, during MV medial costal diaphragm vascular resistance was greater and blood flow lower with high-PEEP MV vs. low-PEEP MV (P ≤ 0.05). Diaphragm perfusion with high-PEEP MV+PTX and low-PEEP MV were not different (P > 0.05). The reduced total and medial costal diaphragmatic blood flow with low-PEEP MV appears to be independent of intrathoracic pressure changes and is attributed to increased vascular resistance and diaphragm quiescence. Mechanical compression of the diaphragm vasculature may play a role in the lower diaphragmatic blood flow at higher levels of PEEP. These reductions in blood flow to the quiescent diaphragm during MV could predispose critically ill patients to weaning complications.NEW & NOTEWORTHY This is the first study, to our knowledge, demonstrating that mechanical ventilation, with low and high positive-end expiratory pressure (PEEP), increases vascular resistance and reduces total and regional diaphragm perfusion. The rapid reduction in diaphragm perfusion and increased vascular resistance may initiate a cascade of events that predispose the diaphragm to vascular and thus contractile dysfunction with prolonged mechanical ventilation.

4E-T-bound mRNAs are stored in a silenced and deadenylated form.

Human 4E-T is an eIF4E-binding protein (4E-BP) present in processing (P)-bodies that represses translation and regulates decay of mRNAs destabilized by AU-rich elements and microRNAs (miRNAs). However, the underlying regulatory mechanisms are still unclear. Here, we show that upon mRNA binding 4E-T represses translation and promotes deadenylation via the recruitment of the CCR4-NOT deadenylase complex. The interaction with CCR4-NOT is mediated by previously uncharacterized sites in the middle region of 4E-T. Importantly, mRNA decapping and decay are inhibited by 4E-T and the deadenylated target is stored in a repressed form. Inhibition of mRNA decapping requires the interaction of 4E-T with the cap-binding proteins eIF4E/4EHP. We further show that regulation of decapping by 4E-T participates in mRNA repression by the miRNA effector protein TNRC6B and that 4E-T overexpression interferes with tristetraprolin (TTP)- and NOT1-mediated mRNA decay. Thus, we postulate that 4E-T modulates 5'-to-3' decay by swapping the fate of a deadenylated mRNA from complete degradation to storage. Our results provide insight into the mechanism of mRNA storage that controls localized translation and mRNA stability in P-bodies.

Transcapillary PO2 gradients in contracting muscles across the fibre type and oxidative continuum.

KEY POINTS: Within skeletal muscle the greatest resistance to oxygen transport is thought to reside across the short distance at the red blood cell-myocyte interface. These structures generate a significant transmural oxygen pressure (PO2 ) gradient in mixed fibre-type muscle. Increasing O2 flux across the capillary wall during exercise depends on: (i) the transmural O2 pressure gradient, which is maintained in mixed-fibre muscle, and/or (ii) elevating diffusing properties between microvascular and interstitial compartments resulting, in part, from microvascular haemodynamics and red blood cell distribution. We evaluated the PO2 within the microvascular and interstitial spaces of muscles spanning the slow- to fast-twitch fibre and high- to low-oxidative capacity spectrums, at rest and during contractions, to assess the magnitude of transcapillary PO2 gradients in rats. Our findings demonstrate that, across the metabolic rest-contraction transition, the transcapillary pressure gradient for O2 flux is: (i) maintained in all muscle types, and (ii) the lowest in contracting highly oxidative fast-twitch muscle. ABSTRACT: In mixed fibre-type skeletal muscle transcapillary PO2 gradients (PO2 mv-PO2 is; microvascular and interstitial, respectively) drive O2 flux across the blood-myocyte interface where the greatest resistance to that O2 flux resides. We assessed a broad spectrum of fibre-type and oxidative-capacity rat muscles across the rest-to-contraction (1 Hz, 120 s) transient to test the novel hypotheses that: (i) slow-twitch PO2 is would be greater than fast-twitch, (ii) muscles with greater oxidative capacity have greater PO2 is than glycolytic counterparts, and (iii) whether PO2 mv-PO2 is at rest is maintained during contractions across all muscle types. PO2 mv and PO2 is were determined via phosphorescence quenching in soleus (SOL; 91% type I+IIa fibres and CSa: ∼21 µmol min-1 g-1 ), peroneal (PER; 33% and ∼20 µmol min-1 g-1 ), mixed (MG; 9% and ∼26 µmol min-1 g-1 ) and white gastrocnemius (WG; 0% and ∼8 µmol min-1 g-1 ) across the rest-contraction transient. PO2 mv was higher than PO2 is in each muscle (∼6-13 mmHg; P < 0.05). SOL PO2 isarea was greater than in the fast-twitch muscles during contractions (P < 0.05). Oxidative muscles had greater PO2 isnadir (9.4 ± 0.8, 7.4 ± 0.9 and 6.4 ± 0.4; SOL, PER and MG, respectively) than WG (3.0 ± 0.3 mmHg, P < 0.05). The magnitude of PO2 mv-PO2 is at rest decreased during contractions in MG only (∼11 to 7 mmHg; time × (PO2 mv-PO2 is) interaction, P < 0.05). These data support the hypothesis that, since transcapillary PO2 gradients during contractions are maintained in all muscle types, increased O2 flux must occur via enhanced intracapillary diffusing conductance, which is most extreme in highly oxidative fast-twitch muscle.

HELZ directly interacts with CCR4-NOT and causes decay of bound mRNAs.

Eukaryotic superfamily (SF) 1 helicases have been implicated in various aspects of RNA metabolism, including transcription, processing, translation, and degradation. Nevertheless, until now, most human SF1 helicases remain poorly understood. Here, we have functionally and biochemically characterized the role of a putative SF1 helicase termed "helicase with zinc-finger," or HELZ. We discovered that HELZ associates with various mRNA decay factors, including components of the carbon catabolite repressor 4-negative on TATA box (CCR4-NOT) deadenylase complex in human and Drosophila melanogaster cells. The interaction between HELZ and the CCR4-NOT complex is direct and mediated by extended low-complexity regions in the C-terminal part of the protein. We further reveal that HELZ requires the deadenylase complex to mediate translational repression and decapping-dependent mRNA decay. Finally, transcriptome-wide analysis of Helz-null cells suggests that HELZ has a role in the regulation of the expression of genes associated with the development of the nervous system.

Molecular basis for GIGYF-Me31B complex assembly in 4EHP-mediated translational repression.

GIGYF (Grb10-interacting GYF [glycine-tyrosine-phenylalanine domain]) proteins coordinate with 4EHP (eIF4E [eukaryotic initiation factor 4E] homologous protein), the DEAD (Asp-Glu-Ala-Asp)-box helicase Me31B/DDX6, and mRNA-binding proteins to elicit transcript-specific repression. However, the underlying molecular mechanism remains unclear. Here, we report that GIGYF contains a motif necessary and sufficient for direct interaction with Me31B/DDX6. A 2.4 Å crystal structure of the GIGYF-Me31B complex reveals that this motif arranges into a coil connected to a ß hairpin on binding to conserved hydrophobic patches on the Me31B RecA2 domain. Structure-guided mutants indicate that 4EHP-GIGYF-DDX6 complex assembly is required for tristetraprolin-mediated down-regulation of an AU-rich mRNA, thus revealing the molecular principles of translational repression.