Remodeling of skeletal muscle myosin metabolic states in hibernating mammals.

Hibernation is a period of metabolic suppression utilized by many small and large mammal species to survive during winter periods. As the underlying cellular and molecular mechanisms remain incompletely understood, our study aimed to determine whether skeletal muscle myosin and its metabolic efficiency undergo alterations during hibernation to optimize energy utilization. We isolated muscle fibers from small hibernators, Ictidomys tridecemlineatus and Eliomys quercinus and larger hibernators, Ursus arctos and Ursus americanus. We then conducted loaded Mant-ATP chase experiments alongside X-ray diffraction to measure resting myosin dynamics and its ATP demand. In parallel, we performed multiple proteomics analyses. Our results showed a preservation of myosin structure in U. arctos and U. americanus during hibernation, whilst in I. tridecemlineatus and E. quercinus, changes in myosin metabolic states during torpor unexpectedly led to higher levels in energy expenditure of type II, fast-twitch muscle fibers at ambient lab temperatures (20 °C). Upon repeating loaded Mant-ATP chase experiments at 8 °C (near the body temperature of torpid animals), we found that myosin ATP consumption in type II muscle fibers was reduced by 77-107% during torpor compared to active periods. Additionally, we observed Myh2 hyper-phosphorylation during torpor in I. tridecemilineatus, which was predicted to stabilize the myosin molecule. This may act as a potential molecular mechanism mitigating myosin-associated increases in skeletal muscle energy expenditure during periods of torpor in response to cold exposure. Altogether, we demonstrate that resting myosin is altered in hibernating mammals, contributing to significant changes to the ATP consumption of skeletal muscle. Additionally, we observe that it is further altered in response to cold exposure and highlight myosin as a potentially contributor to skeletal muscle non-shivering thermogenesis.

Many animals use hibernation as a tactic to survive harsh winters. During this dormant, inactive state, animals reduce or limit body processes, such as heart rate and body temperature, to minimise their energy use. To conserve energy during hibernation, animals can use different approaches. For example, garden dormice undergo periodic states of extremely low core temperatures (down to 4­8^oC); whereas Eurasian brown bears see milder temperature drops (down to 23­25^oC). An important organ that changes during hibernation is skeletal muscle. Skeletal muscle typically uses large amounts of energy, making up around 50% of body mass. To survive, hibernating animals must change how their skeletal muscle uses energy. Traditionally, active myosin ­ a protein found in muscles that helps muscles to contract ­ was thought to be responsible for most of the energy use by skeletal muscle. But, more recently, resting myosin has also been found to use energy when muscles are relaxed. Lewis et al. studied myosin and skeletal muscle energy use changes during hibernation and whether they could impact the metabolism of hibernating animals. Lewis et al. assessed myosin changes in muscle samples from squirrels, dormice and bears during hibernation and during activity. Experiments showed changes in resting myosin in squirrels and dormice (whose temperature drops to 4­8^oC during hibernation) but not in bears. Further analysis revealed that cooling samples from non-hibernating muscle to 4­8^oC increased energy use in resting myosin, thereby generating heat. However, no increase in energy use was found after cooling hibernating muscle samples to 4­8^oC. This suggest that resting myosin generates heat at cool temperatures ­ a mechanism that is switched off in hibernating animals to allow them to cool their body temperature. These findings reveal key insights into how animals conserve energy during hibernation. In addition, the results show that myosin regulates energy use in skeletal muscles, which indicates myosin may be a potential drug target in metabolic diseases, such as obesity.

Remodelling of Skeletal Muscle Myosin Metabolic States in Hibernating Mammals.

Hibernation is a period of metabolic suppression utilized by many small and large mammal species to survive during winter periods. As the underlying cellular and molecular mechanisms remain incompletely understood, our study aimed to determine whether skeletal muscle myosin and its metabolic efficiency undergo alterations during hibernation to optimize energy utilization. We isolated muscle fibers from small hibernators, Ictidomys tridecemlineatus and Eliomys quercinus and larger hibernators, Ursus arctos and Ursus americanus. We then conducted loaded Mant-ATP chase experiments alongside X-ray diffraction to measure resting myosin dynamics and its ATP demand. In parallel, we performed multiple proteomics analyses. Our results showed a preservation of myosin structure in U. arctos and U. americanus during hibernation, whilst in I. tridecemlineatus and E. quercinus, changes in myosin metabolic states during torpor unexpectedly led to higher levels in energy expenditure of type II, fast-twitch muscle fibers at ambient lab temperatures (20°C). Upon repeating loaded Mant-ATP chase experiments at 8°C (near the body temperature of torpid animals), we found that myosin ATP consumption in type II muscle fibers was reduced by 77-107% during torpor compared to active periods. Additionally, we observed Myh2 hyper-phosphorylation during torpor in I. tridecemilineatus, which was predicted to stabilize the myosin molecule. This may act as a potential molecular mechanism mitigating myosin-associated increases in skeletal muscle energy expenditure during periods of torpor in response to cold exposure. Altogether, we demonstrate that resting myosin is altered in hibernating mammals, contributing to significant changes to the ATP consumption of skeletal muscle. Additionally, we observe that it is further altered in response to cold exposure and highlight myosin as a potentially contributor to skeletal muscle non-shivering thermogenesis.

Predominant myosin superrelaxed state in canine myocardium with naturally occurring dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a naturally occurring heart failure condition in humans and dogs, notably characterized by a reduced contractility and ejection fraction. As the identification of its underlying cellular and molecular mechanisms remain incomplete, the aim of the present study was to assess whether the molecular motor myosin and its known relaxed conformational states are altered in DCM. For that, we dissected and skinned thin cardiac strips from left ventricle obtained from six DCM Doberman Pinschers and six nonfailing (NF) controls. We then used a combination of Mant-ATP chase experiments and X-ray diffraction to assess both energetic and structural changes of myosin. Using the Mant-ATP chase protocol, we observed that in DCM dogs, the amount of myosin molecules in the ATP-conserving conformational state, also known as superrelaxed (SRX), is significantly increased when compared with NF dogs. This alteration can be rescued by applying EMD-57033, a small molecule activating myosin. Conversely, with X-ray diffraction, we found that in DCM dogs, there is a higher proportion of myosin heads in the vicinity of actin when compared with NF dogs (1,0 to 1,1 intensity ratio). Hence, we observed an uncoupling between energetic (Mant-ATP chase) and structural (X-ray diffraction) data. Taken together, these results may indicate that in the heart of Doberman Pinschers with DCM, myosin molecules are potentially stuck in a nonsequestered but ATP-conserving SRX state, that can be counterbalanced by EMD-57033 demonstrating the potential for a myosin-centered pharmacological treatment of DCM.NEW & NOTEWORTHY The key finding of the present study is that, in left ventricles of dogs with a naturally occurring dilated cardiomyopathy, relaxed myosin molecules favor a nonsequestered superrelaxed state potentially impairing sarcomeric contractility. This alteration is rescuable by applying a small molecule activating myosin known as EMD-57033.

CaMKK2 is not involved in contraction-stimulated AMPK activation and glucose uptake in skeletal muscle.

OBJECTIVE: The AMP-activated protein kinase (AMPK) gets activated in response to energetic stress such as contractions and plays a vital role in regulating various metabolic processes such as insulin-independent glucose uptake in skeletal muscle. The main upstream kinase that activates AMPK through phosphorylation of α-AMPK Thr172 in skeletal muscle is LKB1, however some studies have suggested that Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) acts as an alternative kinase to activate AMPK. We aimed to establish whether CaMKK2 is involved in activation of AMPK and promotion of glucose uptake following contractions in skeletal muscle. METHODS: A recently developed CaMKK2 inhibitor (SGC-CAMKK2-1) alongside a structurally related but inactive compound (SGC-CAMKK2-1N), as well as CaMKK2 knock-out (KO) mice were used. In vitro kinase inhibition selectivity and efficacy assays, as well as cellular inhibition efficacy analyses of CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) were performed. Phosphorylation and activity of AMPK following contractions (ex vivo) in mouse skeletal muscles treated with/without CaMKK inhibitors or isolated from wild-type (WT)/CaMKK2 KO mice were assessed. Camkk2 mRNA in mouse tissues was measured by qPCR. CaMKK2 protein expression was assessed by immunoblotting with or without prior enrichment of calmodulin-binding proteins from skeletal muscle extracts, as well as by mass spectrometry-based proteomics of mouse skeletal muscle and C2C12 myotubes. RESULTS: STO-609 and SGC-CAMKK2-1 were equally potent and effective in inhibiting CaMKK2 in cell-free and cell-based assays, but SGC-CAMKK2-1 was much more selective. Contraction-stimulated phosphorylation and activation of AMPK were not affected with CaMKK inhibitors or in CaMKK2 null muscles. Contraction-stimulated glucose uptake was comparable between WT and CaMKK2 KO muscle. Both CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) and the inactive compound (SGC-CAMKK2-1N) significantly inhibited contraction-stimulated glucose uptake. SGC-CAMKK2-1 also inhibited glucose uptake induced by a pharmacological AMPK activator or insulin. Relatively low levels of Camkk2 mRNA were detected in mouse skeletal muscle, but neither CaMKK2 protein nor its derived peptides were detectable in mouse skeletal muscle tissue. CONCLUSIONS: We demonstrate that pharmacological inhibition or genetic loss of CaMKK2 does not affect contraction-stimulated AMPK phosphorylation and activation, as well as glucose uptake in skeletal muscle. Previously observed inhibitory effect of STO-609 on AMPK activity and glucose uptake is likely due to off-target effects. CaMKK2 protein is either absent from adult murine skeletal muscle or below the detection limit of currently available methods.

Physical activity impacts resting skeletal muscle myosin conformation and lowers its ATP consumption.

It has recently been established that myosin, the molecular motor protein, is able to exist in two conformations in relaxed skeletal muscle. These conformations are known as the super-relaxed (SRX) and disordered-relaxed (DRX) states and are finely balanced to optimize ATP consumption and skeletal muscle metabolism. Indeed, SRX myosins are thought to have a 5- to 10-fold reduction in ATP turnover compared with DRX myosins. Here, we investigated whether chronic physical activity in humans would be associated with changes in the proportions of SRX and DRX skeletal myosins. For that, we isolated muscle fibers from young men of various physical activity levels (sedentary, moderately physically active, endurance-trained, and strength-trained athletes) and ran a loaded Mant-ATP chase protocol. We observed that in moderately physically active individuals, the amount of myosin molecules in the SRX state in type II muscle fibers was significantly greater than in age-matched sedentary individuals. In parallel, we did not find any difference in the proportions of SRX and DRX myosins in myofibers between highly endurance- and strength-trained athletes. We did however observe changes in their ATP turnover time. Altogether, these results indicate that physical activity level and training type can influence the resting skeletal muscle myosin dynamics. Our findings also emphasize that environmental stimuli such as exercise have the potential to rewire the molecular metabolism of human skeletal muscle through myosin.

Myosin Heavy Chain as a Novel Key Modulator of Striated Muscle Resting State.

After years of intense research using structural, biological, and biochemical experimental procedures, it is clear that myosin molecules are essential for striated muscle contraction. However, this is just the tip of the iceberg of their function. Interestingly, it has been shown recently that these molecules (especially myosin heavy chains) are also crucial for cardiac and skeletal muscle resting state. In the present review, we first overview myosin heavy chain biochemical states and how they influence the consumption of ATP. We then detail how neighboring partner proteins including myosin light chains and myosin binding protein C intervene in such processes, modulating the ATP demand in health and disease. Finally, we present current experimental drugs targeting myosin ATP consumption and how they can treat muscle diseases.

An endogenous inhibitor of angiogenesis downregulated by hypoxia in human aortic valve stenosis promotes disease pathogenesis.

Aortic valve stenosis is the most common valve disease in the western world. Central to the pathogenesis of this disease is the growth of new blood vessels (angiogenesis) within the aortic valve allowing infiltration of immune cells and development of intra-valve inflammation. Identifying the cellular mediators involved in this angiogenesis is important as this may reveal new therapeutic targets which could ultimately prevent the progression of aortic valve stenosis. Aortic valves from patients undergoing surgery for aortic valve replacement or dilation of the aortic arch were examined both ex vivo and in vitro. We now demonstrate that the anti-angiogenic protein, soluble fms-like tyrosine kinase 1 (sFlt1), a non-signalling soluble receptor for vascular endothelial growth factor, is constitutively expressed in non-diseased valves. sFlt-1 expression was, however, significantly reduced in aortic valve tissue from patients with aortic valve stenosis while protein markers of hypoxia were simultaneously increased. Exposure of primary-cultured valve interstitial cells to hypoxia resulted in a decrease in the expression of sFlt-1. We further reveal using a bioassay that siRNA knock-down of sFlt1 in valve interstitial cells directly results in a pro-angiogenic environment. Finally, incubation of aortic valves with sphingosine 1-phosphate, a bioactive lipid-mediator, increased sFlt-1 expression and inhibited angiogenesis within valve tissue. In conclusion, this study demonstrates that sFlt1 expression is directly correlated with angiogenesis in aortic valves and the observed decrease in sFlt-1 expression in aortic valve stenosis could increase valve inflammation, promoting disease progression. This could be a viable therapeutic target in treating this disease.

Baseline Psychological Traits Contribute to Lake Louise Acute Mountain Sickness Score at High Altitude.

Talks, Benjamin James, Catherine Campbell, Stephanie J. Larcombe, Lucy Marlow, Sarah L. Finnegan, Christopher T. Lewis, Samuel J.E. Lucas, Olivia K. Harrison, and Kyle T.S. Pattinson. Baseline psychological traits contribute to Lake Louise Acute Mountain Sickness score at high altitude. High Alt Med Biol. 23:69-77, 2022. Background: Interoception refers to an individual's ability to sense their internal bodily sensations. Acute mountain sickness (AMS) is a common feature of ascent to high altitude that is only partially explained by measures of peripheral physiology. We hypothesized that interoceptive ability may explain the disconnect between measures of physiology and symptom experience in AMS. Methods: Two groups of 18 participants were recruited to complete a respiratory interoceptive task three times at 2-week intervals. The control group remained in Birmingham (140 m altitude) for all three tests. The altitude group completed test 1 in Birmingham, test 2 the day after arrival at 2,624 m, and test 3 at 2,728 m after an 11-day trek at high altitude (up to 4,800 m). Results: By measuring changes to metacognitive performance, we showed that acute ascent to altitude neither presented an interoceptive challenge, nor acted as interoceptive training. However, AMS symptom burden throughout the trek was found to relate to sea level measures of anxiety, agoraphobia, and neuroticism. Conclusions: This suggests that the Lake Louise AMS score is not solely a reflection of physiological changes on ascent to high altitude, despite often being used as such by researchers and commercial trekking companies alike.

Losartan Has No Effect on High Altitude Diuresis or Acute Mountain Sickness in Well-Acclimatizing Individuals.

Delamere, John P., Susie B. Bradwell, Christopher T. Lewis, Alex Clarke, and Arthur R. Bradwell. Losartan has no effect on high altitude diuresis or acute mountain sickness in well-acclimatizing individuals. High Alt Med Biol. 22:96-101, 2021. Introduction: The diuretic response that occurs on ascent to altitude is associated with suppression of aldosterone. We speculated that losartan, an angiotensin II receptor blocker, might further reduce aldosterone activity thereby enhancing the diuresis. Materials and Methods: Twenty subjects (paired for angiotensin converting enzyme genotypes [II:ID:DD] gender and age) were randomized, on a double-blind basis, to either daily losartan, 100 mg, or placebo. During 7 days of motorized ascent from 2,850 to 5,035 m, collections of 24-hour urine output were measured daily with samples taken for sodium (Na+) and potassium (K+) concentrations. In addition, measurements were made of blood gases and aldosterone concentrations. Results: During the main ascent, there were similar progressive increases in 24-hour urine volumes in placebo and losartan groups with no change in Na+ or K+ excretion. There were negative correlations between mean 24-hour urine volumes and PaO2 (r = -0.97, p < 0.03), and the diuretic response and acute mountain sickness scores at 5,053 m (r = -0.51, p < 0.03). There were no significant changes in aldosterone concentrations measured at baseline and at our high point on day 6 within or between the losartan and placebo groups. Conclusion: The high altitude diuretic response was not increased by losartan indicating aldosterone activity was suppressed in individuals on placebo who were acclimatizing well to altitude.

Prehospital haemostatic dressings for trauma: a systematic review.

BACKGROUND: Haemorrhage is a major cause of mortality and morbidity following both military and civilian trauma. Haemostatic dressings may offer effective haemorrhage control as part of prehospital treatment. AIM: To conduct a systematic review of the clinical literature to assess the prehospital use of haemostatic dressings in controlling traumatic haemorrhage, and determine whether any haemostatic dressings are clinically superior. METHODS: MEDLINE and EMBASE databases were searched using predetermined criteria. The reference lists of all returned review articles were screened for eligible studies. Two authors independently undertook the search, performed data extraction, and risk of bias and Grading of Recommendations, Assessment, Development and Evaluation quality assessments. Meta-analysis could not be undertaken due to study and clinical heterogeneity. RESULTS: Our search yielded 470 studies, of which 17 met eligibility criteria, and included 809 patients (469 military and 340 civilian). There were 15 observational studies, 1 case report and 1 randomised controlled trial. Indications for prehospital haemostatic dressing use, wound location, mechanism of injury, and source of bleeding were variable. Seven different haemostatic dressings were reported with QuikClot Combat Gauze being the most frequently applied (420 applications). Cessation of bleeding ranged from 67% to 100%, with a median of 90.5%. Adverse events were only reported with QuikClot granules, resulting in burns. No adverse events were reported with QuikClot Combat Gauze use in three studies. Seven of the 17 studies did not report safety data. All studies were at risk of bias and assessed of 'very low' to 'moderate' quality. CONCLUSIONS: Haemostatic dressings offer effective prehospital treatment for traumatic haemorrhage. QuikClot Combat Gauze may be justified as the optimal agent due to the volume of clinical data and its safety profile, but there is a lack of high-quality clinical evidence, and randomised controlled trials are warranted. LEVEL OF EVIDENCE: Systematic review, level IV.

High altitude arterialised capillary earlobe blood gas measurement using the Abbott i-STAT.

INTRODUCTION: Measurement of physiological parameters in extreme environments is essential to advancing knowledge, prophylaxis and treatment of altitude sickness. Point-of-care testing facilitates investigation in non-specialist and remote settings, as well as becoming increasingly popular at the bedside for real-time results in the clinical environment. Arterialised capillary earlobe blood gases are recommended as a valid alternative to arterial sampling in research. This study aimed to test the feasibility of obtaining and analysing daily earlobe samples at high altitude. METHODS: From 17 to 24 January 2016, 24 participants on a research expedition to Ecuador underwent daily earlobe blood gas measurements including pH, partial pressure of oxygen and partial pressure of carbon dioxide to 5043 m. Samples were analysed using an Abbott i-STAT blood gas analyser and G3+ cartridges. RESULTS: Daily measurements were successfully obtained and analysed at the point of care in 23/24 participants and were well tolerated with no adverse events. 12% (27/220) cartridges failed and required repeat sampling. CONCLUSIONS: Daily earlobe blood gas analysis using the Abbott i-STAT is feasible in a protected environment at high altitude. Participants and equipment should be kept warm before and during testing. Spare cartridges should be available. This methodology may be useful for both research and therapeutic measurements in remote, rural and wilderness medicine.

Prehospital point-of-care lactate following trauma: A systematic review.

BACKGROUND: Serum lactate serves as a surrogate marker for global tissue hypoxia following traumatic injury and has potential to guide resuscitation. Portable, handheld point-of-care monitoring devices enable lactate values to be readily available in the prehospital environment. The current review examines the utility of prehospital lactate (pLa) measurement in the management of trauma. METHODS: MEDLINE and EMBASE databases were searched using predefined criteria (pLa measurement, trauma patients) until March 10, 2016. Studies were examined for lactate measurement as an intervention, prognostic indicator, and utilization in the guidance of goal-directed therapy. The Newcastle-Ottawa Scale was used to assess risk of bias, and quality of evidence was evaluated using the Grading of Recommendations, Assessment, Development, and Evaluation system. Data were unsuitable for meta-analysis and are presented in narrative form. RESULTS: Of 2,415 articles of interest, seven met the inclusion criteria, all of which were observational studies, including 2,085 trauma patients. Lactate sampling techniques, timings, and thresholds were heterogeneous. No studies used pLa to guide intervention. Elevated pLa may be an independent prognostic marker of critical illness in trauma patients, particularly in blunt trauma. Prehospital lactate measurement may be more sensitive than systolic blood pressure in determining need for resuscitative care. Early lactate measurement may be particularly useful in the detection of occult hypoperfusion, with elevated pLa detectable within 30 minutes of injury. All current studies were assessed as being of "low" or "very low" quality and were at risk of bias. Considerable logistical barriers to pLa measurement exist. CONCLUSIONS: Prehospital point-of-care lactate monitoring for trauma has been variably performed. There is a paucity of evidence relating to its use. The limited data available show feasibility and potential clinical utility, and further investigation is warranted to establish whether lactate might give meaningful guidance during prehospital triage and trauma patient resuscitation. LEVEL OF EVIDENCE: Systematic review, level IV.

Draft Genome Sequence of Alternaria alternata ATCC 34957.

We report the draft genome sequence of Alternaria alternata ATCC 34957. This strain was previously reported to produce alternariol and alternariol monomethyl ether on weathered grain sorghum. The genome was sequenced with PacBio technology and assembled into 27 scaffolds with a total genome size of 33.5 Mb.

Draft Genome Sequences of Ralstonia solanacearum Race 3 Biovar 2 Strains with Different Temperature Adaptations.

Ralstonia solanacearum race 3 biovar 2 (R3bv2) causes brown rot of potato in countries with temperate climates. Here, we report two draft genome sequences of R. solanacearum R3bv2 NCPPB909 and CFIA906 with different temperature adaptations. Analysis of these genome sequences will provide detailed insight on virulence, functionality, and plant/pest interactions of this widely distributed and regulated pathogen.

Draft Genome Sequences for Canadian Isolates of Pectobacterium carotovorum subsp. brasiliense with Weak Virulence on Potato.

Pectobacterium carotovurum subsp. brasiliense causes soft rot and blackleg diseases on potato. Here, we report the draft genome sequences of three weakly virulent P. carotovurum subsp. brasiliense strains isolated in Canada. Analysis of these genome sequences will help to pinpoint differences in virulence among P. carotovurum subsp. brasiliense strains from tropical/subtropical and temperate regions, such as Canada and United States. A small number of key factors for adaptation to this bacterium's specific environmental niche were also evaluated.

Draft Genome Sequence of Pectobacterium wasabiae Strain CFIA1002.

Pectobacterium wasabiae, originally causing soft rot disease in horseradish in Japan, was recently found to cause blackleg-like symptoms on potato in the United States, Canada, and Europe. A draft genome sequence of a Canadian potato isolate of P. wasabiae CFIA1002 will enhance the characterization of its pathogenicity and host specificity features.

Draft genome sequences of three arcobacter strains of pig and dairy cattle manure origin.

The genus Arcobacter has been associated with human illness and fecal contamination by humans and animals. Here, we announce the draft genome sequences of three strains of Arcobacter species cultured from pig and dairy cattle manure tanks. This information will assist in the characterization of features related to host specificities and identify potential pathogenic health risks to humans and animals.