bluuebunny/crossref_metadata_2025 huggingface dataset:
α1G T‐type Ca2+ Channel Contributes to the Angiogenic Capacity of Pulmonary Microvascular Endothelial Cells
Authors: Zheng Zhen , Chen Hairu , Zhou Chun , Liu Bao , King Judy A , Alexeyev Mikhail F , Wu Songwei
Published: November 2023

Pulmonary microvascular endothelial cells (PMVECs) characteristically possess intrinsic rapid proliferation and angiogenic capacity, both of which are essential for vascular homeostasis as well as tissue remodeling and repair in response to vascular injury. Although it has long been established that cellular Ca2+ signaling plays an important role in regulating cell proliferation and angiogenesis, the molecular mechanisms underlying such regulation remain poorly understood. In the present study, we demonstrated that α1G T‐type Ca2+ channel, the only voltage‐gated Ca2+ channel expressed in PMVECs, is critical for cell proliferation, adhesion, migration, and in vitro tube formation capability. PMVECs exhibited rigorous in vitro capillary sprout‐like, tube formation in a reconstituted basement membrane extracellular matrix (Matrigel). Pharmacological blockade of the T‐type Ca2+ channel with NNC 55‐0396, a highly selective T‐type channel blocker, dose‐dependently reduced the tube formation capacity, by 26% at 5 μmol/L and 48% at 10 μmol/L (P < 0.001). In stark contrast, pharmacological blockade of TRPC4 or TRPV4, two other Ca2+ permeable channels expressed in PMVECs, had no effect on tube formation. Similarly, α gene suppression by shRNA significantly reduced tube formation capacity compared to the scramble shRNA control. Next, cell proliferation assays revealed that the cell proliferation in PMVECs was significantly reduced by α1G gene suppression with shRNA. Further, pharmacological blockade of the T‐type Ca2+ channel with NNC 55‐0396 dose‐dependently reduced the number of adherent PMVECs in a Matrigel‐coated substratum, by 31% at 5 μmol/L and 66% at 10 μmol/L (P < 0.001). Moreover, pharmacological blockade of this channel attenuated cell migration as revealed by a wound‐healing assay in PMVECs. Taken together, these findings support a novel functional role of the site‐specific endothelial α1G T‐type Ca2+ channel in the control of cell angiogenic capacity in PMVECs.

Support or Funding Information

Supported by HL66299
Reactive Oxygen Species Signaling in the Hypoxia‐Tolerant Turtle and Goldfish Stellate and Pyramidal Neurons Is Species Specific
Authors: Buck Les , Hossien‐Javaheri Nariman , Pillai Varshinie , Hawrysh Peter
Published: November 2023

The majority of vertebrate species are not hypoxia‐tolerant; however, some freshwater turtles and fish from the carp family are tolerant of extreme hypoxia for days or months. Two examples are the western painted turtle and the goldfish which provide excellent models for the study of hypoxia‐tolerance mechanisms in brain. We have previously shown that severe hypoxia results in a natural decrease in reactive oxygen species (ROS) in turtle brain; therefore, here we investigate in a turtle brain sheet and goldfish brain slice model the response of pyramidal and stellate neurons to scavenging of ROS with MPG (N‐2‐mercaptopropionyl glycine) for comparison to the tissue exposed to severe hypoxia. Using single cell fluorescence we first confirm that ROS levels do indeed decrease with severe hypoxia in goldfish brain slices. Then using whole‐cell and perforated patch clamp techniques and ROS scavenging we find that whole cell conductance (Gw) increases from 4.5 to 6.6nS in both turtle cell types and from 3.96±0.95nS to 6.84±0.62nS in both goldfish cell types. In both cases the increased Gw is related to increased GABA‐A receptor conductance since application of the GABA‐A receptor inhibitor gabazine blocks the increase. While goldfish and turtle pyramidal cells remained mostly quiescent the action potential frequency (APf) of goldfish stellate neurons increased with ROS scavenging from 0.03±0.009Hz to 2.04±0.12Hz or 68 fold. Turtle stellate neurons exhibited a unique burst firing pattern where AP's are clustered into discrete bursts of about 2 AP's per burst with quiescent periods in between bursts. When exposed to ROS scavenging overall APf decreased from 0.49 ± 0.14 Hz to 0.33 ± 0.07 Hz but the number of AP's per burst increased from 2.4 ± 0.21 to 3.0 ± 0.12. With ROS scavenging the stellate neuron burst pattern coalesces into a burst every 7–8 s that corresponds with the frequency of giant inhibitory GABAergic current recorded from pyramidal neurons. It remains unclear how these differences in ROS signaling impacts the hypoxia‐tolerance of each species but we conclude that the unique responses of stellate neurons is species specific.

Support or Funding Information

Natural Sciences and Engineering Council of Canada grant to LTB
Caffeine governs molecular mechanism(s) on circadian rhythm and circadian clock gene expression
Authors: Kang Hyunbon , Park Sookyoung , Jin Yunho , Hong Yonggeun
Published: November 2023

Caffeine is one of the most widely used psychoactive substances, and it induces the arousal in the CNS, but also disrupts the quality of sleep. Recent studies reported that caffeine can affect the circadian rhythm, which associates many aspects of physiology, energy metabolism, sleep and homeostasis. Therefore we examined the arousal effects of caffeine via behavioral test, and determined its effects on circadian rhythm by measuring clock gene and clock‐controlled gene expression in mice brain. Male C57BL/6 mice were randomly divided into control, three experimental groups treated with vehicle, caffeine and caffeine with adenosine, respectively. Before administration of caffeine, forced treadmill exercise was applied to mice inducing physiological fatigue. Balance beam test and foot fault test were used to evaluate the balance ability in subjected mice. Caffeine treatment reduced weight gain, suggesting physiological effect of caffeine to inhibit food intake. In addition, average time to cross beam and error number of foot slip after caffeine injection were decreased compared with control. A foot fault was defined as the misplacement of fore or hind foot, thus the foot fault number after caffeine treatment was reduced compared with control. There were differences in expression of circadian clock genes including Clock, Per2, Cry1, and Cry2 by caffeine injection. Thus, these results demonstrate that caffeine can alter circadian rhythm in mice by regulating circadian clock gene expression.

Support or Funding Information

NRF‐2013R1A2A2A01067169 to Y.H., NRF‐2014R1A1A3051724 to S.P., Republic of Korea.
Behavioral Profile Assessment in Offspring of Swiss Mice Underwent to Neonatal Treatment with Fluoxetine
Authors: da Silva Cortes Wellington , Laureano‐Melo Roberto , Conceição Rodrigo Rodrigues , Souza Janaina Sena , Lau Raphael Silva , da Silva Almeida Claudio , Giannocco Gisele
Published: November 2023

Serotonin exerts an important role in the central nervous system embryogenesis of mammals, influencing directly several brain systems ontogeny. Therefore, we investigated the effect of neonatal Fluoxetine (FLU), a selective serotonin reuptake inhibitor, on the behavioral expression of adult male Swiss mice. For this purpose, three groups (n=6 each, and ~35g) of pregnant female Swiss mice were mated. The offspring obtained was treated with FLU (1mg/kg, s.c.) from the 5th at 15th post‐natal day. On the 16th post‐natal day, female puppies were euthanized and hippocampus was dissected for RNA analysis whereas the male offspring, by completing 70 days of life, were underwent a behavioral assessment in open field, light‐dark box and tail suspension test. Statistical analysis was performed by t‐Student and the means were considered significantly different when p<0.05. According to our results, the programmed animals had a decrease in TPH2 (0.07±0.007 vs. 1.07±0.19, p<0.001), 5HT1a (0.08±0.007 vs. 1.02±0.17, p<0.001), SERT (0.35±0.08 vs. 1.09±0.18, p=0.004), BDNF (0.19±0.02 vs. 1.07±0.19, p=0.001) and LMX1B (0.03±0.01 vs. 1.12±0.25, p=0.001) expression. In addition, it was observed less immobility time in tail suspension test (77.6±7.25 vs. 112.1±12.2, p=0.02) and higher grooming time in open field test (7.6±1.4 vs. 3.1±0.9, p=0.01). In light‐dark box test, the animals treated with FLU showed a shorter time in the light side than control (36.69±10.4 vs. 76.2±5.48, p<0.01). These findings suggest that programming with FLU during the neonatal period alters hippocampal serotonergic system, promoting anxiety and depression‐like behaviors in adults.

Support or Funding Information

FAPERJ & CNPq
IBUPROFEN IMPROVES POSTOPERATIVE SPATIAL RECOGNITION
Authors: Schoemaker Regien G , Hovens Iris B , de Haan Jacco J , van Leeuwen Barbara L
Published: November 2023

Background

Ten percent of older surgical patients experience prolonged dementia‐like symptoms after major surgery. This postoperative cognitive dysfunction (POCD) appears to be mediated by surgery‐induced (neuro)inflammation. We aimed to investigate the feasibility of acute anti‐inflammatory treatment prior to surgery as preventive therapy against POCD.

Methods

Young (3 mo, n=16) and aged (23 mo, n=16) male Wistar rats were subjected to major abdominal surgery. Before surgery, rats were fasted overnight and received ibuprofen (15 mg/kg i.p.; IBU) 30 minutes before surgery or no treatment. In the second postoperative week cognitive performance was assessed with the novel object (NO) and novel location (NL) test, and training, probe trials, and reversal training in the Morris water maze (MWM). Brains were collected two weeks after surgery and processed for immunohistochemical analyses of microglial activity and neurogenesis in the hippocampus.

Results

NO recognition was not affected by age nor by IBU. Whereas spatial learning and reversal learning were impaired with age but not affected by IBU, in young, but not in old rats IBU improved spatial recognition in the NL test. This was accompanied by increased hippocampal neurogenesis in young, but not in old rats. Surprisingly, hippocampal microglial activity after ibuprofen treatment appeared concomitantly increased.

Conclusion

Improved spatial recognition accompanied by increased hippocampal neurogenesis, but higher microglial activity suggest mixed effects of preoperative anti‐inflammatory treatment with ibuprofen.

Support or Funding Information

Research was funded by the University Medical enter Groningen
Disruption of Adenylyl Cyclase Type 5 Induces Anti‐Depressive Behavior
Authors: Trifiolis Taylor Lyn , Guers John , Buchholz Bruno , Vatner Dorothy , Vatner Stephen , Gelpi Ricardo
Published: November 2023

It is generally thought that increases in cyclic AMP signaling will induce depressive behavior. We examined the extent to which behavior was modified by disruption of adenylyl cyclase type 5 (AC5), which reduces cyclic AMP levels, and which is prevalent in most organs, including the brain. Accordingly, we assessed behavior using traditional tests, i.e., locomotor activity using an open field, examining total distance traveled, rest time and vertical activity, which were all recorded and analyzed for AC5 knock out (KO) mice and their wild type (WT) littermates. The AC5 KO mice were significantly more active in all aspects of the open field test in comparison to the wild type mice. The average total distance traveled by AC5 KO mice over a two hour testing period was (7,584 ± 749 cm) while WT average total distance was (1,764 ± 99 cm; p<0.05). Rest time was less in AC5 KO. AC5 KO only rested for (3,884 ± 172 s) while AC5 WT remained inactive for (5,745 ± 86 s; p<0.05). AC5 KO moved vertically more often (1008 ± 117 counts) than AC5 WT (125 ± 11 counts; p <0.05). These data, in contrast to most of the literature, indicate that AC5 KO mice, which have lower cAMP signaling, actually exhibit increased locomotion and strongly suggesting an anti‐depressive behavior. Translating this concept to the clinics would be a novel therapeutic approach for patients with depression.

Support or Funding Information

This study is supported by NIH grant: R01 HL106511.
Immunohistochemical Localization of Neuropeptide Arginine Vasopressin and Morphometric Study of Domestic Cat Amygdala
Authors: Kaur Gagandeep , Tang Karena , Seelman Amanda , Shahriar Farshid , Khamas Wael
Published: November 2023

Neuropeptide arginine vasopressin (AVP) is known to modulate social behavior in several species. The AVP and its receptors are broadly distributed in the limbic system, with several studies documenting their involvement in behavior regulation. However, the role of AVP in feline behavior is unknown, due to lack of studies and possible species differences. Here, as the first step, we investigated the presence of AVP in the domestic cat amygdala by using immunohistochemistry on freely‐floating brain tissue sections. The amygdala is an important component of the circuitry underlying social behavior and emotions. Therefore, we also characterized the neuronal morphology of domestic cat amygdala using cresyl violet stained 30μm serial sections. Our results show the presence of AVP immunoreactive neurons in the domestic cat amygdala (4 males, 1 female). The average neuronal diameter was 10.7±0.3μm in the lateral nucleus, 14.2±0.6μm in the medial nucleus, 11.1±0.8μm in the central nucleus, and 14.3±1.4μm in the basal nucleus, within the amygdala. The rostrocaudal length of the amygdala ranged from 6.7 – 7.3mm (3 males, 1 female). Variations in neuronal diameters did not translate to AVP immunoreactivity. This study is the first demonstration of the presence of neuropeptide AVP in the domestic cat amygdala. This might suggest a potential role of AVP in regulating social behavior in domestic cats. The morphometric findings of neuronal diameter might complement the physiological findings, implementing different roles in behavior for different subnuclei. Such studies will lay foundation for future research in feline behavior to improve feline health and welfare.

Support or Funding Information

Western University of Health Sciences, Pomona, CA
Chronic, complete cervical6‐7 cord injury: distinct autonomic, cardiac and vascular deficits
Authors: Lujan Heidi L. , Mulder Rebecca A. , DiCarlo Stephen E
Published: November 2023

Spinal cord injury (SCI) resulting in tetraplegia is a devastating, life‐changing injury causing paralysis and sensory impairment as well as autonomic dysfunction that triggers compromised cardiovascular, bowel, bladder and sexual activity. Life becomes a battle for independence as even routine bodily functions and the smallest activity of daily living become a major challenge. The human physiology, mind and spirit are wonderfully adaptable as individuals with tetraplegia find ways to adjust. However, addressing autonomic losses and cardiovascular consequences would greatly improve the quality of life for individuals living with tetraplegia. Virtually all pre‐clinical investigations of autonomic losses and cardiovascular consequences following SCI induce lesions in the thoracic region. However, cervical lesions produce distinct autonomic, cardiac and vascular deficits and result in the most dramatic autonomic outcomes and cardiovascular consequences. Specifically, although cardiovascular disease (CVD) is the leading cause of morbidity and mortality for individuals living with SCI, the magnitude of CVD risk is dependent on the level of SCI, whereby individuals with tetraplegia have a 16% greater risk of all‐cause CVD than individuals with paraplegia. Accordingly, there is a critical need for a chronic pre‐clinical model of tetraplegia. In this report, we compare resting cardiac sympathetic and parasympathetic tonus, reflex sympathetic effects (heart rate responses to sodium nitroprusside), tonic autonomic effects (responses to autonomic ganglionic blockade), cardiac responses to beta‐adrenergic stimulation and the response to angiotensin‐converting enzyme inhibition in 6 rats with chronic (4 week) complete C6‐7 transection and 6 intact sham‐transected rats. Histological examination of the heart and stellate ganglia support the profound and distinct autonomic and cardiac deficits and reliance on angiotensin to maintain cardiovascular stability following chronic, complete cervical6‐7 cord transection.

Support or Funding Information

R01HL122223
Identification of Inflammatory Biomarkers for Hypertension from Isolated Human Monocytes
Authors: Norlander Allison Elizabeth , Galindo Cristi L , Elijovich Fernando , Laffer Cheryl L , Montaniel Kim Ramil Contreras , Gnecco Juan S , Madhur Meena S
Published: November 2023

Individuals on traditional hypertensive treatments are still more likely to develop cardiovascular and kidney complications, including occurrences of myocardial infarction and chronic kidney disease, than normotensive individuals. In recent years, our laboratory has demonstrated a role for inflammation, while specifically characterizing the contribution of monocytes and dendritic cells, in the development of hypertension. We embarked on the current study in attempts to determine, through an unbiased approach, if individuals on conventional hypertensive therapies have altered monocytic gene expression compared to normotensive humans that could help to explain the maintenance of increased cardiovascular risk. We used next generation RNA sequencing to profile monocytes isolated from the peripheral blood of 5 normotensive and 7 hypertensive subjects. We identified 60 transcripts that were significantly differentially expressed. Regression analysis was performed on these transcripts in order to correlate them with mean arterial pressure (MAP). After multivariate analyses, two genes, LTF (r=0.99, p<0.0001) and PGLYRP1 (r=0.99, p<0.005) significantly correlated with MAP in normotensive individuals. GZMH (r=0.88, p<0.01) significantly correlated with MAP in hypertensive individuals. While, IL18RAP significantly correlated with MAP in normotensive (r=0.93, p<0.03) and hypertensive (r=0.78, p<0.04) humans. Finally, we examined transcript expression of the four genes identified in the regression analyses in combination with several other candidate genes identified as significantly differentially expressed in the original analysis using quantitative RT‐PCR in a separate cohort of 6 normotensive and 9 hypertensive subjects. We were able to validate 6 out of 8 transcripts tested; expression of LTF (3.13 fold, p=0.0128), MMP8 (3.78 fold, p=0.044), ARG1 (1.39 fold, p=0.0248), PGLYRP1 (4.62 fold, p=0.0332), and IL18RAP (2.1 fold, p=0.038) all significantly increased in hypertensives while expression of VSIG4 (1.7 fold, p=0.0248) significantly decreased in hypertensives. These data demonstrate that monocytes from treated hypertensive patients exhibit a pro‐inflammatory gene signature which could potentially serve as biomarkers for disease severity and may explain the residual cardiovascular risk in these individuals.

Support or Funding Information

NIH T32 Training Grant; F31 Predoctoral Individual National Research Service Award; AHA Strategically Focused Prevention Research Network Grant; NIH NHLBI K08 Award
Hydrogen sulfide controls CBS‐CTH feedback regulation by inducing miR‐133a and suppressing SP1 in a dose dependent manner in cardiomyocytes
Authors: Nandi Shyam Sundar , Mishra Paras Kumar
Published: November 2023

Previously we reported that hydrogen sulfide (H2S) donor can mitigate hyperhomocysteinemia (HHcy)‐mediated cardiomyocyte hypertrophy by upregulating miR‐133a. However, the dose dependent effect of Hcy and H2S on miR‐133a level, the effect of homocysteine metabolizing enzyme cystathionine‐β‐synthase (CBS) on cystathionine‐ϒ‐lyase (CTH) (a precursor enzyme for H2S biosynthesis), and the role of H2S in CBS and CTH regulation in cardiomyocytes, are poorly understood. We hypothesized that H2S and homocysteine (Hcy) influence CBS and CTH expression in a dose dependent manner, and H2S controls CBS‐CTH feedback regulation by inducing miR‐133a and suppressing SP1, a transcriptional activator of CTH promoter, in cardiomyocytes. We treated HL1 cardiomyocytes with 0, 5, 25, 50, 75 and 100 μmL−1 doses of Hcy or H2S donor Na2S for 24 hours. Our results demonstrated that 50 μmL−1 Na2S upregulates miR‐133a and downregulates CTH, whereas 25 μmL−1 Hcy downregulates miR‐133a and upregulates CTH. To determine the in vivo effect of CBS on CTH, we measured the cardiac levels of CBS and CTH in CBS+/− mice, a model for HHcy. We found that in CBS+/− hearts, CBS is downregulated but CTH is upregulated, suggesting a negative feedback regulation between CBS and CTH. To assess the effect of H2S on CTH, we measured the levels of SP1 in Na2S‐treated cardiomyocytes. Our results revealed that Na2S treatment suppresses SP1 in a dose dependent manner, suggesting that H2S may downregulate CTH by inhibiting SP1. Moreover, our luciferase reporter assay on CTH 3′UTR demonstrated that miR‐133a targets CTH. Since H2S upregulates miR‐133a, it could be an indirect mechanism for H2S‐mediated regulation of CTH. In conclusion, H2S and Hcy regulate CBS and CTH in a dose dependent manner. CBS negatively regulates CTH. H2S upregulates CBS but downregulates CTH, possibly by suppressing SP1 and inducing miR‐133a in cardiomyocytes.

Support or Funding Information

American Heart Association Postdoctoral fellowship award 16POST30180003 and National Institute of Health grants HL‐113281 and HL‐116205
The Role of Tumor Necrosis Factor Receptor 2 (TNFR2) in a Mouse Model of Valvular Inflammation and Fibrosis
Authors: González‐Torres Mayra I. , Meier Lee , Auger Jennifer , Binstadt Bryce
Published: November 2023

Fibrosis is increased production of extracellular matrix that results from chronic inflammation. Acquired cardiovascular diseases are the most significant source of morbidity and mortality worldwide and virtually all exhibit underlying fibrosis. The K/B.g7 mouse develops mitral valve (MV) inflammation and fibrosis with complete penetrance, spontaneous onset, and with many features that mirror human disease, and thus it provides a powerful tool for studying the effects of chronic inflammation on the cardiovascular system. We have found that fibrosis in this model is dominated by recruitment of circulating inflammatory monocytes rather than the local self‐renewal of resident macrophages that has been observed in other systems. Previous results lend support to a TNF/IL6‐VCAM1‐VLA4 axis mediating inflammatory cell recruitment and subsequent fibrogenesis in the K/B.g7 system. We next sought to determine the role for tumor necrosis factor receptor 1 (TNFR1) vs. TNFR2 in mediating endothelial activation in this pathway. Based on recent studies highlighting a role for TNFR2 in inflammatory monocyte recruitment, we hypothesized a similar role for TNFR2 in this system. We performed inhibition studies using anti‐TNFR2 monoclonal antibody injected biweekly beginning at the onset of valve disease. After 4 weeks of treatment, hearts were collected and frozen in liquid nitrogen. Coronal sections at 10 mm were stained with hematoxylin and eosin. MV thicknesses were measured using ImageJ and compared to IgG control treated animals. Histological analysis showed that TNFR2 inhibition did not reduce K/B.g7 mitral valve thickening and fibrosis. Rather, TNFR2 blockade resulted in a trend toward increased fibrosis and valve thickening. Due to TNFR2 expression in T regulatory cells (Treg), decreased Treg activity is one potential explanation for the trend toward increased fibrosis. Ongoing studies are investigating the roles of TNFR1 using bone marrow chimeras. These studies will enhance our understanding of cardiovascular fibrosis and could inform potential therapeutic strategies that target the inflammatory cell recruitment process in human fibrotic diseases.
The impact of severe burn injury on cardiac mitochondrial function
Authors: Guillory Ashley Nicole , Clayton Robert Patrick , Prasai Anesh , El Ayadi Amina , Bhattarai Nisha , Herndon David N , Porter Craig , Finnerty Celeste C
Published: November 2023

Hypermetabolism, hypercatabolism, inflammation, and muscle wasting are all key characteristics of the pathophysiological response to severe burn injury. Severe burn injury also induces an exaggerated and prolonged release of catecholamines. These catecholamines include norepinephrine and epinephrine, which activate β‐adrenergic receptors (β‐ARs). The over‐activation of β‐ARs leads to increased resting heart rate and cardiac work, which is associated with morbidity and increased risk of mortality. We have previously shown that severe burn injury causes mitochondrial dysfunction in skeletal muscle, which persists for more than a year after injury. We hypothesized that cardiac mitochondrial function would also be altered by burn injury.

We tested this hypothesis using a rat model of 60% of total body surface area scald burn. Hearts were excised at three days and seven days post‐burn. Mitochondrial respiration was determined in saponin‐permeabilized myofiber bundles. Statistical analysis was performed by one‐way ANOVA followed by Tukey's post‐hoc test. Significance was accepted at p<0.05.

Respiration coupled to ATP production was significantly decreased at 3 days post‐burn compared to both the control and 7 days post‐burn groups (control: 194±35 pmol/s/mg; 3 days: 104±53 pmol/s/mg; 7 days: 164±40 pmol/s/mg; p=0.003). The respiratory control ratio for ADP was also significantly decreased 3 days post‐burn but was comparable to controls by 7 days post‐burn (control: 2.79±0.50; 3 days: 1.76±0.57; 7 days: 2.46±0.37; p=0.004). However, the flux control ratio was significantly higher 3 days post‐burn compared to the other groups (control: 0.85±0.06; 3 days: 1.01±0.15; 7days: 0.84±0.05; p=0.009).

Our data verifies that severe burn injury has a significant impact on cardiac mitochondrial function. Respiratory control and thus capacity for ATP production is lost at 3 days post burn. Interestingly, these effects appear temporal.

Support or Funding Information

This study was supported by grants from the National Institutes of Health (P50‐GM60338, R01GM056687, R01GM112936), Shriners Hospitals for Children (80100, 84291, 80500, 84202, 84090, 85410,87300), and the ‘Remembering the 15’ Research Education Endowment Fund.
Identity and Function of a Cardiac Mitochondrial Small Conductance Ca2+‐Activated K+ Channel Splice variant
Authors: Yang Meiying , Camara Amadou K.S. , Kwok Wai‐Meng , Aldakkak Mohammed , Stowe David F
Published: November 2023

Small conductance Ca2+‐activated K+ channels (SKCa) have three isoforms, SK1, 2 and 3, with small unit conductance of 3–30 pS; Ca2+ binding to calmodulin initiates opening of SKCa channels. SKCa channel activator DCEBIO protects against ischemia and reperfusion (IR) injury in isolated hearts but protection is abolished by reactive O2 species scavenger MnTBAP, indicating SKCa channels play an important role in mitochondrial function during IR injury. Here we provide further evidence by showing that: SKCa channel opening via i.v. infusion in intact rats reduces regional infarct size induced by coronary artery occlusion and reperfusion; agonists of SKCa channels improve mitochondrial respiration after IR injury; and CaCl2 applied to isolated mitochondria (m) increases K+ influx. Moreover, we confirm the presence of mSKCa isoform 3 (mSK3) in heart mitochondria of guinea pig, rat and human using Western blotting of inner mitochondrial membrane proteins, immunocytochemical staining of cardiomyocytes, and immunogold labeling of isolated mitochondria. We identify mSK3 splice variants in guinea pig (SK3.1, aka SK3a) and human ventricular cells (SK3.2). Overexpression of full length, N‐terminal truncated and C‐terminal truncated guinea pig SK3.1 in HL‐1 cells demonstrated that N‐terminus is not required for mitochondrial trafficking but the C‐terminus beyond the Ca2+ calmodulin binding domain is required for Ca2+ sensing to induce mK+ influx and to promote mitochondrial localization. Silencing of SK3.1 with siRNA in HL‐1 cells induced a greater fall in membrane potential (DYm), and enhanced cell death with simulated IR injury. Together, these molecular and physiological experiments support the presence of, and a prominent role for, SKCa channel splice variants in mitochondria that afford cardiac protection against oxidative stress injury.

Support or Funding Information

This research study was supported in part by the Veterans Administration (BX‐002539‐01) and the National Institutes of Health (R01 HL089514 and P01‐GM066730).
Stress‐induced Nitration of the Voltage‐Dependent Anion Channel and its Dissociation from Hexokinase and Adenine Nucleotide Translocase: Implications in Cardiac Injury
Authors: Yang Meiying , Camara Amadou KS , Kwok Wai‐Meng , Stowe David F
Published: November 2023

The voltage‐dependent anion channel (VDAC1) is the main conduit for transport of metabolites, substrates, and ions across the outer membrane of mitochondria (OMM). It is also a receptor for pro‐ and anti‐apoptotic proteins. Thus, as a channel and a receptor, VDAC1 plays key roles in modulating mitochondrial function and determining cell fate. We have previously reported on the nitration of VDAC1 following cardiac ischemia‐reperfusion injury. In the current study, we identified and investigated the impact of specific stress‐induced VDAC1 nitrated tyrosine residues on cell survival, and the consequence of stress on VDAC1 association with hexokinase II (HKII), an anti‐apoptotic protein, and with the adenine nucleotide translocase (ANT1) on the inner mitochondrial membrane. Proteomic analysis by LC‐MS/MS using an LQT Orbitrap Velos analyzer identified tyrosine nitration of Y62, Y67 and Y225 on VDAC1 enriched from mitochondria that were isolated from guinea pig hearts subjected to 35 min ischemia + 20 min reperfusion (IR). Transient overexpression of VDAC1 mutants (where tyrosine was substituted by phenylalanine to prevent nitration) in HL‐1 cells revealed that Y225F did not protect against hypoxia‐reoxygenation as measured by LDH release. In contrast, a double mutant of Y62F+Y67F significantly reduced LDH release, indicative of cytoprotection against hypoxia‐reoxygenation. Furthermore, mitochondria isolated from hearts exposed to the IR protocol showed reduced association of VDAC1 and ANT1, as determined by immuno‐precipitation. In another set of experiments, isolated mitochondria that were exposed to peroxynitrite (ONOO−) resulted in decreased association of VDAC1 with HKII. ONOO− treatment also led to an increase in cytochrome c release, which was attenuated by DIDS, a putative VDAC blocker. Our results show that nitration of specific tyrosine residues leads to cell death. Furthermore, stress induced dissociation of VDAC1 from ANT1 can potentially result in the derangement of metabolite transport and the inability of mitochondria to efficiently deliver ATP under pathophysiological conditions. Dissociation of HKII from VDAC1 would also promote cell death. These events are also associated with an increase in OMM permeability via VDAC1. Thus, stress‐induced nitration of VDAC1 likely impairs metabolite transport and promotes dissociation of proteins from VDAC1 that are key to cell function and survival.

Support or Funding Information

This study was supported in part by the Veterans Administration (BX‐002539‐01) and the National Institutes of Health (R01 HL089514 and P01‐GM066730).
Testing the Potential for Nitric Oxide Treatment to Prevent Disuse Atrophy in Muscle During Suspension
Authors: Anderson Judy E , Zhu Antonia , Craig Paige , Letourneau Lucien
Published: November 2023

Disuse atrophy in muscle is progressively debilitating in obesity and exacerbates age‐related atrophy. Effective treatment is challenging in both conditions, and prevention is the best approach to avoiding progressive weakness. Since a nitric‐oxide (NO) stimulus activates muscle stem cells, we tested the hypothesis that treatment with a NO‐donor drug, isosorbide dinitrate (ISDN), during hind limb suspension for 18 days, can alleviate muscle atrophy (UM protocol F11‐029). Muscles in unloaded lower and weight‐bearing upper limbs were weighed to study muscle loss. Western blots were used to measure protein levels in experimental (suspended, ISDN‐suspended) and age‐matched control groups of young (3wk) and young‐adult (6–8wk) mice. Protein levels of atrogin‐1, myostatin, and myosin heavy chain isoforms (MyHCs) were analyzed (relative to beta‐actin) for changes expected with muscle atrophy. Analysis of variance was used to determine if changes were statistically significant. Unloaded muscles (quadriceps and soleus) in suspended mice showed atrophy compared to control. In ISDN‐suspended mice, the weight of these 2 muscles tended to increase toward control. In the unloaded quadriceps of suspended mice, ISDN significantly decreased atrogin‐1, a negative regulator of muscle mass (p<0.05) and myostatin precursor, an inhibitor of myogenesis (p<0.05). ISDN also increased the levels of MyHC‐1 and MyHC‐2b in unloaded muscles of suspended young‐adult mice compared to the untreated suspended group. There were no changes in vascular density in quadriceps or pectoralis major as a result of suspension or ISDN treatment, although density was higher in the 6–8 wk than the 3wk group (p<0.05). Suspension resulted in fatty‐liver changes in the 6–8wk group, and ISDN treatment significantly attenuated that increase (p<0.05). Results suggest that NO treatment has the potential to attenuate muscle‐disuse atrophy, with possible significance to preventing atrophy during aging or space travel.

Support or Funding Information

Supported by the University of Manitoba Research Grant Program (JEA) and NSERC Undergraduate Student Research Award (AZ)
Regulated in Development and DNA Damage 1 (REDD1) Alters the Expression of a Distinct Set of Genes in Skeletal Muscle by Activity/Inactivity
Authors: Gordon Bradley Scott , Steiner Jennifer Lynn , Williamson David , Coen Paul , Rossetti Michael , Shyamalagovindarajan Subramaniam , Eroshkin Alexey
Published: November 2023

Regulated in Development and DNA Damage 1 (REDD1) is a stress induced protein implicated in a variety of physiological and pathological conditions. While the most highly characterized role of REDD1 is its regulation of signaling through the mechanistic target of rapamycin in complex 1, recent work indicates that REDD1 may also act to modulate gene expression patterns in skeletal muscle. Interestingly, REDD1 expression is highly induced in skeletal muscle following a single bout of aerobic exercise; a stimulus known to alter gene expression. To shed further light on the consequence of REDD1 expression by activity/inactivity, REDD1+/+ and REDD1−/− mice were randomized to remain sedentary or subjected to a single bout of aerobic treadmill exercise. Mice subjected to exercise were allowed to recover for 1, 3, or 6 hr. In the plantaris of REDD1+/+ mice, REDD1 protein expression increased 907% and 201% at 1 hr and 3 hr post exercise, respectively, before returning to near baseline levels 6 hrs post exercise. As REDD1 induction was greatest 1 hr post exercise, total RNA of the plantaris from sedentary and 1 hr post exercise REDD1+/+ and REDD1−/− mice was subjected to microarray analysis. Comparison of the gene expression patterns in the muscle of sedentary mice identified 201 genes that were differentially expressed by disruption of REDD1. Specifically, qRT‐PCR analysis confirmed that the gene expression of Park2 and Ucp1, genes implicated in mitophagy and oxidative phosphorylation, were reduced by 50% and 84%, respectively, by the disruption of REDD1. Interestingly, following a bout of exercise, the microarray detected REDD1 as the gene with the greatest exercise‐induced magnitude of change in REDD1+/+ mice. Further analysis of genes altered by the absence of REDD1 in the plantaris of exercised mice revealed significant changes in 23 genes with functions related to muscle endurance, inflammation, and muscle metabolism. Subsequent qRT‐PCR analysis confirmed that the gene expression of Malt1 and Orm1 were decreased by 47% and 53%, respectively, while the gene expression of Arrdc3 was enhanced by 93% by disruption of REDD1 1 hr post exercise. Despite changes in these genes, qRT‐PCR confirmed that the induction of Peroxisome Proliferator Activated Receptor Gamma Coactivator 1 alpha (PGC‐1α), a well‐known gene induced by exercise, was unaffected by the loss of REDD1. In all, these data support a role for REDD1 in the regulation of a distinct set of genes by activity/inactivity.

Support or Funding Information

NIH grant F32 AA023422 (JLS)
Optogenetic Activation of AgRP Neurons Lengthens and Deepens Daily Torpor in the Mouse
Authors: Vicent Maria A , Mook Conor L , Carter Matthew L , Swoap Steven John
Published: November 2023

In response to food scarcity and low ambient temperatures, mice enter bouts of torpor resulting in energy conservation. During a torpor bout, mice exhibit a substantial decrease in body temperature (Tb), heart rate (HR), and metabolic rate. The neurobiology of torpor is largely unknown. It has been shown that peripheral administration of ghrelin, a stomach‐derived hormone released during energy deprivation, to calorically restricted mice deepens and lengthens torpor bouts. It is thought that ghrelin induces its physiological effects by activating Agouti‐related protein (AgRP) containing neurons in the arcuate nucleus of the hypothalamus. Therefore, we hypothesized that direct stimulation of AgRP neurons would decrease the minimum Tb of torpid mice and increase the time spent in torpor. To test this hypothesis, mice were implanted with ECG/Tb telemeters and hypothalamic AgRP neurons were selectively targeted with the light‐sensitive channelrhodopsin‐2 transgene. Mice were calorically restricted daily (65% of normal caloric intake) until regular daily torpor bouts were achieved. On baseline days, when AgRP neurons were not activated, minimum Tb was 25.6 ± 0.8 °C and time in torpor was 233 ± 34 minutes. When AgRP neurons were stimulated for one hour during entry into torpor (20 Hz for 1 second every 4 seconds for 60 minutes), minimum Tb was significantly lower (22.8 ± 0.3 °C) and torpor bouts were significantly longer (435 ± 29 min). These data support the hypothesis that AgRP neurons directly regulate torpor physiology and begin to elucidate the neural circuits responsible for torpor regulation.

Support or Funding Information

Supported by 1R15HL120072‐01A1 to SJS and by 1R15DK105510 to MC
Heat therapy alters the expression of myogenic and angiogenic factors and accelerates functional recovery following exercise‐induced muscle damage in humans
Authors: Kim Kyoungrae , Nie Yaohui , Boersma Doug , Song Qifan , Kuang Shihuan , Gavin Timothy P. , Roseguini Bruno T.
Published: November 2023

Introduction

Exposure to a single session of heat therapy (HT) promotes the mRNA expression of pivotal angiogenic and inflammatory mediators in human skeletal muscle. We investigated the effects of repeated HT on functional recovery and the expression of select genes involved in myogenesis, angiogenesis and inflammation following exercise‐induced muscle damage (EIMD).

Methods

Eleven healthy young adults (23.8±0.6yrs, BMI 23.7±0.6) performed 300 maximal eccentric contractions of the knee extensors with both legs. One randomly selected thigh was treated with HT immediately after the exercise bout and 4 additional consecutive days while the opposite thigh served as a control. A water‐circulating garment perfused with water at ~52°C was used to apply HT for 90 min. Thermoneutral water (33°C) was circulated through the garment in the control thigh. Biopsies from the vastus lateralis muscle were obtained 2 weeks prior to and 1 and 5 days after the eccentric exercise bout. The mRNA expression of myogenic, angiogenic and inflammatory factors was determined using real‐time polymerase chain reaction. Perceived pain (visual analog scale) and fatigue resistance (total work amount during 28 repetition maximums at 180°/s) were assessed before, during and after the intervention.

Results

A similar reduction in fatigue resistance (Control: −27.1±3.7% vs. HT: −21.4±4.6%, p>0.05) and an increase in perceived muscle pain (Control: 4.3±0.7 vs. HT: 4.2±0.7, p>0.05) were observed in both legs 1 day after the exercise bout. However, the thigh treated with HT had greater fatigue resistance than the control thigh (Control: −15.9±2.8% vs. HT: −9.6±1.9%, p=0.04) 4 days after the EIMD protocol. Perceived muscle pain was also lower in the thigh exposed to HT at 4 (Control: 2.9±0.6 vs. HT: 2.3±0.4, p=0.02) and 5 days (Control: 1.8±0.4 vs. HT: 1.3±0.3, p=0.02) after the exercise bout. When compared to the control thigh, the change in mRNA expression from baseline of paired box 7 (Control: 0.9±0.1 vs. HT: 1.3±0.1, p=0.04), vascular endothelial growth factor (Control: 0.8±0.1 vs. HT: 1.2±0.1, p<0.01), and angiopoietin 1 (Control: 0.3±0.1 vs. HT: 0.5±0.1, p=0.02) was higher in the thigh exposed to HT 1 day after the exercise bout. After 5 consecutive days of HT, the change in mRNA expression from baseline of monocyte chemoattractant protein 1 (Control: 1.3±0.3 vs. HT: 0.5±0.1, p<0.01) and myostatin (Control: 0.6±0.1 vs. HT: 0.3±0.1, p<0.01) were significantly lower in the thigh treated with HT.

Conclusion

Taken together, these results suggest that application of HT accelerates functional recovery following EIMD in humans, possibly by enhancing the expression angiogenic and myogenic factors early in the repair process.
Alterations of Mitochondrial Function and Metabolism in Skeletal Muscle, Liver, and White Adipose: Early Evidence for the Lactation Reset Hypothesis
Authors: Hyatt Hayden W , Zhang Yufeng , Hood Wendy R , Kavazis Andreas N
Published: November 2023

Lactation following reproduction serves as one of the largest metabolic challenges a female experiences in their lifetime. As such, the lactation reset hypothesis has been proposed to explain changes in metabolism that confers protective health benefits (e.g., fewer visceral adipocytes, lower fasting glucose, increased insulin sensitivity, etc.) in mothers that breast‐feed their children.

PURPOSE

The aim of the current experiment was to discern mitochondrial function differences and changes in markers of metabolism in female rats that nurse their pups to weaning compared to rats that reproduce but do not nurse their pups or do not reproduce.

METHODS

Female Sprague‐Dawley rats were assigned to one of three groups: 1) gave birth and nursed their pups for 21 days and were sacrificed seven days later (PL), 2) age‐matched that gave birth but had pups removed and thus did not nurse (P), and 3) age‐matched that did not reproduce (NR). Liver and skeletal muscle were dissected immediately following sacrifice and mitochondria were isolated using differential centrifugation for measurements of respiration utilizing high resolution respirometry. Liver, skeletal muscle, and white adipose tissue were stored for western blot analysis.

RESULTS

Liver mass was higher in PL compared to NR (p<0.01) and P (p=0.03). PL and P exhibited higher state 3 respiration in liver mitochondria when utilizing complex I substrates (i.e., pyruvate and malate) compared to NR (p=0.03 and p=0.05, respectively). Noteworthy, PL demonstrated lower respiratory control ratio in liver mitochondria when utilizing complex II substrates compared to NR (p=0.03) and P (p=0.02). No differences in skeletal muscle mitochondria respiration were detected (p>0.05). Protein levels of superoxide dismutase 2 (SOD2) in the liver were higher in PL compared to NR (p=0.01) and P (p=0.05). Peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PGC‐1a) protein was higher in liver of PL compared to NR (p<0.01) and higher in P compared to NR (p=0.05). Catalase (CAT) protein levels in liver were higher in P (p=0.04) and PL (p=0.05) compared to NR. There were no differences in superoxide dismutase 1 (SOD1), glutathione peroxidase (GPX), and peroxisome proliferator activated receptor delta (PPAR‐δ) protein expression (p>0.05) in liver. WAT protein expression of PPAR‐d was higher in PL animals compared to NR (p<0.01) and P (p=0.05). No differences (p>0.05) were observed in WAT protein expression for GPX, CAT, SOD1, SOD2, PGC‐1a.

CONCLUSION

The taxing metabolic event of lactation produces systemic alterations in metabolism and liver mitochondrial function that persist a week after the cessation of nursing in rats, which may, in part, explain the beneficial effects of breast‐feeding.

Support or Funding Information

Funded by NIH R03HD083654
Cardiac baroreflex sensitivity during lower body negative pressure and acute hypoxia: fainters vs. nonfainters
Authors: Aarts Hugo M. , Petersen‐Jones Humphrey G. , Johnson Blair D. , Curry Timothy B. , Joyner Michael J.
Published: November 2023

Background

Hypoxia increases susceptibility to syncope during orthostatic stress. Furthermore, cardiac baroreflex sensitivity (cBRS) is attenuated during lower body negative pressure (LBNP). Recent studies suggest that there is cross‐talk between baroreceptors and carotid body chemoreceptors. With this in mind, hypoxia may further blunt cardiac baroreflex sensitivity (cBRS) during central hypovolemia in individuals susceptible to hypoxic syncope.

Therefore, we hypothesized that participants susceptible to hypoxic syncope (“fainters”) during progressive central hypovolemia would have lower spontaneous cBRS during LBNP when compared to “nonfainters”.

Methods

Ten healthy male participants (35 ± 3 yr, BMI 25 ± 1 kg/m2) were exposed to stepwise lower body negative pressure (LBNP, 5 minutes at 0, −15, −30, −45 mmHg) during both normoxia and hypoxia (FiO2 = 0.12 – 0.15; SpO2 ~85%). LBNP protocols were terminated early if participants developed presyncopal symptoms. Data from uncompleted stages were excluded. Heart rate (HR, 3 lead electrocardiogram) and mean arterial blood pressure (MAP, brachial arterial catheter) were continuously recorded. Spectral cBRS was calculated from the heart rate and blood pressure signals. Following experimentation, participants were stratified as fainters (developed presyncopal symptoms during hypoxic conditions, n=7) and nonfainters (n=3).

Results

MAP was lower in fainters than in nonfainters during hypoxia (interaction with LBNP p<0.05) while HR (p=0.38) and cBRS (p=0.43) were similar – Table 1. Hypoxia had a main effect vs normoxia on MAP, HR and cBRS in fainters (all p<0.02). In nonfainters hypoxia had a main effect vs normoxia on HR and cBRS (all p<0.03) and an interaction effect with LBNP on MAP (p<0.01).

Conclusion

Spontaneous cardiac baroreflex sensitivity is not attenuated in participants susceptible to hypoxic syncope during lower body negative pressure. This suggests that spontaneous cardiac baroreflex sensitivity does not contribute to orthostatic intolerance during hypoxia.

Support or Funding Information

Funding: W81XWH‐13‐2‐0038

Physiological response in fainters and nonfainters to LBNP during hypoxia and normoxia

MAP, mmHg
HR, beats·min−1
cBRS, ms·mmHg−1

Nonfainters
Fainters
Nonfainters
Fainters
Nonfainters
Fainters

Baseline

Normoxia
104 ± 5
102 ± 3
62 ± 5
61 ± 3
13 ± 3
19 ± 2

Hypoxia
106 ± 6
97 ± 4
80 ± 6
72 ± 4
9 ± 3
11 ± 2

LBNP −15 mmHg

Normoxia
109 ± 5
103 ± 3
68 ± 5
64 ± 3
11 ± 3
14 ± 2

Hypoxia
106 ± 6
90 ± 4
88 ± 6
72 ± 4
9 ± 3
14 ± 2

LBNP −30 mmHg

Normoxia
107 ± 5
98 ± 3
79 ± 5
74 ± 3
8 ± 3
11 ± 2

Hypoxia
103 ± 6†
82 ± 5*
95 ± 6
79 ± 5
8 ± 3
9 ± 3

LBNP −45 mmHg

Normoxia
101 ± 5
99 ± 3
92 ± 5
84 ± 4
6 ± 2
5 ± 2

Hypoxia
89 ± 6†
N/A
113 ± 6
N/A
3 ± 3
N/A

Different vs. nonfainters p<0.05

Different vs. normoxia p<0.05