bluuebunny/crossref_metadata_2025 huggingface dataset:
Heart Rate and Heart Rate Variability during the Cold Pressor Test in Recently Concussed Patients
Authors: O'Leary Morgan C , Sackett James R , Schlader Zachary J , Leddy John J , Johnson Blair D
Published: November 2023

Introduction

Autonomic function appears to be altered in concussed patients, especially when a physiological stressor is present. Heart rate variability (HRV) during whole body exercise is attenuated in concussed patients compared to healthy controls. However, HRV during isometric handgrip exercise has been shown to have a similar response between concussed patients and healthy controls. The cold pressor test (CPT) is an exercise‐independent test that could be used to identify shifts in autonomic function in concussed patients. The root mean squared of successive differences (RMSSD) is a metric of HRV that can be used during the CPT to assess the degree of parasympathetic control of heart rate.

Purpose

We tested the hypothesis that heart rate and RMSSD during the CPT in concussed patients is impaired versus healthy controls.

Methods

Four concussed patients who were symptomatic and within 7 days of injury (age: 19 ± 1 years, 2 men) and four healthy controls (age: 27 ± 4 years, 4 men) completed a CPT. The CPT consisted of submerging a hand up to the wrist in an agitated ice/water slurry mixture (~1°C) for 120 seconds. We continuously measured heart rate (ECG) throughout the CPT. Heart rate and RMSSD were analyzed in 60‐second increments at baseline, 60 seconds and 120 seconds during the CPT, and 60 seconds post CPT.

Results

Heart rate in the concussed patients did not change from baseline (67 ± 17 bpm) to 60 seconds (73 ± 20 bpm, P = 0.192), 120 seconds (74 ± 16 bpm, P = 0.187), or post CPT (70 ± 15 bpm, P = 0.412). However, heart rate in the healthy controls increased from baseline (59 ± 8 bpm) to 60 seconds (78 ± 8 bpm, P < 0.001) and 120 seconds (68 ± 10 bpm, P = 0.046) of the CPT and returned to baseline post CPT (55 ± 2 bpm, P = 0.300). RMSSD in the concussed patients did not change from baseline (87 ± 74 ms) to 60 seconds (86 ± 114 ms, P = 0.979), 120 seconds (91 ± 127 ms, P = 0.979), or post CPT (100 ± 134 ms, P = 0.902). RMSSD in the healthy controls also did not change from baseline (71 ± 26 ms) to 60 seconds (48 ± 23 ms, P = 0.325), 120 seconds (41 ± 11 ms, P = 0. 325), or post CPT (104 ± 36 ms, P = 0. 325).

Conclusions

These preliminary data indicate that parasympathetic control of heart rate, represented by RMSSD, is not altered during the CPT in recently concussed patients or healthy controls. However, the blunted heart rate response during the CPT does indicate that cardiac autonomic control is impaired in recently concussed patients.
Local Delivery of β‐Aminopropionitrile Improves Arteriovenous Fistula Maturation and Remodeling
Authors: Hernandez Diana Rosa , Wei Yuntao , Andreopoulos Fotios , Vazquez‐Padron Roberto
Published: November 2023

Introduction

Arteriovenous fistula (AVF) is the preferred vascular access to provide hemodialysis in patients with end stage renal disease (ESRD). The failure of AVF due to improper remodeling is a serious condition that affects not only its prevalence but also the possibility of achieving long‐term patency. β‐aminopropionitrile (BAPN) is an irreversible inhibitor of lysyl oxidase (LOX), the main enzyme responsible for collagen crosslinking. Herein, we hypothesize that inhibition of LOX by local delivery of BAPN improves biomechanical performance in experimental AVF and prevents excessive crosslinking of collagen and other extracellular matrix proteins.

Methods

Poly Lactic‐co‐Glycolic Acid (PLGA) is an FDA‐approved biodegradable highly biocompatible polymer frequently used on deliveries systems. The scaffolds were grown by electrospinning PLGA/BAPN (15:1.5) in hexafluoroisopropanol at 20 keV and 13 cm away from the target with a flow of 2 mL/h. In vitro degradation as well as BAPN release profile was studied during 60 days at 37°C. BAPN concentration was assessed with a Triversa NanoMate® electrospray ionization with direct infusion through triple quadrupole LC‐MS. Two groups of Sprague Dawley rats of both sex received local delivery of scaffold containing BAPN (0.27 mg) or vehicle around the venous limb of the newly created AVF. The rat AVF was created by surgically anastomosing the left epigastric vein to the nearby femoral artery. Pressure myograph (110 P; Danish Myo Technology, Aarhus, Denmark) was used for assessment of the AVF mechanical properties.

Results

In‐vitro release assessment showed that the drug inclusion in the scaffolds affects degradation rate with a maximum difference of 70% at 40 days. LC‐MS showed an excellent sensitivity for the quantification of BAPN with a lower limit of quantification of 30 ng/mL. Both treated and control AVF increased its size more than double that of the contralateral epigastric vein (p<0.05). However, local delivery of BAPN increased lumen volume in treated AVF with respect to control vessels (p=0.0078), Figure 1.

Conclusion

Our results demonstrate that inhibition of collagen cross‐linking by BAPN significantly improved vascular compliance and biomechanical properties of AVF.

Support or Funding Information

This research it is support as a Research Supplements to Promote Diversity in Health under parent award R01DK098511.
Effect of an angiotensin II type 2 receptor agonist on myocardial hypertrophy and fibrosis induced by high salt diet
Authors: Dopona Ellen Priscila Brito , Rocha Veronica Favoni , de Oliveira Ivone Braga , Furukawa Luzia Naoko Shinohara , Heimann Joel Claudio
Published: November 2023

Objective

To evaluated the role of compound 21 (C21), an AT2 receptor agonist, in a model of left (LV) and right (RV) ventricular hypertrophy and fibrosis in response to dietary salt overload.

Methods

Male Wistar rats were fed normal (NS ‐ NaCl = 1,27%, n=15;) or high (HS ‐ NaCl = 8%; n=15) salt diet from weaning up to 18 weeks of age. From the 7th week of age, two subgroups (NS+C21; n=15 or HS+C21; n=13) received compound 21 (1mg/kg per day by gavage). Body weight (BW), tail‐cuf blood pressure (BP), cardiac mass to tibia length ratio (CM/TL), cardiomyocyte transverse diameter (TD), percentage of cardiac fibrosis (FI) were measured.

Results

(mean ± standard error):

At the 8th week of age, BW was lower in HS than in the other groups. BW in HS+C21 did not differ from NS from the 10th week to the 16th week of age. BP in HS+C21 was higher than in NS and lower that in HS from the 7th week to the 18th week of age.

Conclusion

In Wistars rats, compound 21 prevented both left and right ventricular hypetrophy and fibrosis and the increased blood pressure in response to high salt intake.

Support or Funding Information

Supported by FAPESP.

Parameter
Age (weeks)

NS
HS
HS+C21
NS+C21

Body Weigh (g)
3

57.9±2.9
55.6±3.5
60.9±2.99
77.4±1.35

18
571.1±11.2
497.8±12.7*
542.0±12.3
583.8±11.8

BP (mmHg)
6
112.3±3.0
114.0±4.2
111.9±3.4
108±7.9

18
115.6±2.5
143.5±1.2*
135.1±1.8*#
107.7±4.4

CM/TL (g/cm).
18
Total
0.53±0.04
0.51±0.03
0.52±0.05
0.51±0.02

RV
0.08±0.0
0.08±0.00
0.08±0.00
0.07±0.01

LV
0.33±0.02
0.32±0.02
0.35±0.03
0.34±0.01

TD (μm)
18
RV
9.82±0.21
13.80±0.29*
9.79±0.21
8.52±0.19

LV
10.17±0.2
15.60±0.9*
10.41±0.09
9.32±0.5

FI (% area)
18
RV
0.54±0.19
0.74±0.13*
0.43±0.11
0.20±0.06

LV
0.30±0.06
0.64±0.05*
0.45±0.04
0.27±0.06*

p<0.05 vs. NS;

p<0.001 vs. HS.
Regulation of Twist1 and Snail1 Gene Expression in Cardiac Fibroblasts by Scleraxis
Authors: Safi Hamza A , Al‐Hattab Danah S , Nagalingam Raghu S , Czubryt Michael Paul
Published: November 2023

Cardiac fibrosis causes progressive cardiac stiffening due to abnormal deposition of extracellular matrix (ECM) and alters electrical conduction of the action potential, resulting in heart failure and death. Cardiac fibroblasts largely arise from epithelial precursors during development via epithelial‐to‐mesenchymal transition (EMT), and upon injury or stress, are further converted to an activated state – myofibroblasts – which express a variety of pro‐fibrotic genes responsible for ECM synthesis and deposition. Our previous data suggested that the transcription factor scleraxis plays a key role in regulating cardiac fibroblast cell fate, including EMT and myofibroblast conversion. Here, we examined the role of scleraxis in transactivating expression of genes encoding key EMT regulators including the basic helix‐loop‐helix transcription factor Twist1 and the zinc finger transcription factor Snail1. Over‐expression of scleraxis in NIH3T3 fibroblasts up‐regulated Twist1 and Snail1 expression. Using luciferase reporter assays coupled with point mutagenesis, we found that scleraxis is sufficient to directly transactivate the Twist1 and Snail1 gene promoters, and identified the responsible scleraxis binding sites in each promoter. Loss of scleraxis conversely attenuated expression of these genes. Together, our data indicates that scleraxis controls EMT via direct upstream regulation of these key EMT regulatory genes.

Support or Funding Information

Supported by the Canadian Institutes of Health Research (MOP136862 to MPC), Research Manitoba (RSN) and the University of Manitoba GETS Program (HAS, DSA, RSN).
Correlation of Nucleosomes, MMP‐9, and IL‐1α with Relation to Neutrophil Activation in Patients with Atrial Fibrillation
Authors: Liles Jeffrey , Wanderling Christopher , Statz Stephen , Rowe Timothy , Bontekoe Jack , Jabati Sallu , Syed Mushabbar , Hoppensteadt Debra , Fareed Jawed
Published: November 2023

Introduction

Atrial fibrillation (AF) is currently the most common cardiac arrhythmia encountered in clinical practice and a major cause of morbidity and mortality among adults. It is estimated that atrial fibrillation affects approximately 2.7 million people in the United States. Extracellular nucleosomes in plasma (PNs) are complexes of DNA and histones that are released during cell death. Histones have been shown to activate neutrophils by functioning as DAMPs when they are translocated from the nucleus to the extranuclear space. These nucleosomes mediate inflammatory and thrombotic responses and could serve as biomarkers in AF. MMP‐9 degrades type IV collagen and acts as a chemotactic agent for neutrophilic migration. IL‐1α is an acute phase cytokine released by neutrophils that promotes inflammation.

Objective

To examine the relationships of circulating levels of PNs, MMP‐9 and IL‐1α in patients with AF.

Methods

In a prospective study, plasma samples were collected from 50 patients prior to ablation surgery for AF at Loyola University Medical Center. ELISAs kits were used as per kit instructions for PNs, MMP‐9 and IL‐1α.

Results

Circulating levels of PNs were positively correlated with levels of MMP‐9 (p=0.006, r=0.49) and IL‐1α (p=0.008, r=0.50) in patients with AF. Additionally, a positive correlation was seen between circulating levels of MMP‐9 and IL‐1α (p=0.02, r=0.41).

Discussion

Extracellular nucleosomes function as DAMPs by binding to receptors and triggering the activation of multiple signal pathways. Due to the involvement of inflammation and thrombosis in the pathology of atrial fibrillation, the positive correlation of nucleosome levels with the levels of MMP‐9 and IL‐1α in atrial fibrillation imply PNs may activate the immune system and generate the inflammatory response. These results may provide insight into a novel mechanism of thrombogenesis in patients with AF.
Efficacy of Tolerizing Vaccination Strategy for Mitigating Autoimmune‐Mediated Cardiac Injury
Authors: Lust Robert M. , Reece Shaun P. , Van Scott Michael R. , Mannie Mark D.
Published: November 2023

Background

Infectious agents that induce cross‐reactive myocarditic T cell responses may cause autoimmune inflammation of the heart, directly causing cardiac dysfunction, or they may produce an undetected autoimmune background that could manifest later as an exacerbating influence in the setting of future myocardial injury.

Purpose

This study was designed to evaluate the efficacy of GMCSF‐myosin fusion proteins as tolerogenic vaccines capable of inhibiting experimental autoimmune myocarditis (EAM).

Methods

The fusion proteins were comprised of an N‐terminal granulocyte macrophage‐colony stimulating factor (GM‐CSF) domain and a C‐terminal cardiac myosin antigen domain. The antigen domains Myosin 1052–1076 and myosin 614–643 were chosen based on epitopes known to cause EAM in Lewis rats and Balb/c mice respectively. Bioactivity of the cytokine domains for GMCSF‐Myo1052 and GMCSF‐Myo614 was confirmed with bone marrow proliferation assays. Enhanced antigen presentation was confirmed for GMCSF‐Myo1052.

Results

Both fusion proteins were found to inhibit the development of EAM when given as a pretreatment before EAM induction. Myo1052 fusion protein reduced the inducible incidence of EAM by nearly half, from 81.3% (13/16) to 46.7% (7/15), and the severity of EAM by 62%, from a histology severity score of 1.56 ± 1.41 to 0.60 ± 0.83 (mean ± SD, 5 point scale). Myo614 fusion protein reduced the inducible incidence of EAM by 67%, from 76.9% (13/16) to 25% (3/12), and the EAM severity by 85%, from a histology severity score of 2.23 ± 1.42 to 0.33 ± 0.65. Additionally GMCF‐Myo614 showed promise for inhibiting EAM when given after EAM induction. Serial assessment of myocardial performance was completed using echocardiography. Interestingly, despite the histological changes, cardiac function was preserved throughout. A transient increase in wall thickness was observed between days 14 and 17, but had resolved by day 21. Coupled with evidence of pericardial effusion, and in the context of the later histology findings, the change in wall thickness was interpreted to suggest building intramural myocardial edema over the second week that had resolved by the end of the third week. To explore the potential use of knockout mouse strain to identify mechanisms associated with fusion protein tolerizing efficacy, EAM was induced in C57BL/6 mice (the background stain for most knockout lines) using a peptide (myosin 718–736) derived from the murine cardiac myosin heavy chain and theorized to bind to IAd. Myosin 718–736 was capable of inducing EAM in Interferon Gamma receptor knockout mice on the C57BL/6 background. Additional studies revealed nitric oxide was not the primary effector molecule responsible for EAM resistance in C57BL/6 wild type mice.

These studies identify a novel potential means for reducing autoimmune mediated cardiac injury.
Inhibition of Allograft Inflammatory Factor‐1 Attenuates B Cell Development and Infiltration to Cardiac Tissue During the Development of Diabetic Cardiomyopathy
Authors: Sarkar Amrita , Shukla Sanket Kumar , Sikder Kunal , Tsygankov Alexander Y. , Rafiq Khadija
Published: November 2023

Objective

Diabetic cardiomyopathy (DCM) is typified by alterations in cardiac morphology and function, independent of hypertension or coronary disease. The disease is characterized by intramyocardial inflammation, cardiomyocytes apoptosis, and cardiac fibrosis, and it can lead to heart failure. The molecular mechanism that links inflammation to DCM is incompletely understood. This study investigates the novel role of allograft inflammatory factor‐1 (AIF‐1) gene expression over the humoral responses during the progression of DCM.

Methods and Results

Ten‐week‐old wild type male mice were injected with streptozotocin (50 mg/kg for 5 days intraperitoneally) and studied 8 weeks after induction of diabetes mellitus. Mice with elevated blood glucose levels (> 300 mg/dL) were classified as having diabetes mellitus and DCM was confirmed through ECG. Induction of diabetes resulted in an increase in inflammatory cells. This increase correlated with myocyte death and cardiac contractile dysfunction, indicating a possible role of immune cells in the development of heart failure. We therefore assessed which immune cells were involved in myocyte apoptosis. We found that STZ‐induced diabetes leads to an increase of neutrophil infiltration, which is associated with a decrease of B cell growth and function as well as an increased level of AIF‐1. STZ‐induced diabetes also leads to an increase of interleukin 21 (IL‐21), which is known to induce apoptosis in both resting and activated B cells, followed by a decrease of BCl2 and BCl‐xL expression. Our data strongly demonstrated that decreased AIF‐1 expression is associated with downregulation of B cells and that AIF‐1 mediates migration of B cell in vitro.

Conclusions

Downregulation of the B cell/AIF‐1 interaction leads to myocyte apoptosis and contributes to adverse cardiac remodeling after diabetes induction. These findings point to AIF‐1 as a potential therapeutic target for the maintenance of cardiac function and remodeling during the development of DCM.

Support or Funding Information

This work was funded by NIH grant for Dr. Khadija Rafiq.
Modulation of Heme Biosynthesis by Ferrochelatase Inhibition Controls Soluble Guanylate Cyclase Expression and Superoxide Production in Bovine Coronary Arteries
Authors: Alruwaili Norah , Sun Dong , Wolin Michael S
Published: November 2023

Nitric oxide interacts with the Fe2+ heme prosthetic group in soluble guanylate cyclase (sGC) stimulating the conversion of guanosine 5′‐triphosphate to cyclic guanosine 3′,5′‐monophosphate (cGMP). Adequate cGMP production is associated with protective mechanisms against multiple cardiovascular disease processes. Thus, disturbances in this signaling pathway can lead to many pathological consequences. Since there has been minimal investigation on the consequences of altering heme biosynthesis on the cardiovascular system, we investigated if mitochondrial heme biosynthesis is an important factor in controlling the expression and function of sGC and systems influencing superoxide generation and actions. For the purpose of this study, we induced a heme deficiency by inhibiting ferrochelatase (FECH) with N‐methyl protoporphyrin IX (NMPP), potent FECH inhibitor. Bovine coronary arteries (BCA) were treated for 24 hours with 10 μM NMPP. Chemiluminescence (5μM lucigenin) was used to measure superoxide production; western blot analysis was used to measure expression of FECH, sGC and other heme‐containing proteins; and a QuantiChrom heme assay kit was used to measure heme levels. NMPP treatment promoted sGC depletion and attenuation of its activity based on it decreasing protein kinase G‐mediated phosphorylation of VASP and attenuation of relaxation to a nitric oxide donor. NMPP also decreased the expression of heme proteins including cytochrome oxidase subunit 4 and catalase. In addition, superoxide production was significantly elevated in NMPP treated coronary arteries when compared to that in control samples, and scavenging extra mitochondrial superoxide with Tempol and mitochondrial matrix superoxide with MitoTempol attenuated the increase in superoxide. NMPP stimulated increases in superoxide were associated with a decrease expression of mitochondrial FECH expression. Thus, inhibition of FECH increased extra mitochondrial and mitochondrial superoxide and depleted sGC. Our studies suggest that disruption of heme biosynthesis resulting in a loss of cGMP production may serve as a contributing mechanism to the progression of cardiovascular disease.

Support or Funding Information

Funded by NIH grants R01HL115124 & R01HL129797
Repetitive ROS Inhibits Nrf2 Antioxidant Defense in Ischemic Heart Failure
Authors: Benipal Simranjeet Singh , Liu Tingting , Knowlton Anne
Published: November 2023

Nrf2, the master regulator for the antioxidant response, is critical for protecting the heart against reactive oxygen species (ROS). Normally, Nrf2 is bound to Kelch‐like ECH‐associated protein 1 (Keap1) and ubiquitinated in the cytosol. With increased oxidative stress, Nrf2 is released from Keap1, and moves into nucleus, binding to the antioxidant response element (ARE) in target gene promoters. This results in increased expression of antioxidant genes. Ischemic heart failure (IHF), a major cause of cardiovascular morbidity and mortality, is an inflammatory, chronic disease characterized by increased oxidative stress and progressive loss of ventricular function; however, we found Nrf2 expression was decreased by 50% in this disease. The interesting question is does Nrf2 inactivation with corresponding failure of antioxidant response contribute to the progression of IHF? We hypothesized that repetitive ROS exposure in IHF results in depression of Nrf2 activity, which as Nrf2 also regulates its own expression will in turn lead to decreased Nrf2. H2O2 is a major contributor to oxidative stress and damage thus is ideal to investigate the circumstances that occur in vivo due to ROS. Repetitive ROS treatments (modeling the chronic condition) reduced Nrf2 levels by 50% compared to a single treatment. In experiments with isolated rat adult cardiac myocytes treated with repetitive ROS treatments, immunohistochemistry revealed Nrf2 failed to traffic to the nucleus. Downregulation of Nrf2 in IHF and in isolated adult cardiac myocytes after repetitive ROS treatment was associated with decreased Nrf2 nucleus binding activity, suggesting inhibition of the Nrf2 mediated antioxidant defense. Further work evaluated changes in gene expression and mitochondrial function with the reduction of Nrf2.

Support or Funding Information

This work was supported by the following: APS Undergraduate Research Fellowship (UGSRF); NIH RO1 HL079071; VA, Merit Review, 5101BX000839; and NIH R01 HL085844.
Ablation of Toll Like Receptor 4 Mitigates Cardiac Mitochondrial Dysfunction in Hyperhomocysteinemia
Authors: Jeremic Nevena , Metreveli Naira , Tyagi Suresh Chand
Published: November 2023

BACKGROUND

TLR‐4 is a foreign antigen sensor that plays role in innate immune system activation and has recently gained a significant attention in the field of hypertension but still very little or nothing is known regard regulation of TLR expression within the heart, although TLR4 appears to be upregulated in the failing human heart. Mitochondrial dysfunction and mitochondria‐dependent apoptosis have been shown to promote endothelial cell loss leading to endothelial dysfunction that contributes to pathogenesis of hypertension. These events induce mitochondrial dysfunction characterized by excessive mitochondrial fission and mitochondrial apoptosis contributing to cardiac remodeling followed by hypertension.

OBJECTIVE

The objective of this study is to define the mechanisms of homocysteine effect on heart that promote cardiac remodeling and hypertension and explore the role of TLR‐4 mutation in alleviation of homocysteine negative effects.

METHODS

For this study we used 5 groups of mice: C57BL/6J, C3H/HEouJ, CBS+/−; C3H/HeJ, and CBS+/−/C3H. For further analysis we used isolated heart and collected blood. We collected physiological data such as: ultrasonography of heart, cardiomyocyte contractility and blood pressure. We checked expression of mitochondrial fusion and fission proteins, antioxidant markers and expression of collagen/elastin fragments.

RESULTS

Data showed that there were increased values of systolic and diastolic pressure in CBS+/− mice (DP: 128.09±17.11; SP: 162.31±12.55) and C3H/HeJ mice had decreased levels of in comparing to other groups (DP: 51.82±14.82; SP: 111.38±6.40). Tau was increased in C3H/HeJ compared to CBS+/−/C3H mice and time to pick was increased in C3H/HeJ compared to CBS+/− mice. Fusion and fission ratio (Mfn2/DRP1) were increased in C3H/HeJ mice (0.98±0.06).

CONCLUSION

These findings indicate the prevalence of mitochondrial fission over mitochondrial fusion in HHcy may explain possible endothelial cell loss and dysfunction followed by collagen accumulation that contributes to cardiac remodeling.

Support or Funding Information

Grant: NIH (HL74185)
eNOS Regulation of Nrf2 and BNIP3 in Primary Murine Hepatocytes
Authors: Meers Grace M , Naik Radheya Y , Morrison Austin G , Sheldon Ryan D , Rector R. Scott
Published: November 2023

It is well known that endothelial nitric oxide synthase (eNOS)‐derived nitric oxide (NO) stimulates mitochondrial biogenesis in a cGMP‐PGC1α dependent manner in various tissues. However, the importance of eNOS in regulating mitophagy, the clearance of poorly functioning mitochondria, in hepatocytes remains unknown. We hypothesized that eNOS induces mitochondrial turnover through nuclear factor‐E2‐related factor‐2 (Nrf2) mediated BCL2/E1B 19 kDa protein‐interacting protein 3 (BNIP3) induction of mitophagy. To examine the relationship between eNOS, Nrf2, and BNIP3 in primary hepatocytes, we isolated primary hepatocytes from C57/BL6 wild‐type (WT) mice followed by siRNA treatment [Scramble control (Scr), eNOS, or Nfr2] for six hours. Growth media was applied for 12–18 hours after siRNA treatment, then 24 hours of either control media or 500 μM FFA (250 μM oleate + 250 μM palmitate); the 500 μM FFA condition also included one of the following treatments for 16 hours: 1 μM carbonyl cyanide 3‐chlorophenylhydrazone (CCCP, to depolarize mitochondria and induce mitophagy)/10 μM cholorquine (CQ, lysosomal inhibitor), 30 nM CDDO‐Methyl Ester (CDDO‐Me, NRF2 activator), or 50 μM diethylenetriamine (DETA) NONOate/CCCP/CQ. Silencing of eNOS in primary hepatocytes (50% reduction vs Scramble control, p<0.01) knocked down Nrf2 mRNA expression by ~40% and Nrf2 activation (NQO1 mRNA; target gene of Nrf2) and BNIP3 mRNA expression by ~40% following FFA challenge (p<0.05 vs Scr control). In contrast, treating primary hepatocytes with the NO donor DETA NONOate induced Nrf2 activation (NQO1 mRNA) and BNIP3 mRNA by 50% and 300%, respectively (p<0.05 vs Scr control). In support of Nrf2 regulating BNIP3, we provide data, that to our knowledge, is the first evidence that the direct knockdown of Nrf2 in primary hepatocytes by an siRNA approach (−80% knockdown of Nrf2 vs Scr control, p<0.001) not only reduces Nrf2 activity (−80% knockdown of NQO1 mRNA), but also blunts induction of mitophagy flux (BNIP3 induction with CCCP/CQ reduced 50% vs Scr) in primary hepatocytes. In addition, Nrf2 activation with CDDO‐Me treatment (3.5‐fold increase in NQO1 mRNA) induced a ~2‐fold increase in BNIP3 accumulation (w/CCCP+CQ), an effect completely ablated with Nrf2 siRNA. These preliminary observations point to a regulatory role of hepatocyte‐specific eNOS in Nrf2 and BNIP3, and a strong rationale for the further examination of the role of Nrf2 in the regulation of BNIP3 and mitophagy.

Support or Funding Information

Funding: This work was supported by grants from the Missouri Foundation for Veterans Medical Research, the Richard Wallace Faculty Incentive, Department of Medicine Research Council, and a Veterans Affairs VHA‐CDA2 Grant (IK2BX001299).
Effects of WIN 55,212‐2 and AM281 in mitochondrial respiration of skeletal muscle
Authors: Sanchez‐Duarte Elizabeth , Trujillo Xochitl , Huerta Miguel , Rios‐Silva Monica , Sanchez‐Briones Luis Alberto , Ortiz‐Avila Omar , Saavedra‐Molina Alfredo , Montoya‐Perez Rocio
Published: November 2023

The endocannabinoid system is emerging as a critical agent of cellular metabolism regulation, through its effects on the cellular respiration and energy production. Most of the cannabinoid and endocannabinoid effects have been mediated predominantly via the activation of the cannabinoid type 1 (CB1) receptor, which is widely distributed in the brain and peripheral organs as the skeletal muscle and recently were confirmed to be localized on the outer mitochondrial membrane of these tissues. It is known that their activation by synthetic cannabinoids is able to cause changes in mitochondrial respiration of brain mitochondria, however, little is known about its effect in skeletal muscle. Thus, the aim of this work was to explore the effects of the cannabinoid agonist WIN 55,212‐2 and the CB1 antagonist receptor AM281 on oxygen consumption in the state 3 from mitochondrial respiration in isolated mitochondria derived from rat skeletal muscle.

Methods

Mitochondria from hindlimb muscles of male Wistar rats weighing between 300 and 350 were isolated by differential centrifugation. The rate of oxygen consumption was determined in a sealed glass chamber with constant stirring at room temperature, using a Clark‐type oxygen electrode coupled to an oxygen monitor YSI 5300 and a computer for data acquisition. The determinations started after adding 10 mM glutamate/malate as respiratory substrate for complex I (state 4) and 1 mM ADP was added to determine oxygen consumption in the phosphorylating state (state 3). Finally, the inhibitor of complex IV (1mM KCN) were added to inhibit mitochondrial respiration. To explore the effects of the experimental drugs on mitochondrial respiration, a concentration‐response curve was performed for each of them: WIN 55,212‐2 (0.1, 1, 5 and 10 μM) and AM281(1, 5, 10 and 20 μM).

Our results show that both drugs have similar effects by decrease mitochondrial respiration, this inhibitory effects on oxygen consumption is dose‐dependent by WIN 55,212‐2 being more prominent with 10 μM (40%) and to a lesser extent with 0.1 μM (≈ 20%), while with AM281 decreased 50% with 1 μM. These results suggest that the effect by this drugs on mitochondrial respiration exert a similar effect as has been previously reported in brain mitochondria and that could be acting on other respiratory chain components.

Support or Funding Information

We thank to Universidad de Colima, Universidad Michoacana de San Nicolás de Hidalgo (UMSNH) and Consejo Nacional de de Ciencia y Tecnología (CONACyT) the partial financial of this research. ESD was a receipient of a CONACyT PhD fellow. We acknowledge the partial financing to Coordinación de la Investigación Científica of UMSNH: ASM‐CIC‐2.16; RMP‐CIC 182171.
Sex and Metabolic Fuel Preferences Impact Proliferative Capacity of Human Muscle Progenitor Cells
Authors: Riddle Emily S , Roman Heather A , Thalacker‐Mercer Anna E
Published: November 2023

Introduction

Skeletal muscle (SkM) stem cells, satellite cells (SC), are essential for muscle maintenance as well as repair and regeneration post injury. Successful SkM regeneration requires a well‐orchestrated series of events from activation of the quiescent SC, to proliferation [muscle progenitor cells (MPC)], differentiation, and formation of multinucleated cells. During the regenerative process, macronutrients provide energy and substrates for metabolic processes and cellular structures. Defects in macronutrient metabolism and metabolic inflexibility underlie metabolic disease and likely impact the regenerative process. The “phenotype” of human macronutrient utilization and sex specific differences throughout the initial stages of the regenerative process are largely unknown.

Objective

The objective was to characterize metabolic gene expression and signaling throughout human MPC proliferation and identify sex differences that might impact successful regeneration.

Methods

Primary human SC were obtained from the vastus lateralis SkM of healthy young males (male‐MPC, n=6) and young females (female‐MPC, n=6) and cultured. A high‐throughput imaging cytometer was used to determine the confluency, nuclei count, and % of dead cells every 24 hours throughout proliferation. Saturation density was defined as the # of nuclei at proliferation plateau/cm2. Real‐time qPCR was used to measure mRNA abundance of select genes associated with macronutrient uptake and metabolism, mitochondrial biogenesis, inflammation, and stress at the first and third day out of lag. The first day out of lag was defined as the first increase in % confluency of ≥ 5%.

Results

Percent confluency throughout proliferation and saturation density were lower in female‐MPC when compared to male‐MPC. Female‐MPC also had a higher % of dead cells throughout proliferation. PGC1‐α, CD36, and NFkB mRNA levels were higher and IL6, CAT1, and CAT2 mRNA levels were lower in female‐MPC compared to male‐MPC. PGC1‐α and CD36 mRNA levels increased over time while CAT1 mRNA levels decreased over time in both female‐MPC and male‐MPC. No differences in SLC7A5, GLUT1, or GLUT4 mRNA levels were observed between sexes at either time‐point.

Conclusions

Female MPCs proliferated at a slower rate, reached a lower saturation density, and experienced elevated cell death throughout proliferation when compared to males. These differences in markers of proliferative capacity were accompanied by up‐regulation of metabolic genes, CD36 and PGC1‐α. This suggests that female cells may rely more heavily on fatty acids and mitochondrial function during proliferation, which may impair female MPC proliferative capacity.

Support or Funding Information

Cornell University
The Impact of Disuse Muscle Atrophy on Perivascular Stem Cell Quantity and Function
Authors: Munroe Michael R , Dyle Michael C , Adams Christopher M , Boppart Marni D
Published: November 2023

Background

Skeletal muscle atrophy can occur as a result of disuse following limb immobilization, extended bed rest, and spinal cord injury. The mechanisms underlying the duration and severity of disuse atrophy are multifaceted, and as such, require continued study to develop effective therapies which can combat this debilitating condition. Perivascular stem cells are located in a variety of tissue types and have substantial regenerative potential, yet the effect of disuse on their quantity and functionality within muscle is unknown.

Purpose

The purpose of this study was to determine the extent to which hindlimb immobilization, with or without mechanical reload, affects perivascular stem cell quantity within the skeletal muscle of adult mice.

Methods

Cohorts of three month‐old male C57BL/6 mice were immobilized by unilaterally stapling the foot in full dorsotibial flexion in order to atrophy the tibialis anterior (TA) muscle. Mice either underwent 14 days of immobilization (IM) or 14 days of immobilization followed by 14 days of reload (IM+RE). At the end of each time point, both stapled (ST) and unstapled (US) TA muscles were dissected and weighed. For one cohort of mice, each muscle was then processed for multiplex flow cytometry to analyze NG2, CD146, PDGFRα, CD45, and CD31 content within the isolated mononuclear suspension. A second cohort of mice had TA muscles dissected and frozen in precooled isopentane for histological analysis.

Results

Following IM (n=9), TA muscle weight was significantly reduced compared to both contralateral and cage controls (p=0.003). After IM+RE (n=10), TA muscle weights had failed to return to baseline levels and remained significantly reduced compared to controls (p<0.001). The quantity of perivascular stem cells was lower post‐IM (n=4) with a 31% reduction in NG2+CD45−CD31− (p=0.08), a 32% reduction in CD146+CD45−CD31− (p=0.04), and a 51% reduction in PDGFRα+CD45−CD31− (p=0.01) cell quantity compared to the contralateral control. However, no significant differences were detected for any perivascular stem cell population between US and ST limbs following IM+RE (n=5) (p>0.05).

Conclusion

These findings indicate that perivascular stem cell quantity is decreased with disuse atrophy, and normal mechanical reload restores quantity to baseline values. Given the responsiveness of these cells to disuse and mechanical stimuli, perivascular stem cells are viable therapeutic targets in the treatment of skeletal muscle disuse atrophy.
Knockdown of Tim23 In‐Vivo Results in the Activation of the Mitochondrial Unfolded Protein Response
Authors: Oliveira Ashley Nicole , Hood David
Published: November 2023

Mitochondria rely heavily on the tight coordination of the nuclear and mitochondrial genomes. Disruption of this regulation has been shown in lower order organisms to result in a mito‐nuclear imbalance in which holenzymes are unable to assume mature stoichiometry. This lack of coordination between nuclear and mitochondrially encoded subunits resulting in orphaned subunits is an activator of the mitochondrial unfolded protein response (UPRmt). The UPRmt is a highly conserved quality control mechanism that strives to achieve proteostasis by refolding or degrading misfolded proteins as well as misassembled complexes. This process is well characterized in c. elegans however, the UPRmt and its retrograde signals are poorly understood in mammals. Thus we sought to induce the UPRmt in‐vivo‐in a mammalian model to better characterize this stress response. In order to do so, C57BL/6 mice were injected once a day for 3 days with an antisense oligonucleotide (In‐Vivo Morpholino) targeted to Tim23, the major channel of the inner membrane, to perturb the genomic coordination. Following Tim23 Morpholino treatment, Tim23 protein content was significantly reduced by 50%. The consequence was a 60% reduction of protein import into the mitochondrial matrix. As a result, mitochondrial chaperone cpn10 was significantly elevated by 30% and the transcription factor ATF5 also demonstrated a strong trend to increase, suggesting that the UPRmt was activated following Tim23 knockdown. Mitochondrial respiration and ROS emission was also measured to determine whether Tim23 knockdown resulted in mitochondrial dysfunction. However, no differences were found suggesting that the stress imposed is sufficient to induce the UPRmt without disrupting basic mitochondrial functions. The second aim of this study was to investigate the role of mitochondrial proteolytic byproducts in mediating retrograde signaling. Peptides released from the mitochondria following basal proteolysis were isolated and incubated with import reactions. Our results demonstrate a dose‐ and time‐dependent inhibition of import with the addition of peptides, such that import was reduced by 48% with the addition of 6ug of peptides for 30 minutes. This suggests that mitochondrial proteolytic byproducts can exert an inhibitory effect on the protein import pathway, to inhibit excessive protein import as a negative feedback mechanism. The inhibition of import into the organelle may also serve a retrograde function, to modify nuclear gene expression and improve organelle folding capacity, or alter the expression of protein import machinery components.

Support or Funding Information

This work is supported by the Natural Science and Engineering Research Council (NSERC) of Canada.
Deletion of the transcriptional coactivator PGC1α in skeletal muscles is associated with reduced expression of genes related to oxidative muscle function
Authors: Kamei Yasutomi , Hatazawa Yukino , Minami Kimiko , Yoshimura Ryoji , Onishi Takumi , Sawada Naoki , Miura Shinji
Published: November 2023

The expression of the transcriptional coactivator PGC1α is increased in skeletal muscles during exercise. Previously, we showed that increased PGC1α leads to prolonged exercise performance (the duration for which running can be continued), and at the same time, increases the expression of branched‐chain amino acid (BCAA) metabolism‐related enzymes and genes that are involved in supplying substrates for the TCA cycle. We recently created mice with PGC1α knockout specifically in the skeletal muscles (PGC1α KO mice), which show decreased mitochondrial content. In this study, global gene expression (microarray) analysis was performed in the skeletal muscles of PGC1α KO mice and compared with that of wild‐type control mice. As a result, decreased expression of genes involved in the TCA cycle, oxidative phosphorylation, and BCAA metabolism were observed. Compared with previously obtained microarray data on PGC1α‐overexpressing transgenic mice, each gene showed the opposite direction of expression change. Bioinformatic analysis of the promoter region of genes with decreased expression in PGC1α KO mice predicted the involvement of several transcription factors, including a nuclear receptor, ERR, in their regulation. As PGC1α KO microarray data in this study show opposing findings to the PGC1α transgenic data, a loss‐of‐function experiment, as well as a gain‐of‐function experiment, revealed PGC1α's role in the oxidative energy metabolism of skeletal muscles.
MicroRNA in Muscle: Characterising the Powerlifter Phenotype
Authors: D'Souza R. F. , Bjornsen T. , Aasen K. M. , Raastad T. , Mitchell C. J. , Cameron‐Smith D.
Published: November 2023

Skeletal muscle has a remarkable capacity to adapt in response to imposed external loads. Powerlifters are the epitome of this adaptation and are able to generate extremely high muscular forces. The molecular mechanisms underpinning the significant capacity for force generation and hypertrophy are unclear. MicroRNAs (miRs) are short non‐coding RNA sequences that control gene expression via promotion of transcript breakdown and/or translational inhibition. Differences in miR expression at rest may account for phenotypic differences in muscle mass and function between powerlifters and untrained age‐matched controls.

The primary object of this study was to characterize the expression profiles of a subset of miRs known to exert an influence on key aspects of myogenic regulation in the muscles of highly trained powerlifters. Muscle biopsies were obtained from the vastus lateralis of 17 national level powerlifters (25.1 ± 5.8 years) and 17 untrained controls (24.1 ± 2.0 years). The powerlifters were stronger than the controls (isometric knee extensions: 297.9 ± 56.1 Nm, 193.6 ± 46.6 Nm, respectively p <0.001), and also had bigger muscle fibres (type I CSA 8869 ± 1366 μm2 compared to 4196 ± 9632 μm2 p<0.001 and type II CSA 10636 ± 2029 μm2 compared to 5327 ± 1342 μm2 p<0.001). Of the 15 miRs species analysed, 12 were differently expressed between groups. 7 were upregulated in powerlifters; miR ‐15a (p<0.001), ‐16 (p=0.008), ‐23a (p<0.001), ‐23b (p<0.001), ‐451a (p=0.012), ‐30b (p=0.010) and ‐206 (p=0.032). Conversely, 5 were reduced in the powerlifters; miR‐486 (p=0.002), ‐499 (p=0.018), ‐126 (p<0.001), ‐133a (p=0.001) and ‐1 (p=0.013). Whilst, miRs‐208a, ‐208b and ‐145 did not differ between groups.

Powerlifters have larger muscle fibres capable of producing greater contractile force. The unique miR expression profiles of powerlifters compared to the healthy untrained controls indicates miRs may be a component of the post‐transcriptional regulation necessary for the maintenance of increased muscle mass and strength. The miRs of interest have putative roles in muscle atrophy, hypertrophy and angiogenesis indicating a possible involvement in regulation of muscle mass and contractile capacity.
IL‐15 Activates the Jak3/STAT3 Signaling Pathway to Mediate Glucose Uptake in Skeletal Muscle Cells
Authors: Abbott Marcia J , Krolopp James E , Thornton Shantae M
Published: November 2023

Myokines are specialized cytokines that are secreted from skeletal muscle (SKM) in response to metabolic stimuli, such as exercise. Interleukin‐15 (IL‐15) is a myokine with potential to reduce obesity and increase lean mass through induction of metabolic processes. It has been previously shown that IL‐15 acts to increase glucose uptake in SKM cells. However, the downstream signals orchestrating the link between IL‐15 signaling and glucose uptake have not been fully explored. Here we employed the mouse SKM C2C12 cell line to examine potential downstream targets of IL‐15‐induced alterations in glucose uptake. Following differentiation, C2C12 cells were treated overnight with 100 ng/ml of IL‐15. Activation of factors associated with glucose metabolism (Akt and AMPK) and known downstream targets of IL‐15 (Jak1, Jak3, STAT3, and STAT5) were assessed with IL‐15 stimulation. IL‐15 stimulated glucose uptake and GLUT4 translocation to the plasma membrane. IL‐15 treatment had no effect on phospho‐Akt, phospho‐Akt substrates, phospho‐AMPK, phospho‐Jak1, or phospho‐STAT5. However, with IL‐15, phospho‐Jak3 and phospho‐STAT3 levels were increased along with increased interaction of Jak3 and STAT3. Additionally, IL‐15 induced a translocation of phospho‐STAT3 from the cytoplasm to the nucleus. We have evidence that a mediator of glucose uptake, HIF1α, expression was dependent on IL‐15 induced STAT3 activation. Finally, upon inhibition of STAT3 the positive effects of IL‐15 on glucose uptake and GLUT4 translocation were abolished. Taken together, we provide evidence for a novel signaling pathway for IL‐15 acting through Jak3/STAT3 to regulate glucose metabolism.

Support or Funding Information

This work was supported by Chapman University, the American College of Sports Medicine Foundation, and the American Heart Association Grant #16SDG30680003 (MJ Abbott).
Mitochondrial respiration is fiber‐type specific in skeletal muscles
Authors: Lin Cheng‐Hsien , Chen Chiao‐nan
Published: November 2023

Introduction

Mitochondria are the power houses of the cells and they also play critical roles in the cellular oxidative stress and apoptosis. It is known that mitochondrial density and morphology are fiber‐type specific. Based on differential contractile properties of different types of skeletal muscles, we hypothesized that the mitochondria respiration is fiber‐type specific. The aim of the study is to compare and contrast mitochondria respiration between type I and type II muscles.

Method

Soleus (predominantly composed of type I fibers) and plantaris (predominantly composed of type II fibers) muscles were taken from adult rats. Mitochondrial respiration was determined in duplicate by a high‐resolution respirometry OROBOROS Oxygraph‐2k (O2k). The mitochondrial substrate‐uncoupler‐inhibitor titration (SUIT) protocol was used to determine mitochondrial oxygen consumption rate (O2 flux) of permeabilized myofibers. Paired t test was used to compare parameters between soleus and plantaris muscles.

Results

We found that basal mitochondrial respiration (i.e. O2 flux_ complex I plus complex II) and leak of permeabilized muscle fibers was similar between soleus and plantaris muscles. Similarly, mitochondrial coupling control ratio defined by leak divided by basal mitochondrial respiration (i.e. leak/O2 flux_ complex I plus complex II) is similar between soleus and plantaris muscles. Maximal electron transport system capacity (maximal mitochondrial respiration) of soleus muscles was greater than that of plantaris muscles. In addition, mitochondrial respiratory reserve capacity defined by subtracting basal mitochondrial respiration from maximal mitochondrial respiration was greater in soleus muscles than in plantaris muscles.

Conclusion

Basal mitochondrial respiration and leak are similar between type I and type II muscles. However, mitochondria in type I muscles have greater respiratory reserve compared to that in type II muscles. This fiber‐type specific mitochondrial respiration is likely associated with the differential fuel metabolism and contractile properties of muscle fibers.