bluuebunny/crossref_metadata_2025 huggingface dataset:
Skeletal Muscle Mitochondrial Respiratory Capacity and Function Are Diminished in Patients with Advanced Peripheral Artery Disease and Critical Limb Ischemia
Authors: Rontoyanni Victoria G , Lopez Omar Nunez , Fankhauser Grant T. , Cheema Zulfiqar F. , Cheng Charlie , Rasmussen Blake B. , Borsheim Elisabet , Porter Craig
Published: November 2023

Introduction

Peripheral artery disease (PAD) is associated with a marked reduction in functional capacity, where progression to critical limb ischemia (CLI) is the most severe clinical presentation of the disease. Current evidence suggests diminished skeletal muscle oxidative capacity in patients with PAD. To our knowledge, no study has simultaneously assayed mitochondrial oxidative capacity and mitochondrial quality to compressively determine the role of mitochondrial dysfunction in PAD. We set out to compare mitochondrial oxidative capacity and mitochondrial quality in PAD patients with CLI versus healthy controls.

Methods

To date, we have recruited and studied 7 PAD patients with CLI undergoing revascularization procedures or amputation (56±11 y; PAD) and 7 healthy adults (62±6 y; Control). Skeletal muscle samples were collected from the leg, and mitochondrial respiratory capacity and function were determined in permeabilized myofibers by high‐resolution respirometry (Figure 1). Results are presented as means ± SD.

Results

Mitochondrial respiration coupled to ATP production (State 3I+II; Figure 1) and maximal respiratory capacity (State 3u; Figure 1) were diminished in PAD patients with CLI when compared to healthy controls (18.5±12.3 vs. 41.3±12.4 pmol/s/mg, P<0.01 and 27.9±17.4 vs 51.8±16.6 pmol/s/mg, P<0.05, respectively). Respiration coupled to ATP production was highly correlated to maximal respiratory capacity in both PAD patients with CLI (r=0.94, P<0.01) and controls (r=0.97, P<0.001). The flux control ratio (State 3I+II/State 3u), a marker of mitochondrial quality, was significantly lower in PAD patients with CLI compared to controls (O.67±0.11 vs 0.80±0.06, P<0.05), indicating reduced mitochondrial efficiency in CLI.

Conclusion

Our preliminary results show that both skeletal muscle oxidative capacity and mitochondrial efficiency (i.e., quality) are substantially lower in muscle from PAD patients with CLI when compared to healthy controls. Collectively, reduced skeletal muscle respiratory capacity and mitochondrial quality likely contribute to impaired functional capacity in PAD patients with CLI. The mitochondrion may represent a therapeutic target to alter disease progression and/or improve functional capacity in patients with PAD.

Support or Funding Information

Seed funding for this project was supplied by the Department of Surgery, UTMB. This work was also supported by the following grants: NIH RO1 AG‐033761, RO1 AR‐049877, R56 AG05126701, P30 AG024832 and SHC‐84080, and was conducted with the support of the Institute for Translational Sciences at UTMB, supported in part by a Clinical and Translational Science Award (UL1TR000071).
High Glucose Induces Vascular Contraction through Mitochondrial Membrane Potential (MMP)‐Miro‐MLC Pathway
Authors: Xu Jie , Qin Xinghua , Wang Zhen , Zhang Xing , Gao Feng
Published: November 2023

Objective

Although vascular dysfunction is prevalent in diabetes mellitus, the directly effect of high glucose on vascular tone is unclear. This study aimed to examine whether high glucose can induces vascular contraction and the role of mitochondria in this process.

Methods

Vascular ultrasound was used for blood vessel diameter measurement. Wire myograph system 610M was used to record vascular response. Myosin light chain (MLC) phosphorylation and Miro expression level were detected in primary vascular smooth muscle cell isolated from thoracic aorta of rats. mitochondrial membrane potential (MMP) was measured by JC‐1 or TMRM dye.

Results

Arterial blood pressure increased while brachial/abdominal aorta vascular diameter decreased during transient hyperglycemia in healthy human subjects (1.0g/kg glucose, po) and constant hyperglycemia in diabetic rats (2.0g/kg glucose, ip). High glucose (40 mmol/L) induced isolated thoracic aorta contraction which could be enhanced by insulin (10 nmol/L). Importantly, we found that MMP of vascular smooth muscle cells, which was increased by high glucose challenge, induced vascular contraction independently of endothelium. Mechanically, MMP induced vascualr contraction via ROCK‐MLC signaling rather than Ca2+ signaling. Miro, an atypical Rho GTPases located on mitochondrial membrane, serves as an upstream of ROCK signaling.

Conclusion

These data show that high glucose can induces vascular contraction via increasing MMP and resulted activation Miro‐ROCK‐MLC signaling of vascular smooth muscle cells.

Support or Funding Information

This study was supported by the grants from the National Basic Research Program of China (No. 2013CB531204).
The effects of myocardial infarction with or without subsequent reperfusion on respiratory function of mitochondria in rat heart
Authors: Vega‐Lugo Jesus A. , Parodi‐Rullán Rebecca M. , Soto‐Prado Jadira , Chapa‐Dubocq Xavier , Javadov Sabzali
Published: November 2023

Background

Cardiovascular diseases are the leading cause of mortality and morbidity worldwide and coronary heart diseases, particularly myocardial infarction (MI) is the most common heart disease. Cardiac mitochondria play a major role in cell functioning, intracellular Ca2+ regulation, ATP production, and cell death. During MI, the reduction or loss of blood flow induces mitochondrial dysfunction and initiates cell death. Particularly, mitochondria‐mediated cell death during MI is mediated by opening of the mitochondrial permeability transition pores (PTP) in the inner mitochondrial membrane. mPTP opening accompanied by matrix swelling causes ROS production, loss of the membrane potential and ATP depletion leading to cardiac dysfunction. Therefore, PTP inhibition is broadly accepted as a promising therapeutic target for MI. Here we assess the effect of mPTP inhibitor, sanglifehrin A (SfA) on mitochondrial function of the heart during MI with no reperfusion or with subsequent reperfusion.

Methods

MI was induced in female rats (BW=200–250g) by coronary artery ligation (CAL) for 30 min without (permanent ligation) or with subsequent reperfusion for 2 and 28 days. Experiments were conducted in the following 6 groups: i) Sham (S, no MI, n=6 and n=11 for 2 and 28 days, respectively); ii) SS (Sham+SfA, n=5 and n=11 for 2 and 28 days, respectively); iii) MI (no reperfusion, n=4 and n=5 for 2 and 28 days, respectively); iv) MI+SfA (n=4 and n=6 for 2 and 28 days, respectively); v) MI+R (MI+reperfusion, n=4 and n=5 for 2 and 28 days, respectively); and vi) MI+RS (MI+reperfusion+SfA, n=6 and n=7 for 2 and 28 days, respectively). SfA (5mg/kg, IV, Novartis) was administered at 25 min after a start of sham surgery or MI. At the end of each experimental protocol, cardiac mitochondria were isolated from left and right ventricles and oxygen consumption was measured using a Clark oxygen electrode. Results were calculated as nmol O2/min per citrate synthase activity.

Results

Mitochondria in MI and MI+R groups demonstrated reduced state 3 (49±4 and 56±6, respectively, P<0.01 vs. S) compared to the S group (101±7) at 28 days post‐surgery. Indeed, reperfusion had no additional detrimental effect on mitochondrial dysfunction induced by MI (MI and MI+R). SfA had no effect on respiratory function of mitochondria in MI (MI: 49±4 vs MI+S: 51±5) and MI+R (MI+R: 56±6 vs MI+RS: 51±4) groups. Reperfusion for short (2 days) and long (28 days) affected differently the mitochondrial function in right and left ventricles.

Conclusion

MI produce considerable damage to mitochondrial function having no difference between reperfused and non‐reperfused hearts.

Support or Funding Information

NHLBI NIH (SC1HL118669)
Ischemic Postconditioning Transiently Improves Cardiac Mitochondrial Function during Resuscitation in a Porcine Model of Cardiac Arrest
Authors: Matsuura Timothy R , Yannopoulos Demetris , Shekar Kadambari C , Sebastian Pierre S , Bartos Jason A , Olson Matthew D , Douglas Hunter F , Rees Jennifer N , McKnite Scott H , Aufderheide Tom P , Neumar Robert W , Riess Matthias L
Published: November 2023

Introduction

Ischemic postconditioning (IPC) is a technique of brief interruptions in blood flow at the onset of reperfusion to mitigate reperfusion injury after prolonged ischemia. When applied during cardiopulmonary resuscitation (CPR) following ventricular fibrillation (VF) cardiac arrest (CA) in a porcine model, IPC during CPR (IPC‐CPR) reduced adverse events and improved neurologically favorable survival 48 hours after return of spontaneous circulation (ROSC) compared to standard‐of‐care CPR (S‐CPR). IPC‐CPR also increased cardiac mitochondrial function prior to ROSC after 4 minutes of CPR compared to S‐CPR.

Objective

We investigated the effect of IPC‐CPR after prolonged VF CA on cardiac mitochondrial function 6 hours after ROSC.

Methods

Twenty‐four female swine were intubated, anesthetized, and surgically prepared for continuous hemodynamic recording. Eight animals received no CA (Naïve, n=8) as a non‐ischemic control. The remaining animals received 15 minutes of untreated VF CA followed by CPR without (S‐CPR, n=8) or with IPC (IPC‐CPR, n=8). IPC was administered via three 20‐second pauses in chest compressions during the first 2 minutes of CPR. After 4 minutes of CPR, defibrillation was attempted. Six hours after ROSC, cardiac mitochondria were isolated via differential centrifugation for assessment of respiration, rate of ATP synthesis, and calcium retention capacity. ANOVA with Newman‐Keuls posthoc test was used for comparisons between treatments. The null‐hypothesis was rejected for p < 0.05.

Results

ROSC was obtained in all animals that received a CA. Cardiac mitochondrial function did not differ between Naïve, S‐CPR, and IPC‐CPR treated animals.

Conclusions

In a porcine model of prolonged VF CA, cardiac mitochondrial function normalizes within 6 hours of ROSC. The previously documented reduction in adverse events and improvement in survival with IPC‐CPR treatment may be mediated by acute changes in cardiac mitochondrial function earlier than 6 hours post‐ROSC, or by mechanisms independent of cardiac mitochondrial function

Support or Funding Information

Stipend support was provided by the American Heart Association 16PRE27600038 (TRM). Project support was provided by NIH 1R01 OD016727‐01 (DY).
Mitochondrial Abnormalities May Contribute to Skeletal Muscle Weakness and Poor Regeneration in Fukutin‐Deficient Muscular Dystrophy Mice
Authors: Southern William M , Nichenko Anna S , Beedle Aaron , Call Jarrod A
Published: November 2023

The dystrophin‐glycoprotein complex (DGC) provides a link between extracellular and intracellular structures of the muscle cell. We have previously demonstrated that muscle‐specific deletion of the fukutin gene [Myf5/Fktn KO mice (KO)] causes DGC disruption. KO mice present moderate to severe muscular dystrophy characterized by muscle weakness and delayed regeneration following muscle injury. The objective of this study was to determine the extent to which mitochondrial dysfunction contributes to the muscle weakness and delayed regeneration in these mice. We hypothesized that mitochondrial dysfunction contributes to muscle weakness in KO mice and that improving mitochondrial content would benefit muscle regeneration following muscle injury. We administered daily injections of saline or AICAR (500mg/d/kg), an agent previously shown to improve mitochondrial quality in dystrophic muscle, to both KO (saline n = 9, AICAR n = 6) and littermate (LM) control (saline n = 9, AICAR n = 8) mice. Two weeks after onset of treatment, the left hindlimbs (anterior and posterior compartments) of all mice were injured with cardiotoxin to induce muscle regeneration, while the right hind limbs served as contralateral uninjured controls. Mice were sacrificed 2 weeks post‐injury. Body mass and gastrocnemius mass normalized to body mass was 21% and 13% lower in KO mice compared to LM controls (body mass: P<0.001, gastrocnemius: P<0.002). Pre‐injury peak isometric torque about the ankle joint was 38% lower in KO mice compared to LM (P=0.044). Interestingly, muscle strength was not fully recovered in any group of mice 2 weeks post‐injury, but AICAR‐treated mice had 25% greater muscle strength following injury compared to saline‐treated mice, independent of genotype (P=0.036). Mitochondrial respiration of injured and uninjured permeabilized fibers was also assessed 2 weeks post‐injury. Mitochondrial respiration was 23% lower in KO mice compared to LM mice, independent of treatment and injury (P=0.023). Further analysis is required to determine if this is due to lower mitochondrial content or mitochondrial dysfunction. While there was no effect of AICAR on mitochondrial respiration, AICAR‐treated mice had ~14% greater levels of COXIV protein content compared to saline‐treated mice, independent of genotype and injury (P=0.015). This suggests a longer duration of AICAR treatment may be necessary for gains in mitochondrial protein contents to be realized in terms of mitochondrial respiration. In conclusion, this data suggests that fukutin deficiency is associated with mitochondrial abnormalities that may contribute to skeletal muscle weakness and poor regeneration. AICAR treatment may facilitate strength recovery following muscle injury, although it is unclear the extent to which AICAR affects mitochondrial function following injury.
Chronic Binge Alcohol Upregulates Expression of Genes Related to Apoptosis in Skeletal Muscle of Simian Immunodeficiency Virus‐Infected Rhesus Macaques at End‐Stage Disease
Authors: Duplanty Anthony A , Simon Liz , Molina Patricia E
Published: November 2023

Alcohol use disorders are more prevalent in HIV patients than the general population. Both chronic alcohol consumption and HIV‐infection have been linked to mitochondrial dysregulation; and this is considered an important mechanism in the pathogenesis of muscle myopathy. Previous studies from our laboratory have demonstrated that chronic binge alcohol (CBA) administration accentuated loss of skeletal muscle (SKM) mass and promoted a dysfunctional skeletal muscle phenotype which was associated with accelerated progression to end‐stage disease, compared with isocaloric sucrose simian immunodeficiency virus (SIV)‐infected (SUC/SIV) macaques. The mechanisms preceding and leading to accentuated SAIDS wasting in CBA/SIV macaques include localized SKM inflammation, increased proteasomal activity, depletion of SKM antioxidant capacity and increased expression of pro‐fibrotic genes. Moreover, CBA/SIV resulted in decreased expression of peroxisome proliferator‐activated receptor‐γ coactivator (PGC)‐1β, a master regulator of mitochondrial homeostasis.

Purpose

The aim of this study was to investigate the CBA‐associated alterations in expression of selected mitochondrial genes related to mitophagy and apoptosis in the skeletal muscle of SIV‐infected rhesus macaques at end‐stage disease.

Methods

Male rhesus macaques were administered daily CBA (blood alcohol levels ~50 mM) or sucrose (SUC) intragastrically 3 months prior to intravenous SIVmac251 inoculation and continued until animals reached end‐stage criteria. SKM (gastrocnemius) samples were excised at necropsy and total RNA was extracted and mitochondrial gene expression was analyzed by qPCR. SKM tissues excised from a group of uninfected non‐CBA treated animals was used as reference controls.

Results

SIV‐infection resulted in a significant upregulation (P<0.05) of mitophagy‐related gene expression (DRP1, TFEB, PINK1), which was prevented by CBA. CBA suppressed expression of anti‐apoptotic genes (Beclin‐1 and BCL2) and increased expression of pro‐apoptotic genes (BAX and BAK) (P<0.05).

Conclusions

These findings suggest that SIV‐infection disrupts mitochondrial homeostasis and when combined with CBA, results in differential expression of genes involved in apoptotic signaling. We speculate that impaired mitochondrial homeostasis may contribute to the underlying pathophysiology of alcoholic and HIV/AIDS associated myopathy.

Support or Funding Information

This study was supported by grants from the National Institute on Alcohol Abuse and Alcoholism (NIAAA): T32 Postdoctoral Fellowship (AA07577) and the Comprehensive Alcohol Research Center P60 (AA09803) in the department of Physiology, LSU Heath Science Center, New Orleans, LA.
Dietary tributyrin alters satellite cell activity in neonatal swine
Authors: Murray Robert L. , Zhang Wei , Stahl Chad H.
Published: November 2023

Satellite cells (SC), skeletal muscle stem cells, are responsible for all postnatal muscle growth and repair. Recently, we have shown that physiologically relevant concentrations of butyrate altered myogenesis in SC by enhancing terminal differentiation and the upregulation of myogenin mRNA expression. Our objective in this study was to determine the effect of dietary inclusion of tributyrin (Tb), a butyrate pro‐drug, has on neonatal muscle growth. Thirty neonatal piglets (24±8hrs old) were fed a standard milk replacer formula or formula supplemented with 0.50% (dry matter) spray dried Tb for 17d. Skeletal muscle tissue and SC were isolated from the longissimus dorsi (LD) for analysis of muscle growth parameters. Body weights were recorded daily and showed no difference in average daily body weight gain or feed efficiency. Animals that had received the Tb treatment diet had a significant increase (P<0.05) in the DNA:protein ratio in muscle. However, total concentrations of mTOR and the ratio of phosphorylated‐mTOR: total mTOR were not different between treatments. There was no difference in the relative abundance of the transcription factors Pax7 or MyoD in muscle tissue; however, there was an increase in myogenin expression in the LD from the Tb treated group (P<0.15). There was a numerical increase of in vivo proliferation rates of SC from the Tb group as assessed by BrdU staining. For in vitro analysis of SC behavior, the isolated SC were cultured for 48h in proliferation media and then switched to differentiation media for 96h. RNA was collected every 24h and RT‐PCR reveled a 100‐fold increase over the control group in myogenin expression 24h after they were induced to differentiate (P<0.05). Myogenin expression remained elevated in the Tb treatment group during differentiation (P<0.20), while there was no difference in Pax7 or MyoD expression at any time point. After 48h of differentiation, satellite cell fusion rate was assessed by immunofluorescent staining for myosin heavy chain (MyHC). Satellite cells from tributyrin treated animals had doubled the rate of fusion (P=0.10) over the control animals. Butyrate is a known inhibitor of histone deacetylases (HDACs) and when applied to satellite cells in culture it has arrested cell proliferation and enhanced terminal differentiation. In the present study, we have shown using an in vivo rapid muscle growth model that dietary inclusion of Tb can alter SC dynamics by way of HDAC regulation. Further investigation into the epigenetic targets could elucidate candidate genes for disease pathways and pharmacologic manipulation. Furthermore, we have provided evidence of accelerated myonuclear accretion in the neonatal pig; this may result in enhanced life‐time lean muscle growth which would significantly improve the sustainability of the swine production industry.

Support or Funding Information

Maryland Agriculture Experiment Station
Effect of Ethanol Diet on the circulating levels of Myonectin and Irisin
Authors: Hagood Kendra Lyndsey , Peterson Jonathan
Published: November 2023

In this study, we aimed to characterize the link between ethanol consumption and the circulating levels of novel muscle‐produced secreted factors, or myokines. Myonectin (also known as C1q/TNF‐related protein 15) and irisin, the secretory product derived from Fibronectin type III domain‐containing protein 5 (FNDC5) are newly discovered myokines with wide‐ranging effects upon metabolism, inflammation, and tissue survival‐signaling. Alcoholic fatty liver disease (AFLD) is a major co‐morbidity associated with excess alcohol consumption and the leading cause of cirrhosis and liver‐related deaths in the world. AFLD occurs when excess alcohol consumption leads to disruptions in hepatic lipid metabolism resulting in the excess accumulation of lipids in in the liver. Due to the potential role in the activation of hepatic lipid oxidation induced by myonectin and irisin we hypothesized that chronic alcohol consumption will result in reduced circulating myonectin and irisin levels, which would be a potential mechanism for the excessive lipid accumulation. To test this hypothesis, serum was collected from female mice after 6‐weeks of ethanol feeding (Lieber‐Decarli alcohol diet, 5% ETOH by volume) or calorically matched control diet. Our data demonstrated that neither serum levels of myonectin or irisin were reduced in the ethanol‐fed mice. Myokines can control a number of inflammatory and metabolic responses including reducing levels of free fatty acids and lead to the uptake of fatty acids in various tissues such as adipocytes and hepatocytes. Although, our data demonstrate that lower myokine levels were are not associated with ALFD, it remains to be determine whether increasing the levels of these myokines could stimulate lipid metabolism and prevent AFLD.
Inhibition of Platelet‐Derived Growth Factor Receptor Signaling Reduces Postnatal Tendon Growth in Response to Mechanical Loading
Authors: Disser Nathaniel P , Sugg Kristoffer B , Mendias Christopher L
Published: November 2023

Platelet‐derived growth factors (PDGFs) and their receptors (PDGFRs) play an important role in the embryonic formation of many different tissue types. Previous studies have demonstrated the importance of PDGF signaling in the regulation of key cellular processes, including fibroblast differentiation, proliferation and extracellular matrix (ECM) synthesis. However, little is known about the role of PDGF signaling in postnatal tendon growth. We hypothesized that due to its importance in mesenchymal cell function, inhibition of PDGF signaling will suppress the growth of adult tendon tissue in response to a mechanical growth stimulus. Six‐month‐old male C57BL/6 mice were subjected to bilateral Achilles tenectomy to induce rapid growth of the synergist plantaris tendon. Intraperitoneal injections of CP‐673,451, a specific inhibitor of PDGFRα and PDGFRβ, or vehicle were administered at 15 mg/kg BID beginning one day prior to synergist ablation and continued every day until the mice were euthanized. Plantaris tendons were harvested at 3 and 10 days postoperatively. Synergist ablation resulted in the growth of new tendon tissue around the outside of the plantaris tendon in both the control and treatment groups, but the cross‐sectional area (CSA) of this new tendon tissue was significantly lower in the PDFGR inhibitor group compared to vehicle controls. Additionally, immunostaining for Ki67 revealed less proliferating cells in the new tendon tissue of the plantaris tendons from the PDGFR inhibitor group. Compared to the control group, there was significant downregulation of type V collagen and matrix metalloproteinase 14 (MMP14) at 3 days, as well as downregulation of genes involved in fibroblast proliferation at 3 and 10 days in the plantaris tendons treated with the PDGFR inhibitor. The results of this study demonstrate that PDGF signaling plays an important role in postnatal tendon growth in response to mechanical loading. The growth of new tendon tissue after synergist ablation was significantly restricted in the presence of PDGFR inhibition. This work lays the foundation for future studies to determine the specific roles of the PDGF isoforms and their receptors in the growth response of adult tendon tissue.

Support or Funding Information

NIH grants R01‐AR063649 and F32‐AR067086, and an APS Summer Undergraduate Research Fellowship.
Cytokine secretory responses of MYD88 skeletal muscle specific knockout to pathogen exposure in vitro
Authors: Mattingly Alex , Laitano Orlando , Garcia Christian , Robinson Gerard , Van Steenbergen David , Clanton Thomas
Published: November 2023

The role of skeletal muscles in the immune response to pathogens is poorly understood. Though intact skeletal muscles contain toll like receptors (TLRs) and secrete cytokines in response to pathogen exposure, it is unknown whether the cytokines come from muscle fibers or other resident cells. The purpose of this study was to interrupt TLR transduction, only in muscle fibers, in order to isolate their contribution from other cell types. To accomplish this, we created a skeletal muscle specific Myd88 knockout mouse (Myd88skmKO). Myd88 is an adaptor protein, critical to TLR signal transduction in all known TLRs in muscle. To stimulate TLRs, we used lipopolysaccharide (LPS), a potent TLR2 and TLR4 ligand.

Matched solei muscles from MYD88skmKO mice (n=18) and background strain‐matched wild type controls (n=8) were excised in oxygenated (95%O2/5%CO2) Krebs solution and placed under 1 g of tension in baths at 35°C. Following a 30‐min acclimation period (T0), the buffer was replaced and muscles were exposed to 1 μg/ml LPS. Bath samples at T0, 1 hr (T1) and 2 hr (T2) were collected, mixed with a protease inhibitor cocktail and stored at −80°C prior to Luminex multiplex analysis.

Both Myd88skmKO and control mice secreted significant quantities of cytokines in response to LPS. The response was greater in control mice than MYD88skmKOs in IL‐6 (3.9 fold, p=0.0005), IL‐15 (0.7 fold, p< 0.02) GM‐CSF (3.0 fold, p< 0.02) IL‐12p40 (2.7 fold, p< 0.02), IL12‐p70 (0.6 fold, p< 0.02), and MCP‐1 (1.8 fold, p< 0.05). At T2, control mice secreted greater IL‐6 (10.4 fold, p= 0.0002), GCS‐F (8.6 fold, p= 0.002), MCP‐1 (1.7 fold, p< 0.01) IP‐10 (1.4 fold, p= 0.0145) and KC (4.3 fold, p< 0.03). Interestingly, MYD88skmKOs mice secreted greater MIP‐1α at T1 (0.5 fold, p=0.02). Despite the much greater secretion in controls, LPS treated MYD88skmKOs solei still demonstrated significant secretion of some cytokines over T1 to T2 in IL‐6 (p= 0.0001), KC (p= 0.0001), MCP‐1 (P= 0.0001), MIP‐2 (p<0.04) and GM‐CSF (p< 0.04).

In conclusion, solei from MYD88skmKO mice secreted significantly lower amounts of cytokines in response to LPS than controls, with the lone exception of MIP‐1α. This confirms that much of the cytokine secretory response to LPS in whole muscle comes from muscle fibers. However, muscles from Myd88skmKO mice still demonstrated the capacity to secrete some cytokines. This remaining cytokine release may be indicative of remaining MYD88‐independent TLR4 signaling, responses of non‐muscle resident cells or incomplete KO of the MYD88 protein in muscle fibers.

Support or Funding Information

Supported by NIH RO1GM118895‐01 and the BK and Betty Stevens Endowment.
The Effect of Skeletal Muscle AMPK on the Regulation and Localization of the Dystrophin‐Associated Protein Complex
Authors: Dial Athan G , Lally James S , Bujak Adam L , Steinberg Gregory R , Ljubicic Vladimir
Published: November 2023

The dystrophin‐associated protein complex (DAPC) is composed of proteins that are highly expressed along the sarcolemma. The DAPC provides a mechanical link between the intracellular cytoskeleton and extracellular matrix. Additionally, increasing evidence shows the importance of the DAPC as a mechanosensor and signal transducer across the sarcolemma. The signalling molecule AMP‐activated protein kinase (AMPK) is a powerful regulator of phenotypic plasticity. Chronic pharmacological AMPK stimulation induces the expression of DAPC components, whereas reduced AMPK activity results in DAPC dysfunction. However, a more comprehensive understanding of the influence of AMPK on the DAPC is lacking. Therefore, the purpose of this study was to investigate the role of AMPK in the expression of the DAPC in skeletal muscle. Extensor digitorum longus (EDL) and soleus (SOL) muscles representing fast glycolytic and slow oxidative tissues, respectively, were obtained from wild‐type (WT) mice, as well as from mice deficient in both isoforms of the AMPK‐β subunit in skeletal muscle (AMPK‐MKO). Immunofluorescence imaging was performed to measure the expression and localization of DAPC components dystrophin, β‐dystroglycan (β‐DG), γ‐sarcoglycan, (γ‐SG), neuronal nitric oxide synthase (nNOS), and laminin. β‐DG content was found to be ~2.2‐fold higher (p < 0.05) in SOL compared to EDL muscles from WT animals. However, this fiber type difference was not seen in the muscles from AMPK‐MKO mice. The expression of nNOS was significantly higher (+86%) in the SOL relative to the EDL muscles in AMPK‐MKO animals. This fiber type specificity was not observed in WT mice. The levels of dystrophin, γ‐SG, and laminin were similar between muscle types in both WT and AMPK‐MKO mice, and all DAPC components were similar between genotypes. We next investigated potential regulators of DAPC transcription, including myogenin and myoD. Western blot analyses revealed that the expression of both myogenin and myoD were downregulated by 40–60% (p < 0.05) in EDL and SOL muscles from AMPK‐MKO versus WT mice, which suggests that alternative factors may be contributing to the similar level of DAPC expression between genotypes. Indeed, computational analysis of the 3′ untranslated regions of DAPC components revealed multiple regulatory elements that have been shown to affect mRNA stability in skeletal muscle, such as binding sites for RNA‐binding proteins AUF1, HuR, and KSRP, as well as for microRNAs. Our results suggest that the absence of AMPK in skeletal muscle initiates a compensatory cascade of events at multiple levels of gene expression that coordinate to maintain basal levels of DAPC expression. Thus, although chronic AMPK activation induces DAPC content in skeletal muscle, it appears that AMPK is not essential for the maintenance of DAPC expression.

Support or Funding Information

Natural Science and Engineering Research Council

Canadian Research Chairs
Delayed Reinnervation During Muscle Regeneration After NO‐Donor Pre‐Treatment
Authors: Daneshvar Nasibeh , Anderson Judy E
Published: November 2023

Activation of satellite cells (SCs) toward myogenesis leads to structural regeneration of damaged myofibers and reinnervation is essential to muscle function. SC activation is mediated by NO (a signaling and neurotransmitter molecule) and manipulation of the NO signal is shown to enhance muscle repair and promote growth in older mice in combination with exercise. SCs up‐regulate mRNA expression of a neural chemorepellent, semaphorin 3A (Sema3A) that peaks 6–8 days after muscle injury. Since Sema3A expression is implicated in regulating the timing of axon growth toward regenerating fibers, we hypothesized that premature activation of SC before an injury would advance Sema3A synthesis and disrupt the process of reinnervation. The time‐ course of reinnervation in regenerating tibialis anterior muscle (TA) of 6–10‐week‐old C57BL6 mice was examined after injury by crush (a large traumatic injury) or injection of cardiotoxin (CTX, which damages only fibers). We tested whether early activation of SC before injury using a 2‐day daily pre‐treatment with isosorbide dinitrate (ISDN, an NO‐donor drug) affects the process of new‐fiber innervation post‐injury. Muscle was collected 0, 4, 6, 8, 10, and 14 days after unilateral TA injury (n=4–5 per time point in crush, CTX, and respective control groups). The position of the nuclei and the length of the newly‐formed myotubes were used to track muscle regeneration on coded slides. CTX‐injured muscles had noticeably more advanced formation of neuromuscular junctions (NMJs) and faster fiber regeneration compared to crush‐ injured muscles at the same time point post‐injury. Combined staining of NMJs for acetylcholinesterase (AchE, silver staining) and acetylcholine receptors (AchRs, α‐bungarotoxin epifluorescence) on coded slides, revealed immature or mature NMJs (positive for AchE and AchRs) by day 14 in untreated crush‐ or CTX‐injured muscles, respectively. ISDN pre‐treatment in both crush‐ and CTX‐injured muscles delayed NMJ development on myotubes up to day 14 post‐injury. Results are consistent with the action of Sema3A as a neural chemorepellent, since SC activation before injury resulted in delayed myotube reinnervation. Therapeutic manipulation of Sema3A during regeneration, in time and/or concentration, may have potential to improve reinnervation and muscle function in conditions where nerve‐muscle interactions are disrupted by injury or disease.

Support or Funding Information

Supported by a NSERC Discovery grant (to JEA) and a University of Manitoba Graduate Fellowship (to ND)
Effect of a Carbohydrate Mouth Rinse on Plasma Glucose and Plasma Insulin during a 40‐km Cycling Time Trial
Authors: Murray Kevin O
Published: November 2023

Rinsing the mouth with a carbohydrate solution, without ingesting, improves exercise performance of up to an hour in duration. Performance gains are attributed to the activation of brain regions associated with motivation and motor control. In addition to this neural response, a hormonal cascade may also be present, and include a cephalic‐phase insulin response from oral carbohydrate recognition.

PURPOSE

The aim of this study was to examine whether exercise performance gains achieved through a carbohydrate mouth rinse are the partial result of a cephalic‐phase insulin response from oral carbohydrate recognition.

METHODS

Eight trained, competitive cyclists [age (mean ± SEM) = 24 ± 2 y; VO2max = 64.5 ± 2.8 ml/kg/min] completed three simulated, 40‐km time trials comprised of a familiarization trial, carbohydrate mouth rinse trial (CHO) and placebo trial (PLA). Mouth rinse was administered prior to onset of exercise, and every 5‐km throughout exercise. During CHO and PLA trials, plasma glucose and plasma insulin samples were obtained immediately prior to mouth rinsing procedures. Metabolic data and cycling time were collected throughout the exercise session. CHO oxidation rates were calculated based on the equation: g/min = 4.210 VCO2 – 2.962 VO2, while fat oxidation rates were calculated based on: (g/min) = 1.695 VO2 – 1.701 VCO2 (Jeukendrup 2005).

RESULTS

Time to completion was faster for CHO (67.1 ± 1.1 sec) compared to PLA (67.9 ± 1.0 sec). Plasma glucose did not differ between conditions (p = 0.801) and held steady throughout exercise; additionally, respiratory exchange ratio was not statistically different between conditions (p = 0.081). There was no change in plasma insulin either within trials or between trials (p = 0.638). CHO oxidation rates were not statistically different (p = 0.06) between the CHO (3.43 ± 0.80 g/min) and PLA (2.98 ± 0.18 g/min) groups; fat oxidation rates were not statistically different (p = 0.10) between the CHO (0.59 ± 0.09) and PLA (0.76 ± 0.06) groups.

CONCLUSIONS

Rinsing the mouth with a carbohydrate solution prior to exercise and after each 5‐km interval improved 40‐km time trial performance. This finding shows that improved exercise performance occurs independent of changes in plasma glucose, plasma insulin, RER, or substrate oxidation rates. These findings build on previous research to support the notion of a CHO mouth rinse having primarily a neurological response (Chambers 2009), and not acting via a cephalic‐phase insulin response, in order to improve exercise performance.

Support or Funding Information

Indiana University – School of Public Health Student Research Grant Award ‐ March 2016 ($1000)

BK Betty Stevens Award (UF funding for trip to Experimental Biology 2017)
Mathematical Modeling of Oxidative Phosphorylation Predicts in vivo Phosphorus Kinetics
Authors: Lewis Matthew T , Kasper Jonathan D , Bazil Jason N , Meyer Ronald A , Wiseman Robert W
Published: November 2023

Mitochondrial density is a key contributor to exercise capacity. Diseases such as diabetes and age related illnesses lower mitochondrial densities in skeletal muscle and result in a mismatch between energy supply and demand. The precise relationship between mitochondrial energetics and cell metabolism for these conditions is not well described. To address this, a mathematical model of oxidative phosphorylation and in vivo measurements of muscle energetics were used to analyze the role that mitochondrial density has in cellular energy homeostasis during exercise. The mathematical model simulated a broad range of phosphocreatine (PCr) dynamics to mimic exercise at differing intensities in both normal muscle and where mitochondrial densities were reduced through computation. In parallel, experiments were performed on control and chemically‐treated rats using 31Phosphorus Magnetic Resonance Spectroscopy (PMRS). Rat hindlimb muscles were stimulated via electrode implantation of the sciatic nerve with concurrent PMRS and mechanical force acquisition. This setup allows for quantification of contractile ATP costs, mitochondrial ATP synthesis, PCr recovery rates, and intracellular pH. The model accurately simulates the in vivo exercise data in both control and reduced mitochondrial density animals. As the mitochondrial density is decreased, the model correctly predicts a decrease in steady state PCr levels for a given stimulus intensity and an increase in the time to replenish PCr levels after exercise. This result is indicative of decreased mitochondrial ATP production capacity as inferred by model analysis. These results demonstrate the ability of this model to simulate PCr dynamics in Wistar rat skeletal muscle for a range of exercise intensities irrespective of their mitochondrial content. The model serves as a valuable tool to gain insight into future experimental approaches to study the relationship between mitochondrial energetics and various pathological conditions associated with exercise intolerance such as heart failure, COPD and diabetes.

Support or Funding Information

Supported by NIH DK 095210, NIH R00HL121160.
Shortening Heat In Cardiac Muscle
Authors: Tran Kenneth
Published: November 2023

The ‘heat of shortening’ in muscle was first demonstrated by Fenn (1923). Tectanically‐contracting skeletal muscle, released to shorten at a fixed afterload, was shown to produce heat at a rate in excess of the steady‐state isometric heat rate. Subsequent studies in the following decades have provided ample evidence to support the existence of shortening heat in skeletal muscle. The situation is much less clear in cardiac muscle. Using the ‘latency release’ method, Holroyd and Gibbs (1992) showed that no heat was liberated when cardiac muscle was shortened against zero afterload. Adding legitimacy to the latency release protocol, its use showed the existence of shortening heat in both fast‐twitch and slow‐twitch hind‐limb muscles of the rat. (Holroyd et al., 1996). Since that time, the existence of shortening heat in cardiac muscle seems not to have been further investigated, leaving the field in the curious situation that shortening heat is present in skeletal muscle but not in cardiac muscle, despite their micro‐structural similarities. Since it must be imagined that shortening heat is the thermal accompaniment of cross‐bridge cycling, and its attendant thermogenic ATP hydrolysis, we undertake a re‐examination of the issue. We do so using two independent approaches.

Firstly, we quantify shortening heat in isolated preparations (right ventricular trabeculae carneae) from rat hearts. Trabeculae were dissected from the left ventricles of 14–16 week old male Sprague‐Dawley rats and mounted in a flow‐through microcalorimeter at 32 °C, as previously described (Taberner et al., 2011). The muscle was subjected to a series of steady‐state trains of isotonic contractions at progressively diminishing afterloads, and isometric contractions at progressively diminishing muscle lengths. Shortening heat is defined as the difference in twitch heat between these two modes of contractions at a given afterload. Secondly, experimental findings are complemented by predictions of cross‐bridge ATP hydrolysis from a thermodynamically‐constrained, mathematical model of cardiac muscle mechano‐energetics (Tran et al., 2010). In both cases we exploit the difference of enthalpy‐load relations between isometric and isotonic contractions, while ensuring that comparisons are performed at the same peak force.

Our experimental results confirm the existence of shortening heat in rat trabeculae. The magnitude of shortening heat was inversely related to the afterload and was a monotonically‐increasing function of the ‘extent of shortening’. Computational simulations reveal that the heat of shortening arises from the cycling of cross‐bridges, and that, even at very low afterloads where no external work is done, there is always a component of energy that is dissipated as heat.
Nitric Oxide Participation in Thermoregulatory Adjustments During Physical Exercise in Spontaneously Hypertensive Rats
Authors: Drummond Lucas Rios , Campos Helton Oliveira , Pires Washington , de Andrade Lima Paulo Marcelo , Horta Nayara Abreu Coelho , Prímola‐Gomes Thales Nicolau , Coimbra Cândido Celso
Published: November 2023

This study aimed to investigate the nitric oxide participation in thermoregulatory adjustments during physical exercise in spontaneously hypertensive rats. All experimental procedures were approved by the Ethics Committee on Animal Use at Universidade Federal de Minas Gerais (# 21/2015). Eight male Wistar rats (SAP = 119 ± 3 mmHg) and spontaneously hypertensive rats (SHR; SAP = 195 ± 7 mmHg) with 16 weeks of age were used in the experiments. The rats received an implant of a chronic guide cannula in the lateral cerebral ventricle and a temperature sensor into the abdominal cavity. After recovery, the animals were familiarized to run on a treadmill, and then, each animal was subjected to two constant‐speed exercise protocols at 60% of maximal speed attained in the incremental exercise until fatigue. On the day of the experiments, the rats were assigned to receive a 2 μL intracerebroventricular injection of either NaCl (0.15 mol/L) or sodium nitroprusside (SNP; 8.4 mmol) setting four groups, Wistar SAL, Wistar SNP, SHR SAL and SHR SNP. The tail skin temperature (Tskin) and abdominal temperature (Tabd) were recorded every minute throughout the exercise trials. The Tskin and Tabd dynamics were evaluated by the temperature variation (temperature in each minute ‐ the initial temperature). In Wistar groups, the SNP group showed a greater increase in Tskin from the 14th min until the fatigue (3.12 ± 0.33°C vs. 1.60 ± 0.47°C, fatigue; p < 0.05) with the lower increase in Tabd when compared with SAL group from the 18th min until the fatigue (1.28 ± 0.16°C vs. 1.71 ± 0.26°C; fatigue, p < 0.05). In SHR groups, the SNP group showed a greater increase in Tskin from the 8th until the 19th min (1.58 ± 0.83°C vs. −0.40 ± 0.88°C, 15th min; p < 0.05) and lower increase in Tabd when compared with SAL group from the 23th min until the fatigue (1.09 ± 0.13°C vs. 1.70 ± 0.18°C; fatigue, p < 0.05). SHR groups performed a decreased workload during exercise (SAL: 200.1 ± 23.2 J vs. 277.0 ± 27.1 J, p < 0.05; SNP: 206.0 ± 23.2 J vs. 299.0 ± 27.1 J, p < 0.05) when compared with Wistar groups. The SNP injection did not modify the workload performed during the exercise in Wistar and SHR groups (p > 0.05). We conclude that central injection of sodium nitroprusside increased heat dissipation, resulting in a lower increase in abdominal temperature during exercise in Wistar and SHR rats, without modify physical performance.

Support or Funding Information

Financial Support: FAPEMIG, CAPES and CNPq.
Major Metabolic Hormone Responses to Exertional Heat Stroke in Mice
Authors: Robinson Gerard Patrick , King Michelle , Mattingly Alex , Garcia Christian , Laitano Orlando , Van Steenbergen David , Leon Lisa , Clanton Thomas
Published: November 2023

Disordered glucose metabolism has been shown to be a strong prognostic indicator of poor outcomes in heat stroke. In mice, within the first few hours of recovery from exertional heat stroke (EHS), glucose is reduced; however, by 24 h and for up to 4 days, sustained hyperglycemia has also been observed. To gain perspective on the underlying mechanisms that contribute to these responses we looked at circulating metabolic hormones responsible for glucose regulation. Male mice (n= 8, per group) ran in forced running wheels within an enclosed climatic chamber at 37.5°C/35% relative humidity (RH) until loss of cognitive function (approx. 2 h). Animals were sacrificed at 0.5h, 3h, 24h, 4d, 9d or 14d post‐EHS. Plasma samples were collected and metabolic hormones analyzed using Luminex multiplex technology or ELISA (corticosterone). Hormones secreted by the pancreas, amylin (p=0.02), c‐peptide (p=0.003), and insulin (p=0.003) were markedly suppressed at 0.5 h and continued to be suppressed at 3 h. Expected glucagon responses were absent during this period. Cytokines that influence glucose metabolism or uptake, IL‐6 and MCP‐1, displayed a significant increase at 0.5 and 3 h (p=0.0004, p=0.0008 respectively), with peak concentrations appearing at 0.5 h. Resistin (secreted by white adipose) was also significantly elevated at 0.5 h (p=0.003) and then decreased to low levels at 3h (p<0.002). Corticosterone was significantly elevated at the 0.5 h (p=0.0015) and 3 h time points (p=0.0009). These results may reflect, in part, hypoglycemia seen following exercise in the heat but are also consistent with transient organ dysfunction, specifically in the liver (i.e. failure to elevate glucose) and/or pancreas (suppression of insulin secretion without compensating glucagon response). Author views not official US Army or DoD policy. Supported by the Department of Defense W81XWH‐15‐2‐0038
Exercise training reduces TGF‐β mediated epithelial mesenchymal transition in diabetic kidney
Authors: Ren Lu , Sen Utpal , Pushpakumar Sathnur
Published: November 2023

Transforming growth factor‐β (TGF‐β)‐induced activation of Smad2 and Smad3 is implicated in epithelial mesenchymal transition (EMT) and development of glomerular and interstitial fibrosis in diabetic nephropathy. Sirtuin1 (Sirt1), a deacetylase has been shown to reduce TGF‐β induced mesangial cell apoptosis to improve cell survival and also in the attenuation of TGF‐β‐induced renal damage in subtotal nephrectomized rats. Exercise training can affect acetylation of protein residues and counteract systems impairment. Exercise has recently been shown to regulate Sirt1 activity in skeletal muscles. However, it is not known whether Sirt1 regulates TGF‐β signaling in diabetic nephropathy to reduce EMT and thus fibrosis. The purpose of this study was to investigate whether exercise training in type‐II diabetes attenuates TGF‐β‐induced EMT by upregulation of Sirt1 activity. BKS.Cg‐Dock7m +/+ Leprdb/J and Dock7m +/+ Leprdb mice representing type‐II diabetes and non‐diabetic heterozygous control respectively were used in the study. Mice were exercised on a treadmill using an established protocol for 5 weeks. Mice were initially accustomed to exercise at 7m/min and gradually increased to 11m/min for a distance of 300m every day for 5d/week. A 10 min rest period was allowed after every 100m. Sirt1 expression was decreased in diabetic mice compared to non‐diabetic controls. Diabetic mice also exhibited upregulation of TGF‐β and Smad2, 3. mRNA and protein expression of E‐cadherin and claudin‐6 was suppressed and alpha‐smooth muscle actin (α‐SMA) and fibroblast‐specific protein‐1 (FSP1) was upregulated in diabetic mice compared to controls. Moderate intensity exercise reversed the above changes to enhance Sirt1, reduce TGF‐β and Smad2, 3 and α‐SMA and FSP‐1 in diabetic mice. Exercise training was also associated with decreased collagen IV and fibronectin in diabetic kidney. In conclusion, our data suggests that moderate intensity exercise prevents the progression of renal disease by enhancing Sirt1 and regulation of TGF‐β‐induced EMT in type‐II diabetes.

Support or Funding Information

15SDG25840013 to SP, DK104653 to US, HL104103 to US