bluuebunny/crossref_metadata_2025_split huggingface dataset:
Are C1 Neurons Involved in the Sympathetic Overactivity Induced by Chronic Intermittent Hypoxia?
Authors: Moraes Davi J.A. , Bonagamba Leni G.H. , Paton Julian F.R. , Machado Benedito H. 
Published: November 2023

Chronic intermittent hypoxia (CIH) produces expiratory‐related sympatho‐excitation in rats, which might contribute to the development of hypertension. We evaluated the role of sympatho‐excitatory cathecolaminergic medullary C1 neurons in the CIH‐induced expiratory‐related sympatho‐excitation and hypertension in rats. In awake rats in vivo and in in situ perfused preparations of rat, C1 neurons were acutely silenced by application of the insect peptide allatostatin following cell‐specific targeting with a lentiviral vector to express the inhibitory Drosophila allatostatin receptor in control and CIH rats (10 days). In awake rats in vivo, inhibition of ~ 72% of the C1 neurons resulted in a profound and similar fall in arterial pressure and heart rate in control (n=12) and CIH rats (n=12). However, CIH rats still presented elevated arterial pressure after C1 inhibition (p<0.05). In in situ, C1 inhibition resulted in reversible reductions of perfusion pressure, amplitude of inspiratory‐related bursts of thoracic sympathetic nerve and C1 pre‐sympathetic neurons activities in control (n=7) and CIH (n=9) rats. However, CIH‐ (n=9), acute hypoxia‐ (n=7) or CO2‐induced expiratory‐related sympatho‐excitation (n=10) were not affected by C1 inhibition. These data confirm a physiological role of C1 neurons in generating baseline sympathetic activity and arterial pressure. However, C1 neurons apparently are not involved in the expiratory‐related sympatho‐excitation and hypertension induced by CIH.Support or Funding InformationFAPESP and CNPQ
Dysregulation of the Energy‐sensing AMPK Pathway in Skeletal Muscle‐specific ERRα−/− Mice
Authors: Huss Janice , McDonald Marisa , Hamilton Angie 
Published: November 2023

The AMP‐activated protein kinase (AMPK) pathway is involved in both acute control and long‐term metabolic regulation in skeletal muscle. AMPK is activated during exercise and is required for many of the adaptive responses to endurance exercise, mediating its effects via transcription factors that control metabolic gene programs. We previously showed that the Estrogen‐related Receptor α (ERRα) is activated after an exercise bout and in response to AICAR, a pharmacologic AMPK activator. The muscle‐specific ERRα−/− mice (M‐ERRα−/−) exhibit a blunted metabolic response to endurance training. The aim of the present study is to test the hypothesis that AMPK signaling may be altered in ERRα‐deficient muscles and thus contribute to the exercise phenotype in these mice. M‐ERRα−/− and M‐ERRαWT (WT) mice were subjected to a single treadmill exercise bout (1hr) or an acute (30 min) AICAR treatment. M‐ERRα−/− muscles had reduced basal activity of both AMPK and its upstream kinase, LKB1, as assessed by western detection of the phosphorylated proteins. Despite reduced baseline activity, AMPK activation in response to acute exercise was maintained in the M‐ERRα−/− mice. Using IP‐activity assays we showed that AMPKα2 isoform specific activity was reduced at baseline in M‐ERRα−/− white vastus compared to WT. However, AMPKα2 was more robustly stimulated in the M‐ERRα−/− muscle in response to AICAR treatment (6.2‐fold, M‐ERRα−/− vs.2.3‐fold, WT). This is consistent with the greater reduction in blood glucose 30 min post‐AICAR administration observed in the M‐ERRα−/− mice, resulting from AMPK‐stimulated glucose uptake. Analysis of transcripts involved in AMPK signaling revealed that LKB1 and AMPKβ2 isoforms were consistently downregulated in ERRα‐deficient muscles or with ERRα inhibition in myocytes. We demonstrated that the regulatory regions of the Stk11 and Prkab2 genes that encode these transcripts, are directly regulated by ERRα in transient transfection experiments in myocytes. Thus, our data suggest that ERRα is involved in regulating AMPK pathway activity in skeletal muscle which can alter the responsiveness of the pathway to energetic and pharmacologic stimulation.Support or Funding InformationSupport by NIH R01DK74700 and Diabetes and Metabolic Research Institute, City of Hope..
Transcriptional Regulation of Skeletal Muscle Atrophy‐induced Gene Expression by Muscle‐Specific RING Finger 1 (MuRF1)
Authors: Kakareka Karina , Waddell David 
Published: November 2023

Skeletal muscle wasting is a consequence of numerous physiological conditions, including denervation, corticosteroid treatment, immobilization, and aging. The E3 ubiquitin ligases, MuRF1 and MAFbx, are induced under nearly all atrophy conditions and are believed to play a key role in protein degradation in atrophying muscle. However, the preliminary data described in this study provides new evidence that MuRF1 may also act as a transcriptional modulator of neurogenic atrophy‐induced gene activity, including the regulation of MAFbx and MuRF1 expression. To characterize the transcriptional regulation of MuRF1 and MAFbx, reporter gene constructs containing fragments of the proximal promoter regions of these genes were developed, transfected into C2C12 cells with or without a MuRF1 expression plasmid and monitored for differences in reporter gene activity. The MuRF1 and MAFbx reporters each showed repressed activity in cells ectopically expressing MuRF1 compared to cells that did not overexpress MuRF1. Furthermore, ectopic expression of the myogenic regulatory factors (MRF), MyoD1 and myogenin, caused significant activation of the MuRF1 and MAFbx reporter constructs. However, co‐overexpression of MuRF1 with MyoD1 or myogenin resulted in reversal of MRF induction of reporter gene activity. To further characterize the role of the MuRF1 gene product in repression of MuRF1 expression, a MuRF1 RING domain mutant was created. The RING mutant construct was then co‐transfected along with MRF expression plasmids and the MuRF1 or MAFbx reporter gene into C2C12 cells and reporter gene activity was assessed. The MuRF1 RING mutant failed to reverse MRF activation of the reporter gene, suggesting that ubiquitin ligase activity is required for MuRF1 transcriptional regulatory effects. To further investigate the potential regulation of MRFs by MuRF1, Western blot analysis was performed in order to determine the impact on MRF protein levels in C2C12 cells in response to overexpression of MuRF1. Cells with ectopic expression of MuRF1 showed reduced levels of myogenin protein. In addition, cells overexpressing MuRF1 were treated with MG132, a general proteasome inhibitor, followed by Western blot analysis. The results of the Western blot revealed that cells treated with MG132 alone had increased myogenin protein levels, while cells ectopically expressing MuRF1 still exhibited reduced myogenin levels. MuRF1 repression of myogenin expression, with or without 26S proteasome inhibition, suggests that MuRF1 may be acting by a previously uncharacterized mechanism to regulate MRF expression, providing additional support that MuRF1 may act as a transcriptional regulator of atrophy‐induced gene expression. These data offer exciting evidence of a potential new function for MuRF1 as a transcriptional modulator of atrophy‐mediated changes in gene expression.Support or Funding InformationUniversity of North Florida Transformational Learning Opportunity Grant
K (Lysine) Acetyltransferase 6B (Kat6B) is Upregulated during Neurogenic Skeletal Muscle Atrophy and is Differentially Expressed in MuRF1‐null Mice
Authors: West Rita , Waddell David 
Published: November 2023

Skeletal muscle atrophy is characterized by a decrease in muscle size and strength and results from a range of physiological conditions, including denervation, corticosteroid exposure and aging. The K (lysine) acetyltransferase 6B (Kat6B) gene, also known as Myst4, has been identified as a novel gene in skeletal muscle and was found to be differentially regulated in wild‐type mice compared to Muscle RING Finger 1 (MuRF1) knockout mice in response to denervation. Muscle RING Finger 1 (MuRF1), an E3 ubiquitin ligase, has previously been shown to be upregulated during almost all conditions of muscle wasting and is believed to play an important role in targeting proteins for degradation. However, preliminary data described in this study provides new evidence that MuRF1 may also act as a transcriptional modulator of atrophy‐induced gene activity, including the regulation of Kat6B. The Kat6B gene currently has two known and validated isoforms and 8 additional putative isoforms in mice. Microarray data revealed that Kat6B expression is significantly induced at 3 days and 14 days post‐denervation in wild‐type mice, while Kat6B expression is dramatically blunted in MuRF1‐null mice at both 3 days and 14 days post‐denervation. The Kat6B gene codes for a histone acetyltransferase that is believed to be part of the MOZ/MORF protein complex and is thought to be involved in transcriptional regulation. Interestingly, Kat6B has been shown to be required for Runt‐related transcription factor 2 (Runx2) dependent transcriptional activation. Runx2 codes for a transcription factor that plays an important role in bone and cartilage development and is closely related to Runx1. Runx1 is significantly upregulated in response to neurogenic skeletal muscle atrophy and has been shown to be required to sustain muscle mass and integrity following an atrophy‐inducing event. The results presented here in combination with additional research into the transcriptional regulation and function of Kat6B will help contribute new insight into the molecular genetic mechanisms of skeletal muscle atrophy and could provide a more complete understanding of how MuRF1 may transcriptionally regulate neurogenic atrophy‐induced gene expression during muscle wasting.Support or Funding InformationUniversity of North Florida Transformational Learning Opportunity Grant
Mouse Cachectic Skeletal Muscle mTORC1 Signaling in Response to Acute Eccentric Contractions
Authors: Hardee Justin P , Gao Song , VanderVeen Brandon N , Fix Dennis K , Hetzler Kimbell L , Carson James A 
Published: November 2023

Eccentric contraction (EC) induced skeletal muscle hypertrophy involves the activation of protein synthesis through mTORC1 signaling. Cancer cachexia disruption of muscle protein turnover regulation is well established, but whether increased use regulates suppressed muscle protein synthesis warrants further investigation. We recently demonstrated severely cachectic muscle can initiate a growth response to repeated ECs. However, the EC activation of mTORC1 signaling in cachectic muscle has not been investigated. The purpose of this study was to determine if mTORC1 signaling and protein synthesis in cachectic muscle from 2 distinct preclinical cancer cachexia models could respond to EC. Male, ApcMin/+ (N=5; 15% body weight loss) and Lewis Lung Carcinoma (LLC) tumor‐bearing (N=8; 14% body weight loss) mice performed 1 bout (10 sets of 6 repetitions) of EC and were sacrificed 3h post‐contraction. The left TA performed EC while the right TA served as intra‐animal control. C57BL/6 (WT) mice served as controls. In control muscle ApcMin/+ and LLC mice had decreased muscle mass, p70S6K(T389) phosphorylation, and protein synthesis compared to WT controls. Cachexia did not inhibit the acute EC‐induction of p70S6K(T389) phosphorylation in ApcMin/+ or LLC mice. While EC induced protein synthesis in ApcMin/+ mice, protein synthesis remained suppressed compared to WT mice. Interestingly, EC did not stimulate muscle protein synthesis in LLC mice. Although cachexia suppressed muscle anabolic signaling, cachectic muscle maintains the ability to activate mTORC1 signaling by EC. However, in LLC‐induced cachexia there is a disconnect between mTORC1 activation and the induction of protein synthesis. Further studies are required to determine the tumor specific effects that regulate the anabolic response to eccentric contractions in cachectic muscle.Support or Funding InformationSupported by NIH/NCI R01‐CA121249
Proteotyping For Drug Development: Proteomics Impacting Clinical Practice
Authors: Van Eyk Jennifer , Holewinski Ronald , Wang Shengbing , Kirk jonathan , Kass David 
Published: November 2023

The road to medical translation starts with understanding of an individual's history and disease status via the quantification of proteins and their disease‐induced modified forms. Each individual's proteome reflects both their cellular memory and current health status and should allow prediction of their response to environment and therapeutic treatment. Thus, precision medicine maybe feasible but it will require the ability of specific drug targeting at the level of an individual. One example is the targeting of drugs to activate the protein kinase G (PKG) pathway, which in animal studies have been shown to blunt the development of heart failure. Drugs that inhibit the breakdown of cGMP (thus, activating PKG) have had less than expected therapeutic efficacy in numerous clinical trials. We have recently shown that there are multiple unanticipated levels of regulation of the PKG pathway that influence which subproteome is affected by PKG that ultimately leads to varied functional consequences. This includes an individuals’ redox status and heart failure type (e.g heart failure with preserved ejection fraction) that should affect the effectiveness of the current PKG‐targeted drugs. Similarly, resynchronization therapy, one of the few clinically used treatments for heart failure where dual pacemakers are implanted to ensure coordinated cardiac pumping, activates GSK 3 beta altering the contractile subproteome. This kinase is also influenced by the redox status and the oxidation of GSK 3 beta changes its downstream targets and potentially the functional outcome. Methods to assess individual's status prior to treatment selection will increase efficacy and patient selection as well alteration to their basal/pretreatment status could increase an individual's response to therapy.Support or Funding InformationP01 HL77189‐01, R01HL119012, NHLBI‐HV‐10‐05 (2)
Downregulation of myocardial pigment epithelium‐derived factor in mice exposed to cigarette smoke: A possible mechanism for smoking‐related cardiovascular disease
Authors: McHowat Jane , Kuenzel Hannah , Kispert Shannon , Marentette John , Kolar Grant 
Published: November 2023

Cigarette smoking is estimated to cause 10% of cardiovascular disease with approximately 6 million deaths per year worldwide. Despite the well‐established correlation between smoking and cardiovascular disease, many fundamental questions regarding underlying mechanisms remain unanswered. We have shown that smoking is associated with platelet‐activating factor (PAF) accumulation, an inflammatory metabolite that is implicated in ischemia/reperfusion injury and heart failure progression. PAF enhances matrix metalloproteinase (MMP) activation, which may contribute to downregulation of pigment epithelium‐derived factor (PEDF), a glycoprotein with anti‐inflammatory and antioxidative properties that can protect against endothelial damage, platelet aggregation and T –cell activation. We hypothesized that PAF accumulation and/or PEDF downregulation may directly contribute to smoking‐induced cardiovascular disease. We exposed mice to cigarette smoke for up to 6 months and measured biochemical and histological changes in the heart. Smoking resulted in increased calcium‐independent phospholipase A2 activity (3.46 ± 0.21 to 5.76 ± 0.64 nmol/mg protein/min, p<0.01, n=4) and decreased PAF acetylhydrolase activity (2.65 + 0.06 to 1.48 + 0.02 nmol/mg protein/min), changes that indicate increased PAF synthesis and decreased inactivation. Smoking was associated with increased MMP‐2 expression and downregulation of PEDF expression in the heart by immunoblot analysis. Immunohistochemical analysis demonstrated that the majority of PEDF was associated with cardiac myocytes and was reduced in animals exposed to cigarette smoke. Following exposure to cigarette smoking, cardiac vessels exhibited thickening of the vessel walls, particularly in the smooth muscle layer. Changes in the vessel wall were similar to those observed in PEDF knockout mice. These are the first data to implicate PEDF downregulation in smoking‐related cardiovascular disease.Support or Funding InformationSaint Louis University Doisy Research Fund
Phosphorylation and Calcium Antagonistically Tune Myosin‐binding Protein C's Molecular Structure and Function
Authors: Previs Michael J , Mun Ji Young , Michalek Arthur J , Previs Samantha Beck , Gulick James , Robbins Jeffrey , Warshaw David M , Craig Roger 
Published: November 2023

During each heartbeat, calcium activates the sliding of actin‐thin filaments towards the centers of myosin‐thick filaments to shorten the overall length of cardiac muscle cells. Cardiac myosin binding protein C (cMyBP‐C) tunes these interactions throughout the contractile cycle. cMyBP‐C's C terminus is strongly bound to the thick filament backbone and its N‐terminal domains extend away and transiently interact with actin and/or the myosin S2 domain, sensitizing thin filaments to calcium and governing maximal sliding velocities. Both mechanisms are tunable by phosphorylation of 4 serines within an extensible, intrinsically disordered region of cMyBP‐C's N‐terminus, the M‐domain. Does phosphorylation impact the structure and molecular mechanics of cMyBP‐C's N terminus to tune it's function? Atomic force spectroscopy studies of N‐terminal fragments (domains C1–C2), showed that phosphorylation reduced the M‐domain's contour length and extensibility. Rotary shadowing electron microscopy showed that M‐domain phosphorylation caused N‐terminal fragments (C0–C3) to shift from an extended, rod‐like structure to a compact conformation. Taken together, M‐domain phosphorylation and its impact on cMyBP‐C's N‐terminal structure suggest a mechanism for tuning cMyBP‐C's function in motility assays. Interestingly, we found that free calcium (0.1 mM), necessary to fully activate the thin‐filament, mitigated the structural effects of phosphorylation by increasing the free‐extensibility of the phosphorylated M‐domain and shifting the phosphorylated N‐terminal fragments back to the extended, rod‐like state, as if unphosphorylated. Functionally, even though phosphorylation reduced cMyBP‐C's ability to inhibit actin filament sliding velocity in the motility assay (24% vs. 40% inhibition), addition of calcium ablated the impact of phosphorylation, fully restoring cMyBP‐C's inhibitory capacity. We conclude that phosphorylation of MyBP‐C's M‐domain may have its greatest effect on cardiac contractility by tuning cMyBP‐C's ability to sensitize actin‐thin filaments to calcium at the low levels present during the onset of contraction. Importantly, calcium levels at the peak contraction would allow cMyBP‐C to remain a potent contractile modulator, regardless of the cMyBP‐C's phosphorylation state.Support or Funding InformationNIH grants HL124041 and HL059408 supported these studies.
Effect of Hypertrophic Cardiomyopathy‐Associated R282W Mutation of MYBPC3 on M‐domain Phosphorylation in vitro
Authors: Giuffre Allison Mary , Lynch Thomas L. , Sadagopan Nandhini , Sadayappan Sakthivel 
Published: November 2023

Hypertrophic cardiomyopathy (HCM) is a disease of the myocardium characterized by left ventricular hypertrophy and contractile dysfunction. HCM is the most common inherited cardiac disease and is caused by mutations in sarcomeric proteins. Importantly, mutations in MYBPC3, the gene encoding cardiac myosin binding protein‐C (cMyBP‐C), account for 40% of all HCM cases. A missense mutation causing the amino acid substitution R282W within the M‐domain of cMyBP‐C has been previously discovered in HCM patients. This mutation directly alters the protein kinase A (PKA) recognition motif for phosphorylation of the human cMyBP‐C Ser‐284. Indeed, previous studies show that phosphorylation at the homologous murine Ser‐282 residue is a prerequisite for subsequent phosphorylation of the murine Ser‐273 and Ser‐302 residues. Given the importance of cMyBP‐C phosphorylation of these residues in regulating myofilament cross‐bridge kinetics and contraction, the R282W mutation may alter cMyBP‐C phosphorylation within the M‐domain. Therefore, we determined the effect of the R282W M‐domain mutation on cMyBP‐C phosphorylation status. We hypothesized that this mutation diminishes cMyBP‐C phosphorylation at human Ser‐284. To test the hypothesis, H9C2 cells were infected with adenovirus containing cDNA encoding full‐length human WT or R282W cMyBP‐C. After 48 hours of infection, cells were treated with or without isoproterenol, to activate PKA. Western blot analysis of total cell lysates from H9C2 cells expressing R282W cMyBP‐C demonstrated a significant reduction in phosphorylation of Ser‐284, compared to WT cMyBP‐C. In conclusion, these data suggest that the R282W mutation decreases cMyBP‐C phosphorylation at Ser‐284, which may contribute to contractile dysfunction in HCM.Support or Funding InformationThis research was funded by the American Physiological Society.
Essential roles of ANO1 in mouse urethral smooth muscle contraction and urinary continence
Authors: Feng Mei , Liu Donghai , Lu Ping , Bellve Karl , Lifshitz Lawrence M , Fogarty Kevin , ZhuGe Ronghua 
Published: November 2023

Smooth muscle, the major cell type of urethra, generates sufficient intraurethral pressure to prevent voiding of urine from the bladder; its dysfunction contributes to stress urinary incontinence, a pathological disorder affecting up to 30% of women and causing physical discomfort and emotional distress. However, the cellular and molecular mechanisms underlying the contraction of urethral smooth muscle is not well understood. In the present study, we report that mouse urethral smooth muscle expresses type 2 ryanodine receptors, L‐type Ca2+ channels, and the Ca2+‐activated Cl− channel ANO1. In response to a load, mouse urethral strips relaxed and then developed a spontaneous tone. This tone was blocked by ryanodine receptor antagonist ryanodine, ANO1 antagonist benzbromarone, and L‐type Ca2+ channel inhibitor nifedipine; this tone was absent in the urethral strips from smooth muscle specific ANO1 deletion mice. Ryanodine receptor agonist caffeine contracted mouse urethral strips dose‐dependently. This contraction was inhibited by ryanodine, nifedipine, benzbromarone and ANO1 deletion. Caffeine increased global [Ca2+]i in isolated single urethral smooth muscle cells, and this increase in [Ca2+]i was inhibited by ryanodine, benzbromarone and nifedipine. In ANO1 deleted cells, caffeine caused less increase in [Ca2+]i compared to the wild type cells, and this rise in [Ca2+]i was resistant to benzbromarone and nifedipine. Caffeine induced Ca2+‐activated Cl− currents in wild type cells, and failed to do so in the ANO1 deletion cells. The size of urine stains was greater while the number of stains was smaller in wild type mice than in ANO1 deletion mice, implying ANO1 deletion may impair urinary continence. We conclude that ANO1 is functionally coupled with ryanodine receptors and L‐type Ca2+ channels, and this functional coupling is required for spontaneous tone and agonist‐induced contraction in urethra, and its disruption could lead to urinary incontinence.Support or Funding InformationNIH NIDDK098586