bluuebunny/crossref_metadata_2025 huggingface dataset:
Treatment of Hypertensive Obese Zucker Rats with Metformin Enhances Phenylephrine‐Induced Bradycardia and c‐Fos Expression in the NTS
Authors: Chaudhary Parul , Schreihofer Ann
Published: November 2023

With the progression of metabolic syndrome adult obese Zucker rats (OZR) develop impaired baroreflexes that coincide with a reduced ability to activate the nucleus tractus solitarius (NTS). The NTS, which receives baroreceptor afferent inputs, appears to respond less to acute rises in mean arterial pressure (MAP) as suggested by less phenylephrine (PE)‐induced c‐Fos expression compared to age‐matched lean Zucker rats (LZR). In addition microinjection of glutamate into the NTS produces smaller reductions in splanchnic sympathetic nerve activity and MAP in adult OZR versus LZR. We have recently observed that prevention of hypertension improves baroreflexes in OZR, but they are still impaired compared to like‐treated LZR. Because hyperglycemia impairs NTS function and baroreflexes in type I diabetic rats, we hypothesized that improvement of glycemia in OZR would also enhance baroreflexes and the ability to activate the NTS. OZR and LZR were treated with metformin (MET; 300 mg/kg/day in drinking water) for 3 weeks (began at 13 weeks of age) and compared to untreated age‐matched rats. Blood samples were taken in conscious, unfasted rats at 9–10 am. Compared to untreated OZR, MET‐treated OZR had lower blood glucose levels (179±19 vs. 120±8 mg/dl, P<0.05) with no difference between untreated and treated LZR (119±6 vs. 122±6 mg/dl). In contrast, MET did not alter plasma insulin levels (in ng/ml) in either LZR or OZR (5.5±2.1 vs. 2.3±0.7 in LZR and 23±0.1 vs. 19.0±4.7 in OZR). In conscious rats fitted with indwelling femoral catheters and tethers with swivels, MAP was elevated in OZR compared to LZR (136±4 vs. 120±2 mmHg, P<0.05), and these differences persisted in OZR and LZR treated with MET (140±2 vs. 118±4 mmHg, P<0.05). Infusion of PE (iv) to raise MAP by 40 mmHg evoked a smaller bradycardia in untreated OZR vs. LZR (−52±7 vs. −120±7 bpm, P<0.05). Treatment with MET enhanced PE‐induced bradycardia in OZR (−84±12 bpm, P<0.05) but not LZR (−135±12 bpm), and MET‐treated OZR were still impaired compared to MET‐treated LZR (P<0.05). Likewise, PE‐induced c‐Fos expression in the intermediate NTS (total bilateral counts at bregma −14.2 and −13.8 mm) was reduced in OZR compared to LZR (70±8 vs. 117±9, P<0.05), and MET treatment increased PE‐induced c‐Fos expression in OZR (170±18, P<0.05) but not LZR (154±23). These data suggest treatment with MET improves glycemia in OZR without significantly altering plasma insulin or MAP levels in either OZR or LZR. Further, these data suggest that improved glycemia in OZR partially restores baroreflex‐mediated changes in heart rate coincident with an enhanced response of the NTS to an acute rise in MAP. Further study is needed to determine whether combined treatment to prevent hypertension and improve glycemic control in OZR will normalize baroreflexes and NTS function.

Support or Funding Information

AHA GRNT18880005.
Infusion of Losartan into the 4th Ventricle Reduced Cardiac Sympathoexcitation in Heart Failure
Authors: Abukar Yonis , May Clive N
Published: November 2023

Background and Aims

A key feature of heart failure (HF) is an increase in cardiac sympathetic nerve activity (CSNA). We have found that the area postrema (AP), a brainstem circumventricular organ, plays an important role in controlling CSNA and in determining the increase in CSNA in heart failure. We hypothesized that in sheep with HF, 4th ventricular infusions of losartan (angiotensin II type 1 receptor antagonist) will reduce CSNA to a greater extent than infusion into the cisterna magna.

Methods

In sheep (n=6), when ejection fraction (EF) had fallen to ~50%, cannulae were implanted for infusion into the 4th ventricle and cisterna magna. Blood pressure, heart rate, CSNA & renal SNA were recorded when EF had fallen to <40%. Losartan (1.0 mg/h) or artificial cerebrospinal fluid (aCSF) (1.0 mL/h) were infused into the 4th ventricle and cisterna magna for 5 h, at two days after nerve recording electrodes were surgically implanted.

Results

In sheep with HF, there was a significant decrease in EF (from 81 ± 3 % to 37 ± 1 %, P <0.001). During 4th ventricle infusion of losartan, there was a significant decrease in CSNA (89 ± 3 to 48 ± 8 bursts/100 heartbeats, P<0.01). Infusion of losartan into the cisterna magna caused a smaller decrease in CSNA (85 ± 2 to 74 ± 4 bursts/100 heartbeats, P<0.05). Infusion of losartan into the 4th ventricle caused no significant changes in renal SNA or in other parameters. Infusion of aCSF into the 4th ventricle or cistrena magna had no effects.

Conclusions

Infusion of losartan into the 4th ventricle reduced the elevated CSNA in sheep with HF. The findings suggest that angiotensin type 1 receptors in the brainstem have an important role in determining the increased CSNA in heart failure.
High inorganic phosphate diet augments the sympathetic response to exercise pressor reflex activation in rats
Authors: Mizuno Masaki , Crawford Scott , Carnahan Cooper , Mitchell Jere H , Smith Scott A , Vongpatanasin Wanpen
Published: November 2023

Inorganic phosphates are widely used in the food industry as preservatives, flavor enhancers, and color stabilizers. Evidence suggests high inorganic phosphate (Pi) diets induce hypertension in normal rats. Whether alterations in sympathetic function contribute to this manifestation of high blood pressure is unknown. Accordingly, we measured changes in renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) during stimulation of the muscle exercise pressor reflex (EPR) in decerebrated Sprague‐Dawley rats fed a normal (0.6%) or high (1.2%) Pi diet for 12 weeks. Compared to normal Pi diet‐fed rats, a high Pi diet significantly increased 24‐hour urine excretion of phosphate (12±1 vs. 77±5 mg/day, P<0.05) as well as resting MAP (94±5 vs. 120±5 mmHg, P<0.05). There were no differences in sodium and potassium excretion between groups. Comparing control rats to those fed a high Pi diet, contraction‐induced stimulation of the EPR evoked significantly greater increases in RSNA (Δ = 46±9 vs. 126±25 %, P<0.05) and MAP (Δ = 14±2 vs. 44±5 mmHg, P<0.05) in rats fed a 1.2% Pi diet. The data provide the first evidence that excess phosphorus intake induces sympathetic overactivity in response to EPR activation. This finding suggests that the high dietary Pi commonly observed in the normal American diet may lead to sympathetic dysfunction contributing to the pathogenesis of hypertension.

Support or Funding Information

Supported by NIH HL‐113738
Effect of Renal Denervation and Celiac Ganglionectomy on Mean Arterial Pressure in the Hypertensive Schlager (BPH/2J) Mice
Authors: Gauthier Madeline M , Breitenstein Claire , Asirvatham‐Jeyaraj Ninitha , Osborn John W
Published: November 2023

Renal denervation (RDNx) lowers mean arterial pressure (MAP) in a number of models of hypertension. Less well studied is the effect of celiac ganglionectomy (CGx), a procedure that has proven equally effective at lowering MAP in some animal models. The mechanism of action of both surgical interventions remains unknown, however evidence suggests RDNx may affect the presence of inflammatory markers in the kidneys. In this study, we hypothesized that RDNx and CGx would both lower MAP in genetically hypertensive Schlager (BPH/2J) mice and that this would correlate with a decrease in sympathetic activity and a decrease in proinflammatory cytokines. BPH/2J mice underwent radiotelemeter implantation for monitoring of MAP followed by a 7–10 day convalescence period, after which control data was collected for 2 days. Next, mice were randomly subjected to one of three treatments: RDNx, CGx, or sham. MAP and heart rate were recorded for 14 days post‐operatively. Baseline MAP in the three groups was similar (~130mmHg). On post‐operative day 14, MAP in both RDNx (−13 ± 2 mmHg) and CGx (−8 ± 0.3 mmHg) decreased, but was not affected in sham controls (−6 +/− 10 mmHg). The change in heart rate (beats/min) was not different between groups 14 days after surgery (−24 ± 4 in sham, −21 ± 33 in RDNx and −41 ± 18 in CGx). To assess the effect of RDNx on renal cytokines in BPH/2J hypertension, tissues were collected from RDNx and sham‐treated animals two weeks post‐treatment. Tissues were then analyzed by quantitative real time polymerase chain reaction (qRT‐PCR). RDNx decreased mRNA expression of the proinflammatory cytokines interleukin 1 beta and tumor necrosis factor alpha in BPH/2J mice 2 weeks after surgery. Future studies will explore the link between targeted sympathetic ablation, inflammation and change in mean arterial pressure in the hypertensive Schlager mice.

Support or Funding Information

This study was supported in part by the National Institute of Health 5RO1HL11647602 to J.W.O. Other grant support: R01‐HL 067357‐15 and Undergraduate Research Opportunities Program
Impaired Cyclic Nucleotide Signalling in Pre‐Hypertensive Spontaneously Hypertensive Rats: A Dichotomy of Pathways?
Authors: Bardsley Emma , Paterson David J
Published: November 2023

Background

Spontaneously hypertensive rats (SHRs) and young pre‐hypertensive SHRs (pre‐SHR) have enhanced cardiac sympathetic activity; a feature underpinned by abnormal calcium‐dependent exocytosis. The crosstalk between cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in peripheral sympathetic stellate neurons is poorly understood.

Aim

We used Förster Resonance Energy Transfer (FRET) to test whether there is a difference in cAMP and protein kinase A (PKA) activity in post‐ganglionic sympathetic neurons (PGSNs) obtained from 4‐week pre‐SHR compared with normotensive controls. Furthermore, we sought to determine whether activation of the cGMP‐PKG signalling pathway modulates cytosolic cAMP concentrations and its effector PKA, in sympathetic stellate neurons obtained from Wistar and pre‐SHR.

Method

Cardiac stellate neurons were isolated from male Wistar rats and pre‐SHR at 4‐weeks. On the day of culture, stellates were infected with either cAMP or PKA reporter adenovirus particles for 24 hours, and FRET experiments were carried out 3–4 days post‐isolation. Data were obtained from neurons expressing FRET biosensors using dual‐emission imaging, where YFP and CFP emission intensities were measured. During imaging, cells were perfused continuously with Tyrode's, or stimulated with forskolin (0.1–25μM) and saturating concentrations of 3‐isobutyl‐1‐methylxanthine (IBMX; 100μM). In alternative experiments, neurons were perfused with 8‐bromoguanosine (8‐br‐cGMP; 100μM) prior to saturation with forskolin (25μM) and IBMX (100μM). All raw percentage FRET changes were subsequently normalised to the maximal FRET change at saturation.

Results

There was no significant difference between cAMP or PKA activity in response to forskolin administration in 4‐week stellates obtained from Wistar and pre‐SHR; however, cAMP and PKA responses to 8‐br‐cGMP were significantly different in PGSNs from the two strains. In neurons obtained from pre‐SHRs there was no change in PKA in response to 8‐br‐cGMP. Conversely, in Wistar stellate neurons, administration of the cGMP analogue facilitated a rapid increase in PKA activity (66%). Here we show that the responses to 8‐br‐cGMP results in a dichotomy in cyclic nucleotide signalling pathways in PGSNs from young Wistar versus pre‐SHR neurons. These findings suggest that the interaction between the two cyclic nucleotide signalling pathways may differ in PGSNs from the two phenotypes, prior to the onset of hypertension; either as a result of differential phosphodiesterase (PDE) profiles, or physiological uncoupling in response to diverse signalling microdomains.

Support or Funding Information

This work was supported by the Wellcome Trust Ion Channels and Disease (OXION) Initiative
The connectome of rostral ventrolateral medulla C1 neurons
Authors: Le Sheng , Dempsey Bowen , Turner Anita , Allen Andrew , Goodchild Ann , McMullan Simon
Published: November 2023

The transcription factor Phox2b is co‐expressed on a large proportion of rostral ventrolateral medulla (RVLM) sympathetic premotor neurons. This population is thought to play a key role in the transmission of vasomotor sympathetic drive and represents a major site of convergence for multiple descending and reflex pathways that co‐ordinate sympathetic nerve activity. Elucidating the organization of the circuits that control these neurons is a key research objective. Here we present brain‐wide connectomic maps of neurons that provide monosynaptic drive to RVLM C1 neurons, generated using a two‐step restricted trans‐synaptic viral tracing strategy. Phox2b expressing C1 neurons were first transduced with a lentiviral vector to express a cassette containing the cognate avian receptor TVA and rabies G‐protein. TVA‐expressing neurons were then selectively infected with a reporter‐expressing rabies variant, SADΔG (EnvA)‐RFP, which retrogradely and trans‐synaptically labelled neurons that provide monosynaptic drive to Phox2b‐positive neurons.

By precisely targeting rabies injections to the core of the RVLM we were able to restrict rabies ‘seeding’ to small numbers of RVLM C1 neurons immediately caudal to the facial nucleus, and then map the locations of neurons that provide monosynaptic input to the seed population. We observed reproducible patterns of inputs arising from the dorsal, contralateral, and midline medulla, and local RVLM interneurons and neurons likely to reside within the Bötzinger, pre‐Bötzinger, and ventral respiratory groups. Distant inputs were identified in the pons and midbrain, and included previously suspected sites of monosynaptic drive such as the paraventricular nucleus of the hypothalamus.
Maternal protein intake as a determinant of the total number of motor units and muscle fibres in mice
Authors: Vasilaki Aphrodite , Giakoumaki Ifigeneia , Pollock Natalie , Goljanek‐Whysall Katarzyna , McArdle Anne , Sayer Avan Aihie
Published: November 2023

Age‐related sarcopenia is characterized by reduction in the number of myofibres and an additional weakening of the remaining fibres, resulting in a reduction in muscle mass and function. Studies associate these changes with poor maternal malnutrition. However, very little is known about the effect of the maternal protein intake on the number of motor units in offspring. The aim of this study was to examine the effect of maintaining females on a low‐protein diet on the number of muscle fibres in offspring and whether any changes can be modulated by optimization of the nutrition in early post‐natal stages. Studies were undertaken in Thy1‐YFP16 mice that express yellow fluorescent protein only in neuronal cells. Female mice were fed either a normal (20%) or a low‐protein (5%) diet. Newborn pups were cross‐fostered to different lactating dams (maintained on 20% or 5% diet) within 24 h after birth, generating 4 groups: Normal‐to‐Normal (N‐N), Normal‐to‐Low (N‐L), Low‐to‐Normal (L‐N) and Low‐to‐Low (L‐L). Pups were culled at weaning (21 days) by cervical dislocation and the body weight recorded. One EDL was removed and snap frozen, sectioned and the total number of fibres counted. To visualize the neuromuscular junctions (NMJs), the second EDL muscle was removed and fixed in 4% p‐formaldehyde. Muscles were incubated for 30‐min with 5 μg/mL Alexa 594‐conjugated a‐Bungarotoxin (BTX).

Data showed that mice born from dams on 20% diet but fed post‐natally by a foster dam on 5% diet (N‐L) show significant reductions in body size and body weight whereas mice born from dams on 5% diet but fed post‐natally by a dam on 20% diet (L‐N) did not show such changes when compared with pups from dams on 20% diet throughout the in utero and post‐natal periods [(N‐N); N‐N: 6.6±0.3g vs. N‐L: 3.56±0.15g)]. Muscle fibre cross sectional areas (CSAs) and innervation and the total number of muscle fibres were reduced in the N‐L compared with the N‐N group whereas in the L‐N only the total number of muscle fibres where affected (N‐N: 1450±51 vs. N‐L: 1231±67 vs. L‐N: 1270±51). Mice born from dams on 5% diet and fed post‐natally by a dam on a 5% diet (L‐L) show a reduced body size, body weight, muscle size and muscle fibre CSAs compared with the N‐N but these reductions were not as profound as in the N‐L group. Thus, data suggests that, in mice, poor maternal protein intake, either only in utero or only post‐natally, results in a significant loss of muscle fibres and that interventions post‐natally are able to reverse the detrimental effects in utero.

Support or Funding Information

Supported by the British Physiological Society
FGGY Carbohydrate Kinase Domain Containing is Upregulated during Neurogenic Skeletal Muscle Atrophy
Authors: De Las Casas Bella , Waddell David
Published: November 2023

Skeletal muscle atrophy results from a range of physiological conditions, including denervation, corticosteroid exposure and aging. In order to better characterize the molecular genetic events of neurogenic atrophy, the gastrocnemius muscle was isolated from mice following 3 days and 14 days of sciatic nerve denervation. The gene expression profile in the denervated muscle tissue was then analyzed by microarray and compared to control muscle tissue in order to identify novel neurogenic atrophy‐induced genes. The microarray data revealed for the first time that FGGY carbohydrate kinase domain containing (Fggy) is expressed in skeletal muscle and is significantly induced in response to denervation. Bioinformatic analysis of the Fggy gene locus in mice reveals two validated alternative variants with distinct transcription initiation sites. In order to confirm that Fggy is expressed in muscle cells, the cDNA of the two validated alternative splice variants of Fggy were cloned from the C2C12 mouse myoblast cell line. The results presented here in combination with additional research into the transcriptional regulation and function of Fggy will help contribute new insight into the molecular mechanisms of skeletal muscle atrophy. Furthermore the identification and characterization of novel genes that are activated during neurogenic atrophy helps improve our understanding of the molecular and cellular events that lead to muscle wasting and could eventually lead to new therapeutic targets in the future.

Support or Funding Information

University of North Florida Transformational Learning Opportunity Grant
Low‐Frequency Electrical Stimulation (Acu‐LFES) attenuates denervation‐induced muscle atrophy by increasing muscle regeneration
Authors: Wang Xiaonan H , Klein Janet D. , Wang haidong
Published: November 2023

Muscle wasting occurs in a variety of clinical situations including denervation. There is no effective pharmacologic treatment for muscle wasting. In this study, we used a tibial nerve denervation model to test acupuncture plus low frequency electric stimulation (Acu‐LFES) as a therapeutic strategy for muscle atrophy. Acupuncture needles were connected to an SDZ‐II electronic acupuncture device delivering pulses at 20Hz and 1mA, the treatment is 15 minutes daily for 2 weeks. Acu‐LFES prevented soleus and plantaris muscle weight loss and increased muscle cross‐ sectional area in denervated mice. The abundance of Pax7, MyoD, myogenin and embryo myosin heavy chain was significantly increased by Acu‐LFES in both normal and denervated muscle. The number of central nuclei was increased in Acu‐LFES treated muscle fibers. Phosphorylation of Akt was downregulated by denervation leading to a decline in muscle mass; however, Acu‐LFES prevented the denervation‐induced decline by upregulation of the IGF‐1 signaling pathway. Acu‐LFES reduced the abundance of muscle catabolic proteins forkhead O transcription factor and myostatin, contributing to the attenuated muscle atrophy. Acu‐LFES stimulated the expression of macrophage markers (F4/80, IL‐1b and arginase‐1) and inflammatory cytokines (IL‐6, IFNg and TNFa) in normal and denervated muscle. Acu‐LFES also stimulated production of the muscle specific microRNAs miR‐1 and miR‐206. We conclude that increased muscle regeneration is associated with the Acu‐LFES sparing of muscle mass in denervation‐induced skeletal muscle atrophy. Upregulation of IGF‐1, downregulation of myostatin and alteration of microRNAs contribute to Acu‐LFES‐induced myogenesis.

Support or Funding Information

NIH/NIAMS: R01 AR060268
Sarcolipin is Upregulated in Response to Soleus Unloading Caused by Tenotomy and Opposes Muscle Atrophy
Authors: Fajardo Val Andrew , Rietze Bradley A , Chambers Paige J , Quadrilatero Joe , Tupling A. Russell
Published: November 2023

Previous studies have shown that sarcolipin (SLN), a small inhibitor of the sarco(endo)plasmic reticulum Ca2+‐ATPase (SERCA) pump, is upregulated in conditions of muscle atrophy. To date, however, the role of SLN in muscle atrophy remains unknown. In another unpublished study from our laboratory, we mechanically overloaded plantaris muscles by performing soleus and gastrocnemius tenotomy (surgical transection of the tendon), and found that SLN is critical in mediating muscle growth and fiber type remodeling. In this study we questioned the role of SLN in the tenotomized soleus, which is a model of muscle unloading known to cause muscle degeneration, atrophy, and a fast‐glycolytic muscle phenotype. We hypothesized that in the absence of Sln, soleus muscles would exhibit greater muscle atrophy and an accelerated slow‐to‐fast fiber type shift. Our results show that 2 weeks after tenotomy, SLN protein was upregulated 14‐fold (P ≤ 0.05) in WT muscle compared with sham. Furthermore, removing this 14‐fold upregulation led to a greater percent reduction in soleus muscle mass relative to body weight in SlnKO mice compared with WT (WT, −51.0 ± 3.5% vs. SlnKO, −59.0 ± 1.8%, P ≤ 0.05). In addition, significant reductions in myofiber cross‐sectional area were found in WT mice (−29% to −38%, P ≤ 0.05, two‐way repeated ANOVA), and also in SlnKO mice, but perhaps to a greater extent (−33% to −42%, P ≤ 0.05, two‐way repeated ANOVA). With respect to the slow‐to‐fast fiber type shift, the WT mice exhibited a partial shift in response to tenotomy with a significant decrease and trending increase in type IIA (P ≤ 0.05) and type IIX (P = 0.10) fiber percent, respectively. In contrast, in response to tenotomy, SlnKO mice exhibited an accelerated slow‐to‐fast fibre type shift with a significant decrease in type I (P ≤ 0.05) and increase in type IIX (P ≤ 0.05) fiber percent, respectively. Since calcineurin stabilizes the deleterious effects of reduced mechanical loading, we examined the phosphorylation status of its well‐known substrate nuclear factor of activated T‐cell (pNFAT). Our results show that, in response to tenotomy, WT soleus muscles had significantly lower pNFAT compared with sham (−57%, P ≤ 0.05); however pNFAT was not different between SlnKO sham and tenotomized soleus muscles. Thus, our results show that SLN upregulation in tenotomized soleus muscles is required to counteract muscle atrophy and the transition to the fast, glycolytic fiber phenotype, and may do so through its stimulatory effect on calcineurin.

Support or Funding Information

This work was supported by the Canadian Institutes of Health Research (CIHR; MOP 86618 and MOP 47296 to A.R.T).
Spatiotemporal Organization Of Signaling: From Plasma Membrane To Chromatin
Authors: Gingras Anne‐Claude
Published: November 2023

Compartmentalization is essential for all complex forms of life. In eukaryotic cells, membrane‐bound organelles, as well as a multitude of protein‐ and nucleic acid‐rich subcellular structures, maintain boundaries and serve as enrichment zones to promote and regulate protein function. Consistent with the critical importance of these boundaries, alterations in the machinery that mediate protein transport between these compartments has been implicated in a number of diverse diseases. Signaling molecules are no exception, and must be targeted to specific locations for proper activation in time and place.

Understanding the composition of each cellular “compartment” (be it a classical organelle or a large protein complex) remains a challenging task. For soluble protein complexes, approaches such as affinity purification other biochemical fractionation coupled to mass spectrometry provides important insight, including on the temporal regulation of signaling, but this is not the case for detergent‐insoluble components. Classically, both microscopy and organellar purifications have been employed for identifying the composition of these structures, but these approaches have limitations, notably in resolution for standard high‐throughput fluorescence microscopy and in the difficulty in purifying some of the structures (e.g. p‐bodies) for approaches based on biochemical isolations. Prompted by the recent implementation in vivo biotinylation approaches such as BioID, we have begun the systematic mapping of the composition of various subcellular structures, using as baits proteins (or protein fragments) which are well‐characterized markers for a specified location. We report here our low‐resolution map of a human cell (currently defined from BioID profiling of 100 marker proteins), including various membrane compartments, cytoskeletal structures and nuclear subdomains. We will also introduce a higher resolution map of RNA‐containing cellular structures, including the p‐bodies and the stress granules that regulate mRNA stability, and new data on signaling pathways that span insoluble compartments. We demonstrate in vivo biotinylation to be a scalable approach capable of revealing protein complexes as well as their association into larger structures.

Support or Funding Information

Work in the Gingras lab was primarily supported by the Canadian Institutes for Health Research (Foundation grant) and the Natural Sciences and Engineering Research Council of Canada (Discovery grant). Additional funding was provided by Genome Canada through Ontario Genomics.
Overexpression of Muscle Ring Finger 1 Reduces Mitochondrial Volume in Cardiomyocytes
Authors: Parikh Chirag , Kobayashi Satoru , Gerdes Martin , Willis Monte S , Liang Qiangrong
Published: November 2023

Objectives

Muscle Ring Finger 1 (MuRF1) is an ubiquitin E3 ligase that participates in atrophy of cardiac myocytes, and when upregulated, increases risk of dilated cardiomyopathy. While many studies have shown the regressive atrophy that MuRF1 participates in, more recent studies also indicate that increased expression of MuRF1 results in broad disruption of metabolic functions in cardiac tissues and in particular, mitochondria. However, it is unclear what role MuRF1 plays with mitochondrial regulation. Since the energy requirement in cardiac muscle cells is high, mitochondrial homeostasis is tightly regulated by multiple processes which include fusion, fission, mitophagy and biogenesis. This study aimed to determine whether MuRF1 plays a role in regulation of these processes by examining the morphological changes of mitochondria and their relationship with cell viability.

Methods

H9C2 Cardiomyoblast cell line was pretreated with an adenovirus‐encoded mitochondrial‐targeting protein, mtRosella, a construct that results in fluorescence of mitochondria, allowing examination of morphology and mitophagy. Following this, these cells were infected with an adenovirus encoding MuRF1 to increase the protein expression levels of MuRF1. Change in MT morphology was determined with confocal microscopy. Morphological descriptors (count, average size, major and minor axes) were measured using ImageJ analysis software to describe changes in the degree of mitochondria size, fragmentation, and mitophagy. Additionally, propidium iodide staining was used in an identical setup to determine if MuRF1 overexpression affects cell viability.

Results

Morphometric analysis demonstrated that overexpression of MuRF1 in H9C2 cells did not change the count of mitochondria, but decreased the size of mitochondria. Both major and minor axes were decreased in the overexpression group. In addition, mitophagy levels did not differ in the overexpression group versus the control. Finally, propidium iodide staining did not show an increase in cell death in the overexpression group, a finding that agrees with other study findings. Further study is needed to determine if MuRF1 affects mitochondrial biogenesis to reduce the mitochondrial size.

Conclusion

Overexpression of MuRF1 is sufficient to reduce mitochondrial size but it does not have an effect on mitochondrial number and mitophagy. The reduced mitochondrial size may reduce the overall capacity of mitochondrial metabolic function and increase the sensitivity of the cardiomyocyte or the heart to pathological stresses such as ischemia and chemotherapy.

Support or Funding Information

NIH Grant 1R15HL120027‐01A1
Diet Restriction Exacerbates Memory Loss in Alzheimer's Disease Drosophila melanogaster Model Flies
Authors: Damschroder Deena Jan , Paddock Brie
Published: November 2023

Major risk factors for the development of Alzheimer's disease (AD) include aging and diabetes. Although among the top ten leading causes of death in the U.S., there is still no cure for AD (Ho, 2004). It is believed that diet restriction (DR) and caloric restriction (CR) increase lifespan due to the fact that they slow the aging process and oxidation process, two processes which contribute to AD (Cline, 2000). It is unclear whether the extension of lifespan in humans and animal models corresponds with a delay in degeneration associated with aging (Burger et al 2010). This study uses the aversive phototaxis assay (Ali et al., 2011) to examine how diets consisting of reduced sucrose and reduced yeast affect the memory in a Drosophila model of AD. Our results support the hypothesis that nutrient availability plays a key role in the pathogenesis of AD, but that CR is not necessarily protective against neuronal deterioration.

Support or Funding Information

College of Arts and Sciences, Arcadia University
Serotonin Type 3 Receptor B Subunit (5‐HT3BR) and Corticotropin‐Releasing Hormone (CRH) Expression in the Serotonin Transporter Knockout (SERT KO) Rat Model of Visceral Hypersensitivity
Authors: Mecca Andrew , El‐Ayache Nadine , Galligan James
Published: November 2023

Background

Visceral hypersensitivity is a chronic abdominal pain symptom present in functional gastrointestinal disorders such as irritable bowel syndrome (IBS). IBS is a global health issue, and is more prevalent in females than in males suggesting a potential role of female sex hormones in its pathogenesis. Some drug treatments for IBS target the ionotropic serotonin type 3 receptor (5‐HT3R), but these drugs have limited clinical benefit and can be associated with serious side effects. Therefore, a deeper understanding of the pathophysiology of visceral hypersensitivity is crucial for the development of more effective treatments. Environmental stress and altered serotonergic signaling may influence the severity of visceral hypersensitivity in IBS patients. Corticotropin‐releasing hormone (CRH) is an important mediator of the neuroendocrine stress response, but its role in IBS needs to be further studied.

Aim/Methods

We studied the expression of CRH and the B‐subunit of the 5‐HT3BR in the lumbar spinal cord, colon and lumbar dorsal root ganglia (DRG), in a serotonin‐transporter knockout (SERT KO) rat model of visceral hypersensitivity using immunohistochemistry and real‐time quantitative PCR.

Results

Female SERT KO rats have higher proportion of CRH+ neurons in DRG than male SERT KO rats (P < 0.01), and CRH was most strongly expressed by small‐diameter nerve cell bodies that are likely to be involved in the pain pathway. There was no difference in the distribution of 5‐HT3BRs in the dorsal spinal cord or colon between SERT KO and WT animals. Similarly, there was no difference in CRH+ neurons in the spinal cord, and no difference in CRH expression in the colon between SERT KO and WT. However, we found a 10‐fold and 6‐fold increase in CRH mRNA expression in female and male SERT KO rats, respectively, compared with wildtype controls (P < 0.05). Lastly, preliminary data show that there is no difference in the mRNA transcript for the 5‐HT3BR in DRG between SERT KO and WT rats.

Conclusions

We found a novel sex‐specific increase in CRH expression in female SERT KO animals at the level of primary afferent neurons at the protein and mRNA levels. We found no difference in the distribution of the 5‐HT3BR between SERT KO and WT in the colon, DRG and spinal cord. Our results support our hypothesis that environmental stress mediated by CRH may influence visceral hypersensitivity in IBS.
Analysis of Inflammatory Modulation on Term and Preterm Neonatal Gastrointestinal Non‐Human Primate Explants
Authors: Alana Nicholas B , Acevedo Steven J , Carr Nick J , Mustafa Shamimunisa B , Blanco Cynthia L , King Jonathan M
Published: November 2023

BACKGROUND

Dysregulation of inflammatory processes are associated with intestinal injury, necrotizing enterocolitis, and risk of perforation in the newborn. A reduction of Epidermal Growth Factor (EGF) and impaired function of the Epidermal Growth Factor Receptor (EGFR), have been demonstrated in the preterm gastrointestinal system compared to term. Prostaglandin E2 (PGE2) as well as EGF have independently been shown to have a role in the barrier maintenance, recovery from injury, and regulation of perfusion.

OBJECTIVE

Evaluate the regulation of EGF/EGFR/PGE2 pathway in a non‐human primate gastrointestinal explant. Additionally evaluating the roles of inflammatory and anti‐inflammatory agents on this pathway.

DESIGN/METHODS

Tissue explants were created from gastrointestinal regions (ileum through colon) upon necropsy of term and preterm (67% gestation) non‐human primates. Explants were evaluated in conditions of control, pro‐inflammatory (TNF 10 ng/ml), and anti‐inflammatory (indomethacin 50uM) variables. Absolute and relative prostaglandin secretion (PGE2); mRNA expression of Prostanoid (EP) receptor subtypes, EGFR, EPS8, AKT1, AKT2, and MAPK1 were used as markers of EGF/PGE2 pathway regulation at 24 hours post exposure.

RESULTS

PGE2 secretion significantly increases in term colon compared preterm colonic secretion. In addition, the term gastrointestinal explants are significantly more resistant to indomethacin inhibition of PGE2 secretion, noted across ileum, cecum, and colon. There were no significant differences noted in EP receptor subtype expression. Preterm expression of EGFR and its downstream effectors are elevated when compared to term ileum explants. However, there is markedly more variability in response noted to TNF and indomethacin in the term explants when compared to preterm ileum explants.

CONCLUSIONS

A positive gradient of PGE2 secretion in the distal gastrointestinal regions is observed in term explants likely correlated to the advanced differentiation status compared to the preterm. In addition, the up regulation of EGFR along with overall increased expression of downstream targets in the preterm explant is consistent with the expected mitogenic status. However, the decreased response to TNF and indomethacin, suggest a limited ability to modulate inflammation.
Tachykinin Activation on Enteric Glia: A Novel Mechanism of Enteric Nervous System Dysfunction in Irritable Bowel Syndrome
Authors: Delvalle‐Dorta Ninotchska Michelle , Rivera‐Lopez Gretchen , Gulbransen Brian
Published: November 2023

The Enteric Nervous System (ENS) is the major neural regulator of the gastrointestinal (GI) tract. Enteric circuits involve precise interactions between neurons and glia and abnormal ENS activity is considered a key contributor to gut motility disorders like irritable bowel syndrome (IBS). However, the role of neuron‐glia interactions in functional GI motility disorders is not understood. Tachykinins are important excitatory neurotransmitters in the ENS that contribute to IBS pathophysiology. In the spinal cord, substance P (SP) and neurokinin A (NKA) release by hyperactive neurons drives reactive gliosis. We hypothesize that the activation of enteric glia by tachykinins plays an important role in neuroinflammation during IBS. We tested our hypothesis using immunohistochemistry (IHC) and calcium imaging recordings in tissue from 5–8 week old C57BL/6 male mice. Our results show that both NKA and SP are present in varicosities that surround enteric glia in the myenteric plexus. We found that this population of glial cells primarily express neurokinin‐2 receptors (NK2R) (n=3 animals) which preferentially bind NKA over SP. NK1R labeling in the myenteric plexus was primarily neuronal. However, we could not exclude the possibility of expression on glia. We did not find evidence of NK3R expression in the myenteric plexus. We tested the breadth of responsiveness of myenteric glia to tachykinins such as SP and NKA in whole‐mount preparations loaded with the calcium indicator dye Fluo‐4. Our results show that enteric glia produce robust calcium transients in response to stimulation with NKA but not to SP (n=62 glial cells from 3 animals). To determine if tachykinin receptor activation contributes to neurodegeneration during inflammation, we stimulated enteric glia with either SP or NKA and measured neuronal packing density within myenteric ganglia. Our results show that treatment with NKA significantly reduces neuronal survival (p=0.0416, n=4 animals) while treatment with SP had no effect (p=0.7171, n=3 animals). In conclusion, our results provide strong evidence that the activation of glial NK2Rs by NKA is a novel mechanism of neuroinflammation in the ENS.

Support or Funding Information

T32NS44928‐11 and RO1DK103723
Arsenic (As) and Copper (Cu) Serve as Cofactors to Produce Hepatotoxicity With Mitochondrial Damage, Double Strand DNA Breaks and Disruption of ATM Signaling
Authors: Chinnasamy Thirunavukkarasu , Sharma Yogeshwar , Gupta Priya , Gupta Sanjeev
Published: November 2023

Despite increasing exposure of populations to As the role of cofactors in organ toxicity, e.g., reactive Cu ions, is poorly defined. Since As and Cu ions may combine to increase oxphos events and processes directing mitochondrial and cellular DNA damage, we studied metal toxicity in cultured adult and fetal human hepatocytes with IC50 concentrations of As in MTT assays. In even nontoxic conditions by itself Cu dose‐dependently worsened As toxicity. This worsening included alterations related to oxphos activity with mitochondrial and nuclear perturbations (JC‐1 dye assays, acridine orange nuclear stain, 8‐OHdG stain), double strand DNA breaks (Comet assay, γH2AX stain), and replicative stress including loss of cycling cells and/or arrest in G2/M (Ki‐67staining, flow cytometry). In addition, verapamil‐sensitive side populations denoting stem cells were depleted by As toxicity. Use of cells stably transduced lentivirally to express hATM promoter construct with tdT reporter showed that Cu exacerbated cellular DNA damage by perturbing ATM signaling pathways. To further establish whether Cu accumulation may also worsen As toxicity in vivo, we studied Atp7b−/− mice that model Wilson's disease (WD) and accumulate Cu in liver due to absence of hepatobiliary Cu excretion, along with their healthy littermates. After As was given i.p. for 10d, liver necrosis increased significantly in Atp7b−/− mice (H&E tissue staining, serum hyaluronic acid levels). Moreover, we noted increased ATM pathway‐dependent DNA strand breaks (Comet assay, γH2AX stain). This As and Cu toxicity was ameliorated in human hepatocytes cultured with an anti‐oxidant, indole‐3 propionic acid (IPA), as shown by MTT, JC‐1 and hATM promoter assays. Similarly, IPA decreased As toxicity in Atp7b−/− mice.

Conclusions

Cu ions markedly exacerbated As toxicity with mitochondrial and ATM pathway‐regulated DNA damage. These findings will help understand how toxic metals may alter physiological processes and contribute additionally in human diseases, e.g., WD with Cu toxicosis.
Effects of a Broad‐Spectrum Antibiotic on the Siberian Hamster (Phodopus sungorus ) Gut Microbiome
Authors: St. John Emma A. , Sylvia Kristyn E. , Demas Gregory E.
Published: November 2023

The gut microbiome is a diverse, host unique, and symbiotic bacterial environment. Host antibiotic treatment can adversely affect the diversity of the bacterial population and the symbiotic relationship with the host. Although therapeutically beneficial, antibiotics can change the structure, function, and evolution of host microbial communities in the gut, affecting nutrient availability and predisposing infection in the gut by non‐communal species (Archie & Theis, 2011). Previous studies have shown the effects of antibiotics on the host's gut microbiome in many model species, including mice and rats (reviewed in Clarke, O'Mahony, Dinan, & Cryan, 2014), yet, in non‐model species, such as the Siberian hamster, the role of the microbiome and the effects that antibiotics have on gut microbial communities have yet to be fully explored. Studying non‐model species is particularly valuable because it enables us to make meaningful comparisons between various organisms and to understand the strategies they use to cope with environmental pressures. In order to study the various effects of antibiotics on the microbiome and ultimately the consequences it has on behavior in our species, we first had to determine if and how antibiotic treatment affects the gut microbiome of both male and female Siberian hamsters. In order to assess this question, male (n=12) and female (n=12) hamsters were either administered sterilized water or a broad‐spectrum antibiotic (0.3 μl of enrofloxacin (Baytril) 10% oral solution per gram of body mass) via sterile pipette orally once daily for seven days (Romick‐Rosendale et al., 2009). Body mass was monitored, and fecal samples were taken throughout the seven days before treatment (D1–7), the seven days during treatment (D8–14), and the seven days following the treatment period (D15–21). Microbiome analysis was completed to determine the effects of this broad‐spectrum antibiotic on the gut microbiome. At the end of the experiment, all animals were euthanized and organs were harvested and weighed. To determine the bacteria makeup of the gut microbiome, DNA was extracted and purified from the fecal material, and the V4 region of the 16S rRNA gene was amplified using the Illumina MiSeq platform to identify the bacteria present in each sample. Antibiotic treatment decreased the diversity of the microbial communities, and by the end of the recovery period (D21), the host gut microbiome was able to regain much of its microbial diversity. There were no significant differences in body mass, and additionally, the liver, spleen, and reproductive organs showed no significant difference in mass at the end of the study. The results of this work help us to advance our understanding of the gut microbiome's function in host communities, and they will allow us to further investigate the role that the microbiome plays in the gut‐brain axis in non‐model species.
Cardiorespiratory Fitness as a Predictor of Intestinal Microbial Diversity and Distinct Metagenomic Functions
Authors: Estaki Mehrbod , Pither Jason , Baumeister Peter , Little Jonathan P , Gill Sandeep , Ghosh Sanjoy , Ahmadi‐Vand Zahra , Marsden Katelyn R , Gibson Deanna L
Published: November 2023

Background/Objective

An imbalance between harmful and beneficial bacteria in the gut, termed dysbiosis, has been associated with a variety of illnesses. In specific, reduced community diversity has been implicated in various conditions such as inflammatory bowel disease, colorectal cancer, and irritable bowel syndrome amongst others. The role of physical fitness in the context of gut microbial community in humans is currently not known. A recent study of elite athletes suggested a positive association between fitness and microbial diversity in humans, however due to extreme dietary differences amongst the groups, disentangling the respective influences of fitness versus diet was not possible. Hence, we analyzed the gut microbiota of healthy subjects with varying cardiorespiratory fitness (CRF) levels with similar diets in order to isolate the relationship between fitness and microbial composition.

Methods

22 males and 19 healthy females of similar age, BMI, and diets participated in this study. The subjects’ aerobic capacities were determined using a standard peak oxygen uptake (VO2peak) test, considered the gold standard of CRF. High‐throughput sequencing of the 16S rRNA of fecal microbiota were carried using the Illumina platform. Fecal short‐chain fatty acids (SCFA) were analysed using gas chromatography.

Results

The primary findings from this study showed that VO2peak was positively correlated with gut species richness (P=0.01, B‐coefficient=5.35) and can predict over 20% of the observed variability, while keeping all other factors constant, including diet. Further, protein intake but not VO2peak was significantly associated with changes in beta‐diversity, correlating strongly with Bacteroides genus. VO2peak was a significant factor in explaining variability of the microbiomes predicted metagenomic functions, aligning positively with genes related to bacterial chemotaxis and motility, and negatively with lipopolysaccharide biosynthesis. Of all SCFA analysed, VO2peak was positively correlated with only butyrate, known for its role in promoting gut health.

Conclusions

Results from this study show that CRF is correlated with increased microbial diversity in healthy humans and that the associated changes are anchored around a set of functional cores rather than specific taxa. The microbial profiles of those with high CRF levels favor the production of the beneficial SCFA, butyrate. As increased microbiota diversity and butyrate production is associated with overall host health, our findings warrant further research in the use of exercise prescription as an adjuvant therapy in combating dysbiosis‐associated diseases.

Support or Funding Information

This project was funded by the Natural Sciences & Engineering Research Council (NSERC) of Canada
Vitamin D Supplementation and DNA Methylation Patterns during Pregnancy and Lactation in Mothers and Infants
Authors: Anderson Cindy M. , Thiele Doria K. , Ralph Jody L. , Perley Danielle , Ohm Joyce E.
Published: November 2023

The purpose of this study was to identify the temporal influence of maternal vitamin D supplementation during pregnancy and lactation on DNA methylation in mothers and infants. In this double blind randomized control trial, pregnant women planning to exclusively breastfeed were randomized to control (400 IU vitamin D3 daily, n=8) or experimental (3800 IU vitamin D3 daily, n=8) groups from 28 weeks gestation through 4 weeks postpartum. Genome‐wide DNA methylation (Illumina Infinium 450K) was analyzed in white blood cells from mothers at 28 weeks gestation (baseline), 2 days postpartum and 4 weeks postpartum and in their fully breastfed infants at birth and 4 weeks of age. Significant DNA methylation was determined by at least 20% difference in individual CpG methylation between control and experimental groups. In the experimental group, differential methylation in >400 individual maternal CpGs in 280 known genes across the genome was identified with methylation loss present in 2/3 of individual CpGs. Among the infants, differential methylation in 143 CpGs (91 genes) was identified with common methylation loss patterns as in the mothers. These findings provide evidence for the direct influence of vitamin D supplementation on DNA methylation in pregnant and lactating women, in utero on the fetus and via breastmilk in newborns.

Support or Funding Information

1P20GM104360‐01 National Institutes of Health, National Center for Research Resources (NCRR) Centers of Biomedical Research Excellence (COBRE). Center of Excellence in Epigenetics/Epigenomics of Development and Disease. COBRE Epigenomics Jumpstart Pilot Grant, Epigenomic gestational responsiveness to vitamin D Association of Women's Health, Obstetric and Neonatal Nursing (AWHONN). Maternal vitamin D supplementation to correct deficiency in mothers and breastfed infants.