bluuebunny/crossref_metadata_2025 huggingface dataset:
Abnormal tracheal cartilage formation in mice lacking Cav3.2 T‐type Ca2+ channels (540.3)
Authors: Lin Shin‐Shiou , Tzeng Bing‐Hsiean , Lee Kuan‐Rong , Smith Richard , Campbell Kevin , Chen Chien‐Chang
Published: June 2021

AbstractCa2+ influx is crucial in initiating the differentiation of mesenchymal cells into chondrocytes, but which Ca2+ channels are involved remains uncertain. Here we show that the T‐type voltage‐gated Ca2+ channel Cav3.2 is essential for tracheal chondrogenesis. Mice lacking this channel (Cav3.2‐/‐) showed congenital tracheal stenosis because of incomplete formation of cartilaginous tracheal support. Conversely, Cav3.2 overexpression in ATDC5 cells enhanced chondrogenesis, which could be blunted by both blocking T‐type Ca2+channels and inhibiting calcineurin and suggests that Cav3.2 is responsible for Ca2+ influx during chondrogenesis. Finally, the expression of sex‐determination region of Y chromosome (SRY)‐related high mobility group‐Box gene 9 (Sox9) was reduced in Cav3.2‐/‐ tracheas. Cav3.2‐dependent Sox9 upregulation required a putative nuclear factor of activated T cell (NFAT) binding site identified within the mouse Sox9 promoter. The signaling pathway underlying Ca2+‐induced chondrogenesis involves the Cav3.2 T‐type channel and depends on calcineurin and NFAT.
Secondhand smoke exposure leads transition from physiological aging hypertrophy to pathological heart failure (540.5)
Authors: Wu Jia‐Ping , Huang Chih‐Yang
Published: June 2021

The aim of this study was to examine secondhand smoke exposure in age‐related heart failure. The older rats were placed in exposure chamber, and exposure 10 cigarettes for 30 min, for 1 day. Left ventricular (LV) morphological assessed on histopathologic of LV sections were stained with hematoxylin‐eosin staining. Echocardiography assessed LV function. Related molecular mechanisms of LV hypertrophy signaling pathways by western blot. Results showed heart and LV weight, heart (LV)/body weight ratio, heart (LV)/tibia ratio were increased in the aging and aged SHS exposure. In aging heart, we observed LV muscle fiber rearrangement was disorder, interstitial space large and fibrosis. From echocardiography results, we found EF (%) and FS (%) were apparently decreases, LVID, LVPW and IVS at systolic and diastolic diameters were decreased in the aging and aged SHS exposure. In aged SHS exposure, we found myocytes numbers decreased, extracellular space (%), cell length (μm) and collagen occupied (%) were increased, in the aging rats cardiomyocytes width (μm) increased. Indeed, related molecular mechanisms trend to pathological LV hypertrophy (dilation) and heart failure.

Grant Funding Source: Taiwan Department of Health Clinical Trial and Research Center of Excellence (DOH102‐TD‐B‐111‐004)
LHX2: a critical link in the FGF to SHH pathway? (541.2)
Authors: Watson Billy , Pira Charmaine , Oberg Kerby
Published: June 2021

Fibroblast growth factors (FGFs) and Sonic hedgehog (SHH) are morphogens known to be integrally involved in limb outgrowth/development. Published reports assert that for SHH to be expressed, FGFs have to be secreted from the apical ectodermal ridge (AER); however, not much is known about the molecular mechanisms underlying this regulation.

SHH expression can be induced by the application of FGF‐soaked beads in the posterior region of the developing limb bud. DNA microarray analysis, following FGF bead application, revealed that LHX2 (LIM Homeobox 2) was upregulated >5‐fold. LHX2 encodes a transcription factor that is highly conserved across species, is expressed in the distal limb mesenchyme, and implicated in the integration of signaling inputs from the AER (FGFs), ZPA (SHH), and the dorsal ectoderm (WNT7a). Furthermore, LHX2/LHX9 double knockout mice show a marked reduction in SHH expression, confirming a role for LHX2 in regulating the signals that direct limb patterning and growth. Thus, we hypothesize that LHX2 is an intermediate in the FGF‐to‐SHH pathway.

In order to test this hypothesis, we implanted FGF‐soaked heparin sulfate beads in embryonic chicken limb buds (Hamburger ‐ Hamilton stage 23), harvested the embryos, and assessed LHX2 expression in response to FGF induction via whole mount In situ hybridization (WMISH).

Our results show that LHX2 is rapidly upregulated in response to FGF (<3 hrs). Its upregulation persisted through the experimental time course (24 hrs) and overlaps temporally with the upregulation of SHH in response to FGF.

We therefore conclude that LHX2 is a downstream target of FGF and the early induction of LHX2 suggests that its regulation is direct. Further studies are underway to determine whether LHX2 can upregulate SHH and establish LHX2’s role as an intermediate in the FGF to SHH pathway.

Grant Funding Source: Institutional
Exploration of the effects of Doublethumb on neural tube development (541.7)
Authors: Smither Allison , Petersen Juliette , Niswander Lee
Published: June 2021

Neural tube defects (NTDs) occur in approximately 1 in every 1000 births. NTDs have both an environmental and genetic basis. A hypomorphic mutation of the PCP effector protein gene Inturned (Intudtm/dtm) was studied to determine its effects on ciliogenesis, cranial neural tube patterning, and cell proliferation in a mouse model. Heterozygous mice carrying the Intudtm allele were bred and mothers were sacked and dissected for embryos. Embryos (n=6) at embryonic day 10.5 were fixed, embedded in Optimum Cutting Temperature, and cryosectioned into 10 m slices. PCR was used to distinguish between Intu+/+ and Intudtm/dtm embryos. Immunofluorescence was performed then analyzed using confocal microscopy and FIJI analysis software. Ciliogenesis, Sonic Hedgehog expression, certain patterning markers and transcription factors required for embryonic and fetal development, and cell proliferation were all decreased in Intudtm/dtm. Cell differentiation was unaffected in Intudtm/dtm compared to Intu+/+. Thus, there are biological effects of the hypomorphic mutation of Inturned. Future work is needed to increase the number of biological and technical replicates, as well as to quantitate the expression of these and other markers at varying time points in embryonic development.

Grant Funding Source: Supported by the Howard Hughes Medical Institute and the LSU‐HHMI program.
Temporal expression pattern of hLAMP‐1 and BMP‐2 during early heart development in the chick (542.4)
Authors: Abd‐Elhamid Tarek , Conway Marianne , Sinning Allan
Published: June 2021

The heart is the first organ to develop during embryogenesis in mammals and birds. Heart development requires coordinated activity of various factors, the disturbance of which can lead to congenital heart disease. The matrix protein hLAMP‐1 is expressed at Hamburger‐Hamilton stage 4 within the Hensen’s node and subsequently in the lateral mesoderm by stages 5‐6. At stage 7, its expression is enhanced within the left precardiac field. Later, by stages 15‐16, hLAMP‐1 expression has been shown in the atrioventricular (AV) canal and the outflow tract (OT). Also, BMP‐2 expression has been reported in comparable stages of chick embryo. To further elucidate the role of these two molecules in heart development, we examined their expression patterns during stages 8‐14 in chick embryos. In this study, we used antibodies specific to hLAMP‐1 or BMP‐2 to examine the expression pattern of both proteins. We detected hLAMP‐1 at the interface of the splanchnic mesoderm and endoderm at stages 8‐9 where the preendocardial cells delaminate from the precardiac mesoderm and migrate toward the endoderm. In contrast, BMP‐2 was not detected at stages 8‐9. After fusion of the bilateral heart field to form the heart tube at stage 10, both hLAMP‐1 and BMP‐2 expression is restricted to the arterial and venous poles of the heart tube. This expression pattern is maintained until stage 14. This pattern correlates with the time at which the cardiac progenitors being added to both ends of the heart tube. These findings point to a specific role for hLAMP‐1in heart development, possibly in formation of the endocardial tube and the role of BMP‐2 in elongation of the heart tube.
Tracking venous anatomical variation of the upper limb for optimal placement of arteriovenous fistulas in hemodialysis patients (543.10)
Authors: Advolodkina Polina , Di Raimo Robert , Song Mark , Pagano Anthony , Márquez Samuel
Published: June 2021

The National Kidney Foundation Disease Outcomes Quality Initiative (KDOQI) guidelines support placement of arteriovenous (AV) fistulas in hemodialysis patients over grafts. Long‐term outcomes of AV fistulas are variable and access site failure remains a major factor in morbidity of hemodialysis patients. Preoperative sonographic mapping of anatomic variation and vessel diameter may prolong the success of native AV fistulas. This study examines venous anatomy of 96 cadaveric upper limbs from SUNY Downstate medical school anatomy laboratory. The vessel diameters of the cephalic and basilic veins were documented at specific landmarks along their respective courses. Normal and variant venous anatomy were compared and gender differences were evaluated. Results showed the normal anatomical condition in 55% of limbs. Among the remaining 45% of limbs, the duplicate cephalic system was the most frequent variant, representing 23% of the sample. Males exhibited greater frequency of anatomic variants and females had significantly smaller vein diameters than males in both the basilic and cephalic veins (p < 0.05). In all but the upper cephalic location, anatomical variants exhibited smaller vein diameters than those cadavers that had normal anatomy. Further investigation is warranted into relationships among rate of AVF construction failure, venous width, and drainage pattern.
Neuroanatomy and immunohistochemistry of cardiac ventricles in rabbit (543.2)
Authors: Pauziene Neringa , Rysevaite Kristina , Saburkina Inga , Inokaitis Hermanas , Alaburda Paulius , Pauza Dainius
Published: June 2021

Rabbit model is used in cardiac electrophysiology but neuroanatomy of the rabbit heart (RH) is poorly examined. We aimed to ascertain the architecture of intrinsic cardiac nerve plexus of the RH, and the neurochemical phenotypes of ventricular nerves (VNs) and nerve fibers (NFs) using histochemical acetylcholinesterase staining and immunofluorescent labeling for PGP 9.5, TH, ChAT, nNOS, and SP in 18 New Zealand white rabbits. Cardiac nerves (CNs) accessed ventricles of the RH by the left and middle dorsal and the right and left coronary subplexuses. In the rabbit ventricular walls, there were found TH, ChAT, nNOS and SP positive (+) NFs. In VNs, more than 65% of NFs were TH(+), while only 7% were cholinergic ones. On the average, 4% of nNOS(+) and SP(+) NFs were found in VNs. The TH(+) NFs also predominated in the interventricular septum, papillary muscles, and in perivascular nerves. The PGP9.5(+) NFs distributed within the myocardium occupied 4.2% in the left ventricle and 3.8% in the right one. The mean area of NFs in papillary muscle were 2.6% for TH(+), 2.4% for ChAT(+), 1.4% for SP(+) and 0.6% for nNOS(+). Endocardial nerves contained 15.8% TH(+), 6.3% SP(+), 4.2% ChAT(+), and 1.5% nNOS(+) NFs. In summary, the RH corresponds neuroanatomically to hearts of some rodents, but it’s noticeably distinct from the human one.

Grant Funding Source: MIP‐031/2013 from the Research Council of Lithuania
Blood supply of anterior longitudinal ligament: an approach to understand diffuse idiopathic skeletal hyperostosis (543.3)
Authors: Khammash Leena , Hanna George
Published: June 2021

Diffuse idiopathic skeletal hyperostosis (DISH) or Forestier’s disease is a common entity among elderly and is characterized by ossification of the anterior longitudinal ligament (ALL) of the spine and various extraspinal ligaments.

The pathogenesis of abnormal bone growth in DISH is not fully understood. In an attempt to figure out an anatomical explanation of the etiology of such a debilitating disease, the present study investigated the role of blood supply of the ALL.

In cadavers of the anatomy lab in Libyan International Medical University, fine dissection of vessels supplying ALL was performed in the vicinity of azygos, hemiazygos veins and descending thoracic aorta; starting from the level T5 till T12 vertebrae. The current work elucidated that the blood vessels supplying ALL ran in ascending manner ranging from oblique to nearly vertical in direction.

The present study demonstrated that poor blood supply reaching certain regions of the spine caused by antigravity‐directed blood vessels supplying ALL, may lead to ischemic changes caused by hypoperfusion and thereby ossification of ALL leading to DISH disease. The current results open a new era of investigation of the probable etiology of this disease.

Grant Funding Source: Supported by the Libyan International Medical University
Anatomical basis for retrograde perfusion of the medial femoral condyle corticoperiosteal flap (543.5)
Authors: Hirtler Lena , Lübbers Anke , Bürger Heinz
Published: June 2021

Introduction: The descending genicular vessels are widely used for free vascularized corticoperiosteal flaps of the medial condyle in cases with scaphoid nonunions. Due to the small diameter of the artery and the therefor demanding surgical procedure, this study aimed at the possibility of retrograde perfusion of the graft.

Material and Methods: The descending genicular vessels (artery and veins) were extracted from 18 formalin‐fixed specimen of the anatomic institute. The samples were severed proximal at the level of the intermuscular septum and traced as far as possible towards the periosteum of the medial condyle of the femur. The veins were inspected macroscopically with help of a surgical microscope. Occurrence, distance and number of venous valves were documented.

Results: The samples measured 5,1 ± 18 cm, each artery was accompanied by two veins in most cases (one vein in six specimen, three veins in one specimen, 32 veins in total). These veins had several intervenous connections. Of the 32 veins, 13 veins showed a venous valve, situated 1,6 ± 1,4 cm from the proximal end of the specimen. Of the 18 descending genicular vessels, there existed at least on vein in 14 samples without any venous valve.

Conclusion: Provided previous intraoperative examination of the veins, retrograde perfusion of the free vascularized corticoperiosteal flap of the medial condyle and therefor arteriovenous anastomosis seems possible.
Various branching pattern of aortic arch in the Siberian roe deer (543.7)
Authors: Ahn Dongchoon , Sim Jeong‐Ha , Park Byung‐Yong , Kim In‐Shik
Published: June 2021

This study examined the branching pattern of the aortic arch and its major branches in Siberian roe deer (Capreolus pygargus) and compared its branching patterns with those of other ruminants. Silicone casts were taken from 16 carcasses (male 7, female 9) with body weights ranging from 7.70~26.35 kg. Silicone casts of the vessels were made by a retrograde injection into the abdominal aorta. Only the brachiocephalic trunk (Bct) branched from the aortic arch in all carcasses. The left costocervical trunk (LCct) branched off as the first branch of Bct in 2 cases, but, in 9 of the 16 cases, the LCct and the left subclavian artery (LSb) were branched off first at the same level; rest 5 case had LCct from LSb. The right costocervical trunk (RCct) branched separately off before the right subclavian artery (RSb) was exhibited in 7 cases. The case that Bct trifurcated into the bicarotid artery (Bc), the RCct and the RSb was observed in 3 cases; however, Bct bifurcated in to Bc and RSb from which the RCct branched off as the first artery were showed in 5 cases. The right common carotid artery (RCc) branching at same site with RCct so that having no Bc was observed in only 1 case. In 4 cases the highest intercostal artery (Hi) formed the common trunk with the dorsal scapular artery (Ds). The combination in these different branching orders of costocervical trunk in both side and the formation of common trunk of Hi and Ds resulted in 12 types of the branching patterns in Siberian roe deer. These results suggest that the Siberian roe deer has a Bct with various branching patterns and its types are different with those of other ruminants.

Grant Funding Source: Supported by NRF‐2013028138
Interaction of the kallikrein kinin system and vascular endothelial growth factor in left ventricle of mice exposed to intermittent hypoxia (544.1)
Authors: Visniauskas Bruna , Gomes Guiomar , Tufik Sergio , Chagas Jair
Published: June 2021

Intermittent hypoxia (IH) is an experimental model of obstructive sleep apnea syndrome (OSAS). However, the occurrence of cardiac angiogenesis in IH and its related mechanism until now have not yet been clarified. In other models of tissue hypoxia, like ischemia/reperfusion, an interaction between VEGF and the KKS was demonstrated and related to the cardioprotective effects. Aiming to find out evidences for VEGF‐KKS interactions in male mice myocardium after IH, we have performed a RT‐PCR assay in the left ventricle. A significant mRNA level increase in VEGF and B2 bradykinin receptor and a decrease in angiotensin converting enzyme (ACE) were observed after 2 weeks of IH when compared to normoxia group. In models of ischemia/reperfusion it has been suggested that VEGF interaction with B2 receptor agonist is an important step in angiogenesis induction. Our findings allow us to suppose that the increased myocardial expression of VEGF in IH by stimulating the expression of B2 receptor and reducing the expression of ACE could contribute to increase bradykinin, favouring neoangiogenesis in this tissue.

Grant Funding Source: Supported by FAPESP (2012/02123‐0)
Timing and magnitude of prestretch strongly affect cardiac muscle tension development (545.2)
Authors: Tangney Jared , Campbell Stuart , McCulloch Andrew , Omens Jeff
Published: June 2021

Ectopic ventricular pacing leads to regional myocardial prestretch. In‐vivo studies suggest that the timing of prestretch determines the effect on pump function and can lead to regional wall remodeling. Here we measure the direct effects of prestretch timing on the magnitude of tension development in isolated cardiac muscle. The effects of prestretch were simulated using excised murine right ventricular papillary muscles. A servo motor was used to impose precisely timed stretches, while a force transducer measured force output and strain was monitored using a CCD camera and topical markers. A critical stretch timing interval was observed to statistically increase peak tension by as much as 500%, while shortening during this interval inhibited peak tension significantly by as much as 40%. A simple model consisting of experimentally obtained time varying elastance and the force‐velocity relation could not completely explain the results. An estimate of shortening deactivation was measured by performing quick stretches to dissociate crossbridges. The results showed that the timing of shortening deactivation could explain the remaining differences between the model and the experimental results. These evaluations have led us to the conclusion that prestretches performed at slow physiologic velocities cause alterations in twitch kinetics that cannot be explained solely by time varying elastance.
Oxidative stress reduces the expression of neuronal NOS in cardiac tissue and cell (545.3)
Authors: Kar Rekha , Ikeno Yuji , Masters Bettie , Roman Linda
Published: June 2021

Obesity is a major global health concern and greatly increases the risk of developing insulin resistance and type 2 diabetes. Diabetic patients are also predisposed to various cardiovascular diseases; however, the underlying mechanism of the effects of diabetes on cardiomyocyte viability and thus, overall heart function remains to be established. Increased fatty acid oxidation during diabetes results in increased oxidative stress, which likely contributes to the adverse changes in the myocardium. Nitric oxide (NO) is involved in the regulation of cardiovascular homeostasis in experimental animal models and humans. Neuronal nitric oxide synthase (nNOS) knockout in mice resulted in increased oxidative stress in heart. In cardiac myocytes. nNOS was shown to coimmunoprecipitate with xanthine oxidoreductase, a critical enzyme for superoxide production, and to inhibit its production by the formation of peroxynitrite, all of which suggest a critical role of nNOS‐derived NO in regulating oxidative stress in heart. To investigate the effect of oxidative stress or fatty acids on nNOS expression in cardiomyocytes, we treated HL‐1 cardiomyocyte cells with hydrogen peroxide(H2O2) or palmitate. We show here for the first time that H2O2 or palmitate decreases the expression of nNOS in the cardiomyocytes, as assessed by immunoblot analysis. Furthermore, nNOS expression is also reduced in the heart lysates from thioredoxin 2 (TRX 2) knockout mice, which are prone to oxidative stress. The effect of such a decrease in nNOS expression by oxidative stress or saturated fatty acid treatment on cardiomyocyte viability, overall cardiac hypertrophy and cardiac function is under investigation.

Grant Funding Source: Supported by NIH #HL07446 and #GM052419
Role of transient receptor potential vanilloid 2 in exercise tolerance and cardiac function (545.5)
Authors: Naticchioni Mindi , Smith Margaret , Apel Ryan , Jiang Min , Koch Sheryl , Rubinstein Jack
Published: June 2021

Background: We have previously shown that abrogation of the semi‐selective calcium channel Transient Receptor Potential Vanilloid 2 (TRPV2) results in impaired cardiac contractility without any other observable phenotype. To expand on these findings, we investigated the effect of TRPV2 abrogation on exercise tolerance and corresponding changes in cardiac function.

Methods: A group of 8‐12 week male WT and TRPV2‐/‐ mice were subjected to 4 weeks of daily treadmill exercise with weekly endurance training. A second group underwent 4 weeks of voluntary wheel running. Interruptions in treadmill exercise and voluntary running distances were assessed in addition to weekly echocardiographic imaging.

Results: TRPV2 abrogation resulted in decreased tolerance to treadmill exercise (p<0.05 versus WT). Echocardiographic imaging showed that cardiac functional parameters improved in WT mice but not in TRPV2‐/‐ (treadmill) mice in response to exercise as measured by EF, SV, and CO (p<0.05 vs. baseline and in comparison to WT). Ongoing wheel experiments have preliminarily demonstrated a similar pattern.

Conclusions: TRPV2 abrogation results in impaired cardiac function, exercise tolerance and cardiac response to exercise. Associated studies are underway to elucidate the underlying mechanisms involved.
The role of the extracellular matrix protein Perlecan in the arterial wall (546.10)
Authors: Nonaka Risa , Iesaki Takafumi , de Vega Susana , Yamada Yoshihiko , Arikawa‐Hirasawa Eri
Published: June 2021

Perlecan (HSPG2; Perl) is expressed in all basement membranes and cartilage. Perl has been reported to have anti‐atherogenic activity; however, its role in the aortic wall is unknown. In this report, we analyzed the structure of the aortic wall in Perlecan deficient mice (HSPG2‐/‐‐Tg).

Previously, we created the lethality rescued HSPG2‐/‐‐Tg mice model. Aorta samples were prepared from the thoracic aorta of HSPG2‐/‐‐Tg (Perl KO) and control HSPG+/+‐Tg mice. We analyzed the histology of these aortic tissues by Elastica Van Gieson (EVG) staining, immunohistochemistry (IHC), and electron microscope (EM). In IHC, we observed that perlecan is localized at the intersection between elastin and fibrillin‐1. EVG staining showed no difference in aorta histology between Perl KO and control mice, however, EM analysis showed that the elastic fibers in Perl KO mice were partially torn and thinner. In addition, we found that Perl KO mice had an aortic dissection with a frequency of about 15% in 10‐weeks mice. Real‐time PCR analysis showed no significant differences in the expression of genes for the major components of elastic fibers and matrix metalloproteinases (MMPs).

These results suggest that Perl is likely to contribute to the maintainance of elastic fibers strength against mechanical stress, such as blood flow, vasodilation, and contraction.
Oral vitamin C prevents the attenuation in flow mediated dilation during prolonged sitting (546.3)
Authors: Thosar Saurabh , Bielko Sylvanna , Wiggins Chad , Mather Kieren , Klaunig James , Wallace Janet
Published: June 2021

Prolonged sitting has been hypothesized to impair endothelial function via mechanisms involving oxidative stress. This study was designed to determine if Vitamin C prevents the decline in flow mediated dilation (FMD) during 3hr of sitting.

Methods: Eleven inactive, non‐obese, healthy men (24.2±4.4 yrs) participated in two randomized 3 hr sitting trials, performed fasting in the morning on both occasions. In the prolonged sitting (SIT) trial, subjects were seated for 3 hours without moving their lower extremities. In the vitamin C trial (VIT) subjects sat similar to the SIT trial, but were asked to swallow two vitamin C tablets (1000 mg and 500 mg) at 0:30 and 1:30 hr respectively. Superficial femoral artery (SFA) FMD was measured every hour for 3 hrs. Paired sample t‐tests were performed to examine the differences at each time point between SIT and VIT trial. Significance was set at p蠄0.05.

Results: Baseline SFA FMD% did not differ between the study conditions (SIT 5.05±3.77; VIT 2.74±2.32). However, FMD% was significantly different between conditions at 1hr (SIT 0.56±0.88; VIT 4.54±3.96) 2hr (SIT 1.76±1.10; VIT 5.28± 3.32) and 3hr (SIT 2.35±2.19; VIT 6.45±4.03).

Conclusion: Our data demonstrate that acute oral vitamin C supplementation prevents the acute decline in SFA FMD with prolonged sitting, suggesting that oxidative stress may contribute to the associated impairment in endothelial function.

Grant Funding Source: Supported by American College of Sports Medicine Foundation grant
Sustained delivery of androgens triggers the induction of intracardiac interleukin‐6 in adult male rats (547.13)
Authors: Benghuzzi Hamed , Tucci Michelle , Cameron Joseph
Published: June 2021

It is well documented that Inflammatory cytokines such as IL‐6 are thought to be mediators in the initiation of several organ injuries by inducing apoptosis and necrosis. Several reports have indicated that IL‐6 was elevated upon the exposure to supraphysiological levels of androgens. The objective of this study was to investigate the effect of sustained delivery of testosterone (T), dihydrotestosterone (DHT) and androstenedione (AED) on the induction of inracardiac IL‐6 in male rats. A total of 60 adults male rats were randomly divided into three equal groups and implanted with tricalcium phosphate delivery system by following standard aseptic surgical techniques. Group 1 animals were implanted with empty devices (sham) and groups 2, 3 and 4 were implanted with devices loaded with 40 mg of T, DHT and AED, respectively. At the end of 2, 4, and 8 weeks 5 animals from each group were sacrificed following lab protocols and vital, as well as, reproductive organs were retrieved for histopathological evaluation. Results of this study revealed: (1) the wet weights of retrieved hearts from experimental groups were significantly higher (P<0.05) than control animals, (2) conventional morphological screening (H&E) showed occasional myocardial hypertrophy in all experimental groups, (3) immunohistochemical evaluation demonstrated an expression of IL‐6 in all experimental animals with the following ease: AED.DHT>T. In conclusion, results of this study suggest that IL‐6 family plays a central role in the pathophysiology of cardiovascular diseases, and exposure to androgen may contribute to the progression of myocardial damage and dysfunction through the induction of intracardiac IL‐6.
Acute testosterone infusion does not exacerbate arrhythmias in the rat model of ischemia/reperfusion (547.15)
Authors: Dow Joan , Herring Michael , Bhandari Anil , Kloner Robert
Published: June 2021

Introduction: With increasing direct‐to‐consumer marketing, testosterone (T) prescriptions have tripled over the past decade. Clinical literature suggests that exogenous T use may increase various arrhythmias. Therefore, we examined the effect of exogenous T in an acute rat model of arrhythmias.

Methods and Results: Anesthetized female Sprague Dawley rats (250g) were given T (2.5 mg/kg, n=9) or vehicle (n=10) intravenously 15 minutes before 5 minutes of coronary artery occlusion. Rats were then reperfused for 5 minutes and arrhythmias were examined. T levels were measured one minute before occlusion. T levels were different between groups (5.4 ± 1 ng/mL control; 206.9 ± 35 ng/mL T, P < 0.001). The ischemic risk zone as a percentage of the left ventricle did not differ between groups (33 ± 2% control; 29 ± 3% T, P = 0.48). The number of episodes of ventricular tachycardia did not differ between groups (11.7 ± 3.2 control; 8.8 ± 3.0 T, P = 0.3). There was a non‐significant trend toward fewer ventricular premature beats with T (37.8 ± 10.3 control; 16.9 ± 8.9 T, P = 0.06) Conclusion: Despite large differences in T levels between groups, T had little effect on the incidence of arrhythmias, suggesting that T supplementation does not increase risk of arrhythmias.
Dystrophin absence is implicated in the transition from physiological to pathological cardiac hypertrophy (547.4)
Authors: Prado Cibele , Blefari Valdecir , dos Santos Daniele , Prado Fernanda , Silva Carlos , Fazan Rubens , Salgado Helio , Rossi Marcos
Published: June 2021

Hypertension causes cardiac hypertrophy, cardiac dysfunction, and heart failure (HF). The mechanisms implicated in the transition from physiological to pathological hypertrophy are still unknown. Growing evidence points out to the role of inappropriate mechanical coupling in the pathogenesis of HF and the cytoskeletal proteins as the most obvious candidates for intracelular remodeling. We investigated dystrophin and its potential role in the transition from physiological to pathological cardiac hypertrophy in rats submitted to chronic abdominal aorta stenosis. Wistar rats were submitted to abdominal aorta stenosis and killed at 30, 60 and 90 days post surgery (dps). The hearts were collected and Western blot and immunofluorescence were performed for dystrophin and calpain. Blood pressure was evaluated and cardiac function evaluate by echocardiography. Data were considered significant when p<0.05. At 90 dps 70% presented hypertrophied hearts (HH) and 30% hypertrophied+dilated hearts (DH), as evaluated by echocardiography. Blood pressure increased 53,42% (30d), 50,8% (60d) and 64,36% at 90dps. Dystrophin expression at 30, 60 dps and HH animals was the same as control, but decreased in DH animals. The ejection fraction was the same as control at 30 and 60dps, however it was reduced 35% at 90d in HH and 48% in DH. Loss of dystrophin could be partly responsible for the development of heart failure in rats submitted to chronic abdominal aorta stenosis. Thus, it is essential to define the mechanistic aspects of the pathophysiological processes involved in deterioration of cardiac function and design develop strategies to inhibit and/or reverse evolution of heart hypertrophy from compensated to decompensated phase. Grants from FAPESP 09/17787‐8; 10/19216‐5.

Grant Funding Source: Supported by FAPESP 09/17787‐8; 10/19216‐5
Myocardial clefts: do they really exist? (547.8)
Authors: Casier Craig , Rossler Carmen , Tubbs R. Shane , Loukas Marios
Published: June 2021

Myocardial clefts are described as discrete approximately V‐shaped extensions of blood signal penetrating >50% of the thickness of adjoining compact myocardium in long‐axis views on MRI and CTs and the clefts tending to narrow or occlude in systole, without local hypokinesia or dyskinesia.” The terms recesses, fissures, diverticula and crypts have all been used to describe myocardial clefts. According to the literature these diverticula are present in the healthy population approximately from 5‐10% of the individuals. The aim of our study was to explore if these diverticula exist in normal healthy hearts. Dissection of 100 hearts was performed on formalin fixed cadavers. We were unable to find any diverticula as described on CT and MRI studies. This is likely due to the fact that it might be challenging to differentiate the clefts from multiple large trabeculations creating an uneven surface of the endocardial tissue during systole and diastole. Traditionally, clefts are found in individuals with repaired congenital pulmonary valve stenosis, hypertension, hypertrophic cardiomyopathy and in patients with repaired tetralogy of Fallot. Much of the literature to date has made use of vertical long‐axis views to detect these clefts. This might suggest that this plane of view creates a false impression of myocardial cleft over a large septal trabeculations.
Creating targeted double‐stranded breaks in vitro and in vivo using RecA‐dependent endonucleases P1 and φW39 Ref (552.1)
Authors: Olsen Tayla , Gruber Angela , Dvorak Rachel , Cox Michael
Published: June 2021

P1 bacteriophage recombination enhancement function (Ref) protein is an HNH class and RecA‐dependent endonuclease. RecA‐bound oligonucleotides homologous to a specific region on circular dsDNA will catalyze RecA strand‐invasion, creating a displacement loop (D‐loop). Upon Ref addition, double‐stranded breaks (DSBs) are created within the D‐loop. Thus, the Ref protein, in concert with RecA, is a programmable endonuclease. The closely related φW39 Ref protein exhibits increased RecA‐dependent endonuclease activity compared to P1 Ref. P1 Ref protein missing the disordered 76 N‐terminus residues is incapable of DNA binding and exhibits decreased nuclease activity, which indicates the importance of the N‐terminal region for these functions. Characterization of N‐terminal truncations based on charge distribution for φW39 Refs ΔN21, ΔN47, ΔN59, ΔN66, and ΔN74 revealed modulated activity for DSB formation and DNA binding. In addition to these in vitro studies, cells transformed with a plasmid containing P1 Ref and introduced to an oligo homologous to a region in the rpsL gene demonstrate recombination events with the oligo. The Ref/RecA/oligo system acts as an oligo‐targeting inducer of DSBs operating both in vitro and in vivo with potential for genome editing functions and in vitro targeting.

Grant Funding Source: NIH GM32335
Isoaspartyl modification of ZAP70 signaling molecule alters T cell responses (555.9)
Authors: Yang Mei‐Ling , Clarke Steven , Mamula Mark
Published: June 2021

The immune system is designed to protect the host by distinguishing self‐proteins from invading pathogens. However, neo‐epitopes generated from self‐proteins by post‐translational modifications can trigger autoreactive immune cells to break such immune tolerance. One such modification, isoaspartyl (isoAsp), is believed to contribute the onset of autoimmune B and T cell responses and lupus pathology. Protein L‐isoaspartyl (D‐aspartyl) methyltransferase (PIMT) is the only known repair system for abnormal isoaspartyl residues. Previously, we found that the isoAsp content in lupus‐prone MRL autoimmune mice increased and accumulated with age in brain and kidney, the major target organs in systemic lupus erythematosus (SLE). Recently, we found that the content of isoAsp modification in erythrocytes is correlated with PIMT activity in humans with SLE. The aberrant lupus T cell hyperproliferation can be rescued by PIMT overexpression. Of interest, ZAP70 is an essential kinase in T cell signaling and several ZAP70 mutations have been reported for patients with T cell immunodeficiencies. We found that isoAsp modification is present within ZAP70. In addition, ZAP70 is hyperphosphorylated upon TCR engagemant in PIMT deficient mice. Taken together, these studies demonstrate that isoAsp modification of ZAP70 TCR signaling molecule alters T cell immune responses and is associated with lupus T cell defects.

Grant Funding Source: These studies were supported by NIH grant AI‐48120 to MJM.
Regulation of Nrd1 in RNA processing and termination by multiple binding proteins (561.6)
Authors: Kim Kyumin , Heo Dong‐hyuk , Kim Minkyu
Published: June 2021

The yeast Nrd1 interacts with C‐terminal domain (CTD) of RNA polymerase II (RNApII) using its CTD‐interaction domain (CID) and associates with nuclear exosome, acting as a transcription termination and RNA processing factor. Previously, we have found that it is the Nrd1 CID that couples RNApII termination and subsequent RNA processing by recruiting nuclear exosome. However, what bridges the Nrd1 CID to nuclear exosome was not known. In this study, using yeast two‐hybrid assay, we show that two nuclear exosome cofactors Mpp6 and Trf4 directly interact with the Nrd1 CID. Various analyses indicate that these factors regulate the interaction of Nrd1 not only with nuclear exosome, but also with RNApII CTD. Thus, multiple proteins recognize the Nrd1 CID, fine‐tuning Nrd1 in both RNA processing and transcription termination.

Grant Funding Source: Supported by the National Research Foundation of Korea
Replication serves as a trans‐generational epigenetic switch during SV40 infection (563.2)
Authors: Milavetz Barry , Kallestad Les , Woods Emily , Christensen Kendra , Balakrishnan Lata
Published: June 2021

We have recently shown that the repression of early transcription during a Simian Virus 40 (SV40) infection results in different histone modifications depending upon the replicative status of the chromatin (Kallestad, et al., 2013, Frontiers in Genetics). In order to test whether replication affected other aspects of transcriptional regulation, we have analyzed SV40 chromosomes for the presence of the methylated forms of H3K4 and H3K9 following dysregulation of transcription by DRB which inhibits RNA Polymerase II elongation, and sodium butyrate which increases transcription from genes committed to transcription. SV40 chromosomes from treated and control infections were isolated between 2 and 12 hours post‐infection when only transcription was occurring or 48 hours post‐infection when transcription and replication were both occurring, purified, and subjected to ChIP analyses with antibodies to mono‐, di‐, and tri‐methylated H3K4 and H3K9 followed by real‐time PCR quantitation. Consistent with our previous published results we observed that dysregulation of transcription yielded different epigenetic readouts depending upon whether replication was occurring. Treatment with DRB resulted in a large increase in the levels of H3K9me2 and me3 in the presence of replication but had little effect on the levels of these modfications in the absence of replication. Treatment with sodium butyrate had the opposite effects. There was a significant reduction in H3K9me3 during replication and transcription but not when only transcription occurred. Taken together these results indicate that replication acts as a switch to epigenetically define the chromatin structure of newly replicated chromatin which will become new virus particles.

Grant Funding Source: Supported by National Institutes of Health grant AI094441 to B.M.
In vitro determination of the 30 nm chromatin fiber structure (564.1)
Authors: Brandt Anthony , Grilley Dan
Published: June 2021

To be able to fit 6 ft. of human DNA into a cell’s nucleus, an elaborate organizational system is necessary to compact the genomic DNA first into nucleosomes, and then into a 30 nm chromatin fiber. The ability to figure out the spatial conformation between these nucleosomes is vital in understanding gene regulation of underlying DNA. Literature data support two different models of the chromatin fiber that differ in their geometry and possible impact on the accessibility of the underlying DNA sequence. The “zig‐zag” model uses a straight linker DNA region, while the “solenoid” model illustrates a bent DNA linker, greatly influencing which nucleosomes are in close space in relation to one another. However, there is debate about which of these models actually represents the true structure. High resolution in vivo nucleosome maps show that the length of DNA between two nucleosomes is not constant across a genome. Computational studies have shown that changing this linker‐length can drastically change the available compacted structures accessible to an array of nucleosomes. To determine how much the properties of the linker DNA affect chromatin structure, we are developing a quantitative crosslinking assay. In our studies, we use this method to investigate the linker‐length dependence of chromatin structure in synthetic nucleosome arrays. Initial results have shown evidence for both zig‐zag and solenoid models depending on solution conditions.
Identification of a putative promoter element in intron5 of the RUNX1 gene (565.2)
Authors: Gutierrez Soraya , Hinojosa Marcela
Published: June 2021

t(8:21) is one of the most frequent chromosomal translocation found in acute myeloid leukemia (AML) and involves RUNX1 and ETO genes. To date all the break points mapped are located in intron 5 of RUNX1 gene. Interestingly, this intron exhibits DNaseI hypersensitive sites, which has been associated to cis regulatory elements. Therefore, we hypothesize that regulatory elements are harbored in intron5 of the RUNX1 gene. In order to test this hypothesis, we performed bioinformatics analysis of ChIPseq data available from the server ENCODE to identify regions of intron 5 enriched in epigenetics marks associated with regulatory elements. We show that in hematopoietic cells one region is enrich in K4H3me, K4H3me3 and K27H3ac that co‐localizes with DNaseI hypersensitive sites. These marks have been associated with promoter modules. Indeed, when we cloned this region (PPR=putative promoter region) in a reporter vector, we found that it activates expression of the reporter gene in an orientation dependent manner. Moreover, when we analyzed putative transcription factors binding sites, we found one RUNX binding motif. Indeed when we transfected the promoter with RUNX1 expression vector we found that RUNX1 modulates the PPR expression. Using ETS database, we have assembly a putative messenger for this promoter, which is differentially expressed in several cell lines. Taken together, our results shown that a novel promoter is located in intron 5 of RUNX1 gene.

Grant Funding Source: Supported by Fondecyt 1130697
Human eukaryotic initiation factor 4G (eIF4G) binds to eIF3c, ‐d, and ‐e to promote mRNA recruitment to the ribosome (566.9)
Authors: Villa Nancy , Do Angelie , Hershey John , Fraser Christopher
Published: June 2021

Recruitment of mRNA to the 40S ribosomal subunit requires the coordinated interaction of a large number of translation initiation factors. In mammals, the direct interaction between eukaryotic initiation factor 4G (eIF4G) and eIF3 is thought to act as the molecular bridge between the mRNA cap‐binding complex and the 40S subunit. A discrete ~90 amino acid domain in eIF4G is responsible for binding to eIF3, but the identity of the eIF3 subunit(s) involved is less clear. The eIF3e subunit has been shown to directly bind eIF4G, but the potential role of other eIF3 subunits in stabilizing this interaction has not been investigated. It is also not clear if the eIF4A helicase plays a role in stabilizing the interaction between eIF4G and eIF3. Here, we have used a florescence anisotropy assay to demonstrate that eIF4G binds to eIF3 independently of eIF4A binding to the middle region of eIF4G. By using a site‐specific crosslinking approach, we unexpectedly show that the eIF4G‐binding surface in eIF3 is comprised of the ‐c, ‐d and ‐e subunits. Screening multiple crosslinker positions reveals that eIF4G contains two distinct eIF3‐binding subdomains within the previously identified eIF3‐binding domain. Finally, by employing an eIF4G‐dependent translation assay, we establish that both of these subdomains are required for efficient mRNA recruitment to the ribosome and stimulate translation. Our study reveals unexpected complexity to the eIF3‐eIF4G interaction that provides new insight into the regulation of mRNA recruitment to the human ribosome. With this new model, we are currently exploring whether different mRNAs, such as those with upstream open reading frames, are more or less dependent on this interaction for translation.

Grant Funding Source: Supported by grant R01GM092927 from the National Institute of General Medical Sciences
Identification and characterization of a putative FKBP52 regulatory surface on the androgen receptor hormone binding domain (567.9)
Authors: Ramos Paola , Guy Naihsuan , Cox Marc
Published: June 2021

The 52‐kDa FK506‐Binding Protein (FKBP52) plays important physiological and potential pathological roles in mammals through interactions with steroid hormone receptors. FKBP52 specifically regulates the androgen (AR), progesterone (PR) and glucocorticoid (GR) receptors, but not the closely related mineralocorticoid receptor (MR). Previous studies demonstrated that the FK1 domain, containing the proline‐rich loop overhanging the FK1 catalytic pocket is critical for FKBP52’s potentiation of receptor function. Additionally, it likely comprises a functionally important interaction surface. Utilizing receptor sequence alignments, we identified residues conserved in the FKBP52‐regulated receptors (AR, GR, and PR), which implicate a surface region that is within the recently characterized BF3 surface on AR. The recently characterized inhibitor of FKBP52‐regulated AR activity, MJC13, is predicted to bind this surface as well. To further characterize this surface, we systematically mutated residues within the AR BF3 surface and assessed the mutants for activity in the presence or absence of FKBP52. These studies led to the identification of three adjacent residues within BF3 (P723, F673, and C805) that result in a receptor with increased dependence on FKBP52 for normal function when mutated. Thus, the AR BF3 surface, especially the region containing the residues P723, F673 and C805, represents a putative FKBP52 interaction/regulatory surface. Additionally, our data suggests the lack of FKBP52 regulation in MR is due to divergence in this region of the BF3 surface. We are currently using site‐directed and random mutagenesis to generate a gain‐of‐function MR mutant that can respond to FKBP52. These studies will strongly support the BF3 region as an AR‐FKBP52 interaction site and allow us to further improve inhibitors that target with greater specificity.
Vitamin D supplementation in patients with diabetes mellitus type 2 on different therapeutic regimens: a one‐year prospective study (575.4)
Authors: Alkharfy Khalid , Al‐Daghri Nasser , Sabico Shaun , Al‐Othman Abdulaziz , Moharram Osama , Alokail Majed , Al‐Saleh Yousef , Kumar Sudhesh , Chrousos George
Published: June 2021

Background: The objective of this study was to determine the effect of vitamin D3 supplementation in a cohort of Saudi DMT2 population on diet, insulin and/or different oral hypoglycemic agents and compare them with a non‐DMT2 control cohort.

Methods: A total of 499 randomly selected Saudi subjects divided into 8 groups [Non‐DMT2 Control; Rosiglitazone alone; Diet; Insulin alone; Insulin+Orals; Metformin alone; Oral agents combination; Sulphonylurea alone] were included in this interventional study. All DMT2 patients were given 2000IU vitamin D3 daily, while the control group received none but were advised to increase sun exposure. Anthropometrics, glucose, lipid profile and 25‐hydroxyvitamin D (25‐OHVitD) were measured at baseline, 6 and 12 months.

Results: Circulating 25‐OHVitD concentrations improved in all groups. Metformin group showed the highest change in vitamin D levels both at 6 and 12 months than baseline (p< 0.001). The insulin+oral agents group showed significant improvements in triglycerides, total cholesterol, systolic blood pressure and HDL‐cholesterol in males. Significant decreases in triglycerides were observed in the rosiglitazone and insulin+oral hypoglycemic agent groups (p‐values <0.001).

Conclusion: DMT2 patients on insulin+oral agents benefitted the most in improving cardiovascular risk. Metformin but did not seem to confer other added cardiometabolic benefits.