Implantable Telemetry Research Articles

Sex Dependent Exacerbation of Salt Sensitive Hypertension and Cardiac Dysfunction Is Associated With Upregulation of miRNA Targeting ADAM17 and ACE2 in the Brain

Background: The DOCA-salt model has been widely used to study salt-sensitive hypertension and we previously reported that it is associated with ADAM17-mediated ACE2 shedding in the central nervous system. However, previously reported data are mainly from male mice, and it is unclear whether female mice exhibit the same phenotypes. Objective: We aimed to compare sex specific development of salt sensitive hypertension and cardiac dysfunction and investigate miRNA regulation associated with these changes. Methods: C57 mice (male and females, n = 8 -15) underwent uninephrectomy and telemetry implantation (DSI Inc.) for blood pressure (BP) conscious recording. After baseline recording, a DOCA pellet (50 mg, 21 days, Innovative Research of America) was implanted subcutaneously and drinking water was replaced by 1% NaCl solution to induce salt sensitive hypertension. Cerebral Spinal fluid (CSF) was collected at baseline and after 3 weeks of DOCA-salt treatment for exosome miRNA sequencing (Creative Biolab). In another set of mice, cardiac function was assessed by echocardiography (Visualsonics Vevo F2) and invasive left ventricular hemodynamics (Transonic). Mortality from each group was analyzed as well. Results: Baseline BP was not different between male and female mice. Following DOCA-salt treatment for 3 weeks, BP was increased in both sexes, with higher systolic BP observed in males compared to females (active phase: 173.0 ± 19.6 vs. 154.2 ± 19.1 mmHg, p<0.05). Although ejection fraction was not changed in either male or female mice, mitral peak early diastolic filling velocity and tissue doppler showed that E/E’ was increased in both sexes compared to baselines (Male: 33.4 ± 4 vs. 26.6 ± 1.9; Female: 30.3 ± 5.0 vs. 26.4 ± 4), with a greater increase in males (25.3% vs. 14.4%). In addition, hemodynamics revealed increased left ventricular end-diastolic pressure (11.1 ± 1.9 vs. 4.5 ± 0.5 mmHg, p<0.01) and relaxation time constant Tau (6.7 ± 1.0 vs. 4.9 ± 0.5 msec, p<0.01) in DOCA-salt treated males compared to non-treated controls, with no changes shown in female mice. These data suggest worsened cardiac diastolic function in males compared to females following DOCA-salt treatment. Moreover, a 20% mortality was observed in males while no death occured in females, confirming a more severe phenotype in males. Furthermore, miRNAseq analysis of CSF exosomes showed several upregulated miRNA in males but not in females following DOCA-salt treatment. Notably, miR206 associated with increased ADAM17 activity was increased by 8-fold in males but only 3-fold n females. Conclusion: DOCA-salt treatment promotes exacerbated hypertension, impaired cardiac function and mortality in a sex dependent manner, associated with upregulation of key miRNA targeting the brain renin-angiotensin system. The changes of ADAM17 and ACE2 targeted exosome miRNAs in the central nervous system might contribute to the sex difference in the cardiovascular dysfunction phenotypes in this model. This work was supported in part by a research grant from the National Institutes of Health (HL163588). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cardiovascular Autonomic Control During Hibernation in American Black Bears

The dramatic early decrease in HR upon entry into torpor in small hibernators has recently been shown to be associated with a marked increase in cardiac parasympathetic activity as indicated by increased short term beat to beat heart rate variability (RMSSD) normalized to average R-R interval (RMDSSD/RR, Zanetti et al 2023). At steady state torpor, as their body temperature drop to ~2°C, small hibernators benefit from the Q10 effect on cardiac pacemaker cells in maintaining a low HR with a reduced parasympathetic activity. Black bears in hibernation maintain body temperatures between 30-35°C while suppressing metabolism by 75% and with a marked decrease in HR from non-hibernating 55 bpm to hibernating 6-12 bpm including extreme respiratory sinus arrhythmia (Tøien et al 2011). As hibernating bears would not significantly benefit from the Q10 effect on cardiac pacemaker cells to sustain a low HR, we hypothesized that an elevated cardiac parasympathetic tone is needed to suppress HR. Continuous electrocardiogram (ECG) and blood pressure (BP) were recorded with implantable telemetry transmitters (DSI D70-CCP/EET and Konigsberg T28F-14B) in American black bears. A Pan-Tomkins algorithm modified for R-wave detection with long R-R intervals followed by visual inspection was used for the analysis. RMDSSD/RR for a 2 hour period in mid hibernation with no activity was 3 times higher than that during the resting phase in non-hibernation (0.68 vs. 0.23, n=10 and 7 respectively, p<0.001), confirming the presence of a high parasympathetic tone during hibernation. HR decreased from 68.3 ± 5.8 bpm to 12.9 ± 0.9 bpm (±SE, p<0.0001). RMSSD/RR during a 10 min period recovering from anesthesia (Telazol with supplemental Ketamin) during hibernation in mid March was only 0.019 ± 0.008 vs 0.58 ± 0.14 during a 10 min period before disturbance to induce anesthesia and HR 87.0 ± 8.7 b/min vs. 16.3 ± 1.4 b/min, demonstrating a parasympatholytic effect (n=6, p< 0.0006). The longest R-R intervals during extreme sinus arrhythmia detected in mid hibernation ranged from 11.8 to 33.0 s (Mean 19.0 ± 2.7, n=21). BP typically increased to above 200 mmHg during periods of rapid heart beats, and decayed slowly to 50-60 mmHg at the end of the longest R-R intervals. Analysis of data from a single bear from 2 hour periods at 2 week intervals throughout hibernation and recovery indicates that a high parasympathetic activity is maintained throughout hibernation and strongly correlated to the maximum observed R-R interval. We conclude that opposed to small hibernators, hibernating bears sustain low HR in hibernation with high parasympathetic activity controlling the extreme sinus arrhythmia. We suggest that BP is maintained during long R-R intervals by peripheral vasoconstriction. Supported by NIH COBRE grant number [P20GM130443]. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The rat telemetry assay and venous catheter access buttons for use in cardiovascular safety pharmacology assessments – Surgical methods, refinements and colony maintenance

IntroductionRat telemetry is the assay of choice to assess the potential effects of novel drug candidates on cardiovascular parameters during early drug discovery. Telemetry device implantation can be combined with venous catheter and access button implantation when intravenous administration of the drug substance is required. MethodsRats (Sprague Dawley or Han Wistar) were implanted with telemetry devices for arterial blood pressure measurement using either direct aortic catheterisation (n = 131) or aortic catheterisation via the femoral artery (n = 17). Bipolar leads for ECG recording were also implanted in some of the animals (n = 102). Femoral vein catheters and access buttons were implanted as a separate surgery after the initial telemetry implantation (n = 43). Results128 animals (86%) were implanted successfully with telemetry devices without any notable surgical or post-surgical problems. When considering the 2 different catheterisation methods separately, the success rate of the direct aortic approach was 88% compared to 76% with the aortic placement via the femoral artery. Lameness was the most common post-surgical problem. Blood loss during surgery and ischaemic patches on the tail were also observed at a low incidence with the direct aortic approach. Catheter pull-out occurred in some rats before the first signal check reducing the overall success rate for blood pressure measurement using the direct aortic approach to 85%. A 95% success rate was observed for catheter and access button implantation. DiscussionA high success rate is possible when implanting telemetry devices in rats with and without venous catheters and access buttons. We have attempted to provide solutions to problems and describe refinements to the procedure which may further improve surgical outcomes.

Use of Atrial Fibrillation Electrograms and T1/T2 Magnetic Resonance Imaging to Define the Progressive Nature of Molecular and Structural Remodeling: A New Paradigm Underlying the Emergence of Persistent Atrial Fibrillation.

The temporal progression states of the molecular and structural substrate in atrial fibrillation (AF) are not well understood. We hypothesized that these can be detected by AF electrograms and magnetic resonance imaging parametric mapping. AF was induced in 43 dogs (25-35 kg, ≥1 year) by rapid atrial pacing (RAP) (3-33 weeks, 600 beats/min), and 4 controls were used. We performed high-resolution epicardial mapping (UnEmap, 6 atrial regions, both atria, 130 electrodes, distance 2.5 mm) and analyzed electrogram cycle length, dominant frequency, organization index, and peak-to-peak bipolar voltage. Implantable telemetry recordings were used to quantify parasympathetic nerve activity over RAP time. Magnetic resonance imaging native T1, postcontrast T1, T2 mapping, and extracellular volume fraction were assessed (1.5T, Siemens) at baseline and AF. In explanted atrial tissue, DNA oxidative damage (8-hydroxy-2'-deoxyguanosine staining) and percentage of fibrofatty tissue were quantified. Cycle length and organization index decreased (R=0.5, P<0.05; and R=0.5, P<0.05; respectively), and dominant frequency increased (R=0.3, P n.s.) until 80 days of RAP but not thereafter. In contrast, voltage continued to decrease throughout the duration of RAP (R=0.6, P<0.05). Parasympathetic nerve activity increased following RAP and plateaued at 80 days. Magnetic resonance imaging native T1 and T2 times increased with RAP days (R=0.5, P<0.05; R=0.6, P<0.05) in the posterior left atrium throughout RAP. Increased RAP days correlated with increasing 8-hydroxy-2'-deoxyguanosine levels and with fibrosis percentage (R=0.5, P<0.05 for both). A combination of AF electrogram characteristics and T1/T2 magnetic resonance imaging can detect early-stage AF remodeling (autonomic remodeling, oxidative stress) and advanced AF remodeling due to oxidative stress and fibrosis.

Chicken Muscle-Derived ACE2-Upregulating Peptide VVHPKESF Reduces Blood Pressure Associated with the ACE2/Ang (1-7)/MasR Axis in Spontaneously Hypertensive Rats.

This study aims to investigate the antihypertensive effect of four chicken muscle-derived angiotensin (Ang)-converting enzymes (ACE)-regulating peptides: Val-Arg-Pro (VRP, ACE inhibition), Leu-Lys-Tyr and Val-Arg-Tyr (LKY and VRY, ACE inhibition and ACE2 upregulation), and Val-Val-His-Pro-Lys-Glu-Ser-Phe (VVHPKESF [V-F], ACE2 upregulation) in spontaneously hypertensive rats. Rats (12-14 weeks old) are grouped: 1) untreated, 2) VRP, 3) LKY, 4) VRY, and 5) V-F. Blood pressure (BP) is monitored using implantable telemetry technology. Over 18-day oral administration of 15mgkg-1 body weight (BW) per day, only peptide V-F significantly (p < 0.05) reduces BP, decreases circulating Ang II, and increases ACE2 and Ang (1-7) levels, and enhances aortic expressions of ACE2 and Mas receptor (MasR). Peptide V-F also attenuates vascular inflammation (TNFα, MCP-1, IL-1α, IL-15, and cyclooxygenase 2 [COX2]) and vascular oxidative stress (nitrotyrosine). The gastrointestinal (GI)-degraded fragment of peptide V-F, Val-Val-His-Pro-Lys (VVHPK), is also an ACE2-upregulating peptide. Peptides VRP, LKY, and VRY do not reduce BP, possibly due to low bioavailability or other unknown reasons. Peptide V-F is the first ACE2-upregulating peptide, purified and fractionated from food proteins based on in vitro ACE2 upregulation, that reduces BP associated with the activation of ACE2/Ang (1-7)/MasR axis; the N-terminal moiety VVHPK may be responsible for the antihypertensive effect of V-F.

Reversible expansion of tissue macrophages in response to macrophage colony-stimulating factor (CSF1) transforms systemic lipid and carbohydrate metabolism.

Macrophages regulate metabolic homeostasis in health and disease. Macrophage colony-stimulating factor (CSF1)-dependent macrophages contribute to homeostatic control of the size of the liver. This study aimed to determine the systemic metabolic consequences of elevating circulating CSF1. Acute administration of a CSF1-Fc fusion protein to mice led to monocytosis, increased resident tissue macrophages in the liver and all major organs, and liver growth. These effects were associated with increased hepatic glucose uptake and extensive mobilization of body fat. The impacts of CSF1 on macrophage abundance, liver size, and body composition were rapidly reversed to restore homeostasis. The effects of CSF1 on metabolism were independent of several known endocrine regulators and did not impact the physiological fasting response. Analysis using implantable telemetry in metabolic cages revealed progressively reduced body temperature and physical activity with no change in diurnal food intake. These results demonstrate the existence of a dynamic equilibrium between CSF1, the mononuclear phagocyte system, and control of liver-to-body weight ratio, which in turn controls systemic metabolic homeostasis. This novel macrophage regulatory axis has the potential to promote fat mobilization, without changes in appetence, which may have novel implications for managing metabolic syndrome.NEW & NOTEWORTHY CSF1 administration expands tissue macrophages, which transforms systemic metabolism. CSF1 drives fat mobilization and glucose uptake to support liver growth. The effects of CSF1 are independent of normal hormonal metabolic regulation. The effects of CSF1 are rapidly reversible, restoring homeostatic body composition. CSF1-dependent macrophages and liver size are coupled in a dynamic equilibrium.

Combined effects of methadone and quetiapine on respiratory rate, haemodynamic variables, and temperature in conscious rats.

Fatal poisonings where both methadone and quetiapine are detected post-mortem occurs frequently in legal autopsy cases. It is unclear whether quetiapine increases the risk of fatal methadone poisoning or if it is merely detected due to widespread use. We hypothesized that methadone and quetiapine would have additive toxic effects on respiratory rate, blood pressure, and the QTc-interval. To investigate this hypothesis, we used telemetry implants for measurements of respiratory rate, haemodynamic variables, the velocity of blood pressure changes, temperature, and movement in conscious, freely moving male Wistar rats aged 12-13 weeks. The combined effects of three accumulative i.p. doses of methadone (2.5, 10, 15 mg/kg) and quetiapine (3, 10, 30 mg/kg) were compared to rats treated with the same doses of each drug alone, and a vehicle-treated group in a randomized investigator blinded study. No additive effects of quetiapine and methadone on respiratory rate, haemodynamic variables, or movement were observed. However, body temperature was significantly lower by approximately 1.5°C on average in the group treated with both methadone and quetiapine (15 + 30 mg/kg) compared to the other groups. This indicates a synergistic effect of quetiapine and methadone on thermoregulation, which may increase the risk of fatal poisoning. We suggest studying this finding further in human settings.

PO-455617-1 EXPLORING THE INTERPLAY OF FUNCTIONAL AND STRUCTURAL REMODELING PROGRESSION IN ATRIAL FIBRILLATION IN A RAPID ATRIAL PACING CANINE MODEL

In sustained atrial fibrillation (AF) it is still unclear to what extent, at which time points functional and structural remodeling processes develop over time, and which parameters can measure the progression state. We hypothesized that persistent AF progression state induced by rapid atrial pacing (RAP) can be uncovered by measuring electrogram characteristics, OS levels, nerve activity, and the degree of fibrosis with MRI imaging. AF was induced in 15 dogs (25-35 kg, ≥1 year) by RAP (3-17 weeks RAP, 600 beats/min), and 8 controls were used. We performed high-resolution epicardial mapping (6 regions, 130 electrodes, distance 2.5mm) (Figure 1) and analyzed bipolar peak-to-peak voltage, cycle length (CL), and organization index (OI). We quantified OS levels as damaged nuclei against total number in 8-OHdG staining (N=36 samples, 6 animals) and degree of fibrosis (Masson’s trichrome) (N=24, 4 dogs). We also quantified in implantable telemetry recordings (DSI) parasympathetic nerve activity (PNA) (2 hours) assessed as the time between PNA spikes (3 AF dogs, 3 controls). MRI native T1, post-contrast T1, and T2 mapping, and late gadolinium enhancement (LGE) were assessed (1.5T clinical scanner, Siemens Aera) in the posterior left atrium (PLA) (7 AF, 8 at baselines). Voltage, CL, and OI decreased (R=0.9, P<0.05), (R=0.6, P<0.05), (R=0.5, P<0.05) over time of RAP (Figure 1). After 80 RAP days voltage changed, but no more changes in other EGM measures were observed. OS levels in AF were highest in the left atrial free wall and lowest in the appendages (Figure 2). The electrogram measures OI and CL decreased with increased oxidative stress levels (Figure 2). Fibrosis (R=0.6, P<0.05) increased with OS (R=0.6, P<0.05) in all atrial regions (Figure 2). Parasympathetic nerve activity increased by factor 5 after 120 RAP days, (Figure 3). Native T1, T2 times increased with RAP days (R=0.6, P<0.05), (R=0.4, P<0.05) in PLA (<120 pacing days) (Fig. 4). Functional and structural remodeling in persistent AF is the result of an interplay among the development of increased OS, fibrosis, and nerve activity in a RAP canine model and can moderately be uncovered using the electrogram measures CL and OI until the saturation point after 80 RAP days. Voltage also uncovers the AF progression state post 80- RAP days with a strong correlation coefficient. Additional parasympathetic nerve recordings and MRI imaging including T1 mapping may be complementary to detect AF remodeling states.

Chronic Effects of Apelin on Cardiovascular Regulation and Angiotensin II-Induced Hypertension.

Apelin, by stimulation of APJ receptors, induces transient blood pressure (BP) reduction and positive inotropic effects. APJ receptors share high homology with the Ang II type 1 receptor; thus, apelin was proposed to play a protective role in cardiovascular disease by antagonizing the actions of Ang II. In this regard, apelin and apelin-mimetics are currently being studied in clinical trials. However, the chronic effect of apelin in cardiovascular regulation has not been fully investigated. In the current study, blood pressure (BP) and heart rate (HR) were recorded using a telemetry implantation approach in conscious rats, before and during chronic subcutaneous infusion of apelin-13, using osmotic minipumps. At the end of the recording, the cardiac myocyte morphology was examined using H&E staining, and cardiac fibrosis was evaluated by Sirius Red in each group of rats. The results demonstrated that the chronic infusion of apelin-13 did not change either BP or HR. However, under the same condition, the chronic infusion of Ang II induced significant BP elevation, cardiac hypertrophy, and fibrosis. Co-administration of apelin-13 did not significantly alter the Ang II-induced elevation in BP, changes in cardiac morphology, and fibrosis. Taken together, our experiments showed an unexpected result indicating that the chronic administration of apelin-13 did not alter basal BP, nor did it change Ang II-induced hypertension and cardiac hypertrophy. The findings suggest that an APJ receptor biased agonist could be a better therapeutic alternative for treatment of hypertension.

Stress Drivers of Glucose Dynamics during Ozone Exposure Measured Using Radiotelemetry in Rats.

Inhaled irritant air pollutants may trigger stress-related metabolic dysfunction associated with altered circulating adrenal-derived hormones. We used implantable telemetry in rats to assess real-time changes in circulating glucose during and after exposure to ozone and mechanistically linked responses to neuroendocrine stress hormones. First, using a cross-over design, we monitored glucose during ozone exposures (0.0, 0.2, 0.4, and ) and nonexposure periods in male Wistar Kyoto rats implanted with glucose telemeters. A second cohort of unimplanted rats was exposed to ozone (0.0, 0.4 or 0.8 ppm) for 30 min, 1 h, 2 h, or 4 h with hormones measured immediately post exposure. We assessed glucose metabolism in sham and adrenalectomized rats, with or without supplementation of adrenergic/glucocorticoid receptor agonists, and in a separate cohort, antagonists. Ozone () was associated with significantly higher blood glucose and lower core body temperature beginning 90 min into exposure, with reversal of effects 4-6 h post exposure. Glucose monitoring during four daily 4-h ozone exposures revealed duration of glucose increases, adaptation, and diurnal variations. Ozone-induced glucose changes were preceded by higher levels of adrenocorticotropic hormone, corticosterone, and epinephrine but lower levels of thyroid-stimulating hormone, prolactin, and luteinizing hormones. Higher glucose and glucose intolerance were inhibited in rats that were adrenalectomized or treated with adrenergic plus glucocorticoid receptor antagonists but exacerbated by agonists. We demonstrated the temporality of neuroendocrine-stress-mediated biological sequalae responsible for ozone-induced glucose metabolic dysfunction and mechanism in a rodent model. Stress hormones assessment with real-time glucose monitoring may be useful in identifying interactions among irritant pollutants and stress-related illnesses. https://doi.org/10.1289/EHP11088.

Blood pressure and the kidney cortex transcriptome response to high-sodium diet challenge in female nonhuman primates.

Blood pressure (BP) is influenced by genetic variation and sodium intake with sex-specific differences; however, studies to identify renal molecular mechanisms underlying the influence of sodium intake on BP in nonhuman primates (NHP) have focused on males. To address the gap in our understanding of molecular mechanisms regulating BP in female primates, we studied sodium-naïve female baboons (n = 7) fed a high-sodium (HS) diet for 6 wk. We hypothesized that in female baboons variation in renal transcriptional networks correlates with variation in BP response to a high-sodium diet. BP was continuously measured for 64-h periods throughout the study by implantable telemetry devices. Sodium intake, blood samples for clinical chemistries, and ultrasound-guided kidney biopsies were collected before and after the HS diet for RNA-Seq and bioinformatic analyses. We found that on the LS diet but not the HS diet, sodium intake and serum 17 β-estradiol concentration correlated with BP. Furthermore, kidney transcriptomes differed by diet-unbiased weighted gene coexpression network analysis revealed modules of genes correlated with BP on the HS diet but not the LS diet. Our results showed variation in BP on the HS diet correlated with variation in novel kidney gene networks regulated by ESR1 and MYC; i.e., these regulators have not been associated with BP regulation in male humans or rodents. Validation of the mechanisms underlying regulation of BP-associated gene networks in female NHP will inform better therapies toward greater precision medicine for women.

Abstract 028: Toll-Like Receptor 4 Promotes Renal Immune Cell Infiltration And The Development Of Renal Damage During Dahl Salt-Sensitive Hypertension

Previous work in our laboratory has implicated a significant role for the immune system in potentiating the hypertensive and kidney damage phenotypes which occur in the Dahl Salt-Sensitive (SS) rat. Toll-like receptors (TLRs), usually expressed on macrophages, play a key role in the innate immune system. A number of TLRs have been implicated in cardiovascular disease and chronic kidney disease with particular interest in TLR4, which specifically binds gram-negative lipopolysaccharide or endogenous molecules produced as a result of tissue injury. We previously performed a transcriptomic analysis which demonstrated TLR4 upregulation in renal medullary tissue of Dahl SS rats fed a high salt (4.0% NaCl) diet. The present study tests the hypothesis that TLR4 influences high salt diet-induced hypertension and renal damage in the Dahl SS rat. Using CRISPR/Cas9 technology, a mutation was generated in the TLR4 gene on the Dahl SS background, creating a 1-bp frameshift deletion resulting in a premature stop codon. SS TLR4-/- males and parental Dahl SS males (SS TLR4+/+ ), maintained on a 0.4% NaCl chow, underwent carotid artery telemetry implantation at 7 weeks of age. After recovery and baseline mean arterial pressure (MAP) recordings (day 0: 125.6 ± 0.5 vs 124.2 ± 2 mmHg), animals were switched to a 4.0% NaCl chow for 3 weeks. Overnight urine collections occurred once during the low salt period and weekly throughout the high salt challenge. Kidneys were flushed prior to euthanasia. Although SS TLR4-/- MAP was not different from SS TLR4+/+ during the high salt challenge (day 21: 176.8 ± 14 mmHg, n=2 vs 172.3 ± 18 mmHg, n=4), high salt day 21 urinary albumin excretion was significantly reduced in SS TLR4-/- (272.7 ± 65 mg/day, n=5, p&lt;0.01) compared to SS TLR4+/+ (427.5 ± 67 mg/day, n=7), indicating reduced renal damage in TLR4 absence. Furthermore, flow cytometric analysis of isolated renal immune cells indicated a 33% reduction in CD45+ total leukocytes (5.0 ± 1.2 vs 3.4 ± 0.9 x10 6 cells) and a 31% reduction in CD11bc+ monocytes/macrophages (3.9 ± 0.9 vs 2.6 ± 0.7 x10 6 cells) in SS TLR4-/- (n=5) vs SS TLR4+/+ (n=7) kidneys. Together, these data indicate that TLR4 may drive the immune response during the induction and progression of salt-sensitive hypertension and renal disease.

Real-Time Electrocardiogram Monitoring during Treadmill Training in Mice.

Regular physical exercise is a major contributor to cardiovascular health, influencing various metabolic as well as electrophysiological processes. However, in certain cardiac diseases such as inherited arrhythmia syndromes, e.g., arrhythmogenic cardiomyopathy (ACM) or myocarditis, physical exercise may have negative effects on the heart leading to a proarrhythmogenic substrate production. Currently, the underlying molecular mechanisms of exercise-related proarrhythmogenic remodeling are largely unknown, thus it remains unclear which frequency, duration, and intensity of exercise can be considered safe in the context of disease(s). The proposed method allows to study proarrhythmic/antiarrhythmic effects of physical exercise by combining treadmill training with real-time monitoring of the ECG. Implantable telemetry devices are used to continuously record the ECG of freely moving mice over a period of up to 3 months both at rest and during treadmill training. Data acquisition software with its analysis modules is used to analyze basic ECG parameters such as heart rate, P wave duration, PR interval, QRS interval, or QT duration at rest, during and after training. Furthermore, heart rate variability (HRV) parameters and occurrence of arrhythmias are evaluated. In brief, this manuscript describes a step-by-step approach to experimentally explore exercise induced effects on cardiac electrophysiology, including potential proarrhythmogenic remodeling in mouse models.

Repetitive Preload Elevation in Swine with Chronic Coronary Artery Disease Promotes Pro‐Fibrotic Gene Expression and Transmural Fibrosis in Remote Non‐Ischemic Myocardium

ObjectiveUsing implantable telemetry, we recently found that transient elevations in preload are common in swine with multi‐vessel coronary artery disease (CAD), presumably as a result of intermittent demand‐induced regional myocardial ischemia. Based on prior evidence that repetitive preload elevation can lead to interstitial fibrosis and myocardial stiffening in the absence of ischemia, the present study was designed to compare temporal changes in pro‐fibrotic gene expression and transmural variability in interstitial fibrosis in ischemic vs. non‐ischemic areas of the left ventricle (LV) in a porcine model of chronic CAD.MethodsA total of 20 juvenile swine were instrumented with fixed 1.5 mm constrictors on the proximal left anterior descending (LAD) and left circumflex (LCx) coronary arteries and followed for up to 3‐months, during which coronary stenosis severity progressed to &gt;90 % (LAD: 93±2 %; LCx: 91±3 %). Expression of fibronectin (FN1) and lysyl oxidase like‐2 (LOXL‐2) was assessed in ischemic (LAD) and non‐ischemic (RCA) regions of the LV via quantitative PCR 1‐month (n=4) or 3‐months (n=8) after instrumentation and compared to normal controls (n=8). Post‐mortem quantification of interstitial fibrosis was performed on picrosirius red‐stained tissue samples from ischemic and non‐ischemic regions (n=16), with an automated classifier‐based approach to compare regional transmural variability in collagen content to that of normal controls (n=11). Flash‐frozen tissue samples from a sub‐set of animals with CAD (n=4) and normal controls (n=4) were subjected to uniaxial tensile testing to assess passive subendocardial stiffness.ResultsOne month after instrumentation, FN1 expression was 2.5±0.9‐fold higher than control in the ischemic region and 2.7±0.7‐fold higher than control in the non‐ischemic region of the LV (both p&lt;0.05). LOXL2 expression was also significantly higher in the ischemic (2.2 ± 0.6‐fold higher than control) and non‐ischemic (2.4 ± 0.3‐fold higher than control) regions of the LV at this timepoint. Two months later, pro‐fibrotic gene expression had normalized but a significant elevation in interstitial collagen content was observed throughout the LV of animals with CAD (Figure; left panel). Interestingly, subendocardial fibrosis was particularly prominent in the non‐ischemic RCA territory, which coincided with a significant increase in subendocardial passive stiffness in this region (Figure; right panel).ConclusionsIn a porcine model of chronic CAD, repetitive preload elevation is associated with transient changes in pro‐fibrotic gene expression that ultimately lead to transmural fibrosis that is particularly prominent in non‐ischemic areas of the LV. Regional transmural variability in collagen content coincides with changes in the passive stiffness of non‐ischemic myocardium, suggesting that repetitive preload elevation may contribute to fibrosis‐mediated diastolic dysfunction in patients with chronic CAD.

325 Blood pressure and the kidney cortex transcriptome response to high sodium diet challenge in female nonhuman primates

OBJECTIVES/GOALS: The goal of this study was to understand the impact of a high sodium diet on gene networks in the kidney that correlate with blood pressure in female primates, and translating findings to women. METHODS/STUDY POPULATION: Sodium-naïve female baboons (n=7) were fed a low-sodium (LS) diet for 6 weeks followed by a high sodium (HS) diet for 6 weeks. Sodium intake, serum 17 beta-estradiol, and ultrasound-guided kidney biopsies for RNA-Seq were collected at the end of each diet. Blood pressure was continuously measured for 64-hour periods throughout the study by implantable telemetry devices. Weighted gene coexpression network analysis was performed on RNA-Seq data to identify transcripts correlated with blood pressure on each diet. Network analysis was performed on transcripts highly correlated with BP, and in silico findings were validated by immunohistochemistry of kidney tissues. RESULTS/ANTICIPATED RESULTS: On the LS diet, Na+ intake and serum 17 beta-estradiol concentration correlated with BP. Cell type composition of renal biopsies was consistent among all animals for both diets. Kidney transcriptomes differed by diet; analysis by unbiased weighted gene co-expression network analysis revealed modules of genes correlated with BP on the HS diet. Network analysis of module genes showed causal networks linking hormone receptors, proliferation and differentiation, methylation, hypoxia, insulin and lipid regulation, and inflammation as regulators underlying variation in BP on the HS diet. Our results show variation in BP correlated with novel kidney gene networks with master regulators PPARG and MYC in female baboons on a HS diet. DISCUSSION/SIGNIFICANCE: Previous studies in primates to identify molecular networks dysregulated by HS diet focused on males. Current clinical guidelines do not offer sex-specific treatment plans for sodium sensitive hypertension. This study leveraged variation in BP as a first step to identify correlated kidney regulatory gene networks in female primates after a HS diet.

Effects of early exercise training on the severity of autonomic dysreflexia following incomplete spinal cord injury in rodents.

Hemodynamic instability and cardiovascular (CV) dysfunction are hallmarks of patients living with cervical and high thoracic spinal cord injuries (SCI). Individuals experience bouts of autonomic dysreflexia (AD) and persistent hypotension which hamper the activities of daily living. Despite the widespread use of exercise training to improve health and CV function after SCI, little is known about how different training modalities impact hemodynamic stability and severity of AD in a model of incomplete SCI. In this study, we used implantable telemetry devices to assess animals with T2 contusions following 3.5 weeks of exercise training initiated 8 days post‐injury: passive hindlimb cycling (T2‐CYC, n = 5) or active forelimb swimming (T2‐SW, n = 6). Uninjured and non‐exercised SCI control groups were also included (CON, n = 6; T2‐CON, n = 7; T10‐CON, n = 6). Five weeks post‐injury, both T2‐CON and T2‐CYC presented with resting hypotension compared to uninjured CON and T10‐CON groups; no differences were noted in resting blood pressure in T2‐SW versus CON and T10‐CON. Furthermore, pressor responses to colorectal distention (AD) were larger in all T2‐injured groups compared to T10‐CON, and were not attenuated by either form of exercise training. Interestingly, when T2‐injured animals were re‐stratified based on terminal BBB scores (regardless of training group), animals with limited hindlimb recovery (T2‐LOW, n = 7) had more severe AD responses. Our results suggest that the spontaneous recovery of locomotor and autonomic function after severe but incomplete T2 SCI also influences the severity of AD, and that short periods (3.5 weeks) of passive hindlimb cycling or active forelimb swimming are ineffective in this model.

Analyzing Long-Term Electrocardiography Recordings to Detect Arrhythmias in Mice.

Arrhythmias are common, affecting millions of patients worldwide. Current treatment strategies are associated with significant side effects and remain ineffective in many patients. To improve patient care, novel and innovative therapeutic concepts causally targeting arrhythmia mechanisms are needed. To study the complex pathophysiology of arrhythmias, suitable animal models are necessary, and mice have been proven to be ideal model species to evaluate the genetic impact on arrhythmias, to investigate fundamental molecular and cellular mechanisms, and to identify potential therapeutic targets. Implantable telemetry devices are among the most powerful tools available to study electrophysiology in mice, allowing continuous ECG recording over a period of several months in freely moving, awake mice. However, due to the huge number of data points (>1 million QRS complexes per day), analysis of telemetry data remains challenging. This article describes a step-by-step approach to analyze ECGs and to detect arrhythmias in long-term telemetry recordings using the software, Ponemah, with its analysis modules, ECG Pro and Data Insights, developed by Data Sciences International (DSI). To analyze basic ECG parameters, such as heart rate, P wave duration, PR interval, QRS interval, or QT duration, an automated attribute analysis was performed using Ponemah to identify P, Q, and T waves within individually adjusted windows around detected R waves. Results were then manually reviewed, allowing adjustment of individual annotations. The output from the attribute-based analysis and the pattern recognition analysis was then used by the Data Insights module to detect arrhythmias. This module allows an automatic screening for individually defined arrhythmias within the recording, followed by a manual review of suspected arrhythmia episodes. The article briefly discusses challenges in recording and detecting ECG signals, suggests strategies to improve data quality, and provides representative recordings of arrhythmias detected in mice using the approach described above.

Repetitive Preload Elevation and Stretch‐Induced Stiffening of Non‐Ischemic Myocardium in a Porcine Model of Multi‐Vessel Coronary Artery Disease

Objective We have recently shown that a transient elevation in left ventricular (LV) preload causes stretch-induced myocyte injury, with repetitive episodes resulting in interstitial fibrosis and reduced diastolic compliance. The present study was designed to test the hypothesis that this phenomenon contributes to stiffening of remote, non-ischemic myocardium that is subjected to repetitive preload elevation secondary to demand-induced ischemia in the setting of chronic multi-vessel coronary artery disease (CAD). Methods Swine (n=12) were instrumented with fixed 1.5 mm constrictors on the proximal left anterior descending (LAD) and left circumflex (LCx) coronary arteries. One month later, continuous ECG and LV pressure monitoring was initiated via implantable telemetry (PhysioTel Digital, DSI) to assess the frequency of preload elevation (LV end-diastolic pressure (EDP) > 30 mmHg) and tachycardia (heart rate > 170 beats/min). Serial physiological studies were performed for 3 months to assess stenosis severity (angiography), coronary flow reserve (fluorescent microspheres at rest and during adenosine vasodilation), and LV function (echocardiography). Circumferential diastolic stiffness was derived from stress-strain relationships obtained via speckle tracking echocardiography and invasive measurements of LV minimum pressure and EDP. Myocardial tissue samples were subsequently collected from the LAD (ischemic) and right coronary artery (RCA; non-ischemic) perfusion territories for post-mortem quantification of collagen content (hydroxyproline assay) and comparison to normal controls (n=8). Results By the end of the 3 month study period, animals exhibited severe LAD (93 ± 2 %) and LCx (91 ± 3 %) stenoses and significant reductions in subendocardial flow reserve (LAD: 1.4 ± 0.2; LCx: 1.3 ± 0.2) relative to the RCA territory (3.8 ± 0.6). Although LV EDP was only mildly elevated at rest (18 ± 2 mmHg), transient preload elevation occurred frequently (63 ± 10 episodes/day) and resulted in a total of 199 ± 45 min/day during which LV EDP exceeded 30 mmHg. Spontaneous tachycardia was also common (26 ± 5 episodes/day) and associated with transient elevations in LV EDP (28 ± 2 mmHg; p<0.05 vs. rest). Although LV ejection fraction was preserved (58 ± 3 %), radial and circumferential strain was depressed in ischemic and non-ischemic segments of the LV compared with controls (Table). Non-ischemic segments also exhibited increased diastolic stiffness, which coincided with a significant ~40% elevation in collagen content (0.31 ± 0.03 mg/g) vs. normal controls (0.22 ± 0.03 mg/g) that was comparable to that observed in the ischemic area of the LV (0.29 ± 0.02 mg/g). Conclusions Transient elevations in preload are common in swine with multi-vessel CAD and associated with interstitial fibrosis and myocardial stiffening in normally-perfused segments of the LV. Repetitive stretch-induced stiffening of remote myocardium may contribute to diastolic dysfunction and could explain the high prevalence of angiographically-significant CAD in patients with heart failure with preserved ejection fraction (HFpEF).

Alprazolam-induced EEG spectral power changes in rhesus monkeys: a translational model for the evaluation of the behavioral effects of benzodiazepines.

Benzodiazepines induce electroencephalography (EEG) changes in rodents and humans that are associated with distinct behavioral effects and have been proposed as quantitative biomarkers for GABAA receptor modulation. Specifically, central EEG beta and occipital EEG delta activity have been associated with anxiolysis and sedation, respectively. The extent to which nonhuman primates show the same dose- and topography-dependent effects remained unknown. We aimed at establishing a nonhuman primate model for the evaluation of benzodiazepine EEG pharmacology. Four adult male rhesus monkeys were prepared with fully implantable telemetry devices that monitored activity, peripheral body temperature, and contained two EEG (central and occipital), one electromyography (EMG), and one electrooculography channel. We investigated daytime alprazolam-induced changes in EEG spectral power, sleep-wake states, EMG activity, locomotor activity, and body temperature. Alprazolam (0.01-1.8 mg/kg, i.m.) or vehicle was administered acutely, and telemetry recording was conducted for 1 h. Daytime alprazolam dose-dependently increased central EEG power (including beta activity), increased occipital EEG delta power, and decreased occipital EEG alpha, theta, and sigma power. There was an ~8-fold difference in the potency of alprazolam to increase central EEG beta vs. occipital EEG delta activity (based on relative EEG power). The highest dose, which increased both central EEG beta and occipital EEG delta relative power, induced sedative effects (increased time spent in N1 and N2 sleep stages) and decreased peripheral body temperature and locomotor activity. Alprazolam induces dose- and topography-dependent EEG changes in rhesus monkeys and provides a valuable model for studying benzodiazepine pharmacology.

Abstract MP08: Testosterone Contributes To Renal Necrosis In Male Spontaneously Hypertensive Rats (SHR).

We previously published a sex difference in renal necrosis in SHR with males having a maturation induced increase in renal necrosis that is absent in females. Testosterone is known to drive an increase both in blood pressure (BP) with maturation in male SHR and necrosis of renal tubular epithelial cell in vitro. In the current study we tested the hypothesis that testosterone underlies the maturation induced increase in renal necrosis in male SHR. At 4 weeks of age, male SHR were randomly assigned to either sham or gonadectomy (ORX) groups (n=3). To control for the influence of high BP on renal necrosis, a third group was subjected to a sham surgery and treated with the anti-hypertensive medications hydrochlorothiazide (HCTZ; 55mg/kg/day) and reserpine (Res; 4.5 mg/kg/day) in drinking water starting at 9 weeks of age (n=4) to prevent age-related increases in BP. At 8 weeks of age, telemeters were implanted in all groups followed by a recovery for 1 week before BP was recorded. A separate set of rats were randomly assigned to Sham (n=3), ORX (n=4), or HCTZ/Res (n=4) and subjected to the exact same procedures as the previous set except for telemetry implantation. All rats were euthanized at 13 weeks of age and kidneys were collected for the quantification of renal necrosis using flow cytometric analysis of 7AAD + cells. Data were analyzed using one-way ANOVA and presented as mean ± standard error. BP was significantly lower in ODX and HCTZ/Res treated groups compared to sham (mean arterial blood pressure (mmHg): Sham= 139±2; HCTZ/Res= 117±3; ODX= 126±2; p=0.002; n=3-4). Renal necrosis was also significantly less in ORX rats, but not altered in HCTZ/Res treated groups compared to sham (renal necrosis expressed as % total gated kidney cells: Sham= 6±0.3%; HCTZ/Res=5±0.4%; ODX= 4±0.4%; P=0.003; n=6-7). Testosterone contributes to maturation induced increase in renal necrosis in male SHR.