bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P319: Chronic Ischemia Induces Global Changes in Placental Epigenetic Modifications
Authors: Chapman Heather , George Eric M
Published: June 2021

Preeclampsia is a common and serious obstetrical complication, hallmarked by new-onset hypertension and maternal endothelial dysfunction. Believed to result as a consequence of placental insufficiency and chronic placental ischemia, the symptoms of preeclampsia are caused by release of pathogenic factors from the placenta itself. A number of these factors have been identified, and it is likely that there are molecular mechanisms which remain obscure. The molecular regulation of even the known factors is often unclear. Here, we have used an established rodent model of placental ischemia which mimics many of the aspects of the human disorder to determine the direct effects of ischemia on global epigenetic modification in the placenta proper. In response to placental insufficiency, maternal blood pressure and rate of fetal demise increased significantly, while placental and fetal mass were decreased. Whole placental levels of histone H3 acetylation at K9 and K27 were increased ~2 and 3 fold respectively. Global methylation of cytosine from placental DNA was low in both groups (<1%), but there was ~50% increase in 5-mC in response to chronic ischemia. These results suggest that chronic placental ischemia induces global changes in epigenetic modification of chromatin in the placenta. This could be an important factor in the regulation of known and unknown pathogenic factors produced by the placenta in the preeclampsia patient.
Abstract P321: Differential Leptin Levels are Associated with Hypertensive Disorders of Pregnancy and Adverse Pregnancy Outcomes
Authors: Santillan Donna A , Cowman Whitney L , Scroggins Sabrina M , Santillan Mark K
Published: June 2021

During pregnancy, there are normal changes in maternal body weight and blood pressure that occur. Leptin is found in higher levels in obese individuals and has been demonstrated to have an inhibitory effect on myometrial contractility and to regulate blood pressure. Obesity in pregnancy is common and is associated with many complications, including hypertension in pregnancy, failed induction of labor (IOL), and intrauterine growth restriction. Our goal was to determine whether maternal leptin in a pregnant population is indicative of hypertensive disorders in pregnancy, dysfunctional labor, and whether there is a correlation between cord blood leptin and birthweight. We utilized a case control study with samples from the UI Maternal Fetal Tissue Bank (IRB#200910784). In order to analyze labor outcomes, 168 women were selected based on having undergone an IOL, including 54 failed IOL. Maternal/neonatal characteristics were collected from the medical record. Maternal and cord blood plasma leptin and total protein levels were measured using commercially available ELISAs. Bivariate analyses and logistic regression models were constructed using regression identified clinically-significant confounding variables. All variables were tested at significance level of 0.05. Women with hypertensive disorders in pregnancy, including pregnancy-induced hypertension and preeclampsia, had higher maternal leptin levels (14783 vs. 21440 pg/mL, p=0.049). Women with failed IOL also had higher maternal plasma leptin values (0.5 vs 0.3 leptin/protein [pg/ug],
P
= 0.01). Birthweight was also correlated with cord blood leptin (correlation coefficient 0.494
P <
0.001). These data suggest that maternal and fetal leptin levels are associated with the central mechanisms responsible for poor pregnancy outcomes.
Abstract P327: Increased Brain iNOS Contributes to Hypertension in Dahl Salt Sensitive Rats
Authors: Huber Michael J , Zhu Fengli , Larson Robert A , Chen Qing-Hui , Shan Zhiying
Published: June 2021

The hypothalamic paraventricular nucleus (PVN) is one of the key central nuclei to play an important role in regulating arterial blood pressure (ABP) of salt-sensitive hypertension (SSH). However, the detailed molecular mechanism(s) whereby the PVN increases ABP are not well understood. Here, we tested the hypothesis that high salt (HS) loading increases expression of iNOS in the PVN which contributes to SSH. Six-week-old male Dahl salt sensitive (Dahl S) rats and age matched Sprague Dawley (SD) rats were fed either a HS (4% NaCI) or a normal salt (NS, 0.4% NaCl) diet (n=4~7/group). Mean arterial pressure (MAP) was measured via tail cuff method. Five weeks following diet treatment, HS diet induced hypertension in Dahl S rats (HS: 153±9; vs. NS: 122±2 mmHg, P<0.05), but not in SD rats (HS: 107±3; vs. NS: 107±2 mmHg). Rats were then euthanized and PVN tissues were punched out for real time PCR. The HS diet induced dramatic increases in mRNA levels of iNOS (25-fold), and Fra1 (3.6-fold), a chronic neuronal activation marker, in Dahl S rat but not in SD rats. Next, we investigated the effect of intracerebroventricular (ICV) administration of hypertonic saline on PVN iNOS and Fra1 expression in SD rats. Anesthetized adult male SD rats received ICV infusion of isotonic NaCI (0.15 M, 2μl, as control) or hypertonic NaCI (2M, 2μl) (n=7~8/group). Three hours following ICV infusion, rats were euthanized and PVN mRNA levels of iNOS and Fra1 were assayed. ICV hypertonic saline increased mRNA levels of iNOS (9.5-fold) and Fra1 (4.1-fold). We further tested whether these increases in iNOS and Fra1 expression occurred in neurons. Incubation of hypertonic saline (10 mM NaCI) for 3 hours increased iNOS (6-fold) and Fra1 (2.8-fold) mRNA levels in neuronal cultures from the hypothalamus containing the PVN. Finally, we tested whether increased iNOS activity contributes to ABP elevation in Dahl SSH. In anaesthetized Dahl S rats, bilateral PVN microinjection of the iNOS inhibitor, aminoguanidine (250 pmol) significantly decreased MAP in HS treated animals compared to rats with a NS diet (HS: -13±3; vs. NS: -2±2 mmHg, P<0.05) (n=5/group). These observations suggest that HS intake increases iNOS expression in PVN neurons, which may contribute to the central neural mechanism of Dahl SSH.
Abstract P337: Angiotensin 1-7 Mitigates Oxidative Stress, Protects Renal Dopamine D1 Receptor Function and Prevents Development of Hypertension
Authors: Lokhandwala Mustafa , Diaz Diaz Andrea , Banday Anees Ahamd
Published: June 2021

The role of angiotensin in etiology of cardiovascular diseases especially in hypertension is well established. Renin-angiotensin-aldosterone contributes to the development and maintenance of hypertension directly by increases in vascular tone and renal sodium reabsorption or indirectly by increasing oxidative stress and inflammation. Contrary to this pathological arm, angiotensin (Ang) 1-7 via Mas receptors has been reported to protect the cardiovascular function although the exact mechanism is not yet clear. We have previously shown that oxidative stress leads to renal dopamine D1 receptor (D1R) dysfunction which could disrupt sodium regulation and subsequently lead to hypertension. In here we wanted to test whether chronic administration of Ang 1-7 in mice could mitigate oxidative stress, protect renal D1R function and prevent development of hypertension. Mice (C57BL) were implanted with telemetry probes and concomitantly treated with L-buthionine sulfoximine (BSO, in drinking water) and Ang 1-7 (via jugular vein by osmotic pumps). Control (C, no treatment) and shams (implanted with saline filled pumps) exhibited similar behavioral and physiological parameters. Mice treated with BSO alone exhibited increased oxidative stress and high BP as compared to controls. Ang 1-7 treatment did not affect oxidative stress and BP in control mice but prevented the increase in BP and oxidative milieu in BSO treated mice. Mean arterial pressure (mmHg), C: 78.5 ± 2.3*; BSO: 97.3 ± 3.8; Ang 1-7: 80.1* ± 4.1; BSO+Ang 1-7: 83.2 ± 3.4*, *P <0.05 vs BSO. SKF38393, a D1R agonist, increased urine and sodium excretion in control mice but failed to induce diuresis or natriuresis in BSO-treated mice. Treatment with Ang 1-7 protected D1R function as both natriuresis and diuresis was observed in mice treated with BSO plus Ang 1-7. Chronic Ang 1-7 had no effect on D1R function in the absence of BSO. These data show that oxidative stress leads to hypertension by disrupting renal D1R dependent sodium regulation. Ang 1-7 mitigates oxidative stress, protects renal D1R function and prevents increase in BP. This study provides a new insight on how beneficial arm of Ang system could protect renal D1R-mediated sodium regulation and prevent development of hypertension during oxidative stress.
Abstract P346: Endothelial Function and NO Bioavailability are Improved in Angiotensin Ii-treated Transglutaminase-2 Knock-out Mice
Authors: Savoia Carmine , Arrabito Emanuele , Chiandotto Sergio , Nicoletti Carmine , Carletti Raffaella , Di Gioia Cira , Volpe Massimo
Published: June 2021

We hypothesized that transglutaminase-2 (TG2) may contribute to the impaired functional properties of resistance arteries from angiotensin-II-treated mice. TG2-knockout mice (TG2-K/O, 12 weeks old, n=6) and wild type (WT) mice were treated or not with angiotensin-II (400ng/kg/min) for 14 days. Blood pressure (BP) and heart rate (HR) were measured by tail-cuff method. Endothelium-dependent and -independent relaxations were assessed by concentration-response curves to acetylcholine (1nM-to-100μM)±L-NAME (100μM) and sodium nitroprusside (10nM-to-1mM) respectively, in mesenteric arteries pre-contracted with norepinephrine (10μM). The expression of p-eNOS-(S1177)/eNOS, NOSIP (the negative modulator of eNOS), NOX-1, and its positive modulator ERp72 were evaluated in aorta by immunoblotting. Reactive oxygen species (ROS) production in aorta was evaluated by dihydroethidium staining. Plasma nitrate/nitrate were measured by ELISA. BP and HR were higher in TG2-K/O mice compared to WT (116.8±0.9 mmHg vs 89.6±1.5 mmHg, P<0.001; and 595.0±15.0 bpm vs 467.1±14.7 bpm, P<0.001, respectively). In both groups, angiotensin-II increased significantly BP (+28% in WT, and +21% in TG2-K/O) and HR (+33% in WT, and +9% in TG2-K/O). Acetylcholine-induced relaxation was preserved in WT and TG2-K/O and it was significantly impaired by angiotensin-II only in WT (-28%). L-NAME blunted this response in all the groups, although this effect was less evident in angiotensin-II-treated WT. Endothelium-independent relaxation was similar in all the groups. Plasma nitrates/nitrates and p-eNOS-(S1177)/eNOS were similar in WT and TG2-K/O, and they were reduced by angiotensin-II significantly only in WT (-37% and -44%, respectively). NOSIP expression was similar in both WT and TG2-K/O and was significantly increased by angiotensin-II only in WT (+40%). ROS production was similar in WT and TG2-K/O and significantly increased by angiotensin-II only in WT (+9%). NOX-1 and ERp72 were similar in WT and TG2-K/O and were significantly increased by angiotensin-II only in WT (+23% and +29%, respectively). In conclusion TG2 may contribute to endothelial dysfunction through the modulation of ROS production and the reduction of NO bioavailability in angiotensin-II infused mice.
Abstract P604: Astrocyte-dependent Regulation of Vascular Tone: Role in Hypertension
Authors: Ramiro-Diaz Juan Manuel , Kim Ki Jung , Filosa Jessica A
Published: June 2021

Clinical studies support that untreated hypertension (HT) accelerates the development of vascular cognitive impairment (VCI). Yet, the underlying mechanisms for VCI are not known. In a recent study we demonstrated the role of astrocytes in the regulation of parenchymal arteriole (PA) steady-state vascular tone.

Here we hypothesized hypertension results in structural and functional changes to the neurovascular unit resulting in enhanced astrocytic TRPV4 channel-dependent Ca
2+
increases contributing to augmented pressure-induced PA constriction

. Functional studies were conducted in brain slices from angiotensin II (AngII) treated mice (600 ng/Kg/min, 28 days). PA arterioles within brain slices were perfused and pressurized and myogenic-evoked diameter changes measured using video microscopy. In addition, using the
GLAST-CreERT2
;
R26-lsl-GCaMP3
mice we measure myogenic-evoked Ca
2+
changes in perivascular astrocytes. We demonstrate that HT increases pressure-induced PA tone by 11.14% at 30 mmHg and 12.97% at 60 mmHg (10.88 to 22.02 and 15.46 to 28.43% of tone, P<0.05 and P<0.01, respectively). In ANG II-treated mice, PA myogenic-evoked responses significantly increased astrocytic Ca
2+
oscillations frequency (119.4%, 0.0366 to 0.0803 Hz, P<0.0001). A significant increase in astrocytic Ca
2+
oscillation frequency was also observed after 2 min of AngII (500 nM) bath application (44.8%, 0.0366 to 0.053 Hz, P<0.01) in brain slices from AngII treated mice. Furthermore, using the model of spontaneous hypertensive rat (SHR) we observed that HT differentially increases vascular density and the number of vascular pericytes in cortical layers with highest neuronal densities (L III-V). Finally, while aquaporin 4 (AQP4) expression pattern was not different in the gray matter of SHR compared with WKY rats, a significant increase in unpolarized AQP4 expression was observed in the white matter of SHR. Taken together, this evidence indicates that HT induces functional and structural changes to the neurovascular unit favoring the development of regional brain hypoperfusion likely contributing to the development of VCI.
Abstract P612: The AA2-Ratio: Towards Improved Screening for Primary Aldosteronism
Authors: Poglitsch Marko , Ahmed Ashraf H , Stoller Andrea , van Oyen Dunja , Domenig Oliver , Haschke Manuel , Stowasser Michael
Published: June 2021

Background:
Primary aldosteronism (PA) is a widely under-diagnosed, potentially curable and specifically treatable cause of hypertension. PA screening involves measuring the aldosterone-to-renin-ratio (ARR), but false negative results can occur in the setting of medications, which block the renin-angiotensin system (RAS). Withdrawing RAS blockers from patients with resistant hypertension is not without cardiovascular risk. A novel diagnostic approach, the aldosterone-to-angiotensin-II-ratio (AA2-Ratio), has the potential for less drug interference and improved reliability in PA screening and confirmation of diagnosis.

Methods:
Serum samples from 80 patients undergoing PA confirmation testing were analyzed. Sampling was performed in a recumbent (7 a.m.) and in an upright (10 a.m.) position before and after 4 days of oral administration of fludrocortisone and salt loading. The concentrations of renin, aldosterone and equilibrium Angiotensin-II were determined and ARR and AA2-Ratios were calculated. The interference of ACE-inhibition with the AA2-Ratio was investigated in healthy volunteers receiving 10mg enalapril daily for 8 days.

Results:
Renin concentration was undetectable in more than 40% of samples, while equilibrium Angiotensin-II was measurable in 98% of all 320 samples analyzed. Angiotensin-II levels were significantly higher in upright collected samples compared to samples collected in a recumbent position. Comparison of the ARR with the AA2-Ratio revealed a significantly larger diagnostic window for the AA2-Ratio. While the ARR was significantly suppressed by ACE-inhibitor treatment, the AA2-Ratio remained unaffected by ACE-inhibition.

Conclusion:
The AA2-Ratio may be superior to the ARR in PA screening among hypertensive patients. Equilibrium Angiotensin-II levels show expected responses to posture and appear to outperform renin concentration as a marker for RAS activation in terms of sensitivity, giving a measurable readout even in clinical states characterized by markedly suppressed RAS activity. The stability of the AA2-Ratio in the presence of ACE-inhibition points to a potential use of the AA2-Ratio PA screening in hypertensive patients without ACE-inhibitor discontinuation.
Abstract P617: Actions on the Splanchnic Venous System Contribute to 5-HT-induced Hypotension
Authors: Seitz Bridget M , Krieger-Burke Teresa , Garver Hannah , Fink Gregory D , Watts Stephanie W
Published: June 2021

Infusion of serotonin (5-hydroxytryptamine, 5-HT) into conscious normotensive and hypertensive rats causes a sustained reduction in systemic blood pressure. Imaging studies reveal that the blood pressure fall is closely associated with dilation of the large splanchnic veins (mesenteric, portal and abdominal vena cava), suggesting that active venodilation contributes to the fall in blood pressure. In fact, isolated splanchnic veins dilate directly to 5-HT via activation of the 5-HT
7
receptor, and a 5-HT
7
receptor antagonist prevents the 5-HT induced fall in blood pressure. To determine if the splanchnic venodilation caused by 5-HT is active or passive, anesthetized male Sprague Dawley rats were instrumented with arterial and venous lines for pressure measurements and 5-HT administration, respectively, while splanchnic veins were imaged using the Vevo® 2100 Ultrasound system. Measures were made relative to baselines measures. Within 5 minutes of infusion, 5-HT (25 ug/kg/min) caused an initial fall in portal vein pressure (~8-10% reduction) accompanied by dilation of the portal vein (~40% increase). No changes were seen in the dimensions or pressure of the abdominal vena cava at this time. Mean arterial blood pressure was reduced (>40% reduction). All of these changes were prevented by pretreatment with the 5-HT
7
receptor antagonist SB269970. SB269970 during 5-HT infusion also caused an immediate reversal of changes in blood pressure and venous dimensions. Thus, active dilation of the pre-hepatic splanchnic venous system may be an early cause of 5-HT-induced hypotension. A more chronic experiment was performed in rats that were instrumented with a new dual channel radiotelemeter for concomitant measure of systemic and portal pressure in the conscious state. Within one hour after initiation of 5-HT infusion, portal venous pressure was elevated 38±0.2% above baseline (n=3) versus vehicle infused animals (4±0.3% above baseline; n=3), suggesting an action of 5-HT on intrahepatic venous resistance. Within 24 hours, portal pressure elevation resolved but blood pressure remained reduced. Collectively these data highlight the portal venous circulation as an important site of action for 5-HT in causing acute and chronic falls in systemic blood pressure.
Abstract P621: Endothelial Mineralocorticoid Receptor Signaling Mediates Parenchymal Arteriole Hypertensive Remodeling and Cerebral Perfusion
Authors: Diaz-Otero Janice M , Jackson William F , Jaffe Iris Z , Dorrance Anne M
Published: June 2021

Mineralocorticoid receptor (MR) activation causes hypertensive cerebral artery remodeling, but the vascular cell type involved has not been defined. Peripheral endothelial MR (EC-MR) activation mediates hypertensive artery dysfunction; the unique anatomy of cerebral arteries prevents the extrapolation of findings from the periphery to the brain. Parenchymal arterioles (PAs) perfuse the cerebral microcirculation and regulate cerebral perfusion. We hypothesized that EC-MR activation mediates inward PAs remodeling and reduces cerebral perfusion in hypertensive mice. To induce hypertension, male endothelial cell specific MR knockout (ECMRKO) and MR-intact littermates were treated with angiotensin II (AngII) (800ng/kg/min) for 4 weeks (n=6). Control mice were normotensive (n=6). PA structure, in 20-week-old mice, was assessed by pressure myography. Data are presented as mean ± SEM. In the absence of hypertension, EC-MR deletion did not change blood pressure, plasma aldosterone, cerebral perfusion or PA structure. AngII increased mean arterial pressure and plasma aldosterone in MR-intact and ECMRKO mice. Hypertension reduced cerebral perfusion in MR-intact mice but not in ECMRKO mice. In MR-intact mice hypertension caused inward hyprotrophic remodeling evidenced by a decrease in the lumen diameter and wall area. These effects of hypertension where not observed in ECMRKO mice. (*p<0.05, Indicates significantly different from strain control). EC-MR activation mediates hypertensive remodeling in cerebral PAs; this was associated with a in reduction cerebral perfusion that could worsen the outcome of ischemic stroke or cause vascular dementia development.
Abstract P633: Resistance Training Counteracts The Systemic Catecholaminergic Hyperactivation Associated With Experimental Diabetes, But Not Normalize Cardiac Sympathetic Outflow
Authors: Santos Ralmony A , Silva Kleiton A , Perez Juliana D , Schor Nestor , Casarini Dulce E , Cunha Tatiana S
Published: June 2021

Previous studies from our laboratory have demonstrated that chronic diabetes in rats results in cardiomyopathy, associated with sympathetic nervous system (SNS) hyperactivity. On the other hand, it is well known that the beneficial cardiovascular effects of exercise training in diabetes are due in part to normalization of the sympathetic outflow and improvement in the responsiveness of the myocardium to autonomic stimulation. Recently, resistance training (RT) has been recognized as a useful therapeutic tool for the treatment of chronic diseases and similar to aerobic exercise, has been reported to improve metabolic profile and body composition. Therefore, the aim of this study was to evaluate the effect of moderate-intensity RT on circulating and cardiac catecholamines concentration, to understand whether this type of exercise is also associated with cardiovascular protection. Wistar rats (3 months old) were randomized into: control (C), diabetic (D), diabetic + RPT (DR) and diabetic + APT (DA). Animals were made diabetic with a single tail injection of streptozotocin (STZ, 50 mg/Kg). Resistance exercise training was performed on a vertical ladder (5 days/week, 8 weeks) at 40-60% maximal load, and moderate aerobic training was performed on a treadmill (5 days/week, 8 weeks). Diabetes significantly increased plasma concentration of adrenaline (D: 5.3 ± 1.0
vs.
C: 4.1 ± 0.6 ng/mL) and noradrenaline (D: 14.5 ± 0.2
vs.
C: 3.1± 0.8 ng/mL), and both exercise modalities induced a significant reduction of them: adrenaline (DR: 1.1 ± 0.3; DA: 0.7 ± 0.16
vs.
D: 5.3 ± 1.0 ng/mL) and noradrenaline (DR: 1.0 ± 0.2; DA: 0.7 ± 0.1
vs.
D: 14.5 ± 0.2 ng/mL). Cardiac concentration of noradrenaline was also increased in diabetic group (D: 62 ± 7
vs.
CS: 34 ± 6 pg/g) and only aerobic exercise was capable to reduce its concentration in heart tissue (DA: 30 ± 6
vs.
D: 62 ± 7; DR: 55 ± 7 pg/g). The results from the present study show for the first time additional beneficial effects of RT on modulating SNS activity in diabetes. Moreover, considering that RT does not modulate cardiac catecholaminergic secretion, it also highlights the importance of aerobic training in diabetes treatment. Financial Support: FAPESP, CAPES, CNPq
Abstract P640: A Single-Chain Derivative of the Realaxin Hormone (B7-33) Protects Cytotrophoblasts from Hyperglycemia-Induced Preeclampsia Phenotype and Induces the Survival Pathway
Authors: Afroze Syeda H , Pantho Ahmed F , Kalagiri Ram R , Kuehl Thomas J , Bathgate Ross , Hossain Mohammed A , Uddin Mohammad N
Published: June 2021

Background:
Relaxin is a peptide hormone that allows vasodilation and plays an important role in the process of parturition. The literature suggests potential therapeutic role of H2 relaxin in preeclampsia (PreE), however, there is a controversy on hypotensive action of the peptide. Due to the complex insulin-like structure of relaxin (A- and B- chains, 53 amino acids, 3 disulfide bonds), a novel H2 relaxin B-chain-only peptide variant B7-33 (27 amino acids without any disulfide bonds) has recently been developed. This single-chain peptide displayed equivalent efficacy to the natural H2 relaxin in several functional assays both
in vitro
and
in vivo
. Importantly, B7-33 was shown to have H2 relaxin-like RXFP1 specific effects, particularly in endogenously expressing RXFP1 cells, thus we hypothesized that B7-33 could be an alternative and cost-effective treatment option for PreE compared with H2 relaxin.

Methods:
Human CTBs were treated with 100, 150, 200, 300, or 400 mg/dL glucose for 48h and were co-treated with B7-33 (25 nM) with glucose exposure, while some cells were treated with 5, 10, 25 and 50 nM B7-33 alone. Levels of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), and soluble endoglin (sEng) were measured in culture media using ELISA kits. Cell lysates were utilized to evaluate the mTOR, pAKT and total AKT expression by western blotting. Statistical comparisons were performed using analysis of variance with Duncan’s post hoc test.

Results:
Secretion of sFlt-1 and sEng were increased while VEGF and PIGF were decreased in CTBs treated with ≥150 mg/dl of glucose (*p < 0.05 for each). B7-33 co-treatment significantly rescued CTBs from hyperglycemia-induced anti-angiogenic profile (p < 0.05 for each). There is no effect of B7-33 on sFLT-1, sEng and PlGF; however, it increases expression of VEGF, while CTBs were treated only with B7-33. B7-33 also causes increased mTOR and pAKT expression in CTBs without any change in total AKT.

Conclusions:
B7-33 mitigates the hyperglycemia-induced dysfunction of CTBs by attenuating anti-angiogenic phenotype similar to that seen in PreE. This study supports the importance of continuing research of B7-33 in preE prevention.
Abstract 102: Downregulation of Mitochondrial Energetics in Senescent Human Atria
Authors: Emelyanova Larisa , Gupta Anu , Maria Viqar , Yousufuddin Mohammed , Edwards Stacie , Kraft Kelsey , Negmadjanov Ulugbek , Ross Gracious , Rizvi Farhan , Shi Yang , Jahangir Arshad
Published: June 2021

Introduction:
Although alterations in mitochondrial function have been observed in senescent hearts, the molecular basis for aging-associated decline in energy metabolism in the human heart is not fully understood.

Objective:
To determine aging-related changes in functional activity and protein expression level of mitochondrial oxidative phosphorylation (OXPHOS) complexes and expression of genes regulating mitochondrial energetics in the human heart.

Methods:
Right atrial appendages from well-matched adult (29-57 y.o., n=19) and aged (65-85 y.o., n=22) patients undergoing elective CABG surgery were used. Functional activity of OXPHOS complexes was measured spectrophotometrically. Protein expression level of corresponding OXPHOS protein subunits was determined by Western blot. Gene expression profiling was performed using Affymetrix Human Genome U133 Plus 2.0 microarrays.

Results:
Mitochondria from senescent hearts demonstrated a significant decrease in complex I and V functional activity without significant changes in the rest of the complexes (Fig. A). At the protein expression level, there was reduction in complex I, III, and IV (Fig. B). This was associated with downregulation of the majority (86%, 90 of 105) of genes coding mitochondrial proteins (Fig. C). Forty-six percent (41 of 90) of the downregulated genes were confined to pathways regulating mitochondrial energetics.

Conclusion:
With aging, the functional activity of OXPHOS is reduced due to supression of protein expression and downregulation of genes regulating mitochondrial energetics. This provides a substrate for reduced energetic efficiency associated with senescence in the human atria.
Abstract 104: Defective Branched-chain Amino Acid Catabolism Impairs Glucose Metabolism and Sensitizes the Heart to Ischemia/Reperfusion Injury
Authors: LI TAO , Zhang Zhen , Kolwicz Stephen C , Sun Haipeng , Tian Rong
Published: June 2021

The branched-chain amino acids (BCAA) are essential amino acids required for protein homeostasis, energy balance, and nutrient signaling. It has been shown recently that elevations in the circulating BCAAs and related metabolites are associated with insulin resistance and ischemic heart disease, suggesting an important link between BCAA metabolism and cardiac pathology. BCAA catabolism in mitochondria is regulated by the branched-chain keto acid dehydrogenase complex (BCKDC). A mitochondria localized phosphatase 2C (PP2Cm) has been identified to activate BCKDC, serving as a key regulator of BCAA catabolism. Deletion of PP2Cm (KO) increased blood BCAA levels but did not alter cardiac function in unstressed mice. Using
13
C- and
31
P-NMR spectroscopy, we found a significant reduction in the contribution of glucose to oxidative metabolism in KO hearts (16±3 vs. 26±2% for KO and wild-type (WT), respectively,
P
<0.05), which was accompanied by an ~25% reduction in glucose uptake rate (
P
<0.05). Cardiac glycogen content was also reduced (4.4±0.5 vs. 10.9±1.8 μmol glucose/g,
P
<0.05). In isolated mitochondria from KO or WT hearts treated with BCAAs, significant reductions in pyruvate supported respiration were observed. In enzyme kinetics experiment, acute exposure to BCAAs inhibited pyruvate dehydrogenase complex (PDH) activity in a dose-dependent manner. In isolated perfused hearts subjected to ischemia/reperfusion (I/R), KO hearts showed a greater increase of diastolic pressure during ischemia (
P
<0.05), which persisted throughout the reperfusion (
P
<0.05). The contractile function in KO barely recovered while it recovered to ~40% of baseline in WT at the end of reperfusion. Increasing glucose supply via overexpression of GLUT1, an insulin-independent glucose transporter, fully restored the impaired recovery to I/R in KO mice. These results show that BCAA catabolism deficiency, as a result of PP2Cm deletion, disrupts normal glucose metabolism and renders the heart highly sensitive to acute ischemia/reperfusion (I/R) injury. These findings reveal an important role of BCAA catabolism in regulating glucose metabolism and cardiac stress response.
Abstract 107: Acetyl-coa Carboxylase 2 Prevents Cardiomyocyte Hypertrophy by Reducing Glucose Reliance
Authors: Ritterhoff Julia , Shao Dan , Tian Rong
Published: June 2021

In cardiac hypertrophy, the adult heart switches from mainly using fatty acids to increased reliance on glucose to maintain its energetic demands. Reducing fatty acid overload and further increasing glucose reliance has been suggested to be beneficial in the diseased state. Recently, however, it has been shown that increasing fatty acid oxidation (FAO) by cardiac-specific deletion of Acetyl-CoA Carboxylase 2 (ACC2) maintains cardiac energetics and prevents cardiac dysfunction as well as cardiomyocyte hypertrophy during chronic pressure overload. However, it remained unclear, how increased FAO specifically prevented cardiomyocyte hypertrophy. Thus, the goal of this study was to determine the impact of ACC2 deletion on cardiomyocyte hypertrophy
in vitro
.

Adenoviral-mediated knock-down (KD) of ACC2 in adult rat ventricular cardiomyocytes (CMs) resulted in a 70% downregulation of ACC2 mRNA. In standard CM medium (medium M199, 5.5mM glucose) ACC2 KD resulted in a similar increase in CM growth after phenylephrine (PE) treatment as control CMs (+39±10% in control vs. 41±16% in ACC2 KD CMs). Supplementation of 0.4 mM mixed long-chain fatty acids (FA) and 0.1 mU/ml insulin had no effect on cardiomyocyte morphology or hypertrophic response after PE treatment (+42±6%). However, ACC2 KD effectively prevented CM hypertrophy after PE stimulation in the presence of FA/insulin (+9±6%). Whereas PE stimulation in control CMs increased glucose uptake (+28±8%) and reduced fatty acid uptake (-25±6%), both were normalized after ACC2 KD. Inhibiting FAO by etomoxir or increasing glucose utilization by dichloroacetate abolished the beneficial effects of ACC2 KD after PE stimulation. When cultured in glucose-free medium supplemented with FA, ACC2 KD was incapable of preventing cardiomyocyte hypertrophy.

Together, these data indicate that increased FAO after ACC2 deletion prevents cardiomyocyte hypertrophy by reducing glucose reliance, suggesting that rather increasing than reducing FAO is beneficial in cardiac hypertrophy.
Abstract 109: Understanding Multi-allelic Heterozygous Variant Contributions to Cardiomyopathy Using iPSCs
Authors: Happe Cassandra , Tenerelli Kevin , Deacon Dekker C , Tran Vivien , Taylor-Weiner Hermes , Adler Eric D , Chi Neil C , Engler Adam J
Published: June 2021

Dilated cardiomyopathy (DCM) is a multivariate disease with poorly understood mechanisms, but recently hundreds of variants in 30+ genes have been suggested to contribute to disease pathology. As evidence that two seemingly unrelated heterozygous variants could combine to induce disease, we have identified a family with multiple cases of DCM detected across four generations, where DCM cosegregates with heterozygous variants of thin filament-associated α-tropomyosin (TPM1; +/c.G97A) and intercalated disc-associated vinculin (VCL; +/c.659dupA) (Fischer Exact p-value < 10
-3
; LOD score = 3.21). To understand disease mechanism, we generated human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from the family cohort carrying these variants to test the hypothesis that multiple, heterozygous variants can uniquely combine to cause disease where their single heterozygous variant counterparts do not; variants were also isogenically modeled using CRISPR in human embryonic stem cell-derived CMs (hESC-CMs). Proband and affected cousin CMs had half of the protein expressed for both variants with morphological and cytoskeletal abnormalities relative to non-carriers. This resulted in reduced contractile energy, prolonged calcium transients, and more irregular timing compared to non-carrier CMs. Decreased sodium and potassium channel expression accompanied slowed action potential kinetics in affected vs. non-carrier patient-derived CMs, which together suggests that heterozygous VCL and TPM1 mutations may cause unique downstream transcriptome regulation that leads to the observed dysfunction
in vitro
. CRISPR edited hESC-CMs exhibited identical alterations relative to their isogenic controls along with losing gap junction protein connexin43 expression, suggesting that dysfunctional force generation and transmission combine, which would likely lead to more severe disease and DCM observed when the variants co-segregated. Given the lack of disease in single variant carriers, these data provide a unique set of analyses that result in identification of how dissimilar but co-segregating variants can result in disease.
Abstract 111: Cdk5 is Associated in Cardiac Calcium Channelopathy
Authors: Yazawa Masayuki , Song LouJin
Published: June 2021

Cardiac calcium channels play important roles in hearts. Mutations in Ca
V
1.2 channels are associated with cardiac arrhythmias including long QT syndrome type 8 (LQTS8). Using human induced pluripotent stem cells (iPSCs) to generate human cardiomyocytes from LQTS8 patients, we reported that the G406R mutation (LQTS8, Timothy syndrome) affected channel inactivation, action potentials and calcium handling in human cardiomyocytes. We found that roscovitine, a cyclin-dependent kinase (CDK) inhibitor, rescued the phenotypes in LQTS8 cardiomyocytes. However, how roscovitine restores the physiological functions in the patient cardiomyocytes remains unclear. Our recent results using roscovitine analogs and CDK inhibitors suggested that CDK5 is involved in the molecular bases of LQTS8. We found that dominant negative forms and shRNA of CDK5 alleviated the phenotypes of the LQTS8 cardiomyocytes and that the expressions of phosphorylated ERK, EGR1 and CDK5R1, a CDK5 activator, significantly increased in the patient cardiomyocytes. The results reveal that calcium overload due to affected Ca
V
1.2 inactivation activates ERK/EGR1 pathway, resulting in increased expression of CDK5R1. Biochemical assays supported a direct binding and phosphorylation of Ca
V
1.2 channels by CDK5. Overall, the results demonstrated that CDK5 regulates Ca
V
1.2 channels and that CDK5 inhibition could be a new therapeutic for LQTS8. This study demonstrates the role of ERK/EGR1/CDK5 in pathogenesis of cardiac arrhythmia. The outcomes of this study provide new insights into Ca
V
1.2 channel regulation.
Abstract 116: Loss of T-type Ca
2+
Channels Alters the Cardiac Response to Myocardial Infarction
Authors: Harper Shavonn C , Wang Tao , Yang Yijun , Houser Steven R
Published: June 2021

Rationale:
Cav3.1 T-type calcium channels are expressed during development and then re-expressed in ventricular myocytes after pathological stress such as myocardial infarction (MI). Our lab has previously shown that mice with cardiac specific overexpression of Cav3.1 have improved contractility, survival, and preservation of the myocardial mass post-MI.

Hypothesis:
Loss of the Cav3.1 subunit of the T-type calcium channel reduces the ability of the heart to adapt to MI due to alterations in the wound healing response.

Methods and Results:
Ischemia reperfusion (IR) injury was induced in 12-16 week old Cav3.1
-/-
and wild type (WT) mice. Previously collected data from our groups showed that Cav3.1
-/-
mice have decreased survival after permanent occlusion MI. In the present study there was no significant difference in the one week survival of these mice after IR (100% vs 90% for WT vs Cav3.1
-/-
, respectively). Cav3.1
-/-
mice had significantly increased heart weight to body weight (7.066 vs 5.452) and heart weight to tibia length ratios (9.284 vs 7.594) compared to WT mice at 7 days post IR. Echocardiography performed at baseline showed no significant differences between the two groups. At 1 week post-IR, Cav3.1
-/-
mice had significantly lower ejection fraction (11.58% vs 21.77%) and fractional shortening (10.02% vs 17.46%) compared to WT mice. This decrease in function was accompanied by a significantly increased percentage of fibrosis in the Cav3.1
-/-
mice (26.42% vs 11.17%) and this was associated with alterations in the infiltration of proliferative inflammatory cells. The left ventricular internal diameter was also significantly increased in Cav3.1
-/-
mice compared to WT (4.766 mm vs 4.151 mm).

Summary and Conclusions:
Cav3.1
-/-
mice have decreased cardiac function and increased pathological remodeling at 1-week post-IR. The mechanisms resulting in these pathological changes appear to involve alterations in post MI inflammatory cell infiltration and myocyte survival. These results suggest that loss of Cav3.1 T-type calcium channels alters the behavior of inflammatory responses and myocyte survival after MI.
Abstract 120: HIF1 Alpha Can be Inhibited in the Heart by Protein Kinase C Binding Protein 1
Authors: Schunke Kathryn J , Chang Karen H , Walton Chad B , Shohet Ralph V
Published: June 2021

Hypoxia-inducible factor 1 alpha (HIF-1α) is a key transcription factor responsible for the induction of genes that facilitate adaptation to hypoxia. The HIF-1 heterodimer (HIF-1α and HIF-1ß) recognizes a consensus element designated the hypoxia response element (HRE) within the enhancer regions of target genes that are implicated in many different cellular functions that ameliorate hypoxic stress. To study HIF-1 signaling in the heart, our lab developed a mouse model in which an oxygen-stable form of HIF-1α can be inducibly expressed in cardiac myocytes with a tet-off system . Remarkably, expression of the transgene generated two distinct phenotypes. One was the expected expression of HIF-regulated transcripts and associated HIF–related changes in cardiac angiogenesis and contractility. The other was a “resistant phenotype” with much less expression of typical HIF-response genes and substantial expression of a zinc-finger protein, a receptor for protein kinase C called PRKCBP1, also known as ZMYND8. We have demonstrated that this second phenotype is due to an insertion of a fragment of DNA upstream of the PRKCBP1 gene that contains two additional HREs and leads to substantial HIF binding, assessed by ChIP, and transcriptional activation. This inserted allele is found only in the FVB strain of mice that contributed the αMHC-tet binding protein allele to the transgenic strain. In HEK293 cells transfected with oxygen-stable HIF1α and PRKCBP1 we demonstrated inhibition of HIF-1 activity by a luciferase reporter assay. In the MCF-7 breast cancer cell line we have shown that increased expression of PRKCBP1 produces inhibition of HIF degradation in normoxia. This and co-immunoprecipitation of HIF with PRKCBP1 suggests a direct interaction of the two proteins that acts to inhibit HIF action. We have genetic, transcriptional, biochemical, and physiological evidence that PRKCBP1 inhibits HIF activity through direct interaction in a homeostatic mechanism mediated by transcriptional control.
Abstract 124: Integrated Omics Approaches to Elucidate Targets of Cyclic AMP-Epac-Rap1A Signaling in Microvascular Smooth Muscle Cells: Identification of Role in Production and Deposition of Extracellular Matrix Fibrillar Collagen
Authors: Chotani Maqsood A
Published: June 2021

Objective:
Intracellular signaling by cyclic AMP (cAMP) has an essential role in vascular smooth muscle (VSM) physiology, including relaxation of large blood vessels such as aorta and pulmonary artery. Studies support a mechanism of signaling through
e
xchange
p
rotein
a
ctivated by
c
AMP (epac) and Ras-related small GTPase Rap1B down-regulation of RhoA activity. The role of cAMP in small blood vessels however was not examined, and remained unknown. This study elucidated the targets of cAMP signaling in peripheral blood vessels, specifically VSM explanted from healthy human (h) cutaneous arterioles and mouse (m) tail artery (microVSM).

Results:
Global changes in protein expression were assessed by Differential-in-Gel Electrophoresis in quiescent (h)-microVSM treated with the adenylate cyclase activator and cAMP elevating agent forskolin (10 μM, 30 min or 18 hr). Detectable changes were observed in proteins associated with the cytoskeleton, stress-response, protein-synthesis, -folding, -membrane transport, and extracellular matrix. Notably, actin modulating proteins caldesmon, ezrin-moesin, zyxin, gelsolin, and α-adducin, as well as cytoskeletal proteins, tubulin and vimentin were differentially expressed (
P<
0.05; 3-5 replicates). These results agreed with our recent demonstration of cAMP activation of epac-Rap1A, and RhoA-ROCK-F-actin signaling in (h)- and (m)-microVSM to increase expression and cell surface transport of functional α
2C
-adrenoceptors (α
2C
-ARs) that mediate vasoconstriction. Transcriptome analysis of Rap1A-null (knockout) (m)-microVSM transduced with constitutively active Rap1A showed connections to signaling linked to vessel production and deposition of extracellular matrix fibrillar collagen, compared with control cells (
P<
0.05, 4 replicates).

Conclusions:
Rap1 subtypes have a discrete role in the vasculature. This study links cAMP-Rap1A signaling to collagen and α
2C
-AR expression in arteriole VSM. It suggests over-activation of Rap1A-coupled signaling in the peripheral circulation during chronic inflammation could lead to increased α
2C
-AR mediated vessel reactivity, progressive perivascular fibrosis, and dysfunction as seen in human pathologies such as Raynaud’s phenomenon and scleroderma.
Abstract 125: Role of 14-3-3eta Protein in the Heart During Aging: Involvement of MAPK Mediated Endoplasmic Reticulum Signaling in Senescence-accelerated Prone Mice
Authors: Giridharan Vijayasree V , Sreedhar Remya , Arumugam Somasundaram , Karuppagounder VengadeshPrabu , Palaniyandi Suresh S , Krishnamurthy Prasanna , DeQuevedo Joao L , Watanabe Kenichi , Konishi Tetsuya , Thandavarayan Rajarajan A
Published: June 2021

Heart failure is typically related to aging as there is a definite relationship between age-related changes in the heart and the pathogenesis of heart failure. We have previously reported the involvement of p38 mitogen-activated protein kinase protein in cardiac function using animal models of heart failure. To further understand its relationship with aging-induced heart failure, we have compared its expression in the hearts of senescence accelerated-prone (SAMP8) mice and their control (SAMR1) with normal aging behavior. We have identified its activation along with reduced expression of 14-3-3η protein in SAMP8 mice hearts than in SAMR1 mice. To reveal the downstream signaling, we have measured the endoplasmic reticulum stress marker proteins along with some inflammatory and apoptosis markers and identified a significant increase in SAMP8 mice hearts than that of SAMR1. In addition, we have performed comet assay and revealed a significant DNA damage in the cardiomyocytes of SAMP8 mice when compared with SAMR1 mice. All these results demonstrate the role of 14-3-3η protein and the downstream mitogen-activated protein kinase-mediated endoplasmic reticulum stress, and apoptosis and DNA damage in aging-induced cardiac malfunction in SAMP8 mice. Thus targeting this signaling might be effective in treating age-related cardiac dysfunction.
Abstract 127: Phosphorylation of Serum Responsive Factor by p90 Ribosomal S6 Kinase 3 Promotes Concentric Growth of Cardiac Myocytes
Authors: Li Jinliang , Martinez Valencia Eliana C , Passariello Catherine L , Thakur Hrishikesh , Kapiloff Michael
Published: June 2021

p90 Ribosomal S6 Kinase 3 (RSK3) is required for the induction of concentric hypertrophy in hearts subjected to pressure overload and catecholamine infusion, as well as in a model for Noonan Syndrome-associated hypertrophic cardiomyopathy. Serving important roles in both cardiac development and adult function, the transcription factor Serum Responsive Factor (SRF) regulates genes expression involved in myocyte growth and sarcomeric assembly. It has been reported that SRF can be phosphorylated by RSK protein kinases on residue Ser-103, but the function of that post-translational modification has remained uncertain. We now show that SRF is a substrate for RSK3 associated with muscle A-Kinase-Anchoring-Protein (mAKAP) scaffold in cardiac myocytes, contributing to the regulation of concentric myocyte growth. By co-immunoprecipitation assay, SRF and RSK3 were both associated with the mAKAP scaffold in heart extracts, such that a ternary complex could be detected when recombinant proteins were expressed in cells. Silencing RSK3 or mAKAP expression by siRNA transfection reduced phenylephrine-induced SRF(S103) phosphorylation in neonatal cardiac myocytes. Similar results were acquired following expression of a peptide comprising the mAKAP RSK3-binding domain (RBD) that competed the binding of the kinase to the scaffold. Overexpression of either a SRF S103A phosphoablative mutant or the RBD peptide diminished the increase in width of adult cardiac myocytes stimulated by phenylephrine. In contrast, overexpression of RSK3 or a SRF S103D phosphomimetic mutant promoted adult cardiac myocyte concentric hypertorphy in vitro. While baseline SRF S103 phosphorylation in the heart was inhibited by mAKAP or RSK3 gene knock-out in mice, acute transverse aortic constriction significantly increased SRF S103 phosphorylation in the heart. Based upon these results, we porpose that RSK3 phosphorylation of SRF at mAKAP signalosomes is an important regulator of concentric cardiac myocyte growth. These results are consistent with additional findings that an adeno-associated virus gene therapy vector that expresses RBD in the cardiac mycoyte attenuates pathological hypertrophy and prevents heart failure in response to pressure overload.