bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P504: Resistin Mediates Sex-dependent Effects of Perivascular Adipose Tissue on Vascular Function in the SHRSP
Authors: McNeilly Sarah , Small Heather Y , Sheikh Adam , Mary Sheon , Pinel Karine , Delles Christian
Published: March 2022

Background:
Premenopausal women are relatively protected against hypertension compared to males. Estrogen levels have been identified as a potential underlying cause, but the pathophysiological mechanisms remain incompletely understood. We hypothesised that sex-dependent effects of perivascular adipose tissuePVAT mediate altered vascular function in hypertension.

Methods:
The effect of PVAT was investigated on resistance vessels of 16 week old male and female stroke-prone spontaneously hypertensive rats (SHRSP).

Results:
Wire myography was used on 3
rd
-order mesenteric vessels (maximum contraction: male +PVAT 113.3±1.1 vs. female +PVAT 91.4±11.36 %). Noradrenaline mediated vasoconstriction was increased in SHRSP males compared to females. K
ATP
channel-mediated vasorelaxation by cromakalim was impaired in males compared to females (maximum relaxation: male +PVAT 46.9±3.9 % vs. female +PVAT 97.3±2.7 %) A cross-over study assessing function of male PVAT on female vessels and vice versa confirmed the reduced K
ATP
mediated vasorelaxation induced by male PVAT (maximum relaxation: female +PVAT
female
90.6±1.4 % vs. female +PVAT
male
65.8±3.5 %). An adipokine array with subsequent western blot validation identified resistin as a potential modifier of vascular reactivity. Resistin was increased by approximately 2-fold in SHRSP male PVAT. Male and female vessels pretreated with resistin (40ng/ml) showed no difference in response to noradrenaline. However, vasorelaxation in response to cromakalim was significantly impaired in resistin treated female vessels, similar to levels observed in male vessels (maximum relaxation: female +PVAT 97.3±0.9 % vs. female +PVAT +resistin 36.8 ±2.3 %).

Conclusion:
We identified a novel role for resistin in sex-dependent PVAT mediated vascular function in hypertension through a K
ATP
channel mediated mechanism.
Abstract P506: Deoxycorticosterone Acetate-salt Promotes Endothelial-mesenchymal Transition in Human Glomerular Endothelial Cells
Authors: Luo Zaiming , Welch William J , Wilcox Christopher S
Published: March 2022

Endothelial cells (ECs) lose their endothelial specification and gain mesenchymal cell features during endothelial-mesenchymal transition (EndMT). Post-developmental EndMT disrupts EC homeostasis, leading to vascular dysfunction. We found that afferent arterioles from deoxycorticosterone acetate (DOCA)-salt treated mice had > 5 fold upregulation of mRNAs for preproendothelin-1, p47phox ,NOX2 and TGF-β accompanied by microvascular dysfunction. Since these may cause EndMT, we investigated the mechanism in human glomerular endothelial cells (HGECs) treated for 7-21 days with high salt and DOCA. Endothelin-1 (ET-1) in the medium was increased 2.8±0.2 fold by day 7 while the cells gained multiple mesenchymal markers with increased mRNA for alpha-smooth muscle actin (1.78±0.19 and 2.96±0.32 fold, P<0.05 and 0.01, n=3) and transgelin ( 1.96±0.14 and 2.91±0.28 fold, p<0.05 and 0.01, n=3) on day 7 and 21, respectively, and markedly downregulated mRNA for endothelial markers with decreased vascular endothelial cadherin ( 1.99± 0.27 and 2.12±0.24 fold, P<0.05 and 0.005, n=3) and platelet endothelial cell adhesion molecule 1 (1.78±0.26 and 1.94±0.23 fold, P<0.05 and 0.005, n=3). There were parallel changes in protein expression. Dihydroethidium and MitSox fluorescence probes were used to determine intracellular and mitochondria ROS. The fluorescent intensities were increased by 1.89±0.27 and 1.62±0.22 fold (P<0.01, N=6) respectively in the cells treated for 7 days with DOCA-salt accompanied by increased expression of TGF-β and phosphorylated-extracellular signal-regulated kinases (P-ERK 1/2). In conclusion, human glomerular endothelial cells treated with high salt and DOCA for 1-3 weeks have increased cellular and mitochondrial ROS, ET-1, TGF-β and P-ERK that could account for adverse changes of endothelial-mesenchymal transition and associated microvascular dysfunction.
Abstract P508: Inhibition of Big-conductance Calcium-activated Potassium Channels in Vascular Myocytes is a Key Novel Mechanism for Tacrolimus-induced Hypertension
Authors: Zheng Yun-Min , Wang Yong-Xiao
Published: March 2022

Background and Purpose:
Tacrolimus (TAC, also called FK506), a common immunosuppressive drug, causes hypertension. In this study, we tested a novel hypothesis that TAC inhibits big-conductance Ca
2+
-activated K
+
(BK) channels in artery smooth muscle cells (SMCs), leading to hypertension.

Experimental Approach:
Noninvasive and invasive blood pressure measurements were used to determine hypertension; organ bath technique and live videomicroscopy to assess vasoconstriction; fluorescence imaging and confocal microscope to detect whole-cell and local intracellular Ca
2+
concentration; patch-clamp techniques to record channel activity; Western blot analysis and real-time RT-PCR to evaluate channel expression; and gene knockout mice to determine its functions.

Key Results:
Intraperitoneal injection of TAC once a day for a week causes hypertension with a peak at 4 weeks. Application of norepinephrine (NE) induces a larger vasoconstriction in arteries and increase in [Ca
2+
]
i
in artery SMCs from mice treated with TAC for 4 weeks. Whole-cell BK channel activity is significantly decreased in SMCs from TAC-treated mice, whereas Ca
2+
spark activity is increased. The voltage sensitivity, Ca
2+
sensitivity and open time of single BK channels are all decreased in TAC-administrated mouse cells. BK channel β1, but not α, subunit protein and mRNA expression levels are significantly decreased in arteries from TAC-treated mice. Chronic administration of TAC fails to further increase blood pressure in BK channel β1 knockout mice with hypertension.

Conclusion and Implications:
Our findings provide novel evidence that TAC inhibits BK channel β1 subunit expression and functions in artery SMCs, thereby leading to enhanced vasoconstriction and hypertension.
Abstract P513: Seasonal Fluctuations of Novel Arterial Stiffness Indexs in Hypertensive Patients
Authors: Kita Toshihiro , Kitamura Kazuo
Published: March 2022

Seasonal fluctuation of blood pressure (BP) is well known and possible relationship is reported between increases of BP and increases of cardiovascular accidents (CVA) in winter. Parameters of arterial stiffness may have seasonal fluctuation but available data are highly limited. Novel arterial stiffness index, Arterial Velocity pulse Index (AVI) and Arterial Pressure volume Index (API), can obtain through usual maneuver of BP measurement, and thus repeated measurement is easy in regular examination in outpatient office. Here, we analyzed seasonal fluctuation of AVI and API in hypertensive patients for 2.5 years (30 months), where the parameters were measured on every visits. Objects are 60 hypertensive patients under stable treatment and continuously visited our outpatient office for 2.5 years. BP, pulse rate (PR), AVI and API was measured using AVE-1500 (Pasesa, Shisei datum, Tokyo, Japan) in sitting position. Six periods were made like January to February, March to April and so on. Every measurements (at least 12 to over 30 per person) were sorted using the six period and averages of the parameters were used for analysis. Obvious seasonal fluctuations in PR (p<0.001) and AVI (p<0.001) and marginal fluctuations in SBP (p=0.055) and API (p=0.067) were confirmed by repeated ANOVA (Figure). SBP, API and PR were decreased in summer as expected, but, unexpectedly, AVI was increased in summer. Coefficient variations were SBP 5.1%, PR 4.8%, AVI 12.5% and API 10.6%. AVI is affected by reflected wave and closely correlated with augmentation index, and high AVI increases cardiac afterload. Decrease of BP and increase of AVI may relate to another CVA risk in summer like ischemic stroke.
Abstract P515: Vascular Stiffness is Not Changed by Dipeptidyl Peptidase 4 Inhibitor
Authors: Vilela-Martin Jose F , Cosenso-Martin Luciana N , Giollo Luiz T
Published: March 2022

Introduction:
The action of dipeptidyl peptidase-4 (DPP-4) inhibitors on blood pressure (BP) and arterial stiffness is controversial. This study aimed to investigate the effects of DPP-4 inhibitor (vildagliptin) on the BP and arterial stiffness.

Methods:
Fifty patients over 35 years of age with diabetes and hypertension without cardiovascular disease were randomized to vildagliptin (n=25) or glibenclamide (n=25) in a prospective, open, drug-controlled study. Both groups used metformin and renin angiotensin system blockers. BP was evaluated by digital sphygmomanometer and by 24-hour ambulatory BP monitoring with the Mobil-O-Graph PWA® device, which analyzes parameters of arterial stiffness (central systolic BP, pulse wave velocity (PWV) and augmentation index (AIx75). Laboratory evaluation (glycemia and glycated hemoglobin), and BP and arterial stiffness measurements were performed before and after 12 weeks of treatment.

Results:
Glycated hemoglobin reduced non-significantly with treatment in both groups. 24-hour systolic BP decreased significantly in the vildagliptin (123.8±12.5 vs 119.1 ± 1.7 mmHg, P=0.03) and glibenclamide group (125.1 ± 12.2 vs 117.3 ± 8.0 mmHg, P=0.002), but without difference between the groups. There were no changes in central systolic BP and PWV in the vildagliptin group before and after treament. However, in the glibenclamide group, central systolic BP decreased (116.4±12.19 vs 109.0±7.5, mmHg P=0.003), as well as PWV (8.5±1.3 vs 8.1±1.1 m/s, P=0.003), but with no difference between groups (P=0.27 and 0.32, respectively). There was also no difference for 24 hour AIx75 for both drugs. Pearson’s correlation did not demonstrate a correlation of glycemic control with PWV and AIx75. However, office systolic BP and central pulse pressure showed correlation with PWV in the vildagliptin group (P=0.008 and 0.002, respectively) and glibenclamide (P<0.001 and 0.001, respectively).

Conclusions:
Vildagliptin does not alter BP and arterial stiffness in diabetic and hypertensive patients. The observed improvement in 24-hour BP is due the antihypertensive treatment, as it was demonstrated in the 2 groups. Unlike blood glucose, BP correlates to arterial stiffness.
Abstract P520: Low Plasma Levels of Vitamin D are Not Associated With Carotid Artery Stiffness in Hypertension
Authors: Catena Cristiana , Colussi Gianluca , Brosolo Gabriele , Bertin Nicole , Novello Marileda , Frangipane Alessandro , Duratti Andrea , Sechi Leonardo A
Published: March 2022

Objectives:
Recent studies suggest an association between vitamin D deficiency and prevalence of cardiovascular disease in the general population. However, the evidence of a relationship between vitamin D status and vascular functional changes in hypertension is currently controversial. We aimed to investigate if the presence of low serum vitamin D levels is associated with an increased arterial stiffness in essential hypertension.

Design and Method:
In 151 essential hypertensive patients (53±13 y, 71 males) we measured serum 25(OH)-vitamin D levels and performed a carotid ultrasound examination to determine variables of carotid stiffness, such as distensibility, compliance, Young elastic modulus and beta-stiffness. Furthermore, in a subgroup of 86 subjects we evaluated the augmentation index and the pulse wave velocity. The patients were subdivided into two groups according to the 25(OH)-vitamin D levels, lower or higher than 30 nmol/L.

Results:
A plasma vitamin D level lower than 30 nmol/L was found in 83 (55%) of 151 patients. Patients with low vitamin D levels were significantly older than those with a normal value of serum vitamin D. No significant differences in carotid artery distensibility, compliance, Young elastic modulus, and beta-stiffness were observed between patients with plasma vitamin D below or above 30 nmol/L. Also, the augmentation index and the pulse wave velocity were comparable in hypertensive patients with lower or higher plasma vitamin D levels.

Conclusions:
This study does not support the hypothesis of an association between vitamin D deficiency and functional vascular changes in hypertensive patients.
Abstract P522: Endothelial Cell-specific Knockout of Epithelial Sodium Channel Prevents Aortic Stiffness, Cardiac Stiffness and Diastolic Dysfunction in Response to a Western Diet in Female Mice
Authors: Aroor Annayya , Jia Guanghong , Hill Michael , Habibi Javad , Manrique Camila , Lastra Guido , Barron Brady J , Allen Sadie A , Chen Dongqing , Yang Yan , Sun Zhe , Meininger Gerald , Martinez-Lemus Luis , Whaley-Connell Adam , DeMarco Vincent G , Jaisser Frederic , Sowers James R
Published: March 2022

A western diet (WD), high in fructose and fat, is often accompanied by insulin resistance and cardiovascular disease characterized by endothelial cell (EC) dysfunction, increased arterial and cardiac stiffness, and diastolic dysfunction. Although premenopausal non-obese women are protected against cardiovascular disease, arterial stiffness and diastolic dysfunction, in obese women these abnormalities are more pronounced than in men. We have recently developed a clinically relevant, WD fed, murine model that exhibits increased aortic stiffness associated with vascular and cardiac dysfunction. In this model, female mice have high plasma aldosterone levels and increased mineralocorticoid receptor expression (MR) in both the vasculature and heart. One of the mechanisms by which MR activation promotes endothelial stiffness is through increased expression and activation of epithelial sodium channel (ENaC) in ECs (EnNaC). We reported increased aortic EC stiffness associated with increased expression of EnNaC in WD fed mice and suppression of aortic stiffness and improved diastolic function by treatment with a low dose of an MR antagonist or the ENaC inhibitor, amiloride. In this study, we tested the hypothesis that specific deletion of EnNaC, decreases aortic EC stiffness and improves vascular relaxation and diastolic function in WD fed female mice. To produce cell specific deletion of the EnNaC gene,”floxed” EnNaC mice were serially crossed with Tie 2-Cre transgene mice. This resulted in marked suppression of EnNaC expression in ECs. Female KO mice and littermate controls were fed a WD with high in fat (46%) and fructose (17.5%) for 12 weeks. Compared to mice fed a control diet (CD), aortic EC stiffness, measured ex vivo by atomic force microscopy (AFM) was significantly increased in WD fed mice and this was prevented in EnNaC KO mice fed WD. Decreased EC stiffness was associated with improved endothelial-dependent aortic relaxation in response to acetylcholine. Moreover, deletion of EnNaC also prevented WD induced impairment of diastolic function. Taken together, these findings support the notion that a WD promotes ECMR mediated activation of EnNaC and associated aortic stiffness, cardiac stiffness and diastolic dysfunction.
Abstract P525: Mtor Inhibitor Everolimus Decreases Aortic Sensitivity To Phenylephrine In Normotensive Rats
Authors: Warner Alex , Martinez Quinones Patricia , Wencesalu Camilla , McCarthy Cameron , Webb Clinton
Published: March 2022

While targeted chemotherapeutics have improved cancer clinical outcomes, many of these agents have the drawback of cardiovascular complications. It has been established that the mTOR complexes have a pivotal role in many types of cancer, promoting cell growth, proliferation, and survival. Inhibitors of this pathway such as first generation Everolimus, and second generation PP242, have inhibited cancer cell proliferation and cancer progression. When compared with other target chemotherapeutics such as VEGF and tyrosine kinase inhibitors, first generation mTOR inhibitors have demonstrated decreased incidence of cardiovascular complications, however the specific vascular effects of mTOR inhibitors have not been investigated. Therefore, Everolimus, an inhibitor of mTORC1, and PP242, an inhibitor of mTORC1 and mTORC2, were evaluated for their effects on vascular reactivity of rat aorta. Previous studies have demonstrated that mTORC2 displayed cardioprotective effects. Therefore, we hypothesize that Everolimus, which does not inhibit mTORC2, will have a decreased contractile response to Phenylephrine (PE) and increased Acetylcholine (Ach)-induced relaxation. On the contrary, since PP242 inhibits mTORC2, we hypothesize that PP242 will increase contractile response to PE, and will decrease ACh-induced relaxation. Concentration response curves to PE (1nM - 30μM), and Ach (1nM - 30μM) were conducted in isolated aortic rings from male normotensive Wistar rats (12-15 weeks old) on a pin myograph
ex vivo
after 1 hour of incubation with each drug. Aortic rings treated with 31 μM of Everolimus showed decreased sensitivity to PE (LogEC
50
= -7.322 ±0.078 vs. treated: -6.578 ± 0.1116 p<0.05). No differences were observed in the contractile responses to PE for Everolimus (0.1 nM) or PP242 (245 nM) (p>0.05). Similarly, no differences were observed in the endothelium-dependent relaxation response to Ach for all three treatments. These results suggest that Everolimus may be a good therapeutic choice for cancer patients also suffering from cardiovascular dysfunction.
Abstract P540: Distinct Profile of Circulating Extracellular Vesicles in Normotensive Obese Humans
Authors: Erdbruegger Uta , Eichner Natalie Z , Gilbertson Nicole M , Rudy Christine , Musante Luca , Malin Steven K , Le Thu H
Published: March 2022

Hypertension (HTN) is a leading cause of cardiovascular disease. Early and non-invasive biomarkers of vascular dysfunction and end-organ damage in HTN are needed to optimize treatment for patients at risk for HTN. Extracellular vesicles (EVs) are potential candidate biomarkers that reflect occurrence of end-organ damage prior to symptom development. EVs are a heterogeneous population of submicron vesicles that shed from various cell types in the blood and carry markers from their parent cells. We hypothesized that obese adults with normotension have a distinct profile of circulating EVs. Twenty-two obese normotensive patients (Age: 62.5±8.9y, BMI 33.1±6.4 kg/m2) were enrolled in this cross-sectional study. After an overnight fast, systolic and diastolic blood pressure were 126±12.5mmHg and 68.7±8.2mmHg, respectively. Arterial stiffness was assessed by pulse wave velocity and augmentation index. Enumeration and phenotyping of platelet poor plasma EVs was performed using imaging flow cytometry. CD42 positive (platelet), CD31 (PECAM), CD105 (S-endoglin, endothelial), CD45 (leukocyte) positive and Annexin V (AnV) positive EVs were used as surface markers for circulating EVs. Annexin V (AnV) and S-Endoglin positive EVs (CD105+, AnV+) correlated significantly with the diastolic blood pressure (p=0.03, r=0.46). In addition, leukocyte derived EVs (CD45+, AnV+) correlated with pulse wave velocity (p=0.04, r=0.44). Other platelet derived EVs and AnV negative EVs did not correlate with blood pressure level or measures of vascular health. Subgroups of endothelial and leukocyte derived EVs correlate with diastolic blood pressure and arterial stiffness in obese normotensive adults. These findings suggest that the specific EV profile might reflect pathophysiologically significant pre-hypertensive blood pressure.
Abstract P045: Central Cytochrome P450 1B1 Generated 17Beta Estradiol Metabolite 2-Methoxyestradiol Minimizes Angiotensin II-induced Hypertension in Female Mice
Authors: Singh Purnima , Song Chiyoung , Heldt Scott A. , Malik Kafait U.
Published: March 2022

Recently we have shown that 17beta-estradiol (E2)-generated cytochrome P450 1B1 (CYP1B1) metabolite 2-methoxyestradiol (2-ME) protects against angiotensin II (Ang II)-induced hypertension in female mice. The demonstration that central E2 inhibits Ang II-induced hypertension together with the expression of CYP1B1 in various areas in the brain and the production of 2-ME from E2 by microglia cells led us to hypothesize that 2-ME contributes to the inhibitory effect of E2 on the brain on Ang II- induced hypertension in female mice. To test this hypothesis, we examined the effect of intracerebroventricular (ICV) administered E2 in ovariectomized (OVX) wild-type (

Cyp1b1
+/+
)

and OVX

Cyp1b1
-/-

mice on the action of systemic Ang II (700 ng/kg/min) for 14 days. E2 (1.5 μg/2μL/injection) or its vehicle (20% w/v 2-Hydroxypropyl-β-cyclodextrin dissolved in artificial CSF) was injected (400 nL/min every 2
nd
day) via a cannula implanted in the brain. Mean arterial blood pressure (MAP) was measured by radiotelemetry (n=3-4). E2 but not its vehicle attenuated Ang II-induced increase in MAP on Day 12 in OVX

Cyp1b1
+/+

(111±2 vs. 154±5, mmHg, P<0.05). ICV injections of 2-ME but not E2 (1.5 μg/2μL/injection every 2
nd
day) attenuated the increase in MAP by Ang II in OVX
Cyp1b1-/-
mice (111±1 vs. 141±6, mmHg, P<0.05). Administration of ganglionic blocker hexamethonium (30 mg/Kg, IP) on day 14 of Ang II infusion resulted in greater reduction in MAP (P<0.05, n=4) in centrally injected E2 in OVX

Cyp1b1
-/-

, and vehicle-injected OVX

Cyp1b1
+/+

mice (Δ90±1 and Δ91±14, mmHg) than in E2 injected OVX

Cyp1b1
+/+

(Δ68±5 mmHg), and 2-ME injected OVX

Cyp1b1
-/-

mice (Δ60±4 mmHg). Furthermore, in intact

Cyp1b1
-/-

, but not OVX

Cyp1b1
-/-

mice reconstitution of CYP1B1 in the brain by transduction with adenovirus (AdV)-CYP1B1-cDNA (ICV 2μL of 1.0 X 10
12
particle/mL) reduced systolic blood pressure (SBP) measured by tail-cuff on Day12 (136±2 vs. 166±6 mmHg, P<0.05, n=4-5). ICV injection of AdV-GFP-cDNA in these mice did not alter Ang II-induced increase in SBP (168±8 and 171±2 mmHg, n=4). These data suggest that central effect of E2 to attenuate Ang II-induced hypertension is dependent on brain CYP1B1 and is most likely mediated via generation of 2-ME, which decreases sympathetic outflow in female mice.
Abstract P015: Hypoxia Regulates Embryonic Cardiac Myocyte Proliferation and Differentiation
Authors: Zambon Alexander , Stowe Jennifer
Published: March 2022

Mammalian cardiac myocytes (CM) proliferate readily during development but exit the cell cycle perinatally. The molecular mechanism for this is poorly understood could lead to new approaches for cardiac regeneration. We employed a novel bioinformatics approach to identify transcription factors that may control perinatal cell-cycle arrest and identified hypoxia inducible factor 1α (Hif1α) as a potential regulator of CM proliferation and differentiation. To test our hypothesis, e12.5d embryonic heart cells were cultured in atmospheric (17%) or hypoxic (3%) [O2] and then assessed for proliferation over 4 days. Total cell numbers were significantly increased (∼2 fold, P<0.005) in cells cultured in 3% vs. 17% [O2] after 4 days. Hypoxia increased numbers of cTn+ cells labeled with p-Histone-H3 indicating an increase in CM proliferation. Carboxyfluorescein succinimidyl ester staining indicated that the majority (70%) of cells cultured in 3% [O2] divided 2–3 times while the majority (73%) of cells in 17% [O2] divided 0–1 times after 3 days of culture. As expected, hypoxia stabilized the Hif1α protein and induced the expression of Vegf. Messenger RNA for the G1-S phase cyclin dependent kinase Ccnd2 was increased (>2 fold) in hypoxic vs. atmospheric cultured cells indicating that hypoxia my increase proliferation by regulating the expression of cell cycle machinery. Furthermore, early mesodermal transcription factors Brachyury and Mesp1 and the CM progenitor marker Nkx2.5 were induced (>5 fold) in hypoxic vs. atmospheric [O2] cultured cells suggesting that hypoxia positively regulates early mesodermal and CM progenitor cell differentiation. To validate this hypothesis, we differentiated mouse embryonic stem cells in atmospheric and hypoxic [O2] and observed similar increases in the expression of Brachyury, Mesp1 and Nkx2.5 in hypoxic differentiated cells. Transient culture (2 d) of embryoid bodies (EBs) in hypoxia increased the frequency of beating EBs and the % of cTn+ cells compared to cells differentiated in atmospheric O2. Taken together these data suggest that the hypoxic environment in which CMs develop may serve as a key signal that regulates mesodermal progenitor marker expression and CM progenitor cell differentiation and proliferation.
Abstract P027: Preconditioning Cardiosphere-Derived Cells by Hypoxia and Prolyl Hydroxylase Inhibitors to Induce HIF-Related Metabolic Changes and C-Kit Expression
Authors: Tan Suat Cheng , Carr Carolyn A , Yeoh Kar Kheng , Heather Lisa C , Ambrose Lucy , Schofield Chris , Clarke Kieran
Published: March 2022

Cardiosphere-derived cells (CDCs) decrease loss of cardiac function following infarction, but the potential of CDC therapy is limited due to the low retention rate and the time required to obtain sufficient cells for transplantation.

AIM: Here, we aimed to precondition CDCs by culturing under hypoxia, or with prolyl hydroxylase inhibitors (PHIs), to prepare the cells for the hypoxic environment within the infarcted heart.

METHODS: Rat heart explants were cultured under hypoxia (2% O2) or normoxia (21% O2) to generate explant-derived cells (EDC). EDCs were isolated, cultured to form cardiosphere and expanded into monolayer CDCs. At 80% confluency, normoxic CDCs were treated with dimethyloxalyl glycine (DMOG), Ethyl 2-(2,3-dihyroxybenzamido) (KKC226) and FG-2216 for 24 h.

RESULTS: Hypoxia, 1 mM DMOG, 0.5 mM KKC226 and 30 μM FG-2216 treated CDCs showed significantly increased HIF-1α protein expression (3.9-fold, 2.6-fold, 2.1-fold and 1.5-fold, respectively) and reduced oxygen consumption (81%, 32%, 47% and 35%, respectively), compared with normoxic CDCs. Hypoxia and PHI treatment increased protein levels of GLUT-1 (6.7-fold, 3.2-fold, 4.0-fold and 2.1-fold, respectively), resulting in significant increases in glucose uptake and lactate accumulation in the culture medium, compared with controls, commensurate with increased glycolytic metabolism after hypoxia or PHI treatment. Further, hypoxia and KKC226 increased c-Kit mRNA expression 5.1-fold and 1.5-fold, respectively. qRT-PCR confirmed increased CXCR-4 mRNA in all hypoxia and PHI-treated CDCs. EDC and CDC proliferation were 1.7-fold faster under hypoxia, compared with normoxia, but did not increase with PHI treatment.

CONCLUSION: Hypoxia and PHIs stabilized and activated HIF, which induced metabolic changes in CDCs, including GLUT-1 upregulation and a switch to anaerobic glycolytic metabolism. All preconditioned cells had reduced oxygen consumption and were better adapted to survive within an hypoxic infarct scar. HIF-induced upregulation of CXCR-4 may increase the homing of these cells to the infarcted myocardium. Finally, increased proliferation and expression of the cardiac stem cell marker, c-Kit, could decrease the time required for cell expansion prior to therapy by a week.
Abstract P048: The Role of p62 in Cardiac Protein Quality Control
Authors: Zheng Qingwen , Su Huabo , Wang Xuejun (XJ)
Published: March 2022

Rationale:
Two recent studies suggest an important role of autophagy in protection against αB-crystallin-based (CryAB
R120G
) desmin-related cardiomyopathies (DRC) but this has not been demonstrated in a different model of cardiac proteinopathy. Moreover, mechanisms underlying the response of cardiomyocytes to increased expression of misfolded proteins remain incompletely understood.

Objective:
First,to determine whether and how the autophagic activity is changed in a mouse model of desminopathy; second, to investigate the role of p62 in the protein quality control of cardiomyocytes.

Methods and Results:
Using an autophagosome reporter and determining changes in LC3-II protein levels in response to lysosomal inhibition, we found significantly increased autophagic flux in mouse hearts with transgenic overexpression of a DRC-linked mutant desmin. Similarly, autophagic flux was increased in cultured neonatal rat ventricular myocytes (NRVMs) expressing a mutant desmin. Suppression of autophagy by 3-methyladenine increased, whereas enhancement of autophagy by rapamycin reduced, the ability of a comparable level of mutant desmin overexpression to accumulate ubiquitinated proteins in NRVMs. Furthermore, p62 mRNA and protein expression was significantly upregulated in cardiomyocytes by transgenic overexpression of the mutant desmin or CryAB
R120G
both in intact mice and in vitro. p62 depletion impaired aggresome and autophagosome formation, exacerbated cell injury, and decreased cell viability in cultured NRVMs expressing the misfolded proteins.

Conclusions:
Autophagic flux is increased in desminopathic hearts, serving as an adaptive response to overexpression of misfolded proteins. p62 is upregulated in mouse proteinopathic hearts. p62 protects cardiomyocytes against proteotoxic stress by promoting aggresome formation and autophagy activation.
Abstract P049: Molecular Mechanisms of PARP1-Mediated Necrosis
Authors: Douglas Diana L , Baines Christopher P
Published: March 2022

Poly(ADP-ribose) polymerase-1 (PARP1) is a nuclear enzyme associated with DNA replication, transcription, repair, and cell death. Although, PARP1 is most commonly associated with apoptosis, a specific PARP1-mediated caspase-independent necrosis pathway has recently been described. Two main models for this pathway have been proposed: one involving RIP1 and JNK kinases and mitochondrial permeability transition (MPT), the other involving activation of Bax and subsequent release of apoptosis inducing factor (AIF) from mitochondria. However, these models still remain untested. Consequently, the purpose of this study was to determine which (if any) of these molecules are truly involved in PARP1-mediated necrosis. Murine embryonic fibroblasts (MEFs) were treated with either N-Methyl-N’-Nitro-N-Nitrosoguanidine (MNNG) or β-Lapachone, 2 chemically distinct PARP1 activators. To inhibit RIP1, CypD, and AIF, MEFs were transfected with specific siRNAs for 48 hrs prior to treatment. RIP1 and CypD-null cells were also used. To block JNK activity, cells were incubated with the JNK-specific inhibitor SP600125. Inhibition of Bax was achieved using either Bax/Bak-null MEFs or Bcl2 overexpression. Necrosis, MPT, JNK activation, and AIF translocation were then analyzed using a combination of cell death assays, western blotting, and immunocytochemistry. Treatment of MEFs with MNNG and β-Lapachone induced translocation of AIF from the mitochondria to nucleus, and necrotic cell death. However, inhibition of RIP1, CypD, and Bax had no significant effect on MNNG or β-Lapachone- induced necrotic cell death. Moreover, neither PARP1 activator induced MPT. In contrast, the JNK inhibitor SP600125 significantly reduced MNNG- and β-Lapachone-induced JNK activation, AIF translocation, and necrosis. This suggests that JNK plays a key role in the pathway and that it is upstream of AIF. To our surprise, however, depletion of AIF did not influence PARP1-mediated cell death, thus indicating that AIF is more of a marker, rather than a key mediator, of this particular process. We conclude that JNK plays a significant signaling role in PARP1-mediated necrosis. However, RIP1, Bax, CypD, and AIF do not appear to be essential components of the PARP1 death pathway.
Abstract P054: Crosstalk Between Toll-like Receptor 4 and Lipid Metabolism Modulates Macrophage Survival
Authors: Schilling Joel , Machkovech Heather , Schaffer Jean
Published: March 2022

Diabetes and obesity are well-established risk factors for cardiovascular disease, including atherosclerosis and adverse cardiac remodeling after myocardial infarction (MI). Abnormalities of lipid metabolism are a hallmark of diabetes and are strong predictors of the risk of future cardiac events. Macrophages are cells of the innate immune system that play a vital role in the host response to infection and tissue injury. In the cardiovascular system, macrophages are key participants in the pathogenesis of atherosclerosis and post-MI infarct remodeling. Toll-like receptor (TLR)-4 is highly active on macrophages and has been shown to contribute to the injury response in the heart and vasculature. Thus, understanding how lipids modulate TLR4-mediated macrophage responses is particularly relevant to diabetic cardiovascular disease. To investigate this question, we studied the effect of palmitate; an abundant dietary saturated fatty acid, on macrophage viability and inflammatory signaling after stimulation with the TLR4 ligand LPS. Interestingly, LPS or palmitate alone failed to trigger significant cell death; however, the combination of these stimuli markedly reduced macrophage viability. The synergistic death induced by LPS and palmitate required functional TLR4, yet occurred independently of ER stress, reactive oxygen species generation, ceramide production, caspase activation and autophagy. Instead, lysosomal integrity is compromised early and followed by the loss of mitochondrial membrane potential and ultimately cell death. Interestingly, inhibition of the lipid remodeling enzyme iPLA2 attenuates lysosomal instability and cell death following treatment with palmitate/LPS. Thus, we describe a novel cell death response in macrophages that occurs via a mechanism involving iPLA2 activation and lysosomal destabilization. These data provide evidence that the lipid environment modulates macrophage function following inflammatory stimuli. Given the critical role of macrophages in the response to tissue injury, these findings are highly relevant to understanding cardiac injury and repair in diabetics.
Abstract P055: 2-Deoxy-Glucose Reduces Doxorubicin Cardiotoxicity in Tumor-Bearing Mice
Authors: Timm Derek , Phillips Michelle , Haugrud Allison , Zhuang Yongxian , Miskimins Keith , Liang Qiangrong
Published: March 2022

Doxorubicin (DOX) is a potent, commonly used anthracycline antibiotic for a wide range of cancers, but its dose is limited by its strong cardiotoxic effects. The glucose analog 2-deoxy-D-glucose (2-DG) has been shown to enhance the antitumor efficacy of DOX in tumor cell lines and in animals. However, the cardiac effects of 2-DG in these experiments were not determined. In the current study, we examined whether 2-DG could protect from DOX cardiotoxicity in a BALB/c mouse breast cancer model. A total of 32 mice were injected with 10
5
4T1 mouse mammary epithelial tumor cells in the right hind leg. The tumor was allowed to progress for 1 week before dividing the mice into four groups: saline, 2-DG, DOX, and 2-DG with DOX treatment. 2-DG was given 150 mg/kg i.p. Monday through Friday; DOX was given 5 mg/kg i.p. Wednesday. The study was conducted for 3 weeks and echocardiography was performed the day before sacrifice. Two of the eight mice in the DOX group died before echocardiography, while none died in the 2-DG with DOX group. Echocardiography indicated similar cardiac function between saline and 2-DG groups as measured by fractional shortening (36.3±1.31% vs 37.7±0.942%, p>0.05). DOX markedly reduced cardiac function, which was almost completely recovered by 2-DG (25.1±1.06% vs 36.7±1.76%, p<0.01). Serum CK-MB content, a common clinical marker for heart damage, was increased ~6 fold in the DOX group (67.5±19.4 vs 400±145, p<0.023). 2-DG tended to reduce DOX-triggered CK-MB release, but it did not reach significant levels likely due to the small sample size and high variation (279±68.7 vs 400±145, p>0.05). Nonetheless, 2-DG largely eliminated DOX-induced apoptosis in the heart as shown by a DNA laddering assay. 2-DG or DOX alone increased cardiac autophagic flux as measured by the difference in LC3-II protein levels in the absence and presence of the lysosomal inhibitor, bafilomycin A1. Paradoxically, autophagic flux was not notably elevated when mice were treated with 2-DG and DOX simultaneously. These results demonstrate the ability of 2-DG to reduce DOX cardiotoxicity without affecting its antitumor activity, suggesting that 2-DG can improve the therapeutic window for DOX, allowing greater flexibility in designing different regimens for treating cancers.
Abstract P058: Necessity of mTOR Inhibition and Autophagy Induction for Maintaining Cardiac Homeostasis During Caloric Restriction
Authors: Chen Kai , Timm Derek , Jepperson Tyler , Xu Xianmin , Kobayashi Satoru , Liang Qiangrong
Published: March 2022

Caloric restriction (CR) is a robust dietary intervention known to delay age-related heart disease. CR is able to antagonize cardiac remodeling in animal and human under various pathological conditions. However, the direct effect of CR on the heart at basal physiological state is not well characterized. In this study, we examined the impact of CR on cardiac homeostasis and explored the signaling mechanisms that mediate the cardiac effect of CR. Male C57BL/6J mice underwent 20% CR,
i.e.
20% less food than
ad libitum
(AL), for 2 weeks and 40% CR for another 2 weeks. This CR regimen caused a roughly 20% decrease in heart weight and myocyte cross-sectional area. CR diminished the activity of the
mammalian target of rapamycin

(
mTOR)

and accelerated autophagic flux. To determine the functional role of mTOR inhibition in CR-induced heart size reduction, we used transgenic mice that express a constitutively active mTOR (mTORca) in the heart. CR did not inhibit cardiac mTOR signaling in mTORca mice as it did in wild type (WT) mice. As a result, the ability of CR to reduce heart size in WT mice was severely impaired in mTORca mice. Specifically, CR reduced the heart weight by 23.1% in WT mice (p<0.01 vs AL), but only tended to reduce it in mTORca mice (13.1%, p>0.05 vs AL). Strikingly, when animals were treated with the mTOR inhibitor rapamycin, CR decreased heart weight in both WT and mTORca mice to the same extent, strongly suggesting that mTOR inhibition is required for CR to exert its cardiac effect. Autophagy is a cellular degradation pathway that may contribute to CR-induced heart size reduction. Surprisingly, CR can still decrease heart weight in 2 different autophagy-deficient mouse models, namely, beclin 1 heterozygous knockout mice and ATG16L1 hypomorphic mice. Nonetheless, cardiac function in these mice was markedly deteriorated during CR as indicated by decreased fractional shortening (Beclin 1+−: AL 37.9±2.8 vs CR 29.6±2.1, p<0.01; ATG16L1-HM: AL 36.8±1.8 vs CR 30.1±2.1, p<0.05). This was accompanied by increased oxidative injury, suggesting that autophagy is indispensible for a normal cardiac function during CR. In conclusion, both mTOR inhibition and autophagy induction are essential for maintaining cardiac homeostasis in response to CR-triggered mild stress.
Abstract P061: The Possible Apoptosis Pathways Involved in Mechanical Trauma-Induced Secondary Heart Injury
Authors: Yan Zi , Guo Li , Wen Yongjin , Cao Chengzhang , Wang Jie , Zhang Yan , Guo Yong , Liang Feng
Published: March 2022

Background:
Trauma induces not only primary heart injury but also secondary heart injury. Our previous study has demonstrated that mechanical trauma could cause cardiomyocyte apoptosis which contributes to posttraumatic secondary cardiac dysfunction. The aim of the present study was to elaborate the potential mechanism involved in secondary post-traumatic cardiac dysfunction.

Methods:
Male adult rats were subjected to Noble-Collip drum with a total of 200 revolutions at a rate of 40 rpm. The activities of cardiomyocyte Caspase 3, 8, 9, 12 were detected respectively by Fluorometric assays. The cardiac function
in vitro
was determined using a Langendorff isolated heart perfusion system.

Results:
No direct mechanical traumatic injury was observed in the heart immediately after trauma. (1)Compared with the sham group, myocardial Caspase-3 activity in the traumatized rats significantly increased 6h after trauma (51±4
vs.
16±2,
P
<0.01). 12h after trauma myocardial Caspase-3 activity reached the peak (69±4
vs.
Sham,
P
<0.01), which still remained at high level (46±3
vs.
Sham,
P
<0.01) 24h after trauma. (2) Administration of Z-VAD-FMK, a broad-spectrum caspase inhibitor, could reverse post-traumatic cardiac dysfunction evidenced by increased +dP/dt
max
[(4111±189) mmHg/sec,
vs.
sham (3414±208) mmHg/sec,
P
<0.01], and decreased -dP/dt
max
[(-4997±351) mmHg/sec,
vs.
sham group (-3301±458) mmHg/sec,
P
<0.01] 24h after trauma. (3) Compared with the sham group, myocardial Caspase-12 activity significantly increased 3h after trauma (66±8
vs.
27±10,
P
<0.01). 6h after trauma Caspase-12 activity reached the peak (89±16,
P
<0.01
vs.
Sham) , but 12h after trauma it decreased (
P
>0.05
vs.
Sham). Both myocardial Caspase-8 activity and Caspase-9 activity markedly increased 24h after trauma (2312±648
vs.
1449±296,
P
<0.01
vs.
Sham; 875±460
vs.
470±222,
P
<0.01
vs.
Sham, respectively).

Conclusion:
Caspase-dependent apoptotic pathway played an important role in post-traumatic secondary cardiac dysfunction. Moreover, in the early period of trauma, cardiomyocyte apoptosis was induced by activation the Caspase-12, an endoplasmic reticulum (ER)-specific caspase, following by activation of Caspase-8 (extrinsic pathway) and Caspase-9 (intrinsic pathway).
Abstract P069: Interplay Between the Ubiquitin-Proteasome System and Autophagy in the Heart
Authors: Wang Changhua , Tian Zongwen , Zheng Qingwen , Su Huabo , Li Jie , Wang Xuejun (XJ)
Published: March 2022

The ubiquitin-proteasome system (UPS) and autophagy are responsible for the degradation of most cellular proteins. Dysfunctions in the UPS and autophagy are both implicated in cardiac pathogenesis. The UPS and autophagy, thought to be two parallel pathways, seem to interact. Pharmacologically induced proteasome inhibition (PSMI) was shown to increase autophagosomes in cultured cardiomyocytes (CMs) but this observation remains to be demonstrated in intact animals and it is unclear whether the increased autophagosomes are caused by an increased autophagic flux. A recent report shows that chronic autophagic inhibition (AI) impairs the degradation of proteasomal substrates in a p62-dependent manner in cultured cells but this has not been tested in intact animals or CMs. Here we sought to address these important issues. Using the GFP-LC3 reporter, we demonstrate in both cell cultures and intact mice that PSMI upregulates p62 and increases autophagic flux in CMs. This is consistent with the increased autophagic flux in proteinopathic mouse hearts in which proteasome function is inadequate. We then tested the effect of AI on the degradation of a surrogate proteasome substrate (GFPu) in cultured CMs. GFPu was expressed in cultured
n
eonatal
r
at ventricular myocytes (NRVMs) via adenoviral delivery. Compared with the vehicle treatment, a 24-hr treatment but not 6-hr treatment of autophagy inhibitor 3-methyladenine (3-MA) or lysosome inhibitor bafilomycin A1 (BFA) significantly increased GFPu and p62 proteins in cultured NRVMs. Next, we tested the effect of AI on UPS-mediated protein degradation in intact mice. Transgenic mice overexpressing GFPdgn, a previously validated surrogate proteasome substrate, were treated with BFA (1.6 mg/kg/12hr,
i. p.
). BFA induced lysosome inhibition was verified by a significant increase in LC3-II and p62. Myocardial GFPdgn protein levels were significantly increased 24hrs but not
3hrs
after the first BFA injection. The protein level of representative proteasome subunits was not affected by AI. Our data demonstrate that PSMI activates autophagy while chronic AI impairs the degradation of proteasome substrates in the heart. It will be interesting and important to test the role of p62 in the interplay.
Abstract P075: Neuronal Nitric Oxide Synthase Contributes to the Beneficial Cardiac Effects of Exercise
Authors: Roof Steve R , Ostler Joseph , Periasamy Muthu , Ziolo Mark T
Published: March 2022

Exercise results in beneficial adaptations to the heart. These adaptations are observed at the level of the cardiomyocyte as increased Ca2+ cycling through the sarcoplasmic reticulum (SR) and greater fractional shortening. Interestingly, these adaptations are similar to the contractile effects of neuronal nitric oxide synthase (NOS1) signaling. Thus our objective is to determine if the exercise induced adaptations at the level of the cardiomyocyte are NOS1 mediated. After an 8 week high-intensity aerobic interval training program, exercise (Ex) mice had a higher VO2max, greater citrate synthase activity, decreased weight, increased heart-to-body ratio (P<0.05), but similar heart-to-tibia ratio compared to sedentary (Sed) mice. Isolated ventricular myocytes from the Ex mice exhibited larger Ca2+ transient and shortening amplitudes and faster Ca2+ transient decline rates (RT50) compared to Sed myocytes (P<0.05). There was also greater NOS1 expression levels in Ex ventricular myocytes (P<0.05 vs Sed). The greater NOS1 expression was observed functionally in myocyte contraction. That is, acute inhibition of NOS1 by S-methyl-L-thiocitrulline (SMLT) resulted in a greater reduction in Ca2+ transient amplitude, Ca2+ transient RT50, shortening amplitude, SR Ca2+ load, and SR Ca2+ fractional release in Ex versus Sed (P<0.05). Furthermore, acute NOS1 inhibition (SMLT) normalized the Ex induced increase in contraction (Ca2+ transients and shortening) and Ca2+ decline rates to Sed levels. These data demonstrate that the enhanced Ca2+ cycling through the SR observed after a training period is, in part, due to NOS1 signaling. Thus, NOS1 is a major contributor to the increased contractile amplitudes and relaxation observed with exercise. Hence, mimicking the beneficial effects of exercise to the heart may be obtainable by enhancing NOS1 signaling. This pathway may be a novel therapeutic for cardiac patients that are unable/unwilling to exercise.
Abstract P085: Inhibition of Akt Reverses the Isoproterenol-Enhanced Diastolic Calcium Leak in Rabbit Ventricular Myocytes
Authors: Velmurugan Sathya , Curran Jerry , Shannon Thomas
Published: March 2022

Cardiac ryanodine receptor (RyR)-dependent diastolic SR Ca2+ leak is increased in heart failure and during beta-adrenergic (β-AR) stimulation. This increased SR Ca2+ leak could limit the SR [Ca2+] thereby decreasing contractility in HF and could also lead to arrhythmogenic Ca-dependent inward depolarizing current resulting in HF. It has recently been shown in our lab that this leak is dependent on calcium-calmodulin-dependent protein kinase II (CaMKII) and subsequent NOS activation, and that it is independent of either protein kinase A (PKA) activation or an increase in free Ca2+ concentration in the cytosol ([Ca2+]i). Here we investigate whether Akt is also involved in the pathway leading to increased diastolic leak during β-AR stimulation. Methods: [Ca2+]i was measured using fluo-4 in left ventricular myocytes isolated from rabbits. To vary the SR load, myocytes were field-stimulated to steady state at different frequencies. Tetracaine (1mM) was used to rapidly and reversibly block the RyR. The tetracaine-dependent shift of Ca2+ from the cytosol to the SR (decrease in [Ca2+]i and increase in SR Ca2+ content) is proportional to SR Ca2+ leak. To activate β-AR, isoproterenol (ISO; 250 nM) was added to perfuse the myocytes starting 5 min before leak assessment through the end. Results: ISO-induced increase in the SR Ca2+ leak is abolished by the treatment of myocytes with Akt inhibitor X (AIX; 5μM; 30min pre-incubation and perfusion throughout the experiment). When SR Ca2+ load was matched in each group (ISO alone: 155.22 ± 5.1 μM; ISO + AIX: 153.92 ± 3.8 μM), myocytes treated with ISO alone had significantly higher leak (12.43 ± 3.8 μM) vs. those treated with ISO and AIX (1 ± 2.2 μM) (P = 0.01, t-test). This evidence indicates that Akt activation may also be involved in the β-AR-induced SR Ca2+ leak possibly upstream from CaMKII and NOS activation.