bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P202: The Metabolic Syndrome and Shift Work Among firefighters in South Korea
Authors: Choi Jee eun , Bae Mun-Joo , Park Sungha , Park Ki-Soo , Kim Changsoo
Published: July 2021

Background/Aim:
Multiple risk factors including dysipidemia, hypertension and hyperglycemia which cluster together are termed the metabolic syndrome. It means managing the metabolic syndrome is crucial to prevent cardiovascular disease (CVD). Several studies found that CVD is the common disease and the leading cause of on-duty death among firefighters. Although importance of understanding to investigate risk factors that causes CVD among firefighter has been emphasized, research about it is still behind. Thus, to understand risk factor of CVD among firefighters, this study was examined an association between metabolic syndrome and shift work among firefighters.

Methods:
A total of 257 men firefighters were included from Firefighter Research Enhancement of Safety & Heath (FRESH) cohort in Korea. No history of CV related disease including hypertension and diabetes were selected. Weight circumference, systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured. SBP and DBP were measured three times at interval of five minutes in resting and average of the three of SBP and DBP was used in this analysis. Fasting hyperglycaemia, Triglycerides and HDL cholesterol were analysed from blood sample collected from the participants. Metabolic syndrome, using the modified National Cholesterol Education Program (Adult Treatment Panel III) criteria, was defined. The information of shift works, smoking and alcohol consumption were self-reported from the participants and divided into three; no shift work, 24 hour shift work and 2 or 3 shifts works. To analysis the association between metabolic syndrome and shift works, logistic model was used, adjusting for age, BMI, smoking and alcohol.

Results:
26 (10.1%) participants out of 257 were metabolic syndrome in the participants group. The unadjusted prevalence of the metabolic syndrome by shift works is 8.2% in no shift work group, 7.1% in 24 hour shift work group and 15.9% in 2 or 3 shifts work group. Adjusted Odds Ratio (OR) with 95% CI for 24 hours shift work was 1.51 [0.35 6.45] and 4.77 [1.08 20.9] for 3 shifts works.

Conclusions:
There is an association between metabolic syndrome and shift work in Korea firefighters, which implies shift work might be associated with CVD.
Abstract P247: Triiodothyronine Ameliorates Endothelial Dysfunction in Rats Following Acute Myocardial Infarction
Authors: Zhang Kuo , Tang Yi-Da , Zhang Youhua , Li Ying , Savinova Olga , Ojamaa Kaie , Gerdes A. Martin , Carrillo Sepulveda Maria Alicia
Published: July 2021

Background:
Low thyroid hormone (TH) function is recognized as a significant contributor in the pathogenesis after acute myocardial infarction (MI). Endothelial dysfunction contributes significantly to the poor prognosis of MI. We hypothesize that long-term treatment with low dose T3 improves endothelial function and cardiac contractile activity compared to beta blocker, the current recommended therapy for MI.

Methods:
Adult female Sprague-Dawley rats were subjected to left anterior descending coronary artery ligation (MI) or sham surgeries. Survivors were randomly assigned to vehicle (MI, n=11), T3 (MI+T3, n=11) and metoprolol (MI+Meto, n=11). Vehicle, T3 (5 ug/kg/day) and metoprolol (2 mg/kg/day) were supplied in drinking water ad libitum immediately following MI for 2 months. Heart function and LV hemodynamics were measured. Isolated thoracic aortic rings were used to test relaxation response to acetylcholine (ACh) in a wire myograph. The maximal effect elicited by ACh (E
max
) and the sensitivity to ACh (pEC
50
) were analyzed. One-way ANOVA with Bonferroni correction was used for multiple comparisons.

Results:
Serum concentration of free and total T3 were normal in all the experimental groups. T3 and metoprolol improved LV contractile function measured by fractional shortening (21.88±2.06 vs 17.88±1.23%, p<0.01, T3 vs MI; 21.12±3.88 vs 17.88±1.23%, p<0.05, Meto vs MI; 46.86±1.84% for sham) and LV +d
p
/d
t

(7307±1128 vs 5479±810 mmHg/s, p<0.01, T3 vs MI; 7022±695 vs 5479±810 mmHg/s, p<0.05; Meto vs MI; 9160±1881 mmHg/s for sham). Aortas from vehicle-treated group exhibited a marked impairment of endothelial-dependent relaxation measured by pEC
50
(6.65±0.22 vs 7.19±0.16, p<0.001, MI vs Sham), which was significantly improved in the T3 treated group (6.96±0.22 vs. 6.65±0.22, p<0.01, T3 vs MI) but not in metoprolol group (6.85±0.21 versus 6.65±0.22, p=0.22, Meto vs MI). T3 and metoprolol increased maximal relaxation measured by E
max
(90.56±3.55 vs 79.50±3.98%, p<0.001, T3 vs MI; 89.81±6.75% vs 79.50±3.98%, P<0.001, Meto vs MI; 96.93±1.91% for sham).

Conclusion:
Long-term treatment with a physiological dose of T3 following MI is equally effective as metoprolol on LV function while improving endothelial function as an additional benefit.
Abstract P272: Preeclamptic Placental CD4+ T Cells Cause Preeclamptic Like Features in Normal Pregnant Nude Athymic Rats
Authors: Ibrahim Tarek , Harmon Ashlyn C , Cornelius Denise C , Amaral Lorena , Cunningham Mark , Wallace Kedra , Lamarca Basbette
Published: July 2021

Preeclampsia (PE), new onset hypertension during pregnancy, is associated with a proinflammatory profile compared to normal pregnancy (NP). We hypothesize that CD4
+
T cells play an important role to cause much of the pathophysiology associated with PE. To determine if CD4
+
T cells isolated from PE patients cause PE symptoms during pregnancy compared to those collected from NP women, CD4
+
T cells were isolated from placentas of both PE and NP women using magnetic separation with anti-CD4 antibodies, cultured in TexMACS medium with IL-2 at 37°C in 5% CO
2
, and injected intraperitoneally into pregnant, nude-athymic rats on day 12 of gestation. On day 18, carotid catheters were implanted and on day 19 MAP was measured and blood and tissues were collected. MAP was 125±3 mmHg in rats with NP T cells but increased to 140±9 mmHg in rats with PE T cells. Significant differences in circulating cytokines TNF-α, IL-6, IL-17 and sFlt-1 were found with PE vs NP CD4
+
T cells (TNF-α- PE=142.4 pg/mL, NP=79.4 pg/mL; IL-6 - PE=311.6 pg/mL, NP=277.8 pg/mL; IL-17-PE= 7.054 pg/mL, NP=3.185 pg/mL; sFlt-1-PE=90.7 pg/mL, NP=58.2 pg/mL. However, there was no difference in Isoprostane levels or IL-2 between the two groups (Isoprostane-PE=3628 pg/mL, NP=3061 pg/mL; IL-2-PE=125 pg/mL, NP=125 pg/mL). ROS in tissues was measured using chemiluminescence and no difference was found in placenta while renal ROS showed an increased with PE CD4
+
T cells vs NP CD4
+
T cells (ROS-PE=5528 RLU/min/mg protein, NP=3372 RLU/min/mg protein). In addition, no difference in ET-1 mRNA was found in the placenta of nude rats receiving PE CD4+T cells, however, renal cortical ET-1 mRNA expression was increased 4.5 fold in rats with PE CD4+ T cells compared to those receiving to NP CD4+ T cells. These data indicate an important role for placental PE CD4
+
T cells to cause inflammation and stimulate renal vasoactive pathways that may contribute to hypertension during pregnancy. This research is funded by R01HD067541-06
Abstract P381: Understanding the Symptoms of Neurogenic Orthostatic Hypotension: Results From a Survey of Patients and Caregivers
Authors: Hewitt Lawrence A , Adler Charles H , Claassen Daniel O , Gibbons Christopher H , Raj Satish R
Published: July 2021

Objective:
To understand the burden and impact of symptoms of neurogenic orthostatic hypotension (nOH) on patients

Background:
nOH and its symptoms such as dizziness/lightheadedness are common in patients with Parkinson disease (PD) and other forms of autonomic dysfunction.

Methods:
An author-designed, US-based survey was conducted by Harris Poll. Eligible participants were aged ≥18 years with PD, multiple system atrophy (MSA), or pure autonomic failure and ≥1 of the following: orthostatic hypotension, nOH, low BP, OH/nOH symptoms, or were caregivers of eligible patients.

Results:
Most patients (90%) had a diagnosis of PD, and most caregivers (88%) cared for a patient with PD
(Table 1)
. Patients (34%) and caregivers (49%) reported experiencing nOH symptoms before PD or MSA motor symptoms and >40% indicated that nOH symptoms were more troublesome than motor manifestations of PD or MSA. Less than a quarter (22%) of respondents suggested symptoms were most severe in the morning; more (30%) reported a consistent severity throughout the day. Patients (40%) and caregivers (63%) reported trouble managing symptoms during the day. In the past 12 months, a fall due to nOH symptoms was reported by 57% of patients and 80% of caregivers.

Conclusions:
These findings suggest that nOH symptoms may predate the onset of motor symptoms in neurodegenerative conditions linked to alpha-synuclein pathology. Many respondents report nOH symptoms are the same severity through the day. Patients with nOH may have trouble managing symptoms and note an increased risk for falls.
Abstract P437: The Extracellular Domain of Na
+
/K
+
ATPase Serves as a Receptor for Cyclic GMP in Mediating Natriuresis
Authors: Kemp Brandon A , Howell Nancy L , Keller Susanna R , Gildea John J , Hinkle Josh D , Shabanowitz Jeffrey , Hunt Donald F , Carey Robert M
Published: July 2021

Previous studies from our laboratory have shown that extracellular renal interstitial (RI) cyclic guanosine 3’5’-monophosphate (cGMP) inhibits renal proximal tubule (RPT) sodium reabsorption and induces natriuesis via activation of Src family kinase
in vivo
. The cellular mechanisms by which extracellular cGMP regulates this process are unknown. Previous immunofluorescence competitive binding studies in isolated RPTs from normal rat kidneys demonstrated that cGMP and ouabain (OUA) compete for binding to Na
+
/K
+
-ATPase (NKA). We hypothesized that cGMP binds to the extracellular domain of the α1-subunit of NKA on basolateral membranes of RPT cells thereby inhibiting Na
+
transport. In the present study, we cross-linked cGMP to isolated RPTs from normal rat kidneys (N=6). We incubated RPT cells with 4-N
3
;-PET-8-Biotin-11-cGMP (azido cGMP) or 8-N
3
-6-Biotin-10-cAMP (azido cAMP; both at 2 μM) in the presence or absence of UV light to induce cross linking; azido cAMP served as a negative control. Immunoprecipitation was then performed using strepavidin beads followed by Western blot analysis probing for NKA. RPTs with cross linked azido cGMP exhibited a strong signal for NKA that was significantly weaker for non-cross linked azido cGMP (33.4 ± 6%; P<0.0001) and cross linked (45.8 ± 8%; P<0.001) or non-cross linked (35.2 ± 6%; P<0.001) azido cAMP samples. We further demonstrated that in the presence of OUA (10 μM) NKA was reduced in cross linked azido cGMP RPTs, providing confirmation for previous immunofluorescence competition studies. Azido cGMP cross linked samples (N=3) were subsequently separated in a Tris-HCl gel and stained with Coomassie Blue. The protein band corresponding to NKA was excised and processed for mass spectrometry. NKA was identified as the second most ubiquitous protein in that band with 50 unique peptides for NKA represented covering 47% of all the amino acids in NKA. Together, these data demonstrate that cGMP competes with OUA for binding on NKA and that NKA serves as a receptor for cGMP thereby mediating natriuresis.
Abstract P466: Circadian Rhythm Disruptions in a Novel Diabetic

db/db-mPer2
Luc

Mouse Model
Authors: Hou Tianfei , Su Wen , Gong Ming C , Guo Zhenheng
Published: July 2021

Db/db
mouse, which lacks functional leptin receptor, is an extensively used model of obesity and type 2 diabetes. We and others have demonstrated that
db/db
mouse has disruptions in circadian rhythms of behavior, physiology and some clock genes. However, systemic investigations of the alterations in clock gene oscillations in multiple systems with high time resolution in this model are impeded by the impractical demand for large number of animals. To overcome this limitation, we cross bred the
db/db
mouse with

mPer2
Luc

mouse in which the clock gene
Period2
is fused with a luciferase reporter thus allow real-time monitoring of the clock gene
Per2
oscillations. The generated

db/db-mPer2
Luc

mice had the typical diabetic mellitus including obesity, hyperglycemia, hyperinsulinemia, glucose intolerance and insulin resistance. In addition, the

db/db-mPer2
Luc

mice also exhibited disruptions in circadian rhythms in behavior (locomotor activity), physiology (blood pressure) and metabolism (respiratory exchange ratio and energy expenditure). Using the LumiCycle system, we monitored in real-time of the
Per2
oscillations in both the SCN central clock and multiple peripheral tissues
ex vivo
. The results showed no difference in the phase of the central SCN
Per2
oscillation. However, the peripheral tissues that related to metabolism, such as liver and white adipose clocks, displayed 3.28±0.86 and 4.64±1.06 hours of phase advance respectively. Aorta, mesentery artery and kidney, organs play important role in blood pressure homeostasis, showed 0.99±0.37, and 2.12±0.4, and 2.21±0.5 hours phase advance respectively. Interestingly, no difference was observed in the lung and adrenal gland. We then investigated the
Per2
oscillation
in vivo
by using the IVIS imaging system. Consistent with the
ex vivo
results, the liver
Per2
oscillation were phase advanced in vivo. Our findings demonstrated that clock gene
Per2
oscillations were disrupted in multiple peripheral tissues but not in central SCN. Moreover, the extent of phase advance in peripheral tissue varies largely. Our results suggest dyssynchrony of the clock oscillations among various peripheral systems likely contribute to the multiple disruptions in physiology and metabolism in diabetic
db/db
mice.
Abstract P489: Effect of Continuous Intake of Hesperidin on Blood Pressure in 2-kidney, 1-clip Renovasucular Hypertensive Rats
Authors: Yamaoka Ayuna , Segawa Yukiko , Maruyama Saki , Saito Natsumi , Hashimoto Hiroko , Osera Tomoko , Kurihara Nobutaka
Published: July 2021

Objective:
Hesperidin (HES) is a flavonoid which is contained in citrus fruit peel. It has physiological effects on blood vessels such as strengthening capillary vessels. Thus, it is known to be one of the effective ingredients of herbal medicine. Some studies have shown that the intake of HES decreases blood pressure (BP) in spontaneously hypertensive rats. The antihypertensive effect of HES is suggested to be due to vasodilation by nitric oxide (NO). However, its mechanism has not been clarified in detail. In this study, we observed whether HES intake decreases BP in 2-kidney, 1-clip renovasucular hypertensive rats (2K1C) and evaluated endothelial NO synthase (eNOS) mRNA to investigate its role in the mechanism.

Methods:
Male Sprague-Dawley rats (6 weeks old) were treated with sham operation (SHAM) or clipping the left renal artery (2K1C). After surgery, the rats started receiving continuously a control diet (C) or a diet containing 0.1% (w/w) HES for 6 weeks. The systolic BP (SBP) was measured by a tail-cuff method every week. At the end of the protocol, mean arterial blood pressure (MAP) was measured in each rat under anesthesia. Then, the aortas were removed for extracting mRNA. eNOS mRNA expression was evaluated using real-time RT-PCR.

Results:
At the end of the protocol, SBP in 2K1C-C was significantly higher than in SHAM-C (170±6 vs 117±6 mmHg,
p
<0.001). On the other hand, 2K1C-HES was lower in SBP (141±4 mmHg) than 2K1C-C (
p
<0.01). There were no significant differences between SHAM-HES (122±7 mmHg) and SHAM-C. MAP at the end of the protocol were similar to in SBP. ANOVA revealed mRNA expression of eNOS was significantly higher in 2K1C than in SHAM (
p
<0.05), and showed no significant difference between C and HES, nor a significant interaction.

Conclusion:
Continuous intake of HES may suppress BP increase in 2K1C. The role of eNOS mRNA expression may not be involved in the mechanism.
Abstract P016: Human Mesenchymal Bone Marrow--Derived Stem Cells Require Connexin 43 to Form Beating 3-Dimensional Tubes When Cocultured with Neonatal Rat Cardiomyocytes
Authors: Sanina Cristina , Rodrigues Claudia O , Schulman Ivonne H , Margitich Irene , Balkan Wayne , Hare Joshua M
Published: July 2021

Mesenchymal stem cells (MSCs) have begun to manifest themselves as a safe and beneficial therapy for restoring cardiac function in failing hearts. However, the mechanism underlying this function is unclear. The present study was initiated to investigate the role of Connexin 43 (Cx43) in hMSC integration, migration and differentiation in co-culture with neonatal rat cardiomyocyte (NRVMs) in vitro. Cx43 is a gap junction connexin which is required for proper heart development and heart electrophysiology. We generated lentiviral constructs for Cx43 knockdown and Cx43 overexpression and used them to alter the level of Cx43 in hMSCs. The effectiveness of these vectors was confirmed by assessing Cx43 levels in hMSCs by Western Blot analysis and by Real Time-PCR. These hMSCs were co-cultured with NRVMs for up to 1 month on poly-lysine and collagen I coated glass cover slips. Co-cultures containing MSCs overexpressing Cx43 exhibited coordinated beating and three-dimensional tube formation in 10 days, whereas 14 days were required for control cells. The hMSCs were integrated into these beating three dimensional tubes. No tubes were observed in co-cultures with Cx43 knockdown hMSCs, rather we observed the formation of small, unconnected beating spheres after 30 days in co-culture. Human MSC-Cx43 knockdown cells were integrated into these spheres and immunofluorescence staining demonstrated that Cx43 expression in hMSC-Cx43 knockdown remained reduced when compared to the control group. Real Time-PCR analysis using human specific primers showed significant upregulation of KDR, smooth muscle actin, Pecam1, CD34, CDH2, and CaCNA1C. Several genes, not initially seen in hMSCs, including Gata4, CDH5, SCN5A, SLC8A1, and KCNQ1, appeared in co-cultures with hMSCs with normal and elevated expression levels of Cx43. However, in co-culture of cardiomyocytes with Cx43 knockdown MSCs we observed downregulation of KDR, CHD2, SCN5A, SLC8A1, KCNQ1 compare to control. These results suggest a strong correlation between the presence of Cx43 and the ability of hMSCs to differentiate into cardiac and endothelial lineages.
Abstract P026: Development of Autologous Tissue Small-Diameter Vascular Grafts (BIOTUBEs)
Authors: Yamanami Masashi , Watanabe Taiji , Kanda Keiichi , Oie Tomonori , Takamizawa Keiichi , Ishibashi-Ueda Hatsue , Yaku Hitoshi , Nakayama Yasuhide
Published: July 2021

Objectives:
There are actually no small-caliber synthetic vascular grafts (< 6 mm) with acceptable patency rate for the use of coronary bypass or peripheral vascular repair below the knee in case the autologous vessels are not available. We developed autologous small-caliber vascular grafts “BIOTUBEs” using simple, safe and economical in vivo tissue engineering. In this study, we summarize the development of BIOTUBEs.

Methods:
Silicone rod molds (diameter: 1.5∼5 mm, length: 20∼50 mm) were placed into subcutaneous pouches of Wister rats, Japan white rabbits or Beagle dogs. After 1 month, BIOTUBEs formed around the molds were auto-implanted to the aorta (1.5 mm; rats) or the carotid arteries (2 mm; rabbits and 5 mm; dogs) of the respective animals. They were evaluated after determined period of implantation.

Results:
Irrespective of species, BIOTUBEs had thin wall (ca. 0.1mm) and mainly consisted of autologous fibroblasts and collagen fibers. Rats; After 12-week implantation, other than the oriented endothelial layer and smooth muscle layer, multilayered elastin fiber formation was observed in the grafts. Rabbits; Little thrombus was formed on the luminal surfaces completely covered with endothelial cells within 2 weeks. During 2 year-implantation, neither formation of aneurysms nor rupturing was observed in BIOTUBEs. Dogs; Longest follow up is 3 years under arterial pulsatile pressure condition without any degenerative changes in the grafts.

Conclusions:
BIOTUBEs could be ideal small caliber vascular prostheses that greatly facilitate healing process and exhibit excellent biocompatibility.
Abstract P060: VDAC2 and 3, but Not VDAC1, Are Required for HK2-Mediated Protection Against Cell Death
Authors: McCommis Kyle S , Baines Christopher P
Published: July 2021

The glycolytic enzyme hexokinase-2 (HK2) has been shown to protect several cell types including cardiomyocytes against death. However, the mechanisms of this HK2-mediated protection have not been elucidated. HK2 is normally associated with the mitochondrial voltage-dependent anion channels (VDACs) on the outer mitochondrial membrane, but it is unknown if association with VDACs is required for HK2’s protective functions. In addition, Akt has been shown to phosphorylate HK2, resulting in its translocation to the outer mitochondrial membrane. The purpose of this study was to investigate the role of the three VDAC isoforms, as well as Akt signaling, on HK2-mediated protection. Studies were conducted using cultured murine embryonic fibroblasts (MEFs) from wildtype (WT), VDAC1-/-, and VDAC3-/- mice. WT MEFs were also transfected with a VDAC2 siRNA to reduce VDAC2 protein expression. Murine HK2 was overexpressed by infection with adenovirus. Akt signaling was inhibited by 2 μM Akt Inhibitor VIII. Cell death was induced by 500 μM H2O2 for 4 hours, and cells were stained by Sytox green to identify dead cells and Bis-Benzimide to label all cell nuclei. Protein expression as well as subcellular localization was determined by Western Blot. The HK2 adenovirus resulted in 50-60% increased HK2 expression. In WT MEFs, HK2 overexpression resulted in roughly 50% less death compared to control transfected cells. Surprisingly, although VDAC1 appears to bind the greatest percentage of HK2, cells lacking VDAC1 were still protected by HK2 to a similar degree as WT cells. However, cells with reduced levels of VDAC2, or cells lacking VDAC3 were no longer protected from cell death by HK2. Additionally, HK2 overexpression remained protective in cells treated with the Akt inhibitor. In conclusion, association with the less abundant VDAC2 and VDAC3 isoforms, not the more abundant VDAC1 isoform, appears to be central to HK2-mediated cytoprotection. It also appears that active Akt is not essential to HK2-mediated protection against cell death.
Abstract P098: Inhibitory Role of
Notch1
in Calcific Aortic Valve Disease Is Mediated by Sox9
Authors: Hans Chetan P , Acharya Asha , Koenig Sara N , Nichols Haley A , Galindo Cristi L , Garner Harold R , Merrill Walter H , Hinton Robert B , Garg Vidu
Published: July 2021

Introduction:
Aortic valve calcification is the most common form of valvular heart disease; however the mechanism(s) underlying calcific aortic valve disease (CAVD) are unknown.
NOTCH1
mutations are associated with aortic valve malformations and adult-onset calcification in families with inherited disease. The Notch signaling pathway is critical for multiple cell differentiation processes, but its role in the development of CAVD is not well understood.

Objective:
To investigate the molecular changes associated with the calcification of aortic valve that occurs with inhibition of Notch signaling.

Methods and Results:
The expression of Notch signaling pathway members was validated in the aortic valve cusps from adult mice, and examination of diseased human aortic valves revealed decreased expression of NOTCH1 in areas of calcium deposition. To identify downstream mediators of Notch1 signaling, we examined gene expression changes that occur with chemical inhibition of Notch signaling in rat aortic valve interstitial cells (AVICs). We found significant downregulation of many cartilage-specific genes that constitute the valve extracellular matrix (ECM). Analysis of these cartilage-specific genes demonstrated that several were transcriptional targets of Sox9, a master regulator of chondrogenesis, which has been previously shown to be essential for proper valve development and maintenance. Utilizing an
in vitro
porcine aortic valve calcification model system, inhibition of Notch activity resulted in accelerated calcification while stimulation of Notch signaling attenuated the calcific process. Finally, utilizing transfection studies, addition of Sox9 was able to prevent the calcification of porcine AVICs that occurs with Notch inhibition.

Conclusions:
Loss of Notch signaling contributes to aortic valve calcification by a Sox9-dependent mechanism. Further elucidation of the Notch1-Sox9 molecular pathway and its role in the maintenance of the ECM will lead to an improved mechanistic understanding of aortic valve calcification and development of novel therapeutic strategies for CAVD.
Abstract P154: Focal Adhesion Kinase Plays a Critical Role in Upregulating Fibrogenesis in Load-Induced Cardiac Hypertrophy
Authors: Dalla Costa Ana Paula , Clemente Carolina F , Theizen Thais H , Souza José Roberto , Cardoso Leandro , Franchini Kleber G
Published: July 2021

Myocardial fibrosis is maladaptive, accelerating the evolution of diseased hearts to failure. The pathogenesis of myocardial fibrosis is critically dependent on complex processes of activation (i.e. enhanced proliferation, production and secretion of soluble factors, collagen and matrix metalloproteinases) and terminal differentiation of cardiac fibroblasts into myofibroblasts, resultant from the mobilization of numerous signaling molecules by physical and humoral stimuli. Noting that Focal Adhesion Kinase (FAK) is activated in areas of ongoing myocardial fibrosis, we sought to examine whether it is a critical mediator of fibrogenesis in load-induced hypertrophic hearts. Isolated fibroblasts from hypertrophic hearts of mice subjected to transverse aortic constriction (TAC; 1 to 8 weeks) were highly activated as recognized by markers that indicate enhanced proliferation (nuclear Ki67), production of collagen and matrix metalloproteinase-2 (MMP-2) and differentiation into myofibroblasts (expression of α-smooth muscle actin - α-SMA). In these cells, FAK was upregulated, as also were its dowstream pathways Src/ERK1/2 and PI3K/AKT/mTOR. Depletion of FAK (∼80%) after treatment with small interfering RNA (siRNA-FAK) markedly attenuated cardiac hypertrophy and fibrosis, and significantly reduced the number of activated fibroblasts harvested from overloaded hearts. Restoration of FAK function by overexpressing a full-length FAK construct in these cells, selectively enhanced the activity of the downstream PI3K/AKT/mTOR and rescued the activated phenotype of fibroblasts. Transfection with an inactive FAK mutant (Tyr397 substituted by phenylalanine) did not rescue the activated phenotype of fibroblasts harvested from overloaded hearts depleted of FAK. However, cells harvested from overloaded hearts depleted of FAK and treated with the mTOR activating aminoacid leucine showed typical phenotype of activated fibroblasts. These findings uncover a role for FAK in regulating the signaling cascade PI3K/AKT/mTOR in cardiac fibroblasts, which seems to be critical for the pathogenesis of myocardial fibrosis in hypertrophic hearts. Targeting this pathway may provide a novel strategy for treating hypertrophic heart diseases.
Abstract P158: MicroRNA-22 Modulates Cardiac Gene Expression and Controls Compensation to Hemodynamic Stress in Mice
Authors: Gurha Priyatansh , Kelm Robert , Entman Mark , Taffet George , Bradley Allan , Rodriguez Antony
Published: July 2021

Recent evidence suggests that miRNAs play an important role in cardiac morphogenesis and pathophyiology of heart failure. To explore the role of miR-22 in the mouse heart physiology, we generated miR-22 null (KO) mice. Although, miR-22 KO mice showed normal cardiac structure and function at baseline, these mice are sensitized to maladaptive remodeling (cardiac dilation) and decompensation in response to pressure overload by transverse aortic constrictions (TAC) stimulation. Genome-wide molecular analysis of KO hearts revealed attenuated expression of numerous CarG-dependent genes encoding proteins that reside at the sarcomeric Z-disc (including Myh7, Acta1, Mlp, Melusin, MyoZ2) indicating that miR-22 is required for optimum muscle gene expression. Alterations in sarcomeric gene expression is especially interesting as this suggests a primary role of miR-22 in controlling cardiac contractility and adaptation to stress. Targetomics analysis revealed that mechanistically this effect could be modulated in part by miR-22 target PURB (Purine Rich element binding protein B), a transcriptional/translational repressor.

In conclusion we define a critical role of miR-22 in cardiac adaptation to hemodynamic stress. Furthermore, these data provides a previously unseen essential molecular mechanism that underlies homeostatic control of sarcomeric protein expression in the heart.
Abstract P222: T-Type Calcium Channels Regulate NFAT Signaling in Cardiomyocytes and Are Expressed in Chronic Hypoxic Right Ventricle
Authors: Cribbs Leanne L , Pluteanu Florentina
Published: July 2021

Alterations in intracellular calcium (Ca
2+
) have a significant impact on Ca
2+
signaling, an integral part of the cardiac response to pathological conditions. The calcineurin/NFAT signaling pathway is a well established Ca
2+
-dependent mechanism for transcriptional regulation of ventricular remodeling target genes. Although low voltage-activated T-type Ca
2+
channels are not normally present in the adult myocardium, they reappear in conditions of hypertrophy and heart failure, where their functions remain poorly understood. To further investigate T-type Ca
2+
channels, we have employed cultured neonatal rat ventricular myocytes (NRVM) and rat models of pulmonary hypertension that lead to right ventricular (RV) hypertrophy and heart failure. To assay for NFAT activation in cardiomyocytes, NRVM were infected with NFATc1-GFP and nuclear localization of GFP was measured. NFAT activation was markedly stimulated by over expressing the human Ca
v
3.2 T-type Ca
2+
channel cDNA in NRVM, and this activation was inhibited by cyclosporin A (a calcineurin inhibitor), by T-type selective concentrations of mibefradil and NiCl
2
, and in the absence of extracellular Ca
2+
. To investigate the up regulation of cardiac T-type Ca
2+
channels
in vivo
, rats were maintained in chronic hypoxia, where they developed significantly elevated RV systolic pressure, RV hypertrophy and reduced cardiac output. Electrophysiological measurements revealed elevated membrane capacitance in RV myocytes indicative of RV hypertrophy, and T-type current density was also increased. Myocytes isolated from the RV of pulmonary hypertensive rats that displayed elevated T-type currents and RV dysfunction, also showed impaired contractility with decreased sensitivity to Ca
2+
. The results in NRVM suggest that T-type channels contribute to hypertrophic signaling via their interaction with calcineurin/NFAT signaling intermediaries. Furthermore, T-type channels are expressed in the RV during pulmonary hypertension where they influence RV myocyte contractility. Thus, when expressed in pathological conditions, T-type Ca
2+
channels may play a dual role as a signaling intermediary, as well as a modifier of ventricular contractile function.
Abstract P241: Calpain-1 Is Increased in Mitochondria and Contributes to Mitochondrial ROS Generation in High Glucose--Stimulated Endothelial Cells and Endothelial Dysfunction in Mouse Models of Diabetes
Authors: Zhao Qing , Tang Futian , Shan Limei , Cepinskas Inga , Cepinskas Gedas , Peng Tianqing
Published: July 2021

Objectives:
Elevated levels of reactive oxygen species (ROS) are the initial source of endothelial dysfunction in diabetes. Calpain has been implicated in diabetic vascular complications. The present study was to investigate the role of calpain in mitochondrial ROS generation in endothelial cells and vascular dysfunction in diabetic mice.

Methods:
Endothelial cells cultured from human umbilical vein (HUVEC) were stimulated with high glucose. Calpain activity and protein were determined in mitochondria of HUVEC. Intracellular and mitochondrial ROS generation as well as apoptosis were measured. Type I diabetic OVE 26 mice and type II diabetic db/db mice with calpastatin over-expression (OVE26/CAST and db/db-CAST) were generated, respectively. Type I diabetes was also induced in both wild-type and Tg-CAST mice by injection of streptozocin (STZ). The endothelium-dependent relaxation of aortic ring was measured.

Results:
High glucose significantly increased calpain-1 protein, calpain activity and ROS generation in mitochondria of HUVEC. Pharmacological inhibition of calpain or over-expression of calpastatin abrogated high glucose-induced intracellular ROS production, mitochondrial ROS generation and apoptosis in HUVEC. Incubation of isolated mitochondria with calpain-1 protein significantly induced its ROS generation and the membrane potential. In diabetic mice, calpain activity was induced in aortic vessels, which correlated with an increase in ROS production and protein tyrosine nitration. Over-expression of calpastatin prevented calpain activity, reduced ROS production and inhibited protein tyrosine nitration in diabetic mice. Aortic ring segments from diabetic mice exhibited a significant reduction in vascular relaxation to acetylcholine, which was reversed by over-expression of calpastatin in Tg-CAST, OVE26/CAST and db/db-CAST mice.

Conclusions:
This study has demonstrated a novel role of calpain in mitochondrial ROS generation, which contributes to apoptosis in endothelial cells during hyperglycemia. Thus, over-expression of calpastatin inhibits reduces ROS production and ameliorates endothelium-dependent vascular dysfunction in mouse models of diabetes.
Abstract P279: YAP, a Transcriptional Cofactor of the Hippo Pathway, Regulates Cardiomyocyte Growth, Survival, and Proliferation
Authors: Yang Yanfei , Nakano Noritsugu , Sadoshima Junichi
Published: July 2021

Mst1 and Lats2, components of the mammalian Hippo pathway, stimulate apoptosis and inhibit hypertrophy of cardiomyocytes (CMs), thereby mediating reperfusion injury and heart failure. YAP, a transcription factor co-factor, is negatively regulated by the Hippo pathway, and controls cell survival, proliferation and tissue regeneration. The role of YAP in regulating growth and death of CMs is poorly understood. YAP overexpression in CMs induced cardiac hypertrophy, as indicated by increases in cell size (+1.2 fold, p<0.01), protein content (+1.1 fold, p<0.01) and ANF (luciferase reporter activity +1.7 fold, mRNA +2.2 fold, and staining +2.7 fold, p<0.01). Lats2 phosphorylates YAP at Serine 127, which induces cytoplasmic translocation of YAP, whereas YAP(S127A) is localized constitutively in the nucleus. Expression of YAP(S127A) enhanced hypertrophy in cultured CMs compared to that of wild type YAP (+1.87 fold ANF staining, p<0.05), suggesting that the Mst1/Hippo pathway negatively regulates cardiac hypertrophy through YAP. YAP inhibited cell death induced by H2O2 treatment, as evaluated with TUNEL staining (-65%, p<0.05) and CellTiter Blue assays (+34.9%, p<0.01), indicating that YAP plays an essential role in mediating CM survival. Interestingly, YAP also significantly increased Ki67 positive cells in cultured CMs compared to LacZ (+2.65 fold, p<0.05). We used a mouse model of chronic myocardial infarction (MI) to evaluate the function of YAP in the heart in vivo. Although YAP is diffusely localized both in the nucleus and cytosol in CMs in control hearts, CMs in the border zone of MI exhibited nuclear localization of YAP whereas YAP was excluded from the nucleus in CMs in the remodeling area four days after MI (+6.52 fold and +1.28 fold). Some of the YAP positive CMs in the border zone exhibited positive co-staining with Ki67, suggesting that YAP potentially induces CM proliferation. A significant increase in nuclear YAP and Ki67 positive CMs (+2.95 fold, p<0.01 and +2.18 fold, p<0.05) was also observed in neonatal rat hearts whose apex was surgically resected three days before euthanasia. These results suggest that YAP plays an important role in mediating not only hypertrophy and survival, but also proliferation of CMs in response to myocardial injury.
Abstract P281: Bcl-2 Limits Ischemia-Reperfusion Injury of the Naive Myocardium by Preserving Mitochondrial Integrity and Respiratory Complex Functionality and Is Modulated by PKCε
Authors: Liem David A , Zhang Jun , Lotz Christopher , Wang Ding , Ping Peipei
Published: July 2021

Over-expression of Bcl-2 protects against myocardial ischemia/reperfusion (I/R) injury. Nevertheless, the participation of Bcl-2 in
basal
myocardium, and its subcellular targets under such conditions, remains elusive. Using a mouse line with an ablation of the Bcl-2 gene, we found that myocardial infarct size (IS) was exacerbated vs. wild type (WT) mice, demonstrating that Bcl-2 limit IS in
basal
I/R injury. The exacerbated IS in Bcl-2 KO was abolished by in vivo treatment with the selective Mitochondrial Permeability Transition (MPT) inhibitor cyclosporine A (10 mg/kg, iv) , while isolated cardiac mitochondria from Bcl-2 null mice exhibited increased matrix swelling in response to CaCl
2
, showing an increased susceptibility to MPT. However, recombinant Bcl-2 or PKCε were both sufficient to attenuate the increased susceptibility to MPT. Interestingly, spectrophotometric analysis of baseline activities of Mitochondrial Electron Transport Chain Complexes (ETC) I and V (but not of ETC II, III and IV), were increased in cardiac mitochondria from Bcl-2 null mice as compared to mitochondria from normal WT mice, demonstrating an altered mitochondrial respiratory complex functionality. In addition, immunoprecipitation with PKCε in AE-PKCε mouse hearts (i.e. mice with an increased activity of PKCε) followed by immunoblotting for Bcl-2, showed an 2-fold increased interaction between PKCε and Bcl-2. Similarly, immunoprecipitation with Phospho-Serine followed by immunoblotting for Bcl-2 indicated a 2-fold increased Serine residue phosphorylation of Bcl-2. Mass spectrometry analysis further showed that PKCε can phosporylate Bcl-2 at its Serine24 residue site in vitro, indicating that PKCε can directly interact and phosphorylate Bcl-2. These data suggest that Bcl-2 is pivotal in limiting IS in basal I/R injury by counteracting MPT and preserving mitochondrial respiratory complex functionality, and implicate a direct interaction and phosphorylation of Bcl-2 by PKCε in this process.
Abstract P295: The Inflammasome Is Involved in Myocardial Ischemia-Reperfusion Injury
Authors: Takahashi Masafumi , Kawaguchi Masanori , Usui Fumitake , Kimura Hiroaki , Taniguchi Shun'ichiro , Ikeda Uichi
Published: July 2021

Background:
Accumulating evidence indicates that inflammation is involved in the pathophysiology of myocardial ischemia-reperfusion (I/R) injury. However, the mechanism of I/R-initiated inflammation remains to be determined. The inflammasome is a multiprotein complex consisting of nod-like receptor (NLR), apoptosis-associated speck-like adaptor protein (ASC), and caspase-1, and regulates caspase-1-dependent maturation of IL-1beta and IL-18. In the present study, we investigated the role of inflammasome in myocardial I/R injury.

Methods and Results:
Wild-type (WT), ASC−/−, and caspase-1−/− mice were subjected to 30 min LAD ligation, followed by reperfusion. ASC and caspase-1 were expressed at the site of myocardial I/R injury. Deficiency of ASC and caspase-1 reduced inflammatory responses, such as inflammatory cell infiltration and cytokine expression, and subsequent injuries such as infarct development, myocardial fibrosis, and dysfunction in myocardial I/R injury. To determine the contribution of inflammasome in bone marrow cells, we produced bone marrow transplant mice and found that inflammasome activation was critical not only in bone marrow cells but also in myocardial resident cells. Since myocardial damage was observed before the inflammatory cell infiltration after I/R, we hypothesized that myocardial resident cells are responsible for an initial activation of inflammasome. To test this hypothesis, we examined whether hypoxia/reoxygenation (H/R) stimuli could induce inflammasome activation in cardiac fibroblasts and cardiomyocytes in vitro. Interestingly, inflammasome activation was detected only in cardiac fibroblasts, but not in cardiomyocytes, and mediated through reactive oxygen species (ROS) and potassium efflux.

Conclusion:
These findings indicate that inflammasome activation in cardiac fibroblasts is essential for inflammation and injury after myocardial I/R, and suggest that the inflammasome is a potential novel therapeutic target for myocardial I/R injury.
Abstract P327: COPa Library: A Proteomic Knowledge Base for Cardiovascular Biology and Medicine
Authors: Zong Nobel , Li Haomin , Deng Ning , Chen Lingxuan , Wang Yueju , Fang Caiyun , Jimenez Rafael , Xu Tao , Hermjakob Henning , Duan Huilong , Yates John , Apweiler Rolf , Ping Peipei
Published: July 2021

We constructed the Cardiac Organellar Peptide Atlas Library (COPa library) as a targeted and interactive resource to the cardiovascular community. Annotated peptide spectra are hosted using a relational database in a modular fashion based on species (e.g. human, mouse) and organelles (e.g. mitochondria, proteasome). Within this release of COPa library, a total of 108,268 spectra have been disseminated via two avenues. A web portal was established to navigate the library via parallel set of identifiers, such as protein name, accession number, gene symbol, etc. In parallel, a web-service cyber-infrastructure was engineered to aid the annotation of mass spectra submitted via internet. The large raw spectra files are dissected into small data packages at the local PC before submission. This workflow surpasses the limitation of network bandwidth, as well as enables parallel data submission and search. A benchmark test with 897,327 ms/ms spectra showed the library searching covers 93.4% of proteins identified via database searching, as well as additional 23.9% of proteins at the same level of statistical confidence. In addition, a wiki-like web interface was embedded in the library web portal in order to facilitate the synthesis of consensus knowledge among the cardiovascular community on innovations of functional proteomics. Overall, the COPa library search supports targeted proteomic characterization, which complements database search for exploratory survey.

The implementation of the COPa library-based proteomic knowledgebase leverages state-of-the-art technology and annotated datasets among the research community at large. Its application bridges discovery-driven and hypothesis-driven research while fostering translational medicine.
Abstract P342: Mitochondria-Targeted Antioxidant Therapy Prevents Angiotensin II--Mediated Connexin43 Remodeling and Sudden Arrhythmic Death
Authors: Sovari Ali A , Zandieh Shadi , Iravanian Shahriar , Gu Lianzhi , Jeong Euy-Myong , Bonini Marcelo G , Dudley Samuel C
Published: July 2021

Introduction:
Angiotensin II activation and associated elevation in ROS have been implicated in pathogenesis of arrhythmia. Nevertheless commonly used antioxidants have been ineffective in clinical trials. We created a transgenic mouse model of cardiac restricted overexpression of ACE (ACE8/8). These mice show spontaneous VT/ VF, SCD, and a reduction in Cx43 level, which impairs conduction and predisposes to arrhythmia. We sought to determine the role and the major source of ROS by angiotensin II in VT/ VF and Cx43 remodeling.

Method:
Wild type and ACE8/8 mice with and without 2 weeks of treatment with a NOS inhibitor (L-NIO, 25mg/Kg IP injections daily), a mitochondria-targeted antioxidant (Mito-TEMPO, 0.7mg/Kg IP injections daily), a NADPH oxidase inhibitor (Apocynin 80mg/L in drinking water), and ACE8/8 crossed with P67DN were studied. Western blotting (with derivatization to dinitrophenylhydrozone to detect oxidized protein levels), detection of superoxide production in mitochondria by red mitochondrial superoxide indicator and immunohistochemistry staining for Cx43 were performed. EP study was performed by a 1.1F octapolar catheter through pacing the right ventricle using a burst pacing protocol.

Results:
Proteins were more oxidized (increased protein-carbonyl detection), and Cx43 was reduced in ACE8/8 to 33% of control. Treatment with Mito-TEMPO prevented SCD and improved survival in ACE8/8 mice (p=0.0005, hazard ratio 4.76 with 95% CI of 1.96 to 11.53). Inducibility of VT/VF was higher in ACE8/8 mice compare to WT (87.5% vs. 2.3%) and VT inducibility was reduced with Mito-TEMPO treatment (50% in treatment group). Cx43 level was increased by 1.7 fold with Mito-TEMPO treatment. Treatments with L-NIO, Apocynin and crossing with P67DN mice did not prevent VT/VF and SCD in ACE8/8 mice.

Conclusion:
In a model of angiotensin II activation mitochondria-targeted antioxidant, prevents VT/VF/SCD and Cx43 remodeling. Suppression of NADPH oxidase activity by Apocynin and crossing the ACE8/8 mice with P67DN or inhibition of NOS by L-NIO did not prevent the arrhythmic deaths in ACE8/8 mice. This result suggests that mitochondria are the major source of ROS by angiotensin II and mitochondria-targeted antioxidants may be effective antiarrhythmic drugs.
Abstract 105: The Therapeutic Efficacy of Embryonic Stem Cell-Derived Exosomes for Postinfarction Myocardial Repair and Regeneration
Authors: Lambers Erin , Ramirez Veronica , Mackie Alex , Misener Sol , Hoxha Eneda , Krishnamurthy Prasanna , Verma Suresh , Thal Melissa , Gupta Raj , Kishore Raj
Published: July 2021

Although embryonic stem cells (ESCs) hold great promise for regeneration of the heart post myocardial infarction (MI) due to their pluripotent potential, it is this same potential that poses risk for the formation of teratomas. Lately there have been reports that exosomes, 30-100nm membrane bound vesicles, are not simply used by the cell to exocytose unwanted material, but contain intact protein, mRNA, and miRNA important for intercellular communication. Here we hypothesize that ESC derived exosomes can be used to exploit an already established mechanism to shuttle ESC content intercellularly in a cell free system for physiological and anatomical repair of the myocardium following acute myocardial infarction. To test our hypothesis exosomes were isolated by ultracentrifugation from equal numbers of murine ESCs or primary embryonic fibroblasts (MEFs). ESC exosomes had intact pluripotent transcripts similar to their parent cells in contrast the MEF exosomes. Using a double blinded acute myocardial infarction model, immediately after permanent ligation of the coronary artery, mice were injected with saline, ESC exosomes, or MEF exosomes at 3 locations in the border-zone of the left ventricle (LV). To assess the functional recovery of the LV, echocardiographical analysis was performed at Day 7, 14, and 28 following the AMI. Mice were sacrificed at D28 for histological assessments. LV fractional shortening, ejection fraction, and end systolic diameter measurements demonstrate that mice treated with ESC exosomes have improved LV function compared to mice treated with control MEF exosomes or saline alone (P<.05 for all functional parameters). Mice treated with ESC exosomes show less infarct size and apoptosis, greater capillary density, and greater cycling of both cardiomyocytes and ckit+ stem cells. Taken together, these data demonstrate a novel cell free system in which ESC exosomes can exploit regenerative capabilities of ESCs while bypassing the risk of teratoma formation and hold great promise for cardiovascular regenerative medicine.