bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 282: Adropin Enhancement of Cardiac Insulin Sensitivity and Inhibition of Fatty Acid Oxidation are Associated With Improvement of Cardiac Function and Efficiency in Hearts From Fasted Mice
Authors: Altamimi Tariq R , Fukushima Arata , Zhang Liyan , Gao Su , Gupta Abhishek , Lopaschuk Gary D
Published: June 2021

Impaired cardiac insulin signaling and high cardiac fatty acid oxidation rates are characteristics of diabetic cardiomyopathy. Potential roles for liver-derived metabolic factors in mediating cardiac energy homeostasis are underappreciated. Plasma levels of adropin, a liver secreted peptide, increase during feeding and decrease during fasting and diabetes. In skeletal muscle, adropin preferentially promotes glucose over fatty acid oxidation. We therefore determined what effect adropin has on cardiac energy metabolism, insulin signaling and cardiac efficiency. C57Bl/6 mice were fasted to accentuate the differences in adropin plasma levels between animals injected 3 times over 24 hr with either vehicle or adropin (450 nmol/kg i.p.). Despite fasting-induced predominance of fatty acid oxidation measured in isolated working hearts, insulin inhibition of fatty acid oxidation was re-established in adropin-treated mice (from 1022±143 to 517±56 nmol. g dry wt
-1
. min
-1
, p <0.05) compared to vehicle-treated mice (from 757±104 to 818±103 nmol. g dry wt
-1
. min
-1
). Adropin-treated mice hearts showed higher cardiac work over the course of perfusion (p<0.05 vs. vehicle), which was accompanied by improved cardiac efficiency and enhanced phosphorylation of insulin signaling enzymes (tyrosine-IRS-1, AS160, p<0.05). Acute addition of adropin (2nM) to isolated working hearts from non-fasting mice showed a robust stimulation of glucose oxidation compared to vehicle-treated hearts (3025±401 vs 1708±292 nmol. g dry wt
-1
. min
-1
, p<0.05, respectively) with a corresponding inhibition of palmitate oxidation (325±61 vs 731±160 nmol. g dry wt
-1
. min
-1
, p<0.05, respectively), even in the presence of insulin. Acute adropin addition to hearts also increased IRS-1 tyrosine-phosphorylation as well as Akt, and GSK3β phosphorylation (p<0.05), suggesting acute receptor- and/or post-translational modification-mediated mechanisms. These results suggest adropin as a putative candidate for the treatment of diabetic cardiomyopathy.
Abstract 284: Ovariectomy Exacerbates Mitochondrial Dysfunction, Oxidative Stress and Fibrosis to Cause Diastolic Dysfunction
Authors: Hamilton Dale J , Li Shumin , Zhang Aijun , Vedula Indira , Smith Jessie A , Gupte Anisha A
Published: June 2021

Heart failure with preserved ejection fraction, characterized by diastolic dysfunction (DD) is most common in post-menopausal women, suggesting a link with estrogen (E2). However, the underlying mechanisms are not well understood. In this study we tested how ovariectomy (OVX)-induced E2-deficiency exacerbates DD.

Female C57BL6/J mice (12 weeks old) underwent a sham or OVX surgery, and were administered either normal drinking water or L-N
G
-Nitroarginine methyl ester (L-NAME, 0.3 mg/ml in 1% NaCl solution) in the drinking water and Angiotensin II (AngII, 1.2 mg/kg/day) via subcutaneous osmotic pumps for 5 weeks to induce DD. Subsets of the OVX-DD mice were treated with E2, E2-receptor (ER) α agonist PPT, ERβ agonist DPN or G-protein ER-1 agonist G1.

Echocardiography revealed reduced left ventricular (LV) internal diameter, LV volume, cardiac output but increased LV posterior wall thickness in OVX-DD mice compared with sham-DD mice. However, ejection fractions and fractional shortening were normal in all groups. Doppler studies showed E/A ratios dramatically suppressed and E/e’ ratios increased in mice treated with L-NAME+AngII, with larger changes in OVX mice.

Extensive fibrosis was seen in OVX-DD hearts than sham-DD hearts. ADP-supported cardiac mitochondrial function with pyruvate-malate was moderately reduced in OVX-DD mice along with greater production of reactive oxygen species. Ionoptix studies revealed delayed relaxation but maintained contraction in cardiomyocytes isolated from OVX-DD mice, similar to effects of low-dose oligomycin in control mice. These data suggest that E2 deficiency is associated with impaired mitochondrial function to cause small decreases in ATP formation, which impair diastolic relaxation but not contraction. Treatment with E2, PPT, DPN and G1 reduced E/e’ ratios in OVX-DD mice, with the greatest effects of G1. Treatment of mice with L-NAME in this model excludes the previously described action of G1 through nitric oxide.

In conclusion, OVX exacerbates mitochondrial dysfunction, oxidative stress and fibrosis in non-ischemic HF, leading to exacerbated DD. These may be some of the mechanisms by which E2 protects the myocardium in females. Activation of GPER1 offers a novel therapeutic target for DD after menopause.
Abstract 289: Transcriptomic Analysis of Inter- and Intra-patient Variation in Human iPSCs: Platform for Precision Medicine to Predict Drug Toxicity
Authors: Matsa Elena , Burridge Paul W , Yu Kun-Hsing , Wu Haodi , Termglinchan Vittavat , Liu Chun , Shukla Praveen , Sayed Nazish , Karakikes Ioannis , Snyder Michael P , Wu Joseph C
Published: June 2021

Rapid improvements in human induced pluripotent stem cell (hiPSC) differentiation methodologies have allowed previously unattainable access to high-purity, patient-specific cardiomyocytes (CMs) for use in disease modeling, cardiac regeneration, and drug testing. In the present study, we investigate the ability of hiPSC-derived cardiomyocytes (hiPSC-CMs) to reflect the donor’s genetic identity and serve as preclinical functional readout platforms for precision medicine. We used footprint-free Sendai virus to create two separate hiPSC clones from the fibroblasts of five different individuals lacking known mutations associated with cardiovascular disease. Whole genome expression profiling of hiPSC-CMs showed that inter-patient variation was greater than intra-patient variation, thereby verifying that reprogramming and cardiac differentiation technologies can preserve patient-specific gene expression signatures. Gene ontologies (GOs) accounting for inter-patient variation were mostly metabolic or epigenetic. Toxicology analysis based on gene expression profiles predicted patient-specific susceptibility of hiPSC-CMs to cardiotoxicity, and functional assays using drugs targeting key regulators in pathways predicted to produce cardiotoxicity showed inter-patient differential responses in hiPSC-CMs. Our data suggest that hiPSC-CMs can be used
in vitro
to predict and help prevent patient-specific drug-induced cardiotoxicity, potentially enabling personalized patient consultation in the future.
Abstract 295: Importance of the Rate of Platelet Activation by Thrombin for Regulating the Size of Thrombi Formed Under Low Blood Flow Velocity Conditions
Authors: Goto Shinichi , Ayabe Kengo , Kawamura Youth , Tamura Noriko , Goto Shinya
Published: June 2021

Background:
Platelets, coagulation cascade including thrombin and fibrin, and fibrinolysis by plasmin along with blood flow are known to play regulatory roles in thrombogenesis. However, quantitative and interactive contribution of these factors are not fully understood.

Method:
We developed a computer simulation model of thrombi at sites of vessel injury by implementing quantitative parameters of blood flow, platelet, coagulation cascade and fibrinolysis. In this model, we defined thrombi as the area where the amount of activated platelet become 80% or more of the global platelet count of 3.0х10
5
/mm
3
upon vessel damage We have measured the 3-dimensional size of thrombi as defined in various conditions with various parameters of flow velocity, rate of platelet activation by thrombin, rate of thrombin production on the surface of activated platelets, rate of fibrinolysis and rate of plasmin production.

Results:
Rate of platelet activation by thrombin had the largest influence on the size of thrombi under low flow rate condition of 0.5 (cm/sec) (Fig1 A). When blood flow rate increased to 2.0 (cm/sec), the absolute importance of this parameter decreased (Fig1 B).

Conclusion:
Our results show that the rate of platelet activation by thrombin, which are signaled with PAR-1 receptor in human, has a marked effect on the size of thrombi in low flow rate condition suggesting the benefit of blocking this receptor in low blood flow condition such as venous thrombosis.
Abstract 299: Natural Product Derivative Bio Promotes Recovery After Myocardial Infarction via Unique Modulation of the Cardiac Microenvironment
Authors: Kim Yong Sook , Kang Wan Seok , Kang Ju Hee , Ahn Youngkeun
Published: June 2021

Rationale:
Myocardial infarction (MI) induces cardiac remodeling, which is regulated by the cardiac microenvironment and results in scarring and loss of cardiac function. Targeting the microenvironment represents a novel therapeutic approach.

Objective:
To investigate the therapeutic effect of a natural compound derivative, BIO [(2’Z,3’E)-6-Bromoindirubin-3′-oxime], on cardiac microenvironment cells and remodeling post-MI.

Methods and Results:
Using a series of co-culture studies, BIO was shown to induce proliferation in cardiomyocytes and, conversely, inhibit proliferation in cardiac fibroblasts. In addition, BIO produced anti-fibrotic effects in the fibroblasts, such as reduced motility and expression changes in fibrosis-related factors. In macrophages, BIO inhibited the expression of pro-inflammatory inducible nitric oxide synthase. Interestingly, BIO modulated molecular crosstalk between cardiac fibroblasts and differentiating macrophages to increase expression of anti-inflammatory M2 macrophage markers. In the optically transparent zebrafish-based model of heart failure, BIO induced cardiomyocyte proliferation to recover survival rate. BIO is a known glycogen synthase kinase-3β inhibitor, but these effects could not be recapitulated using the classical inhibitor, lithium chloride; indicating novel, potential therapeutic effects of BIO on remodeling. We characterized these novel effects of BIO as differential modulation of p27 expression and potent induction of interleukin-10 in microenvironment cells. In rat MI model, BIO reduced fibrosis and improved cardiac performance. Histological analysis revealed a greater presence of anti-inflammatory M2 macrophages in the infarction zone. BIO treatment also reduced serum levels of pro-fibrotic interleukin-6.

Conclusions:
BIO differentially activates signaling pathways in cardiomyocytes, cardiac fibroblasts and cardiac macrophages to reduce fibrosis, increase cardiomyocyte proliferation and promote recovery post-MI.
Abstract 323: Loss of Gravity Impairs Cardiac Neural Crest Cell Lineage Development and Function
Authors: Hatzistergos Konstantinos E , Valasaki Krystalenia , Jiang Zhijie , Takeuchi Lauro M , Balkan Wayne , Saur Dieter , Seidler Barbara , DiFede Darcy L , Hare Joshua M
Published: June 2021

Introduction:
A multitude of structural, haemodynamic and electromechanical cardiovascular disorders have been observed in humans following space-travel. These abnormalities are thought to emerge from transient alterations in autonomic nervous system (ANS). However, since the ANS is cardiac neural crest (CNC)-derived, whether microgravity-induced cardiomyopathies reflect CNC dysfunction, is unknown.

Hypothesis:
Impairment of CNCs underlies microgravity-induced cardiomyopathies.

Methods:
Myocardial explants from adult

cKit
CreERT2/+
;IRG

mice (n=5/group), as well as

cKit
CreERT2/+
;IRG-

derived (iPSC

Kit-Cre

; n=6/group) and
Wnt1-Cre;tdTomato
-derived (iPSC

Wnt1-Cre

; n=18/group) induced pluripotent stem cells, were cultured under static (SC) or simulated microgravity conditions (rotary cell-culture system; RCCS).

Results:
CNC lineage-tracing in cardiac explants illustrated that, compared to SC, RCCS abolished the pool of
cKit
+
CNCs in adult hearts, indicated by quantitation of

cKit
CreERT2
-

mediated EGFP expression (
p
<0.05). Cardiogenesis modeling experiments with iPSC

Kit-Cre

yielded fewer beating EBs (
p
=0.0005), and ~10-fold reduction in EGFP
+
cardiomyocytes (
p
=0.01), in RCCS
vs
. SC. Microarray analyses suggested that RCCS-mediated alterations in BMP and Wnt/β-catenin pathways, downregulated ANS and CNC-related gene programs, and enhanced vasculogenic differentiation without affecting the expression of cardiac mesoderm-related genes. Differences were verified by quantitative PCR. Modeling CNC development in iPSC

Wnt1-Cre

further confirmed an RCCS-mediated dramatic impairment in development and function of CNCs, indicated by quantitation of tdTomato expression in day-10 and day-21 beating embryoid bodies (
p
<0.0001). Intriguingly, the effect of RCCS in CNCs could be only partially rescued upon transfer to SC.

Conclusions:
Together these data indicate that microgravity negatively regulates the development and function of CNCs, thus partly explaining the cellular and molecular mechanisms of microgravity-induced cardiomyopathies. Moreover, these findings are expected to have important implications in space exploration, since they suggest an essential role for gravity in vertebrate development.
Abstract 36: Application of “Omics” to Assess the Inter Passage Variability in the Immunoprivilege of Mesenchymal Stem Cells
Authors: Sequiera Glen Lester , Sareen Niketa , Sharma Vikram , Chaudhary Rakesh , Abu Al-Rub Ejlal , Moudgil Meenal , Ravandi Amir , Dhingra Sanjiv
Published: June 2021

Introduction:
Bone marrow derived allogeneic mesenchymal stem cells (MSCs) from young healthy donors are immunoprivileged. However recent data from preclinical and clinical studies indicate that allogeneic MSCs provoke immune response in the recipient heart and are rejected after transplantation. We recently observed that stem cell surface oxidized phospholipids (ox-PCs) modulate allogeneic immune cell response. In most of the allogeneic MSCs based clinical trials in cardiac patients, cells from a single donor were expanded in cultures and transplanted. However, the effect of increase in the passage number on stem cell membrane lipidome and its association with immunogenicity of MSCs has not been investigated yet, the present study was carried out to address this

Methods:
MSCs isolated from Wistar rats were allowed to grow for different passages namely Passage 3, 5 and 7 (P 3, 5 and 7). The immunoprivilege of MSCs was assessed by mixed leukocyte reaction assay. Briefly, MSCs were co-cultured with mixed leukocytes derived from SD rats for 72 h. Leukocyte mediated cytotoxicity (lactate dehydrogenase release) and apoptosis (Bax/Bcl-xl ratio) in MSCs was assessed after the co-culture. Furthermore, MSCs at different passages were analyzed by LC/MS/MS -“lipidomic platform” for cell surface ox-PCs levels and “proteomic platform” for intracellular proteins.

Results and conclusions:
We observed a decrease in lymphocyte mediated cytotoxicity and Bax/ Bcl-xl ratio in MSCs at P5 and7 compared to P3. The lipidome analysis showed that the levels of immunomodulatory ox-PCs increased in MSCs at P5 and P7. Inhibition of ox-PCs was associated with the loss of immunoprivilege of MSCs. To find if the ox-PCs were acting in tandem with downstream intracellular protein alterations, we performed proteome analysis. There were significant changes in proteins related to intracellular immune response, lipid metabolism and mitochondrial dynamics. In depth analysis of the ‘omics’ data is now going on to pinpoint associations between cell surface ox-PCs and the candidate intracellular proteins involved in immune modulation. To the best of our knowledge this is the first study integrating membrane lipidomic with intracellular proteins to assess immunogenicity of MSCs.
Abstract 368: Ex vivo Fabrication of Heart Tissue Model With Perfusable Blood Vessels Using Cell Sheet
Authors: Sekine Hidekazu , Shimizu Tatsuya
Published: June 2021

Introduction:
Creation of 3-D tissue model similar to a biological tissue using tissue engineering method are expected as an alternative approach to 2-D culture or animal test in a research for developing a new medicine. We have already developed functional 3-D heart tissue with perfusable blood vessels in vitro using cell sheet based tissue engineering approach. Present study assessed the possibility as 3-D tissue model by performing drug evaluation in the engineered heart tissues.

Methods:
Firefly luciferase positive cardiac cells co-cultured with endothelial cells were harvested as cell sheets from temperature-responsive culture dishes. Triple-layer cell sheets were overlaid on the vascular bed with an artery and a vein in vitro. Layered cardiac cell sheets were maintained in custom made bioreactor system with perfusion medium. After neovascularization within the first triple-layer tissue, the second triple-layer cardiac cells sheets were overlaid on the first graft after cultivation for 3 days and then the whole construct was further perfused for an additional 3 days. After 6-day perfusion culture, isoproterenol or doxorubicin were administrated to the engineered heart tissues from artery side. The sympathetic and toxic effects were measured by in vitro bioluminescence imaging, electrical potential, and blood pressure.

Results:
In case of isoproterenol administration, bioluminescence imaging (BLI) demonstrated that adenosine triphosphate (ATP) of the heart tissue improved. In addition, electrograms demonstrated increasing beating rate and amplitude. Moreover, sphygmomanometer showed decreasing arterial pressure. In case of doxorubicin administration, BLI showed that the ATP of the heart tissue worsened after 24 hours. It is now possible to analyze the pharmacological effects such as metabolism and the blood pressure as well as action potential by using our vascularized heart tissue model.

Conclusions:
We believe that our 3-D heart tissue model offers a new possibilities for evaluation of drug efficacy.
Abstract 377: A Systematic Review of Medication Adherence Among African American Heart Failure Patients
Authors: de Tantillo Lila , Gonzalez-Guarda Rosa M
Published: June 2021

Background:
Heart failure is a leading cause of death in the United States and a condition of particular concern for the African American population. Although effective treatments are available for the disease, medication adherence is a key determinant of survival.

Objective: This systematic review examines articles from the last 15 years that research medication adherence and African American heart failure patients.

Methods:
A systematic review of the published literature was conducted to identify articles examining predictors and outcomes of medication adherence among African Americans and then using Sister Callista Roy’s Health Adaptation conceptual framework - physiological, self-concept, role function and interdependence.

Results:
This review identified 14 articles that met inclusion criteria, most of which focused on physiological predictors, noting that age and comorbid conditions such as dialysis status may play a role in medication adherence. Psychological, cultural and social determinants were also found to impact adherence behaviors.

Conclusions:
More research is needed to examine other components of the Health Adaptation model, especially socioeconomic and healthcare system level predictors.
Abstract 379: The Prognostic Value of Eosinophil to Leukocyte Ratio in Patients Presenting With ST-elevation Myocardial Infarction Undergoing Primary Percutaneous Coronary Intervention
Authors: Konishi Takao , Funayama Naohiro , Yamamoto Tadashi , Hotta Daisuke
Published: June 2021

Background:
Elevated neutrophil to leukocyte ratio in patients with ST-segment elevation myocardial infarction (STEMI) is associated with adverse clinical outcomes. However, whether decreased eosinophil ratio after primary percutaneous coronary intervention (PCI) reflects larger infarct size has not been fully investigated. This study examined the relationship between eosinophil ratio and creatinine kinase-MB (CK-MB) elevation after primary PCI in patients presenting with STEMI.

Methods and Results:
We analyzed the data of 321 consecutive patients who underwent primary PCI for ST-elevation myocardial infarction between January 2009 and August 2015. Total and each type of leukocyte counts 24 hours after admission were measured. The eosinophil/leukocyte ratio (ER) was calculated as the ratio of eosinophil to leukocyte count. The primary end point was major adverse cardiac event (MACE) and the follow-up period was 180 days. The mean ER and max CK-MB was 0.44±0.65 % and 217.3±224.4 IU/l, respectively. ER obtained 24 hours after admission was inversely correlated with CK-MB concentration (r=-0.37, r2=0.14, P<0.001). MACE within 180 days occurred in 68 patients (21%) including death (9%), myocardial infarction (MI) (1%) and target lesion or vessel revascularization (10%). Patients who had MACE within 180 days had lower ER (0.20±0.51 vs 0.49±0.66, P<0.001) at 24 hours after admission.

Conclusions:
The decreased ER after primary PCI in patients presenting with STEMI was associated with increased CK-MB concentration, which might indicate larger infarct size, therefore, poor prognosis.
Abstract 402: Mutation-Specific Activation of CaMK-II in Hypertrophic Cardiomyopathy
Authors: Lehman Sarah J , Tal-Grinspan Lauren , Richards Amanda , Bedrossian Nora , Anderson Mark E , Tardiff Jil C
Published: June 2021

Approximately 5-10% of all Hypertrophic Cardiomyopathy (HCM) mutations reside within cardiac troponin T (cTnT). Residue Arg92 of cTnT represents a mutational hotspot, including Arg92Leu (R92L) and Arg92Trp (R92W). Interestingly, patients expressing these substitutions that differ by only a single amino acid present with distinct phenotypes and disease severity. Studying the differential effects of the R92 mutations on Ca
2+
homeostasis revealed an age-dependent increase in phospholamban (PLB) phosphorylation at Thr17 (targeted by CaMKII) only in R92W mice. Thus, we hypothesized a mutation-specific role of CaMKII in cTnT-linked HCM progression and tested this hypothesis via genetic partial inhibition of CaMKII (AC3I peptide) in both R92W and R92L transgenic mice. A progressive increase in PKA-mediated PLB-Ser16 phosphorylation was observed in the AC3I/RW mice, leading to an increase in SERCA2 activity. This improved Ca
2+
re-uptake activity was coupled with a decrease in atrial mass, indicative of a decrease in diastolic filling pressure. Thus, the R92W mutation initiates downstream pathophysiologic activation of CaMKII, leading to progressive cardiomyopathic remodeling. Partial inhibition of CaMKII in R92W mice resulted in the alteration of the natural history of HCM, indicating a potential therapeutic target for patients expressing the mutation. In contrast, AC3I/RL mice showed no change in SERCA2 activity or atrial morphology, suggesting a CaMKII-independent progression to disease for R92L-linked HCM. Finally, R92L and R92W mice were treated with diltiazem hydrochloride, an L-type Ca
2+
channel blocker, and diastolic function monitored over time. R92W mice showed improvement in diastolic function with treatment while R92L mice showed a progressive decrease in diastolic function. These findings suggest that closely related mutations, even at the same residue, can have discrete molecular effects that result in mutation-specific ventricular remodeling and these observations can be incorporated in targeted disease management.
Abstract 408: The Role of the Chemokine Receptor CXCR3 - Integrin Lymphocyte Function Associated Antigen 1 (LFA1) - Intercellular Adhesion Molecule 1 (ICAM1) Axis in T Lymphocyte Mediated Cardiac Inflammation in Pressure Overload Induced Heart Failure
Authors: Salvador Ane Miren , Nevers Tania , Velazquez Francisco , Aronovitz Mark , Alcaide Pilar
Published: June 2021

Background:
Left ventricular (LV) dysfunction and Heart Failure (HF) are associated in humans and mice with increased circulating chemokines CXCL9, CXCL10 and CXCL11. Expression of their receptor, CXCR3, in circulating T cells is associated with disease progression in HF patients. We recently published that ICAM1 is significantly upregulated in the LV endothelium and contributes to the progression of HF by regulating T cell recruitment to the LV in a mouse model of pressure overload (PO) induced HF. As CXCR3 can signal to integrins upon chemokine recognition and induce T cell adhesion to endothelial cells under flow conditions
in vitro
, we hypothesize that chemokine signaling through CXCR3 contributes to LV T cell recruitment and regulates integrin dependent adhesion to ICAM1 in PO induced HF.

Methods and results:
We used the mouse model of Thoracic Aortic Constriction (TAC) to induce LV remodeling and HF in WT and ICAM1
-/-
mice, cardiac fibroblasts (CFB) isolated from adult C57/BL6 mice, flow cytometry and qPCR in mouse tissues and in cells cultured
in vitro
. The LV expression of CXCL9, 10 and 11 was upregulated in WT mice in response to 2 and 4 weeks TAC as compared to Sham, correlating with ICAM1, IL-6 and IL-1β upregulation and increased CXCR3+ T cell LV infiltration.
In vitro
, CFB cultured with CXCR3+ T cells or with their culture supernatants, induced CXCL9, 10 and 11, as compared to control media cultured CFB, suggesting CFB are a source of these chemokines under stress. 4 weeks post TAC in WT mice, LV recruited CXCR3+ T cells, as well as mediastinal lymph node and circulating CXCR3+ lymphocytes expressed higher levels of the integrin LFA1, the main ligand of ICAM1, than CXCR3- T cells. The same pattern was observed in systemic CXCR3+ T cells in ICAM1
-/-
mice, which, in contrast to WT mice, had reduced LV infiltrated T cells.

Conclusion:
Our data supports a CXCR3-LFA1-ICAM1 axis being involved in non- ischemic HF. Further studies will determine the CXCR3 ligands triggering T cell LFA1 activation through CXCR3 and whether this mechanism regulates the pathology of non- ischemic HF via ICAM-1 mediated adhesion.
Abstract 410: Novel Sulfonylhydrazone Derivative Prevents Cardiac and Vascular Remodeling in Rats With Streptozotocin-induced Diabetes
Authors: Sudo Roberto T , Araujo Josenildo S , Silva Ananssa M , Alencar Allan K , Delgobbo Mateus S , Silva Thiago F , Trachez Margarete M , Lima Lidia M , Barreiro Eliezer J , Zapata-Sudo Gisele
Published: June 2021

Objectives:
Investigate the effects of LASSBio-1773, a ligand of PPAR-gamma receptor with hypoglycemic activity, in a model of diabetic cardiomyopathy (DC) induced by streptozotocin (STZ).

Methods:
Male Wistar rats received a single i.v. injection of STZ (60 mg/kg) for diabetes induction. Experimental groups were: control, STZ + vehicle, and STZ + LASSBio-1773 (50 mg/kg/day i.p.). Animals were treated with vehicle or LASSBio-1773 for 7 days after disease onset. Cardiovascular function and plasma biochemistry evaluations were performed by the end of protocol.

Findings:
Serum glucose, total cholesterol and triglycerides levels were significantly higher in STZ group, and treatment with LASSBio-1773 normalized both hyperglycemia and hypertriglyceridemia (Table 1). Following 8 weeks of STZ injection, echocardiography showed that left ventricular (LV) filling pressures (E/e′) increased in STZ + vehicle group. Treatment with LASSBio-1773 improved diastolic parameters in STZ-injected rats but no changes were observed on LV ejection fraction (Table 1). STZ + vehicle group have developed hypertension which was reduced after treatment with LASSBio-1773 (Table 1). Vascular dysfunction was found by the reduced acetylcholine maximum relaxation (Ach-MR) in isolated aortic rings from STZ + vehicle group which was improved with LASSBio-1773 administration.

Conclusions:
LASSBio-1773 improved cardiovascular function and lipid profile of diabetic rats, indicating that this novel PPAR-gamma agonist is a promising candidate for the treatment of diabetes-related cardiac complications.
Abstract 429: Thoracic Aortic Smooth Muscle Cell Phenotype and Contractility are Altered With Aging
Authors: Wheeler Jason B , Stroud Robert E , Nadeau Elizabeth K , Mukherjee Rupak , Ikonomidis John S , Jones Jeffrey A
Published: June 2021

Background:
Age-related changes in the thoracic aorta, including alterations in medial extracellular matrix (ECM) in terms of increased collagen and reduced medial cellularity, are associated with a loss of contractility in isolated smooth muscle cells (SMCs). SMCs can regulate both the passive ECM content and mechanical properties of the aorta. However, the age-dependent effects on cellular proliferation, migration, adhesion, and gene expression (phenotype) of aortic SMCs remains unknown. Accordingly, the goal of this study was to determine the age-dependent changes in these characteristics of aortic SMCs.

Methods/Results:
Primary cultures of SMCs were established from thoracic aortic explants harvested from 6 month (n=6) and 21 month old (n=6) C57BL/6 mice. Cellular proliferation (by Cyquant assay), migration (Boyden chamber), and adhesion (on a cell culture treated surface) were reduced in the SMCs from old mice compared to those from young mice. Expression of ECM remodeling genes, including ECM proteins, matrix metalloproteinases, and tissue inhibitors of MMPs, produced a distinct genotypic profile between old and young SMCs (Figure).

Conclusions:
Findings from this study demonstrated that thoracic aortic SMCs from old mice are phenotypically distinct compared to those from young mice. Further, the changes in gene expression with age are consistent with the ECM changes observed in the aorta. Future studies will be required to define the role of these age-related changes in aortic SMC phenotype in aortopathies.
Abstract 431: Mitochondrial Fission in the Heart is an Adaptation to Increased Energetic Demands: The Role of Physiologic Fragmentation
Authors: Coronado Michael , Fajardo Giovanni , Nguyen Kim , Zhao Mingming , Bezold Kooiker Kristina , Jung Gwanghyun , Hu Dong-Qing , Reddy Sushma , Sandoval Erik , Stotland Aleksandr , Gottlieb Roberta , Bernstein Daniel
Published: June 2021

Mitochondria play a dual role in the heart, responsible for meeting energetic demands and regulating cell death. Current paradigms hold that mitochondrial fission and fragmentation are the result of pathologic stresses such as ischemia, are an indicator of poor mitochondrial health, and lead to mitophagy and cell death. However, recent studies demonstrate that inhibiting fission also results in cardiac impairment, suggesting that fission is important for maintaining normal mitochondrial function. In this study, we identify a novel role for mitochondrial fragmentation as a normal physiological adaptation to increased energetic demand. Using two models of exercise, we demonstrate that “physiologic” mitochondrial fragmentation occurs, results in enhanced mitochondrial function, and is mediated through beta 1-adrenergic receptor signaling. Similar to pathologic fragmentation, physiologic fragmentation is induced by activation of Drp1; however, unlike pathologic fragmentation, membrane potential is maintained and regulators of mitophagy are downregulated. To confirm the role of fragmentation as a physiological adaptation to exercise, we inhibited the pro-fission mediator Drp1 in mice using the peptide inhibitor P110 and had mice undergo exercise. Mice treated with P110 had significantly decreased exercise capacity, decreased fragmentation and inactive Drp1 vs controls. To further confirm these findings, we generated cardiac-specific Drp1 KO mice and had them undergo exercise. Mice with cardiac specific Drp1 KO had significantly decreased exercise capacity and abnormally large mitochondria compared to controls. These findings indicate the requirement for physiological mitochondrial fragmentation to meet the energetic demands of exercise and support the still evolving conceptual framework, where fragmentation plays a role in the balance between mitochondrial maintenance of normal physiology and response to disease.
Abstract 432: Elamipretide (BendaviaTM) Restores Aldehyde Dehydrogenase-2 Protein Levels and 4-Hydroxy-2-Nonenal Protein Adducts in Left Ventricular Myocardium of Dogs with Heart Failure
Authors: Gupta Ramesh C , Singh-Gupta Vinita , Sabbah Hani N
Published: June 2021

Background:
Reactive aldehydes such as 4-hydroxy-2-nonenal (4-HNE) are generated in the failing heart and contribute to cardiomyocyte injury and death and progressive left ventricular (LV) dysfunction. Aldehyde dehydrogenase-2 (ALDH2) plays a pivotal role in detoxifying mitochondrial (MITO) reactive aldehydes. We previously showed that long-term therapy with Elamipretide (ELA,
Bendavia
TM
), a novel MITO targeting peptide, improves LV systolic function and normalizes MITO function and rate of ATP synthesis in dogs with heart failure (HF). In this study, we examined the effects of long-term treatment with ELA on protein levels of 4-HNE adducts and ALDH2 in LV myocardium of dogs with coronary microembolization-induced HF (LV ejection fraction ~30%).

Methods:
LV tissue from 14 HF dogs randomized to 3 months monotherapy with subcutaneous injections of ELA (0.5 mg/kg once daily, n=7) or saline (control, CTR, n=7) and tissue from 6 normal (NL) dogs was used in the study. Using LV tissue extracts, 4-HNE protein adducts levels were quantified using a commercially available Elisa kit and expressed as ng/mg. In isolated MITO fractions prepared from LV tissue, protein levels of ALDH2 and porin, the latter used as an internal loading control, were determined by Western blotting coupled with Chemiluminescence. Band intensity was expressed in densitometric units (DU).

Results:
No changes in protein level of porin was observed among the 3 study groups (NL: 0.26±0.02; CTR: 0.24±0.01; BEN: 0.26±0.01 DU). 4-HNE protein adducts levels were increased in CTR dogs compared to NL dogs and treatment with ELA partially restored 4-HNE to near normal levels (NL: 185±21; CTR: 399±35*; ELA: 252 ± 18† ng/mg, *=p<0.05 vs. NL; †=p<0.05 vs. CTR). Compared to NL, dogs, ALDH2 protein levels were significantly reduced in HF CTR dogs (0.49±0.02 vs. 1.16±0.10; p<0.05). Treatment with ELA significantly increased ALDH2 protein compared to untreated CTR (0.74±0.02 DU, p<0.05).

Conclusions:
Long-term therapy with ELA normalizes ALDH2 and 4-HNE levels in LV myocardium of dogs with HF. Improved protein levels of ALDH2 in HF after therapy with ELA can account, in part, for the observed improvement of global LV function elicited by long-term therapy with ELA.
Abstract 435: The Mitochondrial Calcium Uniporter is Necessary for Cardiac Energetic Signaling During Chronic Stress
Authors: Luongo Timothy S , Elrod John W
Published: June 2021

The mitochondrial calcium uniporter (MCU) is a multicomponent channel that is the primary mechanism for mitochondrial Ca
2+
uptake (
m
Ca
2+
). We previously reported that the MCU is required for energetic signaling to meet contractile demand during the ‘fight or flight’ response. In addition, we showed that deletion of the pore-forming component (
Mcu
gene) protected against mitochondria permeability transition pore (MPTP) opening and ischemia-reperfusion injury. However, results from our study and others questioned the physiological relevance of MCU-mediated Ca
2+
uptake during chronic stress states featuring sustained intracellular Ca
2+
load (
i
Ca
2+
). To address this, we deleted
Mcu
from cardiomyocytes in adult mice (
Mcu
cKO) and implanted osmotic pumps to deliver the β adrenergic agonist isoproterenol (iso, 70 mg/kg/day for 14d). In contrast to controls,
Mcu
cKO mice lacked contractile responsiveness to chronic βAR stimulation with evidence of LV dysfunction and failure by d14 (
Fig 1
). Next, we crossed the
Mcu
cKO with mice overexpressing the β2a subunit (β2a-Tg) of the L-type Ca
2+
channel (LTCC). This model displays enhanced LTCC activity and cardiac contractility, but with added stress such as iso infusion, Ca
2+
overload eventually leads to MPTP-dependent cell death and heart failure. Surprisingly, loss of
Mcu
in this model was lethal with all mice dying by d13 (
Fig 2
). Baseline echocardiography revealed that loss of
Mcu
ablated all β2a-mediated enhancements in LV contractility and accelerated dysfunction post-iso. These findings demonstrate that MCU-mediated
m
Ca
2+
uptake is critical to meet energetic demand during chronic stress states featuring sustained
i
Ca
2+
load.
Abstract 439: Modeling Diabetic Cardiomyopathy Model Using Human Induced Pluripotent Stem Cell-derived Cardiomyocytes
Authors: Horikoshi Yuichi , Williams Anna , Bai Xiaowen , Yan Yasheng , Kwok Wai-Meng , Bosnjak Zeliko
Published: June 2021

Background:
Diabetic cardiomyopathy (DCM) is one of the major cardiac complications in diabetic patients. However, the underlying mechanisms and pathogenesis of diabetic cardiomyopathy are only partially understood and the specific therapeutic target remains unknown. Here, we developed an
in vitro
diabetic cardiomyopathy model through simple metabolic manipulation using human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs).

Methods:
After 30 days from initial differentiation, iPSC-CMs were cultured with maturation medium (containing fatty acid but no glucose) for 3 days to obtain matured cardiomyocyte properties. Maturation extent of iPSC-CMs was analyzed using patch-clamp to analyze electrophysiological properties and RT-PCR to analyze cardiomyocyte-specific structure proteins and ion channel expression. Matured iPSC-CMs were then treated with 11 mM glucose for 2 days. 11 mM glucose represents mild glucose conditions in diabetic patients. Cardiomyopathy phenotype of iPSC-CMs was evaluated by analyzing brain natriuretic peptide (BNP) secretion from iPSC-CMs using ELISA.

Result:
Differentiated iPSCs-CMs were immature. Following 3 day culture in maturation medium, iPSC-CMs exhibited the cardiomyocyte maturation characteristics: 1) an increase of contractility with diminished automaticity, suggesting progressive maturation toward adult cardiomyocytes, 2) the lower maximal diastolic potential and increased amplitude, indicating functional maturation, and 3) an upregulation of the following gene expression: sarcomeric proteins (e.g., TNNI3, MYL2 and MYL3) and potassium channel α-subunit genes (KCNJ12 and KCNJ4). Matured iPSC-CMs treated with 11 mM glucose showed increase in cell size and BNP secretion (17-fold increase vs. control cells without glucose, p<0.01). These results suggested that cardiomyocytes exposure to the maturation medium followed by diabetic milieu of glucose recapitulate DCM
in vitro
.

Conclusion:
We conclude that the phenotype of DCM can be modeled using cultured iPSC-derived cardiomyocytes through diabetic extracellular milieu. This clinically relevant stem cell model would be a powerful platform to study novel mechanisms of DCM and the specific therapeutic targets.
Abstract 443: Class I Histone Deacetylases Mediate Cardiac I/R Injury and Modify the Mitochondrial Acetylome
Authors: Herr Daniel J , Aune Sverre E , Bethard Jennifer R , Ball Lauren E , Menick Donald R
Published: June 2021

Although rapid reperfusion of ischemic tissue is the treatment of choice for myocardial infarction, a significant amount of damage occurs as a result of reperfusion itself. The role of epigenetic enzymes in modulating this damage has become an area of interest in basic cardiac research. Previously, we have shown that pharmacological inhibition of the class I histone deacetylases (HDACs) with MS-275 (entinostat) preserves left-ventricular (LV) function and substantially reduces the area of infarcted tissue in isolated rat hearts subjected to ischemia-reperfusion (IR) injury. Interestingly, we have also observed that class I HDAC inhibition during 60 minutes of reperfusion alone is sufficient to protect cardiac tissue viability following I/R injury. Therefore, we hypothesized that class I HDACs mediate reperfusion injury by modulating acetylation of non-canonical pathways. To examine this, hearts from male Sprague-Dawley rats were subjected to I/R injury +/- class I HDAC inhibition during reperfusion. We then performed mass spectrometry to analyze the changes in the acetylome between sham and I/R groups with and without class I HDAC inhibition. Unexpectedly, mass spectrometry analysis revealed significant changes in the acetylation state of multiple mitochondrial enzymes. Further biochemical studies show that class I HDACs localize to the mitochondrial fraction of cardiac tissue homogenates and may modulate mitochondrial acetylation by direct or indirect mechanisms. This study emphasizes the importance of exploring class I HDAC inhibitors for protection against ischemia-reperfusion injury.
Abstract 444: Probiotic Administration Mitigates the Detrimental Effects of Myocardial Infarction in Mice
Authors: Konhilas John
Published: June 2021

Advances in sequencing and bioinformatics technologies have allowed unprecedented characterization of the gut microbiome. As a result, there is a growing appreciation that our microbial environment plays a critical role in the maintenance of health and the pathogenesis of disease. Accordingly, recent evidence suggests a role for gut microbiota in modulating cardiovascular disease and cardiac injury. We hypothesized that administration of the probiotic,
Bifidobacterium animalis
subsp.
lactis
420 (B420), to mice will mitigate the pathological impact of ischemic heart disease (IHD), and that anti-inflammatory T regulatory (T
reg
) immune cells are necessary to impart protection against IHD as a result of B420 administration. Pretreatment with B420 for 14 or 35 days attenuated cardiac injury from ischemia/reperfusion or permanent coronary ligation. Infarcted hearts from B420 treated animals displayed a significant reduction in pro-inflammatory markers and an increase in anti-inflammatory regulatory T cells (T
reg
). We further show that T
reg
immune cells are necessary players to communicate this protection by B420 administration. This protection is due, at least in part, to an increase in anti-inflammatory M2 type macrophages in B420 treated animals. These results suggest that administration of the probiotic B420 protects against cardiac injury and that regulatory T cells mediate this effect. Modulation of the inflammatory response by administration of a specific strain of probiotic may offer a rational, safe, and cost effective way to prevent inflammatory damage after cardiac injury.
Abstract 447: Essential Role of Stromal Interaction Molecule 1 in Heart Failure
Authors: Mali Vishal R , Ali Maha , Haddox Samuel , Trebak Mohamed , Matrougui Khalid
Published: June 2021

Background:
Stromal interacting molecule 1 (STIM1) is a calcium sensor in the endoplasmic reticulum (ER). We previously reported that STIM1 plays opposing roles in vascular smooth muscle cells (SMC) vs. endothelial cell in the regulation of vascular reactivity. However, the role of SMC STIM1 in heart failure is yet to be determined.

Methods and Results:
We utilize control (C57/Bl6) and mice lacking STIM1 specifically in SMC (Stim1
SMC-/-
). We subjected all mice to left anterior descending coronary artery (LAD) permanent occlusion for 3 weeks. We performed echocardiography before the LAD ligation and 3 week after. In the end, we sacrificed mice and harvested the heart for biochemical and histology studies. The heart weight, collagen, and infarct area were significantly augmented in control mice subjected to LAD occlusion. The diastolic (Ejection fraction) and systolic functions (fractional shortening) were significantly compromised in control mice subjected to LAD occlusion. Interestingly, the cardiac hypertrophy, the collagen content, the infarct area, the ejection fraction, and the fraction shortening were protected in Stim1
SMC-/-
mice subjected to LAD occlusion. The protective effect of STIM1 disruption in SMC involved the reduction in ER stress activation, the autophagy, and apoptosis mechanisms.

Conclusion:
Our results indicate that the disruption of STIM1 in SMC protects the heart against chronic ischemia through the inhibition of ER stress, autophagy, and apoptosis.
Abstract 449: GRK2-S670A Mice Reveal Cardioprotection Post Ischemia-reperfusion
Authors: Sato Priscila Y , Chuprun J K , Traynham Christopher J , Lucchese Anna Maria , Yuan Ancai , Gao Erhe , Koch Walter J
Published: June 2021

β-adrenergic receptors (βARs) are critical regulators of cardiac contractility whose function are drastically impaired during heart failure (HF). Activated βARs are regulated via phosphorylation by G-protein coupled receptor kinase-2 (GRK2) and subsequent interaction with β-arrestin. Numerous studies have shown that GRK2 is elevated in human HF and animal models demonstrate that it contributes to HF pathogenesis after ischemic injury (IR). Further, our lab has uncovered non-canonical actions of GRK2 that are involved in HF development. One such novel action of GRK2 is within mitochondria where we have shown increased GRK2 localization after oxidative stress. Our lab has shown that in the heart, phosphorylation of GRK2 via Map kinase at residue Ser670 promotes binding to Hsp90 and further enhances translocation of GRK2 to the mitochondria after oxidative stress such as ischemia. To determine the ultimate role of this pathway in the post-ischemic heart we have generated novel GRK2-S670A mice where all endogenous GRK2 cannot be phosphorylated at this residue. Baseline characterization of these mice along with their controls, show a minimal phenotype, however we have found significant differences post-IR. Compared to control mice (NLC), GRK2-S670A have significantly less infarction size 24 hrs after IR and echocardiography and hemodynamics revealed significantly improved cardiac function in GRK2-S670A mice post-IR. GRK2-S670A mice showed improved ejection fraction (52.83%+/- 2.826 S670A vs 42.38%+/- 1.603 NLC) and smaller infarcted areas (9.942%+/- 1.763 S670A vs 20.78%+/- 3.579 NLC) when compared to NLC mice. Further, we found that mitochondrial GRK2 protein levels were lower in the GRK2-S670A mice at the area at risk (0.1428+/-0.02485 S670A vs 0.2327+/- 0.023 NLC). Overall, our data suggest that phosphorylation at Ser670 of GRK2 might act as a “biological switch” in cardiomyocytes, where un-phosphorylated GRK2 is readily available in the cytoplasm to desensitize receptors such as βARs, but upon phosphorylation, GRK2 translocates to the mitochondria leading to detrimental effects including cell death. This mechanism suggests a novel way to develop pharmacological interventions to aid in the treatment of HF.