bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 248: Integrin-β1 Regulates Cardiac Stem Cell Self-renewal and Differentiation
Authors: Florea Victoria , Taylor Christy N , Martinez Paola A , Lamondin Courtney , Hare Joshua M , Rodrigues Claudia O
Published: July 2021

Background:
Adult cardiac stem cells (CSCs) capable of self-renewal and differentiation into cardiac, smooth muscle and endothelial lineages have been shown to promote tissue repair after ischemic injury and improve heart function. Despite their potential role in cardiac regeneration, evidence suggests that resident CSCs are impaired in conditions such as myocardial infarction, limiting their regenerative capacity.

Methods and Results:
The growth rate of CSCs is significantly reduced by 40% (p<0.05) under hypoxic stress (0.5% O
2
). Using an extracellular-matrix (ECM) and adhesion-focused PCR array, we found that incubation of CSCs for 18 hours under hypoxia leads to down-regulation of several genes, including osteopontin, thrombospondin-1 and integrins. Due to their critical role in communicating extracellular signals regulating stem cell mobilization, proliferation, survival, migration and differentiation we decided to investigate the role of integrins in CSCs self-renewal and differentiation. Our gene expression studies showed that integrin-β1 (Itgb1) is the most abundant integrin expressed in CSCs. Therefore, we knocked down (KD) Itgb1 in CSCs and evaluated these cells regarding adhesion, growth and differentiation properties. Our results showed that reduction in Itgb1 levels in CSCs significantly decreased their interaction with extracellular matrix components fibronectin, laminin and vitronectin by 32%, 20% and 24% (p<0.05), respectively. KD of Itgb1 in CSCs reduced growth by 40% (p<0.05), similar to what we observed by with wild-type cells in hypoxia. Assessment of endothelial differentiation showed that KD cells have a higher angiogenic potential vs. NS-control, as demonstrated by upregulation of vWF (3-fold), VEGF (2.5-fold), Pecam1 (2.3-fold) and Nos3 (1.9-fold) (p<0.05). Interestingly, Itgb1 KD per se induced upregulation of these vascular markers in CSCs.

Conclusions:
Our results suggest that Itgb1 plays an important role in CSCs self-renewal, differentiation and cell adhesion. It is possible that loss of self-renewal is a consequence of changes in CSCs fate as knockdown of Itgb1 leads to upregulation of endothelial lineage markers in these cells. This would limit the expansion of CSCs and their regenerative capacity.
Abstract 261: Human IPS-Derived Cardiac Myocytes (hiPSC-CMs): How Good Are They as a Model for Cardiac Pacing and E-C Coupling?
Authors: Morad Martin , Zhang Xiao-Hua , Wei Hua , Cleemann Lars
Published: July 2021

Derivation of cardiomyocytes from human induced pluripotent stem cells has opened a new field of biology where hiPSC-CM are being used as electrophysiological models of human cardiovascular physiology and pathology. Whether hiPS-CM represent also reliable Ca
2+
signaling and pacemaking models for the mammalian myocytes remains to be determined. Here we evaluate the EC-coupling and spontaneous pacemaking properties of human iPS-CM in detail and compare them to those of native mammalian myocytes. iPS-CMs, dissociated after ~40 days of differentiation and voltage-clamped at -50 mV, showed spontaneous beating and Ca
2+
oscillations that activated in-phase I
NCX
oscillations at holding potentials between -60 and +20mV, while I
f
activated negative to -75mV. Withdrawal of [Ca
2+
]
o
and application of NCX-blocker (KBR- 7943) or tetracaine rapidly and reversibly inhibited spontaneous Ca
2+
-oscillations. Nifedipine and NO-synthase inhibitor L-NAME failed to alter spontaneous beating. Identical sets of data as these were also obtained from neonatal rat myocytes (NRM), suggesting that SR Ca
2+
release and uptake, and not I
f
, were responsible for spontaneous beating in NRM and hiPS-CM. Ca
2+
signaling parameters of hiPS-CM were also similar to those of adult atrial myocytes with Ca
2+
currents averaging ~8 pA/pF and I
Ca
-induced Ca
2+
-transients having a bell-shaped voltage-dependence similar to that of I
Ca
, consistent with Ca
2+
-induced Ca
2+
-release (CICR) mechanism. The ratio of I
Ca
-activated to caffeine-triggered Ca
2+
-transients was ~0.3, similar to the value in rat atria, but significantly smaller than the value of >0.8 in ventricle. The gain of CICR was voltage-dependent as in adult cardiomyocytes. Adrenergic agonists enhanced I
Ca
, but elevated diastolic Ca
2+
. Ca
2+
-sparks were sporadic and brief in duration (< 25ms). Our data suggest that hiPS-CM have all the specific characteristics of adult cardiomyocytes and the mechanisms of their spontaneous pacing are similar to those found in NRM, and involve Ca
2+
cross-talk between NCX, RyR/SR, and possibly mitochondria.
Abstract 262: Derivation of Duchenne Muscular Dystrophy Disease Specific Cardiomyocytes From Patient Urine
Authors: Guan Xuan , Mack David L , Moreno Claudia M , Santana Fernando , Murry Charles E , Childers Martin K
Published: July 2021

Introduction:
Human somatic cells can be reprogrammed into primitive stem cells, termed induced pluripotent stem cells (iPSCs). These iPSCs can be extensively expanded in vitro and differentiated into multiple functional cell types, enabling faithful preservation of individual’s genotype and large scale production of disease targeted cellular components. These unique cellular reagents thus hold tremendous potential in disease mechanism study, drugs screening and cell replacement therapy. Due to the genetic mutation of the protein dystrophin, many DMD patients develop fatal cardiomyopathy with no effective treatment. The underlying pathogenesis has not been fully elucidated.

Hypothesis:
We tested the hypothesis that iPSCs could be generated from DMD patients’ urine samples and differentiated into cardiomyocytes, recapitulating the dystrophic phenotype.

Methods:
iPSCs generation was achieved by introducing a lentiviral vector expressing Oct4, Sox2, c-Myc and Klf4 into cells derived from patient’s (n=1) and healthy volunteers’ (n=3) urine. Cardiomyocytes were derived by sequentially treating iPSCs with GSK3 inhibitor CHIR99021 and Wnt inhibitor IWP4. Differentiated cardiomyocytes were subjected to calcium imaging, electrophysiology recording, Polymerase Chain Reaction (PCR) analysis, and immunostaining.

Results:
iPSCs were efficiently generated from human urine samples and further forced to differentiate into contracting cardiomyocytes. PCR analysis and immunostaining confirmed the expression of a panel of cardiac markers. Both normal and patient iPSC derived cardiomyocytes exhibited spontaneous and field stimulated calcium transients (up to 2Hz), as well as action potentials with ventricular-like and nodal-like characteristics. Anti-dystrophin antibodies stained normal iPSC-derived cardiomyocyte membranes but did not react against DMD iPSC-derived cardiomyocytes.

Conclusions:
Cardiomyocytes can be efficiently generated from human urine, through the cellular reprogramming technology. DMD cardiomyocytes retained the patient’s genetic information and manifested a dystrophin-null phenotype. Functional assessments are underway to determine differences that may exist between genotypes.
Abstract 264: Beta3 Integrin Promotes Protein Ubiquitination and Prosurvival Signaling in Cardiomyocytes
Authors: Pleasant Dorea L , Sundararaj Kamala P , Balasubramanian Sundaravadivel , Kuppuswamy Dhandapani
Published: July 2021

Cardiac hypertrophy occurs in response to stress, such as increased hemodynamic load. Upon hypertrophic stimulus, β3-integrin, a cell surface receptor that mediates nonreceptor tyrosine kinases (NTKs) and mammalian target of rapamycin (mTOR) signaling, undergoes activation. Using β3-integrin global knockout mice, our recent work shows that β3-integrin is required for mTORC2/ubiquitin-mediated prosurvival signaling during early pressure overload (PO). Also, attenuation of β3-integrin signaling during PO causes cardiomyocyte (CM) apoptosis and thus compromised ventricular function. Therefore, we hypothesized that β3-integrin is essential to ubiquitin-mediated prosurvival signaling in CM, with the involvement of key downstream players, such as NTKs, mTOR, E3 ligases, and apoptotic proteins. To generate CM-specific β3-integrin KO mice, cardiac troponin-T2 promoter (cTnT)-driven, reverse tetracycline-dependent transactivator (rtTA)-inducible
Cre
mice were crossed with β3-integrin-floxed mice. The resulting
cTnT-rtTA-Cre/β3-integrin-floxed
mice, were administered doxycycline (Dox) at 1 mg/ml for a period of 7 days before transverse aortic constriction (TAC) or Sham surgery. After 72 h PO, protein was isolated from left ventricle tissue and subjected to immunoblotting. Offspring from the above mating have been bred, genotyped and appropriate colonies have been established. PO-induced β3-integrin expression was lost in CM, but not in cardiac fibroblasts of transgenic mice. Furthermore, a robust reduction in protein Ub was also seen in these mice following Dox treatment and PO. Loss of downstream mediators of cardioprotective signaling is being explored in these transgenic mice. In conclusion, we have developed a mouse with CM-specific deletion of β3 integrin. Initial analysis also demonstrates a lack of protein Ub in isolated CM taken from these animals. Further PO studies will identify downstream mediators of β3-integrin essential to survival in cardiomyocytes. The information gained will identify targets and develop novel therapeutic methods to enhance and/or sustain cardioprotection and thereby prevent/delay maladaptive changes in hypertrophying myocardium.
Abstract 265: Assessment Of Oxidative DNA Double Strand Break And Repair In Cardiomyocytes
Authors: Ye Bo , Hou Ning , Xiao Lu , Xu Haodong , Li Faqian
Published: July 2021

Backgrounds:
DNA damage occurs in cardiomyocytes during normal cellular metabolism and is significantly increased under cardiac stresses. How cardiomyocytes repair their DNA damage, especially DNA double strand breaks (DSBs), remains undetermined. We assessed DSBs caused by oxidative stress. More importantly, we investigated the spatiotemporal dynamics of DNA repair protein assembly/disassembly in DNA damage sites.

Methods:
Cultured neonatal rat cardiomyocytes were treated with different doses of hydrogen peroxide (H2O2) for 30 minutes to assess DNA damage response (DDR). To investigate the dynamics of DDR, cells were treated with 200 uM H2O2 and followed up to 72 hours. DSBs were evaluated by counting DNA damage foci after staining with antibody against histone H2AX phosphorylation at serine 139 (g-H2AX). The dynamics and posttranslational modification of DNA repair proteins were determined by Western blotting, immunolabeling, and confocal microscopy.

Result:
g-H2AX was proportionally increased to H2O2 dosage. Discrete nuclear g-H2AX foci were seen 30 minutes after hydrogen peroxide treatment with 50 uM, but became pannuclear when H2O2 was above 400 uM. At 200 uM of hydrogen peroxide, g-H2AX started to increase at 15 minutes and reached to highest levels at 60 minutes with up to 70 nuclear foci, started to decline at 2 hours, and returned to basal levels at 24 hours. DDR transducer kinase, ataxia telangiectasia mutated (ATM) was activated at 5 minutes with increased phosphorylation at serine 1981 (pATM) which started to decrease at 24 hours, but remained elevated up to 48 hours. Another DDR transducer kinase, ATM and Rad3-related (ATR) showed a biphasic activation at 30 minutes and 8 hours. ATM and ATR colocalized with g-H2AX. DNA damage mediator proteins such as MRN complex and p53BP1 were also recruited to sites of DNA damage at g-H2AX foci.

Conclusions:
DSBs and their repair have emerged as a new frontier of stress responses. Newly developed methods for studying g-H2AX and DNA repair protein dynamics can be explored to investigate DDR to oxidative stress in cardiomyocytes.
Abstract 268: Epigenetic Regulation Of Canonical Tnfa Pathway By Hdac1 Determines Survival Of Cardiac Myocytes
Authors: Biala Agnieszka , Kirshenbaum Lorrie A , Gang Hongying , Dhingra Rimpy
Published: July 2021

Gene transcription is regulated by post-translation modifications. Histone deacetylases (HDACs) remove acetyl-groups from histone and non-histone factors inhibiting transcription. Pro-inflammatory cytokines such as TNFα activate the canonical Nuclear Factor -κB (NF-κB) pathway. Earlier we established a cytoprotective role for NF-κB in the heart. Though a causal relationship for HDAC1 and NF-κB has been established, the impact of HDAC1 on TNFα signaling is unknown. Herein, we demonstrate HDAC1 provides a molecular switch for determining cell survival in the TNFα pathway. In contrast to vehicle treated control cells, TNFα treated cells displayed a marked increase in NF-κB gene transcription. Notably, cells treated with TNFα were indistinguishable from vehicle controls cells with respect to viability. Interestingly, HDAC activity was reduced in cells treated with TNFα. Conversely, in the presence of HDAC1, NF-κB gene transcription by TNFα was repressed, resulting in mitochondrial perturbations and widespread cell death. Heterologous fusion proteins comprised of yeast Gal4 DNA binding domain fused in frame to the NF-κB p65 transactivation domain were preferentially repressed by HDAC1. Moreover, transcription mediated by Gal4VP16 protein from herpes virus was unaffected by HDAC1 in cardiac myocytes. Mutations that abrogate known catalytic activities of HDAC1, small interference RNA or pharmacological inhibition of HDAC1, restored NF-κB signaling and suppressed cell death induced by TNFα. These data provide the first evidence for an obligate link between HDAC1 and canonical TNFα pathway for cell survival of cardiac myocytes.
Abstract 271: A Novel Functional Role for Plasma miR-30d in Dyssynchronous Heart Failure
Authors: Danielson Kirsty , Melman Yonathan , Simonson Bridget , Barth Andreas , Chakir Khalid , Kleber Andre , Singh Jagmeet , Kass David , Das Saumya
Published: July 2021

Introduction:
We have previously shown that plasma miR-30d level is an independent predictor of echocardiographic response to cardiac resynchronization therapy (CRT) in patients with dyssynchronous heart failure (DHF). We now test the hypothesis that miR-30d is dynamically regulated in cardiomyocytes (CMs) and plays a functional role in DHF.

Methods:
miR-30d levels were assessed in a canine model of DHF and CRT using qRT-PCR, and potential miR-30d targets were identified using a bioinformatics approach. miR-30d targets were validated in the canine model and in CMs in culture. The regulation and functional role of miR-30d was investigated in CMs in culture using microscopy, western blotting and qRT-pCR.

Results:
miR-30d is enriched in the coronary sinus compared to peripheral blood in human patients, suggesting a cardiac origin (n=7, p<0.05). In tissue samples from the canine model of DHF, miR-30d levels are highest in the lateral wall, in concert with the greatest wall stress, and decreases with CRT (n=5, p<0.05). Bioinformatics analysis using differential gene expression data and in silico miR target prediction algorithms identified integrin and PI3/Akt signaling pathways as targets of miR-30d. Several targets including MAP4K4 and lims1 were further validated in tissue as well as in cultured CMs (n=4, p<0.05). miR-30d appears to be expressed in CMs, packaged into exosomes and micovesicles, and released in response to pathological rotational stress (n=2). Over-expression of miR-30d in CMs induces cellular hypertrophy with a unique expression signature of cardiac hypertrophy markers most consistent with physiological hypertrophy (n=3, p<0.05). Overexpression of miR-30d appears to be cardioprotective by abrogating TNF-induced increase in MAP4K4 expression (n=4, p<0.05).

Conclusions:
miR-30d is dynamically regulated in DHF and appears to play an important role in CM biology. Further insight into the role of ‘stretch’-regulated microRNAs such as miR-30d may pave the way for novel therapeutic and diagnostic strategies.
Abstract 278: Exercise Training Ameliorates LV Dysfunction in Mice with a Calcium Influx Mediated Cardiomyopathy
Authors: Barr Larry A , Zhang Hongyu , Makarewich Catherine A , Berretta Remus M , Molkentin Jeffery D , Houser Steven R
Published: July 2021

Background—
Ca
2+
influx through L-type Ca
2+
channels (LTCCs) induces Ca
2+
release from the SR to induce and regulate cardiac contraction. We have characterized a mouse model with cardiac-specific expression of the β2a subunit of the LTCC (β2a), leading to pathological hypertrophy due to excessive LTCC Ca
2+
entry. Chronic exercise causes physiological hypertrophy in normal hearts. We determined if swim training improved or further deranged the performance of β2a mice.

Methods and Results—
β2a (n=12) and WT (n=20) mice were swim trained for 21 consecutive days. ECHO was performed at beginning and end of swim training. Ejection fraction (EF) and fractional shortening (FS) increased significantly with swim training in WT animals. (EF pre-swim: 57±2.02% vs. post-swim: 70±2.88%; FS pre-swim: 30±1.34% vs. post-swim: 39±2.42%). β2a mice were hypercontractile before swimming and had no significant change after training (EF pre-swim: 70±1.75% vs. post-swim: 74.06±2.72%; FS pre-training: 38±2.38% vs. post-training: 42±2.22%). Contractile performance was significantly greater in β2a mice versus WT before but not after training. There was a significant increase in HW/BW [mg/gm] ratio in trained WT (n=8) versus sedentary WT (n=8) (swim: 5.75±0.19 vs. sed: 4.63±0.2) but training did not cause additional hypertrophy in β2a (n=8) versus sedentary (n=7) (swim: 5.98±0.31 vs. sed: 5.96±0.25). Isolated WT and β2a hearts were placed on a Langendorff apparatus and a balloon was inserted into the LV to record isovolumic pressure. Each heart underwent 15 min of ischemia followed by 30 min of reperfusion. End diastolic pressure (EDP) increased in all hearts during ischemia. There were no protective effects of training on ischemia-induced increases in EDP in WT hearts. EDP increased more in sedentary β2a during ischemia but this effect was eliminated in trained β2a hearts. LV developed pressure (LVDP) fell with ischemia and partially recovered with reperfusion. LVDP recovery was significantly greater in trained versus sedentary WT hearts. Swim training also significantly improved LVDP recovery in β2a hearts.

Conclusions—
Chronic exercise training reduces pathological hypertrophy and damaging effects of ischemia in a murine model of LTCC overexpression.
Abstract 287: Tcf7l2 Expression By Insulin And High Glucose
Authors: Hou Ning , Ye Bo , Xiao Lu , Xu Haodong , Li Faqian
Published: July 2021

Backgrounds:
Genetic variations of TCF7L2, a nuclear transcriptional factor in canonical Wnt signaling are associated with increased risks of Diabetes Mellitus. How TCF7L2 is regulated by diabetic conditions in the heart is poorly understood.

Methods:
TCF7L2 expression was investigated by Western blotting in HL-1 cells treated with 14 mM mannitol (M), 25 mM glucose (G), 10 nM insulin (I), and G plus I (G+I). In addition, TCF7L2 levels were examined by Western blotting in the heart of B6.Cg-m Leprdb/+ +/J (db) and C57Bl/6J (C57) mice 4 hours after intraperitoneal injections of 1.5 IU/kg insulin.

Result:
The expression of TCF7L2 was higher in db than C57 hearts. Insulin treatment decreased TCF7L2 in C57 hearts. Paradoxically, TCF7L2 was further increased by insulin in db hearts. In HL-l cells, TCF7L2 started to decrease at one hour and remained lower up to 24 hours after insulin treatment compared to untreated controls. In contrast, G increased TCF7L2 expression at 1, 6 and 24 hours after treatment relative to M. Insulin could decrease TCF7L2 at 1 and 6 hours, but failed to do so at 24 hours in G+I. At 48 hours, no difference in TCF7L2 expression was observed among different treatment groups.

Conclusion:
Insulin regulation of TCF7L2 is interfered by high glucose in vitro and misregulation of TCF7L2 in the heart of diabetic mice may contribute to the pathogenesis of diabetic cardiomyopathy.
Abstract 289: Non-viral Rna Delivery To The Endothelium
Authors: anderson daniel
Published: July 2021

Dysfunctional vasculature contributes to more disease than any other tissue in the body1. Small interfering RNAs (siRNAs) have the potential to help elucidate the role of endothelial cells in vivo by durably silencing multiple genes simultaneously, but this requires efficient delivery, which has remained challenging in cell types besides hepatocytes. We have developed nanoparticles that deliver siRNA to endothelial cells with high specificity, thereby facilitating the silencing of multiple endothelial cell genes in vivo. These particles do not significantly reduce gene expression in hepatocytes or immune cells even at doses forty times greater than those required for endothelial gene silencing. Optimized formulations achieved the most durable non-liver silencing reported to date, and delivered siRNAs that modified endothelial function in mouse models of vascular permeability, emphysema, primary tumor growth, and metastasis. We believe the nanomaterial described here may improve the ability to study endothelial gene function in vivo, and be used to treat diseases caused by vascular dysfunction.
Abstract 292: Maternal Transfer of Anti-Cardiac Myosin Antibody Induces Dilated Cardiomyopathy and Underdevelopment of Aortic Arch in Newborn Rats
Authors: Heidari-Bateni Giv , Goessling Lisa , Brar Anoop , Cunningham Madeleine , Eghtesady Pirooz
Published: July 2021

Introduction:
Anti-cardiac myosin (CM) antibodies are induced following certain infectious diseases. We evaluated for the first time the effect of maternally-transferred anti-CM antibody on the newborn heart in a rat model.

Methods:
Female Lewis rats were immunized with 1 µg of emulsified CM and boosters (0.5 µg) were administered every 2 weeks, followed by mating; control rats (n=3) were injected with saline. After birth, pups were continually housed with dams. Echocardiography was performed on pups on day 20. Pups were sacrificed on day 21 and hearts were fixed, sectioned, and analyzed for morphological changes. Trichrome staining was performed to detect fibrotic changes.

Results:
All the pups in CM group (n=22) showed anti-CM antibody titers of more than 12800. Echocardiographic study revealed that 45.4% (10/22) of pups in the CM group had evidence of decreased left ventricular systolic function. The affected pups had both thinner anterior and inferior left ventricle walls compared with pups in control group (3.43±0.33 vs. 3.82±0.24, p=0.017 and 3.71±0.32 vs. 4.18±0.31, p=0.009 respectively). There were also evidence of underdevelopment of thoracic aorta and aortic arch (3.1±0.16 vs. 3.3±0.16 for ascending aorta diameter, p=0.002 and 2.6±0.14 vs. 2.8±0.25 for descending aorta diameter, p=0.008). Morphological changes consistent with dilated cardiomyopathy (image) and fibrosis were detected.

Conclusion:
Maternal anti-CM antibodies can cross the placenta and affect the development of the newborn heart; the resulting phenotype is one of dilated cardiomyopathy with associated hypoplasia of vascular structures perhaps secondary to reduced flow.
Abstract 298: Diastolic Dysfunction Predicts Remodeling in Mice Following Transverse Aortic Constriction: A Potential Model of Heart Failure with Preserved Ejection Fraction
Authors: Garry Julie A , Shearer Gregory , O’Connell Timothy D
Published: July 2021

Heart failure (HF) secondary to hypertensive heart disease typically presents as heart failure with preserved ejection fraction (HFPEF), which now accounts for nearly half of all HF diagnoses. HFPEF pathology is characterized by interstitial fibrosis leading to ventricular stiffness, diastolic dysfunction, and ultimately HF. Clinically HFPEF is a significant problem, as standard HF therapies, which target heart failure with reduced ejection fraction, are ineffective in HFPEF. A major challenge to developing new therapies for HFPEF is the lack of an animal model. In mice, although transverse aortic constriction (TAC) induces significant systolic dysfunction, it produces ventricular remodeling similar to chronic, untreated hypertension, which is associated with concentric hypertrophy, interstitial fibrosis, and diastolic dysfunction. Here, we examined pathologic remodeling and ventricular dysfunction following TAC in mice to determine what factors drive remodeling. TAC mice developed significant hypertrophy after 4 weeks (HW/BW, TAC: 7.2±0.4, n=13; Sham: 4.5±0.2, n=3; P<0.05 and Myocyte Area, TAC: 317±88μm2, n=13; Sham: 193±111μm2, n=3, P<0.05). However, TAC mice varied in extent of fibrotic response. Four mice showed minimal fibrotic response (1.47±0.28% of total ventricular area), five developed only perivascular fibrosis (6.87±1.02%), and four developed perivascular and interstitial fibrosis (29.95±5.81%). At four weeks post-surgery, TAC mice developed systolic dysfunction, indicated by reduced EF (TAC: 47±1%, n=13; Sham: 66±2%, n=3 P<0.05) and diastolic dysfunction, indicated by increased E/E’ (TAC: 44.9±4.5, n=13; Sham: 24.8±3.5, n=3; P<0.05). Interestingly, pathologic remodeling following TAC, both hypertrophy and fibrosis, correlated with E/E’ at week 4 (HW/BW: R2=0.343, P=0.036; Fibrosis: R2=0.517, P=0.008), but not EF (P=NS for both). The data suggest that pathologic remodeling following TAC was driven by diastolic dysfunction and interstitial fibrosis, not systolic dysfunction, indicating TAC induced HF might have more similarities to HFPEF than previously recognized.
Abstract 303: HDL Mimetic Peptide 4F Rescues Pre-existing Pulmonary Hypertension via MicroRNA 193-3p
Authors: Sharma Salil , Umar Soban , Wong Gabe , Mai Denise , Navab Mohamad , Reddy Srinu , Eghbali Mansoureh
Published: July 2021

Pulmonary hypertension (PH) is a chronic lung disease associated with severe vascular disorders leading to right ventricular(RV) failure. An HDL mimetic peptide, 4F, has been shown to be effective for the treatment of atherosclerosis and a number of inflammatory disorders. Here, we explored whether 4F can rescue advanced PH by controlling the expression of specific microRNAs (miRs). PH was induced in rats by a single injection of monocrotaline (MCT, 60mg/kg,
s.c
.) or by placing mice in hypoxia chamber(O2≤10%) for 21 days. MCT-rats or hypoxic mice received 4F therapy (50mg/kg/day,
s.c
.,days 21-30 in MCT model and days 14-21 in hypoxia model). We performed microRNA microarray analysis (non-affymetrix) in lung tissues of CTRL, PH and 4F-rescued groups. OE of miR193 was achieved by intratracheal instillation of 20nM dose at days 16, 21 and 26 in MCT model or at days 14 and 18 in hypoxia model.

4F therapy starting after the establishment of PH in both MCT and hypoxia models improved significantly RV pressure (RVP) and RV hypertrophy index (RVP=46±3 vs RVP=74±1 mmHg in PH; RV/LV+IVS=0.38±0.02 vs RV/(LV+IVS)=0.68±0.05 in PH, p<0.05 vs PH and in hypoxia model RVP=22±3.8 vs. 36.91±5.74 in PH and 20.93±2.52mmHg in ctrl, p<0.05 vs PH ). Microarray and qPCR showed downregulation of miR-193 by ~3 fold in MCT model. 4F therapy normalized miR-193 to ctrl levels. MiR-193 OE in both MCT-rats and hypoxic-mice rescued PH (RVP=38±5.5mmHg, RV/LV+IVS=0.37±0.034 in MCT-rats and RVP=25.48±0.88mmHg in hypoxic-mice). Lung sections showed increased arteriolar muscularization and ox-LDL deposition in the PH group, prevented by miR-193 therapy. In vivo, OE of miR-193 suppressed transcription of in-silico targets ALOX5, a lipoxygenase; IGF1R, insulin-like growth factor 1 receptor and ARHGEF12, a Rho guanine nucleotide exchange factor and decreased human pulmonary artery smooth muscle cell (HPASMC) proliferation in vitro in the presence of serum or 12-HETE by >2 folds whereas miR-193 KD increased proliferation.

In conclusion, 4F rescues pre-existing severe PH by targeting genes associated with HETEs and HODEs production, inflammation and growth via inducing miR-193.
Abstract 314: The Interaction Of The Estrogen Receptor Alpha With The Cardiac Myosin Essential Light Chain Isoform 4 As A New Regulatory Mechanism In The Heart
Authors: Mahmoodzadeh Shokoufeh , Schanz Miriam , Pham Hang , Davidson Mercy M , Morano Ingo , Petrov George , Regitz-Zagrosek Vera
Published: July 2021

Estrogen (17β-estradiol, E2) is one of the key regulators of growth, differentiation and physiological function in different tissues, including the heart. The effects of E2 are mainly mediated by estrogen receptor (ER) alpha and beta, which act in concert with many cofactors to mediate estrogenic effects. So far, only a few cofactors of ER have been described in the human heart. To gain a better understanding of E2-mediated ERα action in the human heart, we identified and characterized the novel interaction partners of ERα Yeast two hybrid screening of a human heart cDNA library revealed that ERα interacts with the cardiac myosin essential light chain isoform 4 (ALC1) in presence of E2. ALC1, as a member of contractile proteins, is expressed in the fetal heart and becomes restricted to the atria of the adult heart under physiological conditions, and is reexpressed in ventricle of adult hypertrophic hearts. This switch is accompanied by alteration of contractile performance, thus improving the heart function. Retransformation experiments showed that ALC1 interacts with full-length ERα and ERα-EF domain in presence of E2. The interaction of ERα with ALC1 was also confirmed by Co-IP in human atrium. Double irnmunofluorescence (IF) analysis of paraffin-embedded sections from human atrium tissues showed co-localization of ERα and ALC1 proteins in a striated sarcomeric pattern. Co-localization corresponds most likely to the H-zone of sarcomere. IF analysis of AC16 cells (a human cardiomyocytes cell line) showed that ERα and ALC1 were mainly localized in the cytoplasm in the absence of E2. However, E2 treatment of AC16 cells led to a translocation of ALC1 and ERα into the nuclei of AC16 cells, where they also co-localized. Expression analysis in AC16 cells showed that E2 increases the expression of ALC1 gene (MYL4). Using ERE-based luciferase reporter assays we showed that E2-induced interaction of ERα with the ALC1 represses the transcriptional activity of ERα. We characterize for the first time an E2-regulated interaction of ALC1 with ERα in cardiomyocytes that may be crucial in physiological and/or pathological processes by regulating of transcriptional activity of ERa in the heart and/or by modulating contractile properties of cardiomyocytes.
Abstract 324: Analysis of Transcriptome Profiles Identifies Novel Genes in Pathological Cardiac Hypertrophy
Authors: Chyu Jennifer , Zhang Yiqiang , MacLellan Robb
Published: July 2021

Both the underlying cause and resulting consequence of many cardiovascular diseases is the permanent loss of contractile cardiac myocytes (CMs). Because the vertebrate heart has no innate ability to regenerate, the response to disease stimuli is limited to hypertrophy, an initial response to preserve cardiac function. However, sustained hypertrophy can turn the growth response of myocytes to one of cell death leading to pathological ventricular remodeling and decreased cardiac function. In this study, we are interested in identifying novel therapeutic gene targets that play a role in cardiac hypertrophic progression. To identify differentially expressed genes in hypertrophy, we utilized trans-aortic constriction (TAC) to induce hypertrophy in mice and performed whole transcriptome profiling on isolated CMs and compared to wild-type adult and embryonic CMs. Globally, the correlation coefficients between adult or TAC CM samples ranged from 0.955 to 0.991, suggesting limited early response of CMs in hearts subjected to TAC. However, pathway and ontology analysis revealed that embryonic CMs are far different from both adult and TAC in global gene expression profile. Through bioinformatic analysis, we identified a panel of 12 genes that are differentially expressed between TAC and adult CMs. Pathway and ontology analysis implicated the genes to participate in either oxidative stress pathways or cardiac remodeling processes. Selective validation of the gene panel revealed MMP23 to have increased expression in an in vitro model of HF and in patients with end-stage heart failure (HF) suggesting a previously undescribed link to human HF disease progression. Together, this work identifies individual genes that contribute to pathological hypertrophy and can serve as novel targets for HF interventional therapies.
Abstract 332: Mitochondrial Inner-Membrane Potential Instability Promotes Sarcolemmal Electrical Instability after Ischemia-Reperfusion in Monolayers of Cardiac Myocytes
Authors: Solhjoo Soroosh , O’Rourke Brian
Published: July 2021

Mitochondrial uncoupling due to oxidative stress can, through opening of sarcolemmal KATP channels, alter cellular electrical excitability, and it has been proposed that mitochondrial function is a major factor in arrhythmogenesis during ischemia-reperfusion. Here, we examine the effects of ischemia-reperfusion on mitochondrial inner membrane potential (ΔΨm) and corresponding changes in electrical excitability and wave propagation in monolayer cultures of neonatal rat ventricular myocytes. Changes in ΔΨm were observed using TMRM and changes in the sarcolemmal voltage were recorded with a 464-element photodiode array using di-4-ANEPPS. Ischemia was induced by covering the center part of the monolayer (D = 22 mm) with a coverslip (D = 15 mm). Cell contractions ceased after approximately 6 min of ischemia; however, electrical activity continued for 11.3 ± 4.2 min (N = 5). Amplitude and conduction velocity of the action potentials in the ischemic region decreased over the same time period. ΔΨm was lost in two phases: a reversible phase of partial depolarization, after 11.2 ± 1.3 min of ischemia, and a nonreversible phase, which happened after 30 ± 6 min of ischemia, during which the whole mitochondrial network of the myocyte became depolarized and the cells underwent contracture (N = 4). Reperfusion after the long ischemia resulted in only partial recovery and the observance of oscillations of ΔΨm in the mitochondrial network or rapid flickering of individual mitochondrial clusters and was associated with heterogeneous electrical recovery, and formation of wavelets and reentry (4/5 monolayers). In contrast, mitochondria fully recovered and reentry was rare (1/5 monolayers) for reperfusion after the short ischemia (10-12 min). 4’-chlorodiazepam, an inhibitor of inner membrane anion channels, stabilized mitochondrial function after the long ischemia, and prevented wavelets (5/5 monolayers) and reentry (4/5 monolayers). In conclusion, incomplete or unstable recovery of mitochondrial function after ischemia correlates with reentrant arrhythmias in monolayers of cardiac myocytes. Our findings suggest that stabilization of mitochondrial network dynamics is an important strategy for preventing ischemia/reperfusion-related arrhythmias.
Abstract 351: Downregulation of Connexin43 by MicroRNA-130a Results in Cardiac Arrhythmias
Authors: Kim Gene , Osbourne Appledene , McSharry Saoirse , Broman Michael
Published: July 2021

MicroRNAs (miRs) are endogenous ~22 nucleotide RNA molecules that are now recognized as critical regulators of diverse physiological and pathological processes; however, studies of miRs and arrhythmogenesis remain sparse. Connexin43 (Cx43), a major cardiac gap junction protein, is critical for maintaining normal cardiac electrical conduction. One aspect of arrhythmia pathogenesis that has elicited great interest has been changes in Cx43 expression. Computational analysis of messenger RNAs predicted Cx43 to be a target for miR-130a. We hypothesized that overexpression of miR-130a in adult cardiomyocytes would result in cardiac arrhythmias. To study this, we used an inducible (tetracycline-off) bigenic system to study miR-130a overexpression in the adult mouse heart.

12 weeks after induction of miR-130a, 100% (16/16) of transgenic mice demonstrated a loss of sinus rhythm and irregular ventricular activity suggestive of atrial fibrillation on surface electrocardiograms (ECG). Nonsustained ventricular tachycardia was also observed during ECG recordings. Atrial contractions could not be detected by pulse wave Doppler. Intracardiac electrocardiography confirmed the presence of atrial arrhythmias. Western blot analysis demonstrated a 90% reduction in Cx43 levels and immunofluorescent staining confirmed a near complete loss of Cx43 in transgenic compared to littermate control hearts (n=12). Morphology of the intercalated disc was evaluated by electron microscopy and showed a loss of gap junctions in transgenic hearts. To validate Cx43 as a direct target of miR-130a, we performed in vitro target assays in 3T3 fibroblasts, known to express miR-130a. Using a luciferase reporter fused to the 3’UTR of Cx43 compared to a control (SV40) 3’UTR, we found a 55% ± 6% reduction in luciferase activity with the Cx43 3’UTR (p<0.01, n=9). Addition of an antisense miR-130a inhibitor resulted in a dose dependent loss of inhibitory activity of the Cx43 3’UTR reporter, with no changes seen in the control vector.

In conclusion, we have identified an unappreciated role of miR-130a as a direct regulator of Cx43. Overexpression of miR-130a results in the development of a variety of important cardiac arrhythmias including atrial fibrillation and ventricular tachycardia.
Abstract 354: Acetylation Regulates miR-133a Expression In Cardiac Hypertrophy
Authors: Renaud Ludivine , Mani Santhosh , Baicu Catalin , Van Laer An , Stroud Robert , Jones Jeffrey , Zile Michael , Menick Donald
Published: July 2021

Background:
The expression level of several microRNAs (miRNAs) is affected by pressure overload hypertrophy and has an impact on cardiac function. Histone deacetylases (HDACs) regulate the transcription of many genes whose expression is altered in hypertrophy.

Therefore, we hypothesize that acetylation regulates the expression of some miRs during cardiac hypertrophy
.

Methods:
Transverse aortic constriction (TAC) was performed to initiate cardiac hypertrophy induced by pressure overload. Mice were treated with the Class I and IIb HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) in the drinking water post-TAC for 2 and 4 weeks. miRNAs expression normalized to RNU6B was determined by qRT-PCR. Echocardiography was performed at baseline and post-TAC endpoints to assess physiological parameters. Chromatin immunoprecipitation (ChIP) was used to identify HDACs and transcription factors associated with miR-133a promoter.

Results:
Based on previous studies, we selected 8 miRNAs that play major roles in hypertrophy and assessed the effect of SAHA treatment on their expression level. The expression of one of the miRNAs, miR-133a was significantly (P < 0.05) downregulated at both 2 weeks (1887 ±105, n=8) and 4 weeks post-TAC (1651 ±103, n=9) compared to control group (2972 ±334, n=11). SAHA treatment significantly de-repressed miR-133a expression 2 weeks post-TAC (3317 ±560, n=12) and to a lesser extent after 4 weeks post-TAC (2391 ±455, n=10). As expected, left atrium (LA) diameter, a marker of diastolic pressure, increased 2 weeks post-TAC, while SAHA treatment significantly reduced LA diameter. miR-133a targets connective tissue growth factor (CTGF) and collagen 1a1. Consistent with SAHA-mediated miR-133a de-repression, CTGF expression and collagen volume fraction were decreased compared to TAC alone. Importantly, ChIP analysis revealed that HDAC2 was present on the miR-133a promoter.

Conclusion:
We show that HDAC2, a Class I HDAC, plays a role in the regulation of miR-133a expression in cardiac hypertrophy. HDACs and miRNAs are key regulators of the events mediating cardiac pathology. Our work demonstrates that HDAC inhibition may be an attractive therapeutic strategy for the regulation of some miRNAs in heart disease.
Abstract 359: Human CD34+ Cell-Derived Exosomes Target Endothelial Cells to Deliver MiR-126 Promoting Revascularization and Myocardial Repair
Authors: Sahoo Susmita , Misener Sol , Kim David , Thorne Tina , Kamide Christine , Losordo Douglas W , Vaughan Douglas E
Published: July 2021

Introduction:
Locally transplanted human CD34
+
stem cells have been shown to improve exercise tolerance in patients with myocardial ischemia and promote angiogenesis in animal models. Recently we have demonstrated that CD34
+
cells secrete exosomes, membrane bound nano-vesicles, as a major component of their paracrine secretion which induces angiogenesis. We
hypothesize
that cell-free exosomes from CD34
+
cells (CD34 Exo) mimic the beneficial effects of cells, and promote myocardial revascularization and repair via transfer of pro-angiogenic microRNAs, possibly to endothelial cells.

Methods and Results:
Therapeutic potential of CD34 Exo isolated from equal number of adult human peripheral blood-derived CD34
+
cells was evaluated in a murine model of myocardial ischemia (MI). Similar to cells, treatment with CD34 Exo resulted in significant improvement in ischemia compared to treatment with PBS (ejection fraction, 42±4 v 22±6%; capillary density, 113±7 v 66±6/HPF; fibrosis 27±2 v 48±7%, p<0.05, n=7-12). Interestingly, confocal imaging and flow cytometry analyses revealed that CD34 Exo was selectively internalized by endothelial cells when injected in to ischemic tissue. MicroRNA expression profiling and confirmatory tests indicated that CD34 Exo is significantly enriched with pro-angiogenic miRNAs such as miR126. Treatment of CD34 Exo increased miR126 expression specifically in endothelial cells of ischemic tissue; treatment of CD34 Exo lacking miR126 abolished that increase and diminished its pro-angiogenic function. This, and studies using fluorescent miR126 confirm that miR126 was directly transferred from CD34 Exo to endothelial cells, and that indirect increase of miR126 by other components of CD34 Exo was minimal. This data also suggests that miR126 is important for CD34 Exo function. Ongoing studies will test the role of transferred miR126 on modulation of proangiogenic gene expression pathways in endothelial cells lacking miR126 either by dicer, or, Egfl-7 knockdown.

Conclusion:
CD34
+
exosomes transfer miR126 to endothelial cells to induce angiogenesis and myocardial repair. The functional benefits associated with CD34
+
cell therapy may be mediated by exosomes-mediated transfer of angiogenic microRNAs to endothelial cells.
Abstract 360: IL-10-inhibits Pressure Overload-induced Homing, Proliferation And Differentiation Of Non-resident Fibroblast Progenitors And Improve Heart Function.
Authors: Verma Suresh K , Krishnamurthy Prasanna , Barefield David , Mackie Alexander R , Vaughan Erin E , Abramova Tatian , Ramirez Veronica , Misener Sol , Sadayappan Sakthivel , Qin Gangjian , Kishore Raj
Published: July 2021

Background:
Recently we have shown that IL-10, an anti-inflammatory cytokine, markedly inhibited the pressure overload-induced cardiac fibrosis, however, antifibrotic mechanisms of IL-10 are largely unknown. In most of organs, including heart, extracellular matrix (ECM) remodeling is primarily mediated by excessive proliferation of activated fibroblasts and myofibroblasts. Here we hypothesized that IL-10 inhibits stress-induced homing, proliferation and differentiation of nonresident bone marrow-derived fibroblast progenitor cells and therefore, attenuates cardiac remodeling and improves of heart function.

Methods and Results:
Cardiac hypertrophy was induced in Wild-type (WT) and IL-10-knockout (KO) mice by transverse aortic constriction (TAC). TAC-induced left ventricular (LV) dysfunction and fibrosis were further exaggerated in KO mice compared to WT. TAC significantly increased TGF-β, collagen Iα and IIIα genes expression. Systemic recombinant mouse IL-10 administration markedly improved LV function, inhibited TAC-induced cardiac fibrosis and fibrosis associated genes expression. To identify the role of fibroblast progenitor cells (FPCs), we measured the mobilization of FPCs (Prominin1 positive cells) from bone marrow to heart by FACs. Exacerbated mobilization of FPCs in peripheral blood and heart in IL-10 KO mice were found 3 and 7 days after aortic constriction. Bone marrow transplantation experiments were performed where WT-GFP positive marrow was transplanted in BM depleted IL-10 KO mice. TAC-induced mobilization was significantly reduced in WT-transplanted marrow as compare to TAC-IL-10 KO mice. To identify the role IL-10 on TGFβ-induced endothelial cells trans-differentiation to myofibroblasts, we treated aortic endothelial cells with IL-10 and TGFβ2 for 96 hrs. Both Immunocytochemistry and Western blot analysis results suggested that TGF-β2-induced EndMT was significantly inhibited by IL-10 treatment. To understand the mechanisms, we found that TGF-β2-induced Notch1 signaling was reduced by IL-10.

Conclusion:
Taken together our observations suggest that the anti-fibrotic effects of IL-10 treatment are mediated by reduced proliferation and differentiation of non-resident myofibroblasts.
Abstract 112: Targeted Metabolomics Profiling of Heart Failure Patients Undergoing Mechanical Circulatory Support
Authors: Chan X'avia , Choi J.H. Howard , Ju Chelsea J.-T. , Wang Wei , Zhang Jun , Tabaraki Jason , Liem David , Cadeiras Martin , Deng Mario , Ping Peipei
Published: July 2021

Metabolomics investigations hold promise for the characterization of small molecules, metabolites, which govern the ultimate manifestation of cardiac phenotypes. In this study, we employed a mass spectrometry-based metabolomics approach to identify metabolic marker(s), which dynamically reflect the cardiac performance of heart failure patients amid the implantation of mechanical circulatory support. Using the MRM-based and triple quadrupole technology platform, we have quantified 266 metabolites native to human plasma and collected from thirteen heart failure patients. The temporal profile of these metabolites was sampled from 1 day prior to the implantation of mechanical circulatory support, as well as 1-, 3-, 5-, and 7-day following their surgical interventions. We identified subgroups of these metabolites with coordinated behaviors that are interesting to their diseased phenotypes. In a pair-wise correlation analysis, 36.8% (98 out of 266) of metabolites were significantly correlated. Intriguingly, majority of which (65 out of 98) are representing the functional groups of phosphatidylcholines; several of them are known to have close associations with the pathogenesis of cardiovascular diseases. In addition, there are 33 metabolites contributing to multiple functional groups, including twelve of them belong to sphingomyelines, ten of them in the family of lysophosphatidylcholines, eight amino acids (Gln, Ser, Ala, His, Lys, Gly, Thr, and Arg), as well as three fatty acids (eicosapentaenoic acid, pentadecenoic acid, and heptadecenoic acid). The behaviors of these 266 metabolites have constituted individualized metabolic fingerprints. Delineation of the intrinsic relationships among alterations in distinct metabolite groups and their reflected cardiac function will enable us to identify new metabolic markers aiding stratification and/or prediction on the clinical outcome of each individual patient undergoing the treatment of mechanical circulatory support. This personalized metabolic fingerprint will offer unique prognostic utilities, supporting clinical decision-making process to deliver intervention that is most effective and beneficial to an individual.