bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 45: Cardiomyocytes Undergo Division Postnatally to Generate New Cardiomyocytes in Mouse Models of Aging and Cardiac Injury
Authors: Ali Shah R , Saadat Lily S , Hippenmeyer Simon , Luo Liqun , Weissman Irving L , Ardehali Reza
Published: July 2021

The dogma of the adult mammalian heart as a post-mitotic organ has recently come under question. Radiolabeled isotope studies have demonstrated that the human heart exhibits a low rate of renewal of cardiomyocytes throughout one’s lifespan. Furthermore, a neonatal mouse can regenerate its ventricle if the apex is resected within the first week of birth. However, the field has yet to clonally address whether cardiomyocytes divide symmetrically upon birth, or if a resident progenitor differentiates into cardiomyocytes. Rather than rely on proxies for cell division (e.g. BrdU incorporation studies), we use genetic mouse models in which cell division results in asymmetric, indelible labeling of the daughter cells (“Mosaic analysis of double markers” (MADM)) to identify which cell type(s) generate cardiomyocytes. We made two triple-transgenic mouse strains: actinCreER;MADMGT/TG and Myh6CreERT2;MADMGT/TG. The former model allows us to identify postnatal cardiogenesis from any cell type, whereas with the latter model we identify postnatal cardiomyocytes that arise from existing alpha-myosin heavy chain-expressing cardiomyocytes. Our studies demonstrate limited symmetric division of cardiomyocytes during normal aging up to six weeks, with a significantly high rate of cardiomyocyte division during the first postnatal week, in both models. The rate of generation of cardiomycoytes is very similar in both models, suggesting that the postnatal cardiomyocyte division accounts for all postnatal cardiomyocyte birth. In the injury model, we provide evidence for cell division in the infarcted heart of a myocardial infarction model, but this rate of division is not greater than in the sham animal, suggesting that the inflammatory environment following an MI does not provide additional pro-mitotic signals. Our data suggests that, as in teleost fish such as zebrafish that demonstrate robust myocardial regeneration even in adulthood, the cell of origin for mammalian postnatal cardiogenesis is a differentiated cell - the cardiomyocyte - rather than a multipotent progenitor cell. It remains to be explored whether all cardiomyocyte are able to divide postnatally or whether this phenomenon is restricted to a subpopulation of cardiomyocytes.
Abstract 55: Detection of Atrial Fibrillation Using a Smartphone Camera
Authors: Maitas Oscar , Lee Jinseok , Robotis Dionyssios , Chon Ki H , McManus David D
Published: July 2021

Introduction:
Atrial fibrillation (AF) is the most common sustained dysrhythmia in the U.S. and is associated with significant morbidity and mortality. Although new AF treatment strategies have emerged over the last decade, a major challenge facing clinicians and researchers is the paroxysmal, often short-lived, and sometimes asymptomatic nature of AF. We hypothesized that a smartphone can detect AF based on its ability to record heart beat variability by placing a fingertip on the built-in camera.

Methods:
In this prospective clinical investigation, we recruited 33 participants at a tertiary care medical center who presented with persistent AF prior to elective cardioversion. We recorded 2-5 minutes of pulse readings from participants’ index finger using an iPhone 4S before (AF cases), and after, electrical cardioversion (Sinus rhythm controls). With heart beat interval time series obtained from changes in color signals of a fingertip recording, we compared the results of an AF detection algorithm using Root Mean Square of Successive Difference in combination with Shannon Entropy and Sample Entropy in comparison to the gold-standard of an electrocardiogram.

Results:
The AF detection accuracy was 97.67% (sensitivity of 96.08% and specificity of 99.97%) for each sample when a combination of the three signal processing techniques were applied compared to the gold-standard electrocardiographic recordings. All of the 33 AF episodes were detected. In addition, we tested on 29 healthy subjects and found that the accuracy (specificity) was 99.51%.

Conclusions:
In this paper, we show that a smartphone can accurately detect AF based on its ability to obtain RR time series. More accessible methods to accurately detect AF are necessary, and the use of existing smartphone technologies has potentially revolutionary clinical applications for in-home monitoring for AF.
Abstract 57: Therapeutic Effects of Small Molecule Gβγ Inhibition in Pressure Overload Heart Failure
Authors: Kamal Fadia A , Smrcka Alan V , Blaxall Burns C
Published: July 2021

Heart failure (HF) is a progressive disease with rapidly increasing rates of morbidity and mortality; it is the leading cause of death worldwide. Elevated sympathetic nervous system activity, a salient feature of HF progression, leads to pathologic attenuation and desensitization of β-adrenergic receptors (β-ARs) due in part to Gβγ-mediated signaling. We recently reported that novel small molecule Gβγ inhibitors selectively block specific Gβγ signals and halt HF progression in pharmacologic and transgenic mouse models of HF.

We assessed the
hypothesis
that the Gβγ inhibitor Gallein could be salutary in treating pre-existing HF in a clinically relevant model.

We utilized the pressure-overload HF model of mouse transverse aortic constriction (TAC). Four weeks post-TAC, mice received daily IP injections of vehicle (PBS; group V) or Gallein (10mg/Kg/day; group G) for eight weeks. Gallein treatment improved survival (7 of 9 mice survived vs. 5 of 9 mice in group V) and cardiac function (%EF 75.2 ± 7.5 vs 35.6 ± 17.2 in group V, +dP/dt (mmHg/sec) 7022 ± 485.3 vs. 3584 ± 598.6 in group V), -dP/dt (mmHg/sec) -5826 ± 910.7 vs. -3260 ± 62.3 in group V, LVEDP (mmHg) 11.5 ± 3.7 vs. 29.45 ± 3.6 in group V). In addition, gallein reduced cardiac hypertrophy (HW/BW (mg/g) 5.8 ± 0.3 vs. 8.8 ± 1.1 in group V) and plasma catecholamine concentrations (adrenaline (ng/ml) 1.3 ± 0.3 vs. 6.6 ± 2.8 in group V, noradrenaline (ng/ml) 3.6 ± 0.6 vs. 15.1 ± 3.6 in group V). Reduction of interstitial fibrosis as well as mRNA levels of α-SMA, TNF-α, and IL-6 was observed in the hearts of Gallein treated animals (59.7 ± 14.1%, 43.8 ± 9.3% and 28.5 ± 3.5% relative to group V, respectively). On the molecular level, Gallein treated mice showed less GRK2 and PI3Kγ membrane recruitment, and less Akt activation (42.9 ± 7.1%, 66.7 ± 13.3% and 46.2 ± 7.7% relative to group V, respectively) in myocardial lysates.

In
conclusion
, these data suggest a possible therapeutic role for small molecule Gβγ inhibition in halting the progression of HF, potentially via inhibition of the Gβγ-GRK2-PI3Kγ-Akt pathway. The combined effect of halting HF progression and reducing plasma catecholamines suggests a possible systemic role for small molecule Gβγ inhibition in both the heart and the adrenal gland.
Abstract 6: Therapeutic Angiogenesis by Human CD34+ Hematopoietic Stem Cells via Exosomes-Mediated MicroRNA Transfer
Authors: Sahoo Susmita , Gupta Rajesh , Thorne Tina , Misener Sol , Ito Aiko , Losordo Douglas W , Vaughan Douglas E
Published: July 2021

Introduction:
Locally transplanted human CD34+ hematopoietic stem cells have stimulated neovascularization in preclinical studies and have been associated with functional improvements in phase I and II clinical trials of patients with ischemic cardiovascular diseases. Recently, we have demonstrated that membrane-bound nano-vesicles called exosomes are one of the primary pro-angiogenic components of human CD34+ cell paracrine secretion that induced angiogenesis independently of the cells.

Here, we
hypothesize
that the functional benefits associated with CD34+ cell therapy are primarily mediated by the secretion of exosomes (Exo), and that CD34+ Exo transfer pro-angiogenic microRNAs to promote angiogenesis and ischemic tissue repair.

Methods and Results:
Therapeutic potential of CD34+ Exo isolated from adult human peripheral blood-derived CD34+ cells was evaluated in a mouse model of hind limb ischemia (HLI). Similar to CD34+ cells, administration of CD34+ Exo from equal number of cells induced angiogenesis and tissue repair; it significantly improved perfusion (ratio: 1.01±0.04 v 0.57±0.1, P<0.05), increased capillary density (1.8±0.3/HPF v 0.9±0.1/HPF, p<0.001) and prevented ischemic leg amputation (16% v 100%), compared to Exo from non-angiogenic, CD34+ cell-depleted-mononuclear cells (MNC Exo). Our microarray data and confirmatory tests revealed that unlike MNC Exo, CD34+ Exo are enriched in pro-angiogenic miRNAs such as miR-126. Flow cytometry and live confocal imaging demonstrated that Cy3-tagged miRNA in CD34+ Exo is directly transferred to endothelial cells in vitro and in ischemic tissues in vivo; concurrent loss of function gain of function studies prove that direct transfer of miR-126 is crucial for CD34+ Exo-induced angiogenesis and functional recovery. To address whether exosome secretion is needed for miR-126 transfer, we are exploring the effect of inhibition of exosome secretion from CD34+ cells.

Conclusion:
CD34+ exosomes transfer the pro-angiogenic miR-126 to endothelial cells in the ischemic tissues to induce angiogenesis and ischemic tissue repair. Our study illustrates a mechanism of stem cell communication involving intercellular traffic of miRNAs via exosomes to induce functional recovery.
Abstract 62: Pim-1 Engineering of Human CPCs Increases Telomere Length from Aged Patients with Heart Failure
Authors: Mohsin Sadia , Khan Mohsin , Wallach Kathleen , Cottage Travis , Mcgregor Michael , Sussman Mark
Published: July 2021

Rationale:
Adoptive transfer of human cardiac progenitor cells (hCPCs) can repair damaged myocardium and improve function in a pathologically challenged heart. Aging can exhaust hCPC pool and pose a challenge to effectively regenerating the damaged myocardium. There is a dire need to reverse the senescent phenotype of these hCPCs isolated from elderly patients to target large segment of population suffering from heart failure.

Objective:
Demonstrate that Pim-1 engineering of human cardiac progenitor cells (hCPCs) isolated from elderly patients with heart failure can increase the telomere lengths, telomerase activity, population doubling and reverse their senescent phenotype.

Methods and Results:
hCPCs positive for the putative stem cell marker c-kit were isolated from heart biopsy samples from patients undergoing Left Ventricular Assist Device (LVAD) implantation. hCPCs isolated from patients aged 75-85 years (n=3) with heart failure demonstrate decreased population doubling time concomitant with decreased telomere lengths measured by in situ hybridization and telomerase activity measured by TRAP assay compared to patients aged 60-73 years (n=3). hCPCs were engineered to express Pim-1-GFP (hCPCeP), a fused GFP version of the kinase by using a lentivirus expression system. The cell cycling time measured by population doubling is increased after over expression of Pim-1 in hCPCs isolated from old patients. Pim-1 modification significantly increased telomere lengths and telomerase activity indicative of reversal of senescent characteristics of aged hCPCs.

Conclusion:
Ex vivo gene delivery of Pim-1 enhances telomere length, telomerase activity and decreases population-doubling time. Modification of aged hCPCs from elderly patients augments their ability to regenerate damaged myocardium, making stem cell engineering a viable option to address current limitations associated with senescent phenotype of aged hCPCs.
Abstract 69: Diminished T-Tubule Density in Newborn Human Ventricular Cells Is Associated with Heterogeneity of Calcium Transients
Authors: Saafir Talib , Crawford Brian , Shen Ming , Ding Guoliang , Kirshbom Paul M , Wagner Mary B
Published: July 2021

For children with congenital heart disease (CHD), therapies developed for adults may have different effects on immature myocardium. Thus, it is critical to understand calcium handling in the young human ventricle.

Methods:
Human ventricular cells were isolated from tissue removed as part of the surgical repair for CHDs from two age groups: newborns (<1 week old) and infants (2-12 months old). Developmental changes in t-tubules were examined in live cells with Di-8 ANEPPS which highlights the cell membrane. We measured the T-index (percent of cell interior occupied by t-tubules), averaged for at least 3 confocal z-sections per cell. Changes in calcium transients were measured by confocal line scans in cells loaded with Fluo-4 and field stimulated (37°C).

Results:
Newborn human myocytes have very few t-tubules whereas infant myocytes have a range of t-tubule densities. The T-index in newborn myocytes was significantly less than in infant myocytes (4.4±0.2%, n=8 cells, 3 newborns vs. 9.1±0.9%, n=55 cells, 7 infants). Furthermore, there was sizable heterogeneity in the T-index for each patient in the infant group, in which some cells had few t-tubules and others had many t-tubules. We calculated the coefficient of variation (CV=SD/mean*100) for each patient and compared CV values for newborns vs. infants. CV was significantly greater in infants compared to newborns (46.5±7.4%, n=7, vs. 9.4±1.9%, n=3, p<0.02). In addition, calcium transients from newborn cells had a ‘U’ shaped wavefront with calcium first increasing at the outer edge of the cell then propagating toward the center. In contrast, infant cells had a homogenous calcium wavefront. The delay of peak calcium from the edge to center was significantly longer in newborns compared to infants (61.7±8.9 ms, n=8 cells, 3 newborns vs. 10.6±1.6 ms, n=6 cells, 4 infants, p<0.001).

Conclusions:
There are heterogeneous calcium transients in newborn human myocytes that correspond to a lack of t-tubules. Furthermore, t-tubule development occurs in a heterogeneous manner throughout the first year of life with cells from the same patient exhibiting different degrees of t-tubule development. This is the first study to examine t-tubule development and calcium transients in the very young human ventricular myocardium.
Abstract 90: The Spleen Is an Essential Regulator of Systemic Neovascularization In Vivo
Authors: Ismahil Mohamed Ameen , Hamid Tariq , Kingery Justin R , Prabhu Sumanth D
Published: July 2021

Myocardial neovascularization play key role in the structural remodeling that occurs in heart failure. Prior studies have suggested an important role for splenic monocytes in tissue healing after myocardial infarction; however, the role of macrophages in regulating angiogenesis at the site of tissue injury is complex and not well defined. Moreover, it is unclear how the spleen regulates macrophage-mediated neovascularization. We hypothesized that the spleen plays a critical role in determining macrophage polarity that in turn influences tissue neovascularization. In 7 naïve and 7 splenectomized C57BL/6 mice (splenectomy performed 28 d prior), we performed in vivo Matrigel plug assays. Matrigel (0.6 mL/plug) was injected subcutaneously, either alone or together with peritoneal macrophages (∼20,000 cells/plug) isolated from syngeneic mice, and Matrigel plug neovascularization was assessed after 10 d. Masson trichrome staining and CD31 immunostaining was used to index vascularization. Circulating activated monocytes (CD11b+F480+) and endothelial progenitor cells (EPCs, CD34+Flk-1+) were assessed by flow cytometry. Flow cytometry of peripheral blood revealed significant reductions in circulating activated monocytes (2.20 ± 0.32 vs. 3.95 ± 0.49 %; p=0.002) and EPCs (0.0076 ± 0.0013 vs. 0.018 ± 0.0023; p=0.0002) in splenectomized mice as compared with naïve mice. Splenectomized mice also exhibited markedly reduced neovessel formation in Matrigel plugs indexed by morphometric and immunohistological analyses. The addition of peritoneal macrophages to the Matrigel plug restored vessel formation in splenectomized mice, but did not change neovascularization in naïve mice. Moreover, this restoration of neovascularization occurred with the addition of peritoneal macrophages polarized to an M2 phenotype (using interleukin [IL]-4 or IL-10) but not to an M1 phenotype. We conclude that the spleen is a key determinant of macrophage polarization and function that subsequently impacts the efficiency of tissue neovascularization. Hence, the spleen provides an anti-inflammatory and pro-angiogenic regulatory function in vivo that may be of central importance to the adequacy of myocardial repair and remodeling following tissue injury.
Abstract 93: Sestrin2 Mediates the LKB1-AMPK Signaling Cascade in the Ischemic Heart
Authors: Morrison Alex , Tong Chao , Lee Jun Hee , Karin Michael , Li Ji
Published: July 2021

Background:

AMP-activated Protein Kinase (AMPK) has emerged as a pertinent stress-activated kinase shown to have substantial cardioprotective capabilities against myocardial ischemia/reperfusion (I/R) injury. The regulation of AMPK in the ischemic and reperfused heart is critical to the development of new therapeutic strategies. We hypothesized that a novel stress-inducible protein, sestrin2, mediates AMPK activation in the ischemic heart.

Methods and Results:

C57BL/6 mice were subjected to left anterior descending coronary artery occlusion for different time points of ischemia and I/R in order to detect the signaling activity in the left ventricle. The kinetics of AMPK phosphorylation at Thr
172
of the α catalytic subunit appears brief and bell-shaped, peaking around 10 min (3.6-fold
vs.
sham,
p
<0.01), decreasing with prolonged ischemia, and leveling off to basal levels upon reperfusion. Intriguingly, at 10 min of ischemia, there was also a robust increase in p53 accumulation compared to basal conditions. Consequently, sestrin2 a p53-inducible protein, accumulates at the peak of ischemic AMPK activation (10 min, 2.5-fold
vs.
sham,
p
<0.05). Respective left ventricle lysates were immunoprecipitated with sestrin2 and the immunoblots revealed that sestrin2 forms a complex with AMPK during I/R, moreover, AMPKα2 is the predominant isoform that interact with sestrin2 as compared to AMPKα1 isoform during I/R. Interestingly, LKB1, an upstream AMPK kinase becomes significantly associated with sestrin2 (5 min), while there is a dramatic interaction between sestrin2 and p-AMPK (Thr
172
) (10 min), suggesting the interaction of LKB1 with sestrin2 initiates the phosphorylation of AMPK in this complex. Furthermore, sestrin2 knock out hearts demonstrate impaired ischemic AMPK activation and higher sensitivity to I/R-induced injury as compared to wild type hearts (
p
<0.01).

Conclusions:

Here we show for the first time evidence of a unique mechanism that sestrin2 is a stress-induced scaffold protein that mediates the activation of AMPK in the ischemic myocardium
via
a time-dependent interaction with LKB1. Moreover, sestrin2 deficiency leads to blunted ischemic AMPK activation and increased sensitivity to ischemic insults.
Abstract 014: Bone Marrow Mesenchymal Stromal Cells Rescue Cardiac Function In Streptozotocin-induced Diabetic Rats
Authors: Medei Emiliano , Monnerat Cahli Gustavo , Trentin-Sonoda Mayra , Rodrigues Deivid , Guerra Bárbara , Manso Gabriel , Coutinho Danielle , Cabral-da-Silva Mauricio , Ferreira Andrea , Silva Diorney , Nascimento José , dos Santos Goldenberg Regina , Carneiro-Ramos Marcela , Campos de Carvalho Antonio
Published: July 2021

Cardiac complications are one of the main causes of death in diabetes. Several studies have shown the anti-diabetic effects of bone marrow mesenchymal stromal cells (MSC). In the present study, we investigated whether MSC-transplantation can revert cardiac dysfunction in streptozotocin-induced diabetic rats and the immunoregulatory effects of MSC were examined. The rats were divided in three groups: Non-diabetic, Diabetic and Diabetic-Treated that received 5x106 MSC 4 weeks after establishment of diabetes. Four weeks after MSC-therapy, systemic metabolic parameters, immunological profile and cardiac function were assessed. MSC-transplantation was able to revert the hyperglycemia and body weight loss of the animals, while decreasing to control levels sera corticosterone and the proinflammatory cytokine CINC2 without restoring insulin and leptin plasma levels and oral glucose tolerance. Also, MSC-therapy improved electrical remodeling, shortening the QT and QTc in the ECG and the action potential duration of left ventricular myocytes. No arrhythmic events were observed after MSC-transplantation. MSC-therapy rescued the cardiac beta-adrenergic sensitivity by increasing beta-1 adrenergic receptor expression. Both alpha and beta cardiac AMPK and p-AMPK returned to baseline values after MSC-therapy. However, the total ERK1 and p-ERK1/2 were not different among groups. MSC-therapy reestablished the cardiac control levels of key proinflammatory cytokines/chemokines (IL-6, IL-18, CINC2, CINC3). However MSC-therapy did not modulate toll-like receptor signaling pathways. The results indicate that MSC-therapy was able to rescue cardiac impairment induced by diabetes, normalize cardiac AMPK subunit expression and activity, decrease corticosterone and glycemia and exert local (cardiac) and systemic immunoregulation.
Abstract 020: Cell-sheet-based Bioengineered Human Cardiac Tissue Using Pluripotent Stem Cells For Heart Repair And Disease Models
Authors: Matsuura Katsuhisa , Shimizu Tatsuya , Hagiwara Nobuhisa , Okano Teruo
Published: July 2021

We have developed an original scaffold-free tissue engineering approach, “cell sheet engineering”, and this technology has been already applied to regenerative medicine of various organs including heart. As the bioengineered three-dimensional cardiac tissue is expected to not only function for repairing the broad injured heart but also to be the practicable heart tissue models, we have developed the cell sheet-based perfusable bioengineered three-dimensional cardiac tissue. Recently we have also developed the unique suspension cultivation system for the high-efficient cardiac differentiation of human iPS cells. Fourteen-day culture with the serial treatments of suitable growth factors and a small compound in this stirring system with the suitable dissolved oxygen concentration produced robust embryoid bodies that showed the spontaneous beating and were mainly composed of cardiomyocytes (~80%). When these differentiated cells were cultured on temperature-responsive culture dishes after the enzymatic dissociation, the spontaneous and synchronous beating was observed accompanied with the intracellular calcium influx all over the area even after cell were detached from culture dishes as cell sheets by lowering the culture temperature. The cardiac cell sheets were mainly composed of cardiomyocytes (~80%) and partially mural cells (~20%). Furthermore, extracellular action potential propagation was observed between cell sheets when two cardiac cell sheets were partially overlaid, and this propagation was inhibited by the treatment with some anti-arrhythmic drugs. When the triple layered cardiac tissue was transplanted onto the subcutaneous tissue of nude rats, the spontaneous pulsation was observed over 2 months and engrafted cardiomyocytes were vascularized with the host tissue-derived endothelial cells. These findings suggest that cardiac cell sheets formed by hiPSC-derived cardiomyocytes might have sufficient properties for the creation of thickened cardiac tissue. Now we are developing the vascularized thickened human cardiac tissue by the repeated layering of cardiac cell sheets on the artificial vascular bed in vitro.
Abstract 026: Optical Recording of Cardiac Action Potentials from Human Induced Pluripotent Stem Cell Derived Cardiomyocytes
Authors: Yi B Alexander , Kralj Joel M , Cohen Adam E
Published: July 2021

The electrically excitable properties of cardiomyocytes stem from the activity of ion channels that allow the coordinated entry of ions to generate cardiac action potentials. Disruptions in ion channel function either by drugs or gene mutations can lead to cardiac arrhythmias. The ability to screen drugs or gene mutations rapidly for effects on the cardiac action potential would be of interest for both drug discovery as well as for studies of ion channel function; however, the time-consuming and technically challenging nature of conventional patch clamping can limit the ability to perform high throughput screens. Archaerhodopsin3, or Arch, is an Archaebacterial variant of the membrane protein bacteriorhodopsin that binds a retinal fluorophore whose signal is rapidly responsive to changes in membrane potential. Here, we report the use of Arch to optically record action potentials from human induced pluripotent stem cell-derived cardiomyocytes. Human induced pluripotent stem cells that stably express Arch were generated and then differentiated into cardiomyocytes. As compared to patch clamping, Arch faithfully reproduces many of the key features of cardiac action potentials and may be a tool to be used for high throughput electrophysiological screens of cardiomyocytes.
Abstract 029: Autophagy is Essential For Myofibril Differentiation
Authors: Hiraumi Yoshimi , Huang Chengqun , Andres Allen M , Xiong Ying , Ramil Jennifer , Gottlieb Roberta A
Published: July 2021

Introduction:
Over the last decade, stem/progenitor cell therapy has emerged as an innovative approach to promote cardiac repair and regeneration. However, the therapeutic prospects of are currently limited by inadequate means to regulate cell proliferation, homing, engraftment, and differentiation. Autophagy, a lysosome-mediated degradation pathway for recycling organelles and protein aggregates, is recognized as important for facilitating cell differentiation. Studies have shown that induced pluripotent stem cells (iPCs), which exhibit a predominantly glycolytic metabolism, shift towards oxidative mitochondrial metabolism as a prerequisite for the formation of sarcomeres and differentiation into cardiomyocytes. C2C12 myoblasts are a mouse-derived myogenic progenitor cell line which can be induced to differentiate into myotubes. We hypothesize that autophagy is essential in coordinating transcription factor activity and metabolic reprogramming of mitochondria to support myocyte differentiation.

Methods:
C2C12 myoblasts were cultured in DMEM (10% FBS) and induced to differentiate into myotubes with DMEM (2% horse serum) for 6d. To disrupt autophagy, cells were: 1) transfected with 50nM Atg5 siRNA for 8h twice over a 48h period, or 2) treated with 10nM bafilomycin A1 or vehicle control for a 3h/day for the first 3d of differentiation. GFP-LC3 adenovirus was employed to visualize autophagy. Western blot and real-time qPCR were used to examine proteins and transcripts of interest.

Results:
We observed increased LC3-II levels and GFP-LC3 puncta during the differentiation of C2C12 cells, suggesting the involvement of autophagy in this process. Transient inhibition of autophagy during the early stages of differentiation with either Atg5 siRNA or bafilomycin A1 interfered with myotube formation and attenuated the upregulation of myogenic transcription factors MyoD and myogenin. Differentiation was accompanied by an increase in PGC1α mRNA, mitochondrial mass, and oxygen consumption, all of which were blocked by disruption of autophagy.

Conclusions:
Autophagy coordinates transcription factor expression and mitochondrial turnover essential for cell differentiation.
Abstract 032: Impaired Cardiac Autophagy In Metabolic Syndrome Despite Intact AMPK And mTOR Signaling
Authors: Ravindran Nandini , Bazan Carlos , Ito Bruce R , Gottlieb Roberta A , Mentzer Robert M
Published: July 2021

Objective:
We previously reported that basal cardiac autophagy is lower in the setting of metabolic syndrome (MetS) (obesity, dyslipidemia, insulin resistance) and is associated with attenuation of cardioprotection after ischemic preconditioning. To understand the underlying mechanisms and exclude effects of the extracellular milieu, we investigated the roles of two major cellular energy sensing pathways, adenosine monophosphate-activated protein kinase (AMPK), and mammalian target of rapamycin (mTOR), using adult rat ventricular cardiomyocytes (ARVM) isolated from Sprague Dawley (SD) and Zucker obese (ZO) rats.

Methods:
ARVM were cultured overnight and subjected to nutrient deprivation (1 hr), or treated with 1µM phenformin (Ph), an AMPK activator, or 5µM rapamycin (Rap), an mTOR inhibitor. Immunoblotting was used to measure phosphorylation of AMPK, p70S6 kinase (a downstream target of mTOR), LC3-I, -II, and p62 (autophagic clearance).

Results:
In ARVM from SD rats, starvation or Ph increased p-AMPK and decreased p-p70S6K, accompanied by increased LC3-II and 50% reduction in p62. Rapamycin decreased phosphorylation of p70S6K, increased LC3-II and decreased p62. In ZO ARVM, Ph and Rap also activated AMPK and inhibited mTOR; however, LC3-II was unchanged (Fig.) and p62 clearance was blunted.

Conclusions:
Despite appropriate activation of AMPK and inhibition of mTOR, autophagy is impaired in ARVM from ZO rats. These cells retain the MetS phenotype, facilitating efforts to delineate the molecular basis of impaired autophagy and vulnerability to ischemia/reperfusion injury in MetS. This could lead to new approaches to myocardial protection in at-risk patients.
Abstract 036: The Role Of Spinal Modulation On The Ventricular Electrophysiology In A Porcine Model
Authors: Zhou Wei , Yamakawa Kentaro , Ajijola Olujimi , Yagishita Daigo , Takemoto Mariko , So Eileen , Beharash Peyman , Vaseghi Marmar , Shivkumar Kalyanam , Mahajan Aman
Published: July 2021

Background:
Enhanced cardiac sympathetic tone has been associated with ventricular arrhythmias and sudden cardiac death. The spinal cord is an important integrative region of afferent and efferent pathways that participate in cardiovascular regulation. The purpose of this study is to investigate the role of spinal processing of cardiac afferent information on ventricular electrophysiology during cardiac sympathoexcitation.

Methods:
Female Yorkshire pigs (n=5) underwent surgical exposure of the heart and left stellate ganglion (LSG) through thoractomy as well as the dorsal and ventral roots of the spinal cord through laminectomy. A 56-electrode sock was placed over the ventricles to record epicardial electrograms. Animals underwent LSG stimulation in intact, after dorsal root transaction (DRTx), and followed by ventral root transaction (DVRTx). Activation recovery intervals (ARIs) were measured at each electrode before and during LSG stimulation.

Results:
With intact roots LSG stimulation resulted in significant global ARI shortening by 12.9% (p<0.05). After DRTx, mean global ARI shortened by 7.2%. LSG stimulation after DRTx and DVRTx resulted in greater ARI shortening compared to LSG stimulation with intact roots (21.5 and 18.4 vs 12.9%, p<0.05, see figure 1 below). ARI shortened more during LSG stimulation after DRTx than that after DVRTx (21.5 vs. 18.4%, p<0.05).

Conclusion:
Spinal afferent pathways play an inhibitory role in sympathoexcitation of ventricle induced by LSG stimulation. This finding provides insight into the mechanism underlying the beneficial effects of thoracic epidural anesthesia in reducing ventricular arrhythmias.
Abstract 038: A Novel Histology Based Classification System to Identify Patients at Risk for Postoperative Atrial Fibrillation
Authors: Mirza Mahek , Strunets Anton , Holmuhamedov Ekhson , Sra Jasbir , Werner Paul H , Khandheria Bijoy K , Tajik A Jamil , Kress David C , Jahangir Arshad
Published: July 2021

Postoperative atrial fibrillation (PoAF) is a common complication in up to 40% of patients after cardiac surgery, increasing morbidity, hospital stay and costs. The myocardial substrate underlying PoAF is not fully characterized. The objective was to assess the impact of atrial fibrosis on incident AF and define the fibrosis threshold level predictive of PoAF.

Methods:
Right atrial appendages removed from patients undergoing elective CABG with no history of AF or class III/IV heart failure were used to characterize the ratio of collagen to myocardium (Masson’s trichrome; NIH ImageJ software; Fig A), which was correlated with incident AF. Percentage burden of fibrosis predictive of PoAF with high sensitivity and specificity was determined by ROC curve.

Results:
Of 28 patients (67±10 years, 64% males), 15 had PoAF. There were no age, gender or comorbidity differences between groups. Compared to the group that remained in sinus rhythm, patients with PoAF had a significantly higher ratio of extracellular collagen to myocardium (45±16% vs. 5±4%, p <0.001; Fig B). A threshold ratio of 12.7% collagen to myocardium (ROC area under the curve 0.997; z statistic 137; P<0.0001) with 96% sensitivity and 97% specificity identified those with PoAF (Fig C). A classification system based on histological extent of atrial fibrosis is proposed for identifying patients at risk for PoAF (Fig D).

Conclusion:
Ongoing studies will confirm the predictive value of this new classification system for identifying the atrial substrate predisposing PoAF and correlate with preoperative cardiac imaging and circulatory serum biomarkers to provide a novel noninvasive tool to stratify patients at risk for PoAF.
Abstract 040: Upregulation Of Cyp Epoxygenase In The Connecting Tubule(cnt)/cortical Collecting Duct (ccd) Is Essential For High Potassium (k) Intake-induced Antihypertensive Effect
Authors: Lin Daohong , Zhang Chengbiao , Wang Lijun , Wang Wenhui
Published: July 2021

Cyp epoxygenase is responsible for metabolizing arachidonic acid to epoxyeicosatrienoic acid (EET) in the kidney and vascular tissues. EET has been shown to cause vasodilation by stimulating Ca
2+
-activated K channels in vascular smooth muscles and to have natriuretic effect by inhibiting the epithelial Na channel (ENaC) in the kidney. In the present study we used real time PCR technique to examine the effect of high salt intake or high K intake on Cyp2c44 (a major type of Cyp epoxygenase in the mouse kidney) in the proximal tubule (PxT), thick ascending limb (TAL), distal convoluted tubule (DCT) and the CNT/CCD. An increase in dietary Na content stimulates the expression of Cyp2c4 in TAL, DCT and CNT/CCD but not in PxT while an increase in dietary K intake augments the expression of Cyp2c44 only in DCT and CNT/CCD. Neither high salt intake nor high K intake has a significant effect on the blood pressure (BP) in wt mice. However, high K intake increased BP in CNT/CCD specific conditional knockout (KO) mice. In contrast, the high Na intake did not significantly increase the BP in those KO mice. This suggests that Cyp2c44 in the CNT/CCD plays a key role in preventing hypertension induced by increasing dietary K intake. Administration of amiloride (a ENaC inhibitor) restored the normal BP in KO mice fed high K diet, suggesting that down-regulation of Cyp2c44 may enhance the Na absorption in the CNT/CCD. This notion was also supported by metabolic cage study demonstrating that renal Na excretion was compromised in KO mice. We conclude that Cyp2c44 plays a key role in stimulating renal Na excretion during increasing dietary K intake and that Cyp-epoxygenase is required for antihypertensive effect induced by high K intake.
Abstract 071: Estrogen-related Receptor Gamma Induces Cardiac Hypertrophy By Activating Gata4
Authors: Kwon Duk-hwa , Eom Gwang Hyeon , Kee Hae Jin , Nam Yoonseok , Cho Young Kuk , Kim Don-Kyu , Koo Jayoung , Ko Jeong-Hyeon , Shin Sera , Choe Nakwon , Joung Hosouk , Kim Ha-neul , Kim Hyung Seok , Nam Gwang-Il , Lee In-Kyu , Park Seung Bum , Choi Hueng-Sik , Kook Hyun
Published: July 2021

Introduction—
Estrogen-related receptor gamma (ERRγ) is an orphan nuclear receptor that has biological roles mainly in metabolism and it controls metabolic switching in perinatal heart. In adult heart diseases, however, the functional roles of ERRγ have not yet been elucidated.

Hypothesis—
In the present study, we aimed to characterize the role of ERRγ in cardiac hypertrophy. Here we show that ERRγ provokes cardiac hypertrophy by inducing GATA4 and that its inverse agonist, GSK-5182, prevents cardiac hypertrophy.

Methods and Results—
The functional roles of ERRγ in association with development of cardiac hypertrophy were examined in primarily cultured cardiomyocytes, in animal models, and in heart samples from human hypertrophic cardiomyopathy patients. ERRγ expression was increased in hearts obtained from human hypertrophic cardiomyopathy patients and in both agonist-induced cellular models and aortic banding-induced animal models of cardiac hypertrophy. Transgenic overexpression in mouse heart as well as forced expression of ERRγ in cardiomyocytes induced hypertrophic phenotypes. Knock-down of ERRγ blocked agonist-induced hypertrophic phenotypes. ERRγ directly bound to the proximal ERR-responsive element in the GATA4 promoter in a sequence-specific manner and thereby induced transcription. ERRγ-induced hypertrophy was blocked by inhibition of GATA4. GSK-5182 completely blocked cardiac hypertrophy in cardiomyocytes. It also prevented aortic banding-induced cardiac hypertrophy and fibrosis in mouse heart.

Conclusion—
These findings demonstrate a novel ERRγ/GATA4 signal cascade in the development of cardiac hypertrophy and suggest GSK-5182 as a possible therapeutic.
Abstract 077: Small Heterodimer Partner, An Orphan Nuclear Receptor, Inhibits Cardiac Hypertrophy
Authors: Nam Yoon Seok , Kee Hae Jin Kee , Kim Yoojung , Eom Gwang Hyeon , Shin Sera , Kim Don-Kyu , Choi Hueng-Sik , Joung Hosouk , Cho Young Kuk , Ko Jeong-Hyeon , Choe Nakwon , Kook Hyun
Published: July 2021

Background:
Small heterodimer partner (SHP; NR0B2) is an atypical orphan nuclear receptor lacking DNA binding domain. SHP directly modulates the activities of other nuclear receptors and regulates a variety of cellular events such as cell differentiation, proliferation, and metabolism in various tissues. However, the role of SHP in heart has not yet been elucidated. Thus, in this study, we tried to investigate the functional roles of SHP in heart physiology and in the development of cardiac hypertrophy.

Methods and Results:
We observed that SHP knock-out mice elicited cardiac hypertrophic features determined by heart weight to body weight or to tibia length ratios. Fetal genes, such as atrial natriuretic factor (ANF) or beta myosin heavy chain (βMHC) were significantly up-regulated in SHP knockout mice heart. In neonatal rat ventricular cardiomyocytes (NRVCs), phenylephrine (PE) reduced promoter activation of SHP and decreased protein level of SHP. Adenovirus-mediated over-expression of SHP (Adeno-SHP) significantly reduced hypertrophic responses induced by PE as assayed by [3H]-leucine incorporation, Nppa promoter activity, and cell size measurement. Adeno-SHP significantly reduced hypertrophy-associated proteins. In contrast, knock-down of SHP by small hairpin RNA, decreased both Myh7 and Nppa promoter activities, whereas it up-regulated ANF or α-tubulin expressions. Metfomin (N,N-Dimethylimidodicarbonimidic diamide), an anti-diabetic agent, up-regulated SHP in dose-response fashion. PE-induced activation of Nppa promoter and [3H]-leucine incorporation were completely blocked by metformin. PE-induced down-regulation of SHP was blunted by simultaneous treatment of metformin. Metformin-mediated antihypertrophic action was not observed when the SHP was down-regulated by small interfering RNA against to SHP.

Conclusions:
These results suggest that atypical orphan nuclear receptor SHP prevents cardiac hypertrophy and it mediates metformin-mediated antihypertrophic responses, implicating that theses signal cascades may serve as a novel therapeutic target of treatment of hypertrophic cardiomyopathy patients.
Abstract 079: Estrogen Receptor beta Regulation Of miRNA Expression In The Heart - A Hint For Sex Differences In Hypertrophy
Authors: Queirós Ana , Eschen Claudia , Fliegner Daniela , Regitz-Zagrosek Vera , Sanchez Ruderisch Hugo
Published: July 2021

Sex differences (SD) in cardiovascular diseases are described and have been attributed to the effects of sex hormones, such as estrogen (E2). Our previous studies in a mouse model of pressure overload revealed SD in fibrosis and apoptosis related genes that were abolished in estrogen receptor β deficient (ERβ-/-) mice. Other studies focus on miRNA regulation by E2, but little is known about sex-specific regulation of miRNAs in heart diseases. We hypothesize that E2 and ERβ are regulators of miRNA expression in the heart potentially contributing to the molecular mechanism of SD observed in cardiovascular diseases.

This project aims to identify SD in the expression of miRNAs in a 9-week transverse aortic constriction (TAC) model and the possible role of E2 and ERβ in their regulation in the heart.

A Targetscan analysis of 80 sex-specific dysregulated genes in hypertrophy allowed identifying 157 different miRNAs that could target them. Based on their expression in the heart we selected 60 miRNAs for quantification by qRT-PCR. In WT mice, 23 miRNAs showed SD in their expression in hypertrophy, all of them showing a higher expression in males than in females. All these significant SD were abolished in ERβ-/- mice. A direct comparison of some of these miRNAs in WT and ERβ-/- female animals identified nine miRNAs significantly higher expressed in the knock-out animals (let-7e, miR-106b, miR-130a, miR-133a, miR-20a, miR-24, miR-27b, miR-29a and miR-378). In vitro studies performed using a female cardiomyocyte cell line (AC16) showed a down-regulation of eight of these miRNAs after a 48h treatment with E2, being the exception miR-133a. The latter showed however a down-regulation after 48h treatment with an ERβ specific agonist, as well as let-7e, miR-106b, miR-130a, miR-20a, miR-24, miR-27b and miR-29a. The effect of treatment with ERα was only visible as a down-regulation of miR-24, miR-29a and miR-378. The potential of these miRNAs having targets in the same pathway and acting in a synergistic way is still under investigation.

ERβ is for the first time identified as a major regulator of miRNA expression in the heart. It may play an important role in determining SD in cardiac hypertrophy, being responsible for the inhibition of miRNA expression in the female heart.
Abstract 080: Eya4 Induces Hypertrophy Via Regulation Of p27kip1
Authors: Williams Tatjana , Hundertmark Moritz , Nordbeck Peter , Voll Sabine , Muehlfelder Melanie , Schraut Susanna , Elsner Ines , Schoenberger Jost , Ritter Oliver
Published: July 2021

Introduction:
E193, a truncating mutation in the transcription cofactor Eyes absent 4 (Eya4) causes hearing impairment followed by heart failure. Here we identified the Eya4 dependent molecular mechanisms leading to the cardiac phenotype in the E193 mutation.

Methods and Results:
First we showed in vitro that the cyclin-dependent kinase inhibitor protein p27kip1 is a direct target of Eya4/Six1 and is suppressed upon Eya4 overexpression, whereas E193 has a dominant negative effect, releasing Eya4 mediated suppression of p27. We next generated transgenic mice with cardiac specific constitutive overexpression of full-length Eya4 or the mutant form E193. While E193 transgenic mice developed age-dependent DCM, Eya4 mice displayed cardiac hypertrophy already under basal conditions as judged by increases in heart weight and cardiomyocyte cross-sectional areas along with increases in myocardial dimension and mass. These two distinct cardiac phenotypes were even more aggravated upon pressure overload suggesting Eya4 is a regulator of cardiac hypertrophy. We also observed that the activity of Casein Kinase 2-α and the phosphorylation status of HDAC2 were significantly upregulated in the Eya4 transgenic mice, while they were significantly reduced in E193 mice, under baseline conditions and pressure overload. We were also able to identify a new human mutation (E215) with a phenotype comparable to the one seen in E193 patients.

Conclusion:
Our results implicate that Eya4/Six1 regulates cardiac hypertrophic reactions via p27/CK2-α/HDAC2 and indicate that truncating mutations in Eya4 interfere with this newly established signalling pathway.
Abstract 089: Response Gene To Complement 32 Is Involved In Glucose Metabolism Disorder In Endothelial Cells And Mediated By P53
Authors: Guo Shuzhen , Lian Hongjian , Chuo Wenjing , Feng Xuanchao
Published: July 2021

Response gene to complement 32 (RGC-32) was considered as an apoptosis-promoting factor. To explore the novel functions of RGC-32, a DNA array analysis was performed in Human Microvascular Endothelial Cells (HMEC) treated with SiRNA, which showed that glucose metabolism was one of most dramatically changed biological process. qPCR analysis also proved that many glucose metabolism related genes were changed dramatically in RGC32 o/e HMEC, one of which was Insulin-like growth factor binding protein 4(IGFBP4). We propose that RGC-32 may be a potential factor involved in the cycle of endothelial glucose metabolism besides glucose induced apoptosis. Then we found that expression of RGC-32 was dramatically increased in mice hearts and adipose tissue after high fat diet treatment, as well as in HMEC after high glucose (30mM) treatment. In high fat diet treated endothelial specific RGC-32 transgenic mice (VE-Cad/RGC-32), decreases in area under the curve (AUC) of glucose tolerance test were observed when compared to their wild type littermates. Upregulated P53 and downregulated IGFBP4 mRNA expression level were also observed in the heart of VE-Cad/RGC-32 mice.Furthermore, we found that P53 was upregulated in RGC-32 o/e HMEC and blocking of p53 was able to decrease the RGC-32-related induction of apoptotic cells. We also observed that RGC-32 can not only decrease IGFBP4 but also reverse the decrease of IGFBP4 in high glucose or insulin condition. Interestingly, after treated the cells with pifithrin-a, a specific P53 inhibitor, decreased IGFBP4 by RGC-32 was rescued. These results demonstrate that RGC-32 may have some kind of connections with glucose metabolism besides its well-known apoptosis-promoting characters and P53 may be a mediator of RGC-32 related glucose metabolism disorder as well as apoptosis.These findings will provide some clues for novel insights into the functions of RGC-32.