bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 29: Isolation of Cardiomyocytes Undergoing Mitosis with Complete Cytokinesis
Authors: Milliron Hsiaoyun Y , Weiland Matthew , Bowman Megan , Jovinge Stefan
Published: July 2021

Adult cardiomyocytes (CMs) fail to exit the cell cycle and result in polyploidization and multi-nucleation. This extremely limited renewal potential is a key barrier for cardiac regeneration after heart injury. The use of traditional cell cycle assays such as Ki67 or purine analogue administration does not allow for isolation of viable CMs in late-M phase. We hypothesize that a molecular beacon (MB)-based method could be developed to isolate a highly pure population of hiPSC-derived CMs capable of completing mitosis and cytokinesis. We first differentiated human PSCs into cardiomyocytes using the small molecules CHIR and IWR and then applied MBs targeting
CDC20
mRNAs, followed by fluorescence-activated cell sorting (FACS). Validation of cell cycle-specific and mitosis-associated gene expressions of cell fractions sorted from G2 and M-phase were performed by RT-qPCR prior to transcriptional profiling by single-cell RNA sequencing. Gene set enrichment analysis (GSEA) confirmed the observation of the gene ontology (GO) analysis. The ‘GO_cytokinesis’ and the ‘GO_cell separation after cytokinesis’ gene sets were highly enriched and predominantly upregulated in the
CDC20
(+) (M-phase) population compared with the
CDC20
(-) (G2-phase) population. Furthermore, using these data sets we expect to develop robust surface marker based flow cytometry protocols at a number and purity allowing for statistically significant genome-wide integrative transcriptome and epigenome analysis. In conclusion, the isolation of CMs using
CDC20
MB and DNA content dye proved to enrich for cells that were confirmed by GSEA to be CMs in cytokinesis.
Abstract 302: Chronic Neuropathic Pain Increases Susceptibility to Myocardial Ischemia/Reperfusion Injury by Sirt1 Carbonylative Inactivation
Authors: Li Chen , Yang Jingrun , Yu Lu , Gu Chunhu , Ma Heng
Published: July 2021

Pain is not a symptom that exists alone, but whether chronic pain enhances susceptibility to myocardial ischemia/reperfusion (MI/R) injury and the underlying mechanisms remain unknown. Reactive aldehydes contribute to pain pathologies and cardiac injury, suggesting that aldehyde dehydrogenase (ALDH2), which detoxifies aldehydes, may regulate chronic pain related MI/R injury. In this study, chronic neuropathic pain was induced by chronic compression of the dorsal root ganglion (CCD). CCD for 2 weeks, ALDH2 KO or wild-type (WT) littermates were subjected to in vivo MI/R. CCD-WT mice exhibited heightened nociception and correlated with circulating aldehyde (4-HNE) accumulation and cardiac protein carbonylation. CCD induced 4-HNE overload provoked cardiac SIRT1 carbonylative inactivation and impairment the cardioprotection of LKB1-mediated AMPK activation, which resulting in enhanced MI/R injury and higher mortality compare with pain free WT mice. Chronic neuropathic pain enhanced susceptibility to MI/R injury was further exacerbated by ALDH2 deficiency in which associated with more impaired SIRT1-LKB1-AMPK signaling. However, treatment of CCD-WT mice with ALDH2-selective activator (Alda-1) or cardiac specific ALDH2 upregulation by AAV9-cTNT-mediated gene delivery significantly reduced chronic neuropathic pain-induced SIRT1 carbonylative inactivation and decreased MI/R injury (minor infarct size, less apoptosis, and elevated cardiac function). These results strongly suggest that elevated reactive aldehyde concentration, like that observed in the presence of chronic pain, may render cardiomyocytes more susceptible to MI/R injury by SIRT1 carbonylative inactivation and impairment the cardioprotection of LKB1-mediated AMPK activation. ALDH2 activation blocked reactive aldehyde overproduction induced carbonyl stress and attenuated myocardial ischemic vulnerability in chronic pain individual.

Keywords:
chronic pain; myocardial ischemia/reperfusion; ALDH2; 4-HNE; carbonylation
Abstract 319: Role of Phosphodiesterases 4B and 4D in cAMP Signal Compartmentation Around Calcium Handling Proteins
Authors: Kraft Axel E , Nikolaev Viacheslav O , Conti Marco
Published: July 2021

Phosphodiesterase subfamilies 4B and 4D are critically involved in the regulation of cAMP signaling in mammalian cardiomyocytes. Alterations in activity of these enzymes in human hearts have been shown to result in arrhythmia and heart failure. The aim of this project was to systematically investigate specific roles of PDE4B and PDE4D in regulating cAMP dynamics in three distinct subcellular microdomains formed around Ca
2+
handling proteins, such as L-type calcium channels (LTCCs), sarco/endoplasmic reticulum Ca
2+
-ATPase (SERCA) and ryanodine receptors (RyRs), to understand their impact on heart function and disease.

Transgenic mice expressing three different Förster resonance energy transfer (FRET) based cAMP biosensors targeted to caveolin rich plasma membrane, SERCA and RyR microdomains, were crossed with PDE4B and PDE4D knockout mice. Using FRET imaging in ventricular cardiomyocytes freshly isolated from wildtype and knockout mice, direct analysis of the specific effect of both PDE subfamilies in these microdomains could be performed by measuring the kinetics of local cAMP degradation.

Our results indicate that the cAMP kinetics around the LTCC microdomain is critically regulated by PDE4B and PDE4D. So far was it has been known that the isoform that is associated with the RyR microdomain belongs to the PDE4D family, however we found PDE4B to be involved in regulating the cAMP signaling in this microdomain. PDE4D deletion also revealed the critical role of this subfamily for the control of cAMP dynamics in the SERCA microdomain of adult mouse cardiomyocytes. Basal levels of cAMP were elevated when PDE4B was absent from any of the PDE4B-regulated microdomain, whereas no such alterations were detected for PDE4D knockout cells.

These data demonstrate that all three microdomains are differentially regulated by PDEs. Even within one organelle such as sarcoplasmic reticulum, we could show the existence of at least two distinct cAMP microdomains, i.e. around RyR and SERCA which are preferentially controlled by PDE4B and PDE4D, respectively. In the future, we aim to systematically analyze biochemical composition of the three microdomains, their distinct roles in cardiac function and disease as well as ways of their pharmacological modulation.
Abstract 321: Protein Tyrosine Phosphatase 1B: a Novel Regulator of Proliferation and Apoptosis in the Development of Pulmonary Arterial Hypertension
Authors: Moazeni Shayan , Ruffenach Gregoire , Sarji Shervin , Cunningham Christine , Vaillancourt Mylene , Umar Soban , Eghbali Mansoureh
Published: July 2021

Background:
Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling that leads to an increase in pulmonary arterial pressure resulting in right ventricle failure and death. PAH is driven by pulmonary artery smooth muscle cell (PASMC) proliferation and resistance to apoptosis. Protein Tyrosine Phosphatase 1B (PTP1B), a negative regulator for platelet-derived growth factor (PDGF) and BCL-2, has recently been implicated in PAH in humans. While PDGF and BCL-2 are increased in PAH patients, the pathway for regulating BCL-2 and PDGF is poorly understood. We aim to investigate if PTP1B has a role in proliferation and resistance to apoptosis in PAH in human PACMCs and in the Sugen/Hypoxia/Normoxia (Su/Hx/Nx) PH rat model.

Method:
Adult male Sprague-Dawley rats were treated with single intraperitoneal dose of SU5416 (20 mg/kg) and kept in Hx for 3 weeks followed by Nx for 2 weeks. Saline treated rats kept in Nx for 5 weeks served as control (n=4/group). RV catheterization was performed terminally for recording RV systolic pressure (RVSP). RV, LV, and interventricular septum (IVS) were isolated for Fulton index (FI, RV/IVS+LV). We analyzed gene expression in lungs via qPCR. Healthy hPASMCs were incubated with a PTP1B inhibitor (Ethyl-3,4-dephostatin) at IC50=0.58ug/ml for 24hrs under Nx conditions and cells were stained with Ki67 to assess proliferation.

Results:
Su/Hx/Nx rats had severe PH evidenced by a significantly elevated RVSP compared to control (88.97+/- 13.67 vs 28.47+/- 2.22 mmHg, p<0.05). PH rats also showed severely reduced RV function and increased RV hypertrophy (FI= 0.7+/- 0.063 vs 0.274 +/-0.01, p<0.05). PH lungs exhibited severe pulmonary vascular remodeling with excessive growth of the PASMCs. PTP1B was significantly decreased in PH lungs compared to controls (0.158+/-0.0647 vs 1+/-0.06, P<0.05). BCL-2 expression was significantly increased in PAH compared to control (2.01+/-0.162 vs 1 +/-0.1, P<0.01). Inhibition of PTP1B in cultured hPASMCs increased proliferation by ~2 fold as assessed by Ki67 positive cells (n=3).

Conclusion:
Severe angioproliferative PH in rats is associated with a downregulation of PTP1B and increased expression of BCL-2 and PASMC proliferation.
Abstract 350: The Developmental Origin Of Cardiac Fibroblasts Influences the Efficiency of Direct Reprogramming to Cardiomyocytes
Authors: Ranjbarvaziri Sara , Ardehali Reza
Published: July 2021

Direct conversion of fibroblasts to cardiomyocytes (CM) is advancing the field of cardiac regeneration. Despite advantages of direct reprogramming, the presence of residual epigenetic memory of the original cells may hinder clinical transition. Thus, choosing a starting cell with similar ontogeny to the desired reprogrammed cell may overcome some of the limitations. Expression of key cardiogenic genes shared between cardiac fibroblasts (CFb) and CM in addition to the abundance of these cells, suggest that CFb may be the optimal autologous cell source for therapeutic manipulation in treating heart disease. We hypothesized that progenitors, transiently expressing Mesp1 generate a sub-population of CFb which are more prone to direct reprogramming and adopt a cardiomyocyte gene profile due to their maintained epigenetic memory. We generated a Mesp1CremTmG mouse to label all cells expressing Mesp1 and their progeny and we observed that the majority of the cells in the heart including CFb are derived from Mesp1 cells. We showed that ~80% of resident CFb are derived from Mesp1 while a minor non-Mesp1 subset also exists. We compared the reprogramming efficiency of CFb of Mesp1 origin to CFb of other origin to induced CM (iCM) by overexpression of specific cardiac transcription factors. Results from immunostaining and gene analysis showed higher expression of cardiac muscle markers in CM induced from Mesp1 CFb. To further delineate potential differences between two subsets, we performed RNAseq and our results showed that the non Mesp1 CFb were enriched in neural crest related genes. We generated Pax3CremTmG mice to lineage trace neural crest-derived cells. Our results confirmed a minor contribution of Pax3 cells to CFb. We developed a modified CLARITY technique to transform the heart into an optically-transparent organ for light-sheet fluorescence imaging. We observed that Pax3 CFb were mainly located in the wall of aorta while Mesp1 CFb were distributed throughout the heart. Additionally, we are studying whether each CFb subset has the tendency to generate a specific subtype of iCM (ventricular, atrial, and nodal CM). These results can identify distinct sub-populations of CFb, which can generate functional cardiomyocytes for cardiac-regenerative therapies.
Abstract 359: Autosis is Triggered by Ischemia/Reperfusion in the Heart
Authors: Nah Jihoon , Zhai Peiyong , Levine Beth , Sadoshima Junichi
Published: July 2021

Although autophagy generally acts as a protective function in cardiomyocytes (CMs), uncontrolled or excessive activation of autophagy can be detrimental to CMs under some conditions. However, whether autophagy mediates cell death for CMs remains largely unknown. Recently, it has been shown in HeLa cells that excessive activation of autophagy induces cell death with characteristic morphological and biochemical features, and this form of cell death is termed autosis. Here we investigated whether death of CMs is induced by autosis in the presence of TAT-Beclin1, an autophagy-inducing peptide, and ischemia/reperfusion. TAT-Beclin1 treatment dose-dependently increased the level of LC3-II and decreased that of p62. TAT-Beclin1-treated CMs showed increases in trypan-blue-positive cells suggesting that autophagy-related cell death is induced by TAT-Beclin1. TAT-Beclin1-treated CMs showed the typical morphological features of autosis, including increased autophagic vacuoles and empty vacuoles, perinuclear spaces with cytoplasmic materials, and ballooning under electron microscopic observation. TAT-Beclin1-induced autosis in CMs was not inhibited by inhibitors of apoptosis, necrosis, or lysosomal degradation. Although autosis is known to be inhibited by cardiac glycosides, since cardiomyocyte Na
+
-K
+
-ATPase in rodents is relatively resistant to cardiac glycosides, we generated adenovirus harboring shRNA-α1 subunit of Na
+
-K
+
-ATPase. We found that downregulation of the α1 subunit of Na
+
-K
+
-ATPase inhibits TAT-Beclin1-induced autosis in CMs. We also found that ischemia/reperfusion induces autosis in the mouse heart, as evidenced by the presence of characteristic morphological features of autosis, including the presence of autophagosomes, empty vacuoles, and perinuclear space. Lastly, in order to elucidate the molecular mechanism mediating autosis in the heart, we conducted unbiased screenings of 150 kinase inhibitors and identified several classes of kinase inhibitors suppressing TAT-Beclin1-induced autosis in CMs. These results indicate that autosis occurs in the heart during ischemia/reperfusion injury. Our findings may provide novel therapeutic targets for treatment of cardiac cell death during ischemia/reperfusion.
Abstract 365: Change in Saturation of Oxygen and Patent Foramen Ovale in Postural Orthostatic Tachycardia Syndrome
Authors: AbuAlSha’r Mu’taz F , Abu-Jajeh Ra’ed K , Ahmad Mo’yed N , Ashangari Chandralekha , Hamid Stacy E , Suleman Amer
Published: July 2021

Background:
Postural Orthostatic Tachycardia Syndrome (POTS) is a constellation of signs and symptoms that occurs when the patient stands upright. It affects primarily young women. On the other hand, PFO is a hidden condition that doesn’t cause signs and symptoms in most cases.

Method:
331 POTS patients were randomly selected during the period of June 2014 to April 2016 from our clinic and they underwent PFO test. In addition to that, patients underwent tilt test with SPO2 recordings every 2 minutes until 10 minutes to calculate the change in SpO2 of that period of time.

Results:
Out of 331 patients, 292 patients were females (88%; n=292 age 32.48 ± 10.78), 39 patients were males (12%; n=39 age 28.87 ± 11.69). 136 patients (41%) had no drop in their SPO2 and 17 of 136 patients had positive(+) PFO test (Grade 1 PFO in 4 patients, Grade 2 PFO in 2 patients, Grade 3 PFO in 3 patients and Grade 4 PFO in 8 patients). 77 patients (23%) had a drop of 1% in their SPO2 and 11 of 77 patients had + PFO test (Grade 1 PFO in 3 patients, Grade 2 PFO in 1 patient, Grade 3 PFO in 2 patients and Grade 4 PFO in 5 patients). 46 patients (14%) had a drop of 2% in their SPO2 and 10 of 46 patients had + PFO test (Grade 1 PFO in 2 patients, Grade 3 PFO in 2 patients and Grade 4 PFO in 6 patients). 16 patients (5%) had a drop of 3% in their SPO2 and 3 of 16 patients had + PFO test (Grade 1 PFO in 1 patient, Grade 2 PFO in 1 patient, Grade 3 PFO in 1 patient). 9 patients (3%) had a drop of 4% in their SPO2 and 1 of 9 patients had + PFO test (Grade 2). 5 patients (1.5%) had a drop of 5% in their SPO2 and 2 out of 5 patients had + PFO test (Grade 1 PFO in 1 patient and Grade 4 PFO in 1 patient). 2 patients (0.6%) had a drop of 6% in their SPO2 and both of them had + PFO test (Grade 2 PFO in 1 patient, Grade 4 PFO in 1 patient). 40 patients (12%) had a rise in their SPO2 and 4 of 40 patients had + PFO test (Grade 2 PFO in 2 patients, Grade 3 PFO in 1 patient and Grade 4 PFO 1 patient).

Conclusion:
Our research results demonstrated that 15% of patients with POTS have PFO. Though orthodeoxia is relatively uncommon, this study demonstrated orthodeoxia in 59% of patients. Only 2% patients showed orthodeoxia of >5%, but further studies should be done to define the clinical significance of the study.
Abstract 367: Plasma Based cBIN1 Correlates with Myocardial Health in Heart Failure with Reduced Ejection Fraction
Authors: Baum Rachel , Xu Bing , Xie Yu , Zadikany Ronit , Hitzeman Tara , Agvanian Sosse , Geft Dael , Chang David , Moriguchi Jaime , Hage Antoine , Azarbal Babak , Czer Lawrence , Kittleson Michelle , Patel Jignesh , Kobashigawa Jon , Hamilton Michele , Hong TingTing , Shaw Robin M
Published: July 2021

Background:
Acquired heart failure (HF) originates with failing heart muscle, yet it is currently not possible to assay the biochemical health of cardiomyocytes. A key pathophysiology of HF is weakened calcium transient under the regulation of a membrane deformation protein bridging integrator 1 (BIN1). BIN1 decreases with HF and is blood available. We hypothesized that a recently cloned cardiac BIN1 isoform (cBIN1, BIN1+13+17) can be selectively measured to diagnose heart muscle health.

Methods:
Expression of cBIN1 in human heart and plasma was determined by immunoprecipitation, western blotting, and mass spectrometry analysis. A cBIN1 specific ELISA was developed using the combination of anti-BIN1 exon 17 (clone 99D from Sigma) and 13 (gift from Sarcotein Diagnostics) antibodies. Plasma cBIN1 concentration was then measured in a large clinical cohort of HFrEF (N=180) patients and compared to a sex and age matched cohort of healthy volunteers. Plasma cBIN1 concentration was also compared to NT-proBNP for its ability to detect patients with HFrEF.

Results:
Biochemistry and mass spectrometry confirms that cBIN1 is expressed in human myocardium. An ELISA assay selects for the cBIN1 isoform which significantly reduced in HFrEF (3.0±0.3, n=286, mean±SEM, ng/ml, p<0.001) versus matched healthy volunteers (3.0±0.3, n=340). Low plasma cBIN1 diagnoses HFrEF with a ROC area under the curve of 0.92. cBIN1 ROC characteristics are additive to those of NT-proBNP. In addition, low plasma cBIN1 predicts future cardiovascular hospitalization and death over a 12 month follow-up period.

Conclusions:
A cBIN1 specific ELISA can quantify cBIN1 in human plasma. Low plasma cBIN1 diagnoses diseased heart muscle in HFrEF patients and predicts future hospitalization and death.
Abstract 369: Engineering Rrm2 to Elevate 2-deoxy-ATP and Improve Cardiomyocyte Function
Authors: Murray Jason D , Guan Xuan , Moussavi-Harami Farid , Olafsson Sigurast S , Murry Charles E , Regnier Michael
Published: July 2021

Overexpression of ribonucleotide reductase (RNR) in cardiomyocytes increases the amount of cytosolic 2-deoxy-ATP (dATP), which can be used by myosin and significantly increases contraction of cardiac muscle at all levels of calcium activation. Our group is working to develop enhanced dATP as a therapeutic option for heart failure. We have demonstrated that virally-mediated overexpression of RNR elevates dATP and increases the rate and magnitude of contraction and increases left ventricular contraction in normal hearts as well as rodent models of myocardial infarction and dilated cardiomyopathy. RNR is a heterotetramer containing two subunits, Rrm1 and Rrm2. While cardiomyocyte-specific overexpression of Rrm1 is stable, we have observed high variability in expression levels of the Rrm2 subunit in multiple disease models. We hypothesized that this variability was largely due to protein degradation via the ubiquitin-proteasome complex (UPC). We found that pharmacological inhibition of proteasome activity leads to increased expression of Rrm2 in virally-transduced cardiomyocytes
in vitro.
To confirm the hypothesis that the overexpressed Rrm2 is degraded via UPC-mediated degradation, we engineered mutations in specific ubiquitin-binding degrons of the Rrm2 gene. Transfecting human induced pluripotent stem cell-derived cardiomyocytes resulted in higher levels of Rrm2 than those overexpressing wild-type protein and resulted in higher levels of cytosolic dATP as measured by Liquid chromatography-mass spectrometry. Ongoing and planned experiments will compare the effects of this engineered mutation on Rrm2 overexpression, dATP production, and contractility in cultured cardiomyocytes. Our goal is to develop an improved RNR vector that will be resistant to degradation through the ubiquitin-proteasome pathway and therefore enable more stable and consistent RNR enzyme activity and deoxynucleotide levels of cardiomyocytes transduced
in vivo.
Abstract 378: A Systematic Review of Mobility/Immobility in Thromboembolism Risk Assessment Models for Hospitalized Patients
Authors: Ye Fan , Stalvey Carolyn , Khuddus Matheen , Winchester David , Toklu Hale , Mazza Joseph , Yale Steven
Published: July 2021

Introduction:
Venous thromboembolism (VTE) is a potentially fatal disorder. Prophylaxis is often suboptimal in medical inpatients, attributed to the difficulty in identifying at-risk patients. Simple and validated risk-assessment models (RAMs) are available to assist clinicians in identifying and stratifying patients who have a higher likelihood for developing VTE. Despite the well-documented association of immobility with increased risk of thrombosis, immobility is not consistently defined in clinical studies.

Methods:
We conducted a systematic review of published RAMs, based on objective criteria, to determine how the term immobility is defined in RAMs.

Results:
We identified seventeen RAMs with six being externally validated. The concept of immobility is vaguely described in different RAMs, impacting the validity of these models in clinical practice. The widespread variability in defining mobility in RAMs precluded its accurate clinical application further limiting generalization of published RAMs.

Conclusion:
Externally validated RAMs with clearly defined qualitative or quantitative terms of immobility are needed to assess VTE risk in real-time at the point-of-care.
Abstract 401: Nuclear Remodeling After Mechanical Circulatory Support
Authors: Luo Jun , Farris Stephen , Helterline Deri , Stempien-Otero April
Published: July 2021

Rationale:
Cardiomyocytes increase DNA content in normal growth and in response to stress in humans by both increases in nuclear number and ploidy. This observation complicates analysis of human cardiomyocyte proliferation as DNA content can increase in the absence of cytokinesis. Proliferation has been reported in cardiomyocytes following LVAD unloading which may represent a reversal of this process. However, cardiac recovery from LVAD is rare. Thus, we sought to analyze changes in cardiomyocyte nuclear characteristics for clues to this paradox.

Objective:
We used a novel technique to determine changes in nuclear content to test the hypothesis that adult cardiomyocytes can complete cell cycle progression by mitosis after long-term hemodynamic unloading of the failing heart.

Methods and Results:
The makeup of myocyte nuclear number, ploidy (per cell and per nucleus) and the frequency of cell cycling markers were evaluated by imaging flow cytometry. Hypertrophic hearts from 15 subjects with left ventricular assist device (LVAD) were compared with 8 non-LVAD unloaded hearts. After hemodynamic unloading for 13.7 ± 9.1 months, myocyte nuclear makeup, specifically the average sizes of both cell and individual nuclei, did not significantly change. DNA content per nucleus was significantly decreased (P < 0.01). The frequency of cell cycle markers, i.e. Ki67 and phospho-histon3 (H3P) were not increased after hemodynamic unloading.

Conclusions:
Our data demonstrate that unloading of failing hearts with mechanical ventricular assist devices does not alter nucleation state of cardiomyocytes. However, unloading is associated with decreased DNA content of nuclei independent of nucleation state within the cell. As these changes were associated with a trend to decreased cell size but not increased cell cycle markers, they may represent a regression of hypertrophic nuclear remodeling.
Abstract 402: Vitamin C Deficiency Impairs Cardiac Function and Post-infarction Survival in the Mouse
Authors: Mauro Adolfo G , Kraskauskas Donatas , Mohammed Bassem M , Fisher Bernard J , Mezzaroma Eleonora , Fowler Alpha A , Natarajan Ramesh , Toldo Stefano
Published: July 2021

Introduction:
L-gulonolactone oxidase (Gulo) is the rate limiting enzyme for Vitamin C (VitC) biosynthesis. Humans rely on dietary VitC for collagen synthesis, extracellular matrix formation, and tissue regeneration. VitC deficiency is an unrecognized condition and its role in cardiac homeostasis and post-acute myocardial infarction (AMI) remodeling is unknown.

Hypothesis:
Low levels of VitC impair cardiac function and tissue repair following AMI.

Methods:
Adult male Gulo
-/-
knockout mice (C57BL6 background, N=8) and control C57BL (N=8), which are able to synthesize VitC were used. VitC deficiency was maintained supplying low levels of VitC (30mg/l) to Gulo
-/-
mice in drinking water. Mice underwent M-mode and Doppler echocardiography to measure left ventricular (LV) diameters and wall thicknesses, fractional shortening (FS), E and A waves, E/A ratio, isovolumetric relaxation time (IRT) and myocardial performance index (MPI). Experimental AMI was induced by coronary artery ligation for 7 days. An additional group of Gulo
-/-
were mice supplemented with physiological levels of VitC (330 mg/l) and underwent AMI.

Results:
VitC deficient Gulo
-/-
mice exhibited significantly reduced LV wall thicknesses, reduced FS, and impaired diastolic function, measured as significantly reduced E/A ratio and longer IRT (Panel A, B & C). Following AMI, 100% (8/8) of deficient Gulo
-/-
mice died within 5 days. Supplementation with physiological levels of VitC significantly improved survival after AMI (Panel D).

Conclusion:
VitC deficiency impairs systolic and diastolic function. Moreover, VitC is critical for the post-AMI survival.
Abstract 41: Cardiomyocyte-specific Conditional Deletion of GSK-3β Leads to Cardiac Dysfunction in a High Fat Diet Induced Obesity Model
Authors: Gupte Manisha , Tumuluru Samvruta , Singh Anand P , Umbarkar Prachi , Zhang Qinkun , Ahmad Firdos , Parikh Shan , Force Thomas , Lal Hind
Published: July 2021

Introduction:
Previous studies from our group have demonstrated that cardiac myocyte glycogen synthase kinase-3’s (GSK-3) are required to maintain normal cardiac physiology. Adult mice lacking both isoforms of GSK-3 (α and β) in cardiac myocytes exhibit excessive dilatative remodeling and ventricular dysfunction ultimately leading to death. While high fat diet (HFD) induced obesity is associated with increased risk of cardiovascular disease, the specific role of cardiac GSK-3α or GSK-3β in obesity-associated cardiac dysfunction is unknown.

Objective:
The primary goal of the present study was to investigate the role of cardiomyocyte GSK-3β in cardiac homeostasis in HFD-induced chronic obesity model.

Method:
Cardiomyocyte specific-GSK-3β knock out (CM-GSK-3βKO) and wild type (WT) mice were fed either a chow (11.5% calories from fat) or high-fat (60% calories from fat) for 24 weeks. Cardiac function was accessed by non-invasive transthoracic echocardiography.

Results:
HFD significantly increased body weight, lean and fat mass in the WT and CM-GSK-3βKO compared to chow. However, there was no difference in body weights, lean and fat mass between the two genotypes fed either a chow or HFD. Furthermore, ventricular chamber dimensions and cardiac function were comparable between the WT and CM-GSK-3βKO mice fed a chow diet. In contrast, high fat fed CM-GSK-3βKO hearts exhibit significant cardiac hypertrophy (heart weight/tibia length ratio) and ventricular dysfunction (reduced ejection fraction (EF) and fractional shortening (FS)) compared to the WT. Interestingly cardiomyocytes from HF fed CM-GSK-3βKO exhibit structural abnormalities and increased expression of pro-apoptotic protein Bax and reduced expression of Bcl-2, an anti-apoptotic protein.

Conclusion:
In summary, these data suggests that cardiac GSK-3β is important in the setting of HFD-induced chronic obesity to maintain cardiac function. In the absence of GSK-3β, cardiomyocytes undergo morphometric abnormalities, excessive fat infiltration and apoptosis leading to cardiac dysfunction.
Abstract 422: Small Molecule and Activated Fibroblast Targeting of the Gβγ-GRK2 Interface After Myocardial Ischemia Attenuates Heart Failure Progression
Authors: Travers Joshua G , Kamal Fadia A , Valiente-Alandi Inigo , Nieman Michelle L , Sargent Michelle A , Lorenz John N , Molkentin Jeffery D , Blaxall Burns C
Published: July 2021

Cardiac fibroblasts are a critical cell population responsible for myocardial extracellular matrix homeostasis. Upon injury or pathologic stimulation, these cells transform to an activated myofibroblast state and play a fundamental role in myocardial fibrosis and remodeling. Chronic sympathetic overstimulation, a hallmark of heart failure, induces pathologic signaling through G protein βγ subunits and their interaction with G protein-coupled receptor kinase 2 (GRK2). We hypothesized that Gβγ-GRK2 inhibition/ablation after myocardial injury would attenuate pathologic myofibroblast activation and cardiac remodeling. The therapeutic potential of small molecule Gβγ-GRK2 inhibition alone or in combination with activated fibroblast- or myocyte-specific GRK2 ablation, each initiated after myocardial ischemia/reperfusion (I/R) injury, was investigated to evaluate possible salutary effects on post-I/R fibroblast activation, pathologic remodeling and cardiac function. Small molecule Gβγ-GRK2 inhibition initiated one week post-injury was cardioprotective in the I/R model of chronic heart failure, including preservation of cardiac contractility and reduction in cardiac fibrotic remodeling. Systemic small molecule Gβγ-GRK2 inhibition initiated one week post-I/R in cardiomyocyte-restricted GRK2 ablated mice (also post-I/R) demonstrated additional cardioprotection, suggesting a potential protective role beyond the cardiomyocyte. Inducible ablation of GRK2 in activated fibroblasts (i.e. myofibroblasts) post-I/R injury demonstrated significant functional cardioprotection with reduced myofibroblast transformation and fibrosis. Systemic small molecule Gβγ-GRK2 inhibition initiated one week post-I/R provided little to no further protection in mice with ablation of GRK2 in activated fibroblasts alone. Finally, Gβγ-GRK2 inhibition significantly attenuated activation characteristics of failing human cardiac fibroblasts isolated from end stage heart failure patients. These findings suggest a potential therapeutic role for Gβγ-GRK2 inhibition in limiting pathologic myofibroblast activation, interstitial fibrosis and heart failure progression.
Abstract 433: The Role Of Claudin-5 on Mitochondrial Dynamics in Neonatal Rat Cardiomyocyte
Authors: Luo Tao , Liu Han , Kim Jin K
Published: July 2021

Background:
Claudin-5, an integral transmembrane protein that controls the para-cellular permeability, is severely downregulated in end stage cardiomyopathy and during ischemia-reperfusion injury. Sustaining claudin-5 levels prevented myocardial damage and dysfunction. However, the underlying mechanism behind the cardioprotection associated with claudin-5 is unclear. The aim of this study is therefore to investigate the role of claudin-5 on mitochondria
in vitro
.

Methods and Results:
Western blot showed that claudin-5 was expressed in mouse heart tissue and neonatal rat cardiomyocyte (NRCM). Its protein level was severely decreased after myocardial ischemia/reperfusion (I/R; 30 min/24 hr) and after hypoxia/reoxygenation (H/R; 24 hr/4 hr), respectively. Further
in-vitro
confocal observation showed that claudin-5 was co-expressed on mitochondria in NRCM and its decrease was accompanied by mitochondrial fragmentation. We then detected the mitochondrial fusion and fission proteins in NRCM under the condition of H/R. Claudin-5 siRNA and claudin-5 overexpressing adenovirus were used to knockdown claudin-5 and overexpress claudin-5. The protein level of mitofusin 2 (responsible for mitochondrial outer membrane fusion) was dramatically decreased while the expression of Drp 1 (responsible for mitochondrial fission) was significantly increased after H/R; Claudin-5 siRNA pretreatment further decreased the level of mitofusin 2 and increased the level of Drp 1. However, overexpression of claudin-5 reversed these effects respectively. We further measured the apoptotic protein, cytochrome c, by western blot. The expression of cytochrome c in the cytoplasm of NRCM was significantly increased after H/R, which was reduced by overexpression of claudin-5 by adenovirus.

Conclusion:
In the present study, we first time introduce a new concept that claudin-5 may help maintaining mitochondrial dynamics by upregulating mitochondrial fusion and suppressing mitochondrial fission.
Abstract 446: Activation of γ2-ampk Suppresses Ribosome Biogenesis and Protects Against Myocardial Ischemia-reperfusion Injury
Authors: Cao Yang , Bojjireddy Naveen , Kim Maengjo , Li Tao , Zhai Peiyong , Nagarajan Narayani , Sadoshima Junichi , Tian Rong
Published: July 2021

AMP-activated protein kinase (AMPK) is a heterotrimeric protein that senses cellular energy status and maintains energy homeostasis by switching off biosynthetic pathways and increasing catabolism. The subcellular localization of AMPK has been shown to affect its activation and function. The γ2 subunit has both nuclear localization sequence and nuclear export sequence suggesting that it can shuttle between the two compartments. By overexpressing GFP-tagged γ subunits in COS7 cells followed by glucose deprivation or AMPK activation (A769662), we demonstrated that AMPK containing γ2 but not γ1 or γ3 subunit translocates into nucleus. Nuclear accumulation of AMPK complexes containing γ2-subunit phosphorylates and inactivates Pol I-associated transcription factor TIF-IA at Ser-635, precluding the assembly of transcription initiation complexes and lowering preribosomal RNA (pre-rRNA) level. Down-regulation of rRNA synthesis attenuated expression of ER stress markers (spliced X-box binding protein-1 and C/EBP homologous protein) and ER stress-induced cell death. Deleting γ2-AMPK using CRISPR-Cas9 system led to increases in pre-rRNA level, ER stress markers and cell death during glucose deprivation. To study the function of γ2-AMPK in the heart, we generated a mouse model with cardiac specific deletion of γ2-AMPK (cKO). Although the total AMPK activity was unaltered in cKO hearts due to upregulation of γ1-AMPK the lack of γ2-AMPK sensitizes the heart to myocardial ischemia-reperfusion (I/R, 30min ischemia followed by 24hr reperfusion) injury as evidenced by larger infarct size (Infarct size/area at risk: 34.7±5.7% vs. 50.6±8.9%, for control and cKO respectively, P<0.05). The cKO failed to suppress pre-rRNA level during I/R and showed higher levels of ER stress markers. Conversely, cardiac-specific overexpression (OE) of γ2-AMPK decreased ER stress markers and pre-rRNA level upon I/R injury and the infarct size was reduced (Infarct size/area at risk: 26.8±6.5% for control vs. 15.8±3.7% for OE, P<0.05), suggesting that γ2-AMPK protects against I/R injury and ER stress in the heart. Taken together, our study reveals isoform-specific functions of γ2-AMPK in modulating protein synthesis, cell survival and cardioprotection.
Abstract 467: ATF6B and ATF6A Play Complimentary Roles in Mediating Adaptive and Maladaptive Signaling in Cardiac Myocytes
Authors: Koul Shivsmriti , Jin Jung-kang , Hogan Clifford M , Glembotski Christopher C
Published: July 2021

Rationale:
ATF6α and ATF6β are endoplasmic reticulum (ER) transmembrane proteins that sense the accumulation of toxic misfolded proteins in the ER of cardiomyocytes, which can be brought about by ER stresses as ischemia. Upon ER stress, ATF6α is proteolytically cleaved into a transcription factor that binds to ER stress response elements (ERSEs) and increases expression of cardioprotective genes that restore ER protein folding. If ER proteostasis is not restored, maladaptive signaling is initiated. ATF6β is also proteolytically cleaved during ER stress, binds to the same ERSEs as ATF6α, but does not induce transcription. Hence it is clear from the above studies done in cancer cells that there are some marked similarities and differences between ATF6α and ATF6β. However, the relative roles of ATF6α and ATF6β have not been studied in the heart, where they might work in concert to mediate the dynamic switch from adaptive to maladaptive gene programing during myocardial pathology.

Methods:
We used neonatal rat ventricular myocytes (NRVMs) to explore the effects of ATF6α or ATF6β loss-of-function in cells treated with the ER stressor, thapsigargin (TG), which mimics ischemic heart disease.

Results:
In NRVM treated with TG, knockdown of ATF6β resulted in much more pronounced cell death in isolated myocytes than knockdown of ATF6α. Consistent with this finding, transcriptome analyses showed that compared to knocking down ATF6α, knockdown of ATF6β upregulated much more maladaptive, cell death-inducing genes and downregulated more cardioprotective genes. Surprisingly, knockdown of either ATF6α or ATF6β downregulated some common adaptive ER stress response genes, such as GRP78 and Derlin while also upregulating common maladaptive ER stress response genes, such as CHOP, Bcl2, Bax.

Conclusion:
These data indicate that both ATF6α and ATF6
β
are needed for optimal viability of NRVM subjected to ER stress. There is a common, as well as differential gene regulation program controlled by these two isoforms of ATF6. Importantly, this study demonstrates a novel mechanism by which these two isoforms of ATF6 interact to govern the progression from adaptive to maladaptive ER stress signaling during chronic misfolding of ER proteins that occurs in ischemic heart disease.
Abstract 484: Mir-19b-3p Regulates Autophagy by Targeting Raf-1 During Hypertrophic Cardiomyopathy
Authors: Mittal Anupam , Dhandapany Perundurai S
Published: July 2021

Hypertrophic cardiomyopathy (HCM) is a heterogenous disease predominantly caused by sarcomeric genes. However, 40-50% cases etiology are not known. RAF1 mutations cause syndromic and isolated childhood cardiomyopathies. Functional mutations in untranslated regions (UTRs) of RAF-1 gene are rare, and their role in cardiomyopathy is unexplored. UTRs are important sites for interaction of epigenetic regulators such as microRNAs (miR). miRs have exhibited as crucial regulators of the cardiac remodeling process. We identified a novel mutation in RAF-1 3’-UTR (an important site for binding of miR-19a-3p/19b-3p) in large family members associated with HCM. Whole exome sequencing revealed that these family members are negative for mutations in known cardiomyopathy associated genes. miR-19a/b are known to play a crucial role in cardiac hypertrophy. The aim of this study is to determine the role of miR-19a-3p/19b-3p in regulating RAF-1 expression and delineating the molecular and functional consequences of identified RAF-1 3’-UTR mutation. Our results show that the overexpression of miR-19b-3p, leads to downregulation of RAF-1 expression and regulation. Notably, miR-19b-3p was found to be pro-hypertrophic as its overexpression resulted in increasing hypertrophic markers including ANP and β-MHC expression. Moreover, findings from experiments using RAF1
+/+
and RAF1
-/-
mouse embryo fibroblasts (MEFs) confirms the role of miR-19b-3p interaction and regulation of RAF-1 signaling axis. Interestingly, we also observed many autophagy markers (Atg3, Atg 12-5 complex, and LC3 II) are dysregulated by miR-19-b-3p overexpression. Our study uncovers a novel mechanism through which miR-19b-3p regulates autophagy by targeting RAF-1.
Abstract 70: Protein Phosphatase 1 Contributes to Atrial Stunning in Atrial Fibrillation
Authors: Perike Srikanth , Wehrens Xander , Darbar Dawood , McCauley Mark
Published: July 2021

Background:
Atrial fibrillation (AF) is the most common cardiac arrhythmia, and increases a patient’s stroke risk five-fold. Reduced atrial contractility (stunning) is observed in AF and contributes to stroke risk; however, the mechanisms responsible for atrial stunning in AF are unknown. Recent data from our laboratory indicate that protein phosphatase 1 (PP1) dephosphorylation of myosin light chain 2a (MLC2a) may contribute to atrial stunning in AF.

Objective:
To determine how the PP1 regulatory subunit 12C (PPP1R12C) and catalytic (PPP1c) subunits modify atrial sarcomere phosphorylation in AF.

Methods:
We evaluated the protein expression, binding and phosphorylation among PPP1R12C, PPP1c, and MLC2a in transfected HL-1 cells, murine atrial tissue (Pitx2null
+/–
mice, with a genetic predisposition AF), and in HEK cells. An inhibitor of PPP1R12C phosphorylation, BDP5290, was used to enhance the PPP1R12C-PPP1C interaction.

Results:
In

Pitx2
null

+/–
mice, PPP1R12C was increased by 2-fold (
P
<0.01) and associated with a 40% reduction in S-19-MLC2a phosphorylation versus WT mice (
P
<0.058). BDP5290 increased PPP1R12C-PPP1C binding by >3-fold in HL-1 cells (
P
<0.01). BDP5290 reduced MLC2a phosphorylation by 40% through an enhanced interaction with PPP1R12C by >3-fold in HEK cells (
P
<0.01).

Conclusion:
In

Pitx2
null+/-

mice, increased expression of PPP1R12C is associated with PP1 holoenzyme targeting to sarcomeric MLC2a, and is associated with reduced S19-MLC2a phosphorylation. Additionally, BDP5290 enhances the PPP1R12C-PPP1C interaction and models PP1 activity in AF. Future studies will examine the effects of both AF and BDP5290 upon atrial contractility in vitro.
Abstract 78: Hippo Pathway and Dystrophin Glycoprotein Complex Regulate Cardiomyocyte Proliferation
Authors: Morikawa Yuka , Heallen Todd , Leach John , Xiao Yang , Martin James
Published: July 2021

Regeneration of mammalian heart is limited due to the extremely low renewal rate of cardiomyocytes and their inability to reenter the cell cycle. The Hippo pathway controls heart size during development and represses postnatal heart regeneration by repressing cardiomyocyte proliferation. Our approach for activating adult heart regeneration is to uncover the mechanisms responsible for repression of cardiomyocyte proliferation. We have previously found that deletion of Salv, a modulator of the Hippo pathway, results in myocardial damage repair in postnatal and adult hearts. Deletion of Salv results in activation of the transcription factor, Yap, which positively regulates cytoskeleton and cell cycle genes. We also found that the components of dystrophin glycoprotein complex (DGC) are the target of Yap and DGC regulates heart regeneration. The dystrophin glycoprotein complex (DGC) is essential for muscle maintenance by anchoring the cytoskeleton and extracellular matrix. Disruption of the DGC results in muscular dystrophies, including Duchenne muscular dystrophy, resulting in both skeletal and cardiac myopathies. To explore the connection between DGC and the Hippo pathway, we conditionally deleted Salv in the mdx background, a mouse model of muscular dystrophy. We found that simultaneous disruption of the DGC and the Hippo pathway leads an increased cardiomyocyte proliferation after heart damage. This is associated with increased activity of Yap, suggesting DGC negatively regulate Yap to repress proliferation. We also found that one of the components DGC, dystroglycan directly binds Yap and anchors to the membrane. Our findings provide new insights into the mechanisms leading to heart repair through proliferation of endogenous cardiomyocytes.
Abstract 85: The AAA-ATPase p97 is a Critical Regulator of Cardiac Homeostasis
Authors: Brody Matthew J , Sargent Michelle A , Molkentin Jeffery D
Published: July 2021

p97 is a AAA-ATPase that plays critical roles in a myriad of cellular protein quality control processes, including the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway that targets misfolded proteins in the ER for degradation in the cytosol by the ubiquitin proteasome system. Mutations in p97 cause a multisystem degenerative proteinopathy disorder called inclusion body myopathy with Paget disease of bone and frontotemporal dementia (IBMPFD) that includes pathologies of the nervous system, skeletal muscle, bone, and heart. Previous studies in the laboratory into the mechanisms whereby thrombospondin 4 has its cardioprotective effects and enhanced ERAD activity identified p97 as a direct interacting partner. This observation suggested that p97 itself could be an important cardioprotective effector by benefiting protein quality control in the heart. To address this hypothesis here we generated cardiac-specific transgenic mice overexpressing wildtype p97 or a p97
K524A
mutant with deficient ATPase activity, the latter of which functioned as a dominant negative. Mice overexpressing wildtype p97 exhibit normal cardiac structure and function while mutant p97 overexpressing mice develop cardiomyopathy, upregulate several ERAD complex components, and have elevated levels of ubiquitinated proteins. Proteomics and immunoprecipitation assays identified overwhelming interactions between endogenous p97 and a number of interesting protein complexes that suggest unique functions for this protein in regulating protein quality control in the heart. The results and novel regulatory relationships will be presented, which suggests entirely unique pathways whereby p97 functions in the heart.
Abstract 90: Inimitable Ouabain: The Endogenous Circulating Cardiotonic Steroid that Singularly Stimulates Sodium Potassium Pump Activity at Low Doses
Authors: Kim Yeon Jae , Hamilton Elisha , Hannam William , Liu Chia-Chi , Teh Rachel , Rasmussen Helge H , Garcia Alvaro
Published: July 2021

Rationale:
Cardiotonic steroids (CTS), such as digoxin, have been used to treat heart failure (HF) for over 200 years. They inhibit the sodium-potassium pump (NKP), and increase cardiac contractility by inhibiting efflux of sodium through the pump (“digitalis hypothesis”). CTS possess three structural components: a saturated/unsaturated lactone ring, steroid core, and sugar moiety, each of which may be involved in NKP inhibition/stimulation. It is now known that inhibition of the NKP in patients with HF increases mortality, and all major beneficial treatments increase its activity. Endogenous circulating CTS such as ouabain are generally thought to inhibit the NKP, despite studies sporadically reporting ouabain-induced pump stimulation. This study aims to identify whether ouabain-induced pump stimulation occurs, and if so, which structural components are involved in causing pump stimulation.

Methods & Results:
Cardiac myocytes were isolated from male New Zealand White rabbits, placed in a Tyrode’s solution, and whole-cell patch clamped. They were exposed to 0-30nM ouabain, 0-50nM dihydroouabain (ouabain with a saturated lactone ring) or 0-500nM ouabagenin (ouabain lacking a sugar moiety) for 1 min, followed by a potassium-free solution, with the difference in current yielding the NKP current. Compared to the 0.47±0.05 pA/pF Tyrode’s solution control (n=11), 5nM ouabain significantly increased NKP current to 0.69±0.09 pA/pF (
P
<0.05, n=6). Exposure to dihydroouabain or ouabagenin did not significantly change NKP current in the studied concentration range. Cell viability assays carried out on the breast cancer cell line MCF7, which have an NKP structure extremely similar to that of cardiomyocytes, showed significantly elevated viability above control values (n=2) following 24h treatment with 0-9nM ouabain; maximum viability was 116±5% at 0.28nM (
P
<0.05, n=4). A significant change in viability was not observed for ouabagenin or digoxin in the same concentration range.

Conclusion:
Low-dose ouabain uniquely stimulates NKP activity. Low-dose dihydroouabain and ouabagenin do not, suggesting that a sugar moiety and unsaturated lactone ring are required for pump stimulation. Ouabain in its unaltered form may be a potential treatment for HF.