bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 189: DWORF: Discovery and Characterization of a Cardiac Micropeptide Encoded in a Putative Long Noncoding RNA
Authors: Nelson Benjamin R , Makarewich Catherine A , Reese Austin L , Winders Benjamin R , Anderson Douglas M , McAnally John R , Kavalali Ege T , Bassel-Duby Rhonda , Olson Eric N
Published: July 2021

Background:
Mammalian transcriptomes contain thousands of potential open reading frames (ORFs) with fewer than fifty codons. Most of these ORFs are presumed to be random and therefore noncoding, but recently several genes annotated as long noncoding RNAs (lncRNAs) were shown to encode functional micropeptides.
Objective:
Identify and characterize novel micropeptides encoded in cardiac-expressed lncRNAs using comparative genomics.
Methods and Results:
Using codon substitution frequency, a comparative genomics strategy, we identified several previously unknown small proteins, including a 34-amino acid micropeptide that we have named DWORF (
Dw
arf
O
pen
R
eading
F
rame). DWORF is a tail-anchored transmembrane protein that localizes to the sarcoplasmic reticulum and is expressed only in cardiac ventricle and slow-twitch skeletal muscle by quantitative PCR. We subsequently derived a custom antibody targeting the N-terminal residues of DWORF. Western blots using this antibody revealed a 4 kDa protein that is expressed in cardiac ventricle and soleus muscle, but not other tissues. We further validated specificity of this signal by disrupting the reading frame of DWORF in mice using CRISPR/Cas9 mutagenesis, conclusively demonstrating that the mRNA encodes a small protein. Transgenic overexpression of DWORF in cardiac myocytes resulted in increased peak amplitude of Ca
2+
transients and improved Ca
2+
resequestration kinetics with no apparent adverse effects. These findings suggest that DWORF specifically enhances activity of the sarco/endoplasmic reticulum calcium ATPase (SERCA).
Conclusions:
Some putative lncRNAs encode micropeptides that can be identified using bioinformatics strategies. Micropeptides, though likely too small to have enzymatic activity of their own, may have modulatory functions in important cellular processes such as calcium handling, as we have demonstrated in the case of the muscle-restricted micropeptide DWORF.
Abstract 19: Microrna-19b Enhances Cardiac Cell Survival After Ischemic Injury
Authors: Molenaar Bas , Demkes Charlotte , Ruiter, de Hester , Versteeg Danielle , Gladka-de Vries Monika , Korporaal Ricardo , Deel, van Else , Semus Hillary M , van Rooij Eva
Published: July 2021

Ischemia/reperfusion (I/R) injury in cardiac tissue results in substantial loss of cardiomyocytes, leading to a functional decline. The release of several cytokines increases cardiomyocyte survival after ischemic injury by activating the janus kinase (JAK) and signal transducer and activator of transcription (STAT) pathway. However, this pathway is inhibited by Suppressor of cytokine signaling (SOCS)1 and SOCS3, which act as endogenous negative regulators of cardiomyocyte survival by blocking JAK activity.

MicroRNAs (miRNAs) are small, non-coding RNAs that can repress gene expression by binding to recognition sequences within target genes. In a bioinformatic screen, we identified conserved binding sites in SOCS1 and 3 for miR-19b. Using a luciferase reporter assay, we were able to confirm direct binding of miR-19b to the 3’-UTR of both SOCS1 and 3
in vitro
. In addition, we found that miR-19b downregulates SOCS1 in cardiac-like cells and isolated neonatal cardiomyocytes.
In vivo
, we observed a downregulation of miR-19b in both rodent and human cardiac tissues after ischemic injury, which corresponds to an upregulation of both SOCS1 and 3.

Since inhibition of SOCS1 and 3 could enhance cardiac JAK-STAT signaling, miRNA-based inhibition could lead to an increase in cardiomyocyte survival. Here we show that cardiomyocyte-specific overexpression of miR-19b lowers SOCS1 and SOCS3 expression and enhances JAK-STAT signaling during ischemia reperfusion, which corresponds to a decrease in the pro-apoptotic proteins Bad, Bax and P53. Additional genes that are decreased by miR-19b during ischemic injury are Rnf11 and TNFAIP3, both repressors NF-kB signaling. To further explore the cardioprotective effects of miR-19b, we are currently investigating the therapeutic benefits of administering synthetic miR-19b mimics through intracardiac injection after I/R injury in mice.

Our study indicates a conserved mechanism by which miR-19b targets both SOCS1 and 3 and to increase the activation of JAK/STAT signaling to decrease cardiomyocyte apoptosis. Administration of miR-19b mimics might be of therapeutic interest to enhance cardiomyocyte cells survival and preserve heart function in the setting of ischemic injury.
Abstract 196: Cardiotrophin-1 Promotes Physiologic Cardiac Hypertrophy
Authors: Megeney Lynn A , Abdul-Ghani Mohammad , Suen Colin , Stewart Duncan J
Published: July 2021

The post-natal heart retains a remarkable adaptive capacity, which matches growth of the myocardium to increased physical demands. The beneficial form of myocardial growth is referred to as physiologic hypertrophy, and derives from a compensatory response to conditions that require increased blood volumes such as pregnancy and chronic exercise training. Despite the clear benefits associated with this form of adaptation, the molecular mechanism(s) that initiate or sustain such cardiac remodeling remain obscure. As such, identification of factors that promote physiologic hypertrophy will be of considerable interest to both cardiac biologists and clinicians. Here, we have begun to explore the role of the cytokine, cardiotrophin 1 (CT-1) as a central mediator of physiologic hypertrophy. We demonstrate that primary cardiomyocytes treated with recombinant human CT-1 protein engage a physiologic growth response, adding sarcomeres in series, a cellular/molecular change that is fully reversible with the removal of CT-1. This physiologic hypertrophy response to CT-1 is dominant, as CT-1 administration overrides the pathologic hypertrophy associated with agonists such as phenylephrine. In vivo delivery of human CT-1 protein leads to a rapid (14 days) alteration in the structure of the rat heart, with volume and wall dimension increases characteristic of exercise adaptation, changes that are fully reversible upon cessation of CT-1 treatment. In addition, CT-1 administration mitigates right heart failure in a model of pulmonary arterial hypertension, and sustains cardiac function in the post infarct rat heart. Finally, gene expression analysis revealed that CT-1 treatment leads to up-regulation of angiogenic factors within cardiomyocytes, suggesting that this cytokine engages a paracrine signaling mechanism to ensure that myocardial growth is matched by a robust angiogenesis.
Abstract 201: Enhancing Pgc-1α Activity Improves Heart Function Through Activating Mitochondrial Biogenesis in Chagas Disease
Authors: Wan Xianxiu , Wen Jianjun , Sue-jie Koo
Published: July 2021

Chronic chagasic cardiomyopathy (CCM) is presented with ventricular hypertrophy and contractile dysfunction that can lead to heart failure. I have found that a substantial decline in mitochondrial biogenesis and SIRT1/PGC-1α activity ensue in chronic chagasic mice. It was evidenced by the decline in mitochondrial DNA content as well as mRNA levels of mitochondrial encoded genes and mtDNA replication machinery. Further, the activity of SIRT1 (required for PGC-1α activation) was decreased and associated with decreased nuclear levels of PGC-1-regulated NRF1 transcription factor in chagasic hearts. The mitochondrial size and number were also reduced in chagasic heart, determined by electron microscopy. Therefore, we hypothesized that enhancing the SIRT1/PGC-1α activity by SIRT1 agonist would improve heart function through activating mitochondrial biogenesis in Chagasic disease. Mice were infected with T. cruzi, and beginning at day 90 post-infection (pi), treated with resveratrol (SIRT1 agonist) or metformin (AMPK agonist, can enhance SIRT1 activity) for 21 days; and then heart function was monitored at 150 days pi. We found that treatment with resveratrol partially attenuated the heart dysfunction (stroke volume, cardiac output, ejection fraction, heart rate) and cardiac hypertrophy in chagasic mice. These benefits were associated with improved expression of the mitochondrial DNA encoded genes and mtDNA content though the expression of genes involved in mtDNA replication was not improved. Treatment with metformin was not significantly beneficial in improving the CCM outcomes. The partial beneficial effects of resveratrol could be due to inefficient activation of SIRT1 or delayed start of the treatment. We plan to treat mice with SIRT1 agonist SIRT1720 (10 fold more active than resveratrol) during the indeterminate phase of T. cruzi infection in next set of experiments. This study will improve our understanding of the molecular and immune mechanisms of chagasic heart disease and will provide a novel treatment for chronically-infected chagasic patients.
Abstract 21: Diabetic Ketoacidosis Compromises Cardiac Mitochondrial Quality Control in Humans
Authors: Kobayashi Satoru , Gilliland M.G.F. , Hoffman William H , O’Connor Alice , Liang Qiangrong
Published: July 2021

Mitochondrial injury plays a key role in the pathogenesis of diabetic cardiomyopathy, a risk factor for heart failure in diabetic populations. Dysfunctional mitochondria are eliminated through several coordinated mitochondrial quality control mechanisms including the autophagy-lysosome degradation pathway, a process termed mitophagy. Diabetic ketoacidosis (DKA) is a potentially life-threatening complication of type 1 diabetes. We have shown that adolescents and young adults with uncomplicated DKA mount a robust systemic inflammatory response which is associated with diastolic cardiac abnormality. However, it remains unknown if cardiac abnormality in patients with DKA is correlated with altered mitochondrial quality control processes. In the present study, using immunofluorescent labeling and confocal microscopic imaging, we examined a series of proteins involved in autophagy, mitophagy and mitochondrial dynamics in paraffin-embedded autopsy heart tissues from young people without or with fatal DKA. We found that the expression levels of beclin 1, microtubule-associated protein light chain 3 (LC3) and lysosome-associated membrane protein (LAMP) 1/2 are not changed, but that of p62 is increased in the heart of DKA patients, suggesting a decreased autophagy. Also, the expression levels and localization of Pink1, parkin and Rab9, three potential regulators of mitophagy, are markedly changed, indicating an altered mitophagy in the DKA heart. In addition, mitochondrial fusion and fission proteins (Mitofusin 2, Opa1, Drp1 and Fis1) are all altered to varying degrees in the DKA heart, which is accompanied by increased mitochondrial fragmentation, oxidative injury and apoptosis (TUNEL positive cells). Together, these findings demonstrate that the mitochondrial quality control mechanisms are not operating normally in the heart of patients with DKA. Therefore, therapeutic strategies that aim to improve the efficiency of the mitochondrial quality control may have the potential to reduce diabetic cardiac injury and heart failure in young type 1 diabetic patients.
Abstract 210: Titin Phosphorylation by Protein Kinase G as a Novel Mechanism of Diastolic Adaptation to Acute Hemodynamic Overload
Authors: Almeida-Coelho João , Leite-Moreira André M , Neves João S , Neiva-Sousa Manuel , Castro-Ferreira Ricardo , Hamdani Nazha , Linke Wolfgang A , Falcão-Pires Inês , Lourenço André P , Leite-Moreira Adelino F
Published: July 2021

Titin is the main determinant of myocardial passive tension and its distensibility is increased via phosphorylation by protein kinase G (PKG), activated by nitric oxide (NO) and natriuretic peptides (NP) upon acute overload. We hypothesized whether myocardial stretch led to decreased stiffness, optimizing diastolic filling along with the usual increase in contractility.

Intact rat Langendorff hearts, strips dissected from the LV or right atrium of cardiac surgery patients and rabbit papillary muscles were acutely stretched for 15min. Passive tension (PT) was measured in skinned cardiomyocytes extracted from the LV of control and stretched rat hearts for sarcomere lengths (SL) 1.8-2.3μm before and after incubation with PKG. Rabbit muscles were incubated with a PKG inhibitor or, simultaneously, a NO synthase inhibitor, a NO scavenger, a NP receptor A antagonist. All-total titin phosphorylation was stained with Pro-Q Diamond and indexed to total-protein signals using SYPRO Ruby. Values are given as mean±SEM and statistical significance was set to p<0.05.

After acute stretch there was a progressive decrease in passive tension/diastolic pressure over 15min: 27±8% and 27±6% in human atrium and ventricular muscles, respectively, and 43±2% in rabbit papillary muscles and isolated hearts. This decrease in myocardial stiffness was significantly blunted by PKG inhibition (40%) and NO/NP pathway inhibition (29%). PT of cardiomyocytes was significantly lower (≈60%) in the previously stretched group for all SL. A similar effect, only significant in the control group, was observed after incubation with PKG. Titin phosphorylation increased markedly 15 minutes after acute myocardial stretch in human (atrial: 11±1% vs 41±8%; LV: 27±8% vs 71±21%) and rabbit (13±2% vs 23±3%) myocardium.

The progressive decrease in myocardial stiffness after acute hemodynamic overload is preserved at the myofilamental level and seems to depend on PKG activity, representing a potential therapeutic target in patients with pathologically rigid myocardium. Moreover, blocking PKG activation seems to attenuate this adaptive diastolic response. Therefore, titin phosphorylation by this kinase is probably involved in this new myocardial response to stretch in both animals and humans.
Abstract 218: Chemical Induced Reductive Stress Causes Cardiomyocyte Hypertrophy
Authors: Shelar Sandeep B , Narasimhan Madhusudhanan , Shanmugam Gobinath , Vargees Neelu E , Sakthivel Ramasamy , Brown Charmaine , Sethu Palaniappan , Warner Wayne E , Darley-Usmar Victor , Rajasekaran Namakkal S
Published: July 2021

Background:
Progressive accumulation of misfolded or unfolded proteins is a symbol of impaired proteostasis and proteotoxicity. Such a chronic proteotoxicity is amenable to cell types that are post mitotically matured with lack of further differentiation or proliferation. Our recent discovery using a mouse model of familial human cardiac disease displayed protuberant shift in the redox state towards reductive stress (RS) in association with accumulation of toxic protein aggregates. Further, sustained trans-activation of Nrf2/antioxidant signaling caused RS in the myopathy hearts. Accordingly, we hypothesized that whether profound activation of Nrf2/antioxidant signaling and subsequent RS may cause pathological remodeling in cardiomyocyte. The aim of this study was to investigate the effect of sustained pharmacological activation of Nrf2 on cardiac remodeling. Methods: HL1 cardiomyocytes were used as an in vitro model to study the RS-mediated cardiac remodeling. They were treated with 2-10 μM of potential Nrf2-inducers; sulforaphane (SF), di-methyl fumarate (DMF) and novel small molecules (C-38, C-50, C-63 and C-66) to establish RS by sustained activation of Nrf2/antioxidant signaling. Next, we investigated the implications of RS in cardiomyocyte remodeling by analyzing transcriptional and translational mechanisms using immunoblotting, qPCR, immunofluorescence, GSH and NADPH redox measurements in HL1 cells. Results: Dose dependent effects for individual small molecules including known Nrf2 inducers (SF and DMF) revealed distinct pro-reductive and reductive intracellular (i.e. reductive stress) environments. In fact, the obligatory activation of Nrf2 signaling was associated with significant upregulation of antioxidant enzymes and small molecular thiols including glutathione (GSH). Surprisingly, while pro-reductive condition in HL1 cells was subdued, the RS induced cardiomyocyte hypertrophy was evident from microscopic examination and molecular signature (increased expression of ANF and BNF) after 24-48 hrs of Nrf2 activation. Conclusion: In summary, the chemical induced sustained activation of Nrf2 leading to formation of reductive stress showed hypertrophic remodeling in HL1 cardiomyocytes.
Abstract 219: Diabetes-induced Mitochondrial Dna Damage and Cardiac Dysfunction are Aggravated Due to Low Aldehyde Dehydrogenase 2 (aldh2) Activity in Aldh 2*2 (e487k) Knock-in Mutant Mice
Authors: Palaniyandi Suresh S , Pan Gudong , Deshpande Mandar , Mali Vishal R , Xu Jiang , Yang Xiao-Ping , Giri Shailendra , Chen Che-Hong , Mochly-Rosen Daria
Published: July 2021

A prevalent mutation (E487K) in aldehyde dehydrogenase (ALDH) 2, a cardiac mitochondrial enzyme, in East Asians (ALDH2*2) reduces ALDH2 activity and thereby increases aldehyde toxicity. Decreased ALDH2 activity is associated with cardiovascular diseases in humans and animal models. In this study, we hypothesized that reduction in ALDH2 activity in ALDH2*2 mice is sufficient to increase 4-hydroxy-2-nonenal (4HNE) levels and impair mitochondrial respiration and consequently induce cardiac damage in diabetes mellitus (DM). To test the hypothesis, streptozotocin (150 mg/kg i.p.) injected type-1 diabetic ALDH2*2 and C57BL mice as well as corresponding non-diabetic mice were employed. Four experimental groups were C57BL Control, C57BL DM, ALDH2*2 Control, and ALDH2*2 DM. N=6. Data were presented below in the same order. The mice were sacrificed after 3 weeks of DM. Myocardial ALDH2 activity and levels were reduced and 4HNE protein adducts were increased in ALDH2*2 DM mice relative to C57BL DM mice. Decrease in mitochondrial respiration was higher in ALDH2*2 DM mice compared to C57BL DM. Increase in cardiac hypertrophy (207 ± 8, 355 ± 4, 289 ± 22, 370 ± 20 in μm2; $$P<0.01 ALDH2*2 DM vs C57 DM) and fibrosis (4 ± 0.4, 8 ± 0.5, 6 ± 0.7, 9 ± 2.3 in % area of fibrosis; $$P<0.01 ALDH2*2 DM vs C57 DM) were higher in ALDH2*2 DM mice compared to C57BL DM. But the contractile function (56 ± 0.7, 54 ± 1.6, 43 ± 2.7, 48 ± 1.8 in %FS; $p <0.05 ALDH2*2 DM vs C57 DM) was lower only in ALDH2*2 DM, not WT DM. At the molecular level, increased mitochondrial DNA (mtDNA) damage and resultant decrease in MtDNA-encoded respiratory complex proteins were potentiated in diabetic ALDH2*2 mice compared to C57BL DM mice. Based on our data, we conclude that reduced ALDH2 activity in ALDH2*2 mice aggravated diabetes-induced cardiac mitochondrial respiratory dysfunction, ventricular remodeling and dysfunction.
Abstract 222: CCN5 Overexpression Triggers Early Amplification Followed by Regression of Electrical Remodeling in a Pressure Overload Rat Model
Authors: Hu Jun , Jeong Dongtak , Chaanine Antoine H , Motloch Lukas J , Xie Chaoqin , Hajjar Roger J , Akar Fadi G
Published: July 2021

The matricellular proteins CCN2 & CCN5 play opposing roles in pressure overload hypertrophy (PoH). While CCN2 promotes adverse remodeling, CCN5 suppresses fibrosis and therefore may be an important therapeutic target. Since arrhythmias are prevalent during early stages of PoH, we investigated the electrophysiological (EP) effects of CCN5 gene transfer (GT) in a rat model. We hypothesized that CCN5 GT prevents electromechanical dysfunction in PoH.

Methods:
4 wk old rats underwent aortic constriction and were randomized to receive AAV9 CCN5 or an empty vector (E) 3 wks later. In vivo hemodynamic analysis followed by ex vivo EP measurements using high resolution optical action potential (AP) mapping were performed at 9 or 21 wks of PoH.

Results:
CCN5 GT did not prevent but rather amplified early electromechanical remodeling as fractional shortening was significantly reduced, AP duration prolonged, and conduction velocity (CV) impaired in AAV9 CCN5 compared to AAV9 E hearts (Fig, top). Remarkably EP remodeling in AAV9 CCN5 hearts was largely reversed by 21 wks of PoH (Fig, top). To probe the basis of these unexpected findings, we tested whether CCN5 GT alters the EP substrate in normal (fibrosis free) hearts. Indeed AAV9 CCN5 GT caused significant CV slowing, consistent with a potent effect on excitability that was independent of fibrosis (Fig, bottom).

Conclusions:
CCN5 GT alters myocardial conduction by directly modulating gap junction and/or Na channel function. EP remodeling is amplified during early PoH but undergoes regression at later stages possibly through reversal of fibrosis. CCN5 modulates the EP substrate via fibrosis dependent (late) and independent (early) effects.
Abstract 223: Remodeling of K-ATP Channel Expression and Function in Type-2 Diabetes Mellitus
Authors: Hu Jun , Xie Chaoqin , Akar Fadi G
Published: July 2021

Diabetes mellitus (DM) is associated with enhanced propensity for ventricular tachyarrhythmias (VT). We found that DM hearts exhibit attenuated APD shortening in response to ischemia (Fig). We hypothesized that altered KATP channel expression & function underlie this impaired electrophysiological (EP) adaptation.

Methods:
We investigated differential effects of the SUR2a activator Pinacidil (PIN, 100 uM) and the SUR1 activator Diazoxide (DZX, 30 uM) on EP properties in Zucker Diabetic Fatty (ZDF) and control (ctrl) rats at baseline and in response to no flow ischemia. We measured the mRNA expression of KATP channel subunits (Kir6.1, Kir6.2, SUR1a, SUR1b, SUR2a, and SUR2b) in normal and ZDF animals.

Results:
PIN caused comparable APD shortening in ctrl & ZDF hearts during baseline perfusion and promoted early onset of sustained VT within 6 min of ischemia in ctrl & ZDF hearts. In contrast, DZX did not alter APD (p=NS) or cause arrhythmias in either group during baseline perfusion. However, DZX accelerated APD shortening and promoted VT in all (8/8) ZDF hearts during ischemia (Fig 1). The putative molecular mechanism underlying the differential proarrhythmic sensitivity of ZDF vs ctrl hearts to DZX was investigated. We found marked upregulation of Kir6.1 (by 3.7X p<0.001) and SUR1b (by 2.8X p<0.001) mRNA levels with no changes in Kir6.2, SUR1a, SUR2a, or SUR2b expression in ZDF.

Conclusion:
Remodeling of KATP channel subunits likely underlies the differential sensitivity of ZDF hearts to DZX. Our findings indicate that this classically cardioprotective agent should be avoided in DM patients at risk of ischemic events. Upregulation of Kir6.1 & SUR1b may be markers of proarrhythmia.
Abstract 224: Primary Human Cells Isolated From Atria and Bone Marrow Exhibit Comparable Anti-fibrotic Cell Phenotypes Upon Transfection With MicroRNA-301a
Authors: Müller Alison L , Freed Darren H
Published: July 2021

There are many cell types that can contribute to cardiac fibrosis including atrial fibroblasts (AFs) and bone marrow-derived progenitor cells (MPCs). We have previously shown that MPCs display a myofibroblast phenotype in vitro which is linked to altered microRNA(miR)-301a expression, a miR affiliated with maintaining proliferation in numerous cell types. We have also shown that miR-301a influences a dichotomous phenotype in primary human MPCs isolated from patients undergoing open heart surgery. As both MPCs and AFs display a dichotomous phenotype where each cell type displays a phenotype that pathologically contributes to fibrosis, we transfected both MPCs and AFs with miR-301a. AFs were also isolated from patients undergoing open heart surgery. We observed decreases in levels of both mRNA and protein of collagen I, non-muscle myosin IIA, and EDA-fibronectin. These proteins are expressed in myofibroblasts, the cell type predominantly responsible for causing cardiac fibrosis. In addition, transfection of miR301a caused both cell types to increase proliferation, which was analyzed using MTT proliferation assays. These results indicate that miR-301a could be influencing a non-fibrotic phenotype, which could prove useful in cell therapy trials where progenitor cells are injected into scar tissue in order to help heal patients who have suffered from a myocardial infarction. Over-expressing miR-301a in cells used could prevent them from differentiating into pro-fibrotic phenotypes and encourage their proliferation, thereby potentiating their efficacy.
Abstract 227: The Effect of PHLPP2 Removal on Cardiomyocyte Hypertrophy
Authors: Purcell Nicole H , Moc Courtney , Birrueta Giovanni , Taylor Amy , Koch Walter , Zambrano Cristina
Published: July 2021

Crucial cellular decisions that lead to cell growth, metabolism, proliferation, and survival are all dependent on the precise control of the phosphorylation state of proteins. The serine/threonine phosphatase, PHLPP (PH domain leucine-rich repeat protein phosphatase) has been shown to directly dephosphorylate several members of the AGC family of kinases. Knockdown of PHLPP1 by siRNA in neonatal cardiomyocytes potentiates Akt activity and phosphorylation specifically at Ser473 basally and following agonist stimulation while, the removal of PHLPP2 in cardiomyocytes does not affect Akt phosphorylation as previously reported in other cells. We hypothesize that PHLPP2 may target other AGC kinases in cardiomyocytes to regulate cardiac hypertrophy. Preliminary data suggests that removal of PHLPP2 activates fetal gene re-expression at baseline and potentiates phenylephrine (PE) induced gene expression 2 fold over siControl. Recently, G protein-coupled receptor kinase 5 (GRK5), which is an AGC kinase, has been shown to regulate cardiac hypertrophy through HDAC5 phosphorylation and de-repression of gene transcription. We wanted to determine whether PHLPP2 regulates GRK5 phosphorylation and localization in cardiomyocytes. GRK5 translocates to the nucleus following hypertrophic stimulation and we found that removal of PHLPP2 increased GRK5 translocation to the nucleus at baseline and with PE treatment compared to siControl cells. Also, removal of PHLPP2 increased nuclear export of HDAC5 at baseline and following PE treatment. Conversely, overexpression of PHLPP2 blocked nuclear translocation of GRK5 following PE treatment. Ongoing studies will determine whether PHLPP acts as a scaffold or if its phosphatase activity is necessary for inhibition of GRK5 translocation by directly measuring the phosphorylation of GRK5 in the presence and absence of PHLPP2 following hypertrophic stimulation. Our preliminary data is the first to uncover GRK5 as a novel PHLPP2 target in cardiomyocytes. Since little is known about the non-canonical regulation of GRK5, understanding whether phosphorylation and localization is regulated within the cardiomyocyte by PHLPP has potential for new therapeutic targets in the treatment of cardiac hypertrophy and failure.
Abstract 228: CaMKII as a Link Between Inflammation and Fibrosis Induced by Chronic Isoproterenol and Angiotensin II Treatment
Authors: Willeford Andrew , Heller Brown Joan
Published: July 2021

The Ca2+/calmodulin-dependent kinase, CaMKIIδ, is an established mediator of the development of heart failure and myocardial injury. Cardiac inflammation has been increasingly recognized as an important player in these cardiac pathophysiological changes. We previously demonstrated that CaMKIIδ contributes to cardiac inflammation induced by ischemia/reperfusion through activation of cardiomyocyte NF-kB. In the current study we ask whether angiotensin II (Ang II) and isoproterenol (ISO), both known to activate CaMKII, promote cardiac inflammation through this protein kinase and its effects on NF-kB activation. In addition, chronic ISO and Ang II treatment promote cardiac fibrosis and we hypothesize that this response is initiated through activation of CaMKIIδ and subsequent inflammatory responses. We report on our recent findings that show attenuated inflammatory cytokine expression (e.g. IL-6, MCP1, and TNFα) in response to 7 days Ang II infusion in mice in which CaMKIIδ is specifically deleted in cardiomyocytes (cardiac specific knockout; CKO). In addition the expression of fibrotic markers (e.g. col1a1, col3a1, and CTGF) in response to Ang II infusion is decreased in the CKO mice. This is associated with attenuated fibrosis as evident in histological analysis of CKO vs WT heart sections. Ongoing studies will compare the effects of chronic ISO and Ang II in CKO and WT mice to determine whether inflammation precedes fibrosis and assess the extent to which apoptosis induced by CaMKII activation plays a part in these responses. Currently, we are determining whether Ang II and ISO act through CaMKII to activate NF-kB in the cardiomyocyte compartment to induce proinflammatory and profibrotic factors and whether preventing the expression of these factors block development of further inflammatory and fibrotic responses. Findings from these studies may implicate CaMKII as a promising therapeutic target for attenuating cardiac fibrosis.
Abstract 232: Bendavia, a Novel Mitochondria Targeting Peptide, Improves Mitochondrial Function in Circulating Blood Monocytes From Dogs With Chronic Heart Failure
Authors: Sabbah Hani N , Sing-Gupta Vinita , Gupta Ramesh C
Published: July 2021

Background:
Mitochondrial (MITO) function is abnormal in heart failure (HF) as evidenced by reduced MITO respiration and rate of ATP synthesis. We showed that MITO dysfunction can be normalized in HF dogs after therapy with Bendavia (BEN), a novel MITO targeting peptide. This study tested the hypothesis that BEN will also reverse abnormalities of MITO function present in blood monocytes (MCs) of HF dogs.

Methods:
Blood samples obtained from 6 normal (NL) dogs and 6 dogs with coronary microembolizations-induced HF (LV ejection fraction ~30%) were used to isolate MCs by sequential Ficoll and Percoll density gradients. An XFe/XF96 analyzer (Seahorse Bioscience) was used to measure oxygen consumption rates (OCR) in MCs in the presence and absence of 1 μM oligomycin, 0.5 μM FCCP, or 1 μM each rotenone and antimycin. MITO proton leak, maximal respiration (MAXresp) and spare respiratory capacity (SRC) were measure in the presence and absence of 0.1, 1.0 and 10 μM concentrations of BEN and results expressed in pmols OCR/min/μg protein.

Results:
Proton leak, MAXresp and SRC were abnormal in MCs of HF dogs compared to NL. Incubation with BEN had no effect on measures of MCs MITO function of NL dogs but nearly normalized MITO function of MCs of HF dogs as evidenced by a dose-dependent increase MAXresp and SRC and dose-dependent decrease in proton leak (Table).

Conclusions:
MITO function is abnormal in blood MCs of HF dogs. BEN had no effect on MITO function of MCS from NL dogs but normalized MITO function in MCs from HF dogs. These findings support the use of circulating blood MCs as means of assessing MITO dysfunction in HF and as a marker of potential benefits derived from treatment with MITO targeted therapies.
Abstract 238: Molecular Identity of Cardiac Mitochondrial Chloride Intracellular Channel Proteins and Their Role in Mitochondrial Function
Authors: Ponnalagu Devasena , Gururaja Rao Shubha , Singh Harpreet
Published: July 2021

Introduction:
Mitochondria are primary target organelles for cardioprotection. Rabbit heart and cardiomyocytes showed increased infarction upon treatment with IAA-94, a known chloride intracellular channel (CLIC) inhibitor implicating role of CLICs in cardioprotection. There are six CLIC homologs (CLIC1-6), but the molecular identity of cardiac mitochondrial CLICs and their functional roles are not deciphered. We aim to determine the identity of cardiac mitochondrial CLICs and establish their role in mitochondrial function to understand the mechanism of CLIC-mediated cardioprotection.

Methods:
Hearts were isolated from 2-3 months old SD rats for all the experiments. qPCRs were performed to determine the abundance of CLIC1-6 and corroborated by Western blot using specific antibodies. Immunofluorescence was performed on neonatal and adult cardiomyocytes. Percoll-purified mitochondria were also probed with Western blot, immuno organelle chemistry and Mass spectrometry to establish the presence of CLICs. In order to determine their role in mitochondrial function, spectrofluorimetric analysis were performed to measure the reactive oxygen species (ROS) production using amplex red and calcium retention capacity (CRC) using calcium green.

Results:
We have discovered that CLIC4, CLIC5 and CLIC1 as the most abundant paralogs in rat heart using qRT PCR and corroborated their presence by Western blot. CLIC4 and CLIC5, not CLIC1 were found to be present in mitochondria of neonatal and adult cardiomyocytes. Percoll-purified mitochondria further showed presence of CLIC4 and CLIC5 where they colocalizes with mitochondria (45+/-2 and 74+/-3, n=3, respectively). Over-expressing CLICs in H9C2 cells showed mitochondrial localization of CLIC4 and CLIC5 and not CLIC1 establishing them as mitochondrial anion channels. Blocking of CLICs with IAA-94 modulated the ROS production at both complex I and II/III of electron transport chain of mitochondria. Further IAA-94 reduced CRC of mitochondria by 83+/-6%, n=4.

Conclusions:
We have established that CLIC4 and CLIC5 are the cardiac mitochondrial anion channels and their activity regulates the function of mitochondria hence playing a direct role in cardioprotection from ischemia-reperfusion injury.
Abstract 241: Acetylomic and Metabolomic Alterations in Aged Hearts: Role for Silent Information Regulator (SIRT) 3
Authors: Zhang Jimmy , Urciuoli William R , Brookes Paul S , Porter George A , Nadtochiy Sergiy M
Published: July 2021

Introduction:
SIRT3 is a mitochondrial metabolic regulator, and a decline in function of SIRT3 may play a role in age-related mitochondrial alterations. The aim of this study was to investigate the possible down-regulation of SIRT3 activity in aged hearts, and to identify which metabolic pathways in aged hearts may be impaired due to SIRT3 dysfunction.

Methods:
Mitochondria were isolated from WT adult (7 mo.), SIRT3
-/-
adult (7 mo.) and WT aged (18 mo.) hearts. Acetylated proteins in mitochondrial samples were identified using 2D gels and mass spectrometry. Metabolite concentrations and carbon fluxes through core metabolic pathways were determined using
13
C-labeled substrates and LC-MS/MS.

Results:
Mitochondrial acetylation patterns in the SIRT3
-/-
adult group matched those found in the WT aged group; the level of acetylation was significantly higher than in WT adult. While the SIRT3
-/-
samples exhibited zero SIRT3 protein content, no difference in SIRT3 protein level was seen between adult and aged WT hearts. Mechanistically, this suggests that alterations in mitochondrial acetylation during aging were not caused by lower SIRT3 protein levels, but rather by a lower SIRT3 enzymatic activity. Furthermore, aged myocardium exhibited 40% lower NAD
+
levels, which may underlie compromised SIRT3 activity.

ATP levels were decreased in both SIRT3
-/-
and WT aged hearts, suggesting possible defects in energy metabolism. Using metabolomics, we demonstrated that alterations of TCA cycle intermediates were similar in SIRT3
-/-
and WT aged hearts (relative to WT adult), and included a substantial decline of carbon flux through α-ketoglutarate and malate. Furthermore, regulation of energy production might also be impaired at the level of the electron transport chain, where Complex I was significantly inhibited in both SIRT3 deficient and aged hearts.

Conclusions:
Collectively these data suggested that acetylomic and metabolomic fingerprints observed in SIRT3
-/-
hearts were recapitulated in aged hearts.
Abstract 243: The E3 Ubiquitin Ligase Parkin Mediates Clearance of Damaged Mitochondria via Two Distinct Pathways
Authors: Hammerling Babette C , Cortez Melissa Q , Hanna Rita A , Gonzalez Eileen R , Gustafsson Åsa B
Published: July 2021

Damaged mitochondria release reactive oxygen species and pro-death factors which can lead to loss of cardiac myocytes. To protect against such damage, myocytes have developed several mechanisms of quality control that act both on the protein and organelle levels. We have previously identified the E3 ubiquitin ligase Parkin as an important regulator of mitochondrial clearance via autophagy in the myocardium. Here, we report that Parkin can also mediate clearance of mitochondria via the endosomal-lysosomal pathway. We found that Parkin promotes clearance of damaged mitochondria in both wild type (WT) and autophagy-deficient Atg5 knockout mouse embryonic fibroblasts (MEFs) treated with the mitochondria uncoupler FCCP. Mitochondrial damage leads to rapid activation of the endosomal-lysosomal pathway in both WT and Atg5-/- MEFs. We further observed increased activation of Rab5, a protein involved in early endosome formation, in both WT and Atg5-/- MEFs after treatment with FCCP. In addition, we observed sequestration of damaged mitochondria in Rab5+ and Rab7+ early and late endosomes, respectively. Mitochondria also colocalized with Lamp2+ vesicles in Atg5-/- MEFs indicating that the mitochondria are ultimately being delivered to the lysosomes for degradation. Overexpression of Rab5S34N, a dominant negative of Rab5, reduces FCCP-mediated clearance and increases cell death in Atg5-/- MEFs. Pharmacological inhibition of the endosomal-lysosomal pathway also results in increased FCCP-mediated cell death. Furthermore, we confirmed that FCCP treatment or simulated ischemia reperfusion exposure induces Rab5 activation with subsequent mitochondrial sequestration in early endosomes in neonatal myocytes. Interestingly, the activation of Rab5 is abrogated in the presence of the mitochondrial targeted antioxidant Mito-Tempo, suggesting that mitochondrial ROS is involved in the activation the endosomal pathway. Mitochondrial clearance via this pathway is also dependent on Parkin, as FCCP treatment fails to activate Rab5 and induce mitochondrial clearance in both WT and Atg5-/- MEFS in the absence of Parkin. Thus, our data suggest that Parkin can mediate clearance of damaged mitochondria via two distinct pathways in cells.
Abstract 247: Dose-dependent Role of Inwardly Rectifying Potassium Current on Cardiac Automaticity of Human Embryonic Stem Cell-derived Cardiomyocytes
Authors: Fu Jidong , Dennis Adrienne
Published: July 2021

The inwardly rectifying potassium current (IK1), encode by Kir2 family, is responsible for maintaining the negative resting potential, and contributes to phase 3 repolarization of the cardiac action potential. IK1 was generally thought to suppress cardiac automaticity, while the suppression of IK1 in adult ventricular cardiomyocytes (CMs) could engineer bio-artificial pacemaker-like cells to spontaneously fire action potential. Our studies also showed that overexpressed the gene of Kir2.1 could facilitate the electrophysiological maturing of mouse and human embryonic stem cell-differentiated CMs (ESC-CMs), which have the high degree of automaticity with nearly 50% of cells that can spontaneously fire action potential. In this study, we extensively analyzed the electrophysiology of mouse and human ESC-CMs, and found that the maximum diastolic potential in spontaneously firing ESC-CMs, -72.1±1.3 mV in atrial cells and -75.0±2.1 mV in ventricular cells, were significantly more hyperpolarized than that in quiescent ESC-CMs (-64.4±2.1 mV in atrial cells and -67.1±3.2 mV in ventricular cells). Applying a small amount of IK1 to hyperpolarize the membrane potential could enable those quiescent ESC-CMs to spontaneously fire action potential, indicating the enhancement of cardiac automaticity, while a large amount of IK1 could quiet those spontaneously firing cells down. By combining computational and experimental analyses, we confirmed that the synergistic interaction of IK1 and pacemaker current (If) could efficiently regulate cardiac automaticity during the differentiation. Our studies disclosed a dose-dependent role of IK1 on cardiac automaticity that a small amount of IK1 enhances and a large amount of IK1 suppresses cardiac automaticity in ESC-CMs during differentiation.
Abstract 252: Doxorubicin-induced H9c2 Cell Death is Mediated by Excessive Mitochondrial Fission and Mitophagy
Authors: Catanzaro Michael P , Weiner Ashley , Kaminaris Amanda , Kobayashi Satoru , Liang Qiangrong
Published: July 2021

Doxorubicin (DOX) is a widely used antineoplastic agent that can cause heart failure. DOX cardiotoxicity is closely associated with mitochondrial damage. Mitochondrial fission and mitophagy are quality control mechanisms that help maintain a pool of healthy mitochondria. However, too much mitochondrial fission and/or mitophagy may compromise cell viability. Indeed, Mdivi-1, an inhibitor of the fission protein Drp1, can attenuate DOX-induced cardiac injury, suggesting that mitochondrial fragmentation may play a role in DOX cardiotoxicity. Using genetic gain- and loss-of function approaches, we determined whether mitochondrial fragmentation and/or mitophagy contribute to DOX-induced cardiomyocyte death. H9c2 cardiac myoblast cells were transfected with siRNA targeting Drp-1 before DOX administration. Mitochondrial morphology was examined with confocal microscopy after infection of the cells with the adenovirus encoding mitochondria-targeted fluorescent protein MitoDsRed. Morphometric analysis demonstrated that Drp-1 knockdown markedly diminished DOX-induced mitochondrial fragmentation as shown by form factor, aspect ratio, and mean mitochondrial size. This led to reduced cardiomyocyte death as revealed by the percentage of propidium iodide (PI)-positive cells and the cleavage of caspase-3 and Poly ADP ribose polymerase (PARP). Not surprisingly, Drp-1 knockdown also attenuated DOX-induced mitophagy flux as assessed by the dual fluorescent mitophagy reporter mt-Rosella. Further, knockdown of Parkin, a key regulator of mitophagy, dramatically diminished DOX-induced H9c2 cell death. Although Drp1 overexpression did not markedly increase DOX-induced cell death, Parkin overexpression predisposed H9c2 cells to DOX toxicity. Together, these results suggest that DOX-induced cardiotoxicity may be due to excessive mitochondrial fragmentation and accelerated mitochondrial degradation through autophagy. Strategies that limit mitochondrial fission and mitophagy within the physiological range may help reduce DOX cardiotoxicity. However, further studies are clearly warranted to make sure that these strategies will not compromise the antitumor efficacy of DOX.
Abstract 255: Hur/ppar-gamma Mediates Posttranscriptional Regulation of Hyperglycemia-induced Pro-inflammatory Response
Authors: Suresh Babu Sahana , Thandavarayan Rajarajan A , Joladarashi Darukeshwara , Jeyabal Prince , Krishnamurthy Shashirekha , Garikipati Venkata N , Verma Suresh K , Kishore Raj , Krishnamurthy Prasanna
Published: July 2021

Metabolic disorders including diabetes and obesity are associated with persistent activation of the innate immune response, which leads to low-grade inflammation and increased cardiovascular risk. The RNA-binding protein HuR binds to and post-transcriptionally regulates inflammatory cytokines, mRNA stability and gene expression, although it’s exact role in the diabetic condition remains unclear. Bone marrow-derived macrophages (BMM) exposed to high glucose (HG, 25mM D-glucose) displayed elevated expression of HuR and reduced expression of nuclear receptor peroxisome proliferator-activated receptor-gamma (PPAR-gamma) as compared to low glucose (LG, 5mM D-glucose)-treated cells. The increase in HuR expression was associated with an increase in the gene expression of various inflammatory cytokines including TNF-alpha, MCP-1, IL-1beta and IL-6. At the molecular level, macrophages activated with LPS showed enhancements in expression of inflammatory mRNAs and also promoted their stability. Conversely, knockdown of HuR induced post-transcriptional silencing, reduced the mRNA stability and expression of inflammatory cytokines. Macrophages treated with selective antagonists of the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPAR-gamma), GW9662 displayed increases in HuR expression and of various inflammatory cytokines. In an in vivo model of myocardial infarction, mice receiving intramyocardial injection of HuR-specific shRNA displayed a reduced inflammatory cytokine profile in the myocardium as compared to control shRNA treated mice. Taken together, these data highlight the role of HuR as a homeostatic coordinator of cytokine mRNA that regulates innate inflammatory effects and demonstrates the potential of modulating the effects of HuR for clinical benefit against pathologic inflammation in diabetes/obesity.
Abstract 265: CITED4 Induces Physiologic Hypertrophy and Improves Cardiac Remodeling After Ischemic Injury
Authors: Bezzerides Vassilios J , Platt Colin , Paruchuri Kaavya , Oh Loren , Xiao Chunyang , Mann Nina , Rosenzweig Anthony
Published: July 2021

Cardiac hypertrophy is an adaptive response to increased hemodynamic stresses, which can be either physiologic, as with exercise, or pathologic, as with valvular heart disease. Recent data suggest that physiologic hypertrophy secondary to exercise may be mediated by the transcription factor CEBPβ and the p300-interacting protein CITED4. We sought to investigate the cardiovascular effects of CITED4 expression in vivo.

Using a cardiac-specific and inducible transgenic mouse (Tg) model, we determined the effects of CITED4 expression on cardiac parameters including heart weight, cell size, cardiac function and gene expression. Expression of CITED4 for 3 weeks induced increases in heart weight (22% in HW/TL, p < 0.01) and cardiomyocyte (CM) size (24.5% in cell area, p < 0.001) with normal systolic function and without evidence of fibrosis. Gene profiling demonstrated increased expression of cardiac troponin, a favorable αMHC/βMHC ratio and a reduction in BNP consistent with physiologic hypertrophy. Genome-wide expression profiling of neonatal rat ventricular myocytes (NRVMs) over-expressing CITED4 demonstrated the activation of a unique set of genes including BCL2, ATP12a, Efemp1, Ifi204 and Tcf19.

To further examine the potential beneficial role of CITED4, we induced ischemia by transient occlusion of the left anterior descending (LAD) coronary (30 min) followed by reperfusion for 24 hours, 6 days or 4 weeks. At 24 hrs after ischemia-reperfusion injury (IRI), neither cardiac dysfunction on echo nor infarct sizes were different between CITED4 Tg and controls. However, CITED4 Tgs showed substantial recovery of cardiac function at 4 weeks (FS: CITED4 Tg 51%, Control 34%, p < 0.01) and a 3.4-fold reduction in fibrosis (p < 0.005). Analysis of possible cellular responses responsible for the functional recovery demonstrated enhanced autophagic flux with reduced accumulation of LC3II (down 71%, p<0.05) and p62 (down 54%, p<0.005). Further examination of the involved signaling pathway revealed direct activation of mTORC1 and its effectors consistent with a growth phenotype.

We conclude that CITED4 expression is sufficient to induce physiologic cardiac hypertrophy and improves cardiac remodeling after ischemic injury likely through activation of mTORC1.