bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P309: ZnT-1 Protects HL-1 Cells from Simulated Ischemia-Reperfusion Through Activation of Ras-ERK Signaling
Authors: Dror Shani , Beharier Ofer , Levy Shiry , Mor Merav , Kahn Joy , Katz Amos , Moran Arie , Etzion Yoram
Published: March 2022

BACKGROUD:
ZnT-1 is a protein that confers cellular resistance from heavy metal toxicity, but its function in the myocardium is not clear. Our group recently demonstrated that ZnT-1 regulates calcium influx into cardiomyocytes by interacting with the regulatory β-subunit of the L-type calcium channel. In addition, in non-cardiac cells ZnT-1 was found to interact with Raf-1 kinase leading to downstream activation of MEK/MAPK signaling. In the present study we investigated the role of ZnT-1 in myocardial ischemia\reperfusion injury (I\R).

METHODS AND RESULTS:
Cultured cells of cardiomyocyte origin (HL-1 cells) were exposed to ischemic conditions for 60 min using sodium cyanide and 2-Deoxi Glucose, followed by 60 min of washout mimicking reperfusion. I\R injury was detected by measuring LDH release and staining for pro-apoptotic proteins activation (caspase 3 and 7). Overexpression of ZnT-1 reduced the LDH release following I\R injury to 50.1±2.5% of control (n=6, p<0.01) and markedly reduced caspases staining. Consistently, ERK phosphorylation was increased in the ZnT-1 transfected cells to 266±27.8% of control (p<0.01). Knockdown of endogenous ZnT-1 by shRNA blocked the phosphorylation of ERK and markedly augmented LDH release following I/R injury to 287.4±36.9% of control (n=5, p<0.01). The protective effect of ZnT-1 following I\R injury was apparent following pretreatment of HL-1 cells with the L-type calcium channel blocker nifedipine (1 μM). In contrast, pretreatment with the MEK inhibitor PD98059 (10 μM) completely abolished the protective effect of ZnT-1 following I\R injury (n=3). Moreover, a mutated form of ZnT-1 lacking the ability to bind Raf-1, failed to protect HL-1 cells from I\R injury (n=3).

CONCLUSIONS:
ZnT-1 confers cellular resistance from I\R injury trough its ability to interact with Raf-1 and its ability to stimulate the Raf/MEK/MAPK signaling pathway. Our findings suggest an important new role for ZnT-1 in the myocardium.
Abstract P310: Connective Tissue Growth Factor Antibody Protects from Adverse Left Ventricular Remodeling Following Myocardial Infarction
Authors: Vainio Laura , Szabo Zoltan , Serpi Raisa , Ruskoaho Heikki , Kerkelä Risto
Published: March 2022

Ischemic heart disease (IHD) is the most common cause of death in most Western countries, but the therapies for IHD are lacking. We aimed to determine the potential of connective tissue growth factor (CTGF) antibody in protecting myocardium from long-term deleterious effects of myocardial infarction. Healthy 8-week old mice were subjected to myocardial infarction (MI) and at 1 week post-MI the animals were randomized for treatment with vehicle, metoprolol or CTGF monoclonal antibody. After six weeks of treatment, CTGF antibody treated animals showed significantly better left ventricular (LV) systolic function than the vehicle-treated animals. LV diastolic function or LV dimensions were not affected by treatments. Analysis of cardiac sections revealed no difference in infarct scar size between the experimental groups. However, RT-PCR analysis revealed a decrease in the expression levels of fibrosis-related genes in non-ischemic area in the hearts of CTGF antibody treated animals. In addition, cardiomyocyte size was significantly reduced in LV sections of CTGF antibody treated animals compared to vehicle-treated animals. This was accompanied with a significant decrease in B-type natriuretic peptide mRNA levels in the LV, whereas expression levels of atrial natriuretic peptide or beta-myosin heavy chain were similar in MI groups. There was also a slight decrease in mRNA levels of interleukin-6 and CTGF in the hearts of CTGF antibody treated animals. Treatments had no effect on mRNA levels of transforming growth factor-β1, tumor necrosis factor-α and vascular endothelial growth factor A following MI. In conclusion, CTGF antibody treatment attenuates LV hypertrophy and improves LV systolic function following MI. Blocking CTGF function by a specific antibody may be useful for prevention of adverse myocardial remodeling and heart failure.
Abstract P313: Genetic Deletion of Pim Causes Premature Cardiac Senescence
Authors: Din Shabana , McGregor Michael , Bailey Brandi , Mohsin Sadia , Avitabile Daniele , Gude Natalie , Sussman Mark A
Published: March 2022

Rationale:
Cardiac aging is defined as increased cellular senescence and loss of survival signaling within cardiomyocytes and the cardiac stem cell population. To prevent myocardial, aging it is important to reverse the characteristic aging phenotype. Pim-1 is a cardioprotective kinase downstream of AKT, and has been shown to be pro-proliferative and anti-apoptotic. Pim-1 mediated antagonism of myocardial aging will lead to a decrease in cardiac senescence, maintain contractile performance, and prolong the lifespan of an organism.

Objective:
Demonstrate that myocardial senescence is antagonized by Pim-1 mediated signaling and loss of Pim-1 activity leads to premature aging of the myocardium and associated decline in cardiac performance.

Methods and Results:
Pim Triple KnockOut (PTKO) mice, in which all three isoforms of Pim are genetically deleted, develop premature phenotypic hallmarks of myocardial senescence. Elevated levels of the tumor suppressor p53 and senescence marker p16 are present in hearts of PTKO mice at two months of age. Increased p16 expression is consistent with observed induction of Ets-1, a transcription factor that activates p16 transcription in PTKO mice. Levels of Id1 and Id2, positive transcriptional regulators of the cell cycle, are decreased in hearts of PTKO mice. Hemodynamic analyses reveal impaired cardiac function in PTKO mice at two months of age. Conversely, cardiac specific overexpression of Pim1-kinase attenuates the senescent phenotype. Hypoxia treatment of neonatal rat cardiomyocytes infected with Pim-1 encoding adenovirus results in reduction of p16 accumulation as seen by qRT-PCR. Pim-1 overexpression increases expression of c-myc, thereby upregulating Id2 and inhibits p16 induced senescence. Cardiac Pim-1 transgenic mice show a delay in the onset of senescence as evidenced by decreased frequency of p16 positive cells in the myocardium.

Conclusion:
Pim-1 inhibits cardiac senescence and is important for maintaining cardiac function. Therefore, selective manipulation of Pim-1 should be pursued as a therapeutic target to promote and extend myocardial cellular lifespan and function.
Abstract P316: Cysteine-Independent Interactions of Thioredoxin-1 and Apex1: Implications for Endothelial Cell Apoptosis and Aging?
Authors: Zschauer Tim-Christian , Beissel Lisa , Altschmied Joachim , Haendeler Judith
Published: March 2022

Aging processes are associated with increased intracellular formation of reactive oxygen species. One redox regulator is the ubiquitously expressed oxidoreductase Thioredoxin-1 (Trx). It contains two active site cysteines and has anti-oxidative and anti-apoptotic properties. Trx levels are reduced in senescent endothelial cells (EC), leading to increased apoptosis sensitivity. Several functions of Trx are enhanced by its interaction with AP-endonuclease 1 (APEX1). APEX1 is a multifunctional protein and in concert with Trx-1 a reductive activator of e.g. the transcription factor AP-1, suggesting at least an interaction of Trx and APEX1 in vivo. Recent evidence suggests that also APEX1 levels are decreased in senescent cells. Therefore, the aim of this study is to understand the function of APEX1 in concert with Trx in EC. We first investigated whether APEX1, similar to Trx has anti-apoptotic properties in EC. Indeed, overexpression of APEX1 inhibited apoptosis. To analyze where the Trx/APEX1 interaction occurs, we performed fluorescence microscopy demonstrating a colocalization throughout the whole cell. To study the interaction in more detail, we carried out coimmunoprecipitations from nuclear and cytosolic extracts and demonstrated their association in both fractions. To determine whether disulfide bridges are involved in the Trx/APEX1 association, we mutated APEX1 cysteines 65, 93 and 310 previously shown to interact with cysteines in other proteins, to serines. This mutant bound Trx in the nucleus and cytosol of EC with the same efficiency as APEX1 wildtype. Moreover, a Trx mutant with serine mutations of active site cysteines (Trx(C32S/C35S)) was still able to bind to endogenous APEX1. These data demonstrate that the Trx/APEX-1 interaction is independent of disulfide bridge formation. We are currently analyzing a set of APEX1 deletion mutants for their interaction with Trx-1 and a potential loss of anti-apoptotic properties Exact delineation of the Trx/APEX1 interaction surface will in the future allow the development of small peptides containing the interaction domains of Trx or APEX1 respectively, in order to enhance transcription factor activity and bypass the loss of antioxidative capacity during aging processes.
Abstract P329: The Thoracic and Abdominal Arterial Segments Are Genetically and Functionally Different
Authors: Chung Ick-Mo , Pritchard David K , Santana Luis F , Mahoney Williams M , Schwartz Stephen M
Published: March 2022

Aims:
Because arterial diseases are highly specific to different segments and because smooth muscle cells (SMCs) of different branches have distinct embryological origin, we undertook a systematic effort to determine the extent of differences in gene expression between the thoracic aorta (TA) and the abdominal aorta (AA). We also correlated these expression differences with physiologic function.

Methods and Results:
We utilized whole genome gene expression microarrays to compare expression patterns in the rat TA and AA. We identified 161 differentially expressed genes between these two segments. Functional analysis of differences in expression by Gene Set Enrichment Analysis identified different categories of genes enriched in each section of the aorta. For example, GPCR-related genes are overrepresented in the AA, while the TA showed dramatic enrichment of gene categories linked to mitochondria and energy generation. Because GPCR functions are implicated in many vascular diseases, we confirmed the differential expression of the arterial specific Regulator of G-protein signaling - 5 (RGS5), and extended the analysis to additional members of the RGS-R4 subfamily. RGS5 was the most abundant RGS-R4 mRNA in the AA, and it was the most differentially expressed family member across all vascular beds. To analyze functional correlates to the differential expression of RGS5, we performed aortic ring contraction assays from the TA and AA. Consistent with the hypothesis that RGS5 blocks angiotensin II (Ang II) type 1 receptor signaling via increased Gα-mediated GTPase activity, Ang II-induced force generation was significantly lower in AA relative to TA. Furthermore, whole-cell voltage-gated K
+
current (I
Kv
) analysis revealed that I
Kv
was significantly greater in SMCs from the AA compared to cells from TA. Kv1.5 mRNA was more highly expressed in the AA relative to the TA.

Conclusions:
The differential gene expression pattern between TA and AA suggests distinct differentiation pathways may explain regional specificity of arterial diseases. Specifically, the enrichment of GPCR-related genes in the AA and mitochondrial genes in the TA may account for the diverse physiological function of these two distinct vascular segments.
Abstract P331: Metabolic Dynamics and Phosphometabolomic Fingerprinting Using
18
O-Assisted
31
P NMR and Mass Spectrometry
Authors: Dzeja Petras , Nemutlu Emirhan , Zhang Song , Terzic Andre
Published: March 2022

Evaluation of disease phenotypes and drug effects requires knowledge not only metabolite levels but also their turnover rates from which metabolic fluxes and status of the whole energetic system can be determined. Oxygen-18-assited
31
P NMR and mass spectrometric technique uniquely allow simultaneous measurements of phosphorus-containing metabolite levels and their turnover rates. This includes simultaneous recordings of
18
O-labeling rates reflecting ATP synthesis (γ-ATP) and ATP utilization (Pi), phosphotransfer fluxes through adenylate kinase (β-ATP/ADP), creatine kinase (CrP) and glycolytic pathways (G-6-P) as well as Krebs cycle associated mitochondrial nucleotide turnover (γ/β-GTP), substrate shuttle (G-3-P) and glycogen metabolism (G-1-P). Metabolomic and fluxomic profiling of hearts and body fluids of phosphotransfer enzyme deficient transgenic animals using
18
O-assisted GC/MS,
1
H and
18
O-assisted
31
P NMR indicate selective metabolic perturbations and adaptations in the whole energetic system. Adenylate kinase and creatine kinase deficiencies were associated with altered metabolomic profiles, increased mitochondrial capacities and redistribution of phosphotransfer flux through glycolytic and guanine nucleotide systems. These phosphometabolomic alterations were more expressed when animals were subjected to treadmill exercise, mitochondria-targeting drugs such as metformin and high-fat diet. Metabolite turnover rates are correlated with heart performance unveiling robustness and dynamics rearrangements in cell bioenergetics infrastructure. Glycerophosphate, G-3-P, metabolic dynamics is altered in transgenic animal models indicating defects in substrate shuttle and supply of reducing equivalents to mitochondria. This is of importance since G-3-P metabolic abnormalities and metabolic arrest are linked to human diseases such as sudden death syndrome. Thus, our study demonstrates that phosphometabolomic and fluxomic profiling is a valuable tool for metabolic phenotyping of transgenic animal models of human diseases as well as for plasma biomarker identification and monitoring of treatment efficacy and drug toxicity.
Abstract P334: Comparative Effectiveness of Left Ventricular Assist Devices as a Bridge to Heart Transplantation
Authors: Higgins Robert S , Wissman Sherri , MacBriar Kelly , Bumgardner Erin , Burke Emily , Solanki Pallavi , Hasan Ayehsa , Crestanello Juan , Sai-Sudhakar Chittoor , Haas Gary , Abraham William , Leier Carl
Published: March 2022

Left ventricular assist devices (LVAD) have evolved to be more frequently utilized in patients with advanced congestive heart failure as a bridge to heart transplantation. In the era of limited healthcare resources and diminishing funding for innovative technologies, there is little data to evaluate the comparative effectiveness of LVADs in comparison to more conventional heart failure therapies leading to heart transplantation without mechanical circulatory support.

Objective
. To evaluate the clinical outcomes, complications, length of stay, financial impact and satisfaction of patients with advanced heart failure undergoing heart transplantation as part of a comprehensive heart failure program with or without LVAD as a bridge to transplant (BTT).

Methods
We retrospectively reviewed 25 patients undergoing heart transplantation from 2009–2010 (12 patients without LVAD, 13 with LVAD as BTT). Inpatient hospital, financial and clinic records were reviewed to evaluate length of stay, morbidity, mortality, DRG assignment, insurance status, charges and cost, reimbursement and patient satisfaction. All patients received surveys assessing their satisfaction with the mechanical circulatory support and transplant program.

Results
There were 14 males and 11 females (avg age 54 ).In the non- LVAD group, 9 patients fell into DRG 1 ,3 were in DRG 2;in the LVAD group, 10 patients fell in DRG 1, 3 patients were in DRG 2.Complications occurring in the LVAD patients included pneumonia (2),CVA, depression, rejection and bowel perforation ;in the non-LVAD patients pneumonia, pulmonary embolus, aspergillosis, peri-rectal abscess and failure to thrive; Mortality at 1 year occurred in 2 patients after LVAD; 1 patient after transplant without LVAD. All patients responded that they were very satisfied with the advanced heart failure program whether they received LVAD prior to transplant or not

Conclusions
Transplant recipients with advanced heart failure have comparable clinical outcomes after transplant whether they required LVAD implantation prior to transplant. Perioperative care algorithms focused on reduction of common post-operative complications resulting in significant financial impact (ie pulmonary), should be developed to address these issues.
Abstract P344: A Natural p300-Specific Histone Acetyltransferase Inhibitor, Curcumin, Prevents the Development of Heart Failure in Addition to ACE Inhibitor After Myocardial Infarction in Rats
Authors: Morimoto Tatsuya , Sunagawa Yoichi , Wada Hiromichi , Katanasaka Yasufumi , Takaya Tomohide , Kawamura Teruhisa , Yanagi Shigeki , Marui Akira , Sakata Ryuzo , Shimatsu Akira , Kimura Takeshi , Fujita Masatoshi , Hasegawa Koji
Published: March 2022

Background:
We found that curcumin, a p300 histone acetyltransferase (HAT) inhibitor, prevents deterioration of the systolic function in rat heart failure models in vivo. To clinically apply this novel therapy to humans, it should be clarified whether or not curcumin has additional effects on conventional heart failure therapy comprising angiotensin-converting enzyme inhibitors (ACEI).

Methods and Results:
Rats were subjected to a sham operation or myocardial infarction (MI). One week later, 32 rats were randomly assigned to solvents (control), enalapril (ACEI, 10 mg/kg/day) alone, curcumin (50 mg/kg/day) alone, or curcumin plus enalapril for 6 weeks. ACEI, but not curcumin treatment, decreased the blood pressure in post-MI rats. After treatment, LVFS (fractional shortening) was significantly (
p
<0.05) higher in the ACEI (29%) and curcumin (29%) groups than in the vehicle group (22%). Notably, LVFS significantly (
p
<0.05) increased on ACEI/ curcumin combination therapy (35%) compared with therapy comprising either ACEI or curcumin alone. The LV wall thickness and cardiomyocyte diameter were significantly smaller in the ACEI/curcumin than the ACEI group. Moreover, perivascular fibrosis was significantly reduced in the ACEI and curcumin groups compared with the vehicle group. This reduction was further augmented by the ACEI/ curcumin combination therapy.

Conclusion:
Curcumin, restores the post-MI LV systolic function in rats without affecting the blood pressure. This natural non-toxic dietary compound in addition to ACEI has beneficial effects on LV systolic function.
Abstract P349: Ambient Air Pollution Compromises Nitric Oxide Bioavailability, Leading to Accelerated Endothelial Cell Senescence
Authors: Buechner Nicole , Sydlik Ulrich , Unfried Klaus , Altschmied Joachim , Haendeler Judith
Published: March 2022

Particulate matter (PM) pollution is a burden to public health. Although PM causes a health risk to the lung, the overall evidence indicates that the majority of the PM effects are upon the cardiovascular system. Several studies demonstrated that ultrafine particles can directly enter the circulation and thus may interact with endothelial cells. However, intracellular mechanisms affected by non-cytotoxic, in vivo relevant concentrations of particles have not been investigated in endothelial cells in vitro and in vivo. Therefore, this study aims for the first time to analyze the effects of ultrafine and fine particles of different materials in non-toxic concentrations on human endothelial cells (EC) and in animals. We incubated EC with ultrafine and fine Carbon Black (ufCB and fCB) particles as well as Titaniumdioxide (ufTiO2 and fTiO2) and determined the non-toxic concentrations. MTT measurements revealed that 0.1 and 1 µg/cm2 did not reduce endothelial cell viability. To test whether these concentrations influence endothelial cell function, we measured nitric oxide (NO) bioavailability, which is important for vessel function. Only ultrafine particles reduced the S-NO content of EC, whereas fine particles had no effect at the same concentrations. Interestingly, the effects observed with ufCB and ufTiO2 were more pronounced than with the known reducer of NO bioavailability, H2O2. We previously demonstrated that NO increases activation of Telomerase Reverse Transcriptase (TERT), an enzyme essential for telomere maintenance. TERT activation is required to protect EC from apoptosis and the onset of senescence and TERT is inactivated by the Src kinase under conditions of oxidative stress. Therefore, we investigated the effects of ufCB and ufTiO2 on TERT and Src activation. ufCB and ufTiO2 significantly reduced TERT and increased Src kinase activation. To investigate whether ufCB show also effects in vivo, we instilled ufCB into rats and determined eNOS and TERT expression in the aorta. ufCB reduced eNOS and TERT expression in the abdominal aorta of animals treated with ufCB. Thus, ultrafine nanoparticles, which we inhale every day, seem to reduce endothelial function and thus should be considered a risk factor for cardiovascular disease.
Abstract 101:
FHL2
-null Mice Showing Less Cardiac Remodeling
Authors: Chu Pao Hsien
Published: March 2022

The four and a half Lin11, Isl-1 and Mec-3 (LIM) domain protein 2 (FHL2) is a member of the four and a half LIM domain-only (FHL) gene family, and has been shown to play an important role in cardiac remodeling. Here, we tested the hypothesis that isoproterenol-induced myocardial fibrosis in mice lacking FHL2 is related to resistant to myocardial fibrosis.

We compared the cardiac phenotypes of
FHL2
-null mice. Extra-cellular matrix remodeling and apoptosis are examined to determine the roles of FHL2 on cardiac fibrosis induced by isoproterenol. Compared to wild type littermates,
FHL2
-null mice exhibited lower mortality. Masson’s Trichrome and silver stains showed significantly less severe ventricle fibrosis in FHL2-null mice. In addition,
FHL2
-null mice exhibited lower levels of TGF-β, collagen I/III, but higher membrane-type matrix metalloproteinase-1 (MT1-MMP/MMP-14) during cardiac remodeling. This study shows that alternations of FHL2 might be implicated in the pathogenesis of cardiac fibrosis and suggests FHL2 as novel targets for cardiac fibrosis therapy. Further studies are required to define the mechanism by which FHL2 modulates cardiac remodeling.
Abstract 104: Activated Cardiac Fibroblast Can Deactivate to Become Resting Fibroblasts Upon Injury Resolution
Authors: Kanisicak Onur , Khalil Hadi , Karch Jason , Maliken Bryan , Fu Xing , Molkentin Jeffery D
Published: March 2022

Resident cardiac fibroblasts (CFs) are potential therapeutic targets in treating heart failure given the prominent role that fibrosis plays in this disorder. CFs directly convert to myofibroblasts (MFs) with injury where they mediate both adaptive wound healing after acute myocardial infarction as well as long-standing fibrosis during chronic disease states. However, the fate of activated MFs after injury resolution remains poorly understood, in part because the field has lacked a definitive strategy for identifying and tracing MFs and CFs in vivo. To address this issue we recently generated a novel mouse model that permits lineage tracing of all activated MFs in the heart after injury or stress stimulation. MFs were lineage traced with a tamoxifen inducible periostin allele knockin of the MerCreMer cDNA (PostnMCM) in combination with a Rosa26-eGFP Cre-dependent reporter. PostnMCM x R26-eGFP mice were transiently injured with the combined infusion of angiotensin II and phenylephrine (Ang/PE) for 2 weeks, during which time tamoxifen was also given to permanently label all newly formed MFs with eGFP. Ang/PE infusion was then removed and mice were then allowed to recover for 4 weeks as the fibrotic response regressed. The data show that during the Ang/PE infusion period nearly all the eGFP+ periostin lineage-traced myofibroblasts were αSMA positive with and activated myofibroblast gene expression profile. However, when the fibrotic response regressed weeks later, numerous periostin-lineage traced eGFP+ cells remained in the heart and these cells showed a phenotypic and molecular reversion back to resident CFs with a loss of myofibroblast marker genes. These results suggest that CFs are very unique cell types that can differentiate to MFs then de-differentiate back into resident CFs.
Abstract 105: Cardiomyocyte Secretory Beta-2 Microglobulin Contributes to Myocardial Fibrosis During Pressure Overload
Authors: Hui Gong , Li Yang , zhang Xiaoyi
Published: March 2022

Plasma Beta-2 microglobulin (β2M) level is inversely associated with glomerular filtration rate (GFR) and ejection fraction (EF) in patients with chronic kidney disease. However, few studies have evaluated roles of β2M in cardiac remodeling induced by hypertension. The present data showed that serum β2M level was increased in patients with hypertension as compared to control group, and it was significantly higher in patients with chronic heart failure than in those with hypertension. Serum β2M level was related to diastolic dysfunction in patients with hypertension and heart failure. During 4 weeks after transverse aortic constriction (TAC) in mice, β2M level in serum or heart tissue increased progressively in time-dependent manner. Cardiomyocytes but not fibroblasts secretory β2M was rapidly increased after mechanical stretch (MS)
in vitro
. Exogenous β2M showed pro-fibrotic effects and enhanced migratory capability in cardiac fibroblasts. Conditional medium (CM) from mechanical stretched cardiomyocytes showed the similar effects, but these effects were partly abolished by CM from β2M-downregulated-cardiomyocytes subjected to MS. In vivo, sh-β2M-AAV9 injection greatly improved the cardiac fibrosis and dysfunction induced by TAC. Further analysis indicated that β2M promoted the phosphorylation of epidermal growth factor receptor (EGFR) in fibroblasts and inhibition of EGFR significantly attenuated β2M-induced-activation of cardiac fibroblasts. The present data demonstrated the importance of β2M as a paracrine pro-fibrotic mediator and the relevance of β2M in the cardiac fibrosis associated with hypertension or heart failure. It suggests that inhibition of β2M may have the therapeutic potential for the prevention of cardiac fibrosis or the progression of heart failure associated with hypertension.
Abstract 106: microRNA-33a Regulates Myocardin Expression and Attenuates Cardiac Remodeling in Renal Artery Ligation Model of Heart Failure
Authors: Khullar Madhu , Mittal Anupam , Rana Santanu , Sharma Rajni , Arige Vikas , Khanna Sanskriti , Mahapatra Nitish , Sarkar Sagartirtha , Saikia Uma N , Bahl Ajay
Published: March 2022

Myocardin (MYOCD), a cardiac specific transcriptional co-activator is up-regulated in heart failure (HF). Up-regulation of MYOCD expression has been proposed to be an important adaptive response in cardiac remodelling. However, molecular mechanisms contributing to increased cardiac MYOCD expression in HF are not known. The goal of this study was to identify microRNA(s) regulating cardiac MYOCD expression and to study the effect of cardiac modulation of MYOCD specific miRNA in an animal model of HF. miRNA(s) targeting MYOCD were identified using
in silico
approach and validated by 3’-UTR luciferase reporter assay. Cardiac expression of miRNA was measured in endomyocardial biopsies from idiopathic DCM (IDCM) patients, renal artery ligation rat model of HF (RAL) and in Ang II treated cardiomyocytes by real-time PCR. miRNA-33a, miRNA-33b, miRNA-139 and miRNA-542 were identified with MYOCD as putative target. Cardiac expression of miRNA-33a and miRNA-33b was significantly decreased, whereas expression of miRNA-139 and miRNA-542 was not altered in IDCM. miRNA-33a expression was also decreased in RAL and in Ang II treated cardiomyocytes. Luciferase assay confirmed MYOCD as target gene for miRNA-33a. miRNA-33a overexpression significantly decreased expression of MYOCD, ANP and fibrotic genes in Ang II treated cardiomyocytes. Cardiac specific delivery of miRNA-33a, using a homing peptide conjugated siRNA, attenuated cardiac hypertrophy and fibrosis, decreased expression of ANP, β-MHC and fibrotic genes and ameliorated the impaired diastolic dysfunction in RAL. Our results provide the first evidence that miRNA-33a regulates MYOCD expression and cardiac specific augmentation of miRNA-33a attenuated cardiac remodelling and partially restored left ventricular function. Our results suggest miR-33a as a potential therapeutic target in reversal of cardiac remodelling and improvement in heart function in HF.
Abstract 115: Myocardial Hypertrophy Upregulates the Calcium-activated Chloride Channel TMEM16A in the Heart
Authors: Zhang Yaofang , Ye Lingyu L , Duan Dayue D
Published: March 2022

Background:
Transmembrane member 16A (TMEM16A), or Anoctamin-1, is a protein encoded by the
Ano1
gene. TMEM16A forms a voltage-sensitive calcium-activated chloride (Cl
-
) channel in the cardiac atrial and ventricular myocytes. In this study we tested our

hypothesis

that activation of TMEM16A may be a novel adaptive mechanism for the pressure-overload induced structural and electrical remodeling of the heart
.

Methods:
Wild type C57BL/6 (8-10 week old, male) mice were subjected to minimally-invasive transaortic banding (MTAB) surgery to create a pressure-over load myocardial hypertrophy model. Echocardiography at 2-D and M-mode were performed weekly after MTAB or sham-operation to monitor changes in left ventricular structure (wall thickness and chamber diameters) and cardiac function such as ejection fraction (EF) and fraction shortening (%FS). TMEM16A protein expression in left ventricles of control mice subjected to sham-operation or MTAB was estimated by capillary electrophoresis immunoblotting system (or Simple Western) using Wes platform. Whole-cell voltage- or current- clamp recordings were used to examine the functional expression of TMEM16A in left ventricular myocytes.

Results:
MTAB caused a time-dependent increase the left ventricular wall mass (LVWM) and wall thickness in the mice while no significant changes in the EF and SF, TMEM16A expression was increased significantly, which picked at week 4 and maintained at an expression level higher than normal control at week 10 after MTAB. Electrophysiological recordings followed the similar pattern of changes as the molecular expression of TMEM16A.

Conclusions:
Myocardial hypertrophy caused a significant upregulation of TMEM16A Cl
-
channels in cardiac myocytes and it may provide a novel adaptive mechanism for the structural and electrical remodeling induced by pressure overload.
Abstract 12: M6A Modification in RNA Regulates Cardiomyocyte and Cardiac Function in Heart Failure
Authors: Mathiyalagan Prabhu , Liang Yaxuan , Sassi Yassine , Kohlbrenner Erik , Chen Jiqiu , Lebeche Djamel , Giovanna Trivieri Maria , Ishikawa Kiyotake , Fish Kenneth , Hajjar Roger J , Sahoo Susmita
Published: March 2022

Background:
Adenosine in RNA is a substrate for addition or removal of methyl group. Reported five decades ago, methylated adenosine (m6A), the most abundant and functionally relevant chemical modification in RNA, whose transcriptome-wide mapping became possible only recently due to next generation sequencing (NGS). Coupled with NGS, m6A-methylated RNA capture (MeRIP-seq) identified widespread m6A distribution in ~8000 mRNA and ~1000 lncRNA transcripts in human and mouse transcriptome.

Methods and Results:
In a first of its kind approach, we examined m6A RNA methylation in both failing and non-failing hearts. We discovered that global m6A RNA methylation is significantly higher in left ventricles (LV) of failing human, swine and mouse hearts as compared to non-failing controls. Increase in m6A was associated with significantly lower expression of one of the key m6A demethylases, FTO, in the ischemic heart. siRNA-mediated silencing of FTO resulted in significant arrhythmias, loss of Ca
2+
dynamics such as Ca
2+
transient decay (Tau) and cardiomyocyte relaxation time in isolated adult rat cardiomyocytes. Conversely, FTO gene transfer reduced m6A and improved Ca
2+
transients and contractile function in primary cardiomyocytes under hypoxia. In a mouse model of MI, AAV-mediated gene transfer of FTO significantly improved cardiac function post-MI. We identified transcriptome-wide m6A distribution signatures and conserved methylated sites of several mRNAs and lncRNAs using MeRIP-seq in both human and mouse failing and non-failing LV. Detailed MeRIP map of individual transcripts identified differentially methylated 3’-UTR, 5’-UTR and exon sites within several cardiac mRNAs that are important for cardiac function.

Conclusion:
Our data provide first evidence that m6A modification in RNA is a regulator of cardiomyocyte Ca
2+
dynamics and cardiac function. Our findings on the dynamic nature of the cardiac m6A-epitranscriptome will add another portfolio to mRNA and lncRNA regulation of cardiac remodeling.
Abstract 137: Ghrhr is a Cell-surface Marker of Human Pluripotent Stem Cell-derived Cardiomyogenic Precursors
Authors: Hatzistergos Konstantinos E , Vedenko Anastasia , Valasaki Krystalenia , Balkan Wayne , Hare Joshua M
Published: March 2022

Introduction:
A major roadblock for generating human pluripotent stem cell (hPSCs) derivatives highly enriched in cardiomyogenic precursors (CPCs), has been the lack of CPC-specific cell surface markers.

Hypothesis:
Based on observations that adult CPCs are responsive to growth hormone-releasing hormone (GHRH) signaling, we hypothesized that the GHRH receptor (GHRHR) is a specific cell-surface marker for hPSC-derived CPCs.

Methods:
We performed temporal analysis of GHRHR expression in an
in-vitro
model of human cardiogenesis using induced hPSCs (hiPSCs) and
SOX10::GFP
embryonic hPSCs (hESCs)
;
and mouse (
in-vivo
) cardiogenesis in wild-type (WT),
MEF2c-AHF-Cre, Wnt1-Cre2
and
cKit-CreERT2/+
reporter mice.

Results:
Gene expression and confocal immunofluorescence analyses during chemically-defined, stage-specific, cardiac lineage differentiation indicated that GHRHR is not expressed in undifferentiated hiPSCs or during specification into primitive streak-like Brachyury
+
or Mesp1
+
precardiac cells; but is induced in cardiogenic mesoderm-like cells, at the stage of commitment into NKX2.5
+
and/or ISL1
+
CPCs (
p
=0.001) and persists in Troponin-T
+
cardiomyocytes. Similarly, experiments modeling cardiac neural crest (CNC) with
SOX10::GFP
hESCs indicated that GHRHR is not expressed by GFP
+
CNCs but is induced following differentiation into NKX2.5
+
and/or ISL1
+
derivatives. Importantly, stimulation with 1μm recombinant GHRH during days 5-7 of hiPSCs differentiation increased
NKX2.5
expression 2.5-fold, an effect that was abolished by exposure to 1μM Somatostatin, a GHRH antagonist (
p
=0.0009). Last, in vivo analyses in WT
, MEF2c-AHF-Cre, Wnt1-Cre2
and
cKit-CreERT2/+
reporter embryonic and postnatal hearts corroborated that GHRHR specifically marks NKX2.5
+
mesoderm- and CNC-lineage descendants in vivo, whereas GHRHR is not expressed by
Wnt1-Cre2
and
cKit-CreERT2/+
CNCs descendants that are Nkx2.5
–
.

Conclusions:
Together these findings indicate that GHRHR is universally expressed by NKX2.5
+
/ISL1
+
CPCs and cardiomyocytes of both mesoderm and CNC origin. Therefore, GHRHR appears to be a valuable cell-surface marker for the selection and enrichment of CPCs from hPSCs for biomedical and regenerative medicine applications.
Abstract 142: IL-10 Regulates Inflammation to Improve LV Physiology After Myocardial Infarction by Stimulating M2 Macrophage Polarization and Fibroblast Activation
Authors: Jung Mira , Ma Yonggang , Padmanabhan Iyer Rugmani , Yabluchiansky Andriy , Garrett Michael R , Lindsey Merry L
Published: March 2022

Inflammation resolution is important for scar formation following myocardial infarction (MI) and requires the coordinated actions of macrophages and fibroblasts. In this study, we hypothesized that exogenous interleukin-10 (IL-10), an anti-inflammatory cytokine, promotes post-MI repair through actions on these cardiac cell types. To test this hypothesis, C57BL/6J mice (male, 3-6 months old, n=24/group) were treated with saline or IL-10 (50 μg/kg/day) by osmotic mini-pump infusion starting at day 1 post-MI and sacrificed at day 7 post-MI. IL-10 infusion doubled plasma IL-10 concentrations by day 7 post-MI. Despite similar infarct areas and mortality rates, IL-10 treatment significantly decreased LV dilation (1.6-fold for end-systolic volume and 1.4-fold for end-diastolic volume) and improved ejection fraction 1.8-fold (both p<0.05). IL-10 treatment attenuated inflammation at day 7 post-MI, evidenced by decreased numbers of Mac-3+ macrophages in the infarct (p<0.05). LV macrophages isolated from day 7 post-MI mice treated with IL-10 showed significantly elevated gene expression of M2 markers (Arg1, Ym1 and TGF-β1; all p<0.05). We further performed RNA-seq analysis on post-MI cardiac macrophages and identified 410 significantly different genes (155 increased, 225 decreased by IL-10 treatment). By functional network analysis grouping, the majority of genes (133 out of 410) were part of the cellular assembly and repair functional group. Of these, Hyaluronidase 3 (Hyal 3) showed the largest fold change between groups. IL-10 treatment decreased Hyal3 by 28%, which reduced hyaluronan degradation and limited collagen deposition (all p<0.05). In addition,
ex vivo
IL-10 treatment increased fibroblast activation (proliferation, migration, and collagen production), an effect that was both directly and indirectly influenced by macrophage M2 polarization. Combined, our results indicate that
in vivo
infusion of IL-10 post-MI improves the LV microenvironment to dampen inflammation and facilitate cardiac wound healing.
Abstract 145: Rational Design of a Novel Peptide That Selectively Inhibits δPkc-mediated Pyruvate Dehydrogenase Kinase Phosphorylation Induce Protection From Ischemia and Reperfusion Injury
Authors: Qvit Nir , Mochly-Rosen Daria
Published: March 2022

Rationale:
Protein kinase C delta (δPKC), whose activation induces cardiac injury following heart attack [ischemia andreperfusion (I/R) injury], phosphorylates a number of protein substrates. To determine which of these phosphorylation events is critical to induce cardiac injury, we used a rational approach and identified a novel separation-of-function inhibitor peptide that selectively inhibits specific protein-protein interaction. We showed that following I/R, δPKC-mediated phosphorylation of pyruvate dehydrogenase kinase (PDK) increased PDK-inducedpyruvate dehydrogease phosphorylation, which results in inhibition of ATP regeneration. We therefore determined if selective inhibition of δPKC-mediated PDK phosphorylation alone is sufficient to induce cardiac protection.

Methods:
We designed a short peptide inhibitor, called ψPDK, to selectively inhibit δPKC binding and phosphorylation of PDK without affecting δPKC-mediated functions. The peptide was used in an ex vivo isolated rat hearts model and in vivo rat model of heart attack [by inducing 30 min ischemia followed by reperfusion (I/R)]. I/R-induced injury was determined by infarct size, cell lysis and activation of stress-induced response.

Results:
Our approach identified a selective inhibitor of PDK dockingto δPKC with an in vitro Kd of ∼50 nM and reducing cardiac injury IC50 of ∼5 nM. This inhibitor, which did not affect thephosphorylation of other δPKC substrates even at 1 μM, demonstrated that PDK phosphorylation alone is critical for δPKC mediated injury by heart attack.
Abstract 147: The Valosin-Containing Protein Regulates Mitochondrial Respiration and Membrane Permeability via Inducible Nitric Oxide Synthase in the Mouse Heart
Authors: Stoll Shaunrick A , Zhou Ning , Qiu Hongyu
Published: March 2022

Introduction:
Mitochondrial dysfunction plays a critical role in mediating cell death during ischemia/reperfusion (IR). We previously found that overexpressing valosin-containing protein (VCP), an ATPase associate-binding protein, in a transgenic (TG) mouse heart increases the expression of inducible nitric oxide synthase (iNOS) and reduces infarct size during IR. We tested the hypothesis that VCP mediates cardiac protection by increasing mitochondrial respiration and inhibiting the opening of the mitochondrial permeability transition pore (mPTP) via iNOS.

Methods and Results:
Cardiac mitochondria were isolated from the heart tissues of adult VCP TG and their litter-matched WT mice (N=6/group). VCP TG mice increased the expression of VCP by 3.1 folds and iNOS by 4.5 folds in the mitochondrial fraction vs WT (
p
<0.01). Mitochondrial respiration was determined by oxygen consumption rate (OCR) in an airtight chamber with a Clark-type electrode. Compared to WT, mitochondria from VCP TG showed a significant increase in OCR during ADP-dependent State 3 under Complex I stimulation while no significant change was seen in oligomycin induced-State 4, increasing the efficiency of mitochondrial respiration, as measured by the respiratory control ratio (State 3/State 4). VCP TG mice also displayed a significant increase in their maximum respiration capacity versus WT, measured after the addition of the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone. However, there was no difference between VCP TG and WT on Complex II-dependent respiration rate, suggesting VCP’s predominance on Complex I-stimulated respiration. mPTP opening was induced by Ca
2+
overload in isolated mitochondria and was measured by a swelling assay. Compared to WT mice, VCP TG showed significantly less mitochondrial swelling under the Ca
2+
overload (
p
<0.05). To test whether protection by VCP is mediated by iNOS, a bigenic VCP TG /iNOS knock out mouse was generated. Deletion of iNOS from the VCP TG abolishes the effects of VCP on mitochondrial respiration and mPTP opening.

Conclusion:
VCP prevents the opening of the mPTP and increases mitochondrial respiratory efficiency in an iNOS dependent manner, which may represent a novel mechanism of cardiac protection.
Abstract 148: Gut Microbial Metabolites Modulate Immune Response for Cardiac Repair
Authors: Tang Tony W , Hsieh Patrick C
Published: March 2022

Antibiotic abuse has been a serious issue on human health and homeostasis. Appreciation of the role of gut microbiota in regulating vertebrate tissue repair has exploded recently. However, the effects of gut microbiota on cardiac repair after myocardial infarction (MI) remain to be elucidated. Here, we report that although administration of antibiotics doesn’t influence cardiac function before injury, antibiotic-mediated depletion of the gut microbiota caused high mortality in mice after MI. Since the effect happens only after the mice with MI have received antibiotics, it suggests that the gut microbiota is important for effective cardiac repair, especially in early stage after MI. Because the gut microbiota is an important immune modulator, we speculate that immune response, which plays an important role in cardiac repair, is significantly impaired in mice treated with antibiotics. We have determined that short-chain fatty acids (SCFAs), which are a gut microbiota-derived fermented product, contribute to efficacious cardiac repair after MI. Furthermore, the profile of gut microbiota dynamic changes clues a decisive role of Lactobacillus species in more powerful cardiac repair after MI. Mice with MI that were treated with Lactobacillus showed a significant improvement in left ventricular ejection fraction and smaller infarct size. Finally, the beneficial effects of Lactobacillus come from the alternation of SCFA metabolism which modulates the immune response in the heart. Our study demonstrates that the gut microbiota contributes to cardiac repair after MI via immune response modulation driven by gut microbiota-derived SCFAs. Manipulation of gut microbiota composition or its metabolites may provide opportunities to modulate pathological outcome in the heart after MI.
Abstract 153: Using Enzyme-Catalyzed Proximity Labeling to Define the Interactome of the Insulin-like Growth Factor-1 Receptor
Authors: Bareja Akshay , Hodgkinson Conrad , Payne Alan , Soderblom Erik , Waitt Greg , Dzau Victor
Published: March 2022

Introduction:
Manipulation of
I
nsulin-like
G
rowth
F
actor-
1 R
eceptor (IGF1R) signaling has been proposed to be an important therapeutic for the treatment of heart failure and myocardial infarction. However, activation of this receptor has, under certain conditions, been shown to result in deleterious side-effects such as pathological hypertrophy. Development of therapeutic strategies that avoid these side-effects requires a more comprehensive understanding of the proteins involved in the IGF1R signaling axis. To this end, we have made use of the enzyme-catalyzed proximity labeling technique called BioID.

Objective:
To comprehensively define the interactome of the activated IGF1R.

Methods and Results:
Briefly, BioID makes use of a biotin protein ligase called BirA* that promiscuously biotinylates neighboring proteins. The protein of interest is fused with BirA* and is then expressed in a particular cell line. Following administration of an excess amount of biotin, BirA* will biotinylate those proteins that are adjacent to or interact with the protein of interest. Interacting proteins are identified by tandem mass spectrometry (MS/MS). We have made a HEK 293T cell line that stably expresses the BirA* biotin ligase fused to the C-terminus of IGF1R. We performed BioID using this cell line (N=3) to identify a set of proteins that bind to the IGF1R following stimulation by IGF1. This set included many known interactors of the IGF1R, such as IRS2 and SHC1, thereby validating this technique. Of the many interesting novel interactors, we chose to pursue validation of sorting nexin 6 (SNX6) as some SNX proteins have been shown to be involved in receptor recycling to the plasma membrane following stimulation. We have found that SNX6 knockdown results in a dramatic diminution of IGF1-mediated ERK phosphorylation (2-fold; N=4;
p
<0.05). However, there was no effect on IGF1-mediated Akt phosphorylation (N=4). We are currently assessing the role of SNX6 in IGF1R-mediated function and recycling of this receptor.

Conclusion:
We have demonstrated the ability of the BioID technique to identify a set of proteins that can interact with the activated IGF1R. Moreover, BioID has identified a number of novel interactors that further define IGF1R signaling and function.