bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 310: Beta-adrenergic Receptor/beta-arrestin-mediated MicroRNA Maturation Confers Cardiac Cell Survival
Authors: Teoh Jian-peng , Kim Il-man
Published: June 2021

MicroRNAs (miRs) are small, non-coding RNAs that function to post-transcriptionally regulate gene expression. First transcribed as primary miR transcripts (pri-miRs), they are enzymatically processed in the nucleus by Drosha into hairpin-shaped premature miRs (pre-miRs) and further processed in the cytoplasm by Dicer into mature miRs. MiRs regulate cellular processes upon activation by various signals such as those stimulated by beta-adrenergic receptors (betaARs). Initially discovered to desensitize betaAR signaling, beta-arrestins are now appreciated to regulate diverse effector pathways independent of G protein-mediated second messenger accumulation, a concept known as biased signaling. Using the beta-arrestin-biased betaAR ligand carvedilol (Carv), we test whether Carv could modulate Dicer-mediated miR maturation, thereby providing a novel mechanism for its cardioprotective effects. In mouse hearts, Carv indeed upregulates 3 mature miRs, but not their pre-miRs and pri-miRs, in beta-arrestin1/2- and GPCR kinase 5/6-dependent manners. Interestingly, Carv-mediated activation of miR-466g and miR-532-5p is dependent on beta2AR while the activation of miR-674-5p by Carv is beta1AR-dependent. Mechanistically, beta-arrestins regulate miR maturation by associating with components of Dicer complex on these three pre-miRs (Figure A). Loss-of-function studies in cardiac cells show that miR-466g and miR-532-5p act as gatekeepers of cardiac endothelial cell survival (Figure B). Our findings indicate a novel role for betaAR-mediated beta-arrestin signaling activated by Carv in miR maturation, which may be linked, in part, to its cell survival mechanism.
Abstract 33: Unlocking Reprogramming Capability: Silencing Antiplasticity Gene p63 Enhances the Reprogramming of Fibroblasts into Induced Cardiomyocytes
Authors: Patel Vivekkumar , Cooney Austin , Flores Elsa , Singh Vivek , Mathison Megumi , Sanagasetti Deepthi , Mullany Lisa , Yang Jianchang , Rosengart Todd
Published: June 2021

Objective: In situ cellular reprogramming of cardiac fibroblasts into (induced) cardiomyocytes (iCMs) represents a promising new potential intervention for the treatment of heart failure. Despite encouraging in vivo data in rodent myocardial infarction models, the relative resistance of human cells to reprogramming may be a significant barrier to the clinical application of this new therapy. We hypothesized that knockdown of the anti-plasticity gene p63 could therefore be used to enhance cellular reprogramming efficiency.

Methods:
p63 knockout (KO) murine embryonic fibroblasts (MEFs) and MEFs treated with p63 silencing shRNA were assessed for expression of the cardiomyocyte marker Cardiac Troponin T (cTnT) and pro-cardiogenic genes, with or without the treatment with known cardiac transcription factors Hand2 and Myocardin (HM).

Results:
After 3 wks in culture, expression of the cardiomyocyte marker cTnT (FACS) was significantly greater in p63 KO MEFs than in wild-type (WT) MEFs or WT MEFs treated with transcription factors Hand2 and Myocardin (39% ± 8%, 2.0% ± 1% and 2.7 ± 0.3%, respectively, p < 0.05). Treatment of p63 KO MEFs with Hand2 and Myocardin further increased cTnT expression up to 74% ± 3%. Treatment of WT MEFs with p63 shRNA likewise yielded a 20-fold increase in cTnT expression (qPCR) without HM and a 600-fold increase with HM when compared to non-silencing shRNA treated MEFs. Consistent with these findings, p63 KO or p63 shRNA-treated MEFs demonstrated increased expression (qPCR) of pro-cardiogenic genes Gata4, Mef2c and Tbx5 compared to naïve or non-silencing shRNA treated MEFs. After treatment with p63 shRNA, adult human epidermal cells also demonstrated increased expression of cTnT, myosin heavy chain and pro-cardiogenic genes when analyzed by qPCR.

Conclusions:
Downregulation of the anti-plasticity gene p63 enhances cellular reprogramming efficiency and iCM generation, as reflected in the increased expression of the cardiomyocyte marker cTnT and pro-cardiogenic genes Gata4, Mef2c and Tbx5. Use of such cellular plasticity enhancing strategies may be a useful strategy to overcome barriers to cellular reprogramming in the clinical arena.
Abstract 35: Enhanced Reprogramming of Human Fibroblasts into Cardiomyocytes Using Minimal Transcription Factors
Authors: Zhou Yang , Alimohamadi Sahar , Liu Jiandong , Qian Li
Published: June 2021

The adult human heart has limited regenerative capacity and is thus an important target for novel regenerative approaches to replenish lost cardiomyocytes after cardiac injury. Cardiac reprogramming that converts fibroblasts to contractile induced cardiomyocytes (iCMs) by overexpressing cardiac lineage specific transcription factors holds great promise as an alternative approach for cardiac regeneration and disease modeling. Significant advance has been made to generate mouse iCMs; however, human iCM (hiCM) generation remains challenging and the yield is low for clinical applications. Here, we leveraged the knowledge that we learned from studying mouse iCM reprogramming to define the optimal condition for hiCM induction. We titrated the dosage of the human reprogramming factors systematically and surprisingly found the minimal core components GATA4, MEF2C and TBX5 were sufficient to induce cardiac fate in human primary fibroblasts. This is in sharp contrast to what has been reported in the literature. Subsequently, we cloned these three factors into a polycistronic vector separated by 2A peptides for defined ratio of protein expression. By optimizing the growth condition, we further improved the efficiency of hiCM reprogramming. Mechanistically, we found the balanced expression of this minimal combination of transcription factors with tailored microenvironment enhanced the establishment of cardiac program in non-myocytes. In sum, our study demonstrates that the use of a single vector with only three transcription factors simplifies generation and improves the yield of hiCMs for potential future clinical applications.
Abstract 355: Heart Failure Impairs Bone Marrow Mesenchymal Stem Cell Renewing Capacity and Migration
Authors: Florea Victoria , Sanina Cristina , Difede Darcy , Valasaki Krystalenia , Khan Aisha , Hare Joshua
Published: June 2021

Background:
Clinical trials have shown a sustained beneficial effect of bone marrow mesenchymal stem cells (MSCs) as a therapy for acute and chronic heart failure (HF). Clinical grade cell manufacturing of autologous and allogeneic MSCs is becoming a standard procedure. This study investigates the differences of bone marrow MSC isolation and expansion in favorable in vitro conditions. We compared MSC production from both healthy young donors and chronic HF patients.

Methods:
We analyzed MSC manufacturing records from five clinical trials: TAC-HFT (Ischemic cardiomyopathy (CMP), patient and donors), POSEIDON (Ischemic CMP, donors), POSEIDON DCM (Dilated CMP, donors), TRIDENT (Ischemic CMP, donors), and CRATUS (Frailty, donors).

Results:
All cells expressed CD105, CD90 and lacked hematopoietic marker CD45. The age of healthy donors (25.5 ± 0.7 y.o., N=24) and HF patients (56.1 ± 2.5, y.o., N=23), was significantly different (P<0.0001). Collectively, the number of mononuclear cells (MNCs) isolated from bone marrow (volume range 58-125ml) didn’t differ between the groups. However, plated MNCs from healthy adults generated more MSCs than MNCs from HF patients (p0: 5.9x10^6±0.5x10^6, N=23 vs 3.8x10^6±0.4x10^6, N=24, respectively, P=0.003 and p1: 15.4x10^6±2.5x10^6, N=23 vs 10.8x10^6±1.1x10^6, N=24, respectively, P=0.036). No correlation was found between stem cell growth and age (35 to 78y.o.). Left ventricular ejection fraction (LVEF) in patients with HF had a direct correlation with MSC growth rate (P=0.03), particularly, in patients with severely depressed LVEF (<30%), which had a tendency to generate less MSCs overall. Moreover, MSCs from HF patients demonstrated less migration compared to MSCs from healthy donors at 6, 10 and 24 hours (h) relative to baseline (6 h: 9.5±3.0% vs 30.7±2.1%; 10 h: 19.9±13.7% vs 53.1±2.5% and 24 h: 41.5±15.8% vs 88.9±1.6%, N=4, respectively. P=0.03).

Conclusions:
Despite equivalent numbers of MNCs, healthy young donors generate significantly more MSCs than HF patients, due to increased growth rate and higher number of resident stromal bone marrow stem cells. MSC migration was impaired in HF patients compared to healthy donors. HF appears to be an independent factor for MSC renewal capacity and culture phenotype.
Abstract 357: Role of Combinatorial Extracellular Matrix Proteins in Modulating Endothelial Differentiation of Human Induced Pluripotent Stem Cells
Authors: Hou Luqia , Kim Joseph , Coller John , Natu Vanita , Huang Ngan
Published: June 2021

Human induced pluripotent stem cell (iPSC)-derived endothelial cells (iPSC-ECs) are promising for promoting angiogenesis in patients with cardiovascular disease, but a critical bottleneck is the ability to generate iPSC-ECs at high yields. Since endothelial cells physiologically interact with a milieu of basement membrane extracellular matrix (ECM) proteins, we aimed to examine the role of combinatorial ECMs in modulating endothelial differentiation using engineered ECM microenvironments. ECMs consisting of (gelatin (G), fibronectin (F), laminin (L), heparan sulfate proteoglycan (H), collagen IV (C), matrigel (M)) and the multi-component combinations thereof were conjugated onto glass slides to generate ECM microarrays composed of 63 unique combinatorial ECMs. Human iPSCs were differentiated on the ECM microarray for 5 days, and differentiation was quantified based on the degree of CD31 staining. Heat map analysis of CD31expression was significantly higher on G+M+L, compared to other combinations across 2 iPSC lines (Figure). RT-PCR further confirmed the upregulation in endothelial markers CD31 (>2-fold) and VE-cadherin (> 4-fold) when differentiated on G+M+L, compared to other ECMs. To elucidate the underlying mechanism, upregulation of endothelial genes were associated with upregulation of integrins (α1, α4, α5, αV, β1, β3, and β4) during the differentiation process. This data suggests a role of ECM-mediated integrin expression in modulating endothelial differentiation. Together, this data suggested that combinatorial ECMs differentially promote endothelial differentiation, in part through integrin-mediated pathways.
Abstract 359: The Production of Acellular Cardiac Extracellular Matrix from P3 Murine Heart
Authors: Kren Stefan M , Garry Daniel J , Garry Mary G
Published: June 2021

Introduction:
Understanding the role of extracellular matrix (ECM) in the creation of the cellular microenvironment during tissue formation and regeneration could be vital in extending this capability to injured adult tissue. To date the only confirmed mammalian heart tissue regeneration/regrowth has been in neonatal murine heart, with the regenerative capacity ceasing after day P3. By focusing on ECM from P3 heart tissue we hope to elucidate its contribution to regenerative plasticity and transfer this capacity to injured adult myocardium.

Hypothesis:
Detergent decellularization protocols used in adult tissues can be modified to function on a neonatal scale, serving to remove the cellular components of the neonatal heart, leaving structurally intact ECM to serve as a scaffold for the generation of cardiac tissue equivalents.

Materials and Methods:
Murine P3 hearts were perfused with 1% SDS in water at 20mm Hg for 12 hrs. Following detergent decellularization, perfusion with water, 1% Triton X-100, PBS and culture media restored biocompatibility to the isolated ECM. P1 and P7 primary cardiomyocytes expressing the mCherry red fluorescent protein reporter under control of the alpha myosin promoter were isolated by enzymatic disassociation and cultured in the heart matrix in a perfusion based bioreactor.

Results:
The decellularized ECM demonstrated removal of 97% of native DNA when compared to control by pico-green dsDNA binding assay. Histologic analysis demonstrated an absence of cell nuclei by H & E and DAPI staining. The preservation of the matrix structure and the maintenance of matrix immunoreactivity (collagen IV) were also demonstrated histologically. Following infusion of P1 or P7 mCherry positive cells, contractile behavior of the recellularized heart constructs was observed, and markers of cardiac linage (alpha-actinin in mCherry positive cells) were present.

Conclusions:
Neonatal heart matrix can be effectively decellularized. With appropriate modification of perfusion parameters, pediatric ECM structure can be preserved. This isolated matrix can serve as a scaffold for growth and maintenance of immature and mature cardiomyocytes, supporting continued contractility of cultured cells.
Abstract 364: Cortical Bone Stem Cells Derived Exosomes as Potent Modulator of Cardiac Immune Response and Repair After Injury
Authors: Mohsin Sadia , Troupes Constantine D , Khan Mohsin , Yang Yijun , Johnson Jaslyn , Petovic Jennifer L , Starosta Timothy , Berretta Remus M , Kubo Hajime , Houser Steven R
Published: June 2021

Rationale:
Cortical bone derived stem cells (CBSCs) are known to have improved growth kinetics and myocardial repair properties that are superior to other known stem cell types used. Salutary effects of CBSCs in large are mediated by paracrine secretion. Since exosomes represent an active component of released factors we tested if CBSC derived exosomes (CBSCs-Exo) can recapitulate the beneficial reparative effects of CBSCs.

Objective:
Determine CBSCs derived exosomes and their contents for myocardial repair.

Methods and Results:
Exosomes were isolated from murine CBSCs by ultracentrifugation and had typical size (30-100nm), as validated by electron microscopy and dynamic light scattering. To determine cardiac therapeutic value, CBSCs- Exo (60μg) were injected into the border zone of the mouse heart after myocardial infarction (MI). Animals injected with CBSCs-Exo had reduced infarct size and increased myocyte survival after MI injury. Interestingly, serum levels of pro-inflammatory cytokines were significantly reduced along with decreased expression of CD68+ cells in animals receiving CBSCs-Exo versus control animals. Long term analysis of CBSC-Exo animals showed improved cardiac function and contractility compared to saline treated animals concurrent with enhanced angiogenesis 6 weeks after MI. Salutary effects of CBSC-Exo were confirmed in vitro. CBSCs-Exo increased cardiac protection in NRVMs after hypoxic challenge and enhanced tube formation in HUVECs. Simultaneously, treatment of bone marrow-derived macrophages stimulated with lipopolysaccharide (LPS) and treated with CBSCs-Exo showed increased polarization towards the M2 phenotype, demonstrating an immunomodulatory capacity of CBSCs-Exo. The underlying mechanism for beneficial effects was linked to increased packaging of cardioprotective miRs including miR125, miR20 and miR18a in CBSCs-Exo confirmed by MiRNA array analysis.

Conclusion:
Exosomes derived from CBSCs provide a cell free system that retains the reparative power of CBSC. CBSCs-Exo augment cardiac function after myocardial injury recapitulating earlier findings with CBSCs. The packaging of cardioprotective and immune-modulatory miRs in CBSCs-Exo appears to enhance their reparative effects after MI.
Abstract 367: Combined Effect of Dinitrophenol Preconditioning and Jagged-1 Over-expression in Mesenchymal Stem Cells Improved Cardiomyogenesis and Angiogenesis in Rat Model of Myocardial Infarction
Authors: Salim Asmat , Ali Anwar , Akhter Muhammad A , Khan Irfan , Naeem Nadia
Published: June 2021

Cardiac regeneration following myocardial infarction (MI) largely depends on angiogenesis; an important physiological response that allows the heart to recover. Mesenchymal stem cells (MSCs) can be utilized as a source for cardiac regeneration and angiogenesis using various preconditioning strategies. We investigated the role of cardiomyogenic and angiogenic growth factors combined with preconditioning of MSCs using 2, 4, dinitrophenol (DNP). MSCs were isolated from rat bone marrow and treated with DNP; an uncoupler of oxidative phosphorylation. Based on our earlier report in which we showed that DNP treatment affect the expression of cardiomyogenic and angiogenic factors after different re-oxygenation periods, we selected jagged 1 (Jag1), neuropilin1 (Nrp1), TATA box 20 (Tbx20), and myogenin (Myo) to transfect MSCs. Rat MI models were developed through ligation of left anterior descending coronary artery and confirmed through echocardiographic evaluation 4 weeks post MI. The study was divided into different groups which include control; MI model; and MI groups that received untreated, DNP-treated, transfected-MSCs using the above mentioned growth factors and transfected and DNP-treated MSCs. Functional performance of the hearts was analyzed through echocardiography while regeneration of cardiomyocytes and angiogenesis were observed through histological examination. Significant (p<0.05) improvement in the heart function was observed in case of all treatment groups when compared with the normal MSCs. Histological examination of the heart sections 4 weeks post MI showed that MSCs home towards the site of injury. Most prominent result was observed in case of Jag1-transfected and DNP treated MSCs with heart function comparable to that of the control group. Cardiac regeneration and newly developed blood vessels were prominent in the tissue sections. Significant reduction (p<0.05) in the infarct size was also observed as compared to that in case of Myo, Tbx20 and Nrp1 groups. It is, therefore, concluded that Jag1-transfected MSCs can be a suitable angiogenic factor to be used in combination with DNP for preconditioning of MSCs for future regenerative therapy for cardiomyogenesis.
Abstract 37: Mir-590 Promotes Transdifferentiation of Porcine and Human Fibroblasts Towards a Cardiomyocyte-like Fate by Directly Repressing Specificity Protein 1 (Sp1)
Authors: Singh Vivek P , Mathison Megumi , Patel Vivekkumar , Sanagasetti Deepthi , Yang Lina , Naguib Mostafa R , Somvanshi Sonal , Mullany Lisa K , Gibson Brian W , Yang Jianchang , Rosengart Todd K
Published: June 2021

Objective:
Transdifferentiation of cardiac fibroblasts into cardiomyocyte-like cells (iCMs) represents a promising strategy in treating human heart disease. However, despite encouraging reprogramming data obtained from rodent models, the transgenes used in those models are not sufficient to reprogram human cells. We therefore sought to optimize a cocktail of factors which can efficiently transdifferentiate human cells. A porcine model was included to test its potential as a surrogate for human reprogramming studies.

Methods:
Lentivirus expressing Gata4 (G), Mef2c (M), Tbx5 (T), Hand2 (H), Myocardin (My), or two microRNAs miR-590, miR-199, were transduced into cultured porcine cardiac fibroblasts (PCFs) and human cardiac fibroblasts (HCFs) in different combinations. Two weeks after transduction, qRT-PCR, immunofluorescence (IF), and FACS assays were performed, and the efficiency of iCM production was evaluated by the expression of cardiomyocyte markers cardiac troponin T (cTnT) and α-sarcomeric actinin.

Results:
Combined G, M, T treatment alone was insufficient to transdifferentiate PCFs or HCFs into iCMs, despite the capability of GMT transduction to transdifferentiate up to 7% of treated rat cardiac fibroblasts, as assessed by FACS analysis for cTNT. However, the addition of H, My and miR-590 to GMT resulted in the transdifferentiation of approximately 5% of HCFs and PCFs, as measured by cTnT expression. IF analysis likewise demonstrated high expression of cTNT and α-actinin in these cells. Importantly, the transdifferentiated PCFs exhibited spontaneous contractions when co-cultured with murine cardiomyocytes. qPCR showed that administration of GMT plus either miR-590 or HMy upregulated cardiac genes MYH6 and TNNT2, and downregulated the fibroblast genes Collagen I and Collagen III. Mir-590 also directly suppressed Sp1, a fibrosis inducer and putative inhibitor of cellular reprogramming in the heart.

Conclusions:
Our data suggest that the porcine model can serve as an appropriate surrogate for human fibroblasts reprogramming studies. Enhanced transdifferentiation associated with miR-590-mediated repression of Sp1 suggests a novel pathway that may be targetable to enhance cardiac cellular reprogramming clinically.
Abstract 372: MicroRNA-9 Inhibits Hyperglycemia-induced Cardiac Pyroptosis by Targeting ELAVL1
Authors: Jeyabal Prince , Thandavarayan Rajarajan A , Joladarashi Darukeshwara , Suresh Babu Sahana , Krishnamurthy Shashirekha , Bhimaraj Arvind , Youker Keith A , Kishore Raj , Krishnamurthy* Prasanna
Published: June 2021

Diabetic cardiomyopathy is a common complication in patients with diabetes and is associated with underlying chronic inflammation and cardiac cell death, subsequently leading to left ventricular dysfunction and heart failure. ELAV-like protein 1 (ELAVL1, mRNA stabilizing protein) and NLRP3 activation (inflammasome complex protein)-mediated IL-1beta synthesis play a critical role in the progression of heart failure. However, ELAVL1 regulation of pyroptosis (caspase-1-mediated programmed apoptosis) and associated IL-1beta release in cardiomyocytes, especially under diabetic condition, remains elusive. Human diabetic, non-diabetic heart tissues, human ventricular cardiomyocytes and rat cardiomyoblasts exposed to high glucose (HG) were used for our studies. Our data demonstrates that human ventricular cardiomyocytes exposed to high glucose condition showed significant increase in ELAVL1 and NLRP3 expression with a concomitant increase in caspase-1 and IL-1 beta expression. Furthermore, human cardiac tissue from diabetic patients showed increased ELAVL1, caspase-1 and NLRP3 expression as compared to non-diabetic hearts. Intriguingly, ELAVL1 knockdown abrogates TNF-α induced canonical pyroptosis via NLRP3, caspase-1 and IL-1beta suppression. Interestingly, miRNA-9 expression significantly reduces in high glucose treated cardiomyocytes and in human diabetic hearts. Bioinformatics analysis and target validation assays showed that miR-9 directly targets ELAVL1. Inhibition of miR-9 up regulates ELAVL1 expression and activates caspase-1. Alternatively, miR-9 mimics attenuate hyperglycemia-induced ELAVL1 and inhibit cardiomyocyte pyroptosis. To our knowledge, this is the first report to demonstrate the role of miR-9/ELAVL1 in hyperglycemia-induced cardiac pyroptosis. Taken together our study highlights the potential therapeutic implications in preventing cardiomyocyte cell loss in human diabetic failing heart.
Abstract 375: MCL-1 Regulates Mitochondrial Dynamics and Mitophagy
Authors: Moyzis Alexandra G , Leon Leonardo , Thomas Robert L , Gustafsson Åsa B
Published: June 2021

The anti-apoptotic BCL-2 family protein Myeloid Cell Leukemia-1 (MCL-1) is essential for maintaining mitochondrial integrity and cardiac function in the adult heart.

Recently, we reported that cardiac-specific deletion of MCL-1 in mice leads to rapid mitochondrial dysfunction, hypertrophy, and lethal cardiomyopathy. Surprisingly, MCL-1 ablation does not result in apoptotic cell death, but rather cells show signs of mitochondrial deterioration and necrotic cell death. This suggests that in addition to its anti-apoptotic role, MCL-1 has unidentified roles in maintaining mitochondrial function in cardiac myocytes. MCL-1 exists in two distinct locations in myocytes: one form is localized to the outer mitochondrial membrane (MCL-1
OM
) and a shorter cleaved form exists in the mitochondrial matrix (MCL-1
Matrix
). Interestingly, overexpression of MCL-1
WT
or MCL-1
OM
, but not MCL-1
Matrix
, leads to translocation of Drp1 to mitochondria, which correlates with fission and perinuclear aggregation of mitochondria. We also found that Drp1 co-immunoprecipitates with MCL-1 in heart lysates and that MCL-1-deficient hearts lack mitochondrial Drp1. Additionally, the interaction between Drp1 and MCL-1 and mitochondrial fission increase in response to serum starvation in neonatal myocytes. This suggests that MCL-1
OM
recruits Drp1 to mitochondria to induce fission. Since there is a strong link between mitochondrial fission and degradation, we examined the effect of MCL-1 on mitophagy. Parkin is an E3 ubiquitin ligase that plays an important role in mitophagy. MEFs, which lack detectable Parkin, do not efficiently clear their depolarized mitochondria in response to FCCP treatment. However, overexpression of Parkin or MCL-1 increased the efficiency of mitophagy in MEFs in response to FCCP treatment. MCL-1-mediated mitophagy is abrogated in autophagy-deficient Atg5
-/-
MEFs, confirming that the clearance occurs via enhanced autophagy. Thus, our data suggest that MCL-1
OM
has dual actions in coordinating Drp1-mediated fission and mitophagy, which allows for more efficient removal of damaged mitochondria.
Abstract 391: The Pathophysiological Role of MYBPHL in Dilated Cardiomyopathy
Authors: Barefield David Y , Puckelwartz Megan J , Dellefave-Castillo Lisa , McNally Elizabeth M
Published: June 2021

Background:
Cardiomyopathy is a leading cause of heart failure and is highly heritable. One common form of cardiomyopathy is dilated cardiomyopathy (DCM), which currently has over 70 identified genes that have been described as causative for the disease. Genetic testing for DCM employs gene panels and has a sensitivity of mutation detection less than 50%, indicating that additional genes contribute to DCM. Here, we employed whole genome sequencing (WGS) in a family with DCM and heart block who had previously undergone unrevealing genetic testing. We identified a premature stop codon in the
MYBPHL
gene, a gene that has not previously been linked to DCM as a likely cause of DCM in this family. Myosin binding protein H Like (MyBP-HL) is a muscle-expressed protein bearing structural similarity to myosin binding protein C (MyBP-C), which is commonly mutated gene in cardiomyopathies.

Objective:
Determine the physiological and pathophysiological role of
Mybphl
.

Results:
RNA-seq and qPCR from mouse hearts revealed that
Mybphl
is highly expressed in the right and left atria with lower expression in the ventricle and virtually no expression in skeletal muscle. As MyBP-HL shares a high homology with the myofilament proteins cardiac myosin binding protein-C and H, we investigated if MyBP-HL is also myofilament-associated. We determined that MyBP-HL protein is myofilament-associated in the atria although not clearly so in ventricle. To assess the requirement of MyBP-HL in cardiac function, we used a mouse model with an insertional disruption of the
Mybphl
gene. These mice have deficits in
in vivo
cardiac function, with reduced fractional shortening. In addition, ECG recordings from the
Mybphl
null mice show conduction system abnormalities affecting atrioventricular conduction.

Conclusions:
WGS identified a premature stop codon in
MYBPHL
in human DCM. A mouse model with a disrupted
Mybphl
gene showed similar pathophysiological features as the humans with reduced ventricular function and cardiac conduction system abnormalities. MyBP-HL is an important protein for normal cardiac function.
Abstract 403: Critical Role for Dipeptidyl Peptidase-4 Activity in the Pathogenesis of Experimental Autoimmune Myocarditis
Authors: Maejima Yasuhiro , Ito Yusuke , Tamura Natsuko , Isobe Mitsuaki
Published: June 2021

Autoimmune myocarditis is the most common cause of heart failure among young adults and is a major precursor of dilated cardiomyopathy. We have shown that dipeptidyl peptidase-4 (DPP-4) plays a detrimental role in the pathogenesis of experimental autoimmune myocarditis (EAM) in mice. To elucidate how DPP-4 induces cardiac dysfunction in the EAM heart, mouse EAM models were given normal diet (CONT-group,
N
= 6), or a diet mixed with linagliptin, a potent DPP-4 inhibitor (83 mg/kg chow corresponding to around 3 mg/kg oral dosing) (LINA-group,
N
= 6). After 21-day of EAM induction, left ventricular ejection fraction was significantly higher in LINA-group than in CONT-group (72.6 ± 6.6%* vs. 60.1 ± 9.2%, *
P
< 0.05). Immunostaining demonstrated that the number of RORγt-positive Th17 cells, a subset of T-lymphocytes expressing high level of enzymatically active DPP-4, infiltrated to the EAM hearts was significantly smaller in LINA-group than in CONT-group (46.9 ± 2.4 RFU* vs. 116.8 ± 8.6 RFU, *
P
< 0.05). Consistently, the activity of DPP-4 in the EAM hearts was significantly lower in LINA-group than in CONT-group. Mass spectrometry analysis using lysates from the EAM hearts co-immunoprecipitated with Flag-DPP-4 recombinant protein demonstrated that DPP-4 interacts with cathepsin-G (CTSG), a plasma membrane-bound serine protease, in the EAM hearts. Co-immunoprecipitation assay verified the physical interaction between CTSG and DPP-4. The CTSG activity in the EAM hearts was markedly elevated, and treatment with linagliptin effectively suppressed the CTSG activity in the EAM hearts. We also found that DPP-4 significantly suppressed the activity of α1-antichymotrypsin, a protease which can catalyzes CTSG and is activated in response to EAM. Finally, we demonstrated that the level of angiotensin II, a major product catalyzed by CTSG, in the EAM hearts was significantly decreased in LINA-group than in CONT-group. Thus, these results suggest that DPP-4 expressing on the surface of Th17 cells physically interacts with CTSG, thereby enhancing CTSG activity by suppressing α1-antichymotrypsin, which, in turn, promoting the accumulation of angiotensin II, in the EAM hearts. In conclusion, DPP-4 derived from Th17 cells aggravates cardiac dysfunction during EAM.
Abstract 405: Unprocessed ProBNP Secretion is Regulated by miR30-GALNTs Axis Dependent O-glycosylation in the Pathological Myocardium
Authors: Nakagawa Yasuaki , Kuwahara Koichiro , Nishikimi Toshio , Kinoshita Hideyuki , Yamada Chinatsu , Inazumi Hideaki , Nakao Kazuwa , Kimura Takeshi
Published: June 2021

Background:
It has been reported that biological inactive pro-brain natriuretic peptide(proBNP), precursor peptide of bioactive BNP, is increased in patients with heart failure, as well as mature BNP-32. However, underlying mechanisms of increased plasma proBNP levels in heart failure remain unclear.

Methods:
We used the conventional BNP assay system, which detects both proBNP and BNP-32, and our recently developed specific human proBNP assay system. We assessed cardiac production of proBNP and BNP(=proBNP+BNP-32) in patients with heart failure. To elucidate the molecular mechanisms underlying the processing and secretion of proBNP, we generated lentivirus vectors carrying several mutations in glycosylation sites of human proBNP. We evaluated the proBNP/BNP ratio, as an efficacy of processing of proBNP to BNP-32.

Results:
In coronary sinus, plasma proBNP/BNP ratios were significantly elevated in heart failure patients in accordance with the disease severity. In conditioned medium of cultured ventricular myocytes expressing wild type human proBNP, proBNP/BNP ratio was 37%, while in those of myocytes expressing glycosylation-resistant mutant of proBNP, proBNP/BNP ratio was only 3%, demonstrating the essential contribution of glycosylation to the secretion of proBNP. Among 7 glycosylation sites, we found that glycosylation of Thr48 and Thr71 in proBNP is critical for the secretion of proBNP from ventricular myocytes by attenuating proBNP processing. GalNAc-transferase(GALNT) is the largest glycosyltransferase enyme family, which controll O-glycosylasion. We identified GALNT 1 and 2 mediated the glycosylation-regulated increase in cardiac proBNP secretion by screening with siRNAs. Indeed, GALNT1 and 2-expression was increased in FCS-treated ventricular myocytes and failing hearts. We further found that GALNT1 and 2 expression is suppressed by miR-30 family, and that miR-30s expression is decreased in FCS-treated ventricular myocytes and failing hearts.

Conclusions:
We have elucidated novel molecular pathways that miR-30-GALNTs axis-mediated
O
-glycosylation in proBNP contributes to increased proportion of proBNP among secreted BNP in the pathological myocardium.
Abstract 41: Cell Cycle Activation of Cardiomyocytes Derived From Induced Pluripotent Stem Cells for Cardiac Regeneration
Authors: Zhu Wuqiang , Zhao Meng , Mattapally Saidulu , Gao Ling , Zhang Jianyi
Published: June 2021

Transplantation of cardiomyocytes derived from induced pluripotent stem cells (iPSCs) improves cardiac function in animal models with myocardial infarction. However, the poor number of survived cells and the low proliferation capability of cardiomyocyte derived from iPSCs are bottlenecks in myocardial repair with cell therapy. We hypothesize that increasing the number of surviving iPSC-CMs in the engraftment via cell cycle induction may lead to a transmural replacement of scar tissue and a lasting restoration of cardiac function. Cyclin D2 is a protein that regulates cell cycle transition from G1 to S phase. We transfected MHC-cyclin D2 (designated as MHC-cycD2) cDNA into the iPSCs, and differentiated the iPSCs into cardiomyocytes. Comparing to non-expressing cells, MHC-cycD2-expressing cardiomyocytes displayed increased Brdu incorporation activity, suggesting the enhanced cell cycle in MHC-cycD2-expressing cardiomyocytes. Cell cycle activity was confirmed by increased number of Ki-67 and PCNA positive immunostaining cardiomyocytes and more contractile embryonic body cell mass in MHC-cycD2-expressing culture compared to non-expressing culture. Data from Q-PCR and histology suggested that expression of MHC-cycD2 didn’t alter the pluripotency or cardiomyogenic potential of iPSCs. Thus, we have successfully induced cell cycle in iPSC-derived cardiomyocytes via expression of cyclin D2. We are currently studying if MHC-cycD2-expressing iPSC-cardiomyocytes exhibit superior regenerative potential compared to their non-expressing counterparts following transplantation into chronically infarcted hearts.
Abstract 411: In Utero Exposure to PM2.5 Causes Adult Cardiovascular Dysfunction and Reduced Exercise Capacity
Authors: Tanwar Vineeta , Stanford Kristin I , Wold Loren E
Published: June 2021

Objective:
Exposure to particulate matter 2.5 μm (PM2.5) during intrauterine development is associated with adverse cardiovascular outcomes at adulthood. Deteriorations in cardiac function are observed with increased myocardial demand in PM2.5-exposed individuals. The goal of this study was to determine the effects of in utero PM2.5exposure on exercise training capacity and cardiac function in adult mice.

Methods:
Female FVB mice were exposed either to filtered air (FA) or PM2.5at an average concentration of 73.61μg/m
3
for 6h/day, 7days/wk throughout pregnancy. 12wk old male offspring from exposed dams were assigned to in utero FA (n=5) or PM2.5 (n=5) exposed groups which underwent exercise training for 3 weeks (housed with running wheels for 3 weeks). We measured total distance travelled and performed echocardiography at baseline, 1, 2 and 3 weeks.

Results:
There was a progressive decrease in total distance travelled each week in the in utero PM2.5 exposed mice (Week 1: 12.2±3.46 Km FA, 5.32±2.06 Km PM2.5; Week 2: 41.4±9.62 Km FA, 17.28±6.60 Km PM2.5; Week 3: 61.8±16.59 Km FA, 25.92.±8.62 Km PM2.5) compared to the in utero FA exposed mice. When comparing to their respective sedentary counterparts, the FA exercise group showed increased fractional shortening (%FS), left ventricular end systolic (LVESd) and diastolic (LVEDd) diameters, suggesting eccentric hypertrophy. There was a modest decrease in %FS and marked increase in posterior wall thickness during diastole (PWTd) in the PM2.5 exercise group suggesting concentric hypertrophy. Comparison of in utero FA vs PM2.5 exercise groups after 3 weeks of exercise training showed reduced %FS and marked decrease in LVEDd in the PM2.5 exercise group compared to the FA exercise group. Furthermore, a decrease in PWTs and increased PWTd was also observed in the PM2.5 group compared to FA controls.

Conclusions:
In utero PM2.5exposure reduced exercise capacity at adulthood and the development of both systolic and diastolic dysfunction. Thus, our study showed that individuals residing in high pollution areas are predisposed to develop cardiac dysfunction under conditions of increased myocardial demand.
Abstract 412: Global Phosphopeptide Analyses Identifies a Profile that Distinguishes Advanced Heart Failure Patients Capable of Cardiac Recovery Following Left Ventricular Assist Device Unloading
Authors: Tracy Christopher M , Makaju Aman , Navankasattusas Sutip , McCreath Lauren , Diakos Nikolaos , Selzman Craig , Drakos Stavros , Franklin Sarah
Published: June 2021

The classical view of the human heart characterizes it as an organ with limited ability to recover and restore itself. Consequently, left ventricular assist devices (LVAD), which provide mechanical circulatory support and profound ventricular unloading, have been used as both a destination therapy as well as a bridge to transplantation in heart failure patients. However, evidence from many prospective studies indicates that a subset of patients with LVAD implants can significantly improve the structure and function of their heart (i.e. “responders”) and undergo LVAD explantation. This intriguing phenomenon provides great promise for heart failure patients, although the underlying mechanisms driving this recovery are largely unknown. To identify global changes in signaling pathways operative in the normal and failing human heart and to distinguish patients who respond to LVAD therapy from non-responders we performed phosphopeptide profiling and label-free quantitation of 38 patients. Our analysis utilized cardiac tissue from 10 donor controls, 6 recovered heart failure patients (responder), and 22 patients that did not respond to LVAD therapy (non-responders). Phosphopeptide enrichment and mass spectrometry-based analyses identified 15,816 unique phosphopeptides, 2,017 of which were shared between all samples. Label-free quantitation and bioinformatic analysis further classified 288 peptides [≥ 2 fold change (p<0.01)] within this dataset that distinguish control tissue from those in heart failure. Most intriguing however, was our assessment of the phosphopeptide profile of heart failure patients at the time of LVAD implantation which, using a signature of 67 peptides [≥ 2 fold change (p<0.01)], allowed complete separation of responder and non-responder samples via statistical analysis. Thus, this panel of 67 phosphopeptides enabled us to determine which patients would experience cardiac recovery following ventricular unloading prior to LVAD implantation. Overall this study characterizes unique changes in phosphoproteins and signaling pathways between the normal and failing human heart and specifies those which define hearts capable of cardiac recovery which may guide strategies to improving current heart failure therapies.
Abstract 416: Pluripotent Stem Cell Signaling in Pediatric Heart Failure Patients
Authors: Woulfe Kathleen C , Tatman Phillip , Karimpour-Fard Anis , Jiang Xuan , Nunley Karin , Miyamoto Shelley D , Stauffer Brian L , Sucharov Carmen C
Published: June 2021

Pediatric heart failure is a heterogeneous disease process with congenital heart disease being the most common indication for heart transplantation in infants, and dilated cardiomyopathy (DCM) the most common indication in older children. Due to limitations of studying pediatric populations, there is no specific therapy for children with DCM. Importantly, the application of proven adult therapies to pediatric patients with DCM has resulted in no substantial improvement in survival over the past three decades. Therefore, there is a clear need for new approaches to better understand this disease process.

In this study, we investigated global cardiac transcriptome and circulating serum proteome changes in pediatric patients with idiopathic dilated cardiomyopathy (IDC) and non-failing controls. Changes were identified in cytokine signaling, signal transduction, and transcription in pediatric IDC patients when compared to non-failing controls. Interestingly, these changes closely resemble the induction of stem cell pluripotency and signaling in resting stem cell populations. Importantly, the transcriptome changes are age-specific and were not observed in adult IDC patients. In addition, the protein profile in serum from pediatric IDC patients identified several factors related to inflammation, and treatment of cardiomyocyte-derived induced pluripotent stem cells (iPSC) with pediatric IDC serum recapitulated pathologic changes in gene expression, including expression of genes altered in the failing pediatric heart. These findings suggest that pathologic changes in cardiomyocytes may be triggered by circulating factors present in the serum resulting in pluripotent and stem cell signaling. These results provide several novel targets for future research and therapeutic intervention.
Abstract 423: Kinase Independent Signaling of PI3Kγ Mediates Cardiac Fibrosis
Authors: Mohan Maradumane L , Grove Lisa M , Olman Mitchell A , Naga Prasad Sathyamangla V
Published: June 2021

Phosphoinositide 3 Kinase γ (PI3Kγ) belongs to a family of lipid kinases genetic deletion of which leads to pressure overload induced cardiac fibrosis in mice. However, the mechanism by which PI3Kγ mediates cardiac fibrosis is unknown. Cardiac fibrosis is a key underlying cause of fatal heart failure. A well-known fibrogenic mechanism is the generation of myofibroblasts, which are characterized by overexpression of smooth muscle α-actin (αSMA). Myofibroblast is a fibrosis-effector cell that produces pro-fibrotic cytokines and exuberant extracellular matrix that leads to cardiac fibrosis. To evaluate the role of PI3Kγ in fibrotic phenotype, cardiac tissue lysates from 3 months old WT and PI3Kγ null (PI3Kγ
-/-
) mice were assessed for the expression of αSMA. Interestingly, there is significant up-regulation of αSMA in PI3Kγ
-/-
in comparison to littermate controls (WT) even at baseline suggesting that loss of PI3Kγ predisposes the hearts towards fibrosis. To directly confirm that PI3Kγ
-/-
cardiac fibroblasts (CF) exhibit a myofibroblast phenotype even at baseline, CF were isolated from hearts of WT and PI3Kγ
-/-
mice and assessed for myofibroblast phenotype by immunostaining for αSMA in stress fibers. Fluorescence microscopy on the CF from PI3Kγ
-/-
mice showed intense immunostaining for αSMA with greater number of cells exhibiting αSMA in stress fibers when compared to CF from WT mice. Consistently, immunoblotting showed significantly higher αSMA protein levels in PI3Kγ
-/-
CF compared to WT CF suggesting that PI3Kγ
-/-
fibroblasts are “primed” to undergo myofibroblast differentiation. To determine the role of kinase-independent function of PI3Kγ in vivo, we generated unique mice lines with cardiomyocyte-specific expression of either kinase-dead PI3Kγ (PI3Kγ
inact
) or constitutively active PI3Kγ (
Myr
PI3Kγ) in the global PI3Kγ
-/-
(PI3Kγ
inact
/PI3Kγ
-/-
or
Myr
PI3Kγ/PI3Kγ
-/-
) and measured αSMA. Surprisingly, abundance of αSMA protein is significantly reduced in PI3Kγ
inact
/PI3Kγ
-/-
when compared to WT and PI3Kγ
-/-
mice. These data reveal that kinase-independent function of PI3Kγ is a key component in the myocyte-initiated pathway that ultimately drives CF to become myofibroblasts uncovering a novel mechanism of regulating pro-fibrotic signals.
Abstract 441: Role of the Mitochondrial Translocator Protein (TSPO) in the Proarrhythmic Vulnerability of the Diabetic Heart
Authors: Hu Jun , Koh Won-Joon , Xie Chaoqin , Akar Fadi G
Published: June 2021

In structurally normal hearts, inhibition of the mitochondrial translocator protein (TSPO) prevents ROS-mediated proarrhythmia. Whether and how TSPO modulates electrophysiological (EP) function of diabetic hearts, in which classically cardioprotective pathways are impaired, is unknown. We determined the EP effects of TSPO activation & inhibition in a rat model of type 2 diabetes mellitus (t2DM) and studied the mitochondrial pathway underlying TSPO-related proarrhythmia.

Methods:
TSPO expression & function were determined in Zucker Diabetic Fatty (ZDF) rats with established t2DM (N=16) compared to controls (ctrl, N=15). Optical mapping was performed before & after challenge of hearts with ischemia for 12 min followed by reperfusion. Hearts underwent TSPO activation (FGIN 4.6uM, N=10), blockade (DZP 64uM, N=7) or no treatment (N=14). Dependence of TSPO-related proarrhythmia on ROS and on the PTP a target of TSPO were also determined.

Results:
t2DM hearts exhibited markedly increased TSPO expression (mRNA & protein) compared to ctrl. TSPO activation did not alter EP properties at baseline in ctrl or t2DM. In contrast TSPO activation accelerated action potential (AP) shortening during ischemia in t2DM but not ctrl hearts. Following 8 min of ischemia, FGIN-mediated AP shortening was 2X greater in t2DM compared to ctrl (p=0.008). FGIN-treated t2DM (5/7) but not ctrl (0/5) hearts exhibited VT by 12min of ischemia (p=0.027). AP shortening and VT in FGIN-treated t2DM hearts were not prevented by the PTP blocker Cyclosporin A but rather by the ROS scavenger EUK implicating the inner membrane anion channel in the proarrhythmic activity of TSPO. Upon reperfusion, TSPO activation with FGIN caused VF in 100% of ctrl & t2DM hearts compared to ~50% of untreated hearts. DZP prolonged the AP in t2DM (by 53% p<0.01) but not ctrl (p=0.14) hearts consistent with heightened sensitivity of t2DM to TSPO ligands. DZP blunted AP shortening during ischemia and prevented VF upon reperfusion in ctrl and t2DM hearts.

Conclusion:
t2DM hearts exhibit heightened sensitivity to TSPO ligands. TSPO upregulation may be a key factor in the proarrhythmic vulnerability of the diabetic heart. The cardioprotective efficacy of TSPO inhibition against arrhythmias is preserved in t2DM.
Abstract 45: The Dark Side of Parkin: The Toxic Effects of Enhanced Parkin Levels
Authors: Shires Sarah E , Lampert Mark A , Kubli Dieter A , Gonzalez Eileen R , Purcell Nicole H , Gustafsson Åsa B
Published: June 2021

Parkin is an E3 ubiquitin ligase that facilitates clearance of damaged mitochondria. We have previously shown that Parkin is important for cardiac adaptation to myocardial infarction and that loss of Parkin leads to accumulation of dysfunctional mitochondria. As Parkin is upregulated in response to cardiac stress, we sought to determine the effect of overexpression of Parkin in response to hemodynamic stress. Wild type (WT) and cardiac-specific Parkin transgenic (Parkin-TG) mice were subjected to trans-aortic constriction. Surprisingly, Parkin-TG mice rapidly developed hypertrophy, pulmonary edema, and ventricular dysfunction compared to WT mice. We observed no differences in either mitochondrial content or LC3 levels in Parkin-TG hearts, suggesting that the early progression to heart failure was not due to excessive mitophagy. Cardiac contractility is regulated by intracellular calcium and abnormalities in calcium homeostasis are associated with cardiac dysfunction. To investigate whether Parkin affects calcium homeostasis in the heart, we performed a subcellular fractionation on Parkin
-/-
, WT, and Parkin-TG hearts. We found that Parkin localizes to the sarcoplasmic reticulum where it ubiquitinates proteins under baseline conditions. Furthermore, Parkin-TG mice had increased levels Phospholamban (PLB), but did not alter SERCA, suggesting that Parkin may alter calcium homeostasis via PLB. In addition, loss of-function mutations in Parkin are found in patients with familial Parkinson’s disease (PD). Because Parkin is upregulated in response to stress, we overexpressed either WT Parkin or PD-associated Parkin mutants in HeLa cells to evaluate how elevated Parkin affects cell function. Interestingly, Parkin mutants, but not WT Parkin, caused rapid cell death when overexpressed in HeLa cells. Overexpressing Parkin mutants also triggered elevated autophagic activity, and cells with impaired autophagy were more susceptible to toxicity induced by overexpression of the Parkin mutants. This suggests that cells upregulate autophagy to protect against toxic Parkin mutants. Therefore, in addition to defects in mitophagy, upregulation of mutant Parkin in patients may also detrimentally contribute to cell death and development of disease.