bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 095: Uncoupling The Mitochondria Facilitates Alternans Formation In The Isolated Rabbit Heart
Authors: Tolkacheva Elena , Wothe Jillian , Talkachova Iryna , Smith Rebecca , Visweswaran Ramjay
Published: July 2021

Alternans of action potential duration (APD) and intracellular calcium ([Ca
2+
]
i
) transients in the whole heart are thought to be markers of increased propensity to ventricular fibrillation during ischemia reperfusion injuries. During ischemia, ATP production is affected and the mitochondria become uncoupled. This uncoupling may play an important role in alternans formation in the heart. The aim of our study was to investigate the role of mitochondrial depolarization on the formation of APD and [Ca
2+
]
i
alternans in the isolated rabbit heart. We performed dual voltage and [Ca
2+
]
i
optical mapping of isolated rabbit hearts under control conditions, global no-flow ischemia (n=6), and after treatment with 50 nM of the mitochondrial uncoupler FCCP (n=6). We investigated the formation of alternans of APD, [Ca
2+
]
i
amplitude (CaA), and [Ca
2+
]
i
duration (CaD) in the heart, under different rates of pacing. We found that treatment with FCCP leads to the early occurrence of APD (192 ± 18 ms vs 136 ± 6 ms, p<0.05), CaD (182 ± 20 ms vs 133 ± 6 ms, p<0.05), and CaA (152 ± 5 ms vs 141 ± 3 ms, p<0.05) alternans, and an increase of intraventricular APD (0.44 ± 0.02 vs 0.2 ± 0.05, p<0.05) but not CaD (0.20 ± 0.03 vs 0.16 ± 0.03, p=N/S) heterogeneity, when compared to control. On the other hand, FCCP does not affect the conduction velocity in the heart (0.95 ± 0.06 m/s vs 1.05 ± 0.11 m/s, p=N/S). Furthermore, we demonstrated that FCCP and global ischemia have similar effects on the prolongation of [Ca
2+
]
i
transients, whereas ischemia induces a significantly larger reduction of APD compared to FCCP treatment. Our results demonstrate that uncoupling of mitochondria leads to earlier occurrence of alternans in the heart. Thus, in conditions of mitochondrial stress, as seen during myocardial ischemia, uncoupled mitochondria may be responsible for the formation of both APD and [Ca
2+
]
i
alternans in the heart, which in turn creates a substrate conducive to formation of ventricular arrhythmias.
Abstract 108: TALEN-mediated Gene-Editing in Zebrafish for Studying Titin Biology
Authors: SHIH YU-HUAN , Xu Xiaolei
Published: July 2021

Background:
Titin, the largest protein found in nature, has been found to be crucial for heart and muscle development and mutations on titin caused human diseases. Especially, truncated mutations on TITIN were found to be a major causative gene (more than 25%) for familiar dilated cardiomyopathy (DCM) but the mechanism is lacking. Due to the gigantic size and number of exons, genetic study of titin has been challenging. To facilitate genetic analysis of titin biology, we leveraged the Transcription Activator-Like Effects Nuclease (TALEN) technology in zebrafish to edit the titin gene directly.

Results:
We generated TALEN pairs targeting different domains of zebrafish titin, including Z-disc domain, N2B domain, and A domains. Because zebrafish contain two titin homologues,
ttna
and
ttnb
, TALEN pairs targeting the corresponding loci for both homologues were made. The mutations are efficiently introduced on the
titin
using TALENs. In addition to these truncated mutants, TALEN technology can also create internal deletions. The
titin
mutant fish were identified and further phenotypic analysis has been carried out.

Conclusions:
Combined with TALEN technology, zebrafish can provide a convenient vertebrate model for genetic studies of titin biology. Moreover, this gene-editing technology will facilitate the use of the zebrafish model as a powerful research tool for modeling human diseases and investigating their pathogenesis.
Abstract 114: System’s Biology Approach to Non-Invasive Integrative Analysis of Cardiac Energetics
Authors: Diolez Philippe , Deschodt-Arsac Véronique , Arsac Laurent , dos Santos Pierre , Hocini Mélèze , Jais Pierre , Haissaguerre Michel
Published: July 2021

Our aim is to propose an inovative non-invasive analysis of heart contraction energetics, to be used for diagnosis and therapeutics follow-up in human heart pathologies. The analysis is based on the combination of non-invasive 31P-NMR and IRM techniques and the analytical background of metabolic modular control analysis (MoCA). Metabolic control analysis’ based approaches have been used by us and others to overcome the complexity of intra-cellular regulations and to describe and quantify controls and regulations in complex systems. We previously demonstrated that MoCA does bring new informations on the interplay between ATP demand and supply in heart contraction energetics.

In the present study, MoCA was applied to beating Langendorff perfused guinea pig heart to describe qualitatively and quantitatively the global action of various inotropic effectors effector modifying (Adrenaline) or not (Levosimendan, a calcium-sensitizer; Frank-Starling effect) cytosolic calcium transients. MoCA allows to describe the relative importance of the different routes within the heart by which the inotropic effects are transmitted. The results show that in the absence of calcium transient changes, an energetic feed-back control occurs at the expense of total energy, like in skeletal muscle, while parallel activation of both supply and demand occurs after adrenaline addition, maintaining energy homeostasis (heart energetic paradigm).

In conclusion, clinical application of System’s biology to the study of contraction energetics in patients may help to the understanding of the links between molecular events in pathologies and whole organ function / dysfunction.
Abstract 120: Identification Of A Novel Adult Smooth Muscle-like Stem/progenitor Cell That Facilitates Formation Of Tissue Engineered Vascular Tissue For Use As Vascular Grafts In Vivo
Authors: Turner Elizebeth C , Huang Chien Ling , Sawhney Neya , Govindarajan Kalai , Kumar Arun H S , Clover James P , Martin Kenneth , Leblond Anne-Laure , Wang Sharon , Caplice Noel M
Published: July 2021

The aim of this study was to characterise a recently identified adult smooth muscle stem-like/progenitor cell (SMSPC) and exploit the ability to differentiate contractile smooth muscle cells (SMC) from SMSPCs facilitating tissue engineering of small to medium arteries, an approach which is hampered by inadequate sources of, and scale up methodologies for autologous SMCs. We initially identified a novel SMSPC in the bone marrow (BM) of rats that retained classic stem cell-like characteristics (clonogenicity, unlimited self-renewal, high telomerase activity, expression of stem cell markers) whilst expressing low levels of SMC-like transcripts. Spontaneous differentiation favoured a SMC phenotype where ~20-fold increases in calponin and SM-MHC protein expression were observed 10 days post-embryoid body formation, an effect augmented by TGFβ (P < 0.0001). Differentiation of SMSPCs from an undifferentiated state towards contractile SMCs was Myocd-dependent. Overexpression of Myocd in SMSPCs increased their percentage contraction in response to AngII (P < 0.05) and KCL (P < 0.05) and Ca2+ signalling to levels indistinguishable from rat SMCs. Knockdown of SMC-specific repressors resulted in Myocd-dependent SMC differentiation of SMSPCs. Taking advantage of the fact that SMSPCs possessed unlimited self-renewal capacity ex vivo and could be successfully reprogrammed into functional SMCs, we determined the vascular tissue engineering potential of SMSPCs and showed these cells integrate as viable tissue engineered vascular grafts in vivo. Specifically, after 1 month implantation, a Myocd-GFP+/Isl1+ smooth muscle layer was observed in the grafts along with a vWF+ luminal endothelial layer and multi-layered collagen and elastin fibre formation throughout the graft wall. We have recently isolated cells with similar SMSPC phenotype and differentiation potential from human peripheral blood. This study demonstrates that adult SMSPCs derived from rat BM and human blood can be reprogrammed to efficiently generate large quantities of mature contractile SMCs, displaying great utility as a cellular backbone for tissue engineered vascular grafts, making them an attractive source for vascular cell therapy and surgical applications.
Abstract 121: Cellular Origins of Postnatal Cardiomyogenesis in the Normal, Infarcted and Cell-Treated Heart
Authors: Malliaras Konstantinos , Marbán Eduardo
Published: July 2021

Background:
Controversy surrounds the putative mechanisms of cardiomyocyte (CM) replenishment in adult mammals. Using genetic fate mapping and bone marrow allotransplantation, we investigated the postnatal origins of CMs in the normal, infarcted and cell-treated heart.

Methods & Results:
Tamoxifen pulsing of non-infarcted bitransgenic MerCreMer-ZEG mice (αMHC promoter driving inducible Cre) resulted in specific labeling of CMs by GFP. Comparison of BrdU incorporation (after 5 weeks of BrdU administration) in GFP+ and GFP- CMs (by flow cytometry, immunocytochemistry and histology) indicated that in the normal adult mouse heart, CM turnover occurs predominantly through proliferation of resident small mononucleated CMs at an annual rate of ~1.3%, independent from CM polyploidization, multinucleation, cell fusion or DNA repair. After myocardial infarction (MI), new CM formation in the border zone arises from both progenitor cells (PCs) as well as pre-existing CMs. Transplantation of cardiosphere-derived cells (CDCs) upregulates both host CM cycling and recruitment of PCs. To investigate the identity of endogenous PCs, bitransgenic mice underwent MI, followed by tamoxifen pulsing. One week later, FACS of myocyte-depleted hearts resulted in isolation of small non-myocyte GFP+ cells, which exhibit activated αMHC post-MI (~1.5% of cells in risk area vs <0.2% in sham controls). These novel PCs (Sca1+ [53%]/ nestin+ [11%]/ ckit-/ ABCG2-) express cardiac transcription factors (Nkx 2.5, GATA4, MEF2C, FOXC2), cardiogenic molecules (BMP2) and cardiac structural proteins (at both transcript & protein level), beat spontaneously in vitro and form CMs when injected into recipient hearts. Bone marrow transplantation demonstrated that recruited PCs do not arise from hematogenous seeding. Cell therapy with CDCs stimulates recruitment of PCs (~4.2% of cells in risk area), while improving heart function and increasing viable myocardium.

Conclusions:
The normal adult mouse heart replenishes lost CMs through proliferation of pre-existing CMs. After MI, proliferation of resident CMs increases, but new CMs also arise from recruited PCs. CDCs boost both CM proliferation as well as recruitment of endogenous PCs, resulting in myocardial regeneration.
Abstract 123: Erythropoietin Enhances Abundance of Cardiomyogenic Precursors in vitro and in vivo
Authors: Zafeiriou Maria Patapia , Noack Claudia , Unsoeld Bernhard , Didie Michael , El-Armouche Ali , Bergmann Martin W , Zimmermann Wolfram H , Zelarayan Laura
Published: July 2021

Recent data suggest that erythropoietin (Epo) can improve cardiac function following ischemia reperfusion injury. We found EpoR to be prominently expressed in embryonic rather than adult mouse heart. In the latter EpoR expression was confined to interstitial cells. We isolated the non-myocyte fraction of adult mouse hearts by enzymatic digestion and straining (pore size: 30 µm) and found a 4-fold higher EpoR expression compared to unselected cardiac cells (n=3). Flow cytometry (FC) revealed that 24±3% of this heterogeneous cell pool expressed EpoR (n=3) and 11±2% co-expressed αMHC. Half of the αMHC+ cells stained positive for Ki67, consistent with FC showing that 49±7% αMHC+/EpoR+ cells were in S and G2 phase (n=3; vs. 13±3% in αMHC-/EpoR-; n=3). Interestingly, co-culture with neonatal rat cardiomyocytes yielded EGFP+ cardiomyocyte-like cells. Since αMHC+ proliferated and differentiated in vitro we termed them cardiomyogenic precursors (CMPs). Epo treatment of the non-myocyte pool enhanced Akt phosphorylation (n=6/group) and increased CMP abundance in vitro (2-fold; n=3). Cell cycle arrest abrogated the aforementioned effect, suggesting that Epo influenced CMP proliferation. Finally, we tested the potential of Epo to protect against ischemia by enhancing CMP numbers in a model of myocardial infarction (MI). Following MI mice were treated twice with 2,000 U/kg Epo (i.p.). Ten weeks post MI echocardiography revealed blunted myocardial deterioration in mice receiving Epo (ΔEF=-11.18%, n=5) compared to control (ΔEF=-20.82%, n=6). FC revealed an enhanced αMHC+/cTnT+ cell pool in the Epo treated group (20.3±1.9% vs. 10.6±2.3% in control, n=8/6) 4 weeks post MI. In conclusion, we found that EpoR is expressed in a putative cardiomyogenic precursor cell pool in the adult heart. CMPs appear to proliferate in vitro and in vivo; with further enhanced proliferation under Epo administration. This may contribute to Epo mediated protection after MI and highlights the therapeutic effect of Epo upon ischemic cardiac injury. Whether these cells remain from embryogenesis or just constitute an immature myocyte population in the adult heart deriving from cardiomyocyte dedifferentiation needs to be further investigated.
Abstract 124: Function of the Cytoskeletal Proteins Alpha-catenins in Myocardial Growth Control
Authors: Li Jifen , Carrante Sarah , Yi Roslyn , van Roy Frans , Radice Glenn L.
Published: July 2021

Introduction:
Mammalian heart possesses regenerative potential immediately after birth and lost by one week of age. The mechanisms that govern neonatal cardiomyocyte proliferation and regenerative capacity are poorly understood. Recent reports indicate that Yap-Tead transcriptional complex is necessary and sufficient for cardiomyocyte proliferation. During postnatal development, N-cadherin/catenin adhesion complex becomes concentrated at termini of cardiomyocytes facilitating maturation of a specialized intercellular junction structure, the intercalated disc (ICD). This process coincides with the time cardiomyocytes exit cell cycle soon after birth.

Hypothesis:
We hypothesize that coincident with maturation of ICD α-catenins sequester transcriptional coactivator Yap in cytosol thus preventing activation of genes critical for cardiomyocyte proliferation.

Methods:
We deleted αE-catenin / αT-catenin genes (α-cat DKO) in perinatal mouse heart and knockdown (KD) α-catenins in neonatal rat cardiomyocytes to study functional impact of α-catenins ablation on ICD maturation.

Results:
We previously demonstrated that adult α-cat DKO mice exhibited decrease in scar size and improved function post myocardial infarction. In present study, we investigated function of α-catenins during postnatal heart development. We found increase in the number of Yap-positive nuclei (58.7% in DKO vs. 35.8 % in WT, n=13, p<0.001) and PCNA (53.9% in DKO vs. 47.8%, n=8, p<0.05) at postnatal day 1 and day 7 of α-cat DKO heart, respectively. Loss of α-catenins resulted in reduction in N-cadherin at ICD at day 14. We observed an increase number of mononucleated myocytes and decrease number of binucleated myocytes in α-cat DKO compared to controls. Using siRNA KD, we were able to replicate α-cat DKO proliferative phenotype in vitro. The number of BrdU-positive cells was decreased in α-cat KD after interfering with Yap expression (2.91% in α-cat KD vs. 2.02% in α-cat/Yap KD, n>2500 cells, p<0.05), suggesting α-catenins regulate cell proliferation through Yap in neonatal cardiomyocytes.

Conclusion:
Our results suggest that maturation of ICD regulates α-catenin-Yap interactions in cytosol, thus preventing Yap nuclear accumulation and cardiomyocyte proliferation.
Abstract 132: CD13 and ROR2 Mediated Isoloation of hESC Derived Cardiac Mesoderm
Authors: Brady Bevin , Skelton Rhys J , Elliott David A , Weissman Irving L , Ardehali Reza
Published: July 2021

The human heart has a limited regenerative capacity and there is an unmet demand for improved therapies for myocardial disease. Human embryonic stem cell (hESC) derived cardiomyocytes have potential to facilitate the development of both cell-based and pharmaceutical treatments. To date many studies have focused on the efficient generation of cardiomyocytes from hESCs. However, its clinical use has been limited by technical challenges, including the inability to isolate a pure population of cardiovascular progenitors capable of robust engraftment and regeneration. To this end, we have attempted to identify surface markers that facilitate isolation of cardiac mesoderm and cardiovascular progenitors from differentiating hESCs.

Lineage-specific reporter hESC lines (MIXL1-GFP and NKX2.5-GFP) were utilized to test prospective isolation of early cardiac progenitors using two novel cell surface markers, CD13 and ROR2. A distinct population of cells co-expressing CD13 and ROR2 was isolated after 3 days of hESC differentiation which exhibited a transcriptional profile similar to primitive streak/mesoderm cells. Gene expression analysis and immunostaining confirmed the enrichment of mesodermal cells and near complete removal of endodermal, ectodermal and undifferentiated cells. To quantify the extent of cardiovascular progenitors generated from CD13/ROR2 expressing cells, an NKX2.5-GFP reporter hESC line was used and the double positive cells were sorted after 3 days and maintained in culture for 14 days. The majority of these cells showed enrichment for cardiovascular cells based on gene and protein expression, phenotype, and transplantation studies. By enabling retrieval of early mesoderm precursors from heterogeneous differentiated hESC populations, we anticipate CD13 and ROR2 should have utility in enriching for subsequent cardiac potential_the necessary first developmental step towards creating purified cardiomyocytes from hESC.
Abstract 134: Rigid Microenvironments Promote Cardiac Differentiation Of Mouse And Human Embryonic Stem Cells
Authors: Arshi Armin , Nakashima Yasuhiro , Nakano Haruko , Eaimkhong Sarayoot , Evseenko Denis , Reed Jason , Stieg Adam Z , Gimzewski James K , Nakano Atsushi
Published: July 2021

While adult heart muscle is the least regenerative of tissues, embryonic cardiomyocytes are proliferative, with embryonic stem (ES) cells providing an endless reservoir. In addition to secreted factors and cell-cell interactions, the extracellular microenvironment has been shown to play an important role in stem cell lineage specification, and understanding how scaffold elasticity influences cardiac differentiation is crucial to cardiac tissue engineering. Though previous studies have analyzed the role of the matrix elasticity on the function of differentiated cardiomyocytes, whether it affects the induction of cardiomyocytes from pluripotent stem cells is poorly understood. Here, we examined the role of matrix rigidity on the cardiac differentiation using mouse and human ES cells. Culture on polydimethylsiloxane (PDMS) substrates of varied monomer-to-crosslinker ratios revealed that rigid extracellular matrices promote a higher yield of de novo cardiomyocytes from undifferentiated ES cells. Using an genetically modified ES system that allows us to purify differentiated cardiomyocytes by drug selection, we demonstrate that rigid environments induce higher cardiac troponin T expression, beating rate of foci, and expression ratio of adult α- to fetal β- myosin heavy chain in a purified cardiac population. M-mode and mechanical interferometry image analyses demonstrate that these ES-derived cardiomyocytes display functional maturity and synchronization of beating when co-cultured with neonatal cardiomyocytes harvested from a developing embryo. Together, these data identify matrix stiffness as an independent factor that instructs not only the maturation of the already differentiated cardiomyocytes but also the induction and proliferation of cardiomyocytes from undifferentiated progenitors. Manipulation of the stiffness will help direct the production of functional cardiomyocytes en masse from stem cells for regenerative medicine purposes.
Abstract 138: Thermo-Responsive, Self-Adhesive Injectable Heart Scaffold for Pluripotent Stem Cell-Derived Cardiomyocyte Delivery
Authors: Wang Xintong , Chun Young Wook , Zhong Lin , Hong Charles , Lim Chee , Maltais Simon , Sung Hak-Joon
Published: July 2021

Introduction:
Regeneration of heart tissue post-infarction is hampered by the limited proliferation of cardiomyocytes. Due to their expandability and pluripotency, human induced pluripotent stem cells (iPSCs) are considered an ideal cell source to produce cardiomyocytes and regenerate heart tissue. However, previous clinical trials revealed the quick loss of injected cells (> 90%) after delivery in aqueous solutions (e.g. phosphate buffered saline, PBS), which significantly limited the clinical outcome. To enhance the retention of cells and promote tissue regeneration, we synthesized a temperature-responsive polymer which is water-soluble to encapsulate cardiomyocytes at room temperature, following injection into epicardium, it quickly forms a gel and holds the cells
in situ
at body temperature. Functional peptides can be conjugated to this material and facilitate its adhesion to cardiac tissue for optimal cell integration and cardiac regeneration.

Materials and Methods:
The polymers monomethoxypoly(ethylene glycol) (mPEG) and poly(ε-caprolactone) (PCL) were copolymerized to produce mPEG-PCL. The polymer was further modified with decorin-derived peptide, which can firmly bind to collagenous tissue. The solution-to-gel transition temperature (Ts) was determined by dissolving the polymer in PBS, slowly increasing the temperature from 4 to 40°C and checking the fluidity of the solution. The viability of cardiomyocytes encapsulated in polymer gel at 37°C for 2 weeks was determined by calcein AM. Peptide-modified polymer solution was injected to the epithelium of adult rat heart. Echocardiography was performed to evaluate the heart functions before and two weeks post injection. The tissue response to this material was studied by histological staining.

Results and Conclusion:
The material successfully underwent solution-to-gel transition at 37°C. Human iPSCs-derived cardiomyocytes encapsulated in the gel were still viable after 2 weeks. There was no heart dysfunction 2 weeks post-injection. No significant inflammatory response was induced in vivo. These demonstrate the safety and feasibility of this material to delivery cardiomyocytes to infarct heart.
Abstract 141: Histone Methyltransferase Setdb2 is important for miRNA mediated cardiac reprogramming
Authors: Kirste Imke , Jayawardena Tilanthi M , Payne J. A , Dzau Victor J , Mirotsou Maria
Published: July 2021

Rationale:
Regeneration of damaged cardiac tissue after injury presents a daunting challenge in cardiovascular medicine. Recent developments in reprogramming of somatic cells directly to cells of other lineages have raised the possibility of using this approach for cardiac regenerative therapy. Our group recently demonstrated successful miRNA mediated cardiac reprogramming in vitro and in vivo using a combination of miRNAs 1, 133, 206 and 499. Although, the molecular mechanisms underlying miRNA mediated fibroblast reprogramming to cardiomyocytes are yet unknown, accumulating evidence suggest that reprogramming acts through distinct phases and that histone modifications play an important role in these processes.

Objective:
Identify key genes involved in initiating miRNA mediated reprogramming via histone modifications.

Methods and Results:
For this, we analyzed the expression levels of 81 different genes involved in chromatin modification 4 days after miRNA transfection using PCR arrays. This analysis revealed that 6 of the 81 tested genes showed differential gene expression (≤-1.5-fold and p <0.02). JAK inhibitor-1 treatment, known for increasing reprogramming efficiency, further enhanced gene expression changes in 5 of these 6 genes. Setdb2, an H3K9 methyltransferase, was one of the most down-regulated targets 4 days after miRNA transfection (-1.4 fold, p<0.001). This effect was enhanced further when miRNAs were combined with the JAK inhibitor-1 (-2.6 fold, p<0.001). Silencing of Setdb2 using siRNAs further accentuated miRNA cardiac reprogramming as measured by cardiac transcription factor expression at 3 days and 6 days post treatment. Similar trends were observed by FACS analysis detecting increased percentage of αMHC-positive cells in siRNA treated fibroblasts compared to control treated only with the miRNA combination. Interestingly, our data showed that Setdb2 silencing alone was sufficient to initiate cardiac reprogramming, suggesting that Setdb2 might play a crucial role in defining cardiac cell fate.

Conclusion:
In conclusion our results indicate that Setdb2 down-regulation plays an important role in the direct reprogramming of fibroblasts to cardiomyocyte-like cells.
Abstract 142: Modeling Drug-Induced Long QT Syndrome with Patient-Specific Induced Pluripotent Stem Cell-Derived Cardiomyocytes
Authors: Stillitano Francesca , Karakikes Ioannis , Kong Chi-wai , Martinelli Brett , Li Ronald , Hajjar Roger J , Hulot Jean-Sebastien
Published: July 2021

Long QT syndrome (LQTS) is characterized by prolonged cardiac repolarization time and increased risk of ventricular arrhythmia. LQTS can be either inherited or induced notably after drugs intake. Mutations in genes encoding cardiac ion channels have been reported to underlie inherited LQTS. In contrast, drug-induced LQTS (diLQTS) most frequently arises from altered function of the hERG channel; the risk of developing diLQTS varies largely between subjects and most people who have life-threatening diLQTS have no known genetic risk factors.

We investigated whether the susceptibility to develop diLQTS observed in vivo can be recapitulated in vitro using patient-specific induced pluripotent stem cell (iPSC) technology. We collected skin fibroblasts from ten subjects who developed significant diLQTS after administration of Sotalol and/or Erythromycin. Ten other individuals who displayed no changes in QT interval after administration of the same drugs, were selected. iPSC were generated by retroviral delivery of Oct4, Sox2, Nanog and Klf4 in 17 of the 20 individuals. We report preliminary results obtained from iPSC-derived cardiomyocytes (iPSC-CMs) of two subjects. All experiments were performed in a blinded fashion without knowledge of the associated clinical phenotype. Cardiac differentiation of iPSC resulted in the generation of spontaneously beating embryoid bodies. iPSC-CMs showed positive staining for TNNT2, ACTN2 and Cx43. Gene expression analysis confirmed the expression of NKX2.5, MLC2v, MYH6 and MYH7, and of the relevant KCNH2 gene. The two lines had similar basal electrophysiological properties as assessed by measurements of action potential (AP) by patch-clamp technique and extracellular field potentials (FP) using micro-electrode array (MEA). E4031, a classical HERG blocker, significantly prolonged the FP duration (FPD) in a dose-dependent manner in both lines (EC50: 30.19 and 51.57 respectively). When both Sotalol and Erythromicin were used, FPD was prolonged in one of the two samples in a dose-dependent manner (EC50Sotalol: 100; EC50Erythr: 9.64) while drug response was blunted in the other cell line.

This study suggests that patient-specific iPSC can be used to model the functional abnormalities observed in acquired diLQTS.
Abstract 145: Glucose Depletion Is Essential For The Calorie-restriction-mediated Cardiac Angiogenesis Via Pka/ampk-dependent Autophagy.
Authors: Monji Akio , Bando Yasuko K , Aoyama Morihiko , Mitsui Toko , Kawase Haruya , Murohara Toyoaki
Published: July 2021

Background:
Caloric restriction (CR) promotes beneficial cardiovascular effects; however, its effects on cardiac angiogenesis remain unclear. We thus examined whether CR may modulate cardiac angiogenesis via activation of autophagy and seeked for the essential nutrient factor(s) that may be responsible for the CR-mediated angiogenesis.

Methods:
To confirm whether CR-induced angiogenic effects may be universally observed, we allocated 2 distinct heart failure models; #1 pressure overload (4 weeks) generated by transaortic constriction (TAC) and #2 diet-induced obesity (DIO) mice, as a model of diabetic cardiomyopathy. Male mice (14 w/o) were randomly allocated to a 4-week CR (TAC-CR and DIO-CR) and ad libitum (TAC-AL and DIO-AL). Cultured endothelial cells (ECs) were exposed to culture media of 1) ordinary composition, 2) glucose-depleted, 3) amino acid-depleted, and 4) serum-depleted condition. To visualize cardiac autophagic changes, GFP-LC3 mice were allocated to DIO and TAC treatment. Analyses for the changes in activities of autophagy (LC3-turnover assay and p62 level) and angiogenesis (tube formation and Akt/AMPK/eNOS activity) were evaluated.

Results:
Immunohistochemical analyses revealed that CR enhanced cardiac angiogenesis both in TAC and DIO mice. CR promoted increased in myocardial cyclic AMP concentration with concomitant PKA/AMPK/eNOS activation and the simultaneous increase in autophagic activity both in TAC and DIO hearts. Of note, cardiac Akt activity remains unchanged by CR. In vitro analysis revealed that glucose depletion, as well as forskolin (10 μM) and 8-bromo-cyclic AMP (1 mM), activated PKA/AMPK axis thereby facilitated angiogenesis and autophagy in ECs. The enhanced in vitro angiogenesis induced by glucose depletion and PKA enhancers was abrogated by autophagy inhibitor 3-MA treatment. PKA inhibitors (H89 and RP-cAMP) abrogated the increase in AMPK/eNOS phosphorylation levels induced by glucose depletion. Neither amino acid- nor serum-depletion had no effect on angiogenesis and autophagy.

Conclusions:
Glucose depletion is essential for the CR-mediated activation of cardiac angiogenesis and autophagy in a cAMP/PKA/AMPK-dependent fashion.
Abstract 154: Transcriptomic Analysis Reveals Upregulation of The Pro-Apoptotic, Extracellular Matrix-Degrading Granzyme System in Human Aortic Valve Calcification
Authors: Ohukainen Pauli , Peltonen Tuomas , Näpänkangas Juha , Taskinen Panu , Ohtonen Pasi , Ruskoaho Heikki , Rysä Jaana
Published: July 2021

Background:
Calcific Aortic Valve Stenosis (CAVS) is a major valvular disease that leads to myocardial hypertrophy and heart failure. It shares many hallmarks with atherosclerosis, however, many of the specific mechanisms remain to be discovered. We used a DNA-microarray to study the transcriptomic changes in human aortic valve tissue during different stages of stenosis.

Methods and Results:
RNA was extracted from severely stenotic aortic valves of patients undergoing valve replacement surgery (n=5) and non-sclerotic control valves from patients with aortic insufficiency (n=5). A GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix) was used to study the transcriptomic changes. Among the most upregulated genes were pro-apoptotic Granzymes, which are an integral part of the T-cell-mediated cytotoxic reaction. They have also been shown to be able to degrade the extracellular matrix (ECM). These findings were validated in a larger sample population (n=26) using qRT-PCR. Indeed, significantly increased expression of Granzymes A, B, H, K and M as well as granzyme co-effector Perforin 1 (PRF1) was detected in stenotic valves compared to normal valves. The most significant increase was seen in Granzyme B mRNA levels (7.1-fold; P<0.001) and the gene expression of PRF1 was also markedly upregulated (3.6-fold; P<0.001). Furthermore, the localization of granzymes within the valves and the effect of statin medication on their expression were studied.

Conclusions:
To our knowledge, this is the first time that the upregulation of the Granzyme system has been studied in human aortic valves. Its involvement indicates an active T-cell-mediated cytotoxic response as well as active degradation of the ECM. This opens up novel possibilities for the treatment and diagnosis of CAVS.
Abstract 160: Contribution of IP3 Receptors in the Electromechanical Response of LV Myocytes to Gq-protein Coupled Receptor Agonists
Authors: Signore Sergio , Sorrentino Andrea , Cannata Antonio , Mangiaracina Chiara , Sundman Mark , Ferreira-Martins João , Kannappan Ramaswamy , Shafaie Mehrdad , Webster Andrew , Sanada Fumihiro , Wybieralska Ewa , Ogórek Barbara , Borghetti Giulia , Goichberg Polina , Rota Marcello
Published: July 2021

Gq-protein coupled receptor (GPCR) stimulation promotes PLC function, generating diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). The latter may promote Ca2+ translocation from intracellular stores altering Ca2+ homeostasis in cardiomyocytes. The aim of this study was to establish whether GPCR agonists enhance IP3 receptor (IP3R) activity, affecting the electromechanical properties of LV myocytes. For this purpose, the functional responses of myocytes to GPCR agonists ATP or ET-1 were established. In field-stimulated cells, GPCR activators increased diastolic Ca2+, transient amplitude, and contractility; extra-systolic Ca2+ releases and aftercontractions were promoted. These responses were prevented by inhibition of PLC, or blockade of IP3Rs. Since DAG promotes PKC activity, the effects of GPCR stimulation were tested in the presence of the PKC inhibitor chelerythrine. This compound failed to abrogate the effects of ATP and ET-1, indicating that PKC-independent pathways play a critical role in mediating the observed cellular responses to GPCR stimulation. Additionally, an AAV9 vector carrying EGFP and sh-RNA targeting IP3R type-2 was employed in vivo to downregulate IP3Rs. GPCRs activation failed to increase Ca2+ transients and to induce extra-systolic Ca2+ elevations in EGFP-positive myocytes. Conversely, these responses were preserved in EGFP-negative cells. In patch-clamped myocytes, changes in Ca2+ transient properties following GPCR activation were accompanied by a decrease in resting potential, action potential (AP) prolongation, and emergence of arrhythmic events. Similar electrical disturbances were detected by direct activation of IP3R with IP3 dialysis, or by enhancing the affinity of the receptors to its ligand, with thimerosal. To establish whether Ca2+ mobilized from the sarcoplasmic reticulum (SR) to the cytoplasm via IP3Rs was responsible for the electrical alterations caused by GPCR agonists, experiments were performed in which SR Ca2+ was depleted, or cytosolic Ca2+ was buffered. Under these conditions, ATP and ET-1 failed to prolong the AP and to induce arrhythmias. In conclusion, the GPCR/IP3R axis regulates Ca2+ homeostasis, contractile performance and the electrical stability of LV myocytes.
Abstract 161: TRPC6 Activator Hyperforin Facilitates Arrhythmias Via Activating Store-operated Calcium Entry
Authors: Wen Hairuo , Gordan Richard , Fefelova Nadezhda , Xie Lai-Hua
Published: July 2021

Hyperforin, a major antidepressant constituent of St. John’s wort (
Hypericum perforatum
), exhibits high selectivity in activating the canonical transient potential channel 6 (TRPC6). Our previous studies have shown that TRPC6 may modulate Ca
2+
handling via store-operated Ca
2+
entry (SOCE) in adult ventricular myocytes. In the present study, we aim to test whether hyperforin is arrhythmogenic by facilitating Ca
2+
waves. Intracellular Ca
2+
fluorescence (F/F
0
) was imaged in Fluo-4-AM loaded ventricular myocytes isolated from adult mice (2-4 month old). SOCE was evaluated by increasing the external Ca
2+
concentration ([Ca
2+
]
o
) from 0 to 1 mM after caffeine (10 mM) and thapsigargin (10 µM) were used to completely deplete SR Ca
2+
. Hyperforin (0.1-10 µM) perfusion increased SOCE up to 3.7 folds (F/F
0
= 3.73 ± 0.42, n = 7, compared to the control F/F
0
= 1.69 ± 0.37, n = 9, p < 0.05), in a concentration dependent fashion. Whole-cell currents were recorded using ramp pulses from -110 mV to +50 mV, while K
+
, Na
+
, L-type Ca
2+
, and Na
+
-Ca
2+
exchange currents were pre-blocked. Hyperforin at a lower concentration (0.1 µM) induced a significant increase of inward current (from -1.44 ± 0.23 pA/pF to -2.56 ± 0.36 pA/pF, n = 8, p < 0.05), which was then inhibited by the SOCE blocker Gd
3+
(1 mM) (-0.43 ± 0.22 pA/pF, n = 8, p < 0.05). Hyperforin promotes spontaneous CaWs ([Ca
2+
]
o
4 mM) by increasing the frequency to 180± 14 % (n = 11. p < 0.05), which can be attenuated by Gd
3+
(1 mM), or SKF-96265 (10 μM). In addition, both the amplitudes of Ca
2+
transients (F/F
0
=1.92 ± 0.09 to 2.09 ± 0.10, n = 12, p < 0.05) and calcium content (control: F/F
0
= 2.03 ± 0.11, n=16 to 2.12 ± 0.13, n = 11, p < 0.05) were enhanced by acute hyperforin perfusion (0.1 µM), when the cells were paced at a pacing cycle length of 2 seconds. The proarrhythmic effect of hyperforin was also confirmed in Langendorff-perfused hearts, from which optical membrane voltage mapping and EKG were simultaneously recorded. In conclusion, the TRPC6 activator hyperforin exhibits an arrhythmogenic effect in the heart. Its action is likely to be mediated by generating an inward current and increasing the calcium load in cardiac myocytes through the SOCE pathway. Caution should be taken when prescribing this drug to patients with heart disease.
Abstract 167: Genetic Deletion of p66Shc Adaptor Protein Leads to Increased Myocardial Infarction by Inhibiting RISK and SAFE Prosurvival Pathways
Authors: Akhmedov Alexander , Braunersreuther Vincent , Montecucco Fabrizio , Jakob Philip , Camici Giovanni G , Mach Francois , Luescher Thomas F
Published: July 2021

Background—
Formation of reactive oxygen species (ROS) contributes to many pathophysiological processes. Although ROS production is also involved in some physiological processes, the imbalance between their generation and removal, i.e. oxidative stress, plays a major role in particular in myocardial injury caused by ischemia-reperfusion (I/R). The mammalian Shc locus encodes three Shc isoforms: p46
Shc
, p52
Shc
and p66
Shc
. The p66
Shc
is not involved in mitogenic signals as p46
Shc
/p52
Shc
, but it functions as a critical mediator of intracellular oxidative signal transduction. Various studies relate p66
Shc
to cardiovascular disease; however, few data are available on the role of p66
Shc
in myocardial I/R.

Methods and Results—
8-12-week-old male p66
Shc
deficient (

p66
Shc-/-

) mice and corresponding C57Bl/6 wild-type (WT) control mice were subjected
in vivo
to different durations of ischemia (30, 45 and 60 min) followed by 24h of reperfusion. Infarct size was assessed morphologically and by MRI. After 30 min of ischemia,

p66
Shc-/-

mice developed markedly larger infarcts as compared to WT. This effect was confirmed by in vivo silencing of

p66
Shc

prior to I/R. Both genetic deletion and silencing of

p66
Shc

displayed increased post-ischemic levels of serum cardiac troponin I (cTnI). However, the observed effect on infarct size was limited to 30 min of ischemia since by increasing ischemia duration to either 45 or 60 min infarct size did no longer differ between

p66
Shc-/-

and WT mice. Moreover, differently from WT, infarct size in

p66
Shc-/-

was not significantly larger with increasing duration of ischemia (from 30 to 60 min). On the molecular level the observed effect was linked to the inhibition of activation by phosphorylation of protein kinase Akt and transcription factor STAT3 - two key members of prosurvival pathways RISK and SAFE, respectively.

Conclusions—
Our data suggest that genetic deletion of p66
Shc
leads to an increased sensitivity to myocardial infarction with larger infarcts with shorter, but not prolonged ischemia, and that RISK and SAFE prosurvival pathways are involved. Therefore, activation of p66
Shc
may provide resistance to ischemia and represent a novel therapeutic target in the early phase of myocardial infarction.
Abstract 170: Protein Kinase C regulates alternative splicing transitions during heart development
Authors: Verma Sunil K , Deshmukh Vaibhav , Kuyumcu-Martinez Muge N
Published: July 2021

Introduction:
Highly conserved genetic and molecular pathways coordinate gene expression during heart development. Alternative splicing (AS) regulates gene expression by generating distinct spliced isoforms of proteins such that embryonic variants are expressed only in fetus but not in adult. Importantly, mutations that affect splicing of embryonic genes such as Nkx2.5 and GATA4 can lead to congenital heart defect. Although AS is critical for proper gene expression in developing heart, the signals that regulate AS are not well known. We have previously shown that Protein Kinase C α/β (PKCα/β) phosphorylates splicing regulator CELF1 that controls AS in heart, suggesting that PKC signaling could be responsible for AS regulation during heart development.

Hypothesis:
PKCα/β contributes to embryonic gene expression by regulating AS.

Methods:
We used H9c2 rat embryonic cell differentiation into cardiomyocyte-like cells and rat heart tissues at different stages of development to determine whether PKC is responsible for AS transitions. The localization and levels of proteins were assessed by western blot and immunohistochemistry. Quantitative RT-PCR was performed to determine the splicing pattern of nine developmentally regulated genes using specific primers.

Results:
The results indicate that AS of developmentally important genes transitioned during rat heart development and H9c2 differentiation. PKCα/β regulated embryonic to neonatal AS transitions by phosphorylating splicing regulators CELF1 and Rbfox2. Blocking PKCα/β activity by bisindolylmaleimide IX reduced both the phosphorylation and protein levels of CELF1 and Rbfox2 with concurrent changes in AS transitions. Depletion of PKCα was sufficient to alter a subset of AS events by down-regulating CELF1 and Rbfox2 proteins. Finally, our data suggest that nuclear localization of PKCα/β at embryonic stages could be the mechanism for AS transitions and increased steady state levels of splicing regulators.

Conclusions:
Our results show that PKCα/β regulate splicing regulators and AS during heart development. Changes in PKCα/β subcellular localization is one of the determinants for embryonic to neonatal AS transitions in developing rat heart and during cardiomyocyte differentiation.
Abstract 177: Insulin Resistance and Mechanisms of Heart Failure
Authors: Guo Shaodong , Qi Yajuan , Zhu Qinglei , Xu Zihui , Thomas Candice , Kumar Rajesh , Feng Hao , Dostal David , White Morris , Baker Kenneth
Published: July 2021

A major cause of death in patients with type 2 diabetes is cardiac failure and the molecular mechanism that links diabetes to cardiomyopathy remains unclear. Insulin resistance is a hallmark of type 2 diabetes and intensive insulin therapy on the patients with type 2 diabetes increases the risks of cardiovascular dysfunction. Thus, understanding the mechanisms of insulin actions and resistance, related to cardiac dysfunction, will be critical for development of new strategies treating heart failure in type 2 diabetes. Insulin receptor substrate 1, & 2 (IRS1, IRS2) are major components in insulin signaling pathway regulating metabolism and survival. Here we hypothesized that (1) loss of IRS1 and IRS2 causes heart failure; (2) hyperinsulinemia contributes to loss of IRS1 and IRS2 in type 2 diabetes and promotes cardiac dysfunction; and (3) underlying mechanisms are involved in protein kinase activation. H-DKO mice (Heart Double IRS1 and IRS2 Knock-Out) and L(Liver)-DKO mice were generated using Cre/Loxp system. Cardiac function and ATP content were measured by echocardiograms and ATP assay kit. Protein and gene expressions were detected through western-blot and Q-PCR. Primary cultures of neonatal rat ventricular cardiomyocytes (NRVMs) were prepared from Sprague-Dawley rats with enzymatic method. H-DKO mice reduced ventricular mass, developed cardiac fibrosis and failure, and diminished Akt→Foxo1 signaling accompanied by impaired cardiac metabolic gene expression patterns and reduced ATP content. L-DKO mice decreased cardiac expression of IRS1 and IRS2 proteins with insulin resistance, disrupting cardiac energy metabolism, leading to heart failure and activation of p38α MAPK (p38). Using NRVMs, we demonstrated that hyperinsulinemia degraded IRS1 and IRS2, resulting in insulin resistance and impaired insulin action through activation of p38. In conclusion, myocardial loss of IRS1 and IRS2 causes heart failure and is controlled by p38 during Insulin resistance, revealing a fundamental mechanism of heart failure during insulin resistance and/or type 2 Diabetes Mellitus.
Abstract 178: Metabolomic Approach by
1
H NMR Spectroscopy of Plasma for the Assessment of heart-function in Patients with Chronic Heart Failure
Authors: Juan Wang , Liangtao Luo , Zhongfeng Li , Jianxin Chen , Chan Chen , Huihui Zhao , Shuzhen Guo , Wei Wang
Published: July 2021

Chronic heart failure (CHF) is a progressive clinical syndrome characterized by inability of the heart to adequately pump blood to meet metabolic demands of the body, and assessment of heart function in chronic heart failure patients is needed to make therapeutic decisions. New York Heart Association( NYHA) classification is usually performed to evaluate heart-function of CHF patients. Nevertheless, this does not take into account metabolic perturbations produced by heart-function impairment. In contrast, metabolomics can investigate many metabolic perturbations within biological systems.The purpose of this study was to assess whether metabolomic profiles of plasma, obtained by proton NMR spectroscopy from CHF patients, are affected by the severity of heart-function impairment. An orthogonal projection to latent-structure analysis was performed to compare NMR spectra of 49 chronic heart failure patients according to NYHA classification.The statistical model obtained showed a good explained variance (R
2
X = 0.13 and R
2
Y = 0.92) and a good predictability (Q
2
Y = 0.68). Metabolomic profiles showed significant differences regarding various metabolites depending of severity of heart-function impairment: levels of high-density lipoprotein, glycol-protein, glutamine, glutamate, were significantly higher in patients with mild CHF compared to severe CHF. Other metabolites such as lactate and amino acids were significantly higher in patients with severe CHF than mild CHF. Our conclusion is that metabolomic NMR analysis provides new insights into metabolic processes related to the severity of heart function impairment in chronic heart failure.