bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 77: Fibronectin Signals Through Pim-1 and β1 Integrin in Cardiac Progenitor Cells to Induce Proliferation and Protection
Authors: Konstandin Mathias H , Toko Haruhiro , Volkers Mirko , Quintana Mercedes , Gude Natalie , Sussman Mark
Published: July 2021

Background:
Cardiac Progenitor Cells (CPC) are pivotally involved in cardiac repair. Fibronectin (FN), an extracellular matrix protein, is highly expressed during cardiac development. In adult heart FN is a component of the cardiac stem cell niche and re-appears after myocardial infarction (MI). The role of FN signaling in CPC function and cardiac remodeling following MI has not been elucidated. We demonstrate here proliferative and protective effects of FN signaling in CPC as mediated by β1 integrin receptor and cardioprotective serine/threonine kinase Pim1.

Methods:
Cell death and proliferation of CPCs was measured using propidium iodide and CyQuant assays. Signaling pathways were analyzed by immunoblotting, qRT-PCR and siRNA depletion of targets. FN was localized in heart sections by immunohistochemistry and cardiac function assessed by echocardiography in control and conditional FN knockout (KO) mouse hearts following MI.

Results:
FN inhibits starvation and staurosporine induced cell death in CPCs and promotes proliferation in conjunction with induction of Pim1 expression. Protective and pro-proliferative effects of FN are abrogated by inhibition of Pim1 or deletion of β1 integrin receptor. In vivo CPC expansion correlates with FN expression following MI, and CPC localize to regions of up-regulated FN protein in the infarct. Cardiac function in control and KO mice remains equivalent up to 2 weeks post MI, however by 4 weeks KO heart function worsens compared to control as evidenced by ejection fraction values measuring 16.4 +/- 1.5% vs 26.6 +/- 1.8% at 12 weeks post MI in KO and control hearts, respectively.

Conclusion:
FN provides pro-survival and pro-proliferative effects to CPCs in a Pim1 kinase and β1 integrin dependent manner. CPCs and FN colocalize in vivo in an infarction injury model, while conditional KO of FN in mice leads to further impairment of cardiac function after MI. Taken together these results indicate previously unidentified cardioprotective and regenerative roles for FN in pathologically challenged heart.
Abstract 79: Autophagy Is Necessary in Reversal of Left Ventricular Hypertrophy
Authors: Oyabu Jota , Yamaguchi Osamu , Otsu Kinya
Published: July 2021

Myocardial hypertrophy occurs in response to a variety of stresses as a compensatory mechanism to maintain cardiac output and normalize wall stress. Left ventricular hypertrophy (LVH) in the long term is an independent risk factor for a range of adverse consequences, such as cardiac dysfunction and cardiac mortality. Therefore, prevention or regression of LVH can be a major therapeutic target. Although reversal of LVH occurs after control of etiological factors, the molecular mechanisms remain to be clarified. We have previously reported that inhibition of autophagy, an evolutionarily conserved process for the bulk degradation of cytoplasmic components, induces cardiomyocyte hypertrophy.

In the present study, we investigated the role of autophagy in the course of reversal of LVH. Wild-type mice showed left ventricular hypertrophy by continuous injection of angiotensin II (A-II) for 14 days using osmotic mini-pump, and LVH was reversed 7 days after removal of pump. Autophagy was induced after removal of pump, as evidenced by an increase in LC3-II protein levels. Next, we crossed floxed Atg5 mice with transgenic mice expressing Cre recombinase in cardiac specific manner to generate cardiac-specific autophagy-deficient mice (CKO). We subjected CKO and control mice (CTL) to continuous injection of A-II for 14 days. CKO and CTL showed LVH similarly (LV weight/body weight, CKO 4.60±0.16 versus CTL 4.44±0.08, p=n.s., LV mass index, CKO 69.0±5.5 versus CTL 72.1±2.2, p=n.s.) without showing contractile dysfunction 14 days after injection of A-II. Cross-sectional area of cardiomyocytes was similar in CKO and CTL (CKO 281.0±1.0 μm
2
versus CTL 274.0±1.5 μm
2
, p=n.s.). 7days after removal of pump, CKO showed significantly less reversal of LVH compared with CTL (LV weight/body weight, CKO 4.30±0.13 versus CTL 3.65±0.09, p<0.05, LV mass index, CKO 67.8±3.8 versus CTL 56.2±2.6, p<0.05). Cross-sectional area of cardiomyocytes also showed less reversal of LVH in CKO (CKO 270.6.0±7.0 μm
2
versus CTL 220.9±11.4 μm
2
, p<0.05), indicating that reversal of LVH didn’t occur sufficiently in autophagy-deficient heart. Autophagy was suppressed during reversal of LVH in CKO. These results suggest that autophagy is necessary in reversal of LVH.
Abstract 80: Accumulation of Mitochondrial DNA in Autolysosome Causes Inflammation and Heart Failure Through Toll-Like Receptor 9 (TLR9) Signaling
Authors: Oka Takafumi , Yamaguchi Osamu , Komuro Issei , Otsu Kinya
Published: July 2021

Backgrounds
Nuclear DNA in apoptotic cell is digested by lysosomal deoxyribonuclease II (DNase II) in macrophages. Improper DNA digestion can lead to inflammation. We previously reported that cardiac-specific DNase II-deficient mice (CKO) exhibited heart failure after transverse aortic constriction (TAC). We observed inflammatory response and DNA accumulation in autolysosome in TAC-operated CKO heart. They were considered to be mitochondrial DNA (mtDNA). In present study, we elucidated the mechanism of inflammation integrated by DNA accumulation in TAC-operated CKO hearts. Furthermore we investigated the pathogenesis of inflammation and heart failure in wild-typeTAC-operated mice.

Methods & Results
First, we identified the origin of accumulated DNA in lysosome. To label cardiac mtDNA, EdU (5-ethynyl 2’ deoxyuridine) were injected into mice before TAC. In TAC-operated CKO mice, EdU- and LAMP2a (lysosomal marker) or LC3 (autophagosome marker) positive deposits were observed, indicating that mtDNA accumulated in autolysosome.

Then, we examined the mechanism how the mtDNA accumulation leads to inflammation. mtDNA has similarities to bacterial DNA, which contains inflammatogenic unmethylated CpG motif. TLR9, localized in the endolysosome, senses DNA with unmethylated CpG motifs. Therefore, we hypothesized that undigested mtDNA is sensed by TLR9. We administrated the inhibitory oligodeoxynucleotides against TLR9 to TAC-operated CKO mice. They attenuated the development of cardiomyopathy in CKO mice. Ablation of
Tlr9
also canceled the cardiac phenotype of CKO mice.

Next, we examined the involvement of DNA accumulation and TLR9 signaling in wild-type TAC-operated mice. DNase II activity was up-regulated in hypertrophied hearts, but not in failing hearts. LAMP2a- or LC3- positive DNA accumulation was observed in failing hearts. To determine the significance of TLR9 signaling pathway in the pathogenesis of heart failure, we subjected TLR9-deficient mice to TAC. They showed significant resistance to pressure-overload. TLR9-inhibitory oligodeoxynucleotides also improved the mortality in wild-type TAC-operated mice.

Conclusion
mtDNA-TLR9 axis is involved in inflammation in failing hearts in response to pressure overload.
Abstract 86: Catabolic Remodeling of Branched Chain Amino Acids in Heart Failure
Authors: Sun Haipeng , Zhou Meiyi , Gao Chen , Olson Kristine , Youn Ji-Youn , Wang Zhihua , Huang Ying , Ren Shuxun , Cai Hua , Ping Peipei , Lynch Christopher , Wang Yibin
Published: July 2021

Metabolic remodeling is an integral part of heart failure. Although glucose and fatty acids metabolism have been extensively studied, little is known about the role of amino acids homeostasis in heart physiology and pathology. Branched chain amino acids (BCAAs), including leucine, isoleucine, and valine, are essential amino acids for both protein synthesis and cellular signaling. Elevated levels of BCAAs have been linked with heart failure. However, the underlying regulatory mechanism and functional significance of abnormal BCAA catabolism in heart failure have not been established. We found that genes involved in BCAA catabolism, including a key regulatory protein PP2Cm, are significantly down-regulated at mRNA as well as protein level in pressure-overload induced failing heart in mice. Furthermore, the concentrations of BCAA catabolic products branched-chain keto acids (BCKAs) are also elevated in heart tissues of post TAC mice. Interestingly, the down-regulation of BCAA catabolic genes mimics a similar expression pattern observed in fetal heart, suggesting that decreased BCAA catabolic activity is part of the metabolic remodeling in pathologically stressed heart from an adult to a fetal-like state. Genetic ablation of PP2Cm in mouse leads to defect in BCAA catabolism and accumulation of BCAAs and BCKAs in cardiac tissue and serum. PP2Cm deficient mice had lower cardiac contractility and higher susceptibility to develop heart failure under pressure overload. In addition, BCKAs treatment to isolated mitochondria resulted in lower oxygen consumption rate and ATP production. PP2Cm deficiency as well as BCKAs treatment induced oxidative stress in cardiomyocyte and antioxidant treatment ameliorated the development of heart failure in PP2Cm deficient animals. Together, these data indicated that BCAA catabolic remodeling is likely an integrated component of metabolic remodeling during heart failure. More importantly, mis-regulation of BCAA catabolism in heart promoted heart failure progression, involving direct impact on mitochondrial function and redox homeostasis in cardiomyocytes.
Abstract 89: The Ventriculo-Ventricular Coupling Index: A Novel Approach to Assessing Biventricular Function
Authors: Kapur Navin K , Aronovitz Mark J , Blanton Robert , Mackey Emily , Paruchuri Vikram , Qiao Xiaoying , Daly Gerard H , Kass David A , Karas Richard H
Published: July 2021

Bi-ventricular (Bi-V) function in primary pulmonary hypertension (PPH) or PH secondary to left heart failure (SPH) remains difficult to characterize. As a measure of ventricular efficiency, ventriculo-arterial coupling (VAC) is calculated as the ratio of effective arterial elastance (Ea) to end-systolic elastance (Ees). No measure currently assesses ventriculo-ventricular coupling (VVC). Therefore, we developed a novel catheter-based approach to quantify Bi-V function using pressure-volume loop (PVL) analysis and hypothesized that Bi-V VAC ratios, defined as the VVC index (VVCI), may discriminate PPH and SPH.

Methods:

Adult male mice (n=6/group) underwent constriction of the pulmonary artery (PAC) or thoracic aorta (TAC) to model PPH and SPH respectively. Sham-operated animals underwent a left thoracotomy. Closed chest simultaneous Bi-V catheterization was performed after 7 days in PPH and 10 weeks in SPH. Conductance catheters were used for right and left ventricular PVL analysis via the right external jugular vein and right common carotid artery under steady-state conditions and with variable preload.

Results:

Steady-state Bi-V PVL and changes in VAC ratios and the VVCI are shown below. In sham mice VAC ratios and the VVCI reflect optimal ventricular efficiency. In PPH, the VVCI is significantly increased, while in SPH, the VVCI is significantly decreased compared to controls.

Conclusion:

These results identify a novel method to quantify Bi-V function in mice and further show that the VVCI can distinguish PPH and SPH. These findings have important implications for examining cardiac function in preclinical and clinical studies of left- and right-sided heart failure.
Abstract 91: Pathogenicity of Naturally Produced Fragments of Cardiac Myosin Binding Protein C and Their Effects on Heart Function and Failure
Authors: Razzaque Md. Abdur
Published: July 2021

Background:
Myosin binding protein C (MyBP-C) is a thick filament protein consisting of 1274 amino acid residues (149kD) and mutations in the cardiac isoform (cardiac MyBP-C; cMyBP-C) are responsible for a substantial proportion (20-35%) of identified cases of familial hypertrophic cardiomyopathy (FHC). Recently we found a 40kD fragment is produced from cMyBP-C when the heart is stressed, using a stimulus such as ischemia reperfusion injury. This fragment can be detected in both the mouse and human heart and appears to be stable. Its ability to interfere with normal cardiac function is unexplored.

Methods and Results:
To understand the potential pathogenicity of the 40kd fragment in vivo, we generated cardiac myocyte-specific transgenic mice (TG) using a Tet-Off inducible system to permit controlled expression in cardiomyocytes. When 40kD protein expression is induced by crossing the responder animals with tetracycline transactivator (tTA) mice, the double TG mice show protein expression and, subsequently, sarcomere dysgenesis and altered cardiac geometry. The double transgenic heart fails between 3 to 17 weeks of age. Expression, the fragment in cardiomyocytes led to development of significant cardiac hypertrophy with myofibrillar disarray and fibrosis. Subsequent analyses showed that MEK-ERK hypertrophic signaling pathways were activated. To determine the role of this pathway in the pathogenic response being generated, we subjected an experimental cohort of animals to treatment with the MAPK/ERK kinase inhibitor U0126 during pregnancy. The drug effectively improved heart function and prolonged survival as compared to the untreated control cohort.

Conclusions:
The data show that a 40kD fragment of cMyBP-C, which is generated during the development of heart disease in both the mouse and human, is a pathogenic fragment whose presence leads to hypertrophic cardiomyopathy and heart failure. Blockade of the MEK-ERK pathway was effective therapeutically in decreasing morbidity and increasing lifespan in the face of continued synthesis of the fragment.
Abstract 94: Nitric Oxide--Regulated RGS4 Loss of Function Coordinates Vascular Growth with Cardiomyocyte Growth in the Absence of External Hypertrophic Stimuli
Authors: Jaba Irina M , Li Na , Zhuang Zhen W , Jiang Yifeng , Tirziu Daniela
Published: July 2021

The mechanisms that coordinate an increase in vasculature with cardiomyocyte growth in adult heart are not known. We report that induction of angiogenesis is sufficient to promote cardiomyocyte growth and myocardial hypertrophy through a novel nitric oxide (NO)-dependent mechanism.

Our hypothesis
is that an increase in endothelial-derived NO release promotes the proteosomal degradation of negative regulator of G protein signaling RGS4 in cardiomyocytes (CM) thus relieving repression of hypertrophic stimulation through G proteins.

Methods
include cell culture systems and transgenic mouse models. We generated three conditional cardiac inducible mouse models: (1) to stimulate angiogenesis by transgenic expression of placental growth factor (PlGF); (2) to stimulate angiogenesis by PlGF and compensate for RGS4 degradation by transgenic expression of RGS4 in CM; and (3) to stimulate angiogenesis by PlGF in eNOS
-/-
mice.

Results.
In cultured rat neonatal CM, the treatment with a slow release NO donor resulted in increased degradation of RGS4 through the N-end rule pathway of protein degradation. RGS4 loss of function led to increase in cell size through Gbetagamma/PI3Kgamma-dependent Akt/mTORc1 activation. In mice, PlGF expression induced an increase in capillary/myocyte ratio and arteriolar branches (determined by microCT angiograms). Myocardial hypertrophy was induced subsequent to angiogenic stimulation and concurrent with increased in NO production. Consistent with in vitro data hypertrophic stimulation was associated with reduced RGS4 protein levels and robust activation of Akt/mTORc1 signaling. This hypertrophy was prevented by concomitant transgenic expression of RGS4 in CM, despite the persistence of angiogenesis, arteriogenesis, and increased NO release. Furthermore, the NOS inhibitor L-NAME or mTORc1 inhibitor rapamycin significantly attenuated RGS4 degradation, Akt/mTORc1 signaling, and heart size in PlGF-induced mice. A similar effect was observed upon conditional heart-specific PlGF expression in eNOS
-/-
mice.

In conclusion,
these findings support a novel NO/RGS4/G protein/Akt mechanism that couples vessel growth with myocyte growth and heart size in the absence of traditional hypertrophic stimuli.
Abstract 95: Exercise Training Prevents Attenuation of Nrf2/ARE-Antioxidant Signaling in Aging Myocardium
Authors: Namakkal Soorappan Rajasekaran , Gounder Sellamuthu S , Kannan Snkaranarayanan , Devdoss Dinesh , Firpo Matthew A , Boehme Christoph , Abel E. Dale , Hoidal John R
Published: July 2021

Background:

Aging promotes accumulation of reactive oxygen/nitrogen species (ROS/RNS) in cardiomyocytes, which lead to contractile dysfunction and cardiac abnormalities. These conditions are prominent in elderly human patients with oxidative stress induced CVD due to impaired antioxidant defense. Inducible antioxidant pathways are regulated by nuclear erythroid 2 p45-related factor 2 (Nrf2) through antioxidant response cis-elements (AREs). We find that the Nrf2 levels are impaired in the aging heart. The purpose of this study was to investigate age-dependent regulation of Nrf2-antioxidant mechanisms and redox homeostasis in the heart.

Methods:

Age-matched wild-type (WT) and Nrf2-/- (KO) mice at 2 and >20 months were subjected to endurance exercise stress (EES) and assessed the activation of Nrf2/ARE-dependent transcriptional mechanisms in the heart. Myocardial ROS was measured by electron paramagnetic resonance (EPR) analysis.

Results:

Our recent findings indicate that acute exercise stress (AES) activates Nrf2 signaling and promotes myocardial antioxidant function in the young (∼2 months) mouse. Whereas aging mice (>20 months) exhibit significant (p<0.05) oxidative stress as compared to hearts of the young. Though aged-mice are highly susceptible to develop oxidative stress (OS) on endurance exercise stress (EES), they slowly become adaptive to maintain redox homeostasis after moderate exercise training (MET; 10-12m/min, for 45 min/day) for ∼6 weeks. While transcription and protein levels for most of the ARE-antioxidants were increased (p<0.05) in young mice, their induction was blunted in aging mice on EES. Taken together, age-related Nrf2 dysfunction exacerbates EES mediated oxidative stress, which is likely to increase the vulnerability of the aging heart to develop hypertension and pressure overload type of diseases. Of note, a 6-weeks of chronic MET promoted nuclear translocation of Nrf2 along with its target antioxidants in the aging heart to near normal levels seen in young mice.

Conclusions:

These observations emphasize the importance of enhancing the Nrf2 function and endogenous cytoprotective mechanisms to combat age-induced ROS/RNS and protecting myocardium from oxidative stress diseases.
Abstract 001: The Investigation of Peptide Amphiphile Micelles for Detection of Vulnerable Atherosclerotic Plaques
Authors: Chung Eun Ji , Drews Laurie , Tirrell Matthew
Published: July 2021

Atherosclerosis is an inflammatory disease that is complicated by progressively unstable plaques which are characterized by specific molecular events. While current diagnostic imaging technology aim to determine the stability of plaques by assessing their physical attributes, molecular imaging tools aim to assess the true nature of the plaque through targeting of molecular markers. Furthermore, these existing technologies can hardly meet the sensitivity and accuracy standards that are required for any clinical application for preventive measures.

In order to preventatively intervene during the early pathology of plaque vulnerability, we are engineering monocyte-targeting, fluorescently-labeled peptide micelles (MFPMs) through the incorporation of the chemokine receptor CCR2-binding motif (residues 13-35) of the monocyte chemoattractant protein-1 (MCP-1). Since monocytes are found concentrated in plaques prone to rupture, and our molecular imaging tool binds specifically to monocytes, this nanoparticle system may provide a noninvasive, detection system for plaques of high risk.

The MCP-1 peptide or Cy-7 was conjugated to DSPE-PEG-2000 and mixed to form self-assembled, fluorescently-labeled, monocyte-targeting peptide amphiphiles. The critical micelle concentration of MFPMs was determined to be approximately 1 μM with an average diameter of approximately 15 nm, confirmed via dynamic light scattering and transmission electron microscopy. Chemotaxis assays confirmed the chemoattractive capabilities of MFPMs using murine monocytes at 4 hours in vitro, and MFPMs were confirmed to be biocompatible using murine aortic endothelial cells and smooth muscle cells after 24 hours. Peptide amphiphile micelles can be synthesized with multiple functionality (i.e. targeting element, imaging dye, etc.) and our investigations will lay the ground work for peptide amphiphile micelle-mediated drug delivery at the site of vulnerable plaques.
Abstract 003: Progenitor Cells Suppress T-Lymphocyte Proliferation Via a Paracrine Mechanism
Authors: van den Akker Frederieke , Vrijsen Krijn R , Deddens Janine C , Doevendans Pieter A , Sluijter Joost P
Published: July 2021

PURPOSE:
Limited treatment options are available for heart failure patients. Stem cell therapy has recently become a potential new way of repairing injured cardiac tissue. Different progenitor cell sources have been investigated, but most promising for cardiac therapy are mesenchymal stem cells (MSC) and cardiomyocyte progenitor cells (CMPC). Cardiac stem cell therapy using MSC or CMPC improved cardiac function, despite low engraftment of the cells. Paracrine factors, produced by the injected cells, presumably cause these improvements. Many studies are performed on the paracrine effects, yet modulation of the immune response in cardiac stem cell therapy, especially the strong influence of T-lymphocytes on adverse remodeling, has not been explored extensively.

Methods:
Human fetal MSC and CMPC were characterized and tested for multipotency. The immunosuppressive properties of both cell types were tested in co-culture with allogeneic peripheral blood mononuclear cells (PBMC) or T-lymphocytes stimulated with IL-2 and PMA. Proliferation was measured by CFSE-analysis using flow cytometry.

Results:
Proliferation of PBMC and T-lymphocytes was significantly reduced in the presence of MSC (65 ± 8%) or CMPC (97 ± 0.6%). In addition, production of inflammatory cytokines IFN-gamma and TNF-alpha was strongly downregulated. This effect was observed in both direct cell contact as well as in transwell co-culture systems (MSC: 58 ± 10%; CMPC: 62 ± 9%). Transfer of conditioned medium from these co-cultures to unrelated, activated PBMC or T-lymphocytes abrogated proliferation in these cells to a similar extent as the original co-culture (MSC: 51 ± 8%; CMPC: 97 ± 0.7%). Interestingly, exosomes isolated from the conditioned medium of MSC and CMPC prevented T-lymphocyte proliferation in a dose-dependent fashion. At a concentration of 1.5μg, T-lymphocyte proliferation was significantly suppressed (MSC-exosomes: 73 ± 12%; CMPC-exosomes: 77 ± 10%).

Conclusion:
Both MSC and CMPC have a strong capacity for in vitro immunosuppression, which is mediated by paracrine factors. One potent immunosuppressive factor secreted by both MSC and CMPC are exosomes, which prevented T-lymphocyte proliferation in a dose-dependent fashion.
Abstract 006: An Advance toward Making A Heart: Engineering Functional Human Heart Constructs with Induced Pluripotent Stem Cells and Decellularized Mouse Hearts
Authors: Yang Lei , Lu Tung-Ying , Lin Bo , Kim Jong , Tobita Kimimasa , Salama Guy
Published: July 2021

Background:
Heart disease is the leading cause of death in the world, requiring the development of novel and personalized therapeutic strategies for disease treatment. Heart tissue engineering holds a great promise for those therapies based on the reconstruction of patient-specific cardiac muscle. A bioartificial heart was reported by reseeding rat cardiomyocytes (
CMs
) into decellularized rat heart. Whether such method could be used to make human heart constructs remained unknown.

Objectives:
We sought to rebuild functional heart constructs using human induced pluripotent stem (
iPS)
cells and decellularized whole mouse hearts.

Methods:
We repopulated decellularized mouse hearts with human iPS cell derived multipotential cardiovascular progenitors (
MCPs
). MCPs represent the earliest heart progenitors during human cardiogenesis and can give rise to multiple cardiovascular lineage cells.

Results:
The seeded MCPs differentiated
in situ
into CMs, smooth muscle and endothelial cells, which reconstructed the decellularized mouse hearts. The engineered constructs exhibited myocardium and vessel-like structures, contracted spontaneously with a rate of 40~50 beats/min, exhibited intracellular Ca2+ transients and responded as expected to various drug interventions. In addition, we found heart ECM could promote proliferation and myofilament formation of CMs from the repopulated human MCPs. Therefore this study established a novel strategy of human heart tissue engineering, which could be beneficial to study heart development, and future preclinical applications.
Abstract 021: Extended Proliferation Capacity of Cardiomyocytes is Insufficient to Complete Cardiac Regeneration in Mice with a Mutation of Cardiac Myosin Binding Protein C
Authors: Fomovsky Gregory M , Gannon Joseph B , Schaefer Kimberly K , Jiang Jianming , Wakimoto Hiroko , Sadek Hesham A , Seidman Jonathan G , Seidman Christine E , Lee Richard T
Published: July 2021

Introduction:
Adult mammalian cardiac regeneration rate is inadequate to compensate for the loss of myocardium following injury. One-day old (P1) mice fully regenerate myocardium after ventricular apex resection by division of cardiomyocytes (CMs). This regeneration ability is lost by P7. While CMs of a P1 mouse are mostly mononuclear, CMs withdraw from cell cycle and become mostly binuclear in the first post-natal week. CMs in mice with a mutation of a sarcomeric cardiac myosin-binding protein C (MyBP-C
t/t
) have an extended proliferating capacity. In MyBP-C
t/t
mice, there are more CMs per ventricle and a significant number of CMs remain mononuclear compared to WT.

Hypothesis:
The loss of regeneration potential during the first post-natal week in WT mice is a result of rigid sarcomeric structure of maturing CMs, and a mutation of sarcomeric MyBP-C would extend the regenerative capacity of CMs beyond P1.

Methods:
We performed apical resections on P10 MyBP-C
t/t
and WT mice with sham-operated controls (n=61 of 118 survived surgery). For the resection surgery, neonates were anesthetized on ice, causing transient sedation, apnea and asystole. We assessed cardiac regeneration over two weeks by measuring cell cycle activity: Ki67 and pH3 expressions, and BrdU incorporation.

Results:
All cell cycle activity markers in MyBP-C
t/t
CMs were significantly higher than in WT. At 7 days-post resection (dpr) the number of BrdU-positive CM nuclei per 40X field was (mean±SD): 1.8±1.2 in MyBP-C
t/t
resections (n=4), 0.7±0.3 in MyBP-C
t/t
shams (n=5), 0.3±0.3 in WT resections (n=3), and 0.2±0.2 in WT shams (n=3). However, an increase in cell cycle activity in MyBP-C
t/t
resected hearts was not significant compared to MyBP-C
t/t
sham controls. Interestingly, using VonKossa silver staining, we observed pronounced dystrophic calcifications due to CM necrosis in MyBP-C
t/t
resected hearts only. The calcifications filled the resected area and were positive for cardiac troponin and proliferation markers as early as 3dpr, suggesting that proliferating CMs underwent necrosis and aborted regeneration.

Conclusion:
An extended proliferation capacity of MyBP-C
t/t
CMs beyond the first post-natal week is insufficient for complete cardiac regeneration following apical resection.
Abstract 023: Development Of Molecular Beacons Targeting Atrial Natriuretic Factor Mrna For Live Cell Sorting
Authors: Wile Brian , Jha Rajneesh , Xu Chunhui , Bao Gang
Published: July 2021

Introduction:
Human embryonic stem cell (hESC) differentiation protocols generate both working and nodal cardiomyocytes, however this mixed cell population is not ideal for clinical applications. No surface markers have been used for the separation of live nodal or working cells to date. Molecular beacons are hairpin oligonucleotide probes with a reporter fluorophore at one end and a quencher at the other; they are designed to fluoresce when hybridized to a complementary target and can be used to detect mRNA in live cells. Atrial natriuretic factor (ANF) is expressed significantly more in working cardiomyocytes than nodal cardiomyocytes. We hypothesize that molecular beacons targeting ANF mRNA may be used to separate working cells from differentiated hESCs.

Methods and Results:
We designed molecular beacons to target regions of ANF mRNA predicted to be accessible using RNA secondary structure prediction programs. We then developed a positive control cell line using chinese hamster ovary (CHO) cells to select functional beacons. CHO cells were transfected with a plasmid containing the ANF gene and puromycin resistance. Transfected cells were sorted into separate wells and clonal populations were exposed to puromycin for 2 weeks. Surviving colonies were assayed for ANF expression and used as a positive control for beacon characterization. We used this system to screen 10 molecular beacons targeting ANF and found 3 beacons that increased in fluorescence in more than 60% of ANF-CHO cells but less than 5% of untransfected cells. To examine whether molecular beacons are suitable for differentiated cultures, we carried out cardiomyocyte differentiation from hESCs using growth factors. ANF expression was up-regulated in differentiated cultures compared with undifferentiated cells. Transfection of differentiated hESCs using the Neon system was optimized using fluorophore labeled oligonucleotides, a positive control for beacon delivery. Studies are currently underway to determine ANF beacon sensitivity and specificity in hESCs.

Conclusions:
We have developed several molecular beacons with potential for the use of separating live cells based on the expression of ANF mRNA which could represent a powerful technique for stem cell sorting.
Abstract 024: MicroRNAs and Anesthetic Cardioprotection in Diabetes
Authors: Bosnjak Zeljko J , Olson Jessica , Kriegel Alison , Bai Xiaowen , Liang Mingyu
Published: July 2021

Introduction:
MicroRNAs are endogenous small RNA molecules that regulate a wide range of cellular functions primarily through reduction of target protein expression. Several microRNAs have been shown to play important roles in cardiac injury, and also contribute to the development of diabetic complications and cardiac preconditioning. We utilized a model of the patient-specific induced pluripotent stem cells (iPSCs) differentiated into the cardiac lineage in order to delineate the environmental and cellular mechanisms responsible for overturning anesthetic cardioprotection in diabetes. We hypothesized that miR-21 contributes to cardioprotection conferred by anesthetics in human cardiomyocytes and that diabetic conditions compromise this protection in part via suppression of miR-21.

Methods:
We have developed and validated a clinically relevant model of cardioprotection using human cardiomyocytes differentiated from the iPSCs derived from non-diabetic individuals (N-CM) and patients with type 2 diabetes mellitus (T2-CM).

Results:
Our results indicate that cardiomyocytes derived from type 2 diabetes-specific stem cells recapitulate the phenotypic findings from type 2 diabetic patients. For instance, T2-CM exhibited a suppression of protein kinase B (Akt) and activation of glycogen synthase kinase-3β, compared to N-CM; indicating that this pathway is compromised in cardiomyocytes derived from diabetic individuals. In addition, we examined whether isoflurane could delay oxidative stress-induced mitochondrial permeability transition pore (mPTP) opening in T2-CM and found that the effects of isoflurane were significantly attenuated as compared to N-CM. Finally, isoflurane increased miR-21 abundance in N-CM, but not in T2-CM.

Summary:
Diabetes and hyperglycemia substantially increase perioperative cardiovascular risk, with few mitigating strategies. Our data indicate an important role of miR-21 in isoflurane-induced cardioprotection and its impairment by diabetic conditions that may suggest new therapeutic targets for reducing perioperative cardiovascular morbidity and mortality in high-risk patients.
Abstract 039: Natural Killer T Cells Play Protective Roles in Cardiovirus-Induced Myocarditis by Inducing Anti-Viral and Regulatory Cytokines
Authors: Sato Fumitaka , Omura Seiichi , Martinez Nicholas E , Kawai Eiichiro , Pearson Sadie F , Fernando Viromi , Acharya Madan M , Chaitanya Ganta V , Alexander J. Steven , Ajuebor Maureen N , Taniguchi Masaru , Tsunoda Ikuo
Published: July 2021

Picornavirus infections have been known as a leading cause of viral myocarditis in humans. Theiler’s murine encephalomyelitis virus (TMEV) belongs to the genus Cardiovirus, the family Picornaviridae, and can cause myocarditis in susceptible mice. In viral myocarditis, viral replication in the heart and/or immune responses against virus as well as cardiac antigens (autoimmunity) can contribute to the pathogenesis. Natural killer T (NKT) cells can play a regulatory role in viral infections by producing anti-viral and anti-inflammatory cytokines; interferon (IFN)-γ can contribute to either viral clearance or tissue damage (immunopathology), while anti-inflammatory interleukin (IL)-10 has been suggested to regulate viral clearance or immunopathology. To determine the role of NKT cells in TMEV-induced myocarditis, we infected wild-type (WT) and NKT knockout (NKT KO, Jα18 KO) mice with TMEV. Myocarditis was monitored by echocardiography using the Vevo 770 system. During the acute (day 7) or chronic phase (day 60) of TMEV infection, cardiac pathology was evaluated by hematoxylin and eosin staining, and production of cytokines, including IFN-γ and IL-10, from spleen cells was measured by enzyme-linked immunosorbent assays. During the acute phase, the levels of left ventricular ejection fraction were significantly lower in NKT KO mice than in WT mice. Immunologically, NKT KO mice had lower levels of IFN-γ production than WT mice [IFN-γ (pg/ml): WT, 768 ± 533; NKT KO, 293 ± 190]. During the chronic phase, high intensity cardiac lesions were observed by echocardiography in NKT KO mice, but not in WT mice. Histologically, NKT KO mice developed moderate inflammation with basophilic degeneration and calcification in the heart, while WT mice had only mild inflammation in the heart. Immunologically, NKT KO mice had lower levels of IL-10 production compared with WT mice [IL-10 (pg/ml): WT, 1771 ± 381; NKT KO, 1199 ± 160]. These results suggest that NKT cells play a protective role in viral myocarditis by producing IFN-γ and IL-10, which contribute to viral clearance during the acute phase and the suppression of immunopathology during the chronic phase of disease, respectively.
Abstract 047: Transgenic troponin T in Zebrafish recapitulates a Unique type of Cardiomyopathy
Authors: He Bin , Sun Xiaojing , Liu Weibin , Xu Xiaolei
Published: July 2021

Background:
Cardiomyopathy-like phenotype has been recently reported in adult zebrafish stressed by either anemia or doxorubicin. However, it still remains unclear whether gene-based manipulation in this emerging animal model can faithfully recapitulate cardiomyopathy phenotypes in mammals. Here, we aim to address this question by generating the first transgenic model for cardiomyopathy in zebrafish.

Results:
We generated a panel of three transgenic fish lines, including Tg(cmlc2:Tnnt2) that overexpress wild type Tnnt2 in the whole heart, Tg(cmlc2:Tnnt2c) that drive a C-terminal amino acid truncated Tnnt2 (Tnnt2C) in the whole heart, and Tg(vmhc:Tnnt2c) that drives Tnnt2c expression only in the ventricle. Upon overexpression of the Tnnt2C, but not the full-length control, in the heart caused reduced ventricular size but enlarged atrium. A same morphological phenotype was noted in Tg(vmhc:Tnnt2c). At the cellular level, we found significantly reduced size of cardiomyocyte and increased cell proliferation. The morphological remodeling is accompanied by hallmarks of cardiomyopathy including reduced cardiac function and myofibril disarray.

Conclusion:
Transgenic overexpression of Tnnt2C in zebrafish faithfully recapitulates unique cardiomyopathy phenotypes noted in both human patients harboring the similar mutation and mice transgenic models, underscoring zebrafish as a conserved vertebrate model for cardiomyopathy. Our study further suggested that the dysfunction of Tnnt2C in ventricle is primarily responsible for cardiac phenotypes, while the enlarged atrium is a consequence of a defective ventricle.
Abstract 048: Identifying Genetic Modifiers for Cardiomyopathy via Mutagenesis Screening
Authors: Ding Yonghe , Xu Xiaolei
Published: July 2021

Rationale:
The generation of animal models for human cardiomyopathy creates opportunities to uncover mechanisms of diseases’ pathogenesis and to identify genes that modify the progression of pathogenesis. In contrast to candidate gene-based approaches, the phenotype-based forward genetic screening approach is less effectively employed in a vertebrate, mainly due to the prohibitively enormous colony management efforts.

Objective:
To identify genetic modifiers of cardiomyopathy through developing an unbiased forward genetic approach in the vertebrate
Danio rerio.

Methods and Results:
Using an expression-based insertional mutagenesis screening strategy, we identified 49 Zebrafish Insertional Cardiac (ZIC) mutants from a collection of ~300 gene break-transposon (GBT) lines. By applying doxorubicin (DOX) stress to these heterozygous ZIC mutants, we initially identified four GBT lines that either enhanced or suppressed DOX-induced cardiomyopathy phenotypes. Here, we focus on the GBT0411 line which significantly enhances DOX-induced cardiomyopathy phenotypes. We cloned the gene and identified a single transposon insertion that disrupts the long nuclei isoform of
dnajb6b
in this GBT0411 mutant. Consistent with its cardiomyopathy modifier function, we detected a predominant expression of
dnajb6b
in the heart. While mutations affecting the cytoplasmic functions of DNAJB6 were recently reported to cause limb-girdle muscular dystrophy type 1D, our study suggested that the impaired balance between Dnajb6b long (nuclei) and short (cytoplasmic) isoform accounted for the cardiomyopathy modifier effect.

Conclusions:
Our results demonstrated the feasibility of identifying genetic modifiers of cardiomyopathy using a forward genetic approach in a vertebrate. Specifically, our data implicate
DNAJB6
as a new genetic risk factor for DOX-induced cardiomyopathy. Without any presumption, this forward genetic strategy opens the door to systematically identify genetic factors that modify the progression of cardiomyopathy, which will facilitate the practice of personalized medicine and the development of novel therapies.
Abstract 049: Functional Characterization of KCNQ1 Channel Subunit With an A590T Mutation
Authors: Kinoshita Koshi , Kimoto Katsuya , Komatsu Takuto , Nishide Kohki , Tabata Toshihide , Ichida Fukiko , Yamaguchi Yoshiaki , Nishida Kunihiro , Inoue Hiroshi , Hata Yukiko , Nishida Naoki
Published: July 2021

Background:
KCNQ1
encodes the alpha subunit of the voltage-gated K
+
channel that mediates the cardiac slow delayed rectifier K
+
current (I
Ks
). A mutation, A590T, in
KCNQ1
was incidentally identified in a 40 years old female. She had a mild QTc prolongation in electrocardiogram but has never experienced any cardiac events. A590 is located in the C-terminal domain forming a coiled-coil structure, which has been suggested as a pivotal component for subunit tetramerization and channel trafficking to the cell surface. The previously reported mutations around A590 result in markedly reduced cell surface expression and loss of functional channel. We, for the first time, examined whether and how the A590T mutation affects the I
Ks
channel function.

Methods:
To assess the trafficking and channel function of KCNQ1(A590T) mutant subunit, we performed immunostaining, immunoblotting, and voltage-clamp measurements in HEK-293T cells transfected with wild-type or the A590T mutant
KCNQ1
or their mixture (WT, A590T, and A590T/WT cells, respectively).

Results:
The density of a depolarization-activated current in the A590T cells was smaller than that in the WT cells. The threshold, half-maximal activation, and saturating voltages of the depolarization-activated current in the A590T cells were more positive than those in the WT cells. The immunoreactivity against KCNQ1 subunit on the cell surface in the A590T cells is lower than in WT cells. The A590T/WT cells had a similar density of the depolarization-activated current and a similar level of immunoreactivity against the channel subunit to the WT cells. Furthermore, the immunoblotting detected subunit oligomers in the membrane fraction of the A590T cells while their densities were lower than those of the WT cells.

Conclusion:
Although the A590T mutant subunit can form oligomers for itself, this subunit is not efficiently trafficked to the cell surface without the aid of the WT subunit. Thus, homozygous inheritance of the mutant
KCNQ1
might be pathogenic. By contrast, the cells expressing both the mutant and wild-type KCNQ1 subunit had normal I
Ks
and cell surface expression, indicating that the heterozygous inheritance of the mutant
KCNQ1
might not cause severe cardiac diseases.
Abstract 050: Mutational Spectrum in a Cohort of Chinese Sporadic Dilated Cardiomyopathy by Next-generation Sequencing
Authors: Xu Lei , Sun Aijun , Zou Yunzeng , Hu Kai , Fan Zheng , Ge Junbo
Published: July 2021

Rationale:
Dilated cardiomyopathy (DCM) is a leading cause of heart failure and have a genetic basis in 20-30% of cases. Genetic studies in familial DCM have found mutations identified in more than 30 genes. However, the genetics of sporadic DCM is still unknown.

Objective:
To provide new insights into the pathophysiology of sporadic DCM, a mutational screening on 30 DCM-causing genes in a cohort was performed.

Methods and Results:
Patients diagnosed with idiopathic DCM were recruited into the study and underwent clinical evaluation. Genomic DNA was isolated from the peripheral blood and then 30 genes (
ACTC, DES, SGCD, MYH7, TNNT2, TPM1, TTN, VCL, MYBPC3, CSRP3, ACTN2, PLN, LDB3, MYH6, ABCC9, TNNC1, TCAP, TNNI3, EYA4, TMPO, PSEN1/2, CRYAB, PDLIM3, MYPN, LAMA4, ILK, ANKRD1, RBM20, LMNA
, and
SCN5A
) of every sample were detected by next-generation sequencing. All called variants were compared against National Center for Biotechnology Information SNP Database build 137 and the March 2012 release of the 1000 Genomes project. All variants identified in the cohort were compared in ethnic-matched controls consisting of 197 adult Chinese from the 1000 Genomes. The pathogenicity of the variants absent from controls were evaluated by SIFT software. Sixty-six adult Chinese unrelated DCM patients were tested for up to 30 genes causing familial DCM. The mean age was 49.14±15.97 years. Every patient was detected nonsynonymous variants, and the total 87 variants were composed of 39 novel and 48 known. Among the whole 87 variants, 34 satisfied the criteria (1) absent from ethnic-matched controls and (2) altering evolutionary conservation of amino acid verified by SIFT analysis, which were considered pathogenic mutations, identified in 30 patients accounting for 45.45% of the cohort. The 2 most frequent genes involved in mutation positive patients were
SCN5A
(12.12%) and
MYH7
(10.61%).

Conclusions:
Mutant genes found in familial DCM were also common in sporadic DCM, and the discovery of novel mutations spanned the spectrum of DCM genetics, implicating that genetic etiologies play an important role in pathogenesis of sporadic DCM.
Abstract 051: Growth Plasticity Of The Prenatal Murine Heart Depends On Amino Acid Availability
Authors: Hennig Maria , Dziegelewska Maja K , Magarin Manuela , Thierfelder Ludwig , Drenckhahn Jörg-Detlef
Published: July 2021

Intrauterine development influences the susceptibility to cardiovascular disease in adulthood, although the underlying molecular mechanisms are vastly unknown. We have recently shown that the prenatal mouse heart has an impressive regenerative capacity in response to tissue mosaicism for mitochondrial dysfunction caused by a heart specific knockout of holocytochrome c synthase (Hccs) - an X-linked gene required for mitochondrial respiration. In heterozygous Hccs knockout (Hccs+/-) embryos, hyperproliferation of healthy cardiomyocytes compensates for the functional loss of 50% of Hccs deficient cells, ensuring formation of a normally contracting heart at birth. In order to uncover molecular mechanisms enabling compensatory growth of the prenatal myocardium, we performed microarray RNA expression analyses on neonatal Hccs+/- and control hearts. These data revealed numerous genes involved in amino acid metabolism and protein homeostasis being differentially expressed in the neonatal Hccs+/- myocardium. We, therefore, hypothesized that amino acid availability is crucial for compensatory growth of Hccs+/- hearts to build a regularly sized organ and allow normal postnatal function. Thus, we studied the effects of in utero amino acid restriction on growth and development of Hccs+/- hearts by feeding dams a low protein diet (LPD) throughout pregnancy and keeping the offspring on LPD until adulthood. On standard protein diet heart weight to body weight ratio of Hccs+/- mice (n=26) born at gestational age 20.5 dpc does not differ compared to littermate controls (n=21). In contrast, Hccs+/- offspring on LPD (n=20) were found to have a significantly reduced heart weight to body weight ratio compared to control animals (n=19) at postnatal day 1. Importantly, cardiomyocyte size and proliferation were unaffected in neonatal Hccs+/- hearts on LPD, suggesting that amino acid restriction rather inhibits prenatal cardiac growth. This was in line with normal heart size and function in adult LPD Hccs+/- mice, confirming normal postnatal development. In conclusion, metabolic adaptations regulating amino acid availability might be required for growth plasticity of the fetal heart to prevent postnatal dysfunction after impaired intrauterine development.