bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 218: The Role of Novel Tripartite Motif Proteins in Sarcolemmal Membrane Repair
Authors: Gushchina Liubov V , Alloush Jenna , Bhattacharya Sayak , Xu Zhaobin , Beck Eric X , Weisleder Noah L
Published: July 2021

Tripartite motif (TRIM) proteins are a superfamily of coiled-coil-containing RING E3 ligases that function in many cellular processes, particularly in membrane repair pathways. Mitsugumin 53 (MG53) also known as TRIM72, is primary expressed in skeletal muscle and heart. Our experimental data confirm that during membrane damage, MG53 translocates to the injury site and acts as a molecular glue to reseal the damage area. The role of MG53 in membrane repair has been demonstrated in both in vitro studies using molecular approaches and in vivo using rodent wild type and knockout models. Thus, our data indicate that recombinant human MG53 protein can be directly applied as a therapeutic agent to increase the membrane repair capacity of many cell types, including cardiomyocytes during acute injury or in chronic disease progression. However, the precise mechanism and potential partners by which MG53 executes its membrane repair function are not completely understood. On the basis of the global TRIM family protein alignment, we hypothesize that there are other TRIM proteins that, alone or together with MG53, may facilitate repair by targeting the site of an injury. Moreover, data from our lab demonstrated that MG53 and these TRIM proteins can form homo- and hetero-oligomeric assemblies due to the presence of the coiled-coil region in these proteins and, further, that this may be necessary for the active membrane resealing process. Using E. coli protein expression methodology we can generate and isolate new TRIM recombinant proteins and test if these protein complexes are effective when applied externally to cardiac and non-cardiac cells. These novel proteins will also be tested for their pharmacokinetic properties to determine their efficacy in both acute and chronic applications. Our studies should increase our knowledge of the mechanisms controlling cardiac membrane repair and also provide novel therapeutic targets.
Abstract 229: Hydrogen sulfide donor, the Organic Polysulfide Diallyl Trisulfide Augments Ischemia-induced Angiogenesis by Upregulation of VEGF-Akt-eNOS Pathway
Authors: Hayashida Ryo , Kondo Kazuhisa , Shintani Satoshi , Murohara Toyoaki
Published: July 2021

Introduction:
Hydrogen sulfide (H2S) upregulates anti-oxidant, anti-apoptosis, anti-inflammatory, and cell survival. H2S plays an extremely important role in the homeostasis of the cardiovascular system and in the pathogenesis of cardiovascular disease. The diallyl trisulfide (DATS) is a polysulfide constituent found in garlic oil, and known as H2S donor that is able to release H2S continuously. Peripheral artery disease (PAD) causes considerable morbidity and mortality. The aim of this study was investigate whether the DATS would augment ischemia-induced angiogenesis.

Methods:
The unilateral permanent femoral artery ligation was performed in C57BL/6J mice (8-10 weeks old, n=8-10/group) and eNOS KO mice (8-10 weeks old, n=5-8/group), and vehicle or DATS (500 μg/kg/day) was injected intra-peritonealy up to 1 week following the hind limb ischemia (HLI). We evaluated the blood flow recovery using the laser Doppler perfusion image and capillary density stained with CD31 at 3 weeks following HLI. We assessed VEGF level by qPCR analysis and activations of eNOS and Akt by western blot analysis in ischemic tissues. Moreover, we cultured HUVECs in a hypoxic chamber with serum starvation for 24 hour, and then evaluated apoptosis by tunnel staining and oxidative stress by DHE staining with or without DATS (50 μmol). We also evaluated activations of eNOS and Akt in cultured HUVECs with or without DATS.

Results:
DATS treatment significantly increased blood flow (0.45 vs 0.78,p<0.05) and capillary density (1.57 vs 1.85,p<0.05) at 3 weeks following HLI compared to vehicle. DATS also increased mRNA level of VEGF and activation of Akt and eNOS at 3 days following HLI. On the other hand, DATS treatment failed to increase blood flow and capillary density at 3 weeks following HLI in eNOS KO mice. The DATS treatment inhibited apoptosis and oxidative stress in cultured HUVECs via activation of Akt and eNOS pathway in vitro.

Conclusions:
Our results indicate that DATS treatment could augment ischemia-induced angiogenesis by upregulation of VEGF-Akt-eNOS pathway that leads to inhibitions of apoptosis and oxidative stress. These results suggest that administration of H2S releasing agents might be efficacious for the treatment of PAD.
Abstract 232: Studies of Cardiac transcriptome and Dilated Cardiomyopathy-causative genes in zebrafish
Authors: Shih Yu-Huan , Zhang Yuji , Olson Tim , Xu Xiaolei
Published: July 2021

Background:
Rapid advance of genome technologies accelerate the discovery of genetic basis of cardiomyopathy and heart failure and also enable system biology studies to pinpoint underlying mechanism. However, the limited throughput of mammalian models restricted the number of genes that can be studied in a particular lab. Adult zebrafish has been recently pursued as a new model with higher throughput. However, as a non-mammalian model, its conservation is not tested.

Objective:
To assess the conservation of zebrafish for genetic studies of human dilated cardiomyopathy (DCM) via transcriptome analysis of 51 known DCM-causative genes.

Methods and Results:
By conducting RNA-sequencing (RNA-seq) analysis of larva and adult zebrafish, we identified genes with high expression level in the heart and fetal gene program using differential expression between embryonic and adult stages. We then searched zebrafish orthologues for 51 reported human DCM-causative or associated genes and identified zebrafish orthologues for 49 of them. While 30 genes have a single orthologue, 14 genes have two homologues and the remaining 5 genes have more than three. We then applied the transcriptome data to prioritize these homologues for the 19 DCM causative genes with more than one homologue. Based on the cardiac abundance and cardiac enrichment hypothesis, we are able to recommend a single zebrafish homologue of high priority for 12 out of 19 DCM genes, 2 zebrafish homologues of high priority for
ACTC1.
Interestingly, our expressional data suggested zebrafish othologues for human
MYH6
and
MYH7
, respectively. Similar to that in mammals, these two zebrafish othologues are oppositely expressed during zebrafish embryonic and adult stage.

Conclusions:
Orthologues for the majority of DCM causative genes can be found in Zebrafish, supporting its usage as a conserved vertebrate model for studying DCM. The definition of cardiac transcriptome in zebrafish will facilitate the future system biology studies. This vertebrate model with higher throughput can be further leveraged to validate the novel variants identified from human patients, to understand underlying signaling pathways and to develop novel therapeutics.
Abstract 251: Neuregulin Administration Stimulates Cardiomyocyte Proliferation In Neonatal Mice And In Myocardium From Human Infants
Authors: Polizzotti Brian , Ganapathy Balakrishnan , Walsh Stuart , Choudhury Sangita , Bennett David , Haubner Bernhard , Penninger Joseph , Kuhn Bernhard
Published: July 2021

Background:
Regenerative strategies have the potential to transform heart failure therapies. Cell transplantation strategies have shown effectiveness and safety in clinical trials. A complementary strategy is based on directly stimulating the proliferation of endogenous cardiomyocytes. We have demonstrated that administration of the growth factor neuregulin (rNRG1), which has passed phase 2 testing, stimulates cardiomyocyte proliferation and improves myocardial repair in adult animal models. Since cardiomyocyte proliferation is active during myocardial growth, we hypothesized that NRG1 may be more effective in growing animals and humans.

Methods:
We developed a myocardial injury model (cryoinjury) in neonatal mice that recapitulates scar formation seen in infants with congenital heart disease (CHD). In contrast with ligation of the left anterior descending coronary artery (LAD), cryoinjury reduced cardiomyocyte cell cycle activity. We used this model to evaluate the effectiveness of rNRG1 administration. To assess the ability of rNRG1 to stimulate human cardiomyocyte proliferation, we developed an organotypic culture system for myocardium from infants with CHD.

Results:
Administration of NRG1 resulted in a 2-fold increase in cardiomyocyte cell cycle activity at 10 days post injury (dpi). After neonatal cryoinjury in mice, administration of rNRG1 from birth to 30 days of life significantly improved the ejection fraction, which was sustained for 30 days after the cessation of rNRG1 administration. In contrast, administration of NRG1 four to 34 days after cryoinjury transiently reduced scar formation, and transiently improved myocardial structure and function. In myocardium from infants with Tetralogy of Fallot (ToF) less than 6 months of age (n = 6), rNRG1 induced cardiomyocyte proliferation.

Conclusions:
These results indicate that stimulating cardiomyocyte proliferation may be clinically effective in human infants with heart disease, a population in whom a significant and unmet need exists.
Abstract 260: A miRNA-Hippo Pathway Promotes Cardiac Conduction System Regeneration
Authors: Wang Jun , Martin James F
Published: July 2021

The cardiac conduction system (CCS) is required for initiating and maintaining regular rhythmic heartbeats and CCS defects can give rise to cardiac arrhythmia, a leading cause for morbidity worldwide. Given the poor self-repair potential in the adult human CCS, it is critical to elucidate the molecular mechanisms limiting CCS regeneration to facilitate developing efficient cardiovascular therapies. microRNAs (miRs) are small non-coding RNAs that repress gene expression post-transcriptionally. The miR-17-92 cluster can induce cardiomyocyte proliferation and regeneration. Hippo signaling, an ancient organ size control pathway, represses cardiomyocyte proliferation and regeneration. Here we found that both miR-17-92 and Hippo signaling were active in CCS. Disruption of either miR-17-92 or Hippo signaling in heart gave rise to cardiac arrhythmias in mice. Notably, miR-17-92 regulates Hippo signaling through repressing Lats2, a core Hippo pathway component. In miR-17-92 null mutant hearts, up-regulated Lats2 led to increased Hippo pathway activity. Moreover, we performed chromatin immunoprecipitation deep sequencing (ChIP-Seq) using YAP, the Hippo signaling effector, which suggested that Hippo signaling regulates genes involved in CCS homeostasis. Together, we propose a novel miR-Hippo genetic pathway that promotes CCS regeneration.
Abstract 266: Novel Sites of Angiotension II Type 1 Receptor are Identified for Direct Activation by Mechanical Stretch Independent of Angiotension II
Authors: Gong Hui , Jiang Guoliang , Yang Chunjie , Wang Shijun , Chen Zhidan , Zhang Guoping , Zou Yunzeng
Published: July 2021

The angiotensin II type 1 receptor (AT1R) has a crucial role in cardiac hypertrophy induced by pressure overload. In the previous study, we found a novel mechanism for mechanical stress-induced AT1R activation without the involvement of Ang II. However, few reports focus on how AT1R senses mechanical stress and translates it into biochemical signals inside the cells to induce cardiomyocyte hypertrophy. Here, we constructed different site-directed mutagenesis of AT1R and transfected them to COS7 cells and ATG–/– (Angiotensinogen knockout) cardiomyocytes, respectively, to observe the activation of downstream signaling to identify functional site of AT1R. The results showed AT1R-WT, AT1R-K199Q, AT1R-L212F,AT1R-Q257A and AT1R-C289A plasmids or adenovirus were overexpressed at high level in plasma membrane of COS7 or cardiomyocytes respectively. There was no obvious difference in subcellular expression of wt-AT1R and all the mut-AT1Rs. The further study revealed that Ang II-induced-phosphorylation of ERK, Jak2 and the redistribution of Gαq11 were dramatically decreased in COS7 cells expressing AT1R-K199Q or AT1R-Q257A, while these effects induced by mechanical stretch were greatly suppressed in COS7 cells expressing AT1R-L212F,AT1R-Q257A or AT1R-C289A compared to these in COS7 cells expressing AT1R-WT. We then transfected the adenovirus of wt-AT1R or different mut-AT1Rs to ATG–/– cardiomyocytes to exclude the influence of endogenous Ang II. The results were consistent with these results in COS7 cells. Moreover, ATG–/– cardiomyocytes overexpressing AT1R-K199Q or AT1R-Q257A parlty abolished hypertrophic response induce by Ang II, while the cardiomyocytes overexpressing AT1R-L212F,AT1R-Q257A or AT1R-C289A greatly inhibited the hypertrophic response induced by mechanical stretch. The present study indicated that Leu212, Gln257 and Cys289 are critical sites for AT1R activation by mechanical stretch without Ang II but Lys199 and Gln257 play important role in AT1R activation with Ang II.
Abstract 267: High glucose-induced mitophagy limits Cardiomyocyte Injury
Authors: Liang Qiangrong , Kobayashi Satoru , Huang Yuan
Published: July 2021

Cardiovascular complications are the major cause of death among diabetic patients. Mitochondrial dysfunction has been suggested to play an important role in the pathogenesis of diabetic cardiomyopathy. Therefore, timely elimination of dysfunctional mitochondria is essential for maintaining a healthy mitochondrial network and limiting diabetic cardiac injury. This function is performed by mitophagy, a process that selectively degrades mitochondria through the autophagy-lysosome pathway. In the present study, we investigated the functional role of mitophagy in cardiomyocytes treated with high glucose, an independent risk factor for heart failure in diabetic patients. We found that mitophagy in neonatal rat ventricular myocytes was increased by high glucose (30 mM) as indicated by a novel dual fluorescent reporter as well as the co-localization of mitochondrial protein Tom20 and lysosome-associated membrane protein 1 (LAMP1). Parkin is an E3-ubiquitin ligase that positively regulates mitophagy. Parkin overexpression enhanced high glucose-induced mitophagy and attenuated cardiomyocyte injury as measured by the levels of oxidative injury, mitochondrial damage, ROS generation and cardiomyocyte death. Conversely, Parkin knockdown potentiated the toxic effects of high glucose, suggesting that mitophagy is cardioprotective under hyperglycemic conditions. In addition, high glucose increased mitochondrial fragmentation. By overexpressing or knocking down Drp1, a key regulator of mitochondrial fission, we showed that mitochondrial fragmentation was coupled with mitophagy and negatively related to the toxic effects of high glucose. Together, these findings demonstrate that high glucose induces mitochondrial fragmentation and mitophagy, which are adaptive responses that protect cardiomyocytes against high glucose toxicity. Future studies are warranted to determine if mitophagy plays a similar role in diabetic cardiac damage in vivo.
Abstract 270: Rnd3/RhoE Haploinsufficient Mice are Hypersensitive to Pressure Overload and Develop Apoptotic Cardiomyopathy with Heart Failure
Authors: Yue Xiaojing , Yang Xiangsheng , Lin Xi , Yang Tingli , Yi Xin , Dai Yuan , Chang Jiang
Published: July 2021

Rationale:
Rho family guanosine triphosphatase (GTPase) 3 (Rnd3, also called RhoE), a member of the small Rho GTPase family, has been suggested to regulate cell actin cytoskeleton dynamics, cell migration, and apoptosis through the Rho kinase-dependent signaling pathway. The biological function of Rnd3 in the heart is unknown. The downregulation of small GTPase Rnd3 transcripts was found in patients with end-stage heart failure. The pathological significance of Rnd3 loss in the transition to heart failure remains unexplored.

Objective:
To investigate the functional consequence of Rnd3 downregulation and the associated molecular mechanism.

Methods and Results:
we generated Rnd3
+/-
haploinsufficient mice to mimic the downregulation of Rnd3 observed in the failing human heart. Rnd3
+/-
mice were viable; however, the mice developed heart failure after pressure overload by transverse aortic constriction (TAC). Remarkable apoptosis, increased caspase-3 activity, and elevated Rho kinase activity were detected in the Rnd3
+/-
haploinsufficient animal hearts. Pharmacological inhibition of Rho kinase by Fasudil treatment partially improved Rnd3
+/-
mouse cardiac functions and attenuated myocardial apoptosis. To determine if Rho associated coiled-coil kinase 1 (ROCK1) was responsible for Rnd3 deficiency-mediated apoptotic cardiomyopathy, we established a double knockout mouse line, the Rnd3 haploinsufficient mice with ROCK1-null background (Rnd3
+/-/ROCK1-/-
). Again, genetic deletion of
ROCK1
partially but not completely rescued Rnd3 deficiency-mediated heart failure phenotype.

Conclusion:
Downregulation of Rnd3 correlates with cardiac loss of function as in heart failure patients. Hyperactivation of Rho kinase activity is responsible in part for the apoptotic cardiomyopathy development. Further investigation of ROCK1-independent mechanisms in Rnd3-mediated cardiac remodeling should be the focus for future study.
Abstract 287: Chamber Specific Function of p38 MAP Kinases in Right Ventricle Development, Hypertrophy, and Dysfunction
Authors: Yokota Tomohiro , Ren Vincent , Wang Yibin
Published: July 2021

Background:
Postnatal heart maturation is a well orchestrated process involving chamber specific changes in myocyte proliferation and growth. While the left ventricle undergoes significant growth, the right ventricle regresses relative to the left in terms of both myocyte number and size. While much attention has been focused on the left ventricular myocyte proliferation and hypertrophy, right ventricle specific changes are poorly studied. p38 MAP kinase is a well established signaling pathway for stress response and also plays an important role in cell differentiation, viability and proliferation. Its role in chamber specific postnatal heart maturation, however, has not been demonstrated

Results and Methods:
We observed RV specific induction of myocyte apoptosis and suppression of myocyte proliferation in neonatal hearts, coinciding with a transient induction of p38 activity significantly higher in the RV relative to the LV at P3 and P7 wild-type mouse hearts. In cardiomyocyte specific p38 MAP kinase alpha&beta double KO (p38ab cdKO) mice, echocardiographic and histological analyses revealed RV specific chamber enlargement and dysfunction associated with significant postnatal lethality. Apoptotic activity in the RV was suppressed at P1 and myocyte proliferation and hypertrophy were significantly elevated at P3 and P7 in the p38ab cdKO hearts compared to the wild-type. In contrast, no differences were observed in the LV between the p38ab cdKO and wild-type. Consequently, the surviving p38ab cdKO mice showed significant and RV specific enlargement and dysfunction, leading to eventual RV failure and pulmonary hypertension.

Conclusion:
p38 a&b MAP kinases are essential to RV specific maturation in postnatal hearts. p38ab cdKO mice represent a unique RV-specific hypertrophy and heart failure model for further studies.
Abstract 304: Crystal Structure Of Fak/mef2 Complex Reveals The Role Of Fak In The Regulation Of Mef2 Transcription Factor.
Authors: Cardoso Alisson C , Pereira Ana H , Ambrosio Andre L , Consonni Silvio R , Dias Sandra M , Franchini Kleber G
Published: July 2021

Members of MEF2 (Myocyte Enhancer Factor 2) family of transcription factors are major regulators of cardiac development and homeostasis. Their functions are regulated at several levels, including the association with a variety of protein partners. We have previously shown that FAK (Focal Adhesion Kinase) regulates the stretch-induced activation of MEF2 in cardiomyocytes. But, the molecular mechanisms, involved in this process, are unclear. Here, we integrated biochemical, imaging and structural analyses to characterize a novel interaction between MEF2 and FAK. An association between MEF2 and FAK was detected by co-immunoprecipitation in the extracts of stretched cardiomyocytes (10%, 60Hz, 2 hours). MEF2 and FAK staining were co-localized in the nuclei of stretched cells. Pull down assays indicated that the Focal Adhesion Targeting (FAT) domain is sufficient to confer FAK interaction with MEF2. Gene reporter assays indicated that the interaction with FAK enhances the MEF2C transcriptional activity in cultured cardiomyocytes. Also, we present a 2.9-Å X-ray crystal structure for the FAK_FAT domain bound to MEF2C (1-95), comprised by the MADS box/MEF2 domain. The structural information, when used in combination with biochemical studies, small-angle X-ray scattering (SAXS) data and reporter gene assay, lead to a mechanistic model describing how FAK binds to MEF2C and stimulates its transcription function in cardiomyocytes. We further validated this model by showing that the binding of FAK to MEF2C is essential for the hypertrophy of cardiomyocyte in response to mechanical stress. Our results present FAK as a new positive regulator of MEF2, implicated in the fine control of the signal transduction between focal adhesions and the nucleus of cardiac myocytes during mechanical stress.
Abstract 308: Targeted Inhibition of Cardiac Ankyrin Repeat Protein Disrupts Sarcomeric ERK-GATA4 Signaling at the Titin I-band and Abrogates Agonist-Induced Cardiac Hypertrophy
Authors: Lim Chee , Zhong Lin , Chiusa Manuel , Cadar Adrian , Samaras Susan , Davidson Jeffrey M
Published: July 2021

Hypertrophic cardiomyocyte growth occurs in response to various stress stimuli including biomechanical stress and neurohormonal factors. Accumulating evidence suggest that sarcomere signaling complexes play a pivotal role in the cardiomyocyte hypertrophic response by transmitting signals to the nucleus to induce gene expression. Cardiac ankyrin repeat protein (CARP, Ankrd1) is a transcriptional regulatory protein that also associates with the titin I-band spring domain, however the exact role of CARP in the heart remains to be elucidated. We report that CARP directly interacts with mitogen activated protein kinase ERK1/2 and cardiac transcription factor GATA4. Phenylephrine (PE) stimulation in cardiomyocytes induced ERK1/2 and GATA4 to transiently co-localize with sarcomeric CARP, followed by translocation of CARP and GATA4 to the nucleus. Four-and-a-half-LIM (FHL) domains proteins are part of a sarcomeric ERK2 sensory complex and knockdown of CARP by small interfering RNA (siRNA) resulted in disruption of FHL1 and FHL2. Moreover, loss of CARP attenuated PE-induced phosphorylation of ERK1/2 and GATA4, decreased GATA4 DNA binding, and prevented PE-induced cardiomyocyte growth. Mice lacking CARP have decreased FHL1 levels, and PE stimulation in wild-type mice resulted in elevated GATA4 phosphorylation and a hypertrophic response, which were completely abrogated in CARP-KO mice. We demonstrate that CARP plays an important role in PE-induced hypertrophic signaling by recruiting ERK2 and GATA4 into a titin I-band macro-molecular complex to induce GATA4 activation, followed by translocation of CARP and GATA4 to the nucleus to enhance GATA4 DNA binding and hypertrophic gene expression. Loss of CARP destabilizes FHL1 and FHL2, resulting in disruption of the PE-induced sarcomeric complex and abrogation of the cardiomyocyte hypertrophic response. These data reveal a novel role for sarcomeric titin I-band as a transcription factor activation hub that induces downstream nuclear signaling in response to agonist-induced hypertrophic stimuli.
Abstract 320: Long-term Atorvastatin, But Not Pravastatin, Treatment Leads To Repressed Mitochondrial Gene Expression And Altered Cardiac Ultrastructure
Authors: Zemljic-Harpf Alice , Godoy Joseph C , Niesman Ingrid R , Schilling Jan M , Schwarz Anna , Asfaw Elizabeth K , Alvarez Erika A , Dalton Nancy D , Patel Piyush M , Head Brian P , Drummond John C , Roth David M , Kararigas Georgios , Patel Hemal H
Published: July 2021

Statins reduce low-density lipoprotein cholesterol (LDL-C) and decrease cardiovascular events. Although statins are generally well tolerated, the FDA warns that statins may induce skeletal muscle side effects, cognitive changes and increased fasting glucose levels. Skeletal muscle biopsies from patients with statin myopathy have revealed defects in mitochondrial ultrastructure. Impaired mitochondrial function has been postulated as a key cause of statin-induced myopathy and hepatotoxicity. Long-term statin effects on cardiac muscle are currently unknown.

Wild type mice received atorvastatin, pravastatin or vehicle daily for seven months by oral gavage. Atorvastatin and pravastatin reduced LDL-C compared to vehicle. Echocardiography at two-week intervals showed no differences in %FS, %EF, circumferential fiber shortening and ventricular wall thicknesses between atorvastatin, pravastatin and vehicle treated mice. After seven months of atorvastatin, pravastatin or vehicle administration cardiac muscles (n=21-29) were analyzed, and only atorvastatin treated hearts revealed: A) swollen and misaligned mitochondria and accumulation of protein aggregates by transmission electron microscopy (n=4, each), and B) repression of mitochondrial and endoplasmatic reticulum related genes by genome-wide expression profiling. In cultured ventricular myocytes, atorvastatin, but not pravastatin; 1) down-regulated survival pathways via inhibition of ERK1/2T202/Y204, AktSer473 and mTOR signaling (p70 S6 kinaseThy421/Ser4240 and S6 RPSER 235/236), 2) reduced protein expression of caveolin-1, dystrophin, epithermal growth factor receptor and insulin receptor β, 3) decreased RhoA activation, and 4) induced apoptosis.

LDL-C reduction by atorvastatin, but not pravastatin, was associated with a repression of mitochondrial and endoplasmic reticulum related genes, an accumulation of protein aggregates, and swollen mitochondria. This is the first report demonstrating that long-term atorvastatin treatment causes adverse effects on cardiac muscle with preserved cardiac function. Whether these changes predispose atorvastatin treated hearts to contractile dysfunction after hemodynamic stress needs further investigation.
Abstract 333: Maturation Of ß-adrenergic Receptor (ß-AR) Signaling In Human Induced Pluripotent Stem Cell-derived Cardiomyocytes (hipsc-cms)
Authors: Jung Gwanghyun , Wu HaoDi , Fajardo Giovanni , Kodo Kazuki , Wu Joseph , Bernstein Daniel
Published: July 2021

hiPSC-CMs are a novel model system for cardiovascular diseases, with proven utility in cardiac channelopathies, however, how accurately hiPSC-CMs recapitulate the signaling pathways regulating contractility and remodeling of adult cardiomyocytes remains to be determined. As ß-ARs are key regulators of both contractility and remodeling, we examined ß-AR signaling at different stages of hiPSC-CM maturation. hiPSCs, derived from healthy controls, were studied at 0, 14, 30, and 60d after cardiac induction under isoproterenol (ISO) stimulation ± ß1 or ß2 antagonists. Flag-tagged ß-ARs were transfected into hiPSC-CMs for trafficking studies. Compared to the ß1:ß2 ratio of 70:30 in adult ventricular cardiomyocytes, ß2-AR expression was higher in early stage (d14-30) hiPSC-CMs; ß1-ARs increased with maturation (d14-d60), reflective of the normal developmental pattern. Downstream signaling also matured from d14-60, regulating both function (PLB and TnI) and remodeling (ERK). The majority of cAMP generation and PKA activation at d30 was through ß2-ARs; by d60-90, ß1-AR signaling contributed equally. In contrast to studies in rodent cells, ß2-ARs did not show compartmentalization of PKA signaling. Between d30-60, components of the SR (RYR, PLB and CSQ) and caveolae (Cav3) matured, suggesting development of functional compartmentalization machinery. Finally, hiPSC-CMs showed ß2-AR downregulation and internalization in response to ISO, an important component of the normal cardiomyocyte response to pathologic stimulation. Thus, hiPSC-CM ß-AR signaling matures with time after cardiac induction, recapitulating many of the features of post-natal and several of adult cardiomyocytes. hiPSC-CM models of human cardiomyopathies are a reasonable platform for studying ß-AR signaling. Furthermore, the maturation of ß-AR signaling can be used as a read-out for hiPSC-CM maturity.
Abstract 346: Involvement of Non-canonical Wnt-PKC-JNK Pathway in Angiogenesis Enhanced by MSC Overexpressing Wnt11
Authors: Xu Meifeng , Wang Jingcai , Yu Bin
Published: July 2021

Wnt11 is a key regulator of cardiac muscle cell proliferation and differentiation during heart development. Here, we hypothesized that preprogramming mesenchymal stem cells (MSC) with Wnt11 enhanced angiogenesis in ischemic myocardium.

Methods and Results:
An acute myocardial infarction model in SD rats was developed by ligation of the left anterior descending (LAD) coronary artery. Rat bone marrow derived MSC were transduced with Wnt11 gene (MSC-Wnt11) and transplanted into ischemic border area. The animals treated with MSC-Wnt11 showed a significantly improved cardiac function. Furthermore, fluorescent microsphere and histological studies revealed an increased blood flow and blood vessel density in MSC-Wnt11 transplanted animals. In vitro studies using conditioned medium (CdM) obtained from MSC-Wnt11 (CdM-Wnt11) showed an increased the length and number of capillary-like structure (CLS) formation and promoted migration of human umbilical vein endothelial cells (HUVECs), which was similar to that HUVECs were directly treated with recombinant Wnt11 proteins. However, these effects could be abolished by using a Wnt11 neutralizing antibody. Real-time PCR analysis indicated that the expression of novel PKCs including PKCδ, PKCε, PKCη, and PKCθ was significantly upregulated in HUVECs transduced with Wnt11 (H-Wnt11). Immunostaining and western blotting results showed that the protein levels of p-pan-PKC and p-JNK as well as VEGFA in H-Wnt11 were significantly higher than that in control HUVECs. However, no difference was detected in the expression of either p-p38 or p-ERK between H-Wnt11 and its control. Furthermore, the enhanced CLS formation and migration of HUVECs mediated by CdM-Wnt11 were partially abolished by using JNK inhibitor, SP600125 (5 μM) and PKC inhibitor, Calphostin-C (0.1 μM).

Conclusion:
Our results demonstrated, for the first time, that Wnt11 delivered by MSC improved cardiac function and promotes angiogenesis in ischemic myocardium, which may be associated with the activation of the non-canonical Wnt-PKC-JNK pathway.
Abstract 349: Gastrin and D1 Dopamine Receptor Interact to Induce Natriuresis and Diuresis
Authors: Chen Yue , Asico Laureano D. , Zheng Shuo , Villar Van Anthony M. , He Duofen , Zhou Lin , Zeng Chunyu , Jose Pedro A.
Published: July 2021

Oral NaCl produces a greater natriuresis and diuresis than the intravenous infusion of the same amount of NaCl. Gastrin is the major gastrointestinal hormone taken up by renal proximal tubule (RPT) cells. We hypothesized that renal gastrin and dopamine receptors interact to synergistically increase sodium excretion, an impaired interaction of which may be involved in the pathogenesis of hypertension. In Wistar-Kyoto (WKY) rats, infusion of gastrin induced natriuresis and diuresis, which was abrogated in the presence of a gastrin (CCK
B
R; CI-988) or D
1
-like receptor antagonist (SCH23390). Similarly, the natriuretic and diuretic effects of fenoldopam, a D
1
-like receptor agonist, were blocked by SCH23390, as well as by CI-988. However, the natriuretic effects of gastrin and fenoldopam were not observed in spontaneously hypertensive rats (SHRs). The gastrin/D
1
-like receptor interaction was also confirmed in RPT cells. In RPT cells from WKY but not SHRs, stimulation of either D
1
-like or gastrin receptor inhibited Na
+
-K
+
-ATPase activity, an effect that was blocked in the presence of SCH23390 or CI-988. In RPT cells from WKY and SHRs, CCK
B
R and D
1
receptor (D
1
R) co-immunoprecipitated, which was increased after stimulation of either D
1
R or CCK
B
R in RPT cells from WKY rats; stimulation of one receptor increased RPT cell membrane expression of the other receptor, effects that were not observed in SHRs. These data suggest that there is a synergism between CCK
B
R and D
1
-like receptors to increase sodium excretion. An aberrant interaction between the renal CCK
B
R and D
1
-like receptors (e.g., D
1
R) may play a role in the pathogenesis of hypertension.
Abstract 353: Characterization Of Human Plasma Proteome Dynamics Using Deuterium Oxide
Authors: Wang Ding , Liem David A , Lau Edward , Ng Dominic C , Bleakley Brian J , Cadeiras Martin , Deng Mario C , Lam Maggie P , Ping Peipei
Published: July 2021

Protein turnover half-life is a critical determinant of cardiac homeostasis and can reveal previously unknown disease mechanisms. We recently developed a theoretical method to quantify protein half-life in human using stable isotopes in deuterium oxide (D
2
O). Our goal in the present study is to demonstrate the immediate clinical translation potential of the method by evaluating its safety, feasibility, efficacy, and reproducibility in 10 healthy human subjects.

The enrolled human subjects (4 females/6 males; age 22 - 51 y/o; body weight 51 - 108 kg) were labeled with a tailored protocol (UCLA IRB#12-000899), wherein each subject orally consumed weight-adjusted doses of (~45 mL each) 70% D
2
O daily for 14 days to enrich body water and proteins with deuterium. Throughout labeling, the subjects maintained regular food and fluid intake, and normal daily activities. Vital signs and medical health questionnaires were taken daily till 14 days post-labeling. We followed long-term physical conditions and the physiological clearance of D
2
O from body water for up to 240 days post-labeling, finding no physiological effects or signs of discomfort.

To monitor label enrichment and post-labeling clearance in the subjects, we measured the D
2
O level of plasma and saliva samples with GC-MS. Both body fluids were reliable sources for monitoring label enrichment kinetics, giving consistent values of individual enrichment levels (0.9-2.2%) and rates (0.15-0.40 d
−1
) in all subjects. Post-labeling D
2
O level naturally subsided with a characteristic half-life of ~7 days (0.1 d
−1
). With LC-MS and the in-house informatics platform Proturn, we successfully characterized the turnover dynamics of >600 human plasma proteins, the largest such human dataset to-date. We detected diverse protein half-life in plasma, e.g., from albumin (18.3 d) to IGF2 (8 h). Importantly, the method can quantify protein half-life with only a single time point, suggesting it can be used to study the dynamics of one single cardiac biopsy (e.g., during heart transplant).

In summary, D
2
O labeling is a safe, accessible, and effective technique for widespread clinical investigations of protein turnover dynamics. We further discuss its implications in understanding cardiac disease mechanisms.
Abstract 36: Acute Inhibition Of O-GlcNAc Signaling Rescues Inotropic Responsiveness In Type 1 Diabetic Rat Hearts
Authors: Qin Chengxue , S Sleaby Rochelle S , Delbridge Lea M , Davidoff Amy J , Chatham John C , Ritchie Rebecca H
Published: July 2021

Metabolism of excess glucose is an important component of the aetiology of type 1 diabetes. The cardiac phenotype includes left ventricular (LV) remodelling and LV dysfunction. Increased hexosamine biosythesis (HBP) and downstream upregulation of protein O-GlcNAcylation has been linked to diabetic complications in many organs. Its impact on LV contractile responsiveness is however not well understood. This study aimed to test the hypothesis that acute inhibition of O-GlcNAc signaling protects inotropic responsiveness in type 1 diabetic heart. Hearts isolated from adult Sprague-Dawley male rats were Langendorff-perfused (constant flow, 10ml/min). Baseline and phenylephrine-stimulated (PE, 10μmol/L) LV function was determined in diabetic (8wks post-streptozotocin diabetes, 55mg/kg i.v.) versus non-diabetic sham rats in the presence of pharmacological inhibitors of HBP/O-GlcNAc including 6-diazo-5-oxo-L-norleucine (DON, 20μM) and alloxan (5mM). Diabetic rats exhibited a marked reduction in inotropic responsiveness to PE (Table, mean±SEM, one-way ANOVA, #P<0.05 vs non-diabetic vehicle rats, *P<0.05 vs diabetic vehicle, at 40 mins). Acute interruption of cardiac HBP/O-GlcNAc by DON and Alloxan significantly rescued LV responsiveness to PE in type 1 diabetic rat hearts. These results support further assessment of the impact of upregulated protein O-GlcNAcylation on LV function, particularly in the diabetic heart. Treatment strategies that target HBP may provide significant benefits alone or in combination with current standard treatments, to reduce progression of heart failure and death in type 1 diabetic patients.
Abstract 38: Important Role of Adiponectin in Volume Overload Induced Heart Failure
Authors: Wang Lili , Wanders Desiree , Nanayakkara Gayani , Amin Rajesh , Judd Robert L , Morrison Edward E , Zhong Juming
Published: July 2021

Adiponectin (ADP) has been reported to exert cardiac protective effects during ventricular remodeling following pressure overload and myocardial ischemia. However, the potential role of ADP in the volume overload induced heart failure has not been reported. In this study we examined the effect of ADP in cardiac myocyte contractile dysfunction following sustained volume overload. Rat model of volume overload induced heart failure was created by infrarenal aorta-vena cava (A-V) fistula. Some rats were administered with adenoviral ADP (Ad-ADP) at 2-, 6-, and 9-weeks following fistula surgery. Serum total ADP levels were measured at 3 days before, 5 weeks and 10 weeks after fistula surgery. Myocyte contractility and intracellular Ca2+ transients were evaluated at 10 weeks following fistula. Results indicated a progressive reduction of serum ADP levels. In ventricular myocytes isolated from 10-week fistula rats, protein expression of ADP, AdipoR1/R2 and T-cadherin were decreased, and AMPK phosphorylation was reduced. Consistent with these, myocytes exhibited significant depression in cell shortening and intracellular Ca2+ transient. In vivo overexpression of adenovirus-mediated ADP in fistula rats significantly increased ADP serum levels, and prevented the depression of myocyte contractile performance. Moreover, in vitro treatment with ADP significantly improved myocyte contractility and intracellular Ca2+ transient from 10-week fistula rats, but had no effect on myocyte performance in control and Ad-ADP animals. These results demonstrate a positive correlation of ADP reduction and ventricular remodeling induced by volume overload. Adiponectin plays a protective role in volume overload-induced heart failure.
Abstract 41: Dnajb6b Is A Novel Genetic Modifier For Cardiomyopathy That Regulates ER Stress Response
Authors: Ding Yonghe , Liu Weibin , Gore Beninio , Ekker Stephen C , Xu Xiaolei
Published: July 2021

Background:
Cardiomyopathy and heart failure affect millions of people worldwide. Because genetic modifiers contribute in large part to the highly variable phenotypic expression of cardiomyopathy in patients even with identical disease-causing mutations, the identification of modifier genes for this disease will greatly improve risk stratification, prognostic test development, and personalized therapy. However, only a rather limited number of modifier genes for cardiomyopathy have been identified sporadically.

Objective:
To identify genetic modifiers for cardiomyopathy using a novel insertional mutagenesis screening approach in adult zebrafish.

Methods and Results:
We screened 476 gene break-transposon (GBT) lines and isolated 44 zebrafish insertional cardiac (ZIC) mutants. Employing doxorubicin (DOX) stress to these ZIC mutants, we identified four candidate GBT lines that modified the progression of DOX-induced cardiomyopathy. Here, we report the detailed study of the GBT0411 mutant that exacerbated DOX-induced cardiomyopathy. GBT0411 mutant was tagged to the dnajb6b gene. Mutations in the short (sarcomeric) isoform of its human homologue gene DNAJB6 was recently reported to cause limb-girdle muscular dystrophy type 1D. Interestingly, our data showed that long (nuclei) isoform (dnajb6b[L]) was the major isoform expressed in the heart, and loss-of-function of which deteriorated the progression of DOX-induced cardiomyopathy. We further found that a cardiomyocyte-specific dnajb6b(L) transgene reverted the deleterious modifying effect of GBT0411 mutant, and exerted a cardioprotective function on chronic anemia induced cardiomyopathy. Mechanistically, Dnajb6b(L) could partially localize to endoplasmic reticulum (ER) upon ER stress, and function as an ER stress suppressor. Indeed, inhibition of ER stress by using a chemical chaperon mimics the cardioprotective effect of dnajb6b(L) transgene.

Conclusions:
By conducting an unbiased mutagenesis screening in adult zebrafish, we identified dnajb6b as a novel genetic modifier for cardiomyopathy. A cardioprotective function was identified by overexpressing its long isoform in cardiomyocytes, which might be conveyed by inhibition of ER stress response.
Abstract 49: Over-activation Of Natural Killer Cells Due To Dysfunction Of Cd16-mediated Signaling In Adamts13-deficient Ttp Patients With A History Of Relapse
Authors: ZHOU FANG , Farris Darise
Published: July 2021

Thrombotic thrombocytopenic purpura (TTP) is a rare life-threaten vascular autoimmune disease. There is no effective method to treat it in clinical trials since pathogenesis of TTP has not been fully elucidated. Here we investigate the role of NK cells in relapse development of TTP. Our results showed that the frequencies of CD3
−
CD56
dim
CD16
−
and CD3
−
CD56
bri
CD16
−
NK cells are increased in TTP patients with a history of relapse. Expression of CD107a, granzyme A and IFN-γ by CD3
−
CD56
dim
NK cells following
in vitro
stimulation with PMA/ionomycin / monensin is improved in the relapse group, compared with those on NK cells derived from TTP patients without relapse development. NK cells isolated from TTP patients with a history of relapse indicated stronger cytotoxicity to target K562 cells than those of NK cells derived from TTP patients without relapse development, suggesting prior activation of NK cells
in vivo
. Treatment with anti-human CD16 antibody up-regulates cytotoxicity of NK cells derived from TTP patients without relapse development. However, Anti-human CD16 antibody treatment does not affect cytotoxicity of NK cells isolated from TTP patients with a history of relapse, suggesting inability of CD16-mediated signaling in NK cells derived from TTP patients with relapse development. These data provide evidence of altered NK cell activation and/or licensing in TTP patients with a history of relapse modulated by CD16-mediated signaling and a new avenue of investigation into mechanisms of TTP immunopathogenesis.
Abstract 58: Identification Of miRNA-34 Networks In Pathological Cardiac Remodeling
Authors: Ooi Jenny Y , Bernardo Bianca C , Boey Esther J , Lin Ruby C , McMullen Julie R
Published: July 2021

Introduction:
MicroRNAs (miRNAs) are altered in heart disease and have emerged as promising therapeutic targets. We recently reported that therapeutic inhibition of the miR-34 family (with an 8 mer antimiR) was effective in attenuation of pathological cardiac remodeling. We hypothesized the efficacy of antimiR-34 may be due to its regulation of other miRNAs, producing direct and indirect target effects.

Objectives:
To identify miRNAs regulated by miR-34 and understand miRNA networks involved in miR-34 inhibition therapy for pathological cardiac hypertrophy.

Methods:
MiRNA sequencing (Illumina HiSeq 2000) was performed on hearts of mice subjected to transverse aortic constriction (TAC) for 5 weeks, and subcutaneously administered a locked nucleic acid (LNA)-antimiR-34 for 6 weeks (n=3-4).

Results:
Expression of miR-34 family members (miR-34a, miR-34b, miR-34c) is increased in the hearts of TAC mice compared to Sham controls (1.7, 2.5, 4-fold, P<0.05) and attenuated with antimiR-34 treatment (85% decrease [0.85 of 1], p<0.05). TAC mice developed pathological cardiac hypertrophy (60% increase [4.8 of 8] in heart weight/tibial length ratio compared to Sham controls, P<0.05) and treatment with antimiR-34 significantly attenuated heart size (P<0.05). To uncover the underlying miRNA interactions, we carried out miRNA-Seq to identify other miRNAs that might be dysregulated in response to antimiR-34-based therapy. We identified several miRNAs (e.g., miR-3083 and miR-20b) that were downregulated in hearts from TAC vs Sham but not significantly downregulated in the TAC antimiR-34 group. Conversely, two miRNAs (miR-8103, miR-1933) were increased in response to TAC vs Sham but not significantly upregulated in TAC antimiR-34 treated mice. Our data suggests that miR-34 can regulate other miRNA and that these miRNAs can work together to regulate pathways leading to pathological cardiac hypertrophy.

Conclusion:
The identification of other miRNAs regulated by miR-34 may improve our understanding of miRNA networks for optimization of better therapeutic targets for treatment of complex diseases such as heart failure.
Abstract 73: A Novel Role of E2F1 in Stress-induced Cardiac Fibrosis
Authors: Biyashev Dauren , Boriboun Chan , Qin Gangjian
Published: July 2021

Rationale:
E2F1 transcription factor is best known as a cell cycle regulator. Recent reports indicate the importance of E2F1 in cardiovascular system, though its exact role is not clear. TGF-beta/Smad2,3 signaling pathway, on the other hand, has been long implicated in the regulation of cardiovascular health and numerous disease, including cardiac fibrosis. Interaction between these two major pathways has been reported in the cancer settings.

Objective:
To identify the possible interactions between E2F1 and TGF-beta/Smad2,3 signaling pathways in cardiovascular system and determine the functional outcome of these interactions in cardiac health.

Methods and Results:
E2F1-/- mice developed significantly higher degree of cardiac fibrosis than wild type mice in the Angiotensin II - induced cardiac fibrosis model. The levels of phosphorylated Smad2 and Smad3 were significantly higher in the hearts of E2F1-/- mice, as well as in mouse embryonic fibroblasts derived from E2F1-/- animals. Associated expression of collagen I was significantly increased in mouse embryonic fibroblasts derived from E2F1-/- animals, and treatment with TGF-beta resulted in higher collagen deposition compared to wild type fibroblasts. Treating animals with SB 431542, chemical inhibitor of Smad2,3 signaling, obliterated the difference in the degree of cardiac fibrosis between wild type and E2F1 knockout animals in the Ang II model. We discovered that levels of syndecan-4, heparan sulfate proteoglycan transmembrane protein implicated in fibrosis and known to interact with TGF-beta are significantly increased in both E2F1-/- fibroblasts and hearts. siRNA-mediated knockdown of syndecan-4 using siRNA resulted in decreased Smad2,3 phosphorylation in E2F1-/- MEFs. Similarly, down regulation of syndecan-4 in-vivo using morpholino lead to decreased cardiac fibrosis in E2F1-/- mice in Ang II model.

Conclusions:
E2F1 suppresses activation of TGF-beta/Smad 2,3 pathway. The E2F1-dependent suppression of cardiac fibrosis through TGF-beta/Smad 2,3 pathway is at least partially regulated by syndecan-4.