bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 87: Sestrin2 Promotes LKB1-Mediated AMPK Activation in the Ischemic Heart
Authors: Li Ji , Morrison Alex , Ma Yina , Wang Jinli , Liu Zhenling , Budanov Andrei , Lee Jun-Hee , Karin Michael
Published: July 2021

AMP-activated protein kinase (AMPK) has emerged as a pertinent stress-activated protein kinase shown to have substantial cardioprotective capabilities against myocardial ischemia/reperfusion injury. When activated during ischemia, AMPK produces effects that have been demonstrated to be beneficial to the myocardium by means of increasing GLUT4 translocation and glucose uptake, activating eNOS, decreasing apoptosis, and increasing autophagic flux. However, the molecular basis behind the regulation of AMPK activity in the ischemic and reperfused heart remains incompletely understood. Recent evidence implicates the role of Sestrin2 in the AMPK signaling pathway and it is hypothesized that Sestrin2 plays an influential role during myocardial ischemia in order to promote AMPK activation. Sestrin2 protein was found to be expressed in adult cardiomyocytes and accumulated in the heart during ischemic conditions. Sesn2 KO mice were used to determine the importance of Sestrin2 during I/R injury. Sesn2 KO cardiac phenotype analysis indicated no significant difference in left ventricular function as measured by a pressure-volume loop catheter. When wild type (WT) and Sesn2 KO mice were subjected to in vivo I/R, myocardial infarct size was significantly greater in Sesn2 KO compared to WT hearts. Similarly, Langendorff perfused hearts indicated exacerbated post-ischemic contractile function in Sesn2 KO hearts when compared to WT. Ischemic AMPK activation was found to be impaired in the Sesn2 KO hearts. Immunoprecipitation of Sestrin2 demonstrated an association with AMPK. Moreover, LKB1, a major AMPK upstream kinase was associated with the Sestrin2-AMPK complex in a time-dependent manner during ischemia while this interaction was nearly abolished in Sesn2 KO hearts. Thus, Sestrin2 plays an important role in cardioprotection against I/R injury by acting as an LKB1-AMPK scaffold to initiate AMPK activation during ischemic insults.
Abstract 96: Epigenetic And Transcriptional Regulation Of Induced Pluripotent Stem Cells In Patients With Hypoplastic Left Heart Syndrome
Authors: Kobayashi Junko , Yoshida Masashi , Tarui Suguru , Ishigami Shuta , Okuyama Michihiro , Kasahara Shingo , Ito Hiroshi , Sano Shunji , Oh Hidemasa
Published: July 2021

Background:
Although a number of studies have uncovered heterozygous mutations in cardiac regulatory genes caused hypoplastic left heart syndrome (HLHS), the identified genetic variants may not be directly correlated with the disease development. The aim of this study is to determine the epigenetic and transcriptional network responsible for myocardial patterning and morphogenesis during cardiac development in HLHS by using patient-derived induced pluripotent stem (iPS) cells.

Methods:
Five-independent iPS cell lines were generated from HLHS and biventricle (BV) heart-derived cardiac progenitor cells (CPCs). Global gene expression analysis, real-time RT-PCR, mutation analysis, ChIP assay, and cardiac-specific gene promoter activities were examined during differentiation.

Results:
We found one synonymous single nucleotide polymorphism in NKX2-5 and five in NOTCH1, respectively. Cardiac transcriptional factors such as NKX2-5, HAND1, and NOTCH/HEY, those are known to drive primary heart field and outflow tract development, were significantly reduced in HLHS-derived iPS cells after differentiation compared with BV- and control 201B7 iPS-derived cardiomyocytes. ChIP assay showed that a marked decrease in dimethylated histone H3-lysine 4 and acetylated histone H3 was found within the NKX2-5 promoter regions, whereas a significantly increased trimethylated H3-lysine 27 was identified in differentiated HLHS-derived iPS cells. To specify the target transcripts responsible for cardiac development of HLHS, cardiac troponin-T and natriuretic peptide A promoter analyses were performed. We found that both promoter activities were significantly suppressed in HLHS-derived CPCs and iPS cells compared with those from BV hearts. These repressed promoter activities could be fully restored by transient transfection of NKX2-5, HAND1, and NOTCH1 genes into these stem/progenitor cells by synergistic manner.

Conclusions:
These findings suggest that patient-specific iPS cells provide a potential to dissecting the complex cardiac malformations in human. The epigenetic and transcriptional regulation of NKX2-5, HAND1, and NOTCH1 may mutually participate in the development of myocardial growth, patterning, and morphogenesis in HLHS.
Abstract 97: High mobility group box-1 Promotes Restenosis Via Toll like receptor-4 Signal Pathway
Authors: Cai Jingjing , Yuan Hong , Wang Qingde , Billiar Timothy R. , Chen Alex F.
Published: July 2021

Background:
High-mobility group box 1 (HMGB1) is an endogenous molecule released during cell stress and death termed damage-associated molecular patterns (DAMPs). HMGB1 activates the pattern recognition receptor, toll-like receptor 4 (TLR4), and induces sterile inflammation. However, how HMGB1 and TLR4 affect restenosis, the major complication following balloon and stent intervention clinically, remains unknown. We tested the hypothesis that HMGB1 released following acute arterial injury promotes intimal hyperplasia (IH), a hallmark of restenosis, via TLR4 signaling pathway.

Methods and Results:
Wire injury of the carotid artery in C57BL/6 wild-type (WT) mice significantly increased intima-to-media ratio in 4 weeks. Global deletion of HMGB1 using an inducible knockout mouse strain prevented IH and vessel remodeling. IH decreased by over 50% in WT mice treated with a HMGB1 neutralizing antibody. Of the mouse strains deficient in putative receptors and co-regulator for HMGB1 (TLR4-/-, TLR2-/-, RAGE-/- and CD14-/-), TLR4-/- mice showed the greatest inhibition of IH after injury. Both TLR4 adaptors MYD88 and TRIF synergistically participated in the inflammatory response to vascular injury. HMGB1 antibody-treated mice and TLR4-/- mice showed a marked decrease in monocytic recruitment following injury. Mice with selective depletion of TLR4 from macrophages (TLR4-/--Mø) exhibited similar level of IH inhibition and macrophage infiltration, compared to the global TLR4-/- mice. In vitro, disulfide HMGB1 concentration-dependently promoted smooth muscle cell (SMC) migration and MCP-1/CCR2 expression, which were abolished by treatment with TLR4 inhibitory peptide. Moreover, conditioned media from HMGB1-treated macrophage induced SMC proliferation, which was blunted by blocking TLR4 on macrophage, but not SMCs. Finally, HMGB1 increased cytokine (TNF-α and IL-6), chemokine (MCP-1) and mitogen (PDGF-A) levels in macrophage in a TLR4-dependent manner.

Conclusion:
These findings demonstrate, for the first time, that HMGB1 released following acute arterial injury promotes restenosis via SMC migration and MCP-1/CCR2 production as well as macrophage-released TNF-α, IL-6, MCP-1 and PDGF-A in SMC through the TLR4-MyD88/TRIF signal cascade.
Abstract 100: A Novel Anti-cell Death Mechanism by Targeting the Biosynthesis of Oligosaccharide in ER
Authors: Luo Xiangwei , Zhou Wenjuan , Wei Lingling , Wei Liang , Zhang Fengxue
Published: July 2021

The pathological expression of many of the deadly human diseases such as myocardial infarction, renal failure etc. is cell death. In cancer research, myriads of ways have been found to kill a cell. However, we are still craving for an applicable mechanism to prevent cell death. The majority of the current research is to directly target the apoptosis pathway. While some compounds have been developed, none of them are pharmaceutically important likely due to the inherent toxic nature of the target. Here, we report a novel mechanism to block the cell death pathway by activating the cells’ own protective programs coded for stress. Components of the unfolded protein response (UPS) in ER have been implicated in the protection of ischemia/reperfusion heart in recent research. By targeting the biosynthesis of oligosaccharide, which activated UPS as indicated by the expression of the ER chaperones Grp94 and Grp78, we were able to completely block the Hsp90 inhibitor induced apoptotic cell death as well as the oxidative stress (as exemplified by sodium nitroprusside, SNP) induced necrotic cell death in multiple cell lines. Caspase-3 activation and PARP cleavage were nearly completely inhibited. While awaiting further exploration, the biosynthesis of the oligosaccharide could turn out to be an applicable target for acute conditions such as cardiac injury because of its relatively safe nature.
Abstract 104: Vasonatrin Peptide Inhibits Endoplasmic Reticulum Stress and Attenuates Myocardial Ischemia/reperfusion Injury in Diabetic Rats
Authors: Zhang Haifeng , Xing Wenjuan , Gao Feng
Published: July 2021

Aims:
Diabetes mellitus (DM) increases morbidity/mortality of ischemic heart disease. Although the ability of the natriuretic peptides to modulate cardiac function and cell proliferation has been recognized, their effects on myocardial ischemia/reperfusion (MI/R) injury is still unclear. This study was to investigate the effects of the artificial synthetic natriuretic peptide — vasonatrin peptide (VNP) on MI/R injury in diabetic rats, and underlying mechanisms.

Method:
The high-fat diet-fed streptozotocin induced diabetic rats were subjected to MI/R (30 min/4 h) and VNP treatment (100 μg/kg, i.v., 10 min before R).
In vitro
study was performed using H9c2 cardiomyocytes subjected to hypoxia/reoxygenation (H/R, 3 h/6 h) and incubated with or without VNP (10
-8
mol/L).

Result:
The diabetic state aggravated MI/R injury and showed more severe myocardial functional impairment than normal state. VNP treatment (100 μg/kg, i.v., 10 min before R) significantly improved ± LV d
P
/dt
max
and LVSP, and decreased infarct size, apoptosis index, caspase-3 activity, serum CK and LDH levels (n=8,
P
<0.05). Moreover, VNP inhibited endoplasmic reticulum (ER) stress by suppressing GRP78 and CHOP, and consequently increased Akt and ERK1/2 expression and phosphorylation levels (n=3,
P
<0.05). These effects were mimicked by 8-Br-cGMP (1 mg/kg, i.p., 20 min before R), a cGMP analogue, whereas inhibited by KT-5823 (0.5 mg/kg, i.p.), the selective inhibitor of PKG (
P
<0.05). Pretreated DM rats with TUDCA (50 mg/kg, i.p.), an inhibitor of ER stress, couldn’t further promote the VNP’s cardioprotective effect. Additionally, gene knockdown of PKG1α with siRNA blunted VNP’s inhibition of ER stress and apoptosis, while overexpression of PKG1α resulted in significant decreased ER stress and apoptosis in H/R H9c2 cardiomyocytes (n=6,
P
<0.05).

Conclusion:
We demonstrated that VNP protects diabetic heart against MI/R injury by inhibiting ER stress via cGMP-PKG signaling pathway.
Abstract 108: C1q-TNF Related Protein-9, a Novel Cardioprotective Cardiokine, Requires Proteolytic Cleavage to Generate a Biologically Active Globular Domain Isoform
Authors: Du Yuanhui , Yuan Yuexing , Lau Wayne , Christopher Theodore , Lopez Bernard , Wang Yajing , Ma Xinliang
Published: July 2021

The discovery of the cardiac secretomes, known as “cardiokines”, significantly enhanced appreciation of the local microenvironment’s influence upon disease development. We previously reported cardiomyocytes produce cardioprotective adiponectin (APN). However, cardiac APN levels are several orders of magnitude below adipocytes, thereby making APN an unlikely physiologically relevant cardiokine. C1qTNF-related proteins (CTRP1-15) is a newly descovered family of APN paralogs. However, whether any of the CTRP members may function as a “true” cardiokine remains unknown. Here we demonstrated that several CTRPs (CTRP1,4,7,9,13) were expressed in the heart at levels significantly greater than APN. Most notably, the mRNA level of CTRP9 exceeded APN by >100-fold in cardiac tissue. Adult cardiac cells were isolated and cultured in vitro. Conditioned medium was collected 1-8 hours after culture and CTRP9 protein levels were determined by ELISA. A significant increase of CTRP9 in conditioned medium was detected as early as 1 hour in culture, steadily accumulating thereafter. These results indicate CTRP9 is secreted by cardiac cell at levels that may function as a cardiokine. Addional experiemnt unexpectedly demonstarted that CTRP9 circulates in the plasma primarily in the globular domain isoform (gCTRP9), opposing from APN which circulates as full length multimers. Recombinant full length CTRP9 (fCTRP9) was cleaved when incubated with cardiac tissue extracts, generating gCTRP9, a process inhibited by protease inhibitor cocktail. To gain more insight into the biological significance of gCTRP9 production, isolated adult cardiomyocytes were treated with gCTRP9 or fCTRP9. gCTRP9 rapidly activates cardiac survival kinases, including AMPK, Akt, and eNOS. However, fCTRP9-mediated kinase activation is much less potent, and significantly delayed. Kinase activation by fCTRP9, but not gCTRP9, is inhibited by protease inhibitor cocktail. These results demonstrate for the first time CTRP9 is a novel cardiokine undergoing proteolytic cleavage to generate cardioprotective isoform, gCTRP9. Enhancing cardiac CTRP9 production and/or its proteolytic post-translational modification are of therapeutic potential, attenuating cardiac injury.
Abstract 11: Acquisition of Mitochondrial DNA Mutations Impairs Mitochondrial Function in Cardiac Progenitor Cells
Authors: Orogo Amabel M , Gonzalez Eileen R , Kubli Dieter A , Murphy Anne N , Gustafsson Åsa B
Published: July 2021

Activation of cardiac progenitor cells (CPCs) are critical for effective repair in response to pathologic injury. Stem cell activation and commitment involve increased energy demand and mitochondrial biogenesis. We have previously shown that incubation of c-kit+ CPCs in differentiation medium led to expansion of the mitochondrial network and lineage commitment. CPC function is reduced with age but the underlying mechanism is still unclear. Mitochondria contain their own DNA (mtDNA) which accumulates mutations over time that can impair mitochondrial function. In this study, we investigated the effects of acquiring mtDNA mutations on CPC proliferation, survival, and differentiation. We utilized a mouse model in which a mutation in the mtDNA polymerase gamma (POLGm/m) leads to accumulation of mtDNA mutations, mitochondrial dysfunction, and accelerated aging. Isolated CPCs from hearts of 2-month old POLGm/m mice had reduced proliferation and were more susceptible to oxidative stress and chemotherapeutic agents compared to WT CPCs. Incubation in differentiation medium resulted in fewer lineage committed POLGm/m CPCs compared to WT. In addition, the POLGm/m CPCs failed to activate mitochondrial biogenesis and did not increase levels of proteins involved in mitochondrial oxidative phosphorylation. We measured mitochondrial respiration with the Seahorse XF Analyzer and found that POLGm/m CPCs had undetectable oxygen consumption but still generated similar amounts of ATP as WT CPCs. Interestingly, POLGm/m CPCs produced increased amounts of l-lactate and were more sensitive to 2-deoxyglucose treatment, suggesting that these cells rely on glycolysis for energy production. Both WT and POLGm/m CPCs downregulated expression of glycolytic enzymes during differentiation. However, POLGm/m CPCs failed to undergo the metabolic transition from glycolysis to OXPHOS, which led to activation of cell death during differentiation. These data demonstrate that mitochondria play a critical role in CPC function, and accumulation of mtDNA mutations impairs CPC function and reduces their repair potential.
Abstract 112: An Exercise-induced MicroRNA Pathway That Protects Against Apoptosis and Pathological Cardiac Dysfunction
Authors: Liu Xiaojun , Xiao Junjie , Wei Xin , Platt Colin , Xiao Chunyang , Damilano Federico , Bezzerides Vassilios , Rosenzweig Anthony
Published: July 2021

Background:
Exercise induces physiological cardiac growth and protects against adverse cardiac remodeling. microRNAs (miRNA) are important regulators in cardiovascular pathology and disease. Less is known about miRNA roles in the cardiac effects of exercise, and we investigated their roles in exercise-induced cardiac growth.

Methods and Results:
Based on cardiac miRNA profiling in two exercise models (swimming, running) and functional screening in neonatal rat cardiomyocytes (NRCMs), miR-222 was selected for further study. miR-222 was upregulated ~2-fold in both exercise models, pathological hypertrophic and failing hearts (p<0.05). We found that ERK, JNK and p38 signal pathways are involved in FBS increasing miR-222 in NRCMs. miR-222 increased NRCM proliferation and size (p<0.01) and reduced NRCM apoptosis induced by serum starvation with or without doxorubicin. Four potential miR-222 targets (
p27
,
Hipk1
,
Hipk2
, and
Hmbox-1
) were identified and confirmed as direct targets. siRNA knockdown (KD) of p27 or Hipk2 inhibited NRCM apoptosis.

To investigate miR-222’s cardiac role
in vivo
, we made transgenic mice with cardiac-specific, inducible (TgI-miR-222) or constitutive (TgC-miR-222) expression of miR-222. RT-PCR showed that miR-222 is increased ~6-fold in TgI-miR-222 hearts after induction and ~10-fold in TgC-miR-222 hearts. Surprisingly, both transgenic lines have normal heart size and function, and the 4 miR-222 targets are unaffected at baseline. After ischemia-reperfusion injury (IRI), TgI-miR-222 had no difference in initial infarct size or dysfunction but were protected against adverse remodeling over the next six weeks with better function (p<0.01), less cardiac fibrosis (68%, p<0.05), and reduced CM apoptosis (300%, p<0.05). Two weeks after TAC, TgC-miR-222 had less increase in cardiac mass (HW/TL, p<0.05), better cardiac function ([[Unable to Display Character: ∆]]32%, p<0.01), reduced expression of markers of fibrosis (
Col1a1
,
Col3a1
,
Mmp2,
p<0.05
)
and apoptosis (
Caspase 1, 3, 8,
p<0.05). Four months after TAC, TgC-miR-222 had improved survival (p<0.05) compared to wide type controls.

Conclusion:
Cardiac miR-222 is upregulated by exercise and protects against adverse remodeling and cardiac dysfunction after IRI or TAC.
Abstract 115: A Decrease in Mitochondrial, but Not Cytosolic, Iron Protects Against Cardiac Ischemia-reperfusion Damage Through a Reduction in Ros
Authors: Chang Hsiang-Chun , Wu Rongxue , Ardehali Hossein
Published: July 2021

Introduction:
Iron is essential for the activity of several cellular proteins, but excess free iron can cause cellular damage through production of reactive oxygen species (ROS). Iron accumulation in mitochondria, the major site of cellular iron homeostasis, leads to cardiomyopathy. However, it is not known whether a reduction in baseline mitochondrial iron (as opposed to iron in other cellular compartments) can protect against ischemia-reperfusion (I/R) injury in the heart. We hypothesized that since mitochondria are the major site of iron homeostasis and that mitochondrial iron can lead to oxidative damage, a reduction in mitochondrial iron at baseline would be sufficient to protect against I/R injury.

Results:
Transgenic (TG) mice with cardiomyocyte-specific overexpression of the mitochondrial iron export protein ATP-binding cassette (ABC)-B8 had significantly lower mitochondrial iron in the heart than nontransgenic (NTG) littermates at baseline, but their cardiac function and the expression of key antioxidant systems were similar to NTG littermates. In response to I/R, TG mice displayed significantly less apoptosis and lipid peroxidation products and better preserved cardiac function than NTG littermates, suggesting that a reduction in mitochondrial iron protects against I/R injury. To confirm these results, we next took a pharmacological approach to assess the effects of a reduction in mitochondrial vs cytosolic iron on the response to I/R using 2,2’-bipyridyl (BPD, a mitochondria-accessible iron chelator) and deferoxamine (DFO, an iron chelator that can only reduce cytosolic iron). Mice pretreated with BPD but not DFO are protected against I/R injury. In addition, BPD but not DFO treatment in rat cardiomyoblast H9C2 cells significantly lowered chelatable mitochondrial iron and protected against H2O2 induced cell death. These results suggest that a reduction in baseline mitochondrial, but not cytosolic, iron is sufficient to protect against I/R injury.

Conclusions:
Our findings demonstrate that selective reduction in mitochondrial iron is protective in I/R injury. Thus, targeting mitochondrial iron with selective iron chelators may provide a novel approach for treatment of ischemic heart disease.
Abstract 119: The Scaffolding Protein Enh1 is Essential for the Protein Kinase C or Protein Kinase D-dependent Activation of the Transcription Factor Camp-response Element Binding Protein in Cardiomyocytes
Authors: Ito Jumpei , Maturana Andrés D.
Published: July 2021

PDZ-LIM proteins form a family of the scaffolding protein essential for both embryonic and post-natal development. ENH1 (PDLIM5) is a PDZ-LIM protein, composed of an N-terminal PDZ domain and 3 LIM domains at the C-terminal end. The enh gene encodes for several splice variants that have opposite functions. ENH1 promotes the cardiomyocytes hypertrophy whereas ENH splice variants lacking LIM domains prevents it. At the molecular level, ENH1 interacts with Protein kinase C (PKC) and Protein Kinase D1 (PKD1) both kinases playing a pivotal role in the pathological remodeling of the heart. In addition, the binding of ENH1’s LIM domains to PKC is sufficient to activate the kinase without any stimulation. However, the downstream events of the ENH1-PKC/PKD1 complex remain unknown. PKC and PKD1 are known to phosphorylate the transcription factor cAMP-response element binding protein (CREB) in cardiomyocytes. We therefore hypothesized that ENH1 could be a play a role in the PKC/PKD1-dependent activation of CREB. We first found that ENH1 expression is necessary to induce the phosphorylation of CREB at Ser133 in neonatal rat ventricular cardiomyocytes. On the contrary, the overexpression of ENH3, a LIM-less cardiac-specific splice variant, inhibited the phosphorylation of CREB-Ser133. Concomitantly, both real-time qPCR and promoter assay showed that the overexpression of ENH1 enhanced but ENH3 prevented the transcriptional activation of a CREB-target gene, the immediately early gene c-fos. Finally, we found that ENH1 regulated the translocation of phosphorylated CREB to the nucleus. Taken all together, our results suggest that ENH1 plays an essential role in CREB’s activation and dependent transcription in cardiomyocytes. At the opposite, ENH3 splice variant inhibited the CREB activity in cardiomyocytes. In conclusion, our work describes a new molecular mechanism involving ENH splice variants with opposing functions.
Abstract 121: Hyperglycemia-induced Prorenin Receptor Abundance on Plasma Membrane of Kidney Collecting Duct Cells: is a Novel Mechanism to Explain Diabetic Nephropathy?
Authors: Gogulamudi Venkateswara R , Arita Danielle Y , Bourgeois Camille RT , Satou Ryosuke , Prieto Minolfa C
Published: July 2021

Activation of the renin-angiotensin system (RAS) leads to nephropathy during Diabetes Mellitus (DM). Prorenin levels are increased in the circulation of DM patients and predict microvascular damage. In streptozotocin (STZ)-induced type 1 DM rats, the collecting duct is the major source of prorenin in the kidney. The prorenin receptor (PRR), a new RAS component, elicits intracellular signals linked to fibrosis upon binding of prorenin at the cell plasma membrane (PM). We propose that PRR may contribute to the development of tubulointerstitial fibrosis by being locally activated by prorenin. To support this concept, we tested the hypothesis that hyperglycemia increases membrane bound PRR to collecting duct cells. To address this hypothesis, we used T1DM rats (N=10) induced with a single STZ injection (ip; 200ng/kg x 7 d) as well as mouse collecting duct M-1 cells, treated at different time intervals 0, 5 min, 1, 12, and 24 h with normal glucose (NG; 5mM glucose+20mM mannitol) and high glucose (HG; 25mM) to assess PRR trafficking alterations induced by glucose. After 7-days induction, STZ-rats showed plasma glucose as 428±13 vs.138±9 gr/dl and plasma insulin as 0.07±0.02 vs 2.42 ng/ml; compared to controls. By immunofluorescence (IF) in rat kidney sections, PRR was mainly localized on the apical aspect of collecting duct cells in STZ-rats, while in controls most of PRR signal was observed intracellularly. Although PRR mRNA levels did not differ in the collecting ducts between groups; its protein levels were augmented in STZ-rats. Importantly, compared with NG-treated cells, PRR protein levels (membrane bound) were significantly higher in PM extracts from M-1 cells treated with HG, and a maximum peak was observed at 1 h. Interestingly, in de-convoluted IF images of M-1 cells, PRR was localized mainly in the perinuclear areas during NG conditions; however, in HG-treated cells, PRR was found toward the cell surface. These data suggest that hyperglycemia induces PRR trafficking alterations of PRR. The present results suggest that hyperglycemia-induced PRR abundance on the PM in the collecting duct might be a novel mechanism underlying the development of diabetic nephropathy, particularly tissue fibrosis in DM.

Grant support by the NIH-NIDDK (DK104375-01)
Abstract 125: The E2F1 Transcription Factor Suppresses Cardiac Fibrosis via Downregulating Syndecan-4 Expression and Smad2/3 Phosphorylation
Authors: Biyashev Dauren , Boriboun Chan , Ghosh Asish K , Han Shuling , Kishore Raj , Losordo Douglas W , Qin Gangjian
Published: July 2021

Rationale:
The E2F1 transcription factor is best known as a cell-cycle regulator; recent reports suggest its important role in cardiovascular system.

Objective:
To determine whether E2F1 regulates cardiac fibrosis.

Methods and Results:
Following Angiotensin II (Ang II) administration, E2F1-null (E2F1-KO) mice displayed a more severe cardiac fibrosis and higher levels of cardiac phospho-Smad2 (pSmad2) and pSmad3 than WT mice. Consistently, levels of pSmad2 and pSmad3 were elevated in E2F1-KO fibroblasts, which was associated with a significantly increased expression of collagen I (Col I) and α-smooth muscle actin (α-SMA) and interestingly, cell surface proteoglycan syndecan-4 (Sdc4). Knockdown of Sdc4 significantly attenuated the elevation in Smad2/3 phosphorylation and Col I and α-SMA expression in E2F1-KO fibroblasts. Remarkably, either chemical inhibition of Smad2/3 signaling or morpholino-mediated knockdown of Sdc4 expression abrogated the difference in the degree of Ang II-induced cardiac fibrosis between WT and E2F1-KO animals.

Conclusions:
E2F1 suppresses Ang II-induced cardiac fibrosis by downregulating Sdc4 expression and Sdc4-mediated Smad 2/3 activation.
Abstract 126: High Efficiency Reprogramming of Fibroblasts Into Cardiomyocytes Requires Suppression of Pro-fibrotic Signaling
Authors: Song Kunhua , Zhao Yuanbiao , Londono Pilar , Sharpe Emily , Clair Joshua R , Proenza Catherine , O’Rourke Rebecca , Jones Kenneth L , Jeong Mark Y , Walker Lori A , Buttrick Peter M , McKinsey Timothy A , Cao Yingqiong
Published: July 2021

The mammalian heart is composed of ~30% cardiomyocytes which have limited capacity to regenerate and ~70% non-cardiomyocytes including endothelial cells and cardiac fibroblasts. Direct reprogramming of fibroblasts into cardiomyocytes by forced expression of cardiomyogenic transcription factors, GMT (GATA4, Mef2C, Tbx5) or GHMT (GATA4, Hand2, Mef2C, Tbx5), has recently been demonstrated, suggesting a novel therapeutic strategy for cardiac repair. Despite extensive efforts, the efficiency of direct reprogramming of embryonic or adult fibroblasts into cardiomyocytes has yet to exceed 20%, or 0.1% respectively, leading many in the field to question the clinical translatability of this method.

Here, we demonstrate that pro-fibrotic signaling events governed by transforming growth factor-β (TGF-β) and Rho kinase (ROCK) are concomitantly activated in GHMT-expressing fibroblasts, leading to potent suppression of cardiac reprogramming (
Figure 1
). Remarkably, pharmacological inhibition of TGF-β, or ROCK leads to conversion of ≥ 60% of fibroblasts into highly functional cardiomyocytes, displaying global cardiac gene expression, spontaneous contractility, action potentials and calcium transients. Furthermore, inhibition of TGF-β, or ROCK dramatically enhances the kinetics of cardiac reprogramming, with spontaneously contracting cardiomyocytes emerging in less than two weeks, as opposed to 4 weeks with GHMT alone. These findings provide new insights into the molecular mechanisms underlying cardiac conversion of fibroblasts, and should enhance efforts to generate cardiomyocytes for clinical applications.
Abstract 127: Structure and Function Relationship of Phospholipid Transfer Protein in Lipid Transfer Activity Revealed by Electron Microscopy
Authors: Zhang Meng
Published: July 2021

Human phospholipid transfer protein (PLTP) mediates the transfer of lipids among atheroprotective high-density lipoproteins (HDL) and atherogenic low-density lipoproteins (LDL) by an unknown mechanism. Delineating this mechanism would be an important step toward the understanding and regulation of PLTP for treating cardiovascular diseases, hypoalphalipoproteinemia and hyperalphalipoproteinemia. Using electron microscopy, negative-staining, and single-particle image processing, we discovered that PLTP penetrates each class of HDL, LDL and liposome independently, and also bridges a ternary complex with one of its distal end-domains penetrating into HDL and another distal domain interacting with LDL. These new insights into PLTP interaction with lipoproteins and liposomes provide a molecular basis for analyzing PLTP-dependent lipid transfer between lipoprotein particles.
Abstract 130: Epicardial-to-Myocyte Hypertrophic Signaling is Mediated by Epicardial/Mesenchymal Status and Protein Arginine Methyltransferase-1
Authors: Jackson-Weaver Olan , Sucov Henry , Xu Jian
Published: July 2021

The epithelial-to-mesenchymal transition (EMT) is an important cellular mechanism in a diverse range of biological processes such as development, wound healing, cancer metastasis, and organ fibrosis. Epicardial cells are mesothelial cells lining the outer surface of the heart that are an important progenitor population and a source of growth factors during development. Epicardial cells undergo EMT and invade the myocardium, differentiating into cardiac fibroblasts and coronary smooth muscle cells. Of note, resident fibroblasts of epicardial origin are a major cellular mediator of cardiac fibrosis. Our recent work has established an important function for the protein arginine methyltransferase PRMT1 in EMT. We hypothesized that PRMT1 is required for EMT in a mouse epicardial cell line (MEC-1). We found that PRMT1 is required for a subset of the EMT marker changes in epicardial EMT, such as upregulation of vimentin, fibronectin, and slug, as well as loss of E-cadherin. Furthermore, PRMT1 knockdown reduced MEC-1 migration and invasion, suggesting that PRMT1 is critical for epicardial progenitor function. Epicardial cells secrete a variety of signaling factors that affect cardiomyocyte proliferation and structure. This is a critical component of heart development, and may also affect heart diseases such as hypertophic heart failure. Therefore we assessed whether EMT and PRMT1 affect the paracrine functions of epicardial cells. Co-culture of MEC-1 cells with rat neonatal cardiomyocytes caused cardiomyocyte hypertrophy, and this was enhanced when MEC-1 cells were pre-treated with TGF-β to induce EMT. Interestingly, MEC-1 cells treated with PRMT1 siRNA also induced cardiomyocyte hypertrophy, but TGF-β pre-treatment of these cells did not enhance this effect. In conclusion, epicardial EMT is largely dependent on PRMT1. Epicardial cells also promote cardiomyocyte hypertrophy, which is enhanced in epicardial cells that have undergone PRMT1-mediated EMT towards a mesenchymal fate. These studies establish a role for protein methylation in the EMT process, and could lead to novel treatments for heart failure and other diseases affected by EMT.
Abstract 133: Impaired TNFα Signaling Deregulates Basal Nrf2-Antioxidant System in Myocardium
Authors: Shanmugam Gobinath , Ramasamy Sakthivel , Shelar Sandeep B , Narasimhan Madhusudhanan , Hong Jennifer , Atieno Nancy , Bradley Wayne E , Dell`Italia Louis , Darley-Usmar Victor M , Hoidal John R , Rajasekaran Namakkal S
Published: July 2021

Antagonizing TNF-α and its signaling is proven to be a successful therapeutic strategy in various disease processes. Although blocking TNF-α signaling attenuated the disease activity, the overarching clinical contraindications remains to be a greater concern and reasons for the adverse effects are largely unknown. TNF-α induces ROS and as ROS is pivotal for activation of antioxidant transcription factor, Nrf2, we posit that any impairment in TNF-α signaling could affect Nrf2-dependent basal redox homeostasis. HL-1 cardiomyocytes, TNFR1/2 double knockout mice (DKO) that shows hampered TNF-α signaling were used as experimental models. Electron Paramagnetic Resonance Spectroscopy (EPR) measurements revealed that as low as 2 ng TNF-α/ml, ROS was induced significantly (p10ng/ml in HL-1 cells. TNF-α (2-5 ng /ml, for 2 h) evoked robust (p<0.05, n=3) nuclear translocation of Nrf2 and increased nuclear binding (Trans AM DNA binding analysis) along with significant (p<0.05) induction in trans-activation of Nrf2 targets. Additionally, this was associated with 2-fold increase (p<0.05) in intracellular glutathione (GSH) that is associated with significant cell death only at higher (>10 - 50 ng/ml) and later time points (12 h and 24 h). These results suggest that TNF-α mediated ROS induction activates Nrf2 dependent antioxidant system in cardiomyocytes. In vivo experiments with TNFR1/2 DKO demonstrates that the expression of Nrf2-regulated genes were significantly downregulated indicating a weakened antioxidant system. Also, the acute exercise stress (AES) induced Nrf2 trans-activation observed in WT mice was significantly blunted in TNFR1/2 DKO suggesting a need for TNF signaling in stress induced Nrf2 activation. These results support the concept that complete and/or sustained blockade of TNF signaling may result in compromised Nrf2-dependent antioxidant defense in the myocardium. This further necessitates that during chronic anti-TNF-α treatment, a finely tuned and maintenance of specific threshold of TNF-α signaling is essential to avoid oxidant stress based complications in cardiovascular system.
Abstract 156: Hydrogel Mattress, an in vitro Platform to Enhance Maturation and Evaluate Contractile Function of Individual hiPSC-CMs
Authors: Feaster Tromondae K , Williams Charles H , Cadar Adrian G , Chun Young W , Wang Lili , Bloodworth Nathan C , Merryman W. D , Lim Chee C , Knollmann Bjorn C , Hong Charles C
Published: July 2021

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) have great potential as tools for human heart disease modeling and drug discovery. However, their contractile properties have not been routinely evaluated; as current methods are not accessible for most laboratories. We sought to develop a more efficient method to evaluate hiPSC-CM mechanical properties, at the single cell level. Individual hiPSC-CMs were cultured on a hydrogel based platform, termed the “hydrogel mattress,” and their cellular contractile properties evaluated using video-based edge detection. We found that hiPSC-CMs maintained on the mattress reproducibly exhibited robust cell shortening, in dramatic contrast to hiPSC-CMs maintained in a standard manner. We further found that contraction and peak cell shortening amplitude of hiPSC-CMs on mattress was comparable to that of freshly isolated adult ventricular mouse CM. Importantly, hiPSC-CMs maintained on the mattress exhibited several characteristics of a native CM, in terms of myocyte elongation, calcium handling and pharmacological response. Finally, using this platform, we could calculate the traction force generated by individual CMs. In summary, the Hydrogel mattress platform is a simple and reliable
in vitro
platform that not only enables the quantification of contractile performance of isolated hiPSC-CMs, but also enhances CM maturation. This flexible platform can be extended to
in vitro
disease modeling, drug discovery and cardiotoxicity testing.
Abstract 159: Empowering Cardiac Progenitor Cell-mediated Repair of Injured Myocardium by Overexpressing P2Y2 Nucleotide Receptor
Authors: El-Sayed Farid , Sussman Mark
Published: July 2021

Heart failure is a leading cause of death in the US due to the limited capability of adult mammalian heart to regenerate following injury. Autologous stem cell therapy holds promise for regeneration of injured myocardium after myocardial infarction. However, stem cells derived from diseased organs exhibit impaired proliferative and migratory capabilities and increased susceptibility to cell death. Empowering stem cells from diverse origins, including cardiac progenitor cells (CPCs), with pro-survival genes has been attempted. Despite the well-established roles of purinergic signaling mediated by extracellular nucleotides in regulating diverse cellular responses in cardiovascular diseases, it has not been well-defined in CPCs. Our preliminary data show, for the first time, that the majority of P2 purinergic receptors are expressed and exhibit functional responses to ATP and UTP in mouse and human CPCs. Since previous findings have shown that the G protein-coupled P2Y
2
receptor (P2Y
2
R) induces cardioprotective responses in animal models as well as human cardiomyocytes and regulates a wide range of signaling pathways that are crucial to tissue repair in various experimental models and in stem cells from diverse origins, we aim to determine whether P2Y
2
R plays similar roles in CPCs. Our preliminary data show that the P2Y
2
R agonists ATP and UTP enhance human CPC (hCPC) proliferation, migration and survival. Interestingly, hCPCs that exhibit relatively slower growth kinetics and higher levels of senescence markers show a dramatic decrease in P2Y
2
R expression as compared to fast-growing hCPCs consistent with our hypothesis that overexpressing P2Y
2
R participates in rejuvenating hCPCs and improving their growth capabilities. This hypothesis will be tested
in vivo
by determining whether P2Y
2
R overexpression in hCPCs improves their reparative potential for injured mouse myocardium. We also introduce the novel hypothesis that P2Y
2
R-induced regenerative responses in hCPCs involve the activation of Hippo signaling that is known to be regulated by different GPCRs, which links the extracellular nucleotides released during cardiac ischemia to extracellular matrix sensing and Hippo signaling that have been recently implicated in cardiac regeneration.
Abstract 16: Enhancing Myocardial Repair With CardioChimeras
Authors: Quijada Pearl , Salunga Hazel T , Hariharan Nirmala , Cubillo Jonathan , El-Sayed Farid , Moshref Maryam , Bala Kristin M , Emathinger Jaqueline M , De La Torre Andrea , Ormachea Lucia , Alvarez Roberto , Gude Natalie A , Sussman Mark A
Published: July 2021

Dual cell transplantation of cardiac progenitor cells (CPCs) and mesenchymal stem cells (MSCs) after infarction enhances myocardial repair and performance in large animal models relative to delivery of either cell population individually. However, a single stem cell to support both direct and indirect mechanisms of myocardial repair has yet to be identified. CardioChimeras (CCs), a progenitor cell formed by fusion between CPCs and MSCs were analysed for reparative potential after myocardial infarction (MI) relative to individual parents cell or combined parent cell delivery. Two representative CCs, CardioChimera 1 (CC1) and CardioChimera 2 (CC2) were used for this study. CC1 and CC2 improved left ventricular anterior wall thickness (AWT) at 4 weeks, but only CC1 treatment preserved AWT at 18 weeks relative to no cell treatment (PBS). Ejection fraction was enhanced at 6 weeks post injury in CC1 and CC2 groups, which was maintained in CC1, CC2 and CPC + MSC combined groups up to 18 weeks. Infarct size was decreased by 5% in CC1 and CC2 hearts, whereas CPC + MSC and CPC parent groups remained unchanged when comparing 4 to 12 week change in scar size. MSC and PBS groups displayed marked increases in infarct size (10-15%). CC1 and CC2 showed enhanced engraftment potential by 3-fold relative to CPC + MSC and CPC hearts. In contrast, MSCs were detected at low levels (0.04%). CC1 and CC2 discovered within the myocardium expressed early commitment marker cardiac troponin T relative to controls. CC1 and CC2 treatment increased capillary density within the infarct, indicating that cell persistence facilitates paracrine mediated vasculature stabilization and/or formation. CCs merge the application of distinct cells into a single entity for cellular therapeutic intervention in the progression of heart failure. CCs represent a tractable cellular system that improves upon combinatorial cell therapy approaches and supports myocardial regeneration.
Abstract 169: c-fos and Caspase 8 Mediate Palmitate-induced Cardiomyocyte Apoptosis
Authors: Oh Charles C , Nguy Michael , Karen D’Souza , Migrino Raymond , Schwenke Dawn , Thornburg Kent , Reaven Peter
Published: July 2021

Background:
We previously demonstrated that p38α activation mediates palmitate (PA)-induced cardiomyocyte apoptosis, and that specific p38α siRNA markedly and dose-dependently attenuates apoptosis rates in the presence of PA. We tested the hypothesis that c-fos and caspase 8 would be up-regulated concurrently with p38a.

Methods and Results:
Immortalized human adult ventricular cardiomyocytes (
AC16
cells) were exposed to high physiological levels of PA. The p38α-dependent pathway was evaluated using p38α siRNA knockdown. PA dose-dependently increased transcription of an AP-1 factor, c-fos, in AC16 cardiomyocytes (control:0.34±0.11, 150 μM PA:0.83±0.19, 300 μM PA:1.58±0.45, n=5, p<0.05). c-fos protein expression increased dose-dependently with PA treatment (control:0±0%, 150 μM PA:26%±28%, 300 μM PA:258%±103%, n=4, p<0.05). Phospho-c-fos levels increased dose-dependently as well (control:0±0%, 150 μM PA:73%±41%, 300 μM PA:302%±62%, n=4, p<0.05). Transcription of a putative downstream effector, caspase 8, also increased (control:0.94±0.10, 150 μM PA:1.40±0.10, 300 μM PA:1.42±0.11, n=3, p<0.05) but protein levels did not.

p38α knockdown dose-dependently blocked the PA-induced increase in phospho-c-fos protein level (control siRNA:0±0%, 300 μM PA:206±47%, 300 μM PA+30 pmol siRNA:98±31%, 300 μM PA+60 pmol siRNA:58±40%, 300 μM PA+120 pmol siRNA:-21±19%, n=4, p<0.05), without a clear reduction in total c-fos. Interestingly, caspase 8 level was also dose-dependently reduced by p38α knockdown (control siRNA:0±0%, 300 μM PA:-25±3%, 300 μM PA+30 pmol p38α siRNA:-38±13%, 300 μM PA+60 pmol p38α siRNA:-54±7%, 300 μM PA+120 pmol p38α siRNA:-46±11%, n=3, p<0.05).

Conclusions:
This study in cardiomyocytes demonstrated that 1) PA dose-dependently increases gene expression of c-fos and protein levels of total c-fos and phospho-c-fos, 2) PA increases transcription of caspase 8, and 3) reduced p38α expression dose-dependently attenuates PA-induced increases in c-fos and phospho-c-fos protein levels, and decreases caspase 8. Our results suggest that p38α activated by PA up-regulates the downstream transcription factor, c-fos, and maintains caspase 8 level. These molecules may underlie PA-induced cardiomyocyte apoptosis.
Abstract 170: Mechanism of Cardiotoxicity of Tyrosine Kinase Inhibitors
Authors: Elmadani Manar F , Ulvila Johanna , Khan Suleiman , Alakoski Tarja , Magga Johanna , Aitokallio Tero , Wennerberg Krister , Kerkelä Risto
Published: July 2021

Aim:
It is becoming evident that many of the signaling elements driving cancer cell division, for which kinase inhibitors (KIs) are targeted, are the same as those necessary for cardiomyocyte viability. Adverse cardiac events have been reported for a number of KIs. Aim of this study was to identify KIs that induce toxicity to cardiomyocytes and to elucidate the central molecular mechanisms mediating the toxicity.

Methods:
288 anti-cancer agents (123 are KIs) were screened for their ability to induce cardiotoxicity in cultured primary cardiomyocytes. Cell viability assay was done by measuring the ATP levels in cardiomyocytes after exposure of cells to a 3-log concentration range of each compound for 24 hours. Toxicity data was combined with kinase profiling data to identify protein kinases mediating the toxicity of KIs. In parallel, the molecular mechanism mediating the cardiomyocyte toxicity of dasatinib, a second generation Bcr-Abl and Src family tyrosine kinase inhibitor, was investigated. The role of Src kinase in regulation of cardiomyocyte viability was analyzed by siRNA-mediated Src knockdown. Overexpression of wild type Src and dasatinib- resistant Src in cardiomyocytes was performed to investigate the role of Src in regulating the cardiomyocyte toxicity of dasatinib. Western blotting was used to investigate for downstream signaling targets of Src.

Results:
Of the KIs, 70 compounds decreased the cardiomyocyte viability by 10-80 %. The kinase profiling data showed that IGF1R, MEK/ERK pathway, PI3K and Src kinases are the key kinases regulating cardiomyocyte viability. Dasatinib treatment dose-dependently increased cardiomyocyte death. Depletion of Src by siRNA also reduced cardiomyocyte viability. Dasatinib treatment attenuated FGF induced ERK phosphorylation. Overexpression of dasatinib-resistant mutant of Src, but not wild type Src, protected the cardiomyocyte from dasatinib toxicity and rescued the FGF-induced ERK phosphorylation.

Conclusions:
Cardiomyocyte toxicity of KIs can be attributed to inhibition of IGF1R, MEK/ERK pathway, PI3Ks and Src family kinases. Cardiomyocyte toxicity of dasatinib is mediated by Src kinase inhibition which in turn attenuates MEK/ERK pathway signalling.
Abstract 173: The Role of Bax and Bak in Autophagic Cell Death
Authors: Karch Jason , Schips Tobias G , Brody Matthew J , Kanisicak Onur , Sargent Michelle A , Molkentin Jeffery D
Published: July 2021

In times of energy depletion, a cell will attempt to maintain metabolic homeostasis and viability by degrading and recycling organelles and intracellular components and proteins in a process referred to as autophagy. However, if the energy depletion persists, the cell will be overwhelmed by the autophagic process and will succumb to autophagic cell death. This form of cell death has been implicated in cardiac remodeling during heart failure and damage during ischemic injury. Two proteins that have been previously shown to play a role in virtually every form of regulated cell death, including autophagy, are Bax and Bak. These effectors are responsible for cytochrome-c release during apoptosis and effect mitochondrial permeability transition pore opening during regulated necrosis. Although the expression of either Bax or Bak is required for autophagic cell death to occur, the role of Bax/Bak in this type of cell death is poorly understood, although the lysosome appears to be centrally involved. Here we show that Bax/Bak DKO MEFs subjected to several days of serum starvation contain intact lysosomes compared to WT MEFs. Furthermore, the acidity of the lysosomes in starved DKO MEFs is preserved compared with starved WT MEFs. Bax and Bak are both found in isolated lysosomal preparations and Bax targeted to the lysosome can completely restore autophagic cell death in DKO MEFs. Finally, although Bax oligomerization is required for apoptosis, it is not necessary for autophagic cell death, as DKO MEFs expressing an oligomerization defective mutant of Bax are still susceptible to this form of death, as monomeric Bax can still increase membrane permeability. In conclusion our results suggest that lysosomal membrane permeability through Bax or Bak is required for autophagic cell death to occur and without Bax or Bak the lysosomes remain intact where they can function as an energy source during times of nutrient deprivation.