bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 40: Induced Basal Autophagy in Atg7 Mice Ameliorates Cardiac Proteinopathy
Authors: Bhuiyan M. Shenuarin , Pattison J Scott , James Jeanne , Gulick James , Osinska Hanna , Robbins Jeffrey
Published: July 2021

Backgrounds:

Basal autophagy is a cellular homeostatic mechanism that can eliminate damaged and potentially toxic proteins and organelles. We hypothesized that cardiac proteinopathies, such as desmin-related cardiomyopathy (DRC) that lead to accumulations of misfolded proteins, can trigger alterations in cardiomyocyte autophagy as an adaptive process. However, a mouse model of cardiac specific-induced basal autophagy is lacking and the effects of acute or chronic increased basal autophagy remains unclear.

Hypothesis:

To determine whether induction of basal autophagy in the induced Atg7 mice can ameliorate cardiac proteinopathy in a model of DRC.

Methods and Results:

The protein Atg7 is thought to be essential for autophagy and, in an attempt to induce high levels of basal autophagy we generated an inducible-transgenic (Tg) mouse. This mouse is able to undergo cardiac specific overexpression of the Atg7 gene, where the transgene can be switched on or off by doxycycline. The Atg7-Tg mouse showed 10 fold overexpression of Atg7 protein with normal heart morphology and function being maintained even after 10 months. Overexpression of Atg7 had no detectable effect on other proteins involved in the autophagy-lysosomal pathways. However, the hearts show increased basal autophagy, as measured by increased autophagic flux activity and cardiomyocyte ultrastructural examination. We showed earlier that autophagy can be down-regulated in cardiac proteinopathy as observed in a mutant αB-crystallin (CryAB
R120G
) mouse model of DRC. Therefore, we crossed the CryAB
R120G
mice to transgenic mice with cardiac specific overexpression of Atg7. Sustained Atg7-induced autophagy in CryAB
R120G
hearts prolonged CryAB
R120G
survival by 40% and is associated with the maintenance of cardiac function and decreased cardiac hypertrophy.

Conclusions:

Cardiac-specific Atg7 inducible-Tg mice with increased basal autophagy may serve as a novel model to study the regulatory role of autophagy in the heart. Induced basal autophagy in Atg7-Tg mice can extend the life-span in a model of cardiac proteinopathy. Modulating the overall autophagic state may be useful therapeutically in heart disease.
Abstract 46: RSK4 Is a Novel Negative Regulator of Endothelial Differentiation via Direct Suppression of Protein Kinase A Activity
Authors: Matsunaga Taichi , Yamamizu Kohei , Takeuchi Kazuko K , Kojima Mizuyo , Takeuchi Jun K , Yamashita Jun K
Published: July 2021

Endothelial cells (ECs) differentiation is strictly regulated to generate functional blood vessels. Previously, we demonstrated that Flk1+ cells derived from embryonic stem (ES) cells serve as vascular progenitors and can constructively reproduce the early differentiation of both ECs and Mural cells (MCs; vascular smooth muscle cells and pericytes). We recently reported that cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling enhanced EC differentiation from Flk1+ vascular progenitors via induction of vascular endothelial growth factor (VEGF) receptors expression. To identify a novel molecule involved in EC differentiation, we performed microarray analyses at various differentiation stages from ES cells to vascular cells; undifferentiated ES cells, Flk1+ vascular progenitors, ECs and MCs. Then, we found that mRNA of p90 ribosomal protein S6 kinase 4 (RSK4) was highly expressed in Flk1+ vascular progenitors and almost diminished during EC differentiation, whereas it remained being expressed in MCs. The addition of a broad-spectrum RSK inhibitor, SL0101, to Flk1+ vascular progenitors enhanced VEGF receptor expression and EC differentiation specifically in the presence of the simultaneous cAMP signal activation. SL0101-elicited EC differentiation was abolished by PKA catalytic subunit alpha (PKAc) knockdown. Specific RSK4 knockdown in Flk1+ vascular progenitors reproduced the effects of SL0101 on EC differentiation via increasing Flk1 expression. RSK4 had potential to bind with PKAc and inhibited PKA activity. Furthermore, an ex vivo whole embryo culture assay showed that SL0101 treatment drastically resulted in increased CD31+ vasculature area, enlarged vessel diameter and defected vascular remodeling in yolk sac. Taken together, these results indicate that RSK4 acts as a novel negative regulator for EC differentiation via inhibition of PKA function. Fine tuning of PKA and Flk1 signaling by RSK4 is critical to normal vascular development. Elucidation of the new-mode cell fate determination by RSK4 would provide novel insights in developmental biology, stem cell biology, and regenerative medicine.
Abstract 48: Cardiac-Targeted TRPM7 Deletion Induces Cardiomyopathy, Heart Block and Impaired Ventricular Repolarization via Downregulation of Kcnd2 and Hcn4
Authors: Sah Rajan , Mesirca Pietro , Mason Xenos , Bates-Withers Christopher , Gibson William , Robertson Janice , Mangoni Matteo , Clapham David
Published: July 2021

Transient Receptor Potential Melastatin-7 (TRPM7) is a divalent permeant channel-kinase of unknown function. It is concentrated in myocardium during embryonic development, and TRPM7-like current has been reported in adult ventricular myocytes. In atrial cardiac fibroblasts, TRPM7 has been implicated in fibrogenesis in patients with atrial fibrillation, however its function in ventricular myocardium is unexplored. We tested the hypothesis that TRPM7 is required for ventricular function by crossing TRPM7fl/- mice with the cardiac-targeted αMHC-Cre line (αMHC-TRPM7 KO). We show that TRPM7 forms a functional current in adult murine ventricular cardiomyocytes and demonstrate that ∼50% of αMHC-TRPM7 KO mice develop a cardiomyopathy with left ventricular dysfunction and cardiac hypertrophy, associated with heart block and impaired ventricular repolarization. This phenotype is associated with robust elevations in mRNA of Postn, Nppa, Timp1, Acta and reductions in Hdac9, Kcnv2, Kcnj3, Kcnd2, Lgi1 and Hcn4. Consistent with mRNA expression analysis and the observed electrical remodeling, patch-clamp of αMHC-Cre-TRPM7 KO ventricular myocytes (RV and LV) reveals significant action potential prolongation (2.5 to 4-fold increase in APD50) due to a 2 to 4-fold reduction in transient outward current (Ito, encoded by Kcnd2). Similarly, the funny current (If), encoded by Hcn4, is diminished 4-fold in atrioventricular nodal cells isolated from αMHC-Cre-Trpm7 KO hearts, accounting for the observed heart block. The ∼50% of αMHC-Cre-Trpm7 KO mice that do not develop cardiomyopathy are devoid of heart block, show normal expression levels of hypertrophy genes Postn, Nppa, and Acta, but continue to show significant reductions in Kcnd2, Lgi1, Kcnj3, Kcnv2, and Hdac9 expression. In conclusion, these results demonstrate that cardiac-targeted TRPM7 deletion dysregulates the expression of ion channels important for cardiomyocyte repolarization (Kcnd2) and atrioventricular conduction (Hcn4), leading to impaired ventricular repolarization and heart block in mice. The observed cardiomyopathy may arise from hemodynamic stresses secondary to chronic bradycardia from heart block in the setting of down-regulated histone deacetylase 9 (HDAC9).
Abstract 50: Cardiomyogenesis in Hypertrophic Cardiomyopathy
Authors: Arranto Christian A , Iascone Maria R , Sanarico Anna G , Valle Matteo , Mangiaracina Chiara M , Quaini Federico , Kajstura Jan , Leri Annarosa , Ferrazzi Paolo , Anversa Piero
Published: July 2021

Hypertrophic cardiomyopathy (HCM) is the most prevalent heritable cardiovascular disease and the leading cause of sudden death in young adults. HCM is characterized by regional ventricular hypertrophy in the absence of cavitary dilation. Myofibrillar disarray associated with mutations of sarcomeric proteins and collagen accumulation are considered major determinants of the disease phenotype. Septal hypertrophy has been viewed as the result of an increase in size of preexisting myocytes, but whether myocyte formation contributes to septal thickening has never been determined. Thus, 29 patients with HCM undergoing septal myectomy were studied, and 10 sex-, age-matched septa, collected from patients who died from causes other than cardiovascular diseases, were used as controls. Mutations of genes coding for contractile proteins were present in all cases of HCM; 79% of patients were in NYHA class III, 17% in class II, and 4% in class IV. Arrhythmic events consisting of atrial fibrillation and ventricular tachycardia were found in 11 patients. In 8 cases, HCM was diagnosed in family members as well. Septal thickness is the product of myocyte diameter and the number of myocyte across the septum. The thickness of the septum increased 2.3-fold in HCM (Controls: 11±1 mm; HCM: 25±8 mm, p<0.001). Septal thickening in HCM was mediated by a 69% increase in myocyte number (Controls: 722±120; HCM: 1,217±375, p<0.001) and a 31% increase in cell diameter (Controls: 16±3 λm; HCM: 21±4 λm, p<0.001). Myocyte regeneration was confirmed by the expression of the cell cycle protein, Ki67. The number of Ki67-positive myocyte nuclei increased 15-fold in HCM (Controls: 400 myocytes/10
6
cells; HCM: 6,000 myocytes/10
6
cells, p<0.001). In humans, myocyte formation is regulated by commitment of resident c-kit-positive cardiac stem cells (CSCs). In comparison with control hearts, the number of CSCs increased 2.7-fold in patients with HCM (Controls: 11±5 cells/mm
3
; HCM: 30±17 cells/mm
3
, p<0.01). Additionally, the expression of contractile proteins was detected in differentiating CSCs. In conclusion, myocyte regeneration and hypertrophy contribute to septal thickening in HCM and newly formed myocytes originate from growth activation and lineage specification of resident CSCs.
Abstract 51: Nucleostemin Induced by Pim-1 Kinase Is Critical to Maintain Pluripotency and Enhance Regenerative Potential of Cardiac Progenitor Cells
Authors: Hariharan Nirmala , Joyo Anya Y , Samse Kaitlen M , Avitabile Daniele , Bailey Brandi , Mohsin Sadia , Sussman Mark A
Published: July 2021

Myocardial regeneration and repair in response to injury are governed in part by cell survival, proliferation and pluripotency. Proliferation and survival in cardiac progenitor cells (CPCs) are mediated by Pim-1, a serine threonine kinase, and nucleostemin (NS), a nucleolar stress sensor protein. The role of NS in regulating CPC pluripotency and the molecular mechanism of NS induction and action is largely unknown. The hypothesis of the study is that NS, induced by Pim-1 mediated stabilization of transcription factor c-Myc is critical to maintain CPC pluripotency and inhibits senescence. NS and c-Myc protein levels are increased in cultured CPCs overexpressing Pim-1 (3.1 and 5.5 fold, p<0.01) while knockdown of Pim-1 using sh-RNA decreases c-Myc and NS expression (-60%, -54%, p<0.05), similar to effects mediated by a Pim-1 kinase inhibitor (p<0.01), indicating that Pim-1 regulates both c-Myc and NS. c-Myc is necessary and sufficient for NS regulation, as indicated by the increase (3.1 fold, p<0.01) and decrease (-60.2%, p<0.01) in NS expression upon lentiviral mediated over-expression or knockdown of c-Myc, respectively. Regulation of NS promoter by c-Myc is evident from loss of GFP expression and fluorescence following knock down of c-Myc in CPCs isolated from transgenic mice expressing eGFP driven by the NS promoter. The role of NS in regulating CPC pluripotency is determined by silencing NS. Change in morphology (flat, round cells vs spindle shape control CPCs), decreased expression of stem cell marker c-Kit (-55%, p<0.05), up-regulation of cell cycle inhibitors p53 and p16 (4.2, 3.8 fold, p<0.01) and decreased proliferation (p<0.05) result from loss of NS in CPCs, suggestive of increased senescence and loss of pluripotency. NS-mediated regulation of CPC senescence is p53 dependent, as silencing p53 reverses CPC morphology and pluripotency lost by NS depletion. In conclusion, NS which is induced downstream of Pim-1 kinase maintains pluripotency and enhances regenerative potential in CPCs. These findings are consistent with cumulative evidence that Pim-1 induced cardiac regeneration is mediated in part by NS, providing an additional mechanistic basis for benefits of genetic engineering with Pim-1 to enhance NS expression in cardiac stem cells.
Abstract 53: PTEN Nuclear Export and p53 Inhibition Mediate Free Fatty Acid-Induced Oxidative Stress in Endothelial Cells
Authors: Wu Yong , Zhou Hongci , Liu Xuan
Published: July 2021

Oxidative stress induced by free fatty acids (FFA) contributes to metabolic syndrome-associated development of cardiovascular diseases. In this study, we investigated the mechanisms involved in the p53-mediated regulation of antioxidant enzyme glutathione peroxidase-1 (GPx-1) expression and induction of intracellular ROS levels by palmitic acid (PA). The p53 pathway plays a very important role in regulating ROS production. We exposed human umbilical vein endothelial cells (HUVEC) to PA and demonstrated that p53 acetylation and protein levels decrease in a dose-dependent manner. The p53 downregulation correlated with a decrease in its downstream target GPx-1 and increase in the production of intracellular ROS. Nuclear PTEN was previously identified as an important factor involved in the upregulation of p53 acetylation, transcription activity and protein stability. Here, we show treatment of HUVEC with PA induced increase in PTEN phosphorylation at Ser380 and in interaction between PTEN and HAUSP, a deubiquitylating enzyme that cleaves ubiquitin from its substrates. The increased PTEN-HAUSP interaction leads to a dramatic decrease in PTEN mono-ubiquitination and increase in PTEN nuclear export, which prevents p53 acetylation. Pharmacologic inhibition or genetic ablation of mammalian target of rapamycin (mTOR) or S6K abolished PA-induced PTEN phosphorylation, PTEN-HAUSP interaction and PTEN nuclear export, suggesting that mTOR/S6K pathway is responsible for nuclear PTEN phosphorylation. Importantly, inhibition of mTOR/S6K pathway significantly blocks PA-induced PTEN nuclear export, p53-mediated GPx-1 expression, and increase in ROS production in HUVEC cells. These results demonstrate that PA treatment elevates PTEN-Ser380 phosphorylation through mTOR/S6K pathway. Once phosphorylated, PTEN in turn binds to HAUSP, reduces its mono-ubiquitination and nuclear localization, which prevents p53 from activating GPx-1 to compromise the most important antioxidant defense system and cause oxidative damage to human vascular endothelium.
Abstract 58: Modeling Pathogenesis in Familial Hypertrophic Cardiomyopathy Using Patient-Specific Induced Pluripotent Stem Cells
Authors: lan Feng , Lee Andrew , Liang Ping , Navarrete Enrique , Wang Li , Leng Han , Sanchez Veronica , Yen Michelle , Wang Yongming , Nguyen Patricia , Sun Ning , Abilez Oscar , Lewis Richard , Yamaguchi Yoshinori , Ashley Euan , Bers Donald , Robbins Robert , Longaker Michael , Wu Joseph
Published: July 2021

Background:
Hypertrophic cardiomyopathy (HCM) is a prevalent familial cardiac disorder linked to development of heart failure, arrhythmia, and sudden cardiac death. Molecular genetic studies have demonstrated HCM is caused by mutations in genes encoding for the cardiac sarcomere. However, the pathways by which sarcomeric mutations result in myocyte hypertrophy and contractile abnormalities are not well understood.

Methods:
We aimed to elucidate the molecular mechanisms underlying the development of HCM through the generation of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from dermal fibroblasts of a 10 member family, five of whom carry a hereditary HCM missense mutation (Arg663His) in the MYH7 gene.

Results:
As compared to control iPSC-CMs derived from healthy family members, HCM iPSC-CMs exhibited enlarged cell size, increased atrial natriuretic factor (ANF) expression, nuclear translocation of nuclear factor of activated T-cells (NFAT), and aggravated contractile dysfunction in response to stimulation by β-adrenergic agonists. Interestingly, both video analysis of beating cells and whole cell patch clamping revealed arrhythmia in a significant portion of diseased iPSC-CMs at the single cell level. Ca
2+
imaging demonstrated elevated cytoplasmic Ca
2+
content and irregular transients in HCM iPSC-CMs prior to the onset of cellular hypertrophy, suggesting the HCM phenotype is triggered by dysfunction in Ca
2+
cycling. Treatment of irregular Ca
2+
homeostasis by the Ca
2+
channel blocker verapamil prevented development of cellular hypertrophy and arrhythmia.

Conclusions:
We hypothesize the cellular abnormalities observed in HCM iPSC-CMs are caused by deficiencies in Ca
2+
regulation. We anticipate our findings will elucidate the mechanisms underlying HCM development and identify novel targets for treatment of the disease.
Abstract 59: Preservation of Myocardial Structure Is Enhanced by Pim-1 Engineering of Bone Marrow Cells
Authors: Quijada Pearl J , Toko Haruhiro , Fischer Kimberlee , Bailey Brandi , Reilly Patrick , Gude Natalie , Avitabile Daniele , Sussman Mark
Published: July 2021

Rationale:
Bone marrow derived cells to treat myocardial injury improve cardiac function and support beneficial cardiac remodeling. However, survival of stem cells is limited due to low proliferation of transferred cells.

Objective:
Demonstrate long-term potential of c-kit+ bone marrow stem cells (BMCs) enhanced with Pim-1 kinase to promote positive cardiac remodeling.

Methods and Results:
Lentiviral modification of c-kit+ BMCs to express Pim-1 (BMCeP) increases proliferation and expression of pro-survival proteins relative to BMCs expressing GFP (BMCe). Intramyocardial delivery of BMCeP at time of infarction supports improvements in anterior wall dimensions and prevents left ventricle dilation compared to hearts treated with vehicle alone. Reduction of the akinetic left ventricular wall was observed in BMCeP treated hearts at 4 and 12 weeks after infarction. Early recovery of cardiac function in BMCeP-injected hearts facilitated modest improvements in hemodynamic function up to 12 weeks post infarction between cell treated groups. Persistence of BMCeP is improved relative to BMCe within the infarct together with increased recruitment of endogenous c-kit+ cells. Delivery of BMC populations promotes cellular hypertrophy in the border and infarcted regions coupled with an up regulation of hypertrophic genes. Thus, BMCeP treatment yields improved structural remodeling of infarcted myocardium compared to control BMCs.

Conclusions:
Genetic modification of BMCs with Pim-1 may serve as a therapeutic approach to promote recovery of myocardial structure. Future approaches may take advantage of salutary BMC actions in conjunction with other stem cell types to increase efficacy of cellular therapy and improve myocardial performance in the injured myocardium.
Abstract 60: Hypoxia Preserves the Quiescent Lineage Negative Phenotype of Cardiac Stem Cells Within the Niche
Authors: Sanada Fumihiro , Mangano Emily , Kim Junghyung , Arranto Christian , Ferreira-Martins Joao , Signore Sergio , Fiorini Claudia , Ide-Iwata Noriko , Pesapane Ada , Ricciardi Mario , Zheng Hanqiao , Gurusamy Narasimman , Wybieralska Ewa , Caballero Virginia , Cimini Maria , Goichberg Polina , Rota Marcello , Hosoda Toru , Kajstura Jan , Anversa Piero , Leri Annarosa
Published: July 2021

In stem cell-regulated organs, a subset of niches is characterized by low oxygen tension. This metabolic adaptation offers a selective advantage to stem cells favoring the preservation of their quiescent undifferentiated phenotype. The objective of this work was to determine whether in the mouse heart cardiac niches constitute a heterogeneous compartment composed of hypoxic and normoxic niches, and whether differences in O
2
concentration affect the function of c-kit-positive cardiac stem cells (CSCs).To test this possibility, we studied first the in vivo uptake of the hypoxic marker pimonidazole (PIMO), which identifies intracellular O
2
concentration <10 mmHg. Mice were sacrificed 2 hours after intraperitoneal administration of PIMO, and PIMO-labeling was analyzed. By immunolabeling, 15% of cardiac niches were characterized by a hypoxic microenvironment and more than 20% of isolated CSCs were PIMO-positive, as measured by flow-cytometry. The cell cycle protein Ki67 was restricted to the PIMO-negative CSC class, which contained early committed cells expressing c-kit together with the myocyte specific transcription factors GATA4 and Nkx2.5. Mice were then administered tirapazamine, a compound that kills selectively hypoxic cells. One day later, the fraction of PIMO-positive CSCs was markedly decreased but, at 5 days, this compartment was partly reconstituted. This compensatory response was coupled with increased proliferation of PIMO-negative CSCs, suggesting that normoxic CSCs have the ability to replenish hypoxic niches following injury. Subsequently, the effects of hypoxia were studied in human CSCs (hCSCs) exposed in vitro to 1% O
2
. With respect to cells cultured in normoxia, 1% O
2
led to upregulation of HIF1α in hCSCs which also showed lower levels of BrdU incorporation. These cellular responses were accompanied by an increase in transcripts for the stemness genes c-kit, Oct4, Nanog and Sox2, and a decrease in mRNA for myocyte and vascular genes. Apoptosis, measured by TdT labeling, did not differ in normoxic and hypoxic hCSCs. In conclusion, our data indicate that hypoxic and normoxic niches coexist in the myocardium, and that intracellular hypoxia regulates the quiescent primitive CSC phenotype.
Abstract 61: Dendritic Cell Regulates Monocyte/Macrophage Polarization and Inflammatory Response After Myocardial Infarction in Mice
Authors: Anzai Atsushi , Anzai Toshihisa , Fukuda Keiichi
Published: July 2021

Inflammation and immune responses are integral components in the healing process after MI. We previously reported dendritic cell (DC) infiltration in the infarcted heart. However, the precise contribution of DC in postinfarction healing is unclear. Bone-marrow (BM) cells from CD11c-diphtheria toxin receptor/GFP transgenic mice were transplanted into lethally irradiated WT recipient mice. After reconstitution of BM-derived cells, the recipient mice were treated with either diphtheria toxin (DC ablation) or vehicle (control), and MI was created by left coronary ligation. CD11c
+
GFP
+
DCs expressing CD11b and MHC class II were recruited into the heart, peaking on day 7 after MI in control group. Mice with DC ablation for 7 days showed deteriorated left ventricular function and remodeling. DC-ablated group demonstrated enhanced and sustained expression of inflammatory cytokines such as IL-1β, IL-18 and tumor necrosis factor-α, prolonged extracellular matrix degradation associated with a high level of MMP9 activity, and diminished expression level of IL-10 and endothelial cell proliferation following MI compared with control group. In vivo and in vitro analyses revealed that DC-ablated infarcts had an enhanced capacity of monocytes/macrophages recruitment. Among these cells, augmented infiltration of proinflammatory Ly6C
high
monocytes and F4/80
+
CD206
-
M1 macrophages, and conversely impaired recruitment of anti-inflammatory Ly6C
low
monocytes and F4/80
+
CD206
+
M2 macrophages in the infarcted myocardium were identified in DC-ablated group than in control group. To confirm these results, adoptive transfer experiment was performed. BM cells from WT mice were cultured with recombinant mouse GM-CSF for 6 days and CD11c
+
BMDCs were then positively collected by magnetic sorting. We confirmed that intravenously injected CFSE-labeled BMDCs reached infarcted zone. Adoptive transfer of BMDCs into DC-depleted mice restored LV function and negated the increases of inflammatory Ly6C
high
monocytes and myocardial MMP-9 activity following MI. In conclusion, dendritic cell is a potent immunoprotective regulator during the postinfarction healing process, via controlling monocyte/macrophage homeostasis.
Abstract 63: Missense Mutation Lys18Asn in Dystrophin that Triggers X-Linked Cardiomyopathy Affects Protein Structure, Decreases Protein Stability and Increases Protein Unfolding
Authors: Mallela Krishna , Singh Surinder , Armstrong Geoffrey
Published: July 2021

A genetic mutation in dystrophin that replaces lysine at amino acid position 18 by an asparagine (K18N) results in X-linked cardiomyopathy (XCM), however, the underlying molecular mechanisms are not known. Such knowledge might help in developing effective therapies to treat XCM. The first step in understanding disease-triggering mechanisms is to probe how mutation affects protein structure and function. The K18N mutation occurs in the N-terminal actin binding domain (N-ABD) of dystrophin. Here, we examined the effect of K18N on the structure, stability, and function of N-ABD. Fig. 1A shows the
15
N-
1
H HSQC NMR spectrum before and after the mutation. Each crosspeak in the spectrum represents the structure around an amino acid. Mutation affects the position of nearly all the crosspeaks. These changes are not just located at the mutation site, but were observed in regions far away from the mutation site and also in the three actin binding regions with which N-ABD interacts with actin. In addition, the mutation makes the protein more dynamic. These structural changes result in decreased actin binding function of N-ABD. Also, the K18N mutation decreases the stability by ΔΔG = 5.3 kcal/mol (Fig. 1B) and increases the rate of unfolding by 3.2 times (Fig. 1C), which exponentially increase the relative population of unfolded state that is more prone to proteolysis and hence will result in decreased levels of the functional protein. These results indicate that the physical mechanism by which the K18N mutation triggers the disease might be by affecting protein structure leading to decreased actin binding and by decreasing the net protein levels leading to decreased net function.
Abstract 68: Role of Substance P Signaling in the Pathogenesis of Murine Viral Myocarditis
Authors: Robinson Prema , Firozgary Bahrom , Taffet George , Reynolds Corey , Pham Thuy , Khumbatta Mitra , Firozgary Gohar
Published: July 2021

Viral myocarditis is an important cause of heart failure in adolescents and young adults for which no specific treatment is currently available. Substance P (SP) is a neuropeptide and pain transmitter that activates RhoA, a small G-protein, shown to induce hypertrophy of neonatal rat ventricular myocytes. The current studies were performed to determine if SP signaling contributes to the pathogenesis of encephalomyocarditis virus (EMCV) infection, a well-established murine model of viral myocarditis. Aprepitant (1.2 mg/kg), a SP receptor antagonist, or fasudil (10mg/kg), a Rho A inhibitor, or saline control, was administered daily to mice orally by gavage 3 times for 3 days prior to intraperitoneal infection with 50 PFU of EMCV, and the mice monitored for 14 days. Pretreatment with aprepitant reduced mortality by 40% (25±7% vs. 65±7%), reduced heart-to-body weight ratio by 26% (0.0088±0.0001 vs. 0.0119±0.0001), reduced end systolic diameter (ESD) by 21% (3.0±0.2mm vs. 3.8±0.4mm), improved ejection fraction by 72% (57.2±4.7% vs. 33.2 ±9.3%), reduced chamber volume by 46% (34±5.0 μl vs. 62.7±30.8 μl), increased peak aortic flow velocity by 41% (87.0±24.7 vs. 61.7±20.1), and reduced cardiomyocyte diameter size by13% (11.0±0.1 μM vs. 12.7± 0.2 μM); p<.05 for all, Student’s t-test). Pretreatment with fasudil reduced heart-to-body weight ratio by 18% (0.0097±0.0001 vs. 0.0119±0.0001) and reduced cardiomyocyte diameter size by 12% (11.14±0.51 μM vs. 12.7± 0.2 μM; p<.05 for both), but did not significantly impact heart functional changes or mortality. These findings indicate that SP contributes to cardiac remodeling and dysfunction following ECMV infection and raises the possibility that inhibition of SP signaling may be of benefit in the treatment of patients with viral myocarditis.
Abstract 75: Insulin Modulates Cardioprotective Akt Signaling During Ischemia/Reperfusion Attenuating PHLPP-1 Phosphatase
Authors: Xing Yuan , Gao Feng , Ma Heng
Published: July 2021

Introduction

The recently discovered phosphatase PHLPP-1 directly dephosphorylates and inactivates Akt. Whether the suppression of Akt by PHLPP-1 may lead to myocardial ischemia intolerance in aged heart, has never been previously studied. Moreover, we unveiled that the cardioprotective effects of insulin in limiting ischemia/reperfusion(I/R) injury were Akt activation dependent. However,the role of insulin,regulating the endogenous negative regulator of Akt activity, remains largely unknown. In this study, an important role for insulin in intensifying Akt activity through attenuating PHLPP-1 and ameliorating myocardial injury following I/R in the senescent heart has been described.

Methods and Results

Young (3 M) and aged (20 M) rats were subjected to I (20 min) / R (4 h)
in vivo
. Insulin treatment during reperfusion significantly increased Akt phosphorylation(2.32 times) response to I/R and decreased the infarct size by 18±3%. Simultaneously, insulin treatment significantly suppressed the PHLPP-1 level by 65±5%. Insulin-induced Akt activation was impaired and the myocardial infarct was larger in I/R aged hearts compared with the young ones. The effect of Insulin attenuating PHLPP-1 to intensify Akt activity was impaired in aged ischemic hearts as compared to young ischemic hearts. In cultured rat ventricular myocytes, the inhibitory effects of insulin on cardiac PHLPP-1 were abolished by the proteasome inhibitor MG132. PHLPP-1 knockdown by small interfering RNA significantly enhances insulin-induced Akt phosphorylation and reduced simulated hypoxia/reoxygenation induced apoptosis. Moreover, swimming training (6 w) incresed insulin sensitivity in the senescent heart. The inhibitory effect of insulin on cardiac PHLPP-1 was partially reversed accompanying enhanced Akt phosphorylation. The tolerance of the aged heart to ischemic damage is also enhanced.

Conclusions

We conclude that PHLPP-1 may contribute to the aging-associated ischemic intolerance. Insulin acts as a crucial cardioprotective regulator by inhibiting physiological function of PHLPP-1 and thus enchancing Akt activity. For the first time,our results also indicated a new cardioprotective mechanism of insulin modulating ubiquitin-proteasome pathway of PHLPP-1.
Abstract 8: GTPase-Activating Protein for Rho Associated with FAK (GRAF) Regulates Cardiomyocyte Membrane Integrity
Authors: O'Neill Thomas J , Lenhart Kaitlin , Doherty Jason , Rojas Mauricio , Christopher Mack P , Taylor Joan M
Published: July 2021

Cardiac myocytes are unique in their requirement to sustain continuous repetitive contraction in the setting of intense mechanical stress while simultaneously maintaining high membrane integrity for an appropriate electrical gradient. The consequence of failure of the membrane repair response has been highlighted in recent reports linking cardiomyocyte membrane fragility with cardiac degeneration in patients as well as in their analogous mouse models. Herein, we describe a novel role for GTPase activator for Rho associated with Focal Adhesion Kinase (GRAF) in regulating cardiomyocyte membrane integrity. We previously published that disruption of GRAF in Xenopus laevis resulted in progressive skeletal muscle degeneration. We now show that GRAF-depleted tadpoles exhibit defective cardiac formation and function. Interestingly, damage of muscle cells in vivo and in vitro led to a translocation of GRAF to the sarcolemma, suggesting that GRAF may be an important component of the cardiac membrane repair machinery. To further explore this possibility, we generated GRAF hypomorphic mice that exhibit greater than 99% reduction of endogenous GRAF expression. While GRAF deficient mice show normal Mendelian birth distribution and are viable, they exhibit a modest skeletal muscle pathology. Although baseline cardiac integrity was not compromised in GRAF deficient mice, treatment either with cardiotoxin or intraperitoneal injection of isoproterenol led to elevated cardiomyocyte membrane damage (assessed by Evan’s blue dye uptake) in GRAF deficient compared to control mice (19% vs 2% of myocytes within afflicted ventricular area for cardiotoxin, 18% vs 8% for isoproterenol respectively). Moreover, cultured GRAF null myocytes exhibited a significantly attenuated membrane resealing response following laser-mediated disruption compared to GRAF-containing control cells as assessed by accumulation of the membrane impermeable dye, FM-143. As well, the survival rate after injury of GRAF-deficient cells was markedly attenuated (20% vs 85% in control cells). While cardiac cell membrane damage is likely a frequent and important event, the repair process is currently understudied, and this is the first report to implicate a Rho regulator in this response.
Abstract 81: End-Stage Human Failing Heart Increases Functional Cardiac Progenitor Cells
Authors: Mishra Rachana , Simpson David , Sharma Sudhish , Colletti Evan , Kaushal Sunjay
Published: July 2021

Background:
Human cases of end-stage heart failure provide the rare opportunity to test whether the cells within these hearts exhibit different phenotypic characteristics than those in healthy hearts. We tested whether the failing heart, as it reverses to a well-known fetal reprogramming state, also compensates by increasing the number of functional cardiac progenitor cells.

Methods and Results:
We examined samples from end-stage human failing hearts and also normal human hearts to quantitate the expression of various cardiac progenitor markers, by immunofluorescence, flow cytometry and RT-PCR. We further tested whether the cardiac progenitor cells were functional in a rodent model of myocardial infarction. All the failing end-stage hearts (N=13) reversed to a fetal state by switching their myosin heavy chain isoforms from beta to alpha. Additionally, atrial natriuretic factor was increased. Compared to normal congenital myocardium, failing end-stage hearts had a 2 to 5 fold increase in the number of C-kit+ and ISL-1+ cardiac progenitor cells (P<0.5). The numbers of cardiac progenitor cells was highest in the right atria as compared to other chambers of the end-stage heart. Cardiac progenitor cells isolated from failing hearts expressed several stemness markers that were upregulated compared to normal human hearts. FACS and IF analysis demonstrated significantly (P<0.5) higher c-kit expression in CDCs derived from end stage patients compare to normal congenital myocardium. Also, there was a tendency for increased FLK1 and Sca-1 expression in CM patients respectively. Transplanted cardiac progenitor cells from end-stage hearts promoted greater myocardial regeneration and functional improvement in the infarcted rat myocardium than transplanted cardiac progenitor cells derived from normal congenital patients (EF=57+3 vs. 41.5±3, P<0.05).

Conclusion:
Our results show an increase of progenitor cells within the end-stage heart that have the functional potential to regenerate the myocardium. Stimulating the differentiation and increasing the population of cardiac progenitor cells may provide a novel therapeutic strategy for these end-stage hearts.
Abstract 85: Cardiac Pathology Is Worsened by Loss of the Cell Junction Protein Claudin-5 and Improved by Claudin-5 Gene Therapy
Authors: Delfín Dawn A , Schill Kevin E , Xu Ying , Swager Sarah , Janssen Paul M L , Rafael-Fortney Jill A
Published: July 2021

Claudin-5 is a cell-cell adhesion protein that plays established roles in the blood-brain barrier and cancer. We were the first to demonstrate that it plays a role in the heart with implications for heart failure and cardiomyopathy. Several animal models with cardiac pathology show reduced cardiac claudin-5 levels. Importantly, 60% of human heart failure patients show reduced levels of cardiac claudin-5, independent of changes in other cell junction proteins in the heart, supporting that the loss of cardiac claudin-5 has clinical relevance. To test our hypothesis that claudin-5 plays an important role in protecting the heart, we used mouse models to demonstrate whether circumventing reductions in cardiac claudin-5 is able to improve cardiac histology and physiology, and whether ablation of claudin-5 is able to induce cardiac pathology. We now show that maintaining cardiac claudin-5 levels using gene therapy (cldn5 transduction using an adeno-associated virus vector) significantly reduced cardiac pathology in mice which otherwise demonstrate heart failure and cardiomyopathy indicators. We also present preliminary evidence of the effects of knocking out the cldn5 gene specifically in cardiomyocytes using the inducible cre-loxP system.
Abstract 96: Thioredoxin-1 Is Essential for Hydrogen Sulfide-Mediated Cardioprotection in the Setting of Ischemic-Induced Heart Failure
Authors: Nicholson Chad K , Moody Bridgette F , Hood Rebecca L , Sadoshima Junichi , Calvert John W
Published: July 2021

Background:
Numerous studies have reported the cytoprotective effects of hydrogen sulfide (H2S) in various models of myocardial injury. Here we examined the role that thioredoxin-1 (Trx1) plays in mediating the protective effects of H2S in a model of heart failure.

Methods and Results:
Mice were subjected to 60 min of left coronary artery ischemia followed by 4 wks of reperfusion (R) at which time left ventricular dimensions and function were assessed. Mice received saline (Veh) or H2S in the form of sodium sulfide (Na2S, 100 μ g/kg) at the time of R followed by daily i.v. injections for the first 7 days of R. Mice treated with Na2S experienced less left ventricular dilatation and hypertrophy, displayed improved left ventricular ejection fraction, and displayed improved contractility and relaxation when compared to Veh-treated mice. Studies aimed at evaluating the underlying cardioprotective mechanisms found that Na2S treatment increased the expression of Trx1. Further analysis revealed that this was accompanied by an increase in phosphorylation of apoptosis signaling kinase-1 (ASK1) at serine residue 966 (inhibitory site), as well as a decrease in the phosphorylation of JNK and p38 (downstream targets of ASK1). We also found that Na2S treatment did not improve cardiac dilatation, cardiac dysfunction, or cardiac hypertrophy in cardiac specific Trx1 dominant negative transgenic (Trx1 dnTg) mice when compared to Veh-treated mice.

Conclusion:
These findings provide important information that the upregulation of cardiac Trx1 by H2S in the setting of ischemic-induced heart failure sets into motion events, including ASK1 inhibition, which ultimately leads to cardioprotection.
Abstract 99: Conditional Depletion of CD11c
+
Cells Attenuates Myocardial Ischemia-Reperfusion Injury in Mice
Authors: Li Jun , Zuidema Mozow Y , Zhang Cuihua , Hill Michael A. , Gao Feng
Published: July 2021

Excessive infiltration of pro-inflammatory monocytes /macrophages early after myocardial ischemia/reperfusion (MI/R) contributes to myocardial injury and cardiac dysfunction. We tested the hypothesis that conditional depletion of pro-inflammatory macrophages and dendritic cells may dampen excessive inflammatory response in the infarct myocardium, and thus attenuate MI/R injury. To achieve this, we took advantage of CD11c-diphtheria toxin receptor (DTR) transgenic mice, which allow for selective and conditional ablation of CD11c
+
cells for 2-3 days after diphtheria toxin administration. CD11c-DTR mice with conditional deficiency of CD11c
+
cells and wild type (WT) mice were subjected to MI/R (30 min /48 h). CD11c-DTR mice showed decreased infiltration of CD11c
+
dendritic cells and CD11c
+
F4/80
+
macrophages in the I/R heart, reduced infarct size, attenuated myocardium apoptosis and improved cardiac function compared with those in WT mice. Moreover, inflammatory cytokines, such as TNF-α, MCP-1 and IFN-γ in the infracted myocardium as well as myocardial nitrotyrosine level, an index of oxidative and nitrative stresses, were dramatically decreased in CD11c-DTR mice than those in WT mice. These data indicate that the decreased infiltration of pro-inflammatory macrophages /dendritic cells and subsequent ameliorated inflammation may contribute to attenuated reperfusion injury and better reserved cardiac function in CD11c-DTR mice. This may provide potential novel strategies to target pro-inflammatory cells to reduce detrimental effects while sparing the beneficial wound healing roles of the recruited macrophages.
Abstract 007: Platelet Derived Growth Factor-AA Promotes Migration of Resident Cardiac Stem Cells
Authors: Windmolders Severina , De Boeck Astrid , Koninckx Remco , Daniëls Annick , Hensen Karen , De Wever Olivier , Bracke Marc , Hendrikx Marc , Rummens Jean-Luc
Published: July 2021

Bone marrow mesenchymal stem cells modulate cardiac healing via the release of paracrine factors. Although the exact mechanisms are not yet understood, one promising approach is through the mobilization of endogenous cardiac stem cells. Recently, we reported the existence of a new type of cardiac stem cell, named Cardiac Atrial appendage Stem Cells (CASCs), that is characterized by a high aldehyde dehydrogenase expression. In this study, we hypothesized that conditioned medium from mesenchymal stem cells (MSC-CM) is able to promote migration of resident cardiac stem cells. Therefore, human cardiac fragments were cultured inside a collagen type I matrix and subjected to serum-free DMEM (control) or MSC-CM. By analysis of cell counts, perimeter and area measurements, we found that MSC-CM promoted migration of cells from cardiac fragments throughout the matrix. These migrating cells possessed stem cell properties, as shown by a clonogenicity of 21.6±4.3% (n=3) and pluripotency associated genes like
Oct-4, Nanog, Dppa-3, Tbx-3, c-Myc
and
Klf-4.
Flow cytometrical analysis showed that these cells expressed a CSC-phenotype. Further investigation demonstrated that, comparable to CASCs, migrating cells had a high aldehyde dehydrogenase activity and were able to differentiate towards cardiomyocytes. In addition, we demonstrated an extensive activation of the platelet derived growth factor receptor-α (PDGFRα) in migrating cells subjected to MSC-CM. Subsequent collagen assays indicated that pure PDGF-AA was sufficient to produce migration effects similar to MSC-CM, albeit higher PDGF-AA concentrations than measured in MSC-CM were necessary. Imatinib mesylate, a PDGFR inhibitor, was partially able to attenuate MSC-CM induced migration, pointing out that PDGF-AA might work synergistically with other factors to promote migration. In conclusion, these data demonstrate that MSC-CM stimulates migration of resident CASCs. PDGF-AA is an important paracrine factor in this process, although the synergic role of other growth factors has to be further investigated. These findings contribute to the development of future therapies in which paracrine agents can be utilized to activate the endogenous repair system of the heart.
Abstract 008: Bone Marrow Mesenchymal Stem Cells Prevented Cardiac Arrhythmias Of Infarcted Heart Via Regulating Calcineurin And Potassium Channels Expression
Authors: Cai Benzhi , Chen Nan , Wang Gang , Liu Yanju , Li Xingda , Yang Fan , Lu Yanjie
Published: July 2021

Bone marrow mesenchymal stem cells (BMSCs) emerge as a new promising approach for treating heart diseases. However, the effects of BMSCs-based therapy on cardiac electrophysiological remodeling following myocardial infarction were largely unclear. This study was aimed to investigate whether BMSCs transplantation prevented cardiac arrhythmias in post-infarcted hearts. Myocardial infarction was established in male SD rats, and BMSCs were then intramyocardially transplanted into the infarcted hearts after 3 days. Cardiac electrophysiological remodeling in the border zone was evaluated by western blotting and whole-cell path clamp technique after 2 weeks. We found that BMSCs transplantation significantly ameliorated the increased heart weight index, the impaired left ventricular functions, and the enhanced cardiac fibrosis in infarcted hearts. The survival ratio of infarcted rats was also improved after BMSCs transplantation. Importantly, electrical stimulation-induced arrhythmias were seldom observed in BMSCs-transplanted infarcted rats compared with rats without BMSCs treatment. Furthermore, BMSCs transplantation effectively inhibited the prolongation of action potential duration and the reduction of transient and sustained outward potassium currents in ventricular myocytes from post-infarcted rats. Consistently, BMSCs-transplanted infarcted hearts exhibited the more expression of K
V
4.2, K
V
4.3, K
V
1.5 and K
V
2.1 proteins than infarcted hearts. Moreover, intracellular free calcium level, calcineurin and nuclear NFATc3 protein expression was shown activated in infarcted hearts, which was inhibited after BMSCs transplantation. Collectively, BMSCs transplantation prevented ventricular arrhythmias by reversing the reduction of cardiac potassium channels in post-infarcted heats, which involves attenuated intracellular calcium overload and calcineurin/NFATc3 signal pathway.
Abstract 010: A Separable Upstream Promoter of the Slc8a1 (NCX1) Gene Efficiently Marks Cardiac Cell Populations Derived from Human Pluripotent Stem Cells
Authors: Hidalgo-Gonzalez Alejandro , Ovchinnikov Dmitry A , Hudson James , Cooper-White Justin , Ernst Wolvetang
Published: July 2021

The sarcolemmal Na+/Ca2+ exchanger SLC8A1(NCX) regulates intracellular Ca+ in cardiomyocytes from early developmental stages. The upstream-most SLC8A1(NCX1) promoter is well conserved amongst the homoeothermic animals and contains putative binding sites for transcription factors of the NKX, GATA, STAT and CDX families. We hypothesized that functional cardiac cells with mature cardiac structural markers will express the sarcolemmal Na+/Ca2+ calcium antiporting channel, important for proper functional contractivity of in vitro differentiated human pluripotent stem cells. Pseudotyped lentiviral particles delivering NCX1cp-EGFP reporter cassette were used to confirm the efficiency and specificity of the reporter in rodent foetal cardiac cell isolates, and to establish stable human pluripotent stem cell lines. Cells were differentiated using a 2D induction protocol, and gene expression analysis and protein quantification carried at day 16. Initial NCX1cp-EGFP expression was observed from day 10-11 of cardiac differentiation. Beating foci were visualized 1-2 day after initial NCXCP-EGFP expression, reporter expression was confined to the grouped and individual beating cells, and highly correlated with the efficiency of spontaneously contractile cell production. At later stages, NCX1cp-EGFP expression correlated with clusters of formed spontaneously contractile units harbouring essentially all cardiomyocytes present in cultures, as evidenced by colocalization of high levels of cardiac troponin T (cTnT) and α-actinin proteins. The EGFP+ sorted fraction of differentiated cultures was found to be highly enriched in both early (ISL1, TBX5) and late (cTnT, MYH6) cardiomyocyte markers when compared to the EGFP- fraction. We conclude that a ~3 kb genomic fragment of the distal cardiac-specific promoter of the SLC8A1(NCX1) containing the upstream-most exon of the gene is sufficient to drive the expression of a lentiviral reporter in both rodent heart-derived primary and human (embryonic and induced) pluripotent stem cell-derived cardiac cells. Isolation of a homogenous and functional cardiomyogenic population represents one of the key objectives for cardiac tissue engineering, and in particular in vitro drug screening applications.