bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 205: Feasibility and Testing of a Human Induced Pluripotent Stem Cell Derived Cardiac Patch in a Pre-clinical Swine Model of CHF
Authors: Lancaster Jordan J , Chinyere Ike , Kim Bin Na , Daugherty Sherry , Kim Samuel , Jokerst Clint , Avery Ryan , Larson Brandon , Lancaster Laryenth D , Juneman Elizabeth , Goldman Steven
Published: June 2021

Introduction:
Previously we have demonstrated that a tissue engineered heart patch comprised of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) and fibroblasts improves both left ventricular (LV) systolic and diastolic function in a rat model of CHF. In this study we tested the feasibility of upscaling cardiac patch size and surgical deployment in a swine model of CHF to test clinical utility.

Methods:
Four male Gottingen mini swine 20-25kg and three domestic swine 50-60kg were infarcted using percutaneous methods. Embolizing coils were deployed via catheter distal to the first diagonal branch of the left anterior descending (LAD) coronary artery and animals recovered for 4 weeks. Cardiac patches engineered with bio absorbable polygalactin-910 knitted mesh, dermal fibroblasts and hiPSC-CMs were cultured and implanted on the infarcted epicardium 4 weeks after MI. Cardiac magnetic resonance imaging was performed at baseline, 4 and 8 weeks post MI. All swine were implanted with continuous event recorders to acquire surface electrocardiogram during the entire study. In addition quality of life and functional capacity were assessed through video monitoring and treadmill exertion testing respectively. Infarct size was determined through 2,3,5-triphenyltetrazolium chloride staining.

Results:
LAD occlusion resulted in a significant (P<0.05) decrease EF (15%), and increase in EDV (59%) and ESV (100%). Average TIMI score decreased from 3.0±0 at time of MI to 1.5±0.6 4wks post MI. Cardiac patches were upsized to 6cm diameter for application in the swine. Patches displayed synchronous and spontaneous contractions within 48hrs. The 6cm patches, when implanted effectively covered the infarcted region bridging viable myocardium. Surgical handling and epicardial deployment was successfully accomplished via median sternotomy. The patches were robust in nature and could be deployed via a minimally invasive robotic procedure. No adverse arrhythmic activity was observed. Implantation of the cardiac patch restored activity levels (quality of life) of patch treated swine vs CHF controls.

Conclusion:
Our hiPSC-CM cardiac patch can be constructed in a clinical size, easily handled and implanted on the epicardium of the infarcted heart.
Abstract 207: Electrically-induced Calcium Handling in Human Cardiac Progenitor Cells
Authors: Maxwell Joshua T , Wagner Mary B , Davis Michael E
Published: June 2021

The discovery of a resident population of cardiac stem cells known as cardiac progenitor cells (CPCs) has raised the question of the mechanisms involved in regenerative properties of this niche of cells. The physiology of human CPCs is not well understood, and a thorough understanding of the Ca
2+
handling ability of these cells is crucial in order to fully realize their regenerative potential. Therefore, to characterize the mechanism of electrically-induced Ca
2+
handling in human CPCs, we have used cells loaded with fluo-4 calcium dye and imaged using confocal microscopy to determine the spatial and temporal profile of cytosolic Ca
2+
upon electrical stimulation. Upon activation of electrical stimulation, a large increase in cytosolic Ca
2+
was observed followed by oscillations of the cytosolic Ca
2+
. The electrically-induced Ca
2+
oscillations were found to be dependent on the activity of L-type Ca
2+
channels (LTCC) and the presence of extracellular Ca
2+
. Furthermore, these oscillations could be abolished by pre-treatment of the cells with the inositol 1,4,5-trisphosphate receptor (IP
3
R) antagonist 2-APB. High-speed confocal imaging of the electrically-induced Ca
2+
oscillations in hCPCs revealed that these cells oscillate in a Ca
2+
wave-like fashion with activity originating in isolated regions of the cell and propagating throughout the cytosol and nucleus. In conclusion, we propose that Ca
2+
-dependent regulatory mechanisms can be exploited to influence their differentiation potential leading to improved function in cardiac regenerative medicine.
Abstract 209: Exosomes Derived From c-kit+ Cardiac Progenitor Cells are Essential for Myocardial Repair After Acute Myocardial Function
Authors: Sharma Sudhish , Bigham Grace E , Mishra Rachana , Gruia Flaviu , Brohawn Philip Z , Karanthasis Sotirios , Kaushal Sunjay
Published: June 2021

Background:
Human cardiac progenitor cells (hCPCs), identified by ckit
+
/CD45
-
, provide a promising therapeutic option following myocardial infarction (MI) as their clinical relevance has been validated in the
S
tem
C
ell
I
nfusion in
P
atients with
I
schemic Cardi
o
myopathy (SCIPIO) Phase I clinical trial. The mechanism for their functional recovery of the injured myocardium is unknown.

Hypothesis:
We hypothesized whether CPCs secrete biologically active exosomes and if these exosomes could provide cardioprotection after myocardial infarction (MI).

Methods and results:
Exosomes were isolated from cultured CPCs, generated from the biopsies of right atrial appendage (RAA) from neonatal (nCPCs) and adult (aCPCs) patients with normal functioning myocardium. TEM showed that both CPCs secrete microvesicles, which fall within the same size range as exosomes (80-170nM, diameter). FACS performed for canonical exosomal surface markers CD63, ALIX and CD9 confirmed the presence of exosomes in the secretome of CPCs. Quantification of exosomes by Nanosight NS300 showed that nCPCs produce more than twice the amount of exosomes as compared to aCPCs in 48 hours. Exosomes were internalized by cardiomyocytes, endothelial cells and fibroblasts, within the myocardium. CPCs derived exosomes enhanced angiogenesis as analyzed by HUVEC tube assay formation and proliferation of neonatal rat cardiomyocytes while inhibiting their apoptosis in the presence of oxidative stress and inflammation. Intra-myocardial injection of exosomes into rat myocardium after MI restored ejection fraction (CPCs 63.74±3.68% vs CPCs-exosomes 62 ± 2.97%), attenuated adverse left ventricular remodeling and reduced infarct size which were comparable to CSC therapy at 28 days post MI. CPC exosomes also contain distinctive cargo of miRs and proteins. Immunoblot analysis shows that CPC exosomes are enriched in the paracrine factors VEGFA, ANG1, SCF1 and HGF1, with cardioprotective roles.

Conclusion:
Our findings identify exosomes as the smallest functional unit and potential biomarkers of CPC therapy. CPCs derived exosomes can be utilized as an off the shelf cell-free therapy which eliminates several shortcomings of cell therapy, including cell retention, cell rejection and arrhythmia.
Abstract 210: Mesenchymal Stem Cells Reduce Neutrophil Mediated Inflammation Following Myocardial Ischemia-reperfusion Injury
Authors: Shin Eric , Zemskova Marina , Wang Lanfang , Tirouvanziam Rabin , Garcia Andres , Taylor William R , Levit Rebecca
Published: June 2021

Neutrophils (PMNs) are a central mediator of oxidative stress and tissue necrosis following acute myocardial infarction and reperfusion (MI/R). Currently no effective treatment directed towards PMN mediated injury exists. Cell based therapies may represent a novel strategy to reduce the damaging effects of the early immune response following MI/R injury. In this study, we examine whether mesenchymal stem cells (MSCs) reduce markers of PMN mediated inflammation as a possible mechanism for cardio-protection.
In vitro
analysis of human PMNs was performed following inflammatory stimuli and co-culture with MSCs, MSC conditioned media (MSC-CM), or no treatment. H
2
O
2
production and super oxide (SO) release was measured by amplex red and hydrocyanine based assays respectively. There were significant reductions in H
2
O
2
(p<0.0001, n=4) and SO (p<0.001, n=4) when PMNs were co-cultured with MSCs and MSC-CM compared to PMNs cultured alone.
In vivo
studies were performed in rats following acute MI/R injury. Treatment subgroups included normal saline control or MSCs implanted onto the heart by means of hydrogel encapsulation. One day after MI/R, H
2
O
2
levels were significantly reduced in myocardial tissue when treated with MSCs compared to saline treated hearts (p<0.05, n=5). Furthermore, infiltration of myocardium by CD45+ leukocytes was significantly reduced in animals treated with MSCs compared to saline treated controls as measured by flow cytometry (p<0.05, n=4). The effect of MSCs on functional outcomes was confirmed by use of speckle-tracking echocardiography which demonstrated significant improvements in cardiac function in MSC treated animals compared to saline controls as early as days 7 and persisting to day 28 post injury (p<0.017, n=5).This study demonstrates that MSCs modulate multiple pro-inflammatory functions of PMNs
in vitro
and is the first to demonstrate
in vivo
reductions in ROS production and leukocyte infiltration as a potential mechanism for cardio-protection following MI/R injury, allowing for earlier transition to regenerative processes. Future studies will determine whether PMN infiltration and markers of respiratory burst activity are reduced with MSC therapy.
Abstract 216: Intra-myocardial Injection of a Novel Thermosensitive Hydrogel Inhibits Post-infarct Heart Failure in Rats
Authors: Wen Ying , Li Xiao-yan , Li Ze-yong , Wang Meng-long , Zhang Xian-zheng , Jiang Xue-jun
Published: June 2021

Intra-myocardial injection of hydrogel after myocardial infarction (MI) has been confirmed for preventing post-infarct ventricular remodeling (VR) in numerous studies. Meanwhile, VR or existed heart failure (HF) symptoms of patients with MI was improved after intra-myocardial injection of hydrogels in recent clinical trials. However, whether the injection of hydrogel immediately after MI can prevent post-infarct HF in a long follow-up period is unclear. In present study, MI was induced by permanent coronary artery ligation in SD rats. Immediately, 100μl Dex-PCL-HEMA/ PNIPAAm (DPHP) hydrogel solution was injected into the infarct border myocardium of experimental rats, while phosphate-buffered saline (PBS) was employed in control group. Twelve weeks later, compared with PBS group (n=11), echocardiography and hemodynamics showed left ventricle dilatation was attenuated (end-diastolic diameter 9.30±0.73 mm vs 10.74±0.57 mm,
p
<0.01) and cardiac function was improved (+dp/dt 12351±1158 mmHg/s vs 9488±695 mmHg/s,
p
<0.01) in hydrogel group (n=11). Serum brain natriuretic peptide (107.74±18.21 pg/ml vs 132.17±20.62 pg/ml,
p
<0.05) and cardiac aldosterone production of hydrogel group was suppressed, and so did pulmonary congestion. Histological analysis indicated thicker scar, smaller infarct size, diminished cardiac fibrosis (collagen volume fraction 21.74±3.13% vs 33.38±3.08%,
p
<0.01) and hypertrophy in hydrogel group. Higher Col3α1 mRNA (27.32±0.71 vs 14.29±1.37,
p
<0.01) in infarct zone but lower Col3α1 mRNA (0.97±0.12 vs 9.06±1.04,
p
<0.01) and ANP mRNA (10.03±1.49 vs 24.74±2.40,
p
<0.01) in non-infarct zone (NIZ) were detected by RT-PCR in hydrogel group. These data proves that the intra-myocardial injection of DPHP hydrogel immediately after MI can improve cardiac function including diastolic function, decrease infarct size, and enhance fibrotic healing in the infarct area. On the other hand, this treatment suppresses reactive fibrosis and hypertrophy in NIZ and thus delays post-infarct VR and prevents HF. DPHP hydrogel implantation immediately after MI is an effective strategy to prevent abnormal cardiac remodeling and have a long term beneficial effect even after it has been biodegraded.
Abstract 220: Epigenetic Reprogramming Rescues Diabetic Endothelial Progenitor Cell Dysfunctions and Enhances Their Reparative Activities in Ischemic Tissue Repair
Authors: Zhou Jibin , Garikipati Venkata N , Zhongjian Cheng , Benedict Cindy , Nickoloff Emily , Gao Erhe , Verma Suresh , Khan Mohsin , Kishore Raj
Published: June 2021

Type 2 Diabetes mellitus (DM) is the leading cause of death in the United States with concomitant complications from circulatory disorders including endothelial cell (EC)/endothelial progenitor cell (EPC) dysfunction in a variety of organs including heart. Although clinical trials of bone marrow progenitors/EPCs have shown some promise, however, diabetic dysfunction of EPC limits the functional benefits of stem/EPC-based cellular therapies. Emerging evidence suggests that epigenetic silencing of functional genes leads to dysfunctional EPCs in diabetes. In the present study, we show that epigenetic modulation of diabetic EPCs using small molecules epigenetic modifiers rescue diabetes-induced EPC dysfunction. EPCs from diabetic (db/db) mice were treated with valproic acid (VPA, histone deacetylase inhibitor) and 5’-azacytidine (5’-Aza; DNA methyltransferase, DNMT, inhibitor), and their function was determined both in vitro and in vivo. In contrast to untreated EPCs, treatment with VPA/5’-Aza, enhanced their angiogenic activities including tube formation, survival and proliferation. Expression of multiple pro-angiogenic genes including eNOS, and VEGF/VEGFR2 was upregulated in reprogrammed EPCs and chromatin immunoprecipitation experiments confirmed enhanced histone3-lysine9 acetylation (H3K9ac) of these gene promoters. Reprogrammed EPCs displayed reduced protein expression of DNMT3a and DNMT3b; whereas expression of histone H3K9ac and H3K14ac (gene activating epigenetic mark) was up-regulated. In db/db mouse hind limb ischemia model, transplantation of reprogrammed EPCs restored blood flow, enhanced capillary density and muscle architecture and resulted in significant limb salvage. In a myocardial ischemia model in db/db mice, transplantation of reprogrammed EPCs enhanced left ventricular functions, reduced infarct size and increased capillary density in the border zone. Taken together our data suggests that the reprogramming of db/db-derived EPCs might result in pattern shift of epigenetic modifications, leading to up-regulation of functional genes repressed by hyperglycemia and may rescue dysfunctional EC/EPC phenotype leading to improved functional repair capacity of diabetic stem cells/EPCs.
Abstract 227: Single-blind Study of Q-sweat in Postural Orthostatic Tachycardia Syndrome (POTS)
Authors: Ahsan Muhammad M , Thompson Tara , Ashangari Chandralekha , Suleman Amer
Published: June 2021

Background:
Postural Orthostatic Tachycardia Syndrome (POTS) is a form of dysautonomia that is estimated to impact between 1,000,000 and 3,000,000 Americans and millions more around the world. Symptoms often include orthostatic intolerance such as dizziness, fatigue, excessive sweating and many others. The aim of this study is to determine the Q-sweat study in POTS patients.

Methods:
This study was a randomized, single-blind study. Patients evaluated between October 2014 to June 2015 were included. The skin on the leg and wrist is wiped with acetone, then alcohol and dried, cleaning the skin in preparation for the test. Four electrodes filled with acetylcholine are placed on three areas of the leg and one area on the wrist. A mild electrical current called iontophoresis is then applied to help the drug stimulate the sweat glands. This evokes sweating at the site, but it also allows the body to release its own acetylcholine, resulting in sweat production at nearby sites. After a stimulus (e.g. a deep breath) any deviation from the baseline is reported. If no change is seen, a stronger stimulus is applied (e.g. electrical stimulation) and if there is still no change seen, an “absent response” is reported. The onset latency and magnitude of response can be quantified, although the results are highly variable within and between subjects. The sweat response is measured Q-sweat recordings of Left proximal leg, Left distal leg and Left foot sites were analyzed. Results are categorized as Normal response, no response, hung up and prolong response.

Results:
A total 144 POTS patients were included, Out of 144 POTS patients 88% are females (n=127, mean age 31.93±12.37) and 12% are males (n=17, mean age 31.59±11.14), Left proximal leg site 69/144(48%) Patients had hung up, 8/144(6%) had No response, 29/144(20%) had Normal, 38/144(26%) had Prolong. Left distal leg site 72/144(50%) had hung up, 13/144(9%) had No response, 23/144(16%) had Normal, 36/144(25%) had Prolong. Left foot site 36/144(25%) had hung up, 35/144(24%) had No response, 40/144(28%) had Normal, 33/144(23%) had Prolong response.

Conclusion:
Postural Orthostatic Tachycardia Syndrome had a higher percentage of patients with Hung up response.
Abstract 230: Humoral Immune Response Against
Trypanosoma
c
ruzi
Infection Using Plasmid DNA Immunotherapy in the Experimental Canine Model of Chagas Disease
Authors: Carrillo-Sanchez Silvia C , Martinez-Cruz Mariana , Rodriguez-Morales Olivia , Arce-Fonseca Minerva
Published: June 2021

Chagas disease or American trypanosomiasis is a parasitic chronic infectious disease that is one of the major causes of heart disease in Latin America. However, this disease is now present in non-endemic regions such as Canada, the United States of America, Europe and is one of the most important emerging health problems. The World Health Organization reported recently that approximately 8 million people are infected worldwide. The pBCSP and pBCSSP4 plasmids were evaluated as DNA-antigens to promote the generation of antibodies against
Trypanosoma cruzi
as an option of immunotherapy in a canine model to protect against Chagas disease. We worked with 30 dogs, which were divided into 5 experimental groups, dogs from four of these groups were infected with 3,500 parasites / kg body weight (H8 strain) and at 15 days post infection were treated thrice at 2-week intervals with 500 micrograms of pBCSP and pBCSSP4 plasmids, separately and combined; dogs from control groups were treated with cloning empty vector and saline solution. Specific IgG antibodies detection by ELISA showed that the plasmid-DNA treatment generated a response of increased total IgG significantly with respect to the status preimmune. A significant increase of IgG after the two last doses was generated by the pBCSP plasmid treatment, whereas that the pBCSSP4 plasmid treatment induced a significant IgG rise only after the third dose of the immunotherapy. The combination of both plasmids showed an increase similar to that induced by the infection alone. An unpolarized Th1 or Th2 response after immunotherapy or infection was observed by a similar IgG1 or IgG2 antibodies detection, respectively. This shows that therapy with DNA recombinant plasmids generates a specific humoral immune response against
T. cruzi
in dogs with experimental Chagas disease.
Abstract 233: DJ-1 Gene Therapy Opposes the Development of Ischemic Heart Failure
Authors: Shimizu Yuuki , Sha Di , Chin Lih-Shen , Li Lian , Calvert John
Published: June 2021

Background:
DJ-1 is a cytoprotective protein that is activated in response to various pathological stimuli. The ability of DJ-1 to perform its cellular actions is dependent on a conformational change, as evidenced by previous findings that it is cleaved by oxidative stress and that this cleavage activates its cytoprotective function. Previously, we demonstrated that the loss of functional DJ-1 lead to the enhancement of myocardial infarction and exacerbation of cardiac dysfunction following the onset of acute myocardial ischemia-reperfusion (I/R) injury. Currently, it is not known if the gain of functional DJ-1 has cardioprotective effects. Here, we investigated if the delivery of the cleaved form of DJ-1 using a gene therapy approach protected against the development of ischemic-induced heart failure.

Methods and Results:
We generated a viral vector to express the active form of DJ-1 (AAV9-DJ1WTΔC). To test the efficiency, specificity, and dose-dependence of AAV9-mediated transduction, we injected increasing doses of AAV9-GFP into the femoral vein of mice. A dose-dependent increase in GFP levels was observed in the heart and our analysis found that the 2x10
11
vg/mL dose achieved maximal delivery to the heart with minimal delivery to other tissue. GFP-stained sections of heart indicated that a substantial proportion of cardiomyocytes expressed the transduced gene. Next, we evaluated the protein expression of the full-length and active form of DJ-1 2 weeks after the administration of AAV9-DJ1WTΔC. No changes in the expression of the full-length form of DJ-1 were observed, but a 2-fold increase in the active form of DJ-1 was observed. Finally, mice administered AAV9-DJ1WTΔC displayed significant less left ventricular dilatation, left ventricular dysfunction, and hypertrophy compared to the AAV9-Empty treated following mice 60 minutes of myocardial ischemia and 4 weeks of reperfusion.

Conclusion:
These results indicated that DJ-1 is an endogenous cytoprotective protein that protects against the development of ischemic heart failure. Importantly, these results demonstrate the feasibility of using a gene therapy approach to deliver the cleaved form of DJ-1 to the heart as a therapeutic strategy to protect against ischemic heart disease.
Abstract 234: Physiological Toxicity Assessment of Tyrosine Kinase Inhibitors with Human Induced Pluripotent Stem Cell Cardiomyocytes
Authors: Stohlman Jayna , Schocken Derek , Vicente Jose , Chan Dulciana , Johannesen Lars , Zhang Xiaoyu , Abassi Yama , Strauss David , Blinova Ksenia
Published: June 2021

Tyrosine kinase inhibitors (TKIs) have improved targeted therapeutics for cancer management despite possible cardiac side effects, including decreased contractility. A combination microelectrode and impedance platform was used to assess field potential (FP) electrical activity and contractility, respectively, after acute and chronic exposure of six TKIs on human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs). Four non-TKI drugs were tested to confirm assay sensitivity.

hERG current inhibitors dofetilide and pentamidine increased beat rate (BR) corrected FP duration (FPDc). Dofetilide (6μM) induced transient early afterdepolarizations (EADs), characterized as ectopic beats/notching in the FP signal, and decreased impedance amplitude (BAmp) at 48hrs. Multi-channel blocker amiodarone (2μM) acutely decreased FPDc, with quiescence up to 1hr, and increased BAmp after 24hrs. Doxorubicin decreased BAmp, inducing quiescence starting at 48hrs (5μM and 10μM). Nilotinib, sorafenib and sunitinib, which have varying documented risks of clinical QT prolongation and/or cardiotoxicity, increased FPDc. With highest tested doses, nilotinib (6μM) generated persistent EADs and increased BR, sunitinib (2.5μM) produced tachyarrhythmias with minimal impedance-evident contractions that recovered by 24hrs, and sorafenib (10μM) induced quiescence that recovered by 3hrs. Imatinib, erlotinib and lapatinib, which have fewer clinical cardtiotoxic effects, did not cause rhythm disturbances. Erlotinib and lapatinib had no significant effect on FPDc, lapatinib (2.5μM and 5μM) decreased BR, while imatinib (15μM) acutely increased FPDc and increased BR after 48hrs.

Tandem electrical and impedance screening in hiPSC-CMs for drug cardiotoxicity was consistent with the clinical experience for TKIs, suggesting its potential utility for preclinical cardiotoxicity assessment for proarrhythmia and decreased contractility.
Abstract 236: Myocardial Relaxation is Modified by Fast Stretch but not Titin Isoform Size
Authors: Chung Charles S
Published: June 2021

Fast myocardial relaxation is important in vivo to reduce left ventricular pressure and promote efficient filling after cardiac contraction and ejection. Afterload is theorized to be a modifier of relaxation, but afterload reduction does not appear to help patients with diastolic dysfunction or Heart Failure with preserved Ejection Fraction. We hypothesized that a fast stretch, not afterload, modifies the relaxation rate, and that this relationship is modified by the giant elastic protein titin. A rat model carrying a spontaneous mutation in the RNA binding motif-20 (RBM20) results in long titin isoforms. Cardiac trabeculae from wild-type and RBM20 rats were dissected, paced at 0.5 Hz, and stretched to Lo, the length at which maximum isometric twitch force was developed. Isotonic load clamps maintained a specified afterload in experimental twitches by shortening and (if needed) relengthening the muscles using feedback control. Identical protocols were performed at reduced preload and two temperature conditions (37°C and 25°C). We determined that myocardial relaxation rate (1/tau) was modified by fast stretch (strain rate) just prior to relaxation; preventing stretch eliminated the inverse relationship between afterload and relaxation rate. Computational modeling indicates that fast stretch enhances crossbridge detachment and that passive tension compensates for some of the active force reduction during stretch. Stretch of 1 length•s
-1
causes a 240% increase in relaxation rate (54.6 s
-1
vs 22.5 s
-1
) at 37°C. Relaxation rate was preload independent and was slower but more responsive to stretch at 25°C. Trabeculae from long-titin mutant rats were not statistically different than control animals, but trend (p<0.1) towards faster relaxation and may require faster stretch to maintain a load clamp at 50% peak developed force. The faster stretch may compensate for a reduced passive tension in the long-titin mutant rats that made it harder to maintain the load-clamp during stretch. In all, these data suggest that fast myocardial stretch (strain rate) at end systole is a preload independent mechanism to modify the relaxation rate. The fast stretch appears to enhance crossbridge detachment to accelerate relaxation independently of titin based stiffness.
Abstract 24: Hydrogen Sulfide Potentiates Bone Marrow-derived Angiogenic Progenitor Cell-mediated Ischemic Limb Angiogenesis in Diabetic Animals
Authors: Cheng Zhongjian , Garikipati Venkata NS , Nickoloff Emily , Wang Chunlin , Polhemus David , Zhou Jibin , Benedict Cynthia , Verma Suresh K , Khan Mohsin , Rabinowitz Joseph E , Lefer David , Kishore Raj
Published: June 2021

Rationale:
Critical hindlimb ischemia (CLI) represents an outcome of peripheral artery disease with a high incidence in diabetic patients. Recently, insufficient hydrogen sulfide (H
2
S) production has been implicated in cardiovascular disease.

Objective:
Explore the role of H
2
S in diabetes-induced bone marrow-derived angiogenic progenitor cells (BMAPC) dysfunction and its therapeutic effects on ischemic hindlimb injury of diabetes.

Methods and Results:
Diabetic BMAPCs from db/db mice showed decreased intracellular H
2
S production and blunted cystathionine γ-lyase (CSE) expression-an enzyme required for H
2
S generation. High glucose (HG) inhibited H
2
S production, migration and increased apoptosis of BMPAC that was rescued by H
2
S donor diallyl trisulfide (DATS) or overexpression of CSE. Administration DATS or local injection of diabetic BMAPCs overexpressing CSE significantly potentiated BMAPC-mediated blood flow recovery, capillary and arteriole formation, skeletal muscle architecture preservation, cell survival and decrease of perivascular infiltration of monocytes (CD68
+
) cells in ischemic skeletal muscle. Moreover, overexpression of CSE increased diabetic BMAPCs homing and engraftment in ischemic hindlimb. HG-impaired human cardiac microvascular endothelial cells (HCMVECs) tube formation and migration were rescued by DATS or overexpression of CSE. Mechanistically, HG increased threonine-495 phosphorylation of eNOS (eNOS-pT495) and inhibited nitric oxide (NO) production in HCMVECs which were rescued by DATS or overexpression of CSE. Silencing CSE by siRNA impaired tube formation and increased eNOS-pT495 expression in HCMVECs. Finally, HG-treated BMAPCs impaired mouse microvascular endothelial cell tube formation that was rescued by DATS.

Conclusions:
Our data suggests that CSE downregulation-induced H
2
S insufficient plays a critical role in diabetes-mediated BMAPC dysfunction. Administration of DATS or overexpression of CSE improves diabetic BMAPC-mediated angiogenesis/neovascularization via, at least partially, eNOS inactivation/NO reduction pathways. Our data indicate that H
2
S and overexpression of CSE in diabetic BMAPCs may open novel avenues for cell-based therapeutics of CLI in diabetic patients.
Abstract 243: A Novel
PRKAG2
Mutation (K475E): Infantile-onset Hypertrophic Cardiomyopathy Phenotype and Impact on mTOR Signaling
Authors: Xu Yanchun , Liu Hong , Phornphutkul Chanika , Hardie D. Grahame , Dudley SC , Padbury James , Tseng Yi-Tang
Published: June 2021

Objective:
PRKAG2
encodes the 2 subunit isoform of the 5’ AMP-activated protein kinase (AMPK), a heterotrimeric enzyme with major roles in regulation of energy metabolism in response to cellular stress. Mutations in
PRKAG2
have been implicated in a unique hypertrophic cardiomyopathy (HCM) characterized by cardiac glycogen overload and hypertrophy. We identified a novel
PRKAG2
mutation in a neonate with prenatal onset of HCM noted on a 27-week prenatal ultrasound. Molecular testing for HCM revealed a
de novo
PRKAG2
mutation (K475E). We aim to investigate the signaling pathway of mutation in PRKAG2 gene.

Methods:
HEK-293 and H9C2 cells stably expressing wild type (WT) or K475E FLAG-tagged 2 from a tetracycline-inducible promoter were established. Basal AMPK activities were measured at varying AMP concentrations. Phenformin-stimulated AMPK activity and T172-phosphorylation were also measured. Changes in mTOR signaling pathway were accessed by Western blot. Cells were treated with angiotensin II, rapamycin (RAPA) or both followed by staining or flow cytometry to assess cell area/size. Primary fibroblasts from the K475E patient and non-diseased controls were cultured and compared. Finally, transgenic mice with cardiac-specific overexpression of human wild type (Tg
WT
) or K475E (TgK
475E
) were established for further study of the
PRKAG2
phenotype by histology and echocardiography.

Result:
HEK-293 cells: K475E mutation induced a markedly increase in the basal AMPK activity and T172-phosphorylation, reduced sensitivity to AMP in allosteric activation and loss of response to phenformin. H9c2 cells: K475E mutation induced increases in the phosphorylation of p-P70S6K, p-4EBP1 and a hypertrophy phenotype which was reversed by RAPA. Transgenic mice: Tg
K475E
mice had an early onset hypertrophy and were prone to sudden death. Histology revealed large vacuoles and high glycogen content in Tg
K475E
myocytes throughout the ventricles. Echocardiography confirmed significantly enlarged heart in Tg
K475E
mice.

Conclusions:
We identified a novel infantile-onset
PRKAG2
mutation with a severe cardiac phenotype. In vitro study revealed involvement of mTOR signaling pathway and reversal of the hypertrophy phenotype by mTOR inhibitor.
Abstract 246: Acetylation of Vgll4 Regulates Hippo-yap Signaling and Postnatal Cardiac Growth
Authors: Lin Zhiqiang , Guo Haidong , Zohrabian Sylvia , Cao Yuan , Pu William T.
Published: June 2021

Binding of the transcription co-activator YAP with the transcription factor TEAD stimulates growth of the heart and other organs. Many signaling pathways, including the Hippo kinase cascade, converge to regulate YAP activity. However, less in known about the mechanisms that govern TEAD. YAP overexpression potently stimulates fetal cardiomyocyte (CM) proliferation, but YAP’s mitogenic potency declines postnatally, when mammalian cardiomyocytes largely exit the cell cycle. Here, we show that VGLL4, a CM-enriched TEAD1 binding protein, inhibits CM proliferation by limiting its binding to YAP and by targeting TEAD1 for degradation. VGLL4 antagonism of TEAD1 was governed by its acetylation at K225. Overexpression of VGLL4-K225R, an acetylation-refractory mutant, enhanced TEAD1 degradation, limited neonatal CM proliferation, and caused CM necrosis and heart failure. Our study defines an acetylation-mediated, VGLL4-dependent switch that regulates YAP-TEAD1 activity and restrains CM proliferation. These insights may enable more effective regulation of TEAD-YAP activity in applications ranging from cardiac regeneration to restraining cancer.
Abstract 248: Age-specific Differences in Outcomes Following Isoproterenol-induced Sudden Cardiac Death in Guinea Pigs
Authors: Woulfe Kathleen C , Chau Sarah , Walker Lori A , Tompkins Christine , Sucharov Carmen C , Miyamoto Shelley D , Stauffer Brian L
Published: June 2021

Background:
The pathophysiological mechanisms involved in adult and pediatric heart failure (HF) are unique. One example of clinical differences in these two patient populations is the need for primary prevention implantable defibrillators (ICDs) in adults with HF, whereas pediatric HF patients infrequently have ventricular arrhythmias and rarely require ICDs. To better understand the age-specific molecular mechanisms involved in HF, we are developing a guinea pig model of pediatric HF. We have found age-specific differences in guinea pigs acutely treated with isoproterenol (ISO). Additionally, initial exposure to ISO leads to sudden death in guinea pigs. This sudden death has previously been reported as an interaction with an inhaled volatile anesthesia. We hypothesized that isoproterenol is leading to sustained ventricular arrhythmias in guinea pigs.

Methods:
Adult (n= 11) and young (n= 50) guinea pigs were treated with vehicle, 16 mg/kg/day or 32 mg/kg/day of ISO by osmotic pump (Alzet) implanted under isoflurane anesthesia. Cardiac rhythm was monitored in a subset via simultaneously implanted Linq recorders (Medtronic, n= 12).

Results:
Acute exposure to ISO in the presence or absence of isoflurane resulted in sudden death in adult and young guinea pigs. Four of the 6 adult guinea pigs exposed to ISO died even with attempts at resuscitation. In contrast, 61% (22 out of 36) of the young guinea pigs treated with ISO arrested and 60% (13 out of 22) were rescued with chest compressions. Analysis of the heart rhythms demonstrated that the guinea pigs experienced ventricular fibrillation. The arrhythmia was transient in the young guinea pigs, but sustained in the adult.

Conclusions:
Acute ISO leads to age-dependent differences in arrhythmias and sudden death in guinea pigs. Contrary to prior reports, this response occurs independently of isoflurane. These age-specific differences suggest unique mechanisms in calcium handling, which can lead to arrhythmias. Guinea pigs may be a useful model for the age-related differences in HF arrhythmias seen in humans. A better understanding of these differences may lead to the development of therapies to protect adult HF patients from arrhythmias.
Abstract 249: The Epigenetically Modified Pro-inflammatory Cardiac Fibroblast Drives Cardiomyocyte Dysfunction in Pulmonary Hypertension-induced Right Ventricular Pressure Overload
Authors: Bruns Danielle R , Thoemmes Stephen F , Buttrick Peter M , Walker Lori A
Published: June 2021

Right ventricular (RV) dysfunction is a strong predictor of survival in a variety of clinical contexts including pulmonary hypertension (PH). Thus elucidating mechanisms that contribute to RV dysfunction is of great importance. Persistent inflammation, a central component of PH-induced RV dysfunction, is regulated in part by cardiac fibroblasts (Cfib). We hypothesized that persistent changes in the Cfib pro-inflammatory phenotype may be mediated through epigenetic processes such as DNA methylation and that secretion of inflammatory signals from activated Cfib drives cardiomyocyte dysfunction and contributes to myofibrillar disarray. Therefore the purpose of this investigation was to assess epigenetic changes to pro-inflammatory Cfib and the contribution of the Cfib secretome on myocyte function. We approached this question using a large animal model with significant resonance with human disease- the neonatal calf exposed to hypobaric hypoxia (HH). HH caused RV Cfib global DNA hypomethylation. RNA-seq identified 2115 genes significantly changed in HH including 193 transcriptional regulators and 21 genes involved in DNA methylation. Further, we identified 105 inflammatory genes with enrichment of IL-1β as a central node of inflammatory signaling. Mass spec profiling of conditioned media (CM) from Cfib isolated from the PH calf (PH-CM) also confirmed significant pro-inflammatory protein secretion. We then examined the effects of PH-CM on adult rat ventricular myocytes (ARVM) in culture. Brief exposure to PH-CM caused marked dedifferentiation of ARVM to a neonatal-like phenotype that exhibited spontaneous contractile behavior. This response was not seen in ARVM exposed to CM from Cfib from the normoxic RV. These data suggest the combination of pressure overload and hypoxia causes epigenetic reprogramming of pro-inflammatory Cfib, signaling a phenotypic transformation in a population of myocytes which likely contributes to RV dysfunction. Therapies that target this inflammatory process have the potential to prevent RV dysfunction.
Abstract 25: A Streamlined Technique to Stabilize C-kit Expression in Cardiac Mesenchymal Cells
Authors: Dassanayaka Sujith , Long Bethany W , Zafir Ayesha , Ghafghazi Shahab , Hunt Gregory N , Noble Camille T , DeMartino Angelica M , Brittian Kenneth R , Jones Steven P , Wysoczynski Marcin
Published: June 2021

Adoptive transfer of various reparative cells attenuates cardiac dysfunction in preclinical models of heart failure. Although c-kit sorted cells do not transdifferentiate to an extent sufficient to explain improvements in ventricular function, their adoptive transfer reliably attenuates cardiac dysfunction. Curiously, few studies report maintenance of c-kit expression with passage, and our experience indicates rapid dissipation of c-kit. Here, we asked whether we could stabilize c-kit expression during passage of c-kit sorted cells. To this end, we standardized an approach to isolate c-kit
+
cardiac mesenchymal cells (CMCs) and to preserve c-kit expression over several passages (panel A). This protocol was predicated on the differential adhesion capacity of c-kit sorted CMCs, allowing their stratification into two groups: rapidly adhering (RA) and slowly adhering (SA). After characterization of these cells (panel B), we tested their reparative capacity using echo-guided, percutaneous intracavitary delivery in mice at two days following myocardial infarction (MI). SA, but not RA, CMCs significantly reduced scar area and improved ejection fraction (EF) compared to [cell-free] vehicle (panels C, D, and E). Examination of the post-MI hearts indicated augmented ischemic zone capillary formation in the SA group (panel F), consistent with potential neovascularization. In conclusion, we established a method to stabilize c-kit expression in a reparative subset of CMCs (i.e. SA CMCs). Our results, however, did not reconcile whether such maintenance of c-kit expression is required for the reparative function of the SA CMCs, which is the subject of future investigation.
Abstract 250: Differential Role of Autophagy in Idiopathic versus Ischemic Heart Failure in Humans
Authors: Bugyei-Twum Antoinette , Singh Krishna K , Billia Filio , Connelly Kim A
Published: June 2021

Background:
Autophagy is an evolutionary conserved process that plays a key role in a variety of physiological and pathological processes. Despite its beneficial role, excessive/insufficient autophagic activity is known to contribute to the pathogenesis of cardiovascular disorders, including ischemia/reperfusion injury and heart failure. However, the differential role of autophagy in idiopathic versus ischemic heart failure remains unknown.

Methods:
To investigate the role of autophagy and associated apoptosis in idiopathic versus ischemic heart failure, we obtained LV myocardium biopsies from healthy controls (via 3 commercial sources), and from 10 patients with idiopathic and ischemic end-stage heart failure before the insertion of a left ventricular assist devise. The expression of inducers/markers of autophagy (mTOR, phospho-mTOR, LC3-I/II, p62, Beclin-1, autophagy-related genes ATG4B/ATG5) and apoptosis (Bcl-2 and caspase-3) were assessed at the transcript and protein level using quantitative RT-PCR and Western blotting.

Results:
Autophagy was activated in both idiopathic and ischemic heart failure in comparison to control, as confirmed by a significant reduction in mTOR expression/activation and a 3.4-fold and 2.2-fold increase in LC3 II/I ratio, respectively. An increase in apoptosis, marked by increased caspase3 and Bcl2 expression, was also observed in both groups in comparison to control. Interestingly, autophagy activity—marked by decreased mTOR expression/activation, increased ATG4B, ATG5, and Beclin-1—was significantly higher in idiopathic heart failure, when compared to ischemic heart failure. While we observed increased autophagic activity in idiopathic heart failure, p62 expression was also significantly increased in this group (2.8-fold increase; p<0.05), demonstrating an impairment of autophagic flux in idiopathic versus ischemic heart failure.

Conclusions:
For the first time, we provide a direct comparision of autophagy and apoptosis in idiopathic versus ischemic heart failure. Our data, demonstrating an excessive yet insufficient autophagic activity in idiopathic heart failure, suggests a differential role of autophagy apoptosis in idiopathic versus ischemia-related heart failure.
Abstract 251: Mixed Lineage Kinase 3 Functions as a Protein Kinase G I Alpha Antiremodeling Substrate in the Heart
Authors: Calamaras Timothy D , Baumgartner Robert A , Wang Guang-rong , Lane Angela M , Aronovitz Mark , Davis Roger J , Karas Richard H , Blanton Robert M
Published: June 2021

Background:
cGMP-dependent protein kinase G I α (PKGIα) via its leucine zipper (LZ) domain prevents adverse cardiac remodeling. Identifying and characterizing PKGIα-LZ dependent substrates may reveal novel therapeutic targets in the myocardium. We previously identified the LZ-containing Mixed Lineage Kinase 3 (MLK3) as a potential PKGIα substrate. Further, MLK3 whole body knockout mice had increased LV hypertrophy and dysfunction after pressure overload. In this study we sought to further explore the PKGIα-MLK3 interaction, and to investigate MLK3 in human cardiomyopathy.

Results:
We first tested for a direct interaction between PKGIα and MLK3. Using affinity purified recombinant proteins we observed co-precipitation of PKGIα and MLK3 that was disrupted by mutation of the PKGIα LZ domain (LZ mutation: MLK3 binding decreased by 61.61% ±13.6, n=4). In mouse heart the interaction between native MLK3 and PKGIα was observed by co-immunoprecipitation (n=3). PKGIα phosphorylated MLK3 at the activation loop in vitro, which was attenuated by inhibiting PKGIα kinase function (n=3).

We next tested if MLK3 regulates hypertrophy of cultured cardiomyocytes. Adult rat ventricular cardiomyocytes treated with the MLK3 inhibitor URMC-099 (100 nM, 48 hrs) exhibited increased cell size compared to vehicle treated cells (23.3% increase ± 3.64 SEM vs DMSO vehicle, n=3, 50 cells per treatment).

Finally, we examined MLK3 expression in hearts from human patients with non-ischemic or hypertrophic cardiomyopathy. Compared to normal LV samples (NDRI, n=4), MLK3 expression was markedly elevated in both non-ischemic and hypertrophic cardiomyopathy LV samples (MLK3/GAPDH: non-ischemic: 6.26 ADU ± 0.85, n=9, hypertrophic: 6.97 ADU ± 1.42, n=8).

Conclusion:
These data support a model in which PKGIa directly binds and activates MLK3, leading in the cardiomyocyte to repression of cellular hypertrophy. Our findings in tissue from human failing hearts further suggest that MLK3 upregulation may act as a compensatory anti-remodeling signal in the setting of cardiac dysfunction, which ultimately becomes overwhelmed by pro-remodeling signals. More broadly our findings support that identifying PKGIa LZ-dependent substrates can reveal novel anti-remodeling molecules.
Abstract 256: Ablation of 14-3-3 Protein Exacerbates Doxorubicin Induced Cardiomyopathy: Role of Apoptosis Signal Regulating Kinase-1
Authors: Karuppagounder Vengadeshprabhu , Arumugam Somasundaram , Sreedhar Remya , Giridharan Vijayasree V , Afrin Rejina , Harima Meilei , Suzuki Kenji , Ueno Kazuyuki , Thandavarayan Rajarajan A , Watanabe Kenichi
Published: June 2021

Background:
14-3-3η family members are dimeric phosphoserine-binding proteins that participate in signal transduction and checkpoint control pathways. Anthracycline anticancer drug doxorubicin (Dox) can induce cardiotoxicity, which is believed to be based on inflammatory or oxidative injury. However, the role of 14-3-3η is not clear in Dox induced cardiac injury. We examined the role of 14-3-3η protein and apoptosis signal-regulating kinase-1 (Ask1) and inflammatory signaling by using transgenic mice with cardiac-specific expression of a dominant-negative 14-3-3η protein mutant (DN 14-3-3) in Dox induced cardiac injury.

Methods:
Cardiac dysfunction was induced by a single injection of Dox into wild-type (WT) and DN 14-3-3η mice. By the end of the study, echocardiography was performed to assess the cardiac function. The heart tissues were used for histopathology and western blotting.

Results:
Left ventricular (LV) fractional shortening and ejection fraction were dramatically decreased in DN 14-3-3η mice, when compared to WT mice after Dox injection. Inactivation of 14-3-3η protein significantly increased Dox induced mortality. Significant Ask1 activation in DN 14-3-3η after Dox injection was evidenced by pronounced de-phosphorylation at Ser-967 and intense immunofluorescence observed LV sections. Marked increase in myocardial apoptosis, cardiac hypertrophy, and fibrosis were observed with a corresponding up-regulation of proinflammatory factors and cytokine expression in DN 14-3-3η mice after Dox injection. Furthermore cardiac expression of high mobility group box (HMGB)1 and its cascade protein expressions were significantly up-regulated in DN 14-3-3η mice compared to WT mice after Dox injection.

Conclusion:
Taken together, these findings suggest that depletion of 14-3-3η protein causes reduce survival rate in mice with cardiac dysfunction, presumably via activation of downstream Ask1 signaling pathways. This may provide a novel therapeutic strategy against Dox-induced cardiac injury by regulating Ask1 signaling.