bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 168: Stress Dependent MicroRNA Mediate Crosstalk Between DNA Repair ′ Senescence & Angiogenic Signaling
Authors: Espinosa-Diez Cristina , Wilson RaeAnna , Chaterjee Namita , Hudson Clay , Ruhl Rebbeca , Helms Erin , Anand Sudarshan
Published: March 2022

The endothelium is highly exposed to a myriad of stressors that can lead to endothelial dysfunction if persistent. One of the hallmarks of endothelial stress is increased DNA damage. MRN complex plays a vital role in DNA double strand break repair and replication. Nevertheless, its role in cardiovascular impair has not been studied in depth. Our results show that a cohort of microRNAs, upregulated under genotoxic and oxidative stress conditions, target MRN complex inducing senescent phenotype in endothelial cells. These microRNAs are rapidly transcribed in response to different types of stress, however the transcription factors that induce the expression of these miRs in response to DNA damage in endothelial cells is not known. We have profiled a subset of active transcription factors as c-Myc, Nrf2 and Nrf1 that robustly induce these miRs. Furthermore, our gain and loss-of-function studies indicate that these miRs, miR-99b and miR-494, affect telomerase activity, enable the activation of p21 and Rb pathways and regulate angiogenic sprouting. Ectopic expression of the miR mimics inhibits angiogenesis
in vivo
suggesting that targeting the MRN complex could be a viable anti-angiogenic strategy. Finally, our studies suggest a crosstalk between VEGF signaling and DNA Damage. Genetic and pharmacological disruption of VEGFR-2 signaling and the MRN complex reveal a surprising co-dependency of these pathways in regulating EC senescence and proliferation. Using a pathway focused gene expression array we find that disruption of the MRN complex leads to a robust transcription of CD44, a known driver of senescence as well as a negative regulator of VEGF signaling. Our work identifies a putative miR-facilitated mechanism by which endothelial cells can be insulated against VEGF signaling to facilitate the onset of senescence. We propose that stress response drives specific microRNA expression perturbing canonical VEGF signaling, proliferation and senescence by thresholding DNA damage repair in the vasculature.
Abstract 186: Heart Disease Associated Lamin A/C Mutations and Resultant Defects in Cell and Nuclei Structure
Authors: Core Jason Q , Robinson Zachery R , Mehrabi Mehrsa , McCarthy Linda A , Zaragoza Michael V , Grosberg Anna
Published: March 2022

Changes in cell organelle structure or shape, which can be associated with a variety of diseases, can be caused by inherited genetic mutations. For example, the progeria disease is associated with a mutation to the LMNA gene, and it leads to a devastating early aging and death. However, other LMNA gene mutations do not cause early aging, but instead have a more subtle effect with patients presenting only with heart disease symptoms. One way to study the mechanisms that lead to these differences is in vitro with patient specific cell-lines. Here we will present a study of 10 cell-lines negative for mutations or with four different types of LMNA mutations: 1) Hutchinson-Gilford progeria syndrome LMNA gene mutation, 2) LMNA splice-site mutation (c.357-2A>G , p.N120Lfs*5), 3) LMNA nonsense mutation (c.736 C>T, p.Q246X), and 4) LMNA missense mutation (c.1003C>T, p.R335W) in exon 6. Fibroblasts from each of these cells lines were cultured on either isotropic fibronectin or fibronectin patterned into anisotropic lines. The cells were then fixed and stained to visualize the nuclei, actin fibrils, and the underline remodeled fibronectin. To analyze this data, we applied existing algorithms to quantify the re-organization of actin in response to extracellular matrix cues by calculating both the actin and fibronectin orientational order parameter. Additionally, we have created a new algorithm to automatically identify nuclei defects, which is a known property of the progeria LMNA mutation. Using the new algorithm, it is possible to separately characterize the shape of normal and defective nuclei for each cell-line. Through this algorithm, we show the varied effects of the different mutations on the number of fibroblasts that present with nuclear defects and their properties. The current data is consistent with the known patient symptoms providing further avenues of study into the mechanisms triggered by these mutations. Furthermore, we probe whether the unique mechanical conditions present in the myocardium are responsible for the heart disease symptoms of some of the tested mutations.
Abstract 188: Manipulation of Excitation-contraction Coupling in Cardiomyocytes Using Conductive Polyaniline Scaffolds
Authors: Humphrey Eleanor J , Kapnisi Michaella , Mawad Damia , Peters Nicholas S , Stevens Molly M , Terracciano Cesare M
Published: March 2022

The application of tissue engineered patches made of conductive polymer scaffolds combined with cardiomyocytes (CMs) could provide a dual method of improving the damaged myocardium after an infarction: firstly by introducing functional CMs to the area; secondly the conductive polymer could modulate electrical transmission across the scar tissue. Polyaniline (PANI) scaffolds are one such example, however, the consequences of growing CMs on conductive PANI scaffolds with regards to CM electrophysiology are unknown. In this study we assess the hypothesis that conductive PANI scaffolds affect CM calcium transients and action potential morphology in culture. Neonatal rat ventricular myocytes (NRVMs) and neonatal rat fibroblasts were co-cultured on conductive and non-conductive (sodium hydroxide treated) PANI scaffolds and remained viable after four days of culture, covering the surface of the construct. Compared to those cultured on non-conductive PANI scaffolds, NRVM cultured on conductive PANI scaffolds show faster calcium transients, measured using Fluo-4AM and field stimulated at 1 Hz, with a decrease in the time to peak (t
p
non-conductive=105±6 ms, t
p
conductive= 85±5 ms, p<0.05, n=6) and time to 50% (t
50
non-conductive=212±12 ms, t
50
conductive= 116±7 ms, p<0.001, n=6) and 90% decay (t
90
non-conductive=404±24 ms, t
90
conductive= 266±15 ms, p<0.001, n=6). Action potential morphology, assessed using FluoVolt membrane potential dye and stimulated at 1 Hz, remain unchanged for conductive and non-conductive PANI scaffolds. The PANI scaffolds are compatible with NRVMs and the cells have good viability after four days in culture. The conductive PANI scaffolds have a significant effect on myocyte calcium cycling but this is not caused by a change in action potential morphology. Further work is required to understand the mechanism behind the change in calcium handling in the CMs on the conductive PANI scaffolds.
Abstract 194: Frequency of Mononuclear Diploid Cardiomyocytes Underlies Natural Variation in Heart Regeneration
Authors: Patterson Michaela , Sucov Henry
Published: March 2022

Strong evidence from the literature supports the model that mononuclear, diploid cardiomyocytes (MNDCMs), a relatively rare population in the adult mammalian heart, are a regeneration-competent population, but this has not previously been subjected to experimental analysis. This project explores the hypothesis that the frequency of MNDCMs in the adult mammalian heart and regenerative potential are two interlinked, variable traits determined by multiple genetic parameters. If so, different individuals will have varying capacity to undergo heart regeneration following injury based on their unique genetic backgrounds. By surveying 120 inbred mouse strains, we found that the percentage of mononuclear cardiomyocytes in the adult mouse heart is a polygenic trait that is surprisingly variable (2.3%-17.0%). We confirmed experimentally that the degree of functional recovery and cardiomyocyte proliferation after permanent coronary artery ligation correlates with MNDCM content. Using genome-wide association, we identify Tnni3k as one gene with natural alleles that influence variation in this composition, and show in an isogenic strain background that Tnni3k knockout results in elevated MNDCM content and elevated cardiomyocyte proliferation after injury. Reciprocal to mammals, zebrafish are known to retain an almost pure MNDCM population through adulthood and are able to efficiently regenerate after adult heart injury. Here, we show that overexpression of Tnni3k in zebrafish promotes cardiomyocyte polyploidization and compromises heart regeneration, indicating a commonality in the regenerative process between the two species. Our results provide support for the requirement of the resident mononuclear diploid subpopulation of cardiomyocytes in heart regeneration. Moreover, our results imply that intrinsic heart regeneration is not uniform and limited in all individuals, but rather is a variable trait subject to the influence of multiple genes.
Abstract 201: High Fat Diet Selectively Decreases the Bone Marrow Lin-/c-kit+ Cell Population in Aging Mice Through Increased ROS Production
Authors: Xiao Yichao , Liu Xuanyou , Zhu Qingyi , Jiang Meng , Hao Hong , Ilangovan Govindasamy , Zhu Hua , Liu Qiming , Zhou Shenghua , LIU Zhenguo
Published: March 2022

Bone marrow-derived c-kit-positive (c-kit
+
) cells are a potential source for cell-based therapy and tissue repair and regeneration. The ability of tissue repair and regeneration is significantly decreased in the aging population for various reasons. Western diet usually has increased level of fat. The present study was designed to evaluate the effect of long-term high fat diet on bone marrow c-Kit positive cells and the role of ROS in aging mice. Aging male wild-type (WT) C57BL/6 mice (40 weeks) were fed with high fat diet (HFD) for 3 months with regular diet as the control. To evaluate the role of reactive oxygen species (ROS) in mediating the effect of HFD on bone marrow cell population, a separate group of mice were treated with N-acetylcysteine (NAC) to reduce ROS production. Bone marrow (BM) and blood cells were harvested and prepared in the mice after 3 months of HFD treatment for flowcytometry analysis for Sca-1+, or c-Kit+, or CD133+ cells. The lineage negative (Lin-) and c-Kit positive (Lin-/c-Kit+) cell population was significantly decreased in the BM, not in blood, in the mice with HFD, while no significant change was observed in Sca-1+ or CD133+ cell populations in the BM or blood. The BM Lin-/c-Kit+ cells also exhibited increased intracellular ROS level, increased level of apoptosis, and deceased level of proliferation in HFD-treated mice. NAC treatment significantly decreased intracellular ROS level and normalized the BM Lin-/c-Kit+ cell population in HFD mice. In conclusion, the present study demonstrated that long-term HFD selectively decreased BM Lin-/c-Kit+ cell population in the aging mice through increased ROS production.
Abstract 205: Overexpression of the Mitochondrial F
o
F
1
ATP Synthase regulators ATP12 and ATP5J are Protective against Ischemia-Reperfusion Injury in the Heart
Authors: Karch Jason , Michael Demetria M , Brody Matthew J , Kanisicak Onur , Maliken Bryan D , Sargent Michelle A , Molkentin Jeffery D
Published: March 2022

Loss of cardiomyocytes by necrotic cell death is a substantial underlying problem in progressive heart failure. One crucial step that is required for regulated necrosis to ensue is the opening of the mitochondrial permeability transition pore (MPTP), which is triggered by elevations in mitochondrial matrix calcium and reactive oxygen species levels. Opening of the MPTP causes permeabilization of the inner mitochondrial membrane and collapse of the electrochemical proton gradient required for ATP production. The latest model of the MPTP consists of the F
o
F
1
ATP synthase as the pore forming component within the inner mitochondrial membrane, although the mechanistic aspects of how the ATP synthase generates the pore is unknown. Two regulators of the ATP synthase, ATP12 and ATP5J, were identified in an unbiased calcium overload necrotic cell death screen using a lenti-viral gain-of-function library containing over 13,000 human cDNAs. ATP12 is an assembly factor of the F
1
component of the ATP synthase where it directly binds to the α-subunit and prevents homo-oligomerization. ATP5J is a component of the peripheral stalk that links the F
o
and F
1
portions of the ATP synthase together. To test if overexpression of ATP12 or ATP5J is indeed protective as we identified in the screen, here we generated cardiac-specific transgenic mice expressing each of these proteins. When subjected to ischemia-reperfusion injury, both ATP5J and ATP12 overexpressing mice were significantly protected. Additionally, mitochondria isolated from the overexpressing hearts demonstrated increased mitochondrial calcium uptake, a hallmark of MPTP desensitization. These data provide evidence that ATP12 and ATP5J are novel inhibitors of MPTP opening through their association with the F
o
F
1
ATP synthase and thus protect against necrotic cell death.
Abstract 206: Mitofilin Knockdown Induces Cell Death by Apoptosis via an AIF-PARP-dependent Mechanism and Cell Cycle Arrest
Authors: Bopassa Jean C , Madungwe Ngonidzashe B , Feng Yansheng , Lie Mihaela
Published: March 2022

Introduction:
Mitochondrial activity plays an essential role in the efficient function of cardiomyocytes. Mitofilin is an inner membrane protein that has been defined as a mitochondria-shaping protein in controlling and maintaining mitochondrial cristae structure and remodeling. In this study, we determined the role of mitofilin in H9c2 and C2C12 myoblasts survival by investigating the mechanism underlying mitofilin knockdown-induced cell death by apoptosis.

Methods:
H9c2 and C2C12 cells were cultured and treated with mitofilin siRNA or scramble siRNA for 24 hours. Cell death (apoptosis), caspase 3 activity, and cell cycle phases were assessed by flow-cytometry, cytochrome C release and intracellular ATP production were measured by ELISA. Mitofilin, AIF and PARP expression was measured by Western blot analysis and calpain activity assessed using calpain kit. Mitochondria images were taken using electron microscopy.

Results:
We found that mitofilin knockdown increases cell death by apoptosis mainly via activation of AIF pathway leading to nuclear fragmentation and subsequent PARP1 activation in the nucleus that is correlated with S phase arrest of the cell cycle. Knockdown of mitofilin in H9c2 as well as C2C12 myoblasts with siRNA led to mitochondrial swelling and damage of mitochondrial cristae that is associated with the increase in ROS production, decrease in intracellular ATP production and a marked decrease in mitochondrial membrane potential. Moreover, cells treated with mitofilin siRNA displayed an increase in calpain activity versus scramble siRNA.

Conclusion:
Together, these results indicate that mitofilin knockdown by siRNA increases calpain activity that presumably leads to mitochondrial structural degradation resulting in a critical reduction of mitochondrial function that is responsible for the increase in cell death by apoptosis via AIF-PARP mechanism which is associated with nuclear fragmentation and S phase arrest of cell cycle.
Abstract 207: Cytotoxic Effect of Palmitate is Caused by a Change in Membrane Fatty Acid Composition and ER Stress
Authors: Yamamoto Tsunehisa , Endo Jin , Shinmura Ken , Sano Motoaki , Fukuda Keiichi
Published: March 2022

Backgrounds:
Saturated fat in diet is known to be detrimental to heart health, but its impact on the cardiac muscle remains unknown. We investigated the impact of saturated fatty acid overload on membrane fatty acid composition and survival in neonatal rat cardiomyocytes.

Methods and Results:
(1) In neonatal rat cardiomyocytes, palmitate, a saturated fatty acid (16:0), induced cell death. (2) Palmitate-induced cell death was rescued by either co-administration of oleate (monounsaturated fatty acid; 18:1), EPA (polyunsaturated fatty acids; 20:5), or DHA (polyunsaturated fatty acids; 22:6). Among them, oleate was most effective. (3) Palmitate-overload did not alter the total amount of fatty acids in membrane phospholipid but changed the composition of fatty acids; it increased palmitate and decreased oleate. (4) Palmitate-induced cell death was associated with activation of ER stress sensors, referred as PERK, IRE1. Co-administration of oleate abolished ER stress. (5) Palmitate-induced cell death was significantly inhibited by APY29, an inhibitor of IRE1 phosphorylation pathway, but not by GSK260414 (PERK inhibitor) and by 4μ8c (IRE1-XBP1 inhibitor).

Conclusions:
Palmitate-induced cell death in cardiomyocytes is associated with a change in membrane fatty acid composition and activation of ER stress. Lipotoxic effect of palmitate is attenuated by co-administration of oleate and an inhibitor of IRE1 phosphorylation pathway.
Abstract 208: Excessive Autophagy Contributes to Microvascular Endothelial Dysfunction in Prehypertensive Spontaneously Hypertensive Rats
Authors: Dong Qianqian , Xing Wenjuan , Su Feifei , Gao Feng , Wang Siwang , Zhang Haifeng
Published: March 2022

Autophagy exists in vascular endothelial cells, however, the exact role of autophagy in hypertensive-associated vascular endothelial dysfunction remains largely elusive. We assessed the hypothesis that autophagy is one of the mechanisms of endothelial dysfunction in normotensive spontaneously hypertensive rats (SHRs). Age-matched male SHRs and Wistar Kyoto rats (WKY) aged 4 weeks (young) and 12 weeks (adult) were randomized into 2 groups and treated daily for 2 weeks by a) gavage with vehicle alone (normal saline) or b) intraperitoneally injection of rapamycin (1 mg/kg/day), and the vascular function of their isolated aorta and mesenteric artery was assessed
in vitro
. Furthermore, human umbilical vein endothelial cells (HUVECs) were incubated serum-starved for 6 h to induce excessive cell autophagy, and then incubated with DMEM containing 10% FBS and a) treated with 10 nmol/L insulin-like growth factor 1 (IGF-1) for 12 h, b) treated with 100 nmol/L Rapamycin for 1 h for 37°C. Compared with WKY, young and adult SHRs showed endothelial dysfunction of the aorta and mesenteric artery, along with decreased phosphorylated (p)-Akt, p-mTOR, and autophagic marker protein p62 and increased LC3 II/I in microvascular but not aortic tissues. Treatment of young SHRs with the mTOR inhibitor rapamycin aggravated the endothelial autophagy and subsequent impaired endothelial function. Moreover, IGF-1 significantly activated Akt/mTOR signaling in HUVECs and reduced autophagic levels, while rapamycin inhibited p-mTOR and increased autophagic levels. In conclusion, mesenteric endothelial dysfunction in prehypertensive SHRs is at least partly attributable to excessive autophagy in vascular tissues, and is likely mediated by the underactive Akt/mTOR signaling pathway; however, autophagy appears not to be responsible for the macrovascular endothelial dysfunction.
Abstract 214: Tracking of CD16 Monocytes Following Delivery Into the Ischemic Limb - a Bench to Bedside Study
Authors: Patel Ashish S , Ludwinski Francesca E , Kerr Alexander , Cho Jun , Fisher Christopher , Hope Andrew , Livieratos Lefteris , Saha Prakash , Lyons Oliver T , Smith Alberto , Modarai Bijan
Published: March 2022

Introduction:
The poor retention of cells delivered into ischemic tissue is a hurdle that needs to be addressed for cell therapy to be effective. We have previously shown that CD16+ monocytes expressing upregulated cell adhesion markers are mobilized in critical limb ischemia (CLI) and salvage limbs in pre-clinical studies of hindlimb ischemia (HLI). We hypothesized that these cells are well retained following delivery into ischemic muscle.

Methods and Results:
CD16+ and CD16- monocytes were isolated from CLI patients, fluorescently labeled with QuantumDots and injected into the adductor muscle of nude athymic mice following HLI (n=15/group). Flow cytometric analysis of cell suspensions from injected muscle showed higher 3 and 7 day retention of CD16+ compared with CD16- monocytes (P<0.05). Five patients with CLI underwent leukapheresis and isolation of CD16+ cells (CliniMACS) under GMP conditions. Autologous cells (30x10
7
) were radiolabeled with Indium
111
and injected into ischemic legs. Cells were tracked at 30mins, 2, 24 and 72 hours using whole-body γ-camera imaging: radiodecay-corrected retention percentages were 60.0, 37.4, 29.9 and 22.0% respectively with evidence of “washout” into the liver. Analysis of muscle biopsies showed >85% viability of the cells at 10 days and areas of increased arteriogenesis compared with remote, non-injected muscle.

Conclusion:
CD16+ Mo are preferentially retained within ischemic muscle in experimental HLI. This first in man study shows these cells remain viable in ischemic tissue. Isolation of sufficient numbers of these cells for delivery directly into poorly perfused muscle may provide a more effective cell therapy for CLI patients.
Abstract 219: Impact of Combination Therapy on Cardiovascular Outcomes in Patients with Symptomatic Subclavian Artery Stenosis
Authors: Ye Fan , Epperla Narendranath , Liang Hong , Chyou Po-Huang , Dart Richard , Mazza Joseph , Idris Amr , Sakkalaek Adeeb , Yale Steven
Published: March 2022

Background:
Subclavian artery stenosis (SAS) is narrowing of the subclavian artery most commonly caused by atherosclerosis. It serves as a marker for cerebrovascular and myocardial ischemic events.

Methods:
A retrospective cohort study was conducted to determine the association of treatment via combination therapy (antiplatelet drug plus either by-pass surgery or percutaneous transluminal angioplasty (PTA) with or without stent implantation) versus antiplatelet drug therapy alone on cardiovascular events and all-cause mortality in Marshfield Clinic patients diagnosed with symptomatic SAS from January 1, 1995 to December 31, 2009.

Results:
Of the total 2153 cases, 100 patients were identified as eligible to be included in the study. Of these 100 patients that meet inclusion criteria, 30 underwent combination therapy while 70 were managed only with pharmacological drug treatment. Adverse cardiovascular events occurred in 5/30 (17%) of combination therapy patients compared to 28/70 (40%) of antiplatelet drug therapy only patients (p = 0.0355). Accordingly, all-cause mortality was higher (47%) in the antiplatelet drug therapy only group than the combination therapy group (13%) [hazard ratio = 3.45, p = 0.0218].

Conclusions:
Preliminary findings in this pilot data set suggest that combination therapy (medications plus either surgical or interventional repair) of subclavian artery stenosis is associated with less cardiovascular adverse events and higher survival rates, benefits that extend beyond the target vessel. Prospective randomized studies with larger number of patients are needed to validate these findings.
Abstract 222: Inhibition of Phosphodiesterase 1 Confers Striking Therapeutic Benefit to HFpEF in Mice
Authors: Zhang Hanming , Wang Xuejun
Published: March 2022

Heart Failure with preserved Ejection Fraction (HFpEF) is the most prevalent HF but with virtually no effective treatment proven. Targeted removal of misfolded proteins by the ubiquitin-proteasome system (UPS) is pivotal to protein quality control. UPS function insufficiency contributes to HF genesis, making UPS priming an attractive strategy to treat HF. Previously we reported that protein kinase G (PKG) positively regulates cardiac UPS functioning while others showed PKA stimulates the proteasome. Thus, we propose that simultaneous activation of PKG and PKA might be a powerful strategy to improve cardiac UPS function and thereby benefit HF treatment. Here we provide the first evidence that strongly supports this proposition. We found that myocardial protein and mRNA levels of phosphodiesterase 1 (PDE1), a major cardiac PDE that degrades both PKG and PKA, were markedly elevated in CryAB
R120G
transgenic (tg) mice which are a classic model of cardiac proteinopathy with HFpEF as the major manifestation of its cardiac dysfunction. To test whether PDE1 inhibition (PDE1I) primes cardiac UPS and thereby ameliorates cardiac proteotoxicity, we performed cell cultures and mouse in vivo experiments taking advantage of a proven surrogate UPS substrate (GFPdgn) and a
bona fide
misfolded protein (CryAB
R120
), which demonstrates that pharmacological PDE1I significantly improves cardiac UPS performance and enhances degradation of the misfolded protein. We then treated CryAB
R120G
tg mice with PDE1 inhibitor LSN2790158 (3mg/kg/day x 28 days) or vehicle control via osmotic mini-pumps, starting at 4 months of age when HFpEF has clearly developed in the tg mice as reflected by significant decreases in left ventricular (LV) end-diastolic volume, stroke volume, and cardiac output and increases in end-diastolic LV posterior wall thickness along with unchanged EF, compared to littermate non-tg mice. Echocardiography revealed that that 4 weeks of PDE1 inhibitor treatment resulted in drastic improvement of HFpEF and Kaplan-Meier survival analysis shows striking delay of premature death in the PDE1I group vs. vehicle-treated group. Taken together, our data provide compelling evidence for the first time that PDE1I shall be explored as a new therapeutic strategy for HFpEF.
Abstract 225: Ischemic Cardiomyopathy Perturbs GSK-3β Myofilament Localization and Reduces Function
Authors: Stachowski Marisa J , Papadaki Maria , Martin Jody L , Moravec Christine S , Kirk Jonathan A
Published: March 2022

In ischemic cardiomyopathy (ICM), regions with ischemic damage are weaker than surrounding tissue, leading to contractile heterogeneity (ischemia-induced dyssynchrony, IID) that worsens function and mortality. IID is distinct from conduction abnormality-induced dyssynchrony that is treated with Cardiac Resynchronization Therapy (CRT). Our previous work found CRT reactivates glycogen synthase kinase 3β (GSK-3β) and restores myofilament function. Pacing an infarct cannot strengthen it, so CRT is ineffective in IID. However, we hypothesized GSK-3β may modulate myofilament function in ICM and could be leveraged to treat IID.

We measured GSK-3β in whole tissue and myofilament-enriched samples from human rejected donor (Control), ICM, and dilated cardiomyopathy (no IID, DCM) LV. GSK-3β was detected in all the myofilament samples, but there was a 71±12% reduction in ICM. In whole tissue, there was very little phospho-Y216 GSK-3β, however it was highly enriched in the myofilament. Immunofluorescence on adult human myocytes showed weak co-localization of total GSK-3β and α-actinin at the z-disc compared to a strong correlation with p-Y216 GSK-3β. Furthermore, co-IP of GSK-3β and the myofilament show total GSK-3β had low-affinity to myofilament proteins, while p-Y216 GSK-3β binds with a high affinity. This led us to hypothesize Y216 phosphorylation modulates GSK-3β binding to the myofilament. To remove the confounding effect of antibodies, we created adenoviral constructs of myc-tagged wild-type, Y216F (unphosporylatable) and Y216E (constitutively phosphorylated) GSK-3β and transfected them into rat neonatal ventricular myocytes (NRVMs). The Y216E construct alone associated with the myofilament in co-IP experiments. We then performed skinned myocyte functional studies on human Control and ICM LV. The ICM myocytes were desensitized to calcium compared to Control and this was restored with exogenous GSK-3β treatment, but had no effect on Control myocytes.

While GSK-3β is a promiscuous kinase in the myocyte, we have identified a specific regulatory mechanism involving Y216 phosphorylation, a site with a largely unknown role, that could allow precise therapeutic intervention to improve contractile function in the ICM heart.
Abstract 227: Skinned Myocardial Strips From Non-failing and Failing Human Hearts Produce More Force at Physiological Temperature
Authors: Tanner Bertrand C , Awinda Peter O , Blair Cheavar A , Guglin Maya E , Campbell Kenneth S
Published: March 2022

Introduction:
Heart failure contributes to 1 in 9 deaths in the United States. Contractile deficits at the myofilament level may contribute to the heart inadequately pumping blood throughout the body. Few studies have investigated force production and myosin cross-bridge kinetics at physiological temperature in human myocardium from non-failing and failing hearts.

Hypothesis:
Myosin cross-bridge kinetics speed up with temperature, thereby altering contractility in non-failing and failing human myocardium.

Methods:
Skinned myocardial strips from the left ventricle free wall were Ca
2+
-activated at 2.3 μm sarcomere length. Isometric force production and cross-bridge attachment and detachment rates were measured at 17 and 37°C (4 non-failing hearts, 8 failing hearts; 4-6 strips tested at each condition).

Results:
Maximal Ca
2+
-activated, force was ~35% greater in non-failing vs. failing tissue at both 17 and 37°C (p<0.001 for condition effect). Moreover, increasing temperature from 17 to 37°C increased maximal force by roughly 30% and 40% in non-failing and failing myocardial strips, respectively (p<0.001 for temperature effect). Ca
2+
-sensitivity of the force-pCa relationship was reduced for non-failing vs. failing myocardial strips (p=0.03 for condition effect), with differences in pCa
50
being greatest at 17°C between non-failing and failing samples. These contractile differences between non-failing and failing myocardium, were not driven via differences in myosin cross-bridge kinetics, which were similar among non-failing and failing samples at each temperature. However, faster cross-bridge cycling rates accompanied greater force production at physiological temperature in both non-failing and failing samples.

Conclusions:
These findings suggest that heart failure compromises force production without significantly altering cross-bridge kinetics, both at sub-physiological and physiological temperatures in human myocardium.
Abstract 231: A Hand1 Left Ventricular Enhancer that is Associated With Prolonged QRS Interval
Authors: Vincentz Joshua W , Zhang Wenjun , Shou Weinian , Arking Dan , Sotoodehnia Nona , Rubart-von der Lohe Michael , Firulli Anthony B
Published: March 2022

Throughout the process of cardiogenesis, dynamic changes in cardiomyocyte morphology are defined by gene regulatory networks that drive atrial, ventricular, septal, compact zone or trabecular cardiomyocyte cellular identity. The bHLH transcription factors Hand1 and Hand2 are both critical for heart development. We have isolated a 750 bp evolutionarily conserved non-coding sequence 5’ to the
Hand1
transcription start site that is necessary and sufficient to drive left ventricle (LV)-specific
Hand1
expression. Regulatory
cis
-elements include Gata and T-box binding sights. We show this enhancer as necessary and sufficient for LV expression using CrispR/Cas9 deletion. Mice homozygous for this enhancer deletion (

Hand1
αLV/
αLV

) are viable and fertile. Adult

Hand1
αLV/
αLV

mice exhibit a prolonged QRS interval. Interestingly, human
HAND1
SNPs associated with prolonged QRS are located near the LV enhancer, but these SNP sequences are not conserved in mice. More refined examination of the human sequences revealed additional prolonged QRS-associated human nucleotide changes occurring directly within the conserved enhancer that alter
cis
-element DNA binding. Ventricular conduction system gene expression is altered in
Hand1
enhancer deletion mice, validating
in vivo
a human GWAS association study that links
Hand1
function to the development and perhaps maintenance of the LV purkinje fiber network.

Additionally, using the Hand1 LV-enhancer, we have generated a novel
Cre
transgenic mouse line (

Hand1
LV
-Cre

), in which LV-specific recombination is observed. We use this Cre to delete both cardiomyocytes and Hand genes from the developing LV, discovering that specification of subpopulations of ventricular cardiomyocytes regulates myocardial growth and, thereby, cardiac function.
Abstract 236: Shaping Waves of BMP Inhibition During Vasculogenesis
Authors: Guihard Pierre , Yao Jiyai , Guo Yina , Blazquez-Medela Ana , Jumabay Medet , Yao Yucheng , Garfinkel Alan , Boström Kristina
Published: March 2022

During early embryogenesis, gradients of bone morphogenetic proteins (BMPs) have been shown to be critical in controlling patterning and growth of developing tissues. Two members of the BMP family are well known as strong modulators of vascular development, BMP4 and BMP9. Hereditary hemorrhagic telangiectasia 2 (HHT2) is caused by mutations in a BMP receptor, the activin receptor-like kinase 1 (Alk1) gene, and characterized by abnormal vascular networks and arteriovenous malformations (AVMs). ALK1 expression and signaling are tightly regulated by BMP4 and 9 and their respective inhibitors, matrix Gla protein (MGP) and Crossveinless-2 (CV2), through negative feedback regulation. Aberrant ALK1 signaling disrupting endothelial cell (EC) differentiation is closely related to vascular morphogenesis and AVMs, and depends on normal action of MGP and CV2. We have previously shown that deletion of the
Mgp
gene leads to AVMs in multiple organs (100% penetrance), characterized by large-caliber shunts and abnormal endothelium with increased cellularity and EC marker expression. Using
in vitro
,
in silico
and
in vivo
approaches, we found that the vascular growth, patterning and EC maturation are orchestrated by “shaping waves” resembling those in somatogenesis. In cultures ECs, the waves are seen as temporal oscillations of MGP and CV2 expression in response to BMP9, whereas in tissue such as the retina, the waves are observed as stripes perpendicular to the direction of growth. Thus, lack of MGP or disruptions in BMP9/ALK1/Endoglin signaling, as in HHT, will cause alterations in the expression of the BMP inhibitors and the patterning during vascular development.
Abstract 24: Graphene Oxide Containing Thermosensitive Hydrogel Improves the Survival of Transplanted Stem Cells in Ischemic Myocardium
Authors: Saravanan Sekaran , Abu Al-Rub Ejlal , Sareen Niketa , Sequiera Glen Lester , Moudgil Meenal , Dhingra Sanjiv
Published: March 2022

Bone marrow derived mesenchymal stem cell (MSC) therapy has the potential to preserve cardiac function and prevent heart failure following a myocardial infarction (MI). However, in our recent studies, long term survival of transplanted stem cells was not detected, which was associated with deterioration of cardiac function. Therefore, strategies to enhance survival of transplanted cells would preserve the benefits of MSCs therapy for heart repair. Recently, graphene oxide (GO) nanosheets are being widely investigated for their ability to promote adherence and homing of transplanted MSCs to the extra cellular matrix in the heart. However, the presence of reactive oxygen functional groups (-OH, -COOH) in the structure of GO increases oxidative stress and activate apoptotic pathways in MSCs, which impairs the benefits of GO based biomaterials. Therefore, to nullify these side effects of GO, we synthesized a novel thermosensitive hydrogel by conjugating GO with chitosan (CS). Our Fourier transformed infrared spectrum (FTIR) analysis revealed an interaction between the oxygen functional groups of GO and amino (-NH2) groups of chitosan. Our data also demonstrate that CS masked the reactive functional groups of GO and prevented GO mediated ROS induction. This novel chitosan-graphene oxide (CS-GO) composite exhibited optimal porosity for cell conjugation and retention. Furthermore, this novel biomaterial was cyto-compatible as it exerted negligible toxicity to encapsulated MSCs. Upon exposure to hypoxic/ischemic environment, we observed a significant increase in apoptosis in MSCs as evidenced by an increase in the Bax/Bcl-xl ratio and caspase 3 activation. However, conjugation of MSCs to CS-GO polymer prevented hypoxia induced apoptosis. Therefore, coupling of GO with chitosan prevented GO mediated oxidative stress and downregulated hypoxia induced apoptosis of MSCs. Therefore, the outcome of this study will provide a novel delivery system for MSCs to the heart and improve their long term survival in the infarcted heart.
Abstract 251: Lna- Mediated Mir-92a Inhibition Induces Therapeutic Neovascularization in a Pig Model of Chronic Myocardial Ischemia
Authors: Klett Katharina , Hinkel Rabea , Fischer Ariane , Bonauer A. , Zeiher A. , Dimmeler Stefanie , Kupatt Christian
Published: March 2022

miRNA are post-transcriptional gene regulators which modulate RNA silencing and gene expression. In chronic myocardial ischemia, miRNA are altered and dysregulated. MiR92a inhibition in a mouse model of LAD occlusion led to improved neovascularisation and reduced infarct sizes. We aimed to investigate this therapeutic effect of miR92a- inhibition in a pig model of chronic myocardial ischemia.

Methods:
In pigs (n=5/group) reduction stent graft implantation in the circumflex artery led to a gradual occlusion of the vessel within 28 days (d28) after intervention. Using selective pressure-regulated retroinfusion the nucleic acid-modified antisense miR92a (LNA-92a; 5 mg/KG heart weight) was injected 28 days post implantation. The global myocardial function (Ejection Fraction (EF)) and the left end-diastolic pressure (LVEDP) were obtained at day 28 and 56. At day 56 after stent implantation regional myocardial function (subendocardial segmentshortning) as well as post mortem angiographies for collateral growth were analyzed. For the histological quantification the ischemic tissue was stained for PECAM-1 positive cells (capillaries) and for NG2- positive cells (vessel maturation, pericyte coverage).

Results:
The regionally applied locked nucleic acid-modified anti-sense miR-92a significantly reduced miR-92a expression in the heart and enhanced capillary coverage in the ischemia. Improved collateral growth (3±1 control vs. 6±1 in LNA-92a treated hearts) and distal perfusion of the occluded coronary artery (Rentrop score: 1.3±0.2 control vs. 2.4±0.2 in LNA-92a treated hearts) was seen after miR-92a inhibition. Moreover the increased LVEDP at day 28 (control: 16±1 mmHg vs. LNA-92a 15±1mmHg) was reduced after regional miRNA-92a inhibition (control: 16±1 mmHg vs. LNA-92a 15±1mmHg) and EF clearly increased after LNA-92a application (39±4% vs. 25±2 % in control). Regional myocardial function, obtained in the ischemic area under increased heart rate, was significantly improved after miR-92a inhibition (SES at hear rate 150bpm: 7±3 % of non-ischemic in control vs. 70±11 %% of non-ischemic in LNA-92a treated animals).

Conclusion:
Even single application of LNA-92a was able to reduce ischemia dependent impaired myocardial function
Abstract 254: MicroRNA Regulation of G Protein-Coupled Receptor Kinase 2 After Cardiac Injury
Authors: Lieu Melissa , Chuprun Kurt , Gao Erhe , Koch Walter J
Published: March 2022

The development and progression of heart failure (HF) involves many processes that occur after cardiac stress and/or injury. Compensatory neuro-hormonal activation and chronic stimulation of the sympathetic nervous system (SNS) is accompanied by the pathological increase in G protein-coupled receptor kinase (GRK) 2 expression. Data indicates that elevated GRK2 activity participates in the progression of HF after cardiac injury such as a myocardial infarction (MI) and this includes chronic β
1
-adrenergic receptor (β
1
AR) desensitization. The molecular transcriptional or translational mechanism of GRK2 upregulation in the heart is largely unknown. MicroRNAs (miRNAs) have been discovered as critical regulatory molecules in the heart as their expression profiles become dysregulated after cardiac injury. Further, miRNAs can play key roles in orchestrating many phenotypic changes observed in HF. Previous studies have shown that components of the βAR pathway, including the β
1
AR, are targeted by miRNAs whose levels are aberrant during cardiac injury. This study was started to determine if GRK2 may also be targeted by a miRNA that goes down after injury, participating in the up-regulation of this pathological kinase. MiRNA profiles of 2 week post-MI C57BL/6 mice were examined through miRNA microarray analysis. MiRNAs that were down-regulated after MI and had seed sequence complementarity to GRK2’s 3’UTR were selected as candidates for further testing. MiR-378a-5p (miR-378a) was chosen as a suitable candidate and transfection of a miR-378a mimic into neonatal rat ventricular myocytes was able to significantly reduce GRK2 protein levels compared to a negative control, while mRNA levels seem to be unaffected. This suggests miR-378a may work primarily through translational inhibition rather than transcriptional inhibition. Using a 3’UTR reporter system, we found that miR-378a was able to directly bind to the 3’UTR of GRK2 and inhibit reporter activity. These data support the hypothesis that GRK2 is regulated by a miRNA in the heart and provides a possible mechanism for GRK2 post-transcriptional regulation
. In vivo
studies are currently in progress to determine if miR-378a may be a novel therapeutic strategy to decrease GRK2 levels in the failing heart.
Abstract 256: Xpln a New Target Protein in the Pathogenesis of Cardiac Dysfunction
Authors: Seiz Mirjam , Katus H.a. , Voelkers M.
Published: March 2022

Backround:
mTOR (mammalian target of rapamycin) is an essential multiprotein complex in cells. It consists in two different complexes, mTORC1 and mTORC2, each with distinct cellular functions. mTORC1 is important for protein synthesis, whereas mTORC2 influences cell survival. Both are important for maintaining cell homeostasis. A negative feedback mechanism between the mTORC1/mTORC2 is indispensable for controlled activity of the protein complex. Adrenergic overstimulation of mTORC1 contributes to severity of myocardial damage, including cardiac hypertrophy and heart failure. Thus the regulatory influence of mTORC2 seems to be important for physiological mTORC1 activation.

Hypothesis:
The activation of mTORC2 affects the pathogenesis of cardiac dysfunction. Therefore XPLN, a new found mTORC2 Inhibitor, could prove as a pivotal regulator protein for the mTOR complex and cardiac function.

Methods:
Combined pharmacological and molecular biological methods were used for disruption of the mTORC1/mTORC2 equilibrium. The experiments were performed in cultured cardiac myocytes as well as in human samples. Gain and loss of function of XPLN was used for demonstrating it′s relevance on mTORC2 regulation and the functionally consequence of mTORC1/mTORC2 interconnection. The importance of the mTORC equilibrium was demonstrated by application of pathological growth factors (Phenylephrine). mTORC2 inhibition after XPLN overexpression resulted in mTORC1 activation, hypertrophic cell formation and cardiac stress. Conversely, knockdown of XPLN results in shifting toward mTORC2 activation with significant reduction in BNP expression.

Conclusion:
The regulatory influence of mTORC2 is essential for physiological mTORC1/mTORC2 activation. Disruption of this equilibrium results in myocardial stress. We demonstrate XPLN as a potent mTORC2 inhibitor with significant influence on the mTORC2 regulation. Furthermore we could show it′s relevant consequences in cell survival and the extensive effects on mTORC1 activation. Therefore XPLN is an essential regulator protein in the pathogenesis of cardiac dysfunction and could prove as a new target protein for medical treatment.
Abstract 262: Myofibroblast-Specific Transforming Growth Factor β Suppression Reduces Fibrosis in a Proteotoxic Cryab
R120g
Mouse Model of Heart Failure
Authors: Bhandary Bidur , Meng Qinghang , Gulick James , Osinska Hanna , Shay-Winkler Kritton , Robbins Jeffrey
Published: March 2022

Introduction:
Transforming Growth Factor beta (TGFβ) is an important cytokine in mediating cardiac fibrosis. Cardiomyocyte-specific expression of a mutant αB-crystallin (CryAB
R120G
) that is responsible for human Desmin Related Myopathy results in significant cardiac fibrosis and cardiac remodeling leading to heart failure. Onset of fibrosis is initiated by the activation of a quiescent fibroblast population to an active, “myofibroblast” state and TGFβ binding is thought to mediate an essential signaling pathway underlying this process. Our central hypothesis is that myofibroblast-based TGFβ signaling can result in significant cardiac fibrosis. Here, we have partially ablated TGFβ signaling in cardiac myofibroblasts to observe if cardiac fibrosis is altered.

Objective:
To understand the contributions of myofibroblast-based TGFβ signaling to the development of cardiac fibrosis.

Methods and Results:
To test the hypothesis we partially ablated myofibroblast specific TGFβ signaling by crossing CryAB
R120G
mice with mice containing a floxed allele of TGFβ’s receptor 1 (TGFβr1). The double transgenic animals were further crossed with activated myofibroblast specific Cre mice in which Cre expression was driven off the periostin promoter so that TGFβr1 would be ablated subsequent to myofibroblast conversion as the periostin promoter became active. Echocardiography, Masson’s Trichome staining, the hydroxyproline assay, PCR arrays, immunohistochemistry and western blots were used to characterize fibrosis and cardiac function in mice lacking TGFβr1 in the myofibroblasts were used to characterize the resultant animals.

Conclusion:
Myofibroblast-targeted knockdown of Tgfβr1 signaling resulted in reduced fibrosis and improved cardiac function.
Abstract 266: Phosphodiesterase 9A Deficiency Does Not Attenuate Chronic-hypoxic Pulmonary Hypertension
Authors: Kolb Todd M , Johnston Laura , Srinath Haarika , Damarla Mahendra , Kass David A , Hassoun Paul M
Published: March 2022

Background:
Phosphodiesterase (PDE) inhibition is a cornerstone of therapy for pulmonary arterial hypertension. A novel role for PDE9A in natriuretic peptide-mediated left ventricular cyclic guanosine monophosphate (cGMP) signaling and remodeling has recently been described. Since cGMP-signaling can promote vaso-relaxation and prevent vascular smooth muscle cell proliferation and cardiac myocyte hypertrophy, we hypothesized that PDE9A deficiency would attenuate right ventricular (RV) and pulmonary vascular remodeling during chronic RV pressure overload.

Methods:
Chronic-hypoxic pulmonary hypertension (CH-PH) was induced in mice lacking PDE9A expression (

Pde9a
-/-

) and wild-type littermates (

Pde9a
+/+

) by exposure to normobaric hypoxia (FiO2 = 10%) for 3 weeks. RV systolic pressures were measured to quantify PH, and RV hypertrophy was quantified as the ratio of RV free wall mass to LV/septal mass (RV/LV+S). Heart, lung, and serum were flash frozen for biochemical analyses.

Results:
In wild-type mice, exposure to chronic hypoxia resulted in significant increases in RV pressure (30 ± 2.4 vs. 20 ± 3.2 mm Hg;
P
= 0.0006) and RV hypertrophy (0.34 ± 0.04 vs. 0.25 ± 0.02;
P
< 0.0001). Serum ANP levels were increased 5-fold with CH-PH (
P
= <0.0001), associated with a 72% reduction in lung
Npr3
receptor expression (responsible for ANP clearance;
P
< 0.0001) but no increase in RV
Nppa
expression. After 3 weeks of CH-PH, tissue levels of cGMP were not increased in lung (993 ± 502 pg/mg vs. 667 ± 210 pg/mg;
P
= 0.27) or RV (47 ± 18 pg/mg vs. 49 ± 13 pg/mg;
P
= 0.88) homogenates. Accordingly, serine-239 phosphorylation of vasodilatory-stimulated phosphoprotein (VASP; a surrogate for protein kinase G activation) was not increased by CH-PH in lung or RV. Expression of
Pde9a
RNA was not increased by CH-PH in lung or RV.

Pde9a
-/-

mice did not have attenuated CH-PH induced increases in RVH (
P
= 0.27, 2-way ANOVA) or RVSP (
P
= 0.23, 2-way ANOVA) when compared to

Pde9a
+/+

mice. In both

Pde9a
-/-

and

Pde9a
+/+

mice, CH-PH was associated with significant increases in lung PDE5A expression.

Conclusions:
In a murine model of chronic RV pressure overload, PDE9A deficiency does not attenuate RV remodeling or PH, despite robust increases in circulating natriuretic peptide levels.