bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P336: Constitutive Activation of ROCK1 in Mouse Heart Leads to Fibrotic Cardiomyopathy
Authors: Yang Xiangsheng , Li Qi , Lin Xi , Ma Yanlin , Yue Xiaojing , Tao Zhengyin , Chang Jiang
Published: March 2022

Background:

Our previous study demonstrated that genetic deletion of ROCK1 inhibited stress-induced cardiac fibrosis and ROCK1 was cleaved by caspase 3 into a constitutively active Rho kinase isoform, ROCKΔ1 that led to increase in Rho kinase activity in human failing hearts. However, the function of truncated ROCK1 accumulated in human failing myocardium and the molecular signaling linked between ROCKΔ1 and cardiac remodeling remain obscure.

Methods and Results:

To recapitulate this human pathophysiological situation and define the pathogenic role of ROCKΔ1 in heart, we generated transgenic mice expressing ROCKΔ1 in heart. Significant increase in Rho kinase activity was achieved. The mice developed fibrotic cardiomyopathy along with upregulation of TGFβ1 and NF-κB signaling. Significant increases in fibrotic factors were observed. Echocardiography revealed diastolic dysfunction in the transgenic heart. The transgenic mice were predisposed to angiotensin II treatment that led to massive fibrotic cardiomyopathy. Rho kinase inhibitor Fasudil treatment attenuated the fibrotic maladaption.

To elucidate the molecular mechanism underlying ROCKΔ1-mediated fibrotic cardiomyopathy, wild-type cardiac fibroblasts were co-cultured with transgenic cardiomyocytes. Large amount of fibroblasts were activated and induced to express α-SMA by the transgenic cardiomyocytes but not by the wild-type control cardiomyocytes. Addition of Rho kinase, TGFβR1 and NF-κB inhibitors to the culture media attenuated the fibroblast activation. The results suggested that the pro-fibrotic TGFβ and NF-κB signaling are responsible for ROCKΔ1-mediated fibrotic cardiomyopathy.

Finally, we demonstrated TGFβ1 was a SRF (serum response factor) new target gene. EMSA, luciferase and CHIP assay revealed SRF binding to TGFβ1 promoter. Genetic deletion of SRF in mouse heart resulted in marked decrease in TGFβ1 expression.

Conclusions:

Our studies demonstrated the pro-fibrotic role of truncated ROCK1
in vivo
and provided an animal model that recapitulated human heart failure. The results elucidated that activation of NF-κB and TGFβ1 signaling contributed to Rho kinase-mediated fibrotic cardiomyopathy, the later was regulated by SRF.
Abstract P341: MRTF-A, a Rho-Dependent Coactivator of SRF, Plays a Critical Role in Vascular Remodeling
Authors: Minami Takeya , Kuwahara Koichiro , Nakagawa Yasuaki , Kuwabara Yoshihiro , Nakao Kazuhiro , Kinohita Hideyuki , Usami Satoru , Yamada Chinatsu , Shibata Junko , Nishikimi Toshio , Takaoka Minoru , Sata Masataka , Nagai Ryozo , Nakao Kazuwa
Published: March 2022

Objective:
Rho-ROCK pathway is involved in the progression of pathological vascular remodeling through the regulation of cell proliferation and migration by controlling gene transcription. While a transcriptional co-activator Myocardin-related transcription factor(MRTF)-A transduces Rho-actin signaling to SRF activation in the nucleus, roles of MRTF-A in the process underlying vascular diseases remain unknown.

Methods and Results:
We found that MRTF-A mRNA and protein expression in femoral arteries 2weeks after wire injury was significantly higher than that in sham-operated arteries of mice. In contrast, the expression of myocardin or MRTF-B was significantly decreased in injured arteries. We then evaluated the role played by MRTF-A in pathological vascular remodeling by creating several vascular disease models of mice lacking MRTF-A (MRTF-A
-/-
). Neointima formation induced by ligation in carotid arteries of MRTF-A
-/-
was significantly smaller than that of MRTF-A
+/-
. Neointima formation induced by wire injury in femoral arteries of MRTFA
-/-
was significantly smaller than that of MRTF-A
+/+
. Atherosclerotic lesions of MRTF-A
-/-
;ApoE
-/-
was also significantly smaller than those of MRTF-A
+/+
;ApoE
-/
. The expression of SRF-target genes involved in cellular migration, such as MMP9 and vinculin,within injured arteries of MRTF-A
-/-
was significantly attenuated compared to that of MRTF-A
+/+
. In cultured, de-differentiated rat aortic vascular smooth muscle cells (RAVSMC), the expression of these genes was decreased by knocking down MRTF-A. Indeed, the promoter activity of vinculin gene was controlled by MRTF-A in RAVSMC. In addition, knocking down MRTF-A in RAVSMC resulted in a significant impairment in migration capacity. Furthermore, treatment with a small molecule CCG1423 inhibiting MRTF-A significantly reduced neointima formation induced by wire injury in femoral arteries of mice.

Conclusions:
Our study revealed that in de-differentiated vascular smooth muscle cells, in which myocardin and MRTTF-B expression is decreased, increased expression of MRTF-A contributes to the acquisition of migration capacity by maintaining SRF-target genes transcription, thereby promoting stress-induced vascular remodeling.
Abstract 114: Myocardin-Related Transcription Factors Are Required for Compensatory Signaling in Maladaptive Cardiac Remodeling
Authors: Trembley Michael A , Mickelson Deanne M , Dirkx Ronald , Alexis Jeffrey D , Moravec Christine , Small Eric M
Published: March 2022

Intercellular communication is essential for coordinating the contraction of cardiomyocytes. This synchrony is established by coupling electrical activity and force transmission at specialized junctions called intercalated discs (ICDs). We have recently found that myocardin-related transcription factor A (MRTF-A) and –B (MRTFs) accumulate at ICDs of healthy mouse and human hearts, but are redistributed in cardiac pathology. MRTFs are mechanosensitive transcriptional co-activators of serum response factor (SRF) that regulate the actin cytoskeleton; however, the role of MRTFs in cardiomyocyte homeostasis remains unclear. We conditionally deleted
Mrtfb
in cardiomyocytes of mice harboring a homozygous null allele of
Mrtfa
(called MRTF
cmdKO
). MRTF
cmdKO
mice exhibit no changes in cardiac function for up to a year; however, MRTF depletion results in conspicuous gap junction remodeling at ICDs and reduced cardiomyocyte communication. This phenotype stems from a significant reduction in genes important for trafficking gap junction proteins, including RP/EB family member 1 (
Mapre1
), which we show is a direct transcriptional target of SRF/MRTFs. The absence of a functional phenotype amid gap junction displacement prompted us to model heart failure and mechanical stress at ICDs by subjecting MRTF
cmdKO
mice to transaortic constriction (TAC) surgery. Unexpectedly, MRTF
cmdKO
mice display partially penetrant sudden cardiac death within 2 weeks post-TAC surgery. MRTF
cmdKO
survivors undergo accelerated heart failure with a reduced adaptive gene response compared to controls. Future investigations are underway to determine the mechanisms governing accentuated heart failure in MRTF
cmdKO
mice. Taken together, the subcellular localization of MRTFs to ICDs creates a paradigm in which potent transcription factors are anchored to regions of electromechanical stress, which may represent a signaling circuit for ICD maintenance and remodeling in disease.
Abstract 124: Chronic Beta-Adrenergic Stimulation Prevents Protective Insulin-Induced Increases in Cardiomyocyte Respiratory Capacity
Authors: Pfleger Jessica , Ibetti Jessica , Koch Walter J
Published: March 2022

Cardiac injury or stress increases circulating catecholamines (CA), stimulating cardiac β adrenergic receptors (βAR) and adaptive positive inotropy. Under such conditions of increased energy demand, cardiomyocyte metabolism and bioenergetics are altered to maintain cellular function and survival. During chronic CA stimulation, however, receptor desensitization occurs, contributing to loss of contractile reserve and heart failure (HF) development. This progression to HF is again marked by metabolic and bioenergetic changes that remain incompletely understood. Notably, chronic CA exposure induces insulin resistance (IR) in the heart, which decreases substrate uptake and prevents protective signaling. Our laboratory has shown that G protein-coupled receptor kinase 2 induces IR in the heart following myocardial infarction. However, neither the molecular mechanisms of CA-induced IR, nor its effects on cardiomyocyte bioenergetics are well characterized. We hypothesize that βAR stimulation induces IR by decreasing insulin-stimulated mitochondrial respiratory capacity, thus, compromising cardiomyocyte survival during stress. Bioenergetics were evaluated by measuring cellular respiration under coupled (basal) and uncoupled (maximal) conditions. Our results show that acute insulin treatment selectively increases (25%) maximal and reserve respiratory capacity in the presence of glucose, which positively correlates with cellular survival. This effect is completely inhibited with chronic βAR stimulation using isoproterenol or clenbuterol, as is glucose transporter (GLUT4) translocation to the membrane. A similar effect is reproduced by high glucose and/or palmitate-induced IR. Notably, chronic βAR stimulation does not affect basal or maximal respiration, while acute stimulation increases basal respiration (25%). In short, the data demonstrate a unique insulin-induced increase in mitochondrial respiratory capacity in cardiomyocytes that is antagonized by chronic CA stimulation. We propose that this effect contributes to the detrimental effects of βAR stimulation during HF. Thus, understanding this phenomenon and its mechanisms is critical for inhibiting IR and improving cardiac metabolism and function during HF.
Abstract 138: Identification of a Novel Small Molecule Activator of ATF6 that Confers Protection Against Ischemia/reperfusion Injury in the Heart
Authors: Blackwood Erik A , Plate Lars , Paxman Ryan J , Malter Kyle , Wiseman Luke , Kelly Jeff , Glembotski Christopher C
Published: March 2022

Rationale:
Reactive oxygen species generated during myocardial ischemia/reperfusion (I/R) potentiate myocyte death and cardiac dysfunction. Recently, our lab published a newly described role for the adaptive ER stress sensor and transcription factor, ATF6, as a novel inducer of an adaptive antioxidant gene family. These results highlight the need for the development of small molecule drug candidates that preferentially activate endogenous ATF6 to promote the adaptive effects of ER stress and ameliorate myocardial I/R damage. To this end we used of a cell-based high throughput-screen to identify a novel small molecular activator of endogenous ATF6, herein called compound 147, and tested its efficacy in cardiac myocytes and in the heart.

Objective/Methods:
The ability of compound 147 to activate endogenous ATF6, as measured by nuclear localization of ATF6 and ATF6-specific target gene induction was examined in cultured neonatal rat ventricular myocytes (NRVM). The effects of compound 147 on the viability of NRVM treated with H2O2 to generate ROS, or simulated I/R were assessed. Finally, the effects of compound 147 in the mouse heart were examined
in vivo
by administering the compound to mice and, 24h later, determining the effects of simulated I/R on cardiac myocytes isolated from the mice, or determining the effects of
ex vivo
I/R on hearts isolated from compound 147-treated mice.

Results:
Compared to a control compound, treatment of NRVM with compound 147 specifically and acutely activated ATF6 and primed cells to mount an adaptive response when treated with H2O2 or subjected to simulated I/R. Treatment of both neonatal and adult ventricular myocytes with compound 147 increased survival in cells subjected to simulated I/R. Compared to control, the cardiac myocytes and hearts from mice treated with compound 147 exhibited increased viability and functional recovery in response to I/R, respectively.

Conclusions:
Compound 147 specifically activates ATF6 in cardiac myocytes and confers cardioprotection during I/R,
in vitro
and
in vivo
. Thus, compound 147 represents a potential first- in-class small molecule drug candidate that enhances myocardial recovery from I/R damage, specifically by activating the endogenous adaptive ATF6 gene program in the heart.
Abstract 14: Identification of a Major Role for Cytochrome B5 Reductase 3 in Cardiomyocyte Metabolism and Function
Authors: Straub Adam C , Carew Nolan T , Altmann Helene M , Galley Joesph C , Hahn Scott , Miller Megan P , Shiva Sruti , McNamara Dennis
Published: March 2022

Cytochrome B 5 Reductase 3 (Cyb5R3) also known as methemoglobin reductase regulates redox signaling in erythrocytes and endothelial cells by maintaining heme iron in the reduced (Fe
2+
) state. Knowing the importance of highly regulated redox signaling in other hematopoietic and somatic lineages, we conducted a pharmacological inhibition study using a CyB5R3 inhibitor in mice. Inhibition resulted in dilated cardiomyopathy (DCM) after 2 weeks. To determine the potential for human relevance, a high frequency point mutation (T117S) in African American populations was studied and served as a model to understand the impact of mutated CyB5R3 in human heart failure. We found T117S individuals associated with accelerated time to first acute cardiac events and time to death. With this evidence and the unknown function of CyB5R3 in cardiomyocytes, we created the first CyB5R3, cardiomyocyte specific inducible knockout (i-cKO) (Myh6-Cre
ERT2
- flox/flox) and we observed >50% lethality 15 days post-last tamoxifen injection. These mice recapitulated a DCM phenotype similar to the pharmacological study. Hemodynamic measurements showed increased left and right ventricular stroke volumes and decreased ejection fractions. Histology displayed myocardial inflammation and early stage fibrosis while electron microscopy revealed myofibril dystrophy. With these results, we hypothesized CyB5R3 i-cKO mice develop impaired metabolism and bioenergetics due to loss of CyB5R3 mediated heme reduction. I-cKO animals had smaller mitochondrial size, a 30% loss of total ATP and a rise in lactate production, indicating glycolytic shift from oxidative phosphorylation. RNAseq analysis showed decreased transcription in mitochondrial complexes I, II and IV and decreased complex IV activity. Since oxygen is the final electron acceptor for complex IV, we hypothesized the loss of CyB5R3 impaired oxygen delivery to the mitochondria. Therefore, a “psuedohypoxic state” was created, which was supported via Hypoxyprobe staining, by labeling cardiomyocytes specifically with low pO
2
. Collectively, the results provide an important breakthrough in cardiomyocyte biology by identifying Cyb5R3 as the first heme iron reductase critical for regulating cardiac metabolism.
Abstract 149: Targeting Protein Tyrosine Phosphatase PTP-PEST for Therapeutic Interventions in Acute Myocardial Infarction
Authors: Yang Chiu-Fen , Chen Yi-Yun , Cheng Ching-Feng , Meng Tzu-Ching
Published: March 2022

Introduction:
To date reperfusion remains the only established clinical management to salvage ischemic myocardium. However, reperfusion also results in additional damage. New interventions that prevent myocardial injury caused by ischemia/reperfusion (I/R) are the unmet clinical need. A hallmark of I/R-induced cardiac signaling perturbation is dysregulation of protein tyrosine phosphorylation (pTyr), which is dynamically controlled by protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs). Recent works showed that forced activation of myocardial PTKs reduces I/R injury to the heart, suggesting a detrimental effect of protein tyrosine dephosphorylation by PTPs on the survival of myocardium. However, the role of PTPs in this disease context has never been explored.

Hypothesis:
PTPs may act to promote I/R-induced injury to the heart.

Methods:
Mice received the surgery for myocardial ischemia (1 h) followed by reperfusion (4 h). We measured the myocardial PTP activity, the pTyr level of cardiac proteins, and the degree of heart injury. To suppress endogenous PTP activity, mice were treated with phenyl vinyl sulfone (PVS), a pan-PTP inhibitor.

Results:
We found that the overall myocardial PTP activity was significantly elevated after ischemia and I/R, in line with a decrease of pTyr in cardiac proteins. Interestingly, PTP-PEST, which is abundantly expressed in myocardium, underwent protease-processed activation in the heart exposed to I/R. Consistently, the pTyr of paxillin and p130CAS, both which are the substrates of PTP-PEST, was significantly diminished. Treatment of the mice with PVS, an inhibitor that inactivates PTP-PEST, protected the heart from I/R injury.

Conclusion:
Our finding suggests that PTP-PEST may be a new target against myocardial I/R injury. The current focus is to validate the role of PTP-PEST in cardiac I/R damage and to test the therapeutic effect of drugable PTP-PEST inhibitors. New pharmacological interventions may be designed for improving the outcome of myocardial infarction.
Abstract 159: Eicosapentaenoic Acid and Docosahexaenoic Acid Have Distinct Membrane Locations and Lipid Interactions as Determined by X-ray Diffraction
Authors: Mason R. P , Sherratt Samuel C
Published: March 2022

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) differentially influence lipid oxidation, signal transduction, fluidity, and cholesterol domain formation, potentially due to distinct membrane interactions. We used small angle x-ray diffraction to test EPA and DHA effects on model membrane structure. Vesicles were prepared to model human peripheral cell membranes using 1-palmitoyl-2-oleoyl-
sn
-glycero-3-phosphocholine (POPC) and cholesterol (C) (0.3 C:POPC mole ratio), and treated with vehicle, EPA, or DHA (1:10 mole ratio to POPC). Membrane width (
d-
space) was 59.5 Å at 10°C, decreasing to 54.7 Å at 30°C due to increased acyl chain dynamics. EPA or DHA had no effect on membrane
d
-space (<1 Å change). Electron density profiles from diffraction data were superimposed on corresponding vehicle profiles (Fig. 1). EPA increased membrane hydrocarbon core electron density over a broad area, up to ± 20 Å from the membrane center, indicating an energetically favorable extended membrane orientation for EPA, stabilized by van der Waals interactions. By contrast, DHA increased electron density in the phospholipid head group region starting at ± 12 Å from the membrane center, due to DHA-surface interactions, resulting in a pronounced electron density reduction and increased disorder in the membrane hydrocarbon core centered ± 7-9 Å from the membrane center. DHA disordering effects were less apparent at higher temperatures, likely due to greater rotational dynamics, while EPA effects were stable. The contrasting EPA and DHA effects on membrane structure signify distinct molecular locations/orientations and may contribute to observed differences in biological activity.
Abstract 160: Cardiomyocyte CaMKII Mediates Expression of Pro-inflammatory Chemokines and Cytokines, Macrophage Infiltration and Cardiac Remodeling in Response to Pressure Overload
Authors: Suetomi Takeshi , Willeford Andrew , Miyamoto Shigeki , Heller Brown Joan
Published: March 2022

Background:
There is evidence that inflammation is associated with pressure overload induced cardiac remodeling and heart failure, as well as evidence for a role of CaMKII in remodeling and heart failure development. Whether CaMKII mediates inflammatory responses that contribute to its role in adverse remodeling following TAC has not been established.

Methods and Results:
CaMKIIδ knockout (CKO) mice in which CaMKIIδ was selectively deleted from cardiomyocytes were subjected to pressure overload by transverse aortic constriction (TAC). By 3 days, the earliest time examined, there were marked increases in cardiac mRNA levels for pro-inflammatory chemokines CCL2 (MCP-1, ~15 fold), CCL3 (MIP1α, ~20 fold) and cytokines (IL-6, ~50 fold). These responses were markedly attenuated (by 56%, 43% and 42 % respectively) in the CKO mice. NFkB signaling was also increased in control heart at 3 days of TAC but not in CKO. Immunohistochemical analysis showed increases in CD68+ macrophage by 7 days TAC which further increased by 14days of TAC. Macrophages accumulation was also significantly attenuated in the CKO mice (50% decrease at 7day, 40% decrease at 14 days). Fibrosis was assessed by Masson trichrome staining and increases in collagen gene expression (col1a1 and col3a1 mRNA). Both were clearly elevated after 14 and 28 days TAC and significantly attenuated in the CKO mice.

Conclusion:
Our results indicate that activation of CaMKII localized in cardiomyocytes initiates an inflammatory transcriptional program within cardiomyocyte. We suggest that this drives immune cell recruitment and is in turn associated with development of fibrosis. Early inflammatory responses and their sequelae may thus be responsible for involvement of CaMKII in the progression from hypertrophy to heart failure.
Abstract 185: Genome Editing of Isogenic Human Induced Pluripotent Stem Cells Allows for Functional and Transcriptomic Insights Into Hereditary Dilated Cardiomyopathy Caused by Phospholamban Mutations
Authors: Ceholski Delaine K , Turnbull Irene C , Koplev Simon , Kong Chi-Wing , Stillitano Francesca , Nonnenmacher Mathieu , Haghighi Kobra , Gorski Przemyslaw A , Cohen Ninette , Kolokathis Fotis , Kremastinos Dimitrios T , Babu Arvind , Li Ronald A , Giannarelli Chiara , Karakikes Ioannis , Kranias Evangelia G , Costa Kevin D , Hajjar Roger J
Published: March 2022

Dilated cardiomyopathy (DCM) can be caused by genetic mutations in numerous cardiac proteins, including phospholamban (PLN). PLN mutations are quite rare and obtaining patient samples for mechanistic insights can be challenging. We used genome editing with CRISPR/Cas9 to successfully insert R14del and R9C PLN mutations into a human induced pluripotent stem cell (hiPSC) line from an individual with no cardiovascular disease. HiPSC-cardiomyocytes (hiPSC-CMs) with the inserted R14del PLN mutation recapitulate the phenotype observed in patient-derived R14del hiPSC-CMs, characterized by abnormal intracellular calcium cycling and arrhythmogenicity. Insertion of R9C PLN results in hiPSC-CMs displaying an abnormal response to β-agonists, defective calcium handling, and a hypertrophic phenotype. In human engineered cardiac tissues (hECTs) created from hiPSC-CMs in a 3D matrix, R14del results in a progressive worsening of developed force and R9C PLN demonstrates an abnormal lusitropic response following β-adrenergic stimulation. Further, transcriptional profiling using RNAseq suggests a role for lipid metabolism in R14del. DNA methylation studies showed that differentially expressed genes were enriched for lipoprotein metabolism and chylomicron-mediated lipid transport pathways in R14del PLN. This was also confirmed with the observation of lipid deposition in R14del hECTs and human myocardial tissues from explanted hearts of affected patients. Furthermore, small RNAseq identified 2 miRNAs that were differentially regulated in R14del hiPSC-CMs (miR-449c-5p and miR-483-3p). For R9C PLN, RNAseq suggests that the mutation results in profibrotic signaling, activation of autophagy, and an altered metabolic state. Our findings demonstrate that gene editing of hiPSCs can be used to successfully create models and delineate molecular mechanisms of human PLN mutations associated with DCM.
Abstract 217: Study II - Antinuclear Antibody Levels Study in Postural Orthostatic Tachycardia Syndrome
Authors: Solipuram Swathi , Wayar Fabian , Bhattarai Apeksha , Ashangari Chandralekha , Suleman Amer
Published: March 2022

Background:
Postural Orthostatic Tachycardia Syndrome (POTS) is a constellation of signs and symptoms that occurs when the patient stands upright. It affects primarily young women. The antinuclear antibody (ANA) test measures the amount and pattern of antibodies in blood that work against own body (autoimmune reaction). If there are more antibodies in the blood than normal, the test is positive. Previously, we have conducted a research on increased ANA levels and POTS and found that 41% of them had positive results. Taking that one step ahead, the aim of this study is to demonstrate further pathologic conditions in patients found to have positive ANA values.

Methods:
27 patients who were having positive ANA levels were selected and followed up on with phone calls about their contact with the rheumatologists out of whom 1 patient (4%) declined to participate in the research.

Results:
Out of 27 patients, 26 patients were Females (age 33.84±12.37) and 1 patient was male (age 24). 11 out of 27(40%) patients reported that they never visited rheumatologists, 15 out of 27 (55%) patients reported rheumatologist visit, 1 out of 27 (3.70%) patient reported endocrinologist visit, 2 out of 27(7.40%) patients reported that their ANA level was monitored by Primary Care Physician. 2(7.40%) patients were diagnosed with Systemic Sclerosis, 2(7.40%) patients were diagnosed with Rheumatoid Arthritis, 3(11.11%) patients were diagnosed with Systemic Lupus Erythematosus. 7 out of 15 patients had other diagnosis; 3(11.11%) patients had Ehlers Danlos Syndrome, 2(7.40%) patients had Osteoarthritis, 1(3.70%) patient had Larsen syndrome, 1(3.70%) patient had Hashimoto’s thyroiditis, 1(3.70%) patient had Fragile X Syndrome, 1(3.70%) patient had Fibromyalgia.

Conclusion:
1) 41% of POTS patients had elevated ANA levels. 2) Elevated ANA levels represents a measure of autoimmune mechanism in a subset of POTS patients. 3) Elevated ANA levels incidentally found need to be further studied.
Abstract 221: A Non-invasive Technique for Fast Assessment of Optimal LVAD Outflow Graft Implant Sites
Authors: YU Huidan W , Deb Anurag , Chen Xi , Chen Rou , Wang Dongfang , Wang I-Wen
Published: March 2022

We develop a novel noninvasive technique, named InVascular, for fast hemodynamic assessment of optimal LVAD (left ventricle assist device) outflow-graft (OG) implant suites. LVADs has been a proven and effective therapy for patients with severe end-stage heart failure. Due to the improved pump technology and better patient management, minimally invasive LVAD implantation has emerged as a paradigm shift for clinical surgical techniques. Appropriate OG insertion to either ascending aorta (a), descending thoracic aorta (b), or axillary artery (c) (Fig. 1) is critical in an LVAD surgery. We have recently invented a novel technique that integrates advanced computational modeling based on patient’s CT and ECHO images (Fig. 1d), with cutting-edge GPU (Graphic Processing Units) parallel computing technology to noninvasively quantify 4-D (time+space) flow (Fig. 1e) and pressure in the segment. Comparisons of flow and pressure among 3 OG locations (Fig. 1f) can guide an appropriate OG location for an LVAD implantation. Our results from one study case show that the OG location has important effects on flow in the ascending and descending aorta: OG locations (a) and (b) generate similar flow in the ascending aorta whereas in descending aorta OG location (a) results in larger flow than (b), which is consistent with results in public data. Meanwhile, we found that location (c) produces the smallest flow among the 3 locations in both ascending and descending aorta. Invascular can complete such assessment for an optimal OG site within 30 minutes after a patient’s imaging data are available, which is promising for clinical use in future.
Abstract 239: Transcriptomic Profiling Maps Anatomically Patterned Subpopulations Among Single Embryonic Cardiac Cells
Authors: Li Guang ,   Adele Xu,   Sean Wu
Published: March 2022

Embryonic gene expression intricately reflects anatomical context, developmental stage, and cell type. To address whether the precise spatial origins of cardiac cells can be deduced solely from their transcriptional profiles, we established a genomewide expression database from 118, 949, and 1,166 single murine heart cells at embryonic day 8.5 (e8.5), e9.5, and e10.5, respectively. We segregated these cells by type using unsupervised bioinformatics analysis and identified chamber-specific genes. Using a random forest algorithm, we reconstructed the spatial origin of single e9.5 and e10.5 cardiomyocytes with 92.0% ± 3.2% and 91.2% ± 2.8% accuracy, respectively (99.4% ± 1.0% and 99.1% ± 1.1% if a ±1 zone margin is permitted) and predicted the second heart field distribution of Isl-1-lineage descendants. When applied to Nkx2-5/ cardiomyocytes from murine e9.5 hearts, we showed their transcriptional alteration and lack of ventricular phenotype. Our database and zone classification algorithm will enable the discovery of novel mechanisms in early cardiac development and disease.
Abstract 269: Epicardial Deletion of Myocardin-related Transcription Factors Prevent Pathological Cardiac Remodeling After Ischemic Injury
Authors: Quijada Pearl J , Trembley Michael A , Velasquez Lissette , Dirkx Ronald , Small Eric M
Published: March 2022

Epicardium-derived progenitor cell (EPDC) activation after injury is a cardio-restorative mechanism in the zebrafish heart, but this response is limited in adult mammals. Identification of mechanisms that regulate adult EPDC function is critical to enhance epicardium-mediated cardiac repair. We previously found that myocardin-related transcription factors (MRTFs) regulate EPDC mobilization and coronary vessel maturation during development. Adult mice lacking both MRTF-A and -B in the epicardium (DKO) were evaluated at baseline and after myocardial infarction (MI) in this study. In non-injured DKO hearts, MRTF expression is reduced in epicardium-derived lineages such as fibroblasts and pericytes, but is unchanged in cardiomyocytes and endothelial cells. DKO mice display increased cellular retention in the epicardium as compared to wildtype mice (WT), consistent with observations during embryonic development. While WT mice demonstrate a decline in cardiac function after MI, ejection fraction is preserved in DKO mice, which also display reduced fibrotic scarring and cardiomyocyte hypertrophy. In order to determine the expression levels of pro-fibrotic genes in epicardial-derived lineages, we isolated CD31-NG2+PDGFRβ+ (pericytes), CD31-NG2-PDGFRβ- (fibroblasts) and CD31+ (endothelial cells) populations from sham and 7-day post-MI hearts. Postn (11-fold), Col1a1 (4-fold), Col3a1 (4.8-fold) and Acta2 (5-fold) are similarly activated in pericyte and fibroblast populations after MI. Of note, pericytes express abundant levels of fibronectin (38-fold vs. 16-fold in fibroblasts) and pro-inflammatory interleukin-1β (41-fold vs 12-fold in fibroblasts) suggesting that pericytes may also contribute to inflammation and fibrosis after cardiac injury. Indeed, NG2+ pericytes are dramatically reduced in remote (5-fold) and damaged regions (3.3-fold) of DKO hearts compared to WT. From this data, we hypothesize that MRTF deletion alters pro-migratory and fibrotic gene programs and reduces inflammatory signals in EPDCs decreasing the presence of scar and improving cardiac function after ischemic injury.
Abstract 278: PGC1α Activation Corrects Cardiac Dysfunction Caused by Anti-diabetic Dual-PPARα/γ Therapy
Authors: Kalliora Charikleia , Lieu Melissa J , Yue Yujia , Pol Christine , Valenti Mesele C , Tian Ying , Scerbo Diego , Schulze Christian , Madesh Muniswamy , Civelek Mete , Finck Brian N , Goldberg Ira J , Drosatos Konstantinos
Published: March 2022

Peroxisome proliferator-activated receptor (PPAR) agonists are used in metabolic diseases. PPARα agonists target hyperlipidemia and PPARγ agonists correct hyperglycemia. Dual PPARα/γ agonists were developed to combine the benefits in type 2 diabetes patients. Despite their beneficial effect, PPARα/γ agonists caused cardiac dysfunction. We studied the mechanisms that underlie this toxic effect aiming to improve a failed therapy.

Wild type mice were fed with chow and high fat diets containing Tesaglitazar (TESA) a dual PPARα/γ agonist for 6 weeks. Although, TESA lowered plasma triglyceride and glucose levels compared to controls, 2D-echo revealed severe cardiac dysfunction. Assessment of the expression of cardiac fatty acid metabolism genes that are regulated by PPARs showed that mRNA and protein levels of PPARγ-coactivator (PGC1α), a regulator of mitochondrial biogenesis, had the most profound reduction. Furthermore, we observed increased acetylation of PGC1α levels, which indicates lower activation and lower mitochondrial respiration.

We then examined the mechanisms that underlie lower expression of PGC1α upon activation of PPARα and PPARγ. Thus, we administered C57BL/6 mice with both single PPARα and PPARγ agonists (WY14643 and rosiglitazone). This treatment reproduced the reduction in PGC1α expression that we observed with TESA. Moreover, mitochondria abundance was lower. Luciferase promoter analysis in a human cardiomyocyte cell line showed that PPARα and PPARγ compete for binding on PPAR elements of the human Pgc1a promoter, as well as that PPARα binding compromises PPARγ-mediated activation of the Pgc1α promoter.

Aiming to decipher the mechanism via which dual PPARα/γ activation increases acetylation and thus inhibits PGC1α, we measured cardiac protein levels of the deacetylase sirtuin 1 (SIRT1), which were lower in primary cardiomyocytes from TESA-treated mice.

We then treated mice with diet containing TESA and Resveratrol (RSV), which stimulates SIRT1. RSV attenuated TESA-driven cardiac dysfunction and corrected mitochondrial respiratory rates in primary cardiomyocytes.

We propose that SIRT1-mediated activation of PGC1α blunts the cardiotoxic effect of dual PPARα/γ agonists and improves their therapeutic potential.
Abstract 282: A
Drosophila
Model of Cardiac Lipotoxicity
Authors: Tuthill Bryon F , Searcy Louis A , Yost Richard A , Musselman Laura P
Published: March 2022

Obesity is associated with cardiovascular disease in humans and a number of model organisms, including the fruit fly
Drosophila melanogaster
. We use a fly model of high-sugar-diet-induced metabolic disease that includes hyperglycemia, insulin resistance, obesity, cardiovascular disease, and reduced longevity. High-sugar-fed flies accumulate lipids in a variety of organs, including the heart. We are using tissue-specific metabolomics and loss-of-function genetics to probe the roles of individual organs and lipids in cardiovascular disease. Our goal is to focus on conserved pathways to better understand how overnutrition with high-calorie diets leads to adverse outcomes in patients with complications of type 2 diabetes.
Abstract 298: Circular Rna Mmu_circ_008396 Attenuates Cardiac Remodeling After Myocardial Infarction in Mice
Authors: Garikipati Venkata N , Verma Suresh K , Cheng Zhongjian , Trungcao May M , Liang Dongming , Khan Mohsin , Benedict Cindy , Cimini Maria , Ibetti Jessica , Grisanti Laurel , Schumacher Sarah M , Gao Erhe , Rabinowitz Joseph E , Wilusz Jeremy E , Goukassian David , Houser Steven , Koch Walter J , Kishore Raj
Published: March 2022

Recent studies highlighted that circular RNAs (circRNA) can play an important role in cardiac hypertrophy. However, the circRNAs in cardiac diseases is still limited. Using global circRNA expression profiling, we identified several circRNA transcripts that were differentially regulated post-MI in mice, including mmu_circ_008396 that is significantly down regulated. Cell fractionation experiments indicated that mmu_circ_008396 is highly enriched in endothelial cells in post-MI mice. Interestingly, we found a mmu_circ_008396 circRNA ortholog in humans, which was also significantly down regulated in ischemic cardiomyopathy patients. Further, overexpression of mmu_circ_008396 significantly enhanced tube formation and reduced apoptosis of human umbilical vein endothelial cells. For cardiac overexpression of mmu_circ_008396 circRNA, we created AAV9 viral particles and found that
in vivo
over expression attenuated LV dysfunction post-MI and enhanced neovascularization. Mechanistically, mmu_circ_008396 binds to its potential target miRNAs (mmu-miR-93-3p, mmu-miR-412-3p and mmu-miR-298-5p) and regulate hemeoxygenase-1/ VEGF signaling, thereby enhancing neovascularization and cardiac repair post-MI. These results indicate that mmu_circ_008396 circRNA might be a novel potential target to prevent cardiac remodeling and also highlight the significance of circRNAs in cardiovascular diseases.
Abstract 338: Cardioprotective Action of the Exosome Secreted From TGFbRI Inhibitor-primed Nkx2.5+ Cardiomyoblasts
Authors: Chen Wen-Pin , Hsiao Wei-Feng
Published: March 2022

Using transgenic cardiac Nkx2.5 enhancer-eGFP mice to label Nkx2.5+ cardiomyoblasts, an embryonic Nkx2.5+ cell population representing the common precursor for cardiovascular lineages was identified in the developing heart and a residual small population was also found in the postnatal heart. Our previous study found that A83-01, an inhibitor of TGFb receptor type I (TGFbRI), could improve cardiac function in post-injured adult heart through inducing the proliferation of Nkx2.5+ cardiomyoblasts and enhancing paracrine benefit in the enhancement of myocardial survival. The aim of the present study is to examine whether the exosome secreted from A83-01-primed Nkx2.5+ cell (exo(Nkx-A83)) could increase cardiomyocyte survival and to investigate the cardioprotective mechanism of the exosomal molecules. Adult mice cardiomyocytes were isolated and cultured in IMDM medium
in vitro
. A83-01 alone did not directly affect myocyte viability. The myocyte viability was 33.3±2.0 % on day 5, and could be prominently increased to be 63.8 ±0.9% when co-cultured with Nkx2.5+ cell in the presence of A83-01. The effect was abolished by GW4869 to block exosome secretion. Moreover, treating cardiomyocyte with the purified exo(Nkx-A83) could markedly increase cell viability
in vitro
. The miRNA microarray and qPCR validation were performed to identify the different miRNAs in the exosome secreted from Nkx2.5+ cell treated with or without A83-01. It was found the marked increase of miR-30c, miR-489, miR-92b, and miR-98 in exo(Nkx-A83). Functional study demonstrated miRNA mimics of miR-30c and miR-92b could significantly enhance myocyte survival
in vitro
. Furthermore, administration of the purified exo(Nkx-A83) via intravenous injection in isoprenaline-induced heart injured mice could significantly improve cardiac function monitored by echocardiography. In conclusion, the findings revealed miR-30c and miR-92b in exo(Nkx-A83) could contribute to its cardioprotective action for the enhancement of myocardial survival.
Abstract 355: CardioClusters: Harnessing the Power of Multi-lineage Cardiac Stem Cells
Authors: Monsanto Megan M , White Kevin , Fisher Kristina , Wang Jessica , Sussman Mark A
Published: March 2022

Existing approaches to modify stem cells for myocardial regeneration desperately need innovative solutions that enhance cell engraftment and persistence. Although this deficiency has been attacked through combinatorial stem cell delivery, there is no evidence that these stem cell injections provide for direct cellular crosstalk to promote stem cell survival and proliferation. Therefore, we created a CardioCluster, a 3D microenvironment consisting of three defined cell populations from the human heart: c-kit
+
cardiac progenitor cells (CPCs), CD90
+
/CD105
+
mesenchymal stem cells (MSCs) and CD133
+
endothelial progenitor cells (EPCs). The size of the CardioCluster can be controlled by the quantity of cells used to create the cluster, allowing them to be infused into the heart without being reduced to single cell suspensions as is the case for cardiosphere-derived cells where the structural and cell-cell contact information is lost when delivered. Unlike cardiospheres, these cardiac cells are combined into a rationally designed cluster with MSCs and CPCs in the central core and EPCs forming the outer layer. EPCs play a vital role in forming neovasculature that will connect the CardioClusters to living heart tissue not damaged by ischemia and allow for revascularization of the damaged myocardium.
In vitro
we have shown that EPCs are better able to form tubular networks on matrigel-coated plates compared to either CPCs or MSCs. MSCs reinforce the 3D structure by releasing growth factors that attract and maintain cells within the cluster, as well as release immunomodulatory signaling factors. Upon induction of an oxidative stress by hydrogen peroxide CardioClusters show improved cell survival with a lower percentage of apoptotic and/or necrotic cell populations compared to the three populations individually. Upon myocardial injection CardioClusters have been shown to maintain their 3D structural integrity. Future directions will be to assess long-term engraftment and regenerative potential in a mouse model of myocardial infarction.
Abstract 374: Evidence for Hyper-reductive and Hyper-oxidative Conditions in Heart Failure Patients
Authors: Sairam Thiagarajan , Shanmugam Gobinath , Narasimhan Madhusudhanan , Subramanian Meenu , Patel Amit N , Gopalan Rajendran , Sankaran Ramalingam , Soorappan Rajasekaran N
Published: March 2022

Background:
Oxidative stress has been linked to heart failure (HF) in humans. Antioxidant-based treatments are not always effective. Hence, we tested a hypothesis that some patients with heart failure may have a hyper-reductive state. Identifying this condition may lead to personalized optimization and better outcomes.

Methods:
Blood samples were collected from age and sex matched healthy control (n=50) and HF patients (n=55). Serum was separated and stored immediately in -80°C until analysis. Reduced glutathione (GSH) and its redox ratio (GSH/GSSG) using sigma GSH kit (38185) and malondialdehyde (MDA) levels by HPLC in the serum of HF patients were quantified. Further, the activities of key antioxidant enzymes including catalase, SOD, glutathione peroxidase and glutathione reductase were analyzed using a kinetic spectrophotometer.

Results:
While majority of the HF patients had significantly decreased the glutathione redox status (GSH/GSSG) and increased MDA levels (lipid peroxidation index) indicating a hyper-oxidative state, a subset of HF patients (n=8) displayed a significantly increased GSH/GSSG ratio along with decreased MDA levels in the serum, suggesting a strong association for a hyper-reductive state in the development of HF. Moreover, closer analyses of echocardiography revealed a lower ejection fraction (EF) with substantial diastolic dysfunction (MV E/A) in the HF patients with hyper-reductive state.

Conclusion:
These results suggest that hyper-reductive condition may indicate a worsening clinical course for HF patients. Thus a thorough diagnosis of redox state and personalized approach for antioxidant treatment is warranted.
Abstract 376: Acute Modulation of Sodium Channel Auxiliary Subunit β1-mediated Adhesion: a Novel Antiarrhythmic Strategy
Authors: Veeraraghavan Rengasayee , Hoeker Gregory S , Laviada Anita A , Wan Xiaoping , Deschenes Isabelle , Smyth James , Gorelik Julia , Poelzing Steven , Gourdie Robert G
Published: March 2022

Cardiac impulse propagation is thought to occur by direct cell to cell flow of current via connexin43 (Cx43) gap junctions (GJs). We recently demonstrated that cardiac sodium channels (Na
v
1.5) localized within the perinexus, an intercalated disk (ID) nanodomain adjacent to Cx43 GJ, may enable ephaptic coupling between cardiac myocytes. We hypothesized that β1-mediated adhesion may closely approximate membranes within ID nanodomains, facilitating ephaptic coupling. Super-resolution STochastic Optical Reconstruction Microscopy-based Relative Localization Analysis (STORM-RLA) and immuno-electron microscopy identified two Na
V
1.5 populations within the ID in guinea pig ventricles (GPVs): A perinexal population, accounting for 47% of ID-localized Na
V
1.5 and a plicate population, co-distributing with N-Cadherin, accounting for 29%. β1 was preferentially localized to the perinexus (48% of ID-localized β1) over N-Cadherin-rich plicate regions (8%). βadp1, a novel peptide inhibitor of β1 adhesion, selectively and dose-dependently inhibited barrier function in β1-overexpressing 1610 cells in electric cell-substrate impedance spectroscopy studies. Neither βadp1 nor a scrambled control peptide (βadp1-scr) affected I
Na
or action potentials in isolated GPV myocytes. However, βadp1 reduced peak current recorded from Na
V
1.5 clusters adjacent Cx43-EGFP at cell-cell contacts using scanning ion conductance microscopy-guided patch clamp. In GPVs, βadp1 (100 μM) compromised the diffusion-resistance of the ID as assessed by perfusion of fixable fluorescent dyes. βadp1 (48±4 μm) but not βadp1-scr (22±1 μm) increased perinexal intermembrane spacing compared to control GPVs (17±1μm). Optical mapping revealed that βadp1 but not βadp1-scr slowed conduction in GPVs and iPSC-derived cardiomyocyte monolayers. Importantly, in GPVs, βadp1 increased conduction anisotropy and precipitated spontaneous tachyarrhythmias in a dose-dependent manner. Thus, β1-mediated adhesion generates close apposition between Na
V
1.5-rich perinexal membranes, facilitating ephaptic conduction in the heart. Importantly, β1-mediated adhesion may be a target for novel, mechanistically-driven antiarrhythmic therapy.
Abstract 383: Modulating Calcium Dysregulation in Troponin T Linked Hypertrophic Cardiomyopathy
Authors: Lehman Sarah , Grinspan Lauren , Anderson Mark E , Tardiff Jil C
Published: March 2022

Mutations in cardiac troponin T (cTnT) account for approximately 5-10% of all Hypertrophic Cardiomyopathy (HCM). Residue Arg92 of cTnT represents a HCM-mutational hotspot, including Arg92Leu (R92L) and Arg92Trp (R92W). These mutations, although differing by only a single amino acid, present with varying degrees of cardiac remodeling and disease severity in the human population. Studying the differential effects of the R92 mutations on Ca
2+
homeostasis revealed an age-dependent disruption in down-stream calcium handling in R92W mice, including differential diastolic calcium levels and SERCA2a uptake activity. To determine the effects of treating this differential calcium handling disruption, R92L and R92W mice were treated with diltiazem hydrochloride, an L-type Ca
2+
channel blocker. While R92L mice showed a progressive worsening in diastolic function and remodeling throughout treatment, R92W mice showed improvement in diastolic function but no change in cardiac remodeling, suggesting an improvement but not amelioration of disease with diltiazem treatment. Of note, previous studies also suggested an increase in phospholamban (PLB) phosphorylation at Thr17 (CaMK-II-mediated) over time in R92W mice only. Thus, we hypothesized a mutation-specific role of CaMK-II in cTnT-linked HCM progression. We tested this via genetic partial inhibition of CaMK-II (AC3-I peptide) in both R92W and R92L transgenic mice. Both R92W and AC3-IxRW hearts exhibited a normalization of SERCA2a activity to non-transgenic levels over time. This improved Ca
2+
re-uptake was coupled with a decrease in atrial mass, improved ventricular relaxation, and normalized diastolic function only in AC3-IxRW animals, consistent with blunting of disease progression
in vivo
. In contrast, AC3-IxRL mice showed depressed SERCA2a activity, increased atrial mass and ventricular thickening, and diastolic dysfunction as compared to R92L alone mice, indicating a CaMK-II independent progression to HCM. These results suggest that closely related primary mutations can result in specific ventricular remodeling and targeting these discrete molecular secondary effects represent a novel targeted treatment approach in HCM.