bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 273: Phosphoinositide 3-Kinase γ Regulates Cardio-protective ERK by Kinase Independent Mechanism
Authors: Mohan Maradumane L , Jolly George , Anand Rohit , Naga Prasad Sathyamangla V
Published: July 2021

Phosphoinositide 3-kinase (PI3K) enzymes are critical in many cellular processes including survival. PI3Kγ, a member of the PI3K family activated by G-protein coupled receptor (GPCR), is known to be a critical player in activation of extracellular regulated kinase (ERK) signal transduction cascade, a cell survival pathway. However, the exact mechanism by which PI3Kγ plays a role in ERK activation is not clearly understood. Our studies show that PI3Kγ plays a crucial role in enhancing the tone of ERK activation as use of PI3K inhibitors reduced GPCR stimulated ERK phosphorylation in HEK293 cells. siRNA knockdown of PI3Kγ resulted in loss of ERK phosphorylation through GPCRs (β-adrenergic) as well as receptor tyrosine kinases. The role of PI3Kγ in ERK activation was further corroborated by loss of insulin stimulated ERK phosphorylation in PI3Kγ-knockout (KO) mouse embryonic fibroblasts (MEFs). Surprisingly, ERK activation in KO MEFs post-insulin stimulation was completely rescued by expression of kinase-dead PI3Kγ mutant in KO MEFs suggesting a kinase-independent role of PI3Kγ in regulating ERK function. Indepth mechanistic studies showed that PI3Kγ mediated activation of ERK by inhibiting ERK dephosphorylation following stimulation, thus stabilizing the ERK phosphorylation. PI3Kγ physically disrupts the interaction between ERK and ERK dephosphorylating phosphatase PP2A as evidenced by increase in phosphatase association with ERK in KO MEFs. Consistent with this observation, ERK activation was completely abolished in KO MEFs following carvedilol suggesting an essential role for PI3Kγ in cardio-protective ERK activation pathway. In this context, it is known that transverse aortic constriction (TAC) in mice leads to increase in ERK activation in the hearts and is also associated with concurrent up-regulation of PI3Kγ suggesting a key role for kinase-independent function of PI3Kγ in activating and maintaining the ERK signaling cascade. These indepth cellular studies and observation from our TAC studies led us to believe that kinase-dependent function of PI3Kγ may contribute to pathology while kinase-independent function may be cardio-protective through inhibition of PP2A by PI3Kγ. This novel signaling mechanism by PI3Kγ will be presented.
Abstract 277: High Consistency in Left Ventricular 31P-MR Spectra in Rats in vivo Allows Follow-up Detection of Small Variations in Energy Status and Early Energy Defects
Authors: Deschodt-Arsac Veronique , Magat Julie , Naulin Jerome , Quesson Bruno , Dos Santos Pierre , Arsac Laurent
Published: July 2021

Introduction:
Phosphorus magnetic resonance spectroscopy (31P-MRS) makes a unique and valuable contribution to understand how mitochondrial energy supply responds to ATP demand in beating hearts. Changes in PCr/ATP allow detecting energy dysbalance that often predict disease and mortality. However, MR techniques in vivo are challenged by tissue contamination and poor signal-to-noise (S/N) ratio, especially in hearts of rodent models.

Methods:
Here we optimized acquisition of localized cardiac 31P-MR spectra, obtained with a surface probe at 9.4T in vivo from left ventricle in control and SHR rats. MR spectra (192s) and short-axis cine-MRI frames were obtained during 9-weeks in sinus rhythm and during tachycardia induced by acute injection of isoproterenol (10μg/kg) at week 10.

Results:
Highly reproducible spectra with low S/N ratio helped demonstrating healthy PCr/ATP despite progressive ventricular hypertrophy in SHR rats. Isoproterenol induced similar tachycardia (425bpm ± 7) but higher drop in PCr/ATP (-17.1% vs. -4.7%, P<0.05) associated with higher recovery time constant (23.9 vs. 7.3 min, P<0.05).

Discussion/Conclusion:
Our results indicate that SHR rats maintain energy homeostasis during early development of pathology but present a blunted capacity of mitochondrial energy supply to respond to high ATP demand during strong ß-adrenergic activation of the myocardium. The optimization in localized 31P-NMR analysis offer exciting possibilities to better investigate primary precursors detection of cardiac failure in rodent models.
Abstract 29: Biodegradable Elastomeric Polyurethane Scaffolds Mechanically Matching With Native Heart Muscle
Authors: Xu Cancan , Brazile Bryn , Nguyen Kytai , Liao Jun , Tang Liping , Hong Yi
Published: July 2021

Introduction:
Biodegradable cardiac patches need to be mechanically matching with native heart muscle, in order to provide appropriate mechanical support to rapidly restore heart functions and promote tissue remodeling for myocardial infarction (MI) management. Here, we utilized chemical molecular design to develop biodegradable elastomers with low initial modulus and then process them into porous scaffolds mechanically matching with native heart muscle.

Methods and Results:
We synthesized various amorphous copolymers including poly (δ-valerolactone-co-ε-caprolactone) (PVCL) and poly (ether ester) triblock copolymers with various molecular weights and poly(ethylene glycol) (PEG) molecular weights (PVCL-PEG-PVCL). The polyurethanes were then synthesized from PVCL or PVCL-PEG-PVCL as a soft segment, hexamethylene diisocyanate (HDI) as a hard segment and putrescine as a chain extender. The polyurethane products were presented as PU-PEGx-VCLy, where x and y refer to molecular weights of PEG and PVCL, respectively. Five polymers including PU-VCL
2k
, PU-VCL
6K
, PU-PEG
1K
-VCL
1K
, PU-PEG
1K
-VCL
6K
and PU-PEG
2K
-VCL
6K
were obtained. All polymers gradually degraded in phosphate buffer solution and enzyme solution. The 3T3 fibroblasts can grow and proliferate on all polymer film surface within 5 day culture, indicating the polymers have good cellular compatibility. PU-VCL
6K
, PU-PEG
1K
-VCL
6K
and PU-PEG
2K
-VCL
6K
were further processed into porous scaffolds using thermally induced phase separation (TIPS). The PU-PEG
2K
-VCL
6K
scaffold at wet state had 0.19 ± 0.08 MPa initial modulus, which has no significant difference from initial modulus (0.19 ± 0.04 MPa) of the native porcine heart muscle. But the tensile strength of this scaffold is lower than that of heart muscle, which requies to be improved in the future.

Conclusions:
A new family of biodegradable elastic polyurethanes was synthesized and processed into porous scaffolds. The scaffolds showed promising mechanical match with heart muscle. These biodegradable polyurethane scaffolds would find opportunities to be used as a cardiac patch for heart infarction treatment.
Abstract 307: Quantitative Characterization of the Contractile Architecture of Human Stem Cell Derived Cardiomyocytes
Authors: Pasqualini Francesco S , Sheehy Sean P , Agarwal Ashutosh , Parker Kevin K
Published: July 2021

Human induced pluripotent stem cell derived cardiomyocytes (hiCMs) exhibit a fetal phenotype, but tools to quantify their relative immaturity are scarce. We reasoned that, during myocyte specification, cells progress through myofibrillogenesis as force-generating units, known as sarcomeres, self-assemble along the cell cytoskeleton. Therefore, we developed image processing techniques to quantitatively score myocyte structural phenotypes by the increasing degree of organization and alignment that sarcomeres acquire during myofibrillogenesis. Since this is a highly conserved process, quantifications obtained from α-actinin immunostains in rodent and hiCMs can be compared. Utilizing these metrics we quantitatively showed that hiCMs patterned on square fibronectin islands had significantly under-developed contractile architecture, in agreement with the qualitative observation that these cells retain a more migratory cytoskeleton. Furthermore, we trained thee independent machine learning algorithms on over 100 α-actin immunostains from engineered primary cardiac tissues at 6, 24 and 48 hours after seeding. These preparations were taken to represent differentiated, immature and mature structural architectures, respectively. After training,[[Unable to Display Character: ]]α-actinin immunostains of hiPS-derived cardiac tissues were unbiasedly analyzed by these classifiers. The results indicated that ~30% of cells exhibited cytoskeletal architectures similar to those of mature myocytes and that treatment with commercially available small molecules influence hiCM structural maturation. In conclusion, we provided metrics to assess the organization of the contractile cytoskeleton in primary and stem cell-derived cardiomyocytes and to unbiasedly quantify their maturation.
Abstract 308: An in vitro Model of Cardiac Stem Cell Therapy to Study the Coupling of Primary and Stem Cell-derived Cardiomyocytes
Authors: Pasqualini Francesco S , Aratyn-Schaus Yvonne , Yuan Hongyan , McCain Megan L , Ye George J , Sheehy Sean P , Campbell Patrick H , Parker Kevin K
Published: July 2021

For cardiac cell therapy to be effective, newly formed immature cardiomyocytes need to structurally and functionally integrate with the existing myocardium. Unfortunately, testing the electro-chemo-mechanical coupling of mature and immature cardiomyocytes in vivo is difficult. Here we engineered two cell μtissues containing combinations of mouse neonate, ES-derived, and iPS-derived cardiac myocytes on flexible substrates and utilized ratiometric calcium detection and traction force microscopy to measure excitation-contraction coupling in individual cells and in the pairs. We found that SC-derived cardiac myocytes can structurally couple with neonate cardiomyocytes to functionally support synchronous contraction, yet diastolic calcium levels were reduced in SC-derived cardiomyocytes. Consistently, neonate cardiomyocytes exerted peak systolic forces that were ~1.5-fold higher than that generated by SC-derived myocytes, yielding an imbalance in tension within the pair that was dissipated by focal adhesion-like structures at the cell-cell boundary. Finally we developed a finite element model of two-cell tissue contraction to demonstrate that an imbalance in isometric tension is sufficient to limit force transmission across cell-cell boundaries. Taken together, these results suggest that reduced force transmission between poorly coupled immature and native cardiomyocytes may explain the incomplete repair of ejection fraction observed in several clinical studies of cardiac cell therapy.
Abstract 309: MiR-31a-5p Controls Cardiomyocyte Proliferation in Postnatal Hearts
Authors: Liu Hui , Shi Jing , Wang Hui , Xuan Qingkao , Bei Yihua , Sun Wei , Li Xinli , Xiao Junjie , Kong Xiangqing
Published: July 2021

Backgroud:
MicroRNAs (miRNAs, miRs) are a class of endogenous non-codingRNAs, participating in a variety of essential biological processes including development, differentiation, proliferation and apoptosis. Rodents have the capacity to regenerate their hearts in response to injury while the capacity would be lost 7 day after birth, suggesting that mammals gradually lose their regenerative potential during postnatal development. The roles of miRNAs in regulating cardiomyocyte proliferation in postnatal hearts are largely unclear.

Methods and Results:
Cardiomyocytes were isolated from rat at day 0 and day 10. Agilent rat miRNA arrays were performed to determine the dysregulated miRNAs in cardiomyocytes between day 0 and day 10. A total of 32 miRNAs were found to be dysregulated between day 0 and day 10 (Fold change over 2 and P values less than 0.05). As determined by quantitative reverse transcription polymerase chain reactions and functional assays using EdU staining and Ki-67 staining, miR-31a-5p was found to be able to promote neonatal cardiomyocyte proliferation. Moreover, the expression of proliferation maker- Proliferating Cell Nuclear Antigen (PCNA) was also increased in cardiomyocytes transfected with miR-31a-5p mimics as determined by PCRs and Western blotting analysis. Tumor suppressor RhoBTB1 was found to be negatively regulated by miR-31a-5p in cardiomyocytes and also was responsible for the pro-proliferation effects of miR-31a-5p in neonatal cardiomyocytes.

Conclusions:
These studies demonstrate that miR-31a-5p regulates cardiomyocytes proliferation in postnatal hearts by targeting RhoBTB1. miR-31a-5p represents a therapeutic target for cardiac repair and regeneration.
Abstract 316: Enhanced Cardiac Proteasome Function in the Naked Mole-rat, the Longest-lived Rodent
Authors: Kramer David A , Grimes Kelly M , Buffenstein Rochelle
Published: July 2021

The ubiquitin-proteasome system (UPS) is responsible for the recycling of misfolded proteins. Dysfunction of the UPS has been implicated in the pathophysiology of multiple heart disorders, including heart failure and reperfusion injury, but the basic science of cardiac UPS function remains unclear. An attractive mode of inquiry into the cardiac proteasome is the long-lived naked mole rat (NMR), which maintains intact cardiac reserve and diastolic function exceptionally late into its lifespan; equivalent to a 90 year old human with a 30 year old’s heart. In this study, we investigated whether the long-lived and healthful NMR had upregulated aspects of UPS function in comparison to the short-lived well-characterized mouse. NMR hearts have more than twofold (p<0.001) greater proteasome-mediated chymotrypsin-like activity than mouse hearts. NMR hearts also have significantly greater levels of proteasome subunits than mice, including α7 and Rpt5, suggesting that the greater numbers of proteasomes could contribute to the high chymotrypsin-like activity, alternatively, the naked mole-rat heart may also have more immunoproteasomes which are more efficient. The UPS is energy-dependent, with its activity significantly influenced by available ATP. Interestingly, basal ATP levels were 40 to 50 fold higher in NMR hearts than in those of mice. This is consistent with the much larger pools of mitochondria observed in the NMR heart than in the mouse heart. Considering that both high and low ATP levels are associated with a decline in proteasome activity, we next asked whether the remarkably high basal ATP levels of the NMR heart caused a qualitative difference in UPS function between NMRs and mice. Levels of ubiquitinated protein were significantly lower in the NMR heart than in the mouse heart, suggesting that the NMR cardiac UPS system is more effective at destroying ubiquitin-tagged damaged proteins than that of the mouse, and that the NMR heart’s elevated ATP levels may play a physiological role in maintaining this enhanced UPS functionality. Overall these data suggest a high basal level of proteasome activity in the NMR heart that may be of paramount importance in this animal’s ability to withstand and/or prevent age-related cardiovascular functional declines.
Abstract 317: Physiologic Cardiac Hypertrophy in Mice Reduces mRNA Levels of Myostatin and Autophagy Genes
Authors: Pinto Graziela H , Andrades Michael E , Cohen Carolina , Martinelli Nidiane C , Leitão Santiago A , Mendoza Mariana R , Rohde Luis E , Clausell Nadine O , Biolo Andréia
Published: July 2021

Myostatin and autophagy are involved in muscle growth regulation. However, there are few studies exploring their role in physiologic cardiac hypertrophy. We evaluated myostatin and autophagy in mice subjected to a swimming protocol to induce physiologic cardiac hypertrophy.
Methods:
Adult (8 weeks-old) male BALB/c mice (
n
=52) were divided in sedentary (S) and trained (T) groups, which were evaluated in 7 (S7 or T7, initial hypertrophy) and 28 (S28 or T28, stablished hypertrophy) days after the start of the protocol. Left ventricular/tibial length ratio (LV/TL) and cardiomyocyte diameter were used to assess cardiac hypertrophy. Gene expression was evaluated by RT-qPCR, while protein expression was analyzed by western blot. Bioinformatic analysis was performed by TargetScan to predict potential miRNAs’ targets and Genemania to create an interaction network between miRNAs and genes. All results were expressed as mean ± SEM and comparisons were performed using the Student T test.
Results:
Myocardial hypertrophy was confirmed in trained group both by the increase in LV/TL ratio in 28 days (13%, p=0.0001) and cardiomyocyte diameter in 7 days (20%, p=0.04) and 28 days (30%, p=0.002). There was a decrease in myostatin gene expression levels in T7 compared to S7 group (0.8 ± 0.1
vs
1.2 ± 0.1, p=0.01) without changes at day 28. However, there was no difference in mTOR phosphorylation at T7, although it was increased in T28 compared to S28 (397±95
vs
90±23 AU; p=0.02). Autophagic genes showed reduced expression levels in trained groups at both time points (reductions of 19% and 10% for
Lc3,
22% and 11% for
P62
, 19% and 10% for
Beclin1
in T7 and T28, respectively; p<0.05 for all analyses compared to sedentary groups), but there was no difference at protein level. Bioinformatics analysis showed that miR-30a, - 221, -27a/b and 208a/b are possible regulators of autophagic and myostatin genes.
Conclusions:
Taken together, reduced myostatin during initial hypertrophy and increased mTOR phosphorylation in the established hypertrophic phenotype might favor muscular growth and reduce basal autophagy. Candidate miRNAs identified through bioinformatic analysis might regulate this process, and should be further validated in this scenario.
Abstract 318: Targeting of Tp53inp1 by Mir-221 to Reduce P62-mediated Autophagy is Cardioprotective in Ischemia / Reperfusion Injury
Authors: Wang Peipei , Chen Qiying , Richards Arthur M
Published: July 2021

Purpose:
Tumor protein 53-induced nuclear protein 1 (Tp53inp1) acts as a tumor suppressor by inducing cell death. Tp53inp1 mRNA is a predicted target of miR-221. Whether targeting Tp53inp1 plays a role in miR-221-mediated cardioprotection has not been investigated. We hypothesized that miRNA-221 directly targets Tp53inp1 to reduce ischemia/reperfusion (I/R)-induced autophagy.

Method:
Myoblast H9c2 cells underwent 16 hours 0.2% O
2
hypoxia followed by 2 hours re-oxygenation (H-R, simulating I/R). H9c2 were transfected with miRNA-221 mimic (25 nmol) and scrambled mimic control (miR-221 and MC). Cell count/viability, WST assay, cell injury-induced LDH release, and GFP-LC3 labeled autophagosome formation were measured. Cells were collected for RT-qPCR and western blot (WB) analyses. pCMV-Myc-Tp53inp1 and pcDNA3.1-Flag-p62 plasmids were cloned and transfected into H9c2 for recovery and immuno-precipitation (IP) studies. The effects of miRNA-221 inhibitor in H9c2 were also assessed.

Results:
miR-221 significantly reduced H-R injury as indicated by higher cell count/viability and WST activity, and reduced LDH (miR-221 vs. MC p<0.05). qPCR confirmed that (1) miRNA-221 expression was reduced in H-R; (2) RISC-loaded (IP pull-down Ago-2) miRNA-221 increased by ~80 fold and reduced by 95% following mimic and inhibitor transfection respectively; (3) Increased Tp53inp1 following H-R was reversed by miR-221. miR-221 inhibited H-R induced autophagosome formation (GFP-LC3). WB indicated (1) increase of LC3-I/II ratio and p62, indicators of reduced autophagy, and (2) decrease of Tp53inp1 by miR-221. IP pull-down Myc-Tp53inp1 indicated the formation of p62-Tp53inp1 complex. The protective effect of miR-221 was abolished by Tp53inp1 overexpression (pCMV-Myc-Tp53inp1 and miRNA-221 mimic co-transfection). The protective effect was corroborated in neonatal rat ventricular myocytes (NRVM). MiRNA-221 inhibitor induced reverse effects.

Conclusion:
The cardioprotection of miR-221 entails direct targeting of Tp53inp1 which reducing p62-Tp53inp1 complex formation and inhibiting H-R-induced autophagy.
Abstract 320: Selective Estrogen Receptor Modulator Retards Arterial Senescence and Atherosclerosis via Upregulation of Sirt-1 and Autophagy in Post-menopause Model Mouse
Authors: Ikeda Yoshiyuki , Miyauchi Takahiro , Iwabayashi Masaaki , Akasaki Yuichi , Sasaki Yuichi , Miyata Masaaki , Ohishi Mitsuru
Published: July 2021

Background:
Incidence of cardiovascular disease (CVD) increases with development of arterial senescence and atherosclerosis, which are accelerated after menopause in women. This study aims 1) to clarify the mechanism of which post-menopose facilitates arterial senescence and atherosclerosis, and 2) to develop optical treatment.

Methods and results:
Female Apolipoprotein E deficient (ApoE KO) mice underwent ovariectomy at 8 weeks old were used as post-menopausal model mice (PM). Age-matched female mice with sham operation were used as control (Ctr). We confirmed that serum estrogen concentration was significantly lower in PM than in Ctr. Twelve weeks after surgery, aortas were harvested to examine molecular biological analysis. Sirt-1 and the ratio of LC3II/LC3I protein expressions were significantly lower in PM than in Ctr. Ratio of senescence associated β galactosidase (SA-β Gal) positive cells and expressions of acetyl (Ac)-p53, p21, and PAI-1 were significantly higher in PM than in Ctr. Aortic atherosclerotic lesions assessed by Oil Red O staining was significantly greater in PM than in Ctr. Overexpression of either Sirt-1 or autophagy retarded arterial senescence in vitro. These results suggest that ovariectomy accelerates arterial senescence and atherosclerotic development through downregulation of Sirt-1 and autophagy. Next, we administrated selective estrogen receptor modulator (SERM) to PM mice. Sirt-1 and the ratio of LC3II/LC3I protein expression were significantly higher in SERM-treated PM mice than in untreated mice. Ratio of SA-β Gal positive cells, protein expressions of Ac-p53, p21, and PAI-1, and aortic atherosclerotic lesions were significantly lower in SERM-treated PM mice than in untreated mice. The effect of SERM on augmentation of autophagy and inhibition of arterial senescence and atherosclerotic development was attenuated by administration of sirtinol, Sirt-1 inhibitor.

Conclusion:
SERM retards development of arterial senescence and atherosclerosis by activation of Sirt-1 and autophagy in postmenopause mice.
Abstract 326: Postural Orthostatic Tachycardia Syndrome and its Associated Chronic Pain Symptoms
Authors: Ashangari Chandralekha , Suleman Amer
Published: July 2021

Background and Purpose:
The Postural Orthostatic Tachycardia Syndrome (POTS) affects primarily young women. POTS is a form of dysautonomia that is estimated to impact between 1,000,000 and 3,000,000 Americans, and millions more around the world. The symptoms of POTS are widespread because the autonomic nervous system plays an extensive role in regulating functions throughout the body. The aim of this study is to determine the chronic pain symptoms in Postural Orthostatic tachycardia syndrome (POTS) patients.

Method:
Two hundred fifty-five (255) POTS patients were randomly selected from our clinic (January 2014 to March 2015)
,
reviewed the medical records of 255 POTS patients for chronic pain symptoms and performed data analysis.

Results:
Two hundred thirty-three of the 255 (91%) patients are females (n=233, age 29.20 ± 10.32), Twenty-three of the 255 (9%) patients are males (n=23, age 29.70 ± 14.52).63% (161 of the 255) had Joint Pain/aches, 51% (131 of the 255) had Chronic headache, 40% (102 of the 255) had chest pain, 31% (80 of the 255) had Migraine, 30% (76 of the 255) had Chronic back pain,16% (42 of the 255) had Heartburn,8%(20 of the 255) had Chronic pleurisy, Rheumatoid arthritis, Muscle aches, Chronic regional pain syndrome and Hip aches.

Conclusion:
Our study is the first to characterize that Patients with postural orthostatic tachycardia syndrome (POTS) have a very high prevalence of chronic pain symptoms.
Abstract 327: A Comprehensive Echocardiogram Study in Postural Orthostatic Tachycardia Syndrome
Authors: Ashangari Chandralekha , Suleman Amer , Brown Asheki
Published: July 2021

Background and Purpose:
The Postural Orthostatic Tachycardia Syndrome (POTS) affects primarily young women. POTS is a form of dysautonomia affecting millions around the world. The aim of this study is to determine the echocardiographic findings in POTS patients.

Methods:
173 POTS patients were randomly selected and underwent echocardiogram results were reviewed and performed cumulative distribution of echocardiogram results in to top 10 percentile to see the abnormalities.

Results:
Out of 173 patients, 96% are females (n=166; age 29.20 ± 9.26), 4% are males (n=7; age 28.50 ± 13.64) Our findings (mean ± SD). RVDD (2.37 cm ± 0.30 cm), LVED (4.34 cm ± 0.35 cm), IVS (0.82 cm ± 0.12 cm), LVEF (63.7%±2.61%), LVES (2.93 cm ± 0.25 cm), LVPW (0.83 cm ± 0.13 cm) , LVOT Dia (1.82 cm ± 0.20 cm) AO Root (2.70 cm ± 0.31 cm) , LA Dia ( 3.13 cm ± 0.36 cm) ,AV Vel (1.12 m/sec ± 0.13 m/sec), LVOT Vel (0.83 m/sec ± 0.31 m/sec), Mean Gradient (2.81mmHg ± 0.68 mmHg), Peek Gradient (4.91 mmHg ± 1.40 mmHg), AVA (2.65 cm2 ± 0.44 cm2),E Vel (0.77 m/sec ± 0.13 m/sec), A Vel (0.54 m/sec ± 0.10 m/sec), E/A Ratio (1.45 ± 0.36), Mean Gradient (0.83 mmHg ± 0.29 mmHg), Peak Gradient (2.21 mmHg ± 0.82 mmHg), PHT (48.25 m/sec ± 11.13 m/sec), MVDT (151.40 m/sec ± 31.44 m/sec) , MVA (4.85 cm2 ± 1.12 cm2), MR Mild in 82 % patients ,TV Vel (0.61 m/sec ± 0.13 m/sec), RAP (est) (10 mmHg ± 0 mmHg), TR Vel (20.78 m/sec ± 5.62 m/sec), RSVP (est) (29.04 ± 4.54),TR Mild in 86 % Patients.PV Vel ( 0.72 m/sec ± 0.16 m/sec). Top 10 percentile parameters are follows RVDD 2.75 cm, LVED 4.79 cm, IVS 0.97 cm, LVEF 67.04 %, LVES 3.25 cm, LVPW 0.97 cm, LVOT Dia 2.08 cm, Ao Root 3.10 cm, LA Dia 3.59 cm AV Vel 1.29 m/sec , LVOT Vel 1.23 m/sec , Mean Gradient 3.68 mmHg, Peek Gradient 6.70 mmHG,AVA 3.21 cm2. E Vel 0.94 m/sec , A Vel 0.67 m/sec , E/A Ratio 1.91 , Mean Gradient 1.20 mmHg, Peak Gradient 3.26 mmHg, PHT 62.51 m/sec , MVDT 191.69 m/sec, MVA 6.29 cm2, TV Vel 0.78 m/sec, RAP (est) 10mmHg, TR Vel 27.98 m/sec, RSVP (est) 34.81,PV Vel 0.93 m/sec.

Conclusion:
POTS patients have normal echocardiographic parameters however even the top 10 percentile of these patients had smaller LVEDD. Similarly the internal dimensions of cardiac chambers does appear to be smaller though still within accepted normal parameters. Further studies should be taken to better define this subgroup.
Abstract 328: Effects of Sympatho-excitation on Directional Anisotropy in Ventricular Myocardium
Authors: Ajijola Olujimi A , Khahera Anadjeet , Aliotta Eric , Kwon Oh Jin , Nakamura Keijiro , Irie Tadanobu , Ennis Daniel B , Shivkumar Kalyanam
Published: July 2021

Introduction:
Control of electrical propagation exerted by the sympathetic nervous system is has not been quantified in-depth.

Methods:
High-resolution mapping (64-electrode plaque, 8x8, 1.96cm
2
) of the anterior LV myocardium in porcine model (n=6) was performed before & during left stellate ganglion stimulation (LSGS). Activation times (AT), activation recovery intervals (ARIs), conduction velocities (CV), and CV anisotropy were obtained during pacing. Ex vivo diffusion tensor MRI and histology were performed to define myocardial fiber orientation in the mapped regions.

Results:
LSGS shortened ARI (314.3±7.8ms vs. 287.8±6.6ms, p<0.001). At baseline, longitudinal CV (CV
L
) was greater than transverse CV (CV
T
) (1.2±0.2m/s vs 0.7±0.1m/s, p<0.001). LSGS did not increase CV
L
(1.16±0.16m/s vs 1.17±0.15m/s, p=0.2), or CV
T
(0.67±0.07m/s vs 0.7±0.04m/s, p=0.2). However, CV in the retrograde direction along fiber orientation was significantly increased by LSGS (0.7±0.05m/s vs 0.9±0.06m/s, p<0.01). This resulted in a significant reduction in directional anisotropy (CV
antegrade
/CV
retrograde
) along fiber direction (1.6±0.3 vs 1.3±0.3, p<0.001), but not CV
L
/CV
T
(1.78±0.2 vs. 1.66±0.2 for BL and LSGS, respectively, p>0.2). Heterogeneity of activation (AT
disp
) was greater in the transverse than longitudinal direction (20±2ms
2
/cm
2
vs. 13±1.5ms
2
/cm
2
, p= 0.022). LSGS decreased transverse AT
disp
to 18±1.8 ms
2
/cm
2
(p=0.015), but not longitudinal AT
disp
.

Conclusion:
Cardiac sympatho-excitation modulates CV and AT
disp
in a fiber orientation-dependent manner. These uneven changes may contribute to arrhythmogenic mechanisms seen during sympatho-excitation.
Abstract 33: Prostaglandin E2 Reduces Cardiac Contractility via EP3 Receptor
Authors: Gu Xiaosong , Xu Jiang , Yang Xiao-Ping , Peterson Edward , Harding Pamela
Published: July 2021

Prostaglandin E2 (PGE2) EP receptors EP3 and EP4 are present in the heart and signal via decreased and increased cAMP production, respectively. Previously we reported that cardiomyocyte-specific EP4 KO mice develop a phenotype of dilated cardiomyopathy with reduced ejection fraction. We thus hypothesized that PGE2 decreases contractility via EP3. To test this hypothesis, the effects of PGE2 and the EP1/EP3 agonist sulprostone (sulp) were examined in the mouse langendorff preparation and in adult mouse cardiomyocytes (AVM) using the IonOptix cell contractility system. Isolated hearts of 18-20 wk old male C57Bl/6 mice were mounted and equilibrated for 10 min, then perfused with PGE2 (10
-6
mol/l) or sulp (10
-6
mol/l) for 30 min. Values at the end of equilibration were set to 100%. Compared to vehicle, PGE2 decreased +dp/dt (77.8±3% vs 96.7±3%, p<0.01) and left ventricular developed pressure, LVDP (77.2±2% vs 96.8±3%, p<0.001). Sulp decreased +dp/dt (75.9±2% vs 96.7±3%, p<0.001), -dp/dt (72.2±1% vs 85.7±1%, p<0.01) and LVDP (70.9±1% vs 96.8±3%, p<0.001). The effects of both PGE2 and sulp were reversed by the EP3 antagonist, L789,106 (10
-6
mol/l). Myocyte contractility was evaluated on the IonOptix system with pacing at 1Hz. Treatment with PGE2 (10
-9
M) for 10 min reduced contractility as measured by peak height (3.69 ± 0.48% for vehicle vs 2.00 ± 0.22% for PGE2, p < 0.05 ), departure velocity (-171.9 ± 22.9 um/sec for vehicle vs -106.3± 12.5 um/sec for PGE2, p < 0.05) and return velocity (87.7 ± 16.3 um/sec for vehicle vs 36.7 ± 6.6 um/sec for PGE2, p < 0.05) with similar effects noted for sulp. Sulp reduced change in peak height (4.79 ± 1.15% for vehicle vs 1.81 ± 0.37% for sulp, p < 0.05), departure velocity (-169.1 ± 35.8 um/sec for vehicle vs -59.4 ± 10.3 um/sec for sulp, p < 0.05) and return velocity (86.5 ± 23.8 um/sec for vehicle vs 16.9 ± 14.7 um/sec for sulp, p < 0.05). We then examined the acute effects of PGE2 and sulp on expression of phosphorylated phospholamban (PLN) and SERCA using Western blot. Treatment of AVM for 15min with either PGE2 or sulp decreased expression of phosphorylated PLN corrected to total PLN, by 67% and 43%. SERCA2a expression was unaffected. In conclusion, PGE2 and sulp reduce contractility via the EP3 receptor through effects on PLN.
Abstract 332: Exercise Training Protects Against Acute Myocardial Infarction via Improving Myocardial Energy Metabolism and Mitochondrial Biogenesis
Authors: Tao Lichan , Bei Yihua , Zhang Haifeng , Zhou Yanli , Jiang Jingfa , Chen Ping , Shen Shutong , Xiao Junjie , Li Xinli
Published: July 2021

Acute myocardial infarction (AMI) represents a major cause of morbidity and mortality worldwide. Exercise has been proved to reduce myocardial ischemia-reperfusion (I/R) injury. However it remains unclear whether, and (if so) how, exercise could protect against AMI. Methods: Mice were trained using a 3-week swimming protocol, and then subjected to left coronary artery (LCA) ligation, and finally sacrificed 24 h after AMI. Results: Exercise training reduces myocardial infarct size and abolishes AMI-induced autophagy and apoptosis. MI leads to a shift from fatty acid to glucose metabolism in the myocardium with a downregulation of PPAR-α and PPAR-γ. Also, AMI induces an adaptive increase of mitochondrial DNA replication and transcription in the acute phase of MI, accompanied by an activation of PGC-1α signaling. Exercise abolishes the derangement of myocardial glucose and lipid metabolism and further enhances the adaptive increase of mitochondrial biogenesis. Conclusion: Exercise training protects against AMI-induced acute cardiac injury through improving myocardial energy metabolism and enhancing the early adaptive change of mitochondrial biogenesis.
Abstract 337: Reversal of Pathological Responses to Pressure Overload by Inhibition of Phospholipase Cbeta1b
Authors: Grubb David R , Kiriazis Helen , Du Xiao-jun , McMullen Julie , Woodcock Elizabeth
Published: July 2021

The immediate downstream effector of Galpahq is the early signaling enzyme phospholipase Cbeta1b (PLCbeta1b), which is selectively elevated in failing human myocardium. When delivered to the adult mouse heart, expression of PLCbeta1b causes rapid contractile dysfunction. PLCbeta1b targets to the sarcolemma for activation through an interaction with Shank3/Homer1C/TrpC4α using a specific C-terminal sequence of 32aa. The targeting/activity of PLCbeta1b can be inhibited by expression of a sarcolemmal targeted mini-gene composed of this 32aa sequence (lyn-FLAG-PLCbeta1b-CT, b-CT). rAAV6-b-CT, or blank virus, was delivered IV (1011vg/mouse) and trans-aortic-constriction (TAC) or sham-operation was performed 8 weeks later. TAC induced maximal hypertrophy by 8 weeks after TAC, followed by contractile dysfunction and lung congestion from 16 weeks. Expression of rAAV6-b-CT prior to TAC reduced the hypertrophic response and prevented the contractile dysfunction and lung congestion. Expressing a modified b-CT peptide that does not inhibit PLCbeta1b signaling had no effect on hypertrophy, contractility or lung congestion following TAC. We conclude that PLCbeta1b can be effectively targeted by preventing its binding to the sarcolemma and that this inhibition ameliorates pathological responses following acute pressure overload. The binding of PLCbeta1b to its sarcolemmal scaffold provides the basis for the development of a new class of inotropic agent.
Abstract 338: Cardiac Overexpression of Creatine Kinase Improves Cardiomycytes Function in Heart Failure and During Increased Redox Stress
Authors: Tocchetti Carlo G , Leppo Michelle , Bedja Djahida , Wang Yibin , Weiss Robert G , Paolocci Nazareno
Published: July 2021

Aims:
Several studies suggest that abnormal energy metabolism contributes to heart failure or that the failing heart is energy starved. Here we aim at testing whether an increase in intracellular CK improves myocellular contractility in experimental myocardial dysfunction and protects from increased oxidative conditions.

Methods-Results:
We tested the response to the β-agonist isoproterenol (2.5 nM, ISO) in field-stimulated (.5 Hz, RT) adult cardiomyocytes isolated from wild-type (WT) mice and mice overexpressing cardiac myofibrillar or mitochondrial CK (CK-M or CK-mito) from sham and failing (8 wk transverse aortic constriction (TAC)) hearts, to dissect whether overexpressing CK alters myocyte function at baseline and during increased energetic demand. There were no differences in sarcomere fractional shortening (FS) or Ca2+ transients at baseline and with ISO among sham WT, CK-M or CK-mito myocytes. However, ISO effects were significantly reduced in WT TAC myocytes, consistent with prior reports. Conversely, in CK-M or CK-mito TAC myocytes, ISO-induced inotropy was fully preserved. Interestingly, incubation with the AMPK-stimulator AICAR (1mM for at least 90’) did not have any effect on WT TAC, but increased FS in TAC CK-M (+82%) and CK-mito (+42%) myocytes significantly, supporting the important metabolic role played by enhancing CK in failing hearts. To test whether overexpressing CK-M or CK-mito confer protection against acute oxidative stress, sham myocytes were exposed to H2O2 (50μM, 10’) and the interval (seconds) between the beginning of H2O2 superfusion and the appearance of irreversible arrhythmias was measured. WT and CK-M myocytes had a similar response (416±91s vs 411±68s), whereas in CK-mito this interval was significantly prolonged (600±64s). Similarly, upon acute infusion of the anticancer TKI sunitinib (2μM), whose cardiotoxic properties have been linked also to an increase in ROS, irreversible arrhythmias appeared after 657±43s in CK-mito (p<.5 vs 561±66 for WT and 467±88 for CK-M).

Conclusions:
Overexpressing CK-M and CK-mito under failing-TAC conditions improves myocyte function likely through better preserved Ca2+ handling, whereas only the up-regulation of CK-mito is more effective in buffering ROS effects.
Abstract 339: DWORF: a Novel Cardiac Micropeptide That Enhances SERCA Activity and Cardiomyocyte Contractility
Authors: Makarewich Catherine A , Nelson Benjamin R , Reese Austin L , Winders Benjamin R , Anderson Douglas M , McAnally John R , Kavalali Ege T , Bassel-Duby Rhonda , Olson Eric N
Published: July 2021

Background:
Our lab has discovered a novel conserved micropeptide of 34 amino acids encoded by a muscle-specific RNA that was previously annotated as a putative long non-coding RNA (lncRNA). This micropeptide, which we named DWORF (DWarf Open Reading Frame), shares structural similarity with the known SERCA modulators phospholamban (PLN), sarcolipin (SLN) and myoregulin (MLN).

Objective:
To define the functional role of DWORF in the heart and elucidate its mechanism of action.

Methods and Results:
Immunofluorescence staining and imaging in isolated adult mouse cardiac myocytes indicates that DWORF specifically localizes to sarcoplasmic reticulum (SR) membranes where it co-localizes with SERCA. Co-IP experiments performed in COS or HEK cells co-transfected with DWORF and SERCA show that DWORF forms a stable complex with various SERCA isoforms. Further biochemical analysis indicates that DWORF binds to the same site on SERCA as PLN, and overexpression of DWORF is capable of competing with PLN for SERCA binding. Co-expression of GFP-tagged DWORF and PLN in the presence of SERCA followed by SERCA pull-down and GFP Western indicates that PLN and DWORF have similar affinities for SERCA binding. Transgenic mice with cardiac specific over-expression of DWORF show enhanced cellular contractility as evidenced by increased peak Ca2+ transient amplitude and faster cytosolic Ca2+ decay rates (reduced Tau values). Furthermore, Ca2+-dependent Ca2+-uptake assays performed in homogenates from DWORF transgenic and littermate control hearts show a statistically significant leftward shift of the Ca2+-dependence curve for SERCA indicating a higher affinity of SERCA for Ca2+. Intriguingly, DWORF expression is silenced in the heart in response to pathological calcineurin signaling, implicating this micropeptide in the cellular pathway for calcineurin action.

Conclusions:
We have identified a novel muscle-specific micropeptide, DWORF, which is expressed in the heart at high levels and has the capacity to bind SERCA and modulate its function to increase contractility in transgenic mice. Our findings provide exciting data in support of DWORF as a previously unrecognized endogenous SERCA activator that plays a role in cardiac contractility.
Abstract 34: STIM1 Increases Calcium Stores in the Sarcoplasmic Reticulum of Adult Feline Ventricular Myocytes
Authors: Troupes Constantine , Mohsin Sadia , Berretta Remus , Kubo Hajime , Soboloff Jonathan , Houser Steven
Published: July 2021

Background:
STIM1 is a Sarcoplasmic Reticulum (SR) membrane resident protein implicated in sensing and maintaining SR Ca2+ levels. The role of STIM1 in the regulation of SR Ca2+ stores in the normal and diseased heart is not well described. Previous reports confirm that STIM1 is present at low levels in the healthy adult heart, but expression levels increase after cardiac injury.

Objective:
To determine if increased STIM1 expression after cardiac injury may be involved in the disturbed Ca2+ cycling present within diseased cardiomyocytes.

Results:
We used adenovirus to express either STIM1 or red fluorescent protein (RFP) in freshly isolated adult feline ventricular myocytes (AFMs). After 48 hours in culture, STIM1 induced cell death in 60% of myocytes versus only 5% in RFP controls. Addition of nifedipine rescued the cell death caused by STIM1, but block of transient receptor potential canonical (TRPC) channels was unable to improve viability. AFMs expressing STIM1 exhibited increased fractional shortening and Ca2+ transient amplitude, which was associated with increased SR load. Interestingly, high SR load levels caused by STIM1 were not reduced by nifedipine addition. We also found that baseline L-type channel current amplitude was significantly reduced by 20%, but no difference in current amplitude was found after Bay K8644 addition.

Conclusions:
In this study, we found that STIM1 caused Ca2+ overload leading to cell death. This process was L-type channel dependent, but TRPC channels were not involved. STIM1 also caused increased SR load at rest, which was not altered by nifedipine revealing that STIM1 mediated Ca2+ influx does not require L-type channel activity but Ca2+ influx through L-type channels is essential for STIM1 induced cellular death. Furthermore, STIM1 reduced L-type current activity but did not alter channel availability. These data show that after cardiac injury, STIM1 is likely to play a role in regulating cell survival as well as excitation-contraction coupling.
Abstract 341: The P2Y2 Nucleotide Receptor Regulates Vascular Calcification
Authors: Seye Cheikh , Sheldon Maxwell , Qian Shaomin
Published: July 2021

Objective:
Vascular calcification is widespread in individuals with atherosclerosis, and is associated with inflammatory changes and expression of osteoblast-like cell phenotypes. Recent studies identified extracellular nucleotides and P2Y receptor cascade as important regulators of bone remodeling. We investigated the potential role of the P2Y2 receptor (P2Y2R) in vascular calcification.

Methods and Results:
P2Y2R-null mice were crossed with ApoE-null mice to generate P2Y2R/ApoE double knock-out mice. When fed a standard mouse chow diet for 16 weeks, P2Y2R–/–/ApoE–/– mice showed significant higher intimal calcification as compared to their APOE–/– counterparts. Smooth muscle cells (SMCs) isolated from aortas of P2Y2R +/+ and P2Y2R -/- mice were identical in morphology and stained positively for SM lineage proteins including, desmin, smooth muscle and SM22alpha. When cultured in medium containing high concentrations of inorganic phosphate, an inducer of vascular calcification, a remarkably higher calcification was observed in P2Y2R -/- SMCs compared to P2Y2R +/+ SMCs. Furthermore, retroviral transduction of mouse P2Y2RcDNA into P2Y2R -/- SMCs rescued the calcification phenotype of the cells.

Conclusion:
These results demonstrate that inactivation of the P2Y2R gene regulates vascular calcification both in vivo and in vitro, suggesting that drugs targeting this receptor could prevent complications associated with vascular calcification
Abstract 344: Finding the Achilles’ heel of Muscle Giant---TALEN-mediated Gene-editing in Zebrafish Titin
Authors: Shih Yu-Huan , Xu Xiaolei
Published: July 2021

Background:
TITIN (TTN) has more than 300 exons and encodes a gigantic protein that is crucial for heart and muscle development. Mutations in TTN caused a variety of human diseases including cardiomyopathy and muscular dystrophy. Recently, dilated cardiomyopathy-associated mutations on TTN have been found more frequently in exons encoding A-band domains but less in exons encoding the N-terminal Z-disc domains, suggesting that mutations in different exons of TTN cause distinct consequences. To elucidate the underlying mechanisms, we leveraged the Transcription Activator-Like Effects Nuclease (TALEN) technology in zebrafish to introduce truncating mutations in different exons of ttn, and then study their effects on heart and somites.

Results:
We generated truncational mutations in different exons of zebrafish titins encoding Z-disc, N2B, Novex-3, and A domains, respectively. Because zebrafish contains two titin homologues, ttna and ttnb, we introduced mutations in both genes at the corresponding loci. While both Z-disc and A band mutations on ttna disrupted sarcomere assembly in heart and somites, Z-disc or A band mutations on ttnb only affect somites without affecting the heart. Interestingly, a Z-disc mutation on ttna resulted in milder phenotypes than an A-band mutation, while a Z-disc mutation on ttnb generated severer phenotypes than an A-band mutation. No phenotype was observed in the homozygous fish in either ttna-novex-3 or ttnb-N2B mutant fish.

Conclusions:
A spectrum of truncational mutations in ttna and ttnb has been generated in zebrafish using the TALEN technology. Mutations in different exons result in different phenotypes. Detailed characterization of these mutants and double mutants will be presented, which shall elicit distinct contribution of alternative splicing and exon skipping as two candidate mechanisms during pathogenesis of Titinopathies.
Abstract 346: A Novel Interaction Between E2F and β-adrenergic Pathways Regulates Survival and Growth in Murine Myocardium
Authors: Major Jennifer L , Salih Maysoon , Tuana Balwant S
Published: July 2021

The E2F/Pocket protein (Rb) pathway regulates cell growth, differentiation, and death by modulating gene expression. We previously examined this pathway in myocardium via manipulation of E2F6, which represses gene activity independently of Rb. Mice with cardiac specific expression of E2F6 develop dilated cardiomyopathy (DCM) without any signs of hypertrophic growth. We assessed the mechanisms of the apparent failure of compensatory growth as well as their response to the β-adrenergic agonist isoproterenol (iso). E2F6 transgenic (Tg) mice present with left ventricle dilation and significantly reduced ejection fraction as early as 2 weeks which persists into adulthood, but with no apparent increase in left ventricle weight: body weight (LVW:BW). E2F6-Tg mice treated with iso show double the increase in LVW: BW than their Wt counterparts (32% vs 16%, p-value: 0.007). Western blot revealed a specific activation of the β2-adrenergic pathway in Tg myocardium under basal conditions including a ~2-fold increase in β2-adrenergic receptors (β2-AR) (p-value: 8.9E-05), protein kinase A catalytic subunit (PKA-C) (p-value: 0.0176), activated c-SRC tyrosine-protein kinase (p-value: 0.0002), and an induction of the anti-apoptotic gene Bcl2. In contrast, a ~70% decrease in the cardiac growth regulator: AKT1 (p-value 0.0001) and a 4-fold increase in cyclic AMP dependent phosphodiesterase 4D (PDE4D), the negative regulator of PKA activity, was evident in Tg myocardium. The expression of E2F3 was de-regulated by E2F6, but was restored by iso while Rb expression was down-regulated. Thus deregulation of E2F/Rb pathway by E2F6 altered the β-adrenergic signaling pathway such that survival signaling was activated while hypertrophy was repressed resulting in the development of DCM without any increase in muscle mass. These data reveal a novel interplay between E2F and the β adrenergic pathway which regulate cardiac growth and fate.