bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 14: Pim-1 Preserves Mitochondrial Morphology by Inhibiting Drp1 Translocation
Authors: Din Shabana , Mason Matt , Volkers Mirko , Johnson Bevan , Konstandin Mathias , Quijada Pearl , Nunnari Jodi , Sussman Mark A
Published: July 2021

Rationale:
Mitochondrial morphological dynamics affect the outcome of ischemic heart damage. Mitochondrial fission protein Dynamin Related Protein 1 (Drp1) is a mediator of mitochondrial morphological changes and cell death during ischemic injury. Mitochondrial integrity is maintained by cardioprotective kinase Pim1, which enhances resistance to apoptotic challenge and ischemia reperfusion injury. In this study we examine the relationship between Pim1 activity and Drp1 regulation of mitochondrial morphology in cardiomyocytes challenged by ischemia.

Objective:
To demonstrate that Pim1 inhibits Drp1 translocation to the mitochondria in response to ischemic injury.

Methods and Results:
Simulated ischemia and simulated ischemia reperfusion (sI & sI/R) induced mitochondrial fragmentation and cell death in neonatal rat cardiac myocytes (NRCM), respectively. Mitochondrial fragmentation accompanied Drp1 translocation to the mitochondria in NRCM. Inhibition of Drp1 by mdivi1 preserved mitochondrial reticular morphology and inhibited apoptotic cell death. Mice subjected to sI/R injury displayed Drp1 mitochondrial localization, while exposure to mdivi1 led to reduced infarct size. Interestingly, transgenic hearts overexpressing Pim1 decreased total Drp1 levels, increased phosphorylation of Drp1 at serine 637, and inhibited Drp1 localization to mitochondria while preserving reticular morphology after sI. In contrast, Pim1 dominant negative (PDN) transgenic hearts and NRCM exhibit increased Drp1 translocation to mitochondria and fragmented mitochondria. PDN hearts exhibit decreased phosphorylation of serine 637 and upregulation of BH3 protein PUMA, inducing Drp1 accumulation at mitochondria and increased sensitivity to apoptotic stimuli. In PDN NRCMs, overexpression of Puma dominant negative (PumaDN) attenuated localization of Drp1 to mitochondria and inhibited cell death during sI.

Conclusion:
Pim1 activity prevents Drp1 compartmentalization to the mitochondria and preserves reticular mitochondrial morphology in response to simulated ischemia. Therefore, selective manipulation of Pim1 should be pursued as a therapeutic target to maintain mitochondrial morphology for cardioprotection.
Abstract 149: Effect of Pentraxin 3 on Pressure Overload--Induced Left Ventricular Hypertrophy
Authors: Suzuki Satoshi , Shishido Tetsuro , Funayama Akira , Netsu Shunsuke , Takeishi Yasuchika
Published: July 2021

Backgrounds:
Left ventricular (LV) adverse remodeling is enhanced by inflammatory cytokines. Pentraxin 3 (PTX3), which is one of the pentraxin superfamily same as C-reactive protein, is rapidly produced in response to inflammatory signals, and plasma PTX3 levels are increased in patients with heart failure. Previously we reported that PTX3 deficient mice demonstrated the suppression of hypertrophic cardiac remodeling after pressure overload. This study aimed to examine the cardiac specific influence of PTX3 on LV hypertrophy and dysfunction in response to pressure overload.

Methods and Results:
We created transgenic (TG) mice with cardiac-specific overexpression of PTX3 using alpha-myosin heavy chain promoter. We performed transverse aortic constriction (TAC) or sham operation on TG and wild type (WT) littermate mice. Expression of PTX3 was increased after TAC in WT mice, and immunohistochemistry proved that PTX3 was produced mainly from the interstitial tissue of the heart and infiltrating macrophages. Activation of extracellular signal-regulated kinase (ERK) and nuclear factor kappa-B was enhanced in TG mice compared to WT mice after TAC. Interleukin-6 production in heart tissue was augmented in TG mice compared to WT mice. Echocardiography and cardiac catheterization revealed that adverse remodeling with LV dysfunction was enhanced with increased interstitial fibrosis in TG mice compared to WT mice. Real time RT-PCR demonstrated that gene expressions related to cardiac hypertrophy and fibrosis were augmented in TG mice compared to WT mice. Next we isolated neonatal rat cardiomyocytes and fibroblasts. PTX3 expressions were increased after H
2
O
2
stimulation in cardiomyocytes and fibroblasts. Phosphorylation of ERK by recombinant PTX3 stimulation was more potent in cardiac fibroblasts than in cardiomyocytes.

Conclusions:
The local inflammatory mediator PTX3 directly modulates the hypertrophic response and LV dysfunction following increased afterload, and cardiac fibroblast might be important target cells of PTX3 stimulation.
Abstract 150: Determining Changes in Contractility, Myofilament Expression and Myofilament Sensitivity in the Developing Human Ventricle
Authors: Crawford Brian H , Saafir Talib , Baroi Gitanjali , Shen Ming , Joyner Ronald , Ding Guoliang , Wagner Mary
Published: July 2021

As pediatric cardiac surgery is increasingly performed in the first year of life, the need for understanding contractility regulation becomes increasingly important. We examined contractility and myofilament changes in ventricular biopsies removed as part of the surgical correction of congenital heart defects from newborns (NB) (hypoplastic left heart syndrome, < 1 wk old) and infants (IF) (tetralogy of Fallot, 3-12 mo old). Sarcomere shortening and calcium (Ca) transients were measured in isolated ventricular cells stimulated at 0.5 and 1 Hz. Increasing pacing frequency caused a significant increase in sarcomere shortening (p< 0.05)in only the IF, but the Ca transient amplitude was relatively unchanged in both groups. Consistent with work in the developing rat heart, we found an isoform switch with increasing developmental age in myofilament proteins, troponin T (TnT) and troponin I (TnI). Western blot analysis revealed that total TnI (the sum of cardiac TnI (cTnI) and slow skeletal TnI (ssTnI)) was not significantly different between the two age groups. However, when comparing only the cTnI isoforms, we found that the NB had a significantly lower levels compared to the IF (11556±789 a.u. vs. 21770±1700 a.u., p<0.001). Analysis of the RT-PCR revealed nearly significant lower levels of TnT isoform 1 in the IF group than the NB group (p=0.078), no significant difference in the levels of TnT isoform 2 (p= 0.536), and significantly higher levels of TnT isoform 3 in the IF group than the NB group (p= 0.039). We also observed developmental changes in myofilament sensitivity to calcium as assessed by measuring the gradient of the fura-2 cell length trajectory on phase-plane diagrams during the late relaxation of the twitch contraction. These results collectively suggest that contractility and myofilament changes, or the lack of changes, may serve as targets for clarifying elements associated with cardiac dysfunction in the developing human ventricle.
Abstract 157: Metabolomic Profiling of AMP-Signaling Stress: Effects of Metformin on Wild Type and Adenylate Kinase AK1-/- Transgenic Mice
Authors: Nemutlu Emirhan , Zhang Song , Terzic Andre , Dzeja Petras
Published: July 2021

Metabolic drugs frequently target mitochondrial functions and AMP signaling. Metformin, one of the most prescribed anti-diabetic medications, exerts its glucose lowering effect through mild interference with mitochondrial energy metabolism triggering homeostatic adenylate kinase (AK)-catalyzed AMP and downstream AMPK signaling. However, metabolomic signature of drug-induced “therapeutic mitochondrial toxicity" and energetic imbalance-induced AMP signaling are poorly understood. This work was aimed to determine metabolomic representation of metformin-induced mitochondrial and AMP signaling stress by comparing wild type and AK1-deficient mice with compromised AMP-signaling. Metabolomic profiling of plasma and urine samples from wild type mice treated with metformin revealed distinct metabolic signature suggesting altered energy, Krebs cycle, lipid and ketone body metabolism, with higher levels of creatinine, malate, α-ketoglutarate, FFA, glycerol, glycerol-3-phosphate, 3-hydroxybutyrate and hypoxanthine. Contrary to expectations glucose lowering effect of metformin was greater in energetically and AMP-signaling compromised AK1-/- mice. Metformin action on AK1 deficient mice was characterized by increased levels of creatinine, lactate, alanine, FFA, phosphoserine, acetoacetate, 3-hydroxybutyrate, malate and α-ketoglutarate indicating broader alterations in energy, glycolytic, Krebs cycle, ketone body, lipid and amino acid metabolism. At baseline, livers from AK1-/- mice had higher AMP/ADP and pAMPKα/AMPKα ratios compared to wild type. There were no significant sustained changes in global nucleotide ratios after metformin treatment. Overall metabolomic profile of AK1-/- mice indicate larger disturbances in energetic and substrate metabolic networks after metformin treatment compared to wild type. Thus, this study revealed specific metabolomic signatures of metformin therapeutic action and mitochondrial toxicity indicating that genetic and metabolic background modulate individual response to metformin treatment.
Abstract 158: Regulation of miR-21 Expression by Acetylation in Myocardial Infarction
Authors: Renaud Ludivine , Kasiganesan Harinath , Gao Erhe , Mani Santhosh K , Jones Jeffrey A , Stroud Robert E , Chou James , Menick Donald R
Published: July 2021

Cardiovascular diseases are one of the leading causes of morbidity and mortality in the world, underlining the need for innovative therapies and diagnosis. Recent reports have identified microRNAs (miRNAs) as central players in regulating gene expression and showed that several miRNAs are aberrantly expressed in cardiac arrhythmia, hypertrophy, fibrosis, ischemia, vascular atherosclerosis and heart failure. Gene expression is also regulated at the transcriptional level by histone deacetylases (HDACs) under basal and pathological conditions. We previously demonstrated that 1) class I and class II HDACs play an important role in the basal expression and upregulation of the sodium-calcium exchanger (Ncx1) gene in adult cardiomyocytes and pressure-overloaded ventricle and 2) treatment with class I/IIb HDAC inhibitors trichostatin (TSA) or suberoylanilide hydroxamic acid (SAHA) improved ventricular function by suppressing matrix metalloproteinases (MMPs) gene expression in myocardial infarction (MI).Therefore it is possible that protein acetylation regulates the expression of some miRNAs and

we hypothesize that HDAC inhibition would attenuate the shift in expression of certain miRNAs that are aberrantly expressed post-MI.

In a pilot study, ligation of the left anterior descending (LAD) coronary artery was performed to induce MI with and without SAHA treatment. Because of its misregulation and relevance in cardiac hypertrophy and MI, we examined the expression level of miR-21. qRT-PCR confirmed that miR-21 is increased by 8 fold 7 days post-MI. Interestingly, SAHA treatment significantly attenuated the abnormal expression of miR-21. To our knowledge, it is the first report of the regulation of a miRNA by HDACs in the heart. We anticipate that not only miR-21 but other miRNAs will fall under the same mechanistic control via acetylation. The miR-21 promoter contains binding sites for several transcription factors that get acetylated and we speculate that one or more HDAC mediate the expression of miR-21 by controlling the acetylation state of transcription factors interacting with the miR-21 promoter.
Abstract 159: Beneficial Cardiac Effects of Caloric Restriction in a Murine Model of Obesity Are Attenuated with Age
Authors: AlGhatrif Majd , Watts Vabren L , Sivakumaran Vidhya , Niu Xiaolin , Halushka Marc , Miller Karen L , Vandegaer Konrad , Bedja Djahida , Fox-Talbot Karen , Bielawska Alicja , Paolocci Nazareno , Aja Susan , Gabrielson Kathleen L , Barouch Lili A
Published: July 2021

Introduction:
Obesity is associated with increased myocardial steatosis, cardiac dysfunction, mitochondrial uncoupling, and increased apoptosis. Caloric restriction (CR) in older leptin deficient ob/obobese micefails to reduce cardiac steatosis and apoptosis but improves mitochondrial function. This discordant response to caloric restriction and the role of aging in this process are not well understood.

Methods:
We studied 4 month (younger) and 8 month (older) old ob/ob mice and age matched C57BL/6J controls. In each age group, ob/ob mice were assigned to ad lib (AL-ob) or calorie restriction (CR-ob) for 4 weeks. We performed echocardiograms, myocardial triglyceride assays, Oil Red O staining, LC-MS/MS ceramide species measurements, and Western blots. Whole animal metabolism was measured using indirect calorimetry.

Results:
In younger mice, CR reversed the adverse cardiac effects of obesity by reducing heart weight and ventricular wall thickness, reversing myocardial steatosis measured by myocardial TG assay and Oil red O staining, and upregulating phosphorylation of Akt. This was associated with improvement in diastolic function demonstrated by reduction in isovolumetric relaxation time by echocardiography. In contrast, none of these beneficial changes were observed in the older mice. Whole animal metabolism showed that younger CR-ob mice had decreased respiratory exchange ratio compared to AL-ob, indicating greater reliance on fat as a source of energy. Interestingly, the effect of CR on lipotoxicity measured by the composition of ceramides was similar in both age groups.

Conclusion:
In this model of obesity, aging attenuates the benefits of CR on myocardial structure and function. This may be due to older animals’ decreased ability to reverse myocardial steatosis and/or accumulation of oxidative damage over time, and the loss of Akt activation with age.
Abstract 160: Pim-1 Preserves Cardiac Stem Cell Proliferation with Age
Authors: McGregor Michael , Din Shabana , Gude Natalie , Sussman Mark A
Published: July 2021

Rationale
Cardiac stem cells (CSC) regulate cardiomyogenesis and support regenerative processes in the heart, but aging adversely affects stem cell repair capacity. Aging is a primary cause of impaired cardiac function characterized by accumulation of senescent cells. CSC senescence is associated with permanent growth arrest that decreases survival signaling and cellular replacement, inevitably diminishing the capacity of the heart to maintain tissue homeostasis. Therefore, promoting CSC growth may improve cardiac performance with age. Pim-1 kinase exhibits protective and proliferative effects in the myocardium but the role of Pim-1 in cardiac aging has not been thoroughly studied.

Objective
Demonstrate that Pim-1 promotes stem cell growth in the aged myocardium correlating with increased expression of centromere protein A (CENP-A), a kinetochore-associated protein known to support cell proliferation in numerous species and cell types.

Methods & Results
CENP-A expression levels were evaluated from murine myocardial tissue samples ranging in age from 11 days post coitum to 4 months of age with analysis by immunoblot as well as quantitative PCR. CENP-A expression was colocalized with c-kit as a marker of CSC by immunohistochemical labeling, revealing a decline in CENP-A expression over the time course of postnatal myocardial maturation. The impact of Pim-1 upon CENP-A level was assessed by comparative analysis of non-transgenic mice versus genetically modified transgenic mouse lines expressing either Pim-1 (wild type) or a dominant negative functionally dead Pim-1 mutant. Pim-1 overexpression increases persistence of CENP-A in CSCs with age, as well as the prevalence of cycling CSCs as marked by phosph-H3 expression, while the functionally dead mutant accelerates CENP-A diminution and decreases CSC proliferation.

Conclusion
CENP-A decline in c-kit positive cells with age provides intriguing evidence of a potential mechanism for the diminished capacity of CSCs to maintain tissue homeostasis. Pim-1 mitigates CENP-A diminution, demonstrating the promising potential of Pim-1 to promote cardiac growth and repair with age.
Abstract 165: Palmitate Induces Dose-Dependent Apoptosis in Chick Embryo Cardiomyocytes
Authors: Oh Charles C , Levy Jennifer , Ma Wuqiong , Reaven Peter , Migrino Raymond , Franco Daniel , Thornburg Kent
Published: July 2021

BACKGROUND:
In morbid obesity and type 2 diabetes saturated fatty acid (SFA) levels are markedly increased. Palmitate, a SFA, crosses the placenta easily and may affect embryos of diabetic mothers. Palmitate induces inflammation and apoptosis in many cell types including cardiomyocytes. Transient cardiomyopathy can develop in offspring of diabetic mothers. We are testing the hypothesis that palmitate will increase apoptosis in chick embryo cardiomyocytes, making it vulnerable for later disease.

METHODS:
Hearts from chick embryos at Hamburger Hamilton stage 31 were harvested and digested into single cells using trypsin then plated. After 48 h of growth, cells were exposed to free palmitate (at ∼2.3:1, FFA:albumin ratio) at high physiological concentrations for 24 h. Apoptosis was measured with Annexin V and cell necrosis with propodium iodide using flow cytometry.

RESULTS:
Apoptosis was markedly elevated in chick embryo cardiomyocytes exposed to palmitate in a dose dependent manner (see Figure). Percentage of apoptotic cells increased from 11.9% in 0 μM palmitate to 32.0% and 44.1% in 150 μM and 300 μM respectively. The percentage of cells demonstrating non-apoptotic necrosis was low and did not change with increasing palmitate concentration.

CONCLUSIONS:
In chick embryo cardiomyocytes apoptosis is increased in a dose-dependent manner by brief exposure to palmitate. This may have important implications for intrauterine cardiac development in pregnant diabetic mothers and needs to be further explored.
Abstract 169: Angiotensin-Converting Enzyme 2: Species-Specific Features and Pharmacologic Characterization by RAS Fingerprinting
Authors: Poglitsch Marko , Domenig Oliver , Schwager Cornelia , Loibner Hans , Schuster Manfred
Published: July 2021

The carboxypeptidase Angiotensin Converting Enzyme 2 (ACE2) is a crucial player of the Renin-Angiotensin-System (RAS) as it balances the ratio between vasoconstrictive and fibrotic Angiotensin II and vasodilative and anti-fibrotic Angiotensin 1-7. This balance is affected by ACE2 either by direct conversion of Angiotensin II to Angiotensin 1-7, or by cleavage of Angiotensin I to Angiotensin 1-9, which is further converted to Ang 1-7 by Angiotensin Converting Enzyme (ACE) or Neutral Endopeptidase (NEP). An increased activity of ACE2 leads to a shift of the RAS towards an Angiotensin 1-7 dominated pattern, which was shown to be protective in various models of cardiovascular and fibrotic diseases.

We performed a comparative pharmacologic characterization of recombinant murine and human ACE2 using natural peptide substrates for the evaluation of the substrate specific ACE2 activity in whole blood. Furthermore, we investigated the impact of ACE2 activators and inhibitors on murine and human ACE2 in whole blood using an LC-MS/MS based approach allowing the simultaneous quantification of 10 different angiotensin peptides (RAS-Fingerprinting).

The investigation of inhibitors and activators of ACE2 using our experimental setting revealed the presence of species-specific enzymatic features, which was indicated by differences between the human and murine enzyme regarding the susceptibility to pharmacologic manipulation and the specificity for different endogenous peptide substrates.

Our studies demonstrate that sequence diversity observed between recombinant human and murine ACE2 significantly affects substrate specificity and pharmacologic enzyme properties. The utilization of Angiotensin I by ACE2 generates Angiotensin 1-7 via Angiotensin 1-9 and represents a pathway of alternative RAS activation, which might have been systematically underestimated in previous studies. The strongly reduced affinity of murine ACE2 for Angiotensin I results in a lack of production of Angiotensin 1-7 via this pathway, which might be of particular importance during anti-hypertensive treatments with ACE inhibitors, where the formation of Angiotensin II is prevented while increased levels of Angiotensin I are observed in circulation.
Abstract 173: Expanded Role for Thrombin/PAR1 Signaling in Cardiac Fibroblast Activation
Authors: Snead Aaron N , Insel Paul A
Published: July 2021

Cardiac fibrosis is a feature of numerous types of heart disease that lead to disability and death and is characterized by the transformation of cardiac fibroblasts (CFs) to myofibroblasts (myofibs) that synthesize and release large amounts of collagen-rich extracellular matrix (ECM), thereby resulting in reduced cardiac function and eventually heart failure. CFs are the most abundant cell type in the heart and regulate the production of ECM as a normal part of wound repair but disease can result if CFs become overactive, at least in part by increased transformation to myofibs. Cardiac fibrosis can occur spontaneously in advanced age, after acute injury (e.g., myocardial infarction) and in disease states (e.g., diabetes mellitus and heart failure). We sought to identify novel therapeutic targets for cardiac fibrosis by using an unbiased approach (RT-PCR array) to define the most abundantly expressed G protein-coupled receptors (GPCRs) predicted to regulate the CF/myofib transformation. Certain GPCRs, e.g. angiotensin and endothelin receptors, can promote cardiac fibrosis but few others are known to do this. We hypothesized that previously unrecognized GPCRs play an important role in regulating the function of CFs. Using the RT-PCR GPCR array, we found that the protease-activated receptor 1 (PAR1) is the most abundant GPCR in adult rat CFs (rCFs). Thrombin activation of PAR1 in rCFs elevates (with distinct kinetics) the expression of a variety of pro-fibrotic markers, induces morphological changes to the myofib phenotype and stimulates synthesis of collagen, the primary ECM component without affecting cell proliferation. The thrombin-induced initiation of a proliferation-independent pro-fibrotic response in adult rCFs contrasts with effects of thrombin in neonatal rCFs, suggesting a differential wound repair response in the adult versus the still-developing heart. By defining the level of expression of GPCRs in rCFs and revealing an expanded role for PAR1 in the wound repair response of the heart, the data provide a proof-of-principle that our approach can identify novel functional and disease-relevant GPCRs in CFs-- and likely in other cardiovascular cells as well.
Abstract 225: HSF-1 Knockout Induces Multidrug Resistance Gene (MDR1b) and Enhances Drug Extrusion in the Heart
Authors: Krishnamurthy Karthikeyan , Vedam Kaushik , Kanagasabai Ragu , Druhan Lawrence J , Ilangovan Govindasamy
Published: July 2021

Heat shock factor-1 (HSF-1), a transcription factor of heat shock proteins, is known to interfere with the transcriptional activity of many oncogenic and anti-inflammatory factors. In the present work we have discovered that HSF-1 ablation induced multidrug resistance gene (MDR1b) in the heart and increased the expression of P-glycoprotein (P-gp, ABCB1), an ATP binding cassette that is usually associated with multidrug resistant cancer cells, and it could successfully extrude doxorubicin (Dox) to alleviate Dox-induced heart failure and reduce mortality in mice. Cardiac MRI showed that Dox-induced left ventricular dysfunction was significantly reduced in HSF-1
-/-
mice. NF-B nuclear translocation and DNA binding activity were higher in Dox-treated HSF-1
-/-
mice. IB, the NF-B inhibitor, was depleted due to enhanced IKK-α activity. In parallel, MDR1b gene expression and a large increase in P-gp, and lowering Dox loading were observed in HSF-1
-/-
mouse hearts. However, application of the P-gp antagonist, Verapamil, increased Dox loading in HSF-1
-/-
cardiomyocytes, deteriorated cardiac function in HSF-1
-/-
mice, and decreased survival. MDR1 promoter activity (as determined by luciferase reporter gene assay), was higher in HSF-1
-/-
cardiomyocytes, whereas a mutant MDR1 promoter with heat shock elements (HSE) mutation showed increased activity only in HSF-1
+/+
cardiomyocytes. However, deletion of both HSE and NF-

κB binding motifs diminished the luciferase luminescence, suggesting that HSF-1 competitively inhibits MDR1 promoter activity. Thus, since high levels of HSF-1 is attributed to poor prognosis of cancer, systemic down regulation of HSF-1 before chemotherapy is a potential therapeutic approach to ameliorate the chemotherapeutics-induced cardiotoxicity and enhance cancer prognosis.
Abstract 227: Cardiomyocytes from Human Pluripotent Stem Cells Predict Chemotherapy-Induced Cardiotoxicity
Authors: Mioulane Maxime , Foldes Gabor , Harding Sian
Published: July 2021

The potential of human pluripotent stem cells-derived cardiomyocytes (hPSC-CM) to mimic human cardiac toxicity tests is largely unknown. Here we investigate the interaction between the chemotherapeutic agents Doxorubicin (DOX) and Lapatinib (LAP) and the protective effect of beta2 adrenergic signalling in hPSC-CM and murine myocytes. We hypothesized that death and survival pathways in hPSC-CM differ from those in animal-derived myocytes and thereby may predict better clinical outcome of chemotherapeutics. Human induced pluripotent or embryonic stem cell H7 cell lines were differentiated into cardiomyocytes (hiPSC-CM and hESC-CM, respectively), treated with drugs and stained with fluorescent dyes (Hoechst 33342, mitochondrial probes, effector caspases activation and impermeant nuclear dyes). Cell death profile was generated from high content screening and analysis (Cellomics). In isolated hPSC-CM, the anthracycline DOX induced apoptosis in a concentration-dependent manner (1-100µM), whereas LAP did not produce any toxicity. However, the combination of the two chemotherapeutic agents worsened the toxicity profile compared to DOX alone (p<0.001, n=12), consistent with clinical observations. In contrast, rat neonates were sensitive to LAP and showed a different cell death profile in DOX and drug combination experiments. Activation of apoptotic caspases in hESC-CM clusters was coincident with the dissipation of the mitochondrial membrane potential, while the potential was preserved in isolated cells during this phase. Beta2 adrenergic stimulation did not reverse DOX pro-apoptotic effect but modified toxicity-related nuclear events. In conclusion, we have shown that hPSC-CM reproduce the clinical adverse cardiac effects after treatment with anti-cancer drugs, making these cells a more relevant system for future drug development than animal-derived cells. However, our data suggest that cell death pathways involved may depend on culture condition for cardiomyocytes.
Abstract 231: Cardiac Myosin Binding Protein C Couples Cross-Bridge Cycling to Calcium Transients to Provide Normal Cardiac Function
Authors: Liu Yang , Abdalla Mahamed , Alluri Himakarnika , Jupiter Daniel , Glaser Shannon , Dostal David , Moss Richard , Tong Carl
Published: July 2021

A 40-year old American has a 20% chance of developing heart failure (HF) in his/her lifetime. Although cross-bridge cycling forms the basis for contraction and relaxation of the heart, mechanisms that modulate cross-bridge cycling are not completely understood, nor are the alterations in these processes in HF. Cardiac myosin binding protein-C (MyBPC3) is a component of heart muscle that is believed to regulate cross-bridge cycling kinetics. We hypothesize that MyBPC3 tunes cross-bridge cycling to the [Ca2+]i transient to produce normal cardiac function, which we tested using our MyBPC3 knockout (KO) mouse. Echocardiography showed that MyBPC3(KO) hearts exhibited abbreviated aortic ejection durations (AED) and AED/(period between heart beats) ratio compared to WT. MyBPC3(KO) hearts also had smaller velocity-time integrals which indicate smaller stroke volumes as a result of abbreviated ejection. Brain natriuretic peptide expression level, heart weight/body weight, and lung weight/body weight were all increased in MyBPC3(KO) mice, consistent with HF. MyBPC3(KO) mice also exhibit increased 1-year mortality. We used simultaneous twitch force and intracellular calcium [Ca2+]i measurements on intact papillary muscles to evaluate the consequences of losing cross-bridge cycling regulation on force response to changing [Ca2+]i transients. MyBPC3 (KO) myocardium exhibited abbreviated contractions despite slower calcium transients and an inability to accelerate relaxation with respect to peak contraction. Therefore, loss of MyBPC3-mediated coupling of cross-bridge cycling to the [Ca2+]i transient was associated with contractile dysfunction and HF.
Abstract 233: Cardiac Dysfunction Is Associated with Diminished Cardiac Neuropeptide Expression in Patients Undergoing Coronary Artery Bypass Surgery
Authors: Pradhan-Nabzdyk Leena , Ejaz Asma , LoGerfo Frank W
Published: July 2021

Background:
Neuropeptide Y (NPY) and its receptors, NPY1R, NPY2R, and NPY5R, Substance P (SP) and its receptor Neurokinin 1R (NK1R) and calcitonin gene related peptide (CGRP), are important regulators of cardiac physiology, including: vasomodulation, cardiomyocyte hypertrophy, ischemia-induced angiogenesis, modulation of Protein Kinase C activity and calcium homeostasis.[[Unable to Display Character: ]]

Objective:
To determine the relationship between cardiac function and neuropeptide expression in human subjects.

Methods:
38 consecutive patients undergoing elective, on pump, coronary artery bypass surgery (CABG) for coronary artery occlusive disease were studied. A Transesophageal Echo was performed immediately following induction of anesthesia and Left Ventricular Ejection Fraction (LVEF) was determined by AHA criteria as Normal, >55% (NLVEF, n = 26) or Low, <50% (LLVEF, n = 12). Patients with HbA1c>6.0 were considered diabetic. Right atrial tissue was obtained at the time of cannulation and the mRNA expression of Pre-Pro-NPY, NPY1R, NPY2R, NPY5R, Pre-Pro-SP, NK1R and CGRP was determined with qRT-PCR. Data are expressed as fold change relative to the NLVEF group.

Results:
The results of this study are summarized in the table below.

Conclusions:
1.
There is a strong association of LLVEF with diminished expression of SP, NK1R and NPY5R, with a similar trend for NPY2R.
2.
LLVEF was not associated with HbA1c or diabetes status.
3.
The consequences of diminished cardiac neuropeptide expression, the mechanisms involved, the relationship to cardiac autonomic neuropathy, with or without diabetes, warrant priority as an investigational initiative.
Abstract 234: Transcriptional Profiling of Neuregulin-Induced Myocardial Recovery After Infarction in Rats and Swine
Authors: Galindo Cristi L , Murphy Abigail , Hill Michael , Cleator John , Kasasbeh Ehab , Caggiano Anthony O , Parry Tom , Sawyer Douglas B
Published: July 2021

Neuregulin-1 (NRG-1) mediates cell-cell interactions and is a critical growth and developmental signaling molecule in the heart. We have been examining whether the recombinant NRG-1 isoform known as glial growth factor 2 (GGF2) has therapeutic potential for heart failure. In rats and swine with experimental myocardial infarction we have found that GGF2 treatment improves myocardial function and limits progressive myocardial remodeling. To understand potential mechanisms for this effect, we compared gene expression in these animals using microarrays. In rats we compared Sham operated, MI treated with vehicle, and MI treated with GGF2 at a single dose. We found that GGF2 treatment was associated with correction of mitochondrial and metabolic genes altered by MI compared to Sham-operated rats. When compared to 9 published datasets of ∼400 samples from rodents and human heart failure, we identified 563 genes associated with heart failure that were also reversed in expression in response to GGF2. Ingenuity pathway analysis demonstrated clusters of genes associated with energy production and cardiovascular tissue development as particularly enriched in GGF2-treated versus untreated MI rats. In swine our analysis was confined to animals with MI +/- GGF2 treatment at two doses. There were 527 genes altered by GGF2 at both doses compared to untreated controls, with a clear GGF2 dose response. Transcripts altered in response to GGF2 treatment were mainly those associated with extracellular matrix structure and function, MAPK signaling, and p53-mediated apoptosis. Electron microscopy of remote infarct left ventricular tissue from swine confirmed extreme morphological differences in mitochondria from GGF2-treated and vehicle-treated control pigs. Most striking was recovery of intercalated discs in response to GGF2, compared to severe disruption of intercalated disc structures in vehicle-treated control animals.
Abstract 239: Nitric Oxide Suppresses the Angiogeneic Potential of Mesenchymal Stem Cells
Authors: Gomes Samirah A , Rangel Erika B , Premer Courtney , Dulce Raul , Cao Yenong , Rodrigues Claudia O , Balkan Wayne , Hare Joshua M
Published: July 2021

Introduction:
Mesenchymal stem cells (MSCs) participate in blood vessel formation which is crucial for tissue regeneration. However, the mechanism involved in regulating MSC angiogenesis remains elusive. We examined the role of Nitric Oxide (NO) in this process by comparing angiogenesis by MSCs derived from wild type mice and mice lacking S-nitrosoglutathione reductase (GSNOR-/-), an enzyme that increases intracellular GSNO and enhances endogenous S-nitrosylation (a ubiquitous redox-related modification of cysteine thiol by NO)

Hypothesis:
Nitric oxide signaling pathways are mediated via small molecular thiols that regulate MSC angiogenesis.

Method:
Bone marrow-derived MSCs were isolated from wild type and GSNOR-/- mice and grown for 7 days in endothelial growth media followed by 24h in Matrigel (in vitro angiogenesis assay) in the presence of either vehicle, 15µM L-NAME (a nitric oxide synthase (NOS) inhibitor) or 500µM SNAP (a nitric oxide donor). Human (h)MSCs were similarly treated with vehicle, 15µM L-NAME and 100µM SNAP. MSC NO production and NOS expression were assessed.

Results:
Both types of MSCs exhibited similar levels of NO production and NOS expression, however, in the Matrigel Assay, MSCs from GSNOR-/- mice formed fewer (29.9±12.0 vs.. 50.0±15.4, p<0.001) and shorter (62.8 ± 34.6 µm vs. 105.9±57.0 µm, p<0.001) tubes than MSCs derived from wild type mice. L-NAME treatment normalized GSNOR-/- tube number (49.9 ± 14.8, p<0.001) and length (82.8±42.5 µm, p<0.001). Treatment of wild type MSCs with SNAP impaired tube formation (15.6±9.0, p<0.001) and length (57.1±24.9 µm, p<0.001). Similarly, hMSCs treated with SNAP exhibited impaired tube formation, both in number (25.0±4.9 vs. 49.2±8.1, p<0.001) and length (81±29.1 vs. 128.0±52.5 µm, p<0.001) compared to vehicle treated MSCs. L-NAME had no effect on hMSCs .

Conclusion:
These findings are consistent with a paradoxical negative influence of NO/GSNO on vascular formation by MSCs and reveal a novel mechanism whereby NO/GSNO inhibits MSC dependent angiogenesis. These findings have implications for disease states characterized by excessive or dysregulated angiogenesis; moreover NO deficient environments may trigger MSC mediated angiogenesis.
Abstract 242: Cardiac Remodeling Induced by Chronic β-Adrenergic Simulation Is Blocked by Myocyte-TGF-β Signaling
Authors: Koitabashi Norimichi , Kurabayashi Masahiko , Takimoto Eiki , Kass David A
Published: July 2021

[Background]
Emerging evidence from cell-specific conditional gene manipulation models has revealed complex interactions with differential roles of cardiomyocyte (CM) and non-CM signaling in the evolution of pathological cardiac remodeling. A major factor involved in such cross talk is transforming growth factor-beta (TGFβ), which exists in CM,fibroblasts, and vascular cells. Both mechanical overload and prolonged catecholamine stimulation are critical determinants of hypertensive heart disease. Here, we tested the role of cell-specific TGFβ signaling in chronic pressure-overload or isoproterenol (Iso)-induced cardiac remodeling using CM-specific gene suppression of TGFβ type2 receptor in mice.

[Methods and Results]
αMHC-driven tamoxifen-inducible Cre (MCM) x Tgfbr2 floxed mice (MCMR2), which achieved CM-specific knockdown of TGFβ signaling, showed a striking suppression of cardiac dilatation and dysfunction induced by chronic pressure-overload. Chronic Iso infusion induced modest cardiac hypertrophy with moderate myocardial fibrosis. Interestingly, in contrast to TAC, myocardial fibrosis induced by the chronic Iso exposure was not inhibited, rather worsened in MCMR2 mice. Systemic treatment with TGFβ neutralizing antibody (NAb) for Chronic Iso significantly inhibited myocardial fibrosis, yet cardiac function and hypertrophy were not improved. In Iso-treated cultured cardiomyocytes, profibrotic genes were up-regulated by TGFβ-receptor inhibition, while they were inhibited in cultured cardiac fibroblasts.

[Conclusion]
CM-specific TGFβ signaling inhibition has marked protective effect for pressure-overload induced cardiac remodeling but not for Iso-induced cardiac remodeling. These results suggest that the role of TGFβ signaling may be determined by targeted cell types and pathological stresses in a development of heart failure.
Abstract 259: Histone Deacetylase1 and 2 Are Increased in Myofibroblasts/Fibroblasts After MI
Authors: Nural Hikmet , Zakharova Liudmila , Mastroeni Diego , Gaballa Mohamed
Published: July 2021

Myofibroblast activation after myocardial infarction (MI) is one of the major mechanisms of myocardial fibrosis and subsequent heart failure. Elucidating the mechanisms of myofibroblast activation will provide a strategy to treat heart failure. Histone Deacetylases (HDAC) were reported to regulate myofibroblast differentiation and extracellular matrix deposition in idiopathic pulmonary fibrosis. Furthermore, HDAC inhibitors were shown to retards myocardial remodeling and improve cardiac function after MI. Therefore, here we examined whether HDAC1/2 are up-regulated after MI, and identify which myocardial cell type is predominately expressing HDAC. MI was created in rats by ligation of the left coronary artery. First, we assessed expression of HDAC 1 and 2 following MI in heart sections. Both HDACs uniformly expressed in myocardium in right ventricular (LV) and left ventricular (LV) in sham hearts. On the other hand, we observed up-regulation of both HDAC1 and 2 in the infarcted LV following MI. In addition, HDAC1 and 2 were co-localized with fibroblast marker Vimentin in the infarcted area. Next, we performed western blot analysis to investigate protein levels of HDAC1 and HDAC2 in right ventricular (RV), septum (S) and LV (infarcted and remote area). We found up-regulation of HDAC1 and HDAC2 only in the infarcted area compared to sham rats. Altogether these results suggest HDAC1 and 2 play a role in fibrosis following MI.
Abstract 261: Endothelial Cells Contribute to RAS Activation in Microvascular Smooth Muscle Cells
Authors: Miyata Kayoko , Satou Ryousuke , Navar L Gabriel
Published: July 2021

Introduction:
We have demonstrated that Ang II augments angiotensinogen (AGT) expression in rat preglomerular vascular smooth muscle cells (VSMCs). However, it is unclear if endothelial cells (ECs) are involved in augmentation of AGT in renal afferent arterioles.

Hypothesis:
We assessed the hypothesis that the ECs respond to paracrine signals that Ang II contribute to AGT augmentation in VSMCs. Objective: We established primary cultures of preglomerular ECs and examined the effects of Ang II and/or culture medium from ECs on AGT expression in preglomerular VSMCs.

Methods and Results:
We established primary cultures of preglomerular ECs, isolated from afferent arterioles of Sprague-Dawley rats. The cells were identified as ECs by being positive for a marker, CD34 and endothelial NOS and negative for alpha-SMA (a marker for VSMCs) and P4H-b (a marker for Fibroblasts) by immnostaining. The expression levels of AGT mRNA and renin mRNA in preglomerular ECs were examined by real-time RT-PCR. Ang II (100 pmol/L) increased AGT mRNA levels (1.34 +/- 0.16, by 100 pmol/L, N=4) and Renin mRNA levels (6.16 +/- 0.96, by 100 nmol/L, N=4) in ECs. On the other hand, the same dose of Ang II suppressed Renin mRNA expression in isolated Juxtaglomerular cells (JGs). These results indicate that preglomerular ECs are respond to Ang II and exclude the possible contamination of JGs into ECs. 100 pmol/L of Ang II increased AGT mRNA expression levels (1.37 +/- 0.03, relative ratio, N=4) in preglomerular VSMCs and the culture medium of ECs without Ang II treatment also more increased AGT mRNA expression (1.62 +/- 0.13, relative ratio, N=4) in preglomerular VSMCs. The AGT mRNA expression augmentation was enhanced when preglomerular VSMCs were treated with culture medium of Ang II-treated preglomerular ECs (2.39 +/- 0.41, relative ratio, N=4). The synergistic effects of Ang II and preglomerular ECs were also observed in PAI-1 expression in preglomerular VSMCs.

Conclusion:
These data demonstrate that preglomerular ECs contribute to Ang II-upregulation of AGT in renal afferent arterioles leading to further Ang II augmentation, which leads to increases in inflammatory and sclerotic factors in preglomerular VSMCs.
Abstract 265: Genetic Polymorphisms in Angiotensinogen Gene as Potential Modifiers of Hypertrophic and Dilated Cardiomyopathy Phenotypes
Authors: Rani Bindu , Bahl Ajay , Khullar Madhu
Published: July 2021

Background:
Hypertrophic Cardiomyopathy (HCM) and Dilated cardiomyopathy (DCM) are diseases of mutant sarcomeric proteins. However, there is marked variation in disease severity and progression, even among patients with identical causal mutation. The renin- angiotensin system plays a major role in the pathophysiology of heart failure and genetic variations in these genes may modulate the risk of disease and be partly responsible for the disease heterogeneity and severity.

OBJECTIVE:
To evaluate the association of angiotensinogen (AGT) gene polymorphisms (T174M and M235T) with risk of developing severe disease phenotype in HCM and DCM patients.

MATERIAL AND METHODS:
275 prospectively enrolled patients (122 HCM and 153 DCM) and 200 normal controls were genotyped for T174M and M235T polymorphisms of AGT gene. Effect of AGT genotypes on interventricular septum thickness and left ventricular ejection fraction (LVEF) were analyzed using linear regression model.

RESULTS:
We observed significantly higher prevalence of 235T allele in DCM patients which was associated with increased risk of DCM (OR 2.37, CI 1.07-5.25, p=0.04), however T174M polymorphism did not show a significant association with risk of DCM (OR 1.1, CI 0.65-1.84, p=0.79). The frequency of 174M allele was significantly higher in HCM patients as compared to controls and associated with increased risk of HCM (OR 1.95, CI 1.16-3.25, p=0.01), but no significant association of M235T polymorphism was observed with HCM (OR=1.10, CI 0.54-2.22,p=0.8). We did not observe any significant difference in mean LVEF in DCM patients carrying either M235 allele or 235T allele (M235: 27.22±7.13; 235T: 28.60±10.40; p=0.6) or carrying T174 allele or 174M allele (28.83±10.34 vs 28.09±9.93; p=0.7). No significant difference in left ventricular hypertrophy (LVH, mean septal thickness) was observed between 235T and M235 allele carriers [(24.07±5.16mm vs 23.26±6.04mm, p=0.6] or between 174M and T174 allele carriers (T174: 23.41±5.12mm, 174M: 22.83±7.17mm; p=0.6) in HCM patients.

CONCLUSION:
The variant AGT M235T and AGT T174M alleles confer increased risk of DCM and HCM respectively, but do not appear to be associated with disease severity or progression in these patients.
Abstract 269: Cardiac Aging in Rats: Do Female and Male Hearts Age Differently?
Authors: Dedkov Eduard I , Pingitore Alessandro
Published: July 2021

Background:
In recent years, the use of middle-aged and old animals of both sexes has become preferential in experimental studies involving the models of cardiovascular diseases to better reflect a human population. However, the evidence showing the extent of sex-related differences in cardiac aging of laboratory animals remains obscure. Accordingly, we designed our study to determine whether the male and female rats differ during aging with regard to left ventricular (LV) geometry and systolic function.

Methods:
We performed transthoracic echocardiographic examinations in 12-month-old (12mo) and 24 month-old (24mo) male and female Sprague-Dawley rats (Charles River, Wilmington, MA) under 1.5% isoflurane anesthesia using a linear (Matrix) array transducer probe GE M12L (5.5-14.0 MHz) and a GE Vivid 7 Dimension ultrasound system. Two-dimensional short-axis views and M-mode tracings of the LV at the papillary muscle level were recorded and used to estimate various LV parameters.

Results:
We found that 24mo-Female rats had a significant increase in LV chamber dimensions associated with a reduction in its systolic performance as compared to 12mo-Female rats (Table 1). In contrast, LV parameters remained similar between 12mo- and 24mo-Male rats. Table 1. Echocardiographic parameters of LV chamber geometry and systolic performance Values are means ± SE. Arrow indicates a significant difference between age groups (
P
≤ 0.05).

Conclusion:
Our data demonstrate that during cardiac aging female rats have a different pattern of LV changes compared to males that must be considered in the design of future experimental studies.
Abstract 275: Berbamine Confers Cardioprotection Against Ischemia-Reperfusion Injury by Attenuating Ca
2+
Overload and Preventing Calpain Activation Through Protection of Mitochondria
Authors: Yang Huang-Tian , Zhang Cai-Mei , Gao Ling , Zheng Yan-Jun
Published: July 2021

Reperfusion is an essential treatment to salvage ischemia myocardium from necrosis. However, the reperfusion can also lead to additional damage. Ischemic preconditioning is a powerful way to initiate intrinsic adaptive responses protecting the heart from subsequent severe ischemia/reperfusion (I/R) injury that can be mimicked by pharmacological preconditioning. However, so far few agents are clinically available for patients with ischemia heart disease. There is a growing interest in exploring effective cardioprotective components extracted from Traditional Chinese Medicine because they have been practiced for thousands of years, providing a vast source of pharmaceutical materials. Berbamine, a kind of bisbenzylisochinoline alkaloids derived from the roots, barks and stems of
Barberry
, was reported to protect myocardium from ischemia/reperfusion (I/R) injury, but the underlying mechanisms are largely unknown. Here, we investigated whether berbamine confers cardioprotection via preventing calpain activation and protein degradation. We identified here that berbamine pretreatment from 10 to 100 nM concentration-dependently improved post-ischemic myocardial function in isolated Sprague-Dawley rat hearts subjected to 30 min ischemia/45 min reperfusion. Similar protection was confirmed in isolated cardiomyocytes characterized by the attenuation of I/R-induced intracellular Ca
2+
overloading and the depression of Ca
2+
transients and cell shortening. Moreover, I/R-induced decreases of Ca
2+
handling proteins were significantly attenuated by berbamine. Further analysis showed that those protections were related to the suppression of I/R-induced calpain activity and the protection of mitochondrial function by berbamine. These findings suggest that berbamine confers cardioprotections against I/R injury by attenuating Ca
2+

overloading and preventing calpain activation; and the protection of mitochondria is critical for the cardioprotection.