bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 248: Elamipretide Reverses Dysregulation of mRNA Expression Levels of FoxO Transcription Factors 1, 3a and 4 in Left Ventricular Myocardium of Dogs with Advanced Heart Failure
Authors: Gupta Ramesh C , Singh-Gupta Vinita , Zhang Kefei , Xu Jiang , Sabbah Hani N
Published: July 2021

Background:
FoxO (Forkhead box) family of transcription factors play important roles in regulating the expression of genes involved in physiologic cellular functions that include 1) cell proliferation and growth, 2) oxidative stress and DNA repair, 3) apoptosis and autophagy, 4) energy metabolism and 5) immune system function. Many components of all 5 cellular functions are abnormal in the failing heart. FoxO1, FoxO3, and FoxO4 are members of the FoxO family and are expressed in adult cardiomyocytes. We previously showed that elamipretide (ELAM), a novel mitochondria-targeting peptide, reverses many of the abnormalities of the above captioned cellular function in dogs with advanced heart failure (HF). Abnormalities of expression and/or phosphorylation of FoxO1, FoxO3a and FoxO4 have been described in HF.

Objective:
This study examined the effects of chronic therapy with ELAM on levels of mRNA expression of FoxO1, FoxO3a and FoxO4 in LV myocardium of dogs with coronary microembolization-induced HF (LV ejection fraction ~30%).

Methods:
LV tissue from 14 HF dogs randomized to 3 months monotherapy with subcutaneous injections of ELAM (0.5 mg/kg once daily, n=7) or no therapy at all (control, CON, n=7) and tissue from 6 normal (NL) dogs was used in the study. Using specific primers, mRNA levels of FoxO1, FoxO3a, and FoxO4 normalized to β-actin, an internal control, were measured using real-time PCR in isolated RNA from LV tissue.

Results:
There were no differences in the levels of β-actin among the 3 study groups. mRNA expression levels of FoxO1 and 3a were significantly decreased and FoxO4 increased in HF-CON dogs compared to NL dogs (0.16, 0.19, and 2.48 fold change from NL respectively, p<0.05). Treatment of HF dogs with ELAM restored the expression of all 3 FoxO transcription factors to near NL (0.53, 0.65, and 1.21 fold change from NL respectively, P<0.05 vs. CON).

Conclusions:
mRNA levels of FoxO1 and FoxO3a are reduced and that of FoxO4 is increased in LV myocardium of HF dogs. Chronic therapy with ELAM normalizes expression of all 3 FoxO transcription factors. This improvement in FoxO expression is consistent with the observed reduction of oxidative stress, apoptosis and cytokines and improved energy metabolism in HF dogs following chronic therapy with ELAM.
Abstract 255: Sex Differences in Survival, Ventricular Remodeling, and Cardiomyocyte Contractility After Isoproterenol
Authors: Peter Angela K , Trexler Christa L , Lugo Kimberly R , Perry Amy R , Leinwand Leslie A
Published: July 2021

Introduction:
Significant sexual dimorphisms have been demonstrated to be important modifiers of cardiovascular disease (CVD). However, women have traditionally been omitted from clinical trials and female animals have been excluded from most preclinical research studies, ultimately leading to therapeutics that are not as effective, or with different side effects, in women relative to men. In light of the effects that sex has on CVD, the National Institutes of Health has called for the inclusion of both male and female animals in preclinical CVD research.

Methods and Results:
Through recent RNAseq experiments, we found both the α- and β-adrenergic receptors were significantly increased in isolated female adult rat ventricular myocytes (ARVMs) when compared to male ARVMs. In order to test whether this difference in gene expression translated to functional differences, the β-adrenergic receptor agonist, isoproterenol (ISO), was utilized to induce pathological cardiac hypertrophy in male and female rats. Survival of both sexes following a 7 day ISO treatment was significantly lower compared to vehicle. In addition, male ISO treated rats had significantly lower survival compared to female ISO treated rats (44% survival versus 77% survival, respectively). Both sexes developed significant cardiac hypertrophy compared to vehicle controls, but male ISO treated rats developed greater cardiac hypertrophy compared to ISO treated females. ARVM contractility experiments revealed no significant increases in peak shortening in response to ISO treatment in either sex. However, in both sexes, ARVMs isolated from ISO treated animals took less time to reach peak shortening and displayed increased departure velocity. Conversely, only male ISO treated ARVMs took less time to relax, exhibiting an increase in return velocity.

Conclusions:
Chronic
in vivo
β-adrenergic receptor stimulation revealed dramatic sex differences both in the intact heart and at the cellular level including increased mortality, increased cardiac hypertrophy, and altered myocyte contractility in males compared to females. These findings suggest that 7 day ISO treatment in rats may serve as a reliable, reproducible animal model for testing current and future CVD therapeutics in both sexes.
Abstract 26: Tcf7l1 Intrinsically Contributes to Cardiac Lineage Establishment
Authors: Liang Rui , Liu Yu
Published: July 2021

T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) proteins are critical downstream effectors of the canonical Wnt pathway. Tcf7l1 is the most abundant among the four TCF/LEF proteins and acts as a key regulator of self-renewal in ESCs. It negatively modulates the expression level of pluripotent genes, and prepares the epiblast for transition to lineage specification. Tcf7l1-/- embryos show a range of defects related to axis misinduction. Some severely affected embryos lack the heart and somites. Because of defective axis induction, whether Tcf7l1 intrinsically contributes to cardiac development is unknown. Earlier work in our lab established that TCF/LEF proteins cooperate with Oct4 in driving the transcription of the cardiac mesoderm factor Mesp1, but the responsible TCF/LEF protein is not identified.

Here, we have built ESC systems to conditionally manipulate Tcf7l1 expression in a Tcf7l1-/- background. A combination of tTA/tet-O-Tcf7l1 transgenes allows us to temporally “knockout” Tcf7l1 with doxycycline, while a combination of rtTA/tet-O-Tcf7l1 transgenes allows us to temporally activate Tcf7l1 expression with doxycycline. We found that “knockout” of Tcf7l1 on day 4 during ESC differentiation caused decreased expression in cardiac genes including Tbx5, Nkx2-5, and α-MHC, without affecting the expression of Goosecoid, Eomes, and Brachyury, markers of mesendoderm development. Tcf7l1-/- ESCs showed slower progression toward mesoderm formation, but temporally activated Tcf7l1 induced cardiac gene expression after mesoderm was formed. Tcf7l1 ChIP-PCR demonstrated that Tcf7l1 bound to cis-regulatory regions of core cardiac transcription factor genes. Our data suggest that Tcf7l1 intrinsically contributes to cardiac lineage development through activating Mesp1 and downstream cardiac transcription factors.
Abstract 285: Osteopontin Deficiency Ameliorates Heart Failure with Preserved Ejection Fraction  Pathology by Upregulating Mitochondrial 2-Oxoglutarate Dehydrogenase Like  Enzyme
Authors: Yousefi Keyvan , Ding Wen , Shehadeh Lina A
Published: July 2021

HFpEF is an increasingly prevalent syndrome associated with impaired myocardial energetics, for which no etiologic therapy is available. Osteopontin (OPN) is a matricellular protein that is upregulated in the circulation of HFpEF patients, and reported to induce mitochondrial stress in rodent cardiomyocytes. Here we evaluate the role of circulating OPN in regulating myocardial function in the nephrotic

Col4a3
-/-

mouse model of HFpEF. We performed extensive cardiac, biochemical and mitochondrial analyses of the

Col4a3
-/-

mouse and found a striking HFpEF phenotype. We showed OPN levels were elevated in

Col4a3
-/-

mice (FC=2.1, n=6; p<.01).

Col4a3
-/-

mice were hypertensive, had diastolic dysfunction, myocyte hypertrophy and interstitial fibrosis - all of which were ameliorated in

Col4a3
-/-

OPN

-/-

mice (n=5-20; p<.05).

Col4a3
-/-

hearts had dysmorphic mitochondria (EM), lowered antioxidant capacity as a 50% reduction in GSH/GSSG ratio (n=6; p<.05) and lower protein levels of mitochondrial respiratory complexes I, II and IV (p<.05). Flux assay in adult cardiomyocytes showed that maximal respiration was reduced in

Col4a3
-/-

hearts (575.84±37.6 vs 322.34±25.48 pmol/min in WT, n=9; p<.0001). Microarray data (validated by mitochondrial blot) implicated OGDHL as decreased in

Col4a3
-/-

hearts but increased in double knockout

Col4a3
-/-
OPN
-/-

hearts compared to WT (n=3; p<.05). OGDH activity was also lower in

Col4a3
-/-

hearts (17.1±7.3 vs 2.5±1.1 mU/mg in WT; n=6; p<.05). In

Col4a3
-/-

mice, heart-specific AAV9-mediated overexpression of OGDHL, similar to global OPN KO, improved survival by ~50-100% (p<.0001). Isovolumetric relaxation time, a marker of diastolic dysfunction, which is prolonged in

Col4a3
-/-

mice (26.17 vs 15.30±1 ms, n=26; p<.001) was decreased in

Col4a3
-/-
OPN
-/-

mice (18.1±1 ms, n=37; p<.01) as well as in AAV9-cTnT-OGDHL-treated

Col4a3
-/-

mice (16.7±2.5 ms, n=8; p<.05). In conclusion, we present a new mouse model for HFpEF in which diastolic function and lifespan can be improved by genetic deletion of OPN or cardiac OGDHL gene therapy. Our results elucidate for the first time the pivotal roles of circulating OPN and cardiac OGDHL in HFpEF pathophysiology and present two related potential therapeutic targets for HFpEF.
Abstract 286: ATPase Inhibitory Factor 1 Regulates Glycolysis in Cardiomyocytes
Authors: Zhou Bo , Qiu Yanhua , Kolwicz Stephen C , Tian Rong
Published: July 2021

It is known that pathological cardiac hypertrophy is associated with decreased fatty acid oxidation (FAO) and increased reliance on glycolysis. This metabolic remodeling is generally recognized and considered ultimately maladaptive for sustaining myocardial energetics and function. The mechanisms responsible for the switch are poorly understood but appear to be coupled with impaired mitochondrial function. We found that mitochondrial ATPase inhibitor factor 1 (ATPIF1) was up-regulated (by 4-fold, p=0.01) in the failing heart induced by TAC surgery as well as in hypertrophied adult rat cardiomyocytes (CMs) induced by 10 uM phenylephrine (PE, by 3-fold, p=0.01), but not in the physiological hypertrophied heart (p=0.9). ATPIF1 is an inhibitor of ATPase in the mitochondrial ATP synthase, or Complex V. It was shown that upregulation of ATPIF1 increased glycolysis in non-cardiomyocytes. Here we wish to test the hypothesis that ATPIF1 stimulates glycolysis in the heart undergoing pathological hypertrophy. Overexpress ATPIF1 (OE) by adenovirus vector in CMs, which mimics the up-regulation of ATPIF1 induced by PE, increased glycolysis in CMs (control: 8.54±0.61 mpH/min, OE: 11.46±0.48 mpH/min; p=0.006), while ATPIF1 knockdown (KD) by shRNA in PE treated CMs abolished the upregulation of glycolytic capacity (control: 29.27±1.31 mpH/min, KD: 28.61±1.81 mpH/min, control-PE: 40.12±1.33 mpH/min, KD-PE: 32.51±2.09 mpH/min; p=0.003). ATPIF1OE or pathological hypertrophy induced by PE increased the expression of glycolytic enzymes, e.g. GAPDH, GLUT1, LDHA and PKM2, which were abolished by ATPIF1KD. We also found that elevation of ATPIF1 decreased mitochondrial oxygen consumption rate (OCR) and promoted mitochondrial reactive oxygen species (mtROS) production, while the generation of mtROS caused by PE was abrogated in the ATPIF1 deficient CMs. Blockade of mtROS production by expressing mitochondria localized catalase suppressed the increased glycolysis in ATPIF1-OE CMs. In conclusion, our results identify ATPIF1 as a regulator of glycolysis in pathological cardiac hypertrophy, which may provide a mechanism for the regulation of glycolysis through mitochondrial bioenergetics.
Abstract 300: Topical Application of IcyHot Reduces Myocardial Infarct Size in Rodents by a TRPA1-dependent Mechanism
Authors: Wu Yun , Lu Yao , Gross Eric R
Published: July 2021

Toxic reactive aldehydes are formed during ischemia-reperfusion. The ion channel transient receptor potential ankryin 1 (TRPA1) is irreversibly modified by reactive aldehydes which can cause calcium influx and cell death. Here we tested whether topically applied creams containing a reversible TRPA1 agonist could reduce myocardial infarct size.

Male Sprague-Dawley rats 8-10 weeks age were subjected to an
in vivo
myocardial ischemia-reperfusion model of 30 minutes of left anterior descending (LAD) coronary artery ischemia followed by 2 hours reperfusion. Prior to ischemia, rats were untreated or had 1g of cream applied to the abdomen. The creams tested were IcyHot, Bengay, Tiger Balm, or preparation H (Fig. 1A). Hearts were negatively stained for the area at risk and the infarct size was determined by using TTC staining (Fig. 1B). A subset of rodents prior to receiving IcyHot also received an intravenous bolus of the TRPA1 antagonist TCS-5861528 (1mg/kg) or AP-18 (1mg/kg).

Interestingly, both IcyHot and Bengay reduced myocardial infarct size compared to untreated rodents (Fig. 1C and 1D IcyHot: 41±3%*, Bengay: 50±2%* versus control 62±1%, n=6/group, *P<0.001). Both preparation H and Tiger Balm failed to reduce myocardial infarct size (Tiger Balm: 63±2%, preparation H 59±2%). Giving a TRPA1 antagonist prior to IcyHot also blocked the reduction in infarct size. Our additional data also indicates the methyl salicylate (mint) in IcyHot and Bengay is the agent that limits myocardial infarct size.

Since IcyHot and Bengay are safely used by humans, targeting TRPA1 by using products such as these could be quickly translatable and widely used to reduce ischemia-reperfusion injury.
Abstract 301: CITED4 Gene Therapy Alters Maladaptive Cardiac Remodeling After Ischemia-reperfusion Injury
Authors: Lerchenmüller Carolin , Rabolli Charles P , Hu Dongjian , Bezzerides Vassilios J , Platt Colin , Liu Laura , Most Patrick , Rosenzweig Anthony
Published: July 2021

Introduction:
Cardiac hypertrophy is an adaptive response to increased physiologic or pathologic hemodynamic stress. Previous work from our laboratory suggested that the CEBPβ/ CITED4 pathway plays an important role in exercise-induced cardiac hypertrophy. Consistent with this model, our laboratory recently found that inducible cardiac expression of CITED4 in adult mice increases in heart weight and cardiomyocyte size with normal systolic function and a gene expression profile consistent with physiologic growth. After ischemia-reperfusion injury (IRI), induced CITED4 mice show significant functional recovery and evidence for decreased adverse remodeling.

Hypothesis:
Here, we assessed the hypothesis that CITED4 gene therapy delivered in a clinically relevant time frame after IRI in a mouse model, will also lead to improved systolic function and favorable cardiac remodeling.

Methods and Results:
Cardiomyocyte-specific CITED4 gene delivery via intravenous AAV9 (CITED4 and GFP control) injections in young wild type (WT) mice led to a steady 4-fold increase in cardiac CITED4 expression. After four weeks, CITED4 treated animals developed physiologic cardiac hypertrophy with increased heart weights (heart weight to tibia length controls 6.54±0.17g/mm vs. CITED4 7.31±0.12g/mm), as well as increased left ventricular mass index and wall thickness with unchanged systolic function evaluated by echocardiography. CITED4 gene therapy in the setting of IRI, delivered 20min. after reperfusion, promoted decreased maladaptive remodeling with improved systolic function (%FS controls 37.5±3.6 vs. C4KO 47.9±1.6), a smaller scar size (% fibrotic area controls 9.1±1.9 vs. C4KO 2.4±0.5) and a favorable gene expression profile eight weeks after IRI. After injury, CITED4 gene therapy led to a 6-fold overexpression already after one week post-IRI, responsible for less apoptosis, fibrosis and inflammation when compared to control mice.

Conclusion:
Taken together, our data identify CITED4 as a regulator of physiologic cardiac growth that protects against adverse remodeling after ischemic injury in a clinically relevant therapeutic intervention after IRI. CITED4 may represent a novel therapeutic target to mitigate adverse ventricular remodeling.
Abstract 308: CTRP9 Regulates the Fate of Implanted Mesenchymal Stem Cells and Mobilizes Their Protective Effects Against Ischemic Heart Injury via Multiple Novel Signaling Pathways
Authors: Yan Wenjun , Lau Wayne , Christopher Theodore , Lopez Bernard , Gao Erhe , Koch Walter , Wang Yajing , Ma Xinliang (Xin)
Published: July 2021

Cell therapy remains the most promising approach against ischemic heart failure. However, the poor survival of engrafted stem cells in the ischemic environment limits their therapeutic efficacy for cardiac repair post-MI. CTRP9 is a novel pro-survival cardiokine with significantly downregulated expression after MI. Here, we tested a hypothesis that CTRP9 might be a cardiokine required for a healthy microenvironment promoting stem cell survival and cardioprotection. Mice were subjected to MI and treated with adipose-derived mesenchymal stem cells (ADSCs, intramyocardial transplantation), CTRP9, or their combination. Administration of ADSCs alone failed to exert significant cardioprotection. However, administration of ADSCs in addition to CTRP9 further enhanced the cardioprotective effect of CTRP9 (P<0.05 vs. CTRP9 alone), suggesting a synergistic effect. CTRP9 significantly increased ADSCs survival and migration after implantation. Conversely, the number of engrafted ADSCs was significantly reduced in the CTRP9KO heart. CTRP9 promoted ADSCs proliferation and migration in vitro, and protected ADSCs against hydrogen peroxide-induced cellular death. Discovery-drive approaches followed by cause-effect analysis identified that CTRP9 enhances ADSCs proliferation/migration by ERK1/2-MMP-9 signaling. CTRP9 promotes anti-apoptotic/cell survival via ERK-Nrf2/anti-oxidative protein expression. Mass spectrometry, immunocytochemistry, and immunoprecipitation identified N-cadherin as the novel CTRP9 binding partner on ADSC. N-cadherin knockdown completely abolished the above noted CTRP9 biological effects. Finally, CTRP9 promotes Sod-3 expression and secretion from ADSCs, protecting cardiomyocytes against oxidative stress-induced cell death. We provide the first evidence that CTRP9 promotes ADSCs proliferation/survival, stimulates ADSCs migration, and attenuates cardiomyocyte cell death by previously unrecognized signaling mechanisms (N-cadherin-ERK/MMP-9 and N-cadherin-ERK/Nrf2-SOD). These results suggest that CTRP9 is a cardiokine critical in maintaining a healthy microenvironment facilitating stem cell engraftment in infarcted myocardial tissue, thereby enhancing stem cell therapeutic efficacy.
Abstract 317: Synthetic HDL Inhibits Endotoxin-induced Inflammatory Cytokine Production in Macrophages Through Neutralization of Endotoxins and Suppression of TLR Signaling
Authors: Guo Ling , Morin Emily , Yuan Yaxia , Zhan Chang-guo , Schwendeman Anna , Li Xiang-an
Published: July 2021

HDL plays critical roles in the regulation of inflammatory signaling in immune effect cells and its plasma levels are negatively correlated with the occurrence of cardiovascular diseases. Numerous efforts have been made to increase circulating HDL levels or improve HDL functions. In this study, we assessed the effect of synthetic HDL (sHDL) ETC642, a second generation of sHDL, on endotoxin-induced inflammatory cytokine production in macrophages and the underlying mechanism. We first calculated the binding of ETC642 to endotoxins via computer simulation which shows that ETC642 is capable of binding to both LPS and LTA. We next investigated the effect of ETC642 on LPS/LTA-induced NF-κB activation using HEK-Blue cells stably expressing either human TLR-4 or TLR-2. We observed that ETC642 effectively suppressed LPS/LTA-induced NF-κB activation in a dose-dependent manner. We further observed that pre-incubation of the HEK-Blue cells with ETC642 partly suppressed LPS/LTA-induced NF-κB activation, indicating that ETC642 not only neutralized LPS/LTA but also suppressed TLR4- or TLR2-NF-kB signaling. We demonstrated that ETC642 regulates the TLR signaling through modulation of TLR4- or TLR2 to lipid rafts. We finally confirmed the effect of ETC642 in RAW cells and in bone marrow-derived macrophages.
Abstract 320: Apoa1 Binding Protein Regulates Lymphangiogenesis by Control of Caveolin-1
Authors: Mao Renfang
Published: July 2021

ApoA-1 binding protein (AIBP) plays a vital role in zebrafish angiogenesis by targeting cholesterol effux, which reduces caveolae abundance, inhibits VEGFR2 activation. Although reduced caveolae abundance often attenuates receptor signaling, it has not bee studied whether AIBP affects VEGFR3 signaling and then plays a role in lymphangiogenesis. Thus, we studied the experesion of AIBP in lymphedema patients skin tissue. Interestingly, we found increased AIBP experession in human lymphedematous tissue and it is expressed in the lymphatic vessels as perilymphatic distribution, which indicated that AIBP plays a role in lymphangiogenesis. We then generated
Aibp
-/-
mice, which are viable and fertile and compared dermal lymphatic development in
Aibp
-/-
mice and wild-type control mice by whole mount immunostaining. Our data show that AIBP deficiency in mice results in significantly decreased lymphangiogenesis. In vitro lymphangiogenesis also show that AIBP/apoA1 significantly promote human lymphatic endothelia cell (HLEC) tube formation. These data suggeste that caveolae abundance controls lymphangiogenesis. Because caveolin1, a core protein in caveolae, inhibits VEGFR3 signaling, we thus determined the effect of cav1 deficiency, which results in loss of caveolae, on HLEC tube formation and showed cav1 knockdown promotes tube formation. We also found that VEGFR3 located in caveolae and binds to cav1 in LEC. What’s more, MβCD, which disrupts caveolae by sequestering cholesterol, enhances VEGFR3 activation. Therefore, our study demonstrated that AIBP promotes lymphangiogenesis. Mechanically, AIBP enhances cholesterol efflux from HLEC to apoA1, which might disrupt caveolae and then abolish cav1 inhibition of VEGFR3 signaling in lymphangiogenesis.
Abstract 323: Metabolic Transformation of Fatty Acids Defines Resolving Versus Non-Resolving Inflammation in Obesity-Associated Heart Failure
Authors: Halade Ganesh V , Kain Vasundhara , Rosa Xavier de la , Norris Paul C , Ingle Kevin A , Prabhu Sumanth D , Serhan Charles N
Published: July 2021

Whether and how fat intake contributes to persistent inflammation in obesity is unclear. Here, we report the differential metabolic transformation of fatty acids in heart failure (HF) pathology in the setting of obesity. We used permanent coronary ligation to induce myocardial infarction (MI) leading to acute (d1), healing phase (d5) and subsequent chronic HF phase (d28) in male C57BL/6J mice, while maintaining naïve controls at day (d)0. Prior to MI, 100 two month-old mice were subjected to n-6 fatty acids (safflower oil; SO; 22Kcal) for 3 months, with subsequent randomized allocation of 50 mice to docosahexaenoic acid (DHA; 22Kcal) enriched n-3 fatty acids for the next 2 months, while maintaining SO control in the remaining 50 mice. Both groups gained significant weight and fat mass independent of lean mass confirmed by magnetic resonance imaging. Post-MI survival and left ventricular (LV) function was decreased in SO-fed mice with downregulation of multiple inflammation resolution mechanisms in the spleen, infarcted LV, and kidney. DHA increased Ly6C
low
macrophages,
MRC, Arg-1, YM1
and
FFAR4 (GPR120)
during the acute and resolving phase from d1 to d5 post-MI as compared with SO-fed mice. DHA-improved post-MI survival at d28, together with increased levels of D-series resolvins (RvD1, RvD2, RvD4, RvD5), maresin 1, and RvE3, and lower levels of prostaglandins (PGD
2
, PGE
2
, PGF2
α
), leukotriene B
4
(LTB
4
), 20-OH-LTB
4
, thromboxane B2 and lipoxin A
4
in the resolving phase. During chronic HF at d28 post-MI, DHA increased CD4
+
/CD127
-
with increased expression of FOXp3 in the LV. DHA reduced
FFAR1-3
, neprilysin activity and simultaneously increased
FFAR4
in the kidney as compared with SO post-MI. In contrast, SO-induced overactivation of pro-inflammatory
FFAR1 (GPR40)
and reduction of anti-inflammatory
FFAR4
tracked with increased serum creatinine and
IL-1β,
and decreased ALX/
FPR2
in kidney at d28 post-MI. Overall, DHA intake modulates the splenocardiac resolving phase with generation of proresolving mediators, augmentation of Ly6C
low
macrophages, and suppression of prostaglandins post-MI. Thus, the metabolic transformation of n-3 and -6 fatty acids can regulate resolution versus non-resolution of inflammation in HF post-MI in the setting of obesity.
Abstract 342: Serum Albumin Hydrogels Alter Excitation-Contraction Coupling in Neonatal Rat Myocytes and Human Induced Pluripotent Stem Cell Derived Cardiomyocytes
Authors: Humphrey Eleanor J , Mazo Manuel M , Amdursky Nadav , Peters Nicholas S , Stevens Molly M , Terracciano Cesare M
Published: July 2021

Tissue engineering provides a promising method of introducing functional cardiomyocytes (CMs) to damaged myocardium after myocardial infarction; however, finding a biocompatible construct with the chemical and mechanical properties capable of supporting CM function is challenging. Serum albumin hydrogels are novel autogenic scaffolds with elastic properties that can be tailored to mimic the stiffness of native adult myocardium. We assessed the hypothesis that culturing immature CMs on these serum albumin hydrogels would affect CM gene expression and calcium handling. Neonatal cardiomyocyte (NRVM) viability was maintained for at least 14 days on the hydrogels, with clear sarcomeric striations. Cardiac gene expression was quantified using RT-qPCR and demonstrated an up regulation in many genes of cells cultured on hydrogels compared to glass (e.g. relative expression (log 2-ΔΔCt) of ryanodine receptor 2: glass= -2.3±0.5, hydrogel= -0.3±0.1,p<0.01; connexin 43:glass= -1.7±0.5, hydrogel= 0.3±0.1,p<0.01,n=4-6). Compared to glass, NRVMs on hydrogels have an increased time to peak of the calcium transients measured using Fluo-4AM and field stimulated at 1 Hz (tp glass=38±3 ms, tp hydrogel= 54±2 ms, p<0.01,n=4-6). Compared to glass the hydrogels also have a reduced time 50% decay (t50 glass=108±13 ms, t50 hydrogel=78±6 ms, p<0.05,n=4-6) and 80% decay (t80 glass=217±19 ms, t80 hydrogel= 152±10 ms,p<0.05,n=4-6). Human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) were cultured on the hydrogels for up to 28 days. Calcium handling was faster in the iPSC-CMs cultured on the hydrogels in comparison to glass with a reduced time to peak (tp glass=281±43 ms, tp hydrogel= 186±8 ms, p<0.05, n=4) and time to 50% decay (t50 glass=269±15 ms, t50 hydrogel=204±10 ms,p<0.01,n=4) and 90% decay (t90 glass=535±33 ms, t90 hydrogel=397±19 ms, p<0.01,n=4). The serum albumin hydrogels are compatible with NRVM and iPSC-CM culture for at least 28 days. We demonstrate that the serum albumin hydrogels have significant effects on CM calcium cycling and have the potential for use in myocardial repair. Further study is required to determine the mechanisms involved in calcium handling alterations and then assess this engineered patch
in vivo
for cardiac repair.
Abstract 343: Embryonic Extracellular Matrix and Cardiomyocyte Maturation
Authors: Kren Stefan M , Garry Daniel J , Garry Mary G
Published: July 2021

Background:
The progression of cell maturation is governed by intrinsic factors, but also by interaction with the milieu in which they reside. The extracellular matrix (ECM) from rapidly developing tissue should form a rich signaling environment for cellular development.

Hypothesis:
Murine embryonic ECM can be prepared by detergent decellularization that is morphologically preserved, biocompatible for cell culture, and at E13.5, substantial enough to permit vascular catheterization and recellularization by perfusion. When used as a scaffold for cell culture, it can be shown to have a salutatory effect on the morphologic and physiologic maturation of post-natal cardiomyocytes (CM).

Methods and Results:
To test the contribution of embryonic ECM to the performance of cardiomyocytes in culture, we undertook the isolation of ECM from developing murine embryos. Using a detergent decellularization protocol, we established an acellular scaffold that was shown to be free of native cells and returned to a biocompatible state, to serve as a scaffold for cell culture. Primary P7 mCherry expressing cardiomyocytes were isolated and introduced by perfusion into the embryonic ECM heart. The same cells were also plated, and the performance of the two culture modalities was compared at 3 and 28 days. Histologic comparison demonstrated the maintenance of the rod cellular morphology. Analysis of cellular contractile performance by video microscopy demonstrated improved contractile performance of CMs when cultured on ECM and paced at 1 Hz (3 Day plated contraction velocity/relaxation velocity (μm/sec) 1.18±0.3/0.91±0.2 and ECM contraction velocity/relaxation velocity (μm/sec) 6.65±1.0/4.52±0.7). At 28 days plated contraction velocity/relaxation velocity was (μm/sec) 2.71±0.2/1.59±0.1 and ECM contraction velocity/relaxation velocity was (μm/sec) 8.83±4.2/6.20±2.6).

Conclusion:
Biocompatible, acellular morphologically preserved embryonic ECM can be extracted from E13.5 murine embryos. By E13.5 the structural integrity of the acellular matrix can sustain vascular perfusion for delivery of P7 cardiomyocytes to internal organoid structures. These ECM preparations support the maturation and physiologic performance of cardiomyocytes.
Abstract 358: Factor Xa Deteriorates Atherosclerosis by Facilitating Inflammasome Formation via PAR-2-mediated Autophagy Suppression
Authors: Maejima Yasuhiro , Ito Yusuke , Tamura Natsuko , Konishi Masanori , Hirao Kenzo , Isobe Mitsuaki
Published: July 2021

Background:
It is known that an endogenous blood coagulation factor Xa (FXa) plays a critical role in facilitating atherosclerosis by activating protease-activated receptor-2 (PAR-2). However, the precise mechanism how FXa-mediated PAR-2 activation promotes atherogenesis remains to be elucidated.

Purpose:
The aim of this study is to explore how FXa promotes atherosclerosis through PAR-2-associated signaling pathway.

Methods & Results:
Administration of direct FXa inhibitor rivaroxaban (Riv; 120 mg/kg/day) to the mice significantly suppressed the plasma FXa activity compared with untreated mice. Administration of Riv to ApoE knockout mice fed with high fat diet (ApoE-KO-HFD) significantly reduced atherosclerotic area in the aorta compared with those in the untreated ApoE-KO-HFD. The plaque size of ApoE-KO mice crossed with PAR-2 knockout mice fed with HFD was similar to those of Riv-treated ApoE-KO-HFD. Ultrastructural examinations of atherosclerotic lesions revealed that the number of autophagosomes in the plaque-resident macrophages of Riv-treated ApoE-KO-HFD was significantly smaller than those of the untreated ApoE-KO-HFD. Immunostaining of NLRP3 revealed that Riv attenuated the inflammasome formation in the atherosclerotic lesion in ApoE-KO-HFD.
In vitro
experiments demonstrated that treatment of 7-ketocholesterol (7KC) markedly enhanced autophagy activity in the murine macrophages. The addition of FXa significantly promoted mTOR (Ser
2448
) phosphorylation and blocked autophagy activity induced by 7KC, which was reversed in the presence of Riv (1 μM). Furthermore, immunoblot analyses demonstrated that FXa administration significantly accelerated inflammasome formation induced by 7KC, which was blocked in the presence of Riv. On the other hand, treatment with FXa failed to inhibit 7KC-induced autophagy and inflammasome activation in PAR-2-KO mice-derived macrophages.

Conclusion:
These results suggest that FXa worsens atherogenesis through PAR-2-mediated pathway by inhibiting macrophage autophagy which, in turn, promoting inflammasome activation.
Abstract 362: Restoring Diabetes-induced Autophagic Flux Arrestment in Ischemic/Reperfused Heart by Adiponectin Receptor Activation Involves Both AMPK-Dependent and AMPK-Independent Signaling
Authors: Wang Yajing , Lau Wayne , Lopez Bernard , Christopher Theodore , Gao Erhe , Koch Walter , Ma Xinliang (Xin)
Published: July 2021

Autophagy is increasingly recognized as an important regulator of myocardial ischemia-reperfusion (MI-R) injury. However, whether and how diabetes may alter autophagy in response to MI-R remains unknown. Deficiency of adiponectin (AdipoQ), a cardioprotective molecule, markedly increases MI-R injury. However, the role of diabetic hypoadiponectinemia in cardiac autophagy alteration after MI-R is unclear. Utilizing normal control (NC), high-fat-diet-induced diabetes, and
Adipoq
knockout (
adipoq
-/-
) mice, we demonstrated that autophagosome formation was modestly inhibited and autophagosome clearance was markedly impaired in the diabetic heart subjected to MI-R.
adipoq
-/-
largely reproduced the phenotypic alterations observed in the ischemic-reperfused diabetic heart. Treatment of diabetic and
adipoq
-/-
mice with AdipoRon, a novel adiponectin receptor agonist, stimulated autophagosome formation, markedly increased autophagosome clearance, reduced infarct size, and improved cardiac function (P<0.01 vs vehicle). Mechanistically, AdipoRon caused significant phosphorylation of AMPK-BECN1 (Ser93/Thr119)-PtdIns3K (Ser164) and enhanced lysosome protein LAMP2 expression both in vivo and in isolated adult cardiomyocytes. Pharmacologic AMPK inhibition or genetic AMPK alpha2 mutation abolished AdipoRon-induced BECN1 (Ser93/Thr119)-PtdIns3K (Ser164) phosphorylation and AdipoRon-stimulated autophagosome formation. However, AdipoRon-induced LAMP2 expression, AdipoRon-stimulated autophagosome clearance, and AdipoRon-suppressed superoxide generation were not affected by AMPK inhibition. Treatment with MnTMPyP (a superoxide scavenger) increased LAMP2 expression and stimulated autophagosome clearance in simulated ischemic-reperfused cardiomyocytes. However, no additive effect between AdipoRon and MnTMPyP was observed. Collectively, these results demonstrate that hypoadiponectinemia impairs autophagic flux, contributing to enhanced MI-R injury in the diabetic state. Adiponectin receptor activation restores AMPK-mediated autophagosome formation and antioxidant-mediated autophagosome clearance, representing a novel intervention effective against MI-R injury in diabetic conditions.
Abstract 368: The Effect of Long-term Administration of PGE1 on the Morphological Change of Ductus Arteriosus
Authors: Iwaki Ryuma , Matsuhisa Hironori , Oshima Yoshihiro , Hoshino Masato , Minamisawa Susumu , Akaike Toru , Morita Kiyozo , Shinohara Gen , Kaneko Yukihiro , Morishita Hiroyuki , Takahashi Masashi , Yagi Naoto , Tsukube Takuro , Okita Yutaka
Published: July 2021

Introduction:
Plostagrandin E
1
(PGE
1
) is essential for maintaining the ductus arteriosus (DA) in ductal-dependent congenital heart disease. Recently, relatively long-term administration of PGE
1
became common, especially in patient with hypoplastic left heart syndrome (HLHS) . Staged Norwood approach which consists of bilateral pulmonary artery banding, PGE
1
infusion, and Norwood procedure beyond newborn period provided excellent survival benefit. However, microstructural changes of DA after long-term PGE
1
administration remain unknown. Therefore, we sought to investigate their features of DA after long-term PGE
1
administration using synchrotron based X-ray phase-contrast tomography (XPCT), a novel modality to inspect biological soft tissue, and pathological scrutiny including DA specific immunostaining.

Methods:
Seventeen DA tissues of HLHS were obtained during Norwood procedure. The median duration of PGE
1
was 48 days (Range 3-123). Radiographic microstructural analysis of DA was performed using X-ray phase contrast tomography at SPring-8 (Hyogo, Japan). Histological changes focused on elastic fiber and smooth muscle formation were evaluated by Elastica van Gieson staining, and expression of prostaglandin E2 receptor EP4 was evaluated by immunohistochemical analysis.

Results:
XPCT study showed a sclerotic changes of ductal media. There was a significant correlation between the duration of PGE
1
infusion and the mass density of ductal media (R: 0.723, p=0.001). Histological study showed that the duration of PGE1 infusion was positively correlated with the elastic fiber formation (R: 0.795, P=0.002), and negatively correlated with smooth muscle formation (R: -0.83, P<0.001). The EP4 expression was disappeared in all DA.

Conclusion:
Long-term administration of PGE
1
induced elastogenesis and density growth of the ductal media. In addition, downregulation of EP4 was observed. These results suggested that dosage of PGE
1
could be decreased after definite period of administration.
Abstract 381: Ex vivo Heart Function Analysis in Adult Zebrafish
Authors: Dvornikov Alexey V , Zhang Hong , Xu Xiaolei
Published: July 2021

Zebrafish (
Danio rerio
) is an efficient vertebrate model of human cardiomyopathy which is amenable to the medium throughput screening approaches opening opportunities to search new genetic modifiers via mutagenesis screening and assessing compound-based therapies at larger scale. The advent of genome editing technology enables the generation of a panel of genetic models of cardiomyopathy with mutations in leading cardiomyopathy genes. However, one of the major bottlenecks for adult zebrafish as a cardiomyopathy model is the lack of appropriate cardiac functional assays. Due to small heart size,
in vivo
methods such as those based on echocardiography, are limited by their insufficient resolution. Here, we report the development of an
ex vivo
approach aimed to facilitate phenotyping in adult zebrafish. We show that our method is able to quantify parameters of pump function of the heart, including end-diastolic/systolic length/volumes, ejection and shortening fractions, and velocities of contraction/relaxation. We defined the basic parameters of these indices using different wild-type strains, age, and sex, and then demonstrated that our method can be useful in definition of progression of pathogenesis of both acquired (doxorubicin-injected) and inherited cardiomyopathy models. We conclude that our novel approach shall facilitate cardiac phenotyping in adult zebrafish models of heart diseases.
Abstract 397: Chronic Infusion of Growth Hormone Releasing Hormone Prevents Heart Failure with Preserved Ejection Fraction Phenotype Development in Murine Cardiomyocytes by Reducing Myofilament Sensitivity to Calcium
Authors: Dulce Raul A , Kanashiro-Takeuchi Rosemeire M , Takeuchi Lauro M , Salerno Alessandro G , Balkan Wayne , Cai Renzhi , Schally Andrew V , Hare Joshua M
Published: July 2021

Introduction:
Heart failure with preserved ejection fraction (HFpEF) represents ~50% of heart failure cases and is characterized by impaired relaxation, ventricular stiffening and fibrosis. Growth hormone releasing hormone agonists (GHRH-A) reduce fibrosis in rat and swine models of ischemic myocardial injury. However, their effect on failing cardiomyocytes (CMs) is unknown. We hypothesized that activation of GHRH receptor signaling targets proteins associated with excitation-contraction coupling, reduces affinity of myofilaments for Ca
2+
and prevents the development of HFpEF.

Methods:
CD1 mice, implanted with mini-osmotic pump (Alzet) to deliver angiotensin-II (Ang-II) for 4 weeks, received daily injections of GHRH-A (MR-356; n=8) or vehicle (n=8). CMs were isolated and Ca
2+
and sarcomere length recorded. Expression and phosphorylation of Ca
2+
handling and sarcomeric proteins were assessed. Unmanipulated CD1 mice (n=7) acted as normal controls.

Results:
Ang-II-treated CMs exhibited reduced sarcomere length consistent with shorter cell length, indicating an inability to completely relax. These CMs also exhibited impaired contractility that correlated with reduced myosin binding protein C (cMyBPC) expression with no changes in phosphorylation. Response of [Ca
2+
] transient amplitude to increasing pacing rate was depressed and Ca
2+
decay was delayed and associated with lower expression of SERCA2 and NCX1, increased SR Ca
2+
leak but no change in phospholamban phosphorylation (p-PLB) at Ser16. Slower sarcomere re-lengthening and reduced phospho-cTnI (p-cTnI) at Ser 23/24 were observed in HFpEF CMs. MR-356 treatment maintained resting sarcomere length as well as sarcomere shortening at control values, and completely abrogated Ang-II-induced delay in Ca
2+
decay and sarcomere relaxation. SR Ca
2+
leak was reduced. p-PLB was further enhanced by MR-356, and cMyBPC and p-cTnI were maintained at control levels.

Conclusion:
Our findings demonstrate that chronic administration of Ang-II mediates functional changes in CMs consistent with HFpEF and suggest that activation of the GHRH receptor signaling pathways desensitizes myofilaments and prevents HFpEF-associated alterations in Ca
2+
handling and dysfunctional CM relaxation.
Abstract 421: Cardiac Hypertrophy Induced by Swimming Exercise in Mice and the Cardiac Renin-Angiotensin System: The More, the Better?
Authors: Tobar Santiago Alonso L , Soares Douglas S , Pinto Graziela H , Caetano Daniel S , Lopes Amanda , Andrades Michael E , Clausell Nadine O , Rohde Luis Eduardo P , Biolo Andréia
Published: July 2021

Cardiac hypertrophy is an adaptive process which is triggered by different mechanism in order to improve blood flow to organism and it may progress as physiological or pathological. Physical exercise offers a wide range hemodynamic stimulus; consequently it may modulate several molecular mechanisms associated to cardiac hypertrophy, for instance the local cardiac renin-angiotensin system (RAS). Thus, the aim of this study was to analyze the classical (ANGII/AT1) and alternative (ANG1-7/MAS) axis of the RAS in the cardiac muscle of mice submitted to exercise with different volumes/intensity training for the development of cardiac hypertrophy. Therefore, male Balb/c mice were divided in three groups: (i) Sedentary (SED), (ii) swimming training twice a day (T2), and (iii) swimming training three times a day with 2% of body weight overload (T3), for six weeks of training. The cardiac hypertrophy was assessed by the left ventricle weight and tibial length (LV/mm) ratio and cardiomyocytes cross-sectional area. Angiotensin peptides were analyzed by HPLC and angiotensin receptor measured by western blotting. We have also analyzed fibrosis by masson’s tricrome and the fetal genes reactivation was assessed by qRT-PCR. Both swimming training induced cardiac hypertrophy, the CHI for groups was T2 (6.34±0.44 mg/mm) and T3 (6.74 ± 0.70 mg/mm) compared to SED (5.55±0.5 mg/mm, p = 0.002). There was no observed change in the levels of angiotensin peptides ANG-I, ANG-II, and ANG1-7 between training groups and sedentary, however when we analyze angiotensin receptors, group T3 showed higher levels of AT1 when compared to SED (p=0.004), while MASR levels was higher in T2 compared to SED (0.017). Further, there was moderate reactivation of fetal genes as evidenced by increased in MHC-β expression observed in T3, but without fibrosis in either group. Our results suggest that increasing volumes/intensity of exercise beyond moderate does not influence the magnitude or the structural phenotype of physiological cardiac hypertrophy. However, it might promote the activation of molecular mechanisms involved in pathological cardiac hypertrophy.
Abstract 441: Role of Nitric Oxide and S-nitrosylation in Cardiomyogenesis by iPSCs
Authors: Salerno Alessandro , Hatzistergos Konstantinos , Dulce Raul , Wanschel Amarylis , Balkan Wayne , Hare Joshua
Published: July 2021

Introduction:
The mechanism by which signaling pathways, such as Wnt and BMP interact and modulate each other’s function is crucial to our understanding of cardiomyogenesis and cardiomyocyte proliferation. Nitric oxide (NO) is a signaling molecule that can trigger cardiac differentiation of stem cells, suggesting a cardiogenic function of NO synthase(s) (NOS).

Hypothesis:
NO modulates transcription factor function during pluripotency and differentiation toward a cardiac phenotype.

Methods:
Induced pluripotent stem cells (iPSCs) were derived from fibroblasts from wildtype mice and mice lacking S-nitrosoglutathione reductase (GSNOR
-/-
), a denitrosylase that regulates protein S-nitrosylation. iPSCs were differentiated into functional cardiomyocytes from embryoid bodies (EBs) via the hanging-drop method.

Results:
During differentiation into cardiomyocytes, GSNOR
-/-
iPSC-derived cardiomyocytes exhibited reduced expression of mesoderm induction-related (
Brachyury
), cardiac mesoderm
(Kdr
,
Isl-1
) and cardiac progenitor genes (
Nkx2.5
,
GATA4
). Axin-1, an inducer of apoptosis and negative regulator of the Wnt signaling pathway and MAPK pathways, specifically p38, were increased on EB-Day (D)4. In contrast, SMAD1/5/8, members of the BMP canonical signaling pathway, were reduced beginning on EB-D8. Increased p38 is associated with reduced GATA4 expression and differentiation of human ES cells into cardiomyocytes. Decreased SMAD1/5/8 is likely at least in part responsible for the reduced expression of
Nkx2.5.

Conclusions:
Our findings support that the absence of GSNOR modulates Wnt/β-catenin and BMP signaling pathways during cardiogenesis, resulting in reduced expression of mesoderm, cardiac mesoderm and cardiac progenitor genes. These findings are expected to have important implications for regenerative medicine and can provide new targets for iPS cell-based therapy.
Abstract 453:
FADS2
Overexpression Exacerbates Myocardial Ischemia-reperfusion Injury in Mice: Role of Mitochondria
Authors: Li Puma Lance C , Specht Kalyn S , Chicco Adam J
Published: July 2021

Common
FADS2
haplotypes associated with hyperactivity of its gene product, delta-6-desaturase (D6D), predict cardiovascular morbidity and mortality in humans. D6D is the rate-limiting enzyme in essential polyunsaturated fatty acid (PUFA) metabolism, but its role in the pathogenesis of cardiovascular disease is unclear. To investigate this, we generated mice with global (CMV promoter) transgenic overexpression of
FADS2
and evaluated their cardiometabolic phenotype.
FADS2
-tg mice exhibit mild glucose intolerance and aortic stiffening with advancing age, but no overt cardiac pathology. However, when challenged with ischemia-reperfusion (I/R)
ex vivo
,
FADS2
-tg mouse hearts have greater infarct sizes compared to their wild-type (WT; FVB) counterparts. Mitochondrial phospholipid analyses revealed a 65% increase in the membrane arachidonic/linoleic acid (AA/LA) ratio of
FADS2-
tg versus WT hearts, consistent with previous work in our lab linking D6D hyperactivity with mitochondrial phospholipid remodeling and disease progression in heart failure. Thus, we hypothesized that
FADS2
might exacerbate myocardial injury by altering mitochondrial responses to ischemia-reperfusion. To examine this further, mitochondria were isolated from
FADS2
-tg or WT mouse hearts for functional studies following 45/90 minutes of global I/R
ex vivo
.
FADS2-
tg
-
I/R mitochondria exhibited lower ADP-stimulated respiration and higher H
2
O
2
emission per O
2
consumed compared to WT-I/R and non-ischemic controls
,
suggesting impaired cardiac energetics and greater oxidant burden following ischemia. I/R augmented Ca
2+
-induced swelling in both WT and
FADS2
-tg mitochondria. This was attenuated in the presence of K
+
in WT, but not
FADS2
-tg, implicating
FADS2
-dependent alterations post-ischemic in mitochondrial K
+
handling. Interestingly, ischemia led to a 38% depletion of mitochondrial membrane AA in
FADS2
-tg, but not WT hearts, suggesting an interaction of
FADS2
and mitochondrial membrane AA liberation following ischemia. Ongoing studies in our laboratory are investigating the potentially novel links between
FADS2
expression with mitochondrial phospholipases, AA signaling, and mitoK
ATP
channel activity in the pathogenesis of myocardial I/R injury.