bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 221: Chronic Alterations of the Tissue Dendritic Cell and Macrophage Network Underlies the Th1 Response in the Failing Heart
Authors: Ismahil Mohamed Ameen , Kingery Justin R , Hamid Tariq , Zhou Guihua , Guo Shangzhi , Prabhu Sumanth D
Published: July 2021

Little is known about changes in the immune cell network that underlie pro-inflammatory cytokine elaboration in chronic heart failure (HF). Dendritic cells (DCs) and macrophages are antigen-presenting cells that play a central role in inflammation and immune tolerance. We investigated the pattern of infiltrating cardiac DCs and macrophages in chronic post-ischemic HF. Myocardial infarction (MI) was induced by left coronary ligation in male C57BL/6 mice. Pathological remodeling and HF were confirmed by hemodynamics, gravimetry, and echocardiography at 8 weeks post-MI. Sham-operated mice served as controls. At 8 weeks, cardiac infiltrating immune cells were isolated by collagenase digestion followed by density gradient centrifugation. Trypan blue-negative viable cells were quantified using flow cytometry. DCs were characterized using specific markers for classical (cDC) (CD11C+B220-), plasmacytoid (pDC) (CD11C+B220+), and immunogenic (CD11c+B220+CD86hi) and tolerogenic (CD11c+B220+CD86low) DCs. Macrophages were classified as activated (CD11b+F480+), pro- or anti-inflammatory (CD11b+F480+Gr1hi or low), and M1 (CD11b+F480+CD206-) or M2 (CD11b+F480+CD206+). Significant increases in cDCs (1.4 fold; p=0.016) and pDCs (4.3 fold; p=0.0016) were observed in failing myocardium. Surface expression of the maturation marker CD86 was increased 8.6 fold (p=0.009), whereas immature DCs exhibited a robust 24.4 fold (p=0.0005) increase. Activated macrophages were also significantly elevated in the failing heart as compared with sham-operated hearts (19.2 ± 1.9 vs 10.9 ± 2.9%; p=0.033), with marked infiltration of pro-inflammatory and M1 macrophages (6.2 ± 0.6 vs 3.8 ± 0.7%; p=0.019). In contrast, levels of anti-inflammatory and M2 macrophages were unchanged. We conclude that in chronic HF, there is marked upregulation of cDC and pDC subsets and pro-inflammatory/M1 macrophages that alter immune homeostasis, contribute to the recruitment and activation of immune cells, and underlie the chronic Th1 response in failing myocardium that promotes pathological remodeling. This suggests that targeting or altering immune cell subsets in the failing heart, rather than specific cytokines, may be a better approach to modulating inflammation in HF.
Abstract 224: Crosstalk Between Mitochondrial and ER Death Pathways in the Heart
Authors: Biala Agnieszka , Wang Yan , Dhingra Rimpy , Gang Hongying , Kirshenbaum Lorrie
Published: July 2021

Alternative gene splicing provides a versatile mechanism by which cells generate proteins with different or even antagonistic properties. Previously we determined the hypoxia-inducible protein Bnip3 is integral component of the mitochondrial death pathway that can signal apoptosis and autophagy but the precise mechanisms that differentially regulates these divergent processes remains cryptic. Herein, we provide novel evidence that inclusion or skipping of exon3 of Bnip3 mRNA by alternative splicing generates proteins with distinct and opposing actions on autophagy and cell survival. Metabolic stress imposed by hypoxia or nutrient deprivation resulted in the synthesis of two Bnip3 mRNA isoforms in post-natal ventricular myocytes in vitro and in vivo. Notably, one Bnip3 mRNA comprised of exons 1 through exon 6 encoded a protein of 26kDa, while a second mRNA generated by the fusion of exon2 and exon4 encoded a truncated Bnip3 protein of 8.2kDa. Sequence analysis revealed the truncated isoform encodes a conserved C-terminus domain that exclusively targets Bnip3 to the endoplasmic reticulum and not mitochondrion. While the 26kDa Bnip3 induced mitochondrial perturbations and autophagy, the spliced variant suppressed Bnip3- induced mitochondrial defects and autophagy. Furthermore, genetic knock-down or mutations within the C-terminus of the spliced variant defective for ER targeting sensitized cardiac myocytes to mitochondrial ROS production and death. To our knowledge our data provide the first direct evidence for a novel survival mechanism whereby the metabolic status of the cell programs autophagy or apoptosis by preferentially targeting Bnip3 isoforms to mitochondria or ER during metabolic stress.
Abstract 23: Characterization of MitoTimer, a Novel Tool for Monitoring Mitochondrial Turnover
Authors: Thornton Christine A , Andres Allen M , Hernandez Genaro , Sin Jon , Gottlieb Roberta
Published: July 2021

Fluorescent Timer, or DsRed1-E5, is a mutant of the red fluorescent protein, dsRed, developed by Terskikh and colleagues. Its fluorescence shifts over time from green to red as the protein matures. This molecular clock gives temporal and spatial information on protein turnover. To visualize mitochondrial turnover, we targeted Timer to the mitochondrial matrix with a mitochondrial targeting sequence (coined “MitoTimer”) and cloned it into a tetracycline-inducible promoter construct to regulate its expression. Here we report characterization of this novel fluorescent reporter for mitochondrial dynamics. Tet-On HEK 293 cells were transfected with pTRE-tight-MitoTimer and induced production with doxycycline. Mitochondrial distribution was demonstrated by fluorescence microscopy and verified by subcellular fractionation and western blot analysis. Doxycycline addition for as little as 1hr was sufficient to label mitochondria. MitoTimer was detected as early as 4hr following doxycycline addition, and persisted in mitochondria for at least 72hr. The color-specific conformation of MitoTimer was stable after fixation with 4% paraformaldehyde. MitoTimer matured to red fluorescence within 48hr, at which time a second pulse of doxycycline induced expression of green (immature) MitoTimer which was selectively incorporated into a subset of mitochondria actively engaged in protein import. The extent of new protein incorporation during a second pulse was increased under conditions of mito-biogenesis and reduced if mitochondrial membrane potential was dissipated. We conclude that MitoTimer can be used to monitor mitophagy and biogenesis.
Abstract 230: Gender Differences in the Development of Experimental Pulmonary Hypertension and Associated Right Ventricular Remodeling: The Role of Estrogen Rescue
Authors: Umar Soban , Nadadur Rangarajan , Eghbali Mansoureh
Published: July 2021

17-beta estradiol or Estrogen (E2) pre-treatment has been shown to attenuate the development of pulmonary hypertension (PH). Here, we aim to investigate the gender differences and the effect of ovariectomy on the development of experimental PH and adverse right ventricular (RV) remodeling. We also evaluate the therapeutic potential of E2 in rescuing PH and adverse RV remodelling in male and female rats. Male and female (intact and ovariectomized (OVX)) rats were treated with a single injection of monocrotaline (MCT, 60mg/kg, s.c.) to induce PH. Twenty one days after MCT, rats developed severe PH. At this point, one set of rats from each group was left untreated to develop PH-induced right ventricular failure (RVF), whereas the other set received E2 (42.5 ug/kg/day, 10-day slow release s.c. pellets). E2-treated rats were observed for an additional 12 days after cessation of therapy to check whether the effects of therapy were long-lived. Saline treated rats served as control (CTRL). Serial echocardiography was performed to monitor cardiopulmonary hemodynamics. Direct RV catheterization was terminally performed to record RV peak systolic pressure (RVPSP). The expression of novel extracellular matrix (ECM) enzymes A Disintegrin And Metalloproteinase (ADAM15 and 17) and Osteopontin (OPN) were assessed by RT-PCR. Intact females developed less severe PH than males and OVX females at day 30 (RVPSP: Females=62±3, Males=74±3, OVX=77±3 mm Hg, p<0.05 vs. respective CTRL). E2 therapy resulted in rescue of PH in all groups (RVPSP: Females=36±4, Males=38±2, OVX=44±2 mm Hg, p<0.05 vs. respective PH-RVF). Intact females also developed less severe RV remodeling compared to males and OVX females. Expression of OPN increased ∼7-9-fold in PH-RVF males and OVX but only ∼2-fold in intact females, (p<0.05 vs. CTRL for all). E2 reversed OPN upregulation in all groups (p<0.05 vs. PH-RVF). Novel ECM-degrading Disintegrin-Metalloproteinases ADAM15 and ADAM17 transcripts were also elevated ∼2-fold in all PH-RVF animals (p<0.05 vs. CTRL). E2-therapy reversed RV remodeling in all groups (p<0.05 vs. PH-RVF). In conclusion, intact females are more protected against the development of PH compared to males and OVX females. E2 rescues PH and adverse RV remodeling in all groups.
Abstract 232: Role of Transient Receptor Potential Melastatin 2 in Cardiomyocyte Apoptosis Induced by TNF-α
Authors: Li Shuzhuang , Liu Xuan , Yu Deqin , Chen Chong , Chen Xiaolong
Published: July 2021

Mechanical trauma, such as that induced by motor vehicle crashes, represents a major medical and economic problem in the world. Identifying the mechanisms responsible for post-traumatic secondary myocardial injury is critical in order to reduce overall mortality and improve quality of life after trauma. We have previously demonstrated that mechanical trauma-induced overproduction of TNF-α plays a causative role in cardiomyocyte apoptosis via oxidative/nitrative stress. Transient receptor potential melastatin 2 (TRPM2) is a Ca
2+
permeable non-selective cation channel activated by oxidative stress, expressed in the cardiomyocytes. The present study attempted to identify whether TRPM2 is involved in TNF-α-induced cardiomyocyte apoptosis. Cardiomyocytes were isolated from adult male Sprague Dawley rats and cultured with TNF-α (10 ng/ml) for 12h. RT-PCR and semi-quantitative immunohistochemistry were used to quantify TRPM2 mRNA and protein levels respectively. Significant increases in TRPM2 mRNA and protein expression were observed in TNF-α-treated cardiomyocytes, suggesting that TRPM2 may contribute to TNF-α-induced cardiomyocyte apoptosis. To identify the effect of TRPM2 on TNF-α-induced cardiomyocyte apoptosis, cardiomyocytes were cultured with TNF-α or TNF-α + TRPM2 inhibitor (flufenamic acid (FFA) 100uM or clotrimazole 30uM), respectively. Exposure of cardiomyocytes to TNF-α for 12h induced significant apoptosis as determined by caspase-3 activation (1.7-fold increase vs. control, P < 0.01). In contrast, TNF-α-induced caspase-3 activity increases were significantly depressed by FFA and clotrimazole, respectively (P < 0.05). To further confirm the effect of TRPM2 on TNF-α-induced cardiomyocyte apoptosis, we tested the effects of TRPM2-specific small interfering RNA (siRNA). As a result, impressively, TNF-α-induced increases of caspase-3 activity and lysate nucleosomes were significantly reduced in TRPM2-specific siRNA-treated cardiomyocytes (P < 0.01). These results indicate that TRPM2 plays an important role in TNF-α-induced cardiomyocyte apoptosis. We propose functional inhibition of TRPM2 channels as a new therapeutic strategy for treating mechanical trauma-induced secondary myocardial injury.
Abstract 235: Ischemia-Reperfusion Injury Decreases Myocardial Hydration Potential Without Changing Flow Resistance in the Interstitial Matrix
Authors: McGee Maria P , Morykwas Michael , Jordan James , Argenta Louis
Published: July 2021

Interstitial edema is an early response to myocardial ischemia, leading to fibrosis and remodeling in several heart failure conditions. We aimed to clarify whether osmotic, frictional, or mechanical forces drive fluid accumulation. Equilibrium and dynamic interstitial hydration parameters were determined, compared, and analyzed using osmotic stress approaches in explants from ischemic and nonischemic myocardial regions of pig heart. They were isolated after injury induced by ligating 3-4 branches of the left anterior descending coronary artery, for 85 min followed by 3 hours’ reperfusion. Their volume changed (Δ

V
max

) linearly with colloidosmotic pressure in both ischemic and nonischemic areas, yielding interstitial compliance values of 1.04 ± 0.09 and 1.08 ± 0.05 µl/g/ mmHg , which do not differ significantly, and hydration potentials from the abscissa intercepts at Δ

V
max

= 0, of -121.4 ± 28 and -14.7 ± 7.6 mmHg, which do (mean ± SE, n = 5 , P-value = 0.001). These hydration potential differences manifest
ex-vivo
influx rates 8.5 ± 2.7- fold slower in ischemic than nonischemic myocardium. Surprisingly, interstitial flow resistance values derived from net-flow rates at an imposed pressure difference of 216 mmHg were 0.23 ± 0.08 and 0.19 ± 0.01 µl
-1
. g. min and did not differ significantly between the areas. The similarity in interstitial compliance and fluid resistance indicates that the more negative hydration potential and faster efflux rates in at-risk regions after reperfusion are due to increased hydrostatic pressure rather than decreased osmotic or frictional forces. Tissue distends due to interstitial fluid accumulation against matrix mechanical forces, including elastic recoil of the collagen elastin mesh and fibroblast action, consistent with impaired drainage and persistent diastolic-like conditions during reperfusion of at-risk areas
in vivo
. These results indicate changes in pressure gradient magnitude and may have clinical and therapeutic implications; for example, reversal of paracrine interstitial flows during early remodeling
Abstract 237: Antioxidant and Ryanodine Receptor Stabilizing Therapy Prevent Statin-Induced Contractile Dysfunction in a Tissue-Engineered Skeletal Muscle Model
Authors: Tiburcy Malte , Engel Guenther , Sanders Sonka J , Zimmermann Wolfram H
Published: July 2021

Rationale:
Skeletal muscle toxicity of HMG-CoA-reductase inhibitors (statins) ranges from reversible myalgia to irreversible rhabdomyolysis. The underlying molecular mechanisms are not well defined. Tissue engineering models may help to gain insight into these clinically limiting pathologies.

Objective:
Here we aimed to model a phenotype of reversible myalgia in vitro to decipher mechanisms that contribute to the early stage of statin toxicity.

Methods and Results:
Engineered skeletal muscle (ESM) was generated from rat myoblasts, matrigel, and collagen. Isometrically suspended ESM developed 1.2±0.1 mN force under tetanic field stimulation (80 Hz; 200 mA; n=25). Exposure of ESM to statins for 5 days resulted in a loss of force and increased fatiguability in a concentration dependent manner. Cerivastatin was identified as the most potent statin with respect to muscle toxicity with a TC50 (=50% force reduction) of 0.02 µmol/L (n=25/group). Interestingly, at low cerivastatin concentration (0.01 µmol/L) contractile force of ESM was impaired without obvious signs for structural muscle damage (sarcomeric actin content, CK activity unchanged, n=4). Importantly, ESM dysfunction was fully reversible if challenged with TC50 statin concentrations (n=12-14/group). We reasoned that contractile dysfunction with increased fatiguability resulted from calcium leak via the ryanodine receptor. To test this hypothesis we co-administered S107 and observed a concentration-dependent inhibition of statin-induced force reduction (n=12) and calpain activitiy (a calcium-dependent protease, n=4-6). We further argued that RYR destabilization may have been caused by reactive nitrogen (RNS) and/or reactive oxygen species (ROS). Interestingly, the antioxidant N-acetyl cysteine (1 mmol/L, n=3/group), but not L-NAME (10 mmol/L; NO-synthase inhibition, n=6-12/group) prevented contractile dysfunction.

Conclusion:
We utilized a novel tissue engineered skeletal muscle model to decipher mechanisms of statin-induced muscle toxicity and provide evidence for a central role of ryanodine receptor leak, possibly caused by oxidative damage. Our data suggest that antioxidant and RYR-stabilizing approaches may be useful in counteracting statin myopathy.
Abstract 252: Dysregulation of β-Adrenergic Signaling in Hearts with Hyperinsulinemia
Authors: Fu Qin , Abel Evan , Xiang Y Kevin
Published: July 2021

Introduction:

Epidemiological studies have demonstrated an increased risk of heart failure in diabetics, but the mechanisms linking diabetes mellitus (DM) to heart failure remain unclear. A uniform metabolic disturbance that characterizes type 2 diabetes is hyperinsulinemia, which accelerates adverse left ventricular (LV) remodeling in pressure overload hypertrophy.

Hypothesis:

We hypothesize that hyperinsulinemia might affect LV contractility by directly impairing β-adrenergic receptor (βAR) signaling.

Methods and Results:
Insulin receptor and β
2
AR form a cell surface complex, which dissociates upon insulin stimulation in the heart and cardiomyocytes. Insulin dose-dependent significantly inhibits PKA activities induced by a βAR agonist isoproterenol, and subsequently impairs isoproterenol-induced PKA phosphorylation of phospholamban and contractile responses in myocytes. Mechanistically, insulin action is mediated via IR and β
2
AR dependent manner. Insulin induces both PKA and GRK2-mediated phosphorylation of the β
2
AR, leading to enhanced β
2
AR/Gi coupling that inhibits adenylyl cyclase-mediated cAMP production. Pretreatment with Gi inhibitor pertussis toxin abolishes the insulin-mediated impairment of β-adrenergic stimulation of cAMP signaling. Further studies reveal that chronic insulin stimulation increases phosphodiesterase 4D protein levels in cardiac myocytes, and that the expression of phosphodiesterase 4D is also increased in animal models with high fat feeding or after 2-weeks of transaortic constriction. This leads further attenuation of βAR-induced cAMP activity for contraction response. Inhibition of phosphodiesterase 4 with rolipram rescues cAMP signaling induced by isoproterenol after insulin treatment. Last, PDE4D protein levels are significantly induced in the myocardium in diabetic patients with heart disease compared to those non-diabetic patients with heart disease.

Conclusions:

This is the first evidence showing that hyperinsulinemia might adversely impact cardiac contractility by inhibiting βAR signaling, which is due to IR-induced β
2
AR/Gi coupling and phosphodiesterase 4 expression in the βAR signaling cascades.
Abstract 254: Losartan Improves Disease Outcome in a Double-Mutant Model of Severe Hypertrophic Cardiomyopathy
Authors: Shephard Rhian E , Tsoutsman Tatiana , Bagnall Richard D , Ritchie William , Semsarian Christopher
Published: July 2021

INTRODUCTION:
Hypertrophic cardiomyopathy (HCM) is an inherited disease of the myocardium, caused by sarcomere mutations. Multiple causal mutations in HCM patients correlates with disease severity, including increased LV hypertrophy, early disease onset and elevated incidence of sudden cardiac death events. There are currently no pharmacological therapies that have definitively been shown to prevent or cause regression of HCM.

OBJECTIVE:
To investigate losartan as a preventative therapy in a severe mouse model of HCM.

METHODS:
This study used the TnI-203/MHC-403 mouse model, which expresses two HCM-causing mutations resulting in severe disease phenotype and 100% mortality by 21 days. This model recapitulates a subgroup of HCM patients who progress to a dilated phenotype and heart failure. TnI-203/MHC-403 and non-transgenic mice were treated with losartan or water from a pre-disease time-point. Measures of outcome included survival analysis, cardiac function, whole heart morphology, fibrosis and hypertrophy. miRNA and mRNA profiling was performed on ventricle RNA of the TnI-203/MHC-403 mice, and miRNAs with modified expression were cross-referenced to predicted, differentially expressed target mRNAs.

RESULTS:
Losartan treatment improved disease outcome in the TnI-203/MHC-403 mice, including increased survival by 35% (mean, 23.3±1.4 days vs 17.3±6 days; P<0.0001), improved cardiac function (fractional shortening, 54±4% vs 40±14%, P<0.01), normalized gross heart morphology and heart-weight:body-weight ratios, decreased interstitial fibrosis at 14 days (0.84±0.12% vs 7.81±2.25%, P<0.01) and 17 days (2.30±0.36% vs 8.98±1.73%, P<0.0001), and reduced expression of pro-fibrotic- and pro-hypertrophic- molecules. Expression profiling showed six miRNAs (miR-30c, miR-203, miR-21, miR-214, miR-31, miR-199a-3p) were significantly altered with treatment, and their predicted targets within the differentially expressed mRNAs were identified.

CONCLUSION:
Early treatment with losartan improves disease outcome in this double-mutant mouse model of severe HCM. miRNAs that play a potentially beneficial role in this improvement, have been identified and may be promising as new therapeutic targets.
Abstract 257: PP2Cm Regulation Links Branched Chain Amino Acid with Hypertrophy Response
Authors: Zhou Meiyi , Wang Zhihua , Jeyaraj Darwin , Pan Bangfen , Huang Ying , Yuan Yuanyang , Jain Mukesh , Wang Yibin , Sun Haipeng
Published: July 2021

[Introduction]
Branched Chain Amino Acids (BCAAs), including leucine, isoleucine, and valine, modulate mTOR activity that controls protein synthesis and cell growth. PP2Cm is a mitochondrial protein phosphatase that regulates the rate limiting step of BCAA degradation. Loss of PP2Cm leads to BCAA accumulation.

[Hypothesis]
PP2Cm downregulation by hypertrophic signal impairs BCAA catabolism and thus hypertrophy response in heart.

[Methods and Results]
High level of PP2Cm is expressed in heart through all development stages. PP2Cm expression was reduced in hypertrophic heart induced by pressure overload, corresponding with increase of BCAA level and mTOR activity. KLF15 and microRNA22 are both key regulators of hypertrophy. It has been shown that hypertrophy signal s upregulated microRNA22 while decreasing KLF15 expression. We found that KLF15 increased PP2Cm promoter activity and miRNA22 regulated PP2Cm mRNA level and protein expression via 3’UTR . Together, our data indicated that hypertrophy inducers down-regulates KLF15 while increasing miRNA22, resulting in decreased PP2Cm level and thus repressed BCAA catabolism, which in turn can impact on mTOR mediated signaling and activity.

[Conclusions]
Pathological stress may affect the expression of PP2Cm and influence the outcome of hypertrophic response, suggesting an important role of BCAA nutrient and PP2Cm expression in heart.
Abstract 273: P2Y2 Receptor-Mediated Lymphotoxin-α Secretion Regulates ICAM-1 Expression in Smooth Muscle Cells
Authors: Seye Cheikh , Derbigny Wilbert , Qian Shaomin
Published: July 2021

Rationale: Single nucleotide polymorphism (SNP) in the LGASL2 galectin-2 (Gal-2) gene leads to altered secretion of lymphotoxin-α (LT-α) and is associated with coronary artery disease.

Objective:Our aim was to determine whether factors other than genetic variations in LGASL2 regulate LT-α release and to define the role of this pro-inflammatory in vascular smooth muscle cells (SMC).

Methods and results: The proinflammatory cytokine lymphotoxin-alpha (LTA) is thought to contribute to the pathogenesis of atherosclerosis. However, the mechanisms that regulate its expression in VSMC are poorly understood. The ability of exogenous nucleotides to stimulate LTA production was evaluated in VSMC by ELISA. The P2Y
2
nucleotide receptor (P2Y
2
R) agonist UTP stimulates a strong and sustained release of LTA from wild-type but not P2Y
2
R
-/-
SMC. Assessment of LTA gene transcription by LTA promoter-luciferase construct indicated that LTA levels are controlled at the level of transcription. We show using RNAi techniques that knockdown of the actin-binding protein filamin-A (FLNa) severely impaired nucleotide-induced Rho activation and consequent Rho-mediated LTA secretion. Re-introduction of FLNa in FLNa RNAi SMC rescued UTP-induced LTA expression. In addition, we found UTP-stimulated LTA secretion is not sensitive to brefeldin A (BFA), which blocks the formation of vesicles involved in protein transport from the ER to the Golgi apparatus, suggesting that P2Y
2
R/filamin-mediated secretion of LTA is independent of the ER/Golgi secretory vesicle route. Furthermore, UTP selectively induces ICAM-1 expression in WT but not SMC expressing a truncated P2Y
2
R deficient in LTA secretion.

Conclusion: These data suggest that P2Y
2
R recruits FLNa to provide a cytoskeletal scaffold necessary for Rho signaling pathway upstream of LTA release and subsequent stimulation of ICAM-1 expression on VSMC.
Abstract 277: Development of Anxiety- and Depression-Like Behavior in Mice After Myocardial Infarction
Authors: Frey Anna , Popp Sandy , Post Antonia , Lehmann Marc , Sirén Anna-Leena , Ertl Georg , Lesch Klaus-Peter , Frantz Stefan
Published: July 2021

Objective: Anxiety and depression are common and independently predict mortality in patients with heart failure. The mechanisms of these interrelations are still unclear. Consequently, we developed a model in C57BL/6 mice with experimental myocardial infarction (MI) and heart failure to study anxiety- or depression-like behavior.

Methods: Heart failure was achieved after coronary artery ligation in 13 mice; 16 sham operated mice acted as controls. Left ventricular (LV) remodeling was assessed by echocardiography, infarct size by histology. Sucrose preference test was performed over a period of 8 weeks to assess depression-like behavior. The elevated plus maze (EPM), the light-dark box (LDB) and the open field (OF) tests were subsequently applied to determine general disinterest and anxiety-like behavior. Finally, the histological and immunohistochemical evaluation of the brain was performed.

Results: Mice with MI size of at least 30% of LV (averaged 50±3%; increase in diastolic LV diameter from 0.40±0.02 cm to 0.62±0.03 cm) showed diminished intake (p=0.029) and preference (p=0.029) for sucrose solution. Besides, MI mice exhibited reduced exploratory behavior and markedly lower interest in unfamiliar environments, indicated by an increase in center time (p=0.016) and a reduced number of vertical rears (p=0.037) in the EPM. An increased latency to the first rear (p=0.018), covered shorter distances (p=0.048) and spent less time moving (p=0.028) in the OF were found in MI mice. MI did not affect anxiety-related measures in all three tests (all p>0.05). MI mice showed normal brain morphology with normal neuronal morphology and neuropil structure, also confirmed by normal expression of selected neuronal markers. Markers for neurodegeneration, apoptosis or inflammation showed no abnormalities in MI mice.

Conclusions: Mice with MI exhibited anhedonia-like behavior, which was accompanied by other characteristics of depression-like behavior, such as decreased exploratory activity and interest in novelty. Hence, MI caused distinct behavioral changes in mice comparable to symptoms observed in humans with heart failure and comorbid depression, but did not affect anxiety-like behavior. The model is suitable for further mechanistic studies.
Abstract 28: In Vivo Knockdown of Mirna-15b Results in Increased Cardiac Hypertrophy and Fibrosis in Response to Pressure Overload of the Mouse Heart
Authors: Tijsen Anke J , van der Made Ingeborg , van Deel Elza D , Hiller Monika , Reckman Yolan J , Duncker Dirk J , Pinto Yigal M , Creemers Esther E
Published: July 2021

MiRNAs play an important role in the control of diverse aspects of cardiac function. MiR-15b is highly expressed in the heart and is found consistently upregulated in hypertrophic and failing hearts. To investigate the function of miR-15b in the heart we set out two experiments. In the first experiment we generated two independent transgenic mouse lines that drive miR-15b expression under the αMHC-promotor and show a three and four fold overexpression of miR-15b. Strikingly, both lines show a decrease in heart weight/tibia length of 20% in adult and aged mice when compared to littermate controls. We investigated the response of these transgenic mice to thoracic aorta constriction (TAC) and found no differences in the hypertrophic response or in cardiac function measured by echocardiography between wild-type and transgenic mice. In a second experiment, we inhibited miR-15b using LNA-based antimiRs. In these mice, TAC resulted in an increased hypertrophic response and increased cardiac fibrosis when compared to a negative control antimiR.

A wide range of predicted targets of miR-15 belong to the pathways of the TGFβ-superfamily and using a smad-dependent reporter we show that miR-15b inhibits TGFβ-induced Smad activity in HepG2 cells. One of the predicted targets in the TGFβ pathway is TGFβ receptor 1 (TGFβR1), of which the 3’UTR contains six predicted miR-15 binding sites. This suggests that the phenotype in the transgenic mice and after knockdown of miR-15b may be (partly) mediated by repression of TGFβR1. Indeed, in the adult miR-15b transgenic hearts we found a downregulation of TGFβR1 mRNA and protein and we confirmed binding of miR-15 to the TGFβR1 3’UTR by luciferase assays.

In conclusion, miR-15b causes a cardiac hypotrophic phenotype at baseline in transgenic mice and inhibition of miR-15b leads to a stronger hypertrophic and fibrotic response after TAC. Furthermore miR-15b inhibits the TGFβ pathway by targeting the TGFβR1 and possibly other targets in this pathway.

This research is funded by the Dutch Heart Foundation (NHF grant #2007B077).
Abstract 283: The Coupling of Tumor Endothelial Marker-8 and Capillary Morphogenesis Gene-2 Receptors to Contractile-Related Signaling Pathway in Cardiac Myocytes
Authors: Nizamutdinov Damir , Golden Honey B , Feng Hao , Gerilechaogetu Fnu , Foster Donald M , Tong Carl W , Glaser Shannon S , Dostal David E
Published: July 2021

Recent molecular and pharmacological studies performed on non-cardiac tissues suggest that tumor endothelial marker-8 (TEM-8), also known as anthrax toxin receptor 1 (ANTXR1) and capillary morphogenesis gene-2 (CMG-2), known as anthrax toxin receptor 2 (ANTXR2) regulate survival, proliferation, cell shape, as well as polarity, adhesion, migration and differentiation. However, downstream signaling mechanisms and function of these molecules have not been studied. Recent studies indicate that activation of protein kinase B (Akt) and c-Jun N-terminal kinases (JNK) have been associated with hypertrophic growth, as well as regulation of contractility in cardiac myocytes (CM).

In this study we’ve investigated the role of ANTXR1 and ANTXR2 receptors in intracellular pathways of contractility regulation in neonatal rat ventricular myocytes (NRVM).

Primary culture of NRVM was used to determine the effects of ANTXR1/ANTXR2 activation on Akt and JNK phosphorylation by Western blot analysis. Flow cytometry and immunostaining of alive CM were used to determine expression levels and distribution of anthrax receptors throughout the cells. Furin-activated form of anthrax protective antigen (PA
63
) was used in order to selectively stimulate ANTXR1/ANTXR2 receptors and get understanding of downstream signaling mechanisms coupled to them. The treatments with angiotensin II, type1 receptor (AT
1
) inhibitors were used in order to separate responses of distinct regulatory pathways of contractility. All treatments were done at variable time courses started in seconds and finished in half of hour to determine a sequence of phosphorylation reactions going from cellular membrane to deep inside of cell. As a result, abundant expression of both receptors was observed in cardiac myocytes. Activation of one phospho-site of focal adhesion kinase (FAK
861
) was started in 15 sec with subsequent phosphorylation of JNK and Akt
473
in 5 and 10 minutes. Involvement of new molecules was observed in cascade chain reaction of contractility regulation.

In conclusion, ANTXR1/ANTXR2 were demonstrated to couple to mechano-sensor molecules: FAK, Akt and JNK, as well as interact with the AT
1
receptors to mediate downstream signaling events responsible for regulation of contractility.
Abstract 284: The Role of Cardiomyocyte Proliferation in Pediatric Heart Growth
Authors: Kevin Bersell , Mollova Mariya , Walsh Stuart , Savla Jainy , DasLala Tanmoy , Park Shin-Young , Silberstein Leslie , dosRemedios Cris , Graham Dionne , Colan Steven , Kuhn Bernhard
Published: July 2021

Introduction
Cardiomyocyte proliferation is an active cellular mechanism in systems that effectively regenerate myocardium, such as zebrafish, newt, and neonatal mice. However, this cellular growth mechanism has not been examined systematically in humans < 20 years of age. Examining the role of cardiomyocyte proliferation in post-natal heart growth in humans may provide a new cellular mechanism that could be targeted therapeutically.

Hypothesis
We hypothesized that cardiomyocyte proliferation contributes to myocardial growth in humans during post-natal development.

Methods
We examined 20 human hearts (3 weeks to 20 years of age) that were free from myocardial disease. We used confocal microscopy and laser-scanning cytometry to quantify cell cycle activity and associated cellular changes (cardiomyocyte ploidy and cellular volume).

Results
Measurements of mitosis, made by automated quantification of phosphorylated histone-3-positive cardiomyocytes, showed that during the first year of life the mean percentage of cardiomyocytes undergoing mitosis was 0.04 ± 0.01% (n = 6), declining to 0.008 ± 0.001% (n = 3) between 10 and 20 years of age. Cardiomyocyte cytokinesis, visualized by an antibody against MKLP-1 (a component of the centralspindlin complex), was detectable up to 20 years of age (n = 8), but not later in life (n = 4). The number of cardiomyocytes, quantified with stereology, increased four-fold between birth and 20 years. The mean cardiomyocyte volume increased concomitantly 12-fold. By relating these mechanistic data to left ventricular mass (determined by echocardiography), we modeled myocardial growth between birth and 20 years, which showed that the mean contribution of cardiomyocyte proliferation was 37%.

Conclusions
Our findings show that cardiomyocyte proliferation is a significant mechanism of developmental heart growth in humans after birth. This suggests that young humans may have the capacity to regenerate myocardium since the underlying mechanism, cardiomyocyte proliferation, is active under the age of 20 years. Cardiomyogenic stem cells may be involved in this process.
Abstract 294: Atrogin-1 Is Required for Atrophic Remodeling of the Heart
Authors: Baskin Kedryn K , Salazar Rebecca , Chen Wenhao , Taegtmeyer Heinrich
Published: July 2021

The heart adapts to changes in load by remodeling both metabolically and structurally. During this process, cardiomyocytes break down unnecessary or damaged proteins and use the resulting amino acids for the synthesis of new proteins and/or energy provision. Protein degradation via the ubiquitin proteasome system is controlled by ubiquitin ligases, which determine the specific proteins to be degraded. Atrogin-1 is a muscle specific ubiquitin ligase required for skeletal muscle atrophy, and over-expressing Atrogin-1 inhibits the development of cardiac hypertrophy. We tested the hypothesis that Atrogin-1 is required for atrophic remodeling of the unloaded heart. Hearts from wild type (WT) and Atrogin-1 -/- mice were subjected to mechanical unloading by heterotopic transplantation. In WT hearts, seven days of unloading significantly reduced heart weight and myocyte cross-sectional area, while hearts lacking Atrogin-1 significantly hypertrophied. Conventional markers of atrophic remodeling, such as the reactivation of the fetal gene program were detected in both WT and Atrogin-1 -/-transplanted hearts. Proteasome activity and markers of autophagy were increased after unloading, although not significantly different between WT and Atrogin-1 -/- hearts. Pathways regulating protein synthesis were enhanced in the absence of Atrogin-1; there was an increase in activated Akt and its downstream pathway including mTOR, 4E-BP1, and p70 S6 kinase. Additionally, calcinuerin, a known target of Atrogin-1 involved in hypertrophy and protein synthesis, was upregulated in unloaded Atrogin-1 deficient hearts. Consequently, μunloaded” cardiomyocytes lacking Atrogin-1
in vitro
exhibit increased basal rates of protein synthesis. While inhibition of calcineurin decreased rates of protein synthesis in unloaded cardiomyocytes in the absence of Atrogin-1, protein synthesis rates were still higher than in WT unloaded cardiomyocytes. These results suggest that more than one pathway regulating protein synthesis is controlled by Atrogin-1 in the heart. Furthermore, the data provide evidence that Atrogin-1 not only enhances protein degradation, but also keeps protein synthesis in check. Thus Atrogin-1 has a duel role in regulating cardiac mass.
Abstract 297: Effects of p53 Stabilization During Heart Failure Progression on Telomere Repeat Binding Factor 2 and Peroxisome Proliferator-Activated Receptor-γ Coactivator-1α in the SHHF Rat
Authors: Nelson Christopher B , Chicco Adam J , Bailey Susan M
Published: July 2021

The tumor suppressor protein p53 is a cellular stress sensor and transcription factor classically accepted to target the transcription of genes that induce senescence and apoptosis. Diverse stressors including genomic or telomeric DNA damage, telomere uncapping, hypoxia, oxidative stress, and mechanical stress increase stable cellular p53. In models of heart failure and in human failing hearts, p53 is increased. High p53 has also been suggested to be necessary for the transition from hypertrophy to heart failure.

In recent years it has become clear that senescence and apoptosis may not be the only significant deleterious effects of increased cellular p53. It has been suggested that p53 can decrease mitobiogenesis and cellular ATP production by direct repression of PGC1-a expression. Additionally, P53 has been shown to stimulate the expression of Siah1, an E3 ubiquitin ligase that mediates degradation of the telomere binding protein TRF2. TRF2 loss is a form of telomere uncapping that, in cell culture models, causes a subsequent DNA damage response including further p53 stabilization. The relative importance of these p53 linked pathways has yet to be explored in the progression of hypertensive heart failure.

Spontaneously Hypertensive Heart Failure rats (SHHF) rats were sacked at four stages during heart failure progression and LV removed for western blot analysis. Compared to young (2-4 month) rats, 14-15 month rats showed significantly increased p53 levels (2.3 fold), normal TRF2 levels, and slightly decreased PGC1-a (.98 fold). Old SHHFs (20-22mo) had p53, TRF2, and PGC1-a levels similar to 14-15 month rats (2.1, 1.09, and.83 fold, respectively). Rats with end stage heart failure showed a significant 4.5 fold increase in p53 expression, with a corresponding decrease in TRF2 (.74 fold, p=.087), and increase in PGC1-a (1.3 fold, nonsignificant).

These data are in agreement with previous suggestions that increased p53 is most important during the transition from hypertrophy to heart failure, and suggest that levels of p53 encountered in the SHHF model have little effect on PGC1-a and mitobiogenesis, but may mediate an increase in telomere uncapping during heart failure.
Abstract 298: CaMKII-Mediated Phosphorylation of InsP3R2 Activates Hypertrophic Gene Transcription
Authors: Natesan Sankar , Maxwell Joshua T , Hale Paul R , Mignery Gregory A
Published: July 2021

Inositol 1,4,5-triphosphosphate receptors are a family of intracellular Ca2+ channels, modulated by the ligands InsP3 and Ca2+ in response to an array of neuro-hormonal stimuli to release Ca2+ from internal stores. In cardiac myocytes, the InsP3R2, CaMKII and CaM are components of a macromolecular signalplex. However, modulation of InsP3R’s Ca2+ channel and the underlying mechanism(s) that converge and integrate nuclear hypertrophic signals to initiate transcription is not fully established. Here, we delineate the mechanistic regulation of nuclear InsP3R2’s bidirectional Ca2+ channel activity and its effect on the activation of nuclear hypertrophic signaling. We show that, endothelin-1 (ET1) stimulates elevated InsP3 induced Ca2+ release into the nucleus from perinuclear InsP3 receptors. This elevated nuclear Ca2+ release is due to CaMKIIδ mediated phosphorylation of cytoplasmic oriented InsP3R2 inhibiting Ca2+ release to the cytoplasm. This increase in nuclear Ca2+ activates Ca2+ sensitive transctiption factors thereby activating gene expression. The elevated nuclear Ca2+ and gene expression can be attenuated by CaMKII inhibitor KN-93 and InsP3R antagonist 2-ABP. We also show that, exogenous InsP3R2 was phosphorylated by both cytoplasmic (CaMKIIδC) and nuclear (CaMKIIδB) isoforms of CaMKIIδ. Intriguingly, CaMKIIδC phosphorylates endogenous InsP3R2 in cardiomyocytes more efficiently than CaMKIIδB and not by its kinase-dead derivatives. In conclusion, our findings demonstrate that, InsP3R2 mediated Ca2+ signaling integrates and vectors the GPCR activated hypertrophic signals to the nucleus triggering gene transcription underlying cardiac hypertrophy.
Abstract 305: Nebivolol Confers Mitochondria-Targeted Cardioprotection in Isoproterenol-Induced Acute Stressor State
Authors: Vaikunth Sumeet S , Weber Karl T , Bhattacharya Syamal K
Published: July 2021

Introduction:
Isoproterenol-induced acute stressor state simulates injury from burns or trauma, and results in Ca
2+
overloading and oxidative stress in diverse tissues, including cardiac myocytes and their subsarcolemmal mitochondria (SSM), overwhelming endogenous Zn
2+
-based antioxidant defenses.

We hypothesized

that pretreatment with nebivolol (Nebi), having dual beta-1 antagonistic and novel beta-3 receptor agonistic properties, would prevent Ca
2+
overloading and oxidative stress and upregulate Zn
2+
-based antioxidant defenses, thus enhancing its overall cardioprotective potential in acute stressor state.

Methods:
Eight-week-old male Sprague-Dawley rats received a single subcutaneous dose of isoproterenol (1 mg/kg) and compared to those treated with Nebi (10 mg/kg by gavage) for 10 days prior to isoproterenol. SSM were harvested from cardiac tissue at sacrifice. Total Ca
2+
, Zn
2+
and 8-isoprostane levels in tissue, and mitochondrial permeability transition pore (mPTP) opening, free [Ca
2+
]
m
and H
2
O
2
production in SSM were monitored. Untreated, age-/sex-matched rats served as controls; each group had six rats and data shown as mean±SEM.

Results:
Compared to controls, isoproterenol rats revealed:
(1)
Significantly (*p<0.05) increased cardiac tissue Ca
2+
(8.2±0.8 vs. 13.7±1.0*, nEq/mg fat-free dry tissue (FFDT)), which was abrogated (
#
p<0.05) by Nebi (8.9±0.4
#
);
(2)
Reduced cardiac Zn
2+
(82.8±2.4 vs. 78.5±1.0*, ng/mg FFDT), but restored by Nebi (82.4±0.6
#
);
(3)
Two-fold rise in cardiac 8-isoprostane (111.4±13.7 vs. 232.1±17.2*, pmoles/mg protein), and negated by Nebi (122.3+14.5
#
);
(4)
Greater opening propensity for mPTP that diminished by Nebi;
(5)
Elevated [Ca
2+
]
m
(88.8±2.5 vs. 161.5±1.0*, nM), but normalized by Nebi (93.3±2.7
#
); and
(6)
Increased H
2
O
2
production by SSM (97.4±5.3 vs. 142.8±7.0*, pmoles/mg protein/min), and nullified by Nebi (106.8±9.0
#
).

Conclusions
: Cardioprotection conferred by Nebi, a unique beta-blocker, prevented Ca
2+
overloading and oxidative stress in cardiac tissue and SSM, while simultaneously augmenting antioxidant capacity and promoting mPTP stability. Therapeutic potential of Nebi in patients with acute stressor states remains a provocative possibility that deserves to be explored.
Abstract 313: Changes in Autophagic Flux in Patients Undergoing Cardiac Surgery
Authors: Jahania Salik , Sengstock David , Vaitkevicius Peter , Mahmood Redah , Sabagh Fahed , Hagood Kristyn , Ito Bruce , Andres Allen , Gottlieb Roberta A , Mentzer Robert M
Published: July 2021

Autophagy is an endogenous survival mechanism and adaptive response to cellular stress. We hypothesized that autophagy plays an important cardioprotective role in humans and would be accelerated during the ischemic stress of conventional heart surgery. In a prospective study to test this hypothesis, we measured key autophagy proteins (Beclin-1, Atg5-12, p62) in cardiac tissue from 5 patients undergoing cardiac surgery. Right atrial biopsies were obtained before and after cessation of cardiopulmonary bypass (CPB). In 4 of these patients, CPB was associated with a marked decrease in cardiac Beclin-1, Atg5-12, and p62. We believe that depletion of these factors, particularly p62, reflects a brisk increase in autophagic flux (Fig. 1). In one patient (#02), autophagic flux appeared to be impaired. This occurred in an aged patient with characteristics of metabolic syndrome (MetS) who had the highest predicted operative morbidity/mortality. While the number of patients studied is small and insufficient to reach any definitive conclusions, these preliminary data confirm the feasibility of studying autophagy in the human heart and suggest that ischemic stress during heart surgery is associated with a marked upregulation of autophagic flux. A better understanding of the role of autophagy could lead to the development of new cardioprotective strategies.

[Figure 1: Changes in cardiac autophagy proteins in patients undergoing heart surgery before and after cross-clamping the aorta (after restoration of myocardial blood flow).]
Abstract 320: Activation of Protein Kinase G Enhances Proteasome-Mediated Degradation of Misfolded Proteins
Authors: Ranek Mark J , Martin Douglas A , Kost Curtis , Wang Xuejun
Published: July 2021

The Ubiquitin Proteasome System (UPS) plays a pivotal role in the intracellular protein homeostasis, thereby regulating a variety of cellular processes including protein quality control (PQC). Inadequate PQC and proteasome functional insufficiency are associated with and may play an important role in adverse cardiac remodeling and cardiac failure (CHF). This is best reflected by the increased presence of misfolded proteins and protein aggregates in failing human hearts. While the regulatory mechanisms of these processes remain poorly understood they carry significant clinical implications. Reduced protein kinase G (PKG) activity is well documented in CHF. Increasing the activity PKG, during various cardiovascular diseases improves patient prognosis. We hypothesize that PKG plays a role in regulating UPS function. Using both genetic and/or pharmacological manipulation of PKG activity in cultured cardiomyocytes and intact mice and taking advantage of a previously validated surrogate misfolded protein (GFPu or GFPdgn) as well as a bona fide misfolded protein (R120G-CryAB) that is known to cause proteinopathy in human and mice, our studies have revealed a significant role for PKG in UPS regulation. In cultured cardiomyocytes, PKG inhibition was achieved by PKG-specific siRNA transfection or administration of PKG pharmacological inhibitor while PKG activity was induced by adenoviral delivery of a constitutively active PKG or by the treatment of phosphodiesterase 5A inhibitor Sildenafil. PKG activation significantly stimulated UPS activity in cultured cardiomyocytes as indicated by a reduced protein level of GFPu and increased proteasome activities; while PKG inhibition reduced UPS proteolytic activity. Furthermore PKG activation resulted in enhanced removal of R120G-CryAB. These findings from cell culture studies were further corroborated by in vivo findings from manipulating the muscarinic (M) receptor in GFPdgn transgenic mice. The M receptor signals through PKG. Activating the M receptor deceased, while antagonizing the M receptor increased, the steady protein level of GFPdgn in the heart. Hence, we conclude that PKG positively regulates the UPS thereby supporting cardiomyocyte PQC.