bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 208: Defining the functions of mitochondrial Telomerase Reverse Transcriptase
Authors: Buechner Nicole , Goy Christine , Rabanter Lothar Ludwig , Jakob Sascha , Altschmied Joachim , Haendeler Judith
Published: July 2021

Over the last few years it has become clear that mitochondria are not only the powerhouses of the cells, having their sole function in producing ATP. Proteome-analyses of mitochondria from different organisms and organs revealed that more than 1000 proteins are localized in and or on the mitochondria. This by far exceeds the number of proteins required for classical mitochondrial functions, e.g. the respiratory chain, the tricarboxylic acid cycle, fatty acid oxidation and apoptosis. This suggests that many of these proteins have other, as yet unknown functions. We identified Telomerase Reverse Transcriptase (TERT), an enzyme that, in the nucleus, protects telomeres, the ends of the chromosomes, against shortening, also in the mitochondria. However, up to now only (TERT) deficient cells and mice or TERT overexpressing cells were used to study nuclear and mitochondrial functions of TERT. In this context, we demonstrated that reduction of endogenous TERT leads to increased reactive oxygen species (ROS) production in the cytosol as well as in the mitochondria. To analyze now specifically the functions of mitochondrial TERT, we isolated TERT deficient fibroblasts and transduced them with lentiviruses containing mitochondrially targeted TERT. We first validated these newly generated cells for transgene insertion and expression. Next, we analyzed compartment specific ROS production and apoptosis in the cells. We observed a reduction specifically in the levels of mitochondrial, but not cytosolic ROS. Moreover, apoptosis induction was significantly reduced in these cells compared to TERT deficient cells as well as cells containing endogenous TERT. Thus, mitochondrial TERT specifically reduces mitochondrially produced ROS and protects cells against apoptosis. In comparison to nuclear TERT, which has the main function to prolong telomeres, mitochondrial TERT specifically protects mitochondria from damage. Currently, we are creating mice that carry mitochondrially targeted TERT on an otherwise TERT deficient background for in vivo experiments.
Abstract 209: Resistin Regulates Dynamin-Related Protein 1, a Mitochondrial Fission Protein in Vascular Smooth Muscle Cells
Authors: Scott Takara A , Francis Sharon
Published: July 2021

Recent studies indicate increased mitochondrial fission and organ dysfunction in models of obesity. While the mechanisms for this morphological alteration are not completely understood it is likely that the altered adipokine profile that occurs during obesity may play a permissive role in enhanced mitochondrial fragmentation. In this study, we investigated the role of resistin, a pro-inflammatory adipokine that is elevated in the plasma of obese individuals, on regulation of the mitochondrial fission protein, dynamin-related protein 1(DNM1L) in vascular smooth muscle cells (VSMCs). We hypothesized that serum and glucocorticoid inducible kinase 1 (SGK1) plays a role in resistin-induced mitochondrial fission expression in VSMCs. In dose and time response studies, we found that resistin stimulated DNM1L protein levels in both rat and human aortic smooth muscle cells suggesting a role for resistin in regulation of mitochondrial fission. In addition, we observed that resistin enhances expression and activity of SGK1. To determine whether SGK1 regulates resistin-mediated expression of DNM1L, we examined the effect of SGK1 over-expression, or inhibition on resistin-induced expression of DNM1L. Over-expression of wildtype SGK1 potentiated resistin-mediated DNM1L expression, while dominant-negative SGK1 inhibited resistin-mediated expression of DNM1L in human VSMC. In conclusion, we have demonstrated for the first time that SGK1 is a potential downstream mediator of resistin. Interestingly, SGK1 activity has been linked to pathological vascular remodeling in various cardiovascular disease settings. Our data suggests that the elevated plasma levels of resistin that occur in obese individuals may lead to activation of SGK1. This in turn, could exacerbate mitochondrial fragmentation in VSMC and predispose to the development of vascular dysfunction that is evident during obesity. Together with further mitochondrial fission studies, additional elucidation of how resistin also alters components of mitochondrial fusion machinery may uncover novel mechanisms of potential theraputic targets for obesity-mediated vascular dysfunction.
Abstract 216: Sex Chromosome Protection Again Myocardial Ischemia/reperfusiong Injury In Mice: One X Is Better Than Two
Authors: Li Jingyuan , McClusky Rebecca , Chen Xuqi , Arnold Arthur P. , Eghbali Mansoureh
Published: July 2021

Sex differences in susceptibility to ischemia/reperfusion (I/R) injury have been demonstrated both in basic and clinical settings. Female hearts show better functional recovery after I/R injury compared with males. Most of these sex differences in the I/R injury model have been attributed to sex hormones. Here we used a unique mouse model (XY*) to investigate the role of sex chromosomes on cardiac I/R injury. The XY* model produces the equivalent of XX and XO gonadal females, and XY and XXY gonadal males. A comparison between mice with one X chromosome (XO or XY) to those with two X chromosomes (XX or XXY) reveals an effect of X chromosome number, whereas comparing mice with a Y chromosome (XY, XXY) with those without Y (XO, XX) shows the effect of presence/absence of the Y chromosome. Four to six weeks before the experiment, mice were gonadectomized (GDX) to eliminate the effects of gonadal hormones. Isolated mouse hearts were subjected to 30 min global normothermic ischemia followed by 60 min reperfusion. Hemodynamic parameters were continuously recorded with a catheter (1.4F Millar SPR-671) connected to a pressure transducer (Power Lab, ADInstruments). At the end of reperfusion, the hearts were cut into four transverse slices and myocardial necrosis was assessed by measurement of the infarct size using triphenyltetrazolium chloride (TTC) staining. The cardiac function was similar among all 4 groups of XY* mice before ischemia. However, postischemic functional recovery of mice with two copies of the X chromosome was significantly lower than mice with a single copy, irrespective of gonadal sex. Rate pressure product (RPP) recovery was markedly lower in XX gonadal females (34.0±10.3%, n=6) and XXY gonadal males (28.0±12.1%, n=5) compared to XO females (60.3±13.5%, n=4) and XO males (60.5±13.8%, n=5, p<0.05). Infarct size was also markedly larger in mice with two X chromosomes (41.4±8.9% in XX gonadal females (n=6) and 46.3±9.5% in XXY gonadal males (n=5)) than mice with one X chromosome (23.7±3.9% in XO females, n=4 and 26.6±6.9% in XO males, n=5, p<0.05). In conclusion, the number of X chromosomes in gonadectomized mice plays a key role in susceptibility to myocardial ischemia/reperfusion injury regardless of the presence or absence of the Y chromosome.
Abstract 220: Role of ECM Stiffness in Microvascular Inflammation
Authors: Ghosh Kaustabh , Scott Harry , Yang Xiao , Ardekani Soroush
Published: July 2021

Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) is a critical regulator of inflammation as it inhibits leukocyte adhesion to vessel endothelium. Notably, reduced eNOS activity/NO bioavailability and chronic inflammation are key features, and often major determinants, of atherosclerosis and diabetic cardiovascular complications. Past approaches aimed at normalizing eNOS activity have focused primarily on the role of soluble, genetic or shear stress-mediated physical factors. However, extracellular matrix (ECM) mechanics also plays a crucial role in endothelial cell (EC) activation and function. Importantly, tissue mechanics is altered in both atherosclerosis and diabetes that are marked by chronic inflammation and impaired eNOS activity. Thus, we investigated whether there exists a causal relationship between ECM stiffness and eNOS/NO-dependent endothelial inflammation. To test this hypothesis, we cultured ECs on fibronectin-coated elastic substrates of varying stiffness (220 Pa/soft, 1000 Pa/intermediate and 4000 Pa/stiff). EC mechanosensitivity was first confirmed by quantifying the density of actin cytoskeletal filaments, which increased with increasing ECM stiffness. EC-leukocyte adhesion (inflammation) on these substrates, however, followed a biphasic trend where ECs on both 220 Pa (soft) and 4000 Pa (stiff) substrates exhibited greater leukocyte adhesion than those cultured on intermediate 1000 Pa substrates. This biphasic effect of ECM stiffness on endothelial inflammation correlated inversely with levels of eNOS-activation and NO production on these substrates. Consistent with these data, NF-kB activation was enhanced on 220 Pa and 4000 Pa while being suppressed on 1000 Pa substrates. Preliminary data implicate differential levels of calcium influx via mechanosensitive calcium ion channels in this ECM stiffness-dependent eNOS activation and endothelial inflammation. These new findings can potentially lead to superior cardiovascular disease management as they identify ECM stiffness both as a novel therapeutic target for immunomodulatory strategies as well as a key physical design criterion for the development of instructive biomaterials for cardiovascular tissue engineering.
Abstract 225: The Mitochondria-localizing Peptide Bendavia Reduces Cardiac Injury By Targeting Cardiolipin
Authors: Brown David A , Harris Mitchel , Moukdar Fatiha , Patel Hetal D , Shaikh Saame R
Published: July 2021

Bendavia is a cardioprotective mitochondria-targeting peptide that is currently being utilized in the EMBRACE-STEMI clinical trial for acute coronary syndromes (ACS). Across a variety of pre-clinical models examining ACS and heart failure, administration of Bendavia reduces infarction and coronary “no-reflow”, improves function, preserves mitochondrial energetics, and lowers ROS-dependent cell death. Despite the clear effects in mitigating reperfusion injury, Bendavia’s molecular targets and underlying mechanisms are not fully understood. The objective of these studies was to determine Bendavia’s molecular mechanism of action and pharmacokinetic profile. In diabetic animals, Bendavia treatment reversed the decline in 18:2 cardiolipin composition observed in diabetic heart. In vivo (i.v.) administration of 3H-Bendavia showed tissue distribution that correlated with tissue mitochondrial volume density. 3H-Bendavia levels in heart mitochondria peaked 1 hour after treatment, and by 24 hours there was no discernible 3H-Bendavia in the myocardium. Biologic processing of Bendavia results in two major metabolites, and neither of these metabolites showed cardioprotective efficacy, suggesting that the cardioprotection is unique to the intact, tetra-peptide. In isolated heart cells, 3H-Bendavia uptake was found to be independent of mitochondrial membrane potential, and respiratory studies revealed no discernible uncoupling activity. We then tested the hypothesis that Bendavia selectively targeted cardiolipin biophysical organization by synthesizing lipid vesicles of differing phospholipid composition. Fluorescence quenching and electrostatically sensitive probes revealed Bendavia preferentially associated with cardiolipin-enriched lipid vesicles. We modeled changes in inner membrane lipids during cardiac ischemia/reperfusion. Bendavia augmented membrane microviscosity in proportion to the cardiolipin content. As a number of disease states (heart failure, diabetes, ischemia/reperfusion) are characterized by low membrane microviscosity, Bendavia’s putative ability to restore the lipid microenvironment represents a novel paradigm for preserving tissue bioenergetics.
Abstract 230: Specific Mitochondrial Adaptation In Chronic Hypoxic Hearts Results In An Improved Intrinsic Energetic Homeostasis
Authors: Deschodt-Arsac Veronique , Calmettes Guillaume , Arsac Laurent , Muller Bernard , Dos Santos Pierre , Hocini Meleze , Jais Pierre , Haissaguerre Michel , Diolez Philippe
Published: July 2021

Hypoxic states of the cardiovascular system are often associated with severe cardiac diseases. In spite of impairments in contractile activity, mitochondrial content, cell energy status and calcium transients, we recently demonstrated an improved energy homeostasis in chronic hypoxic hearts explained by a higher mitochondrial sensitivity to ATP/PCr changes, based on combined 31P-NMR spectroscopy and the analytical framework of metabolic control analysis.

In the present study, we describe the role of calcium in co-variations in cellular energy status (PCr, ATP) and cardiac work (RPP), in perfused hearts from mice adapted for three weeks to a simulated altitude of 5500 m in a barochamber.

Under basal conditions, lower cardiac work and PCr concentration confirmed energetic impairments in chronic hypoxic hearts. At high calcium concentration, cardiac work increased by 40% both in control and hypoxic hearts while a smaller PCr drop in hypoxic hearts, indicating an improved energy homeostasis and higher mitochondrial sensitivity to PCr changes. Stimulating calcium transients by adrenaline and thereby parallel activation of supply and demand components increased cardiac work twice as much only in control hearts (RPP +100%) without any significant PCr variation. The blunted effect of adrenaline in hypoxic hearts is likely the consequence of attenuated ß1-adrenergic receptors stimulation. Inhibition of mitochondrial calcium uniporter by ruthenium red which abolished the mitochondrial-mediated adrenergic regulation was not effective in chronic hearts in contrast of control.

We conclude that although chronic hypoxic hearts failed to use calcium transients to optimize their inotropic response to a specific adrenergic stimulation, they present specific adaptation of the energetic homeostasis based on improved energy supply sensitivity. Interestingly this better regulation of the energetic system is not explained by a direct effect of calcium on mitochondria but rather by an optimized metabolic feedback.
Abstract 232: A Genome-wide Association of Murine Echocardiographic Measures Implicates Muscle A-type Lamin-interacting Protein (MLIP) in Isoproterenol-induced Interventricular Septal Hypertrophy
Authors: Wang Jessica J , Rau Christoph , Ren Shuxun , Cattin Marie-Elodie , Burgon Patrick G , Wang Yibin , Lusis Aldons J
Published: July 2021

Genome-wide association studies of heart failure in humans have yielded limited results, largely due to highly heterogeneous environmental and genetic backgrounds. The Hybrid Mouse Diversity Panel consists of inbred strains that are either fully sequenced or densely genotyped and as a whole displays significant natural variations. We assessed the hypothesis that precise environmental control, a well-defined genetic background, and a careful and uniform phenotyping scheme enable high-resolution mapping of cardiac remodeling in an isoproterenol-induced heart failure mouse model. Eight to ten-week old females from 105 inbred mouse strains (average N per strain = 6.7) were divided into control (average N per strain = 2.5) and treated (average N per strain =4.1) cohorts. Treated mice received 20 μg/g/day of isoproterenol through an abdominally implanted Alzet micropump for 3 weeks. All mice underwent echocardiography at baseline and at weekly time points. Phenotypic data were analyzed using the Efficient Mixed-Model Association (EMMA) algorithm to correct for population substructure. Our study showed that chronic isoproterenol administration resulted in marked inter-strain variations in echocardiographic measurements across the mouse panel. EMMA analysis of the data revealed many significant association peaks across echocardiographic measures. The most significant association signal is in the change of diastolic interventricular septal wall thickness between baseline and 1 week after isoproterenol treatment, a marker of early isoproterenol-induced left ventricular hypertrophy. The peak SNP rs13480288 (p-value of 4.0580e-09) and its surrounding SNPs (within correlation r2 > 0.8) span across 3 genes. Of these, Mlip alone harbors a missence variant and a splice-site variant across our panel. Mlip, also known as muscle A-type lamin-interacting protein, is expressed abundantly in the heart and interacts directly with A-type lamins in the nuclear envelope. Mutations in A-type lamins have been shown to cause both hypertrophic and dilated cardiomyopathy. In conclusion, our study results provide strong evidence that genetic variations in Mlip contribute to differential responses in interverntricular septal hypertrophy to isoproterenol.
Abstract 242: Bone-derived Stem Cells Repair The Heart After Myocardial Infarction Through Transdifferentiation And Paracrine Signaling Mechanisms
Authors: Duran Jason M , Makarewich Catherine A , Sharp Thomas E , Starosta Timothy , Zhu Fang , Hoffman Nicholas E , Chiba Yumi , Husain Sharmeen , Muniswamy Madesh , Berretta Remus M , Kubo Hajime , Houser Steven R
Published: July 2021

Rationale:
Autologous bone marrow- or cardiac-derived stem cell therapy for heart disease has demonstrated safety and efficacy in clinical trials but has only offered limited functional improvements. Finding the optimal stem cell type best suited for cardiac regeneration remains a key goal toward improving clinical outcomes.

Objective:
To determine the mechanism by which novel bone-derived stem cells support the injured heart.

Methods and Results:
Cortical bone stem cells (CBSCs) were isolated from EGFP+ transgenic mice and were shown to express c-kit and Sca-1 as well as 8 paracrine factors involved in cardioprotection, angiogenesis and stem cell function. Wild-type C57BL/6 mice underwent sham operation (n=21) or myocardial infarction (MI) with injection of CBSCs (n=57) or saline (n=59). Cardiac function was monitored using echocardiography with strain analysis. EGFP+ stem cells in vivo were shown to express only 2/8 factors tested (basic fibroblast growth factor and vascular endothelial growth factor) and this expression was associated with increased neovascularization of the infarct border zone. CBSC therapy improved survival, cardiac function, attenuated adverse remodeling, and decreased infarct size relative to saline-treated MI controls. By 6 weeks post-MI, EGFP+ cardiomyocytes, vascular smooth muscle cells and endothelial cells could be identified on histology. Isolated EGFP+ myocytes were smaller, more frequently mononucleated, and demonstrated fractional shortening and calcium currents indistinguishable from EGFP- myocytes from the same hearts.

Conclusions:
CBSCs improve survival, cardiac function, and attenuate remodeling by 1) secreting the proangiogenic factors bFGF and VEGF (stimulating endogenous neovascularization), and 2) differentiating into functional adult myocytes and vascular cells.
Abstract 244: Characterization Of Endogenous C-kit Positive Cells From Right Atrial Appendage Of Neonate And Adult Heart Patients
Authors: Deshmukh Savitha , Mishra Rachana , Sharma Sudhish , Simpson David , Kaushal Sunjay
Published: July 2021

Background:
Stem cell therapy provides novel regenerative strategy for congenital heart patients. We have previously identified two distinct subpopulations (c-kit+ cells and cardiosphere derived cells (hCDCs)) from right atrial appendage (RAA) of neonate and adult heart patients. We have characterized hCDCs from neonate and adult heart patients and found strong regenerative capacity in hCDC derived from neonatal hearts. The aim of this study is to characterize c-kit+ cells derived from neonate and adult heart patients.

Methods:
C-kit+ cells were isolated and expanded from RAA heart samples, ranging in weight from 20 mg to 300 mgs of tissues using the standard protocol. The atrial tissue was minced and suspended in Ham’s F12 medium containing collagenase NB 6. After digestion, cells were plated in c-kit growing media. C-kit+ cells were isolated using MACS column with >90% homogeneity. Cells were characterized using FACS and immunofluorescence techniques.

Results:
The characterization of c-kit+ cells showed that these cells were co-expressing nkx2.5, GATA4 and FLK1 and negative for CD45, Lin and tryptase. The growth potential of c-kit+ cells was determined by use of doubling times which was never greater than 36 hours, computed by use of linear regression of log2 value of cell number. We observed neonate derived c-kit+ cells have more (2x) proliferation capacity compared to adult derived c-kit+ cells. During subsequent passages, c-kit expression was lost more in adult derived c-kit+ cells when compared to neonate derived c-kit+ cells.

Conclusion:
Neonate derived c-kit+ cells retain their stemness and have more proliferative capacity compared to adult derived c-kit+ cells. Based on our previous study and present data we believe that neonate derived c-kit+ cells may have higher regenerative capacity compared to adult derived c-kit+ cells.
Abstract 246: Transdifferentiation of Human Fibroblasts to Endothelial Cells: Role of Innate Immunity
Authors: Sayed Nazish , Wong Wing Tak , Cooke John P
Published: July 2021

Cell fate is fluid, and may be altered experimentally by the forced expression of master regulators mediating cell lineage. Such nuclear reprogramming has been achieved using viral vectors encoding transcription factors. We recently discovered that the viral vectors are more than passive vehicles for transcription factors, as they participate actively in the process of nuclear reprogramming to pluripotency. Viral vectors, by activating innate immunity, cause global changes in the expression of epigenetic modifiers, favoring an open chromatin state (Cell, 151 2012). Based on the recognition that activation of innate immunity increases epigenetic plasticity, we hypothesized that small molecule activators of toll-like receptor 3 (TLR3), together with external microenvironmental cues that drive EC specification, might be sufficient to induce transdifferentiation of fibroblasts into ECs (iECs). Here we show that TLR3 agonist Poly I:C, combined with exogenous EC growth factors, transdifferentiated human fibroblasts into ECs (in the absence of viral vectors or transcription factors). These iECs were comparable to HMVEC in immunohistochemical, genetic and functional assays, including the ability to form capillary-like structures and to incorporate acetylated-LDL. Furthermore, iECs significantly improved limb perfusion and neovascularization in the murine ischemic hindlimb compared to parental fibroblasts. Finally, using genetic knockdown studies, we find that the effective transdifferentiation of human fibroblasts to endothelial cells requires innate immune activation. This study suggests that manipulation of innate immune signaling may be generally used to modify cell fate. Our observations raise the question as to whether epigenetic plasticity and cell fate fluidity generally participate in the immune defense against pathogens. Because similar signaling pathways are activated by damage associated molecular patterns, epigenetic plasticity induced by innate immunity may play a fundamental role in transdifferentiation during wound healing and regeneration. Finally, this study is a first step toward development of a small molecule strategy for therapeutic transdifferentiation in vivo.
Abstract 282: Phospholamban Truncation Mutations Including Heart Failure Mutant L39stop Disrupt Membrane Localization.
Authors: Abrol Neha , Smolin Nikolai , Stefonowicz Chris , Robia Seth L
Published: July 2021

Introduction:
Phospholamban (PLB) is an integral sarcoplasmic reticulum (SR) membrane protein, which directly regulates cardiac Ca
2+
handling and contractility by reversibly inhibiting SR Ca
2+
ATPase (SERCA). Our previous studies have suggested that the naturally occurring human heart failure mutation of PLB, L39X disrupts membrane localization.

Hypothesis:
We hypothesize that the membrane localization of PLB is a prerequisite for PLB oligomerization and interaction with SERCA. The truncation mutations in C-terminus of PLB will disrupt membrane localization, PLB oligomerization, and SERCA regulation.

Results and Methods:
To identify the minimum length of PLB required for membrane localization and function, we generated a series of C-terminal transmembrane truncation mutants of PLB (tagged N-terminally with Cer or YFP) including L51X, M50X, V49X, I48X, I38X, I33X, and the heart-failure mutant L39X. Confocal microscopy revealed that progressive truncation of the C-terminal residues of PLB resulted in escalating increase in mislocalization of PLB to the cytoplasm and nucleus. In addition, we observed an increased solubilization of PLB as indicated by loss of YFP fluorescence after selective permeabilization of the plasma membrane by saponin. As expected, there was no change in localization of Cer-SERCA upon saponin permeabilization. Next, western blot analysis exhibited a decrease in molecular weight corresponding to the relative sizes of truncation mutants compared to full length PLB, indicating that protein degradation is not the cause of membrane mislocalization. Fluorescence resonance energy transfer analysis revealed that truncating the C-terminal residues of PLB results in a progressive decrease in apparent affinity of PLB oligomerization and interaction with SERCA. Finally, molecular dynamics simulations exhibited that the heart failure mutant L39X was unstable compared to full length PLB pentamer and started protruding out of the bilayer until complete solubilization.

Conclusions:
Truncating only two C-terminal residues of PLB resulted in significant mislocalization, while deleting five or more residues profoundly disrupted membrane localization, PLB oligomerization and SERCA regulation.
Abstract 284: Disruption of Nrf2 Promotes Atrial Remodeling and Fibrillation on Aging
Authors: Abu-Rmaileh Naser , Kannan Sankaranarayanan , Tsushima Kensuke , Whitehead Kevin , Abel E Dale , Marrouche Nassir F , Namakkal Soorappan Rajasekaran
Published: July 2021

Background:
Heart failure is a leading health problem with over 500,000 new cases and 275,000 deaths annually in the United States. Recent reports indicate that atrial fibrillation (AF) is linked to various forms of ventricular dysfunction (VD). Over 2 million Americans have AF, which is more prevalent in people over 65 years of age. However, the molecular “cause and effect” relationship between AF and VD has not been elucidated. We hypothesize that abrogation of nuclear erythroid-2 like factor-2 (Nrf2), a master antioxidant transcriptional regulator, induces atrial remodeling and fibrillation on aging.

Methods:
Age and sex matched WT and Nrf2-/- mice were used in this study. Atrial mass, remodeling, antioxidants and molecular redox signaling were studied at 2 and >20 months of age. Echocardiography, immunoblotting, quantitative real-time PCR and immunofluorescence (fibrosis) analyses were performed in the atrial tissue.

Results:
At 2 months of age, WT and Nrf2-/- mice had comparable levels of ROS in the atrium. On aging, the ROS levels were significantly increased in atria of Nrf2-
null
when compared to wild-type (WT) mice at 20 months of age. While decreased Nrf2-antioxidant signaling in response to increased ROS generation in atria of Nrf2-
null
mice was observed, ventricular function appeared to be normal at 20 months of age. However, upon endurance exercise stress (EES), hypertrophy markers including ANF, BNF, PLN and SERCA2A were significantly (p<0.05) altered in Nrf2-
null
when compared to age-matched WT mice. Further, activation of fibrotic process was evident in the Nrf2-
null
mouse atrium as indicated by significantly (p<0.05) increased markers of tissue remodeling (i.e. MMP2/9) on aging. These results indicating an early onset of atrial hypertrophy/remodeling due to age-induced oxidative stress, which cause AF in Nrf2-
null
mice. Age-dependent decline in Nrf2 and sustained progression of AF could lead to ventricular dysfunction and heart failure.

Conclusion:
Our findings indicate that atria are primary targets to age-associated oxidative stress and exhibit fibrosis, which promotes atrial fibrillation and remodeling. Thus, activation of Nrf2 signaling to prevent oxidative stress could be a potential therapeutic target for AF.
Abstract 300: Protein Kinase G Positively Regulates Proteasome-mediated Degradation of Misfolded Proteins
Authors: Ranek Mark J , Terpstra Erin J , Li Jie , Kass David A , Wang Xuejun
Published: July 2021

Objectives:
Proteasome functional insufficiency is implicated in a large subset of cardiovascular diseases and may play an important role in their pathogenesis, evidenced by increased protein aggregates and ubiquitinated proteins. The regulation of proteasome function is poorly understood, hindering the development of effective strategies to improve proteasome function. We sought to establish the role of protein kinase G (PKG) in these debilitating conditions, for which there is currently no cure.

Methods and Results:
PKG was manipulated genetically and pharmacologically in cultured cardiomyocytes. Activation of PKG increased proteasome peptidase activities, facilitated proteasome-mediated degradation of surrogate (GFPu) and bona fide misfolded proteins (CryABR120G), and attenuated CryABR120G overexpression-induced accumulation of ubiquitinated proteins and cellular injury. PKG inhibition elicited the opposite responses. Differences in the abundance of the key 26S proteasome subunits Rpt6 and β5 between PKG manipulated and the control groups were not statistically significant, but the isoelectric points of were shifted by PKG activation. In transgenic mice expressing a surrogate substrate (GFPdgn), PKG activation by sildenafil increased myocardial proteasome activities and significantly decreased myocardial GFPdgn protein levels. Sildenafil treatment significantly increased myocardial PKG activity and significantly reduced myocardial accumulation of CryABR120G, ubiquitin conjugates, and aberrant protein aggregates in mice with CryABR120G-based desmin-related cardiomyopathy. No discernible effect on bona fide native substrates of the ubiquitin-proteasome system was observed from PKG manipulation in vitro or in vivo.

Conclusions:
PKG positively regulates proteasome activities and proteasome-mediated degradation of misfolded proteins likely through posttranslational modifications to proteasome subunits. Improved protein quality control is liekly a new mechanism underlying the benefit of PKG stimulation in treating cardiac diseases. Stimulation of PKG by measures such as sildenafil administration is potentially a novel therapeutic strategy to treat cardiac proteinopathies.
Abstract 302: Neuregulin-1β Inhibits TGFβ-induced Cardiofibroblast to Myofibroblast Transition
Authors: Galindo Cristi L , Truc-Tran Linh , Ryzhov Sergey , Feoktistov Igor , Sawyer Douglas
Published: July 2021

Neuregulin (NRG) is a growth and survival factor critical for cardiac development and repair. Our laboratory recently demonstrated decreased fibrosis with NRG treatment in post-infarcted swine. We subsequently hypothesized that NRG inhibits transition of fibroblasts to myofibroblasts (myoFbs), the cells primarily responsible for collagen deposition after cardiac injury. Immunohistochemistry of post-infarcted swine hearts confirmed reduced presence of myoFbs in NRG-treated pig ventricles, compared to untreated animals. Further investigation revealed that NRG acts directly upon primary cultures of rat cardiac fibroblasts, with subsequent phosphorylation of both AKT and p38. Based on quantitative RT-PCR, primary fibroblasts isolated from both rats and mice express ErbB2 and ErbB3, which are known to form heterodimers and thereby transduce the NRG signal. NRG treatment also reduced myoFb percentages by ~40% in primary rat and mouse cardiac cultures, compared to untreated cells, as demonstrated by immunohistochemistry, Western blot, and flow cytometric analyses of the myoFb marker, alpha smooth muscle actin. NRG pre-treatment of cardiac fibroblasts likewise inhibited TGFβ-induced fibroblast-to-myoFb transition, including suppression of SMAD3 phosphorylation and reduction of the down-stream pro-fibrotic proteins, collagen I and periostin. Considered together, these data strongly support the hypothesis that NRG modulates fibroblast signaling directly. These results demonstrate that NRG inhibits the activation of pro-fibrotic cells and suggests a novel mechanism by which NRG treatment might improve cardiac remodeling.
Abstract 312: STIM1 And The Development Of Pressure-overload Induced Cardiac Hypertrophy In Rodents
Authors: Bénard Ludovic O , Matasic Daniel S , Keck Mathilde , Lompré Anne-Marie , Hajjar Roger J , Hulot Jean-Sébastien
Published: July 2021

STromal Interaction Molecule 1 (STIM1), a membrane protein of the sarcoplasmic reticulum, has recently been proposed as a positive regulator of cardiomyocyte growth by promoting Ca2+ entry through the plasma membrane and the activation of Ca2+-mediated signaling pathways. We studied the role of STIM1 in a pressure-overload induced cardiac hypertrophy model in mice. We observed that STIM1 cardiac expression is increased during left ventricular hypertrophy (LVH) induced by Transverse Aortic Constriction (TAC). We then used recombinant Associated Adenovirus 9 (AAV9) to perform cardiac-targeted gene silencing in vivo. C57Bl/6 mice were injected with saline (noAAV) or with AAV9 expressing shRNA against STIM1 (shSTIM1) at the dose of 1e+11 viral genome which resulted in 70% decrease of STIM1 cardiac expression compared to control mice. Three weeks later, TAC was performed and mice were studied three other weeks later.

We found that TAC-shSTIM1 treated mice did not develop LVH compared to noAAV despite the same increase in aortic pressure. Echocardiographic and hemodynamic measurements (see table) showed that TAC-shSTIM1-treated mice had LV dilation and a decreased left ventricular contractile function in line with the absence of compensatory LVH in these mice. Immunohistochemistry demonstrated that LVH prevention was observed at the cellular level with cardiac myocytes cross-section area comparable to sham littermates however with a trend towards more interstitial fibrosis.

This study reveals the essential role of STIM1 in the development of compensatory LVH in mice.
Abstract 317: Conditional Knockout Of Phd2 Ameliorates High-fat Diet-induced Cardiac Dysfunction Via Inhibition Of Tlr4-nfκB Pathway In A Hifa-independent Manner
Authors: Zeng heng , chen Jian-Xiong
Published: July 2021

Oxygen sensors prolyl hydroxylases (PHDs) plays an important role in the regulation of hypoxia-inducible factor (HIFα) as well as nuclear factor-κ B (NF-κB) activity. Our previous study demonstrated that activation of vascular Toll-like receptor TLR-MyD88-IRAK4 signaling pathway contributes to vascular dysfunction in high-fat-diet (HFD)-induced diabetes. The present study investigates whether conditional knockout of prolyl hydroxylase-2 (PHD2) improves cardiac function via suppression of TLR-NF-κb signaling in a HFD-induced diabetic cardiomyopathy mouse model. Wild type (WT-Cre
+
), PHD2 conditional knockout (PHD2CKO) and PHD2
+/-
mice were fed with either HFD or normal chow diet (NFD) for 18 weeks.
The
levels of PHD1-3, HIF, angiogenic growth factors, MyD88, IRAK-4, TLR4, NF-κb and Wnt7b gene expression were measured. Cardiac hypertrophy, fibrosis and cardiac function were analyzed
at 18 weeks after PHD2 deactivation
. Feeding of WT-Cre
+
mice with HFD for 18 weeks resulted in a significant reduction of cardiac functions. The basal expression of PHD2 was significantly increased in the heart of HFD mice. The expression of PHD2 was significantly reduced in the heart of PHD2CKO and PHD2
+/-
mice fed with HFD whereas PHD1 and PHD3 expression remains unchanged. Conditional knockout of PHD2 resulted in suppression of MyD88, IRAK-4, TLR4, NF-κb and Wnt7b expression. Deactivation of PHD2 had little effect on the expression of HIF1α, HIF2α and angiogenic growth factor in the hearts of HFD-induced diabetic mice. Conditional knockout of PHD2 further suppressed HFD-induced cardiac hypertrophy and significantly improved HFD-induced cardiac dysfunction.
In contrast, conditional knockout of PHD2 resulted in an increase in HIFα expression and cardiac dysfunction in NFD mice.
These results suggest that deactivation
of PHD2
attenuates HFD-induced diabetic cardiomyopathy via suppression of TLR4-MyD88-IRAK4-NF-κb-Wnt7b signaling pathway in a HIF-independent manner.
Abstract 323: Arginine Methylation Regulates Epicardial Cell Plasticity
Authors: Xu Jian , Oses-Prieto Juan , Burlingame Alma , Derynck Rik
Published: July 2021

Kinase activation and substrate phosphorylation commonly form the backbone of signaling cascades. TGFbeta family ligands induce activation of their signaling effectors, the Smads, through C-terminal phosphorylation by transmembrane receptor kinases following type I and type II receptor complex formation. Here we show that arginine methylation, which regulates gene expression, yet also modifies some signaling mediators, initiates TGFbeta-induced Smad signaling. TGFbeta-induced receptor complex formation promotes presentation of the methyltransferase PRMT1 to the inhibitory Smad7, resulting in Smad7 methylation at a specific arginine as revealed by mass spectrometry analysis and confirmed with a mutagenesis approach. Antibodies specific for asymmetric di-methylated arginine further confirmed Smad7 methylation and Smad7 recruitment to the cell membrane upon TGFbeta stimulation. TGFbeta-induced and PRMT1-mediated Smad7 methylation leads to Smad7 dissociation from TGFbeta type I receptors, allowing activation of effector Smads through phosphorylation. This PRMT1-mediated Smad7 methylation is required in TGFbeta-regulated epicardial plasticity, as apparent by the role of PRMT1 in TGFbeta-induced epithelial-to-mesenchymal trans-differentiation and transcriptional reprogramming in mouse epicardial cells.
Abstract 345: R403Q Mutation Increases the Rate of Force Redevelopment in 2 Month Mice
Authors: Birch Camille , Konhilas John P
Published: July 2021

Familial hypertrophic cardiomyopathy is a primary disease of the sarcomere. The R403Q mutation resides at the actin-interaction site on myosin and leads to progressive hypertrophic cardiomyopathy which progresses towards heart failure. Along with deteriorating cardiac function, these hearts experience an overall change in metabolic landscape, suggesting altered energetic function in hearts that express the R403Q mutation. We tested the hypothesis that the R403Q mutation intrinsically increases the energetic cost of contraction. To do this, we determined myofilament function in demembranated cardiac trabeculae from male wild-type (WT) and R403Q mice at 2 months of age, prior to overt signs of cardiac pathology. Firstly, steady-state Ca2+ sensitivity of force generation was not significantly different between male R403Q (n=4) and WT counterparts (n=2) consistent with previous findings. Secondly, the rate of force redevelopment (ktr) in skinned cardiac tissue was measured following unloaded isotonic shortening and a rapid re-stretch to 15% of the original muscle length at a sarcomere length of 2.0μm. R403Q mice display an increased rate of force redevelopment (49.89 s-1 ± 8.13, n = 4) compared to WT counterparts (24.52 ± 4.29, n = 6) at maximal activation indicating an increase in the apparent rates of crossbridges entering and leaving force-generating states (p < 0.05). In conclusion, the R403Q mutation does not impact steady-state Ca2+ sensitivity of force but increases total crossbridge cycling rate suggesting a higher energy cost of force generation. Future studies are aimed at determining the energetic cost of contraction in R403Q hearts and how this increased energetic cost leads to hypertrophic cardiomyopathy.
Abstract 107: MicroRNA-31: A Novel Therapeutic Target for Ischemic Heart Disease
Authors: Martinez Eliana C , Lilyanna Shera , Vardy Leah A , Armugam Arunmozhiarasi , Jeyaseelan Kandiah , Richards Arthur M
Published: July 2021

MicroRNAs (miRNA), small sequences of non-coding RNA which interact with complementary sequences on the 3’untranslated region of target messenger RNAs to modulate translation, have a pivotal role in the development of the heart and its response to injury. Myocardial infarction (MI) triggers a dynamic miRNA response with the potential of yielding therapeutic targets. Following miRNA array profiling in rat hearts 2, 7 and 14 days after MI induced by coronary ligation, we identified a progressive time-dependent up-regulation of miR-31 compared to sham rats. Increase of miR-31 in heart tissue in the acute and subacute phases after MI (up to 90-fold) was also detected by Real-Time PCR (P=0.02 at day 2; P<0.0001 at days 7 and 14, vs. sham). We found that miR-31 has a repressive effect on tissue mRNA expression of cardiac troponin-T (TNNT2), E2F transcription factor 6 (E2F6) and mineralocorticoid receptor (NR3C2). Reporter gene assays showed that miR-31 targets the 3′UTR of these genes, with a marked repressive effect on TNNT2. In vitro, exposure to hypoxia significantly induced the expression of miR-31 in neonatal rat cardiomyocytes (nRCM), rat cardiac fibroblasts (nRCF) and cardiomyoblasts (H9C2) and suppressed the expression of TNNT2, E2F6 and NR3C2 in nRCM and H9C2 cells, and of E2F6 and NR3C2 in nRCF. LNA-based oligonucleotide inhibition of miR-31(miR-31i) in vitro reversed its repressive effect on translation from target genes. Therapeutic modulation of miR-31 expression in vivo after MI via subcutaneous administration of miR-31i (25mg/Kg/q2w) in rats, led to cardiac repression of miR-31 and subsequent enhanced expression of target genes. Also, miR-31i led to preservation of cardiac function and structure by day 14 after treatment. An absolute 10% improvement in left ventricular (LV) ejection fraction (EF) was observed in miR-31i-treated rats from day 2 to 16 after MI, while control rats that received scrambled LNA inhibitor or placebo displayed 23% deterioration in EF (n=6-8/group, P<0.0001). We conclude that miR-31 induction after MI is deleterious to cardiac function and plays an important role in adverse remodeling, while its therapeutic inhibition in vivo ameliorates cardiac dysfunction and prevents the development of post-ischemic heart failure.
Abstract 110: Anesthetic Preconditioning and Mitochondrial Slo K
+
Channel Activity Require
Slo2.1
Authors: Wojtovich Andrew P , Smith C O , Nadtochiy Sergiy M , Urciuoli William R , Xia Xiao-Ming , Jonas Elizabeth , Lingle Christopher J , Nehrke Keith , Brookes Paul S
Published: July 2021

Introduction:
Volatile anesthetic preconditioning (APC) protects the heart from ischemia-reperfusion (IR) injury. APC elicits evolutionarily-conserved protective signaling pathways that converge at the mitochondrial level, where the
Slo
family of K
+
channels is thought to mediate protection through an unknown mechanism. Recent work in
C. elegans
has focused attention on the
Slo2
gene product as a transducer of APC effects on hypoxic survival. In mammals,
Slo2
has diverged into two paralogs,
Slo
2.1
(KCNT2; Slick) and
Slo2.2
(KCNT1; Slack). These genes code for Na
+
-activated K
+
channels and are highly expressed in brain, but their function in cardiomyocytes and/or mitochondria is unknown.

Methods:
The contribution of
Slo
channels to cardiac physiology was characterized using knockout mice, including
Slo1
and two novel
Slo2.x
alleles. APC was assessed through
ex-vivo
cardiac IR injury. Isolated mitochondrial K
+
channel activity was assessed using a Tl
+
flux assay, and by patch-clamp of cardiac mitoplasts. Electron microscopy was used to assess mitochondrial morphology in primary cardiomyocytes and Seahorse extracellular flux analysis used to assess bioenergetics.

Results:
The
Slo2.x
(
double KO
) and the
Slo2.1
single KO mice could not be protected from cardiac IR injury by APC. Physiologic approaches demonstrated that
Slo2.1
is present in mitochondria and
Slo2.1
-dependent mitochondrial K
+
transport can be triggered directly by volatile anesthetics. Cardiomyocytes from
Slo2.x
dKO
mice exhibited profound metabolic remodeling and electron microscopy revealed that
Slo2.1
knockouts had enlarged circular mitochondria. In contrast,
Slo1
KO mice responded normally to APC and exhibited wild type mitochondrial physiology.

Conclusion:
Slo2.1
activation protects against cardiac IR, and is required for APC.
Slo2.1
also contributes to mitochondrial metabolic homeostasis. As a molecular target for APC, identification of
Slo2.1
may facilitate development of targeted therapeutic molecules that can protect the heart and minimize the side effects of volatile anesthetics.
Abstract 118: Epigenetic Regulation of Ventricular Development
Authors: Lee Youngsook , Cho Eunjin , Mysliwiec Matthew
Published: July 2021

Perturbations of the epigenetic machinery can lead to the deregulation of cardiac gene expression, resulting in defective cardiac development and cardiac hypertrophy. Due to a groundbreaking discovery of histone demethylases such as Jumonji (Jmj) family factors, histone methylation is now considered as a reversible epigenetic mark. Jarid2/Jumonji is the founding member of the JMJ family. Jarid2 is enzymatically inactive, but functions as a transcriptional regulator. Jarid2 critically regulates cardiovascular development as well as ES cell differentiation and generation of iPS cells. Jarid2 knockout (KO) mice exhibit cardiac defects including hyper-trabeculation with noncompaction of the ventricular wall. Although Jarid2 interacts with Polycomb Repressor Complex in ES cells, the precise function of Jarid2 in cardiac development remains to be determined. Therefore, we set out to determine molecular mechanisms of Jarid2 critical for cardiac development. To identify cardiac-specific roles of Jarid2, we generated deletion of Jarid2 in early cardiac progenitors using Nkx2.5-Cre Knock-in mice (Jarid2Nkx-KI). Jarid2Nkx-KI mice recapitulate partial phenotypic defects observed in Jarid2 KO including hyper-trabeculation, thin myocardium and ventricular septal defects. By overlapping ChIP-chip and microarray analyses, we have identified potential transcriptional targets of Jarid2, which are occupied by Jarid2, SETDB1, H3K9me3 or H3K27me3, and upregulated in Jarid2 mutant hearts. 174 genes including Isl1 were identified as dysregulated genes that showed accumulation of Jarid2 and H3K27me3. 172 genes including Bmp10 were identified as dysregulated genes and were occupied by Jarid2, SETDB1 and H3K9me3. Isl1, Bmp10/p-Smad1/5/8, and Ifgbp2 were upregulated in Jarid2Nkx-KI hearts by qRT-PCR and Western blotting. Islet1 plays crucial roles in early cardiac development and a marker for cardiac progenitors. Jarid2 occupancy was observed at the Isl1 promoter region by ChIP assays in WT hearts, which was reduced in Jarid2Nkx-KI. All together, our data indicate that Jarid2 regulates target gene expression by interacting with different histone modifiers depending on the cell/promoter context, which is critical for ventricular wall maturation.