bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P346: Regulation of
GRK5
in Myocytes Is Mediated by Nuclear Transcription Factor [ê]B: A New Clue in Heart Disease
Authors: Islam Kazi N , Koch Walter J
Published: March 2022

G protein coupled receptor kinase-5 (GRK5) plays a key role in the heart and it’s expression is increased in heart failure. Moreover, a human mutation within its amino-terminus appears to play a role in therapeutic responses to β-blockers in heart failure patients. Nuclear transcription factor kappa B (NF-κB), a ubiquitous transcription factor, is involved in the regulation of numerous genes in various tissues and activation of NF-κB has also been shown to be associated with heart disease. Here, we investigated the role of NF-κB signaling in the regulation of the
GRK5
gene and GRK5 expression in neonatal rat ventricular myocytes (NRVMs). In analyzing the 5’-flanking DNA of
GRK5
, the presence of a potential NF-κB binding site was observed in the promoter. Phorbol myristate acetate (PMA), a known stimulator of NF-κB, increased the levels of GRK5 in NRVMs. Conversely, treatment of NRVMs with N-acetyl cysteine (NAC), a known inhibitor of NF-κB, decreased the levels of GRK5 compared to control cells. Utilizing electrophoretic mobility shift assay (EMSA) with nuclear extracts from NRVMs treated with or without PMA, it was found that proteins bound to the GRK5 promoter containing the putative NF-κB binding site. When an antibody specific for NF-κB was utilized, a super-shift of the proteins binding to the putative NF-κB binding site was observed. Furthermore, interaction of NF-κB with GRK5 was confirmed by shift Western blot. Chromatin immunoprecipitation (ChIP) showed dynamic recruitment of both p50 and p65 to the NF-κB site of GRK5 promoter after treatment of NRVMs with PMA. Treatment of myocytes with siRNA and subsequent knock-down of NF-κB p65 decreased the levels of GRK5 as judged by real time quantitative PCR. Finally, adenovirus mediated overexpression of a dominant negative IκB-α in NRVMs inhibited the levels of GRK5. Taken together, our present studies suggest that NF-κB plays a potential role in the regulation of GRK5 transcription in myocytes and this may translate to key expressional changes seen in heart disease.
Abstract 103: Role of miRNA-7 in Cardiac Function
Authors: Gupta Manveen K , Xi Wang , Martelli Elizabeth , Naga Prasad Sathyamangla V
Published: March 2022

Studies in cancer have established clearly the role of epidermal growth factors receptors as targets of microRNA-7 (miR-7) but there are no studies so far to elucidate the role and function of miR-7 in human heart. Our studies show that ERBB2 (a member of epidermal growth factors receptor family) is targeted by miR-7 in human heart failure as well as in mice hearts undergoing stress. In order to understand the role of miR-7 in the heart, we generated miR-7 transgenic (Tg) mice with cardiac myocyte-specific overexpression of miR-7. miR-7 Tg mice have age dependent deterioration in cardiac dysfunction associated with cardiac dilation as measured by echocardiography (3 months - 60% FS, 6 month -52% FS and 12 months - 24%FS) and yet, they survive well for more than a year. To investigate whether pathological stress would accelerate the deterioration in cardiac function, miR-7 Tg mice were subjected to transverse aortic constriction (TAC) for two weeks. In contrast to the wild type littermates which undergo hypertrophic response following TAC, miR-7 Tg mice have accelerated cardiac dysfunction and dilation within two weeks. Histological analysis shows increased fibrosis in miR-7 Tg mice hearts as shown by Picro Sirius red and Meson Trichome staining which is further accelerated after TAC stress in hearts of the miR-7 Tg mice as compared to their sham controls. We also find that there is a difference in the mitochondrial morphology and structure in miR-7 Tg mice as compared to Wildtype controls as seen by transmission Electron Microscopy (TEM) explaining the reason for deteriorated cardiac function in terms of energy generation. Our study will discuss the mechanism underlying the deteriorated cardiac function in the miR-7 Tg mice after pathological stress as compared to wildtype littermates.
Abstract 109: PI3Kγ Regulates Release of Myocyte-derived Factors Responsible for Myofibroblast Differentiation and Cardiac Fibrosis
Authors: Mohan Maradumane L , Grove Lisa M , Papay Robert S , Martelli Elizabeth E , Willard Belinda B , Olman Mitchell A , Naga Prasad Sathyamangla V
Published: March 2022

Phosphoinositide 3 Kinase γ (PI3Kγ) is a lipid kinase that regulates downstream anti-apoptotic Akt signaling. Thus, pressure overload in PI3Kγ null (PI3Kγ
-/-
) mice leads to significant cardiac fibrosis, a key underlying cause of fatal heart failure. Classical hallmark of tissue fibrosis is differentiation of fibroblasts to myofibroblasts characterized by smooth muscle α-actin (αSMA) overexpression. However, less is known about the role of PI3Kγ in cardiac myofibroblast differentiation. Assessment of αSMA expression in cardiac lysates from WT and PI3Kγ
-/-
showed significant baseline upregulation in PI3Kγ
-/-
showing that loss of PI3Kγ predisposes the hearts towards fibrosis. To directly confirm that PI3Kγ
-/-
cardiac fibroblasts (CF) exhibit a myofibroblast phenotype, CF were isolated from hearts of WT and PI3Kγ
-/-
and assessed by immunostaining for αSMA in stress fibers. Greater number of CF from PI3Kγ
-/-
exhibited αSMA in stress fibers than CF from WT. Correspondingly, immunoblotting showed significantly higher expression of αSMA in PI3Kγ
-/-
CF compared to WT showing enhanced myofibroblast differentiation by PI3Kγ
-/-
fibroblasts. Surprisingly, abundance of αSMA protein is significantly reduced in the hearts of mice with cardiomyocyte-specific expression of kinase-dead PI3Kγ (PI3Kγ
inact
) in the PI3Kγ
-/-
(PI3Kγ
inact
/PI3Kγ
-/-
) suggesting that myocytes derived factors responsible for myofibroblast differentiation are regulated by kinase-independent function of PI3Kγ. To directly evaluate the PI3Kγ-dependent cardiomyocyte derived factors responsible for myofibroblast differentiation; fibroblasts were treated with conditioned media derived from primary adult cardiomyocytes from WT, PI3Kγ
-/-
and PI3Kγ
inact
/PI3Kγ
-/-
mice. Conditioned media derived from PI3Kγ
-/-
showed pro-fibrotic effects, while that from PI3Kγ
inact
/PI3Kγ
-/-
showed fibrosis protective biological activity compared to WT. These findings reveal that kinase-independent function of PI3Kγ is a key regulator of the myocyte-initiated pathway that ultimately drives myofibroblast conversion. Proteomic analysis of conditioned media identified several pro-fibrotic factors that are regulated by PI3Kγ, the results of which will be discussed.
Abstract 11: Lamin A/C Mutations Epigenetically Dysregulate Scn5a Gene Expression, Perturbing Action Potential Properties in IPSC-derived Cardiomyocytes
Authors: Crasto Silvia , Salvarani Nicolò , Miragoli Michele , Paulis Marianna , Kunderfranco Paolo , Carullo Pierluigi , Forni Alberto , Faggian Giuseppe , Condorelli Gianluigi , Di Pasquale Elisa
Published: March 2022

Mutations of the LMNA gene, encoding the nuclear lamina proteins Lamin A/C, are a common cause of dilated cardiomyopathy, typically manifesting in association with cardiac conduction defects. LaminA/C regulate various nuclear activities, including maintenance of the nuclear structure, gene transcription and chromatin organization. Most studies on the consequences of Lamin A/C defects were conducted on fibroblasts, while studies on human cardiomyocytes (CMs) are scarce. We therefore generated a cardiac model of laminopathy obtained by differentiation of CMs from induced pluripotent stem cells (iPSCs) of patients carrying the K219T Lamin A/C mutation. In vitro, these cells recapitulate the morphological features of dilated cardiomyopathy, specifically sarcomeric disorganization and increased size. Using this model, we performed a comprehensive analysis of the electrophysiological properties of LMNA-CMs both at single cell level and in a multi-cellular setting. Using patch-clamp technique, results revealed significant changes in maximal upstroke velocity (dV/dt
max
), action potential amplitude (APA) and overshoot (OV) in LMNA-CMs compared to those obtained from family-matched healthy controls (CNTR); these defects were associated with a reduction of the peak sodium currents and a diminished conduction velocity, measured in strands of electrically-coupled CMs. Biochemical studies showed a significant reduction of both the sodium channel Nav1.5 protein and its transcript in LMNA-CMs, accompanied by an increased binding of LaminA/C to the promoter of its coding gene, SCN5A. Binding of the Polycomb group protein SUZ12 and of the H3K27me3 histone repressive mark was also increased. Consistently, 3D-FISH experiments also indicated a preferential localization of SCN5A genomic loci at the nuclear periphery in LMNA-CMs. As a whole, our findings support a model in which mutated Lamin A/C perturb SCN5A gene expression by favouring PRC2 (Polycomb Repressive Complex 2) binding to its promoter, leading to decreased sodium current peak and slower conduction velocity. This mechanism may eventually sustain the conduction abnormalities inevitably occurring in patients with LMNA-cardiomyopathy.
Abstract 111: Reduced MK5 Expression Alters Migration, Proliferation, and Collagen Biosynthesis in Murine Ventricular Fibroblasts
Authors: Sahadevan Pramod , Nawaito Sherin A , Calderone Angelo , Gaestel Matthias , Allen Bruce G
Published: March 2022

MAP kinase-activated protein kinase-5 (MK5), a protein serine/threonine kinase expressed in the heart, has been identified as a substrate for p38α/β and ERK3/4 MAPKs. However, the interacting partners and physiological function of MK5 are just beginning to be understood. This study examined the role of MK5 in murine cardiac ventricular fibroblasts.

Confocal immunocytofluorescence microscopy (CIFM) revealed that MK5 immunoreactivity localized primarily to the nucleus whereas ERK3 immunoreactivity was in the cytoplasm. Following serum stimulation, phospho-MK5 immunoreactivity was observed in the cytoplasm and appeared to be associated with the cytoskeleton and pseudopodia. ERK3 immunoreactivity redistributed to membrane ruffles and/or lamellipodia. ERK3 has been reported to be unstable in the absence of MK5: in cardiac fibroblasts, ERK3 immunoreactivity was unaffected by acute knockdown of MK5 with siRNA (MK5-kd), suggesting an as-yet unidentified binding partner may stabilize ERK3 in these cells.

MK5 immunoprecipitates from fibroblast lysates contained ERK3 immunoreactivity and proximity ligation assays indicated the presence of ERK3-MK5 complexes in the cytoplasm: these complexes were less abundant in MK5-kd fibroblasts. Cell migration, in response to serum and/or Ang-II was reduced significantly in MK5-kd fibroblasts. In addition, cell proliferation was decreased in fibroblasts isolated from MK5
-/-
mice compared to fibroblasts from wild-type litter mate mice (MK5
+/+
). Surprisingly, both the abundance of type 1 collagen (COL1A1) mRNA and the secretion of soluble COL1A1 were increased in MK5-kd fibroblasts whereas the ability of Ang-II to increase collagen secretion was unaffected. CIFM revealed staining for COL1A1 was diffuse in MK5
+/+
fibroblasts but condensed in the perinuclear region of MK5
-/-
cells. A similar pattern of subcellular distribution of COL1A1 immunoreactivity was observed in MK5-kd fibroblasts and was observed in passages 0 through 3. Taken together, these data suggest 1) one or more proteins in addition to MK5 serve to stabilize ERK3 in fibroblasts and 2) MK5 may be involved in cardiac fibroblast proliferation, migration, collagen biosynthesis and, possibly, myocardial remodelling.
Abstract 127: Vinculin Mediated Increase in Cardiac Function Confers Systemic Metabolic Efficiency and Increases in Healthspan and Lifespan
Authors: Sessions Ayla , Min Peter , Cordes Thekla , Metallo Christian , Engler Adam
Published: March 2022

Despite limited regenerative capacity, compensatory mechanisms in cardiomyocytes may help maintain their function as we age, e.g. vinculin expression and localization to intercalated discs and costameres in aged organisms. This molecular mechanism, which is conserved from flies to non-human primates, helps sustain the sarcomere lattice structure and maintains cardiomyocyte function. In
Drosophila melanogaster
cardiac-restricted vinculin overexpression can extend lifespan up to 150%, but connections between cytoskeletal structure and metabolism are not apparent. We found that by prolonging heart function, the local and systemic energy requirements are dramatically altered; increased contractility resulted in sustained O
2
consumption with age but without localized metabolic changes in cardiomyocytes. However, systemic changes resulting from vinculin-mediated increases in cardiac function improved physical activity levels in
Drosophila
and energy metabolism. This work is the first to identify systemic metabolic improvements from a cytoskeletal perturbation in cardiomyocytes that result in extended organismal lifespan.
Abstract 139: Deep Sequencing Identifies MicroRNA Let-7 Regulating Cardiomyocyte Apoptosis in a Porcine Model of Early Myocardial Infarction
Authors: Chen Chen-Yun , Hsieh Patrick C
Published: March 2022

Myocardial infarction (MI) is a leading cause of mortality and heart failure worldwide. Several animal models have been utilized to mimic human MI. Porcine is considered one of the most representative models. Here, we take advantage of a porcine MI model and next-generation sequencing techniques to discover differential microRNA expression occurring in early MI. We found that microRNAs let-7a and let-7f were enriched in heart tissues and showed significant downregulation within 24 hours post-MI. Inhibition of let-7 by Tough Decoy (TuD) RNA
in vivo
enhanced cardiomyocyte apoptosis. Overexpression of let-7 downstream target GeneA activated p38 MAPK and led to cardiomyocyte apoptosis. Inhibition of p38 MAPK rescued GeneA induced activation of Caspase 3. We therefore hypothesized that downregulation of let-7 promotes cardiomyocyte death through GeneA and p38 MAPK signaling pathway. In support of this, overexpression of let-7 or inhibition of GeneA reduced p38 MAPK activity and rescued cell death induced by serum starvation
in vitro
. Finally, we confirmed that in pigs with acute MI, the plasma level of let-7 was significantly reduced and the plasma level of GeneA was significantly increased. This study identifies differential expression of microRNAs in early MI, uncovers the roles of let-7 and GeneA in cardiovascular diseases, and also provides potential biomarkers and therapeutic targets for further investigation.
Abstract 140: Role of miR-181c in Mitochondrial Matrix Calcium Accumulation During Ischemia/Reperfusion Injury in the Heart
Authors: Solhjoo Soroosh , Kannan Sangeetha M , Ashok Deepthi , O’Rourke Brian , Steenbergen Charles , Das Samarjit
Published: March 2022

Background:
We have identified a microRNA, miR-181c, which can translocate into the mitochondria of cardiomyocytes, and regulate the mitochondrial gene mt-COX1. Recently, we have also demonstrated that miR-181c/d
-/-
mice are protected against ischemia/reperfusion (I/R) injury by attenuating oxidative stress in the heart. Previous data also suggest that overexpression of miR-181c in the heart can activate Ca
2+
entry into mitochondria. Here, we investigate the mechanism by which miR-181c regulates Ca
2+
influx into the mitochondrial matrix.

Methods and Results:
We found both Mitochondrial Calcium Uptake 1 (MICU1) and mitochondrial respiratory complex IV (COX IV) expression are markedly higher in the miR-181c/d
-/-
mouse heart. Immunoprecipitated with MICU1, and then immunoblot for different sub-units of COX IV confirmed a protein-protein interaction between MICU1 and COX IV. We have also found significantly less Pyruvate Dehydrogenase (PDH) activity in neonatal mouse ventricular myocytes (NMVMs) isolated from miR-181c/d
-/-
mouse compared to C57BL6 (WT), suggesting significantly lower mitochondrial Ca
2+
-concentrations in the miR-181c/d
-/-
group. Utilizing a coverslip induced I/R-model, we observe that siRNAs against MICU1 (si-MICU1) during the ischemic phase significantly increase Ca
2+
-entry into the mitochondria of the NMVMs. Lowering MICU1 also significantly increases Ca
2+
-entry into the mitochondria after 30 min of ischemia in miR-181c/d
-/-
NMVMs. Furthermore, 30 min ischemia followed by 30 min reperfusion in NMVM monolayers led to significantly less oscillatory instability in mitochondrial inner membrane potential (ΔΨ
m
) in miR-181c/d
-/-
NMVMs compared with WT NMVMs. However, using si-MICU1 in the miR-181c/d
-/-
NMVM group attenuated mitochondrial protection against I/R-injury.

Conclusions:
MICU1 is directly associated with complex IV. Thus, miR-181c can regulate mitochondrial Ca
2+
-entry by targeting mt-COX1 during I/R injury.
Abstract 143: The Inner Mitochondrial Membrane Protein Mpv17 is Crucial for Myocardial Recovery From Ischemia Reperfusion Injury
Authors: Madungwe Ngonidzashe B , Feng Yansheng , Lie Mihaela , Bopassa Jean C
Published: March 2022

Introduction:
The MPV17 mutation in humans is an autosomal recessive disorder that results in mitochondrial DNA depletion syndromes (MDS) leading to hepatocerebral defects. Previously attributed to unknown functions, the MPV17 protein has recently been shown in the liver and kidney to be a non-selective channel in the inner membrane of mitochondria whose function is tied to membrane potential (ΔΨ
m
), pH and reactive oxygen species (ROS) generation. These mitochondrial functions are critical in ischemia/reperfusion (I/R) injury caused by myocardial infarction, but despite this, however, Mpv17’s role in the myocardium is still unknown. We hypothesized that Mpv17 plays a critical role in cardiac responses to I/R injury.

Methods:
To investigate the role of cardiac Mpv17 protein in response to I/R injury, we measured cardiac functional recovery and myocardial infarct size in isolated hearts from in Mpv17
-/-
and wildtype mice subjected to I/R. Additionally, we assessed mitochondrial ROS production, calcium retention capacity (CRC) required to induce mitochondrial permeability transition pore (mPTP) opening, ΔΨ
m
and cristae morphology in isolated mitochondria from the same mice.

Results:
We found that normal Mpv17
-/-
mice exhibit similar cardiac and mitochondrial characteristics to wildtype, but after I/R, knockout mice show less cardiac functional recovery and increased myocardial infarct size. Mitochondria in these mice had more damaged cristae and reduced CRC, but surprisingly less ROS production compared to wildtype mice.

Conclusion:
These results shed more light onto the function of the Mpv17 protein and suggest it is critical in cardioprotection against I/R injury, and the cardiac mitochondrial response to I/R stress.
Abstract 150: Irisin Enhances Cardiac Progenitor Cell-induced Cardiac Repair in the Infarcted Heart
Authors: Zhao Yu T , Wang Hao , Dubielecka-Szczerba Patrycja , Zhao Ting C
Published: March 2022

Background:
Our recent evidence has demonstrated that irisin, a newly identified cardiokine, is important for the development of cardiac protection. We and others have shown that engraftment of cardiac progenitor cells (CPCs) into infarcted hearts led to myocardium regeneration and neovascularization. The purpose of this study is aimed at investigating the functional role of irisin, a cardiokine, in facilitating engrafted Nkx2.5/CPCs to promote cardiac repair and preserve cardiac performance in infarcted hearts.

Methods:
Nkx2.5 CSCs were isolated from mouse embryonic stem cells and re-introduced into the infarcted myocardium in which the mouse MI model was created by permanent ligation of the left anterior descending artery. Nkx.2.5 CPCs were treated with or without irisin (5 ng/ml) for 24 hours to precondition CPCs. CPCs were transplanted into the infarcted heart through the pegylated fibrin delivery approach. Myocardial functions were evaluated by serial echocardiographic measurements. Histological analysis was employed to assess newly formed cardiogenesis and cardiac remodeling.

Results:
Eight weeks after engraftment, the retention of CPCs in the infarcted heart was increased significantly following pegylated fibrin delivery of CPCs as compared to the direct injection of cells. Engrafted CPCs demonstrated newly formed and proliferative cardiomyocyte structures, which was enhanced by treatment with irisin. Echocardiography showed improvements in ventricular function following the engraftment of CPCs, which was promoted by irisin in association with the attenuation of remodeling.

Conclusion:
Our results indicate that irisin-promoted, CPC-derived cardiac regeneration improves the restoration of cardiac function in infarcted hearts, suggesting that irisin holds promise in developing a potentially new therapeutic strategy in myocardial repair and suppressing remodeling.
Abstract 155: ATF6 is a Mediator of Cardiac Hypertrophy During Pressure Overload in the Mouse Heart
Authors: Correll Robert N , Lynch Jeffrey M , Sargent Michelle A , York Allen J , Molkentin Jeffery D
Published: March 2022

The endoplasmic reticulum (ER) stress response is one of many signaling events activated in the heart in response to injury and/or cardiac hypertrophy, although the role that this response pathway plays in such events remains under investigation. Recently, our laboratory demonstrated that overexpression of thrombospondin-4 (Thbs4) resulted in a protective ER stress response via activation of activating transcription factor 6 (ATF6), and that mice gene-deleted for Thbs4 demonstrated compromised ER stress signaling and decreased survival after transverse aortic constriction (TAC) or myocardial infarction (MI) surgery. Here we confirm that Thbs4-mediated expansion of the ER compartment requires ATF6 and examine whether the protective ER stress response mediated by transgenic overexpression of Thbs4 is eliminated when crossed with gene-deleted mice lacking ATF6. We also examined cardiac-specific transgenic mice overexpressing the transcriptionally-active ATF6 N-terminus, as well as mice gene-deleted for ATF6, in combination with disease stimuli including TAC and MI surgery. We find that ATF6 proteins are required for compensatory hypertrophy in the mouse heart and that loss of these proteins results in accelerated decompensation and failure, likely due to reductions in ER folding capacity that is required for the increased protein production necessary for cardiac hypertrophy. These results firmly position ATF6 as an essential regulator of compensatory cardiac hypertrophy during disease.
Abstract 16: A Minipig Genetic Model of Hypertrophic Cardiomyopathy
Authors: Green Eric M , Weiss Robert M , Divekar Abhay , Bartholomew Ingle Sadie R , Henze Marcus , Kawas Raja , Gifford Lindsey , Davis Melissa K , Rohret Frank , Thedens Daniel R , Rodriguez Hector M , Evanchik Marc J , Anderson Robert L , Sieren Jessica , Rogers Christopher S , Meyerholz David K , Ahmad Ferhaan
Published: March 2022

Introduction:
Hypertrophic cardiomyopathy (HCM) is a heritable disease of heart muscle associated with increased risk of heart failure and sudden death. Mutations in genes encoding sarcomere proteins are commonly associated with HCM. However, the mechanisms by which these mutations lead to molecular, cellular and organ-level pathophysiology are uncertain, partly because of the lack of model systems amenable to integrated translational studies.

Methods:
Using homologous recombination and somatic cell nuclear transfer, we generated Yucatan minipigs with a heterozygous knock-in of the R403Q mutation in
MYH7
, a well-characterized human HCM mutation. We conducted deep phenotyping with biomechanical studies of myocardial tissue samples, circulating biomarker analysis, cardiac imaging and histologic and multi-omic analysis of LV biopsy samples.

Results:
We followed a cohort of 22 R403Q pigs and 6 WT herdmates. Juvenile animals (3 months) showed early signs of HCM with elevated serum troponin I, increased myocardial contractility in muscle fibers and hearts and interstitial fibrosis and myocyte disarray. At late adolescence (9 months), disarray and fibrosis had progressed, but contractility had normalized with some pigs progressing to systolic dysfunction. Across the cohort, end-diastolic pressure was increased with evidence of diastolic dysfunction and elevation in B-type natriuretic peptide. Transcriptomic analysis at both 3 and 9 months showed dysregulation of metabolic modules and an upregulation of pro-fibrotic pathways. By one year of age, 11 of 22 R403Q pigs had suffered sudden cardiac death, whereas all wildtype pigs survived.

Conclusions:
We have developed the first large-animal genetic model of HCM. Young pigs with the
MYH7
R403Q mutation show functional and histologic features of the preclinical human phenotype, and late adolescent animals have signs of advanced disease with an increased rate of sudden cardiac death. These data suggest that our minipig model may yield insights throughout the natural history of HCM from preclinical to end-stage disease. This model will thus be invaluable for advancing understanding of HCM and for the development of novel therapeutics.
Abstract 161: β-Arrestin-Biased Agonism of β-Adrenergic Receptor Regulates Dicer-mediated MicroRNA Maturation to Promote Cardioprotective Signaling
Authors: Teoh Jian-Peng , Kim Il-man
Published: March 2022

MicroRNAs (miRs) are small, non-coding RNAs that function to post-transcriptionally regulate gene expression. First transcribed as primary miR transcripts (pri-miRs), they are enzymatically processed by Drosha into premature miRs (pre-miRs) and further processed by Dicer into mature miRs. Initially discovered to desensitize β-adrenergic receptor (βAR) signaling, β-arrestins are now well-appreciated to modulate multiple effector pathways independent of G protein-mediated signaling, a concept known as biased signaling. Using the β-arrestin-biased βAR ligand carvedilol (Carv), we previously showed that β-arrestin1-biased β1AR cardioprotective signaling stimulates Drosha-mediated processing of a subset of miRs. Here, we investigate whether Carv could regulate Dicer-mediated miR maturation, thereby providing a novel mechanism for its cardioprotective effects. In mouse hearts, Carv indeed upregulates 3 mature miRs, but not their pre-miRs and pri-miRs, in a β-arrestin1/2-dependent manner. Interestingly, Carv-mediated activation of miR-466g and miR-532 is dependent on β2AR, while the activation of miR-674 by Carv is β1AR-dependent. Mechanistically, β-arrestins regulate maturation of 3 newly identified βAR/β-arrestin-responsive miRs (β-miRs) by associating with the Dicer complex as well as hnRNPK and dyskerin on 3 pre-miRs (Fig. A). Cardiac cell approaches uncover that β-miRs act as gatekeepers of cardiac cell function by repressing detrimental target genes (Fig. B). In conclusion, our findings indicate a novel role for βAR-mediated β-arrestin signaling activated by Carv in miR maturation, which may be linked, in part, to its protective mechanism.
Abstract 165: Role of the Gut Microbiome in Obesity-Related Vascular Dysfunction
Authors: Battson Micah , Lee Dustin , Hou Shuofei , Jarrell Dillon , Ecton Kayl , Weir Tiffany , Gentile Christopher
Published: March 2022

Vascular dysfunction, characterized by arterial stiffness and endothelial dysfunction, represents an important link between obesity and cardiovascular disease. Deleterious changes to the gut microbiome, termed dysbiosis, are associated with obesity and may contribute to the development of vascular dysfunction by promoting systemic and arterial inflammation. We tested the hypothesis that antibiotic treatment in diet-induced obese mice would improve vascular function by suppressing potentially harmful gut-derived bacterial products to reduce dysbiosis-related inflammation. Male C57BL/6 mice were fed either a standard diet (SD) or Western diet (WD) for 5 months. Obese WD-fed mice were then randomized to receive 8 weeks of antibiotic treatment via drinking water. Arterial stiffness was measured via
in vivo
aortic pulse wave velocity and endothelial function assessed via
ex vivo
pressure myography in isolated mesenteric arteries. Plasma and arterial tissue samples were collected for measures of endotoxemia and inflammation and fecal samples were collected for microbial sequencing. Our results showed that mice fed a WD for 5 months had significantly higher body weight and arterial stiffness compared to SD. Antibiotic treatment significantly reduced arterial stiffness in WD-fed mice without affecting body weight. Endothelial function was impaired in WD-fed mice, whereas WD-Abx mice had similar endothelial function to SD-fed mice. Plasma levels of the pro-inflammatory cytokine IL-6 and endotoxemia marker LPS binding protein (LBP) were significantly increased in WD vs SD, but reduced in WD-Abx vs WD. Gut microbial suppression with antibiotic treatment was confirmed using quantitative PCR and ongoing analyses are being conducted to determine specific diet-induced changes to the composition of the gut microbiome. Overall, our results support the hypothesis that gut dysbiosis contributes the development of vascular dysfunction in obesity and provide evidence that a gut-targeted therapy can improve endothelial function and arterial stiffness in diet-induced obese mice. Future studies will further examine the mechanisms involved in, and help identify potential therapeutic targets for, obesity-related vascular dysfunction.
Abstract 166: Anti-cachectic Role of Neuregulin-1β in Heart Failure
Authors: Galindo Cristi L , Gupte Manisha , Tikhomirov Oleg Y , Manda Pranay , Cleator John H , Murphy Abigail , Kasasbeh Ehab , Sawyer Douglas B
Published: March 2022

Neuregulin-1β (NRG-1β) is a growth and differentiation factor with pleiotropic systemic effects. Because NRG-1β has therapeutic potential for heart failure and known growth effects in skeletal muscle, we hypothesized that it might affect heart failure-associated cachexia, a severe co-morbidity characterized by a loss of muscle mass. We therefore assessed NRG-1β’s effect on skeletal muscle gene expression in a swine model of heart failure using recombinant Glial Growth Factor 2 (USAN - cimaglermin alfa), a version of NRG-1β currently being tested in humans with systolic heart failure. Animals received one of two intravenous doses (0.67 or 2 mg/kg) of NRG-1β bi-weekly for 4 weeks, beginning one week after infarct. Skeletal muscle was collected at the time of euthanasia from the intercostal space and paired-end RNA sequencing performed. NRG-1β treatment altered expression of 583 transcripts including 242 transcripts altered at both doses. These included genes required for myofiber growth, maintenance and survival such as
MYH3, MYHC, MYL6B, KY and HES1
. Importantly, NRG-1β altered the directionality of at least 85 genes associated with cachexia, including myostatin, which negatively regulates myoblast differentiation by down-regulating
MyoD
expression. Consistent with this, MyoD was increased in NRG-1β treated animals.
In vitro
experiments with myoblast cell lines confirmed that NRG-1β induces skeletal muscle differentiation with an absolute requirement for ERBB signaling on myoblast differentiation. These findings suggest a NRG-1β-mediated anti-atrophic, anti-cachexia effect that may provide additional benefits to this potential therapy in heart failure.
Abstract 167: Inhibition of Endoplasmic Reticulum Stress Improves Vascular Function in Type II Diabetic Mice
Authors: Lee Dustin M , Battson Micah L , Jarrell Dillon K , Hou Shuofei , Ecton Kayl , Gentile Christopher L
Published: March 2022

Cardiovascular disease is the leading cause of death in the United States with type 2 diabetes (T2D) representing a major risk factor in its development. Vascular dysfunction, characterized by arterial stiffness and endothelial dysfunction occur prior to overt cardiovascular disease and predict future cardiovascular events and mortality in diabetic individuals.

Dysfunction of the endoplasmic reticulum (ER), or ER stress, is associated with the development and progression of chronic metabolic diseases such as obesity and T2D. However, the role of ER stress in the development of vascular dysfunction observed in T2D is unclear. We hypothesized that inhibiting ER stress would improve both measures of vascular dysfunction observed in diabetic mice.

Male C57BL/6J Leprdb (DB) male mice lacking the leptin receptor were used as a model of T2D. Starting at 4 months of age DB mice were given intraperitoneal injections of ER stress inhibitor, tauroursodeoxycholic acid (TUDCA) at 250mg/kg/day for 4 weeks. C57BL/6J mice were used as controls (n=8 for all groups). Pulse wave velocity (PWV) was measured at baseline (prior to treatment) and after TUDCA treatment. Secondary order mesenteric resistance arteries (MRA) were used to determine endothelial dependent dilation (EDD). DB mice not treated with TUDCA were used for acute studies of EDD by incubating the MRA for 1hr with 0.5mM TUDCA.

At baseline, DB mice displayed increased arterial stiffness compared to C57BL/6J controls as measured by PWV (457±25 vs 348±26 cm/s, p<0.05). TUDCA treatment significantly decreased PWV (402±12 cm/s, p<0.05). Both acute and chronic TUDCA treatment improved DB EDD compared to untreated controls (58.4±13.8 and 49.1±6.1%, vs 19.8±9.6%, p<0.05). Expression of ER stress-associated genes CHOP and ATF4 were significantly elevated in DB perivascular adipose tissue and treatment with TUDCA significantly decreased ATF4 compared to DB. In the aorta, TUDCA significantly decreased GRP78 and XBP1 compared to DB.

These data support the hypothesis that ER stress contributes to the vascular dysfunction observed in T2D and suggest that ER stress may be a potential target in the treatment of T2D related vascular stiffening and dysfunction.
Abstract 17: A Critical Role of TRAF2 in Myocardial Survival and Homeostasis by Suppressing Necroptosis
Authors: Guo Xiaoyun , Yin Haifeng , Li Lei , Chen Yi , Li Jing , Doan Jessica , Steinmetz Rachel , Liu Qinghang
Published: March 2022

Programed cell death, including apoptosis and necroptosis, is critically involved in ischemic cardiac injury, pathological cardiac remodeling, and heart failure progression. Whereas apoptosis signaling is well established, the regulatory mechanisms of necroptosis and its significance in the pathogenesis of heart failure remain largely unkown. Here we identified TNF receptor-associated factor 2 (Traf2) as a key suppressor of myocardial necroptosis, which critically regulates myocardial survival and homeostasis. It has been shown that transgenic expression of Traf2 protects the heart against ischemia-reperfusion injury, but the underlying mechanisms remain unclear. Moreover, the role of Traf2 in myocardial necroptosis and pathological remodeling has not been investigated in a loss-of-function approach. By generating cardiac-specific
Traf2
knockout mice, we found that ablation of Traf2 in the heart induced pathological remodeling and heart failure by promoting necroptotic myocyte death. Importantly, plasma TNFα level was significantly elevated in
Traf2
-deficient mice, and genetic ablation of
TNFR1
(TNF receptor-1) largely abrogated pathological cardiac remodeling and dysfunction associated with Traf2 deficiency. Mechanistically, our data revealed that Traf2 critically regulates RIP1-RIP3-MLKL necroptosis signaling, with the adaptor protein TRADD (TNFR1-associated death domain protein) as an upstream regulator and TAK1 (TGFβ-activated kinase-1) as a downstream effector. Moreover, Traf2 prevents the degradation of key pro-survival signaling proteins TAK1, FLIP, cIAPs, and NFκB-p65 via the ubiquitin-proteasome pathway. Lastly, genetic deletion of
RIP3
largely rescued the cardiac phenotype triggered by Traf2 deletion, further validating a critical role of necroptosis in regulating pathological remodeling and heart failure propensity. Taken together, these results identify an important Traf2-mediated, NFκB-independent, pro-survival pathway in the heart by suppressing necroptosis signaling, which may serve as a new therapeutic target for pathological remodeling and heart failure.
Abstract 171: Promoter Negative Elongation Factor a (NelfA) Occupancy Required for Gene Transcription in Heart During Hypertrophy
Authors: Severinova Elena , Yang Zhi , Abdellatif Maha , Sayed Danish H
Published: March 2022

Adaptive change in gene expression is one of the earliest responses of heart to hypertrophy. Gene transcription is tightly regulated by systematic recruitment of factors including positive and negative elongation complexes of RNA Pol II. Recently we embarked on the task of deconstructing the transcriptional machinery in the heart, by examining Pol II and TFIIB dynamics across the genome in sham and hypertrophied hearts. In this study, we continue examining the active transcriptional code by measuring occupancy of two interdependent factors that belong to positive (PTEFb) and negative (NELF) elongation complexes, Cdk9 and NelfA, respectively. Studies in non-cardiac cells have implicated these two factors in the regulation of promoter (prom) clearance of pol II, where Cdk9 mediated phosphorylation of NelfA results in its release from paused pol II and initiation of productive elongation. As expected, we observed increase Cdk9 (>1.5) association with proms in 59.7% of expressed genes (8257 of 13816), while 6% show decrease in prom Cdk9 (<0.75) in TAC vs. sham hearts. In contrast to previous reports, we did not see any correlation between Cdk9 and NelfA occupancy, where 93.2% and 68% of genes did not show change in NelfA with increased or decreased Cdk9 levels in TAC hearts, respectively. Interestingly, highest enrichment of NelfA (av. density 53.5) was observed on genes regulated by prom clearance of pol II (av. prom den 9.5 vs. 7.4; av. Ingene den 3.0 vs. 3.6, sham vs. TAC; housekeeping genes) with no change in NelfA (89.8% of genes). On other hand, 3.3% of genes showed increase in NelfA (av. den 4.6 vs. 8.4, sham vs. TAC), along with increase in pol II recruitment (av. prom den 3.3 vs. 4.04; av. ingene den 1.8 vs. 2.4, sham vs. TAC; cytoskeletal, cell cycle genes). We confirmed increase in mRNA abundance of these genes, (eg. Acta 1) in TAC vs. sham hearts. Intriguingly, genes that were associated with reduced NelfA (av. den 21.7 vs. 14.8, sham vs. TAC) were mostly cardiac specific genes (e.g. cardiac muscle contraction), which show increase in pol II, but no change in mRNA levels with TAC (eg. Actc). While we are investigating the role of NelfA in heart, ChIP- Seq data suggest that NelfA might be required for productive elongation and increase in global gene expression during hypertrophy
Abstract 191: Paracrine Impact of Cardiac Progenitor Cells on Macrophage Phenotypes and Human Ipsc-derived Cardiomyocyte Survival
Authors: Noseda Michela , Constantinou Chrystalla , Samari Sara , Chaves Patricia , Cheng Kevin , Beretta Chiara , Rito Tiago , Abreu-Paiva Marta , Pombo Ana , Schneider Michael D
Published: March 2022

The clinical progression of myocardial infarction, the foremost cause of death globally, to heart failure is proportionally higher with increasing infarct size. Thus, preventing cardiomyocyte loss at time of injury and stimulating self-repair are fruitful approaches to counteract the severity of cardiac damage. We previously found that intramyocardial injection of cardiac progenitor cells (CPCs) at time of ischemia improves cardiac function 12 weeks later. Given poor long-term engraftment in our studies and many others’, the consensus is that early paracrine effects are the probable cause of the benefits observed, including direct effects on cardiomyocytes but also macrophages as key players in the response to injury. Here, using transwell and conditioned medium experiments, we show that CPCs suppress the death of cardiomyocytes after oxidative stress. In this highly tractable model system, CPCs protect not only mouse cardiomyocytes, but also human iPSC-derived cardiomyocytes. Moreover, we show that CPC-conditioned medium affects macrophage differentiation and polarization. Cardioprotective CPCs disrupt the pro-inflammatory (“M1”) programme induced by GM-CSF/LPS/IFNγ, and promote instead the formation of anti-inflammatory (“M2”) macrophages as shown by single cell qRT-PCR. To identify the effectors of the cardioprotective paracrine cocktail, we performed RNA-Seq on unfractionated Sca1
+
cardiac stromal cells, identified wound healing and immune response enriched GeneOntologies, then mapped noteworthy hits by single-cell qPCR to the PDGFRα
+
CPC subpopulation. By a combination of single-cell gene expression profiling in protective versus non-protective cells, plus highly multiplexed immunoassays (LEGENDplex, O-Link), we identified 6-8 candidates as potential mediators of cardiomyocyte protection and M2 macrophage induction. In summary, our data: (1) Define a consistent single-cell paracrine gene signature in adult CPCs; (2) Resolve specific benefits conferred by CPCs in the M1 versus M2 macrophage phenotypes; (3) Demonstrate the utility of iPSC-derived cardiomyocytes as a human platform for paracrine protection studies.
Abstract 192: Persistence of First Generation Adenovirus in the Myocardium: Refuting Old Dogma
Authors: Olive Jacqueline K , Mathison Megumi , Pinnamaneni Jaya Pratap , Singh Vivek P , Sanagasetti Deepthi , Yang Jianchang , Rosengart Todd K
Published: March 2022

Introduction:
Adenovirus (Ad) vectors do not integrate into host genomes and thus offer clinical benefits as non-mutagenic, acute-term gene delivery vehicles. One limitation to Ad vector use is the transient duration of transgene expression, supposedly resulting from immune-mediated cytolysis of Ad-infected cells. Our recent observation of Ad-induced transdifferentiation of infected cardiac fibroblasts into induced cardiomyocytes over 28 days in vivo called this premise into question. Therefore, we hypothesized that Ad remains in the myocardium for over 28 days and, to our knowledge, are the first to suggest alternate mechanisms for this persistence.

Methods and Results:
Ad encoding green fluorescent protein (AdGFP) or mock was administered via intramyocardial injection into Sprague-Dawley rats, and Ad genome and GFP expression were analyzed 3 or 28 days later (n=6 per group, 3 euthanized at each time point). Polymerase chain reaction typing and amplicon sequencing confirmed the presence of Ad DNA in all 6 treated animals. Quantitative real time polymerase chain reaction assays further demonstrated more than a three-fold increase in Ad DNA levels at both days 3 and 28 compared to untreated control animals (3.41±1.40 and 3.37±1.23, respectively). Thus, at 28 days Ad DNA persisted at a high level comparable to that which was present at 3 days. Next, AdGFP expression and host immune response were characterized by fluorescence microscopy and histology. We found that GFP-expressing cells decreased five-fold by 28 days (49±9 v. 3±0 cells). In addition, hematoxylin and eosin staining showed minimal infiltrating mononuclear inflammatory cells in AdGFP and mock-treated animals at both time points.

Conclusions:
Ad DNA and infected host cell persistence suggests that host-mediated cytolysis may not be responsible for the downregulation of transgene expression, indicating that Ad vectors may consequently be useful for therapies like cellular reprogramming wherein host cell persistence is required.
Abstract 193: CRISPR-mediated Introduction of the Sodium-iodide Symporter to Enable Non-invasive Monitoring of Macaque Induced-pluripotent Stem Cell-derived Cardiomyocytes
Authors: Ostrominski John , Yada Ravi Chandra , Sato Noriko , Palisoc Maryknoll , Pittaluga Stefania , Lin Yongshun , Zou Jizhong , Peng Kah-Whye , Hong So Gun , Dunbar Cynthia
Published: March 2022

Due to the limited regenerative capacity of mature cardiomyocytes, cardiac cell therapies constitute an exciting strategy for myocardial repair. However, there is limited understanding of the spatio-temporal distribution and survival of transplanted cells. Hence, there is demand for technologies enabling long-term non-invasive tracking of transplanted cellular therapeutics. Sodium-iodide symporter (NIS)-based
in vivo
imaging has many potential advantages, including predicted safety and immunotolerance due to reliance on an endogenous species-specific gene and on widely available imaging technologies. We believe that non-human primates represent ideal models for investigating the biology of allogenic or autologous cellular grafts, because of close physiologic similarity to humans. We report the development of NIS-based
in vivo
imaging to detect and track rhesus induced pluripotent stem cell (RhiPSC)-derived teratomas as a proof-of-concept model tested in mice, and characterization of NIS-positive RhiPSC (NIS-RhiPSC)-derived cardiomyocytes (CM). NIS-RhiPSCs were generated by CRISPR/Cas9-mediated integration of the rhesus NIS cDNA within the
AAVS1
safe harbor locus. NIS was stably expressed and radiotracer uptake by NIS-RhiPSCs was demonstrated
in vitro
. To evaluate viability of NIS-mediated imaging in RhiPSCs, undifferentiated NIS-RhiPSCs were introduced intramuscularly into immunodeficient mice, and NIS imaging was performed via PET/CT at 2, 4, and 6-weeks post-injection. NIS-positive teratomas were readily detectable as early as 2 weeks post-injection, prior to development of any palpable mass. Using our previously established differentiation protocol, NIS-RhiPS-CMs were derived with high purity, exhibited spontaneous beating in culture, and were similar in all aspects to parental RhiPS-CMs. NIS-RhiPS-CMs maintained stable NIS expression that was comparable to undifferentiated NIS-RhiPSCs, suggesting that
in vivo
imaging of transplanted NIS-RhiPS-CMs should be feasible. Further functional characterization of NIS-RhiPS-CMs, including
in vitro
radiotracer uptake, post-transplantation imaging in a mouse myocardial infarction model, and electrophysiologic analysis is ongoing and data will be presented.
Abstract 197: Heat Shock Factor 1 Enhances Therapeutic Potential of c-kit+ Cardiac Progenitor Cells Derived Exosomes
Authors: Sharma Sudhish , Bigham Grace E , Mishra Rachana , Korutla Laxminarayana , Vallabhajosyula Prashanth , Karanthasis Sotirios , Kaushal Sunjay
Published: March 2022

Background:
Cardiac Progenitor cells (CPCs, identified by ckit+/CD45-/lin-) are being studied in 21 clinical trials (recruiting and completed) to restore heart function and promote tissue regeneration in response to injury. We have recently identified that CPCs derived exosomes (EXO) are critical for their functional activity.

Hypothesis:
We hypothesized that heat shock factor 1 (HSF1) modulates EXO biogenesis from CPCs and their enrichment with therapeutic miRs.

Methods and Results:
EXOs were isolated from cultured CPCs, generated from the biopsies of right atrial appendage (RAA) at the time of cardiac surgery from neonatal (nCPCs, < 1 month) and adult (aCPCs, > 40 years) patients with normal functioning myocardium using size exclusion chromatography (CL-2B). EXOs were analyzed by transmission electron microscopy, Nanosight (LM10) and the flow cytometry for EXO markers as CD63 and ALIX. Our results showed that chronological ageing affects EXOs biogenesis and their functional potential since nCPCs generate significantly more EXOs than aCPCs. HSF1 overexpression in aCPCs significantly enhanced their EXO biogenesis. nCPCs derived EXOs (nEXOs) are significantly more effective (nEXOs 62.14±2.9% vs aEXO 56 ± 3.6%, p<0.05) to improve cardiac function and tissue repair in a myocardial infarction (MI) model in rats as compared to aCPCs derived EXOs. In a rat MI model, using florescent labelled HLA-A antibodies, nEXOs were retained for longer duration in rat serum as compared to aEXOs, which correlated with myocardial functional recovery. miRNA sequencing of EXOs identified significantly higher expression of cardio protective miRs like miR199b, 146a, 454, 590, 21 in nEXOs as compared to aEXOs. We also identified that HSF1 overexpression in aCPCs significantly enriched them with miR21 and miR590, suggesting that HSF1 can affect EXO biogenesis and its miR Cargo.

Conclusion:
We showed that nEXOs are functionally more active and modulation of HSF1 can affect EXOs biogenesis and its cargo from CPCs. These results also suggest that nEXOs has the potential to be utilized as an off the shelf cell-free therapy.