bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 65: Desmin Preamyloid Oligomers in Experimental Heart Failure and Cultured Cardiac Myocytes
Authors: Rainer Peter , Lee Dong I , Sorge Matteo , Guarnieri Carlo , Glabe Charles G , O’Rourke Brian , Tomaselli Gordon F , Kass David A , Van Eyk Jennifer E , Agnetti Giulio
Published: July 2021

Background:
Heart Failure (HF) is one of the main causes of morbidity and mortality in westernized countries but the molecular mechanisms underlying its development are still unclear. A paradigm-shifting view focuses on the accumulation of preamyloid oligomers (PAOs), similar to those observed in Alzheimer disease, as a potential mechanism of cardiac toxicity. We reported that differential desmin phosphorylation at serines (S) 27 and 31 could drive the formation of PAOs in the heart, in the absence of genetic mutations. We sought to establish the identity of the molecular seed triggering the nucleation of cardiac PAOs in an experimental model of HF and in cultured cardiac cells.

Methods:
Mice were subjected to transverse aortic constriction (TAC) for 4 weeks (FS% = 29.3±2.6, P=0.0001). Alternatively, neonatal rat ventricular myocytes were transduced with lentiviral vectors carrying alanine (A) or phospho-mimetic aspartate (D) desmin double mutants at S27 and S31, fused with GFP. Protein homogenates were subjected to western blot analysis with fluorescent co-staining using the A11 anti-PAOs and anti-desmin antibodies. Transduced cells were also subjected to live imaging to assess phenotype.

Results:
Co-western blot analysis of both TAC mice and phospho-mimetic mutant cells revealed the colocalization of PAOs with desmin modified (potentially cleaved) forms. Preamyloid oligomers and a desmin fragment were both increased in TAC mice vs. controls (2.8-fold, P=0.023 and 1.8-fold, P=0.038, respectively). The DD mutant, mimicking the doubly phosphorylated desmin that we hypothesized is the physiological form, showed a healthier phenotype in terms of number of spontaneously contracting cells (P=0.041) and incorporation of GFP-desmin at the Z-discs (P=0.0027), whereas the mono-phosphomimetic mutant (AD) resulted in the increase of desmin positive aggregates (P=0.0014).

Conclusions:
This preliminary evidence suggests that desmin modified forms represent the seed triggering the formation of cardiac PAOs, in the absence of genetic mutations. The accumulation of desmin PAOs could therefore represent an overarching mechanism underlying the deterioration of cardiac function in HF.
Abstract 79: Nrf2 Protects From Intermittent Hypoxia-induced Cardiomyopathy via Metallothionein-dependent and Independent Mechanisms
Authors: Zhou Shanshan , Yin Xia , Jin Jingpeng , Xin Ying , Tan Yi , Zhang Zhiguo , Sun Weixia , Cui Taixing , Cai Jun , Zheng Yang , Cai Lu
Published: July 2021

Background:
We recently reported that cardiac expression of the antioxidant metallothionein (MT) was increased by acute expsorue to intermittent hypoxia (IH), and protected from chronic IH-induced cardiomyopathy. However, how IH stimulates MT in the heart remains unclear. Here we tried to define whether nuclear factor erythroid 2-related factor 2 (Nrf2), a critical redox-balance controller in the body, protects from IH-induced cardiomyopathy and its relationship with MT, and whether Nrf2 and MT are indispensable for sulforaphane (SFN) prevention of IH-induced cardiomyopathy.

Methods and Results:
Mice were exposed to IH for 3 days to 8 weeks. Like MT, cardiac Nrf2 expression was significantly increased in response to 3-day IH, but decreased in response to 4- or 8-week IH. Mice with cardiac overexpression or global deletion of the Nrf2 gene (Nrf2-TG or Nrf2-KO) were completely resistant or susceptible to IH-induced cardiomyopathy. Cardiac protection from IH by endogenous Nrf2 and MT are indispensable each other. Mechanistically 4-week exposure to IH significantly decreased cardiac Nrf2 binding to the promoter of MT, lowing its transcription and translation. MT stimulated Nrf2 function via activation of PI3K/Akt/GSK-3β/Fyn signaling pathway in a feedback manner. Furthermore, Nrf2 inducer SFN prevented IH-induced cardiomyopathy in both WT and MT-KO mice, but not Nrf2-KO mice.

Conclusions:
These results suggest that a reciprocal regulation between Nrf2 and MT is necessary for an efficiently antioxidant response to and protection from IH. However, the protective effect of SFN on IH-induced cardiomyopathy is Nrf2-dependent, instead of MT.
Abstract 93: Genetic Deletion of Slc8b1, the Mitochondrial Sodium/Calcium Exchanger, Causes Sudden Cardiac Death and Overexpression Protects Against Myocardial Infarction and Pressure-Overload Heart Failure
Authors: Luongo Timothy S , Nwokedi Mary , Lambert Jonathan P , Gao Erhe , Carpenter April C , Madesh Muniswamy , Molkentin Jeffery D , Elrod John W
Published: July 2021

Mitochondrial calcium (
m
Ca
2+
) signaling is critical for both energy production and the activation of cell death pathways in the heart. Further,
m
Ca
2+
overload is hypothesized to be a significant contributor to the development and progression of heart failure (HF). The mitochondrial sodium/calcium exchanger (mNCX)

is hypothesized to be the primary mechanism of
m
Ca
2+
efflux, but to date no study has genetically confirmed its identity or function in an
in vivo
system. To investigate the role of mNCX in HF, we generated mutant mice with loxP sites flanking exons 5-7 of the candidate gene,
Slc8b1 (also known as NCLX)
, and crossed them with a tamoxifen (tamox)-inducible cardiac-specific Cre mouse to delete mNCX in the adult heart (mNCX-cKO). Cardiomyocytes isolated from mNCX-cKO mice displayed a significant reduction in
m
Ca
2+
efflux rate and Ca
2+
uptake capacity. Tamoxifen-induced ablation of mNCX resulted in sudden death with only 54% of mice surviving 8d post-tamoxifen treatment (Fig 1). Assessment of mNCX-cKO hearts 2d post-tamox revealed significant remodeling characterized by dilation and a decrease in %EF (Fig 2). Next, we generated a conditional, cardiac-specific mNCX overexpression mouse model (mNCX-Tg) to evaluate if increased
m
Ca
2+
efflux would alter the progression of HF. mNCX-Tg and controls were subjected to both myocardial infarction (LCA ligation) and pressure-overload induced HF (transverse aortic constriction). mNCX-Tg mice displayed preserved LV function, structure and a reduction in HF indices in both models. For the first time we show that mNCX is essential for
m
Ca
2+
efflux in cardiomyocytes and that mNCX represents a novel therapeutic target in HF.
Abstract 95: Pharmacological Characterization of Adenosine Effects on Spontaneously Beating Human-IPSC-Derived Cardiomyocytes
Authors: Imredy John P , Balasubramanian Bharathi , Lis Edward V , Lagrutta Armando , Sannajust Frederick
Published: July 2021

Purified human-Induced Pluripotent Stem Cell-derived cardiomyocytes (hIPSC-CMs: iCells®, Cellular Dynamics) form electrically coupled, spontaneously beating cell monolayers (syncytia) upon plating and continuous culture. In this study, we investigate the pharmacology of the hIPSC-CM adenosine (ADO) response via the use of selective A1 and A2A-receptor antagonists and equilibrative nucleoside transporter type-1 (ENT1) inhibitors. After a 14-day culture in 96-well impedance electrode-plates (ACEA Biosciences), the spontaneous iCell beating rates are highly stable from well to well, with a mean beat period of 1.7 sec at 37°C, and a coefficient of variation (CV) < 1% . Extracellularly applied ADO inhibits the spontaneous beating rate of iCells with an IC
50
= 2.2 μM, and a saturation value of near 50% inhibition. ADO also destabilizes the beat period (CV EC
50
= 1.2 μM) with a maximum CV of 20% at saturating ADO concentrations. The ADO IC
50
is shifted to 248 μM upon pre-incubation of the iCells with 1 μM of an A1-selective antagonist DPCPX (A1 K
i
=3.9 nM), but not the A2A-selective antagonist ZM241385 (A2A K
i
=1nM). The duration of the depressive effect of ADO on beating rate is transient, even at super-saturating ADO concentrations, and depends on the well ADO concentration. The ENT1 inhibitor Draflazine (ENT1 pK
i
=9.5) pre-applied at 0.1 μM increases ADO sensitivity by 4-fold one hour after ADO application. Moreover, at any given ADO concentration, the duration of beating rate depression can be prolonged in a concentration-dependent manner by ENT1 inhibitors, with the prolongation-response curve nearly matching the ENT1 IC
50
s for tested ENT1 inhibitors (Draflazine, NBTI, Dilazep, and Dipyridamole). These findings suggest that the pool of ADO in the test well is cleared by uptake via endogenous ENT1 and support the role of equilibrative ADO uptake in limiting the duration of action of ADO on A1 receptors in cardiomyocytes. We are extending our investigation to the downstream target of A1 receptor activity, and the putative role of IK
ACH/ADO
as the underlying mechanism of beat rate slowing and irregularity. In conclusion, the hIPSC-CM model provides a useful model for investigation of ADO pharmacology of the heart.
Abstract 1:
Sprr2b
Drives Proliferation of Cardiac Fibroblasts by Relieving p53-mediated Cell Cycle Inhibition
Authors: Burke Ryan M , Lighthouse Janet K , Quijada Pearl J , Dirkx Ronald , Trembley Michael A , Small Eric M
Published: July 2021

Pathological cardiac remodeling is initially a compensatory attempt to increase cardiac output, but ultimately leads to the development of fibrosis, a form of scarring that contributes to heart failure (HF). In contrast, physiological cardiac remodeling in response to exercise is not associated with the development of fibrosis and typically remains compensatory. Understanding how cardiac fibroblasts (CF), the primary source of extracellular matrix in the heart, respond to pathological and physiological cues might lead to novel approaches to limit the maladaptive effects of pathological cardiac remodeling. We performed RNA sequencing to define genes that are differentially regulated in CF during physiological (swimming) or pathological (pressure overload) remodeling. This study revealed that cardiac expression of the
s
mall
pr
oline
r
ich 2b (
Sprr2b)
gene is restricted to CFs and is significantly elevated in disease and lost in exercise. We demonstrate that SPRR2B drives CF proliferation, but not myofibroblast differentiation, in response to pathological cues. SPRR2B facilitates an interaction between MDM2 and USP7, a nuclear deubiquitinase that leads to proteasomal degradation of p53. SPRR2B-USP7-MDM2 complex formation and p53 degradation is at least partially dependent upon phosphorylation of SPRR2B by Src-family NRTKs. SPRR2B thus relieves p53-mediated constraints on cell cycle progression in response to Src-dependent signaling, leading to CF accumulation. Importantly,
SPRR2B
expression is elevated in cardiac tissue from human HF patients relative to individuals without heart disease and positively correlates with a proliferative, activated gene expression profile in HF patient CF. Treatment of human HF fibroblasts with IGF-1/H
2
O
2
to mimic physiological cues significantly abrogated
SPRR2B
expression and increased expression of p53-dependent cell cycle checkpoint genes, which correlated with a less activated phenotype. Taken together, this study defines a unique tissue-specific role of
Sprr2b
in driving pathological CF cell cycle progression that may underlie the development of cardiac fibrosis.
Abstract 121: Development of Enzyme Replacement Therapy in Mammalian Models of Barth Syndrome
Authors: Dinca Ana , Chien Wei-Ming , Chin Michael
Published: July 2021

Barth Syndrome (BTHS) is caused by a single gene mutation in the mitochondrial transacylase, tafazzin (TAZ), which results in impaired lipid metabolism leading to dysfunction in highly energetic tissues such as the heart and skeletal muscle. TAZ remodels the signature mitochondrial phospholipid, cardiolipin (CL), which is responsible for providing support to the electron transport chain. BTHS patients suffer from growth deficiencies, cardiomyopathy, hypotonia and neutropenia. Currently, treatment for patients with BTHS is supportive, seeking to ameliorate rather than prevent heart problems, skeletal muscle problems and recurring infections. Protein therapy, on the other hand, might treat and even prevent cardiac, skeletal muscle as well as infection-related morbidities.

We designed a recombinant TAZ protein containing a cell penetrating peptide in its C-terminus, which enables the recombinant TAZ to penetrate cells and then treated TAZ-deficient cells with it. We tested the permeability of the recombinant protein by direct delivery to H9C2 cardiomyoblasts and found that the protein is successfully taken up by the cells. We have generated a CRISPR-mediated TAZ knock out cardiomyoblast cell line and we found that TAZ knock out cells show a decrease in oxygen consumption as compared to the wild type cells; this is consistent with data from BTHS patient-derived cells. We are using this cell line to assess the enzymatic activity of the delivered protein by conducting mitochondrial respiration measurements. We have also acquired a mouse model of BTHS and are testing the recombinant TAZ in vivo. Preliminary data shows an augmentation in oxygen consumption following treatment with TAZ.

These results indicate that the protein is able to reach the mitochondria, where it is enzymatically active and able to enhance respiration. As the protein is able to rescue respiration in cells in which tafazzin was absent, this suggests that our approach should not only be able to prevent onset of symptoms, but also rescue the phenotype in already affected tissues.
Abstract 126: Peroxisomal Proliferator Activated Receptor Alpha Overexpression in Hypertrophied Cardiomyocytes Restores Mitochondrial Integrity and Function
Authors: Sarkar Sagartirtha , Rana Santanu
Published: July 2021

Cardiac tissue engineering is an interdisciplinary field that engineers modulation of viable molecular milieu to restore, maintain or improve heart function. Myocardial workload (energy demand) and energy substrate availability (supply) are in continual flux to maintain specialized cellular processes, yet the heart has a limited capacity for substrate storage and utilization during pathophysiological conditions. Damage to heart muscle, acute or chronic, leads to dysregulation of cardiac metabolic processes associated with gradual but progressive decline in mitochondrial respiratory pathways resulting in diminished ATP production. The Peroxisome Proliferator Activated Receptor Alpha (
PPARα
) is known to regulate fatty acid to glucose metabolic balance as well as mitochondrial structural integrity. In this study, a non-canonical pathway of PPARα was analyzed by cardiomyocyte targeted
PPARα
overexpression during cardiac hypertrophy that showed significant downregulation in p53 acetylation as well as GSK3β activation levels. Targeted
PPARα
overexpression during hypertrophy resulted in restoration of mitochondrial structure and function along with significantly improved mitochondrial ROS generation and membrane potential. This is the first report of myocyte targeted
PPARα
overexpression in hypertrophied myocardium that results in an engineered heart with significantly improved function with increased muscle mitochondrial endurance and reduced mitochondrial apoptotic load, thus conferring a greater resistance to pathological stimuli within cardiac microenvironment.
Abstract 129: Smyd1 is an Essential Regulator of Adaptive Response to Glucose Starvation in Cardiomyocytes
Authors: Warren Junco S , Sabry Amira , Cawley Keiko , Makaju Aman , Szulik Marta W , Cox James , Nix David , Zaitsev Alexey V , Franklin Sarah
Published: July 2021

Smyd1, a muscle-specific histone methyltransferase, has been implicated in global metabolic remodeling in cardiac hypertrophy and failure. We previously showed that cardiac-specific ablation of Smyd1 in mice led to metabolic perturbations prior to overt cardiac dysfunction, suggesting that Smyd1 positively regulates cardiac metabolism. However, the role of Smyd1 in adaptive response to nutritional stress (NS) in cardiomyocytes is largely unknown. Here, we found that glucose deprivation-induced NS led to upregulation of Smyd1 in cultured rat neonatal ventricular myocytes (NRVMs) (FC=1.87, p<0.05), which was associated with the increased mRNA level of PGC-1α, a key regulator of mitochondrial energetics (FC=2.71, p<0.05). Strikingly, siRNA-mediated knockdown of Smyd1 (Smyd1-KD) in NRVM prior to glucose starvation led to extensive cell death not observed in control NRVMs (scrambled siRNA), suggesting that Smyd1 is required for cell survival in NS. To elucidate the mechanism how Smyd1 is involved in adaptive response to NS, we performed unbiased proteomic and metabolomic screening of Smyd1-KD NRVMs. Bioinformatic analysis of proteins and metabolites that were differentially expressed in Smyd1-KD NRVM revealed that oxidative phosphorylation was the most perturbed metabolic pathway in Smyd1-KD NRVMs, concomitant with a reduction in mitochondrial substrates (BCAAs; pyruvate; lactate, all p<0.05). Gene expression analyses using RT-PCR and RNA-seq in Smyd1-KD NRVMs further identified PGC-1α and Perm1 (the muscle-specific PGC-1α and ESRR induced regulator) as potential downstream targets of Smyd1 in regulation of cardiac energetics (FC=-1.92 and -1.66, respectively, both p<0.05). Consistent with downregulation of Perm1, the known Perm1-target genes (Tfb1m; Ctp1b; Glut4; Myl2) were all downregulated at the mRNA levels in Smyd1-KD NRVMs (p<0.05). Lastly, Smyd1-KD NRVMs exhibited accelerated loss of mitochondrial membrane potential during hypoxia, revealing an increased vulnerability to metabolic stress. Taken together, these results show that Smyd1 is an essential regulator of adaptive response and cell survival during metabolic insults, presumably through regulating PGC-1α/Perm1 gene expression.
Abstract 146: Adenosine Production by Biomaterial-supported Mesenchymal Stromal Cells Reduces the Innate Inflammatory Response in Myocardial Ischemia/Reperfusion Injury
Authors: Shin Eric Y , Wang Lanfang , Zemskova Marina , Deppen Juline , Xu Kai , Garcia Andrés J , Tirouvanziam Rabindra , Levit Rebecca D
Published: July 2021

Rationale:
During myocardial ischemia/reperfusion (MI/R) injury there is excessive release of immunogenic metabolites that activate cells of the innate immune system. These metabolites include adenine nucleotides such as adenosine triphosphate (ATP) and adenosine monophosphate (AMP) which up-regulate chemotaxis, migration, and effector function of early infiltrating inflammatory cells. These cells subsequently drive further tissue devitalization. Mesenchymal stromal cells (MSCs) are a potential treatment modality for MI/R due to their powerful anti-inflammatory capabilities; however, the manner in which they regulate the acute inflammatory milieu requires further elucidation. Surface membrane ecto-5’-nucleotidase CD73 may play a critical role in inflammatory regulation by converting pro-inflammatory AMP to anti-inflammatory adenosine (ADO). We hypothesized that the MSC-mediated conversion of AMP into ADO reduces inflammation in early MI/R, favoring a micro-environment that attenuates excessive innate immune cell activation and facilitates earlier cardiac recovery.

Objective:
To determine the contribution of MSC-mediated production of ADO in regulating the innate inflammatory response following MI/R.

Methods and Results:
Adult rats were subjected to 30 minutes of MI/R injury. MSCs were encapsulated within a hydrogel vehicle and implanted onto the myocardium. A subset of MSCs were treated with the CD73 inhibitor, α,β-methylene adenosine diphosphate, prior to implantation. Using liquid chromatography/mass spectrometry, we found MSCs increase myocardial ADO availability following injury via CD73 activity. We also demonstrated that MSCs reduce innate immune cell infiltration as measured by flow cytometry and hydrogen peroxide formation as measured by Amplex Red assay. These effects were also dependent on MSC-mediated CD73 activity. Furthermore, through echocardiography we found CD73 activity on MSCs was critical to optimal protection of cardiac function following MI/R injury.

Conclusion:
MSC-mediated conversion of AMP to ADO by CD73 ecto-5’-nucleotidase activity exerts a powerful anti-inflammatory effect following MI/R injury.
Abstract 15: Glucose-Mediated Remodeling of Cardiac DNA Methylation
Authors: Pepin Mark E , Crossman David K , Barchue Joseph P , Pamboukian Salpy V , Pogwizd Steven M , Wende Adam R
Published: July 2021

To identify the role of glucose in the development of diabetic cardiomyopathy, we had directly assessed glucose delivery to the intact heart on alterations of DNA methylation and gene expression using both an inducible heart-specific transgene (glucose transporter 4; mG4H) and streptozotocin-induced diabetes (STZ) mouse models. We aimed to determine whether long-lasting diabetic complications arise from prior transient exposure to hyperglycemia via a process termed “glycemic memory.” We had identified DNA methylation changes associated with significant gene expression regulation. Comparing our results from STZ, mG4H, and the modifications which persist following transgene silencing, we now provide evidence for cardiac DNA methylation as a persistent epigenetic mark contributing to glycemic memory. To begin to determine which changes contribute to human heart failure, we measured both RNA transcript levels and whole-genome DNA methylation in heart failure biopsy samples (n = 12) from male patients collected at left ventricular assist device placement using RNA-sequencing and Methylation450 assay, respectively. We hypothesized that epigenetic changes such as DNA methylation distinguish between heart failure etiologies. Our findings demonstrated that type 2 diabetic heart failure patients (n = 6) had an overall signature of hypomethylation, whereas patients listed as ischemic (n = 5) had a distinct hypermethylation signature for regulated transcripts. The focus of this initial analysis was on promoter-associated CpG islands with inverse changes in gene transcript levels, from which diabetes (14 genes; e.g. IGFBP4) and ischemic (12 genes; e.g. PFKFB3) specific targets emerged with significant regulation of both measures. By combining our mouse and human molecular analyses, we provide evidence that diabetes mellitus governs direct regulation of cellular function by DNA methylation and the corresponding gene expression in diabetic mouse and human hearts. Importantly, many of the changes seen in either mouse type 1 diabetes or human type 2 diabetes were similar supporting a consistent mechanism of regulation. These studies are some of the first steps at defining mechanisms of epigenetic regulation in diabetic cardiomyopathy.
Abstract 169: miR-21-5p Regulation of Lipid Content and Peroxidation in H9C2 Cells
Authors: Chuppa Sandra , Kriegel Alison J
Published: July 2021

Cardiovascular pathologies are the leading single cause of death in chronic kidney disease (CKD) patients. We have found that the 5/6 nephrectomy model of CKD leads to an upregulation of miR-21-5p in the left ventricle 7 weeks after surgery, targeting peroxisome proliferator-activated receptor alpha (PPARα). PPARα is a regulator of fatty acid uptake and metabolism. In our model we find that suppression of miR-21-5p alters the expression of numerous genes involved with fatty acid oxidation and glycolysis, presumably through its regulatory action on PPARα and/or additional targets. We also find that 5/6Nx rats exhibit dyslipidemia and increased left ventricular lipid content at this time. In this study we evaluated the potential for knockdown or overexpression of miR-21-5p to regulate lipid content and peroxidation in H9C2 cells. Cells were transfected with anti-miR-21-5p (40nM), pre-miR-21-5p (20nM) or appropriate scrambled oligonucleotide controls. After 24 hours medium was changed and half of the cells from each transfection group were treated with lipid (0.66 mM oleic acid and 0.33 mM palmitic acid) for 48 hours (n=6/treatment group for each set of experiments). Lipid content, measured by AdipoRed assay (Lonza) was significantly increased with lipid treatment (nearly two-fold). Overexpression of miR-21-5p significantly attenuated this increase (228.0 ± 9.7 vs. 198.2 ± 8.9% of untreated control), while suppression of miR-21-5p augmented lipid content (235.8 ± 11.2 vs. 328.1 ± 12.3% of untreated control). These results were supported by imaging of Oil Red O stained cells. We found that the abundance of malondialdehyde (MDA), a product of lipid peroxidation, was significantly increased in response to lipid treatment. Overexpression of miR-21-5p reduced MDA content in untreated and lipid treated cells, suggesting that miR-21-5p reduces oxidative stress. Suppression of miR-21-5p had no effect on MDA levels. These results indicate that overexpression of miR-21-5p attenuates both lipid content and lipid peroxidation in H9C2 cells. Ongoing studies aimed at evaluation of alterations in fatty acid oxidation and oxidative stress will further aid in determining the functional impact of miR-21-5p on associated pathways in cardiac tissue.
Abstract 195: Engineering Shear-thinning Injectable Biomimetic Hydrogel to Repair Damaged Cardiac Tissue
Authors: Paul Arghya , Waters Renae , Pacelli Settimio , Chakravarti Aparna
Published: July 2021

Rationale:
In acute myocardial infarction (AMI), prognosis and mortality rate are closely related to the infarct size and the progression of post-infarction cardiac failure. Delivery of stem cell derived secretome (miR, growth factor, exosome) has presented a new approach for the treatment of AMI. However, protection of bioactivity of delivered secretome and its retention at target site remains a challenge that can lead to sub-optimal results.

Methods and Results:
Here, we present a shear-thinning nanocomposite tissue adhesive hydrogels composed of synthetic 2% silicate nanoplatelets and 5% gelatin as injectable platform for effectively deliver adipose stem cell-derived secretome. This nanogel forms a highly crosslinked gel which could sustain high strain and recover its original stiffness once the strain is removed. This property is essential to prevent the material from flowing out of the therapeutic site once injected and thus enabling longer retention of the secretome. We used a novel microfluidic technology to harness high concentration secretome from stem cell spheroids before loading it to the hydrogel. The hydrogel-secretome (HS) demonstrated significantly enhanced in vitro angiogenic activity and cardioprotection. We hypothesized that the HS might promote angiogenesis cooperatively with the delivered secretome. To evaluate our hypothesis, we employed a rat AMI model and injectable HS gel to the heart. A significant increase in capillary density and reduction in infarct sizes were noted in the infarcted hearts with HS treatment compared with ctrl infarcted hearts treated with saline, hydrogel or secretome only groups. Furthermore, the HS showed significantly higher cardiac performance in echocardiography (62.8±3 of ejection fraction vs 45.2±5 ctrl, P < 0.05, n=6) 3 weeks post MI. In combination with bioprinting and microscale technologies, we have also sucessfully fabricated an elastomeric scaffold with this HS gel that can be used as a cardiac patch.

Conclusion:
The combination of injectability, rapid mechanical recovery, cargo retention ability, bioprintability and ability to attenuate progression of cardiac dysfunction makes this HS gel a versatile platform for cardiac biotherapeutics and tissue engineering applications.
Abstract 211: Myocardial Hypertrophy and Circulating RNAs
Authors: Shah Ravi V , Ziegler Olivia , Tanriverdi Kahraman , Rong Jian , Larson Martin , Pico Alexander , Levy Daniel , Vasan Ramachandran S , Das Saumya V , Freedman Jane
Published: July 2021

While increased left ventricular mass (LVM) is strongly associated with incident heart failure (HF), events during transition from increased LVM to HF remain unclear. Extracellular non-coding RNAs (ex-RNAs) have been implicated in cardiac hypertrophy, though whether these ex-RNAs reflect important pathways in HF in humans is underexplored. In >2,000 individuals with concomitant M-mode echocardiography and ex-RNA measurements in the Framingham Heart Study, we found that lower circulating concentrations of three ex-RNAs—miR-20a-5p, miR-106b-5p, miR-17-5p—were associated with (1) greater LVM (+ one other pre-clinical phenotype, e.g., left atrial dimension or LVEDV) and (2) greater incident HF risk over a median follow-up 7.7 years (
Fig. A
). These 3 miRNAs were members of a tight cluster, regulating 883 mRNAs in common, associated with “hypertension” (OMIM) and biological process relevant to HF, including TGF-β signaling. We observed an increase in myocardial expression of these miRNAs during different phases of hypertrophy/HF development (
Fig. C, D
). Using gain and loss of function
in vitro
, our preliminary results suggest up-regulation of cardiomyocyte miR-106b expression abrogates expression of pathologic hypertrophy markers (ANP and BNP) during phenylephrine treatment, consistent with
in silico
results suggesting broad connections between miR-106b targets and natriuretic peptide signaling (
Fig. B, E-F
). These results provide translational evidence that circulating miRNAs associated with hypertrophy in patients may be protective in the transition from hypertrophy to HF at the molecular level.
Abstract 218: Nuclear Remodeling Following Mechanical Circulatory Support
Authors: Luo Jun , Farris Stephen , Helterline Deri , Stempien-Otero April
Published: July 2021

Rationale:
Cardiomyocytes increase DNA content in normal growth and in response to stress in humans by both increases in nuclear number and ploidy. This observation complicates the analysis of human cardiomyocyte proliferation as DNA content can increase in the absence of cytokinesis. Proliferation has been reported in cardiomyocytes following LVAD unloading which may represent a reversal of this process. However, cardiac recovery from LVAD is rare. Thus, we sought to analyze changes in cardiomyocyte nuclear characteristics for clues to this paradox.

Objective:
We used a novel technique-imaging flow cytometry-to determine changes in nuclear content to test the hypothesis that adult cardiomyocytes can complete cell cycle progression by mitosis after long-term hemodynamic unloading of the failing heart.

Methods and Results:
Cardiomyocytes were isolated from 8 subjects undergoing primary heart transplantation and 15 subjects following unloading with left ventricular assist device (LVAD, mean unloading time 13.7 ± 9.1 months). Myocyte size, nuclear number and size, DNA content (per cell and per nucleus) and the frequency of cell cycling markers were evaluated by imaging flow cytometry. Myocyte size and nuclear morphology was not significantly different between the groups. However, DNA content per nucleus was significantly decreased (P < 0.01) and the correlation between nuclear size and DNA content lost. The frequency of the cell cycle markers, Ki67 and phospho-histone3 (H3P) were not increased after hemodynamic unloading.

Conclusions:
Our data demonstrate that unloading of failing hearts with mechanical ventricular assist devices does not alter nucleation state of cardiomyocytes. However, unloading is associated with decreased DNA content of nuclei independent of nucleation state within the cell. As these changes were associated with a trend to decreased cell size but not increased cell cycle markers, they may represent a regression of hypertrophic nuclear remodeling.
Abstract 228:
FADS2
Regulates Cardiometabolic Risk Phenotypes in Mice
Authors: Chicco Adam J , Mulligan Christopher M , Le Catherine H , Routh Melissa A , Nemr Dina , Li Puma Lance C , Linde Peter E , Bouma Gerrit J , Regan Daniel P , Nakamura Manabu T , Moulton Karen S
Published: July 2021

Single nucleotide polymorphisms of the
FADS2
gene associate with cardiometabolic risk in humans. Additionally, serum fatty acid profiles reflecting hepatic hyperactivity of the
FADS2
gene product, delta-6 desaturase (D6D), correspond to cardiometabolic syndrome (CMS) phenotypes in humans and animal models. D6D catalyzes rate-limiting steps in essential polyunsaturated fatty acid (PUFA) metabolism, but its role in the pathogenesis of CMS has not been defined. In the present study, we employed pharmacological and genetic gain- and loss-of-function approaches to investigate the links between D6D activity and CMS phenotypes in mice. Transgenic overexpression (TG) of
FADS2
in normal (FVB) mice modestly increases hepatic D6D protein expression and serum PUFA product/precursor ratios reflecting greater enzyme activity
in vivo
.
FADS2
TG mice develop a mild, but progressive obesity and insulin resistance with age compared to WT mice, as well as elevated serum triglycerides and LDL/HDL and hepatic macrophage infiltration, but not hepatic steatosis. Global
FADS2
ablation prevents obesity/insulin resistance and hyperlipidemia induced by high-fat feeding in C57Bl/6J mice, but promotes severe hepatic steatosis. Pharmacological D6D inhibition
in vivo
with SC-26196 (100 mpk 4-8 weeks) ameliorates hepatic inflammation and glucose intolerance in
FADS2
TG mice and leptin-deficient (
ob
) mice, and prevents severe hyperlipidemia and atherosclerosis in
ldlr
-/-
mice fed an atherogenic diet; despite augmenting hepatic steatosis in all cases. Tissue phospholipid analyses across these models revealed consistent positive relationships between D6D activity, pro-inflammatory eicosanoid accumulation, and a higher phosphatdiylcholine/phosphatidylethanolamine (PC/PE) ratio previously linked to increased hepatic VLDL synthesis and release. These studies establish an important role of D6D activity in the development of CMS and inflammation, and reveal novels links with tissue phospholipid class distribution and metabolism relevant to the development an atherogenic serum lipid profile, hepatic lipid homeostasis, and perhaps other aspects of cardiovascular risk currently under investigation in our laboratory.
Abstract 238: S-nitrosoglutathione Reductase (GSNOR) Plays a Critical Role in Placental Vascularization Working Through the VEGF-NO Pathway
Authors: Kulandavelu Shathiyah , Bellio Michael A , Fritsch Julia , Balkan Wayne , Hare Joshua M
Published: July 2021

Introduction:
Preeclampsia (PE), a leading cause of maternal and fetal mortality and morbidity, is characterized by increased levels of reactive oxygen species (ROS) and S-nitrosylated protein, and decreased levels of the antioxidant, ascorbate (Asc), which is required for the release of nitric oxide (NO) from nitrosylated proteins. Mice lacking S-nitrosoglutathione reductase (GSNOR
–/–
), a denitrosylase that regulates protein S-nitrosylation, exhibit a PE-like phenotype including maternal hypertension, cardiac concentric hypertrophy and impaired placental vascularization. We hypothesized that impaired placental vascularization, one of the primary causes of preeclampsia is mediated by alteration in S-nitrosylation of the VEGF-NO pathway, and ascorbate treatment rescues this pathologic phenotype.

Methods:
Pregnant GSNOR
–/–
and control (WT) mice (n=5-7) were studied at late pregnancy (day 17.5). Ascorbate was provided in drinking water beginning at day 0.5. Fetoplacental capillary density was determined from isolectin staining and reactive nitrosative stress determined from nitrotyrosine staining in placental sections. S-nitrosylation of VEGF was determined using SNO-Rac and eNOS levels by Western blot analysis.

Results:
Fetoplacental capillary density was reduced 19% in GSNOR
–/–
fetuses compared to WT (P<0.001). GSNOR
–/–
placentas exhibited higher nitrotyrosine staining than WT placentas, indicating the presence of nitrosative stress. These increases were associated with reduced level of eNOS protein (P<0.05) and decreased S-nitrosylation of VEGF (P<0.05) in the GSNOR
–/–
placentas as compared to WT. Ascorbate treatment decreased nitrotyrosine staining, and increased fetoplacental capillary density ~10% (P<0.001), eNOS protein levels (P<0.05) and S-nitrosylation of VEGF (P<0.05) in the GSNOR
–/–
placentas as compared to WT.

Conclusion:
These findings suggest that GSNOR plays an essential role in promoting placental vascularization in part working through the VEGF-NO pathway. Ascorbate treatment rescued the nitrosative stress and improved placental vascularization, suggesting that it can be used therapeutically to treat or prevent preeclampsia.
Abstract 249: Chronic Neuregulin-1β Treatment Mitigates the Progression of Post-myocardial Infarction Heart Failure in the Setting of Type 1 Diabetes Mellitus
Authors: Gupte Manisha , Lal Hind , Ahmad Firdos , Zhong Lin , Sawyer Douglas B , Hill Michael F
Published: July 2021

Aim:
Neuregulin-1β (NRG-1β), a growth factor critical for cardiac development as well as maintenance of heart function after injury has been shown to significantly improve heart function in preclinical rodent models. Importantly, number of studies are ongoing to test the efficacy of NRG-1β as a treatment for patients with chronic heart failure. However, the efficacy of recombinant NRG-1β in a typ1 diabetic model of heart failure due to myocardial infarction (MI) has not been investigated. The aim of the present study was to determine the efficacy of exogenous NRG-1β to improve residual cardiac function after MI in type1 diabetic rats.

Methods and Results:
Sprague Dawley rats were induced type 1 diabetes by a single injection of streptozotocin (STZ) (65 mg/kg). Two weeks after induction of type 1 diabetes, rats underwent left coronary artery ligation to induce MI. STZ-diabetic rats were treated with saline or NRG-1β (100 ug/kg) twice a week for 7 weeks, starting two weeks prior to experimental MI. Residual left ventricular (LV) function was significantly greater in the NRG-1β-treated STZ-diabetic MI group compared to the vehicle-treated STZ-diabetic MI group 5 weeks after MI as assessed by high-resolution echocardiography. Furthermore, NRG-1β treatment in STZ-diabetic MI rats reduced myocardial fibrosis and apoptosis as well as decreased gene expression of key oxidant-producing enzymes.

Conclusion:
This study demonstrates that augmentation of NRG-1β signaling in STZ-diabetic post-MI rats via therapy with exogenous recombinant NRG-1β will alleviate subsequent HF through improvements in residual LV function via protection against adverse remodeling and apoptosis.
Abstract 250: Canonical Transient Receptor Potential Channel 6 Ameliorates Increased Cardiac S-nitrosylation in Duchenne Muscular Dystrophy
Authors: Kim Grace E , Chung Heaseung S , Holewinski Ronald J , Zhu Guangshuo , Venkatraman Vidya , Bedja Djahida , Van Eyk Jennifer E , Kass David A
Published: July 2021

Duchenne muscular dystrophy (DMD) is an X-linked disorder that markedly weakens skeletal and cardiac muscle to cause early death. Its elimination of dystrophin disrupts nitric oxide (NO) signaling and amplifies intracellular Ca
2+
responses to mechanical load. We have shown the latter is linked to hyperstimulated transient receptor potential canonical 6 (TRPC6) cation channels. As Ca
2+
also activates NO synthase, we hypothesized TRPC6 couples to redox-dependent nitrosative stress to broadly impact protein S-nitrosylation (SNO). Using an unbiased, dual-labeling proteomic strategy we identified 1276 SNO sites on 491 proteins in DMD hearts (dystrophin/utrophin
+/-
), of which 102 sites among 69 proteins were unique to DMD. Many of the targeted proteins were mitochondrial or metabolic regulators and sarcomere proteins - including titin, myosin binding protein-C, α-myosin heavy-chain, and tropomyosin α1 - that were hyper-nitrosylated. A key redox regulator peroxiredoxin1 was also hyper-nitrosylated at Cys173, a site previously shown to be a requisite regulator of its dimerization and enzymatic activity. DMD mice were then crossed into a Trpc6
-/-
background, and proteomic analysis now found 70% of SNO targeted residues in DMD were reversed towards normal (p<0.01, χ
2
). Trpc6 deletion improved left ventricular dilation (13.7±1.2mm, 22.4±3.9mm, 15.3±2.3mm; p<0.01), fractional shortening (58.5±0.5%, 50.3±1.0%, 59.6±1.2%, p<0.001), and fibrosis (2.3±0.9%, 6.2±0.9%, 3.7±0.6%; p<0.0001) in WT, DMD and DMD-TRPC6
-/-
respectively (1-way ANOVA), and reversed pro-fibrotic gene activation (connective tissue growth factor, fibronectin1 and osteopontin). These results provide the first broad-based SNO analysis of the DMD heart, and support linkage between abnormal calcium via TRPC6, nitrosative stress and cardiac disease.
Abstract 265: Deficiency of Mir-1954 Promotes Cardiac Remodeling
Authors: Gupta Sudhiranjan , Takano Ana P , Guleria Rakeshwar
Published: July 2021

Cardiac remodeling due to hemodynamic overload is associated with significant morbidity and mortality. In response to stress, cardiomyocyte (CM) become hypertrophied whereas cardiac fibroblasts convert into myofibroblasts. The phenomenon leads to the development of cardiac hypertrophy, fibrosis and impair cardiac function. Previously, we have shown the pivotal role of miRNA (a new class of post-transcriptional regulators) in cardiac remodeling, but, loss of miRNA contributing to the onset of cardiac remodeling remains elusive. Using next generation miRNA sequencing, we discovered a panel of novel dysregulated miRNAs from read-data, secondary structure and miRPara classification score analysis in wild-type mice (WT) infused with Angiotensin II (Ang II). Among them, one was identified as miR-1954, a novel miRNA which was significantly reduced in Ang II-infusion and transverse aortic constriction (TAC). Following an unbiased approach, we confirmed that Sp1-Gata4-Col I-Tsp1-axis is the bona-fide targets. Our hypothesis is that deficiency of miR-1954 exacerbates cardiac remodeling leading to hypertrophy and fibrosis through paracrine mechanism; and overexpression of miR-1954 mitigates the cardiac damage and abrogates remodeling by modulating Sp1-Gata4-Col I-Tsp1-axis. Our data demonstrated that depletion of miR-1954 in CM triggers hypertrophic response by modulating Sp1 and Gata4; releases soluble factors (Tgfβ1) that triggers cardiac fibroblasts proliferation; upregulation of thrombospondin 1 (Tsp1) and collagen I (Col I). Overexpression of miR-1954 in CM reverses these processes implicated a cellular cross-talk. Cardiac-specific overexpression of pre-miR-1954 transgenic mice (miR-1954 Tg) showed reduced cardiac mass and improved function compared to WT littermate after Ang II treatment. Inhibition of miR-1954 by locked nucleic acid of anti-miR-1954 exacerbates cardiac hypertrophy and fibrosis. Our findings provide evidence that loss of miR-1954 promotes cardiac remodeling by targeting Sp1-Gata4-Col I-Tsp1-axis and, overexpression of miR-1954 reverses the process. We conclude that miR-1954 could be a triggering factor in cardiac remodeling and providing new mechanistic information for therapeutic benefit.
Abstract 268: Strain-specific Cardiac Fibroblast Response to Isoproterenol-induced Cardiac Fibrosis
Authors: Park Shuin , Ranjbarvaziri Sara , Lay Fides , Zhao Peng , Lusis Aldons J , Ardehali Reza
Published: July 2021

Fibroblasts are a heterogeneous population of cells that function within the injury response mechanisms across various tissues. Despite their importance in pathophysiology, the effects of different genetic backgrounds on fibroblast contribution to the development of disease has yet to be addressed. It has previously been shown that mice in the Hybrid Mouse Diversity Panel, which consists of 110 inbred mouse strains, display a spectrum in severity of cardiac fibrosis in response to chronic treatment of isoproterenol (ISO). Here, we characterized cardiac fibroblasts (CFbs) from three different mouse strains (C57BL/6J, C3H/HeJ, and KK/HIJ) which exhibited varying degrees of fibrosis after ISO treatment. The select strains of mice underwent sham or ISO treatment via intraperitoneally-implanted osmotic pumps for 21 days. Masson’s Trichrome staining showed significant differences in fibrosis in response to ISO, with KK/HIJ mice demonstrating the highest levels, C3H/HeJ exhibiting milder levels, and C57BL/6J demonstrating little to no fibrosis. When CFbs were isolated and cultured from each strain, the cells demonstrated similar traits at the basal level but responded to ISO stimuli in a strain-specific manner. Likewise, CFbs demonstrated differential behavior and gene expression
in vivo
in response to ISO. ISO treatment caused CFbs to proliferate similarly across all strains, however, immunofluorescence staining showed differential levels of CFb activation. Additionally, RNA-sequencing analysis revealed unique gene expression profiles of all three strains upon ISO treatment. Our study depicts the phenotypic heterogeneity of CFbs across different strains of mice and our results suggest that ISO-induced cardiac fibrosis is a complex process that is independent of fibroblast proliferation and is mainly driven by the activation/inhibition of genes involved in pro-fibrotic pathways.
Abstract 279: Mitochondrial Bioenergetic Signaling Drives Myofibroblast Transdifferentiation
Authors: Lombardi Alyssa A , Arif Ehtesham , Luongo Timothy S , Elrod John W
Published: July 2021

When the heart is injured, quiescent fibroblasts differentiate into contractile, synthetic myofibroblasts. Initially fibrosis is reparative, but when chronic it becomes maladaptive and contributes to HF. Intracellular Ca
2+
(
i
Ca
2+
) signaling is reported to be necessary for myofibroblast transdifferentiation yet the role of mitochondrial Ca
2+
(
m
Ca
2+
) exchange has not been explored. The
Mcu
gene encodes the channel-forming subunit of the
m
Ca
2+
uniporter channel (MCUc) and is required for acute
m
Ca
2+
uptake. To examine the contribution of
m
Ca
2+
in cardiac fibrosis, we generated conditional, fibroblast-specific knockout mice by crossbreeding
Mcu
fl/fl
mice with Col1a2-CreERT mice (Col1a2-
Mcu
-/-
), permitting tamoxifen-inducible gene deletion in adult mice. Col1a2-
Mcu
-/-
mice and controls were subjected to ligation of the left coronary artery and cardiac function was examined by echocardiography. Loss of fibroblast
Mcu
worsened LV function and increased fibrosis, as evaluated by Mason’s trichrome staining and qPCR analysis of fibrotic gene expression. To examine the cellular mechanisms responsible for the increased fibrosis we isolated mouse embryonic fibroblasts (MEFs) from
Mcu
fl/fl
mice and deleted
Mcu
with Cre-adenovirus. When challenged with pro-fibrotic ligands (TGF-β and AngII),
Mcu
-/-
MEFs exhibited decreased
m
Ca
2+
uptake and enhanced
i
Ca
2+
transient amplitude. Loss of
Mcu
promoted myofibroblast transdifferentiation: increased α-SMA expression and contractile function (gel retraction) and decreased migration and proliferation.
Mcu
-/-
MEFs were more glycolytic with increased phosphorylation (inactivation) of pyruvate dehydrogenase. Genetic activation of glycolysis with a
Pfk2
mutant in WT MEFs promoted myofibroblast differentiation. Conversely, genetic inhibition of glycolytic flux ablated the increased transdifferentiation observed in
Mcu
-/-
MEFs. Further, TGF-β and AngII altered the expression of regulatory MCUc components in WT MEFs. Our results suggest that alterations in
m
Ca
2+
uptake and bioenergetic pathways are necessary for myofibroblast transdifferentiation. Thus, energetic signaling represents a novel therapeutic target to impede HF progression and other progressive fibrotic diseases.