bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 42: In Vivo Differentiation of Epigenetically Reprogrammed Human Endothelial Progenitor Cells into Cardiomyocytes Enhances Functional and Anatomical Postinfarct Myocardial Repair
Authors: Thal Melissa A , Krishnamurthy Prasanna , Mackie Alexander R , Hoxha Eneda , Lambers Erin , Verma Suresh , Ramirez Veronica , Kishore Raj 
Published: July 2021

Currently, bone marrow derived endothelial progenitor cells (human CD34+ cells, EPC) are being used clinically to improve vascularization in patients with ischemic heart disease. While it is generally accepted that CD34+ cells predominantly work through a paracrine mechanism, there exists no convincing evidence that these cells trans-differentiate into functional cardiomyocytes (CMC). Since ischemic heart disease leads to substantial loss of CMC, improving cardiomyogenic plasticity of an existing autologous cell therapy is of obvious import. EPC and CMC both differentiate from a common mesodermal progenitor however; during EC-specific lineage differentiation, CMC specific genes are epigenetically silenced. We hypothesized that reprogramming of CD34+ cells using small molecules targeting key epigenetic repressive marks may recapitulate their cardiomyogenic potential.
Human CD34+ EPCs were treated with inhibitors of histone deacetylases (valproic acid) for 24 hours followed by an additional 24 hours with the DNA methyltransferase inhibitor (5-Azacytidine). This forty-eight hour treatment led to the reactivation of pluripotency associated and CMC specific mRNA expression while EC specific gene expression was maintained. Intra-myocardial transplantation of a sub-therapeutic dose of reprogrammed CD34+ cells in an acute myocardial infarction mouse model showed significant improvement in LV function compared to the same number of control CD34+ cells that are therapeutically equivalent to no treatment at all. This was histologically supported by de novo CMC differentiation. In addition to increased cardiomyogenic plasticity, drug treatment also enhanced the inherent therapeutic capacity of the CD34+ cells as shown by reduced fibrosis, increased capillary density, increased proliferation, increased cell survival and increased secretion of angiogenic factors.
Taken together, our results suggest that epigenetically reprogrammed CD34+ cells are “super-CD34+ cells” that have an enhanced paracrine effect, display a more plastic phenotype and improve post-infarct cardiac repair by both neo-cardiomyogenesis and neovascularization.
Abstract 092: Transgenic Expression of Heat Shock Protein 60 Results in Neonatal Death and Adult Heart Failure
Authors: Liu Shan-Wen , Chen Tsung-Hsien , Chen Yen-Ling , Chen Mei-Ru , Hsu Ching-Han , Lin Kurt M 
Published: July 2021

Heat shock proteins (HSPs) are known for their enhanced expression and pro-survival role in cells under noxious stress. Only recently was it discovered that aberrant expression of HSPs was linked to the onset of cardiovascular diseases. HSP60, originally identified as one of the mitochondrial chaperonins, was shown involved in autoimmune, metabolic, and cardiovascular diseases, but the roles of HSP60 remain unclear in part due to the lack of HSP60 transgenic mouse models. This study aims to investigate the outcome of expressing HSP60 in cardiac tissues to elucidate the role of HSP60 in initiating apoptotic cardiomyopathy.
By using a conditional HSP60 transgenic mouse line (G-Lox-HSP60) and a Cre mouse line, human HSP60 expression in most mouse tissues, driven by the ubiquitous CAG promoter, was achieved. The ubiquitous expression of HSP60 was found to cause neonatal death during postnatal day one. In HSP60 expressing newborn mice, hemorrhagic cardiac myopathy was found, and some of these mice exhibited atrium septal defects. A greatly increased rate of apoptosis was found in cardiac tissues with HSP60 overexpression, suggesting a pro-apoptotic role for HSP60. A tamoxifen-inducible Cre mouse (Myh6-Cre/ESR) was used to induce HSP60 expression in the adult mouse heart, and the results showed that dilated heart failure developed within 8 weeks after the induction of HSP60 expression. By employing an isolated perfusion model, the basal contractility was found to be significantly enhanced by HSP60, and a cardiac protection against simulated ischemia and reperfusion injury was found in mice with the HSP60 induction for 4 weeks. Both increased contractility and cardiac protection were lost in mice 6 to 8 weeks after HSP60 induction.
Our results showed that HSP60 expression led to heart failure in neonatal mice and likely involved developmental defects and induced apoptosis. While expressing HSP60 in adult cardiac tissues only temporally improved cardiac function and exerted cardiac stress protection, hypertrophy and heart failure eventually occured in these mice. The G-Lox-HSP60 model that achieves expression of HSP60 in an inducible and tissue-specific manner will be useful in studies to investigate the roles and molecular details of HSP60 in various diseases.
Abstract 205: Recovery from Ischemia-Reperfusion Injury Is Metabolic Substrate-Dependent
Authors: Nadtochiy Sergiy M , Brookes Paul S 
Published: July 2021

Introduction:
The adult heart utilizes mostly fat for energy production, with adaptation to different fuels (“metabolic plasticity”) being a hallmark of the healthy heart. However, metabolic maladaptation is known to occur in heart failure. As such, the ability of the heart to metabolize specific substrates could impact the outcome of pathological insults, such as ischemia-reperfusion (IR) injury. The aim of this study was to develop a system whereby adult mouse cardiomyocytes (AMC) subjected to IR injury could be supplied with different fuels, and metabolism measured in real-time.

Methods:
AMC were divided in 3 groups, supplied either with glucose (GLU, 5mM), palmitate/fat free BSA (FAT, 100µM) or GLU+FAT. A previously developed method for in-vitro IR injury using a Seahorse XF24 [1], was adopted for ACM. IR comprised 60 min. ischemia and 60 min. reperfusion, and additional metabolic parameters were measured separately using mitochondrial inhibitors and uncouplers [2]. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were simultaneously measured during the IR protocol, followed by a cell death assay.

Results:
FAT cells showed higher baseline OCR and lower ECAR rates compare to GLU cells, although uncoupled OCR was lower in FAT group, suggesting a lower metabolic reserve capacity for cells respiring on fat. Upon IR, the drop in pH was significantly greater in GLU compare to FAT, indicating faster lactate production. During reperfusion, both OCR and ECAR recovered to pre-ischemic levels in GLU cells but failed to do so in FAT cells. Post-IR cell death was significantly higher in FAT vs. GLU. Surprisingly, GLU+FAT (modeling a “physiologic” substrate mix) replicated the same metabolic profile and cell death as GLU.

Conclusions:
(i) AMC had better recovery from IR injury using glucose as fuel. (ii) Lower cell viability in FAT (vs. GLU) correlated with smaller metabolic reserve capacity and with a smaller pH drop during ischemia. This is consistent with a known protective role for acidification during IR injury. (iii) Mixed substrates (GLU+FAT) gave a similar response to glucose alone, suggesting that fat may not be toxic, rather glucose is protective, in IR injury.

[1] Circ Res. (2012), 110. 948-57.
[2] J Vis Exp. (2010), 46. pii: 2511.
Abstract 303: HDL Mimetic Peptide 4F Rescues Pre-existing Pulmonary Hypertension via MicroRNA 193-3p
Authors: Sharma Salil , Umar Soban , Wong Gabe , Mai Denise , Navab Mohamad , Reddy Srinu , Eghbali Mansoureh 
Published: July 2021

Pulmonary hypertension (PH) is a chronic lung disease associated with severe vascular disorders leading to right ventricular(RV) failure. An HDL mimetic peptide, 4F, has been shown to be effective for the treatment of atherosclerosis and a number of inflammatory disorders. Here, we explored whether 4F can rescue advanced PH by controlling the expression of specific microRNAs (miRs). PH was induced in rats by a single injection of monocrotaline (MCT, 60mg/kg,
s.c
.) or by placing mice in hypoxia chamber(O2≤10%) for 21 days. MCT-rats or hypoxic mice received 4F therapy (50mg/kg/day,
s.c
.,days 21-30 in MCT model and days 14-21 in hypoxia model). We performed microRNA microarray analysis (non-affymetrix) in lung tissues of CTRL, PH and 4F-rescued groups. OE of miR193 was achieved by intratracheal instillation of 20nM dose at days 16, 21 and 26 in MCT model or at days 14 and 18 in hypoxia model.

4F therapy starting after the establishment of PH in both MCT and hypoxia models improved significantly RV pressure (RVP) and RV hypertrophy index (RVP=46±3 vs RVP=74±1 mmHg in PH; RV/LV+IVS=0.38±0.02 vs RV/(LV+IVS)=0.68±0.05 in PH, p<0.05 vs PH and in hypoxia model RVP=22±3.8 vs. 36.91±5.74 in PH and 20.93±2.52mmHg in ctrl, p<0.05 vs PH ). Microarray and qPCR showed downregulation of miR-193 by ~3 fold in MCT model. 4F therapy normalized miR-193 to ctrl levels. MiR-193 OE in both MCT-rats and hypoxic-mice rescued PH (RVP=38±5.5mmHg, RV/LV+IVS=0.37±0.034 in MCT-rats and RVP=25.48±0.88mmHg in hypoxic-mice). Lung sections showed increased arteriolar muscularization and ox-LDL deposition in the PH group, prevented by miR-193 therapy. In vivo, OE of miR-193 suppressed transcription of in-silico targets ALOX5, a lipoxygenase; IGF1R, insulin-like growth factor 1 receptor and ARHGEF12, a Rho guanine nucleotide exchange factor and decreased human pulmonary artery smooth muscle cell (HPASMC) proliferation in vitro in the presence of serum or 12-HETE by >2 folds whereas miR-193 KD increased proliferation.
In conclusion, 4F rescues pre-existing severe PH by targeting genes associated with HETEs and HODEs production, inflammation and growth via inducing miR-193.
Abstract 351: Downregulation of Connexin43 by MicroRNA-130a Results in Cardiac Arrhythmias
Authors: Kim Gene , Osbourne Appledene , McSharry Saoirse , Broman Michael 
Published: July 2021

MicroRNAs (miRs) are endogenous ~22 nucleotide RNA molecules that are now recognized as critical regulators of diverse physiological and pathological processes; however, studies of miRs and arrhythmogenesis remain sparse. Connexin43 (Cx43), a major cardiac gap junction protein, is critical for maintaining normal cardiac electrical conduction. One aspect of arrhythmia pathogenesis that has elicited great interest has been changes in Cx43 expression. Computational analysis of messenger RNAs predicted Cx43 to be a target for miR-130a. We hypothesized that overexpression of miR-130a in adult cardiomyocytes would result in cardiac arrhythmias. To study this, we used an inducible (tetracycline-off) bigenic system to study miR-130a overexpression in the adult mouse heart.
12 weeks after induction of miR-130a, 100% (16/16) of transgenic mice demonstrated a loss of sinus rhythm and irregular ventricular activity suggestive of atrial fibrillation on surface electrocardiograms (ECG). Nonsustained ventricular tachycardia was also observed during ECG recordings. Atrial contractions could not be detected by pulse wave Doppler. Intracardiac electrocardiography confirmed the presence of atrial arrhythmias. Western blot analysis demonstrated a 90% reduction in Cx43 levels and immunofluorescent staining confirmed a near complete loss of Cx43 in transgenic compared to littermate control hearts (n=12). Morphology of the intercalated disc was evaluated by electron microscopy and showed a loss of gap junctions in transgenic hearts. To validate Cx43 as a direct target of miR-130a, we performed in vitro target assays in 3T3 fibroblasts, known to express miR-130a. Using a luciferase reporter fused to the 3’UTR of Cx43 compared to a control (SV40) 3’UTR, we found a 55% ± 6% reduction in luciferase activity with the Cx43 3’UTR (p<0.01, n=9). Addition of an antisense miR-130a inhibitor resulted in a dose dependent loss of inhibitory activity of the Cx43 3’UTR reporter, with no changes seen in the control vector.
In conclusion, we have identified an unappreciated role of miR-130a as a direct regulator of Cx43. Overexpression of miR-130a results in the development of a variety of important cardiac arrhythmias including atrial fibrillation and ventricular tachycardia.
Abstract 126: Pregnancy Hormones Induce An Increase Of Capillary Density In The Mouse Heart
Authors: Hesse Michael , Korzus Daniel , Thaben Kristina , Fleischmann Bernd K 
Published: July 2021

During pregnancy the maternal organism undergoes a variety of adaptive physiological changes. The cardiovascular system is strongly affected and its response is characterized by cardiac hypertrophy. The molecular mechanisms underlying these adaptations are still poorly understood. The recent postulation of resident organotypic stem cells in the cardiovascular system prompted us to explore the potential involvement of these cells and the related signaling cascades involved in this adaptation process in the heart.
We have analyzed cell proliferation using immunostainings in mouse hearts at different stages during pregnancy. The average percentage of proliferating cells in cardiac sections increased from virgin controls to gestational day 3 (GD3), peaked at GD14, and immediately stopped after delivery. Co-staining revealed that the majority of proliferating cells were fibroblasts and endothelial cells (ECs). The lack of cardiomyocyte proliferation was corroborated using BrdU pulse chase experiments analyzed in single cells upon Langendorff perfusion. The proliferation of fibroblasts and ECs reflects angiogenesis, which was underscored by an increase of capillary density, and extracellular matrix remodeling known to occur during pregnancy induced hypertrophy. To mimic the action of pregnancy hormones we implanted pellets with constant release of hormone for 21 days subcutaneously in ovariectomized mice. Capillary density was significantly increased in mice receiving progesterone-pellets alone or in combination with estrogen-pellets without induction of hypertrophy. Thus, increase of capillary density is directly induced by pregnancy hormones. Assessment of differentially expressed genes after hormone treatment revealed induction of angiogenetic factors and their receptors.
We are currently investigating the mechanisms underlying the pregnancy hormone-induced proliferation in cardiac ECs and fibroblasts.
Abstract 14: The Cytoskeletal Architecture of Myocytes Derived from Human Cardiac Progenitor Cells
Authors: Pasqualini Francesco , Di Sante Moises , Pereira João D , Anversa Piero , Rota Marcello , Leri Annarosa 
Published: July 2021

The stem cell antigen c-kit characterizes a heterogeneous pool of human cardiac progenitor cells (hCPCs) that exhibit a remarkable degree of regenerative potential and are currently employed in clinical trials. While this hCPC pool contains distinct subpopulations of c-kit+ cells that preferentially differentiate into muscular or vascular cardiac cells, we hypothesize that hCPCs may be coerced to specify only along the cardiomyogenic lineage by manipulating the Wnt/β-catenin pathway.
We report that pharmacological inhibition of the non-canonical Wnt pathway facilitated the commitment of more than >95% c-kit+ hCPCs to the cardiomyocyte lineage after 4 days in-vitro: this constitutes a substantially more homogeneous population than previously reported with dexamethasone treatment.
The hCPC-derived myocytes stained positive for Nkx2.5, a transcription factor that orchestrates cardiomyogenic differentiation, and for the contractile protein sarcomeric α-actin. To test if we could push the cells towards a more mature phenotype, we mimicked the cyclic modulation of the Wnt pathway observed during development. While activation of Wnt signaling resulted in widespread cell death and reduction in cell size, subsequent Wnt inhibition prompted the spared cells to proliferate. With this protocol, hCPC-derived myocytes increased in size and displayed more mature cytoskeletal architectures. In contrast with dexamethasone treated cells, where the localization of α-sarcomeric actinin is mostly diffuse in the cytoplasm, here we observed both Z-bodies and Z-disks like structures. The latter exhibited a periodicity of ~1.6 um and were clustered in larger, more aligned actin bundles. This finding suggests that the tension developed along these cytoskeletal components may play a role in the recruitment of sarcomeric proteins.
In conclusion, Wnt signaling inhibition in hCPCs may be sufficient to obtain a homogeneous population of cells with features of myocytes, characterized by improved cytoskeletal organization than dexamethasone treated cells and similar to that observed in myocytes derived from human induced pluripotent stem cells.
Abstract 220: Transcriptional Analysis Of Caveolin And Cavin In The Male And Female Ageing Mouse Heart Following Ischemic Stress
Authors: Kiessling Can J , Reichelt Melissa , Headrick John , Ashton Kevin 
Published: July 2021

Cardioprotection against infarction and dysfunction in the myocardium involves G-protein-coupled receptor signalling orchestrated by specialised membrane microdomains termed caveolae. The caveolin protein family consist of three subtypes: caveolin-1, −2 and −3 (Cav1-3) and are responsible for the formation of caveolae and hypothesized to orchestrate cardioprotective signalling. Caveolin-3 deficiency and overexpression has been shown to attenuate and restore cardioprotection, respectively. Recently, a family of four related proteins known as cavins (Cavin1-4) have been implicated as regulators of caveolae formation and function. The roles and expression distribution of the cavin family is currently unknown in cardiac tissue. In this study hearts were isolated from 8, 16, 32 and 48 week male and female mice and subjected to normoxic perfusion (80 min) or ischemic stress (20 min global ischemia, 60 min reperfusion). RT-qPCR was used to assess differential gene expression of caveolin and cavin subtypes across these ages in both sexes.
Decreased post-ischemic pressure development and increased LDH release were observed in 32 and 48 week old relative to 8 week old male hearts hearts, indicative of age-related loss of ischemic tolerance. Females showed greater tolerance to ischemia at 32 and 48 week old hearts when compared to male counterparts. In normoxic male 48 week old hearts, Cav1,-2,-3 and Cavin1 were significantly repressed, whilst post-ischemic male 48 week old hearts demonstrated significant repression of Cav3 and Cavin1 only. Normoxic female hearts showed no significant changes in caveolin and cavin transcript expression over the aging time course. However, post-ischemic female 48 week old hearts showing significant down-regulation of Cav3 only. Taken together, alterations in caveolin and cavin expression may contribute to the age-related loss of ischemic tolerance and G-protein-coupled receptor-mediated protection in aging male and female mice hearts.
Abstract 238: miR-222 is Necessary for Exercise-induced Cardiac Growth and Protects Against Pathological Cardiac Remodeling
Authors: Liu Xiaojun , Xiao Junjie , Zhu Han , Wei Xin , Platt Colin , Damilano Federico , Xiao Chunyang , Bezzerides Vassilios , Rosenzweig Anthony 
Published: July 2021

Exercise induces cardiac growth, protects against adverse remodeling, and may also induce a regenerative response. Since microRNAs (miRNA) play important roles in cardiovascular disease, we investigated their role in the cardiac exercise response.
We used the TaqMan rodent miRNA array to profile cardiac miRNA expressed at three weeks in two exercise models (swimming, running) compared to sedentary controls. Sixteen concordantly regulated miRNAs were identified and validated in both models, and examined for functional effects in neonatal rat ventricular cardiomyocytes (CMs). miR-222 was upregulated ~two-fold in both models and increased CM size (22%, p<0.01) and proliferation markers (EdU and Ki67, p<0.01). Bioinformatic and expression analyses identified four potential miR-222 targets (p27, Hipk1, Hipk2, and Hmbox-1) in CMs. These were confirmed as direct targets by luciferase assays, site-directed mutagenesis, and immunoblotting. siRNA knockdown (KD) of p27 or Hipk1 induced neonatal CM proliferation, while siRNA KD of Hmbox-1 increased CM size.
To examine miR-222’s role in vivo, LNA-antimiR-222 was injected via tail vein or subcutaneously and shown to reduce cardiac miR-222 levels to 1.5% (p<0.01). Untreated animals subjected to three weeks of swimming had the expected increase in heart size (15% in HW/TL, p<0.05), CM size (26%, p<0.05), and markers of CM proliferation (Ki67 and pHH3, p<0.05). Increases in heart and CM size were unaffected by control LNA-antimiR but completely blocked by LNA-antimiR-222, while CM proliferation markers decreased (60%, p<0.05). To see if miR-222 is sufficient to mediate the benefits of exercise, we made transgenic mice with cardiac-specific, regulated expression of miR-222 (Tg-miR-222). Tg-miR-222 have normal heart size and function at baseline. After ischemia-reperfusion injury (IRI), Tg-miR-222 had similar initial dysfunction but were protected against adverse remodeling over the next six weeks with better function (p<0.01), less cardiac fibrosis (68%, p<0.05), and increased CM proliferation markers (63%, p<0.05).

Conclusion:
Cardiac miR-222 is upregulated by exercise, necessary for exercise-induced cardiac growth, and protects against pathological cardiac remodeling after IRI.
Abstract 279: A Deletion In The N2A Region Of Titin Carried By Muscular Dystrophy With Myositis (mdm) Mice Severely Affects Skeletal Muscle, But Not The Heart
Authors: Lunde Ida G , Wakimoto Hiroko , Burke Michael A , Linke Wolfgang , Christensen Geir , Seidman Jonathan G , Seidman Christine E 
Published: July 2021

20% of dilated cardiomyopathy patients carry mutations in the giant protein titin. Mutations are predominant in A band but also occur in I band, a domain that regulates passive tension and myocyte signaling. A recessive mouse mutation in titin I band N2A region (mdm) causes early onset muscular dystrophy with myositis and death. We assessed cardiac morphology, function, and transcriptional profiles (RNAseq) in mdm mice.
Young homozygous mdm mice (n>6) have reduced body weight (7gms) vs. heterozygous (20gm) or WT (17gm) littermates, with severe skeletal muscle dystrophy. Four-week old homozygous mdm mice have higher left ventricular (LV): body weight ratios. Echocardiography revealed thinner LV posterior wall and septum (LVPWd and IVSd) and normal LV diameter (LVDd); when normalized for body weight, cardiac dimensions were increased compared to WT or heterozygous mdm mice. Fractional shortening was reduced in homozygous Mdm mice (35%) vs. WT (40-41%, p<0.01); histology showed neither overt pathology nor fibrosis. Titin gels showed lack of difference in cardiac titin isoform pattern, consistent with RNAseq, which showed the mdm titin transcript excluded exons 107 and 108, deleting in frame 48 amino acids. 240 transcripts (0.8%) were differentially expressed (fold change >1.5 and <0.75, p<0.001) in homozygous vs. heterozygous mdm hearts; ANP and BNP were mildly upregulated (2- and 1.2-fold). Altered transcripts participated in extracellular and immune signaling pathways. Among titin binding partners, only calpain-3 that interacts with N2A was changed (0.6-fold), consistent with previous reports in skeletal muscle. As humans have heterozygous mutations, we stressed adult heterozygous mdm and WT mice (2 weeks of angiotensin II infusion): both had comparable hypertrophic responses (increased LVPWd and IVSd). Aged (89 week old) unstressed heterozygous mdm mice had normal cardiac dimensions and function.
The N2A region, I-band titin mdm mutation causes minimal cardiac dysfunction in mice, unlike the severe skeletal muscle phenotype. Human I-band mutations are unlikely to cause dilated cardiomyopathy.