bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 255: A Fibroblast Population Shares A Developmental Origin With Cardiovascular Lineages
Authors: Ranjbarvaziri Sara , Ali Shah , Talkhabi Mahmood , Zhao Peng , Nam Young-Jae , Ardehali Reza
Published: July 2021

Rationale:
The traditional definition of “cardiovascular” lineages describes the eponymous cell types - cardiomyoctes, endothelial cells, and smooth muscle cells - that arise from a common mesodermal progenitor cell during heart development. Fibroblasts are an abundant mesenchymal population in the mammalian heart which may have multiple, discrete developmental origins. Mesp1 represents the earliest marker of cardiovascular progenitors, contributing to the majority of cardiac lineages. To date no link between Mesp1 and fibroblast generation has been reported.

Objective:
We hypothesized progenitor cells expressing Mesp1 can also give rise to cardiac fibroblasts during heart development.

Methods and Results:
We generated Mesp1cre/+;R26RmTmG reporter mice where Cre-mediated recombination results in GFP activation in all Mesp1 expressing cells and their progeny. To explore their developmental potential, we isolated GFP+ cells from E7.5 Mesp1cre/+;R26RmTmG mouse. In vitro culture and transplantation studies into SCID mouse kidney capsule as wells as chick embryos showed fibroblastic adoption. Results showed that at E9.5 Mesp1+ and Mesp1- progenitors contributed to the proepicardium organ and later at E11.5 they formed epicardium. Analysis of adult hearts demonstrated that the majority of cardiac fibroblasts are derived from Mesp1 expressing cells. Immunohistochemical analysis of heart sections demonstrated expression of fibroblast markers (including DDR2, PDGFRα and Col1) in cells derived from both Mesp1+ and Mesp1- progenitors. Additionally, we investigated whether the two distinct fibroblast populations have different potency towards reprogramming to cardiomyocytes. Results showed no significant difference between Mesp1 and non-Mesp1 isolated fibroblasts to convert to cardiomyocyte fate.

Conclusions:
Our data demonstrates that cardiovascular progenitors expressing Mesp1 contribute to the proepicardium. These cells, as cardiovascular progenitors, also give rise to the highest portion of cardiac fibroblasts in the mouse heart.
Abstract 259: A Microrna Pathway That Promotes Cardiomyocyte Proliferation And Cardiac Regeneration By Inhibiting Hippo Signaling
Authors: Tian Ying , Chen Li , Wang Tao , Zhou Ning , Kong Jun , Liu Ying , Snitow Melinda , Morley Michael , Li Deqiang , Petrenko Nataliya , Lu Minmin , Stewart Kathleen , Morrisey Edward
Published: July 2021

In contrast to lower vertebrates, the mammalian heart has limited capacity to regenerate after injury in part due to ineffective reactivation of cardiomyocyte proliferation. While evidence exists for a low level of cardiomyocyte proliferation in the adult heart, it remains unclear whether increasing the rate could be used to therapeutically promote cardiac regeneration. In this study, we show that the microRNA cluster miR302-367 is important for cardiomyocyte proliferation during development and is sufficient to induce cardiomyocyte proliferation in the adult and promote cardiac regeneration. Loss of miR302-367 leads to decreased cardiomyocyte proliferation during development. In contrast, increased miR302-367 expression leads to a profound increase in cardiomyocyte proliferation, in part through repression of the Hippo signal transduction pathway. Postnatal re-expression of miR302-367 leads to reactivation of the cell cycle in cardiomyocytes resulting in reduced scar formation after infarction. However, long-term expression of miR302-367 leads to cardiomyocyte de-differentiation and dysfunction, suggesting that persistent reactivation of the cell cycle in postnatal cardiomyocytes is not desirable. Importantly, this limitation can be overcome by transient systemic application of miR302-367 mimics, leading to increased cardiomyocyte proliferation and mass, decreased fibrosis, and improved function after injury. Our data demonstrate the ability of microRNA based therapeutic approaches to promote cardiac repair and regeneration through the transient activation of cardiomyocyte proliferation.
Abstract 262: Deletion Of E2f1 Promotes Bone marrow Progenitor Cell Differentiation And Ischemic Cardiac Repair
Authors: Xu Shiyue
Published: July 2021

Background:
We have previously shown that knockout of E2F1 in mice enhances angiogenesis following induction of hind limb ischemia. Recent studies suggest that suppression of E2F1 enhances oxidative phosphorylation in a variety of cell types. Since an increase in oxidative phosphorylation in stem/progenitor cells is often associated with cell differentiation, we hypothesize that E2F1-deficiency may promote bone marrow (BM) progenitor cell differentiation thereby impact on ischemic cardiac repair.

Methods and Results:
We cultured bone marrow (BM) Lin- progenitor cells under hypoxic and normxic conditions for 24 h, then measured the expression of metabolism associated genes and evaluated cell proliferation and differentiation. We also performed adoptive BM transplantation to reconstitute BM of WT mice with E2F1-/- or WT BM, followed by surgical induction of myocardial infarction (MI), to compare the role of BM E2F1 in the cardiac repair in vivo. Notably, we found that the expression levels of pyruvate dehydrogenase kinase (PDK)-4 and PDK2, two critical inhibitors of mitochondrial oxidative phosphorylation, in the E2F1-/- BM Lin- progenitor cells is markedly lower than those in WT cells (P<0.01, n=4). After culture in the EPC differentiation medium for 7 days, the expression levels of EPC markers, CD31 and KDR , were significantly higher in E2F1-/- cells than in WT control cells (P<0.05, n=3). Although there was no significant difference in the proliferating rate between WT and E2F1-/- BM Lin- progenitor cells cultured in normoxia, when cultured under hypoxic condition the proliferating rate of E2F1-/- cells were markedly greater than that of WT cells (P<0.05, n=3). Consistently, the infarcted size in mice transplanted with E2F1-/-eGFPTg BM was significantly smaller than in mice transplanted with WTeGFPTg BM (P<0.01, n=5).

Conclusions:
Genetic deletion of E2F1 in the BM progenitor cells enhances oxidative metabolism that may result in enhanced differentiation towards endothelial lineage and increased proliferation in the ischemic/hypoxic tissue environment. Therefore, inhibition of E2F1 in BM progenitor cells may improve the recovery from cardiac ischemic injury.
Abstract 265: Uroetensin II Protects Cardiomyocytes from Apoptosis Induced by Oxidative Stress through Cystathionine-γ-Lyase (CSE)/Hydrogen Sulfide Pathway
Authors: Gong Hui , Chen Zhidan , Zhang Xiaoyi , Zhang Jie , Li Yang , Ye Yong , Zhang Guoping , Yang Chunjie , Zou Yunzeng
Published: July 2021

Plasma UII has been observed to be raised in patients with acute myocardial infarction, a lower UII response is associated with more severe injury of myocardium, suggesting a possible cardioprotective role for this peptide. In the present study, we studied plasma UII concentration of thirty patients admitted to the Cardiology Department with acute myocardial infartion.The results showed that plasma UII was sharply increased in patients compared to that in health control within one week after admission. We then explore whether UII could protect cardiomyocytes from injury induced by oxidative stress. Cultured cardiomyocyte were treated with H2O2 to induce oxidative stress, and the influence of UII on H2O2-induced apoptosis was observed. The results showed that UII pretreatment significantly reduced the number of TUNEL-positive cardiomyocytes induced by H2O2, and it partly abolished the upregulation of pro-apoptotic protein Bax and the down-regulation of anti-apoptotic protein Bcl-2. siRNA targeted to urotensin receptor (UT) greatly inhibited these effects. H2S has been reported to exert protective effect on cardiomyocytes, we detected the effect of UII on H2S production and CSE (Major H2S-producing enzyme) expressions in cardiomyocytes exposed to H2O2.The present data revealed that UII increased the H2S production by enhancing the expression of CSE by activating the ERK signaling in cardiomyocytes exposed to H2O2. Si-CSE or ERK inhibitor not only greatly inhibited the upregulation of CSE or the phosphorylation of ERK induced by UII but also reversed UII-induced-upregulation of H2S production and anti-apoptosis in cadiomyocytes exposed to H2O2.

In conclusion, UII rapidly promoted the phosphorylation of ERK, increased CSE exression and induced H2S production, which in turn enhanced the p-ERK level to protect cardiomyocytes from apoptosis under ischemic or oxidative stress. The increased plasma UII level in patients may be critical for cardiac protection in patients at early-phase of acute myocardial infarction.
Abstract 27: A Novel Variant Cell Cycle-related Kinase Provides Cardioprotection Upon Pressure Overload By Promoting Autophagy
Authors: Qiu Hongyu , Lai Lo
Published: July 2021

Background:
One approach to discovering novel targets for heart failure (HF) therapy is to determine which genes are down-regulated in HF. We found previously that cardiac cell cycle-related kinase (cCCRK), a novel variant of CCRK in heart, is down-regulated by 50% in failed heart vs control, and over-expression of the cCCRK decreases the susceptibility of cardiac myocytes to pro-apoptotic agents, indicating that cCCRK might be a potential target of HF. We tested here our hypothesis that cCCRK provides cardiaoprotection upon stress of pressure overload.

Methods and Results:
A transgenic (TG) mouse model with cardiac-specific over-expression of cCCRK was generated. Both wild type (WT) and TG mice were submitted to aortic banding for two weeks. In basal condition, no significant differences were found between TG and WT in left ventricular (LV) /body weight (3.5±0.3 in TG vs. 3.6±0.2 in WT) and contractility (LV ejection fraction (EF): 72.7±1 in TG vs 72.7±1 % in WT). After two weeks banding, EF was significantly impaired in the WT (62±2%, P<0.05 vs sham), but maintained in the TG (71.1±2 %, P<0.05 vs WT). Lung /tibial length ratio, an index of pulmonary congestion and heart failure was lower (p<0.05) in banded heart of TG (9.9±1) vs WT (13.1±1). A cyclin-dependent kinase inhibitor p21, which is also an important regulator of autophagy, was significantly upregulated in TG by 5 folds vs WT (P<0.05). Overexpression of cCCRK in rat neonatal cardiomyocyte (RNC) stimulated autophagy in a dose-dependent manner representing by the enhanced LC3-II (autophagy marker) (P<0.05 vs β-gal). To test its effect in response to stress, glucose starvation was induced to in RNC. cCCRK induced an early response to cardiac stress via the activation of autophagy by increasing LC3-II at 0.5 hrs vs the control at 2 hrs after starvation.

Conclusions:
over-expression of cCCRK in heart provides protection against pressure overload via activation of autophagy signaling pathways.
Abstract 272: Sex Differences in β-Adrenergic Responsiveness of Excitation-Contraction Coupling in Isolated Rabbit Hearts
Authors: Hoeker Gregory , Hood Ashleigh , Katra Rodolphe , Poelzing Steven , Pogwizd Steven
Published: July 2021

Introduction:
Sex differences in β-adrenergic receptor (β-AR) responsiveness are associated with female cardioprotection. We hypothesize that female (F) rabbits have reduced responsiveness to β-AR stimulation vs males (M), and that the degree and type of sex differences vary with the β-AR subtypes that are activated.

Methods:
Ventricular action potentials (AP) and intracellular calcium transients (CaT) were optically mapped from the epicardial surface of rabbit hearts during 3 Hz pacing. Spontaneous calcium release (SCR) and ectopic activity were elicited at 1, 3, and 5.5 Hz. β-responsiveness was assessed with the nonselective β-agonist isoproterenol (Iso, 1-316 nM), or β2-AR selective agonist zinterol (Zin, 10 nM).

Results:
At baseline, the time constant of CaT decay (τ) was faster in F than M (54.0±1.7 vs 62.1±3.0 ms; n=14, 14; p < 0.05), with no sex difference in CaT duration (CaD80). AP duration (APD90) was shorter in F than M (202.5±5.0 vs 218.2±5.7 ms; p < 0.05). Iso decreased τ, CaD80, and APD90 in a dose-dependent manner in both sexes (n = 5, 5 for F, M). Iso decreased τ to a lesser extent in F than M for 1 and 32-316 nM Iso (F = 11-32 ms, M = 23-48 ms; p < 0.05). The Iso-induced decrease in CaD80 was not significantly different in F than M at any dose. The Iso-induced decrease in APD90 was significantly less in F than M only at 316 nM Iso (75.5±8.7 ms vs 103.9±6.2 ms, p < 0.05). In contrast, there were no sex differences in the response to Zin for τ, CaD80, or APD90 (n = 6, 6 for F, M). Zin decreased τ by 7.2±2.0 ms in F vs 12.7±3.7 ms in M; CaD80 by 18.0±5.3% in F vs 21.1±8.0 ms in M; and APD90 by 24.9±8.5 ms in F vs 21.9±8.9 ms in M. SCR was observed in 50% (6/12) of hearts treated with Zin, whereas Iso elicited SCR in all hearts (10/10) with a dose threshold of 32 nM. No ectopic beats were observed with Zin (0/36 trials in 12 hearts). With Iso, ectopic activity was less frequent in F hearts (16%, 12/75 trials in 5 hearts) than in M hearts (41%, 26/68 trials in 5 hearts, p < 0.05).

Conclusions:
These results suggest that sex differences in AP and CaT depend on the dose of the agonist used and the β-AR subtypes that are activated. Elucidating nuances of sex differences in β-AR subtype physiology will provide a better understanding of the mechanisms of reduced β-responsiveness in F and its cardioprotective effects.
Abstract 274: Spatiotemporal Regulation of β Adrenergic Signaling In Heart failure
Authors: Xiang Yang K , Barbagallo Federica , Xu Bing , Fu Qin
Published: July 2021

Our long-term goal is to understand mechanisms that govern spatiotemporal regulation of cAMP/PKA signaling in cardiac myocytes under physiological and pathophysiological conditions, and their implication in cardiac disease therapy. Here we use a series of biosensors to measure cAMP/PKA activity under βAR subtype regulation. In failing cardiac myocytes, the cAMP and PKA activity are shifted from the plasma membrane to the intracellular sarcoplasmic reticulum and the myofilaments. Meanwhile, β2AR displays an increased role in signaling to the myofilaments in failing myocytes when compared to the control myocytes. Moreover, we show that an increased βAR association with phosphodiesterases promotes the alteration in spatiotemporal propagation of cAMP/PKA signaling in failing myocytes. These observations and the underlying mechanisms and functional implications will be discussed.
Abstract 28: Increased Expression Of Mitochondrial Inducible Nitric Oxide Synthase By Heat Shock Protein Hsp22/h11 Kinase Promotes Oxidative Phosphorylation In Mammalian Heart
Authors: Qiu Hongyu , Rashed Eman , Depre Christophe
Published: July 2021

Aims:
Stress-inducible heat shock protein 22 (Hsp22) confers protection against ischemia through induction of the inducible isoform of nitric oxide synthase (iNOS). Hsp22 over-expression
in vivo
significantly stimulates cardiac mitochondrial respiration, whereas Hsp22 deletion
in vivo
shows a reciprocal effect. It has also been shown in
Drosophila
that Hsp22 is expressed in the mitochondria that depends on its N-terminal domain. We hypothesized that Hsp22-mediated regulation of mitochondrial function is dependent upon its mitochondrial translocation together with iNOS.

Methods and Results:
Adenoviruses harboring either the full coding sequence of Hsp22 (Ad-WT-Hsp22) or a mutant lacking a 20 amino acid putative N-terminal mitochondrial localization sequence (Ad-N20-Hsp22) were generated, and infected in rat neonatal cardiomyocytes. Compared to β-Gal control, Ad-WT-Hsp22 accumulated in mitochondria by 2.5 fold (P<0.05), reduced chelerythrine-induced apoptosis by 60% (P<0.01), and increased oxygen consumption rate by 2-fold (P<0.01). This latter effect was abolished upon addition of the specific iNOS inhibitor, 1400W. Ad-WT-Hsp22 significantly increased global iNOS expression by about 2-fold (P<0.01), and also increased its mitochondrial localization by 2.5 fold vs β-gal (P<0.05). Upon comparable over-expression, the Ad-N20-Hsp22 mutant did not show significant mitochondrial translocation, protection against apoptosis or stimulation of mitochondrial respiration. Although Ad-N20-Hsp22 did increase global iNOS expression by 6-fold it did not significantly promote iNOS mitochondrial translocation.

Conclusion:
Translocation of both Hsp22 and iNOS to the mitochondria is necessary for the stimulation of oxidative metabolism and protection against apoptosis.
Abstract 283: Nppa Marks A Subset Of Embryonic Cardiomyocytes Predestined To Form Trabeculae In Zebrafish
Authors: Arnaout Rima , Stainier Didier Y , Coughlin Shaun R
Published: July 2021

The development and maintenance of cardiac trabeculae is required for proper ventricular function. Aberrant trabeculation can cause heart failure, arrhythmia, and death. Yet the mechanisms controlling initiation of trabeculation are incompletely understood, partly due to a lack of markers for trabeculation during early embryogenesis. We hypothesized that natriuretic peptide A (nppa) marks trabeculae before cardiomyocytes leave the single-celled ventricular layer. While nppa has been studied in mammals, the external development of the translucent zebrafish embryo offers new insight into the earliest stages of trabeculation. We have found by in situ hybridization, and by creating a fluorescent transgenic zebrafish line, that nppa exclusively marks zebrafish cardiac trabeculae from their onset through adulthood. Live spinning disc confocal video microscopy shows that nppa:GFP-positive cells move into the trabecular layer starting at 60 hours post fertilization (hpf). GFP-positive trabeculae are seen by 5 days post fertilization (dpf) in normal larvae. However, in cardiac troponin T2a mutants and in erbB2 mutants _ both known not to trabeculate _ nppa is still expressed, but the nppa:GFP positive cells fail to form trabecular projections. In embryos injected with a morpholino against atrial myosin heavy chain 6, where weakened atrial contraction leads to weaker blood flow without affecting the ventricle directly, GFP-positive cells form trabeculae similar to uninjected controls. Using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs), we created nppa mutants causing frameshift deletions. Mutants have poorly contractile hearts at 3 dpf and have been crossed into transgenic reporter lines in order to specifically assess trabeculation. Together, these data suggest that nppa marks a subset of embryonic cardiomyocytes destined to form trabeculae, and that nppa loss of function causes cardiac defects.
Abstract 293: Role Of G-protein Coupled Receptor Signaling In Cardio-renal Injury
Authors: Kamal Fadia A , Travers Joshua G , Ma Qing , Devarajan Prasad , Blaxall Burns C
Published: July 2021

The kidneys play an important role in cardiovascular disease (CVD), where renal co-morbidities accompany CVD in a large proportion of patients thus complicating their treatment regimen. Moreover, the incidence of acute renal injury after cardiac surgery plays an important role in disease progression. Emerging data suggest the importance of understanding the mechanisms of cardio-renal injury and the development of novel therapies that can be safely used with cardiovascular and renal co-existing pathologies. Although the role of G-protein coupled receptors (GPCRs) in CVD has been broadly recognized, their role in renal injury remains poorly understood. We have found, in a chronic mouse model of heart failure, attenuated renal fibrosis and attenuated pathologic RAAS activation by the small molecule GPCR-Gβγ inhibitor “gallein”. To investigate the direct effects of GPCR-Gβγ inhibition on renal injury, we utilized an acute renal ischemia-reperfusion (RIR) mouse model. Gβγ inhibition by gallein pretreatment attenuated the histopathological profile of RIR, including attenuation of tubular hypertrophy, apoptosis, cast formation, and tissue Lipocalin2 expression. This was accompanied by attenuated inflammation, reflected by reduced CCL2 and ICAM1 gene expression and cellular infiltration, in addition to reduced Collagen III gene expression. These preliminary results suggest a promising protective role for Gβγ inhibition in renal injury and remodeling. Future mechanistic investigation of this possible protective effect will provide better understanding of the role of GPCR-Gβγ signaling in cardio-renal injury and remodeling and possible novel therapeutic targets.
Abstract 299: Pulmonary Hypertension Activates Cardiac Fibroblasts To Release Paracrine Factors Regulating Cardiac Myocyte Dedifferentiation
Authors: Walker Lori A , Brown R. D , Stenmark Kurt R , Buttrick Peter M
Published: July 2021

In virtually all models of heart failure, prognosis is ultimately determined by right ventricular (RV) function. Thus understanding the unique cellular mechanisms that contribute to RV dysfunction is of critical importance. We have approached this question by studying a large animal model that has significant resonance with human disease: exposure of the neonatal calf to hypobaric hypoxia. This model demonstrates rapid onset of severe pulmonary hypertension (PH) and significant RV dysfunction. Strikingly, the RV of these animals shows a chamber-specific increase in markers of inflammation and local infiltration of activated pro-remodeling macrophages that are proportionate to the rise in PA pressure. In order to test the hypothesis that this inflammatory milieu directly contributes to myocyte dysfunction, we examined the effects of conditioned media (CM) from cardiac fibroblasts isolated from the PH calf on normal adult rat ventricular myocytes (ARVM) in culture. Even brief exposure (<2 days) to RV-CM results in rapid and marked dedifferentiation of ARVM to a neonatal-like phenotype that exhibits spontaneous contractile behavior. Dedifferentiated cells maintain viability for over 30 days with continued expression of cardiomyocyte proteins including TnI, sarcomeric actinin, and desmin. This response is not seen in ARVM exposed to CM from fibroblasts collected from the hypoxic LV or from the normoxic RV. We have characterized the factor(s) that are present in the RV-CM and have established that the active components are proteins with a mol wt >30 kDa and that inflammatory cytokines such as IL-18 are necessary but not sufficient to effect this phenotypic change. These data suggest that local and perhaps focal inflammation in the RV, induced by the combination of pressure overload and hypoxia, has the capacity to signal a phenotypic transformation in a population of RV cardiocytes, which likely contributes to chamber specific dysfunction. Therapies that target and interrupt this inflammatory process either by preventing in-migration of inflammatory cells or by blocking cell-cell signaling have the potential to prevent RV dysfunction.
Abstract 30: Nub1l Suppresses Neddylation And Enhances Misfolded Protein Clearance In Cardiomyocytes
Authors: Su Huabo , Li Jie , Ma Wenxia , Hou Ning , Li Faqian
Published: July 2021

Protein modification by ubiquitin (Ub) or Ub-like proteins such as NEDD8 (neddylation) constitutes a fundamental regulatory mechanism of protein function. In contrast to well-recognized role of Ub in protein degradation, little is known about the role of NEDD8 in protein quality control. We have previously revealed that CM-restricted inactivation of deneddylation, a process that removes NEDD8 from modified proteins, accumulates neddylated proteins and impairs proteasomal and autophagic proteolysis. Here we report that proteasome inhibitors, simulated ischemia/reperfusion and H2O2 significantly increase NEDD8 conjugates in cardiomyocytes (CMs). Immunoprecipitation analysis reveals mixed modification of these proteins by Ub and NEDD8. Expression of NEDD8 but not the conjugation-deficient mutant increases neddylated proteins and accumulates a proteasome surrogate substrate GFPu in a dose-dependent manner, suggesting that excessive neddylation disrupts proteasomal proteolysis. We further targets to NUB1L, a UBL (Ub-like domain)-UBA (Ub associating domain) family protein that was shown to negatively regulate neddylation. NUB1L expression markedly reduces free NEDD8 by promoting its degradation, and abrogates proteasome inhibition-induced neddylation in CMs. Suppression of neddylation by NUB1L expression enhances GFPu degradation at baseline, and attenuates GFPu accumulation upon sI/R and H2O2 treatment. Furthermore, NUB1L expression promotes, while down-regulation of NUB1L impairs, the clearance of a bona fide misfolded protein in CMs. NUB1L expression also ameliorates proteotoxic stress- and sI/R-induced CM injury. Finally, increased NEDD8 conjugates are evident in the mouse hearts of a number of cardiac disease models as well as in human failing hearts. Together, our findings suggest that excessive neddylation disrupts protein quality control and that antagonizing neddylation by NUB1L promotes misfolded protein degradation. Targeting neddylation/NUB1L could be a novel therapeutic strategy for prevention and treatment of insufficient protein quality control-associated cardiac disease.
Abstract 301: Deletion of Sam68 Attenuates Myocardial Hypertrophy
Authors: Zhou Junlan , Boriboun Chan , Biyashev Dauren , Qin Gangjian
Published: July 2021

The Src-associated in mitosis 68 kDa (Sam68) belongs to the STAR (signal transducer and activator of RNA) family of RNA-binding proteins and has recently been shown to regulate angiotensin II (Ang II) signaling in vascular smooth muscle cells. In this study, we investigated the role of Sam68 in cardiac hypertrophy. Wild-type (WT) and Sam68 knockout (KO) mice were administered continuously with Ang II for 14 days via osmotic minipumps. Echocardiography analyses reveled that basal left-ventricular posterior wall thicknesses (LVPTs and LVPTd) and basal chamber sizes (LVIDs and LVIDd) are similar between the 2 groups of mice, that Ang II induced a significant elevation in LVPTs and LVPTd and reduction in LVIDs and LVIDd in both groups of mice, and that the changes of all these hypertrophic parameters were significantly attenuated in Sam68 KO than in WT mice (p<0.05 at day 14, n=15). The increase in the heart weight (HW) / body weight (BW) ratio was also lesser in Sam68 KO mice than in WT mice (p<0.05 at day 14, n=15). Then, we performed transverse aortic constriction (TAC) model. Echocardiography analyses confirmed significantly lower levels of LVPTs/d and LVIDs/d in Sam68 KO mice than in WT mice (LVPTs/d: p<0.05 at weeks 2 and 4, p<0.01 at weeks 8; LVIDs/d: p<0.05 at weeks 2, 4, and 8; n=12), however, left ventricular ejection fraction (LV EF) remained not significantly different at all these time points between the 2 groups. Histological analyses of fibrosis by Masson’s trichrome staining demonstrated a ~ 2-fold less in collagen deposition areas in Sam68 KO than in WT mice (p<0.05 at weeks 8, n=12). The attenuated hypertrophic responses of Sam68 KO mice to both Ang II treatment and TAC surgery was further supported by a lower expression of fetal genes, including β-myosin heavy chain, atrial and brain natriuretic peptides in the heart of Sam68 KO mice than in WT mice (qRT-PCR). Collectively, our results suggest that Sam68 may modulate the cause of cardiac hypertrophy
Abstract 302: Sex Differences In Nonlinear Dynamics And Their Circadian Variation In Control Dogs And In A New Arrhythmogenic Canine Model Of Heart Failure
Authors: Zhu Yujie , Pogwizd Steven M
Published: July 2021

Introduction:
Females can be more arrhythmogenic than males, and this sex difference can persist with development of chronic heart failure (CHF). The aim of this study was to investigate sex differences in the arrhythmogenic substrate in control dogs and in a new arrhythmogenic canine model of CHF.

Methods:
CHF was induced in 30 dogs by aortic insufficiency and aortic constriction. Holter monitoring assessed VT and PVCs from 30 dogs, as well as traditional HRV measures and nonlinear dynamics (including correlation dimension (CD), detrended fluctuations analysis α1 (DFAα1), and Shannon entropy (SE)) at baseline, 240 days (240d) and 720 days (720d) after CHF induction.

Results:
At baseline, females had lower LF/HF (0.27±0.03 vs 0.33±0.02, p=0.04), CD (1.60±0.17 vs 2.21±0.15, p=0.01), DFAα1 (0.62±0.03 vs 0.72±0.03, p=0.03), and SE (2.99±0.02 vs 3.10±0.03, p=0.03 vs males). Females lacked circadian variation in LF/HF, DFAα1, and SE while males had circadian variation in all of these. Of 11 dogs with frequent runs of VT and PVCs, 95% and 91% of total VT runs and total PVCs, respectively, were in females. With CHF, all these linear and nonlinear parameters progressively declined in males and females. CHF females had less decline in LF/HF than males so that by 720 days there was no more sex difference (0.24±0.06, 0.17±0.03 in females vs 0.22±0.05, 0.18±0.01 in males at 240d, 720d). However, for nonlinear parameters of CD, DFAα1, and SE, CHF females had lower values than males (CD: 1.56±0.21, 0.99±0.32 vs 1.87±0.24, 1.50±0.34; DFAα1: 0.51±0.05, 0.43±0.04 vs 0.54±0.07, 0.48±0.04; and SE 2.93±0.08, 2.76±0.08 vs 3.01±0.11, 2.91±0.04 in females vs males at 240d, 720d). With CHF, circadian variation in CD, DFAα1, and SE were lost in both males and females.

Conclusions:
There are sex differences in the arrhythmogenic substrate in control dogs and in this new arrhythmogenic canine model of moderate CHF. At baseline, females have lower sympathetic stimulation, reduced cardiac chaos, and loss of circadian variation in nonlinear dynamics. With CHF, sex differences in nonlinear dynamics persist; this reflects a loss of complexity and fractal properties that could contribute to increased arrhythmias in female CHF dogs.
Abstract 303: Sexual Dimorphisms in Cardiomyocytes Exposed to a Pathological Stimulus
Authors: Blenck Christa , Harvey Pamela , Leinwand Leslie
Published: July 2021

Background:
Sex is an important modifier of the development of cardiovascular disease; women consistently display less hypertrophy, fewer changes in pathologic gene expression, and less cardiac dysfunction compared to men exposed to the same pathological stimuli. The signaling pathways responsible for these sexual dimorphisms, especially within the cardiomyocyte, are not well understood.

Objective:
To investigate sexual dimorphisms in activation of signaling pathways and expression of genes related to cardiomyocyte contractility in response to a pathological stimulus in isolated adult rat ventricular myocytes (ARVMs).

Methods:
ARVMs from both sexes were isolated using a Langendorff perfusion apparatus and treated with the β-adrenergic agonist isoproterenol (ISO) or vehicle (saline) for 24 hours. Activated signaling molecules were detected by performing a phospho-kinase antibody array (R&D Systems) in ARVMs isolated from two rats of each sex treated with ISO or vehicle. Total RNA was isolated from these cells, and real-time PCR was used to determine expression levels of calcium handling genes, including Cav1.2 and sodium calcium exchanger (NCX1).

Results:
A distinct profile of activated signaling molecules was observed between male and female ARVMs in response to ISO. Female ARVMs displayed activation of kinases such as Akt, ERK1/2, p38α, GSK3α/β and MSK1/2. Male ARVMs were characterized by an activation of signaling molecules such as PLCγ1, RSK1/2/3 and c-Jun. ARVMs of both sexes displayed activation of signaling molecules such as eNOS, STAT3 and mTOR. Because the activated signaling molecules identified with the array can modulate expression of calcium handling genes, expression of these genes was analyzed. In response to ISO treatment, female ARVMs displayed an increase in NCX1 expression normalized to vehicle (1.63 ± 0.079) as well as Cav1.2 expression (1.48 ± 0.072). However, these increases were not observed in male cells.

Conclusions:
Cardiomyocytes isolated from rats of different sexes display distinct responses to the pathological stimulus ISO. Understanding the mechanisms responsible for these sexual dimorphisms can lead to the development of more effective treatment options for both men and women with cardiovascular disease.
Abstract 305: Pressure Overload-Induced Myocardial Hypertrophy Causes an Electrical Remodeling of CLC-3 Chloride Channels in Mouse Heart
Authors: Duan Dayue D , Yu Ying , Wang Guanlei , Ye Lingyu L , Li Yi-gang
Published: July 2021

Backgrand:
Myocardial hypertrophy causes an increase in myocyte volume and constitutive activation of a volume-sensitive outwardly-rectifying anion channel (VSORAC). The underlying molecular mechanisms and function of VSORAC in the electrical remodeling during myocardial hypertrophy and heart failure remain undefined. We tested whether cardiac CLC-3 chloride channels play a role in the hypertrophy-induced electrophysiological remodeling.

Methods and Results:
The age-matched CLC-3 knockout (Clcn3-/-) mice and their wild-type (Clcn3+/+) littermates were subjected to minimally-invasive transverse aortic banding (MTAB). In 77% (44/57) left ventricular (LV) myocytes isolated from MTAB-Clcn3+/+ mice a large VSORAC current was activated under isotonic conditions. Hypotonic cell-swelling caused no changes in the VSORAC but hypertonic cell-shrinkage significantly inhibited it. This constitutively-activated VSORAC had an anion selectivity of I->Cl->Asp-, and was inhibited by tamoxifen, PKC activation and intracellular application of anti-CLC-3 antibody. In the age-matched MTAB-Clcn3-/- mice, a significantly smaller outwardly-rectifying current was present in only 38% (36/94, P<0.05 vs MTAB-Clcn3+/+) LV myocytes. This current was neither increased by hypotonic stress nor inhibited by tamoxifen, PKC or anti-CLC-3 antibody, indicating not a VSORAC or CLC-3 current. Expression of CLC-3 protein was significantly increased in the LV tissues of MTAB-Clcn3+/+ mice but not in Sham-Clcn3+/+ and MTAB-Clcn3-/- mice. Both surface and intracardiac electrophysiological recordings revealed more atrial or ventricular arrhythmias in MTAB-Clcn3-/- mice than in MTAB- and Sham-Clcn3+/+ mice.

Conclusions:
Pressure-overload-induced myocardial hypertrophy causes an upregulation of CLC-3 expression and constitutive activation of CLC-3 may serve as a novel protective mechanism against the electrical remodeling during myocardial hypertrophy and heart failure.
Abstract 306: A Novel PKCalpha Splicing Isoform in Signaling for Cardiac Hypertrophy
Authors: GAO CHEN , Gong Jianli , Steinberg Susan F , Wang Yibin
Published: July 2021

RNA splicing is a significant post-transcriptional regulatory step to enrich the overall transcriptome complexity, yet its specific contribution to cardiac development and pathogenesis remains underexplored. Here, we performed a comprehensive deep RNA-seq analysis in murine heart to investigate the cardiac transcriptome at single exon-resolution. From the dataset, we have identified a previously un-annotated novel exon in the PKCα transcript ---PKCα-NE (Novel Exon). The novel exon encode a 16 amino acid insert adjacent to the PKCα turn motif. Interestingly, the splicing event is detected only in cardiac and skeletal muscle. Based on bioinformatics analysis, we have identified two highly conserved binding motifs for a cardiac splicing regulator_RBFox1. We further demonstrated that RBFox1 indeed functions as a positive regulator for the splicing event of this novel exon.

In vitro studies showed that the NE converts PKCα from a lipid-dependent enzyme to a constitutively active kinase with high levels of autocatalytic activity and activity toward peptide substrates. The NE also increases PKCα’s Km for peptide substrate. These changes in steady state kinetic parameters suggest that PKCα-NE would exert it’s cellular actions most prominently when co-localized with target substrates, either on protein scaffolds or in subcellular signaling compartments. In fact, immune-precipitation for individual PKCα spicing variants followed by Mass Spectrometry exposed a specific interaction between PKCα-NE and elongation factor (eEF1A1). PKCα-WT and PKCα-NE both stimulated cardiac hypertrophy to a similar level based on myocyte size measurement, but PKCα-NE selectively increased eEF1A1 phosphorylation in association with elevated protein translational activity in cultured cardiomyocytes.

In summary, we have identified a novel cardiac specific splicing variant of PKCα that is dynamically regulated during development and heart failure. Our finding has revealed a novel signaling pathway in cardiac hypertrophy involving a novel alternative splicing event of PKCα and its specific downstream effects on protein synthesis regulation.
Abstract 307: Structural Characterization of Calcineurin A-Calsarcin 1 Assembly
Authors: Koscky Paier Carlos R , Cardoso Alisson C , Brenneli Tatiani L , Honorato Rodrigo V , Gozzo Fábio C , Oliveira Paulo S , Franchini Kleber G
Published: July 2021

Signaling by the calcium-dependent phosphatase calcineurin (Cn) plays key roles in regulating cardiac development, hypertrophy, and pathological remodeling. Cn binds to and is negatively regulated by calsarcins (CS), a family of muscle-specific proteins. However, the molecular mechanisms involved in the inhibition of Cn by CS remain unclear. Understanding the architecture and structure of Cn-CS complex is critical to unravel the regulation of Cn by CS. Here we combined biochemical assays, chemical cross-linking coupled to mass spectrometry experiments (MS/MS), mutational analysis and a modeling strategy for structural characterization of CnA-CS1 assembly. The MS/MS data obtained from the cross-linked peptides of both proteins were used to guide an in silico docking of their polypeptide models. The protein complex models with the smallest estimated binding energy were clustered according to structural similarity and submitted to molecular dynamics simulation. The interacting surface of CnA was mapped in a pocket between the 1st and 3rd α-helixes and surrounding loops, while the corresponding surface of CS1 was mapped to the carboxyterminal loops within the Leu179-Phe185, Phe195-Ser199 and Thr250-Leu264 regions. Notably, the region of CnA that interacts with CS1 was found to be located in close proximity, but not coincident, to the β-sheet 14, the main binding site for the PxIxIT sequence of NFAT. Experiments performed with several CnA (FLAG-CnA) and CS1 (myc-CS1) mutants were used to validate the structural model of the CnA-CS1 assembly. The Lys40 (CnA) and Glu254 (CS1) residues were identified as critical for the complex stability. The model that emerges from this study supports the notion that CS1 interacts with an allosteric site to inhibit the activity of CnA. Alternatively, the close proximity of the CS1 to NFAT interacting site supports an interference of CS1 on the ability of CnA to bind and activate NFAT.
Abstract 313: Elucidation Of The Fibrotic Transcription Factors Gene Network Leading To Chagas Heart Disease
Authors: Nde Pius N , Udoko Aniekanabassi N , Johnson Candice A , Dykan Andrey , Rachakonda Girish , Villalta Fernando , Lima Maria F , Pratap Siddharth
Published: July 2021

Background:
Trypanosoma cruzi
the causative agent of Chagas heart disease (CHD) remains incurable. The major pathology induced by the parasite is cardiac fibrosis leading to heart failure followed by death. The mechanisms of
T. cruzi
induced cardio-pathology remains largely unknown.

We hypothesize that
T. cruzi
infection regulates the expression of profibrotic genes in human cardiac myocytes (HCM), tilting the heart towards a profibrotic phenotype seen in CHD patients.

Methods and Results:
To elucidate the molecular mechanisms of
T. cruzi
induced cardiac fibrosis, we challenged primary HCM with
T. cruzi
for two hours and purified total RNA for microarray. We investigated changes at the whole transcriptome level on an affymetrix platform. The arrays were done in triplicates at different time points; changes in gene expression greater than 2-fold and having a Benjamini and Hochburg false discovery rate corrected p-value <0.05 were considered significant. The microarray data was validated using real-time PCR followed by PCR array and immunoblotting, to evaluate changes in the protein expression levels of fibrotic transcription factors. Protein expression levels were evaluated in triplicate and analyzed by ANOVA. The fibrotic interactome induced by
T. cruzi
in HCM was elucidated using Cytoscape.

Our results indicate that exposure of HCM to
T. cruzi
upregulates the transcript levels of two transcription factors associated with fibrosis, SNAI1 (more than 2 fold up-regulated) and Early Growth Response protein 1, EGR1, (about four fold up-regulated). SNAI1 and EGR1 were increased at the protein level. Furthermore, we identified the first interactome regulating fibrosis in primary HCM induced by
T. cruzi
.

Conclusions:
This is the first report showing that
T. cruzi
upregulates the expression of profibrotic transcription factors in HCM early during the process of cellular infection and the operational fibrotic interactome. Thus, abnormal sustained expression of SNAI1 and EGR1 upregulate the expression of genes essential for conversion of HCM towards a profibrotic phenotype in CHD. Elucidation of the molecular mechanisms by which
T. cruzi
induces cardiac fibrosis will lead to the identification of new therapeutic targets for CHD.
Abstract 318: Identification of Novel Cardiac Fibroblast Enriched Genes
Authors: Swonger Jessica M , Tallquist Michelle D
Published: July 2021

The leading cause of death in the United States is heart disease. While current therapies have reduced mortality, patients surviving the initial stages of cardiac injury are left with long-term disruption of heart function including fibrosis. Pathological fibrosis in the heart is caused by excess proliferation and deposition of extracellular matrix primarily by cardiac fibroblasts (CFs). One factor required for cardiac fibroblast formation is the basic helix-loop-helix transcription factor, TCF21 (epicardin/Pod1/capsulin). Previous studies from our lab have shown that
Tcf21
null embryos lack CFs. Our current work focuses on identifying genes downstream of TCF21. Deep-sequencing identified over one hundred differentially expressed genes when comparing embryonic hearts from
Tcf21
nulls to wild types. We have verified a subset of these differentially expressed genes by qPCR and have demonstrated that these genes are also expressed in cultured primary cardiac fibroblasts. Future work will focus on determining the function of these genes during fibroblast activation and determine which of these genes are directly regulated by TCF21. The elucidation of cardiac fibroblast specific genes and their function will provide much needed information for identification of therapeutic targets aimed at cardiac fibroblast activation.
Abstract 32: LincRNA-p21 Regulates Neointima Formation and Atherosclerosis by Enhancing p53 Activity
Authors: Wu Gengze , Cai Yue , Yang Yujia , Xu Zaicheng , Wang Wei Eric , Chen Ken , Zeng Chunyu
Published: July 2021

Background:
Long non-coding RNAs (lncRNAs) recently have been implicated in many biological processes and diseases. Atherosclerosis is a major risk factor for cardiovascular disease. However, the functional role of lncRNAs in atherosclerosis is largely unknown.

Methods and Results:
The downstream targets of p53 also been detected by un-bias mRNA array and specific qPCR. Furthermore, bioinformatics prediction, RNA-Immunoprecipitation, biotin-labeled RNA-pulldown and deletion mapping were performed to test the potential interaction between lincRNA-p21 and MDM2. Co-IP, genome-wide ChIP-seq and ChIP-qPCR were preformed to verify the possible influence on p53 transcriptional activity. We further investigated the function of lincRNA-p21 in vivo, therefore recombinant lincRNA-p21 knockdown lentivirus were injected into the injured area of carotid arteries injury mouse model. Then the expression of lincRNA-p21 in vascular samples of clinical coronary disease patients were also detected. We identified lincRNA-p21 as a key regulator of atherosclerosis. Expression of lincRNA-p21 was dramatically down-regulated in atherosclerotic plaques of ApoE-/- mice, an animal model for AS. Through loss- and gain-of function approaches, we showed that lincRNA-p21 represses cell proliferation and induces apoptosis in VSMCs in vitro. Moreover, we found that inhibition of lincRNA-p21 results in neointimal hyperplasia in vivo in a carotid artery injury model. Mechanistically, we revealed that lincRNA-p21, which is a transcriptional target of p53, feeds back to enhance p53 transcriptional activity via binding to mouse double minute 2 (MDM2), an E3 ubiquitin-protein ligase. The association of lincRNA-p21 and MDM2 releases MDM2 repression of p53, enabling p53 to interact with p300 to regulate apoptosis, cell proliferation and suppress neointima formation.

Conclusions:
Our studies identified lincRNA-p21 as a novel regulator of cell proliferation and apoptosis and suggest that this lncRNA could serve as a therapeutic target to treat atherosclerosis.
Abstract 321: Obesity Causes Enhanced Sensitivity Of The Mitochondrial Permeability Transition Pore
Authors: Bernal-Ramírez Judith , Riojas-Hernández Adriana , Morales-Marroquín Flor E , Domínguez-Barragán Elvía M , Rodríguez-Mier David , García-Rivas Gerardo , Altamirano Julio , García Noemí
Published: July 2021

Several mechanisms have been implicated in heart failure (HF) development due to obesity, including altered Ca2+ homeostasis and mitochondrial increased reactive oxygen species (ROS).

Besides their metabolic role, mitochondria are important cell death regulators, since their disruption induces apoptosis. The mitochondrial permeability transition pore (MPTP) formation is key in this process. Ca2+ and ROS are known inducers of MPTP, and mitochondria are the main ROS generators. However, it has not been demonstrated that MPTP formation is involved in cardiac cell death due to obesity. Therefore, the aim of this work was to determine whether Ca2+ alterations and/or MPTP opening underlie cardiac dysfunction.

We used obese Zucker fa/fa rats (32 weeks old), displaying concentric hypertrophy and cardiac dysfunction. We measured: i) Systolic and diastolic Ca2+ signaling in isolated myocytes, in basal conditions and upon β-adrenergic stimulation (β-AS), and ii) in vitro mitochondrial function: respiration, ROS production and MPTP opening.

We found that the main alteration in Ca2+ signaling in fa/fa myocytes was a decrease in SERCA Ca2+ removal capacity, since Ca2+ transient amplitude and spark frequency were unchanged. Furthermore, in fa/fa myocytes, β-AS response was preserved. On the other hand, fa/fa mitochondria respiration, in state 3 decreased, but was unchanged in state 4, when glutamate/malate were used as substrate, resulting in an small decrease in respiratory control. In addition, fa/fa mitochondria were more sensitive to MPTP opening, induced by Ca2+ and carboxyatractiloside (CAT). Moreover, fa/fa mitochondria showed increased H2O2 production, and in exposed thiol groups in the adenine nucleotide translocase, a regulatory MPTP component.

Since Ca2+ signaling is relatively normal in fa/fa cells, it does not seem to be the main contributor to the cardiac contractile dysfunction. However, given that fa/fa mitochondria showed decrease respiratory performance, were more susceptible to MPTP opening, and showed enhanced H2O2 production. We conclude that fa/fa mitochondria were more vulnerable to enhanced oxidative stress, causing MPTP opening, which could be exacerbated by SERCA slower Ca2+ removal capacity, leading to myocyte apoptosis.