bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 165: The 18 kDa FGF-2 Prevents the Doxorubicin-induced Cardiac Damage and Amp-activated Kinase Activation, in vitro and in vivo
Authors: Koleini Navid , Santiago Jon Jon , Nickel Barbara E , Fandrich Robert , Jassal Davinder S , Kardami Elissavet
Published: July 2021

Introduction:
Protection of the heart from chemotherapeutic (Doxorubicin, DOX) drug-induced toxicity is a desirable goal, to limit side effects of cancer treatments. DOX toxicity has been linked to the activation (phosphorylation) of the AMP-activated kinase, AMPK. The 18 kDa low molecular weight isoform of fibroblast growth factor 2 (Lo-FGF-2) is a known cardioprotective and cytoprotective agent. In this study we have tested the ability of Lo-FGF-2 to protect from DOX-induced damage in rat cardiomyocytes in vitro, and in transgenic mouse models in vivo, in relation to AMPK activation.

Methods:
Rat neonatal cardiomyocytes in culture were exposed to DOX (0.5 μM) in the presence or absence of pre-treatment Lo-FGF-2 (10 ng/ml). Compound C was used to block phosphorylation (activity) of AMPK. Levels of cell viability/death (using Calcein-AM/Propidium iodide assay), phospho -and total AMPK, and apoptotic markers such as active caspase 3 were analyzed. In addition, transgenic mice expressing only Lo-FGF2, and wild type mice, expressing both high molecular weight (Hi-FGF2) as well as Lo-FGF2 were subjected to DOX injection (20 mg/kg, intraperitoneal); echocardiography was used to examine cardiac function at baseline and at 10 days post-DOX.

Results:
DOX-induced cell death of cardiomyocytes in culture was maximal at 24 hours post-DOX coinciding with significantly increased in activated (phosphorylated) AMPK. Compound C attenuated DOX-induced cardiomyocyte loss. Pre-incubation with Lo-FGF-2 decreased DOX induced cell death, and also attenuated the phosphorylation of AMPK post-DOX. Relative levels of phospho-AMPK were lower in the hearts of Lo-FGF2-expressing male mice compared to wild type. DOX-induced loss of contractile function (left ventricular ejection fraction and endocardial velocity) was negligible in Lo-FGF2-expressing mice but significant in wild type mice.

Conclusion:
Lo-FGF-2 protects the heart from DOX-induced damage in vitro and in vivo, by a mechanism likely involving an attenuation of AMPK activity.
Abstract 179: Targeted Inhibition of Plasminogen Activator Inhibitor-1 Attenuates Weight Gain and Prevents Vascular Dysfunction Following a High Fat Diet
Authors: Khan Sadiya S , Mackie Alexander , Beussink-Nelson Lauren , Kamide Christine E , Henkel Anne S , Place Aaron T , Eren Mesut , Lloyd-Jones Donald , Shah Sanjiv J , Miyata Toshio , Vaughan Douglas E
Published: July 2021

Introduction:
Elevated plasminogen activator inhibitor-1 (PAI-1) is associated with obesity, but there is controversy whether PAI-1 causes or is a consequence of obesity. We sought to determine whether targeted PAI-1 inhibition with a novel small molecule antagonist (TM5441) alters the development of obesity and/or obesity-induced vascular dysfunction in a diet-induced obesity model.

Methods and Results:
C57BL/6J mice were fed control, high fat diet (HFD), or high fat diet with TM5441 (HFD+TM5441) for 12 weeks. The HFD had marked weight gain (77±5%) as compared with control (32±2%). TM5441 significantly attenuated weight gain (49±8%, p=0.0075, Figure). HFD-induced hepatic triglyceride accumulation was attenuated by TM5441 (116±31 vs. 76±35 mg trig/g liver, p=0.03). Energy expenditure was reduced in the HFD compared to control (11.1±0.4 vs. 12.9±0.4 kcal/h/kg, p=0.005). However, HFD+TM5441 maintained a level of energy expenditure that was similar to control (13.2±0.6 kcal/h/kg, p=NS). The HFD group demonstrated higher systolic and diastolic blood pressure (141±3; 112±3 mm Hg) compared with control (122±7, 94±8; P<0.05 for both), while administration of TM5441 prevented diet-associated increase (120±6; 93±7 mm Hg, p=NS compared to control) at week 12. Pressure myography of mesenteric arteries in the HFD showed a significant rightward shift in the constrictor response to phenylephrine as compared to control (EC50: 14.5uM vs. 25.1uM, p=0.002). The HFD+TM5441 was similar to control (p=NS).

Conclusions:
Inhibition of PAI-1 with TM5441 attenuates weight gain, enhances energy expenditure, and prevents obesity-related vascular dysfunction in a murine model of obesity.
Abstract 199: Dissecting the Shox2-nkx2-5 Antagonistic Mechanism in the Pulmonary Vein Myocardium and Sinoatrial Node
Authors: Ye Wenduo , Wang Jun , Song Yingnan , Yu Diankun , Sun Cheng , Liu Chao , Chen Fading , Zhang Yanding , Wang Fen , Harvey Richard , Schrader Laura , Martin James , Chen YiPing
Published: July 2021

Atrial fibrillation is often triggered by ectopic pacemaking activity in the myocardium sleeves of the pulmonary vein (PV) and systemic venous return. However, the genetic programs that abnormally reinforce pacemaker properties at these sites and how this relates to normal sinoatrial node (SAN) development remain uncharacterized. We have identified a Shox2-Nkx2-5 antagonistic mechanism that primes the pacemaking cell fate in the PV myocardium and SAN in the embryonic stage. Specifically, Shox2 deletion in the Nkx2-5+ domain of the SAN caused sick sinus syndrome, associated with the loss of pacemaker program. Nkx2-5 hypomorphism rescued the requirement for Shox2 for the expression of genes essential for SAN development in Shox2 mutants. Similarly, the pacemaker-like phenotype induced in PV myocardium in Nkx2-5 hypomorphs reverted back to a working myocardial phenotype when Shox2 was simultaneously deleted. Shox2 interacts with Nkx2-5 directly, and a substantial genome wide co-occupancy of Shox2, Nkx2-5, and Tbx5 suggest a balanced transcription output is essential for the pacemaker cell fate determination. Further characterization of mice carrying allelic series of Shox2 and Nkx2-5 revealed an essential role of both Shox2 and Nkx2-5 in maintain the normal development of pulmonary vein myocardium and the structures in the venous pole, suggesting the importance of transcriptional precision of Shox2 and Nkx2-5 and a role of Shox2-Nkx2-5 antagonistic mechanism in integrating the transcriptional accuracy of Shox2 or Nkx2-5.
Abstract 200: Novel Cardiac Dyssynchrony Metrics Based on Ultrasound Images and Fibrosis
Authors: Shearer Greg , Hanson Todd , Eclov Julie , Courtier Sam , Ortmeier Steve , Ewert Dan
Published: July 2021

Ventricular stiffening is a component of heart failure with preserved ejection fraction. This study focuses on building a dyssynchrony metric based on ultrasound images obtained from normal and fibrotic mouse hearts. The objective is to evaluate the association between regional fibrosis and ventricular wall dynamics as an indicator for the gradient of cardiac dyssynchrony.

Ultrasound images were obtained from a previous study in which mice were given thoracic aortic constriction to induce heart failure that included ventricular stiffening and fibrosis. Ultrasound video (frame rate 4.3ms) along the short axis of the mouse heart was captured using commercial software. On each short axis frame, six points were selected and traced on the endocardial wall. Each radial displacement pair is plotted in a loop diagram against one another in 15 unique combinations.

A loop whose points are close to the
ideal line
u = v
corresponds to synchronous wall movement, while the area enclosed by the loop is proportional to time dyssynchrony. The inferior free wall and the section nearest the interventricular septum had 35% (26,48) interstitial fibrosis compared to the IS itself which had 9% (7, 13) in hearts with 25% fibrosis, and the contrast had the most variance (8.7%, P<0.0001). Strong predictors of the contrasting fibrosis included the trace perimeter and the maximal normalized displacement, but more pragmatically, the differences between the two conditions are apparent and consistent.

Figure 1 is the set of (u,v) pairs measured over the course of a heartbeat where u is the radial displacement of heart segment x measured when segment y had radial displacement v.
Abstract 202: Genetic Ablation of Interleukin-18 Does Not Attenuate Hypobaric Hypoxia-Induced Right Ventricular Dysfunction
Authors: Bruns Danielle R , Buttrick Peter M , Walker Lori A
Published: July 2021

Interleukin-18 (IL-18), a pro-inflammatory cytokine, has been implicated in pathogenic left ventricular hypertrophy and is elevated in plasma of heart failure patients. However, IL-18 blockade strategies in animal models of heart disease have been conflicting. Accordingly, the purpose of these experiments was to determine whether genetic ablation of IL-18 would protect male and female mice against hypobaric hypoxia-induced right ventricular (RV) dysfunction. We hypothesized that IL-18 knockout mice (KO) would be protected while wild type (WT) mice would show significant right ventricular dysfunction in response to exposure to hypobaric hypoxia (HH). KO and WT mice were exposed to HH for 7 weeks, and control (CO) mice were exposed to normoxic, ambient air. Following echocardiography, the RV was dissected and flash frozen for biochemical analyses. 7 week HH exposure trended toward an increase in IL-18 mRNA (p=0.08) in RV from WT mice. However, contrary to our hypothesis, IL-18 KO mice were not protected against HH-induced RV dysfunction, as evidenced by higher RV weights, elevated RV systolic pressure, and increased RV anterior wall thickness compared to normoxic KO mice. Importantly, these measurements were not significantly different from WT HH mice. Biochemical analyses suggest HH RV underwent early remodeling, as no changes were observed at the molecular level in the RV of HH mice compared to CO in either KO or WT animals. Compensatory upregulation of the other pro-inflammatory cytokines IL-2 and SDF-1 may have contributed to the lack of protection in IL-18 KO animals. These data suggest IL-18 signaling is not necessary for hypobaric hypoxia induced RV dysfunction, and blockade of IL-18 is not a viable therapeutic strategy in this model.
Abstract 208: Parkin Contributes to the Development of Cardiac Hypertrophy in Response to Cardiac Pressure Overload
Authors: Shires Sarah E , Kubli Dieter A , Gonzalez Eileen R , Purcell Nicole H , Gustafsson Åsa B
Published: July 2021

Parkin is an E3 ubiquitin ligase known to mediate mitochondrial clearance by marking damaged mitochondria for autophagy. Our lab has previously shown that Parkin is important for stress adaptation following myocardial infarction, and that loss of Parkin leads to accumulation of dysfunctional mitochondria. However, whether Parkin plays a role in cardiac adaptation to pressure overload is currently unknown. Here we investigated the functional importance of Parkin in cardiac hypertrophy and development of heart failure in response to hemodynamic stress. Wild type (WT), Parkin knock out (Parkin
-/-
), and cardiac-specific Parkin transgenic (Parkin-TG) mice were subjected to trans-aortic constriction (TAC). Cardiac anatomy and function was evaluated by histology and echocardiography. Inflammation and hypertrophy gene expression profiles were assessed using qPCR and immunohistochemistry. We discovered that after 2 weeks of TAC, cardiac hypertrophy markers were not increased in hearts from Parkin
-/-
mice, and there was no increase in the heart weight to body weight ratio (HW/BW). However, after 8 weeks of TAC, Parkin
-/-
mice showed similar cardiac hypertrophy and loss of function as WT hearts. Parkin deficient hearts also displayed increased interstitial and perivascular fibrosis compared to WT hearts after 8 weeks of TAC. This suggests that there is a delay in activating the hypertrophy program in the absence of Parkin, and that lack of Parkin leads to excessive fibrosis. In contrast, Parkin-TG mice showed a rapid development of hypertrophy and progression to heart failure compared to WT mice. Interestingly, we observed no differences in either mitochondrial content or LC3 levels after two weeks of TAC in Parkin-TG hearts, suggesting that the rapid development of hypertrophy and early progression to heart failure was not due to excessive mitophagy. These data suggest that Parkin plays an important role in the activation of the cardiac hypertrophy program and that this function may be independent of its role in regulating mitophagy. Thus, this study provides novel insight into the functional importance of Parkin in the heart. Additional studies are needed to determine the mechanism of how Parkin regulates cardiac hypertrophy.
Abstract 211: Intercellular Adhesion Molecule 1 Regulates Cardiac Remodeling and Function and T Cell Recruitment to the Heart in Pressure Overload Induced Heart Failure
Authors: Salvador Ane Miren , Nevers Tania , Aronovitz Mark , Blanton Robert , Alcaide Pilar
Published: July 2021

Background:
Left ventricular (LV) dysfunction and Heart Failure (HF) are associated in humans with systemic inflammation, including increased circulating levels of pro-inflammatory cytokines and soluble intercellular cell adhesion molecule-1 (ICAM-1). Endothelial ICAM-1 regulates leukocyte recruitment into tissues, which in the heart can result in altered cardiac function. We
hypothesize
that ICAM-1 regulates cardiac remodeling by mediating leukocyte recruitment to the LV and thus contributing to worsening of cardiac function during pressure overload induced HF.

Methods and results:
We used the mouse model of Thoracic Aortic Constriction (TAC) to induce LV remodeling and HF in WT and ICAM-1 deficient mice (ICAM-1
-/-
). Immunohistochemistry, flow cytometry, qPCR, echocardiography and hemodynamics were used to investigate leukocyte infiltration into the LV, cardiac function, hypertrophy and fibrosis mechanisms taking place in response to TAC. Endothelial ICAM-1 was upregulated in WT mice in response to TAC as compared to Sham, correlating with LV T cell infiltration. In contrast, CD3+ and CD4+ T cell recruitment into the LV was significantly reduced in response to TAC in ICAM-1
-/-
mice as compared to WT mice. Further, indices of sistolic and diastolic function were preserved in ICAM-1
-/-
mice (dP/dt
max
= WT TAC 5,627±549 vs. ICAM-1
-/-
TAC 8,396±1,495 ; dP/dt
min
= WT TAC -5,614±1,195 vs. ICAM-
1-/-
TAC -8,832±2,274) and the End Diastolic Pressure was significantly lower than in WT TAC mice (31.0±7.0mmHg in WT TAC vs 8.1±7.8mmHg in ICAM-1
-/-
TAC). Despite increased LV weight, ICAM-1
-/-
did not develop fibrosis in response to TAC, with blunted collagen deposition and lack of mRNA upregulation of fibrotic markers Collagen-I, TGFβ and SMAα four and ten weeks after TAC when dilated cardiomiopathy is established in WT mice.

Conclusion:
Our data indicate that ICAM-1 regulates LV T cell infiltration, cardiac function and fibrosis in HF induced by TAC. Further studies will determine whether ICAM-1 contributes to HF pathogenesis exclusively by regulating T cell interactions with the LV endothelium or participating in novel mechanisms regulating cardiac cell function, which could represent new targets for the treatment of this deadly syndrome.
Abstract 217: Anti-fibrotic Function of Relaxin
Authors: Lindner Diana , Becher P. Moritz , Hinrichs Svenja , Schwarzl Michael , Fluschnik Nina , Blankenberg Stefan , Westermann Dirk
Published: July 2021

Introduction:
The RELAX in Acute Heart Failure (RELAX-AHF) trial was done in patients admitted for acute decompensated heart failure to evaluate the efficacy of Serelaxin (human recombinant relaxin-2) on dyspnoea relief, as well as its safety and tolerability. Interestingly, the 180-day mortality was significantly better in the Serelaxin group compared to placebo. The pathophysiology of these beneficial effects remains elusive.

Methods and results:
To induce heart failure in mice, they were treated with Angiotensin II in absence or in presence of relaxin. After 21 days hemodynamic measurements revealed an improved hemodynamic function in relaxin treated animals with AngII-induced heart failure compared to mice without relaxin treatment.

To further investigate the , cardiac fibroblasts were stimulated with pro-fibrotic TGF-β in the presence or in the absence of relaxin. Additionally as a control cardiac fibroblasts were treated with relaxin alone. The treatment with TGF-β induced an increased gene expression of connective tissue growth factor (CTGF) as well as monocyte chemotactic protein 1 (MCP-1) in cardiac fibroblasts, whereas treatment with relaxin alone decreased the gene expression level of CTGF as well as MCP-1 . To investigate a potential inhibitory function of relaxin during TGF-β induced pro-fibrotic gene expression regulation, cardiac fibroblasts were pre-incubated with relaxin 1 hour prior to TGF-β treatment. After the following 6 hours of TGF-β treatment no decreased gene expression was observed in relaxin pre-treated cardiac fibroblasts compared to TGF-β stimulated fibroblasts without relaxin treatment.

Conclusion:
In this study we could demonstrate no inhibitory effect of relaxin during the pro-fibrotic TGF-β-induced signaling pathway. Nevertheless, relaxin alone induced anti-fibrotic function. Therefore, we conclude that the anti-fibrotic function is not due to an influence on the TGF-β induced pro-fibrotic signaling.
Abstract 226: p21-activated kinase-1 (Pak1) Mediates Exercise-induced Cardiac Remodeling Through Calcineurin Signaling
Authors: Davis Robert T , Simon Jillian N , Ke Yunbo , Wolska Beata M , Solaro R. John
Published: July 2021

Aims:
Despite its known cardiovascular benefits, the intracellular signaling mechanisms underlying physiological cardiac growth remain poorly understood. Therefore, the purpose of this study was to investigate a novel role of p21-activated-kinase-1 (Pak1) in the regulation of exercise-induced cardiac hypertrophy.

Methods & Results:
Wild-type and Pak1 KO mice were subjected to six weeks of treadmill endurance exercise-training (ex-training). Cardiac function was assessed via echocardiography, in situ hemodynamics, and the pCa-force relations in skinned fiber preparations at baseline and at the end of the training regimen. Post-translational modifications to the sarcomeric proteins and expression levels of calcium-regulating proteins were also assessed following ex-training. HW/TL and echocardiography data revealed there was marked hypertrophy following ex-training in the WT mice, which was not evident in the KO mice. Additionally, following ex-training, WT mice demonstrated an increase in cardiac contractility, myofilament calcium sensitivity, phosphorylation of cMyBP-C, cTNT, and TM compared to KO mice. The improvement in contractility with ex-training was accompanied by increased protein levels of SERCA2a and calcineurin along with increased phosphorylation of phospholamban.

Conclusions:
Our data suggest that Pak1 is essential for adaptive physiological cardiac remodeling and support previous evidence that demonstrate Pak1 signaling is important for cardiac growth and survival.
Abstract 230: Cartilage Intermediate Layer Protein-1 Attenuates Pressure Overload-induced Cardiac Fibrosis by Targeting Transforming Growth Factor-β1
Authors: Li Li , Zhang Cheng-Lin , Wu Dan , Wu Li-Ling
Published: July 2021

Background:
Cartilage intermediate layer protein-1 (CILP-1), a monomeric extracellular matrix glycoprotein expressed mainly in the middle zones of articular cartilage, interacts directly with transforming growth factor-β1 (TGF-β1). Recent studies showed that CILP-1 was upregulated in the heart tissue following cardiac ischemia reperfusion injury. However, the role of CILP-1 in pathological cardiac remodeling is poorly defined.

Aims:
To explore the effect of CILP-1 on myocardial interstitial fibrosis and reveal the possible molecular mechanism.

Methods and Results:
We found that CILP-1 was mainly expressed in mouse cardiac fibroblasts (CFs) by using western blot analysis and immunofluorescence. Myocardial expression of CILP-1 was upregulated in mice subjected to transverse aortic constriction (TAC) for 2, 4, and 8 weeks. AAV-9-mediated delivery of CILP-1 into mice increased the binding of CILP-1 with TGF-β1, attenuated interstitial fibrosis, and improved cardiac function. In cultured adult mouse CFs, CILP-1 overexpression inhibited myofibroblast differentiation and expression of profibrotic molecules induced by TGF-β1. Furthermore, CILP-1 attenuated TGF-β1-induced Smad3 phosphorylation and nuclear translocation.

Conclusions:
CILP-1 alleviates pressure overload-induced cardiac fibrosis and dysfunction. CILP-1 exerts its anti-fibrotic effect through targeting TGF-β1 signaling. This study will offer a new therapeutic strategy for preventing and treating myocardial interstitial remodeling.
Abstract 234: Mitofilin Directly Interacts With Cyclophilin D in the Inner Membrane of Heart Mitochondria
Authors: Bopassa Jean C.
Published: July 2021

Introduction:
We recently found that acute estrogen treatment delays the mitochondrial permeability transition pore (mPTP) opening and reduces ROS production after ischemia/reperfusion, suggesting that estrogen promotes mitochondrial integrity. As mitochondrial inner membrane protein (mitofilin) has been found to control mitochondrial cristae morphology and function, and cyclophylin-D is a mitochondrial peptidyl-prolyl isomerase known to modulate the opening of the mPTP, we investigated whether a direct interaction between these two proteins exist in the inner membrane of mitochondria.

Methods:
Western blot analysis and immunoprecipitation were performed in isolated heart mitochondrial fractions from male WT (C57BL/6NCrL) mice using mitofilin and cyclophilin-D monoclonal antibodies of. Mitofilin and cyclophylin-D distribution in isolated cardiomyocytes as well as in single mitochondria was also visualized using high resolution microscopy.

Results:
Western blot analysis shows that both mitofilin and cyclophilin-D proteins were abundantly expressed in the heart mitochondria. Immunoprecipitation with mitofilin antibody pulled down cyclophilin-D, which was detected by Western blot analysis. Co-immunoprecipitation with cyclophylin-D antibody confirms its direct association with mitofilin as mitofilin was detected by Western analysis in the immunoprecipitated product. High resolution microscopy revealed a higher degree of colocalization between mitofilin and cyclophylin-D in both cardiomyocytes and mitochondrial fractions.

Conclusion:
Together, these data indicate that mitofilin directly interacts with cyclophylin-D in the inner membrane of heart mitochondria. As mitofilin plays an important role in controlling mitochondrial cristae morphology and cyclophylin-D, known to modulate the opening of the mPTP, this interaction between mitofilin-cyclophilin-D may have an impact in the opening of the mPT pore.
Abstract 236: Cardiac Overexpression of Thioredoxin-1 Delays Mitochondrial Damage and Prolongs Survival in Septic Mice
Authors: Sanchez-Villamil Juana P , D’Annunzio Veronica , Rebagliati Ines , Finocchietto Paola , Peralta Jorge , Morales Celina , Gelpi Ricardo J , Poderoso Juan J , Carreras Maria C
Published: July 2021

Introduction:
Current evidence suggests a main role of thioredoxin-1 (trx1) in the protection against oxidative stress. However, it is unknown yet a putative role of trx1 in the regulation of contractility and mitochondrial function, dynamics and biogenesis in sepsis-induced myocardial dysfunction in which oxidative stress is an underlying cause.

Methods:
Transgenic male mice (Tg-trx1) and its wildtype (wt) were subjected to cecal ligation and double puncture or sham operation. After 6, 18 and 24 h, antioxidant enzymes, protein carbonylation and mitocondrial function were evaluated. Hearts were isolated and perfused using the Langendorff technique. Inotropism was evaluated through left ventricular developed pressure (LVDP) and contractile reserve after β-adrenergic stimulus by addition of isoproterenol (ISO) (1 μM). Evaluation of mitochondrial fusion-fission dynamics (Drp1, Mfn2, Opa1) and biogenesis (PGC-1α, NRF-1 and TFAM) were made by real-time qPCR.

Results:
Over the time course of sepsis, there was an improvement in average life expectancy in Tg-trx1 (Tg-trx1: 36, wt: 28 h; p=0.0204), and 15 percentages points higher β -adrenergic response at 6 h compared to wt (22.8 vs 7.8%, means of relative percentage differences respectively). Inhibition of complex I activity, protein oxidation, and loss of membrane potential was lower in Tg-trx1, with a sustained MnSOD activity at 24 h. mRNA levels of Opa1 were significantly reduced in sepsis, accompanied by ultrastructural alterations in mitochondrial cristae, while significant Drp1 activation (measured by Ser P616 phosphorylation) was observed in wt mice at 24 h. PGC-1 α mRNA decreased at 6 h in both groups, and then, showed a 2.5-fold increase in Tg-trx1 at 24 h. Autophagy was demonstrated by increased LC3-II/LC3-I ratios in both groups during the progression of sepsis, although the levels were early higher in Tg-Trx1. No indicators of apoptosis were observed.

Conclusions:
The results show that mice with cardiac-specific overexpression of Trx1 have higher survival. Trx1 provides protection against oxidative stress, which seems to preserve contractile reserve and keep mitochondrial biogenesis. Associated with mitochondrial autophagy-processes allow mitochondrial renewal and life extension.
Abstract 242: Mitophagy is Required for Mitochondrial Biogenesis and Myogenic Differentiation of Myoblasts
Authors: Sin Jon , Andres Allen , Taylor David J , Stotland Aleksandr , Huang Chengqun , Gottlieb Roberta A
Published: July 2021

Myogenesis is a crucial process governing muscle development and homeostasis. Differentiation of primitive myoblasts into mature myotubes requires a metabolic switch to support the increased energetic demand of contractile muscle. Skeletal myoblasts specifically shift from a highly glycolytic state to relying predominantly on oxidative phosphorylation (OXPHOS) upon differentiation. We have found that this phenomenon requires dramatic remodeling of the mitochondrial network involving both mitochondrial clearance and biogenesis. During early myogenic differentiation, autophagy is robustly upregulated and this coincides with DNML1/DRP1-mediated fragmentation and subsequent removal of mitochondria via p62/SQSTM-mediated mitophagy. Mitochondria are then repopulated via PPARGC1A/PGC-1α-mediated biogenesis. Mitochondrial fusion protein OPA1 is then briskly upregulated, resulting in the reformation of mitochondrial networks. The final product is a myotube replete with new mitochondria. Respirometry reveals that the constituents of these newly established mitochondrial networks are better primed for OXPHOS and are more tightly coupled than those in myoblasts. Additionally, we have found that blocking autophagy with various inhibitors during differentiation results in a blockade in myogenic differentiation. Together these data highlight the integral role of autophagy and mitophagy in myogenic differentiation.
Abstract 27: Psychometric Testing of Instruments for Use in Women’s Cardiovascular Recovery Globally
Authors: Gonzales Lucia , Anne Koci , Gee Rose Mary , Noji Ariko , Glaser Dale , Marsh Allison , Marsh Kathy K , Altman Amara M , Al Salmi Nasser , Al Sabei Sulaiman
Published: July 2021

Worldwide, 3.4 million women die each year from cardiovascular disease. After experiencing a cardiovascular event, a woman’s physical health, the women’s likelihood of being treated with coronary artery bypass graft surgery, likelihood for referral for cardiac rehabilitation are less favorable than men. An established conceptual model depicts psychosocial stressors and the influence of behavioral risk factors on the pathogenesis of atherosclerosis and the occurrence of CV events. The woman’s social stressors of role quality and behavioral risk factors of low self-efficacy for physical activity are associated with physical and mental health outcomes following cardiovascular crises. The study aimed to evaluate the reliability of the translated versions (Japanese, Ukrainian, Tagalog, Hispanic and Arabic) of the worker, partner, mother Role Quality and the Self Efficacy of Lifestyle Physical Activity indices among 282 women (aged 35-92 years) representing seven cultures. The study was performed in a multi-center, multicultural context. Translations followed an established process. Results showed reliability was strong (coefficient alphas of 0.93 and 0.88). These instruments underwent first-time confirmatory factor analyses with acceptable, though borderline fit, thus providing valuable input for strengthening in future studies. Understanding a woman’s role quality and self-efficacy for lifestyle physical activity provides valuable information that assists health-care professionals to co-develop with the woman an individualized plan to reduce role stress and to initiate increased physical activity following cardiovascular episodes.
Abstract 270: The Eya4/six1 Signalling Cascade is Activated in Acquired Heart Disease
Authors: Williams Tatjana , Oppelt Daniel , Nordbeck Peter , Voll Sabine , Schoenberger Jost , Ritter Oliver
Published: July 2021

Rationale:
We previously identified a mutation in the human transcriptional cofactor Eya4 as cause of familial dilated cardiomyopathy (DCM). We now provide evidence that the Eya4/Six1 signalling cascade also is crucial in acquired heart disease.

Hypothesis:
We hypothesize that the transcriptional complex Eya4/Six1 regulates targets relevant in normal cardiac function. We speculate it, amongst others, regulates expression of p27kip1, a known inhibitor of hypertrophy in adult cardiomyocytes, upon hypertrophic stimuli.

Methods and results:
We first examined the correlation of Eya4 and p27 in regards to phosphorylation and cellular distribution in failing and normal human hearts. Immunhistology revealed Eya4 is mainly distributed in the cytoplasm while p27 predominantly resides in the nucleus of healthy myocardial tissue. In sections of failing human hearts, Eya4 accumulated in the perinuclear and nuclear region; nuclear p27 levels were significantly diminished, phosphorylated p27 was evenly distributed in the cytoplasm. In a murine model of MI, IH showed Eya4 translocates in a time-dependent manner. WB analyses for p27 showed an age dependent decrease in p27 protein levels upon MI compared to control littermates. We generated transgenic mice with constitutive myocardial overexpression of Eya4 and E193. As judged by MRI, hemodynamic and morphometric analysis both transgenic mouse models developed cardiac phenotypes compared to age-matched wildtype littermates already under basal conditions in an age dependent manner. p27 expression and downstream factors were also altered in both transgenic lines as a result of Eya4, and accordingly, E193 overexpression. In summary, we provide evidence that the Eya4/Six1 signalling cascade is not only relevant in a rare version of heritable DCM but also in more common forms of acquired heart disease. Eya4/Six1 seems to regulate p27, which was shown to be an important regulator of cardiac physiology in postmitotic cardiomyocytes.
Abstract 271: Nos1ap Alters Qtc Intervals Upon Overexpression in Mice
Authors: Williams Tatjana , Oppelt Daniel , Arias-Loza Anahi Paula , Abesser Marco , Schmitt Joachim , Schuh Kai , Ritter Oliver
Published: July 2021

Rationale:
The QT interval duration (QTc) reflects cardiac depolarization. It may predispose individuals to ventricular tachycardia and sudden cardiac death if prolonged (long QTc), shortened (short QTc) or otherwise unregularly. Whole-genome association studies have linked genetic variations in the neuronal nitric oxide synthase adapter protein NOS1AP to variations in QTc intervals and sudden cardiac death.

Hypothesis:
We hypothesize NOS1AP functions as an L-type-Ca2+ channel modulator via its interaction with the neuronal nitric oxide synthase NOS1. Therefore, alterations in myocardial NOS1AP expression should temper with QTc intervals and increase susceptibility to rhythm disorders.

Methods and results:
We generated conditional double transgenic mice by crossbreeding pTRE-6xHN-Nos1AP animals with α-MHC-tTA mice; NOS1AP expression is therefore restricted to cardiomyocytes and under control of doxycycline (Tet-Off system). Double transgenic animals were investigated with the main focus upon electrical alterations. Heart rates were similar in NOS1AP overexpressing and non-induced animals. Atrial programmed stimulation repeatedly caused atrial tachycardia, while ventricular programmed stimulation caused VT in NOS1AP overexpressing mice. There was a clear decrease of QTc intervals in NOS1AP overexpressing mice paralleld by a significantly reduced survival (only 56% after 12 weeks vs 100% in non-induced mice. Induced QTc alterations and accompanied deaths subsided upon readministration of doxycycline.

We also investigated the functional effect of the human SNP rs16847548 (T/C). We found that this SNP decreased NOS1AP transcriptional activity in vitro and therefore suggest this leads to a decrease in NOS1AP expression in humans.

Conclusion:
Myocardial overexpression of NOS1AP leads to short QTc syndrome with increased susceptibility to atrial and ventricular rhythm disorders and cardiac death. SNP rs16847548 in NOS1AP resulted in less NOS1AP promoter activity in vitro which could explain the alteration in QTc intervals.

In summary, not only mutations in ion channels themselves but also genetic alterations in the expression of ion channel modulators such as NOS1AP, have an impact on QTc intervals.
Abstract 274: Carvedilol Stimulated Gαi-β-Arrestin Biased β1 Adrenergic Receptor Signaling
Authors: Wang Jialu , Xiao Kunhong , Rockman Howard A
Published: July 2021

The β1 and β2 adrenergic receptors (βARs) are the predominant G-protein-coupled receptors (GPCRs) subtypes expressed in the heart. It is now appreciated that ligands can induce multiple distinct “active” receptor conformations with unique downstream functional signaling profiles or efficacies. Our current understanding of GPCR signaling is that ligands can be biased toward activating either a G protein or a β-arrestin-signaling pathway, a concept known as biased ligand signaling. To identify novel biased signaling pathways mediated by β1- and β2ARs, we performed a proteomic interactome study of β1AR and β2AR using stable isotope labeling by amino acids in cell culture (SILAC). We identified several hundred proteins that distinctly bind to the β1AR or β2AR following stimulation with the unbiased full agonist, isoproterenol, or the β-arrestin-biased ligand carvedilol. We found that stimulation by the β-arrestin-biased agonist carvedilol of only β1ARs resulted in the recruitment of Gαi. No of the other ligand tested promoted Gαi recruitment, suggesting that carvedilol may be unique in its ability to activate Gαi-biased signaling. The Gαi inhibitor pertussis toxin blocked β-arrestin-dependent extracellular signal-regulated kinase (ERK) activation and epidermal growth factor receptor (EGFR) transactivation stimulated by carvedilol, suggesting the involvement of Gαi in carvedilol-induced β-arrestin biased signaling of β1ARs. Using β1AR/β2AR chimeric receptors we show that the C-terminal tail of the β1AR is required for carvedilol stimulated Gαi recruitment, but is unable to rescue the lack of Gαi recruitment by the β2AR. Since our current conceptual framework for biased signaling is based on the “bar code hypothesis”, ongoing phosphoproteomic experiments are testing whether recruitment of Gαi by carvedilol induces a distinct phosphorylation pattern on the c-tail of the β1AR. Our study shows that activation of β-arrestin-biased signaling requires G proteins for signaling and provides a new mechanistic understanding of how biased β-blockers can activate signaling.
Abstract 275: The Effect of High Salt-diet on Pioglitazone Treated-db/db-/- mice
Authors: Obih Patience O , Ezebuenyi Michael C , Jimoh Akeem P , Obih John-Clifford A
Published: July 2021

The objective of this study was to evaluate the impact of high-salt-diet on pioglitazone treated db/db mice. Groups of 6 weeks-old db/db mice obtained from Jackson Laboratory were given either pioglitazone (0.02%) in diet or pioglitazone (0.02%) plus high salt-diet (8% NaCl) for 6 to 12 weeks. Control groups received either pioglitazone or normal salt diet. During the course of treatment, urine volume, urine sodium, creatinine and blood glucose were measured in the animals. For urine collection, mice were placed in metabolic cages. In order to evaluate whether differences exist between diabetic animals that received pioglitazone or not in the handling of a sodium load and to characterize the transport mechanisms involved, at the end of experimental period the animals were given an acute sodium load (physiological saline), 1.25 ml/100 g body weight by intraperitoneal route. The sodium load was repeated in mice that was treated 15 minutes earlier with hydrochlorothiazide (40 mg/kg i.p.) or furosemide (8 mg/kg i.p.), or amiloride (1.65 mg/kg). The animals were placed in metabolic cages and urine voided was collected over 5 hr. for determination of urine volume and sodium. Sodium was determined by flame photometer. No significant changes were observed in mean arterial blood pressure in all the groups (at p 0.05). There was significant increase in UNav with all the diuretics in db/db mice that received high salt diet at 6 weeks. The blood pressure did not increase with the sodium diet. This might be due to natriuresis resulting from polyurea in the diabetic condition of the mice.
Abstract 278: Cardiac Med1 is Necessary for Postnatal Survival in Mice
Authors: Spitler Kathryn M , Ponce Jessica M , Hall Duane D , Grueter Chad E
Published: July 2021

Alterations in gene transcription are commonly associated with cardiovascular disease pathogenesis characterized by cardiomyocyte hypertrophy. The phenotypic responses result in diminished cardiac contractility, ventricular dilation, fibrosis and ultimately sudden death. The mediator complex is a crucial facilitator of gene transcription; however, few studies have investigated the role of mediator in cardiovascular disease initiation and progression. A key subunit of the Mediator complex, MED1, interacts with nuclear receptors to target gene-specific transcription. To determine the role of MED1 in regulating cardiac function, we generated a heart specific knockout of MED1 (cMED1KO). Postnatal deletion of MED1 in mice results in lethality between 3 to 6 weeks of age. The cMED1KO mice display a marked increase in heart mass compared to floxed controls. Furthermore, echocardiography and histological analysis of hearts taken at 3 weeks showed that the cMED1KO animals had decreased cardiac function, increased fibrosis and a dilated left ventricle. Transcriptional changes were observed for key markers of cardiac disease including MYH7, ANF, ACTIN1. We performed RNAseq analysis to identify changes in the transciptome between cMED1KO and floxed control hearts. The analysis unveiled changes in expression of genes regulating cardiac development, metabolism and function. Taken together these results reveal a critical role for MED1 in postnatal cardiac growth and development due to altered gene expression in adult cardiomyocytes.
Abstract 280: Cardiac Disease Status Dictates Functional mRNA Targeting Profiles of Individual microRNAs
Authors: Matkovich Scot J , Dorn Gerald W , Grossenheider Tiffani C , Hecker Peter J
Published: July 2021

Background:
Gain- and loss-of-function experiments have demonstrated that microRNAs are key players in cardiac stress responses, but the mRNAs whose abundance and/or translational potential are primarily affected by changes in cardiac microRNAs are not well characterized. Stimulus-induced, large-scale alterations in the cardiac transcriptome, together with consideration of the law of mass action, further suggest that the mRNAs most substantively targeted by individual microRNAs vary between unstressed and stressed conditions. To test the hypothesis that microRNA target profiles differ in health and disease, we traced the fate of miR-133a and miR-378 targets in mouse hearts undergoing pressure-overload hypertrophy.

Methods and Results:
Ago2 immunoprecipitation with RNA-sequencing (RISC-sequencing) was used for unbiased definition of microRNA-dependent and -independent alterations occurring amongst ~13,000 mRNAs in response to transverse aortic constriction (TAC), in response to the microRNA overexpression needed to define miR-133a and miR-378 targets and to track their fate, and in response to the combined stimuli. Of 51 direct targets of miR-133a defined in unstressed hearts (fold-change ≥25%, FDR<0.02), only 8 (16%) continued to be targeted by miR-133a during TAC, while for miR-378 direct targets, 17 of 28 (61%) targets were maintained during TAC. Similarly, only 9% (for miR-133a) and 58% (for miR-378) of hundreds of indirectly affected mRNAs underwent comparable regulation, demonstrating that the significant effect of TAC on microRNA direct target selection resulted in wider alterations of signaling function. Numerous microRNA-mediated regulatory events occurring exclusively during pressure overload revealed signaling networks likely responsive to the endogenous decreases in miR-133a and miR-378 during TAC.

Conclusions:
Pressure overload-mediated changes in overall cardiac RNA content alter microRNA targeting profiles, reinforcing the need to define microRNA targets in tissue-, cell- and status-specific contexts. Such considerations need to be taken into account for tailoring of therapeutic manipulation of microRNAs toward the most appropriate microRNAs active under defined stimuli.
Abstract 281: Loss of Krueppel-like Factor 15 (KLF15) Leads to Altered Wnt-dependent Gene Regulation in Hearts With Systolic Dysfunction
Authors: Noack Claudia , Iyer Lavanya M , Zafiriou Maria-Patapia , Zimmermann Wolfram-Hubertus , Zelarayán Laura C
Published: July 2021

Background & Aim:
Developmentally conserved pathways, such as the Wnt/β-catenin pathway, are upregulated in adult heart diseases. Here, we aimed to elucidate the role of Wnt regulation via KLF15 for adult heart homeostasis.

Results:
We identified the transcription factor KLF15, a factor previously identified to contribute to cardiac fibrosis and hypertrophy, as a cardiac specific Wnt/β-catenin inhibitor. Accordingly, a constitutive Klf15 KO mouse model revealed specific cardiac upregulation of the Wnt target genes Tcf7l2 and cMyc (n=6, p<0.001). Serial echocardiography showed reduced systolic function starting at 16 weeks in the Klf15 KO hearts (n=10, p<0.05). Genome-wide sequencing studies of Klf15 KO vs. WT hearts at the postnatal age day P10, week 4 and 20 showed that gene expression differs increasingly at 4 and 20 weeks of age, respectively. This is in line with the increasing KLF15 expression after birth, reaching significant level at P13 (n=3/embryonal, fetal, and neonatal stage; each 5-8 pooled hearts). Accordingly, gene set enrichment analysis revealed activation of the Wnt signaling in Klf15 KO hearts at 4 and 20 weeks but not at P10 (p<0.001). This activation was milder in 20 weeks vs. 4 weeks, which may be explained by a concomitant upregulation of Wnt repressors, as Shisa3 and Dact3 with so far unknown function in the mammalian heart (n=9, p<0,01). Wnt activation was accompanied by upregulation of developmental genes and transcriptional regulators at 4 weeks and followed by upregulation of stress factors such as ANP, BNP, and Ankrd1 at 20 weeks. Moreover, we found the Wnt targets TCF7L2 and AXIN2 significantly upregulated in human ventricular biopsies from dilated (n≥5, p<0.05) and ischemic cardiomyopathy (n≥5, p<0.001) vs. non-failing, further underscoring the relevance of the Wnt pathway for human cardiac cellular homeostasis as well.

Conclusion:
A Wnt-dependent gene signature may precede the expression of stress factors and functional deterioration due to deletion of its cardiac repressor KLF15. Our data indicate that KLF15 is an important regulator of the Wnt pathway and is relevant for cardiac homeostasis and function in the postnatal heart.
Abstract 310: Vascular Endothelial Growth Factor and Endothelial Nitric Oxide Synthase-mediated Regulation of Cardiomyocyte Proliferation During Development in Humans and Mice
Authors: Gentile Carmine , Chuang Christine Y , Drake Christopher J , Davies Michael J
Published: July 2021

Our previous studies in early mouse embryonic development (E8.2) showing that vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) are expressed in embryonic endothelial cells, but not in embryonic cardiomyocytes, together with the findings by others indicating that NO is required for cardiomyocyte proliferation in mice, led us to investigate the relevancy of the VEGF/eNOS signaling pathway during cardiovascular development. First, wild type and NOS3 knockout mouse embryos between E8.0 and E17.0 were stained with antibodies against smooth muscle α-actin, phospho-histone H3 (PH3), VEGFR2 and PECAM, markers of cardiomyocytes, proliferating, progenitor and endothelial cells, respectively. Our confocal analysis showed hearts of E8.0 NOS3 nulls develop normally. However, E8.5 and E9.5 NOS3 nulls have reduced cardiomyocyte proliferation and impaired heart development. As consequence, hearts of E17 NOS3 nulls were approximately 20% smaller compared to wildtype hearts. To translate our findings to humans, we stained human heart specimens with antibodies against VEGFR2, eNOS, PH3 and sarcomeric α-actinin. Confocal analyses showed for the first time that VEGFR2 is highly expressed in the perinuclear region of human cardiomyocytes of a young donor. They also showed a correlation between eNOS expression and cardiomyocyte proliferation in humans. Consequently, we developed an in vitro three-dimensional co-culture model of human endothelial cells, cardiomyocytes and fibroblasts: “human cardiac tissue spheroids” (HCTSs). Our data showed that laminin and collagen type IV synthesis is increased in VEGF-treated HCTSs generated using CMs from an older donor compared to untreated cultures, suggesting a role for VEGF and eNOS in postnatal human heart development. In conclusion, our data showed that VEGF and eNOS play a similar role in mediating cardiomyocytes proliferation and heart regeneration in both mice and humans. Current studies are focusing on evaluating molecular targets of the VEGF/eNOS signalling pathway in human proliferating cardiomyocytes, which may have significant therapeutical impact for stem cell differentiation, and prevention of cardiovascular complications such as myocardial infarction.