bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 398: Alternative Splicing of Titin Restores Diastolic Function and Improves HFpEF Associated Symptoms
Authors: Bull Mathew , Methawasin Mei , Strom Joshua , Nair Pooja , Hutchinson Kirk , Granzier Henk
Published: June 2021

Greater than half of all heart failure (HF) patients suffer from increased diastolic stiffness and impaired relaxation of the left ventricle while their ejection fraction (EF) is preserved (HFpEF). HFpEF is a complex disease characterized by diastolic dysfunction, exercise intolerance, and concentric hypertrophic remodeling. No effective therapy exists for treating this pervasive disease due in part to our limited understanding of the underlying pathophysiology. Titin, the largest known protein and molecular spring in the heart, has emerged as a prime candidate for therapeutic targets aimed at restoring compliance to the sarcomere in order to improve diastolic function.

Titin has two main cardiac isoforms; the smaller N2B isoform (~3.0 MDa) and the larger more compliant N2BA isoform (~3.3 MDa). Diastolic stiffness of the left ventricle is in large part dependent upon the N2BA:N2B isoform ratio. Modification of these two primary isoforms can be achieved post-transcriptionally through inhibiting the known titin splicing factor Rbm20. Alternative splicing of messenger RNA occurs primarily in the spring region of titin and therefore regulates the elastic property of titin through changes in contour length of the spring.

The current study evaluates whether reduction of titin splicing factor Rbm20 has a beneficial effect on the diastolic function of mice with restrictive cardiomyopathy. A previously published mouse model deficient in titin’s IA junction (

Ttn
ΔIAjxn

) places increased strain on the spring region of titin and acts as a mechanical analogue of the titin-based increase in passive myocardial stiffness found in HFpEF patients. This model exhibits a HFpEF-like phenotype apparent as early as 3 months in the adult mouse that becomes exaggerated with age apart from pharmacological or surgical intervention. We report for the first time a rescue of HFpEF in the mouse through inhibiting Rbm20. We demonstrate that Rbm20 reduction restores diastolic function, improves exercise tolerance and attenuates afterload induced pathologic remodeling of the left ventricle. No specific FDA approved therapies exist to treat the pathologically stiff left ventricle, therefore our findings on Rbm20 inhibition are clinically significant.
Abstract 400: Role of the 17beta-Estradiol/beta-Catenin Axis in Heart Failure
Authors: Kararigas Georgios , Zelarayan Laura C , Toischer Karl , Hasenfuss Gerd , Jarry Hubertus , Regitz-Zagrosek Vera
Published: June 2021

The role of β-catenin and the actions of 17β-estradiol (E2) in cardiovascular (patho)physiology remain unclear. In the diseased heart, E2 is expected to be cardioprotective. However, in the absence of prior disease, complications in pregnancy or transgender individuals indicate that E2 might lead to cardiovascular disease. We aimed at the characterization of the effects of the E2/β-catenin axis in the healthy C57Bl/6N heart. Two-month-old female C57Bl/6N wild-type (WT) and cardiac-specific β-catenin-deleted (β-cat
Δex2-6
) mice were ovariectomized and randomized to an E2-containing or soy-free (control, CON) diet (
n
= 7-13/group). The 3-month physiological dose of E2 led to a significant repression of nuclear β-catenin levels vs. CON (
P
< 0.001) in WT mice. This was underlain by a significant decrease in the heart weight to tibia length ratio and cardiomyocyte cross-sectional area (E2 vs. CON WT mice
P
< 0.001). Echocardiography revealed a significant decrease in septum width (
P
< 0.001), posterior wall thickness (
P
< 0.01) and fractional shortening (
P
< 0.05) in E2 vs. CON WT mice. Deletion of β-catenin abrogated these E2-mediated deleterious effects on cardiac structure and function. Mechanistically, we found a significant increase in the levels of the ubiquitin ligase and key regulator of proteasome-dependent protein degradation muscle-specific RING finger protein 1 (MuRF1) in E2 vs. CON WT mice (
P
< 0.05), while there was no effect in β-cat
Δex2-6
mice. Although we also hypothesized increased autophagic activity, we found no effect on the autophagy-related protein LC3 in WT or β-cat
Δex2-6
mice. In a translational approach, we profiled the left ventricular transcriptome of female patients (
n
= 5) with end-stage non-ischemic dilated cardiomyopathy (DCM) vs. healthy controls (
n
= 8). We found a significant induction in the expression of the genes coding for
SFRP5
, a negative regulator of Wnt signaling,
DACT2
, an antagonist of β-catenin, and
APC2
, which is required for targeted degradation of β-catenin (
P
< 0.01), in DCM samples. Our mouse data indicate that repression but not deletion of β-catenin leads to heart failure and our human data suggest that targeting Wnt/β-catenin signaling in heart failure may be of therapeutic value.
Abstract 401: The γ2-AMPK Regulates Glucose and Glycogen Metabolism in the Heart by Modulating Cytosolic Hexokinase II Level
Authors: Kim Maengjo , Garcia-Menendez Lorena , Choi Yong Seon , Roos Birthe , Wamelink Mirjam M , Gu Haiwei , Abell Lauren , Raftery Daniel , Palmiter Richard D , Tian Rong
Published: June 2021

Mutations in the γ2-subunit of AMPK (encoded by PRKAG2 gene) cause glycogen storage cardiomyopathy. We generated a mouse model with cardiac specific deletion of γ2-AMPK (cKO) to define its function in the heart. The cKO mice were born normal and remained indistinguishable from control mice under baseline conditions but developed greater cardiac hypertrophy and dysfunction after transverse aortic constriction (TAC). The total AMPK activity was normal in cKO hearts and it increased after TAC to the same extent as that of the controls. However, the γ2-AMPK activity was abolished in the cKO and the overall AMPK activity was maintained by a compensatory increase in the γ1-AMPK activity. The lack of γ2-AMPK selectively reduced hexokinase II in the cytosol resulting in decreases in glycogen content and NADPH levels. Knock-down of γ2-AMPK subunit in neonatal rat ventricle cardiomyocytes resulted in increased oxidative stress and exacerbated response to phenylephrine and H
2
O
2
, both were rescued by HKII overexpression. Taken together, these data identify isoform-specific functions of γ2-AMPK in regulating glucose and glycogen metabolism that are critical for cardiac response to stress. The results also indicate that the specific activation of γ2-AMPK is responsible for the glycogen storage cardiomyopathy associated with PRKAG2 mutations.
Abstract 404: FK506-binding Protein 38 Protects the Heart From Pressure Overload-induced Cardiac Dysfunction via Endoplasmic Reticulum-associated Apoptosis
Authors: Misaka Tomofumi , Murakawa Tomokazu , Nishida Kazuhiko , Otsu Kinya
Published: June 2021

Backgrounds:
FK506-binding protein 38 (FKBP38) is a membrane chaperone with its peptidyl-prolyl cis-trans isomerase activity, localized to mitochondria and endoplasmic reticulum. It has been reported that chaperone systems in the heart play a protective role against the toxicity of misfolded proteins during the development of heart failure, but the pathophysiological effect of FKBP38 in the heart has not been investigated.

Methods and Results:
To study the role of FKBP38 in the heart, we have generated cardiac-specific FKBP38 knockout (KO) mice. KO mice had no effect on cardiac function compared to wild type (WT) littermates under unstressed conditions at 8 to 10 weeks old. The mice were subjected to transverse aortic constriction (TAC) or Sham surgeries. Echocardiography demonstrated that KO mice showed significantly decreased fractional shortening and increased end-diastolic left ventricular dimension compared to WT mice 1 week after TAC (20.7
+
2.6 vs 39.3
+
2.4%, 3.92
+
0.4 vs 3.32
+
0.4 mm, P<0.01, respectively). TAC-operated KO mice exhibited similar heart weight/tibial length (11.1
+
0.2 vs 11.8
+
0.3 mg/mm, P=NS) but significantly higher lung weight/tibial length (13.7
+
1.0 vs 9.4
+
0.3 mg/mm, P<0.01) compared to TAC-operated WT, indicating that KO mice had more progressive left ventricular dysfunction with pulmonary congestion in response to pressure overload. TUNEL staining revealed that the numbers of TUNEL positive nuclei of cardiomyocytes were dramatically increased in KO mice compared to WT mice after TAC (140
+
13 vs 25
+
9 per 10
5
nuclei, P<0.01). To understand further mechanisms of apoptosis, we estimated the activation of caspase 12, which is responsible for ER stress-induced apoptosis. The expression levels of cleaved caspase 12 were significantly elevated in KO mice compared to WT mice after TAC. Moreover, the expression levels of ER stress markers such as 94 kDa glucose-regulated protein, 78 kDa glucose-regulated protein and protein disulfide-isomerase were significantly upregulated in KO mice compared to WT mice after TAC.

Conclusion:
Our findings demonstrate for the first time that FKBP38 plays protective effects in the heart against pressure overload-induced cardiac dysfunction via endoplasmic reticulum-associated apoptosis.
Abstract 407: Overexpression of the Valosin-containing Protein in vivo Prevents Cardiac Hypertrophy and Heart Failure by Chronic Pressure Overload via Suppressing the Activation of mTORC1 Pathway
Authors: Zhou Ning , Ma Ben , Hays Tristen T , Qiu Hongyu
Published: June 2021

Aims:
Pressure overload induced cardiac hypertrophy is a key risk factor for heart failure. Although several defined interventions result in a significant inhibition of cardiac hypertrophy, the functional consequences are controversial. Identification of novel targets modulating the cardiac hypertrophy without adversely affecting cardiac function is particularly crucial to the treatment of heart failure. Here we test our hypothesis that the valosin-containing protein (VCP) is a novel mediator of cardiac protection against cardiac hypertrophy and heart failure by pressure overload.

Methods and Results:
Pressure overload was induced by transverse aortic constriction (TAC) in a mouse model to mimic the progression of cardiac hypertrophy and heart failure. Cardiac structure and function were measured by echocardiography and hemodynamic analysis. VCP expression was significantly reduced in wild type (WT) mice after 2 weeks TAC at both the mRNA and protein levels by 40% and 45 % respectively and even more markedly reduced after 5 weeks TAC (68% in mRNA and 73% in protein, all,
P
<0.01 vs sham). Cardiac overexpression of VCP in a transgenic (TG) mouse did not alter either cardiac structure or function at baseline condition. However, compared to 2 week TAC WT mice, VCP TG mice showed a significant repression of cardiac hypotrophy, evidenced by a significant reduction in the ratio of left ventricle (LV) /tibial length (TL) by 36%, LV posterior wall thickness by 20%, and cardiomyocyte cross sectional area by 39% (all
P
<0.05 vs WT). After 5 weeks of TAC, while WT mice progressed to cardiac failure, VCP TG mice exhibited preservation of cardiac function in terms of ejection function (EF,72±1% vs 52±4.1% in WT) and Lung weight /TL ratio (8.0±0.8mg/mm vs 9.8±0.8 mg/mm in WT) (
P
<0.05 vs WT). Induction of fetal cardiac genes in TAC WT, e.g. ANP and BNP, was significant suppressed in VCP TG mice (
P
<0.05 vs WT). TAC induced activation of mammalian target of rapamycin complex 1 (mTORC1), e.g., an increase of phosphorylation of mTOR and S6K1, was significantly blunted in VCP TG mice vs WT after TAC (
P
<0.05 vs WT).

Conclusion:
Overexpression of VCP in vivo prevents the progression of cardiac hypertrophy and dysfunction upon pressure overload by modulating mTORC1 signaling pathways.
Abstract 414: Putative Role of MicroRNA-143 in Modulating Natriuretic Peptide Signaling via Down-regulation of the Expression of Natriuretic Peptide Receptor 3
Authors: Wang Juan , Wong Lee L , Richards Arthur M , Chen Yei-Tsung
Published: June 2021

Cardiac natriuretic peptides (NPs) play important roles in the regulation of intravascular blood volume and vascular tone. Among other clearance mechanisms, bio-active circulating NPs are removed by the clearance receptor, Natriuretic Peptide Receptor 3 (NPR3). We hypothesized that the level of NPR3 could be modulated by microRNAs (miRNAs) resulting in changes in the bioactivity of NPs. We have previously reported a cluster of miRNAs potentially regulating NPR3 expression. To extend these findings, expression of the microRNAs concerned was examined in multiple platforms, including plasma from a clinical heart failure cohort, in the rat myocardial infarction model, and in a human cardiac derived cell line subjected to hypoxic challenge.

Results:
miR-143 was up-regulated in peripheral blood in heart failure patients compared with controls. The binding of miR-143 to the 3’UTR of NPR3 m RNA was verified by luciferase reporter assay. Antagomir-based silencing of miR-143 enhanced NPR3 expression in human derived cardiac cells. Elevation of miR-143 and down-regulation of NPR3 levels were observed in hypoxia treated cells and in the myocardium from the rat myocardial infarction model. Taken together, these findings suggest miR-143 may be involved in the down-regulation of NPR3 which in turn may provide more cardiac protective bioactivity from NPs in heart failure, myocardial hypoxic stress and in myocardial infarction. In summary, NPR3 is negatively regulated by miR-143, pointing to the therapeutic potential of miR-143 to beneficially enhance NP responses.
Abstract 417: BET Bromodomain Inhibition Blunts Heart Failure Progression and Fibrosis in Genetic Cardiomyopathy
Authors: Burke Michael A , Wakimoto Hiroko , Gorham Joshua M , Conner David A , Seidman Jonathan G , Bradner James E , Haldar Saptarsi M , Brown Jonathan D , Seidman Christine E
Published: June 2021

Pathologic gene network activation is a hallmark of DCM and HF. We have used a chemical genetic strategy to study the role of the bromodomain and extraterminal (BET) family of epigenetic reader proteins in the regulation of gene expression in DCM. Transgenic mice carrying a human DCM mutation in phospholamban (PLN
R9C
) develop profound fibrosis and DCM that progresses to symptomatic HF and early death. Temporal RNA-seq and pathway analysis identified pro-fibrotic gene expression as a key early driver of DCM progression. PLN
R9C
and age-matched wild type (WT) mice were treated daily with the potent and specific BET inhibitor JQ1 or vehicle from 8-weeks-of-age (preDCM) to 20-weeks-of-age. Vehicle-treated PLN
R9C
mice developed DCM with severely reduced LV function and negative cardiac remodeling. JQ1-treated PLN
R9C
mice had substantially better fractional shortening (27% vs. 15%, p=7x10
-10
) and less negative remodeling (LV end diastolic diameter 3.9mm vs. 4.5mm, p=1x10
-5
; LV wall thickness 0.69mm vs. 0.62mm, p=6x10
-6
) compared to PLN
R9C
vehicle-treated mice at 20-weeks (n=15). Myocardial fibrosis was also significantly reduced by JQ1 (5.8% PLN
R9C
-JQ1 vs. 18.4% PLN
R9C
-vehicle, p=3x10
-5
; n=3). RNA-seq in preDCM mice treated with JQ1 for 2 weeks demonstrated a decrease in pathologic gene activation. JQ1-treated PLN
R9C
mice versus vehicle treated mice had reduced expression of natriuretic peptides (
Nppa
-31%,
Nppb
-29%; p<0.001), less myosin heavy chain and actin isoform switching (ratio
Myh6
/
Myh7
PLN
R9C
-JQ1=12.2; PLN
R9C
-vehicle=8.8; ratio
Actc1
/
Acta1
PLN
R9C
-JQ1=39.3; PLN
R9C
-vehicle=12.1; p<0.001), and lower pro-inflammatory gene expression (
Tgfb2
-48%,
Tgfb3
-41%,
Ctgf
-21%,
Gdf15
-56%; p<0.001). At 20-weeks-of-age, continued treatment with JQ1 resulted in sustained suppression of cytokine expression with 2-fold lower levels of
Tgfb2
and
Tgfb3
compared to vehicle-treated PLN
R9C
mice. These data suggest that early induction of pro-fibrotic signaling is a key driver of pathologic remodeling in PLN
R9C
DCM. This study also identifies epigenetic control of pathologic gene activation by BET bromodomain proteins as a critical mechanism for cardiomyopathy progression and supports BET inhibition as a possible novel therapeutic strategy for HF.
Abstract 422: CITED4 is Sufficient to Induce Physiologic Cardiac Hypertrophy and Necessary to Protect Against Pathological Hemodynamic Stress
Authors: Lerchenmueller Carolin , Bezzeridis Vassilios J , Platt Colin , Xiao Chunyang , Rosenzweig Anthony
Published: June 2021

Cardiac hypertrophy is an adaptive response to increased physiologic or pathologic hemodynamic stress. Previous work from our laboratory suggested that the CEBPβ/ CITED4 pathway plays an important role in exercise-induced cardiac hypertrophy. Consistent with this model, our laboratory recently found that inducible cardiac expression of CITED4 in adult mice increases heart weight and cardiomyocyte size with normal systolic function and a gene expression profile consistent with physiologic growth. After ischemia-reperfusion injury (IRI), induced CITED4 mice show significant functional recovery and evidence for decreased adverse remodeling. Next, we sought to investigate the role of CITED4 in the setting of physiologic (forced swimming exercise) and pathological (transverse aortic constriction, TAC) cardiac hypertrophy. Cardiomyocyte-specific CITED4 knockout mice (C4KO) undergoing a three week swimming exercise protocol showed modestly but significantly reduced systolic function when compared to control animals (%FS controls 55.4±1.09 vs. C4KO 51.75±0.86; p=0.025). C4KO mice exposed to TAC demonstrated a more rapid and severe decline in cardiac function after TAC (at 6 weeks post surgery, %FS controls 41.55±2.06 vs. C4KO 32.51±2.67; p=0.024). Both in vitro and in vivo we demonstrate that CITED4 is necessary and sufficient for activation of mTOR signaling. Of note, mTORC1 inhibition by rapamycin abrogated the beneficial effects of CITED4 expression after IRI. Taken together, our data identify CITED4 as a novel regulator of mTOR signaling. Moreover they demonstrate that CITED4 is sufficient for physiologic growth and to protect against adverse remodeling after ischemic injury. CITED4 is also necessary for adaptive responses to pathological biomechanical stress and may represent a novel therapeutic target to mitigate adverse ventricular remodeling.
Abstract 425: Doxorubicin Upregulation of the Fibrotic Transforming Growth Factor-beta Pathway
Authors: Ramirez Trevi A , Aune Greg
Published: June 2021

Childhood cancer survivors are at an increased risk of heart disease as a result of their cancer treatments. Drugs like doxorubicin (DOX) are an effective part of treatment regimens, but have been proven to cause acute and chronic cardiotoxicity (DOX tox). An under-investigated aspect of DOX tox is the interstitial fibrosis that the majority of patients develop. This project aims to better understand the pathology of DOX-induced cardiac fibrosis and the role of the pro-fibrotic transforming growth factor-beta (TGFb) signaling pathway. Research in the area of fibrosis and the effect of DOX on cardiac fibroblasts will increase our understanding of DOX tox. This understanding will allow for improved treatment of pediatric cancer patients by reducing the cardiotoxic sequelae of many standard chemotherapy regimens.

Cardiac fibroblasts, isolated from 3 week old mice and treated with 5 μM DOX, showed an increase in nuclear pSMAD compared to control cells via fluorescent immunocytology (2.06 ± 0.26 vs 1.13 ± 0.15, p<0.05). Mice treated with 3 mg/kg DOX injections from 2 weeks to 6 weeks of age showed increased TGFb staining in the left ventricle (1.83 ± 0.34 vs 0.87 ± 0.28, p<0.05) a week after treatment ceased. A subset of mice were followed into old age and sacrificed at 80 weeks. A clear increase in TGFb was seen with age. However, 80 week mice that were exposed to DOX early in life showed a greater increase in TGFb staining compared to untreated 80 week old mice (44.50 ± 2.48 vs 30.93 ± 2.30, p<0.001).

Early DOX exposure causes chronic molecular changes as evidenced by acute and chronic changes in signaling molecules in cardiac tissue. Changes in collagen seen in earlier studies and increases in MMP-2 from the literature suggest a cardiac remodeling phenotype in DOX-exposed animals. This project demonstrates that DOX initiates changes to pro-fibrotic pathways, seemingly driven by the TGFb signaling pathway.
Abstract 426: Laminin Downregulation Facilitates Cardiomyocyte Coupling
in situ
to Increase Contractile Function
Authors: Sessions Ayla , Kaushik Gaurav , Engler Adam
Published: June 2021

Aging is associated with extensive remodeling of the heart, including basement membrane extracellular matrix (ECM) components that surround cardiomyocytes. Remodeling is thought to contribute to impaired cardiac mechanotransduction, but the contribution of specific basement membrane ECM components to age-related cardiac remodeling is unclear, owing to current model systems being complex and slow to age. To investigate the effect of basement membrane remodeling on mechanical function in genetically tractable, rapidly aging, and simple model organisms, we employed
Drosophila melanogaster,
which has a simple trilayered heart tube composed of only basement membrane ECM.

We observed differential regulation of collagens between laboratory
Drosophila
strains
,
i.e.
yellow-white
(
yw
) and
white-1118
(

w
1118

), leading to changes in muscle physiology, which were linked to severity of dysfunction with age. Therefore, we sought to understand the extent to which basement membrane ECM modulates lateral cardiomyocyte coupling and contractile function during aging. Cardiac-restricted knockdown of ECM genes
Pericardin
,
Laminin A
, and
Viking
in
Drosophila
prevented age-associated heart tube restriction and increased contractility, even under viscous load. Most notably, reduction of
Laminin A
expression decreased levels of other genes that co-assemble in ECM, leading to overall preservation of contractile velocity and extension of median organismal lifespan by 3 weeks or 39%. These data provide new evidence of a direct link between basement membrane ECM homeostasis, contractility, and maintenance of lifespan.
Abstract 428: Pharmacologic and Activated Fibroblast-specific Gβγ-GRK2 Inhibition is Protective Following Ischemia Reperfusion Injury
Authors: Travers Joshua G , Kamal Fadia A , Nieman Michelle L , Sargent Michelle A , Molkentin Jeffery D , Blaxall Burns C
Published: June 2021

Heart failure is a devastating disease characterized by chamber remodeling, interstitial fibrosis and reduced ventricular compliance. Cardiac fibroblasts are responsible for extracellular matrix homeostasis, however upon injury or pathologic stimulation, these cells transform to a myofibroblast phenotype and play a fundamental role in myocardial fibrosis and remodeling. Chronic sympathetic overstimulation induces excess signaling through G protein βγ subunits and ultimately the pathologic activation of G protein-coupled receptor kinase 2 (GRK2). We hypothesized that Gβγ-GRK2 inhibition plays an important role in the cardiac fibroblast to attenuate pathologic myofibroblast activation and cardiac remodeling. To investigate this hypothesis, mice were subjected to ischemia/reperfusion (I/R) injury and treated with the small molecule Gβγ-GRK2 inhibitor gallein. While animals receiving vehicle demonstrated a reduction in overall cardiac function as measured by echocardiography, mice treated with gallein exhibited nearly complete preservation of cardiac function and reduced fibrotic scar formation. We next sought to establish the cell specificity of this compound by treating inducible cardiomyocyte- and activated fibroblast-specific GRK2 knockout mice post-I/R. Although we observed modest restoration in cardiac function in cardiomyocyte-specific GRK2 null mice, treatment of these mice with gallein resulted in further protection against myocardial dysfunction following injury, suggesting a functional role in other cardiac cell types, including fibroblasts. Activated fibroblast-specific GRK2 knockout mice were also subjected to ischemia/reperfusion injury; these animals displayed preserved myocardial function and reduced collagen deposition compared to littermate controls following injury. Furthermore, systemic Gβγ-GRK2 inhibition by gallein did not appear to confer further protection over activated fibroblast-specific GRK2 ablation alone. In summary, these findings suggest a potential therapeutic role for Gβγ-GRK2 inhibition in limiting pathologic myofibroblast activation, interstitial fibrosis and heart failure progression.
Abstract 43: SIRT1 Enhances Oxidized Low-density Lipoprotein Induced Autophagy in Human Valve Interstitial Cells
Authors: Li Huadong , Shi Jiawei , Qiao Weihua , Dong Nianguo , Qin Gangjian
Published: June 2021

Objectives:
Sirt1, an NAD
+
-dependent class III deacetylase, has been shown to regulate autophagy during cardiac remodeling. Its role in calcific aortic valve disease has not been studied.

Methods:
Aortic valve samples were collected from patients with calcific aortic valve diseases complicated with hyper-lipidemia (at the time of heart valve replacement) (CAVD group), and from patients with dilated cardiomyopathy with non-calcified valves and a normal blood lipid profile (at the time of heart transplantation) (control group); the expression and distribution of calcification and Sirt1 in the aortic valves were analyzed by immuno-histochemical staining and Western blotting. In addition, primary human valvular intersititial cells (VICs) were isolated from normal valves in control group, and their response to DiI-ox-LDL treatments was analyzed in the presence of adenovirus-mediated Sirt1 over-expression (Ad-SIRT1) and Sirt1 inhibitor EX527, followed by assessments of the levels of reactive oxygen species (ROS), autophagy, apoptosis and calcification with biochemical methods.

Results:
The H&E, Masson, Von Kossa and α-SMA immuno-fluorescence staining showed that the aortic valves in CAVD group were thickened by excessive elastin, collagen expression and calcium deposition, which was associated with an elevated expression of osteocalcin and a reduced expression of Sirt1, as compared to control group. In vitro, the primary VICs were incubated with different concentrations (25, 50 and 100μg/mL) of ox-LDL for 48 h. ROS levels increased in an ox-LDL dose dependent manner. In addition, ox-LDL treatment led to a slight increase in the level of LC3 and Beclin-1, autophagic death-related hMOF and acetyl-histone H4, and a significant decrease in the level of Sirt1 through Western blotting. Interestingly, co-treatment of VICs with EX527 further increased ROS generation and expression of Caspase 3, Bax, and β-Catenin, while Sirt1 over-expression by Ad-SIRT1 resulted in increased expression of LC3 and Beclin-1, decreased expression of Caspase 3, Bax, Wnt3a and β-Catenin and reduced ROS generation.

Conclusion:
Our data suggest that Sirt1 may play a protective role in ox-LDL-induced VIC autophagy and heart valve calcification.
Abstract 433: Mitochondrial Nadp-dependent Isocitrate Dehydrogenase is a Key Regulator in Vasomotor Function
Authors: Kim Cuk-Seong , Jung Saet-byel
Published: June 2021

Mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDH2) plays an essential role protecting cells against oxidative stress-induced damage. A deficiency in IDH2 leads to mitochondrial dysfunction and the production of reactive oxygen species (ROS) in cardiomyocytes and cancer cells. However, the function of IDH2 in vascular endothelial cells is mostly unknown. In this study the effects of IDH2 deficiency on mitochondrial and vascular function were investigated in endothelial cells. IDH2 knockdown decreased the expression of mitochondrial oxidative phosphorylation (OXPHOS) complexes I, II and III, which lead to increased mitochondrial ROS (mtROS). In addition, the levels of fission and fusion proteins (Mfn-1, OPA-1, and Drp-1) were significantly altered and MnSOD expression also was decreased by IDH2 knockdown. Furthermore, knockdown of IDH2 decreased eNOS phosphorylation and nitric oxide (NO) concentration in endothelial cells. Interestingly, treatment with Mito-TEMPO, an mtROS-specific scavenger, blunted mitochondrial fission, fusion and mtROS production, and reduced the IDH2 knockdown-induced decrease in MnSOD expression, eNOS phosphorylation and NO production in endothelial cells. Endothelium-dependent vasorelaxation was impaired, and the concentration of bioavailable NO decreased in the aortic ring in IDH2 knockout mice. These findings suggest that IDH2 deficiency induces endothelial dysfunction through the induction of dynamic mitochondrial changes and impairment in vascular function.
Abstract 434: Sirt4 Accelerates Ang II-induced Pathological Cardiac Hypertrophy by Inhibiting Mnsod Activity
Authors: Liu Depei , Luo Yu-Xuan , Tang Xiaoqiang , An Xi-Zhou , Xie Xue-Min , Chen Xiao-Feng , Zhao Xiang , Hao De-Long , Chen Hou-Zao
Published: June 2021

Aims:
Oxidative stress contributes to the development of cardiac hypertrophy and heart failure. One of the mitochondrial sirtuins, Sirt4, is highly expressed in the heart, but its function remains unknown. The aim of the present study was to investigate the role of Sirt4 in the pathogenesis of pathological cardiac hypertrophy and the molecular mechanism by which Sirt4 regulates mitochondrial oxidative stress.

Methods and results:
Male C57BL/6 Sirt4 knockout mice, transgenic mice exhibiting cardiac-specific overexpression of Sirt4 (Sirt4-Tg) and their respective controls were treated with angiotensin II (Ang II). At 4 weeks, hypertrophic growth of cardiomyocytes, fibrosis and cardiac function were analyzed. Sirt4 deficiency conferred resistance to Ang II infusion by significantly suppressing hypertrophic growth, and the deposition of fibrosis. In Sirt4-Tg mice, aggravated hypertrophy and reduced cardiac function were observed compared with non-transgenic mice following Ang II treatment. Mechanistically, Sirt4 inhibited the binding of manganese superoxide dismutase (MnSOD) to Sirt3, another member of the mitochondrial sirtuins, and increased MnSOD acetylation levels to reduce its activity, resulting in elevated reactive oxygen species (ROS) accumulation upon Ang II stimulation. Furthermore, inhibition of ROS with MnTBAP, a mimetic of SOD, blocked the Sirt4-mediated aggravation of the hypertrophic response in Ang II-treated Sirt4-Tg mice.

Conclusions:
Sirt4 promotes hypertrophic growth and cardiac dysfunction by increasing ROS levels upon pathological stimulation. These findings reveal a role of Sirt4 in pathological cardiac hypertrophy, providing a new potential therapeutic strategy for this disease.
Abstract 436: Mfn2 is a Nodal Point Regulatory Step That Coordinates Angiogenic Phenotype in the Vascular Endothelium
Authors: Nchaw Gladys A , Papanicolaou Kyriakos N , Parker-Duffen Jennifer L , Bonegio Ramon G , Walsh Kenneth
Published: June 2021

Recent published data suggests that endothelial metabolism regulates angiogenesis. The key regulators of mitochondrial dynamics, mitofusins (Mfn1 and Mfn2) have been linked to cellular metabolic profile in tissues with high energetic requirements but their role in endothelial cells, were mitochondria function as signaling organelles is unknown. Here, we found that Mfn2 modulates postnatal neovascularization. Endothelial deletion of Mfn2 (Mfn2
ecko
) impaired, whereas, endothelial Mfn1 deletion (Mfn1
ecko
) did not affect clinical recovery, limb blood flow reperfusion, and capillary density compared with wild-type mice. These effects were associated with reduced eNOS activation in the ischemic leg of Mfn2
ecko
mice compared with wild-type mice. Transplantation of bone marrow cells from WT to Mfn2ecko mice did not significantly improve blood perfusion in the ischemic limb of Mfn2
ecko
mice. In agreement with these results, salient features of angiogenesis such as eNOS phosphorylation, cell migration, proliferation and capillary sprout formation were significantly inhibited in Mfn2 null primary mouse lung endothelial cells (Mfn2
-/-
MLECs), whereas ablation of Mfn1 did not affect angiogenic signaling following VEGF stimulation. Because Mfn2 modulates the unfolded protein response (UPR) and the UPR machinery has been shown to generate angiomodulatory signals, we ascertained the effects of Mfn2 loss on UPR-mediated VEGF angiogenic signal transduction as potential mechanism. Mfn2 ablation sensitized Mfn2
-/-
MLECs to proapototic UPR activation following VEGF treatment according to CHOP levels. Augmented CHOP levels corresponded with reduced eNOS phosphorylation. Inhibition of the UPR PERK signaling axis with PERK siRNA or reconstitution of Mfn2 (AdMfn2) reduced CHOP levels and significantly improved eNOS activation, EC proliferation and migration in Mfn2
-/-
MLECs. Moreover, PERK siRNA or reconstitution of Mfn2 rescued the defect in capillary sprout formation in Mfn2
ecko
mice. These data suggest that Mfn2 regulate angiogenic signaling in a UPR PERK axis-dependent manner and extend the role of Mfn2 beyond mitochondrial dynamics, metabolism and ER stress to angiogenesis and reparative vascular remodeling.
Abstract 437: Increasing Cardiac Fatty Acid Oxidation Protects Against High Fat Diet Induced Cardiomyopathy in Mice
Authors: Shao Dan , Roe Nathan , Tomasi Loreta D , Braun Alyssa N , Mattos Ana , Kolwicz Stephen , Tian Rong
Published: June 2021

In the obese and diabetic heart, an imbalance between fatty acid uptake and fatty acid oxidation (FAO) promotes the development of cardiac lipotoxicity. We previously showed that cardiac specific deletion of acetyl CoA carboxylase 2 (ACC2) was effective in increasing myocardial FAO while maintaining normal cardiac function and energetics. In this study, we tested the hypothesis that ACC2 deletion in an adult heart would prevent the cardiac lipotoxic phenotype in a mouse model of diet-induced obesity. ACC2 flox/flox (CON) and ACC2 flox/flox-MerCreMer+ (iKO) after
tamoxifen
injection were subjected to a high fat diet (HFD) for 24 weeks. HFD induced similar body weight gain and glucose intolerance in CON and iKO. In isolated Langendorff-perfused heart experiments, HFD feeding increased FAO 1.6-fold in CON mice which was increased to 2.5-fold in iKO mice compared with CON on chow diet. Fractional shortening was significantly decreased in CON-HFD (32.8±2.8% vs. 39.2±3.2%, p< 0.05, n=5-6), but preserved in iKO-HFD mice (42.8±2.3%, vs. 38.5±1.4%, n=6), compared to respective chow fed controls. Diastolic function, assessed by E’/A’ ratio using tissue Doppler imaging, was significantly decreased in CON-HFD mice (1.11±0.08 vs. 0.91±0.09, p<0.05 n=5-6), while no difference was observed in iKO-HFD compared to iKO-chow (1.10±0.03 vs. 1.09±0.04, n=6). Heart weight /Tibia length ratio was significantly higher in CON than iKO mice after HFD feeding (7.19±0.22 vs. 6.47±0.28, p<0.05, n=6). Furthermore, HFD induced mitochondria super complex II, III and V instability, which was attenuated in iKO-HFD mice. These data indicate that elevated myocardial FAO per se does not cause the development of cardiac dysfunction in obese animals. In fact, enhancing FAO via ACC2 deletion prevents HFD induced cardiac dysfunction and attenuates pathological hypertrophy. These effects may be mediated, in part, by maintenance of mitochondrial integrity. Taken together, our findings suggest that promoting cardiac FAO is an effective strategy to resist the development of cardiac lipotoxicity during diet-induced obesity.
Abstract 442: Intralipid Protects the Heart in Late Pregnancy Against Ischemia/Reperfusion Injury by Reducing Cardiomyocyte Apoptosis via Mir122 Induction
Authors: Li Jingyuan , Motayagheni Negar , Barakati Neusha , Eghbali Mansoureh
Published: June 2021

The prevalence of coronary artery disease in late pregnancy (LP) has increased recently due to significant changes in women’s lifestyle patterns (age, stress, smoking, diabetes and chronic hypertension). Myocardial infarction during LP and the peripartum is associated with significant maternal mortality and morbidity compared to non pregnant women for unclear reasons. We have recently demonstrated that cardiac vulnerability to I/R injury drastically increases in LP rodents, leading to myocardial infarct size ~4 fold greater than in non-pregnant controls. We also discovered that administration of intralipid (an emulsion of soy bean oil, egg yolk phospholipids and glycerol) at reperfusion resulted in ~60% reduction in infarct size of the heart in LP rat subjected to I/R injury. However, the molecular mechanisms underlying intralipid-induced cardioprotection in late pregnancy is not clear. Here we hypothesized that intralipid protects the heart in late pregnancy by regulating the levels of specific microRNAs. The left anterior descending coronary artery was occluded in LP rats (21-22 days of pregnancy) for 45 min followed by 3 hr of reperfusion. One single bolus of PBS (control group) or 20% intralipid (intralipid group) was applied through the femoral vein 5 min before the reperfusion. The hearts of control and intralipid groups were used for microRNA microarray analysis (Ocean Ridge Biosciences). MicroRNA-microarray analysis identified MiR122 as a novel micro-RNA which its expression was strikingly upregulated more than 10 fold in the heart of LP rats in intralipid group compared to control group. miR122 regulates apoptosis in cardiomyocytes subjected to hypoxia/reoxygenation since miR122-overexpression resulted in reduced apoptosis, whereas knockdown of miR122 enhanced apoptosis. Pyruvate kinase isoform M2 (PKM2), which is known to regulate cell apoptosis in the liver, is a direct target of miR122. Our data show that PKM2 and caspase 3 are two targets of miR122 since the expression of PKM2 and capase-3 in the heats subjected to I/R was significantly lower in intralipid group compared to control group in LP. In conclusion intralipid protects the heart in late pregnancy against ischemia/reperfusion injury via inducing miR122 by targeting PKM2.
Abstract 448: The Loss of Rad GTPase Protects Against Infarction-induced Myocardial Remodeling, Scar Formation, and Decompensatory Dilation
Authors: Manning Janet R , Levitan Bryana M , Haroun Ayman , Withers Catherine N , Nagareddy Prabhakara R , Smith Jeffrey D , Haggerty Christopher M , Fornwalt Brandon , Gao Erhe , Andres Douglas A , Satin Jonathan
Published: June 2021

Rationale:
Myocardial infarction (MI) is a leading cause of death in the U.S. A non-contractile infarct compromises the overall mechanical function of the heart, reducing cardiac output and triggering decompensatory ventricular dilation. Rad GTPase, a member of the small GTPase RGK (Rem, Gem, Kir) family, is a calcium channel blocker that is endogenously expressed in the myocardium. We have previously shown that Rad deletion in mice results in increased Ca
2+
handling and a sustained non-pathological improvement in left ventricular function compared to wildtype.

Hypothesis:
Rad-ablation attenuates post-ischemic loss of function, resulting in reduced remodeling and improved long-term contractility.

Methods and Results:
We subjected Rad-deficient mice to ligation of the left anterior descending (LAD) coronary artery, and monitored cardiac function using echocardiography. We found that Rad deletion reduces both mortality and contractile dysfunction after MI, as well as ventricular dilation over five weeks. This improvement is also accompanied by preserved calcium handling in isolated myocytes. Histological and MRI examination of both
ex vivo
global ischemia and
in vivo
24 hour LAD ligated myocardium revealed that initial infarct size is comparable between knockout and wildtype. We found that Rad loss reduced scar development and elongation independent of preserving tissue viability. Investigation of inflammatory pathways to account for this revealed increased expression of the anti-inflammatory protein thrombospondin accompanied by a reduction in neutrophil infiltration into the myocardium after MI.

Conclusion:
Rad deletion results in reduced cardiac remodeling, diminished myocardial inflammation, and improved contractile function after MI.
Abstract 452: Effect of Exercise Training on Sleep Quality and Quality of Life in Post Myocardial Infarction Patients
Authors: Vohra Jayati , Madan Kushal , Sharma Manish , Sawhney J.P.S
Published: June 2021

Objectives:
To study the effect of four weeks of aerobic exercise training, on sleep quality and quality of life parameters like anxiety, depression and physical functioning in Post Myocardial Infarction (MI) patients.

Methods:
ST elevated MI patients (n=21, <65years age) were voluntarily divided into 2 groups, control group (n=11) and exercising group (n=10). The patients in experimental group followed an exercise program of 3 sessions per week for 4 weeks. The subjective quality of life, anxiety and depression levels were assessed by the SF 36 questionnaire and Hospital Anxiety & Depression Scale (HAD scale) and the sleep quality was assessed using Pittsburgh Sleep Quality Index (PSQI). Poor sleep quality and short sleep duration was defined as PSQI > 5 and total sleep time <6hrs. respectively. After 4 weeks of the program the questionnaires were repeated for all 21 patients. Wilcoxon rank order test and Student T test were used. P<0.05 was considered statistically significant

Results:
Intergroup analysis showed statically significant reduction in anxiety score (9vs0.5) and depression score (3vs0) in exercise group as compared to control group respectively. As compared to control group, exercise group also showed statistically significant improvement in the quality of life parameter score [physical functioning (750vs1000), role functioning physical (0vs400) & emotional (0vs300), energy level (215vs400), emotional well-being (400vs500), social functioning (120vs200), general health (230vs462.5) & health changes (50vs100)]. Intra group analysis also showed significant changes only in exercise group. After 4 weeks of exercise training, PSQI score improved in the experimental group from 9.5±4.11 to 1.13±0.35 (p=0.00029) indicating better sleep quality and duration. While it remained comparable to the baseline values in the control group i.e. from 9.9±4.70 to 10.33±3.87 (p value= 0.514).

Conclusion:
Four weeks of exercise program improves the quality & duration of sleep and quality of life in MI patients after 2 weeks of index event.
Abstract 468: Identification of a New Component of the Na
v
1.5 Complex: αB-crystallin Interacts with Na
v
1.5 and Regulates Ubiquitination and Internalization of Cell Surface Na
v
1.5
Authors: Huang Yuan , Wang Zhijie , Liu Yinan , Chen Qiuyun , Wang Qing k.
Published: June 2021

Mutations in cardiac Na
+
channel Na
v
1.5 cause cardiac arrhythmias and sudden death. The cardiac Na
+
channel functions as a protein complex, however, its complete components remain to be fully elucidated. A yeast two-hybrid screen identified a new candidate Na
v
1.5-interacting protein, αB-crystallin. GST-pull-down, co-immunoprecipitation and immunostaining analyses validated the interaction between Na
v
1.5 and αB-crystallin. Overexpression of αB-crystallin significantly increased peak sodium current (

I
Na

) density, and the underlying molecular mechanism is the increased cell surface expression level of Na
v
1.5 via reduced internalization of cell surface Na
v
1.5 and ubiquitination of Na
v
1.5. Knockout of αB-crystallin expression significantly decreased the cell-surface expression level of Na
v
1.5. αB-crystallin interacted with Nedd4-2, however, a catalytically inactive Nedd4-2-C801S mutant reduced the interaction between αB-crystallin and Nedd4-2 and also blocked the increase of peak I
Na
density by αB-crystallin. Na
v
1.5 mutation V1980A at the interaction site for Nedd4-2 eliminated the effect of αB-crystallin on reduction of Na
v
1.5 ubiquitination and increases of

I
Na

density. The data suggest that the interactions between αB-crystallin and functionally active Nedd4-2 and between αB-crystallin and Na
v
1.5 are critical to increased

I
Na

density by αB-crystallin. Multiple mutations in αB-crystallin have been associated with human diseases. Two mutations, R109H and R151X, eliminated the effect of αB-crystallin on

I
Na

.

This study identifies αB-crystallin as a new binding partner for Na
v
1.5. αB-crystallin interacts with Na
v
1.5 and increases

I
Na

by modulating the expression level and internalization of cell surface Na
v
1.5 and ubiquitination of Na
v
1.5, which requires the protein-protein interactions between αB-crystallin and Na
v
1.5 and between αB-crystallin and functionally active Nedd4-2.
Abstract 51: A Unique Combination of SMarTR Bioinformatics and Cardiomyocyte Screening Technology Allows for Development of Novel Cardio-protective Drugs
Authors: Pike Nirupama , Kim Kevin , Park Eunhye , Armstrong Chris , Pham Lisa , Cadag Eithon
Published: June 2021

Anthracyclines are a class of highly effective chemotherapy drugs used in the treatment of many cancers, including leukemias, lymphomas, breast, uterine, ovarian, bladder and lung cancers. Approximately 50% of childhood cancer treatment regimens include anthracyclines. Dose dependent cardiotoxicity is a major side effect of anthracyclines. Cancer survivors receiving anthracycline therapy are 15 times more likely to develop heart failure and 8 times more likely to die of cardiovascular disease. Dexrazoxane is the only clinically approved cardio-protectant that has been shown to reduce the risk of cardiotoxicity. However, it is only indicated for metastatic breast cancer and has been linked to secondary malignancies. It is, therefore, critical to develop safer and more effective cardio-protectant drugs to address this major unmet medical need. The recent advancement in generation of human cardiomyocytes from induced pluripotent stem cells (hiPSC-CMs) enables a biologically relevant setting for compound screening because hiPSC-CMs possess many similar functional characteristics as primary human cardiomyocytes and can be generated in significant quantities. We have established a platform to screen small compounds and natural product for efficacy and safety across multiple donor lines using hiPSC-CMs. Compounds to be screened were selected in collaboration with Capella Biosciences who used their SMarTR computational analysis platform to identify potential candidates for further screening in hiPSC-CMs. Using this novel drug discovery engine that combines bioinformatics-driven in-silico screening and hiPSC-CM-based compound screening platforms, we discovered four drugs that protect hiPSC-CMs from doxorubicin-induced cardiotoxicity. We used two cancer cell lines to confirm that these candidates do not interfere with doxorubicin’s anti-cancer activity. We also demonstrated that these candidates do not show acute cardiotoxicity mediated through ion channels. Thus, a unique platform combining SMarTR computational approaches and hiPSC-CMs screening technology allows for rapid identification of highly effective cardio-protective drugs.