bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 258: Progressive Diastolic Dysfunction, Perivascular Fibrosis and Left Ventricular Hypertrophy in LDL-Receptor Deficient Mice
Authors: Nachar Walid , Lee Candace , Maafi Foued , Shi Yanfen , Mihalache-Avram Téodora , Rhéaume Éric , Tardif Jean-Claude
Published: June 2021

Background and Aim:
Left ventricular diastolic dysfunction (LVDD) refers to abnormal filling of the left ventricle (LV) due to its impaired relaxation or increased stiffness. Animal models of LVDD are limited and underlying mechanisms remain largely unknown. We aimed to assess LVDD in
Ldlr
-/-
mice using imaging and gene expression measurements.

Methods:
Sixty-nine
Ldlr
-/-
mice were fed with a western-type diet supplemented with vitamin D
2
(30 U/g/day) for 20 weeks to induce LVDD. Eight normal mice were fed with a normal diet and used as control group. Serial echocardiograms were used to assess cardiac structure and function, histological analyses were done on LV sections, and RT-PCR was performed on LV samples.

Results:
Echocardiographic results show the development of LVDD over time (P values <0.05), with a modest increase in LV mass/BW and preserved systolic function. There was an increase of perivascular fibrosis (14.9%±1.0% (14.9 to 100) vs 9.0%±1.6%; (9 to 100), respectively, P<0.01) in
Ldlr
-/-
mice compared to controls as detected by Masson’s trichrome staining, which was correlated with increased mRNA expression for TGFß1 and Smad2 and 3 (P<0.05 for all). This was accompanied by numerical increases in Type I and III collagen mRNAs (
Col I
: 1.33 (1.03; 1.97) vs 1.03 (0.90; 1.11), P=0.06;
Col III
: 1.49±0.30 vs 1.00±0.10, P=0.14). The angiotensin II precursor
Agt
mRNA level was also higher (1.07±0.08 vs 0.72±0.05; P<0.01) in
Ldlr
-/-
mice compared to controls.

Conclusion:
Taken together,
Ldlr
-/-
mice fed with a western diet supplemented with vitamin D
2
develop progressive LVDD, perivascular fibrosis and mild left ventricular hypertrophy, which represents a new model of lipid-mediated diastolic dysfunction that may be used in future studies for the evaluation of novel treatments.
Abstract 260: Systemic Loss of p62 Reduces Pressure Overload Induced Cardiac Hypertrophy, Dysfunction and Apoptosis
Authors: Auger-Messier Mannix , Rezvani Khosrow , Pattison Scott
Published: June 2021

Introduction:
p62 is a pleiotropic protein with defined roles in TNFα signaling, protein aggregate formation, and protein degradation processes. Current data suggest that p62 is a stress-response protein, with increased protein levels reported in TAC, MI, I/R, and protein aggregation models of cardiac disease. To date, there has been little study on the gain- or loss- of p62 function in cardiomyocyte/cardiac pathology. Our preliminary data found that adenoviral overexpression of p62 caused cardiomyocyte hypertrophy and cytotoxicity and that p62 was upregulated by pressure-overload stress.

Hypothesis:
Loss of p62 will be cardioprotective against pressure-overload pathology.

Methods:
Systemic p62 knockout mice underwent sham or transverse-aortic constriction surgery and were studied longitudinally to 8 weeks post-surgery by echocardiography.

Results:
Hearts from p62-null mice had significantly preserved cardiac function (%Fractional Shortening) over wild-type controls. p62-deficient mice had significantly less cardiac hypertrophy (heart weight/body weight ratios and myofiber cross-sectional areas) and showed no chamber dilation (LVED) in response to pressure-overload stress, unlike wild-types. Hearts from wild-type mice showed pronounced fibrotic remodeling and induction of apoptosis (TUNEL), while p62 knockouts had significantly less collagen staining and no evidence of apoptotic stimulation. Overexpression of p62 in rat neonatal cardiomyocytes significantly inhibited proteasomal catalytic activities (>50%) and showed increased indices of cardiomyocyte cell death.

Conclusion:
Our data show that induction of p62 is deleterious
in vitro
and that loss of p62 imparts cardioprotection against hemodynamic stress
in vivo
. The beneficial phenotype observed in hearts from p62-deficient mice may be due to p62-dependent mechanisms responsible for proteasomal dysfunction and apoptosis activation.
Abstract 261: G3bp1-Gas5 Axis Regulates Glucocorticoid Receptor Signaling During Cardiac Hypertrophy
Authors: Yang Zhi , He Minzhen , Sayed Danish
Published: June 2021

Congestive heart failure is one of the leading causes of mortality in US. With persistent work overload, pathological hypertrophy progresses into an irreversible state of dysfunction and failure. Understanding the mechanisms involved in these advances is crucial to prevent adverse changes in the heart. Studies show that Glucocorticoid (Gc) signaling and -dependent gene transcription promotes cardiac hypertrophy and fibrosis. High serum cortisol levels have been shown as independent risk factor for increased mortality in patients with cardiac failure. Gc activates cytosolic Glucocorticoid receptor (GR) that translocates to nucleus and regulates gene transcription by binding to genomic GR-response element (GRE). We confirmed nuclear translocation of GR in hypertrophing cardiac myocytes
in vitro
and
in vivo
. Our aim was to examine GR signaling in cardiac myocytes. Using
in vitro
biotin labeling and RNA immunoprecipitation we show that a non-coding RNA Gas5, which harbors decoy GRE sequence associates with GR in cardiac myocytes and competes with genomic GRE for DNA binding domain of GR, thus inhibiting the transcriptional effects of activated GR. Interestingly, this association decreases with hypertrophy or Gc agonist (dexamethasone) stimulation. Conversely, Gas5 association with G3bp1, a RNA binding protein increases in cardiac myocytes with similar stimulations, suggesting that increase in G3bp1-Gas5 binding might play a role in release of activated GR from Gas5. In accordance, expression of exogenous Gas5
in vitro
and
in vivo
restricted hypertrophy, suggesting critical role in progression of hypertrophy. We recently reported that G3bp1 is required for development of cardiac hypertrophy. Moreover, in situ hybridization revealed sequestration of Gas5 transcripts to perinuclear focal regions with growth stimulus, which resemble cytoplasmic protein-RNA aggregates seen with hypertrophic stimulation or G3bp1 over expression in cardiac myocytes. These results suggest that G3bp1 regulates Gas5 cellular compartmental dynamics and hence its GR-repressor function in cardiac myocytes. Thus, we conclude that G3bp1-Gas5 axis regulates GR -dependent gene transcription and progression of pathological cardiac hypertrophy and onset of failure
Abstract 264: Inhibiting Fibronectin Improves Cardiac Function in a Mouse Model of Heart Failure
Authors: Valiente-Alandi Iñigo , Nieman Michelle , Blaxall Burns C.
Published: June 2021

Heart failure (HF) is a devastating disease with poor prognosis. Hallmarks of HF include pathological fibrosis, remodeling and reduced function. In response to cardiac injury, cardiac fibroblasts (CF) undergo pathologic transition to a myofibroblast (MF) phenotype, characterized by excess production of collagen and other myocardial extracellular matrix (ECM) components, thus exacerbating HF. The ECM protein fibronectin (FN) plays an essential role in pathologic remodeling of the ECM in HF. FN polymerization tightly regulates the assembly of collagens and promotes cell proliferation, growth, migration and contractility. We hypothesize that inhibiting FN polymerization utilizing novel peptides will attenuate cardiac remodeling by limiting CF activation and fibrosis. To investigate this hypothesis, we administered daily injections of the FN polymerization inhibitory peptide pUR4 into wild type animals after ischemia-reperfusion (IR) injury for 1 week, and hearts were harvested 4 weeks post-IR. Mice receiving pUR4 demonstrated significant improvement in cardiac function compared to control peptide-treated animals. In addition, pUR4-treated animals showed a robust reduction of fibrosis, inflammatory cell infiltration, pro-inflammatory cytokines, apoptosis and hypertrophy. The information gleaned from the
in vitro
data obtained from isolated CF, from unchallenged or injured hearts, treated with pUR4 or control peptide suggested attenuation in cell migration and proliferation cell functions. To examine the impact of FN genetic ablation in cardiac function and cardiac fibrosis, we investigated the fibroblast-mediated role of FN in pathologic cardiac remodeling utilizing our fibroblast-restricted Periostin
mERCremER
;FN
Flox/Flox
(Periostin FN-KO). Analysis of cardiac function by echocardiography of Periostin-FN-KO 4 weeks post-IR revealed limited cardioprotective effect compare to the control group (FN
Flox/Flox
). This data suggest that inhibiting FN polymerization may be cardioprotective following cardiac injury, attenuating the pathological effects of FN overproduction and polymerization in the activated CF after injury. Limiting FN polymerization will possible lead to the generation of novel treatments for HF.
Abstract 265: Identification of MicroRNA in an Ovine Model of Heart Failure
Authors: Wong Lee Lee , Saw Eng Leng , Lew Kar Sheng , Rademaker Miriam T , Ellmers Leigh J , Charles Christopher J , Richards Arthur M , Wang Peipei
Published: June 2021

Introduction:
The sheep (
Ovis aries
) provides a large animal model in cardiovascular research including heart failure (HF). However, microRNA (miR) related work in the sheep model has been limited due to a paucity of information regarding oar-miR. The aim of this study was to identify novel oar-miRs in myocardium and examine their regulation in HF and HF recovery.

Methods:
Heart tissue was harvested from sheep undergoing 1. HF induced by rapid left ventricular (LV) pacing at 225bpm for 14 days, 2. HF recovery (HF-R) after discontinuation of pacing for an additional 14 days and 3. Sham. LV miRs were examined using next generation deep sequencing (NGS), miR array and stem-loop qPCR. Sequences were aligned with miRBase v20.0 and mapped to ovine genome and miRBase Mature BLAST search engine. Plasma was collected to assess natriuretic peptides (ANP and BNP).

Results:
Three miR libraries were generated from NGS and a total of 619 miRs were detected. Of these, 93 were oar-miRs; 49 novel miRs (high confidence) mapped to ovine genome and perfectly aligned to mature miRs in other organisms (miRBase v20.0); 69 putative novel miRs (high confidence) that mapped to ovine genome and aligned partially to mature miRs of other species in miRBase v20.0; 168 miRs (low confidence) that mapped to ovine genome but unaligned to any known mature miRs; and 240 miRs (low confidence) that were unmapped to the sheep genome but aligned to either miRBase mature hsa-/mmu-/rno-miRs. Plasma BNP and ANP increased 19-folds and 18- folds respectively in HF, and returned to baseline in HF-R. MiR levels in HF model were examined using miR array. About 1000 miRs were detected from array and at least 301 of them were overlapped with NGS data. Using miR array profiling followed by stem-loop qPCR validation, we found that myocardial enriched miR-133b-3p, miR-208b-3p, miR-21-5p and miR-125a-5p, -125b-5p, -126-3p, -210-3p, and 29a-3p were significantly upregulated in HF (p<0.05 vs. Sham). All trended downwards towards baseline levels during recovery (HF-R) but only miR-210 was significant (p<0.001 vs. HF).

Conclusion:
We identified 118 novel oar-miRs with high confidence and 408 potential oar-miRs, which is a solid foundation for miR function studies using sheep models. We also identified miR changes in HF and HF-R.
Abstract 266: Is the Col4a3

-/-

Alport Mouse a Novel Model for Heart Failure With Preserved Ejection Fraction?
Authors: Yousefi Keyvan , Ding Wen , Singh Jayanti , Shehadeh Lina
Published: June 2021

Heart failure with preserved ejection fraction (HFpEF) accounts for more than 50% of all HF cases. HFpEF patients manifest normal or mildly reduced left ventricle (LV) ejection fraction (LVEF), LV hypertrophy, diastolic dysfunction, myocardial stiffness, cardiac fibrosis, hypertension, nephropathy and sudden death. There is no appropriate animal model for HFpEF. The Alport Col4a3
-/-
mouse, a model of nephropathy, hypertension and early death, develops a cardiovascular pathology that is yet to be well characterized. We elected to analyze the cardiovascular pathology of the Alport mouse and investigate any commonalities with HFpEF. Male Alport and wild type (WT) littermates of mixed background at 2 months of age were subjected to echocardiographic and 2D speckle tracking analyses and heart tissues were used for histopathological examinations (N=6 mice per group). Circulating Galectin-3, a marker of HFpEF, was measured by Elisa in plasma samples. Data are shown as mean±SEM. Normalized heart weight increased in Alport relative to WT mice - p<0.01, indicative of cardiac hypertrophy. Consistently, echocardiography showed interventricular septum (IVS) thickening -p<0.05. Reduced stroke volume p<0.01, and impaired global longitudinal and circumferential strain (GLS and GCS) indicated systolic dysfunction in Alport mice. No significant reduction in LVEF was observed. Alport mice developed diastolic dysfunction evidenced by a prolonged Isovolumetric relaxation time -P<0.05, and a reduced E/A, a marker of LV relaxation and stiffness -p<0.01. Elevated LV filling pressure and pulmonary artery wedge pressure were demonstrated by an increase in E/E’ - p<0.01. Galectin-3 increased in Alport relative to WT plasma (P<0.01). Alport hearts had more Fibronectin protein - P<0.05 and increased number of fibroblasts with “activated” phenotype as demonstrated by increased mass of rough endoplasmic reticulum in EM cross sections. Our study suggests that the cardiovascular pathologies of the Alport mouse are similar to HFpEF, specifically preserved ejection fraction, diastolic dysfunction, hypertension, early death and cardiac stiffness and fibrosis. Further study of this multi-factorial pathology may render the Alport mouse as a useful novel model for HFpEF.
Abstract 267: Activation of a Novel Estrogen Receptor Ameliorates Experimental Pulmonary Hypertension in Male Rats
Authors: Zapata-Sudo Gisele , Alencar Allan K , Montes Guilherme C , Martinez Sabrina T , Fraga Aline G , Wang Hao , Groban Leanne , Sudo Roberto T
Published: June 2021

Introduction:
Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling that leads to uncompensated RV failure, and premature death. Preclinical studies have demonstrated that activation of the G protein-coupled estrogen receptor (GPER) is cardioprotective in male rats, and that a selective agonist G1, elicits vascular relaxation in rats of either sex. This work investigated the effects of G1 in male rats with monocrotaline (MCT)-induced PAH.

Methods and Results:
Male Wistar rats received a single intraperitoneal injection of MCT (60 mg/kg) for PAH induction. Experimental groups were: control, MCT + vehicle, and MCT + G1 (400 μg/kg/day s.c.). Animals were treated with vehicle or G1 for 14 days after the onset of disease (n = 5 per group). Treadmill test and transthoracic echocardiography were performed to access exercise capacity and cardiac function, respectively. RV systolic pressure (RVSP) and mean arterial pressure (MAP) were measured. Time to exhaustion in the treadmill (s) was reduced from 1042.0 ± 66.4 to 188.0 ± 53.1 (MCT + vehicle) and recovered to 809.4 ± 61.5 after G1 treatment. Pulmonary acceleration time (PAT) (ms) was reduced from 44.7 ± 1.4 to 24.7 ± 1.2 in MCT + vehicle group and restored to 41.8 ± 1.0 in MCT + G1 group. RVSP (mmHg) was increased from 24.6 ± 0.6 to 41.1 ± 1.4 (MCT + vehicle) and was reduced to 27.5 ± 0.7 (MCT + G1). MAP (mmHg) was reduced from 100.9 ± 1.1 to 77.1 ± 2.4 (MCT + vehicle), indicating HF induced by the RV dysfunction, and G1 normalized it to 92.4 ± 1.4. G1 decreased pulmonary vascular remodeling and normalized endothelial nitric oxide enzyme levels in lungs from PAH rats. PLB/SERCA2a ratio increased, as a sign of contractility dysfunction, in PAH rats and tumor necrosis factor alpha upregulation was significantly correlated with these Ca
2+
handling proteins impairment. Activation of GPER beneficially reduced PLB/SERCA2a ratio and TNF-α levels in RV tissue of MCT-induced PAH rats.

Conclusions:
G1 was effective to reverse PAH-induced RV dysfunction and exercise intolerance in male rats, a finding that may have important implications for ongoing clinical evaluation of new drugs for the treatment of the disease.
Abstract 271: NF-kB Mediated Modulation of mir-23b/-130a-axis is Critical for Cardiac Remodeling
Authors: Gupta Sudhiranjan , Li Li
Published: June 2021

Cardiac remodeling is a process that alters structural and functional determinants of myocardium in response to hemodynamic stress resulted in left ventricular hypertrophy, fibrosis leading to heart failure. Pressure overload that induces cardiac remodeling involves both cardiomyocytes (CM) and cardiac fibroblasts (CF) cross-talk for the development of adverse remodeling. The miRNAs are a new class of post-transcriptional regulator capable of repressing gene expression by base pairing to the 3' UTR of mRNA targets; involved in diverse cardiac diseases. However, the function of miRNAs in cellular cross-talk leading to cardiac remodeling remains elusive. Previously, we showed that inhibition of NF-kB prevents cardiac remodeling; however, NF-κB mediated miRNAs’ role in cardiac remodeling remains elusive. We will test the hypothesis that NF-κB dependent miR-23b/-130a is a pathogenic niche regulating the cardiac remodeling; and inhibition of miR-23b/-130a abrogates the adverse remodeling by restoring PPARg-PTEN-axis. We have identified a panel of novel dysregulated miRNAs in the left ventricle of wild type mice subjected to thoracic aortic constriction (TAC). The dysregulated miRNAs were restored in cardiac-specific IκBα triple-mutant transgenic mice (3M) subjected to TAC indicated NF-kB dependent regulation. We observed that miR-130a and miR-23b were significantly upregulated in TAC and Ang II infusion and were inhibited in 3M-TAC mice. We identified PPARg-PTEN-axis, a
bona-fide
target for miR-23b/-130a after unbiased
in vitro
screening. Inhibition of NF-kB normalized miR-23b/-130a expression and the target genes in Ang II and TGFb1 stimulated CM and CF. Inhibition of miR-130a showed reduction of cell sizes in CM and decrease cellular proliferation in CF indicated a cellular cross-talk. The
in vivo
inhibition of miR-130a in wild-type mice after Ang II infusion significantly reduced cardiac remodeling, restoring PPARg-PTEN level and improved cardiac function. Our findings provide evidence that miR-23b/-130a displays a critical role in the pathogenesis of cardiac remodeling. We conclude that miR-23b/-130a could be a triggering factor in cardiac remodeling and providing new mechanistic information for therapeutic benefit.
Abstract 275: The Role of Fibroblast-specific Canonical TGFβ Signaling in Cardiac Fibrosis
Authors: Khalil Hadi
Published: June 2021

Heart failure is a progressive disease characterized by cardiomyocyte loss, interstitial fibrosis, and chamber remodeling. During physiological conditions cardiac fibroblasts contribute to the homeostatic maintenance of myocardial structure as well as the maintenance of biochemical and electrical properties of the heart. Injury and/or cytokine stimulation activate fibroblasts which transdifferentiate into myofibroblasts. These newly formed cells secrete extracellular matrix (ECM) for wound healing and tissue remodeling through their contractile activity. Fibrosis mediated by these cells can initially be a beneficial response that acutely scarifies areas after an infarct to prevent wall rupture. However, during chronic disease states such as heart failure, persistent recruitment and activation of fibroblasts leads to excessive deposition of ECM that results in stiffening and pathological remodeling of the ventricles. During chronic heart disease, cardiomyocytes, immune cells and fibroblasts secrete the cytokine transforming growth factor-TGFβ, which activates fibroblasts and promotes their conversion to myofibroblasts. Previous work from Kass lab showed that manipulation of TGFβ by deletion of
Tgfbr
1 (type I TGFβ receptor) in cardiomyocytes reduced the fibrotic response after pressure overload. However heart failure was not improved because deleterious TGFβ signaling in fibroblasts persisted. Here we utilized a novel myofibroblast-specific inducible Cre-expressing mouse line (Periostin-MerCreMer) to examine canonical (Smad2/3) TGFβ signaling to determine how these cells and their activation mediate disease in heart failure. Our data indicate that myofibroblast-specific deletion of Smad3 but not Smad2 was sufficient to significantly inhibit myocardial fibrosis after pressure overload, but not ultimately prevent it. Also, myofibroblast specific Smad2/3 double nulls and Tgfbr1/2 double nulls were generated and analyzed. Data from all these myofibroblast-specific mouse models with inhibited TGFβ signaling indicated that TGFβ initiates myofibroblast transformation and myocardial fibrosis with injury to the heart, but that ultimately other pathways can fully compensate and fibrosis eventually occurs.
Abstract 277: Microrna-33 Promotes Cardiac Fibrosis by Maintaining Cellular Lipid Homeostasis
Authors: Nishiga Masataka , Horie Takahiro , Kuwabara Yasuhide , Baba Osamu , Nakao Tetsushi , Nishino Tomohiro , Hakuno Daihiko , Nakashima Yasuhiro , Nakazeki Noriko , Ide Yuya , Koyama Satoshi , Hasegawa Koji , Kita Toru , Kimura Takeshi , Ono Koh
Published: June 2021

Background:
A highly conserved microRNA, miR-33 is considered as a potential therapeutic target for atherosclerosis, because recent reports, including ours, indicated miR-33 has atherogenic effects by reducing HDL-C. However, the functions of miR-33 in heart failure remain to be elucidated.

Methods and results:
To clarify the functions of miR-33 involved in cardiac hypertrophy and fibrosis in vivo, we investigated the responses to pressure overload by transverse aortic constriction (TAC) in miR-33 deficient (KO) mice. When subjected to TAC, miR-33 expression level was significantly up-regulated in wild-type (WT) left ventricles, whereas miR-33 KO hearts displayed no less hypertrophic responses than WT hearts. However, interestingly, histological and gene expression analyses showed ameliorated cardiac fibrosis in miR-33 KO hearts compared to WT hearts. Furthermore, we generated cardiac fibroblast specific miR-33 deficient mice, which also showed ameliorated cardiac fibrosis when they were subjected to TAC. We also found that cardiac fibroblasts were mainly responsible for miR-33 expression in the heart, because its expression was about 4-folds higher in isolated primary cardiac fibroblasts than cardiomyocytes. Deficiency of miR-33 impaired cell proliferation in primary fibroblasts, which was considered due to altered lipid raft cholesterol content by up-regulated ATP-binding cassette transporter A1/G1.

Conclusion:
Deficiency of miR-33 impaired fibroblast proliferation in vitro, and ameliorated cardiac fibrosis induced by pressure overload in vivo.
Abstract 280: Cardiac Fibroblast Specific Deletion of Gsk3α Alleviate From Cardiac Dysfunction and Fibrotic Remodeling in Ischemic Heart
Authors: Verma Vipin K , Ahmad Firdos , Zhang Qinkun , Force Thomas L , Lal Hind
Published: June 2021

Heart failure is the leading cause of mortality, morbidity and healthcare expenditures worldwide. Numerous studies from our lab and others have implicated GSK-3 as a promising therapeutic target for cardiovascular diseases. Recently, we reported that cardiomyocyte-specific deletion of GSK3α limits adverse remodeling and preserves cardiac function, post-MI. However, the role of cardiac fibroblast GSK3α (CF-GSK-3α) in myocardial biology is unknown. To determine the role of CF-GSK-3α in MI-induced fibrotic remodeling, we created CF-GSK-3α KO mice in which Cre recombinase is driven by Postn (periostin) promoter. WT and CF-GSK-3α-KO mice were subjected to MI or sham surgery at 2 months age and cardiac function were monitored by serial echocardiography. Interestingly, at four weeks post-MI, CF-GSK-3α-KO mice showed preserved chamber dimensions and LV functions as reflected by preserved ejection fraction and fractional shortening as compared to littermate controls. The preserved LV dimensions and cardiac function were remained significantly better up to the end of the study i.e., 8 weeks post-MI. At 8 weeks post MI, hearts were excised, and Masson trichrome staining was performed. LV scar circumference was measured and expressed as a percentage of total area of LV myocardium. Scar tissue percent circumference was significantly reduced in CF-GSK-3α-KO hearts. Furthermore, GSK3α KO heart scars were thicker with much higher numbers of viable cardiomyocytes as compared to WT. To gain the mechanistic insights of observed GSK-3α mediated fibrotic remodeling, we examined profibrotic TGFβ signaling and myofibroblast transformation. WT and GSK-3α KO mouse embryonic fibroblasts (MEFs) were treated with TGF-β1 (10 ng/mL) for 1 hour, and phosphorylation of SMAD-3 (Ser423/25) was determined. Indeed, TGF-β1 induced activation of SMAD3 was significantly reduced in GSK3α KO cells. Consistently, deletion of GSK-3α leads to reduced myofibroblast transformation as reflected by significantly reduced expression of α-SMA in GSK-3α KO cells. These findings suggest that CF-specific deletion of GSK3α is protective in ischemic heart and GSK3α could be a novel therapeutic target for management of adverse fibrotic remodeling in the diseased heart.
Abstract 285: Endoplasmic Reticulum Stress Induces Mitochondrial ROS Overproduction and Contributes to Electrical Remodeling in Heart Failure
Authors: Liu Man , Shi Guangbin , Terentyev Dmitry , Zhou Anyu , Dudley Samuel C
Published: June 2021

Introduction:
Heart failure (HF) is associated with endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR), which has three branches (PERK, IRE1 and ATF6α). UPR inhibits protein translation, and we hypothesized UPR contributes to electrical remodeling in HF.

Methods:
HF was induced in C57BL/6 mice 6-7 weeks after unilateral nephrectomy, deoxycorticosterone acetate pellet implantation, and salt water substitution. Sham operated mice were used as controls. Isolated ventricular myocytes were utilized for whole-cell patch clamp recording and ROS measurement by DCFDA with confocal imaging. Heart tissue was used for mRNA and protein measurements. GSK2606414, a specific inhibitor of PERK, was applied to myocytes at 4-30 nM for 6-20 h.

Results:
The action potential duration was prolonged in HF myocytes (204±26 vs. 100±16 ms of sham, P<0.05). The mRNA and protein levels of the UPR effectors indicated that the PERK and IRE1 branches were activated in HF heart tissue. The peak I
Na
, I
to
, I
K1
, and I
Kslow
were decreased significantly in HF group, while I
CaL
and other K
+
currents were not affected. A PERK inhibitor, GSK2606414, restored I
Na
and I
K,slow
. Current changes were similar to those observed in myocytes under tunicamycin-induced ER stress. Significant reduction of channel protein levels were observed for K
v
4.3, Kir2.1, K
v
1.5, and Ca
v
1.2, but not Na
v
1.5, suggesting that I
Na
reduction was mediated by PERK but not by translational downregulation. Significant ROS production was observed in mouse cardiomyocytes under ER stress, which was suppressed by GSK2606414. Confocal imaging of ROS indicates the source to be mitochondria.

Conclusions:
TM-treated cardiomyocyte changes indicated that reductions of I
Na
, I
to
, I
K1
and I
K,slow
were mediated by ER stress. Proportionally decreased protein and current levels of K
v
4.3, Kir2.1, and K
v
1.5 suggested direct effects of ER stress. The downregulation of Na
v
1.5 and K
v
1.5 could be prevented by PERK inhibition. Nevertheless, the effect of PERK inhibition on I
Na
was indirect and mediated by a PERK-dependent elevation in mitochondrial ROS. In conclusion, the UPR contributes to electrical remodeling in HF by direct and indirect means. Targeting the UPR may be a novel antiarrhythmic strategy.
Abstract 291: MANF, a Structurally Unique ER Stress-inducible Protein, Restores ER-protein Folding in ER Stressed Cardiac Myocytes and in the Ischemic Heart
Authors: Arrieta Adrian , Stauffer Winston T , Pentoney Amber N , Blackwood Erik A , Doroudgar Shirin , Glembotski Christopher C
Published: June 2021

The sarco/endoplasmic reticulum (SR/ER) of cardiomyocytes is a critical site of protein synthesis and folding, as most secreted and membrane proteins including receptors, growth factors, ion channels, and calcium-handling proteins are made at this location. Myocardial ischemia induces ER stress, during which toxic, misfolded proteins accumulate in the SR/ER and contribute to cardiomyocyte death. The branch of the ER stress response mediated by the transcription factor, ATF6, induces ER chaperones that restore SR/ER protein folding. We found that ATF6 also induces mesencephalic astrocyte-derived neurotrophic factor (MANF), a novel, ubiquitously expressed, ER-luminal protein of unknown function. MANF is structurally unique, thus its function cannot be inferred by structural analogy to known proteins. Since it is ATF6-inducible, and resides in the ER lumen, we hypothesized that MANF is an ER chaperone required for optimal viability of cardiac myocytes during ER stresses, including ischemia. The characteristics of MANF gene induction by ER stress, and the effects of MANF knockdown on the ER stress response and cell viability were determined in cultured neonatal rat ventricular myocytes (NRVM). The ability of recombinant MANF to restore structure and function to model misfolded proteins was also examined. Finally, the effects of MANF loss-of-function in the ischemic heart,
in vivo
, were determined by generating a transgenic mouse model that expresses a cardiomyocyte-specific MANF-targeted microRNA. MANF induction and functional characteristics phenocopied those of a well-studied ER chaperone, glucose-regulated protein 78 (Grp78). Like Grp78, MANF was induced by ER stress in an ATF6-dependent manner. Like knockdown of Grp78, knockdown of MANF in NRVM increased myocyte death in response to ER stress. Like recombinant Grp78, recombinant MANF exhibited a robust ability to restore structure and function to model misfolded proteins,
in vitro.
Finally, MANF knockdown in the heart,
in vivo
, increased damage in a mouse model of myocardial infarction. These results suggest that MANF is an SR/ER-resident chaperone required for restoration of SR/ER protein folding during the adaptive ER stress response, and decreasing tissue damage in the ischemic heart.
Abstract 294: Therapeutic Silencing of miR-375 Attenuates Post-MI Inflammatory Response and Left Ventricular Dysfunction in Mice With Myocardial Infarction
Authors: Garikipati Venkata N , Verma Suresh K , Jolardarashi Darukeshwara , Cimini Maria , Ibetti Jessica , Yue Yujia , Benedict Cindy , Nickoloff Emily , Gao Ehre , Krishnamurthy Prasanna , Koch Walter J , Kishore Raj
Published: June 2021

MicroRNAs are known to be dysregulated in the ischemic heart disease and have emerged as potential therapeutic targets for treatment of myocardial infarction (MI). Our preliminary data indicated elevated MicroRNA-375 levels in failing human heart tissue. Therefore, we assessed whether inhibition of the miR-375 using a s.c.-delivered locked nucleic acid (LNA)-modified anti-miR (LNA-antimiR-375) can provide therapeutic benefit in mice with myocardial infarction (MI). After the induction of acute myocardial infarction, mice were treated with either control or LNA based LNA-anti-miR-375, and inflammatory response, cardiomyocyte apoptosis, capillary density and LV functional and structural remodeling changes were evaluated. LNA-anti-miR-375 therapy significantly reduced inflammatory cell infiltration, expression of pro-inflammatory cytokines and cardiomyocyte apoptosis in the myocardium. Further, our cell sorting experiments revealed that within the myocardium, LNA-anti-miR-375 was taken up by cardiomyocytes, endothelial cells and macrophages and repressed miR-375 levels, thereby activating 3-phosphoinositide-dependent protein kinase 1 (PDK-1) and downstream AKT phosphorylation on Thr-308. LNA anti-miR-375 therapy significantly improved LV functions, enhanced neovascularization and reduced infarct size. Corroborating with our in vivo findings, our in vitro studies demonstrated that knock down of miR-375 in macrophages enhanced the expression of PDK-1 and revealed reduced pro-inflammatory cytokines expression following LPS challenge. Taken together, our studies demonstrate that anti miR-375 therapy reduced inflammatory response, cardiomyocyte death, improved LV function and enhanced angiogenesis by targeting multiple cell types via activation of PDK-1/AKT signaling.
Abstract 302: Cathepsin D Haploinsufficiency Exacerbates Post Myocardial Infarction Cardiac Remodeling and Malfunction by Impairing Autophagosome Removal
Authors: Wu Penglong , Zhu Wei , Li Jingbo , Wang Xuejun “XJ”
Published: June 2021

Background:
A marked decrease in the protein level and activity of cathepsin D (Ctsd), a major lysosomal protease, has been observed in failing human hearts; however, the pathophysiological significance of Ctsd downregulation in the heart remains undefined. Given the increasingly recognized role of the autophagic-lysosomal pathway in cardiac pathobiology, we sought to determine the impact of Ctsd haploinsufficiency on post myocardial infarction (MI) cardiac remodeling and explore the underlying mechanism.

Methods and Results:
MI was induced in 2-3 months old mixed-sex wild type (WT) or heterozygous Ctsd knockout (Ctsd
+/-
) mice via permanent ligation of the left anterior descending (LAD) coronary artery. Myocardial Ctsd protein levels are reduced by ~50% in Ctsd
+/-
mice, compared with WT mice. Echocardiography (Echo) did not detect discernible difference between Ctsd
+/-
and WT mice immediately before and 1 week (wk) after MI; however, compared with the WT MI group, significantly greater left ventricular (LV) internal diameters at end systole and at end diastole and significantly smaller ejection fraction and fractional shortening were detected in the Ctsd
+/-
MI group at 2, 3, and 4 wks post-MI (p<0.05 ~ 0.001). LV pressure-volume relationship analyses at 4 wks post-MI also revealed that LV maximum and minimum dP/dt values were significantly reduced in the WT MI group, compared with the WT sham control group (p<0.05, 0.01); these post-MI reductions were more pronounced in the Ctsd
+/-
MI group (P<0.05, 0.01). The infarct size at 4 wks post-MI measured with trichrome staining was significantly enlarged by Ctsd
+/-
(p<0.002). These results indicate that Ctsd haploinsufficiency exacerbates post-MI cardiac remodeling and malfunction. Post-MI myocardial Ctsd protein levels were increased in WT mice but this increase was not detectable in Ctsd
+/-
mice. However, the myocardial protein levels of LC3-II, beclin1, and Atg5 were further elevated in Ctsd
+/-
MI mice, compared with the WT MI mice, indicative of impairment of autophagic flux in Ctsd
+/-
MI mice.

Conclusion:
Ctsd haploinsufficiency exacerbates maladaptive post-MI cardiac remodeling and malfunction likely through impairing autophagosome removal.
Abstract 303: Optical Tissue Interrogation Technology Incorporated in Irrigated Radiofrequency Ablation Provides Real Time Monitoring of Ablation Lesion Depth
Authors: Alzahrani Talal , Aljishi Mohammed , Mercader Marco
Published: June 2021

Background:
Radiofrequency ablation (RFA) is an effective therapy to treat cardiac arrhythmias. The success rate depends on the location and size of ablation lesions. Electroanatomic Mapping and Contact force-sensing catheters have been shown to enhance the success rates of RFA. We used real-time optical tissue interrogation within the NADH fluorescence (fNADH) range to assess the progression of ablation lesion depth in real time during RF delivery (LuxCath catheter).

Methods:
Multiple RFA lesions (n=20) were made on an intubated and mechanically ventilated canine (Mongrel) thigh muscles using 7 Fr quadripolar RFA catheter with a 3.5 mm tip open 3.5 mm irrigated electrode incorporating imaging optic. Light was delivered at (360+/-25nm), and fluorescence acquired through fiberoptic bundle at (370-660 nm) by a spectrometer and analyzed in real time. Two types of plots were created for each lesion: intensity (in counts) vs. wavelength (nm), and 465nm peak amplitude (normalized) vs. time (sec). The lesions were made at room temperature with fixed power (15W) and four different durations (10, 20,30,40 sec). After ablation the muscle was stained with 2,3,4-triphenyl-2H-tetrazolium chloride (TTC), bisected at the center and measured the with and depth of the lesion using imageJ software.

Results:
We performed a total of twenty lesions that were divided into four groups based on the duration of the ablation (10, 20, 30, 40 seconds). The mean lesion depth for those groups was 4.05+/-1.28, 4.99+/-0.9, 6.9+/-1.06 and 7.14+/-1.41 mm, respectively. Lesion depth was correlated with the duration of ablation and fNADH signal intensity with a correlation coefficient of 0.93 and 0.99, respectively. Finally, there was a significant correlation between lesion size and fNADH signal intensity with a correlation coefficient of 0.96.

Conclusion:
Real-time optical tissue characterization can provide an excellent assessment of lesion progression during RF delivery. Lesion depth was directly correlated to the decrease in fNADH signal intensity. This information may be used to optimize the selection of RF power and RF application time to maximize RF lesion formation and improve the success of ablation procedures.
Abstract 306: Targeting of Cdc42-Interacting Protein 4 - Calcineurin Aβ Complexes Ameliorates Pressure Overload-induced Cardiac Remodeling
Authors: Li Xiaofeng , Li Jinliang , Valencia Eliana C , Passariello Catherine , Thakur Hrishikesh , Kapiloff Michael S
Published: June 2021

Pathological cardiac remodeling is the primary mechanism underlying the development of heart failure. The Ca
2+
/calmodulin-dependent protein phosphatase calcineurin is a key regulator of pathological myocyte hypertrophy. Previously, it was shown that the β-isoform of the calcineurin catalytic A-subunit (CaNAβ) was uniquely required for hypertrophy. In order to determine the mechanism of CaN A-subunit specificity, we performed a yeast two-hybrid screen using the CaNAβ-specific N-terminal polyproline domain. We now show that a new binding partner for CaNAβ is the F-Bar (Fes-CIP4 homology [
F
CH] -
B
in/
A
mphyphysin/
R
vs) scaffold protein Cdc42-interacting protein 4 (CIP4/TRIP10). The CaNAβ polyproline domains binds the SH3 domain of CIP4. Using a new CIP4 conditional knock-out mouse, we show that CIP4 contributes to the hypertrophy induced in vivo by pressure overload. After induction of conditional gene deletion using tamoxifen,

CIP4
fl/fl

;Tg(Myh6-cre/Esr1*) mice exhibited decreased hypertrophy and improved cardiac function over a period of 16 weeks following transverse aortic constriction survival surgery when compared to control tamoxifen-treated Tg(Myh6-cre/Esr1*) and

CIP4
fl/fl

mice. In order to show the relevance of CaNAβ-CIP4 binding, we also developed an adeno-associated virus (AAV) gene therapy vector that expresses a CaNAβ polyproline domain - GFP fusion protein under the control of the cardiac myocyte-specific troponin T promoter. Disruption of CIP4-CaNAβ binding in vivo by the AAV vector attenuates cardiac hypertrophy following two weeks of pressure overload and improved systolic function. Together these results both demonstrate the role of a F-Bar scaffold protein in cardiac remodeling and reveal a novel mechanism for specificity in calcineurin signaling. Identification of this new signaling pathway also suggests a new therapeutic approach to heart failure with reduced ejection fraction.
Abstract 312: Arterial CEPT1 Expression Correlates With Diabetes and Atherosclerotic Occlusive Disease
Authors: Zayed Mohamed A , Naim Uzma , Park Kyoung-mi , Belaygorod Larisa , Semenkovich Clay
Published: June 2021

Choline/ethanolamine phosphotransferase-1 (CEPT1) is a ubiquitously expressed enzyme that catalyzes the final step in synthesis of the majority of mammalian phospholipids, that are known to also impact atheroprogression. We previously found that in the setting of diabetes CEPT1 expression and downstream phospholipid biosynthesis are critically altered in hepatocytes. We therefore hypothesized that in diabetes CEPT1 expression is also altered in arterial tissue, and correlates with disease severity. To test this hypothesis we evaluated CEPT1 protein expression in murine and human arterial tissue in the presence or absence of diabetes, and/or advanced occlusive disease. CEPT1 expression in the aorta of
Db/Db
mice was 43% increased relative to Wt controls (n=7 per mouse group;p=0.03). On the other hand, CEPT1 expression was not significantly altered in
Db/Db
myocardial or skeletal muscle (p=0.2 and p=0.09, respectively). To determine whether our findings translate to similar CEPT1 expression patterns in human arterial tissue, we harvested carotid artery intima from 11 diabetic and 7 non-diabetic patients undergoing carotid endarterectomy (CEA). We also harvested peripheral arterial segments with minimal and maximal atherosclerotic occlusive disease from 8 diabetic and 4 non-diabetic patients undergoing lower extremity amputation. Relative to non-diabetic patients, patients who were diabetic had increased CEPT1 expression in CEA tissue (53% increase;p=0.05), and peripheral arterial segments (36% increase;p=0.04). Interestingly, relative to peripheral arteries with minimal atherosclerotic occlusive disease, segments with maximal disease had a two-fold increase in CEPT1 expression (p<0.01). Analysis of maximally diseased peripheral arteries demonstrated significantly higher CEPT1 expression among diabetic patients (34% increase;p=0.03). There was no difference in CEPT1 expression in minimally diseased arteries of diabetic and non-diabetic patients (p=0.1). These findings highlight that increased CEPT1 expression correlates with diabetes and extent of arterial occlusive disease. Furthermore, they argue that CEPT1 is a unique arterial tissue biomarker for occlusive disease severity.
Abstract 313: Nonionic Acid is a Target Metabolite of Diabetic Microvasculopathy
Authors: Bando Yasuko K , Kawase Haruya , Nishimura Kazuyuki , Monji Akio , Murohara Toyoaki
Published: June 2021

Aim:
The purpose of this study is to screen the target metabolites of diabetic microangiopathy in heart by use of whole heart metabolome analysis.

Methods:
Diet-induced type 2 diabetic mouse were divided into two groups; control and those treated with diabetic remedy GLP-1 receptor agonist Ex-4 for 5 weeks. Mice were euthanized and analyzed at the age of 16 week-old.

Results:
The capillary density of the T2DM was reduced as compared to those non-diabetic counterparts, which was restored by Ex4 treatment. Changes in angiogenic signals detected by immunoblotting analysis revealed that the phosphorylation levels of eNOS and AMPK were elevated by Ex-4, but those Akt remained unchanged. Tube formation assay revealed that Ex-4 increased tube length and branch points in HUVECs. Consistently with the trend that was observed in vivo experiment, AMPK and eNOS phosphorylation levels were enhanced by Ex4 without affecting Akt phosphorylation. To screen the candidate metabolites that is responsible for the diabetic microvasculopathy in GLP-1-dependent fashion, we performed metabolome analysis by using the whole heart of each mouse. The hierarchical cluster analysis revealed that nonanoic acid (NNA) was the only metabolite that increased in type 2 diabetic mice with concomitant decline by Ex-4 treatment. We next examined the impact of nonanoic acid on in vitro angiogenesis and found that NNA suppressed tube length and branch points in HUVECs in a dose-dependent fashion. Interestingly, NNA canceled eNOS and AMPK phosphorylation that was enhanced by Ex4.

Conclusion:
GLP-1 ameliorated diabetic microvasculopathy via the AMPK and eNOS axis. NNA is presumably one of the novel anti-angiogenic metabolites that causes diabetic microangiopathy.
Abstract 314: Sensory Innervation of the Papillary Muscle: A Neural Link Between Mitral Valve Prolapse and Malignant Ventricular Arrhythmias?
Authors: Patel Krishan J , Ajijola Olujimi A , Fishbein Michael , Shivkumar Kalyanam
Published: June 2021

Background:
Malignant mitral valve prolapse (MVP) identifies a subset of patients with MVP associated with ventricular arrhythmias (VAs), including sudden cardiac death (SCD). We hypothesized that papillary muscles, which see significant mechanical stress in MVP, are richly innervated with sensory nerves, which induce severe autonomic imbalance on the heart and may trigger malignant VAs.

Methods and Results:
Longitudinal sections of the anterior and posterior left ventricular papillary muscles, right ventricular outflow tract (RVOT), ventricular septum, and basolateral left ventricular wall (LV) of Yorkshire pigs (n=8) were excised, formalin-fixed, paraffin-embedded, and sectioned. Immuno-staining for protein gene product 9.5 (PGP9.5), a pan-neuronal marker, and calcitonin-gene related peptide (CGRP), a sensory afferent neuron marker, was performed. Areas of immunoreactivity (IR) for CGRP were verified by direct comparison to PGP9.5 IR. The density of CGRP was then compared across anatomical regions. The mean CGRP IR area was 1229.44 ± 116.65 μm
2
/nucleus in papillary muscles, while in the septum, basolateral LV, and RVOT, the CGRP stained areas were 699.36 ± 88.28 μm
2
/nucleus, 681.51 ± 81.90 μm
2
/nucleus, and 381.98 ± 31.14 μm
2
/nucleus, respectively (p<0.001). There was no significant difference between CGRP IR area in the anterior papillary muscle (1091.36 ± 189.82 μm
2
/nucleus) and the posterior papillary muscle (1347.79 ± 140.50 μm
2
/nucleus) (p>0.3).

Conclusions:
There is a significantly greater amount of afferent innervation in the papillary muscle compared to the septum, basolateral LV, and RVOT. This enrichment of afferent innervation warrants further study to understand how afferent neurotransmission during abnormal mitral valve function may impact ventricular electrophysiology.
Abstract 317: Med12 is a Transcriptional Mediator of Cardiac Contractile Function
Authors: Baskin Kedryn K , Makarewich Catherine A , DeLeon Susan , Bassel-Duby Rhonda , Olson Eric N
Published: June 2021

The Mediator complex serves as a central governor of gene transcription by linking the basal transcriptional machinery with DNA-bound transcription factors. The activity of the Mediator complex is modulated by a kinase submodule comprised of four proteins: MED12, MED13, CDK8, and Cyclin C. The kinase submodule components are ubiquitously expressed, however recent studies support the hypothesis that these proteins differentially regulate gene expression in a tissue-specific manner. We previously demonstrated that MED13 acts in the heart to modulate systemic energy homeostasis through signaling to extracardiac tissues, but the role of the other kinase components in the heart has not yet been investigated. MED12 regulates development in a variety of cell types, and MED12 null mice are embryonically lethal. To investigate the cardiac functions of MED12, we generated mice with conditional cardiac-specific deletion of MED12. MED12 mutant mice displayed postnatal cardiomyopathy seven days after birth with a continual decline in cardiac function with age. RNAseq experiments identified specific dysregulation of genes involved in calcium homeostasis and contractility in the ventricles as early as postnatal day one. Defects in calcium handling were detected in cardiomyocytes isolated just after birth, suggesting that perturbed calcium transients lead to altered cardiac contractility and dysfunction. The consequences of cardiac deletion of MED12 and MED13 are clearly distinct, underscoring the unique functions of these Mediator components. These findings highlight the importance of the kinase submodule of the mediator complex in the control of cardiac function and reveal highly specific and distinct transcriptional actions of MED12 and MED13.