bluuebunny/crossref_metadata_2025 huggingface dataset:
Catheter Ablation of Ventricular Tachycardia in Patients With Arrhythmogenic Right Ventricular Cardiomyopathy/Dysplasia: A Sequential Approach
Authors: Mathew Shibu , Saguner Ardan M. , Schenker Niklas , Kaiser Lukas , Zhang Pengpai , Yashuiro Yoshiga , Lemes Christine , Fink Thomas , Maurer Tilman , Santoro Francesco , Wohlmuth Peter , Reißmann Bruno , Heeger Christian H. , Tilz Roland , Wissner Erik , Rillig Andreas , Metzner Andreas , Kuck Karl‐Heinz , Ouyang Feifan
Published: February 2019

Background

It has been suggested that endocardial and epicardial ablation of ventricular tachycardia (
VT
) improves outcome in arrhythmogenic right ventricular cardiomyopathy/dysplasia. We investigated our sequential approach for
VT
ablation in patients with arrhythmogenic right ventricular cardiomyopathy/dysplasia in a single center.

Methods and Results

We included 47 patients (44±16 years) with definite (81%) or borderline (19%) arrhythmogenic right ventricular cardiomyopathy/dysplasia between 1998 and 2016. Our ablation strategy was to target the endocardial substrate. Epicardial ablation was performed in case of acute ablation failure or lack of an endocardial substrate. Single and multiple procedural 1‐ and 5‐year outcome data for the first occurrence of the study end points (sustained
VT
/ventricular fibrillation, heart transplant, and death after the index procedure, and sustained
VT
/ventricular fibrillation for multiple procedures) are reported. Eighty‐one radiofrequency ablation procedures were performed (mean 1.7 per patient, range 1–4). Forty‐five (56%) ablation procedures were performed via an endocardial, 11 (13%) via an epicardial, and 25 (31%) via a combined endo‐ and epicardial approach. Complete acute success was achieved in 65 (80%) procedures, and partial success in 13 (16%). After a median follow‐up of 50.8 (interquartile range, [18.6; 99.2]) months after the index procedure, 17 (36%) patients were free from the primary end point. After multiple procedures, freedom from sustained
VT
/ventricular fibrillation was 63% (95%
CI
, 52–75) at 1 year, and 45% (95%
CI
, 34–61) at 5 years, with 36% of patients receiving only endocardial radiofrequency ablation. A trend (log rank
P
=0.058) towards an improved outcome using a combined endo‐/epicardial approach was observed after multiple procedures.

Conclusion

Endocardial ablation can be effective in a considerable number of arrhythmogenic right ventricular cardiomyopathy/dysplasia patients with
VT
, potentially obviating the need for an epicardial approach.
Abstract 100: PRDM16 is a Novel Regulator of Cardiac Hypertrophy, Remodeling and Mitochondrial Dynamics
Authors: Pires Karla Maria , Boudina Sihem
Published: February 2019

Elucidation of the mechanisms that cause cardiac hypertrophy is essential for developing novel therapeutic strategies to prevent or reverse these prevalent conditions. Prdm16/Mel1 (PRDM16) belongs to the Prdm family of transcription factors involved in cell fate determination and development. PRDM16 deletion, found in humans with 1p36 deletion syndrome, has been associated with two main cardiomyopathies: left ventricle non-compaction (LVNC) and dilated cardiomyopathy. Nonetheless, its role in the heart and the underlying mechanisms by which these mutations contribute to the development of cardiomyopathy are unknown. We sought to directly assess the role of Prdm16 in the developing and adult heart by generating both a germline (cPRDM16) and a tamoxifen-inducible (ciPRDM16) cardiomyocyte-specific Prdm16 knockout mice. cPRDM16KO mice died shortly after birth, showing grossly abnormal ventricles that were reduced in size and often exhibiting a cleft at their apex, consistent with LVCN. Both heterozygous mice (cPRDM16KOhet) and ciPRDM16KO are viable and displayed markedly increased heart size, cardiomyocyte cross sectional area, collagen deposition and expression of genes involved in cardiac hypertrophy and ECM production. We observed an increased in TGF-β receptors mRNA expression as well as activation of Smad signaling in the hearts of cPRDM16KO neonates, cPRDM16 heterozygous, and ciPRDM16 KO mice. Interestingly, we observed a systematic reduction in Drp1 phosphorylation on Ser616 (activation) in the hearts of cPRDM16KO and cPRDM16het mice. The reduction in Drp1 phosphorylation was associated with more tubular mitochondria in cPRDM16KO mice. Interestingly, by 25 weeks, cPRDM16het mice accumulated more mitochondrial proteins, despite exhibiting no change in PGC1α. Together, these data reveal previously unknown roles for Prdm16 in protection against cardiac hypertrophy and fibrosis through the suppression of TGF-β/Smad signaling in the heart. They also reveal roles for PRDM16 in the modulation of mitochondrial dynamics and clearance in the heart, a process that likely involves the fission protein Drp1.
Abstract 102: An Internal Pool of b-Adrenergic Receptors Activates PLC-mediated PI4P Hydrolysis in Cardiac Myocytes
Authors: Smrcka Alan V , Nash Craig A , Wei Wenhui
Published: February 2019

Cardiac hypertrophy and subsequent heart failure is a leading cause of morbidity and mortality in the USA. Previously, our laboratory identified a prohypertrophic pathway where PLCε, scaffolded to mAKAPβ at the nuclear envelope, hydrolyzes phosphatidylinositol 4-phosphate (PI4P) at the Golgi to locally generate DAG and drive hypertrophic gene expression. This pathway is activated by multiple upstream signals including the Epac-selective cAMP analogue, cpTOME. However, stimulation of β-adrenergic receptors with Isoproterenol (Iso) does not activate this pathway despite strongly raising cAMP. We were able to uncover Iso stimulated PI4P hydrolysis only in the presence of a cAMP phosphodiesterase 3 inhibitor.

To determine if internal β-adrenergic receptors can stimulate PI4P hydrolysis, we treated NRVMs with the membrane permeable β-adrenergic agonist, dobutamine. In contrast to the membrane impermeable Iso, dobutamine induced rapid and sustained PI4P hydrolysis that was at least partially blocked by the cell permeable β-adrenergic antagonist, metoprolol. The cell impermeable βAR antagonist, sotalol could not block dobutamine effects, suggesting that a pool of internal β-adrenergic receptors can induce PI4P hydrolysis. Over expression of the RA1 domain of PLCε, which competes PLCε away from the mAKAPβ scaffold, also inhibited dobutamine stimulation of PI4P hydrolysis. These data suggest that β-adrenergic stimulation in internal membranes, potentially at the Golgi apparatus or nuclear envelope, can stimulate PI4P hydrolysis. These observations demonstrate a novel mechanism for PLC activation through an internal GPCR, which may participate in development of cardiac hypertrophy and heart failure. Targeting internal β-adrenergic receptors may allow for the development of selective therapies to treat heart failure.
Abstract 105: Tuning EC-Coupling:
β
-Adrenergic Receptor Activation Stimulates Dynamic Augmentation of Ca
V
1.2 Channel Sarcolemmal Abundance and Cooperativity
Authors: Ito Danica W , Hannigan Karen I , Santana Luis F , Dixon Rose E
Published: February 2019

Voltage-gated L-type Ca
V
1.2 channels play an essential role in cardiac excitation-contraction coupling. These channels decorate the z-lines of ventricular myocytes where they form discrete clusters on the t-tubule membrane. We have previously reported that groups of channels within these clusters are capable of influencing each other’s gating behavior and undergo dynamic, cooperative interactions. This occurs via Ca
2+
-calmodulin-dependent, physical interactions between the C-terminal tails of adjacent channels. The interactions increase the open probability (
P
o
) of adjoined channels, and thus amplify Ca
2+
influx into the myocytes. It has long been known that activation of
β
-adrenergic receptors during the ‘fight or flight’ response, leads to increased mean open time and
P
o
of Ca
V
1.2 channels, and thus enhanced Ca
2+
influx. Given our previous finding that Ca
V
1.2 channel cooperativity is triggered by Ca
2+
-influx, we predicted that
β
-AR stimulation could affect Ca
V
1.2 channel cooperativity in ventricular myocytes. To test this hypothesis, we investigated the effect of the
β
-agonist isoproterenol (ISO) on Ca
V
1.2 channel clustering and cooperativity using a combination of optical and electrophysiological approaches. Ground State Depletion super-resolution nanoscopy revealed ‘super-clustering’ of Ca
V
1.2 channels in ISO-stimulated adult mouse ventricular myocytes. Channels within these super-clusters display enhanced physical interactions compared to control unstimulated cells as revealed by Bimolecular fluorescence complementation. To visualize this process, we performed live cardiomyocyte imaging and observed a robust, dynamic increase in sarcolemmal Ca
V
1.2 channel abundance in response to ISO. Functionally, optical and electrophysiologicaI recordings of Ca
V
1.2 channel activity revealed that ISO-stimulated cells display enhanced Ca
V
1.2 channel cooperativity, and amplified Ca
2+
influx. On the basis of these data, we propose that stimulation of
β
ARs initiates dynamic augmentation of Ca
V
1.2 channel abundance, and enhanced cooperative gating of Ca
V
1.2 channels, in the sarcolemma of ventricular myocytes, to amplify Ca
2+
influx into these cells and tune EC-coupling in times of high metabolic or hemodynamic demand.
Abstract 113: Steering Fibroblast Phenotype to Promote Left Ventricular Scar Maturation and Function Post-MI
Authors: de Castro Brás Lisandra E
Published: February 2019

The prevalence for myocardial infarction (MI) in the US is ~7.9 million. Although current therapies have significantly improved survival rate, adverse left ventricular (LV) remodeling still occurs in nearly 40% of MI patients. The size, composition, and mechanical properties of the scar that forms post-MI determines patient outcomes. Scar formation is mostly driven by inflammatory factors and fibroblast differentiation into myofibroblast. Suppression of inflammation and fibrosis, however, have led to unexpected results, including infarct expansion and LV wall thinning. Recently our lab uncovered that extracellular matrix (ECM) degradation post-MI releases collagen derived fragments that are biologically active. Using a synthesized peptide mimetic of one such fragment - p1158/59 - we found that p1158/59 dynamically interacts with fibroblasts regulating cell migration and the type and amount of collagen deposited post-MI. We hypothesize that p1158/59 binds to fibroblasts to delay or prevent myofibroblast differentiation, which promotes scar stabilization by controlled secretion of ECM and scar maturation. Fibroblasts were isolated from mice LV and incubated with vehicle or p1158/59 in serum free media. Complete media (10% FBS) and TGFb were used as positive controls respectively for migration and myofibroblast activation. We assessed fibroblast phenotype and ECM secretion. Mice were treated with p1158/59 post-MI and mid- and long-term LV remodeling and function assessed. p1158/59 promoted fibroblast migration to levels similar to positive control. Surprisingly, this was not accompanied by increased alpha smooth muscle actin (i.e. myofibroblast activation). Mice treated with p1158/59 for 7 days (D7) post-MI presented less collagen deposition with decreased LV dilation compared to controls. At D14 post-MI, p1158/59 repressed expression of pro-angiogenic factors and promoted fiber alignment and cellular apoptosis, which resulted in improved cardiac function, providing proof-of-concept that p1158/59 reduces adverse LV remodeling. These data suggest that p1158/59 modulates scar formation and remodeling post-MI by delaying or partially inhibiting fibroblast phenotype transition and promoting scar stabilization and maturation.
Abstract 118: NLRP3 Inflammasome Mediates the Obesity-induced Pathogenesis of Atrial Fibrillation
Authors: Scott Larry , Yao Changqun , Li Na
Published: February 2019

Background:
Obesity increases the risk of atrial fibrillation (AF), the most common cardiac arrhythmia; but it is unknown what molecular pathway(s) underlying obesity promotes AF pathophysiology. ‘NACHT, LRR & PYD Domains-containing Protein 3’ (NLRP3) inflammasome can activate Caspase-1 (Casp1) and mediate inflammatory response associated with obesity. Prior studies revealed that NLRP3 activity is enhanced in atrial tissues of AF patients, and our preliminary studies demonstrated a positive correlation between obesity and NLRP3 activation. Thus, we hypothesized that diet-induced obesity promotes AF pathogenesis via activation of the NLRP3 inflammasome.

Methods and Results:
Wildtype (WT) and NLRP3 knockout (KO) mice were fed high fat diet (HFD, 60% kcal) or normal chow (NC, as control) for 10 weeks to establish the obese but non-diabetic phenotype. At 10 weeks, WT+HFD and KO+HFD mice showed a significant increase in body weight, compared to WT+NC and KO+NC mice (P<0.001). Echocardiography showed ventricular function were similar among all groups at 10 weeks. Western blotting revealed increased levels of mature Casp1 in atria of WT+HFD mice (P<0.05 vs WT+NC mice), suggesting activity of NLRP3 is enhanced by HFD. Programmed intracardiac stimulation was performed to assess the inducibility of AF. While only 3 of 14 (21.3%) WT+NC mice showed AF evoked by atrial pacing, 7 of 8 (87.5%) WT+HFD mice developed pacing-induced AF (P<0.05). Atrial effective refractory period (AERP) was reduced in WT+HFD mice (P<0.05 vs WT+NC), suggesting that HFD promotes an electrical remodeling substrate for AF development. In contrast, AF incidence was reduced to 33.3% (3 of 9; P<0.05 vs WT+HFD) and AERP was normalized in KO+HFD mice, suggesting NLRP3 is required for AF development in obese mice. Compared to WT+NC mice, Ca
2+
sparks were increased in atrial myocytes of WT+HFD mice (4.5±0.6 vs 6.4±0.7 sparks/100μm/s, P<0.05), which was normalized in KO+HFD mice (4.4 ±0.4 sparks/100μm/s, P<0.05).

Conclusion:
Our data suggests that obesity promotes NLRP3 activation and inhibition of NLRP3 can prevent the development of substrate for AF development. Our study potentially establishes a mechanistic link between obesity-induced inflammatory signaling and AF pathogenesis.
Abstract 200: Engineering Evolution: Tetraploidization of Human Cardiac Stem Cells to Enhance Functional Activity
Authors: Broughton Kathleen M , Yaareb Dena , Esquer Carolina Y , Pompilio Giulio , Sussman Mark A
Published: February 2019

Introduction:
Reparative and regenerative capacity is consistently observed in lower vertebrate species, but translational implementation has failed to yield comparable results in the clinical setting. Cardiac stem cells (CSC) isolated from rodent or swine heart possess capacity for tetraploidization resulting in 4n chromatin content associated with override of cell cycle arrest and senescence, but comparable tetraploid CSC are not present in human samples. Furthermore, diploid:tetraploid CSC ratio in rodent myocardium increases following infarction injury suggesting ploidy is a dynamic cellular condition influencing response to pathologic damage. Since polyploidization correlates with enhanced regenerative throughout evolution, ploidy differences raise provocative questions regarding translational applicability of experimental studies of myocardial regeneration.

Hypothesis:
Mononuclear chromatin duplication in human stem cells improves functional capabilities by inhibiting cellular senescence and increasing stress resistance.

Methods and Results:
Tetraploidy was artificially induced in human CSCs, ESCs and MSCs from multiple patient samples by transient exposure to demecolcine that blocks mitotic spindle formation and chromosome segregation. Mononuclear tetraploid content confirmed by karyotype analysis was consistently induced in hCSCs and hEPCs, unlike hMSCs which underwent apoptosis. Mononuclear tetraploid content in hCSCs and hEPCs was stable for continued multiple passages demonstrated by flow cytometry with proliferation rates equivalent to the parent diploid line. Stress induction through hydrogen peroxide demonstrate tetraploid hCSCs respond equivalent or better than parental lines measured through apoptosis and necrosis. Double strand DNA breaks in tetraploid hCSCs were reduced by half compared to the parental line, measured by gamma H2AX per total DNA.

Conclusion:
Tetraploid content induction in human CSCs and EPCs enhances functional activity by increased resistance to stress and inhibition of senescence without promoting oncogenic transformation. Ongoing studies focus upon potential of tetraploid hCSCs to exert enhanced capabilities for mediating myocardial repair and regeneration.
Abstract 222: Estrogen Protects Artery From Senescence via Sirt1/AMPK/Rab9—Mediated Mitophagy
Authors: Sasaki Yuichi , Ikeda Yoshiyuki , Akasaki Yuichi , Uchikado Yoshihiro , Ohishi Mitsuru
Published: February 2019

Background:
Estrogen protects artery from senescence. Mitochondria plays a crucial role to regulate cellular senescence. Recently it is reported that mitochondria undergo quality control by mitochondria-targeted autophagy, referred to as mitophagy. This study aims to examine whether estrogen modulates mitophagy, and to clarify the potential mechanism.

Methods and Results:
We used HUVEC and vascular smooth muscle cells. Estradiol treatment (E2) decreased the protein expressions of p21 and p53, senescence-associated beta galactosidase activity, ubiquitinated and depolarized mitochondria, and reactive oxygen species production compared to untreated-control cells, suggesting that estrogen maintains mitochondrial function and retards cellular senescence. E2 significantly increased the number of autophagy formation assessed by electron microscopy and Lamp2 puncta which co-localized with TOMM20 assessed by Immunohistochemistry, suggesting that E2 induces mitophagy. E2 did not moderate the ratio of LC3II/LC3I, whereas upregulated membrane trafficking protein Rab9 expression. The number of Rab9 puncta significantly increased and co-localized with Lamp2 puncta in E2-treated cells. The effects of E2 on increasing autophagy, maintaining mitochondrial function and retarding cellular senescence were abolished by knock-down of Rab9. Taken together, these results suggest that E2-mediated mitophagy was derived from Rab9-dependent alternative autophagy. E2 upregulated protein expression of Sirt-1 and AMPK activity. Administration of either Sirt-1 inhibitor, sirtinol, or AMPK inhibitor, compound c, abrogated upregulation of Rab9 and induction of mitophagy. These results indicate that the effect of E2 on inducing Rab9 is mediated by Sirt-1/AMPK pathway. Finally, we performed in vivo experiment. Apolipoprotein E knockout mouse (ApoE-KO) with ovariectomy showed lower number of autophagy formation, mitochondrial function, and Rab9 expression compared to ApoE-KO with sham operation.

Conclusion:
Mitophagy derived from Sirt1/AMPK/Rab9-mediated mitophagy plays a key role in the effect of estrogen on retarding vascular senescence.
Abstract 224: Reducing Fluoroscopy Times and Contrast Volume Significantly in the Therapy of Acute Coronary Syndromes by Performing Angioplasty Through Cordis 6F Diagnostic Catheters - Changing the Interludes
Authors: Arokiaraj Mark C
Published: February 2019

Aims:
To reduce fluoroscopy times and contrast volume in the therapy of acute coronary syndromes by performing angioplasty and stenting through 6F Cordis diagnostic catheters in acute coronary syndromes.

Methods:
414 patients underwent angioplasty and stenting with acute coronary syndromes with 6F Cordis diagnostic catheters. Primary angioplasty was seen in 124 cases. Baseline renal failure or elevated creatinine >2mg/dl was seen in 15 cases. Baseline creatinine more than 4mg/dl was seen in 6 cases. N acetyl cystenine was given to patients orally for 2 days when creatinine was >1.3mg/dl. All procedures were performed through femoral route only using 6F Cordis diagnostic catheters only. The details of the patients and fluoroscopy times were analysed retrospectively. All patients were followed up at 30 days after discharge.

Results:
In total 511 lesions and 575 stents were used in the angioplasty procedures. Retrospective analysis showed the mean contrast volume per patient was 27±8 ml. Also, the mean fluoroscopy time was 5.8±2.7 min/patient including the angiogram prior to the angioplasty. In 11% cases where the procedure time was >10 mins, and if these cases were eliminated from analysis the mean fluoroscopy time was 4.4±1.8 min/patient including the angiogram before the angioplasty. There were 4 patients who expired, and 3 of these patients had cardiogenic shock, one patient expired due to possible acute stent thrombosis after discharge. In total, 14 cardiogenic shock patients were treated. Two patients had acute stent thrombosis and were treated with an additional stent. One patient required dialysis who had a baseline creatinine of 5.6mg/dl at presentation. Congestive cardiac failure was seen in 21 cases who were treated with oxygen and early BIPAP ventilation, and frusemide injections and infusion. A variety of stents from various companies could be used in the procedures. Buddy wires were used in 12 patients. Proximal mild edge dissection was seen in two patients after stenting and it was treated with a additional stent. Also, it is feasible to perform IVUS during the procedures.

Conclusion:
It is feasible to safely reduce the fluoroscopy times and contrast volume by performing angioplasty through 6F Cordis diagnostic catheters in acute coronary syndromes.
Abstract 227: Inhibition of Histone Deacetylase Catalytic Activity Improves Cardiac and Pulmonary Function in a Feline Model of Heart Failure with Preserved Ejection Fraction
Authors: Wallner Markus , Eaton Deborah , Berretta Remus , Wu Jichuan , Jeong Mark , Lin Ying , Baker Sandy , Oyama Mark , von Lewinski Dirk , Mohsin Sadia , Post Heiner , McKinsey Timothy , Wolfson Marla , Houser Steven R
Published: February 2019

Rationale:
Heart Failure with preserved Ejection Fraction (HFpEF) accounts for about 50% of all heart failure cases but has no effective therapies.

Hypothesis:
Inhibition of histone deacetylase (HDAC) catalytic activity exerts beneficial effects on the cardiopulmonary system in a feline model of HFpEF.

Methods and Results:
Male domestic short hair cats (n=21, aged 2mo), underwent either a sham procedure (n=5) or aortic constriction (n=16) using a customized pre-shaped band, resulting in slow progressive pressure overload during growth. 2 months post-banding, banded cats were treated daily with either 10mg/kg suberoylanilide hydroxamic acid (
b+SAHA
) (n=8), a pan-HDAC inhibitor, or vehicle (
b+veh
) (n=8) for 2 months. At 4 months post-banding,
b+SAHA
cats showed significantly reduced LV wall thickness and LA size (LA/Ao) compared to
b+veh
cats (Fig). Invasively measured left ventricular end-diastolic pressure (Fig. LVEDP) at 4 months was significantly elevated in
b+veh
cats compared to
b+SAHA
and sham cats (Fig). After dobutamine infusion, the increase in dp/dt
max
was significantly attenuated in
b+veh
, but not in
b+SAHA
cats (Fig). In vivo measurements of pulmonary function at 4 months post-banding demonstrated marked improvement in
b+SAHA
cats, reflected by an increase in lung compliance and a decrease in alveolar-arterial oxygen gradient (A-aDO
2
) and intrapulmonary shunt (Fig). Efficacy of SAHA in vivo correlated with enhanced ex vivo myofibril relaxation.

Conclusion:
HDAC inhibition rescues the established HFpEF phenotype by reducing LV hypertrophy, LVEDP, LA size, and enhancing myofibril relaxation, which ultimately results in improved pulmonary function.
Abstract 247: Mitochondrial Damage Associated Molecular Patterns Promotes Endothelial Dysfunction in the Microcirculation
Authors: Ait-Aissa Karima , Kadlec Andrew O , Chabowski Dawid S , Hockenberry Joseph C , Linn Jasmine M , Gutterman David D , Beyer Andreas M
Published: February 2019

Vascular endothelial dysfunction is an early event in the development of atherosclerosis. Flow-mediated dilation (FMD) can be used to identify such endothelial dysfunction which is characterized by a decrease of nitric oxide (NO). Indeed, in subjects with coronary artery disease (CAD), microvascular FMD switches from a NO-mediated to a H
2
O
2
-mediated mechanism with corresponding elevations in mitochondrial reactive oxygen species (ROS). Recent evidence has shown an increase circulating mitochondrial DNA Damage-Associated Molecular Patterns (
mt
DAMPs) in Coronary Heart Disease subjects and has been linked to vascular dysfunction.
mt
DAMPs signaling can activate the inflammatory response directly through activation of Toll Like Receptors 9 (TLR9). The effect of
mt
DAMPs on the endothelial function in the human microcirculation and the potential role for TLR9- mtDNA signaling axis to affect microvascular FMD, is unknown. In this study, we hypothesized that increased levels of
mt
DAMPs result in changes in microvascular redox environment that promote a loss of NO mediated dilation and elevation in mitochondrial derived ROS

The incubation of cultured endothelial cells (ECs) with
mt
DAMPs decreased the ratio peNOS:teNOS in a time-dependent manner supporting the idea that
mt
DAMPs reduce NO bioavailability. This effect was eliminated when the ECs were pre-incubated with TLR9-targeted si-RNA. Prolonged (15-20H) ex vivo exposure to low dose of
mt
DAMPs (1ug/mL) changed the mechanism of FMD from NO to compensatory increase in H
2
O
2
to preserve overall dilator capacity

in non-CAD human adipose micro-arterioles (% Max Diameter: Control 84.7±8.2; + L-NAME: 89.4±5.5; + Peg-Cat 35.4±10.8*; N=5, *p<0.05). With higher dose of
mt
DAMPs (2.5ug/mL), the magnitude of the FMD was however significantly altered (% Max Diameter: high dose: 42.8 ±9.1* vs. low dose of
mt
DAMPs 84.7±8.2; N=4; p<0.05).

Our data suggest a potential pathogenic role of
mt
DAMPs in microvascular function through TLR9 to reduce NO bioavailability and induce endothelial dysfunction.
Abstract 250: Pulmonary Hypertension Induced by 15-HETE is Reverse by Apoai Mimetic Peptide 6f Administration
Authors: Ruffenach Gregoire , O’Connor Ellen I , Vaillancourt Mylene , Sarji Shervin , Cao Nancy , Aryan Laila , Cunningham Christine , Grijalva Victor , Umar Soban , Reddy Srininivasa T , Eghbali Mansoureh
Published: February 2019

Pulmonary arterial hypertension (PAH) is characterized by pulmonary arterial occlusion leading to increased pulmonary arterial pressure. Recently, a growing body of evidence demonstrated a robust increase in oxidized lipids, including 15-hydroxyeicosatetraenoic acids (15HETE), in the lungs and plasma of PAH patients and animal models of pulmonary hypertension (PH). Recently, our group demonstrated that 15HETE induce PH. Nonetheless, the precise mechanism by which 15HETE participates in PH development remains elusive. In order to investigate this mechanism, we fed wild type mice with a diet rich in 15HETE and examine whether ApoA-I mimetic peptide can rescue PH induced by 15HETE.

Methods:
Wild type male mice (C57BL/6) were fed for 3 weeks for 3 weeks with regular chow (n=16-21 mice/group), 15HETE (5μg/day), 15HETE diet supplemented with either empty vector or 6F for the last week of 15HETE diet. PH development was assessed every week via serial echocardiography. Right ventricular systolic pressure (RVSP) was measured via heart catheterization. RV hypertrophy index (RV/[IVS+LV]) was measured. Lung morphology and lipid accumulation were assessed using H&E and Oil red O staining.

Results:
Echocardiography revealed the first sign of PH as early as one week after starting 15HETE diet and a significant decrease in the pulmonary arterial acceleration time (PAAT) after 2 weeks of treatment (16.6±1.9 vs. 20.6±1.4 msec, p<0.05). At the end of three weeks, mice on 15HETE diet had significantly higher RVSP (31.3±1.1 vs. 38.4±2.3 mmHg, p<0.05) and RV hypertrophy index (0.26 ± 0.02 vs. 0.33 ±0.02, p<0.05). This increased pressure was concomitant with a significant increase in pulmonary arteriolar thickness in mice on 15-HETE diet compared to regular diet (35.1±0.8 vs 53.4±1, p<0.05). Furthermore, these WT mice did not develop atherosclerosis as there was no detectable plaque in aorta of the mice on 15HETE diet. At the end of three weeks mice treated with 6F showed a PAAT similar to control value(19±0.5 msec) concomitant with a significantly lower RVSP than mice fed with 15HETE +empty vector (33.6 ±5.7 vs 40.3±4.6, p<0.05).

Conclusion:
Our data demonstrates that APOA-I mimetic peptide 6F is able to rescue pre-exisiting PH induced by 15-HETE diet in wild type mice.
Abstract 251: The Role of Adenosine Derived From Mesenchymal Stromal Cell on Neutrophil Extracellular Trap Formation in Myocardial Ischemia Reperfusion
Authors: Xu Kai , Shin Eric , Wang Lanfang , Deppen Juline , Levit Rebecca
Published: February 2019

Introduction:
In response to tissue damage or infection, neutrophils expel their chromatin into the extracellular space in a thrombotic and inflammatory process called neutrophil extracellular traps(NETs). There is increasing evidence that NETs play a role in myocardial ischemia/reperfusion (MI/R) injury. NETs are found in thrombi aspirated from coronary arteries, atherosclerotic plaques, and deep vein thrombosis. However, nothing is known about the regulation of NETs in vivo. We hypothesized that adenosine, well know for anti-inflammatory effects, reduces NET formation. After MI/R, adenosine is made from conversion of ATP released from damaged cells.

Methods:
Neutrophils were isolated from human venous blood and stimulated to produce NETs by PMA, a PKC agonist and is well known to induce robust NET formation and serves as a positive control. NETs were quantified by Sytox green fluorescent to quantify extracellular DNA and immunohistochemical staining for citrullinated histone H3, a specific marker of NETs.

Results:
Neutrophils treated with PMA increased NET production (1.81fold vs unstimulate groups, p<0.001, n=27-28). Neutrophils treated with adenosine (10uM) had a 30.2% decrease NET formation (p<0.001, n=15). There are four adenosine receptors called A1, A2a, A2b, and A3. We used agonists and antagonists’ strategy to check probable receptors involved in suppression NETs signal. We find both A2a receptor agonist-CGS 21680 and A2b agonist-BAY 60-6583 can suppress NETs formation (29.6% and 29.3% respectively p<0.001, n=5-6). But only A2a antagonist-ZM241385 can reverse adenosine’s inhibition (13.8%v.s.24.7%, p<0.05, n=6).

Conclusion:
Adenosine can downregulate NET formation in vitro likely through A2a receptor activation. Future studies will investigate A2a receptor using genetic knock out, as well as in vivo significance of NET inhibition in MI/R.
Abstract 252: Downregulated Striated Muscle Preferentially Expressed Protein Kinase Enhances Susceptibility to Post-Operative Atrial Fibrillation
Authors: Campbell Hannah , Ni Li , Reynolds Julia , Alsina Katherina , Veleva Tina , Abu-Taha Issam , Quick Ann , Donoviel Zachary , Li Na , Dobrev Dobromir , Wehrens Xander
Published: February 2019

Post-operative atrial fibrillation (poAF) is the most common perioperative arrhythmia associated with increased hospital morbidity and mortality. It is known that atrial myocyte Ca
2+
handling changes can play a crucial role in AF pathogenesis, although the specific mechanisms involved in poAF have not been elucidated. Our lab has previously identified Striated Muscle Preferentially Expressed Protein Kinase (SPEG) as a binding partner of the major cardiac Ca
2+
release channel RyR2 and its negative regulator JPH2, which are critical for cardiac Ca
2+
homeostasis. We hypothesized that decreased SPEG levels may create an arrhythmogenic substrate promoting poAF development.

SPEG protein and mRNA levels were assessed using Western blotting and qRT-PCR, respectively. To establish a mouse model of poAF, we performed thoracotomy in C57Bl/6J mice followed by rapid atrial pacing three days post-operatively to assess for poAF susceptibility. To test for a cause-effect relationship between decreased SPEG and AF susceptibility, we used a cardiac specific tamoxifen-inducible SPEG conditional knockout (cKO) mouse. Rapid atrial pacing was performed in SPEG cKO mice two weeks post-tamoxifen injection prior to onset of heart failure as assessed by echocardiography.

In human atrial samples from patients with poAF we found 41±9% lower
SPEGβ
mRNA expression (n=7, p=0.04) compared to patients with normal sinus rhythm (NSR, n=8), while
SPEGα
expression was unchanged. Similarly, SPEGβ protein was 45±7% lower in poAF vs NSR patients (n=6, p=0.04). In contrast,
SPEGβ
mRNA expression was not decreased in patients with persistent or paroxysmal AF. Similar to poAF patients, our novel mouse model exhibited 90±4% lower SPEGβ protein levels (n=4, p=0.03) compared to mice with NSR (n=4) after rapid atrial pacing three days post-thoracotomy. Furthermore, SPEG cKO mice had increased AF duration after rapid atrial pacing (19.43 ± 15.16 s, n=3) compared to controls (0.27 ± 0.27 s, n=6, p=0.01), suggesting that loss of SPEG is sufficient to drive AF.

In conclusion, we discovered reduced SPEGβ mRNA and protein in both humans and mice with poAF and established that a loss of SPEG might causally contribute to poAF pathogenesis.
Abstract 254: Mitochondrial Permeability Transition Pore, Calcium Uniporter, and Iron Overload in the Heart
Authors: Gordan Richard , Wongjaikam Suwakon , Fefelova Nadezhda , Siri-Angkul Natthaphat , Gwathmey Judith K , Chattipakorn Nipon , Chattipakorn Siriporn C , Xie Lai-Hua
Published: February 2019

Although iron (Fe) overload-induced cardiomyopathy/arrhythmias remain a significant clinical challenge, the underlying mechanism is not well defined. In the present study, we assessed the involvement of mitochondrial permeability transition pore (mPTP) and Ca uniporter (mCU) in Fe overload-induced mitochondrial Ca mishandling and cardiac contractile dysfunction. Firstly, we tested the hypothesis that Fe overload promotes reactive oxygen species (ROS) production and mitochondrial membrane potential (Δ
Ψ
m
) depolarization that may open the mPTP and promote intracellular Ca dysfunction. In isolated mouse ventricular myocytes, both cytosolic and mitochondrial Fe levels (monitored by using specific Fe dyes) were elevated following acute perfusion with Fe
3+
/8-Hydroxyquinoline (8-HQ) (1-100 μM). Mitochondrial superoxide generation (by MitoSOX Red) and cytosolic ROS levels (by DCF) were significantly enhanced by Fe overload. Furthermore, Fe overload induced Δ
Ψ
m
depolarization as measured by TMRM. The mitochondrial maximal respiration rate and reserve capacity (using the Seahorse XF
e
96 analyzer) were significantly reduced by Fe overload. Fe overload significantly increased the rate of Ca waves as imaged with Fluo-4. Fe overload-promoted Ca waves were significantly reduced by the mPTP inhibitor cyclosporine A (CsA). In Langendorff-perfused hearts subjected to programmed S
1
-S
2
stimulation, Fe significantly increased arrhythmia scores compared to the hearts pretreated with CsA, indicating mPTP inhibition can ameliorate the proarrhythmic effects of Fe-overload. These observations were confirmed in cyclophilin D knockout mice, in which mPTP opening is impaired. Furthermore, the involvement of the mCU was evaluated in a chronic Fe overload model (Fe dextran injection,
i.p.
) by using conditional mCU knockout (mCU KO) mice. We observed that mCU KO prevented mitochondrial Fe uptake and conferred a significantly higher resistance to Fe-induced mitochondrial ROS generation, Ca mishandling and cardiac contractile dysfunction. In conclusion, mitochondrial Fe uptake and mitochondrial Ca dysregulation, presumably mediated by both mCU and mPTP, account for the molecular/cellular mechanism for Fe-induced cardiomyopathy and arrhythmias.
Abstract 262: GRK5 is a Novel Regulator of Fibroblast Activation
Authors: Eguchi Akito , Koch Walter J
Published: February 2019

Pathological remodeling of the heart is a hallmark of heart failure (HF) and these structural changes of the heart further perpetuates the disease. Cardiac fibroblasts are the critical cell type that is responsible for maintaining the structural integrity of the heart. Stress conditions, such as a myocardial infarction, can activate and induce the transdifferentiation of quiescent fibroblasts into synthetic and contractile myofibroblasts. G protein-coupled receptor (GPCR) kinases (GRKs) are important mediators of cardiovascular homeostasis through dampening of GPCR signaling. Recently, non-canonical activities of these GRKs have been elucidated. GRK5 is one isoform of these kinases that is expressed in the heart and has been shown to be up-regulated in human HF. Of note, GRK5 has been demonstrated to translocate to the nucleus in cardiomyocytes, promoting hypertrophic gene transcription through activation of nuclear factor of activated T-cells (NFAT) and nuclear factor κB (NFκB). Interestingly, these transcription factors are also involved in fibroblast activation and transdifferentiation. GRK5 is highly expressed and active in cardiac fibroblasts, however its physiological and pathological role in these crucial cardiac cells is unknown. Utilizing adult mouse cardiac fibroblasts
in vitro
, our preliminary data suggests a relevant role for GRK5 in the activation and function of fibroblasts. We observed that genetic deletion of GRK5 in cardiac fibroblasts attenuated α smooth muscle actin (α-SMA) expression, a classical myofibroblast marker, after stimulation with Transforming Growth Factor β (TGFβ) and Angiotensin II (AngII). In addition, deletion of GRK5 diminished the expression of myofibroblast specific genes and proteins such as Collagen I and II after AngII stimulation and were refractory to TGFβ and AngII mediated collagen gel contraction. Conversely, GRK5 overexpression led to an increase in differentiation markers such as α-SMA. These data support the hypothesis that GRK5 is an essential regulator of fibroblast activation. GRK5 fibroblast knockout mice are currently being examined to determine if GRK5 regulates fibroblast transdifferentiation
in vivo
.
Abstract 263: Cytosolic RBFox1 in Cardiac Pathological Remodeling
Authors: Gao Chen , Hsiao Yun-Hua (Esther) , Wang Menglong , Xiong Zhaojun , Ren Shuxun , Rau Christoph D , Li Katelyn , Xiao Xinshu(Grace) , Wang Yibin , Xing Yi
Published: February 2019

Background:
RBFox1 is known to be an RNA splicing regulator with enriched expression in cardiac muscle. Loss of RBFox1 expression is a molecular hallmark associated with heart failure in mouse and human. Genetic manipulation of RBFox1 reveals a major function of RBFox1 in the pathogenesis of cardiac hypertrophy, fibrosis and dysfunction under pathological stresses. However, much of our current knowledge about RBFox1 focuses on the nuclear RBFox1 with a major impact on global alternative splicing changes. Yet, RBFox1 gene also generates a cytosolic isoform-RBFox1c through alternative splicing, and the specific function of RBFox1c has not been characterized.

Goal and Methods:
This study investigated the functional impact and the underlying mechanism of the RBFox1c in cardiac pathological remodeling and targeted gene regulation.

Results:
RBFox1c expression is significantly repressed in stressed cardiomyocytes in vivo and in vitro. RNA-Seq combined with IPA analyses from cardiomyocytes revealed RBFox1c but not the nucleus RBFox1 specifically suppressed pro-inflammatory genes. In the cardiac specific RBFox1 knockout mice, enhanced cardiac fibrosis is observed following I/R injury associated with elevated expression of pro-inflammatory genes. In contrast, cardiac specific expression of RBFox1c reduced cardiac fibrosis and inflammatory gene expression following pressure-overload and myocardial infarct injury associated with improved ejection fraction. Motif enrichment analysis identified significant enrichment of the RBFox1 binding motif in the 3’UTR of the RBFox1c regulated genes. We performed CLIP analysis followed by RT-PCR and observed RBFox1c interacted with inflammatory gene 3’UTR. Lastly, we explored the interactome of RBFox1c and found RBFox1c specifically interacted with a component of RNA decay machinery-Upf1. Indeed, while expression of RBFox1c repressed inflammatory gene expression, inactivation of Upf1 abolished this effect in cardiomyocytes.

Conclusion:
RBFox1 regulates cardiac transcriptome reprogramming at two post-transcriptional steps. The RBFox1 nuclei isoform regulates RNA splicing reprogramming, and the RBFox1c represses proinflammatory genes via recruitment of Upf1 mediated RNA degradation.
Abstract 269: RBM20 Knockout Ameliorates Angiotensin II Induced Hypertension and Cardiac Hypertrophy
Authors: Zhu Chaoqun , Nair Sreejayan , Ren Jun , Guo Wei
Published: February 2019

Introduction:
RNA binding motif 20 (RBM20) is a muscle specific splicing factor that has been implicated in heart failure (HF). RBM20 regulates mRNA splicing of titin, a giant muscle protein that is expressed in cardiac and smooth muscles and plays a major role in passive stiffness of them. RBM20 knockout (KO) results in less myocardial stiffness by inducing large titin isoform expression. Hence, we hypothesize that RBM20 decreases arterial stiffness through large titin isoform expression, and thus reduces blood pressure (BP) in hypertension. The aim of this study was to investigate whether RBM20 could be a therapeutic target to reduce BP in patients with hypertension and prevent HF progression.

Methods:
Male wild type (WT) (Sprague-Dawley
х
Brown Norway) and
Rbm20
KO rats (n=10 each) were subcutaneously implanted with osmotic mini-pump to continuously release angiotensin II (Ang II) at a rate of 400 ng/kg/min for 28 days. BP was measured by noninvasive tail-cuff system weekly. Pulse wave velocity (PWV) was measured by Doppler system. Cardiac remodeling was evaluated by echocardiography. Biochemistry and histological analyses were performed on aorta and heart tissues.

Results:
Larger titin isoform was increased and no difference of elastin and collagen expression was observed in RBM20 KO blood vessels when compared to WT. Basal BPs were the same in WT and KO rats (diastolic BP, KO 91.75 ± 1.83
vs.
WT 94.33±1.88 mmHg; systolic BP, KO 127.6 ± 0.97
vs.
WT 128.0 ± 1.35 mmHg). Ang II increased BP in both WT and KO rats, but KO had a significantly lower BP than WT (diastolic BP, KO 141.7 ± 5.00
vs.
WT 182.0±4.84 mmHg; systolic BP, KO 195.4 ± 3.671
vs.
WT 226.4 ± 3.093 mmHg, P<0.0001). PWV is lower in KO rats than in WT (KO 4.035 ± 0.21 mm/s
vs.
WT 5.128±0.37 mm/s, P<0.05). Cardiac hypertrophy was developed in both WT and KO hearts treated with Ang II, but it was alleviated in the KO heart (left ventricular wall, KO 0.324 ± 0.01 cm
vs.
WT 0.368 ± 0.013 cm, P<0.05).

Conclusions:
RBM20 KO reduces arterial stiffness through increased larger titin isoform expression, decreases Ang II-induced hypertension, and prevents HF progression. These findings suggest that RBM20 could be a potential novel target for the treatment of hypertension and hypertension-induced cardiac hypertrophy.
Abstract 270: Long term Caloric Restriction and Myocardial Aging in Non Human Primates
Authors: Natarajan Niranjana , Kiehm Spencer H , Das Jishnu , Wang Annie C , Mattison Julie A , Colman Ricki J , Lee Richard T
Published: February 2019

Caloric restriction (CR) has been shown to decrease aging and increase the lifespan of several model organisms. However, in two on-going independent, long-term CR studies in non-human primates (NHP) at the National Institute of Aging (NIA) and University of Wisconsin, Madison (UW) the outcome has been more complex. While CR monkeys in the UW study have experienced a significant increase in lifespan relative to their study controls, this was not the case for the NIA study. There, CR did not increase lifespan compared to the already long-lived controls. Yet, in both studies CR was associated with a decrease in the occurrence of age-related diseases. Left ventricle (LV) samples were provided from the rhesus monkeys from both studies to investigate the effect of long-term CR on myocardial pathology and aging in these two cohorts of non-human primates (NHPs). Although CR did not reduce cardiac fibrosis or hypertrophy in either study, there was a 5.9-fold increase in fibrosis (p<0.0001) in LV tissue from the UW study compared to NIA, irrespective of CR treatment. Myocardial mTOR levels were reduced with CR in the UW study, only. These findings indicate that the severity of cardiac fibrosis may not be susceptible to the mechanisms by which CR is affecting disease progression, and myocardial mTOR is apparently differently affected dependent on the study paradigm. Interestingly, the diet administered by the UW study contained a much higher sucrose content (28.5% sucrose by weight), compared to that of the NIA diet (3.9% sucrose by weight). This raises the intriguing possibility that myocardial fibrosis in NHPs may be driven by a high sucrose diet.

Abbreviations:

CR: Caloric Restriction

NHP: Non-human primate

mTOR: mechanistic Target of Rapamycin

NIA: National Institute on Aging

UW: University of Wisconsin, Madison
Abstract 277: Enhanced Mitochondrial Calcium Uptake Promotes Deleterious Remodeling and Impaired Left Ventricular Function During Chronic Adrenergic Stimulation
Authors: Garbincius Joanne F , Luongo Timothy S , Lambert Jonathan P , Elrod John W
Published: February 2019

The mitochondrial calcium uniporter (MCU) constitutes the pore-forming unit of the mitochondrial calcium uniporter channel (mtCU) and is necessary for mitochondrial Ca
2+
(
m
Ca
2+
) uptake. We previously reported that MCU is required to increase cardiac energetics and contractility in response to acute β-adrenergic stimulation. However, deletion of MCU has no detrimental effect on basal cardiac function or metabolism. While these findings support a role for MCU-dependent Ca
2+
flux in functional adaptations to acute stress, little is known about the involvement of MCU in cardiac responses to sustained stress. We compared mice with adult, cardiomyocyte (ACM)-specific loss (
Mcu
fl/fl
;
Mcu
-cKO) or gain (CAG-CAG-MCU; MCU-Tg) of MCU function to explore the role of
m
Ca
2+
uptake in a model of chronic catecholamine overload. Biophysical characterization confirmed that MCU overexpression enhanced and MCU deletion inhibited ACM
m
Ca
2+
uptake. Neither loss nor gain of MCU had a significant impact on baseline contractile function. In αMHC-MCM control mice, fractional shortening was transiently increased after 2 days of isoproterenol infusion (Fig 1). This early increase in contractility was attenuated in
Mcu
-cKO mice. MCU-Tg mice exhibited decreased fractional shortening at 7 and 14 days of isoproterenol infusion. This detrimental effect on LV function was associated with increased LV dilation, HW/BW ratio, and lung edema as compared to αMHC-MCM controls. These data suggest that although mtCU-dependent
m
Ca
2+
uptake is essential for early energetic adaptations to high adrenergic load, under conditions of chronic adrenergic stress it is maladaptive and predisposes to heart failure.