bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 185: Role of Substance P in Pathogenesis of Chemotherapy Associated Cardiotoxicity.
Authors: Legi Ashiq , Robinson Prema
Published: October 2019

Doxorubicin (DOX), is broadly considered the most active single agent available for many cancers. Doxorubicin has been known to induce, cardiotoxicity and life-threatening heart failure or acute coronary syndromes in some patients. There is an urgent need to develop new non-cardiotoxic therapies in cancer. Substance P (SP) is a neuropeptide involved in pain transmission. We had previously determined that aprepitant, an antagonist of SP receptor (neurokinin1 receptor (NK-1R)), that is routinely used to treat chemotherapy associated nausea, prevents DOX induced cardiomyocyte death in vitro. In the current studies, we determined if SP receptor antagonism could prevent chemotherapy induced cardiotoxicity in vivo. Six week old, male C57BL/6 mice were administered DOX (5 mg/kg of body weight, intraperitoneally (IP) once a week for 5 wks. In the aprepitant treated group, five days before the first injection of DOX, aprepitant (0.5 mg/kg) was administered in the drinking water, and then consequently every day until the end of the experiment. The control groups were treated with vehicle alone (1X PBS) instead of DOX without or with aprepitant pretreatment. Mice were euthanized 6 weeks after the first dose of DOX. We determined heart functions by echocardiogram in all mice. The mean ejection fraction (EF) and fractional shortening (FS), percentage, following treatment with DOX respectively decreased significantly by 25.85% and 30.73% compared to that in the control vehicle group (EF: 40.428 ± 6.68 (DOX;
n
= 4) vs 54.526 ± 11.15% (Control Vehicle group;
n
= 4) (FS: 19.51 ± 3.66% (DOX;
n
= 4) vs. 28.16 ± 7.38% (Control Vehicle group;
n
= 4) (both,
P
< 0.05, ANOVA). Importantly, treatment with aprepitant in DOX treated mice normalized the EF and FS values to that present in the control vehicle group (EF; 56.80 ± 6.81 (DOX+aprepitant;
n
= 4) vs 54.526 ± 11.15% (Control Vehicle group;
n
= 4) (FS; 29.64 ± 4.46% (DOX+aprepitant;
n
= 4) v 28.16 ± 7.38% (Control Vehicle group;
n
= 4) (both,
P
< 0.05, ANOVA). These findings indicate that SP contributes to chemotherapy associated cardiac-dysfunction via interaction with its high affinity receptor, NK1R.
These studies suggest that SP-receptor antagonism may be a novel option to prevent chemotherapy associated cardiotoxicity in cancer.
Abstract 199: The Role of miR-987 in Homeostasis of the Aging Heart in
Drosophila
Authors: Hohman Alyssa M , McNeill Elizabeth M
Published: October 2019

Background:
microRNAs (miRNAs) have emerged as ideal candidates to regulate the genes involved in tissue homeostasis of the aging heart. Our work suggests a novel role for miR-987 in heart tissue maintenance during aging.
Drosophila
miR-987 has a highly conserved seed sequence with human miR-95 and miR-545. Both miR-545 and mir-95 have been identified as possible biomarkers in cardiovascular disease and are known to be dysregulated following cardiac events. The physiological consequences of their dysregulation, their molecular mechanism of action and specific gene targets are unknown.

Methods and Results:
We have characterized the impact of miR-987 loss on homoeostasis of heart morphology and function during aging using a combination of null mutants and heart specific knock down of miR-987 expression. Using SOHA (semi-automated optical heartbeat analysis), we have identified an increase in heart area and diameter around middle age (20 days post eclosion), but no significant change in arrhythmicity index, diastolic interval, systolic interval, and fractional shortening in miR-987 null mutants. We have examined the effect of miR-987 loss on lifespan and found a significant impact on male survival starting at 20 days. Using
in silico
analysis we have identified multiple (130) human conserved putative miRNA-target gene interactions to further characterize the molecular mechanisms underlying the observed phenotypes.

Conclusions:
Our results suggest that miR-987 plays and important role in heart tissue homeostasis during aging and may impact the lifespan of mutant animals. The high conservation of
Drosophila
miR-987 with human miR-95 and miR-545 and their conserved predicted gene targets provides us with an excellent platform to facilitate the discovery of the molecular mechanisms underlying miRNA dysregulation in cardiovascular disease.
Abstract 212: Hypertrophic Cardiomyopathy Mutations With Opposite Effects on ß-myosin Biomechanics Show Similar Structural and Biomechanical Phenotypes in Human Induced Pluripotent Stem Cell Derived Cardiomyocytes (hipsc-cms)
Authors: Schroer Alison , Jung Guanghyun , Kooiker Kristina , Adhikari Arjun , Song Linda , Liu Chao , Ruppel Kathleen , Wu Sean , Pruitt Beth , Spudich James , Bernstein Daniel
Published: October 2019

Introduction:
Hypertrophic cardiomyopathy (HCM) is the most prevalent heritable cardiovascular disease, commonly caused by mutations in beta cardiac myosin heavy chain (βMYH). We have measured the kinetics of isolated myosin proteins with different HCM mutations in βMYH and found an intriguing heterogeneity in kinetics and force production: some mutations increase whereas others decrease force and velocity; others result in no change. How these divergent molecular alterations converge to the final HCM phenotype: hypercontractility and cellular hypertrophy is still unknown. hiPSC-CMs provide a powerful tool for studying human cardiomyocyte biology including contractility, hypertrophic growth, and intracellular organization, but are limited by immature phenotypes and population heterogeneity in traditional culture environments.

Methods and Results:
We developed a micropatterned hydrogel platform that enhances force generation and promotes both structural and molecular hiPSC-CM maturation. Using CRISPR/Cas-9 gene editing in an isogenic hiPSC line, we created hiPSCs with two different βMYH mutations (P710R and D239N) that result in opposite effects on force at the molecular level, using in vitro motility and laser trap methods. Cell morphology was quantified by both fluorescence and transmission electron microscopy (EM) and force generation measured by traction force microscopy. Despite having opposite effects at the single molecule level, both HCM lines showed increased contractile force and cellular hypertrophy compared to isogenic controls. Immunostaining for βMYH and EM revealed organizational and microstructural changes including sarcomere and myofibrillar disarray, and thickened z-discs in cells containing these mutations. Both mutations had alterations in AMPK, MAPK, and calcineurin signaling shown to regulate hypertrophy.

Discussion:
Divergent biomechanical alterations due to HCM mutations at the single molecule level lead to common cellular-level structural and biomechanical phenotypes through activation of common signaling pathways.
Abstract 214: Effect of Matrix Stiffness on Adult Cardiomyocytes Using Dynamic, Tunable, and Reversible Magnetorheological PDMS Substrates
Authors: Vite Alexia , Caporizzo Matthew A. , Corbin Elise , Bedi Kenneth , Chen Christina Yingxian , Prosser Benjamin L. , Margulies Kenneth B.
Published: October 2019

The physical properties of the extracellular matrix (ECM), including stiffness and viscoelasticity, play a key role in the development of hypertensive and hypertrophic cardiac diseases at the cellular level, affecting the morphology and function of heart tissue. The ECM is a dynamic structure that is continuously changing in both normal and diseased tissue. In an extension of prior studies examining how substrates of fixed stiffness affect cardiac cells, we introduce a novel experimental model that allows exploration of cardiomyocyte functional alterations in response to rapid and reversible matrix stiffness modulation. We fabricated a biocompatible magneto-rheological elastomer (MRE) culture substrate by adding iron particles to a PDMS elastomer. In the presence of an applied magnetic field, this substrate stiffens reversibly and nearly instantaneously from 10 kPa to 50 kPa: the range observed in normal and diseased myocardium, respectively. Adult cardiomyocytes isolated from non-failing human hearts (unused organ donors) or adult rats were cultured for 24 - 48 hours on the tunable MREs. Cardiomyocytes cultured on a stiffened MRE (~50 kPa) exhibited time-dependent reductions in cell shortening and peak Ca
2+
transient amplitude compared with cardiomyocytes cultured on a soft MRE (~10 kPa). Rates of cardiomyocyte shortening and re-lengthening were also slowed by culture on 50 kPa substrates. Using super-resolution imaging, we found that cardiomyocytes cultured on a stiffened MRE exhibited an increased microtubule network density and demonstrated increased cellular stiffness and viscoelasticity, as measured by nanoindentation. These studies indicate that adult human and rat cardiomyocytes are acutely sensitive to changes in extracellular stiffness within the pathophysiological range and tend to adapt their own stiffness to match that of their surroundings via dynamic changes in microtubule architecture. These mechanisms may contribute to the regulation of mechanical homogeneity within the myocardium. Our method provides a unique
in vitro
platform for studying mechanosensing, mechanotransduction and mechanical memory in isolated adult cardiomyocytes.
Abstract 218: Pathophysiological Significance of Browning of Perivascular Adipose Tissue in the Development of Atherosclerosis
Authors: Adachi Yusuke , Ueda Kazutaka , Ito Kaoru , Takimoto Eiki , Komuro Issei
Published: October 2019

Background:
Perivascular adipose tissue (PVAT) possibly plays a pivotal role in the development of atherosclerosis through its direct local action to the vessels. However, its physiological and molecular mechanisms are less understood. Visceral and subcutaneous white adipose tissues exert a brown adipose tissue-like phenotype under specific conditions such as cold exposure, i.e. browning. We here investigated the pathophysiological role of browning of PVAT against the development of atherosclerosis after endovascular injury.

Methods and Results:
Endovascular injury was generated by wire insertion into the femoral artery of C57BL/6 female mice. Transcriptome analysis revealed robust upregulation of brown adipose tissue markers such as
Ucp1
,
Elovl3
,
Cox8b
and
Cidea
in injured arteries. Notably, administration of an atheroprotective agent, 17-beta estradiol, significantly inhibited these changes;
Ucp1
was the most down-regulated gene by 17-beta estradiol administration in the entire gene set (log2-fold change = -6.58, false discovery rate = 0.003). Consistently, the upregulation of browning markers after endovascular injury and these inhibitions by 17-beta estradiol were confirmed in PVAT by quantitative real-time PCR, western blot and immunohistochemical staining. The present study also demonstrated that vascular injury promotes macrophage infiltration in PVAT accompanied with upregulation of inflammatory cytokine production. Furthermore, we confirmed spatiotemporal synchronicity between browning and inflammation in PVAT by immunohistochemical staining.

Conclusions:
We observed that endovascular injury elicits browning in PVAT accompanied with exacerbated inflammation, and that atheroprotective 17-beta estradiol strongly inhibits this phenomenon. These findings may provide novel insights into pathogenesis of the atherosclerosis.
Abstract 226: Apoai Mimetic Peptide 6f Prevent Pulmonary Hypertension Induced by Oxidized Lipids
Authors: Ruffenach Gregoire , O'Connor Ellen , Vaillancourt Mylene , Sarji Shervin , Cao Nancy , Aryan Laila , Cunningham Christine , Grijalva Victor , Umar Soban , Reddy Srininivasa , Eghbali Mansoureh , Moazeni Donya
Published: October 2019

Introduction:
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). Nonetheless, the the causal role of 15HETE in promoting 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 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). 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 229: TWEAK-Fn14 Axis: A Potential Therapeutic Target for Treating Heart Failure
Authors: Unudurthi Sathyadev , Thomas Evelyn , Patel Nehal , Winkle Alexander , Gratz Daniel , Hund Thomas
Published: October 2019

Heart failure (HF) is a complex disease characterized by compromised cardiac structure and function. Previous work has identified a link between upregulation of pro-inflammatory cytokines, and HF. There is a critical need to gain better understanding of the inflammatory pathways in heart and identify novel therapeutic targets to regulate cardiac inflammation and HF

Recent studies have shown that under stress macrophages are recruited to heart. These macrophages are mainly implicated in the release of inflammatory cytokines that promote cardiac fibrosis, eventually resulting in heart failure. Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a multifunctional member of the TNF superfamily, which binds to fibroblast growth factor inducible 14 (Fn14), a ubiquitously expressed cell-surface receptor. The TWEAK-Fn14 axis has been previously implicated in promoting inflammation in multiple autoimmune disorders, such as rheumatoid arthritis, however, its role in heart in not well known. The objective of this study is to test the hypothesis that TWEAK-Fn14 pathway promotes cardiac inflammation under stress, and blocking this pathway could be a potential therapeutic strategy to reduce stress induced cardiac inflammation and HF. Wild type (WT) and Fn14 knock out (Fn14
-/-
) mice were subjected to pressure overload induced stress (Trans aortic constriction or TAC) for 1 or 6 weeks. A subset of WT TAC animals were treated with the Fn14 inhibitor [L524-0366] (9mg/kg, IP daily) or vehicle beginning at day 3 following TAC. Macrophage infiltration and cardiac fibrosis were quantified in ventricular sections using F4/80 and Masson Trichrome staining respectively, and cardiac function was measured by echocardiography. Levels of monocyte chemoattractant protein-1 (
mcp-1
) were measured by quantitative PCR. Genetic or chemical inhibition of Fn14 reduced fibrosis [40% lower compared to WT TAC] and improved cardiac function [Δ EF of 21.7%, n=6 for WT TAC; Δ EF of 14.2%, n=6 for Fn14
-/-
TAC; Δ EF of 48.5%, n=3 for vehicle; Δ EF of 27.9%, n=5 for drug] compared to WT. Fn14 inhibition also reduced
mcp-1
expression and macrophage infiltration [53.5% lower compared to WT TAC] .These results support TWEAK/Fn14 axis as a potential therapeutic target for treating HF
Abstract 238: Critical Role of Ryanodine Receptor Bound Calmodulin to Prevent Catecholaminergic Polymorphic Ventricular Tachycardia
Authors: Nakamura Yoshihide , Yamamoto Takeshi , Kobayashi Shigeki , Tamitani Masaki , Hamada Yoriomi , Fukui Go , Xu Xiaojuan , Nishimura Shigehiko , Kato Takayoshi , Uchinoumi Hitoshi , Oda Tetsuro , Okuda Shinichi , Yano Masafumi
Published: October 2019

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by a single point mutation in the cardiac type 2 ryanodine receptor (RyR2). Using knock-in mouse (KI) model (R2474S/+), we previously reported that a single point mutation within the RyR2 sensitizes the channel to agonists, primarily mediated by defective inter-domain interaction within the RyR2 molecule and subsequent dissociation of calmodulin (CaM) from the RyR2. Here, we examined whether CPVT can be genetically rescued by enhancing the binding affinity of CaM to the RyR2. We first determined whether there is a possible amino-acid substitution at the CaM-binding domain in the RyR2 (3584-3603) that can enhancethe binding affinity of CaM, and found that V3599K substitution showed the highest binding affinity of CaM to CaM-binding domain. Hence, we generated a heterozygous KI mouse model (V3599K/+) with a single amino acid substitution in the CaM-binding domain of the RyR2, and crossbred it with the heterozygous CPVT -associated R2474S/+ KI mouse to obtain a double heterozygous R2474S/V3599K KI mouse. The CPVT phenotypes—bidirectional or polymorphic ventricular tachycardia, spontaneous Ca2+ transients, and Ca2+ sparks—were all inhibited in the R2474S/V3599K mice. Thus, enhancement of the CaM binding affinity of the RyR2 is essential to prevent CPVT-associated arrhythmogenesis.
Abstract 240: Mistrafficking of Integrin β1d Contributes to Ca2+handling Disorder and Catecholaminergic Polymorphic Ventricular Tachycardia Under Cardiac Stress
Authors: Wang Yihui , Li Chunyan , Ling Shi , Song Long-Sheng , Zhao ShiHua
Published: October 2019

Background:
RyR2 hyper-phosphorylation has been identified as a contributing mechanism to arrhythmogenesis. The integrin β1D colocalized with RyR2 and stabilized phosphorylation of RyR2, which maintains the function of the RyR2 channel. Our objective is to investigate whether the alteration of β1D integrin is a novel mechanism to arrhythmogenesis under cardiac stress.

Methods and Results:
Using transgenic mouse model β1D-/- mice, which results in more uncoupling of β1D and RyR2, showed significantly increased the RyR2 ser2808 phosphorylation and Ca2+handling disorder and CPVT. Translocation of the β1D and uncoupling with RyR2 were discovered in the cardiac disease in human and animal model, instead of decreased β1D. KIF5B mediated β1D redistributed from the center to the periphery with hyper-phosphorylation of ser2808 and Ca2+handling dysfunction.

Conclusions:
These results discover that translocation of β1D and disassociation from RyR2, which leads to hyper-phosphorylation of the RyR2 ser2808, is a novel pathway to trigger CPVTunder cardiac disease.
Abstract 241: Pharmacological Evaluation of Novel Anti-thrombospondin-1 Decoy Strategy for Prophylaxis of Atrial Fibrillation-associated Ischemic Stroke
Authors: Elbayoumi Tamer , Sturdivant Jalicia , Ganguly Samayita , Yao Mingyi
Published: October 2019

Hemostatic abnormalities mainly decreased blood flow and increased stasis, and platelet hyper-aggregation increase thrombotic risk subsequent atrial fibrillation (AF) episodes. Excess elevation of extracellular glycoprotein thrombospondin 1 (TSP1) in circulation has emerged to predict prognosis of ischemic stroke. Moreover, TSP1 is found strongly associated with platelet hyper-aggregability and impaired nitric oxide response, through its cognate receptor CD47, thus exacerbating ischemic progression in AF patients. TSP1 arises as a prospective target of our candidate medicine.

Our work provides “proof-of-concept” of a novel pharmacological strategy, relying on soluble recombinant human CD47 peptide (rh-CD47p) to serve as a decoy receptor to specifically bind TSP1, and consequently neutralize TSP1-impaired vasorelaxation.

Our pilot studies were initially conducted with isolated mouse thoracic aorta. A pre-treatment with rh-CD47p (added 15 min prior to TSP1 incubation) showed a significant amelioration of TSP1-impaired endothelium-dependent vasodilation (
p
<0.0001, rh-CD47p+TSP1 vs. TSP1, n=6), indicating a prophylactic effect by rh-CD47p against vasoconstriction enhanced by excess TSP1. A post-treatment set-up, where TSP1 incubation was started 15 min prior to rh-CD47p addition to mimic pre-existing high level of TSP1 in ischemic stroke, exhibited a marked reversal of TSP1-inhibited vasodilation back to the same level as controls (
p
<0.0001, rh-CD47p+TSP1 vs. TSP1, n=4). Dose titration of rh-CD47p in molar ratios to TSP1 in both set-ups identified the dose range of rh-CD47p for prevention/recovery from TSP1-induced vascular dysfunction.

Binding of rh-CD47p and TSP1 was first verified as the primary mechanism via Western Blot, and further quantified with a modified ELISA assay. A linear correlation was found between % of rh-CD47p bound to TSP1 and dose of rh-CD47p added (r
2
=0.8).

Current results established the ground of rh-CD47p as a unique pharmacologic moiety for drug development to limit vasoconstriction and improve vasodilation against TSP1 in excess, thus showing great potential for proposed novel decoy CD47-coated formulation to abrogate/mitigate TSP1-associated cardiovascular complications in AF patients.
Abstract 242: Sk Channels in the Vascular Endothelial Cells
Authors: Nam Young Woo , Aldakhil Taibah , Viegas Adam , Vu Hannah , Lee Rachel , Wong Pammie , Zhang Miao
Published: October 2019

Small-conductance Ca
2+
-activated K
+
(SK) channels are a unique group of K
+
channels, that play important roles in the vascular system and central nervous system. Four subtypes in the SK channel family are encoded by
KCNN
mammalian genes: including
KCNN1
for SK1 (K
Ca
2.1),
KCNN2
for SK2 (K
Ca
2.2),
KCNN3
for SK3 (K
Ca
2.3) and
KCNN4
for SK4 (also IK or K
Ca
3.1) channels. It is well known that the activation of SK channels expressed on the endothelial cell surface can reduce vascular tone. In our study, we found that SK channels expressed on the endoplasmic reticulum (ER) membrane can protect endothelial cells from apoptosismediated by ER stress. Among the four subtypes of SK channels, Ca
2+
sensitivity and subcellular localization significantly varies by subtype in endothelial cells. Ca
2+
sensitivity of the SK1 channel subtype is negatively modulated more than that of the SK2 subtype, whereas the cell surface expression of the SK2 channel subtype is negatively modulated more than that of the SK1 subtype. The functions of SK channels to reduce vascular tone and to protect against apoptosis are greatly affected by their Ca
2+
sensitivity and subcellular localization. It is critical to understand how each SK channel subtype is modulated, which may become promising therapeutic targets for various cardiovascular conditions.
Abstract 253: Pharmacological and Genetic Inhibition of Transient Receptor Potential Canonical 6 (TRPC6) as a Novel Treatment for Heart Failure and Duchenne Muscular Dystrophy (DMD) Pharmacological and Genetic Inhibition of Transient Receptor Potential Canonical 6 (TRPC6) as a Novel Treatment for Heart Failure and Duchenne Muscular Dystrophy (DMD)
Authors: Lin Brian L , Mishra Sumita , Muller Grace K , Bedja Djahida , Zhu Guangshuo , Yang Jinying , Anderson Mark E , Pullen Steve S , Kass David A
Published: October 2019

Intro:
Duchenne muscular dystrophy (DMD) patients suffer from spinal deformities (scoliosis and kyphosis) and die prematurely in their thirties, mostly due to heart disease. DMD is caused by the lack of the dystrophin protein, but calcium overload contributes to pathology as a secondary insult. Inhibiting hyperactive calcium channels in DMD, such as TRPC6, is a novel therapeutic strategy for treating DMD, and we test the efficacy of pharmacological and genetic TRPC6 inhibition
in vivo
. Therefore, my objectives are: 1) determine the efficacy of chronic pharmacological TRPC6 inhibition in heart disease 2) determine the therapeutic efficacy of genetic TRPC6 inhibition in DMD and 3) identify novel mechanisms of TRPC6 hyperactivity.

Results:
1) Using pressure-overloaded mice as our model of heart disease, we applied the first and only-known TRPC6 inhibitor (BI 749327, 30 mg/kg/day) that is both specific and bioavailable. Chronic pharmacological TRPC6 inhibition blunted a pathological calcineurin/NFAT-TRPC6 feedforward signaling pathway
in vivo
, resulting in improved fractional shortening, normalized cardiac hemodynamics, and potently reduced fibrotic signaling and fibrosis. 2) We generated dystrophin-utrophin-deficient mice, or double-knock-out (DKO) mice, a severe model of DMD that exhibits muscle weakness, spinal deformities, and premature death at ~8 weeks of age. We also generated dystrophin-utrophin-TRPC6-deficient mice, or triple-knock-out (TKO) mice, which exhibited dramatically improved kyphosis, striated muscle function, and nearly three-fold improvement in lifespan. 3) We demonstrate CaMKII mediates TRPC6-mediated contractility combining a custom cardiomyocyte stretch assay with pharmacological and genetic inhibition of TRPC6 and CaMKII.

Conclusion:
TRPC6 inhibition potently reduces fibrosis in heart disease, and dramatically improves life expectancy in DMD. CaMKII is a novel TRPC6-activating kinase, necessary for TRPC6-mediated mechanosensitive activation and force response. The striking therapeutic effects of TRPC6 inhibition in disease may be due to interruption of a kinase imbalance in which CaMKII hyperactivates TRPC6. Future studies will determine the efficacy of pharmacological TRPC6 inhibition in DMD.
Abstract 254: Kinase-dead Pi3kγ Expression in the Cardiac Myocytes Regulates Fibroblast Signaling and Myofibroblast Differentiation
Authors: Mohan Maradumane L , Grove Lisa M , Sahu Anita , Papay Robert S , Olman Mitchell A , Naga Prasad Sathyamangla V
Published: October 2019

Phosphoinositide 3 Kinase γ (PI3Kγ) is an anti-apoptotic molecule acting through Akt pathway. Even though, role of PI3Kγ in cardiac fibrosis has been established, the mechanistic details by which PI3Kγ regulates cardiac myofibroblast differentiation are not clear. Myofibroblasts are hallmark of tissue fibrosis, characterized by smooth muscle α-actin (αSMA) over-expression. We have previously shown that αSMA abundance in cardiac lysates from PI3Kγ null mice (PI3Kγ
-/-
) showed significant baseline and pressure overload [Transverse Aortic Constriction (TAC)] induced upregulation compared to wildtype (WT), indicating that loss of PI3Kγ predisposes the hearts towards fibrosis. Furthermore, isolated cardiac fibroblasts (CF) from PI3Kγ
-/-
exhibited a myofibroblast phenotype with αSMA in stress fibers. Moreover, cardiomyocyte-specific over-expression of kinase-dead PI3Kγ (PI3Kγ
inact
) in the global PI3Kγ
-/-
(PI3Kγ
inact
/PI3Kγ
-/-
) reduced αSMA abundance and myofibroblast differentiation suggesting unique kinase-independent function of PI3Kγ in myocyte-initiated pathway that drives CF to become myofibroblasts. Conditioned media experiments showed that PI3Kγ
-/-
myocytes release pro-fibrotic factors and PI3Kγ
inact
/PI3Kγ
-/-
myocytes release fibrosis protective factors. We have previously observed that PI3Kγ regulated MAPK signaling in fibroblasts in a kinase-independent manner by sequestering PP2A association and activity. Previous studies have shown that fibroblast growth factor mediated activation of the signaling pathway downregulates αSMA and that this inhibition of αSMA expression is through negative regulation by extracellular regulated kinase (ERK). Consistent with these previous observations, PI3Kγ possibly mediates αSMA and myofibroblast differentiation through regulation of ERK signaling in the fibroblasts. Intriguingly, we observed presence of PI3Kγ when lysates of isolated CF from PI3Kγ
inact
/PI3Kγ
-/-
were immunoblotted for PI3Kγ. These data indicate that a unique communication between myocytes and fibroblasts regulated by PI3Kγ, leads to a compensatory mechanism that results in expression of PI3Kγ in the fibroblasts, thereby regulating fibroblast signaling in myofibroblast differentiation.
Abstract 259: Resistin Accelerates Fibroblast-Myofibroblast Differentiation and Induces Myocardial Fibrosis
Authors: Singh Rajvir , Kaundal Ravinder K , Zhao Baoyin , Bouchareb Rihab , Hajjar Roger , Lebeche Djamel
Published: October 2019

Background:
Cardiac fibrosis (CF) is a major cause of heart failure in patients with ischemic insult, obesity, and diabetes. CF, characterized by excessive deposition of extracellular matrix proteins and myofibroblast differentiation, leads to profound changes in ventricular architecture and geometry, and thereby reduces cardiac function. Resistin, an adipokine has been linked to obesity, insulin resistance and diabetes. Our previous findings have implicated resistin in CF, however the molecular mechanisms underlying this process are still unclear. In this study, we investigated whether resistin regulates the transdifferentiation of fibroblasts into myofibroblasts as well as the molecular pathways leading to resistin-induced fibrotic response.

Methods:
Fibroblast-to-myofibroblast differentiation was induced with resistin, TGFβ1 or FBS for 48hours in isolated cardiac fibroblasts from adult mouse and mouse embryonic fibroblasts (NIH3T3 cells). Resistin knockout mice were challenged with high fat diet for 16 weeks to stimulate cardiac lipotoxicity. Fibrotic response and markers were determined in harvested hearts.

Results:
Adult cardiac fibroblasts stimulated with resistin displayed increased fibroblast-to-myofibroblast differentiation, with significantly increased levels of collagen, fibronectin, and connective tissue growth factor. Mechanistically, we demonstrated that resistin promotes fibrogenesis via Jak-Stat and c-Jun signaling pathways, independently of TGFβ1. In-vivo study confirmed the role of resistin in cardiac fibrosis. Resistin knockout mice, when challenged with high fat diet to stimulate cardiac lipotoxicity, showed a decrease in fibrosis paralleled with reduced mRNA levels of various markers of fibrosis such as collagen, fibronectin and connective tissue growth factor compered to wild type hearts. A significant improvement in heart function was also observed in resistin knockout mice.

Conclusions:
To our knowledge, these findings are the first to demonstrate a role of resistin in the process of cardiac fibroblast-to-myofibroblast conversion through Jak-Stat and c-Jun signaling. These results bring new insights into the mechanisms by which resistin may contribute to cardiac fibrosis.
Abstract 261: Excessive O-GlcNAcylation Causes Heart Failure
Authors: Umapathi Mahaa , Mesubi Olurotimi , Granger Jon , Wang Qinchuan , Wu Yuejin , Luczak Elizabeth , Hart Gerald , Anderson Mark
Published: October 2019

Increased protein
O
-GlcNAcylation (OGN) is a common feature of failing heart muscle. However, it is unknown if excessive OGN contributes to cardiomyopathy and heart failure. OGN levels are determined by the net activity of two enzymes: OGT (
O
-GlcNAc transferase, adds OGN) and OGA (
O
-GlcNAcase, removes OGN). We hypothesized that excessive myocardial OGN is a cause of cardiomyopathy. To test for a role of OGN in cardiomyopathy we developed new transgenic (TG) mouse models with myocardial overexpression of OGT or OGA. The OGT-TG hearts showed progressive decline in left ventricular (LV) systolic function, dilation, increased mass (Figure A, B) (Statistical Analysis ANOVA - *** = p<0.001) and increased OGN (Figure C) . OGT-TG mice showed premature mortality compared to WT littermates (Figure D). In contrast, OGA-TG mice exhibit normal contractility, do not have significantly different OGN and have normal lifespan compared to WT littermates. Hearts from OGT-TG and OGA-TG interbred mice have marked improvement of LV systolic function, lower OGN and normal lifespan. We next tested if attenuation of myocardial OGN was beneficial in acquired cardiomyopathy by performing transverse aortic constriction surgery (TAC) on OGA-TG and WT littermates. The OGA-TG hearts had lower OGN, improved LV systolic function, less hypertrophy, and lower expression of heart failure genes compared to WT littermates after TAC. Our data identify excessive OGN as an independent mechanism for cardiomyopathy, and suggest attenuation of OGN may be an effective therapy for heart failure.
Abstract 266: Sex Differences in Anthracycline-Induced Cardiotoxicity in Young Mice
Authors: Grant Marianne , Zordoky Beshay
Published: October 2019

Introduction:
The role of sex as a risk factor for Anthracycline-Induced Cardiotoxicity (AIC) is not well defined. In pediatric cancer patients, most studies report that female sex is a significant risk factor for AIC. Conversely, in adult preclinical rodent studies, there is clear evidence that female sex is protective against AIC, with a possible protective role of female sex hormones and/or a detrimental role of male sex hormones. However, sex differences in AIC have not been reported in young experimental animals. Therefore, the current work aims to characterize sex differences in AIC in young male and female mice.

Methods:
Five-week old male and female C57Bl/6 mice were injected with doxorubicin (DOX) 4 mg/kg/week IP for 3 to 6 weeks. After a 5-week recovery period, a cohort of control and DOX-treated mice were administered daily IP injections of isoproterenol (10 mg/kg) for 14 days to determine the effect of catecholamine stress on cardiac function and morphometry in DOX-exposed mice. Another cohort of gonadectomized and sham-operated male and female mice were injected with DOX 4 mg/kg/week for 5 weeks. Cardiac function and morphometry were measured by trans-thoracic echocardiography 5 weeks after the last DOX injection and after 14 days of isoproterenol injections.

Results:
In male mice only, DOX 4 mg/kg/week for 3 to 5 weeks caused cardiac atrophy without decline in cardiac function, whereas DOX administration for 6 weeks caused cardiac atrophy and dysfunction. Female mice were protected from both cardiac atrophy and dysfunction at the 3-6 weeks regimens. Isoproterenol caused cardiac dysfunction in male and female control and DOX-treated mice. Male, but not female, DOX-exposed mice failed to develop cardiac hypertrophy when challenged by isoproterenol. Intriguingly, castration of male mice exacerbated DOX-induced cardiac atrophy, while ovariectomy had no significant effect.

Conclusion:
In contrast to most clinical studies, young female mice are protected against DOX-induced cardiac atrophy and dysfunction. Juvenile exposure to DOX modulated the cardiac response to isoproterenol in a sex-dependent manner. Deprivation of sex hormones by gonadectomy did not reverse this sexual dimorphism, suggesting that other mechanisms are implicated.
Abstract 281: Single Cell Analysis of Postnatal Heart Development and Disease
Authors: Zhao Juanjuan , Lupino Katherine , Pei Liming
Published: October 2019

A fundamental challenge in understanding cardiac biology and disease is that the remarkable heterogeneity in cell-type composition and functional states have not been well characterized at single-cell resolution in maturing and diseased mammalian hearts. Massively parallel single-nucleus RNA sequencing (snRNA-Seq) has emerged as a powerful tool to address these questions by interrogating the transcriptome of tens of thousands of nuclei isolated from fresh or frozen tissues. snRNA-Seq overcomes the technical challenge of isolating intact single cell from complex tissues including the maturing mammalian hearts, reduces biased recovery of easily dissociated cell types and minimizes aberrant gene expression during the whole-cell dissociation. We have recently applied sNucDrop-Seq, a droplet microfluidics-based massively parallel snRNA-Seq method, to investigate the transcriptional landscape of postnatal mouse hearts in both healthy and mitochondrial disease states. By profiling the transcriptome of nearly 20,000 nuclei, we identified major and rare cardiac cell types and revealed significant cellular heterogeneity in the postnatal developing heart. When applied to a mouse model of mitochondrial cardiomyopathy, we uncovered profound cell type-specific modifications of the cardiac transcriptional landscape at single-nucleus resolution. Here, we expanded these earlier studies and used our dataset to further decipher the cardiac cell type-specific gene regulatory networks. Our analysis reveals novels insights into the key nodes of gene networks that control the postnatal development and disease-associated changes of different cardiac cell types. Our ongoing work is using genetic mouse models to validate these findings from single cell analysis.
Abstract 312: SGLT1 is Essential for Cardioprotection During Ischemia-Reperfusion Injury via Enhanced Glucose Utilization in Diet-induced Obese Mice
Authors: Yoshii Akira , Tomohisa Nagoshi , Kashiwagi Yusuke , Kimura Haruka , Tanaka Yoshiro , Oi Yuhei , Ito Keiichi , Yoshino Takuya , Tanaka Toshikazu D , Yoshimura MIchihiro
Published: October 2019

Recent large clinical trials have shown that sodium-glucose cotransporter 2 (SGLT2) inhibitors reduced cardiovascular events in diabetic patients. However, the regulation and functional role of cardiac SGLTs (SGLT1 is the dominant isoform) compared with those of other glucose transporters (insulin-dependent GLUT4 is the major isoform) remain incompletely understood. Given that glucose is a preferential substrate for myocardial metabolism during ischemia-reperfusion injury (IRI), we hypothesized that SGLT1 contributes to cardioprotection during IRI via enhanced glucose transport, particularly in insulin-resistant phenotypes. The hearts from mice fed a high-fat diet (HFD) for 12 weeks or a normal-fat diet (NFD) were perfused with either non-selective SGLT-inhibitor phlorizin (to inhibit SGLT1) or selective SGLT2-inhibitors (tofogliflozin, ipragliflozin, canagliflozin) during IRI using Langendorff model. After ischemia-reperfusion, HFD impaired left ventricular developed pressure (LVDP) recovery compared with NFD. Although phlorizin-perfusion impaired LVDP recovery in NFD, a further impaired LVDP recovery and dramatically increased infarct size (indicated by CPK release into perfusate and TTC staining) were observed in HFD with phlorizin-perfusion. Meanwhile, none of the SGLT2-inhibitors significantly affected cardiac function or myocardial injury after ischemia-reperfusion under either diet condition. The plasma membrane expression of GLUT4 was significantly increased after IRI in NFD but was substantially attenuated in HFD, the latter of which was associated with a significant reduction in both myocardial glucose uptake and cardiac tissue ATP content. In contrast, regardless of the diet condition, SGLT1 expression remained constant during IRI, whereas SGLT2 was not detected. Of note, phlorizin considerably reduced myocardial glucose uptake and decreased cardiac tissue ATP content after IRI, particularly in HFD. In conclusion, cardiac SGLT1 but not SGLT2 plays a compensatory protective role and contributes to cardiac energy metabolism during acute phase of IRI via enhanced glucose utilization, particularly under insulin-resistant conditions, in which IRI-induced GLUT4 upregulation is compromised.
Abstract 322: Identification of Novel Long Non Coding RNAs Involved in Pitx2>Wnt>microRNA Signaling Pathways Leading to Atrial Fibrillation
Authors: Franco Diego , Garcia-Padilla Carlos , Aranega Amelia E
Published: October 2019

Atrial fibrillation is the most prevalent cardiac arrhythmia in humans. Genetic and genomic analyses have recently demonstrated that the homeobox transcription factor Pitx2 plays a fundamental role regulating expression of distinct growth factors, microRNAs and ion channels leading to morphological and molecular alterations that promote the onset of atrial fibrillation. We now address the plausible contribution of a novel class of non-coding RNA in this Pitx2>Wnt>miRNA signaling pathway, i.e. long non coding RNAs (lncRNAs).
In silico
analyses of annotated lncRNAs in the vicinity of Pitx2, Wnt8 and Wnt11 chromosomal loci identified five novel lncRNAs with differential expression during cardiac development. Among them, Wnt11_45188, Wnt11_44934 and Wnt8_2010Rik displayed preferential atrial-specific expression during embryogenesis. In addition, Wnt11_44653 displayed moderate expression during embryogenesis but peaked preferentially in the right atrium vs left atrium and ventricle in adulthood. Wnt11_45188, Wnt11_44934 and Wnt8_2010Rik were distinctly regulated by Pitx2, Wnt8 and Wnt11 and Wnt8_2010Rik is severely up-regulated in conditional atrial-specific Pitx2 deficient mice. We also demonstrate that arrhythmogenic microRNAs such as miR-1, miR-133 and miR-29 distinctly regulate these lncRNAs. Furthermore, angiotensin II administration to atrial cardiomyocyte cell cultures leads to significant up-regulation of these three newly identified lncRNAs, supporting a link to AF cardiovascular risk factor, i.e. hypertension. Overall, we have identified three novel lncRNAs that are distinctly regulated in the Pitx2>Wnt>miRNA signaling pathways which might be therefore implicated in the gene regulatory network leading to atrial arrhythmogenesis.
Abstract 331: Loss of Myosin Binding Protein H-Like Causes Cardiac Conduction Abnormalities
Authors: Barefield David Y , Yamakawa Sean , Tahtah Ibrahim , Sell Jordan J , Broman Michael , Laforest Brigitte , Pickelwartz Megan J , Wilsbacher Lisa D , Wasserstrom Andrew , Fishman Glenn I , McNally Elizabeth
Published: October 2019

A premature truncation (R255X) in
MYBPHL
associates with human dilated cardiomyopathy (DCM) and arrhythmias. Loss of
Mybphl
in mice causes DCM and arrhythmia.
MYBPHL
encodes myosin binding protein H-like (MyBP-HL) and is expressed highly in the atria. We hypothesize that MyBP-HL is required for proper conduction system function. Immunofluorescence microscopy on normal human atria showed MyBP-HL staining in all atrial cardiomyocytes with a sarcomere A-band pattern. Atria from the heterozygous (het)
MYBPHL
R255X mutant carrier lacked MyBP-HL staining. Human induced pluripotent stem cell-derived cardiomyocytes from the het
MYBPHL
R255X carrier and control cell lines were also examined. MyBP-HL was found in a subset of control cardiomyocytes, whereas R255X cells showed no MyBP-HL, suggesting that the R255X allele exerts a dominant-negative effect on the normal
MYBPHL
allele. Immunofluorescence microscopy in wild-type (WT) mouse ventricles identified MyBP-HL-positive ventricular cardiomyocytes that co-localized with the ventricular conduction system marker contactin-2 near the atrioventricular node and in a subset of Purkinje fibers.
Mybphl
het ventricles have 10% as many MyBP-HL-positive cells compared to WT. Surface telemetry revealed atrioventricular block and atrial bigeminy and intracardiac pacing revealed a shorter atrial relative refractory period and inducible atrial tachycardia in
Mybphl
-null mice. Ca
2+
transients measured with confocal microscopy revealed that isolated
Mybphl
-null atrial cardiomyocytes had an increased occurrence of triggered Ca
2+
waves and more heterogenous Ca
2+
release than WT controls. Super-resolution microscopy revealed ryanodine receptor disorganization in
Mybphl
het and null atrial cardiomyocytes compared to WT controls. Abnormal Ca
2+
release, shorter atrial refractory period, and dilated atria could account for the observed atrial arrhythmias, bigeminy, and atrial tachycardia.
Abstract 332: Pharmacologic Characterization of the Cardiac Myosin Inhibitor, CK-3773274: A Potential Therapeutic Approach for Hypertrophic Cardiomyopathy
Authors: Hwee Darren T , Hartman James J , Wang Jingying , Wu Yangsong , Schaletzky Julia , Paliwal Preeti , Lee Ken , Taheri Khanha D , Wehri Eddie , Chuang Chihyuan , Morgan Bradley P , Malik Fady I , Chin Eva R
Published: October 2019

Hypercontractility of the cardiac sarcomere appears to underlie pathological hypertrophy and fibrosis in select genetic hypertrophic cardiomyopathies. Here, we characterize the small molecule, CK-3773274, as a novel cardiac myosin inhibitor that decreases contractility
in vitro
and
in vivo
. In bovine cardiac myofibrils, CK-3773274 decreased myosin ATPase activity in a concentration-dependent fashion (IC
50
:1.26 μM). CK-3773274 specifically inhibited myosin activity, as it reduced myosin ATPase activity in a concentration-dependent manner in the absence of other sarcomere proteins, including actin, troponin, and tropomyosin. CK-3773274 (10 μM) reduced fractional shortening by 84% in electrically paced, isolated adult rat cardiomyocytes relative to control without any effect on the calcium transient. The effect of CK-3773274 on cardiac contractility
in vivo
was assessed in healthy male Sprague Dawley (SD) rats using single oral doses ranging from 0.5 to 4 mg/kg. Fractional shortening (FS) and left ventricular dimensions were determined by echocardiography at select time points over a 24-hour period. One hour after dose administration, CK-3773274 significantly reduced fractional shortening in a dose-related fashion by 20-70% relative to vehicle treatment (FS %: vehicle: 47.9± 1%; 0.5 mg/kg: 39 ± 2%; 4 mg/kg: 15 ± 4%; mean ±SEM, p<0.05 vehicle vs. all doses) without any changes to heart rate. Lastly, the effect of CK-3773274 was evaluated by echocardiography in healthy beagle dogs. Left ventricular ejection fraction (LVEF) was evaluated following single oral doses ranging from 0.75-3 mg/kg over a 48 hour period. 2 hours after dosing, CK-3773274 decreased LVEF in a dose-related fashion by approximately 15-50% relative to vehicle treatment (LVEF vehicle: 74.6 ± 3 %; 0.75 mg/kg: 62.5 ± 3%; 2 mg/kg: 44.9± 3%; 3 mg/kg: 36.8 ± 2%; mean ±SEM, p<0.05 vehicle vs. all doses). In conclusion, CK-3773274 is a novel, small molecule, cardiac myosin inhibitor that reduces cardiac contractility
in vitro
and
in vivo
. Cardiac myosin inhibition may be a viable approach to treat the underlying hypercontractility of the cardiac sarcomere in hypertrophic cardiomyopathies.
Abstract 337: Cell-Substrate Adhesion Regulates Early Steps of Myofibrillogenesis in Human Cardiomyocytes
Authors: Neininger Abigail C , Taneja Nilay , Burnette Dylan T
Published: October 2019

Forces generated by myofibrils within cardiomyocytes must be balanced by adhesion to the extracellular matrix proteins and to other cardiomyocytes for proper heart function. Loss of this force balance results in cardiomyopathies and heart failure. Using a sarcomere assembly assay we previously developed utilizing human induced pluripotent stem cell-derived cardiomyocytes, we show coupling of focal adhesions to myofibrils during early steps of
de novo
myofibrillogenesis is essential for myofibril maturation. Increasing the extent of adhesion by inhibition of Focal adhesion kinase (FAK), a known regulator of adhesion dynamics, or by increasing the concentration of fibronectin on which the cardiomyocytes are cultured, led to precocious myofibril formation. Decreasing the extent of adhesion by siRNA-mediated FAK knockdown or by decreasing the concentration of fibronectin attenuated myofibrillogenesis. In each case, increased or decreased adhesion extent inversely correlated with rate of retrograde flow of myofibril precursors known as muscle stress fibers (AKA, non-muscle stress fiber-like structures or pre-myofibrils). This suggests a relationship between cell-substrate adhesion and substrate coupling to muscle stress fibers, facilitating their maturation into myofibrils. Taken together, our findings implicate a role for classical mechano-transduction in the assembly of sarcomere containing myofibrils.