bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 393: Cronos Titin in Human Heart Development
Authors: Zaunbrecher Rebecca J , Yang Xiulan , Reinecke Hans , Pabon Lil , Regnier Michael , Murry Charles E
Published: March 2022

Titin is a giant myofibrillar protein that provides passive tension and facilitates sarcomere formation in striated muscle, where it spans from the Z-disk to the M-line. Recently, an alternative start site has been identified in the titin gene that is predicted to allow for expression of a novel isoform, Cronos, that is ~2/3 the size of full-length titin and lacks the Z-disk and most of the I-band domains. Because the start site for this isoform is spliced out of full-length titin transcripts, we are able to determine the expression levels of Cronos compared to full-length titin. Reverse-transcription followed by quantitative PCR using primers specific to this region indicate Cronos transcript levels are higher in human fetal ventricle and atrial tissue (days 90-105) than in adult samples, while full-length titin transcripts are higher in adult than fetal cardiac tissue. Additionally, human ventricular tissue stained with a custom antibody raised to recognize this specific region of Cronos titin indicated high levels of protein expression and integration into myofibrils in early fetal samples (days 54 and 81), while this novel isoform was nearly undetectable in later fetal (day 130) and adult tissue. The Cronos titin transcript and protein were also detected in high quantities in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), which are believed to have a fetal level of maturity. Based on these data, we conclude that Cronos is predominantly a fetal isoform of titin. Future experiments will investigate the effects of a Cronos titin knockdown in hiPSC-CMs during and after differentiation to establish its functional role in human heart development.
Abstract 394: Medium Chain Acyl-Coenzyme a Dehydrogenase Gene Therapy Induces Physiological Cardiac Hypertrophy and Protects Against Pathological Remodeling
Authors: Bernardo Bianca C , Weeks Kate L , Pongsukwechkul Thawin , Gao Xiaoming , Kiriazis Helen , Tham Yow Keat , Du Xiao-Jun , Gregorevic Paul , McMullen Julie R
Published: March 2022

Introduction:
Gene-based therapies represent a realistic and feasible avenue for the treatment of cardiac disease. We previously demonstrated that activation of phosphoinositide 3-kinase [P13K(p110α)] was beneficial in heart failure mouse models and identified PI3K(p110α)-regulated mRNAs. We found that gene expression of medium chain acyl-coenzyme A dehydrogenase (MCAD) to be i) elevated in hearts with increased PI3K activity, ii) reduced in hearts with decreased PI3K activity, iii) reduced in settings of cardiac injury, and iv) positively correlated with cardiac function.

Aim:
The aim of this study was to determine whether gene delivery of MCAD could attenuate pressure overload-induced pathological remodeling.

Methods:
Recombinant adeno-associated viral vectors (rAAV) encoding MCAD or a control (2x10
11
vector genomes) were delivered to normal adult mice (n=9) and mice with pre-existing pathological hypertrophy and cardiac dysfunction due to pressure overload, induced by transverse aortic constriction (TAC, n=16). Cardiac function was assessed by echocardiography. Molecular/histological analyses were performed on heart tissue.

Results:
rAAV6:MCAD delivery promoted physiological growth of the heart in normal mice. Treated mice had larger hearts (Heart weight/tibia length [HW/TL] of rAAV6:control 8.3±0.5 mg/mm
vs.
HW/TL of rAAV6:MCAD 10.1±0.5 mg/mm, n=4-5/group, P<0.05), thicker left ventricular walls (rAAV6:control 0.82±0.03mm
vs.
rAAV6:MCAD 1.00±0.05mm, n=3-4/group, P<0.05), preserved systolic function, and no evidence of cardiac fibrosis, lung congestion or re-expression of the fetal genes atrial- and B- type natriuretic peptides. In a setting of pressure overload, mice that received rAAV:MCAD had less left ventricular fibrosis (3.4-fold lower
vs.
control, n=6-10/group, P<0.05), higher levels of SERCA2a gene expression, and a more favorable metabolic gene expression profile (increased gene expression of
Pgc1α
,
Cpt1b
and
Glut4
, n=6-10/group, P<0.05) than control mice at the end of the treatment period.

Conclusion:
In summary, we show that MCAD can promote physiological cardiac hypertrophy in normal mice, and can attenuate fibrosis in a mouse model of cardiac injury.
Abstract 40: Investigation into Heart Aging
Authors: Chen Kai , Wang Shirley , Chen Jianglei , Sun Wilton , Ullha Fnu M , Sun Zhongjie
Published: March 2022

Background:
Aging is a well-known contributing factor for heart failure, which is the leading cause of death world-wide. However, the underlying mechanisms of heart aging are less clear.

Methods and Results:
We found that the overall heart size, heart weight, and cardiomyocyte size were increased in aged mice (24 months). Cardiac fibrosis was also found in old mice. Therefore, aging induced cardiac hypertrophy and remodeling. Concomitantly, heart function declined significantly in aged mice, as evidenced by decreases in fraction shorting, ejection fraction, left ventricular stroke volume, and cardiac output. Renal and circulating Klotho levels were dramatically decreased, suggesting that there may be association between a decrease in Klotho and heart failure in aged mice. Using Klotho mutant homozygous (KL-/-) mice, we found that Klotho deficiency caused cardiac hypertrophy and heart failure. Interestingly, treatment with secreted Klotho prevented cardiac hypertrophy, remodeling and heart failure in aged mice and KL (-/-) mice. Secreted Klotho treatment attenuated excessive cardiac oxidative stress and apoptosis in old mice and KL (-/-) mice. In this study, all KL (-/-) mice were fed with low-phosphate diet which maintained serum phosphate at the normal level, suggesting that the cardioprotective effects of secreted Klotho were independent of phosphate metabolism. Mechanistically, Klotho deficiency suppressed glutathione reductase (GR) expression and activity in the heart through downregulation of a transcription factor Nrf2. Furthermore, heart-specific overexpression of GR prevented aging- and Klotho deficiency-induced heart failure, excessive cardiac oxidative stress and apoptosis in old mice and KL (-/-) mice.

Conclusions:
Klotho deficiency plays an important role in aging-related heart failure. Secreted Klotho protects against heart aging
via
activation of glutathione reductase which attenuates cardiac oxidative stress, apoptosis and fibrosis.
Abstract 403: TNFα-receptor Associated Factor 2 (TRAF2) Interaction With GRK2 Mediates βAR Desensitization to TNFα
Authors: Mukherjee Sromona , Maradumane Mohan L , Naga Prasad Sathyamangla V
Published: March 2022

β- Adrenergic receptor (βAR), a prototypical G-protein coupled receptor (GPCR) is a key regulator of cardiac function. Loss of surface receptors (downregulation) and impaired G-protein coupling (desensitization) of βARs are hallmarks of heart failure. Desensitization occurs by phosphorylation of βAR by GRK2 in response to sympathetic overdrive leading to β-arrestin binding and its endocytosis into endosomes. Our studies indicate that TNFα induces βAR phosphorylation/desensitization in HEK 293, neonatal and adult cardiomyocyte cells via GRK2 recruitment. The TNFα induced GRK2 increase is independent of agonist or Gβγ pathway, suggesting a non-canonical mechanism of GRK2 recruitment to the plasma membrane. In conditions of obesity and type 2 diabetes which are clear risk factors for heart failure are consistently associated with significant increase in TNFα which is a known cardio-depressant. Correspondingly, β-blockers are known to be contra-indicative in obesity which we believe is due to βAR desensitization through non-canonical GRK2 recruitment to the βAR in response to elevated TNFα. However the underlying mechanisms for βAR desensitization are not known. We hypothesized that TRAF2 (TNFα-receptor associated factor 2) recruits GRK2 to the plasma membrane causing receptor phosphorylation. Using co-immunoprecipitation studies, we identified that scaffolding protein TRAF2 (responsible for TNFR signaling) interacts with GRK2 in untreated whole cell lysate. Furthermore, co-immunoprecipitation and immunoblotting show that TRAF2-GRK2 interaction is increased in the plasma membrane after 30 minutes of TNFα treatment. Immunoblotting also shows that TNF receptor 2 (TNFR2) engages more robustly with GRK2-TRAF2 complex than TNFR1, suggesting GRK2-TRAF2 dependent phosphorylation of βARs with TNFα is TNFR2 driven. Furthermore, TNFα induced βAR phosphorylation/desensitization is GRK2 dependent as significant loss in βAR phosphorylation is observed with GRK2 inhibitor paroxetine despite presence of TNFα. These observations suggest that cardiac dysfunction mediated by TNFα in conditions of obesity could occur through a novel GRK2-TRAF2-TNFR2 axis that could mediate agonist-independent βAR phosphorylation.
Abstract 406: Dantrolene, a Ryanodine Receptor Stabilizer, Can Significantly Attenuate Atrial Fibrillation Inducibility in a Rat Myocardial Infarction-Heart Failure Model Under Sympathetic Stimulation
Authors: Nofi Colleen , Wong Ada , Li Ying , Zhang Youhua
Published: March 2022

Background:
Compared to sympathetic stimulation (SS), normal hearts are more sensitive to vagal stimulation induced atrial fibrillation (AF). In contrast, we have recently demonstrated that failing hearts are less sensitive to vagal stimulation induced AF and more sensitive to SS induced AF. The exact mechanism underlying this SS enhanced AF in heart failure (HF) remains to be investigated. We now hypothesize that SS can worsen ryanodine receptor dysfunction already present in HF, leading to enhanced AF vulnerability. Consequently dantrolene, by stabilizing ryanodine receptors, can decrease SS enhanced AF inducibility in HF.

Methods and Results:
A rat myocardial infarction (MI)-HF model was used. MI was produced in rats by coronary artery ligation. HF was confirmed in 17 rats 2 months after the surgery. These animals were randomized into control (vehicle treated, n=9) and dantrolene (10mg/kg, IP, n=8) groups. In vivo atrial electrophysiology and AF inducibility tests under SS were studied using a catheter approach in all animals 30 minutes after the respective treatments. SS was mimicked by intravenous isoproterenol (0.1ug/min) infusion. Compared with the control group, dantrolene treatment significantly reduced AF inducibility and AF duration under SS (figure).

Conclusion:
The ryanodine receptor stabilizer dantrolene can significantly attenuate SS enhanced AF inducibility in a rat MI-HF model, suggesting that ryanodine receptor dysfunction may play a critical role in enhancing AF inducibility under SS in HF. The ryanodine receptor stabilizer dantrolene may be a new treatment option in reducing AF in HF.
Abstract 412: Galectin-3 Promotes Pro-Hypertrophic Communication Between Fibroblasts and Cardiomyocytes
Authors: Bustamante Mario , Oyarzun Ingrid , Mancilla Georthan , Quiroga Clara , Verdejo Hugo E , Castro Pablo
Published: March 2022

Galectin-3 (gal-3) is a β-galactoside-binding protein used as a prognostic biomarker in chronic heart failure (CHF) patients. Genetic and pharmacologic studies show that gal-3 is required for cardiac remodeling in animal models of CHF, suggesting an active role for gal-3 in the progression of the disease, but the mechanism involved is largely unknown. We assessed the hypothesis that gal-3 induces cardiac remodeling, causing both cardiomyocyte hypertrophy and fibroblasts activation through a mechanism involving intercellular communication. As a model, we used neonatal rat ventricular cardiomyocytes (NRVM), and fibroblasts (NRVF) stimulated with recombinant gal-3. We assessed cell death and proliferation by flow cytometry and MTT assays. To elucidate the signaling pathways involved, we quantified relative changes in protein levels by Western blot. To determine cell size and sarcomerization, we used confocal microscopy. Changes in mRNA and microRNA expression were analyzed by qPCR. Our results show that gal-3 does not induce hypertrophy or sarcomerization on isolated NRVM culture. On the other hand, NRVF treated with gal-3 exhibit an increase in ERK1/2 (n=3, p<0.05) and AKT (n=3, p<0.05) phosphorylation, as well as a marked increase in cell proliferation (n=4, p<0.05), without changes in αSMA and collagen. Interestingly, gal-3 increased tgfb1 expression in cardiac fibroblasts (n=5, p<0.05) and induced an increase of miR-21 and miR-23a levels in the culture media (15- and 6-folds over basal, respectively, n=4, p<0.05). To test a possible paracrine communication between cardiomyocytes and fibroblasts, we stimulated NRVM with fibroblast-derived, gal-3-stimmulated conditioned medium. Our results show that in this condition there is an increase in both cardiac remodeling markers (Nppa and Myh7), as well as cardiomyocyte area. In conclusion, gal-3 activates pro-survival and proliferation signaling pathways in cardiac fibroblasts without a direct effect on cardiomyocytes. However, gal-3 can induce cardiomyocyte hypertrophy by a mechanism involving paracrine communication between fibroblasts and myocytes, which likely includes TGFβ1 and the hypertrophy-related miR-21 and miR-23a.
Abstract 426: Cx43 Isoform GJA1-20k Promotes Microtubule Dependent Mitochondrial Transport
Authors: Zhang Shan-Shan , Xiao Shaohua , Fu Ying , Basheer Wassim A , Baum Rachel , Naami Robert , Naami Edmund , Hong TingTing , Shaw Robin M
Published: March 2022

Traditionally, the role of Connexin 43 (Cx43, encoded by GJA1) has been studied in the context of forming gap junctions to mediate cell-cell communication. Cx43 has been implicated in multiple non-canonical roles including metabolic health. It was recently found that GJA1 mRNA undergoes alternative translation to generate truncated C-terminal isoforms, of which GJA1-20k is the most abundant. We examined the relationship between GJA1-20k and mitochondria. Using live cell imaging and organelle markers, we report that, unlike full length GJA1-43k, GJA1-20k has a powerful tropism for mitochondria of all the cell types examined. Mitochondrial enrichment of GJA1-20k was confirmed using biochemical fractionation in transfected cells. High-resolution imaging and immunogold electron microscopy revealed that GJA1-20k is enriched at the interface between mitochondria and microtubules suggesting a role in mitochondrial trafficking. The interaction between GJA1-20k and the microtubules was further confirmed using co-immunoprecipitation. Exploring function, we found that exogenous GJA1-20k is sufficient to rescue mitochondrial localization to the cell periphery upon exposure to hydrogen peroxide, effectively limiting network fragmentation with oxidative stress. Furthermore, we found that the GJA1-20k microtubule-binding domain is unnecessary for mitochondrial localization, but is essential for mediating mitochondrial trafficking and maintaining the integrity of the mitochondrial network and its localization at the cell periphery upon oxidative stress. GJA1-20k enriches in mitochondria and facilitates microtubule-based mitochondrial trafficking, preserving organelle network integrity upon oxidative stress. GJA1-20k provides a means by which otherwise traditional Cx43 can play diverse non-canonical roles. GJA1-20k has therapeutic potential to prevent mitochondrial fragmentation in situations of cellular stress.
Abstract 428: Interventions Targeting Mitochondrial Function as Therapies to Reverse Cardiac Aging
Authors: Chiao Ying Ann , Ting Ying Sonia , Quarles Ellen , Otero Gabriel , Campbell Matthew , Yue Yongping , Duan Dongsheng , Marcinek David , Szeto Hazel H , MacCoss Michael , Rabinovitch Peter S
Published: March 2022

Aging is associated with a higher incidence of cardiovascular disease and a significant decline in cardiac function. In individuals without overt cardiovascular disease, aging results in impaired diastolic function, reduced myocardial performance and increased prevalence of cardiac hypertrophy. Our laboratory has previously shown that transgenic mice expressing mitochondrial catalase (mCAT) have reduced mitochondrial oxidative damage and display delayed cardiac aging. This study aims to determine whether short-term treatments targeting mitochondrial function can reverse cardiac aging in old mice.

The mitochondrial protective SS-31 peptide (elamipretide) has previously been shown to provide similar protection as mCAT in models of pressure overload-induced cardiac hypertrophy and failure. SS-31 binds to cardiolipin and improves the electron carrying function of cyt
c
, while reducing its peroxidase activity. We administered SS-31 to 24-month-old mice via osmotic minipump for 8 weeks, and found it to reduce cardiac hypertrophy and improve diastolic function and myocardial performance. SS-31 treatment attenuated the age-related increase in cyt
c
oxidation at Met-80, which has been associated with the disruption of Fe-S bond and activation of peroxidase activity of cyt
c
. SS-31 treatment also improved mitochondrial ultrastructure and induced AMPK activation in old hearts.

To investigate whether SS-31 and mCAT protect cardiac aging by overlapping mechanisms, we administered SS-31 to old mCAT mice to study the effects of combined treatments. We found that SS-31 treatment cannot further improve the cardiac function of old mCAT mice. Concordantly, proteomic analysis revealed that changes in the cardiac proteome induced by SS-31 were partially overlapping with changes mediated by mCAT expression, particularly in the mitochondrial proteome, suggesting a convergence of molecular mechanisms of these two treatments. In addition, we showed that mCAT expression transduced by AAV9 in late-life also improved diastolic function, supporting the similar therapeutic effects of short-term mCAT and SS-31. In summary, these results support the therapeutic potential of mitochondrial-targeted interventions to reverse the effects of cardiac aging.
Abstract 430:
In Utero
Exposure to Diesel Exhaust Reprograms the Metabolic Phenotype of Neonatal Cardiomyocytes
Authors: Goodson Jamie M , MacDonald Jamie W , Bammler Theo K , Chien Wei-Ming , Chin Michael T
Published: March 2022

Epidemiological studies have revealed that exposure to particulate matter air pollution is harmful to cardiovascular health. Recent studies have shown that exposure during development can affect fetal outcomes through alterations in blood pressure and decreased birth weight. Our lab has shown that
in utero
exposure to diesel exhaust (DE) in mice causes later life susceptibility to heart failure after transverse aortic constriction surgery, including increased cardiac hypertrophy, fibrosis and apoptosis.

To elucidate the cause behind this increased susceptibility to heart failure, we performed RNA-sequencing on neonatal cardiomyocytes isolated from
in utero
DE or filtered air (FA) exposed p0 pups. This revealed changes in many metabolic pathways, including fatty acid β-oxidation. We analyzed changes in metabolic utilization using the Seahorse Extracellular Flux Analysis platform, and discovered that DE neonatal cardiomyocytes show a significant decrease in both glucose and glutamine dependence and capacity, as well as decreased maximal potential for oxidative phosphorylation. We are currently performing targeted mass spectrometry to identify changes in the metabolic profile of various pathways, including glycolysis and fatty acid oxidation.

Metabolic dysregulation is a hallmark of heart failure. By identifying pathways showing dysregulation in DE-exposed cardiomyocytes, we aim to discover targets for therapeutic intervention.
Abstract 431: Upregulation of Small Conductance Ca
2+
Activated K
+
Channels Promotes Formation of Mitochondrial Supercomplexes and Increases Respiration Reserve
Authors: Hamilton Shanna , Terentyeva Radmila , Roder Karim , Polina Iuliia , Koren Gideon , Clements Richard T , Terentyev Dmitry
Published: March 2022

Introduction:
Small conductance calcium (Ca
2+
)-activated K
+
(SK) channels are present in the inner mitochondria membrane of ventricular cardiomyocytes. We recently reported that pharmacological enhancement of mito-SKs protects from Ca
2+
dependent arrhythmia by reducing production of reactive oxygen species (ROS) by mitochondria thereby normalizing oxidation state and activity of Ca
2+
release channels ryanodine receptors in hypertrophic rat hearts. Recent advances demonstrate that respiratory components of the mitochondrial electron transport chain can organize into supercomplexes (SCs) resulting in more efficient electron transfer and reduced generation of ROS.

Hypothesis:
Functional upregulation of mito-SK channels protects from Ca
2+
-dependent arrhythmia via promotion of mitochondrial SC formation and improved respiration.

Methods:
H9C2 myoblasts were infected with adenoviral vectors to overexpress SK2 and Cox7rp. After 48 hours in culture, cell respiration was measured using Seahorse XFe Analyzer; ROS was measured using general ROS indicator CFDA; and mitochondria were isolated to assess SC formation using Blue-Native electrophoresis (BN-PAGE). The effects of SK enhancer NS309 (2 μM, 10 min.) on SC formation were studied in Langendorff perfused rat hearts with hypertrophy induced by thoracic aortic banding (TAB).

Results:
rSK2 overexpression in H9C2s reduced intracellular ROS ~ 30%, enhanced maximum respiration rate vs. controls (0.74±0.04 and 0.58±0.03 pM/min/μg respectively, p<0.05); and caused a robust shift (>2 fold) of complex IV towards large mitochondrial SCs (~1.2 mDa). As a control, similar results were obtained with overexpression of Cox7rp protein that promotes SC formation. In addition, BN-PAGE experiments in isolated rat hearts demonstrated that robust decrease in high MW Complexes I and IV in hypertrophy was fully restored to control levels upon treatment with SK enhancer NS309.

Conclusion:
Upregulation of SK channels reduces mito-ROS by an increase in respiration reserve which is linked to enhanced mitochondrial SC formation. Our data suggest that targeting mito-SK channels has therapeutic potential to treat cardiac arrhythmia in conditions accompanied by oxidative stress.
Abstract 434: Cardiomyopathy-associated Point Mutation in the ADP-ATP Carrier Reveals Translation-dependent Regulation of Cytochrome
c o
xidase Activity in Yeast
Authors: Ogunbona Oluwaseun B , Baile Matthew G , Claypool Steven M
Published: March 2022

The interaction between the ADP/ATP carrier and the respiratory supercomplexes is evolutionarily-conserved, is dependent on cardiolipin, and is functionally significant as yeast lacking Aac2p have reduced activity of respiratory complex IV (
cytochrome c oxidase
or COX). We have leveraged a transport-dead pathogenic
AAC2
point mutant discovered in a patient with hypertrophic cardiomyopathy and mild myopathy to determine whether the reduction in complex IV activity in the absence of Aac2p reflects the absence of the interaction between Aac2p and components of the electron transport chain and/or the absence of nucleotide transport (i.e. Aac2p function). Importantly, the pathogenic A137D allele of
AAC2
is expressed normally and still interacts with components of the yeast respiratory supercomplex but is unable to support growth on respiratory media due to its inability to transport ADP/ATP. Amazingly, in the absence of Aac2p function, the expression levels of complex IV subunits that are encoded by the mitochondrial genome (and which form the catalytic core of the complex IV holoenzyme) are specifically reduced, even though assembly and interaction with the respiratory supercomplexes are preserved in the mutant Aac2p. This reduction in the levels of complex IV subunits is not caused by a reduction in either the mitochondrial genome copy number or the steady state level of mitochondrial DNA transcripts. Instead, there is a noticeable alteration in the pattern of the mitochondrial translation in the absence of Aac2p activity. Our results suggest that Aac2p function is important for normal translation of the mitochondrial encoded complex IV subunits and that Aac2p activity plays a significant role in regulating oxidative phosphorylation.
Abstract 437: Mitochondrial Cardiomyopathies Feature Enhanced Mitochondrial Calcium Signaling
Authors: Sommakia Salah , Houlihan Patrick , Deane Sadiki , Simcox Judith , Villanueva Claudio , Clapham David , Chaudhuri Dipayan
Published: March 2022

Mitochondrial diseases often feature early onset of dilated cardiomyopathy, due to the large energetic demand placed by the heart. Among regulators of mitochondrial respiration, we focus on calcium, which potently stimulates ATP synthesis. We assessed the hypothesis that cells boost mitochondrial calcium to stimulate respiration as this declines in mitochondrial cardiomyopathies. To this end, we studied mice that develop a neonatal, severe cardiomyopathy caused by cardiac-specific knockout (KO) of mitochondrial transcription factor A (
Tfam
). Such deletion impairs transcription of mitochondrial DNA, which encodes multiple subunits of the electron transport chain (ETC).
Tfam
KO mice exhibited a dilated cardiomyopathy, with decreased fractional shortening and increased LV diameters, and had marked inhibition of multiple ETC complexes. We determined that
Tfam
KO mitochondria took up calcium twice as fast as mitochondria from littermate controls, while calcium efflux was approximately 70% slower. Whole-mitoplast voltage clamp revealed that the enhanced calcium uptake was due to an increase in the current carried by the uniporter. Furthermore, the larger uniporter current reflected increased amounts of uniporter subunit proteins. This occurred despite a reduction in the transcripts of genes encoding these subunits, suggesting a post-translational mechanism for the enhanced uniporter stability. Finally, we found that the rate of ADP-stimulated oxygen consumption in calcium-free solution was 50% less in the
Tfam
KO mitochondria compared to controls, but increased substantially more, nearly to control levels, when calcium was present (2.5-fold increase for KO versus 1.7-fold for control). In conclusion, enhanced mitochondrial calcium signaling in a mitochondrial cardiomyopathy model may serve to compensate for energetic failure.
Abstract 44: NSF is Required for Plasma Membrane Blebbing Occurring in Necrotic Cell Death
Authors: Lombardi Alyssa A , Lambert Jonathan P , Molkentin Jeffery D , Elrod John W
Published: March 2022

The loss of cells that occurs during MI and HF is largely due to necrotic cell death, yet the molecular mechanisms underlying necrosis, specifically plasma membrane rupture, are not well defined. A genome-wide, shRNA loss-of-function screen identified components of SNARE-mediated membrane fusion as potential facilitators of Ca
2+
and ROS-induced membrane rupture. Here, we targeted
Nsf
(N-ethylmaleimide sensitive factor) due to its requirement in SNARE recycling, redox sensitivity, and lack of gene homologs. Deletion of
Nsf
from 3T3 fibroblasts, using CRISPR-Cas9n (

Nsf
-/-

), inhibited membrane rupture and improved cell viability following Ca
2+
overload (ionomycin) and ROS (H
2
O
2
) induced cell death. Further, stable overexpression of
NSF
augmented membrane rupture and decreased cell viability following necrotic insults. Next, using high-resolution live cell microscopy we examined membrane blebs - herniations of the plasma membrane that precede rupture and necrosis.

Nsf
-/-

and WT cells were treated with ionomycin or H
2
O
2
and imaged for 4h with automated Z-stack images taken every 5m. Ionomycin and H
2
O
2
induced prominent blebs in nearly every WT cell followed by membrane rupture. Strikingly, loss of
Nsf
ablated bleb formation (Fig 1). Studies are ongoing in cardiac-specific
Nsf
-/-
mice to define if this novel mechanism contributes to myocyte death during IR injury. Deletion of
Nsf
in adult cardiomyocytes does not result in a baseline phenotype, increasing the translational potential of NSF targeted therapy. In summary, our results identify a new molecular component required for membrane blebbing that occurs during pathogenic cell death.
Abstract 443:
l-cis-
Diltiazem Ameliorates Impaired Calcium Channel Inactivation in a Patient-specific Stem Cell Model of Long-QT Syndrome with a Calmodulin Mutation
Authors: Yokoi Fumika , Makiyama Takeru , Yamamoto Yuta , Harita Takeshi , Hayano Mamoru , Nishiuchi Suguru , Wuriyanghai Yimin , Kohjitani Hirohiko , Hirose Sayako , Yoshida Yoshinori , Toyoda Futoshi , Horie Minoru , Kimura Takeshi
Published: March 2022

Background:
Calmodulin is a ubiquitous Ca
2+
sensor molecule encoded by three distinct calmodulin genes,
CALM1-3
. Recently, mutations in
CALM1-3
have been reported to be associated with severe early-onset long-QT syndrome (LQT) due to impaired Ca
2+
dependent inactivation of L-type Ca
2+
channel (LTCC). However, pharmacotherapeutic agents for the treatment of CALM-related LQT have not been established.

Objective:
We aimed to assess new pharmacotherapies for the treatment of CALM-related LQT using human induced pluripotent stem cell (hiPSC) model.

Methods and Results:
We used CALM-LQT-hiPSC derived cardiomyocytes (CMs) with a
CALM2-
N98S mutation which exhibited prolonged action potential durations. First, we confirmed impaired LTCC inactivation in the CALM-LQT-hiPSC-CMs: the ratio of current remaining after 100 ms depolarization (r100) of LTCC currents was significantly larger in CALM-LQT-hiPSC-CMs (r100; CALM-LQT: 0.333 ± 0.016, control: 0.181 ± 0.025 at 0 mV; P < 0.05). Next, we assessed candidate drugs,
l-cis-
Diltiazem (the stereoisomer of clinically used
d-cis
-Diltiazem) and Cyclosoprin A, which were reported to enhance voltage-dependent inactivation of LTCC for the treatment of impaired LTCC inactivation. We found that 10 μM
l-cis
-Diltiazem attenuated impaired LTCC inactivation in CALM-LQT-iPSC-CMs (r100; baseline: 0.333 ± 0.016, with l-cis: 0.294 ± 0.013 at 0 mV; P < 0.05; Figure A and B). On the other hand, Cyclosporine A had less effect compared to
l-cis-
Diltiazem (Figure A).

Conclusion:
Using patient-derived hiPSC model, we identified that
l-cis
-Diltiazem is a promising candidate drug for treating CALM-LQT resulting from impaired LTCC inactivation.
Abstract 447: Chronic Neuropathic Pain Elicits Cardioprotection via Anterior Nucleus of Paraventricular Thalamus in Mice
Authors: Cheng* Yi-Fen , Chang* Ya-Ting , Chen Wei-Hsin , Shih Hsi-Chien , Chen Yen-Hui , Shyu Bai-Chuang , Chen Chien-Chang
Published: March 2022

Myocardial infarction is the leading cause of death worldwide. Restored blood flow quickly rescues myocardium but also causes ischemia-reperfusion (IR) injury. Chronic pain affects up to 25% of population worldwide. Whether there is any correlation between IR injury and chronic pain is unknown. Here we show that chronic neuropathic pain reduces IR injury via an anterior nucleus of paraventricular thalamus (PVA)-dependent parasympathetic pathway in mice. Intra-PVA infusion of U0126, a MEK inhibitor, prevents the activation of ERK and abolishes spared nerve injury (SNI)-induced cardioprotection. We also demonstrate that activation of PVA neurons by phorbol 12, 13-dibutyrate (PDBu) or optogenetic stimulation is sufficient to induce cardioprotection. The PVA-dependent cardioprotection is abolished by hexamethethonum and glycopyrrolate but not propranolol. Furthermore, SNI surgery and optogenetic stimulation of PVA neurons reduce the heart rate. These results suggest that the parasympathetic nerve mediates this cardioprotection. Thus, we reveal a novel mechanism linking chronic neuropathic pain and cardioprotection via a PVA-dependent parasympathetic pathway.
Abstract 45: HDAC6 Inhibitor Attenuates Autophagy During Chronic Angiotensin-II Induced Hypertrophic Human Cardiomyocytes
Authors: Rajasingh Sheeja , Czirok Andras , Isai Dona G , Samanta Saheli , Zhou Zhigang , Dawn Buddhadeb , Rajasingh Johnson
Published: March 2022

Introduction:
Autophagy is playing a crucial role in cellular functions, and maintaining homeostasis during cardiac hypertrophy, but the mechanism is poorly understood. Studies have shown that pressure overload and chronic ischemia-induced stress leads to an increase in autophagic activity in cardiomyocytes. Reports have demonstrated that STAT3 and Bcl2 participate in the process of autophagy. We plan to study the possible contribution of epigenetic enzymes that mediate transcriptional repression and activation of genes related to autophagy. Here, we have examined the role of histone deacetylase-6 (HDAC6) inhibitor (Tubastatin A- TBSA) in protecting chronic angiotensin II-induced autophagy in human iPSC-derived cardiomyocytes (hiCMCs).

Hypothesis:
We hypothesize that TBSA treatment mediated protection is through STAT3-Bcl2 signaling in angiotensin II-induced autophagy in hypertrophic hiCMCs.

Methods and Results:
We have evaluated the TBSA treatment mediated effects in contractility, cell size and mitochondrial membrane potential (
Δ
Ψm) in response to angiotensin II-induced autophagy in hiCMCs. Our immunofluorescence data show that treatment with TBSA significantly reduced the number of autophagosomes in angiotensin II-induced hiCMCs (
Fig. A)
. Moreover, the flow cytometry data show the fluorescence intensity ratio of red and green in JC1 staining suggest that TBSA restores and maintains the angiotensin II-induced loss of
Δ
Ψm in hiCMCs (
Fig B, C
).

Conclusions:
We report for the first time that the role of HDAC6 in regulation of pathological autophagy, thus can acts as a potential therapeutic target for treatment of chronic heart diseases.
Abstract 452: Primary Left Ventricular Unloading Reduces Infarct Size by Increasing Myocardial Levels of Stromal Derived Factor One Alpha (SDF1a) in Acute Myocardial Infarction
Authors: Zhang Yali , Esposito Michele , Qiao Xiaoying , Paruchuri Vikram , Mackey Emily , Morine Kevin , Schnitzler Gavin , Reyelt Lara , Boggins Courtney , Josephy Noam , Karas Richard H , Kapur Navin K
Published: March 2022

Stromal derived factor 1 alpha (SDF1a) is a constitutively expressed cardioprotective chemokine that is rapidly degraded by proteases. We reported that compared to Primary Reperfusion (PR), first reducing myocardial oxygen demand by activating a trans-valvular pump (Impella CP) while delaying coronary reperfusion (Primary Unloading; PU) reduces infarct size in acute myocardial infarction (AMI). We now hypothesize that PU reduces proteolytic degradation of SDF1a thereby increasing SDF1a signaling via the receptor CXCR4 in AMI.

Methods:
AMI was induced by occlusion of the left anterior descending artery (LAD) for 90 min in male swine (n=4/group). In the PR group, the LAD was reperfused for 120 min. In the PU group, after 90 min of ischemia an Impella CP was activated and the LAD left occluded for an additional 30 min, followed by 120 min of reperfusion. Whole-transcript expression analysis was performed on RNA from the infarct zone using Porcine 1.0 ST microarrays and ConsensusPathDB programs. Quantitative polymerase chain reaction, western blots, and activity assays determined expression and activity of the SDF1a signaling pathway. Sham operated LV samples served as controls.

Results:
Compared to PR, PU reduced fibrotic and inflammatory gene expression including reduced transcript levels of matrix-metalloprotease-2 (MMP2), MMP9 and dipeptidyl peptidase-4 (DPP4). Compared to PR, PU increased SDF1a protein levels within the infarct zone. Gel zymography confirmed reduced activity levels of MMP2 and MMP9 within the infarct zone after PU, not PR. Compared to PR, PU attenuated DPP4 protein levels and activity and protein levels of CXCR7, an SDF1a sequestration receptor, within the infarct zone. To explore a functional role for SDF1a in PU, adult male swine received intra-coronary injections of AMD3100, a CXCR4 receptor antagonist, after Impella CP activation. Loss of CXCR4 activity attenuated cardioprotective signaling via Akt, Erk and GSK3b and increased infarct size compared to vehicle treated controls.

Conclusion:
We introduce a novel mechanism by which PU limits proteolytic degradation and CXCR7 mediated sequestration of SDF1a, thereby increasing cardioprotective signaling via SDF1a and overcoming a critical barrier to SDF1a therapeutics in AMI.
Abstract 454: Oxidative Stress Mediated Myocardial Lysophosphatidic Acid Signaling
Authors: Panchatcharam Manikandan , Chandra Mini , Escalante-Alcalde Diana , Orr Anthony W , Kevil Christopher , Bhuiyan Shenuarin , Wu Joseph C , McCarthy Kevin , Miriyala Sumitra
Published: March 2022

The bioactive lysophosphatidic acid (LPA) plays a well-known role in atherosclerotic disease, whereas, its role in myocardial function remains virtually unexplored. Following acute myocardial infarction, serum LPA concentration rises by six-fold over control human subjects, suggesting LPA may contribute to the pathogenesis of myocardial infarction. LPA production involves hydrolysis of lysophosphatidylcholine by the secreted enzyme autotaxin, whereas lipid phosphate phosphatase-3 (LPP3) catalyzes LPA dephosphorylation to generate lipid products that are not receptor active. We present the first evidence that cardiac ischemia/reperfusion (I/R) injury enhances myocardial autotaxin levels and decreases myocardial LPP3 expression, and this is associated with increased serum LPA levels. Upon reperfusion, reactive oxygen species production arises as a burst of superoxide from mitochondria following I/R injury. The redox-sensitive transcription factor NFAT has been shown to bind to the autotaxin promoter and induce its expression. Therefore, we looked at the autotaxin and LPP3 regulation in mice following I/R injury in the myocardium. After 1h ligation followed by 3h reperfusion in the myocardium, we observed a 3 fold increase in the autotaxin protein levels, whereas LPP3 protein levels were significantly downregulated as observed through Western blot analysis in these myocardial ischemic tissues. Autotaxin and miR-92a mRNA expression levels were significantly upregulated, whereas KLF2 and LPP3 mRNA expressions were significantly downregulated following I/R injury at 24 hours. Western blot analysis showed a 3 fold increase autotaxin protein levels and immunohistochemistry of human infarct tissues at 24 hours showed disruption of the sarcomere with decreased LPP3 staining. We found that I/R injury transactivates miR-92a, and inhibit KLF2, an upstream activator of LPP3. Taken together, our in vivo data, from the myocardial I/R injury and human infarct tissues, suggest that regulation of autotaxin and LPP3 activity might cause the rise in serum LPA levels as reported with acute myocardial infarct patients.
Abstract 471: Kinase-independent Function of PI3Kγ Enables ERK Activation
Authors: Mohan Maradumane L , Chatterjee Arunachal , Ganapathy Swetha , Mukherjee Sromona , Naga Prasad Sathyamangla V
Published: March 2022

Phosphoinositide 3-kinase (PI3K) enzymes are critical in many cellular processes including cell survival. PI3Kγ, a member of the PI3K family, is activated in response to G-protein coupled receptor (GPCR) stimulation leading to extracellular regulated kinase (ERK) signal transduction cascade, a cell survival pathway. However, less is known about the underlying mechanisms of PI3Kγ-directed ERK activation. Knockdown of PI3Kγ showed that PI3Kγ not only regulates ERK phosphorylation in response to GPCR stimulation but also to receptor tyrosine kinase activation in HEK 293 cells. The key role of PI3Kγ in ERK activation was further validated by loss of insulin-stimulated ERK phosphorylation in PI3Kγ-knockout (KO) mouse embryonic fibroblasts (MEFs). Surprisingly, ERK activation in KO MEFs post-insulin stimulation was completely rescued by expression of kinase-dead PI3Kγ mutant in KO MEFs demonstrating a kinase-independent role of PI3Kγ in regulating ERK function. Mechanistic studies showed that PI3Kγ regulates ERK activation by inhibiting ERK dephosphorylation following stimulation thereby, sustaining ERK phosphorylation and activation. Critically, PI3Kγ regulates ERK dephosphorylating phosphatase PP2A by interacting and sequestering PP2A from ERK maintaining ERK phosphorylation, which is evidenced by increased PP2A association with ERK in KO MEFs. Consistently, ERK activation was completely abolished in KO MEFs following carvedilol or insulin suggesting an essential role for PI3Kγ in ERK activation pathway. Correspondingly, primary cardiac fibroblasts isolated from KO mice showed complete loss of insulin-stimulated ERK phosphorylation compared to WT mice. This is intriguing given that GSK3 phosphorylation and not ERK phosphorylation is regulated by inhibition of PP2A through kinase-independent mechanism of PI3Kγ in the total cardiac lysates. Even though GSK3 and ERK are substrates for PP2A, our findings that ERK is regulated by kinase-independent function PI3Kγ suggest the existence of this unique regulation in fibroblasts and not in cardiomyocytes. Thus, kinase activity of PI3Kγ may contribute to cardiac-pathology while kinase-independent function could be beneficial and will be discussed in presentation.
Abstract 474: Combined Polyphenols Synergistically Inhibit Tumor Necrosis Factor-alpha-Induced Monocytes Adhesion to Human Endothelial Cells
Authors: Zhang Lijuan , Wang Xiaoyong , Si Hongwei
Published: March 2022

Monocyte recruitment and adhesion to the endothelium is a crucial step the development of endothelial dysfunction and therefore induces cardiovascular disease (CVD), the number one killer of Americans. Emerging evidence shows that polyphenols, the secondary plant metabolites present in a large variety of foods, have the potential ability in reducing risk of CVD. The aims of this study are to investigate the synergistic anti-inflammatory effects of combined polyphenols at physiological levels and define relevant molecular mechanisms. Our results indicate that resveratrol, curcumin and luteolin dose-dependently inhibited tumor necrosis factor-alpha (TNF-α)-induced monocytes adhesion to human endothelial cells. The half maximal effective concentration (EC50) values of curcumin, luteolin and resveratrol are 15 μM, 8 μM and 26 μM respectively. Particularly, combined luteolin (4μM) and curcumin (8 μM) or combination of curcumin (8 μM) and resveratrol (13 μM) synergistically inhibited TNF-α-induced monocytes adhesion to endothelial cells while the individual chemicals did not have such effect at the physiological concentrations. The combination index(CI)value of curcumin plus luteolin and curcumin plus resveratrol at the selected concentrations are 0.7 and 0.82, respectively, indicating these two combinations have synergistic anti-inflammatory effects. We also found that these phytochemicals ameliorated the TNF-α-enhanced protein expressions of vascular cell adhesion molecule 1 (VCAM-1) and monocyte chemotactic protein-1 (MCP-1), two major indicators of vascular inflammation in endothelial cells. These data suggest that these combined polyphenols maybe a novel strategy to diminish vascular inflammation and therefore prevent/treat cardiovascular disease.
Abstract 56: Individual Susceptibility to Drug-induced QTc Prolongation Did Not Correlate With Subject-specific Induced Pluripotent Stem Cells in a Small Sample of Healthy Subjects
Authors: Blinova Ksenia , Patel Dakshesh , Stohlman Jayna , Schocken Derek , Kondratovich Marina , Johannesen Lars , Vicente Jose , Strauss David
Published: March 2022

Background:
Induced pluripotent stem cells (iPSCs) carry patient-specific genetic information and have the potential to become an important tool for predicting individual clinical response to therapies in an
in vitro
assay. Here, we investigate if subject-specific iPSC cardiomyocytes (iPSC-CMs) derived from healthy volunteers could be used to predict individual response to 2 QT-prolonging drugs which they received in a clinical trial.

Methods and Results:
Independent stem cell manufacturer (Stem Cell Theranostics) used commercially available Sendai virus encoding for
Oct4
,
Sox2
,
Klf4
, and
c-Myc
to reprogram subject’s peripheral blood mononuclear cells (PBMCs) into iPSCs. They then applied chemically defined growth factors, and serum-free protocols to successfully differentiate iPSCs into iPSC-CMs for 17 out of 20 subjects (one batch per subject) enrolled in a randomized, double-blind, placebo-controlled, cross-over trial of dofetilide and moxifloxacin. Effects of the same 2 drugs on cellular action potential duration (APD) were recorded for 16 iPSC-CMs lines using a high-throughput optical imaging system (CellOPTIQ, Clyde Biosciences) using consistent
in vitro
protocols, including reproducible drug doses and exposure, cell culture and recording conditions. Both dofetilide (0.5-8 nM) and moxifloxacin (10-200 μM) induced dose-dependent prolongation of rate-corrected action potential duration (APD) in each iPSC-CM line. iPSC-CM drug responses were compared with individual clinical response represented by the slope of the linear regression fit through baseline and placebo-controlled drug-induced changes in QTc interval vs. plasma drug concentration. We found no statistical significant correlation between subject-specific iPSC-CMs drug-induced APD prolongation and individual QTc prolongation to either moxifloxacin [Spearman’s ρ = 0.191] or dofetilide [Spearman’s ρ = 0.068].

Conclusion:
This study failed to find correlation between clinical and stem cell drug responses in a small sample of healthy subjects. More experiments with a larger sample size and iPSC-CMs batch-to-batch variability controls are required to further investigate the potential of patient-derived stem cells to predict individual clinical response.
Abstract 7: Chromatin Organization in Diseased and Healthy Mouse Heart
Authors: Anene Nzelu Chukwuemeka George , Lee Dominic , Tan Wilson , Tiang Zenia , Ilmaris Matias A , Li Peter , Fullwood Melissa , Foo Roger
Published: March 2022

Background:
The three-dimensional chromatin structure regulates transcription by bringing regulatory elements such as enhancers in close spatial proximity with target genes. This arrangement helps to ensure cell type-specific gene expression profiles. We employed Hi-C, Chip-seq and CRISPR knock out to provide a detailed genome-wide view of the 3D chromatin structure in healthy and diseased mouse heart tissue and also to link regulatory elements with their target genes.

Results:
The 3D chromatin organization of Sham and TAC adult ventricular cardiomyocytes (CMs) as well as atrial-origin HL1 cardiomyocytes showed high degree of similarity. While topological associated domains are stable across all cell types, the A and B genomic compartments showed important variations, which correlated well with differential gene expression profiles. Analysis of the interactions in regions enriched with the histone enhancer mark H3K27ac identified putative regulatory elements responsible for cell-type specific gene expression. In addition, CRISPR deletion of a regulatory region upstream of Nppb and Nppa loci led to a downregulation of these genes in HL1 cells, confirming results obtained via Hi-C and 4C

Conclusion:
We show evidence of the relationship between genome organization and gene expression by comparing atrial and Sham and TAC ventricular cardiac cells. Importantly, this is the first detailed report of genome-wide 3D chromatin interactions in cardiac cells, revealing cardiac-relevant genomic compartments that associate with cardiac-specific gene expression.