bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P2050: Mixed Lineage Kinase 3 Kinase Function Opposes Left Ventricular Hypertrophy And Transcriptional Activity Of Nuclear Factor Of Activated T Cells In The Cardiac Myocyte.
Authors: Raymond Loveth , Martin Gregory , Blanton Robert M
Published: October 2023

Background:
LV hypertrophy (LVH), dysfunction, and remodeling contribute to heart failure (HF). The signaling molecule Mixed Lineage Kinase 3 (MLK3) opposes stress-induced LV dysfunction and remodeling in a kinase-dependent, blood pressure-independent fashion. However, the required downstream MLK3 kinase effectors in the cardiac myocyte (CM) which oppose LVH and remodeling remain poorly understood. The Nuclear Factor of Activated T Cells (NFAT) promotes pathological remodeling, but its regulation in the CM by MLK3 has not been investigated. We hypothesized that MLK3 opposes NFAT in the CM through kinase-dependent mechanisms.

Method:
In reporter mice with a CM-specific luciferase transgene under the control of NFAT response elements (αMHC-NFAT-Luc mice), we administered the MLK3 inhibitor URMC-099 (10 mg/kg twice daily) or vehicle for 5 days (n=7 vehicle, 8 URMC-099) and 14 days (n=9 vehicle, 12 URMC-099). We measured cardiac chamber masses, performed immunoblotting for MLK3, and measured myocardial luciferase fluorescence as a readout for CM NFAT transcriptional activity.

Result:
At 5 days, URMC-099 had no effect on LVH or cardiac hypertrophy and did not induce NFAT activation. However, URMC-099 treatment reduced LV MLK3 protein expression (normalized to β-actin) compared with vehicle-treated littermates. After 14-day administration, URMC-099 induced significant increases in LV and heart mass, normalized to tibia length (TL), (LV/TL males: 4.21 ± 0.03 mg/mm vehicle vs 5.20 ± 0.10 mg/mm URMC-099; p<0.01; females: 3.73 ± 0.14 mg/mm vehicle vs 4.30 ± 0.13 mg/mm URMC-099; p<0.01). LV tissue from URMC-099-treated mice displayed a 7-fold increase in luciferase fluorescence (p<0.001), compared with vehicle-treated, indicating increased NFAT activation by URMC-099.

Conclusion:
Systemic MLK3 inhibition with URMC-099 causes reduced MLK3 expression, LVH, and increased CM NFAT activity. Because URMC-099 does not raise blood pressure, our findings support that MLK3 functions as a tonic brake on NFAT activation in the CM and opposes cardiac hypertrophy through kinase-dependent mechanisms.
Abstract P2051: Fibroblast-specific TGF-b Signaling Promotes Interstitial Fibrosis, Cardiomyocyte Hypertrophy And Cardiac Dysfunction In The Diabetic Heart Through Smad3 Activation
Authors: Tuleta Izabela , Alex Linda , Venugopal Harikrishnan , Humeres Claudio , Hernandez Silvia C , Kubota Akihiko , Hanna Anis , Frangogiannis Nikolaos G
Published: October 2023

Objectives:
Patients with diabetes exhibit a high prevalence of HFpEF associated with interstitial fibrosis and cardiomyocyte hypertrophy. However, the cellular and molecular mechanisms involved in diabetic heart disease remain poorly understood. TGF-bs are potent fibrogenic mediators that are activated in diabetic hearts, and act through a single type 2 receptor, TbRII. We hypothesized that fibroblast-specific actions of TGF-bs may mediate fibrosis and dysfunction in diabetic hearts.

Methods and Results:
To test the hypothesis, we developed lean and db/db type 2 diabetic TbRII KO mice (FTbRII2KO) using the Col1a2
CreER
driver. Fibroblast-specific loss of TGF-b signaling attenuated hypertrophy in db/db mouse hearts. Speckle-tracking echocardiography with strain imaging showed that fibroblast-specific TbRII loss improved systolic and diastolic cardiac function in diabetic animals. In contrast, fibroblast TbRII disruption caused a worsening of both cardiac systolic and diastolic function in lean animals. Attenuated dysfunction in diabetic mice lacking fibroblast TGF-b signaling was associated with reduced collagen fiber thickness and with decreased cardiomyocyte size, suggesting paracrine effects of fibroblasts on cardiomyocytes. To study the downstream cascade responsible for the effects of TGF-b, we generated lean and diabetic mice with fibroblast-specific Smad3 loss. In db/db mice, fibroblast-specific Smad3 loss phenocopied the effects of fibroblast-specific TbRII disruption, suggesting that in diabetic cardiac fibroblasts TGF-b acts through Smad3 signaling. The bioinformatic analyses of transcriptomic profiles of cardiac fibroblasts identified thrombospondin 4 as a candidate mediator of Smad3-dependent diabetes-associated activation.

Conclusions:
Fibroblast-specific TGF-b actions in diabetes promote cardiac dysfunction through effects on collagen deposition and via paracrine actions on cardiomyocytes. The effects of TGF-b on diabetic cardiac fibroblasts are Smad3-dependent. In contrast to the deleterious effects of fibroblast TGFb/Smad3 signaling in diabetic hearts, TGF-b-activated Smad3-dependent signaling is important to maintain homeostatic function and geometry in normal lean animals.
Abstract P2056: The Transcription Factor TFEB Protects Against Cardiac Hypertrophy By Increasing Lysosomal Capacity And Activating AMPKα
Authors: Das Gupta Subhajit , Daou Daniel , May Herman , Jiang Nan , Nguyen Nicholas , Anand Anip , Iansen Irion Camila , Garrido-Moreno Valeria , Olivares-Silva Francisco , Diwan Abhinav , Gillette Thomas G , Hill Joseph A
Published: October 2023

Introduction:
Pathological cardiac remodeling in response to elevated afterload can eventually progress to heart failure. Lysosome-assisted processes such as autophagy play an important role in this remodeling. The lysosome is also a nexus for some of the same signaling pathways that are involved in hypertrophic growth. The transcription factor EB (TFEB) has emerged as a key regulator of lysosomal genes in multiple tissues, especially in response to external stress.

Methods and Results:
Here, we report that TFEB is rapidly activated and translocates to the nucleus in cardiomyocytes under hypertrophic stress conditions, activating a lysosomal gene program independent of autophagy gene changes. We engineered a cardiomyocyte-specific TFEB knockout mouse (CTKO). At baseline, contractile function measured by echocardiography appears normal in these mice compared with their Cre-negative littermates. However, in the setting of pressure-overload stress induced by TAC (thoracic aortic constriction), the CTKO mice manifest an exacerbated hypertrophic response leading quickly to heart failure. Unlike the WT hearts, CTKO hearts fail to increase lysosomal capacity after TAC and manifest an increase in steady-state levels of autophagosome-associated proteins such as LC3II and p62, as well as accumulation of ubiquitinated proteins, suggesting a decrease in protein turnover. Surprisingly, AMPK signaling, while normal at baseline is significantly decreased in the CTKO hearts after TAC. This reliance on TFEB for PE-induced AMPK signaling is also observed
in vitro
, as are the anti-hypertrophic effects of TFEB activation, supporting a direct role of TFEB in this process. Finally, we show that exogenous activation of AMPK in the absence of TFEB can completely rescue the exacerbated hypertrophic response both
in vitro
and
in vivo
, independent of lysosomal function. Surprisingly, blunting of the hypertrophic response had no impact on the decreased cardiac function observed in the TAC-treated CTKO mice.

Conclusion:
Overall, our findings suggest that TFEB antagonizes pathological hypertrophic cardiac remodeling through up-regulation of lysosomal capacity and AMPK signaling.
Abstract P2063: Cellular Fusion Drives Polyploidization In Human Cardiomyocytes
Authors: Sivankutty Indu , Jung Lucy , Huang August Y , Araten Sarah , Hilal Nazia , Walsh Christopher , Lee Eunjung Alice , Chen Ming Hui , Choudhury Sangita
Published: October 2023

Polyploidization is the roadblock to cardiac regeneration. Although endoreplication has been considered to be the main source of polyploidization in cardiomyocytes, recent evidence indicates transient cardiomyocyte fusion and its potential role in cardiac development in zebrafish. To understand polyploidization, it is essential to trace the lineage of cardiomyocytes. The existing lineage method is not suitable for studying the lineage in human heart cardiomyocytes. However, the accumulation of clonal somatic DNA mutation can be traced, and the clonal structure of these somatic mutations can be used for lineage reconstruction of human cardiomyocytes as these non-inherited clonal somatic mutations record the unique history of each somatic cell originating from a zygote and can be used as “endogenous barcodes.” Our analysis of ultra-deep targeted sequencing of ≈500 single cardiomyocyte nuclei using a panel of 253 validated clonal sSNVs revealed that 75.7% of the sequenced cardiomyocytes could be classified into one of the nine clades categorized based on the presence or absence of these mutations. Interestingly, we found that cardiomyocytes can have mutations from more than one clade, indicating the possibilities of cellular fusion in cardiomyocytes. Our results suggest that at least ~10% of tetraploid cardiomyocytes are generated from the fusion mechanism during development, whereas 60% higher ploidy cardiomyocytes are generated from the fusion. Further, we performed ATAC Seq and transcriptome analysis to find out the types of cells that are fused to generate polyploid cardiomyocytes in the human heart. Analysis of the transcriptome and chromatin peaks of tetraploid and higher ploidy cells unraveled a population of cells (cluster 7) that expressed chromatin peaks for endothelial markers (EGFL7) as well as cardiomyocyte-specific markers like TBX5, PLN, etc. Our study demonstrates cellular fusion in the adult human heart and the cell types involved in this fusion event. Understanding the generation of polyploid cardiomyocytes in the human heart might enable us to advance the mechanistic understanding of cardiomyocyte regeneration with the long-term goal of stimulating therapeutic cardiac regeneration.
Abstract P2064: A Specialized Centrosome-Proteasome Axis Mediates Proteostasis And Influences Cardiac Hypertrophy
Authors: McLendon Jared M , Zhang Xiaoming , Boudreau Ryan L
Published: October 2023

Mature cardiomyocytes (CMs) lack centrosomes. Instead, centriolar and satellite proteins redistribute into specialized perinuclear organelle-like compartments, where they likely serve multifunctional roles, e.g., in organizing microtubules and establishing localized clearance of aggregated/misfolded proteins via the ubiquitin-proteasome system (UPS). Taxilin-beta (Txlnb) is a CM-enriched protein that localizes perinuclear and belongs to the family of taxilin proteins, which are centrosomal proteins that have been implicated in protein quality control. We hypothesize that Txlnb plays key roles maintaining specialized centrosomal-proteasomal crosstalk in CMs. We first examined proteostatic roles for Txlnb in neonatal rat CMs treated with phenylephrine and found that Txlnb overexpression coordinately increased protein synthesis and proteasome activity, while decreasing ubiquitin-protein conjugate accumulation. We generated Txlnb knockout (Txlnb-KO) mice, which show robust accumulation of ubiquitinated proteins and decreased 26SB5 proteasome activity in heart tissues, as well as lower cardiac mass with aging. To test if Txlnb influences proteotoxic UPS dysfunction, we crossed Txlnb-KO mice to Cryab-R120G transgenic mice, a model of severe cardiac proteotoxicity with progression to heart failure and early death. Loss of Txlnb resulted in worsening of heart failure, evidenced by lower ejection fractions in Txlnb-KO/Cryab-R120G mice versus Cryab-R120G littermates. By contrast, AAV-mediated Txlnb overexpression in Cryab-R120G mice resulted in only slight reduction in myocardial wall thickness but was overall insufficient to slow heart failure progression. To test if Txlnb influences hypertrophic UPS dysfunction, Txlnb-KO and wildtype (WT) mice were subjected to cardiac pressure-overload induced by transverse aortic constriction (TAC). At 6-weeks post-TAC, Txlnb-KO mice showed reduced ejection fraction, increased heart mass, and elevated accumulation of ubiquitinated proteins, relative to WT mice. Together, these data provide initial evidence connecting Txlnb to proteostatic functions in cardiomyocytes, hinting at the potential importance of functional bridging between specialized centrosomes and UPS in CMs.
Abstract P2065: Mice Under Transverse Aortic Constriction And Treated With Sodium Oxalate May Develop Cardiac Dysfunction
Authors: Silva Amanda A , Marques Juliana R , Nascimento-Carvalho Bruno , Souza Leandro E , Silva Maikon B , Benetti Acaris , Waked Dunia , Veras Mariana M , Irigoyen Maria C
Published: October 2023

Background:
Experimental models are important for studying the mechanisms of cardiovascular disease. The transverse aortic constriction model (TAC) is among the most used in the literature for the study of cardiac hypertrophy. The soluble sodium oxalate (OXA) model generates renal injury but is also associated with cardiac hypertrophy through hypertensive-like lesions. There is little information in the literature on direct hemodynamic recording in both models mentioned, as well as there is no data on the changes caused by the association of these two models. Therefore, our objective is to evaluate whether TAC associated with OXA gavage could be a new model of cardiac dysfunction.

Methods and Results:
Male C57BL mice were randomized as follows: SHAM animals that received gavage with vehicle or OXA solution, and animals submitted to TAC and gavage with vehicle or OXA solution. Echocardiography and metabolic cage assessment were used to quantify cardiac and renal function. Direct blood pressure (BP) recording was also performed to assess baroreflex sensitivity and histological analysis. The TAC+OXA group had the highest BP values (mean BP:SHAM:100±5.0 vs. TAC+OXA:132±4.8mmHg, p<0.01), the highest pulmonary congestion index, an increase in the double product, an important reduction in autonomic modulation (AM), heart rate variability (HRV) (SD-PI: SHAM+OXA:12.5±1.8 vs. TAC+OXA:2.8±0.2ms, p<0.01) and alpha index (SHAM:3.6±1.0 vs. TAC+OXA:0.6±0.1, p=0.04). The TAC group showed the greatest weight of the ventricles and the greatest increase in blood pressure variability (DP-SBP). The SHAM+OXA group showed an increase in AM and HRV. The three groups showed a significant reduction in reflex tachycardia.

Conclusion:
TAC surgery provided cardiac hypertrophy, reduction in reflex tachycardia and changes in autonomic modulation. In the absence of TAC, the treatment with OXA for 15 days increased autonomic modulation. TAC surgery in conjunction with OXA gavage caused greater cardiac dysfunction.
Abstract P2067: Loss Of Cardiomyocyte β2 Spectrin Promotes Cardiac Dyad Dysfunction And Accelerated Heart Failure
Authors: Scott Shane S , Schwendeman Ethan , Matzko Anna , Tonniges Jeffrey , Schwieterman Neil , Derbala Mohamed , Smith Sakima A
Published: October 2023

Background:
The cardiac dyad is composed of the transverse-tubules, junctional sarcoplasmic reticulum (jSR), and its associated calcium (Ca
2+
) release units, which are integral for cardiac excitation-contraction (E-C). Proper E-C coupling requires Ca
2+
influx via L-type Ca
2+
channels (LTCC) which triggers Ca
2+
release from adjacent ryanodine receptor 2 (RyR2) receptors located in jSR. Loss of the βII-spectrin scaffolding protein alters RyR2 localization; however, its contribution to cardiac dyad organization and E-C coupling remains unclear.

Hypothesis:
We hypothesized that βII-spectrin is required for cardiac dyad organization and proper cardiomyocyte calcium signaling.

Methods:
Cardiomyocyte-specific βII-spectrin knockout (βII-cKO) and control (βII-flox) mice were generated and hearts were harvested for immunoblotting, qRT-PCR, and transmission electron microscopy (TEM). Cardiomyocytes were isolated and stained using proximity ligation assay (PLA) and visualized by confocal microscopy. To assess heart response to pressure overload, control and βII-cKO mice were subjected to transaortic constriction surgery followed by treatment with or without, diltiazem and monitored by echocardiography, and electrocardiography.

Results:
Cardiac dyad structure and components are significantly altered in βII-cKO hearts. Ultrastructural analysis of βII-cKO hearts showed fragmented cardiac dyads, dilated jSR and reduced jSR contact with T-tubules. Quantitative RT-PCR and immunoblotting analysis revealed altered expression of dyad mRNAs and proteins. Preliminary PLA demonstrated reduced density of LTCC-RyR2 complexes. Furthermore, pressure-induced overload of βII-cKO hearts promoted accelerated heart failure (HF), which can be rescued by treatment with diltiazem.

Conclusions:
We demonstrate that βII-spectrin is required for cardiac dyad integrity, and that βII-spectrin-mediated HF can be attenuated by diltiazem.
Abstract P2070: The Microprotein Another-Regulin Regulates Serca2a In Response To Neurohormonal Signaling
Authors: Hassel Keira , Gibson Aaron , Makarewich Cat A
Published: October 2023

Objective:
Calcium (Ca
2+
) regulation is a key component of proper heart function and is largely controlled by the sarco/endoplsmic reticulum Ca
2+
ATPase 2a (SERCA2a). Ca
2+
dysregulation is a hallmark feature of heart failure, yet few advances have been made to reestablish Ca
2+
homeostasis during disease. Another-regulin (ALN) inhibits SERCA2a, though little is known about how ALN functions in heart physiology and disease. Here, we determine a mechanism by which ALN is regulated via phosphorylation at serine 19 (S19), resulting in dissociation from SERCA2a.

Methods and Results:
ALN phosphorylation at S19 was confirmed via Phos-tag gel migration assay and site directed mutagenesis (SDM), whereby serine to alanine (S19A) mutation abolished the migrational shift which was confirmed with a custom S19 phospho-specific antibody. Effects of S19 phosphorylation on ALN-SERCA2a affinity were investigated using co-immunoprecipitation (IP) and fluorescence resonance energy transfer (FRET) in HEK293 cells. In addition to phosphodeficient S19A, SDM was used to mutate S19 to phosphomimetic aspartate (S19D) and glutamate (S19E). IPs of SERCA2a and hemagglutinin (HA)-tagged ALN WT, S19A, S19D, or S19E revealed less interaction between SERCA2a and ALN-S19D/E, suggesting S19 phosphorylation causes ALN dissociation from SERCA2a. Acceptor sensitization and fluorescence lifetime measurements show that ALN-S19D decreases ALN/SERCA2a binding affinity. To investigate signaling upstream of ALN phosphorylation, adult mouse cardiomyocytes were stimulated with various agonists and levels of pALN measured. Phosphorylation was significantly induced with the neurohormonal stimuli endothelin-1 and phenylephrine compared to DMSO and the β-adrenergic stimulus isoproterenol, suggesting Gαq/PKC pathway involvement. In addition, pALN levels are elevated in whole heart lysates of mice subject to transverse aortic constriction which is a model of pathological hypertension.

Conclusions:
These results indicate that ALN is phosphorylated at S19 in response to neurohormonal stimuli, resulting in dissociation from SERCA2. Future directions will investigate the impact of ALN phosphorylation on SERCA2 activity and heart function.
Abstract P2078: Vasohibin Inhibition Improves Myocardial Relaxation In A Rodent Model Of Heart Failure With Preserved Ejection Fraction
Authors: Eaton Deborah M , Lee Benjamin W , Caporizzo Matthew A , Chen Yingxian , Iyengar Amit , Meldrum Danika , Dominic Jessica , Weingarten Noah , Hached Khaled , Bedi Kenneth , Atluri Pavan , Van Der Laan Siem , Prosser Benjamin L , Margulies Kenneth B
Published: October 2023

Heart failure with preserved ejection (HFpEF) is a complex syndrome with few therapies available to patients. Diastolic dysfunction is a common clinical feature among HFpEF patients, and strategies to mitigate impaired relaxation may have a therapeutic benefit. Recognizing that increased stabilization of the microtubule network induced by detyrosination of α-tubulin contributes to increased cardiomyocyte stiffness

ex vivo

, we explored if inhibition of detyrosination with a small molecule vasohibin inhibitor (VASHi) could improve

in vivo

diastolic function in obese/hypertensive (ob) ZSF1 rats, compared to lean/hypertensive ZSF1 and non-hypertensive Wistar Kyoto (Wky) rats as controls. Animals received two intravenous injections of VASHi (6.6mg/kg) or vehicle (veh) at 30 weeks old, when ob have a HFpEF phenotype. Echocardiography and invasive hemodynamics were performed after the first and second injection, respectively. Compared to ob veh-treated animals, ob VASHi-treated animals had a significant improvement in invasively measured time constant of early LV relaxation, tau (veh: 14.73ms ± 0.73 vs. VASHi: 11.02ms ± 0.45; P < 0.05) and echo-derived deceleration time of early LV filling, indicative of improved early relaxation (Fig. A, B). VASHi improved early relaxation in cardiomyocytes isolated from non-failing (NF, N=3) and failing (HF, N=4) human hearts (Fig. C), providing the first evidence of small molecule VASHi improving human myocyte function. These studies indicate that excess detyrosination contributes to impaired early myocyte and myocardial relaxation in a clinically relevant HFpEF model and supports a potential therapeutic role for VASHi in HFpEF.
Abstract P2084: Adipose Tissues Thermogenesis Provides Cardioprotection Against Diastolic Dysfunction Developed In An Obesity-Induced HFpEF Model
Authors: Jousma Jordan , Park Jooman , Han Zhenbo , Yan Gege , Nukala Sarath Babu B , Kwon Youjeong , Lee Won Hee H , Jiang Yuwei , Ong Sang-Ging
Published: October 2023

Background:
Heart failure with preserved ejection fraction (HFpEF) is a major source of morbidity/ mortality in the United States. A systemic inflammatory state established through underlying comorbidities, such as obesity, is thought to develop cardiac dysfunction in HFpEF. Clinical observations associate adiposity with diastolic dysfunction. Herein we demonstrate that adipose tissue is a direct source of diastolic dysfunction and reveal a cardioprotective role of adipose tissue thermogenesis in obesity related HFpEF.

Methods and Results:
Mice were treated with a β3-AR agonist, CL-316243 (CL), to induce thermogenesis, followed by HFpEF treatment consisting of HFD and L-NAME. Echocardiography revealed that this protected against HFpEF-induced diastolic dysfunction. Whole heart extracts and AT were then evaluated for the signature of β3-AR signaling. In the heart, no signature of β3-AR signaling was observed. In AT, though, various aspects of β3-AR induced thermogenesis were observed. Histological analysis revelated that CL treatment conferred resistance against obesity-induced whitening of interscapular brown adipose tissue (iBAT). Gene and protein expression changes of thermogenic regulators and lipid metabolism enzymes suggested greater thermogenic activity in CL-treated mice. Accordingly, indirect calorimetry showed enhanced energy expenditure, while regression analysis revealed that AT uniquely contributed to this. Transplantation experiments involving CL-treated donor AT and wild-type donors followed by echocardiographic measurements confirmed that the effects of CL were mediated through AT. This approach was complemented with a genetic model we previously used (
Ucp1
-Cre
ERT2
; Cdkn2a
fl/fl
) to promote the expansion of beige AT. Here, beige AT expansion protected against diastolic dysfunction and cardiomyocyte hypertrophy developed during HFpEF.

Conclusion:
Our study indicates enhanced energy expenditure in AT is cardioprotective in obesity related HFpEF. Furthermore, identifying obese AT as a direct source of diastolic dysfunction highlights the therapeutic potential of targeting AT metabolism as a relevant source of diastolic dysfunction.
Abstract P2092: The Late Sodium Current Conditions Myocardial Repolarization And Relaxation Of The Aged Heart
Authors: Cervantes Daniel O , Pizzo Emanuele , Ketkar Harshada , Parambath Sreema P , Di Stefano Valeria , Singh Kanwardeep , Ripa Valentina , Kouril Anna , Mohler Peter J , Hund Thomas J , Jacobson Jason , Jain Sudhir , Rota Marcello
Published: October 2023

Diastolic dysfunction and delayed ventricular repolarization are observed in the elderly, but whether these defects are intimately associated in the progressive manifestation of the aging myopathy remains to be determined. Interestingly, aging in experimental animals is coupled with increased late Na current (INaL) in cardiomyocytes, raising the possibility that INaL conditions the modality of electrical recovery and relaxation of the aged heart. Thus, male wild-type (WT) and genetically engineered mice with phosphomimetic (gain-of-function, GoF) or ablated (loss-of-function, LoF) mutations of the sodium channel Nav1.5 at Ser571, a phosphorylation site mediating INaL enhancement, were studied. With respect to WT mice at ~5 months of age (m), animals at ~18 m had 20% prolongation of the QT interval of the electrocardiogram and declined diastolic left ventricular filling, consisting of a 23% increase in isovolumic relaxation time (IVRT). These defects were reversed by INaL inhibition with GS967. Prolonged repolarization and impaired LV filling occurred prematurely in GoF mice (QT, +20%; IVRT, +8%, at ~5 m vs. WT), whereas age-related alterations were largely attenuated in LoF animals. At ~24 months, QT interval was not different in WT (66±4 ms) and GoF (70±5 ms) mice but was shorter in LoF (64±5 ms) animals, with respect to GoF. Similarly, IVRT was comparable in WT (22±2 ms) and GoF (22±2 ms) mice, but shorter in LoF (18±3 ms). In field stimulated cells, with respect to WT myocytes at ~5 m, WT cells at ~24 m had prolonged time to peak (+26%) and time to 50% relaxation (+44%). Delayed cell contraction/relaxation was apparent in GoF myocytes at ~5 m (+12% and +27%, with respect to WT), and further deteriorated with age. In contrast, LoF myocytes at ~5 m had contractile kinetics comparable to adult WT cells, and modest alterations occurred in cells at ~24 m. Importantly, inhibition of INaL was effective in correcting kinetics of cell mechanics in old WT mice and adult and old GoF animals but had no effect in LoF myocytes and WT cells at ~5 months. Collectively, these results document that the late Na+ current modulates the modality of myocyte relaxation, constituting the mechanism linking delayed ventricular repolarization and diastolic dysfunction in the elderly.
Abstract P2094: The Regulatory Role Of Sirtuin 6 In Coronary Microcirculation In Hfpef
Authors: Wang Yang , Enrick Molly , Gadd James , Clark Alyssa , Yin Liya
Published: October 2023

Coronary vasculature is essential to support the supply oxygen and nutrients to the heart and maintain the normal cardiac function. Obstruction of large vessels of coronaries (atherosclerosis) is well studied, but coronary microvascular dysfunction (CMD), which is associated with diabetic cardiomyopathy, Takotsubo cardiomyopathy, andheart failure with preserved ejection fraction (HFpEF), is understudied. CMD is characterized by impaired endothelial-dependent vasodilation, but detailed mechanisms have yet to be elucidated. Sirtuin 6 (Sirt6) has been implicated in obesity, insulin resistance, type 2 diabetes mellitus, and cardiovascular diseases. Sirt6 protects EC from premature senescence, oxidative stress, and atherosclerosis by sustaining high eNOS levels and preserving cell replication. A recent publication showed that overexpressed Sirt 6 in EC mitigated heart failure with preserved ejection fraction (HFpEF) in diabetes. We hypothesize Sirt 6 deficiency causes CMD in HFpEF How Sirt6 regulates coronary microvascular function remains to be determined. Inducible Sirt6 endothelial-specific knockout and wild-type (WT) mice were fed a chow or high fat and sugar (HFHS) diet. Coronary arterioles were isolated, and endothelial-dependent vasodilation (EDD) was assessed using myography (DMT). Echocardiography and treadmill exercise exertion tests were performed to evaluate cardiac function. Myocardial blood flow (MBF) was measured by doppler. Cardiac fibrosis was detected using trichrome staining, and molecular pathways were elucidated via gene and protein analysis. Our preliminary data show that the EDD of coronary arterioles of Sirt6 endothelial-specific knockout treated with HFHS was impaired, and the mediator of coronary vasodilation switched from NO to H
2
O
2
in the Sirt6 knockout mice. Compared to the WT mice, myocardial blood flow and cardiac function were changed in Sirt6 knockout mice. Our data suggest that Sirt6 regulated coronary microvascular function through endothelial cells, and ablation of Sirt6 in endothelial cells caused CMD and diastolic dysfunction, eventually HFpEF. Further genetic profiling will elucidate the pathways and mechanisms converging with Sirt6 to regulate microvascular function in HFpEF.
Abstract P2096: Characterization Of Unique Dystrophin Isoforms In Epicardial Cells
Authors: Ware Sarah , Gemelli Terry , Rothermel Beverly A , Tong Dan , Mammen Pradeep P
Published: October 2023

Duchenne muscular dystrophy (DMD) is caused by mutations that abolish dystrophin expression. Dilated cardiomyopathy, the leading cause of death in DMD patients, is characterized by the development of severe subepicardial fibrosis that precedes cardiac dysfunction. While the cellular mechanisms governing this process remain unknown, a recent study in
mdx/mTR
mice suggested that inflammation, in response to cardiomyocyte damage caused by dystrophin deficiency, activates the epicardium to undergo epithelial-to-mesenchymal transition into fibroblasts, thereby contributing to subepicardial fibrosis. Whether dystrophin plays an epicardial-autonomous role in this process has not been investigated. The
DMD
gene produces at least eight dystrophin isoforms from seven independent, tissue-specific promoters (
Fig. 1
). We used an immortalized mouse embryonic epicardial cell line to determine which isoforms are present. We found transcripts for the full-length muscle isoform of dystrophin (Dp427m) in addition to the ubiquitous, more abundant Dp71 and Dp40 isoforms. RT-PCR and western blot analysis revealed the presence of three different Dp71 splice variants. Immunofluorescence and subcellular fractionation determined that Dp71 localizes primarily to the nucleus. Studies are ongoing to understand the role of Dp71 in epicardial cell proliferation and gene expression, as well as defining changes in dystrophin expression during the transition from the activated epicardium to myofibroblasts. Further investigation is needed to determine the role of these shorter isoforms in DMD pathology and how gene editing of full-length dystrophin may impact their function.
Abstract P2098: The Effect Of Cyclic Stretch On Cardiac Fibroblasts
Authors: Bekedam Fjodor , Smit Marisa , Smal rowan , Vonk Noordegraaf Anton , Bogaard Harm J , Goumans Marie J , De Frances S , Llucia-Valldeperas Aida
Published: October 2023

Introduction:
Cardiac fibrosis is the accumulation of extracellular matrix proteins in the interstitium and contributes to heart failure. The main source of cardiac fibrosis is cardiac fibroblasts (cFBs), which are activated as a result of pressure overload. Activated fibroblasts, also known as myofibroblasts, express α-smooth muscle actin (α-SMA) and have an increased production of collagen. Here, the objective was to study the behavior of induced pluripotent stem cell (iPSC) derived cFBs in response to pro-fibrotic cytokines and mechanical stress.

Methods:
Confluent iPSC monolayers were differentiated into cFBs using the protocol in Figure 1A. Afterwards, the differentiated cells were exposed to 10 ng/ml transforming growth factor β (TGFβ) and 10% cyclic stretch at 1 Hz for 3 days using the Flexcell FX-6000 system.

Results:
iPSC-cFBs had a fibroblast-like morphology. Furthermore, the presence of cardiac (
GATA4, TCF21
) and fibroblast (
VIM, PDGFRα, DDR2
) markers at gene and protein levels confirmed the cFB identity. TGF-β stimulation resulted in increased mRNA and protein levels of α-SMA. Additionally, stretching reduced collagen 1 mRNA and protein expression (Fig. 1B/C). Furthermore, stretching was able to protect the cells from TGF-β stimulation, significantly reducing α-SMA and collagen 1 protein and mRNA levels. This was associated with decreased mRNA expression of TGF-β downstream targets (
PAI1).

Conclusion:
Cyclic mechanical stretch reduces cFB activation and can protect against TGF-β stimulation, resulting in reduced collagen production and transdifferentiation into myofibroblasts.
Abstract P2099: Small Proline-rich Protein 1a Is A Key Downstream Effector Of Microrna-150 During Both Maladaptive Cardiac Remodeling In Mice And Human Cardiac Fibroblast Activation
Authors: Sepúlveda Marisa , Hayasaka Taiki , Kim Il-man
Published: October 2023

Background & Objectives:
MicroRNA-150 (miR-150) is conserved between rodents and humans, is significantly downregulated during heart failure (HF), and correlates with patient outcomes. We reported that miR-150 is protective during myocardial infarction (MI) in part by decreasing cardiomyocyte (CM) apoptosis and that proapoptotic small proline-rich protein 1a (
Sprr1a
) is a direct CM target of miR-150. We also showed that
Sprr1a
knockdown in mice improves cardiac dysfunction and fibrosis post-MI and that
Sprr1a
is upregulated in pathological mouse cardiac fibroblasts (CFs) from ischemic myocardium. However, the direct functional relationship between miR-150 and SPRR1A during both post-MI remodeling in mice and human CF (HCF) activation was not established.

Methods & Results:
Here, using a novel miR-150 knockout (KO);
Sprr1a
-hypomorphic (

Sprr1a
hypo/hypo

) mouse model, we demonstrate that
Sprr1a
knockdown blunts adverse post-MI effects caused by miR-150 loss. MiR-150 KO;

Sprr1a
hypo/hypo

mice also exhibit decreased profibrotic markers in the infarct areas of hearts as compared to miR-150 KO mice. Moreover, primary adult HCF studies reveal that
SPRR1A
is upregulated in hypoxia/reoxygenation-treated HCFs and is downregulated in HCFs exposed to the cardioprotective β-blocker carvedilol, which is inversely associated with miR-150 expression. Significantly, we show that the protective roles of miR-150 in HCFs are directly mediated by functional repression of profibrotic
SPRR1A
(See Figure).

Conclusions:
These findings delineate a pivotal functional interaction between miR-150 and SPRR1A as a novel regulatory mechanism pertinent to CF activation and ischemic HF.
Abstract P2102: The Basic Helix-loop-helix Transcription Factor Tcf21 Regulates Fibroblast Cell States In The Adult Murine Heart
Authors: Johansen Anne Katrine Z , Vagnozzi Ronald J , Kidder Bowers Stephanie K , Grimes Kelly , Kuwabara Yasuhide , Sargent Michelle , Lin Suh-chin , Quaggin Susan E , Molkentin Jeff D
Published: October 2023

Objective:
Transcription factor 21 (Tcf21) is a basic helix-loop-helix protein that is essential for normal mesodermal organogenesis and its loss is incompatible with life. In the developing heart, epicardial progenitor cells differentiate to either vascular smooth muscle cells or fibroblasts, the latter of which is dependent on Tcf21 expression. In the adult heart, Tcf21 is expressed by the majority of fibroblasts but is downregulated in response to fibrotic stimuli as the fibroblast differentiates to a myofibroblast. In atherosclerosis, smooth muscle cells that undergo phenotypic modulation towards a fibroblast-like cell acquire expression of Tcf21. These findings led to the hypothesis that Tcf21 could be a regulator of fibroblast cell-fate in the adult mammalian heart and contribute to cardiac fibrosis.

Methods and Results:
To test this hypothesis, we generated tamoxifen-inducible genetic mouse models to study cardiac fibroblast Tcf21 deletion and overexpression
in vivo
. Acute and long-term Tcf21 ablation in the adult heart did not promote differentiation to myofibroblasts. After long-term ablation, no differences were detected on cardiac interstitial cell populations, cardiac structure or function and extracellular matrix deposition. In contrast, in response to fibrotic stimuli, overexpression of Tcf21 suppressed differentiation to myofibroblasts with reduced expression of contractile proteins. This was accompanied by reduced cardiac fibrosis.

Conclusions:
The expression of Tcf21 in the healthy fibroblast is not required to maintain a ‘resting’ fibroblast cell-state. In disease, Tcf21 functions as a suppressor of fibroblast differentiation to the contractile myofibroblast and its overexpression reduces the fibrotic burden on the heart.
Abstract P2103: Cardiac Fibroblast-specific Role Of PTBP1 In Maintaining Heart Function
Authors: Ricketts Shea N , Chen Shufen , Qian Li
Published: October 2023

Cardiac fibroblasts (CFs) are the primary nonmyocyte in the heart that maintain cardiac homeostasis by regulating myocardial function and cardiac remodeling. CFs secrete an extracellular matrix to stabilize the myocardial wall while maintaining the heart’s mechanical integrity during development and pathogenesis. Developmental and pathogenic transitions are directed through CFs by transcriptional and post-transcriptional modifications, such as alternative splicing. Alternative splicing is a central element in post-transcriptional gene regulation and proteomic diversity. Impaired and aberrant splicing is known to cause developmental defects and drive cardiac pathogenesis. RNA binding proteins (RBPs), which function as alternative splicing factors, are upregulated in cardiovascular disease states. While nearly a third of all CF gene regulation changes during cardiac fibrosis are subject to transcriptional regulation via RBPs, the role of CF specific splicing RBPs in the adult heart and during cardiac fibrosis is unknown. Our group previously identified high expression of well-known RBP and splicing factor polypyrimidine tract-binding protein (PTBP1) in CFs when compared to cardiomyocytes in the adult heart. With single cell transcriptomics we found PTBP1 regulates CF identity and plasticity, suggesting an emergent role of PTBP1 within CF biology. We hypothesize PTBP1 is essential for maintaining cardiac fibroblast function in the adult heart. Preliminary
in vitro
data suggests partial
Ptbp1
knockdown impacts mechanical function by decreasing overall CF migration. To further characterize PTBP1
in vivo
, we have validated our novel mouse model to specifically delete PTBP1 from CFs in adult mice. Following PTBP1 deletion, we will utilize an established model of fibrosis by Angiotensin II osmotic minipumps to characterize the CF specific PTBP1 loss during fibrosis. We will evaluate CF biology with our
in vitro
and
in vivo
models to examine PTBP1’s physiological and mechanistic role in maintaining CF biology during cardiac homeostasis and cardiac fibrosis pathogenesis.
Abstract P2105: Store-Operated Channels Mediate Metabolic Adaptations In Activated Fibroblast During Cardiac Fibrosis
Authors: Mancarella Salvatore , Pantoja Patricia , Saxena Sheena , Bajwa Amandeep
Published: October 2023

Endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) are associated with developing fibrotic disorders in multiple organs, including the heart. Many ER resident chaperones require calcium (Ca2+) as a co-factor to mediate protein folding. Store-operated Ca2+ entry (SOCE) mediated by the Orai1 channels primarily maintains the ER- Ca2+ homeostasis. However, the role of the Orai1 channels in the modulation of ER-stress/UPR and downstream cellular processes that lead to cardiac fibrosis is not entirely understood. To investigate the role of Orai1 channels on the ER-stress/UPR signaling we utilized a multilayered approach that includes both genetic and pharmacological inhibition of Orai and a combination of long-term real-time cell imaging combined with genetically encoded sensors for several metabolites signaling. Our results showed that Orai1 expression was upregulated in the fibrotic mouse heart. Transcriptome analysis of fibrotic tissue from a fibroblast-specific Orai1 knock-out mouse model revealed the dysregulation of many genes associated with cell metabolism and collagen turnover. Pharmacological inhibition of Orai1 channel activity in cultured cardiac fibroblast decreased ER-Ca2+ and mitochondrial Ca2+ levels and was sufficient to trigger ER-stress/UPR and promote mitochondrial dysfunction. Like the in vivo fibrotic model, comparative transcriptome and pathway analysis between cells treated with Orai1 blocker (YM-58483, 48 hours) and controls identified dysregulated metabolic pathways and ER-stress/UPR signaling. Bioenergetic analysis showed that Orai1 inhibition induced an overall decrease in oxygen consumption rate (OCR), and mitochondrial membrane potential and reduced cellular ATP levels. Accordingly, we found that Orai1 loss-of-function increased glycolysis and reduced fatty acid oxidation (FAO), substantially reducing fibroblast fuel flexibility. This is a remarkable novel function of Orai1 channels because perturbations in fibroblast metabolic dynamic are the initial steps in the injury response. Our results suggest that Orai1 could be a novel target for developing treatments for fibrosis.
Abstract P2107: Latent Transforming Growth Factor Binding Protein - 2 Deficiency Improves Cardiac Function Post-Myocardial Infarction
Authors: Ehsan Fahad , Dewar Michael , Wang Tao , Izumi Aliya , ZHOU YU-QING , Zhang Hangjun , Heximer Scott P
Published: October 2023

Cardiac fibrosis and myocardial stiffening are key factors contributing to deteriorating cardiac function during heart failure, especially after myocardial infarction (MI). In the myocardium, cardiac fibroblasts (CFs) are the main cell type driving excess extracellular matrix (ECM) protein deposition, leading to pathological remodelling of the heart. Latent Transforming Growth Factor Binding Protein 2 (LTBP2) is one of the ECM proteins released as part of the fibrotic response and is significantly upregulated in all types of heart failure. Although LTBP2 belongs to a family of proteins that regulate the release of Transforming Growth Factor - β; (TGF-β;) in the ECM, it doesn’t interact with TGF-β;. Nonetheless, LTBP2 is shown to play a role in the progression of fibrosis following heart failure by contributing to CFs activation. Therefore, we hypothesized that in the absence of LTBP2, the heart would have improved cardiac function following MI. We used LTBP2 knockout (KO) C57BL/6 mice and modelled MI by ligating the left anterior descending artery. Echocardiography was performed to analyze the cardiac function in KO and wild-type (WT) infarcted hearts at 7 and 28 days post-MI. KO mice showed significantly better ejection fractions (KO = 24.1±4.9% vs. WT = 9.2±1.4%, p-value < 0.01) and fractional shortening 28 days post-MI compared to WT mice. In conclusion, our findings show that LTBP2 contributes to the deteriorating cardiac function following MI suggesting that targeting LTBP2 may have therapeutic potential. As LTBP2 is known to be a part of the fibrotic response, future studies will investigate the fibrotic profile of WT and KO infarcted hearts using histology and CFs-specific RNA sequencing to highlight the underlying differences leading to the observed cardiac function.
Abstract P2114: Chromatin Accessibility Governs Mechanosensitive Gene Expression In Cardiac Fibroblasts
Authors: Morningstar Jordan , Beck Tyler , Gensemer Cortney , Biggs Rachel , Petrucci Taylor , Guo Lilong , Norris Russell A
Published: October 2023

Objective:
We recently demonstrated regionalized myocardial fibrosis in patients undergoing surgical repair for Mitral Valve Prolapse. The location of fibrosis suggests a mechanically driven process wherein mechanical stress alters the phenotype of ventricular fibroblasts to promote an increase in tissue stiffness, eventually causing establishment of a fibrotic scar. We sought to better understand how mechanical stiffness affected cellular programs.

Methods:
We cultured primary human cardiac fibroblasts derived from the same patient on a mechanically stiff (TC Plastic, ~2GPa), as well as a mechanically soft (2kPa) substrate and then performed immunofluorescence, western blotting, RNA Seq, and ATAC Seq. Following differential analysis of omics data, we performed an integration analysis by filterinig differentially accessible chromatin to only include sites that fell within 10 kB of opened/closed chromatin. Finally, we performed a motif enrichment analysis using the online bioinformatics package Homer.

Results:
Immunofluorescence and western blotting revealed that cells cultured on a stiff substrate expressed canonical markers of fibroblast activation compared with soft substrates (alpha SMA, COL1A1, P<0.0001). RNA Sequencing revealed differential changes in 1615 genes in fibroblasts (FDR adjusted P<1.6*10
-6
), several of which were subsequently validated using western blot. ATAC Seq identified 5855 regions across the genome which opened/closed in response to different mechanical environments in fibroblasts (adjusted P<0.05). Integrating these datasets revealed 803 Chromatin changes that fell within 10kb of differentially expressed genes, with AP1 and TEAD family transcription factors predicted to be master regulators of closing and opening chromatin, respectively. Proof-of-concept motif validation conducted on differentially expressed chromatin within intron 12 of the MYH10 gene demonstrated presence of a transcriptional repressor with evidence of JunD as a binding partner for this site.

Conclusions:
The mechanical environment plays a major role in cellular phenotype and regulates chromatin organization and expression of pro-fibrotic genes through changes in chromatin accessibility.
Abstract P2120: Alteration Of Left Ventricular Load Impacts Exosomal And Cardiac MicroRNA Expression In A Porcine Model Of Myocardial Ischemia
Authors: Marx Jonas M , Mazurek Renata , Bikou Olympia , Sakata Tomoki , Mavropoulos Spyros M , Sahoo Susmita , Ishikawa Kiyotake
Published: October 2023

Background:
Mechanical unloading of the left ventricle (LV) after myocardial infarction (MI) exhibits cardioprotective effects. The underlying molecular mechanisms of these benefits are not fully elucidated. MicroRNAs (miRNA) are vital regulators of cellular processes. When released into circulation within exosomes, they participate in inter and intra-organ communications. We hypothesized that LV load influences exosomal and myocardial miRNA expression.

Methods:
Anterior MI was induced in Yorkshire pigs. After one week, pigs underwent mechanical LV unloading per Impella or LV overloading by percutaneous aortic valve destruction (n = 5 each). Two hours later, coronary sinus blood was collected, animals were euthanized and infarct border zone tissue was harvested. Exosomal miRNAs isolated from blood were studied using next-generation sequencing. Myocardial tissue expression of select differentially regulated exosomal miRNAs was assessed by quantitative PCR.

Results:
In coronary sinus blood from LV unloaded animals exosomal miR-19b, miR-20a, miR-21, miR-144, and miR-146b showed higher expression compared to LV overloaded pigs (log2 FC = 3.47, 2.64, 1.86, 2.62, 1.58; p<0.05). Opposite relationships were observed in myocardial tissue. MiR-19b and miR-20a showed lower expression in LV unloaded compared to overloaded animals (FC = 0.36 ± 0.11 and 0.37 ± 0.12, p<0.05). A similar trend was found for miR-21, miR-144, and miR-146b expression.

Conclusion:
Exosomal miRNAs in coronary sinus blood are affected by changes in LV load. Inverse expression patterns in myocardial tissue suggest increased cardiac release or decreased uptake of exosomes as a possible mechanism.