bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 182: CTRP9 - A Novel Cardiac Derived Secreted Factor With Anti-hypertrophic Properties
Authors: Breitbart Astrid H , Brandes Florian , Müller Oliver , Froese Natali , Korf-Klingebiel Mortimer , Schrameck Ulrike , Wollert Kai C , Bauersachs Johann , Heineke Jörg
Published: July 2021

Background:
CTRP9 (also called C1qtnf9) is a newly discovered secreted protein and a paralog of adiponectin. The biological functions of CTRP9, however, are still largely unknown.

Results:
Although previous data from a semi-quantitative real-time PCR had suggested that CTRP9 is mainly secreted by adipose tissue, we found its mRNA to be predominantly expressed in the heart by quantitative real-time PCR. Interestingly, we identified CTRP9 mRNA as significantly upregulated in hypertrophied mouse hearts (after 2 weeks of aortic constriction, TAC) as well as in hypertrophied human hearts (24±4-fold versus healthy human myocardium; p<0.01). LacZ staining in myocardial sections of C1qtnf9
tm1(KOMP)Vlcg
mice (knock-out for CTRP9, containing a lacZ cassette to replace exon 1-3 of the gene) revealed exclusive expression of CTRP9 in capillary and venous endothelial cells. Adenoviral overexpression of CTRP9 or recombinant CTRP9 strongly inhibited cardiomyocyte hypertrophy (assessed as cell size, protein/DNA-ratio, expression of skeletal α-actin) after stimulation with phenylephrine (PE). Accordingly, myocardial overexpression of CTRP9 via a cardioselective adeno-associated virus (AAV9-CTRP9) in mice dramatically reduced cardiac hypertrophy after two weeks of pressure overload (heart weight/body weight ratio, HW/BW in mg/g: AAV9-control 6.5±0.2 versus AAV9-CTRP9 5.6±0.2; p<0.01). In turn, downregulation of CTRP9 by a specific siRNA aggravated cardiomyocyte growth in response to PE
in vitro
and CTRP9 knock-out (KO) mice exerted an enhanced hypertrophic response after two weeks of TAC
in vivo
(% increase in HW/BW versus sham: wild-type 77±13, KO 106±9; p<0.05). Mechanistically, we found that CTRP9 binds to the adiponectin receptor 1 (AdipoR1) and inhibits prohypertrophic mTOR signalling in cardiac myocytes. SiRNA mediated downregulation of AdipoR1 or mTOR in neonatal rat cardiomyocytes abolished the anti-hypertrophic effect of CTRP9.

Conclusion:
Endothelial cell derived CTRP9 inhibits cardiac hypertrophy through binding to AdipoR1 and inhibition of the mTOR pathway in cardiomyocytes. Therefore, myocardial application of CTRP9 could be a novel strategy to combat pathological cardiac hypertrophy.
Abstract 186: The Cardiac Specific Splicing Regulator Rbm20 decreases in Specific Forms of Acquired Heart Disease
Authors: van den Hoogenhof Maarten , Beqqali Abdelaziz , van der Made Inge , Duncker Dirk , van der Velden Jolanda , Pinto Yigal , Creemers Esther
Published: July 2021

Introduction:
Rbm20 is a RNA-binding protein enriched in heart- and skeletal muscle. Mutations in Rbm20 have been described to cause heart failure. Furthermore, Rbm20 has been shown to regulate alternative splicing of titin (Ttn). This demonstrates that RBM20 has a major role in cardiac biology. However, it is unknown how Rbm20 expression changes in acquired heart disease. Therefore, we investigated expression of Rbm20 on mRNA and protein level and the degree of alternative splicing of Ttn in various forms of heart disease.

Methods:
We measured mRNA expression and protein levels of Rbm20 by western blotting and qPCR in transverse aorta constrictions (TAC) in mice and pigs, and in human heart disease including hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), aortic stenosis (AoS) and pulmonary artery hypertension (PAH). We measured cardiac function in mice with echocardiography, and measured stress markers with qPCR. Furthermore, we analyzed Ttn splicing using qPCR and RT-PCR.

Results:
Real-time PCR and western blotting revealed that Rbm20 is selectively downregulated in mice after TAC and in patients with aortic stenosis. In contrast, we did not observe a difference in Rbm20 levels in patients with DCM, HCM and PAH. Furthermore, we measured functional parameters such as ejection fraction (EF), systolic left ventricular internal diameter (LVIDsys), and fractional shortening (FS), as well as ANF level in TAC mice, and correlated these to Rbm20 level. We found that the level of downregulation of Rbm20 is correlated with heart failure severity in mice after TAC. Also, we found that Ttn is differentially spliced after loss of Rbm20.

Conclusions:
Here we show for the first time that Rbm20 decreases in acquired forms of heart failure such as TAC in mice and pigs, and AoS in men. Furthermore, in experimental HF in mice, Rbm20 levels correlate to HF severity. Moreover, loss of Rbm20 leads to skipping of intron retention in Ttn in the region between exon 50-70. However, the biological effect of this event remains unclear. Given its major role in the heart, loss of Rbm20 may play an important role in the development of heart disease.
Abstract 192: Roles of Phosphodiesterases 2 and 3 at the T-tubules of Adult Mouse Cardiomyocytes
Authors: Perera Ruwan K , Nikolaev Viacheslav O
Published: July 2021

PURPOSE:
cAMP is a key regulator of proper heart function. Its functional effects are controlled and regulated by spatial and temporal signal segregation at the subcellular level. In cardiomyocytes, transverse (T)- tubules form a highly organized network of membrane structures which are crucial for normal cAMP signaling. Morphological changes, such as loss of T-tubules, are associated with cardiac disease. This study aimed to gain insight into localized cAMP dynamics and its regulation in the T-tubular microdomain in health and disease.

Methods:
Generation of a transgenic mouse expressing a targeted Förster resonance energy transfer (FRET)-based biosensor (pmE1). Confocal microscopy for co-localization of pmE1 with T-tubular structures. Histological, morphometric and ultrasound analysis for characterization of heart function. Transverse aortic constriction (TAC) surgery for hypertrophy induction. FRET-based cAMP measurements and PDE activity assay with single freshly isolated adult ventricular myocytes of healthy and diseased mice.

Results:
The transgene did not alter heart function per se. We performed FRET measurements in freshly isolated cardiomyocytes to compare T-tubular with cytosolic cAMP responses and their regulation by beta1- and beta2-adrenergic receptors (β-ARs) and phosphodiesterase (PDE) isoforms. As expected, T-tubular β2-AR signals were higher than cytosolic ones (>2-fold). Comparison of PDE activity profiles between the two compartments showed marked differences with regard to the β2-cAMP signal. In particular, T-tubular β2-cAMP is mainly controlled by PDE3. Strikingly, in hypertrophied myocytes (8 weeks post-TAC) we could detect a switch from PDE3 to PDE2-dependent regulation of these cAMP signals.

Conclusions:
We could establish transgenic mice expressing a FRET-biosensor to measure local cAMP levels in the T-tubular compartment and uncover its differential regulation by β1- and β2-ARs and several PDE families, especially by PDE3. The switch from PDE3 to PDE2 regulation of β2-cAMP in diseased cardiomyocytes could represent a novel mechanism of local cAMP signaling and cGMP/cAMP cross-talk in cardiomyocytes.
Abstract 196: Adult-onset Cardiac Hypertrophy In LEOPARD Syndrome Is Caused By Both Cell Autonomous And Non-autonomous Effects That Occur During Development
Authors: Lauriol Jessica , Keith Kimberly , Zhang Boding , Bronson Roderick , Lee Kyu-Ho , Kontaridis Maria I
Published: July 2021

Loss of function mutations in
PTPN11
, the gene encoding the protein tyrosine phosphatase SHP2, cause LEOPARD Syndrome (LS), an autosomal dominant disorder with multiple cardiac defects, including hypertrophy. However, the mechanisms by which the LS mutants cause cardiac disease and regulate development remain unclear. Here, we utilized our inducible LS mouse model to determine whether LS mutations affect onset of cardiac hypertrophy via aberrant regulation of developmental processes. We examined hearts from wildtype (WT) or ubiquitously-expressing heterozygous LS (LS/+) and homozygous LS (LS/LS) embryos. As compared to WT, both LS/+ and LS/LS developing hearts showed diminished trabeculation, valvular hyperplasia and ventricular septal defects. In addition, LS/LS embryos had dextraposition of the aorta, leading to embryonic lethality. Gene expression analysis revealed that LS mutant hearts had decreased Foxp1/Fgf and Notch1/EphB2 signaling, suggesting perturbation of the reciprocal cross-talk between the developing endocardium and myocardium

To assess this directly and to determine the
in-vivo
effects of lineage-specific LS expression during development, we generated, endothelial (Tie2)-, myocardial (Nkx2.5)- and neural crest (Wnt1)- specific LS/+ expressing mice. Surprisingly, the majority of LS cardiac defects, including the adult-onset cardiac hypertrophy, were recapitulated by expression of endocardial-specific LS alone, suggesting a functional necessity for the endocardium in hypertrophy of the heart. Moreover, though myocardial-specific expression of LS showed only minor developmental effects, these mice still recapitulated the adult-onset cardiac hypertrophy. Interestingly, no cardiac abnormalities were observed in the neural crest-specific expressing LS mice. Taken together, our data indicate that the LS mutations affect both cell autonomous and non-autonomous pathways necessary for the reciprocal cross-talk between endocardium and myocardium, and, importantly, that the adult-onset hypertrophy in LS is caused by aberrant signaling effects that occur during cardiac development.
Abstract 197: Overexpression of the Na+/K+ ATPase a2 But Not a1 Isoform Attenuates Pathological Cardiac Hypertrophy and Remodeling
Authors: Correll Robert N , Eder Petra , Burr Adam R , Despa Sanda , Davis Jennifer , Bers Donald M , Molkentin Jeffery D
Published: July 2021

The Na+/K+ ATPase (NKA) directly regulates intracellular Na+ levels, which indirectly regulate Ca2+ levels by controlling flux through the Na+/Ca2+ exchanger (NCX1). Elevated Na+ levels have been reported during heart failure, which permits some degree of reverse mode Ca2+ entry through NCX1 and less efficient Ca2+ clearance. To determine if lower intracellular Na+ levels would enhance forward-mode Ca2+ clearance and prevent reverse-mode Ca2+ entry through NCX1 as a protective measure, we generated cardiac-specific transgenic mice overexpressing either the NKA-α1 or α2 isoform and subjected them to pressure overload hypertrophic stimulation. We found that while increased expression of the NKA-α1 isoform had no protective effect, overexpression of NKA-α2 significantly decreased cardiac hypertrophy after pressure overload at 2, 10 and 16 weeks of stimulation. Remarkably, total NKA protein expression was not altered in either of these 2 transgenic models, as increased expression of one isoform led to a concomitant decrease in the other endogenous isoform. While total NKA ATPase activity and intracellular Na+ levels were unchanged in either overexpression model, and both showed reduced Ca2+ transient amplitudes and sarcoplasmic reticulum Ca2+ load, only NKA-α2 overexpression led to faster removal of bulk Ca2+ from the cytosol in a manner requiring NCX1 activity. This increased NCX1 activity, though correlated with improved outcome after pressure overload, did not affect signaling through Ca2+-sensitive signaling pathways such as calcineurin/nuclear factor of activated T-cells, Ca2+/calmodulin-dependent kinase II, or protein kinase Cα. Overexpression of NKA-α2 did, however, result in reduced expression of phospholemman (PLM), an inhibitor of NKA activity (when dephosphorylated) and NCX1 activity (when phosphorylated). Our results suggest that the protective effect produced by increased expression of NKA-α2 after pressure overload is likely due to: 1) Na+ regulation in a unique signaling microdomain distinct from NKA-α1, and 2) downregulation of PLM expression that removes a negative regulator of NCX1 activity, both leading to preservation of forward-mode NCX1 activity during disease, in association with optimized cardiac function.
Abstract 200: Cardiomyocyte-specific Deletion Of Rhoa Accelerates Heart Failure But Prevents Cardiac Fibrosis In Response To Pressure-overload
Authors: Lauriol Jessica , Keith Kimberly , Goonasekera Sanjeewa A , Ke Qingen , Kang Peter , Molkentin Jeffery D , Kontaridis Maria I
Published: July 2021

Heart failure is a progressive disease characterized by loss of cardiac contractility, interstitial fibrosis, and chamber remodeling. Though several signaling pathways have been identified, the precise mechanisms involved in regulating pathological cardiac responses remain unclear. The Ras-related small G protein RhoA is an identified mediator of in vivo pathological hypertrophy. However, it also promotes cell survival and is cardioprotective after ischemia/repurfusion injury. Therefore, to determine the molecular mechanisms that underlie these opposing roles for RhoA in the myocardium, we generated mice with cardiomyocyte-specific deletion of RhoA (RhoAfl/fl-αMHC-Cre). As compared to wildtype mice, hearts from RhoAfl/fl-αMHC-Cre mice showed similar functional, structural, and growth parameters, both at baseline and in response to acute transverse aortic constriction (TAC) (2wks), suggesting RhoA plays no major role in either homeostatic or acute stress-induced cardiac conditions. However, after exposure to chronic TAC for 8wks, hearts from RhoAfl/fl-αMHC-Cre mice developed an accelerated dilation, with significant loss of contractile function. Mechanistically, cardiomyocyte-specific loss of RhoA reduced ERK1/2 activation and perturbed agonist-evoked calcium cycling, through aberrant IP3 and DAG signaling, indicating that RhoA mediates its cardioprotective effects through regulation of these pathways. Despite this, and surprisingly, hearts from RhoAfl/fl-αMHC mice also showed significantly decreased TAC-induced cardiac fibrosis, with a demonstrated decrease in transcriptional activation of genes involved in the fibrotic response, including the serum response factor (SRF), as well as the secreted factor BMP7. Taken together, our data reveal an interesting, yet highly complex signaling and regulatory mechanism for RhoA, whereby RhoA signaling is both cardioprotective against development of heart failure in response to chronic stress, but also cardio-deleterious through its capacity to activate pro-fibrotic genes.
Abstract 202: Aging Alters the Electrical and Mechanical properties of LV Myocytes
Authors: Sorrentino Andrea , Signore Sergio , Sundman Mark , Kannappan Ramaswamy , Mangiaracina Chiara , Cannata Antonio , Webster Andrew , Isobe Kazuya , Shafaie Mehrdad , Kim Junghyun , Wybieralska Ewa , Matsuda Alex , Borghetti Giulia , Goichberg Polina , Rota Marcello
Published: July 2021

Aging results in delays in the electrical recovery of the heart, enhancing the risk of malignant ventricular arrhythmias and sudden death. But whether altered electrical properties affect myocardial mechanical performance remains unclear. The aim of this study was to establish the ionic basis for the protracted repolarization of the senescent heart, and to determine whether these electrical changes impact on myocardial contractility. For this purpose, electrophysiological analyses were conducted in vivo and in isolated LV myocyte preparations from mice ranging from 3 to 30 months of age. Electrocardiographic parameters were preserved from 3 to 12 months of age, whereas animals 2 years or older presented prolonged PR, QRS and QT intervals. These in vivo results were confirmed in the isolated organ, using Langendorff perfused hearts. Myocytes from 30 months old mice showed longer early and late repolarization phases of the action potential (AP), in comparison with cells from animals at 3 months. By voltage-clamp, the density of the transient outward K+ current (Ito) was significantly reduced in old, whereas the late Na+ current (INaL) was increased, changes consistent with the prolonged AP of old cells. Additionally, by Western blotting, the SCN1B subunit, involved in Na+ channel gating, was reduced with age. Using field stimulation, old myocytes presented slower Ca2+ transient decay and relaxation, in comparison to young. Blockade of INaL with mexiletine in old myocytes shortened the AP duration, and fastened the decay of Ca2+ transients and relaxation. To assess the functional impact of INaL on the myocardium, papillary muscles were studied in an isometric system. Using tension-length protocols, old muscles presented elevated diastolic tension with respect to young. Importantly, inhibition of INaL in old muscles reduced diastolic tension, and attenuated the active developed force. Conversely, increasing INaL in the myocardium of young mice with anemonetoxin had opposite effects. Overall, these data indicate that aging results in a prolongation of the AP mediated, at least in part, by enhanced INaL. The prolonged repolarization of the AP contributes to the slower clearance of diastolic Ca2+ from the cytoplasm, resulting in protracted relaxation phase.
Abstract 204: Loss Of Akt Caused Aging-induced Cardiac Systolic Dysfunction But Is Independent From Comorbid Diastolic Dysfunction In Aging.
Authors: Aoyama Morihiko , Bando Yasuko K , Monji Akio , Mitusi Toko , Kawase Haruya , Murohara Toyoaki
Published: July 2021

PURPOSE:
Aging is one of the primary factors causing cardiac dysfunction. Diastolic dysfunction (DD) is primary characteristics for aging-induced heart failure. Clinical evidences demonstrate a sustained exercise (EX) ameliorates DD; however, it remains unclear whether EX may ameliorate the aging-related DD and the underlying molecular mechanism. We thus evaluated whether EX may ameliorate cardiac dysfunction in aging and the role of Akt in the EX-induced effects on aging heart.

Methods:
Male senescence-accelerated (SAM) mice (P10) and its aging-resistant control (R1) were allocated to exercise [EX; 60-min running on treadmill every single day for 6 months (P10-EX and R1-EX)] and exercise-free control groups (P10-ctl and R1-ctl). Age-matched C57BL6 mice were subjected to the same EX protocol (C57-EX and C57-ctl) to compare any influence of genetic background. To elucidate the role of Akt in aging-induced changes in heart, analysis of aged Akt knockout mice (AktKO) were conducted.

Results:
At baseline, cardiac geometry of R1 strain revealed normal, whereas P10 strain exhibited reduced LV wall thickness and DD. The s-LVF of both strains was preserved. After EX, the body and heart weight of R1 mice were increased (BW; +6.7% and HW; +3.5% versus R1-ctl); however, EX had no influence on BW and HW of P10. EX promoted LV hypertrophy in R1, which was absent in P10-EX. The d-LVF of R1-EX was impaired but their s-LVF was unchanged. In contrast, s-LVF of P10-EX turned impaired [EF(%) 68.9±1.5 vs 74.3±1.2 for P10-ctl], whereas the underlying DD of P10 was unaffected by EX. In C57-EX, cardiac function exhibited the similar trends observed in R1-EX. Cardiac Akt activity of R1-EX and C57-EX were enhanced compared to controls, which was diminished in P10-EX. Aged AktKO exhibited impaired s-LVF [EF(%)61.3±1.0], nonetheless their DD remained unchanged [E/A=2.5±0.3, Dct (msec)= 35.0±2.4].

Conclusions:
Our study demonstrates that #1 Akt is essential for adaptive response of LV hypertrophy to EX, #2 aging impairs Akt signaling in heart, leading to systolic dysfunction, and #3 Akt is independent from modulation of cardiac DD induced by aging. Clinical implications are drawn that the benefit of EX on DD may be irrelevant to the aged population.
Abstract 207: Mitochondrial Calcium Flux is Arrhythmogenic in Nonischemic Cardiomyopathy
Authors: xie an , Wang Qiongyin , Liu Hong , Gu Lianzhi , Liu Man , Rutledge Cody , Dudley Samuel C
Published: July 2021

Introduction:
Mitochondria participate in Ca2+ homeostasis and Ca2+ oscillations are thought to be involved in arrhythmogenesis. Nevertheless, the role of mitochondria in arrhythmogenesis is unclear. We have reported spontaneous and induced arrhythmias in a mouse model of nonischemic cardiomyopathy, so we investigated the role of mitochondrial Ca2+ handling in arrhythmias in this model.

Methods:
Nonischemic cardiomyopathy was induced in C57BL/6 mice by 6 weeks of hypertension evoked after unilateral nephrectomy, deoxycorticosterone acetate (DOCA) pellet implantation, and 1% salt water substitution. Sham operated mice were used as controls. ECG telemetry recording was used to monitor arrhythmias from DOCA mice and 7 sham mice at 18 weeks of age. Action potentials (APs) were recorded by perforated current-clamp in isolated mouse ventricular cells. Potassium and L-type Ca2+ currents were measured by voltage-clamp. Changes in cytoplasmic and mitochondrial Ca2+ were determined by fluorescent imaging using Fluo-4 and Rhod-2, respectively, in isolated ventricular cells.

Results:
Seven DOCA mice and two sham mice had isoproterenol (0.5 mg/kg)-induced premature ventricular contractions (PVCs, p<0.05). Three DOCA mice and no sham mice had isoproterenol (2.5 mg/kg)-induced ventricular fibrillation. QTc was significantly prolonged from 41.9 ±1.4 in sham mice to 52.0 ± 2.1 ms in DOCA mice. At the cellular levels, myocytes had a prominent increase of APD90 from 66.6 ± 21.1 to 311.8 ± 44.9 ms, explained by augmented L-type Ca2+ current and decreased total K+ currents. Two of 12 sham mice ventricular cells and 8 from 12 DOCA mice ventricular cells had EADs (p<0.05). Three of 12 DOCA mice but none of the sham myocytes had PVCs with stimulated APs. Trigger activity could be abolished by 10 µM Ru360, a mitochondrial calcium uniporter specific antagonist. Ru360 had no effect on L-type Ca2+ current, total K+ currents, or APD90. Ru360 inhibited mitochondrial Ca2+ uptake in DOCA mice ventricular cells, implicating mitochondrial Ca2+ release in modulating the arrhythmic risk.

Conclusions:
Mitochondrial Ca2+ handling appears to play an important role in triggered activity and may be important in the incidence of ventricular arrhythmias in nonischemic cardiomyopathy.
Abstract 210: Thioredoxin-2 Inhibits Mitochondrial Ros Generation And Ask1 Activity To Maintain Cardiac Function
Authors: Min Wang , Huang Qunhua
Published: July 2021

Background:
Increasing evidence indicates that mitochondrial-derived reactive oxygen species (ROS) and cellular apoptosis contribute to the pathogenesis of cardiac dysfunction. Mitochondrial thioredoxin (Trx2) is a key protein regulating cellular redox and survival, However, but its role in normal cardiac growth has not been determined.

Methods and Results:
We have generated cardiac-specific Trx2 knockout mice (Trx2-cKO) to determine the physiological importance of the Trx2 system in the heart. Trx2-cKO mice developed a spontaneous dilated cardiomyopathy at 1 month of age with increased heart size, fibrosis, reduced ventricular wall thickness, and progressive contractile dysfunction, resulting in death due to heart failure by 4 months of age. Cardiac changes in Trx2-cKO mice were accompanied by disruption of mitochondrial integrity and function, as evident by alterations in mitochondrial number, ultrastructure, membrane potential and ATP production. Increases in ASK1 signaling and ROS production preceded mitochondrial damage, cellular apoptosis and contractile dysfunction in both Trx2-cKO hearts and isolated cardiomyocytes. Moreover, deletion of ASK1 attenuates ROS production, mitochondrial dysfunction and cellular apoptosis in Trx2-deficient cardiomyocytes. These data indicate that ASK1 is a major target of Trx2 and that activation of ASK1 is causally associated with mitochondrial dysfunction, ROS production and cellular apoptosis. We also detected reduced Trx2 expression and increased ASK1 activity in human hearts from patients with cardiomyopathy, suggesting that this mechanism is clinically important.

Conclusion:
Our data support an essential role for mitochondrial Trx2 in preserving cardiac function by suppressing mitochondrial ROS production and ASK1-dependent apoptosis. These results suggest that pharmacological inhibition of ASK1 may provide a therapeutic strategy for the treatment of dilated cardiomyopathy and heart failure.
Abstract 212: Cd4+ T-cell Activation By T-cell Receptor Engagement Contributes To Myocardial Ischemia-reperfusion Injury
Authors: Hofmann Ulrich , Mathes Denise , Weirather Johannes , Beyersdorf Niklas , Kerkau Thomas , Frantz Stefan
Published: July 2021

Background:
We have recently shown that CD4
+
but not CD8
+
T-cells contribute to ischemia-reperfusion injury of the myocardium. We therefore hypothesized that CD4
+
T-cells become activated by autoantigen recognition via their T-cell receptor during reperfusion.

Methods and Results:
Infarct size as percent of the area-at-risk was determined by combined Evans` blue and triphenyltetrazolium (TTC) staining after 30 minutes of in vivo ischemia followed by 24 hours reperfusion in mice. After 24 hours of reperfusion there was a significantly increased population of CD4
+
T-cells which expressed the surface protein CD40L in comparison to sham operated mice [n≥7; p<0.05; WT 10.8 ± 0.2% vs. sham 6.4 ± 0.5%]. CD40L is typically expressed in T-cells activated by T-cell receptor engagement. OT-II mice carry a transgenic T-cell receptor with specificity for an ovalbumin-derived peptide. These mice have a limited T-cell receptor repertoire, dominated by specificity for the irrelevant antigen ovalbumin. After 30 minutes of ischemia and 24 hours of reperfusion OT-II mice showed significantly reduction in infarct size [n≥4; p= 0.02; infarct/area at risk: OTII mice 38.9 ± 2.4% vs. WT mice 63.7 ± 6.6 % ]. Administration of a CD40L blocking antibody to wildtype mice also reduced infarct size when compared to administration of isotype-matched antibodies [n≥6; p = 0.03; infarct/ area at risk: anti-CD154 treatment 60.4 ± 3.4% vs. control 75.3 ± 4.1%]. CD4
+
CD25
+
Foxp3
+
T-cells (natural T-regulatory cells) have a low activation threshold and constitute a T-cell subset with reactivity against autoantigens. Depletion of these cells by diphtheria-toxin application in a mouse model expressing the diphtheria-toxin receptor under the Foxp3 promotor also resulted in a significant reduction of infarct size when compared to diphtheria-toxin treated wildtype mice [n≥4; p=0.03; infarct/ area at risk: T
reg
-depleted DEREG mice 51.9± 3% vs. WT littermates 72.3± 2%].

Conclusion:
Our results indicate that CD4
+
T-cells that have been activated by an MHC class II/ T-cell receptor dependent mechanism, presumably by autoantigen recognition, contribute to myocardial ischemia-reperfusion injury.
Abstract 214: Role Of Sirtuin 6 In Macrophage Polarization In Diabetes
Authors: Velmurugan G V , Verma Suresh K , Mackie Alexander R , Vaughan Erin E , kishore Raj , Krishnamurthy Prasanna
Published: July 2021

Clinical and experimental studies provide evidence that metabolic and inflammatory pathways are functionally interconnected to cardiovascular diseases. Dynamic changes in macrophage activation [classical M1 activation (promote inflammation) or alternative M2 activation (promote wound healing)], in response to various signals, modulate tissue pathology in diabetes. Sirtuin 6 (SIRT6), a NAD-dependent nuclear deacetylase plays an important role in genomic stability, cellular metabolism, stress response and aging. However, the mechanism by which SIRT6 activity affects macrophage function in diabetes is still unclear. Mouse bone marrow-derived macrophages (BMM) exposed to high glucose (HG, 25mM D-glucose) showed reduced expression of SIRT6 as compared to low glucose (LG, 5mM D-glucose)- and osmotic control (OC, 5mM D-glucose+20mM D-mannitol)-treated cells. SIRT6 knockdown in RAW 264.7 cells exaggerated inflammatory response in macrophages exposed to high glucose (HG) and in contrast, IL4-induced alternative macrophage (M2) phenotype was defective in SIRT6 deficient BM-macrophages under high glucose condition. SIRT6 protein expression was low in failing (MI-induced) and diabetes-affected hearts. Interestingly, mice receiving intramyocardial injection of SIRT6-deficient macrophages showed further deterioration in LV function, post-MI. Taken together, these data highlighting a role for SIRT6 in regulating both M1 and M2 polarization might have broad implications for numerous inflammatory disease states including insulin resistance and wound healing.
Abstract 218: Research On Myocardium Proteome Of Chinese mini-pigs With Myocardial Ischemia
Authors: Chuo Wenjing , Guo Shuzhen , Wang Yong , Li Chun , Lian Hongjian , Feng Xuanchao , Wang Wei
Published: July 2021

Objective:
To establish a stable myocardial ischemia animal model, and to find the characteristics of its myocardium proteome and also to seek the biomarkers for myocardial ischemia.

Methods:
Establishment and assessment of a stable model with myocardial ischemia Chinese experimental mini pigs (25±4 kg) were instrumented with a size-matched Ameroid constrictor on the anterior descending branch under general anesthesia in sterile conditions. The sham group didn’t place the Ameroid constrictor, the other were the same to the model group. After the operations, we dynamically observed the pathophysiology indicators from the two group animals for 12 weeks. Myocardium samples were obtained from the animals with myocardial ischemia and the sham operated animals after operation 12 weeks. Myocardium samples were separated by two-dimensional electrophoresis (2-DE) to identify differentially expressed proteins. A cluster of protein spots differentially expressed were selected and identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS).

Results:
Compared with the sham group, the differential expressed proteins in the model group 5 up-regulated protein spots and 3 down-regulated protein spots were detected in the 2-DE separation of pooled samples. Albumin, aldose reductase, troponin T, isocitrate dehydrogenase subunit alpha, myosin light chain 3 were significantly highly expressed in the myocardium samples, while NADH-ubiquinone oxidoreductase, ropomyosin alpha-1 chain, Hsp27 were decreased in the myocardium proteome.

Conclusion:
The detected differential expressed myocardium proteins of animals with myocardial ischemia were associated with the related proteins of energy metabolism obstruction, cytoskeleton proteins, cell damaged and apoptosis - related proteins. Thus, we supposed that energy metabolism obstruction may be the most important changes of physiological and biochemical process in myocardial ischemia, accompanied by cytoskeletal damage, apoptosis and a series of molecular level changes.
Abstract 219: The Formin mDia1 Is A Novel Mediator Of Injury In Ischemia/Reperfusion
Authors: O’Shea Karen M , Ananthakrishnan Radha , Li Qing , Quadri Nosirudeen , Wang Lingjie , Zirpoli Hylde , Schmidt Ann Marie , Ramasamy Ravichandran
Published: July 2021

mDia1, a member of the formin family and an effector of Rho GTPases, has emerged as a downstream effector of RAGE signaling. RAGE has been shown to stimulate the deleterious effects of ischemia/reperfusion injury. The goal of this study was to elucidate the role of mDia1 in the heart after ischemia/reperfusion injury. In mouse hearts subjected to left anterior coronary artery occlusion (LAD) for 30 minutes followed by 48 hours of reperfusion, there is a ten-fold increase in mDia1 protein expression compared to sham. We thus tested the effects of deletion of mDia1; adult male mDia1-/- mice and wild type littermates (WT) were subjected to LAD or sham surgery. Infarct size in mDia1-/- mice was significantly reduced by 15% compared to WT after LAD (32.6 vs. 27.6% infarct area, respectively; p=0.008). LV function after LAD was improved in mDia-/- mice, as evidenced by a significant increase in fractional shortening compared to WT (34.6 vs. 27.1%; p=0.005). ChIP studies of cardiac tissue post-LAD revealed that there was an interaction between mDia1 protein and Egr1, Serca2a, and SRF genes in WT mice subjected to LAD but not in sham mice. Furthermore, EGR1 protein levels were reduced in the nuclear fraction of mDia1-/- mouse hearts after LAD compared to WT and SERCA2A protein levels were elevated in mDia1-/- mice after LAD compared to WT. These effects were confirmed in mDia1-expressing AC16 human ventricular myocytes subjected to hypoxia for 30 minutes followed by reoxygenation for 60 minutes (H/R). Compared to normoxia, mDia1 protein was increased in H/R cells by three-fold, and there was an interaction between mDia1 protein and genes for Egr1 and Serca2a in the H/R cells but not in normoxic cells. mDia1-/- and WT hearts were also isolated and subjected to 30 minutes of global ischemia followed by 60 minutes of reperfusion. In mDia1-/- hearts, there was improved LVDP recovery compared to WT (63.3 vs. 33.7%, respectively). Taken together, these data suggest that mDia1 deletion mediates cardioprotection in ischemia/reperfusion injury through downstream interactions with SRF, SERCA2A, and EGR1.
Abstract 223: Histone Deacetylase Inhibition Mediated Macrophage Polarization Suppresses Matrix Metalloproteinase-9 Upregulation Post Myocardial Infarction
Authors: Kimbrough Denise M , Mani Santhosh K , Kern Chistine B , Kasiganesan Harinath , Menick Donald R
Published: July 2021

Background:
Following a myocardial infarction (MI) the extracellular matrix (ECM) undergoes massive remodeling to prevent rupture and maintain cardiac output. Large increases in matrix metalloproteinase-9 (MMP-9) are associated with adverse ECM remodeling. We found that treatment with an HDAC inhibitor repressed post-MI upregulation of MMP-9. Significant sources of MMP-9 in the post-MI LV are M1 macrophages. Both phenotypes (M1 and M2) can contribute to MMP expression, but this is dependant on the phase of ECM remodeling.
We hypothesize that HDAC inhibition regulates the post-MI expression of MMP-9 by mediating the M1 to M2 macrophage polarization.

Methods:
CD1 and MMP-9 β-gal reporter mice were induced with MI by LAD ligation then administered HDAC inhibitors: trichostatin A (TSA; class I and IIb), PD106 (class I), or Tubastatin A (HDAC 6) until termination at 5 or 7 days post-MI. Heart function evaluated by echocardiogram and cells or tissue by immunohistochemistry and immunoblotting.

Results:
The post-MI change in LV end-diastolic volume (49±9%) is significantly lower and ejection fraction (-44±8%) is improved with treatment of TSA vs. control (69±12%; -59±6%) respectively [n=28]. Immunohistochemical analysis revealed that infiltrating macrophages express MMP-9 at 5 and 7 days post-MI. HDAC inhibition decreases this expression and did so without reducing presence of macrophages within infarct. Immunoblotting shows that expression of all class I HDACs are increased following MI; however, in cultured macrophages only HDAC2 and HDAC3 are increased. TSA and PD106, inhibit control levels and lipopolysacharide (LPS) stimulated upregulation of MMP-9 in cultured RAW264.7 and bone marrow derived macrophages. Immunofluorescence revealed that treatment with PD106, Tub A, and TSA leads to M1 to M2 morphology specific polarization and maintenance of anti-inflammatory, M2, phenotype even with LPS stimulation in culture. However, only PD106 and TSA reduced MMP-9 expression in cultured macrophages.

Conclusions:
Macrophage mediated secretion of MMP-9 contributes to adverse ECM remodeling and loss of LV function post-MI. Class I HDAC inhibition promotes both M2 macrophage polarization and attenuates adverse remodeling by reducing MMP-9 expression.
Abstract 224: Mitochondrial Complex I Deficiency Promotes Oxidative Stress During Ischemia Reperfusion Of Cardiac Myocytes
Authors: Gong Guohua , Karamanlidis Georgios , Lee Chi Fung , Tian Rong , Wang Wang
Published: July 2021

Normal cardiac function relies on highly coordinated intracellular events, such as calcium cycling and contraction, with adequate mitochondrial energy metabolism. However, mitochondrial respiration unavoidably produces reactive oxygen species (ROS) as electrons leak from the electron transport chain (ETC). Complex I of the ETC is believed to be the major site for ROS generation in the mitochondria. However, suppression of Complex I activity by chemical inhibitors leads to oxidative cell damage. In this study, we used a genetic model of Complex I deficiency, in which a key component of Complex I, Ndufs4, was deleted in the heart, to determine the causal role of Complex I in ischemia-reperfusion-induced oxidative stress in adult cardiac myocytes. Germline deletion of Ndufs4 in the heart (Ndufs4H-/-) leads to a ~75% decline of Complex I activity in cardiac mitochondria without obvious disease phenotype in the mice. As predicted, the mitochondrial respiration-coupled superoxide production events, superoxide flashes, were significantly decreased at baseline in the Ndufs4H-/- myocytes. Respiration substrate (pyruvate, 20 mM) failed to stimulate mitochondrial superoxide flash production in Ndufs4H-/- myocytes. This is accompanied by the slightly decreased steady state intracellular and mitochondrial ROS levels determined by the targeted H2O2 indicator, Hyper. The intracellular redox homeostasis is also tilted toward more reduced state, since the NADH/NAD ratio increased 67%. Surprisingly, ischemia reperfusion mimetic treatment of the myocytes caused dramatic increase in mitochondrial ROS production in Ndufs4H-/- groups, which contributed to the elevated overall cellular oxidative status. Overexpression of catalase in the mitochondria prevented these effects. Mechanistically, increased reducing equivalent (NADH) contributed to the dramatic ROS production during ischemia and reperfusion in Ndufs4H-/- myocytes. In summary, mitochondrial Complex I plays a critical role in controlling mitochondrial and cytosolic ROS homeostasis under normal conditions, and compromised Complex I function leads to accumulation of electron donors that paradoxically promote ROS production during ischemia reperfusion.
Abstract 226: Human Cardiomyocytes Are Protected From Stress-induced Stiffening By Binding Of Small Heat Shock Proteins To Titin Spring Elements
Authors: Kotter Sebastian , Unger Andreas , Hamdani Nazha , Linke Wolfgang A
Published: July 2021

Introduction:
Small heat-shock proteins (sHSPs) generally participate in cellular protein quality-control mechanisms. They are abundant in cardiomyocytes where they bind under diverse stress conditions preferentially to myofilament proteins. The functional role of this association is unclear.

Hypothesis:
HSP27 and αB-crystallin bind to human cardiac titin spring elements and exert a protective effect on cardiomyocyte passive stiffness under stress conditions.

Methods:
Binding of sHSPs to recombinant human titin constructs was characterized by GST-pulldown assay and confirmed by immunofluorescence staining of human donor and failing (DCM) cardiomyocytes. Sedimentation-velocity centrifugation, photometric and chromatographic methods were used to test for titin aggregation and protection from it by sHSPs. Passive force was measured in isolated human cardiomyocytes or single myofibrils, in search for a possible protective effect of sHSPs on mechanical function.

Results:
HSP27 interacted with distinct domains of the human titin-spring region
in vitro
and in cardiomyocytes, and independent of the presence of actin filaments in the sarcomeres. The binding sites on the elastic titin segment resemble those for αB-crystallin and include proximal Ig-domains, the N2-B and N2-A regions, but not the PEVK-domain.
In-vitro
assays revealed a monomeric organization of these titin-spring elements; however, unfolded N2-A domain (mainly composed of Ig-domains) aggregated in pH-6.6 buffer but not in normal-pH buffer, whereas αB-crystallin protected from this effect. The intrinsically disordered N2-Bus titin domain did not aggregate. Single skinned human cardiomyocytes showed greatly increased passive stiffness when pre-stretched under acidic stress (pH 6.6), but αB-crystallin or HSP27 corrected the stiffening. In failing patient heart tissue, both HSP27 and αB-crystallin frequently associated with the elastic I-band region, in contrast to a cytosolic and Z-disk location of these sHSPs in donor heart.

Conclusions:
In cardiomyocytes sHSPs bind to mechanically active titin domains under stress conditions such as intracellular acidosis (
e.g.
, ischemia), protecting the titin springs from aggregation and helping reverse diastolic stiffening.
Abstract 227: Phosphorylation Patterning Determined by AMP-Activated Kinase, the LKB1/MO25/STRAD Complex, and Protein Phosphatase 1 Alters Contractile Function in Cardiac Rat Trabeculae
Authors: Behunin Samantha , Konhilas John P
Published: July 2021

Post-translational modifications (PTM) of myofilament proteins alter contractile function of the heart in healthy as well as diseased myocardium and the patterning of PTMs can influence cardiac disease progression. PTM of the thin filament regulatory protein cardiac troponin I (cTnI) is known to modify contractile properties, including steady-state Ca2+ sensitivity of force and crossbridge cycling rates. Accordingly, the purpose of this study was to determine the effect of cTnI PTM patterning on myofilament function. Therefore, I hypothesize that the impact of cTnI PTM on contractile function will depend on the relative phosphorylation levels. To do this, demembranated rat cardiac trabeculae from 2 month-old male Sprague-Dawley rats were treated with AMP activated kinase (AMPK) (0.005 U/ µL), Protein Phosphatase 1 (PP1) (1U/µL), and the upstream AMPK kinase, the LKB1/MO25/STRAD complex (0.2 mU/µL). Fibers that were incubated with activated AMPK displayed an increase in Ca2+ sensitivity compared to untreated control fibers (EC50 1.41±0.08 μM [n=2] vs. 2.52±0.43 μM [n=9] p<0.001). PP1 treatment, previously shown to decrease cTnI phosphorylation, tended to increase Ca2+ sensitivity compared to untreated fibers (EC50 2.31 μM [n=1] vs. 2.52±0.43 μM [n=9]). Interestingly, PP1 treatment also increased passive tension generation by 27% compared to control fibers (P<0.001 [n=3]). Surprisingly, the LKB1/MO25/STRAD complex decreased overall tension development (14.18±2.27 mN/mm2 [n=2] vs 37.03 ± 16.72 mN/mm2 [n=9] p=0.002), and desensitized the myofilament to Ca2+ (EC50 4.18 ±0.0001 µM [n=2] vs 2.52±0.43 μM [n=9] p<0.001). In conclusion, I have shown that the functional outcomes are determined by the differential PTM patterning of cTnI. Furthermore, we have identified the LKB1/MO25/STRAD complex as a potential novel regulator of myofilament function.
Abstract 235: Global RNA Splicing and Regulation in Cardiac Maturation and Diseases
Authors: GAO CHEN , Ren Vincent , Lee Jae-Hyung , Xiao Xinshu (Grace) , Chen Jau-nian , Wang Yibin
Published: July 2021

Background:
The complexity of transcriptome and proteome is contributed by alternative splicing of mRNA. Altered mRNA splicing is also implicated in many human diseases including cancer. However, the global pattern of alternative mRNA splicing during cardiac development and diseases is unknown, and the regulatory mechanisms remain unexplored.

Methods and Results:
Using deep RNA-Sequencing, we have identified global alternative splicing changes associated with both cardiac development and pathological remodeling in mouse heart following pressure-overload induced heart failure. The alternative RNA splicing events observed in failing hearts mimics the profile in fetal hearts, suggesting a fetal-like RNA splicing program induced in diseased hearts. Using RNA-Seq database and real-time PCR analysis, we examined the expression profile of a large number of known alternative splicing regulators. Among them, we identified Fox1 as a significantly induced regulator during cardiac development in zebrafish, mouse and human, and down-regulated in both mouse and human failing hearts. Morpholino mediated Fox1 knockdown in zebrafish embryos led to lethal phenotype associated with reduced cardiac function and defects in chamber specificity. This phenotype could be rescued by re-expressing both zebrafish and mouse Fox1 gene, suggesting a highly conserved cardiac function of Fox1 for normal cardiac development and function in vertebrates.

Conclusion:
Our study provided the first comprehensive analysis of mRNA splicing regulation in heart during post-natal development and heart failure, and identified Fox1 as a key regulator for alternative RNA splicing in heart. This study expands our current understanding to the complexity of cardiac transcriptome, and reveals the functional importance of RNA-splicing in cardiac development and diseases.
Abstract 237: Sex-specific Regulation Of Collagen I And III Expression By 17β-estradiol In Rat Cardiac Fibroblasts: Role Of Estrogen Receptors
Authors: Dworatzek Elke , Mahmoodzadeh Shokoufeh , Kunze Sandra , Regitz-Zagrosek Vera
Published: July 2021

Clinical and animal studies showed in female pressure-overloaded hearts less cardiac fibrosis and collagen I and III gene expression compared to males, suggesting an inhibitory effect of 17β-Estradiol (E2) on collagens. Therefore we investigated the role of E2 and estrogen receptors (ER) on collagen I and III expression in isolated rat cardiac fibroblasts from both sexes.

Cardiac fibroblasts were isolated from adult male and female Wistar rats, and treated with E2 (10-8M), vehicle, ERα and ERβ-agonist (10-7M) and/or pre-treated with ICI 182,780 (10-5M) for 24h. Cellular localization of ER in cardiac fibroblasts with/without E2 was detected by immunofluorescence staining, and expression of both ER was determined by western blot. Expression of collagen I and III was determined by qRT-PCR and western blot.

E2-treatment led to a nuclear translocation of ERα and ERβ in cardiac fibroblasts, suggesting the functional activity of ER as transcription factors. Furthermore in cardiac fibroblasts from female rats E2 led to a significant down-regulation of collagen I and III gene and protein expression. In contrast there was a significant increase of collagen I and III levels in fibroblasts isolated from male rat hearts by E2. E2-effect could be inhibited by ICI 182, 780 indicating the involvement of ER. In cardiac fibroblasts from female rats, ERα-agonist treatment led to a significant down-regulation of collagen I and III mRNA level, but ERβ-agonist had no effects. In contrast, ERβ-agonist treatment of cardiac fibroblasts from males increased collagen I and III mRNA, but no changes with ERα agonist-treatment were detected. ERα protein levels displayed no sex differences at basal level. After E2-treatment ERα protein was up-regulated in male cells, but decreased in cardiac fibroblasts from females. ERβ protein was higher in female cells compared to males, but the expression was not regulated by E2 in both sexes.

Sex-specific regulation of collagen I and III expression by E2 in cardiac fibroblasts might be responsible for sex-differences in cardiac fibrosis. This might be due to sexually dimorphic ER expression and regulation. Understanding how E2 and ER mediate sex-differences in cardiac remodeling may help to design sex-specific pharmacological interventions.