bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 382: p300 Mediated Acetylation of the Cardiac Calcium Pump Reduces Its Activity and Contributes to Cardiac Dysfunction
Authors: Gorski Przemyslaw A , Lee Ahyoung , Oh Jaegyun , Hajjar Roger J , Kho Changwon
Published: March 2022

Impaired calcium uptake resulting from reduced expression and activity of the cardiac calcium ATPase (SERCA2a) is a hallmark of heart failure (HF). The calcium sequestering activity of SERCA2a is tightly regulated by the endogenous protein phospholamban that lowers its apparent affinity for calcium. However, other mechanisms of SERCA2a regulation, including post-translational modifications (PTMs), have recently emerged. Our lab has identified SUMOylation as a PTM that enhances the stability and activity of SERCA2a. Our latest analysis of SERCA2a PTMs has identified lysine acetylation as another PTM which might play a significant role in regulating SERCA2a activity. Indeed, we have demonstrated that SERCA2a is acetylated, and that this acetylation is more prominent in failing human hearts. In line with these observations, we determined the expression levels of key acetyltransferases to also be elevated in human HF tissues. Using a HEK293 cell-based co-expression screen, we found that p300 directly interacts with and acetylates SERCA2a. This interaction was confirmed in human cardiac tissues and using
in vitro
acetylation of purified SERCA2a by p300. Moreover, we have shown that p300 mediated acetylation profoundly reduced SERCA2a ATPase activity. Analysis of
in vitro
acetylated SERCA2a by mass spectrometry revealed several lysine residues in SERCA2a susceptible to acetylation by p300. We functionally characterized acetylated (Lys-to-Gln) and deacetylated (Lys-to-Arg) mimicking mutants of SERCA2a and found the identified acetylation sites on SERCA2a, specifically Lys492 located in the nucleotide binding pocket, to be essential for SERCA2a function. This was also corroborated by our structural models of acetylated forms of SERCA2a. Finally, we have demonstrated that adenovirus-mediated overexpression of acetyl-mimicking mutants of SERCA2a in cardiomyocytes isolated from SERCA2 conditional knockout mice does not restore normal cardiomyocyte function. Taken together, our data demonstrate that p300 mediated acetylation of SERCA2a is a critical post-translational mechanism that regulates calcium pump function and provides invaluable information necessary to develop new therapies to improve cardiac function in HF.
Abstract 396: GRK5-mediated Exacerbation of Ischemic Heart Failure Involves Cardiac Immune-Inflammatory Responses
Authors: de Lucia Claudio , Grisanti Laurel A , Ibetti Jessica , Lucchese Anna M , Gao Erhe , Tilley Douglas G , Leosco Dario , Ferrara Nicola , Rengo Giuseppe , Koch Walter J
Published: March 2022

Rationale:
Coronary artery disease and subsequent myocardial ischemia (MI) are the most common cause of heart failure (HF) in the US. G protein-coupled receptor (GPCR) kinase 5 (GRK5) has been shown to be upregulated in failing human myocardium. While the canonical role of GRKs is to desensitize receptors via phosphorylation, it has been shown that GRK5 can also locate to the nucleus of cardiomyocytes where it can exert GPCR-independent effects that promote maladaptive cardiac hypertrophy after hypertrophic stress. Despite numerous data indicating the importance of GRK5 in hypertrophy, it is still unknown if GRK5 has a role in ischemic heart disease.

Objective:
In this study, we investigated the critical role that GRK5 plays after myocardial ischemic injury with a novel aspect being the regulation of immune and inflammatory responses including recruitment of immune cells to the injured heart.

Methods and Results:
Cardiac-specific GRK5 transgenic mice (Tg-GRK5) and non-transgenic littermate (NLC) control mice were subjected to MI. Tg-GRK5-HF mice showed decreased cardiac function (both global and segmental contractility) as well as augmented left ventricular diameters and volumes compared to NLC-HF mice. Heart weight (HW) to body weight or tibia length ratios as well as mRNA expression of all major adverse remodeling-associated biomarkers (ANF, BNP, b-MHC) were increased in the TgGRK5-HF compared to NLC-HF. Cardiac fibrosis in the border zone (BZ) area as well as mRNA levels of Collagen-1 (Col-1), Col-3, MMP2 and CTGF were higher in TgGRK5-HF compared to NLC-HF, strongly suggesting increased adverse remodeling. Inflammatory Cytokines (IL-6 and IL-1beta) were augmented in TgGRK5-HF compared to NLC-HF and contribute at least in part to increased immune cell recruitment in the heart. In fact, we found both Neutrophils and T-lymphocytes (T- cells) augmented in the BZ and infarct zone.

Conclusions:
Our study shows that cardiac GRK5 has a detrimental effect during ischemic HF. GRK5 overexpression causes reduced cardiac function and increased immune cell recruitment/inflammation. Further, these results suggest GRK5 as a potential therapeutic target to limit HF development after ischemic injury.
Abstract 4: Regulation of Mitochondrial Complex I Assembly
Authors: Owusu-Ansah Edward
Published: March 2022

Mitochondrial CI (NADH: ubiquinone oxidoreductase) is the first and largest of the electron transport chain (ETC) complexes involved in oxidative phosphorylation, and has a molecular mass approaching 1 MDa. Mitochondrial Complex I (CI) is composed of 44 distinct subunits, but only 14 of these subunits are directly required for catalysis. A fundamental question in mitochondrial biology is to elucidate the roles of the additional 30 or so accessory subunits. In addition, CI can form different supercomplexes with other ETC complexes. It is unclear how these supercomplexes are assembled in the human heart, or any organism. We have established
Drosophila
flight muscles as a suitable system for studying CI assembly as well as the assembly of CI-containing supercomplexes. We show that many of the 30 accessory subunits regulate specific steps of CI assembly
in vivo
; and that CI biogenesis in flight muscles proceeds
via
the formation of ~315-, ~370-, ~550-, and ~815 kDa CI assembly intermediates as has been reported in mammalian systems. A specific accessory subunit (dNDUFA5) is required for the formation or stabilization of the ~315 kDa assembly intermediate. Additionally, we define a specific role for the CX
9
C-containing accessory subunit (dNDUFS5); by showing that it is required for converting a transient CI assembly intermediate (an ~700 kDa assembly intermediate) into the ~815 kDa assembly intermediate. Finally, we are performing genetic screens to identify genes that specifically regulate supercomplex assembly. Our findings highlight the potential of studies of CI biogenesis in
Drosophila
to uncover novel mechanisms of CI assembly in a living organism, and establish
Drosophila
as a suitable model organism for addressing questions relevant to CI biogenesis in humans.
Abstract 405: Bcl-xL Serine 14 Phosphorylation is Critical for Pressure Overload-induced Cardiac Hypertrophy
Authors: Nakamura Michinari , Fefelova Nadezhda , Xie Lai-Hua , Zhai Peiyong , Ikeda Shohei , Sadoshima Junichi
Published: March 2022

Cardiac hypertrophy is an adaptive response to various stresses, wherein Ca
2+
serves as a messenger to induce hypertrophy. We found that phosphorylation of Bcl-xL at Serine (Ser) 14 is significantly increased in the heart within one hour after transverse aortic constriction (TAC)-induced pressure overload. However, the functional significance of the increased Ser14-phosphorylation in hypertrophy is unknown. To address this question, wild-type (WT) and phosphorylation-resistant knock-in (KI) mice with an Alanine mutation at Ser14 in the Bcl-x gene were subjected to TAC. The KI mice had significantly higher mortality than WT mice (p=0.001), accompanied by heart failure (LVEDP, 34.6 vs 16.5 mmHg, p<0.05) with contractile dysfunction (EF, 38.2% vs 67.5%, p<0.001) and increased fibrosis (1.6-fold, p<0.001). Although the level of apoptosis was similar between the KI and WT mice one week after TAC, as assessed by TUNEL staining and cleaved caspase 3 and 9 expressions, cardiomyocyte and cardiac hypertrophy were inhibited in the KI mice (cardiomyocyte size, 0.32-fold; heart weight/tibia length, 0.61-fold, both P<0.001). Consistent with this finding, ANP and BNP gene expressions were significantly suppressed in the KI mice despite worse cardiac function (0.17-fold (p<0.001) and 0.69-fold (p<0.05), respectively). Adult cardiomyocytes isolated from the KI mice two days after TAC showed significantly reduced contractility compared to those isolated from WT mice (0.32-fold, p<0.001). Mechanistically, gene set enrichment analysis using the RNA-seq data obtained from the mouse heart one day after TAC revealed significantly altered channel activity in the KI mice. Although angiotensin II (AngII) increases Ser14-phosphorylation and cytosolic Ca
2+
level in WT-MEFs
in vitro
, MEFs isolated from the KI mice showed a significantly lower elevation of cytosolic Ca
2+
against AngII stimulation. Taken together, these data suggests that Bcl-xL-Ser14 phosphorylation promotes Ca
2+
release from the sarcoplasmic reticulum in response to pressure overload or AngII stimulation, leading to compensatory hypertrophy. Our findings indicate that targeting the Bcl-xL-Ser14 phosphorylation could be a potential therapeutic strategy for acute heart failure.
Abstract 408: Spliced X-box Binding Protein 1 Promotes Cardiac Hypertrophy through Activation of mTORC1
Authors: Wang Xiaoding , Li Jianping , Tran Diem Hong , Xu Lin , Gillette Thomas G , Jiang Xuejun , Wang Zhao V
Published: March 2022

Background:
Spliced X-box binding protein 1 (Xbp1s) is a key signal transducer and the most conserved branch of the unfolded protein response (UPR). Its role in cardiac hypertrophy and heart failure however remains poorly understood.

Aims:
To explore the role of Xbp1s in the development of pathological cardiac hypertrophy and remodeling.

Methods and Results:
Here, we showed that the expression of Xbp1s was highly and acutely induced by pressure overload in mouse hearts. Transgenic overexpression of Xbp1s in vivo led to cardiac hypertrophy, and exacerbated the hypertrophic response in response to transverse aortic constriction. At the mechanistic levels, we found that FK506 binding protein 11 (Fkbp11) is a bona fide transcriptional target of Xbp1s. Overexpression of Xbp1s stimulated Fkbp11 at both in vitro and in vivo levels. In so doing, Xbp1s directly augmented the mTORC1 signaling, the master regulator of cell growth. Indeed, siRNA-mediated knockdown of Fkbp11 significantly diminished Xbp1s-induced mTORC1 activation and hypertrophic growth in cardiac myocytes.

Conclusions:
Our results uncovered a novel link between protein-folding and cell growth with Xbp1s, Fkbp11 and mTORC1 as the key mediators, which may provide insights about our understanding of pathological cardiac remodeling in pressure overload.
Abstract 409: LRRC10 Associates With and Regulates Cardiac Ca
V
1.2 L-type Ca
2+
Channels, and I195T LRRC10 Variant is Linked to Dilated Cardiomyopathy
Authors: Woon Marites T , Long Pamela A , Reilly Louise , Evans Jared M , Keefe Alexis M , Lea Martin R , Beglinger Carl J , Balijepalli Ravi C , Lee Youngsook , Olson Timothy M , Kamp Timothy J
Published: March 2022

Leucine-rich repeat containing (LRR) proteins facilitate protein-protein interactions critical in a number of cellular functions including ion channel regulation. Leucine-rich repeat containing protein 10 (LRRC10), a cardiac-specific protein expressed in zebrafish, mice and humans, is essential for normal cardiac physiology with loss of
Lrrc10
in

Lrrc10
-/-

mice resulting in dilated cardiomyopathy (DCM). However, the mechanism by which LRRC10 contributes to DCM is unknown. Further, the functional role of LRRC10 in the regulation of ion channels in the heart has not been explored. Here we recorded L-type Ca
2+
channel currents (I
Ca,L
) from isolated ventricular myocytes of WT and

Lrrc10
-/-

mice, demonstrating a significant reduction in I
Ca,L
in

Lrrc10
-/-

myocytes (-4.9 ± 0.19 pA/pF, n=10) compared to WT (-6.5 ± 0.24 pA/pF, n=8). Co-immunoprecipitation (co-IP) experiments showed an association between LRRC10 and Ca
v
1.2 in WT, but not

Lrrc10
-/-

mouse lysates, with immunocytochemistry studies further demonstrating colocalization of LRRC10 and Ca
v
1.2 in isolated ventricular myocytes from wild-type (WT) mice. Additionally, whole exome sequencing revealed a novel homozygous recessive missense variant in
LRRC10
, I195T, found in a pediatric DCM patient. To determine whether the I195T variant impacts Ca
v
1.2 L-type Ca
2+
channels, whole-cell patch clamp experiments were performed using HEK293 cells transiently expressing the L-type Ca
2+
channel complex (LTCC) alone or with WT or I195T LRRC10. These electrophysiology experiments demonstrated a significant increase in I
Ca,L
with WT LRRC10 coexpression (-81 ± 5.3 pA/pF, n=12), but a decrease in I
Ca,L
when I195T (-18.2 ± 3.3 pA/pF, n=9) was coexpressed compared to the LTCC alone (-34.1 ± 2.2 pA/pF, n=17). Parallel surface biotinylation experiments demonstrated that WT and I195T LRRC10 did not alter L-type Ca
2+
channel expression on the plasma membrane, while co-IP experiments using lysates prepared from the transiently transfected HEK293 cells showed the association between Ca
v
1.2 and I195T LRRC10 variant. Overall, these findings newly identify LRRC10 as a cardiac-specific component of the Ca
v
1.2 macromolecular complex and demonstrate dysregulation of I
Ca,L
by the DCM associated I195T LRRC10 variant.
Abstract 414: Role of Red Blood Cell Derived Extracellular Vesicles in Cardiac Remodeling After Myocardial Infarction in a Transgenic Murine Model
Authors: Das Avash , Ziegler Olivia , Danielson Kirsty , Simonson Bridget , Lu Shulin , Toxavidis Vasilis , John Tigges , Ghiran Ionita , Das Saumya
Published: March 2022

Introduction:
Extracellular vesicles (EVs) function as novel mediators of intercellular communication. Here, we describe a novel, fluorescence switch-based, experimental model to study EV-mediated communication between RBCs and the heart that permits characterization of cross-talk between RBCs and cardiomyocytes at homeostasis and after myocardial infarction.

Methods:
Mice with RBC-specific expression of cre (Erythropoietin Receptor (EpoR) Cre) were crossed with reporter mTmG Rosa26 mice to yield EpoRCre/mTmG off-springs with membrane GFP expression in RBCs and RBC-derived EVs. Cultured dermal fibroblasts from mTmG mice and a mT/floxed/mGFP HEK 293 reporter cell line were used to assess transfer of functional cre in RBC-derived EVs. To determine targets of RBC-EVs, organs from i)EpoRCre/mTmG (n=3), ii) mTmG (n=3) or iii) mTmG mice transfused with RBC-EVs from EpoR-cre mice and targets of RBC-EVs (determined by mGFP expression due to cre-recombination) were assessed by confocal microscopy. Finally, ischemia-reperfusion-infarction (30 min. LAD ligation) was done in EpoRCre/mGmT mice (n=3) and their blood and organs harvested after a span of 4 weeks to analyze changes in quality and quantity of RBC-EV targets following MI.

Results:
1. RBC-EVs (mGFP positive) in plasma accounted for about 9% of total fluorescent EVs as detected by nano-flow cytometry and microscopy. 2. RBC-EVs contained cre protein by EM, and
in vitro
dermal fibroblasts from mTmG mice or mT/floxed/mGFP HEK 293 reporter cells showed mGFP expression with EpoRCre RBC-EVs, suggesting EV-mediated transfer of functional cre. 3. Cre-mediated recombination was noted in diverse organs in EpoRCre/mTmG mice and mTmTG mice transfused with EpoRCre- EVs with the bone marrow, heart, lungs, kidney and spleen showing the largest degree of recombined cells. 4. Target profile of RBC-EVs demonstrates a distinct pattern of EV-mediated communication among the organs at baseline that may be altered in different disease models.

Conclusion:
We show proof-of-concept for a novel model to study origin and targets of EV-mediated intercellular communication with significant EV-mediated communication between RBCs and cardiomyocytes under homeostatic conditions.
Abstract 417: Syndecan-4 Regulates the Effect of Osteopontin on Cardiac Fibroblast Function and Phenotype
Authors: Herum Kate M , Romaine Andreas , Melleby Arne Olav , Strand Mari E , Braathen Bjørn , McCulloch Andrew D , Carlson Cathrine R , Christensen Geir
Published: March 2022

Pressure overload of the heart induces cardiac fibrosis due to excessive production of extracellular matrix by activated cardiac fibroblasts. This compromises cardiac function and may lead to heart failure. The transmembrane proteoglycan syndecan-4 is upregulated in response to pressure overload and can bind extracellular signaling proteins via its heparan sulfate glycosaminoglycan chains. The matricellular protein osteopontin is associated with fibrosis and contains a heparan binding domain that overlaps with its thrombin cleavage site. We here examine the role of the extracellular part of syndecan-4 in regulating proteolytic cleavage and pro-fibrotic activity of the matricellular protein osteopontin. Syndecan-4 and osteopontin mRNA displayed similar regulation patterns in response to left ventricular pressure overload induced by aortic banding in mice. Syndecan-4 and osteopontin bound to each other in left ventricles and fibroblasts as shown by native western blots, immunoprecipitation and immunofluorescent staining. Osteopontin was protected from cleavage by thrombin when pre-incubated with the extracellular domain of syndecan-4. Thrombin-cleaved osteopontin induced a pro-fibrotic phenotype when given to fibroblasts, whereas full-length osteopontin favored a more quiescent phenotype. Thus, our data suggest that the extracellular domain of syndecan-4 protects against the pro-fibrotic effects of osteopontin. Following the initial stage of extracellular remodeling, the extracellular part of syndecan-4 is shed from the cell surface. Accordingly, increased cleaved osteopontin was detected in hypertrophic pressure-overloaded left ventricles three days after aortic banding. Cleaved osteopontin was also detected in plasma from patients with aortic stenosis and displayed a higher cleaved/full-length ratio in the coronary sinus than the radial artery, suggestion that osteopontin is in fact cleaved locally in the heart. Our results indicate an anti-fibrotic effect of the extracellular part of syndecan-4 when it is still attached to the cell surface. Shedding of syndecan-4 from the cell surface at later stages of remodeling exposes osteopontin to cleavage by thrombin, enabling pro-fibrotic effects on cardiac fibroblasts.
Abstract 423: The Valosin-containing Protein Attenuates Chronic Pressure Overload-induced Cardiac Remodeling and Heart Failure via Activating mTORC2 Pathway
Authors: Zhou Ning , Ma Ben , Stoll Shaunrick , Shu Hongyang , Hays Tristan , Qiu Hongyu
Published: March 2022

Aims:
The pressure overload induced cardiac hypertrophy is a key risk factor for heart failure. However, the mechanism involved remains largely unknown. We previously showed that overexpression of valosin-containing protein (VCP) promotes cardiomyocytes survival
in vitro
. Here we tested the hypothesis that VCP protects heart against pressure overload-induced heart failure via promoting pro-survival signaling in cardiomyocyte
in vivo
.

Methods and Results:
VCP expression were found to be notably decreased in left ventricle (LV) tissues of dilated cardiomyopathy patients compared to healthy donor at both the mRNA and protein levels. A transgenic mouse (TG) with a cardiac specific overexpression of VCP was generated and compared to its litter-matched wild-type (WT). Pressure overload was induced by transverse aortic constriction (TAC) in mice for 5 weeks and sham control was included. Cardiac structure and function were measured by echocardiography and hemodynamic analysis. After 5 week TAC, compared to sham control, WT mice developed a dilated cardiac hypertrophy and cardiac dysfunction, reflected by a significant increase in LV chamber diameter, wall thickness and the ratio of LV weight/ tibia length, and a decrease in ejection fraction (71% vs 51%) as well as an increase in ratio of lung weight /tibia length, a marker of heart failure (all, P<0.01 vs WT sham). However, these pressure overload induced cardiac remodeling and dysfunction were significant suppressed in VCP TG TAC mice (P<0.05 vs WT TAC). Mechanistically, compared to sham control, VCP expression in LV tissues was found to be significant decreased in WT TAC mice, but not in VCP TG mice. VCP TG TAC mice showed a significant decrease in apoptosis of cardiomyocytes vs WT TAC mice (p<0.05). In addition, overexpression of VCP increased the phosphorylation of AKt at 473 and the expression of protein kinase C α in VCP TG mouse heart
in vivo
and in isolated cardiomyocytes
in vitro
which was abolished by the deletion of Rictor, indicating an activation of mammalian target of rapamycin complex 2 (mTORC2) in cardiomyocytes.

Conclusion:
Overexpression of VCP prevents TAC-induced cardiac remodeling and heart failure via a pro-survival mechanism involving mTORC2 signaling pathway.
Abstract 436: Targeting Fatty Acid Oxidation by Acetyl-CoA Carboxylase 2 Deletion in Pathological Hypertrophy
Authors: Ritterhoff Julia , Villet Outi , Kolwicz Stephen C , Shao Dan , Tian Rong
Published: March 2022

We have previously shown that preserving fatty acid oxidation (FAO) by cardiac-specific deletion of Acetyl-CoA Carboxylase 2 (ACC2) prevents the shift of substrate preference towards glucose, reduces cardiomyocyte hypertrophy and preserves cardiac function during chronic pressure overload.

To determine whether maintaining FAO specifically prevented cardiomyocyte hypertrophy, we treated adult rat cardiomyocytes (CMs) with and without adenoviral-mediated ACC2 knock-down (KD) with phenylephrine (PE, 10 μM). ACC2 KD effectively prevented CM hypertrophy after PE stimulation compared to control CMs (+9±6% vs. 42±6%) in medium supplemented with fatty acids (FA) (5.5 mM glucose, 0.4 mM mixed long-chain FAs and 0.1 mU/ml insulin). Whereas PE stimulation in control CMs increased glucose uptake (+28±8%) this was normalized after ACC2 KD. Inhibiting FAO by etomoxir or increasing glucose utilization by dichloroacetate abolished the beneficial effects of ACC2 KD after PE stimulation. When cultured in glucose-free medium supplemented with FA, ACC2 KD was incapable of preventing cardiomyocyte hypertrophy. However, replacing glucose with pyruvate or propionate restored the anti-hypertrophic effect of ACC2 KD.

To determine the therapeutic effects of increasing FAO
in vivo
, male mice were subjected to transverse aortic constriction (TAC) and sham surgery. Three weeks after surgery, TAC mice had a significant increase in left ventricular (LV) mass as determined by echocardiography compared to sham operated mice (130 vs. 92 mg). At this time point, cardiac-specific ACC2 deletion (iKO) was induced by tamoxifen (tam) administration. ACC2 protein was effectively deleted in iKO sham and TAC hearts compared to controls (CON) 2 weeks post tam injection. FAO was 2-fold higher in iKO TAC vs. CON TAC hearts as assessed by isolated heart perfusion and 13C NMR spectroscopy. CON and iKO mice will be followed until 12 weeks after TAC to determine cardiac function and assess hypertrophy.

Together, these data indicate that increasing FAO via inactivation of ACC2 exerts anti-hypertrophic effect in adult cardiomyocytes. Deleting ACC2 in the hypertrophic heart
in vivo
can increase FAO and represents a valid target to treat pathological cardiac hypertrophy.
Abstract 445: Autophagy is Essential for Remote Preconditioning Induced Cardioprotection and May Involve JAK-STAT Pathway for Autophagy Activation
Authors: Billah Muntasir , Ridiandries Anisyah , Khachigian Levon , Dona Anthony , Bhindi Ravinay
Published: March 2022

Autophagy is a cellular process by which mammalian cells degrade and recycle damaged organelles and proteins. Remote ischemic preconditioning (RIPC) protects the myocardium from ischemia-reperfusion (I/R) injury and is thought to involve upregulation of autophagy. In this study we aim to ascertain the role of autophagy in RIPC induced cardioprotection. Sprague Dawley rats were subjected to transient blockade of left anterior descending coronary artery to simulate I/R injury, with or without prior RIPC at the hind limb. RIPC prior I/R decreased myocardial infarct size (51.5 ± 3.5% vs. 30.2 ± 5.16%; n=7, p<0.05), PARP cleavage and upregulated autophagy in the myocardium compared to I/R alone (p<0.05). RIPC alone increased autophagy in a time dependent manner in the myocardium. To further examine the effect of autophagy, H9C2 rat cardiomyoblast cells were exposed to RIPC following I/R simulation. 3-methyladenine (10mM) was used to inhibit autophagy, which ameliorated the cardioprotective effect of RIPC in H9C2 cells, producing significant increases in apoptosis and Bax protein expression, and decreasing cell viability compared to the untreated cells (p<0.05). In contrast, autophagy stimulation with rapamycin (1000nm) significantly reduced apoptosis and Bax protein expression, and improved cell viability in H9C2 cells compared to H/R group mimicking the preconditioning effect (p<0.05). In addition, RIPC of H9C2 cells upregulated IL-6 protein release into the preconditioned media. H9C2 cells exposed to this preconditioned media prior I/R were protected against apoptosis and upregulated autophagy. Recombinant IL-6 treatment prior I/R upregulated autophagy in H9C2 cells in a dose dependent manner, activating JAK-STAT pathway (p<0.05 vs. I/R) without affecting the other kinase pathways including p38 MAPK, GSK-3β pathways. Blocking JAK-STAT pathway with AG-490 (50 μM) prior I/R reduced autophagy upregulation despite recombinant IL-6 treatment (p<0.05 vs. IL-6 treated I/R control). These results suggest that, autophagy is essential for RIPC induced cardioprotection and RIPC may upregulate autophagy through IL-6/JAK-STAT dependent mechanism, thus identifying a potentially new therapeutic option for the treatment of ischemic heart disease.
Abstract 448: Protective Role of Crataegus Oxycantha in Myocardial Ischemia/Reperfusion Injury by Activating Angiogenesis
Authors: Dasagrandhi Divya , Muthuswamy Anusuyadevi , Kesavan Swaminathan Jayachandran
Published: March 2022

Rationale:
Resuscitation of infarcted myocardium during ischemia/ reperfusion (I/R) injury is challenging process due to intrinsic complex mechanism. Reperfusion or revascularization is the best therapy to limit amount of infarction.

Objectives:
The current study was to explore the protective role of ethanolic extract of
Crataegus oxycantha
(COC) in myocardial I/R injury and to find its effect on angiogenesis.

Methods and Results:
Male guinea pigs were randomized into four groups control (con; n=6), high fat diet (hfd; n=6), high fat diet + drug (hfd+coc; n=6) and drug (coc; n=6). Myocardial I/R injury was induced by creating ischemia for 30 minutes through left anterior descending artery ligation followed by 24 h of reperfusion. Electrocardiogram analysis showed delayed RR interval and qrs complex in hfd group compared to hfd+coc; whereas, echocardiogram confirmed the dilation of left ventricle. LVIDs was increased in hfd compared to hfd+coc (0.40±0.13 cm vs 0.31±0.12cm; p<0.05) and IVSd: hfd 0.19±0.12cm vs hfd+coc 0.14±0.08 cm (p<0.05). Histopatology of left ventricle showed increased cellular death in hfd animals compared to hfd+coc and reduced intima-media thickness in aorta was seen in hfd±coc. Immunofluorescent analysis of ICAM showed decreased expression levels in hfd+coc group compared to hfd. Immunoblot of whole infarcted left ventricular lysate showed upregulation of EGF and VEGF in hfd+coc showed increased angiogenesis thereby protecting infracted myocardium.

Conclusion:
Treatment of hfd animals with ethanolic extract of
Crataegus oxycantha
(COC) during I/R injury improved cardiac function and myocardial viability by activating angiogenesis upon upregulation of VEGF and EGF expression levels in acute phase of injury.
Abstract 449: The H2 Domain in the Connexin43 Carboxyl Terminus Regulates Cardioprotection From Ischemia Reperfusion Injury
Authors: Jiang JIngbo , Palatinus Joseph A , He Huamei , Lyyathurai Jegan , Jourdan L Jane , Hoagland Daniel , Bultynck Geert , Wang Zhen , Schey Kevin , Poelzing Steven , McGowan Francis X , Gourdie Robert G
Published: March 2022

Cardiac ischemia-reperfusion (IR) injury occurs in patients with acute coronary syndromes undergoing percutaneous intervention to re-establish blood flow in blocked or narrowed arteries. Presently, there are no therapies approved to prevent the loss of heart muscle, scar tissue formation and contractile dysfunction that result from IR injury. We have previously reported that
α
CT1, a Connexin 43 (Cx43) mimetic peptide currently in Phase III testing in humans (GAIT1; NCT02667327) as a therapy for chronic skin wounds (PMID:27856288; 25072595), improves contractile function of mouse hearts following injury in association with increases in PKCε-mediated phosphorylation of Cx43 at serine 368 (S368) (PMID: 21273554). Here, we report that induction of S368 phosphorylation and cardioprotection from IR injury by
α
CT1 involves direct protein-protein interaction of
α
CT1 with a short alpha-helical sequence within the Cx43 carboxyl terminus (CT) termed the H2 domain. The interacting protein domains involved in Cx43 S368 phosphorylation were identified and validated by a combination of techniques including tandem mass spectroscopy, surface plasmon resonance, immunoprecipitation and thermal shift assays. These studies indicated that
α
CT1 induced S368 phosphorylation (pS368) via a novel mechanism involving interaction between a pair of positively charged lysines (K344, K345) in the Cx43 CT H2 domain and a cluster of negatively charged amino acids i
α
CT1. It is further shown that the known ability of
α
CT1 to interact with Zonula Occludens-1 (ZO-1), has no direct role in either the induction of S368 phosphorylation or protection from IR injury. Finally, we identify a small, novel variant of
α
CT1 (
α
CT11 - RPRPDDLEI, Mr 1110) that penetrates myocyte membranes, induces S368 phosphorylation and robustly preserves ventricular contractile function and cardiac muscle when administered after ischemic injury. Data on Cx43 hemichannel dependence of
α
CT11 uptake by cells will be presented. Our results indicate that selective targeting of the secondary structure and phospho-status of the Cx43 CT by small peptidergic drugs may provide a novel and potent pharmacological approach to preservation of cardiac muscle following IR injury.
Abstract 451: Beta Blocker Use in Asymptomatic Patients with Sepsis and Elevated Troponin is Associated with Lower Risk of Mortality
Authors: Jogu Hanumantha R , Chevli Parag A , Bose Abhishek , Vasireddy Amit , Sompalli Sreeja , Gopireddy Guru Anirudh Reddy , Dutta Abhishek , Maludum Obiora , Yarra Pradeep , Amjad Aysha , Menon Suma , Sunkara Padageshwar R , Qureshi Waqas T
Published: March 2022

Background:
In septic patients with elevated Troponin (e-Tn) levels, beta-blockers (BB) are usually avoided to prevent chronotropic incompetence. We examined the unclear association of BB at discharge with mortality in patients with sepsis and e-Tn levels.

Methods:
We assessed 936 consecutive patients with e-Tn levels and sepsis on admission. Those with septic shock were excluded. BB use was determined by their presence at the time of discharge. Cox proportional hazard models adjusted for demographics and clinical covariates examined the association of BB use with all-cause mortality.

Results:
417 (44.6%) were on BB therapy with mean peak Tn level of 1.0 ± 0.09 ng/ml. Over a median follow-up of 5 years, 108 (36.5%) versus 236 (40.3%) deaths were reported in the BB versus the non-BB group respectively. On adjusting the Cox model for demographics, peak Tn, baseline cardiovascular disease risk factors, use of anticoagulants, antiplatelet and statins during hospitalization and discharge, BB use associated with a lower risk of mortality (HR 0.72; 95% CI 0.58-0.90, p = 0.004). In addition, BB use had a significant interaction with ischemic EKG changes (interaction p = 0.049) leading to a lower risk of mortality among patients without ischemic EKG changes (HR 0.66; 0.51-0.84, p = 0.0008 vs. HR 1.33; 0.75-2.36, p = 0.32, Figure 1).

Conclusions:
In septic patients with e-Tn levels, use of BB on discharge led to a lower risk of mortality and the protective effect was more pronounced in patients without ischemic EKG changes. Future research should focus on its protective mechanism and its use in septic patients with e-Tn in a randomized trial.
Abstract 463: The Orphan G Protein-Coupled Receptor C5B is Regulated by Hyperglycemia and Activates Pro-Inflammatory Signaling in
Endothelial Cells
Via the Tyrosine Kinase Fyn
Authors: Freundt Greta , Nitz Jan Thorge , Frey Norbert , Hippe Hans-Jörg
Published: March 2022

Background:
Atherosclerosis is driven by an inflammatory process of the vascular wall in which endothelial cells (EC) are decisively involved. The orphan G protein-coupled receptor GPRC5B activates inflammatory pathways in adipocytes and regulates the glucose and lipid metabolism in
Drosophila melanogaster
.

Hypothesis:
We hypothesize that GPRC5B is involved in inflammatory signaling of EC, particularly in hyperglycemia.

Methods & Results:
Stimulation of mouse brain endothelial cells (bEnd.3) and primary human umbilical vein endothelial cells (HUVEC) with high glucose concentration (25 mM), Tumor Necrosis Factor α (TNFα 0,1 μg/ml) or Lipopolysaccharides (LPS 1 μg/ml), respectively, leads to a significant upregulation of GPRC5B mRNA and protein level compared to unstimulated cells (mRNA / protein: gluc +254% / +86%, p<0.001; TNFα +203% / +124%, p<0.001; LPS +195%, p<0.05, n=6). Similar results are obtained in HUVECs after stimulation with high glucose and cytokines.

Overexpression of GPRC5B in EC induced a significant increase in Interleukin 6 (IL6), Intercellular Adhesion Molecule 1 (ICAM1) and Vascular Cell Adhesion Molecule 1 (VCAM1) mRNA and protein expression. Furthermore, activation of NFκB (luciferase reporter assay) and phosphorylation of extracellular signal–regulated kinases 1/2 (Erk 1/2) were significantly increased compared to controls
(mRNA: IL6 +42%, p<0.01; ICAM1 +37%, p<0.01; VCAM1 +40%, p<0.001, n=8. Protein: ICAM1 +48%, p<0.05; VCAM1 +52%, p<0.05; pErk 1/2 +67%, p<0.01, n=5. NFκB: +165%, p<0.001, n=18).

Conversely, the siRNA-mediated GPRC5B knockdown resulted in diminished expression of IL6 and VCAM1 as well as decreased NFκB activity compared to controls
(IL6 -55%, p<0.05, n=3, NFκB -36%, p<0.05, n=6)
. The knockdown results were confirmed in HUVECs.

Co-immunoprecipitations in EC revealed the tyrosine kinase Fyn as interaction partner of GPRC5B.

Conclusion:
Our data suggest that GPRC5B is not only regulated by glucose and pro-inflammatory cytokines but also activates pro-inflammatory and pro-atherogenic pathways in mouse and human endothelial cells. Therefore, GPRC5B might play an important role in hyperglycemia-accelerated vascular inflammation and pathogenesis of atherosclerosis.
Abstract 468: TAB2 is Molecular Switch That Critically Regulates Myocardial Survival and Necroptosis
Authors: Yin Haifeng , Guo Xiaoyun , Chen Yi , Steinmetz Rachel , Liu Qinghang
Published: March 2022

Our recent study identified a key role for TAK1 (transforming growth factor β activated kinase-1) in regulating myocardial remodeling and heart failure propensity by antagonizing necroptosis, also termed programmed necrosis (
Circulation
2014;130:2162-2172). TAB2 (TAK1 binding protein-2) is an adaptor protein that regulates TAK1 activation and downstream signaling. Here we tested the hypothesis that TAB2 functions as a critical regulator of myocardial necroptosis and pathological remodeling. We found that both TAB2 and TAK1 were up-regulated during compensatory cardiac hypertrophy but down-regulated in end-stage heart failure. Genetic ablation of TAB2 in cardiomyocytes, but not other TAK1 binding proteins TAB1 or TAB2, markedly increased necroptosis in response to TNFα stimulation. Genetic deletion of RIP3 (receptor interacting protein-3), a key regulator of necroptosis, also largely blocked cell death in TAB2-deficent cells, further confirming the role of TAB2 in regulating necroptotic cell death. Mechanistically, our data showed that TAB2 functions as a molecular switch that tightly regulates TAK1-RIP1 interaction (pro-survival) and the RIP1-RIP3 necrosome formation (pro-necroptosis). We further showed that loss of TAB2 promotes necroptosis in cardiomyocytes via two independent mechanisms: acutely through the induction of RIP1-RIP3 necrosome and later through inhibition of the NFκB pathway. Importantly, cardiac-specific ablation of TAB2 in mice showed enhanced pathological remodeling with massive fibrosis and necroptosis, and the TAB2-deficient mice gradually developed dilated cardiomyopathy and heart failure starting 2 months old. Moreover, the TAB2-deficient mice are also predisposed to heart failure after pathological stress by pressure overload or myocardial infarction, suggesting a protective role of TAB2 in cardiac stress response. These results strongly indicate that TAB2 functions as a molecular switch that critically regulates the pro-survival TAK1 signaling and the necroptosis machinery, thus playing a critical role in myocardial survival and stress response. The TAB2-TAK1 signaling network may serve as a novel therapeutic target in heart disease and other pathological conditions driven by necroptosis.
Abstract 472: The mTOR-Dependent Translatome Adapts Early Changes in Gene Expression During Cardiac Growth
Authors: Riechert Eva , Doroudgar Shirin , Ho Thanh Cao , Malone Brandon , Burghaus Jana , Katus Hugo A , Dieterich Christoph , Völkers Mirko
Published: March 2022

Introduction:
The mTOR kinase links nutrient and growth factor signaling to cell growth by controlling mRNA translation, but the role of translationally controlled networks during cardiac stress remains unknown. We hypothesized that activation of mTOR controls translation of networks of mRNAs, independent from transcription, to rapidly adapt gene expression to physiologic and pathologic stress. We tested this hypothesis by monitoring the transcriptome and the translatome after pharmacologic and genetic inhibition of mTOR in cardiac myocytes.

Methods and Results:
To define the mTOR-dependent translatome, we performed ribosome profiling after pharmacologic mTOR inhibition by Torin1. We found 166 decreased and 199 unchanged mRNAs that we used for further analysis. Among the decreased mRNAs, 17% have a known TOP or TOP like motif in the 5’UTR, explaining why mTOR inhibition selectively suppresses their translation. Gene ontology analyses of Torin1-suppressed mRNAs showed enrichment for genes involved in translation and metabolism.
P
roline
-r
ich
A
KT
s
ubstrate of
40
kDa (Pras40) inhibits mTORC1 activity.
In vivo,
mTOR inhibition by overexpression of Pras40 blocked pathologic growth, and improved cardiac function after transverse aortic constriction (TAC). Conversely, in a cardiomyocyte-specific Pras40 knock-out mouse, heart function was reduced and growth response attenuated after TAC surgery. Similarly, physiological heart growth was inhibited after Pras40 deletion. Mechanistically and in line with the pharmacologic results, global translation rates were decreased after Pras40 depletion. Mass-spectrometric analysis identified ribonucleoproteins, ribosomal proteins and chaperones as Pras40 interaction partners, suggesting a direct function in the protein synthesis and folding machinery downstream of mTOR. Monitoring the translatome
in vivo
after Pras40 depletion using a ribosome tagging approach, followed by ribosome profiling, identified the mTOR-dependent translatome.

Conclusions:
mTOR-dependent translational control might represent a crucial way of controlling gene expression during stress in the myocardium. Once elucidated, mTOR-based therapies could be a powerful approach to prevent worsening of cardiac function.
Abstract 478: Cell-type Specific Surface Markers Effectively Isolate Cardiac Cell Sub-populations
Authors: Prahl Jordan D , Weiland Matthew , Milliron Hsiaoyun , Kort Eric J , Jovinge Stefan
Published: March 2022

Cardiomyocytes comprise only 20-30% of the cells in the heart. To enable downstream applications requiring live cells (including clinical application), identification of surface markers unique to all cardiac subpopulations is needed. Development of such strategies would create a powerful technique to tease apart the multiple cell populations that make up the heart and their role in complex heart diseases. Our hypothesis was that surface markers could be identified in neonatal mice to distinguish smooth muscle cells (SMC), cardiomyocytes (CM), fibroblasts (FB) and endothelial cells (EC), enabling sorting by flow cytometry and analysis of these subpopulations of heart cells. Hearts were surgically removed from one litter of neonatal (Day 0-2) C57BL/6J mice and three litters of transgenic mice with mCherry α-myosin heavy chain promoter (αMHC-mCherry), then digested with trypsin overnight followed by collagenase/dispase and gentle mechanical dissociation. Surface marker staining for Cadherin-2 (Cdh2) showed congruence with αMHC-mCherry positivity. Cells were sorted using fluorescent activated cell sorting with antibodies against the following surface markers: Cdh2 (CM), CD31 (EC), CD90.2 (FB) and CD49a (SMC). Enrichment of these cell populations was then evaluated by qRT-PCR for known intracellular and extracellular markers specific to each cell: EC (CD31 + vWf), CM (TNNT2 + Myh6), FB (CD90) and SMC (Myh10/11 + Tagln). Our panel of surface markers enabled enrichment of four subpopulations of heart cells which will be verified further in coming experiments. In the future, this strategy will enable sorting of cells from each subpopulation for downstream analysis.
Abstract 55: Use of a Novel Electrogram Filter to Visualise the Conduction Tissue Signals in the Ventricle in Sinus Rhythm and Arrhythmia: Canine Studies
Authors: Padmanabhan Deepak , Witt Chance , Sugrue Alan , Killu Ammar , Naksuk Niyada , Desimone Christopher , Ladewig Dorothy , Powers Joanne , Suddendorf Scott , Asirvatham Samuel
Published: March 2022

Background:
Conduction tissue in the ventricles is closely intertwined with myocardium. Owing to high velocity of conduction, there is near simultaneous activation , making it difficult to discern. Restricted sampling rates and limited dynamic range make real time signal processing challenging for these signals in routine recording systems.

Objectives:
We aimed to use novel signal processing techniques in a new system to isolate the conduction tissue signals from the rest of the myocardial activation.

Methods:
We used the PURE-EP™ (BioSig Technologies, MN) signal processing system to record endocardial and epicardial signals from 6 anesthetized canines in an acute study setting. A novel filter based on proprietary algorithms to isolate the high frequency signal was applied at sites where the electrogram signals were thought to have a conduction tissue component to them. . In addition, ventricular fibrillation (VF) was induced at the end of the experiment and signals were then recorded from the endocardium and the conduction system.

Results:
This filter was successfully able to isolate conduction tissue signals in the myocardium from those of the ventricular muscle. It did over sense atrial signals at the basal left ventricular septum owing to the high frequency components of the atrial signal at this location. When applied to epicardial signals, it did not pick up any conduction tissue signals. In VF, we could discern signals originating from the conduction system endocardially. (Figure 1)

Conclusions:
This filter can isolate conduction tissue signals from those of the myocardium and may prove to be a useful adjunct in mapping arrhythmias originating near the conduction system.
Abstract 63: A Novel Inotropic Gene Therapy Approach for Aging Induced Cardiac Dysfunction
Authors: Shettigar Vikram , Zhang Bo , Tikunova Svetlena , Biesiadecki Brandon , Janssen Paul , Weisleder Noah , Davis Jonathan P , Ziolo Mark T
Published: March 2022

We have developed a novel approach to increase cardiac contractility without the deleterious effects of existing inotropes (beta agonists, Ca2+ sensitizers). Using the strengths of protein engineering, we have shown that cardiac function can be directly controlled and strongly influenced by manipulating the Ca2+ dependent switch in cardiac muscle, troponin C (TnC). Our smartly formulated construct, L48Q TnC, is a strong Ca2+ sensitizer. Although effective acutely, long-term inotropic support is detrimental. We transduced mice with AAV9 L48Q TnC at 3 months of age and serially performed echocardiography for 2 years. The current dogma would suggest that chronic Ca2+ sensitization with L48Q TnC should result in a hypertrophic cardiomyopathy and compromised function. Astonishingly, the L48Q TnC mice maintained ejection fraction unlike the progressive decline observed in the controlled, age-matched mice. Furthermore, L48Q TnC mice had an increased rate of survival. Considering cardiovascular disease is an elderly issue, we reasoned L48Q TnC would be beneficial in other age-related cardiovascular problems (i.e., hypertension and pressure-overload). We tested this in two models, transverse aortic constriction (TAC, pressure-overload) and angiotensin II infusion (AngII, hypertension). Consistent with the aging results, L48Q TnC mice displayed enhanced cardiac function (ejection fraction, contractility, relaxation), performance (VO
2max
, distance run before exhaustion), less adverse remodeling, and enhanced survival. These studies provide a promising approach (i.e., inotropic support via L48Q TnC) that can be used therapeutically to enhance heart function in a variety of cardiomyopathies associated with aging.
Abstract 77: The Role of Vinculin in Cardiomyocyte Adhesion and Mechanical Continuity
Authors: Merkel Chelsea D , Kwiatkowski Adam V
Published: March 2022

Cardiomyocytes are joined end-to-end by a complex adhesive structure known as the intercalated disc (ICD). The ICD is composed of mechanical and electrical junctional complexes including the adherens junction (AJ). The core of the AJ is the cadherin/catenin complex, which links the actin cytoskeletons of neighboring cells to provide mechanical continuity. Myofibrils – the contractile machinery of the heart – are coupled to the ICD, presumably through the AJ; however, this mechanical linkage is poorly defined. Tension applied to the AJ is sensed in part through alpha-catenin, which connects the cadherin/catenin complex to actin. Force alters the conformation of alpha-catenin to reveal binding sites for actin-binding ligands, including vinculin and afadin, which are both found at the ICD. We investigated the role of alpha-catenin and its ligands in linking the AJ to actin in cardiomyocytes. Using a Förester resonance energy transfer tension-sensing module inserted into N-cadherin, we found that N-cadherin is under tension at the ICD. We then measured protein mobility of EGFP-tagged N-cadherin and found that N-cadherin dynamics were reduced at sites of myofibril integration, suggesting that myofibrils stabilize N-cadherin. To examine the composition of AJs in cardiomyocytes, we used immunofluorescence and found that vinculin and afadin were preferentially recruited to myofibril integration sites at the ICD. To further examine the linkage between AJs and myofibrils, we built a series of EGFP-tagged N-cadherin:alpha-catenin fusion constructs to delineate the individual and combined roles of alpha-catenin, vinculin and afadin. A constitutively active alpha-catenin increased vinculin recruitment and decreased the mobility of the N-cadherin fusion, suggesting that vinculin recruitment stabilizes the AJ. In contrast, restricting the AJ-actin interface to afadin reduced stability, indicating afadin alone cannot replace vinculin in linking AJs to the myofibril network. Together, our data support a model in which alpha-catenin, vinculin and afadin cooperate to anchor myofibrils at the ICD to provide mechanical continuity between cells.