bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 187: Identification of Cardiac-specific Downstream Substrates of Protein Kinase G I as Potential Novel Anti Cardiac Remodeling Targets
Authors: Blanton Robert M , Lane Angela , Aronovitz Mark , Wang Guang-rong , Thoonen Robrecht , Davis Roger , Mendelsohn Michael , Kass David , Karas Richard
Published: July 2021

We recently reported that mutation of the cGMP-dependent Protein Kinase G I alpha (PKGIα) N-terminal leucine zipper (LZ) domain (in the PKGIα LZ mutant, or LZM, mouse) accelerates LV remodeling and heart failure after TAC, and prevents the anti-remodeling effect of sildenafil. We therefore hypothesized that PKGIα attenuates remodeling by regulating cardiac signaling pathways that are dependent on substrate interactions mediated by its LZ domain. As a first step to identifying cardiac proteins downstream of PKGIα, we screened myocardial lysates for PKGIα LZ domain-interacting proteins.

Our previous work revealed a requirement for the PKGIα LZ domain for the activation of anti-remodeling myocardial JNK activity after LV pressure overload. MLK3 is an MAPKKK that contains an LZ domain and activates JNK. We now demonstrate, by immunoprecipitation, that MLK 3 interacts with the PKGIα LZ domain in myocardial lysates. We show further that 8-Br-cGMP induces MLK3 phosphorylation on Threonine 277 and Serine 281 in WT, but not LZM myocardial lysates. And, in 293 cells transfected with FLAG-MLK3, 8Br-cGMP induced PKGIα-MLK3 co-precipitation, and increased phosphorylation of MLK3 on Thr277/Ser281. Co-transfection of MLK3 and PKGIα also induced MLK3 phosphorylation at the same sites. We next examined the cardiovascular effect of MLK3 deletion in vivo. Male 8 week old MLK3 -/- mice display basal bi-ventricular hypertrophy compared with littermate controls (LV/Tibia length 42.8 + 0.6 mg/cm in WT, 52.9 + 1.8 in MLK3 -/-;
P
<0.01; RV/TL 10.8 + 0.1 mg/cm in WT, 13.3 + 0.3 in MLK3 -/-;
P
<0.01; n= 7 WT, 5 MLK3 -/-). By 14-16 weeks of age, LVH progressed in the MLK3 -/- mice (LV/TL 47.7 + 1.3 mg/cm in WT, 59.8 + 7.5 in MLK3-/-; n= 6 WT, 9 MLK3-.-;
P
<0.01). Arterial blood pressure was modestly increased, though still normal, in the MLK3 -/- mice (SBP 93 + 1 in WT, 113 + 1 in MLK3 -/-). And, 14-16 week MLK3 -/- mice have impaired LV diastolic function (tau 3.2 + 0.1 ms WT, 3.7 + 0.1 MLK3-/-;
P
0.06).

Our studies reveal a previously unknown function of MLK3 as a myocardial PKGIα effector and inhibitor of LVH. Together these results support the strategy of exploring LZ-dependent PKGIα substrates in the myocardium to identify novel therapeutic targets for cardiac remodeling.
Abstract 211: Regulatory T cells improve healing after myocardial infarction
Authors: Frantz Stefan , Weirather Johannes , Vogel Benjamin , Beyersdorf Niklas , Kerkau Thomas , Hofmann Ulrich
Published: July 2021

Background:
The proinflammatory activation of innate immunity by myocardial ischemic injury has been recognized for long time. Our recent data have indicated that activation of CD4+ T cells, presumably by auto-antigen recognition, is a prerequisite for formation of a stable scar and prevention from left ventricular dilation after experimental myocardial infarction in mice. We here hypothesized that regulatory CD4+CD25+Foxp3+CD4+ T cells might improve left ventricular wound healing and prevent from adverse remodeling after myocardial infarction.

Results:
Experimental myocardial infarction in mice induced the proliferation and activation of CD4+CD25+Foxp3+ regulatory T cells, as demonstrated by intracellular expression of the Ikaros family transcription factor Helios, in heart draining lymph nodes. Pretreatment of mice with an anti-CD25 antibody before myocardial infarction efficiently depleted CD4+CD25+Foxp3+ regulatory T cells and increased mortality after myocardial infarction as compared to mice treated with an isotype-matched control antibody of irrelevant specificity, i.e., 25% survival in anti-CD25 treated mice vs. 55,9% survival in control antibody treated animals. Therapeutic activation of regulatory CD4+CD25+Foxp3+ T cells by a superagonistic anti-CD28 antibody (CD28-SA) applied at day 2 after myocardial infarction prevented, compared to mice treated with an isotype-matched control antibody, from left ventricular rupture and resulted in improved survival (47.1% survival in the control group vs. 76.6% survival in the CD28-SA treated group). CD28-SA treatment lead to expansion of CD4+CD25+Foxp3+ T-cells in the peripheral blood and increased their frequency in the infarcted myocardium. This was associated with increased expression of several molecules known to facilitate wound healing by promoting the formation of a stable scar such as osteopontin and coagulation factor XIII.

Conclusion:
CD4+CD25+Foxp3+ regulatory T-cells are a prerequisite for proper myocardial wound healing and can be therapeutically activated to improve outcome after experimental myocardial infarction.
Abstract 222: Inhibition Of Class I But Not Class IIb Lysine Deacetylases Is Responsible For Cardioprotection From Ischemia-Reperfusion.
Authors: Aune Sverre E , Mani Santhosh K , Menick Donald R
Published: July 2021

Background:
While dual inhibition of class I/IIb KDACs protects the mammalian heart from ischemia-reperfusion (IR) injury, class-specific effects have not been examined.

Hypothesis:
We hypothesized that specific inhibition of class I KDACs would preserve left ventricular (LV) function following IR in isolated hearts.

Methods:
Male Sprague-Dawley rats (n=4 per group) were injected with vehicle, the class I KDAC inhibitor entinostat, the class IIb inhibitor tubastatin A, or the class I/IIb inhibitor trichostatin A (TSA). After 18 h, hearts were isolated and perfused in Langendorff mode. A saline-filled balloon was placed in the LV to monitor contractile function. Following IR (30/120 min), LV free wall tissue was assayed for activation of pro-survival proteins. Group results are reported vs vehicle as mean±SE.

Results:
There were no differences between groups in LV function before ischemia. At the end of reperfusion, rate pressure product (mm hg/min) was improved with entinostat (27392±3474, p<0.001) and TSA (22980±3226, p<0.05) vs vehicle (11,352±1977), as were +dP/dt
max
and -dP/dt
max
. Most notably, entinostat alone blunted the rise in end diastolic pressure (mm Hg) during reperfusion (18.6±1.4 vs. 45.3±4.1, p<0.001), and completely prevented diastolic contracture (mm Hg) in reperfusion for up to one hour (12.4±2.0 vs pre-ischemia=8.7±1.7, p=0.21). Contractile function was not significantly preserved in hearts treated with tubastatin A. Entinostat reduced phosphorylation at the eNOS inhibitory site, and increased phosphorylation at the eNOS activation site, and Src activation was dramatically reduced. TSA increased activation of Src, p38, and ERK 1/2, and increased phosphorylation of eNOS at both the activation (Ser1177) and inhibition site (Thr495). Tubastatin A reduced activation of p38, ERK 1/2, and eNOS, but not Src.

Conclusions:
Inhibition of class I KDACs was most effective at preserving contractile function following IR in isolated hearts, notably through prevention of LV contracture during reperfusion. Class I KDAC inhibition may preserve LV function by differentially modulating Src and eNOS activities. These results suggest that the protective effects of KDAC inhibitors in IR can be ascribed to specific inhibition of class I KDACs.
Abstract 238: Class I HDAC inhibition prevents Ncx1 upregulation by stabilizing an HDAC5-HDAC1/Sin3a Repressor Complex during cardiac hypertrophy.
Authors: Wang Sabina , Harris Lillianne G , Mani Santhosh , Menick Donald
Published: July 2021

Cardiac hypertrophy is often associated with the activation of signaling pathways that perpetuate altered calcium efflux and influx. One gene that is upregulated and contributes to altered intracellular calcium concentrations and worsening contractility during cardiac hypertrophy is the Sodium Calcium Exchanger (
Ncx1
). Molecular studies implicate histone deacetylases (HDACs) in possibly regulating the expression of this gene. Our recent work reveals that HDAC1, HDAC5 and Sin3a interact and are recruited to the
Ncx1
promoter through the Nkx2.5 transcription factor. Interestingly, we observed greater associated/interaction of the HDAC1-HDAC5/Sin3a repressor complex upon broad HDAC inhibition. Taken together,
we hypothesized
that HDAC inhibition, stabilizes an HDAC1-HDAC5/Sin3a repressor complex during cardiac hypertrophy. We addressed this hypothesis by treating isolated adult cardiomyocytes with class specific HDAC inhibitors since HDAC1 is a Class I HDAC and HDAC5 is a Class IIa HDAC. Co-Immunoprecipitation (Co-IP) revealed a greater association of repressor complex molecules in the presence of Entinostat, a Class I HDAC inhibitor compared to both non-treated control and TSA, a broad HDAC inhibitor (n=3). These works show enhanced recruitment Sin3a (co-repressor) at the proximal promoter of NCX1 as demonstrated by Chromatin-Immunoprecipitation (ChIP) (n=3). To test whether these observations translated into
in vivo
models, we subjected mice to transaortic constriction (TAC) to induce hypertrophy. In this model, Co-IP revealed results that similar to our
in vitro
studies with greater immuno- detection of repressor complex component, Sin3a after immune-precipitation with HDAC1. Furthermore, our ChIP data showed a greater PCR product amplification of proximal Ncx1 promoter, from experimental groups that were subjected to Entinostat (n=3). Our cumulative data suggests that Class I HDAC inhibition stabilizes a repressor complex on the
Ncx1
promoter that hinders hypertrophy- mediated
Ncx1
upregulation. Class specific HDAC inhibition may be useful in the stabilization and repression of aberrantly expressed genes that contribute to poor clinical outcomes in cardiac hypertrophy.
Abstract 299: Simultaneous Cardiac And Adrenal Small Molecule GPCR-Gβ? Inhibition Halts The Progression Of Pressure Overload Heart Failure
Authors: Kamal Fadia A , Mickelsen Deanne , Moalem Jacob , Hammes Stephen R , Smrcka Alan V , Blaxall Burns C
Published: July 2021

Heart failure (HF) is a progressive disease with rapidly increasing rates of morbidity and mortality. Elevated sympathetic nervous system activity, a salient feature of HF progression, leads to pathologic attenuation and desensitization of β-adrenergic receptors (β-ARs) due in part to Gβγ-mediated signaling.

In the current study, we assessed the hypothesis that the small molecule Gβγ inhibitor “gallein” is salutary in treating pre-existing HF in a clinically relevant model (pressure-overload HF model of mouse transverse aortic constriction (TAC)) by simultaneously normalizing adrenergic receptor signaling in the heart and the adrenal gland. Four weeks post-TAC, mice received daily i.p. injections of vehicle or gallein for eight weeks (n=6-8 per group). Serial echocardiography was performed through out the study. At the end of the experiment, hemodynamic studies were performed, mice were sacrificed, blood, heart, and adrenal glands were harvested for further analysis. Gallein treatment improved survival and cardiac function and reduced cardiac hypertrophy, remodeling, and fetal genes expression in TAC mice. On the molecular level, gallein recovered membrane β-AR density and attenuated GRK2-PI3Kγ membrane recruitment, and Akt-GSK-3β signaling in TAC hearts. A salutary adrenal effect of gallein was obtained in cultured mice adrenal glands and human pheochromocytoma tissue (n=3), where direct gallein treatment restored α2-AR feedback inhibitory function and concurrently reduced catecholamine production. Moreover, gallein treatment attenuated adrenal hypertrophy in TAC mice and downregulated tyrosine hydroxylase and chromogranin A protein expression in adrenal glands from TAC mice and cultured pheochromocytoma tissue as well.

In summary, our data suggest gallein as a systemic pharmacologic therapy with substantial therapeutic benefit in HF by simultaneously normalizing pathologic Gβγ-GRK2 signaling and recovering AR signaling in both the heart and the adrenal gland. In the heart, gallein mediated attenuation of cardiac remodeling probably involves inhibiting GRK2-PI3K-Akt signaling. Our data also suggest a role for small molecule Gβγ inhibition in other diseases of elevated catecholamine release, such as pheochromocytoma.
Abstract 350: Interaction Of Methicillin-resistant Staphylococcus Aureus With Endothelial Cells Increase Intercellular Permeability By Activating Vasopressin Receptor-2.
Authors: Lopez Ernesto , Zhu Yong , Fujiwara Osamu , Enkhbaatar Perenlei
Published: July 2021

More than 700,000 people a year suffer from sepsis in the United States. From sheep studies, we reported that methicillin-resistant Staphylococcus aureus (MRSA) sepsis is associated with vascular hyperpermeability and that vasopressin receptor-2 (V2R) might be involved in this complication. Vascular hyperpermeability is associated with high mortality and new pharmacological targets are required to design new treatments. We hypothesized that in MRSA induced vascular hyperpermeability, peroxynitrite stimulates V2R to release potent angiogenic growth factor angiopoietin-2.

Methods:
Human lung microvascular endothelial cells (HMVECs) were incubated with MRSA plus peroxynitrite decomposition catalyst (FeTMPyP), angiopoietin-2 antibody or tie-2 antibody as well as with MRSA only. Permeability assay (FITC-Dextran quantification across the confluent cell monolayer) was then performed after the different treatments. To strengthen the hypothesis, HMVECs were then treated with MRSA, peroxynitrite or V2R selective agonist (desmopressin). Cell lysates from each treatment were screened for levels of V2R mRNA using RT-PCR.

Results:
1) Permeability was significantly higher in cells treated with MRSA compared to control. 2) Permeability was significantly lower in cells treated with MRSA plus FeTMPyP, angiopoietin-2 antibody, and tie-2 antibody compared to cells treated with MRSA only. 3) Cells treated with MRSA, peroxynitrite and desmopressin had significant higher levels of V2R mRNA compared to control.

Conclusion:
When MRSA interacts with endothelial cells, intercellular permeability is increased suggesting disruption of tight junctions. Peroxynitrite is a player of this phenomenon by activating V2R. Angiopoietin-2 also has an important role disrupting intercellular junctions with tie-2 receptor as the mediator. Further studies have to be performed to know whether V2R activation release angiopoietin-2.
Abstract 1: Hypoxia-Induced Decrease In Cardiac Stem Cell Proliferation Is Associated With Downregulation Of Sirtuin 1
Authors: Bellio Michael , Rodrigues Claudia O , Florea Victoria , Balkan Wayne , Hare Joshua M , Schulman Ivonne H
Published: July 2021

Background:
Myocardial infarction (MI) produces severe hypoxia within regions of the myocardium, resulting in the formation of scar tissue and significant cardiac cell death. Adult hearts contain endogenous cardiac stem cells (CSCs) that have regenerative capacity and participate in myocardial tissue homeostasis and repair post-MI, but are insufficient to promote complete regeneration. The histone deacetylase Sirtuin 1 (SIRT1) likely mediates this hypoxia-induced decrease in regeneration via roles in cell cycle progression and conferring protection from senescence and oxidative damage.

Hypothesis:
Hypoxia decreases CSC proliferation through reduced SIRT1-mediated deacetylation.

Methods and Results:
Murine CSCs were grown at room air (21% O2), physiologic (5% O2), and ischemic hypoxic (0.5% O2) conditions for 72 hours. Ischemic hypoxia, but not physiologic, reduced CSC proliferation and DNA synthesis to 25±2.0% (N=3; p<0.05) and 54±7.0% (N=7; p<0.05), respectively, relative to cells grown in room air. SIRT1 protein expression was decreased by 58±10.0% (N=4; p<0.05) and acetylation of Histone H3 Lys9 (2.4-fold; N=5; p<0.05) and p53 Lys379 (1.64-fold; N=4; p<0.05) were increased after 72 hours of growth in 0.5% compared to 21% O2. However, SIRT1 mRNA transcripts remained unchanged. Furthermore, SIRT1 protein was 59±6.0% less stable (N=4; p<0.05) following 8 hours of cyclohexamide treatment in CSCs exposed to 0.5% O2 for 72 hours compared to CSCs exposed to 21% O2. SIRT1 knockdown by RNA interference significantly reduced proliferation of CSCs grown in room air (N=3 and, p<0.05), similar to that observed in un-transfected cells grown under ischemic hypoxia conditions.

Conclusion:
The decrease in CSC proliferation with hypoxia is in part due to a reduction in SIRT1 protein stability. These results suggest that SIRT1 expression is regulated post-translationally and support a role for SIRT1 in preserving CSC self-renewal under hypoxic conditions.
Abstract 111: Paradoxical Exacerbation of Arrhythmias by the Cardioprotective Mitochondrial K-ATP Channel Agonist Diazoxide in Type 2 Diabetes Mellitus
Authors: Xie Chaoqin , Karam Basil S , Akar Fadi G
Published: July 2021

Type 2 diabetes mellitus (T2DM) is a major risk factor for cardiovascular complications including ischemia reperfusion injury (IR). Activation of mitochondrial KATP channels by Diazoxide (DZX) promotes beta cell rest and suppresses glucose production in patients. We hypothesized that DZX prevents IR arrhythmias in T2DM owing to its dual cardioprotective & antidiabetic property.

Methods:
Obese Zucker Diabetic Fatty (ZDF) rats (n=17) with established T2DM were studied. Control groups consisted of lean ZDF (n=6) and normal Sprague Dawley (n=10) rats. High resolution optical action potential (AP) mapping was performed in hearts before and after challenge with no flow ischemia for 12min followed by reperfusion.

Results:
Basal properties including rate dependence of conduction velocity (CV) and AP duration (APD) were not significantly (p=NS) altered in T2DM. Remarkably, ischemia uncovered major differences between groups as APD in T2DM failed to adapt to the ischemic challenge. Unlike APD, CV was reduced in all groups. DZX paradoxically promoted arrhythmias as all DZX (30uM) treated T2DM hearts exhibited ischemia related VT. In contrast, untreated T2DM (0/5) and control (0/9) hearts did not exhibit VT during ischemia. Underlying arrhythmic vulnerability of DZX treated T2DM hearts was a pronounced reduction (by 75% p<0.01) of the cardiac wavelength (WL) caused by accelerated APD shortening in response to ischemia. Upon reperfusion, T2DM and control groups exhibited a high (60% T2DM vs 44% control, p=NS) rate of VT, although the VT cycle length in T2DM was significantly longer (72 vs 44ms, p<0.01) suggesting a more adverse substrate modification by IR injury. Blocking the inner membrane anion channel (IMAC) fully abrogated reperfusion VT in T2DM.

Conclusion:
Ischemia uncovers a paradoxical resistance of T2DM hearts to APD adaptation. DZX reverses this property resulting in accelerated APD & WL shortening. This promotes reentrant VT during ischemia. Therefore, the anti-diabetic agent DZX should be avoided in T2DM patients at risk of ischemic events. Instead IMAC is an effective antiarrhythmic target for these patients.
Abstract 117: RhoC Regulates VEGF-induced Signaling in Endothelial Cells
Authors: Hoeppner Luke , Sinha Sutapa , Wang Ying , Bhattacharya Resham , Dutta Shamit , Gong Xun , Bedell Victoria , Suresh Sandip , Chun Chang zoon , Ramchandran Ramani , Ekker Stephen , Mukhopadhyay Debabrata
Published: July 2021

Vascular permeability factor/vascular endothelial growth factor A (VEGF) is a central regulator of angiogenesis and potently promotes vascular permeability. VEGF plays a key role in the pathologies of heart disease, stroke, and cancer. Therefore, understanding the molecular regulation of VEGF signaling is an important pursuit. Rho GTPase proteins play various roles in vasculogenesis and angiogenesis. While the functions of RhoA and RhoB in these processes have been well defined, little is known about the role of RhoC in VEGF-mediated signaling in endothelial cells and vascular development. Here, we describe how RhoC modulates VEGF signaling to regulate endothelial cell proliferation, migration and permeability. We found VEGF stimulation activates RhoC in human umbilical vein endothelial cells (HUVECs), which was completely blocked after VEGF receptor 2 (VEGFR-2) knockdown indicating that VEGF activates RhoC through VEGFR-2 signaling. Interestingly, RhoC knockdown delayed the degradation of VEGFR-2 compared to control siRNA treated HUVECs, thus implicating RhoC in VEGFR-2 trafficking. In light of our results suggesting VEGF activates RhoC through VEGFR-2, we sought to determine whether RhoC regulates vascular permeability through the VEGFR-2/phospholipase Cγ (PLCγ) /Ca
2+
/eNOS cascade. We found RhoC knockdown in VEGF-stimulated HUVECs significantly increased PLC-γ1 phosphorylation at tyrosine 783, promoted basal and VEGF-stimulated eNOS phophorylation at serine 1177, and increased calcium flux compared with control siRNA transfected HUVECs. Taken together, our findings suggest RhoC negatively regulates VEGF-induced vascular permeability. We confirmed this finding through a VEGF-inducible zebrafish model of vascular permeability by observing significantly greater vascular permeability in RhoC morpholino (MO)-injected zebrafish than control MO-injected zebrafish. Furthermore, we showed that RhoC promotes endothelial cell proliferation and negatively regulates endothelial cell migration. Our data suggests a scenario in which RhoC promotes proliferation by upregulating -catenin in a Wnt signaling-independent manner, which in turn, promotes Cyclin D1 expression and subsequently drives cell cycle progression.
Abstract 119: Cardiac Transcription Factor EB Sumoylation Deficiency Exacerbates Age-associated Reduction In Autophagy
Authors: Ma Heng , Zhang Le , Yu Lu , Li Yan
Published: July 2021

Background:
Aging-dependent decline of autophagy contributes to cardiac dysfunction and ischemic intolerance. Transcription factor EB (TFEB) is a master transcriptional regulator of the autophagy-lysosome pathway. The present study aimed to characterize the role of TFEB in the autophagic decrease with aging.

Methods and Results:
We analyzed age-associated autophagic changes in male C57BL/6 young (4-6 mo) and aged (22-24 mo) mice. The results demonstrated that TFEB expressed predominantly as a SUMOylated form in cardiomyocyte nuclei and this SUMOylation of TFEB declined in aged heart associated with autophagy reduction. Interestingly, SUMOylation of TFEB was unaffected by rapamycin. Rapamycin induced translocation of TFEB into nucleus but lower level of nuclear TFEB in aged hearts than that seen in young hearts (P<0.05). SUMO1 downregulation by adeno-associated-virus-mediated small hairpin RNA (rAAV9-shSUMO1) significantly reduced nuclear TFEB levels (P<0.05), depressed cardiac autophagy and accelerated cardiomyocyte contractile dysfunction with worse hypoxia/reoxygenation (H/R) injury (all P<0.05). Therefore, impaired SUMOylation decreased nuclear TFEB during aging. By contrast, SUMO1 restitution significantly augmented nuclear SUMOylated TFEB with enhanced autophagy and ultimately reduced infarct size in aged heart. However, knockdown of cardiac TFEB blocked the protective effect of upregulation of SUMO1 in aged hearts, resulted in decline of autophagy and worse in vivo I/R injury.

Conclusions:
The present study newly demonstrates that SUMOylation is a critical post-translational modification that regulates cardiac TFEB. Impaired SUMOylation of TFEB aggravates decline of autophagy in the senescent heart. Targeting SUMO1 may provide a novel therapeutic strategy for the treatment of aging-related loss of cardioprotection.
Abstract 128: Pim1 kinase Overexpression Enhances ckit+ Cardiac Stem Cells Cardioreparative Ability After Intramyocardial Delivery
Authors: Karantalis Vasileios , Suncion Viky Y , McCall Frederic C , Bagno Luiza L , Wang Bo , Golpanian Samuel , Mohsin Sadia , Rodriguez Jose E , Valdes David , Rosado Marcos , Balkan Wayne , Mushtaq Muzammil , Susman Mark A , Hare Joshua M
Published: July 2021

Background:
Pim-1 kinase plays an important role in cell division, survival and commitment towards myocardial lineage. We hypothesized that Pim-1 overexpression in ckit+ cardiac stem cell (CSCs) enhances cardioreparative effects.

Methods:
Immunosuppressed Yorkshire swine (n=31) received human ckit+ CSCs (n=9), Pim1 modified human ckit+ CSCs (n=9) or PBS (n=13) two weeks after myocardial infarction. Cardiac MRI and PV loops were obtained before and after cell administration.

Results:
At 8 weeks post transplantation, scar mass (Fig. 1A), viable tissue (Fig. 1B), ejection fraction (Fig. 1C) and stroke work (Fig. 1D) was significantly improved in Pim-1 modified ckit+ CSC compared to control ckit+, while both cell groups showed partial recovery compared to placebo (two way ANOVA, p<0.05). Both cell types similarly reduced preload (end diastolic pressure; Fig. 1E) and afterload (Arterial elastance; Fig 1F) compared to placebo, while only administration of Pim-1 CPCs improved regional contractility at both the infarct (Fig. 1G) and border zones (Fig. 1H). Collectively, mechanoenergetic recoupling was superior in the Pim-1 group compared to ckit+ controls (Cardiac Efficiency; Fig. 1I).

Conclusions:
Cardioreparative potential of CSCs delivered by intramyocardial injection to infarcted porcine hearts is significantly enhanced by overexpress Pim1, supporting translational development of Pim-1 as a validated genetic modification of CSCs for incorporation into clinical trials.
Abstract 143: Evaluation of the Paracrine Effect of Encapsulated Mononuclear Stem Cells in the Acute Myocardial Rat Model
Authors: Tobar Santiago Alonso L , Olsen Virgílio , Phaelante Amanda P , Silvello Daiane , Biolo Andréia , Rohde Luis E , Clausell Nadine O , Andrades Michael
Published: July 2021

Many studies have considered stem cell-based therapy as a treatment option for acute myocardial infarction (AMI). Paracrine signaling has been proposed as an underlying mechanism, in which released cytokines and chemokines may promote angiogenesis and activation of resident stem cells. In order to characterize this paracrine action, bone marrow mononuclear stem cells (BM-MNC) collected from femur of adults GFP+ Wistar rats were encapsulated in 1.5% of sodium alginate. Animals were randomized in three groups: SHAM (n=3); Empty capsules (n=8); BM-MNC capsules (n=8). AMI was induced by permanent occlusion of the left anterior descending artery after anesthesia with 100mg/kg ketamin and 10 mg/kg xylazine. Soon after the AMI, capsules (empty or BM-MNC) were delivered intra-thoracically. SHAM group was submitted to the same surgical procedures without permanent artery occlusion or treatment given. Troponin I (cTnI) was measured 24h after AMI in order to evaluate the success of the procedures; echocardiography was also performed to assess heart morphofunctional parameters 48h and 7 days after AMI. At day 7, after echocardiography, the animals were euthanized under profound anesthesia (isoflurane 5%) and their hearts were withdrawn for biochemical analysis. Plasma and tissue levels of TNF-α and IL-6 were measured by ELISA. All technical procedures were performed by blinded operators. Statistical comparisons were made using ANOVA analysis followed by Tukey post-test or using t-Student test when appropriated. BM-MNC were viable after day 7 since GFP+ cells were detected by fluorescence microscopy. Nevertheless, the empty capsules groups showed lower levels cTnI compared to BM-MNC group (25 vs. 40 ng/mL, respectively; p=0.03). There was no difference in the shortening fraction (24% VS. 18%, respectively; p=0.08) and in the infarcted area (32% vs. 43%, respectively; p=0.10) when both AMI groups (Empty and BM-MNC) were compared. Also TNF-α and IL-6 levels showed no difference between all groups. We concluded that paracrine effects of cell-based therapy with BM-MNC was unable to modulate events associated to AMI in rat in spite of cell viability after 7 days of implantation.
Abstract 148: Parkin Mediates Clearance of Dysfunctional Mitochondria via Multiple Pathways
Authors: Hammerling Babette C , Cortez Melissa Q , Hanna Rita A , Jimenez Rebecca E , Gustafsson Åsa B
Published: July 2021

The ability to clear damaged mitochondria is critical to prevent unnecessary death. Studies have found that dysfunctional mitochondria are rapidly sequestered by autophagosomes and subsequently delivered to lysosomes for degradation. The E3 ubiquitin ligase Parkin has been identified as an important regulator of mitochondrial autophagy. We have previously shown that Parkin plays an important role in clearing dysfunctional mitochondria via autophagy in the heart after myocardial infarction. In this study, we have discovered that Parkin also induces clearance of mitochondria via an autophagy-independent pathway. We found that Parkin mediated clearance of damaged mitochondria in both wild type (WT) and autophagy-deficient Atg5 knockout mouse embryonic fibroblasts (MEFs) treated with the mitochondria uncoupler FCCP. Interestingly, mitochondrial clearance in both cell types was dependent on the presence of Parkin, suggesting that Parkin represents a rate-limiting step. Immunofluorescence analysis revealed that FCCP-treatment resulted in activation of the Vps34-Rab5 complex with subsequent sequestration of mitochondria inside Rab5-positive endosomes and LAMP2-positive lysosomes in Atg5-/- MEFs. The presence of mitochondria inside endosomes in Atg5-/- MEFs was confirmed by transmission electron microscopy (TEM). Pharmacological inhibition of the endosomal-lysosomal pathway with 3-methyladenine or Bafilomycin A1 caused a significant increase in FCCP-mediated cell death in Atg5-/- MEFs. Also, although BNIP3 functions as an autophagy receptor on mitochondria by interacting with LC3II on the autophagosome, it induced mitochondrial clearance in Atg5-/- MEFs via activation of the endosomal pathway. Finally, we confirmed that mitochondrial clearance occurs via both the autophagy and endosomal pathways in neonatal cardiomyocytes subjected to simulated ischemia/reperfusion (I/R). TEM analysis revealed the presence of mitochondria inside endosomes rat hearts subjected to ex vivo I/R. These data demonstrate that both autophagy and endosomal pathways contribute to clearance of damaged mitochondria in cells, and represent potential future therapeutic targets to enhance or preserve mitochondrial clearance in patients after MI.
Abstract 169: Elucidating the role of GRK5 in Physiological Hypertrophy
Authors: Traynham Christopher J , Cannavo Alessandro , Hullmann Jonathan , Gold Jessica L , Koch Walter J
Published: July 2021

Cardiac function is dynamically regulated by various G protein-coupled receptors (GPCRs). GPCR kinases (GRKs) are important in cardiac GPCR regulation through phosphorylation and desensitization of these receptors. GRK2 and GRK5 are the predominant GRKs in the heart, and the most widely characterized as they are both up-regulated in the failing heart. Prior studies from our Lab have determined that GRK5 plays a crucial role in pathological cardiac hypertrophy. Another type of hypertrophy termed, “physiological hypertrophy” occurs with exercise training and is defined as an enlargement in cardiac myocyte size leading to favorable cardiac adaptations. At present, it is unclear if GRK5 is a regulator of physiological hypertrophy in addition to its role in maladaptive hypertrophy. We hypothesize that GRK5 will not regulate physiological hypertrophy such that mice with cardiac-specific overexpression of GRK5 (TgGRK5) will yield a similar post-exercise cardiac physiological hypertrophic response as that of control wild-type (WT) mice. To test this hypothesis, TgGRK5 and WT mice were exposed to a 21 day high-intensity swimming exercise protocol. For each line, sham mice, which did not swim served as appropriate controls. At the conclusion of this protocol, mice were sacrificed and heart weight (HW), body weight (BW), and tibia length (TL) measured. TgGRK5 and WT mice both exhibited a characteristic 10-15% increase in HW/BW and HW/TL ratios, which are standard measures of cardiac hypertrophy. In addition, hearts were sectioned and H&E stained to evaluate myocyte size. Both TgGRK5 and WT mice exhibited a significant increase in myocyte size. Cardiac function was evaluated via echocardiography both prior to and after exercise training, and no changes were observed between TgGRK5 and WT mice after training. These data were re-affirmed in H9C2 cells and neonatal rat ventricular myocytes overexpressing either GFP or GRK5, which exhibited similar increases in cell size and AKT phosphorylation after IGF-1 treatment, a physiological hypertrophy stimulus. Taken together, these data suggest that physiological hypertrophy is similar in both control and TgGRK5 mice, confirming that GRK5 is solely a regulator of pathological cardiac hypertrophy.
Abstract 17: The Comparison of Adipose Derived Regenerative Cells and Bone Marrow Mononuclear Cells as Transplanted Cells for Therapeutic Angiogenesis
Authors: Shintani Satoshi , Hao Changning , Shimizu Yuuki , Kondo Kazuhisa , Murohara Toyoaki
Published: July 2021

Background:
Transplantation of adipose-derived regenerative cells (ADRCs) enhances ischemia-induced angiogenesis, but the underlying mechanism remains unknown. Here, we compared the efficacy of ADRC transplantation with bone marrow mononuclear cell (BM-MNC) treatment using a hindlimb ischemia (HLI) rabbit model, and examined if the anti-inflammatory phenotypic polarization of macrophages regulates postnatal neovascularization.

Methods and Results:
ADRCs and BM-MNCs were isolated from New Zealand White (NZW) rabbits and C57BL/6J mice. In the rabbit studies, ADRCs were incorporated into the existing vascular formation in vitro and in vivo. ADRC-conditioned media (CM) and BM-MNC-CM similarly enhanced the migratory ability and prevented apoptosis induction in human umbilical vein endothelial cells. Four weeks after treatment, NZW rabbits administered with either ADRCs or BM-MNCs revealed enhanced collateral vessel formation and functional blood flow recovery. In mice, lipopolysaccharide and/or hypoxic stress increased the level of prostaglandin E2 (PGE2), and led to the polarization of M2 macrophages in cultured ADRCs. Gene expressions of several angiogenic cytokines were amplified in macrophages cultured in ADRC-CM rather than BM-MNC-CM. The expression of interleukin (IL)-10 was increased in ischemic muscles of mice treated with ADRCs compared with those from BM-MNC- treated and control groups. The blockade of IL-10 using a neutralizing antibody attenuated ischemia-induced angiogenesis in vivo.

Conclusions:
The therapeutic angiogenesis potential of ADRCs was comparable with those of BM-MNCs in healthy animals. Anti-inflammatory phenotypic polarization of macrophages plays an important role in the regenerative action of ADRCs through the PGE2-EP2/4 axis.
Abstract 171: T Cell Mediated Immune Responses Regulate Cardiac Remodeling and Survival in Pressure Overload Induced Heart Failure
Authors: Alcaide Pilar , Nevers Tania , Salvador Ane , Grodecki-Pena Anna , Knapp Andrew , Aronovitz Mark J , Velazquez Francisco , Kershaw Tanya , Kapur Navin K , Blanton Robert , Karas Richard
Published: July 2021

Background:
Clinical data support that inflammation and the improper regulation of the immune response are intimately associated with Heart Failure (HF), however, the type of immune response involved and whether it regulates cardiac remodeling remains largely unexplored. We hypothesize that T cell mediated immune responses and their recruitment into the heart influence cardiac remodeling and contribute to the pathogenesis of pressure overload induced HF.

Methods and Results:
Using quantitative flow cytometry we found that T cells infiltrated the heart as Wild-type mice (WT) developed systolic dysfunction and LV hypertrophy in response to transverse aortic constriction (TAC) (p<0.01 TAC
vs
Sham). Real time imaging demonstrated that T cells from TAC mice adhered to activated heart endothelial cells in higher numbers than T cells from Sham mice under physiological flow conditions in vitro (P<0.05) indicating a systemic T cell activation to pressure overload induced by TAC. Similarly, circulating T cells from patients with HF adhered more to activated human umbilical vein endothelial cells (HUVEC) than T cells from healthy volunteers. Based on these findings, we performed similar TAC studies in T cell deficient mice (TCRα
-/-
). In contrast with WT TAC mice, TCRα
-/-
had preserved LV systolic and diastolic function (p<0.01) determined by echocardiography and hemodynamic studies, reduced LV fibrosis (p<0.001) and TGFβ1, collagen Iα and αSMA gene expression (p<0.05), and reduced LV hypertrophy and gene expression of ANP and BNP (p<0.05), but unaltered expression of SerCA. Remarkably, TCRα
-/-
had improved survival after 4 weeks of TAC [100%(16/16) TCRα
-/-
vs
73.7%(14/19) WT, p=0.023]. Ongoing studies will determine the mechanisms regulating T cell recruitment into the heart, the type of T cell response involved and its contribution to pathological remodeling of the heart.

Conclusion:
Our studies demonstrate that T cell immune responses and their recruitment into the LV contribute to the pathogenesis of pressure overload induced HF by mechanisms involving T cell regulation of cardiac hypertrophy and fibrosis, and open a window to develop novel therapeutic strategies to improve the structural, functional and molecular deficits of the failing heart.
Abstract 194: Relaxin Receptor-Ligand Expression in a Fibrotic Environment
Authors: Haney Jamie L , Schwartzman David S , Shroff Sanjeev G
Published: July 2021

The renin-overproducing transgenic rat (mREN2) has recently been proposed as a model of left ventricular (LV) hypertension and fibrosis. Comparing mREN2 (n=7) to sex- and age-matched Wistar-Han control rats (WHAN, n=11), we have confirmed hypertension (mean blood pressure: 174±9 vs. 107±3 mmHg, P<0.001), hypertrophy (LV weight:body weight: 4.0±0.1 vs. 2.7±0.1 mg/g, P<0.001), and cardiac fibrosis. At the protein level, LV from mREN2 exhibited significantly higher percentage of interstitial collagen per total protein area versus WHAN (5.95±1.01% vs. 1.96±0.06%, P=0.02). At the level of LV function, mREN2 hearts demonstrated significant diastolic dysfunction (increased stiffness coefficient from stress-strain relationship: 198±30 vs. 78±10, P=0.01, and reduced magnitude of dP/dtmin: 1254±125 vs. 2000±110 mmHg/s, P<0.001), and impaired relaxation (increased relaxation time constant: 0.048±0.001 vs. 0.043±0.001 s, P=0.002). The naturally occurring hormone relaxin is known to have antifibrotic properties in multiple organ systems and it affects both synthesis and degradation aspects of collagen homeostasis. It is known that relaxin (RLN1) and receptor (RXFP1) are expressed locally in LV tissue, yet the biological function of the endogenous relaxin message-receptor system is not fully understood. Interestingly, the relative mRNA expressions of relaxin and relaxin receptor were increased in this model of LV fibrosis (2-ΔΔCT by qRT-PCR for RLN1: 3.04±0.56 (mREN2) vs. 1.0±0.18 (WHAN), P=0.001; and RFXP1: 7.06±1.57 (mREN2) vs. 1.00±0.20 (WHAN), P=0.003). Thus, the unregulated endogenous relaxin-receptor system in the mREN2 fibrosis model seems to be an adaptive response which is trying to keep the fibrosis in check. However, this endogenous adaptive response is insufficient to completely block collagen accumulation and prevent LV diastolic dysfunction. We are currently conducting experiments to examine whether exogenous administration of relaxin can assist the endogenous adaptive response and reverse LV fibrosis and normalize diastolic function in this model.
Abstract 203: Differences Between Mammalian and
Drosophila
Mitochondrial Calcium Handling Help Identify Mechanisms Regulating the Permeability Transition.
Authors: Chaudhuri Dipayan , Clapham David E
Published: July 2021

Studies of cardiomyocyte death during calcium overload induced by ischemia-reperfusion injury or heart failure have implicated the mitochondrial permeability transition as a key pathway. During the permeability transition, an opening of a channel in the inner membrane leads to mitochondrial depolarization and swelling. Despite extensive studies in mammalian systems, the machinery responsible for this phenomenon remains only partially identified. If present in non-mammalian species, the components of the permeability transition may be further elucidated, given potential advantages within these systems for high-throughput screens. However, the existence of a permeability transition remains controversial in non-mammalian organisms. In
Drosophila
, prior studies have documented calcium-induced mitochondrial depolarization, but no obvious swelling. Here we show that
Drosophila
S2R+ cells do possess the machinery for permeability transition, but that the threshold for a calcium trigger is significantly higher than in mammalian systems. Using a calcein-loading method, we show that
Drosophila
permeability transition can be triggered by calcium overload, using ionomycin, and by cysteine oxidation, using phenylarsine oxide. As in mammalian systems, blockade of mitochondrial cyclophilin or the ATP/ADP transporter appears to inhibit the
Drosophila
permeability transition. Finally, we examine three alternative hypotheses that may explain these differences in permeability transition. First, we test if perturbing the pathways for calcium influx into S2R+ mitochondria can trigger this phenomenon. Second, we test if the discrepancy in the calcium threshold is due to structural differences in the key regulators, particularly the mitochondrial cyclophilin. Third, we compare
Drosophila
and human genomes to see if any novel molecules may be responsible for setting the lower threshold for calcium-induced permeability transition in mammalian cells. Since the
Drosophila
cells possess such significant resistance to permeability transition, the results of our investigations suggest potential new strategies for the development of therapeutics inhibiting mitochondrial permeability transition in cardiac calcium-induced injury.
Abstract 206: Accumulation of Mitochondrial DNA Mutations Impairs Survival, Proliferation, and Differentiation of Cardiac Progenitor Cells
Authors: Orogo Amabel M , Kubli Dieter A , Murphy Anne N , Gustafsson Åsa B
Published: July 2021

Activation and participation of cardiac progenitor cells (CPCs) in regeneration are critical for effective repair in the wake of pathologic injury. Stem cell activation and commitment involve increased energy demand and mitochondrial biogenesis. To date, little attention has been paid to the importance of mitochondria in CPC survival, proliferation and differentiation. CPC function is reduced with age but the underlying mechanism is still unclear. Mitochondrial DNA (mtDNA) is more susceptible to oxidative attacks than nuclear DNA due to its proximity to the mitochondrial respiratory chain and lack of protective histone-like proteins. With age, mtDNA accumulates mutations that can impair mitochondrial respiration and increase ROS production. In this study, we examined the effects of accumulating mtDNA mutations on CPC proliferation and survival. We have found that incubation of uncommitted c-kit+ CPCs in differentiation medium increased mitochondrial mass and expansion of the mitochondrial network, which correlated with increased cell size and expression of cardiac lineage commitment markers. Differentiation activated mitochondrial biogenesis, increased mtDNA copy number, and enhanced oxidative capacity and cellular ATP levels in CPCs. To investigate the effect of mtDNA mutations and aging on CPC survival and function, we utilized a mouse model in which a mutation in the mtDNA polymerase γ (POLG
m/m
) leads to accumulation of mtDNA mutations, mitochondrial dysfunction, and accelerated aging. Isolated CPCs from hearts of 2-month old POLG
m/m
mice had reduced proliferation and were more susceptible to oxidative stress and chemotherapeutic agents compared to WT CPCs. The majority of POLG
m/m
CPCs contained fragmented mitochondria as shown by immunostaining. Incubation in differentiation medium resulted in fewer GATA-4 positive POLG
m/m
CPCs compared to WT CPCs. The reduced differentiation in these POLG
m/m
CPCs correlated with reduced PGC-1α expression and OXPHOS protein levels, suggesting that mitochondrial biogenesis is impaired. These data demonstrate that mitochondria play a critical role in CPC function, and accumulation of mtDNA mutations impairs CPC function and reduces their repair potential.
Abstract 217: Use of Magnetic Resonance Imaging Method For Quantification Of Myocardial Salvage In A Rat Model Of Ischemia-reperfusion Injury.
Authors: Grieve Stuart M , Mazhar Jawad , Callaghan Fraser , Kok Cindy Y , Bhindi Ravinay , Figtree Gemma A
Published: July 2021

Introduction:
Quantification of myocardial “area-at-risk” (AAR) and infarct (MI) zone is critical for assessing novel therapies targeting myocardial ischemia-reperfusion (IR) injury. The current standard method involves perfusion with Evan’s Blue (EB), staining with TTC and manual slicing and analysis. We have developed an MRI method for quantifying MI and AAR in whole hearts which provides superior 3D resolution to the standard approach.

Methods:
Rats were given an IR injury, recovered for 24 hours then infused with Gd-DTPA via the tail vein. The coronary artery was then religated, and a solution containing both iron oxide microparticles and EB was infused. For comparison, hearts were then harvested and transversally sectioned for TTC staining. Some hearts were kept intact for MRI only analysis. Ex-vivo MRI T2* and T1 images were acquired on a 9.4T magnet. The AAR (red) was quantified by comparing the T2* signal loss in perfused regions (blue) and high T1-signal in infarcted zones (white) from Gd-DTPA retention.

Results:
MRI and EB/TTC measures on the same slice for both AAR and MI were highly correlated (r=0.92-0.94;p<0.05). 3D MRI acquisition and analysis of whole hearts reduced intra-observer variability, and automated segmentation and analysis further reduced inter-observer variation.

Conclusion:
This novel MRI technique allows precise assessment of infarct and AAR zones and is highly suited to automation of both analysis and acquisition. This method could remove the need for tissue slicing, and via a centralised MRI facility, could permit 3D digital analysis of hearts at high anatomical resolution, accessible for all laboratories already performing IR experiments.