bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 213: Deletion of 12/15 Lipoxygenase and Fatty Acids Interaction Alters Leukocyte Kinetics Leading to Improved Healing Following Myocardial Infarction
Authors: Halade Ganesh V , Kain Vasundhara , Ingle Kevin A , Kabarowski Janusz H , Barnes Stephen , Prabhu Sumanth D
Published: July 2021

The 12/15 lipoxygenase (LOX) enzyme catalyzes oxygenation of fatty acids to form lipid mediators leading to non-resolving inflammation. However, how 12/15LOX interacts with PUFA (polyunsaturated fatty acids) in post-myocardial infarction (MI) cardiac healing is unclear. Here we assessed the role of 12/15LOX in post-MI cardiac remodeling in a PUFA (10% w/w, 22 Kcal) enriched environment. C57BL/6J wild-type (WT) and 12/15LOX null (12/15LOX
–/–
) male mice of 8-12-weeks age were fed PUFA-enriched diet for 1 month and subjected to permanent coronary artery ligation. Post-MI mice were monitored for day (d)1 or d5 along with standard diet (SD)-fed MI controls. No-MI surgery mice served as d0 controls. 12/15LOX and PUFA+12/15LOX
-/-
mice reduced infarcted wall thinning with decreased lung edema index at d5 post-MI (all p<0.05) despite comparable infarct area (49-52%). PUFA+WT and 12/15LOX
-/-
mice showed improved ejection fraction with reduced left ventricle (LV) hypertrophy index than SD+WT at d5 post-MI (p<0.05). The neutrophil density was decreased in both PUFA+WT and 12/15LOX
-/-
mice at d1 post-MI (P<0.05). The neutrophil clearance was rapid in 12/15LOX
-/-
alone and PUFA+12/15LOX
-/-
mice, with increases in expression of
formyl peptide receptor 2
at d5 post-MI. The macrophage density was decreased in PUFA+WT and 12/15LOX
-/-
mice compared with their respective SD+WT control at d5 post-MI. PUFA+12/15LOX
-/-
mice displayed an increased expression of
ccl2
(all p<0.05) in the infarct area. 12/15LOX deletion stimulated 5-LOX and heme oxygenase-1 in PUFA+12/15LOX
-/-
mice with increased levels of 9-and 13-hydroxyoctadecadienoic acid indicating improved resolution of inflammation at d5 post-MI. The PUFA+12/15LOX
-/-
mice displayed higher expression of proresolving macrophages
ym-1, mrc-1
and
arg-1
compared with SD+12/15LOX
-/-
mice at d5 post-MI (all p<0.05). Further, 12/15LOX
-/-
mice with or without PUFA showed reduced collagen deposition at d5 post-MI compared to SD+WT. In conclusion, deletion of 12/15LOX and short-term PUFA exposure attenuated cardiac remodeling via shortening the pro-inflammatory window, thus leading to an improved resolution of inflammation and LV function post-MI.
Abstract 221: miR-19b Protects Myocardial Ischemia Reperfusion Injury
Authors: Lv Dongchao , Ding Shengguang , Chen Ping , Bei Yihua , Zhong Chongjun , Xiao Junjie
Published: July 2021

Ischemia-reperfusion injury (IRI) following acute myocardial infarction (AMI) has no effective treatment and a poor prognosis. microRNA (miRNA)-19b is a key functional member of miRNA-19-72 cluster family, regulating cellular proliferation, apoptosis, differentiation, and metabolism. Dysregulation of the miR-19b cluster is critically involved in a spectrum of cardiovascular diseases. However, the role of miR-19b in myocardial IRI is unknown. In this study, we found that miR-19b was downregulated in a mouse model of IRI. Meanwhile, about 50% downregulation of miR-19b was detected in H2O2-treated H9C2 cells mimicking myocardial IRI. We also found that overexpression of miR-19b decreased H2O2-induced apoptosis (36.02%±3.92% vs 29.34%±0.79% in nc-mimics vs miR-19b-mimics, respectively) and necrosis (23.11%±1.64% vs 18.76%±0.71% in nc-mimics vs miR-19b-mimics, respectively), and increased proliferation of H9C2 cells in vitro, while downregulation of miR-19b had reverse effects. Furthermore, PTEN, a previously validated target gene of miR-19b, has been found to be negatively regulated by miR-19b at protein levels in H9C2 cells. These data reveal the potential of miR-19b as a therapeutic target for myocardial IRI.
Abstract 231: Bendavia, a Novel Mitochondria-Targeting Peptide, Improves Contraction and Relaxation of Failing Cardiomyocytes Isolated From Dogs With Chronic Heart Failure
Authors: Sabbah Hani N , Gupta Ramesh C
Published: July 2021

Background:
Mitochondria (MITO) of humans and dogs with heart failure (HF) manifest functional abnormalities characterized by poor respiration, reduced rate of ATP synthesis and increased production of reactive oxygen species (ROS) that adversely impact LV systolic and diastolic function. We previously showed that chronic therapy with Bendavia (BEN, MTP-131), a novel mitochondria-targeting peptide, improves global LV function in dogs with HF without affecting heart rate or blood pressure. This improvement was associated with a reversal of MITO abnormalities and normalization of rate of ATP synthesis. This study tested the hypothesis that the improvement in global LV function seen in dogs with HF during treatment with BEN results primarily from enhanced function of constituent LV cardiomyocytes.

Methods:
Cardiomyocytes were isolated from the LV free wall of 8 untreated dogs with coronary microembolization-induced HF (LV ejection fraction <30%). A collagenase-based enzymatic process was used for the isolation and yielded ~70% viable rod-shaped cardiomyocytes that excluded trypan blue. Extent of cardiomyocytes shortening, shortening velocity and lengthening velocity were assessed during 1.0 Hz electrical field stimulation delivered via a MyoPacer (ION Optix). Measurements were made at baseline and were repeated after one hour of gradual exposure of the same cardiomyocytes to BEN at a concentration of 0.1 μM.

Results:
At baseline, the extent of cardiomyocyte shortening was 3.7 ± 0.8 μm, shortening velocity was 62.8 ± 16.9 μm /sec and lengthening velocity was -53.8 ± 16.5 μm/sec. Exposure of cardiomyocyte to BEN significantly increased the extent of cardiomyocyte shortening to 5.4 ± 1.1 μm (p<0.012), shortening velocity to 94.5 ± 21.9 μm/sec (p<0.020) and lengthening velocity to -96.8 ± 21.1 μm/sec (p<0.016) compared to baseline.

Conclusions:
Exposure of failing isolated cardiomyocytes to BEN elicits improvements in the rate of cardiomyocyte shortening and re-lengthening indicative of improved cell contractility and relaxation. This improvement is likely mediated by increased availability of ATP along with reduced ROS production both secondary to improved mitochondrial function elicited by treatment with BEN.
Abstract 237: The First Direct Measure of Chylomicron Metabolism in Heart Reveals a Reciprocal Balance Between Mitochondrial Oxidation of Fats From Chylomicron vs Albumin Sources
Authors: O’Donnell J. Michael , Lewandowski E. Douglas , Subbaiah Papasani V
Published: July 2021

Nearly all studies of fat metabolism in healthy and diseased heart are based on albumin sources of fat, even though lipoproteins (chylomicrons, VLDL) are thought to be a major source of fat. If there are distinct differences in uptake, oxidation, and storage of fatty acids (FA) from lipoproteins compared to albumin sources, then our understanding of disease is far from complete. This study provides the very first investigation of the fate of 13C labeled FA from chylomicrons (CM) in heart, with quantified results of the direct competition between CM-FA vs albumin-FA to support mitochondrial ATP synthesis. Chylomicrons were biosynthetically labeled with 13C-oleate by delivering to the duodenum of rat either (a) emulsified in olive oil, or (b) incorporated into micelles. The labeled chylomicrons were collected as lymph via the thoracic duct for six hours. The olive oil approach, used traditionally to prepare CM to contain radiolabeled fats, resulted in a trace fraction of 13C-labeled FA (<2%). The micelle approach enabled 50% enrichment of CM-FA store. Metabolic studies: Isolated rat hearts were perfused with KH buffer containing 13C-chylomicrons, FA free albumin, and glucose; or 13C-chylomicrons, unlabeled oleate complexed to albumin (equimolar FA from CM and albumin; 0.4 mM), and glucose. Hearts were freeze-clamped, and FA oxidation was assessed via high resolution 13C NMR of heart samples. CM-FA provided 50 ± 5 % of the acetyl CoA to support citric acid cycle synthesis of ATP when FA free albumin was used. In the presence of albumin-FA, only 12 ± 2% of acetyl CoA entering the cycle was from the CM-FA. These direct observations of the competition between CM-FA vs albumin-FA counter prior interpretations based on indirect measures. Those studies suggested no change in CM-FA oxidation when albumin-FA was added. Our novel approach enables direct measurements of CM-FA and albumin-FA oxidation in the mitochondria of intact hearts. The uptake and distribution of fat from either exogenous source were found to contribute to a common intracellular source of activated FA, with mitochondrial oxidation being proportional to the availability of the FA source. Importantly, this new evidence for a common pool significantly simplifies our metabolic paradigm of cardiac disease.
Abstract 244: A Novel Method for the Generation of Induced Pluripotent Stem Cells From Human Peripheral Blood
Authors: Yang Han-Mo , Kim Ju-Young , Kwon Yoo-Wook , Kim Hyo-Soo
Published: July 2021

Background:
In terms of the generation of induced pluripotent stem(iPS) cells, one of the important issues for clinical applications is cell source. Human peripheral blood is one of the easily accessible cell sources. However, isolated peripheral blood cells have shown low gene transfection efficiency and inconveniences requiring specific methods to isolate. Here, we report a novel population of peripheral blood-derived stem cells, which can be easily reprogrammed to iPS cells.

Methods and Results:
We cultured peripheral blood mononuclear cells (PBMC) from human peripheral blood and seeded on the fibronectin-coated plate. We observed adherent cells from as early as 5 days after the start of culture and those cells gradually formed colonies. We were able to isolate these cells with very high efficiency. Furthermore, we have also confirmed that these cells can be differentiated to osteogenic, adipogenic, and myogenic-lineage cells. Therefore, we named these cells circulating multipotent adult stem cell. We were successful in generating iPS cells with these cells. These cells showed enhanced efficiency of gene transduction, compared to the human dermal fibroblast. We obtained reprogrammed colonies in 8 days after 4 factor virus transduction without feeder cells. We identified our iPS cells had similar features to embryonic stem cell in morphology, gene expression, epigenetic state and ability to differentiate into the three germ layers. We obtained more than 46 iPS cell lines from PBMC of patients with cardiovascular disease and normal volunteers.

Conclusions:
Our study showed new methods to isolate stem cells from peripheral blood and to generate iPS cells with high efficacy. This result suggests that our new approach could be one of ideal methods for clinical application of iPS cells in future.
Abstract 246: Human Induced Pluripotent Stem Cells Reveal Mitophagy as an Essential Process Against Diabetic Cardiomyopathy
Authors: Ong Sang-Ging , Lee Won Hee , Kodo Kazuki , Wu Haodi , Wu Joseph C
Published: July 2021

Diabetic cardiomyopathy is a common consequence of diabetes and associated with mitochondrial pathology. Using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) as an in vitro model of diabetes, we sought to understand the role of mitophagy, a process that selectively degrades mitochondria through the autophagy-lysosome pathway as a crucial quality control pathway against diabetic cardiomyopathy. We first showed that iPSC-CMs exposed to a diabetic milieu demonstrated increased hypertrophy, impaired calcium signaling, and higher oxidative stress. Flow cytometry analysis of iPSC-CMs subjected to diabetic conditions revealed two distinct population of cells (normal and hypertrophied), suggesting a heterogeneous response to hyperglycemia. In these cells, hypertrophied iPSC-CMs were found to have reduced mitophagy compared to normal cells when exposed to hyperglycemia. In addition, we showed that mitochondrial fragmentation was also decreased in the hypertrophied iPSC-CMs compared to normal cells upon exposure to hyperglycemia, demonstrating a link between mitochondrial fragmentation and mitophagy. Surprisingly, pretreatment of iPSC-CMs with a non-selective antioxidant, N-(2-mercaptopropionyl)-glycine, not only failed to limit the deleterious effects of hyperglycemia, but actually led to increased hypertrophy and cell death. We found that mitophagy was significantly reduced in iPSC-CMs following antioxidant treatment, suggesting the need of mild oxidative stress as a trigger for mitophagy. Future studies are warranted to further investigate the association between oxidative stress, mitochondrial fragmentation, and mitochondrial fission as targets against diabetic cardiomyopathy.
Abstract 250: Elucidating the Role of Platelet Derived Growth Factor Receptor Alpha Signaling in Cardiac Fibroblasts
Authors: Ivey Malina J , Tallquist Michelle
Published: July 2021

Cardiac fibrosis is a major component of heart disease and is a hallmark of decreased cardiac function. Currently, there are no treatments that attenuate fibrosis directly. This major hurdle can be overcome by targeting the resident fibroblast. Preliminary data demonstrates that loss of PDGFRα expression in the adult cardiac fibroblast lineage results in loss of over half of resident fibroblasts. A time course experiment revealed that in as little as 4 days after PDGFRα gene deletion fibroblast loss can observed. Based on the basal level of fibroblast proliferation (0.8%+/-0.9, i.e. 4 of 398 cells), we hypothesize that PDGFRα signaling is essential for fibroblast maintenance and that fibroblasts undergo rapid turnover. We have begun to elucidate which downstream signals of PDGFRα are involved the different roles of the fibroblast. Using a PDGFRα-dependent-PI3K-deficient mouse model, preliminary data indicates that PDGFRα-dependent PI3K signaling is involved in this cell survival response. Future studies will investigate cardiac fibroblast maintenance signals by determining which cell types secrete PDGF ligands. We will also investigate the role of PDGFRα signaling after myocardial infarction. Our lab has genetic tools that enable us to follow fibroblasts after injury, and we have determined both the number of proliferating fibroblasts at different time points, as well as the fraction of fibroblasts that make up the total population of proliferating cells after LAD ligation. Our preliminary data in control hearts shows that fibroblasts reach their peak of proliferation within a week after infarction, although they remain one of the most proliferative cell types as long as three weeks after induction. Our studies will illuminate the role of the fibroblast in tissue homeostasis and after infarction and identify how these cells contribute to overall cardiovascular function and delineate the fine balance between the essential and detrimental functions of the fibroblast.
Abstract 253: Restoring GDF11 to Normal Levels in Old Mice Has no Effect on Cardiac Structure and Function
Authors: Smith Shavonn , Zhang Xiaoxiao , Zhang Xiaoying , Gross Polina , Starosta Tim , Franti Michael , Gupta Priyanka , Hayes David , Myzithras Maria , Ahlberg Jennifer , Chen Xiongwen , MacDonnell Scott , Houser Steven R
Published: July 2021

Rationale:
GDF11 (Growth Differentiation Factor 11) is a member of the TGFβ super family of secreted factors, which play an important role in the regulation of cell processes including proliferation, differentiation, death, adhesion, and migration. Recently it was shown that circulating GDF11 levels fall with aging and this change is associated with pathological cardiac hypertrophy (PCH). Restoring GDF11 to normal levels was shown to rescue PCH.

Objective:
To determine if we can confirm the hypothesis that correcting the levels of a single factor, GDF11, determines aging related PCH.

Methods and Results:
We used the study design described by Loffredo et al, 2013. Investigators were blinded to treatment group. 24 month old C57BL/6 male mice were given a daily injection of recombinant GDF11 at 0 .1mg/kg or vehicle for 28 days. GDF11 bioactivity was confirmed in-vitro. After treatment, GDF11 levels were significantly increased but there was no difference in heart weight (HW) to body weight (BW) ratio (4.74 vs 4.70) between GDF11 and vehicle treated old mice. HW/BW ratios of old mice were not different from12 week-old animals (4.56) and the PCH markers ANP and BNP were not different in young verses old mice. Before GDF11 treatment there were no significant differences in ejection fraction (46 versus 46 %), internal ventricular dimensions (4.33 vs 4.28 mm), and septal wall thickness (0.72 versus 0.78 mm) between GDF11 and vehicle treated groups. All structural and functional parameters remained unchanged at 1, 2 and 4 weeks of treatment. Strain analysis showed no significant difference in radial or longitudinal strain. Invasive hemodynamics performed at time of sacrifice also showed no significant differences in max dP/dt (6499.17 vs 6011.95 mmHg/s) or min dP/dt (-6551.73 vs -6214.33 mmHg/s) between GDF11 and vehicle treated animals respectively.

Conclusions:
There is no significant age-related PCH in C57Bl6 mice. GDF11 injections had no effects on cardiac structure or function in old male C57BL/6 mice. Our results question the idea that there is age-related PCH in disease free C57BL6 mice and question the findings that restoring GDF11 in old mice has any effect on cardiac structure or function.
Abstract 256: Renal Denervation Preconditions the Myocardium by Promoting Nitric Oxide Signaling and Attenuating Oxidative Stress in Spontaneously Hypertensive Rats
Authors: Polhemus David J , Gao Juan , Kapusta Daniel R , Lefer David J
Published: July 2021

Background:
Recent clinical studies suggest that renal denervation (RDN) not only decreases blood pressure (BP) and sympathetic nerve activity, but may also exert additional systemic cardioprotective actions. We investigated whether RDN preconditions the heart against subsequent myocardial ischemia/reperfusion (MI/R) injury in spontaneously hypertensive rats (SHR).

Materials and Methods:
SHR (20w old) received either bilateral radiofrequency (RF) RDN or sham RDN (Biosense Webster Stockert 70 RF generator). After 4w of BP recording, Plasma and left ventricle (LV) tissue were collected for measurement of nitric oxide (NO) metabolites, nitrite (NO2-, ion chromatography) and S-Nitrosothiol (RSNO). RT PCR was used to examine transcriptional changes. Colorimetric assays were used to quantify malondialdehyde (MDA) and carbonyl content. In additional studies, 4w after RDN (n = 8) or Sham (n = 9) treatment, SHR were subjected to 30m of left coronary artery ischemia followed by 24h reperfusion and myocardial infarct/area-at-risk (AAR) was determined.

Results:
4w after treatment, mean BP was significantly decreased in RDN compared to Sham SHR (157±2 vs 142±2 mmHg, p < 0.05). Plasma NO2- levels were elevated 4w following RDN (p < 0.01). Cardiac NO2- levels increased from 2.6 to 3.2 nmol/mg (p < 0.05) and RSNO levels increased from 0.5 to 1.1 nmol/mg (p < 0.05) following RDN. Moreover, myocardial oxidative stress was markedly attenuated as measured by carbonyl content (p < 0.05) and MDA levels (p < 0.01). Greater transcription of antioxidants, SOD1 (p < 0.05) and GPX-1 (p < 0.05) were also observed in the RDN treated group. SHR receiving RDN therapy exhibited a trend in infarct size reduction (42% per AAR reduction, p = 0.09) compared to sham following MI/R. We observed 2-fold greater survival in the RDN treated group (88%, 7 of 8) compared to sham (44%, 4 of 9) following MI/R.

Conclusions:
RDN produced a sustained elevation in NO bioavailability and signaling in the heart and blood. Additionally, RDN attenuated myocardial oxidative stress and augmented the antioxidant defense system in SHR. Although further studies are warranted, preliminary results indicate that these mechanisms may promote myocardial preconditioning and improve survival in the setting of MI/R injury.
Abstract 258: Nanoparticle-mediated Targeting of a Chemical Inhibitor of Drp1 to the Mitochondria Induces Cardioprotection From Myocardial Ischemia-reperfusion Injury
Authors: Ishikita Ayako , Matoba Tetusya , Ikeda Gentaro , Egashira Kensuke
Published: July 2021

Background:
Mitochondria (MITO) injury including MITO permeability transition pore (mPTP) opening plays a major role in the mechanism of ischemia-reperfusion (IR) injury. Intravenous administration of an inhibitor of mPTP opening, cyclosporine A, can reduce IR injury in animals and patients with acute myocardial infarction (MI), however; the power of cardioprotection by cyclosporine A is insufficient. We tested the hypothesis that nanoparticle-mediated targeting of Mdivi1, a chemical inhibitor of Drp1, to MITO enhances cardioprotection from IR injury.

Methods and Results:
We formulated poly(lactic acid/glycolic acid) (PLGA) nanoparticles containing Mdivi1 (Mdivi1-NP) or FITC (FITC-NP). In neonatal rat cardiomyocytes, PLGA nanoparticles accumulated in MITO after the addition of hydrogen peroxide (H2O2) that represents oxidative stress during IR (Fig.A). Treatment with Mdivi1-NP reduced H2O2-induced MITO division and cardiomyocyte death (Fig.B). This Mdivi1-NP cardioprotective effect was not seen over adenovirus harboring Drp1 shRNA transduced cardiomyocyte.

In an in vivo and ex vivo murine model, treatment with Mdivi1-NP at the time of reperfusion reduced infarct size more effectively than Mdivi1 alone (Fig.C). Interestingly, Mdivi1-NP inhibited the leakage of cytochrome c to cytosol and MITO swelling, and reduced IR injury in both wildtype and cyclophilin D (a key regulatory molecule for mPTP opening)-KO mice (Fig.D).

Conclusions:
Mdivi1-NP enhanced cardioprotection against IR injury through mechanisms independent of mPTP opening. Mdivi1-NP can be a novel cardioprotective strategy in acute MI.
Abstract 262: Knockout of Type VI Collagen Preserves Mitochondrial Structure and Function Following Myocardial Infarction
Authors: Meszaros J G , Luther Daniel J , Kang Patrick T , Chen Yeong-Renn , Miller R Lance , Bonaldo Paolo , Chilian William M , Thodeti Charles K
Published: July 2021

Cardiac remodeling is a dynamic process that is accelerated after myocardial infarction (MI), and the traditional focus of key extracellular matrix proteins that mediate remodeling has been on the fibrillar types I and III collagen. We have previously reported that knockout mice of the lesser-known, non-fibrillar collagen VI (Col6-/-) are protected from MI injury as evidenced by significantly reduced infarct size, fibrosis and apoptosis leading to preserved long-term cardiac function. Determining the mechanisms underlying this cardioprotection is the goal of this study. Interestingly, the Col6-/- mice are a model for Bethlam Myopathy, a rare skeletal muscular dystrophy that is characterized by mitochondrial dysfunction leading to premature apoptosis of skeletal myocytes. We hypothesized that alterations in mitochondrial structure and function in the myocardium of Col6-/- mice may play key mechanistic roles responses to ischemic injury. Mitochondrial morphology was visualized by transmission electron microscopy to compare pre- and post-MI changes. Mitochondria from uninjured Col6-/- LV tissue had similar morphology as WT, and at 3 days post-MI the mitochondrial morphology was similarly compromised in both WT and Col6-/- mice. However, at 14 days post-MI the Col6-/- mitochondria were less swollen (43 ± 5.1% decrease in overall volume) and displayed improved orientation/organization over WT (continuous strands). We measured basal O2 consumption and 24 hours post-MI in mitochondria isolated from the infarcted zones of both genotypes. The respiratory control index (RCI) of the Col6-/- mitochondria was lower in the basal, uninjured hearts (7.2 ± 0.9 in WT vs. 4.9 ± 0.6 in Col6-/-). However, the RCI of mitochondria in the infarcted region of Col6-/- hearts at 24 hours post-MI declined less than WT (post-MI values of 2.7 ± 0.5 in WT vs. 2.8 ± 0.7 in Col6-/-) . These data indicate that Col6-/- mice have preserved mitochondrial morphology and a smaller decline in respiration following MI, which may represent a novel homeostatic mechanism underlying protection from ischemic injury in the Col6-/- heart.
Abstract 263: Therapeutic Potential of a Novel Necrosis Inhibitor in Myocardial Ischemia-reperfusion Injury
Authors: Cho Hyun-Jai , Hwang In-Chang , Kim Ju-Young , Lee Hak Seung , Lee Jaewon , Park Jonghanne , Yang Han-Mo , Kwon Yoo-Wook , Kim Soon-Ha , Kim Hyo-Soo
Published: July 2021

Background:
Reperfusion, although essential for salvage of ischemic myocardium, paradoxically causes a wide variety of injuries. Opening of mitochondrial permeability transition pore (mPTP) and Ca
2+
overload contribute to myocardial ischemia-reperfusion (I/R) injury. We aimed to investigate the protective role of a novel necrosis inhibitor (NecroX-7; NecX) against myocardial I/R injury, using in vitro and in vivo models.

Methods and Results:
In H9C2 rat cardiomyoblasts exposed to hypoxia-reoxygenation stress, the main mechanism of cell death was not apoptosis but necrosis, which was prevented mainly by NecX, the necrosis inhibitor, but not by Z-VAD-fmk, the apoptosis inhibitor. The protective effect of NecX was based on its potent ROS scavenging activity, especially on mitochondrial ROS which is one of the major inducers of mPTP opening. NecX preserved mitochondrial membrane potential, mitochondrial structure, through prevention of Ca
2+
influx and inhibition of the opening of mPTP. Inhibition of necrosis by NecX was accompanied by reduction of phospho-p38 MAPK and phospho-JNK, and decrease of HMGB1. Using Sprague-Dawley rats exposed to myocardial ischemia for 45 minutes followed by reperfusion, we compared therapeutic efficacies of NecX and Ciclosporin A (CsA) with 5% dextrose (control), each administrated 5 minutes before reperfusion. NecX markedly inhibited myocardial necrosis, reduced fibrotic area and attenuated the release of cardiac enzymes, compared to dextrose and CsA. Additionally, NecX preserved systolic function and prevented pathologic dilatory remodeling of left ventricle.

Conclusion:
The novel necrosis inhibitor has a significant protective effect against myocardial I/R injury, indicating that it is a promising candidate for cardioprotective adjunctive measure on top of reperfusion therapy.

Clinical implication:
We are trying to translate this experimental data into patients. A phase I clinical trial confirmed the safety profiles of NecX [NCT01737424] and a phase II trial for STEMI patients (NEXsteMI trial) is ongoing [NCT02070471].
Abstract 300: A Novel Targeted Angiogenesis Technique Using Magnetic Nanoparticles
Authors: Arokiaraj Mark
Published: July 2021

Aims:
To develop a novel technique for angiogenesis which could be potentially useful for therapeutic purposes using magnetic nanoparticles.

Methods:
Magnetic nanoparticles were synthesized and were conjugated with vascular endothelial growth factor. The particles were tested in tissue culture microfluidic chips for angiogenesis. Four layers were generated for the experiment respectively - the nutrient layer, hydrogel layer, HUVEC spheroids and another hydrogel layer in the bottom. The particles were inserted in a layer of collagen hydrogel and were guided to desired location using a magnet. The extent and the direction of angiogenesis were studied using 3D confocal microscopy.

Results:
Angiogenesis was observed compared to the controls when nanoparticles were interfaced between the nutrient and HUVEC layer. When the nanoparticles was placed below the HUVEC spheroids angiogenesis occurred in the basal layer predominantly, which showed the effect of the nanoparticles with growth factor on angiogenesis more than the nutrients. In another experiment in microfluidic chip, the nanoparticles were placed in nutrient layer and HUVEC cell monolayer was placed underneath the nutrient layer. The nanoparticles was observed to cross endothelial cells and reach lower hydrogel layer, which was observed by Z stack confocal microscopy.

Conclusion:
Angiogenesis happen by the effect of magnetic nanoparticles conjugated with growth factors. These nanoparticles can be controlled with a magnet. Also, these nanoparticles have potentials to cross endothelium.
Abstract 313: Twist Contributes to Cardiomyocyte Renewal and is Required for Maintenance of Adult Cardiac Function
Authors: Min Yi-Li , Bezprozvannaya Svetlana , Šošic Drazen , Nam Young-Jae , Sadek Hesham , Bassel-Duby Rhonda , Liu Ning , Olson Eric N
Published: July 2021

Cardiomyocyte renewal occurs very slowly in adult mammals, and little is known of the genetic basis of cardiac regeneration. Twist is a highly conserved bHLH transcription factor responsible for Drosophila mesoderm formation during embryogenesis. Recent studies have shown that Twist protein is essential for muscle regeneration in adult Drosophila, but the potential role of Twist in the mammalian heart has not been explored. There are two Twist genes in vertebrates, Twist-1 and -2. We show that Twist-1 and -2 are expressed in epicardium and interstitial cells but not in differentiated cardiomyocytes in mice. To understand the potential function of Twist-dependent lineages in the adult heart, we generated inducible Twist2CreERT2; ROSA26-tdTomato reporter mice. By treating these mice with tamoxifen at 8 weeks of age, we observed progressive labeling of various cell types, such as epithelial cells, cardiac fibroblasts, and cardiomyocytes in the heart. We isolated Tomato-positive nonmyocytes from these mice and found that these cells can differentiate into cardiomyocytes and other cell types in vitro. Furthermore, cardiac-specific deletion of both Twist1 and Twist2 resulted in an age-dependent lethal cardiomyopathy. These findings reveal an essential contribution of Twist to long-term maintenance of cardiac function and support the concept of slow, lifelong renewal of cardiomyocytes from a Twist-dependent cell lineage in the adult heart.
Abstract 322: Reversal of Intramuscular Lipotoxicity is Mediated by a Novel p62-lipophagy Pathway
Authors: Lam Truong N , Harmancey Romain , Vasquez Hernan , Patel Nina , Gilbert Blaine , Taegtmeyer Heinrich
Published: July 2021

Background and Hypothesis:
In the mammalian heart, autophagy is considered an adaptive mechanism promoting the removal of protein aggregates or damaged cell organelles. A common feature of non-ischemic heart failure is lipid accumulation. We hypothesize that autophagy is impaired in the muscle in chronic lipid overload. Therefore, we investigated whether activation of autophagy protects myocytes from lipotoxicity.

Methods:
We incubated rat L6 myocytes over a period of 6 days with or without long chain fatty acids (equimolar mixture of oleate and palmitate, 1.0mM). At day 6, an autophagic inhibitor (bafilomycin A1, 200 nM), or an autophagic activator (rapamycin, 1 μM), or both, were added for 48 hours. Following the pharmacologic treatments, glucose uptake was measured using [3H] 2-Deoxy-D-glucose, and insulin sensitivity was assessed. Intracellular triglyceride (TG) accumulation was assessed by Oil Red O staining and immunofluorescence and was quantified enzymatically. Protein markers of autophagic flux (LC3 and p62) and cell death (Caspase 3 cleavage) were measured by immunoblotting.

Results:
Inhibition of autophagy using bafilomycin increased TG accumulation and also increased fatty acid-mediated cell death. Conversely, activation of autophagy using rapamycin reduced both intracellular lipid accumulation and cell death. Unexpectedly, treatment with both rapamycin and bafilomycin resulted in a decrease in lipid accumulation. Immunoblotting indicated p62 degradation (autophagic flux), while immunofluorescence revealed the colocalization of p62 with lipid droplets in these cells. These findings indicate the potential association of p62 with lipid droplet turnover, which is a novel pathway for the breakdown of lipid droplets in muscle cells. In the same cells, rapamycin treatment increased glucose uptake, in response to insulin (100mM).

Conclusions:
Autophagy promotes the clearance of lipids from myocytes, improves insulin sensitivity, and switches to an alternative p62 mediated pathway of lipophagy in the context of chronic lipid overload. Moreover, lipophagy promotes metabolic adaptation and myocyte survival.
Abstract 324: Knockout of the Pro-apoptotic Er-stress Gene Chac1 in Mice Results in Embryonic Lethality and Activation of the Notch Pathway
Authors: Mungrue Imran , Prescott Eugenia T , Sylvester Charity F , Crawford Rebecca R
Published: July 2021

The ER-stress pathway is activated by oxidized phospholipids in human aortic endothelial cells (HAECs), and may play a role in the development of atherosclerosis. Systems genetics screens of transcriptome wide RNA co-expression in populations of human cells can broadly define novel functional gene-gene relationships. Using this method, we predicted a role for the human CHAC1 gene downstream of ATF4 in the ER-stress pathway in HAECs. Targeted cell culture and genetic perturbations refined the ATF4 dependent ER-stress induction of CHAC1 and a role in regulating apoptosis. Herein we report the generation of a mouse model with the Chac1 gene deleted (Chac1-KO), and heterozygous insufficiency (Chac1-Het). We obtained transgene positive C57BL6 founders that were bred to subsequent generations producing Chac1-Het mice. Intercrossing Chac1-Het mice revealed embryonic lethality, as no Chac1-KO progeny were generated from 25 litters. Chac1-KO embryos were produced at 13 days post coitus and used to make Mouse Embryonic Fibroblasts (MEFs), and the absence of Chac1 expression in these cells was validated. Since Chac1 is an ER-stress inducible gene activated by ATF4, we examined the expression of this pathway in Chac1-KO MEFs, versus controls. We noted increased basal and ER-stress induced expression of ATF4 and its target CHOP accompanying Chac1 knockout, highlighting a novel role for Chac1 in feedback regulation of the ATF4 branch of the ER-stress pathway. We also noted increased expressions of Parp1 and Caspase3, in Chac1-KO MEFs, supporting a role in the regulation of apoptosis pathways. Additionally, Chac1 cells had a higher proliferation rate, displayed a distinct morphology, and grew to a higher density. Since recent reports show a role for Chac1 in blocking Notch maturation, we examined expression of the Notch pathway in Chac1-KO MEFs, versus controls. We noted increased expression of the Notch target gene Hes1 in Chac1-KO MEFs at baseline. Following treatment with the proteasome inhibitor MG132, Chac1-KO MEFs had increased expressions of Hes1, Hey2 and Nrarp, target genes of the Notch pathway. These data reveal activation of the Notch pathway in Chac1-KO cells treated with MG132, a hyper proliferative phenotype and embryonic lethality at dpc 13.
Abstract 336: Conservation of Cardiac L-type Ca2+ Channels and Their Modulation in Drosophila: a Novel Genetically Pliable Channelopathic Model
Authors: Limpitikul Worawan B , Viswanathan Meera C , Cammarato Anthony , Yue David T
Published: July 2021

Misregulation of L-type Ca
2+
channels (LTCCs) underlies numerous cardiac pathologies. For example, heart failure features blunted protein kinase A (PKA) upregulation of the LTCCs. However, studying LTCC regulation in mammalian systems is challenging due to complex physiology and lack of genetic manipulability while recombinant channels expressed in heterologous systems, which lack key auxiliary elements, poorly represent the native context.
Drosophila
, however, has a simple heart with many conserved genes and is genetically pliable. Thus, we pinpointed the signature of Ca
2+
channels (CCs) in the fly cardiac tubes. First, RNAi-mediated selective knockdown of Dmca1D, homologous to the human LTCCs, abolished cardiac contraction (B, as compared to ctrl in A), establishing this channel isoform as the primary CC in fly hearts. Second, we successfully isolated viable single fly cardiomyocytes (C, inset) and recorded robust Ca
2+
currents using patch clamp electrophysiology (C), a feat never before accomplished for the fly cardiac system. Moreover, recording Ca
2+
currents in distinct hypomorphic CC lines, we confirmed the CC type in the fly heart. We also observed pharmacological responses of the CCs that were strikingly similar to those seen in humans. Current through Dmca1D is blocked by a dihydropiridine, nifedipine (C), and is readily upregulated by forskolin, a downstream activator of the PKA pathway (C). In all, we have established the existence of a conserved compendium of cardiac CCs in fly heart tubes, suggesting that
Drosophila
may serve as a robust and effective platform to study the pathophysiology of diseases involving cardiac CCs and to devise therapeutic strategies.
Abstract 353: Mast Cell Inhibition Attenuates Cardiac Remodeling and Diastolic Dysfunction in Ovariectomized Female Fischer344хBrown Norway Rats
Authors: Wang Hao , da Silva Jaqueline , Alencar Allan , Lin Marina , Sun Xuming , Groban Leanne
Published: July 2021

Background:
The incidence of left ventricular hypertrophy (LVH) and diastolic dysfunction (LVDD) increases in postmenopausal women, but no proven pharmacologic interventions exist. Mast cells have an important role in the pathological processes of a variety of cardiac diseases. We determined the cardioprotective effects of chronic mast cell inhibition by the mast cell stabilizer, cromolyn sodium, in middle-aged, female Fischer344хBrown Norway (F344BN) rats after estrogen (E2) loss.

Methods and Results:
Bilateral ovariectomy (OVX) or sham-operations were performed in 18-month-old female F344BN rats. Four weeks after surgery, cromolyn (30 mg/kg/day), or vehicle (saline) was administered s.c. by osmotic minipump (n=7/group). Four weeks after treatment, systolic blood pressure (SBP) increased by 20% in OVX vs. sham rats, and cromolyn attenuated this effect. Myocardial relaxation (e’) was reduced (P<0.01), left ventricle (LV) filling pressures (E/e’) were increased (P<0.01), and LV mass, wall thicknesses, and percent interstitial fibrosis were increased in OVX vs. sham rats. All of these adverse effects of E2 loss on LV function and structure were mitigated by cromolyn treatment. While no differences in plasma angiotensin (Ang) II levels were observed between OVX and sham, Ang II levels were reduced in cromolyn-treated OVX rats (P<0.05). Cardiac chymase protein expression was increased after E2 loss, irrespective of cromolyn. Yet, treatment with cromolyn prevented the increase in cardiac Ang II type 1 receptor (AT1aR) mRNA expression with OVX.

Conclusions:
Mast cell inhibition with cromolyn attenuates adverse LV remodeling and LVDD in OVX-F344BN rats possibly through a chymase/Ang II-mediated mechanism. Cardiac mast cells could be a potential therapeutic target for cardiovascular diseases in postmenopausal women.
Abstract 375: Mechanotransduction via Titin’s N2B Element Contributes to Cardiac Remodeling
Authors: Bull Mathew , Nair Pooja , Strom Joshua , Gotthardt Michael , Granzier Henk
Published: July 2021

Pathological remodeling is responsible for the functional deficits characteristic of heart failure patients. Understanding mechanotransduction is limited, but holds potential to provide novel therapeutic targets to treat patients with heart failure, especially those with diastolic dysfunction and preserved ejection fraction (HFpEF). Titin is the largest known protein and is abundant in muscle. It is the main contributor of passive stiffness in the heart and functions as a molecular mechano-sensor for stress and strain in the myocyte. Titin is composed of four distinct regions, (N-terminal Z-line, I-band, A-band, and C-terminal M-line), and acts as a molecular spring that is responsible for the assembly and maintenance of ultrastructure in the sarcomere. The elastic N2B element found in titin’s I-band region has been proposed as a mechano-sensor and signaling “hot spot” in the sarcomere. This study investigates the role of titin’s cardiac specific N2B element as sensor for stress and strain induced remodeling in the heart. The previously published N2B knock out (KO) mouse was subjected to a variety of stressors including transverse aortic constriction (TAC), aorto-caval fistula (ACF), chronic swimming, voluntary running and isoproterenol injections. Through chronic pathologic stress, pressure overload (TAC) and chronic volume overload (ACF), we found that the N2B element is necessary for the response to volume overload but not pressure overload as determined by changes in cardiac remodeling. Furthermore, the response to exercise either by chronic swimming or voluntary running was reduced in the N2B KO mouse. Finally, unlike the wild-type (WT) mouse, the N2B KO mouse did not respond to isoproterenol injections with hypertrophic remodeling. Ongoing work to elucidate the molecular pathways involving the N2B element and response to stress, is focused on its binding protein Four-and-a-half-LIM domains 2 (FHL2) and the mitogen activated protein kinase (MAPK) pathway. Taken together our data suggest that the N2B element contributes significantly to mechanotransduction in the heart.
Abstract 390: End-stage Human Heart Failure is Characterized by a Deficit in Energetic Lipids in Both Diabetic and Non-diabetic Patients
Authors: Bedi Kenneth C , Snyder Nathaniel W , Javaheri Ali , Brandimarto Jeffery , Mesaaros Clementina , Blair Ian A , Margulies Kenneth B , Rame J. E
Published: July 2021

Introduction:
Animal models and human studies have identified increased intramyocardial lipid accumulation and a lipotoxicity hypothesis has been emerging as a mechanism of myocardial dysfunction in diabetes. We have identified a significant

decrease

in energetic lipids in chronic advanced

non-diabetic

heart failure. We hypothesized that intramyocardial lipid species would be increased in diabetic as compared to non-diabetic end-stage heart failure patients.

Methods:
Left ventricular samples procured at the time of orthotopic heart transplantation from non-diabetic (IDCM n=8) and diabetic (DCMDM n=8) patients as well as organ donors with (NFDM) and without a history of diabetes (Donor) were quantitated for lipids with high-resolution mass spectrometry . Stable isotope labeled essential nutrient in cell culture internal standards for acyl-CoAs were generated using [
13
C
3
15
N
1
]-pantotheonate in Hepa1c1c7 cells.

Results:
The lipidomic signature of end-stage failing myocardium marked by a significant decrease in energetic lipids, a decreased myocardial Succinyl CoA (p-value 0.0079 for failing versus non-failing diabetic subjects) and a decreased ratio of [Succinyl CoA]/[Acetyl Coa] consistent with deficient TCA cycling, is indistinguishable in diabetic and non-diabetic patients (Figure 1).

Discussion:
Despite the known bioenergetic deficits in insulin-resistant diabetes, we have not identified any significant differences between diabetic and non-diabetic subjects in the lipidomic signature of end-stae failing myocardium. Future studies are needed with focused metabolomics to elucidate differences in the diabetic phenotype of human heart failure.
Abstract 400: Regulated Levels of Sarcolemmal Membrane Protein Isoform 3 Impact Cardiac Function in Mice
Authors: Mlynarova Jana , Trentin-Sonoda Mayra , Salih Maysoon , Gaisler da Silva Fernanda , Major Jennifer L , Sorelli Carneiro-Ramos Marcela , Tuana Balwant S
Published: July 2021

The sarcolemmal membrane-associated proteins (SLMAPs) are a family of tail-anchored membrane proteins generated by alternative splicing of the SLMAP gene. A ubiquitously expressed SLMAP isoform 3 encompasses an N-terminal FHA domain with extended coiled-coil structure and has been implicated in cell cycle control. Heart function in transgenic mice with cardiac-specific overexpression of SLMAP3 cDNA driven by α myosin heavy chain promoter was evaluated by echocardiography. qPCR and western blot were used to analyze gene and protein expression respectively. Structure and fibrosis was analyzed by H&E and Masson’s Trichrome staining. Function analysis showed a 15% (p<0.05) decrease in ejection fraction and 19% (p<0.05) decrease in fractional shortening in transgenic mice as early as 5 weeks and persisted into old age at 44 weeks. Transgenic mice presented a mild systolic dysfunction and a trend towards dilated cardiomyopathy without any premature death. Natriuretic peptide ANP and BNP levels were not changed and there was no difference in left ventricular mass or activation of the hypertrophic factor Akt1 in SLMAP3 expressing myocardium. However, significant changes in calcium handling proteins with a significant decrease in phosphorylation of phospholamban ser16 (p<0.05) along with a down-regulation of sarco-endoplasmic reticulum Ca2+ ATPase protein and increased ryanodine receptor 2 phosphorylation ser2808 (p<0.05) were noted at 5 weeks of age in transgenic hearts. These data indicate that increased SLMAP3 levels did not influence cardiac remodeling or hypertrophic growth but did impact membrane biology of calcium transport systems in myocardium leading to depressed contractility. Thus regulated levels of SLMAP3 are important to support normal heart function.
Abstract 404: High-throughput Screening Reveals the Mitochondrial Complex I Inhibitor Nornicotine is Cardioprotective in Ischemia-reperfusion Injury When Delivered at Reperfusion
Authors: Zhang Jimmy , Karcz Marcin K , Nadtochiy Sergiy M , Brookes Paul S
Published: July 2021

Background:
To date, there are no FDA-approved therapies for the reduction of infarct size in acute myocardial infarction. Previously, we developed a cell-based phenotypic assay of ischemia-reperfusion (IR) injury, which was used to identify novel cytoprotective agents delivered prior to ischemia. Herein, we sought to identify cytoprotective agents in a more clinically relevant model: drug delivery at reperfusion, and to investigate possible underlying mechanisms of protection.

Methods:
Primary adult mouse cardiomyocytes were subjected to simulated IR injury using a modified Seahorse XF24 apparatus with drug addition at the onset of reperfusion. Cell death was estimated using LDH release. Drugs which protected cardiomyocytes in vitro were tested in a Langendorff model of IR injury, measuring functional recovery and infarct size. In separate experiments, metabolites extracted from perfused hearts were resolved by HPLC.

Results:
Nornicotine was identified as a cardioprotective agent in the screen. In perfused hearts, 10 nM nornicotine injected at the onset of reperfusion improved functional recovery and decreased in infarct size (13.1% ± 2.4 vs 49.2% ± 2.5 in non-treated hearts, p<0.05, n=16-20). Nornicotine also exhibited profound inhibitory effects on mitochondrial complex I activity.

Succinate is known to accumulate in ischemia, and its rapid consumption during early reperfusion exacerbates reperfusion injury via ROS generation from electron backflow through complex I [PMID: 25383517]. In non-treated hearts, we confirmed that high post ischemic levels of succinate rapidly declined during the first 2 min of reperfusion. In contrast, nornicotine slowed post-ischemic succinate consumption, suggesting that electron backflow through complex I is the major pathway driving succinate consumption.

Conclusions:
Herein, we demonstrated that nornicotine was cardioprotective when delivered at early reperfusion in vitro and ex vivo. The mechanism of cardioprotection may be due to inhibition of rapid succinate consumption during early reperfusion via reverse electron flow back through complex I.