bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 280: Cardiac Deletion of the Mitochondrial Pyruvate Carrier Results in Dilated Cardiomyopathy with Preservation of Fatty Acid Catabolism
Authors: McCommis Kyle S , Gierasch Carrie M , Kovacs Attila , Weinheimer Carla J , Koves Timothy R , Muoio Deborah M , Finck Brian N
Published: July 2021

Pyruvate is an important metabolic substrate for the heart that is formed in the cytosol by glycolysis or conversion of lactate, and then must be transported into the mitochondrial matrix for further metabolism. The mitochondrial pyruvate carrier (MPC) is composed of MPC1 and MPC2 proteins that are each required for complex stability and transport activity. Indeed, mice with cardiac-specific knockout of MPC2 (CS-MPC2-/- mice) exhibited concomitant MPC1 degradation and marked reduction in pyruvate-stimulated mitochondrial respiration. While cardiac function and heart size was normal in 6 week old CS-MPC2-/- mice, serial echocardiograms demonstrated drastic increases in heart size, chamber dilation, and loss of contractile function at 10 and 16 weeks of age. Gene markers of heart failure, hypoxia, and fibrosis were markedly increased in CS-MPC2-/- hearts. Mitochondria isolated from 16 week old failing CS-MPC2-/- hearts exhibited normal respiration on glutamate/malate, succinate, palmitoylcarnitine, and 3-hydroxybutyrate/malate, indicating preservation of mitochondrial energetics with anaplerotic malate, or substrates to produce acetyl-CoA independent of pyruvate. Expression of genes encoding fat and ketone oxidation enzymes was not down-regulated in failing CS-MPC2-/- hearts as is typically observed in heart failure, suggesting these hearts may rely on fat or ketone body oxidation for ATP production. However, targeted metabolomics of hearts from 6 week old CS-MPC2-/- chow-fed mice suggested TCA cycle dysfunction due to decreased acetyl-CoA levels that are insufficient to condense with oxaloacetate, causing an accumulation of oxaloacetate/aspartate, malate, and fumarate. To determine whether increasing the availability of usable substrates (fatty acids and ketones) would rescue the cardiac dysfunction, CS-MPC2-/- mice were fed a high fat, low carbohydrate (ketogenic) diet. Ketogenic diet strikingly decreased hypertrophy and improved functional parameters in 10 week old mice. In conclusion, loss of mitochondrial pyruvate utilization leads to altered cardiac substrate metabolism and inability to maintain TCA cycle flux, resulting in dilated cardiomyopathy that can be corrected by administration of a ketogenic diet.
Abstract 312: Visualization of Cardiomyocyte cGMP Dynamics in Hypoxia/Reoxygenation
Authors: Bork Nadja I , Nikolaev Viacheslav O
Published: July 2021

The second messenger cyclic guanosine 3′5′-monophosphate (cGMP) plays an important role in the regulation of multiple physiologic processes in the cardiovascular system for example it is known to inhibit hypertrophy and to protect against ischemia-reperfusion injury. As a consequence of hypoxia in the heart, maladaptive signaling cascades are activated that can result in cardiac damage and finally lead to heart failure. However, cellular responses to hypoxia/reoxygenation (H/R) are still incompletely understood and almost nothing is known about cGMP dynamics in the context of H/R.

The aim of this project was to investigate the effects of H/R on cGMP dynamics in mammalian cardiomyocytes. Therefore transgenic mice with cardiomyocyte-specific expression of the cytosolic Förster resonance energy transfer (FRET)-based cGMP sensor red cGES-DE5 were used to study cGMP dynamics in single adult cardiomyocytes exposed to H/R. In addition, a Langendorff system was used for FRET measurements in whole-heart.

Basal cGMP levels in single adult cardiomyocytes exposed to H/R were increased. This increase was generated already during the hypoxic period and was maintained during reoxygenation. PDE3 protein expression and activity were significantly downregulated after H/R, however RNA expression level of PDE3 was not significantly changed. At the same time, protein levels of the soluble guanylyl cyclase β-subunit showed a tendency towards downregulation during H/R.

In whole heart measurements, we could show that cGMP levels increase during anoxia and decrease during reoxygenation. This indicates that there is also an influence of other cell-types on cGMP dynamics or on cGMP cell-cell transfer.

In conclusion, we developed FRET-based cGMP measurements in single cardiomyocytes and whole hearts in context of H/R and found an increase of intracellular cGMP in hypoxia. This offers great opportunities to dissect the molecular mechanism of cGMP signaling regulation during ischemic injury and should help to distinguish between direct protective effects on cardiomyocytes and indirect mechanisms such as cell-cell interactions in cGMP signaling during and after H/R.
Abstract 390: Actin Arcs are Essential Templates for Sarcomere Assembly in Cardiomyocytes
Authors: Burnette Dylan T , Fenix Aidan , Taneja Nilay , Williams Annabelle , Bader David , Gutzman Jennifer , Tyska Matthew
Published: July 2021

The sarcomere is the basic contractile unit within cardiomyocytes. The proper assembly of sarcomeres during development and their maintenance during homeostasis are critical for the contraction of the heart. How molecular components of sarcomeres assemble remains a major unanswered question. Here we use newly plated
h
uman
i
nduced pluripotent stem cell-derived
c
ardio
m
yocytes (hiCM) combined with high-resolution microscopy to elucidate the steps of
de novo
sarcomere assembly. We found that sarcomere formation was preceded by bundles of actin filaments resembling the so-called “actin arcs” prevalent in migrating non-muscle cells. Live-cell imaging revealed that sarcomeres appeared along the length of actin arcs; suggesting they are acting as a template for sarcomere assembly. Actin arc formation in non-muscle cells is dependent on the actin filament nucleator, formin, and the molecular motor,
n
on-muscle
m
yosin
II
(NMII). Inhibiting formin with the small molecule SMIFH2 in hiCM stopped the formation of actin arcs and subsequent sarcomere assembly, but had no effect on pre-assembled sarcomeres. We found that two isoforms of NMII, NMIIA and NMIIB, localized to the actin arcs in hiCM. Knockdown of NMIIB, but not NMIIA, in hiCM resulted in a loss of sarcomere assembly, but, much like formin inhibition, did not affect pre-assembled sarcomeres. To test if loss of NMIIB resulted in less sarcomere assembly
in vivo
, we knocked down NMIIB in zebrafish embryos, and found a significant loss of sarcomeres within both the atrium and ventricle. Finally, we use super-resolution microscopy to show that NMII and the muscle isoform,
β
m
yosin
II
(βMII), are found in the same filaments in hiCM and
in vivo
in neonatal mice and in human patients with hypertrophic cardiomyopathy, suggesting individual molecular components within actin arcs (e.g., NMII) could be acting as seeds for their muscle counterparts (e.g., βMII). Taken together, our data supports a model in which contractile systems in cardiomyocytes evolved from non-muscle contractile systems. Furthermore, cardiomyocytes still use some non-muscle contractile components for sarcomere assembly during development, and potentially during aberrant sarcomere formation during hypertrophic cardiomyopathy.
Abstract 407: Divergent Functions of Thrombospondin Genes in Mammals
Authors: Schips Tobias G , Vanhoutte Davy , Brody Matthew , Correll Nathan , Tjondrokoesoemo Andoria , Sargent Michelle , Maillet Marjorie , Molkentin Jeffery D
Published: July 2021

Thrombospondin (Thbs) proteins are multidomain, matricellular proteins comprised of 5 genes that each share similar domains and have been largely ascribed the same functional characteristics. The Thbs protein family is divided in 2 subgroups based on multimerization as trimers, (group A: Thbs1 and 2), or as pentamers (group B: Thbs3, 4 and 5). Thbs proteins modulate various aspects of cell- matrix interactions. Thbs1 and 2 can also alter angiogenesis and modulate MMP- as well as TGF beta activity. More recently, we have shown that they can also serve an intracellular chaperone function along the secretory pathway in response to ER stress (Lynch et. al. Cell 2012). The overall aim of this study is to functionally compare the two Thbs subgroups and elucidate their involvement in cardiac homeostasis and disease. Hence we analyzed gain and loss of function mouse models for Thbs1 as a representative of group A and Thbs3 for group B. Our overall conclusion was that Thbs4 serves a protective function in the heart while Thbs1 and Thbs3 are either overtly maladaptive or they predispose to worsening heart disease with injury stimulus. Taken together, this is the first study comparing Thbs subfamilies, unraveling previously unrecognized functions of Thbs1 and 3 in the mammalian heart, which appears to oppose the protective function of Thbs4.
Abstract 415: Transgenic Expression of Dimethylarginine Dimethylaminohydrolase Attenuates Exercise-induced Fatigue in Duchenne Muscular Dystrophy Carrier Mice
Authors: Garbincius Joanne F , Merz Lauren E , Cuttita Ashley J , Bayne Kaitlynn V , Schrade Sara , Armstead Emily A , Converso-Baran Kimber L , Whitesall Steven E , D’Alecy Louis G , Michele Daniel E
Published: July 2021

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by mutations in dystrophin and characterized by muscle degeneration, cardiomyopathy, and impaired muscle nitric oxide (NO) production that disrupts muscle blood flow regulation and leads to excessive post-exercise fatigue. Interestingly, circulating levels of the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) are elevated in dystrophin-deficient subjects. Therefore, we hypothesized that excessive circulating ADMA impairs muscle NO production and thus negatively impacts exercise tolerance in DMD. The objectives of this study were to determine whether increased circulating ADMA is itself sufficient to affect exercise performance, and whether transgenic modulation of ADMA metabolism could improve exercise-induced fatigue in the
mdx
mouse model of DMD. Although infusion of exogenous ADMA did impair exercise performance in healthy, wild-type mice, transgenic expression of dimethylarginine dimethylaminohydrolase 1 (DDAH), the enzyme that degrades ADMA, did not affect exercise-induced fatigue in dystrophin-deficient male
mdx
mice. Surprisingly, DDAH transgene expression did attenuate exercise-induced fatigue in dystrophin-heterozygous female
mdx
carrier mice. This improvement in exercise tolerance was associated with reduced heart weight, improved cardiac contractile function, and improved chronotropic responsiveness to beta-adrenergic stimulation in DDAH-transgenic female
mdx
carriers. In contrast, DDAH transgene expression did not significantly affect skeletal muscle pathology in female
mdx
carriers. We conclude that DDAH transgene expression improves exercise tolerance in dystrophin-heterozygous females, possibly by limiting pathological cardiac remodeling and helping to maintain heart performance. These findings emphasize the importance of methylated arginines to the development of cardiomyopathy in female DMD carriers, and have interesting implications for current genetic therapies that may only partially restore dystrophin expression in the hearts of male DMD patients.
Abstract 42: Lysosomal Membrane Permeabilization Contributes to Hyperglycemia-Induced Cardiomyocytes Dysfunction
Authors: Kobayashi Satoru , Huang Yuan , Kobayashi Tamayo , Liang Qiangrong
Published: July 2021

Hyperglycemia is an independent risk factor for diabetic heart failure. However, the mechanisms that mediate hyperglycemia-induced cardiac damage remain poorly understood. Previous studies have shown an association between lysosomal dysfunction and diabetic heart failure. Lysosomal membrane permeabilization (LMP) is a prominent feature of lysosomal dysfunction. We examined if high glucose (HG) induces LMP in cardiomyocyte. Immunofluorescent microscopy showed the release of cathepsin D (CTSD) from lysosome under HG, suggesting that HG induces LMP. To determine whether LMP and the ensuing CTSD expression play a role in HG-induced cardiomyocyte injury, we treated cells with siRNA targeting CTSD or low dose of CTSD inhibitor pepstatinA (0.5ng/mL). Remarkably, both CTSD down-regulation approaches reduced HG-induced myocyte injury, as shown by elevated cleavage of Caspase3 and by propidium iodide staining. These results suggest that LMP and the ensuing CTSD expression mediate HG-induced cardiotoxicity. In addition, by using LMP/lysophagy reporter tfGalectin3, we found that HG-induced LMP was accompanied by impaired lysophagy, suggesting a failed elimination of damaged lysosomes by lysophagy, which may exacerbate HG-induced LMP. Indeed, the lysophagy inducer L-leucyl-L-leucine methyl ester (LLOMe) stimulated lysophagy flux, attenuated HG-induced LMP, and restored the beating frequency of HG-treated cardiomuyocytes. Together, these findings demonstrate a novel mechanism by which high glucose impairs lysophagy, induces LMP and triggers the release of CTSD, which collectively contribute to hyperglycemic cardiotoxicity.
Abstract 444: Association Between Cannabis Use and Acute Myocardial Infarction
Authors: Alliu Samson , Lamikanra Olaoluwatomi , Adejumo Adeyinka , Adegbala Oluwole , Akinjero Akintunde , Durojaiye Modupeolowa
Published: July 2021

Background:
Cannabidiol (CBD)-a component of cannabis with no psychoactive or cognitive effect has been proven in animal models to have a vasodilatory, anti-oxidant and anti-inflammatory effect on the blood vessels. However, it is unclear if cannabis users - while being exposed to its CBD constituents - benefit from its vasodilatory and anti-inflammatory effect in the prevention of acute myocardial infarction (MI).

Objective:
To investigate if there is a difference in the odds of MI among cannabis users when compared to nonusers.

Methods:
We used data from the Nationwide Inpatient Sample on patients ages 45 years and older admitted between 2012 - 2014. The main study outcome was clinical diagnosis of MI, and the main exposure variable was cannabis use identified using ICD-9 codes. Cannabis use was categorized into non-use, non-dependent, and dependent use. Multivariable logistic regression models were used to estimate the odds of MI and In-hospital mortality in relation to cannabis use adjusting for demographics, comorbidities, and use of other recreational drugs.

Results:
Of the 7, 995,162 hospitalized patients who were > 45 years, 532,112 (6.7%) had a diagnosis of MI, 56,836 (0.7%) were non-dependent cannabis user and 5,417 (0.1%) were dependent cannabis users. We observed a significant inverse association between cannabis use and MI (non-dependent OR: 0.86, 95% CI: 0.83-0.90; dependent OR 0.26, 95% CI: 0.21-0.31). After adjusting for confounding variables, the association was attenuated for non-dependent cannabis users (OR: 1.03, 95% CI: 0.99-1.06]). However, among dependent cannabis users, there was 66% decreased odds of MI when compared to nonusers. Also, cannabis use was associated with 32% decreased odds of in-hospital mortality among patients with MI when compared to nonuse.

Conclusions:
Using the largest national data, our study showed cannabis use was not a risk factor for MI and alternatively may point to a protective benefit in the diagnosis of MI and in-hospital mortality. Future prospective studies may aid in further exploring this association to maximize the therapeutic advantage of the cannabinoid system in MI prevention.
Abstract 458: Moderate Exercise Training Attenuates Isoproterenol-Induced Myocardial Injury by Activating Nrf2-Signaling
Authors: Shanmugam Gobinath , Narasimhan Madhusudhanan , Conley Robbie , Radhakrishnan Rajesh Kumar , Litovsky Silvio H , Rajasekaran Namakkal-Soorapppan
Published: July 2021

Background:
Nuclear erythroid-2 like factor-2 (Nrf2), a master transcriptional regulator of antioxidants, is critical to maintain cellular redox homeostasis. We recently reported that exercise training activates Nrf2/antioxidant signaling in the heart. Isoproterenol (ISO) mediated structural, and functional changes in the heart involve oxidative stress. Here, we tested a hypothesis that moderate exercise training will protect the myocardium from isoproterenol-induced injury by augmenting Nrf2-dependent antioxidant defense system.

Methods:
Age- and sex-matched WT (C57/BL6) mice (6-8 months old) were subjected to moderate exercise training (MET) on a treadmill for 6 weeks (60 min/day; 10m/min; 0% grade). Randomly assigned untrained (UNT) and trained (MET) animals were intraperitoneally injected (at the start of 6
th
week) with 50 mg of isoproterenol/kg.bw./day for 7 consecutive days. MET was continued during ISO administration and the animals (UNT + PBS, UNT + ISO; MET + ISO) underwent echocardiography analysis. Heart tissues were collected for histopathology, Nrf2-ARE promoter binding assay (Active-motif TransAM kit), antioxidant gene (qPCR) and protein (Immunoblotting) levels, and glutathione redox status.

Results:
ISO administration significantly reduced the Nrf2 promoter activity (p<0.05) and downregulated the expression of cardiac antioxidant genes (
Gclc, Nqo1, Cat, Gsr and Gst-μ
) in UNT mice. Further, increased oxidative stress with severe myocardial injury was evident in UNT+ISO when compared to UNT mice receiving PBS under basal condition. Interestingly, MET stabilized the Nrf2-promoter activity and promoted the expression of Nrf2-dependent antioxidant genes and proteins animals receiving ISO, and thereby attenuated the oxidative stress-induced myocardial damage. Echocardiography analysis showed impaired systolic/diastolic ventricular volumes coupled with decreased cardiac output in UNT+ISO mice, but this was normalized in exercise-trained animals.

Conclusion:
Thus moderate exercise training conferred protection against ISO-induced myocardial injury by augmentation of Nrf2-antioxidant signaling and attenuation of redox perturbations.
Abstract 470: Raf Kinase Inhibitors Activate ERK1/2 in Cardiomyocytes, Promoting Cardiac Hypertrophy
in vitro
and, in the Context of Angiotensin II-Induced Hypertension in Mice,
in vivo
Authors: Meijles Daniel N , Hardyman Michelle A , Fuller Stephen J , Rostron Kerry A , Leonard Sam J , Sugden Peter H , Clerk Angela
Published: July 2021

Introduction:
ERK1/2 promote hypertrophy and are protective in the heart, but cause cancer in dividing cells. Raf kinases lie upstream of ERK1/2 and Raf inhibitors (e.g. SB590885 (SB), dabrafenib (Dab)) are in development/use for cancer. Paradoxically, in cancer cells, low concentrations of SB/Dab stimulate (rather than inhibit) ERK1/2.

Hypothesis:
Our hypothesis is that the heart is primed for Raf paradox signaling. Raf inhibitors have potential to activate ERK1/2 in cardiomyocytes and promote cardiac hypertrophy.

Methods:
Neonatal rat ventricular cardiomyocytes (NRVMs) were exposed to inhibitors. Dab or SB (3 or 0.5 mg/kg/d) were studied in 12 wk male C57Bl6 mice in vivo in the presence of angiotensin II (AngII, 0.8 mg/kg/d) (n=6-11) using osmotic minipumps. Effects were compared with vehicle controls. Echocardiography was performed (Vevo2100). M-mode images (short axis view) were analyzed; data for each mouse were normalized to the mean of 2 baseline controls. Kinase activities were assessed by immunoblotting or in vitro kinase assays.

Results:
SB (0.1 μM) or Dab (1 μM) activated ERK1/2 (2.3±0.1 fold; n=4) in NRVMs consistent with Raf paradox signaling. An explanation is that Raf kinases dimerise and submaximal inhibitor concentrations bind one Raf protomer, locking it in an active conformation but activating the partner. In accord with this, 0.1 μM SB increased Raf activities. High SB concentrations (1-10 μM) initially inhibited ERK1/2 in NRVMs, but ERK1/2 were then activated (1 - 24 h) and promoted hypertrophy. In vivo (24 h), Dab and SB activated the ERK1/2 cascade, increasing ANF (17.3 ± 3.1 fold) and BNP (4.5 ± 0.8 fold) mRNA (n=4/5). Over 3 d, Dab and SB increased fractional shortening in the presence of AngII (1.22±0.06; 1.17±0.08), relative to AngII alone (0.95±0.04), increased systolic left ventricular (LV) wall thickness, and reduced systolic LV volume and internal diameter (0.83±0.03 cf 0.97±0.02 for AngII alone).

Conclusions:
The heart is primed for Raf paradox signaling and Raf inhibitors activate ERK1/2 in cardiomyocytes, promoting hypertrophy. In vivo, Raf inhibitors enhance ERK1/2 signaling and hypertrophy in the context of hypertension, and cardiac hypertrophy may be increased in hypertensive cancer patients receiving Raf inhibitors.
Abstract 482: A Novel Small Molecule Inhibitor of Acetyltransferase p300 Ameliorates Hypertension-Induced Cardiac and Renal Fibrosis
Authors: Rai Rahul , Verma Suresh K , Kim David , Ramirez Veronica , Li Chengjin , Sahoo Susmita , Qin Gangjian , Kishore Raj , Wilsbacher Lisa D , Vaughan Douglas E , Quaggin Susan E , Ghosh Asish K
Published: July 2021

Rationale:
Fibrosis is a common pathophysiological manifestation of chronically injured or stress-affected organs. Additionally, cardiac and renal fibrosis remain leading causes of global morbidity and mortality. Meanwhile, no effective therapy is available to halt the onset and progression of cardiac and renal fibrogenesis. We have previously demonstrated that p300 with intrinsic factor acetyltransferase (FATp300) activity is an essential epigenetic regulator of profibrogenic signal-induced matrix protein synthesis.

Objective:
Here, we attempted to delineate the therapeutic efficacy of a small molecule inhibitor of FATp300, L002, in blocking i) TGF-b-induced profibrogenic processes in cardiac and renal cells in vitro, and ii) Angiotensin II-induced cardiac and renal fibrogenesis in mice.

Methods and Results:
Analysis of cardiac and renal cells demonstrates that L002 inhibits specific histone acetylation and blunts TGF-b induced profibrogenic processes, including cellular proliferation, migration, myofibroblast differentiation and extracellular matrix protein synthesis. The mRNA analysis further indicates that matrix protein, collagen, and myofibroblast marker, a-SMA are inhibited at the transcriptional level. L002 also prevents TGF-b-induced FATp300 upregulation suggesting that TGF-b-induced FATp300 expression is partly self-regulated. However, TGF-b-induced Smad activation is unaltered in the presence of L002, demonstrating its target specificity. Further, mice infused with Angiotensin II show increased blood pressure, cardiac hypertrophy and increased heart weight. Conversely, co-administration of L002 reduces Angiotensin II-induced cardiac hypertrophy and left ventricular wall thickness. Most importantly, L002 administration ameliorates Angiotensin II-induced cardiac and renal fibrosis in mice.

Conclusions:
Taken together this study demonstrates that specific pharmacological inhibition of elevated factor acetyltransferase activity of p300, a major epigenetic regulator, is an ideal approach to control hypertension- or other stress-induced cardiac and renal fibrogenesis as seen in a wide variety of cardiovascular and renal diseases.
Abstract 59: Inflammasome Activation Promotes the Arrhythmogenesis of Atrial Fibrillation
Authors: Li Na , Scott Larry , Veleva Tina , Dobrev Dobromir , Wehrens Xander
Published: July 2021

Background:
Inflammation is a known risk factor of atrial fibrillation (AF), the most common arrhythmia; however, the mechanistic link between the inflammatory signaling and the pathophysiology of AF has not been established. ‘NACHT, LRR and PYD domains-containing protein 3’ (NLRP3) inflammasome is a signaling platform that is responsible for the activation of caspase-1 and interleukin (IL)-1b release. The activity of NLRP3 inflammasome is enhanced in atrial tissues of paroxysmal or long-lasting persistent AF patients. Thus, we tested the
hypothesis
that activation of NLRP3 inflammasome promotes the development of AF.

Results:
To elucidate the role of NLRP3 inflammasome in cardiomyocytes (CMs) and AF development, a CM-specific knockin murine model expressing a constitutive active NLRP3 (aMHC:NLRP3
A350V/+
, CKI) was developed. At 3-month old, telemetry recordings showed that 100% of CKΙ mice (n=5) developed premature atrial contractions (PACs), whereas only 25% of control mice (n=4, P<0.05) had PACs. Rapid atrial pacing induced AF in 89% of CKI mice (n=9), a much higher incidence than control mice (20%, n=5, P<0.05). Level of the active caspase-1 was increased in atrial tissues of 3-month old CKI mice, prior to a detectable increase in the level of macrophage marker at 7-month old, suggesting that the onset of PACs and AF vulnerability is not associated with the activated macrophages. Compared to the control mice, 3-month old CKI mice exhibited atrial hypertrophy, abnormal Ca
2+
release via RyR2, and shortening of atrial effective refractory period, which were associated with the upregulation of
Mef2c
,
Ryr2
,
Kcna5
(encoding Kv1.5),
Kcnj 3
(encoding Kir3.1) and
Kcnj5
(encoding Kir3.4) mRNA. Lastly, inflammasome inhibitor MCC950 (i.p., 10mg/kg, 10 days) reduced the AF inducibility in CKI mice (0%, n=3, P<0.05 vs vehicle-treated CKI).

Conclusion:
Activation of NLRP3 inflammasome promotes structural and electrical remodeling, permissive to the AF development. In addition to its canonical function, NLRP3 inflammasome may exhibit alternative function in regulating gene transcription. Our study establishes a mechanistic link between the inflammatory signaling and the pathogenesis of AF, and the inhibition of NLRP3 may become a novel anti-AF therapy.
Abstract 64: HDL Glycosylation is Associated with HDL’s Anti-Inflammatory Function and is Responsive to Dietary Intervention
Authors: Zivkovic Angela M , Rhodes Christopher , Sacchi Romina , Beals Elizabeth , Sawrey-Kubicek Lisa , Zhu Chenghao , Muchena John K , Luxardi Guillaume , Maverakis Emanual , Lebrilla Carlito B
Published: July 2021

Although HDL cholesterol (HDL-C) levels are protective against cardiovascular disease (CVD) risk, raising HDL-C pharmaceutically has not led to improvements in cardiovascular outcomes. It is becoming clear that HDL composition and function are more important than HDL concentrations in determining risk. This study set out to determine whether glycosylation differences in HDL-associated glycoproteins affect HDL’s anti-inflammatory function. HDL were purified from healthy subjects (n=10), who consumed in randomized order a fast food (FF) diet and a Mediterranean (Med) diet for 4 days, with a 4-day washout between arms. All foods were provided in this isocaloric cross-over intervention trial, which was approved by the UC Davis IRB. Concentrations of HDL-bound serum amyloid A (SAA), apolipoprotein A-I (ApoA-I), apolipoprotein C-III (ApoC-III), α-1-antitrypsin (A1AT), and α-2-HS-glycoprotein (A2HSG); and the site-specific glycosylation of ApoC-III, A1AT, and A2HSG were measured. Secretion of TNF-α in stimulated monocytes was measured to assess HDL anti-inflammatory function. HDL glycosylation was altered by the dietary interventions and correlated with changes in the amount of TNF-α secreted by stimulated monocytes. HDL glycosylation profiles were different in response to the FF diet vs. the Med diet. HDL with a diminished capacity to suppress TNF-α secretion were enriched in ApoC-III and desialylated A2HSG, depleted in A1AT, and had lower levels of sialylation across glycoproteins. Our results demonstrate that HDL glycoprotein composition, including site-specific glycosylation, is responsive to dietary intervention and correlates with HDL’s ability to modulate TNF-α response in stimulated monocytes. These data suggest that the measurement of HDL glycosylation profiles may be useful in stratifying CVD risk and detecting individuals with impaired HDL anti-inflammatory function.
Abstract 8: Effectiveness of Combination Allogeneic Stem Cells in a Novel Large Animal Model of Chronic Kidney Disease-induced Heart Failure With Preserved Ejection Fraction (HFpEF)
Authors: Castellanos Rieger Angela , Tompkins Bryon A , Natsumeda Makoto , Florea Victoria , Collon Kevin , Rodriguez Jose , Rosado Marcus , Balkan Wayne , Hare Joshua M , Schulman Ivonne H
Published: July 2021

Background:
Chronic Kidney Disease (CKD) is an independent risk factor for cardiovascular morbidity and mortality. Left ventricular (LV) hypertrophy and heart failure with preserved ejection fraction (HFpEF) are the primary manifestations of the cardiorenal syndrome in 60 to 80% of CKD patients. Therapies that improve morbidity and mortality in HFpEF are lacking. Stem cell therapy reduces fibrosis, increases neovascularization, and promotes cardiac repair in ischemic and non-ischemic cardiomyopathies. We hypothesized that stem cell treatment ameliorates HFpEF in a CKD model.

Methods:
Yorkshires pigs (n=27) underwent 5/6 nephrectomy via renal artery embolization and 4-weeks later received either: allogeneic (allo-) MSC (10х10
6
), allo-kidney c-kit
+
cells (c-kit; 10х10
6
), combination (MSC+c-kit; 1:1 ratio [5х10
6
each]), or placebo (each n=5). Cell therapy was delivered via the patent renal artery. Kidney function, renal and cardiac MRI, and PV loops were measured at baseline, and at 4- and 12-weeks (euthanasia) post-embolization.

Results:
The CKD model was confirmed by increased creatinine and BUN and decreased GFR. Mean arterial pressure (MAP) was not different between groups from baseline to 4 weeks (p=0.7). HFpEF was demonstrated at 4 weeks by increased LV mass (20.3%; p= 0.0001), wall thickening (p<0.008), EDP (p=0.01), EDPVR (p=0.005), and arterial elastance (p=0.03), with no change in EF. Diffuse intramyocardial fibrosis was evident in histological analysis and delayed enhancement MRI imaging. After 12 weeks, there was a significant difference in MAP between groups (p=0.04), with an increase in the placebo group (19.97± 8.65 mmHg, p=0.08). GFR significantly improved in the combination group (p=0.033). EDV increased in the placebo (p=0.009) and c-kit (p=0.004) groups. ESV increased most in the placebo group (7.14±1.62ml; p=0.022). EF, wall thickness, and LV mass did not differ between groups at 12 weeks.

Conclusion:
A CKD large animal model manifests the characteristics of HFpEF. Intra-renal artery allogeneic cell therapy was safe. A beneficial effect of cell therapy was observed in the combination and MSC groups. These findings have important implications on the use of cell therapy for HFpEF and cardiorenal syndrome.
Abstract 96: Inactivation of Neddylation Causes Left Ventricular Noncompaction Cardiomyopathy Through Suppressing YAP Signaling
Authors: Zou Jianqiu , Ma Wenxia , Li Jie , Littlejohn Rodney , Kim Il-man , Zhou Jiliang , Su Huabo
Published: July 2021

Rationale:
Cardiac development is orchestrated by a number of growth factors, transcription factors and epigenetic regulators, perturbation of which can lead to congenital heart diseases and cardiomyopathies. However, the role of novel ubiquitin-like protein modifiers, such as NEDD8 (neural precursor cells expressed developmentally downregulated 8), in cardiac development is unknown.

Objectives:
The objective of this study was to determine the significance of NEDD8 modification (neddylation) during perinatal cardiac development.

Methods and Results:
Neddylated proteins and NEDD8 enzymes were highly abundant in fetal and neonatal hearts but downregulated in adult hearts. We employed an αMHC
Cre
transgene to delete NAE1, a subunit of the NEDD8 E1 enzyme, in the perinatal mouse heart. Cardiac-specific deletion of NAE1 (NAE1
CKO
) significantly decreased neddylated proteins in the heart. The NAE1
CKO
mice displayed cardiac hypoplasia, ventricular non-compaction and heart failure during late gestation, which became more pronounced by postnatal day 1 and led to perinatal lethality. Mechanistically, genetic deletion or pharmacological inhibition of NAE1 resulted in accumulation of Hippo kinases Mst1 and LATS1/2, which in turn phosphorylated and inactivated YAP, a transcription cofactor necessary for cardiomyocyte proliferation, leading to dysregulation of a number of cell cycle-regulatory genes and blockade of cardiomyocyte proliferation
in vivo
and
in vitro
. Reactivation of YAP signaling by overexpression of a constitutively-active YAP mutant (YAP
5SA
), but not its wild-type counterpart, overcame the blockade of cardiomyocyte proliferation induced by inhibition of NAE1.

Conclusions:
Our findings establish the importance of neddylation in the heart, more specifically, in ventricular chamber maturation, and identify neddylation as a novel regulator of Hippo-YAP signaling to promote cardiomyocyte proliferation.
Determination of Plasma Renin Concentration by Angiotensin I Immunoassay
Authors: STOCKIGT JAN R. , COLLINS R. DENNIS , BIGLIERI EDWARD G.
Published: July 2021

Radioimmunoassay of angiotensin I was applied to the estimation of renin concentration after incubation of human plasma with an excess of renin substrate from plasma of anephric sheep. With this method markedly subnormal plasma renin levels were accurately measured. The technique was used in the study of 18 patients with proven aldosterone-producing adenoma (APA), 5 with hyperaldosteronism associated with bilateral adrenal hyperplasia, and 23 with essential hypertension.

Plasma renin concentration (PRC) was extremely low in APA, and a significantly higher mean basal level of PRC was seen in hyperplastic aldosteronism (
P
<0.01). Postural responses were seen in both groups, and PRC was least variable during recumbency. Comparison of basal recumbent PRC with urinary aldosterone after three days of desoxycorticosterone acetate (DOCA) accentuated the distinction between APA and hyperplasia.

It is suggested that the prediction of a distinct APA can best be made in patients with hyperaldosteronism when the exact extent of renin suppression is taken into account and when DOCA is used to assess suppressibility of aldosterone. While such evaluation may defer surgery in some patients with correctable hypertension, it may reduce the number with persistent hypertension after bilateral adrenalectomy.
Homocysteine and cerebral white matter lesions: the Rotterdam Scan Study
Authors: van Dijk Ewoud J , Vermeer Sarah E , den Heijer Tom , Oudkerk Matthijs , Hofman Albert , Koudstaal Peter J , Clarke Robert , Breteler Monique Mb
Published: July 2021

31

Background
White matter lesions are frequently observed on cerebral MRI scans. There is evidence that white matter lesions are involved in the pathophysiology of cognitive decline, dementia and late-onset depression. Cardiovascular risk factors are associated with these lesions. A high homocysteine level is associated with an increased risk of cardiovascular disease. Therefore, we studied the association between homocysteine and white matter lesions.
Method
This study forms part of the Rotterdam Scan Study, a prospective population-based cohort study among 1077 subjects, aged 60 to 90 years. All participants underwent MRI scanning of the brain. White matter lesions were defined as hyperintense lesions on both proton-density and T2-weighted images, without prominent hypointensity on T1-weighted images. Periventricular white matter lesions were rated on a nine-point scale. For subcortical white matter lesions an approximated total lesion volume was calculated. Homocysteine levels were determined in plasma samples, obtained within 3 weeks before scanning. The association between total plasma homocysteine and white matter lesions was assessed through multiple linear regression analysis, adjusted for age and gender.
Results
The total plasma homocysteine levels ranged from 3.4 to 70.7 μmol/l with a mean of 11.7 μmol/l [95%CI 11.4–12.0]. We found a mean value of 2.4 [95%CI 2.3–2.5] for periventricular white matter lesion severity and of 1.4 ml [95%CI 1.2–1.6] for subcortical white matter lesion volume. Periventricular white matter lesions increased with 0.4 [95%CI 0.1–0.6] and subcortical white matter lesions with 0.5 ml [95%CI 0.1–0.8] per 10 μmol/l increase in homocysteine level.
Conclusion
Higher homocysteine levels are associated with an increased severity of both periventricular and subcortical white matter lesions.
’To feed or not to feed’ - can decisions be improved for
individual
patients following a stroke?
Authors: Williams Jane E
Published: July 2021

55

This research study ascertains whether the sharing of knowledge between doctors, nurses and relatives of stroke patients can assist the ‘feed or not to feed’ decision making process. The research questions are:- a) What factors do doctors and nurses believe influence the ‘feed or not to feed’ decision? b) What factors emerge when asking the views of relatives of stroke patients regarding their involvement in the decision and personal knowledge of the patient? c) What information/involvement would relatives of stroke patients (who are currently being enterally fed in long term care settings) have welcomed when the decision to commence feeding was made? d) Could analysis of these factors be utilised to influence practice? Analysis of qualitative interviews with these groups indicate which factors emerge and influence decision making. Greater depth of knowledge and understanding of the issues for each group enables information to be utilised to make the best decision for each individual patient. The study focusses on stroke patients aged 65 years+ who cannot swallow food safely and are unable to participate in decision making. The study population is comprised of doctors, nurses and relatives of patients within the first week of stroke. The relatives of patients who have survived more than six weeks are included in the study. Advances in stroke treatments within the acute phase are being made. As more people survive their stroke, ethical dilemmas have increased. A major ethical dilemma encountered is the ‘feed or not to feed’ issue. Commencing or withholding tube feeding in an individual unable to swallow is a highly emotionally charged decision. Currently there is no evidence or consensus of opinion to indicate when or if feeding should be instigated. Research is being undertaken in a large international trial and will provide evidence with regard to timing and type of nutritional support but the issue of decision making for the individual has not been studied. The study proposes that the
combination
of specialist clinical and personal individual knowledge of the patient will assist decision making. Preliminary findings will be presented.
Human-Albumin Neuroprotection in Acute Ischemic Stroke: Marked Efficacy at Moderate Doses, with a Broad Therapeutic Window.
Authors: Ginsberg Myron D , Belayev Ludmila , Liu Yitao , Zhao Weizhao , Busto Raul
Published: July 2021

58

We have previously shown that high-dose human albumin (Alb) is markedly neuroprotective in focal and global cerebral ischemia and in traumatic brain injury. In this study, we examined the efficacy of moderate, clinically achievable Alb doses and defined the therapeutic window in focal cerebral ischemia. Sprague Dawley rats (n=62) were anesthetized with halothane/nitrous oxide and received 2-h middle cerebral artery occlusion (MCAo) by intraluminal suture. Neurological status was evaluated during occlusion (60 min) and daily for 3 days after MCAo. In the dose-response study, animals were given either 25% Alb in doses 0.63 or 1.25 g/kg , or 0.9% saline vehicle, i.v. immediately after suture removal (n=5 each). In the therapeutic window study, 1.25 g/kg Alb was administered at either 2 h, 3 h, 4 h, or 5 h after onset of stroke (i.e., 0 to 3 h after onset of reperfusion) (n=9–10 ea.). Three days after MCAo, brains were perfusion-fixed, and image-processing was used to compute infarct volumes and brain swelling. Both moderate Alb doses (0.63 and 1.25 g/kg) significantly improved the neurological score compared to vehicle rats at 24h, 48h and 72h; and markedly reduced cortical infarct volume (by 66±14% and 95±4%, respectively), striatal infarct volume (by 54±8% and 52±14%), and total infarct volume (by 58±5% and 67±9%, respectively). Brain edema was virtually eliminated by Alb treatment. In the therapeutic window study, even when treatment was initiated as late as 4 hours after onset of MCAo, 1.25 g/kg Alb led to significantly improved neurological score and highly significant reductions of infarct areas in cortex (68% reduction), subcortical regions (52% reduction), and total infarct (61% reduction). The striking efficacy and broad therapeutic window of moderate-dose Alb therapy in experimental focal ischemia strongly supports the feasibility of initiating early-phase clinical trials of this promising agent in patients with acute ischemic stroke. This work was supported by NIH Grant NS05820 (MDG) and by AHA Initial Investigator Award (LB).
Microglia Proliferate in Striatum and Cortex but not Hippocampus Following Brief Periods of Ischemia that do not Kill Neurons
Authors: Sharp Frank R , Lu Jialing , Bartels Matthew , Lu Aigang
Published: July 2021

61

Because ischemia induced tolerance induces the birth of new neurons following global ischemia, this study determined whether 2.5 minutes of global ischemia and ischemia-induced tolerance would induce the birth of glia in the brain.Following 0, 2.5, or 5 minutes of global ischemia, adult male gerbils were injected with BrdU (50mg/kg) and dividing cells detected using immunocytochemistry. At four days following 2.5 minutes of global ischemia, when cell proliferation was maximal, BrdU labeled cells were detected in striatum and in neocortex, but not in hippocampus. The majority of the BrdU labeled cells were double-labeled with iso-lectin B4, showing that these dividing cells were mainly microglia. Similarly, BrdU labeled microglia were found in striatum and cortex but not in hippocampus of most animals four days following ischemia-induced tolerance (2.5 minutes of global ischemia followed three days later by 5 minutes of global ischemia). Some of the animals subjected to 2.5 minutes of ischemia showed decreased myelin associated glycoprotein (MAG) immunostaining and increased numbers of APC stained oligodendrocytes in the lateral striatum without apparent neuronal loss. Though the CA1 pyramidal and hilar interneurons of hippocampus are the most vulnerable neurons to ischemia, this data suggests that non-neuronal elements in the striatum may be more sensitive to ischemia than the neurons. In some animals proliferation of microglia in striatum following brief global ischemia may occur in response to injury to white matter and oligodendrocytes.
ESTROGEN AFTER ISCHEMIC STROKE: Effect of estrogen replacement on risk of recurrent stroke and death in the Women’s Estrogen for Stroke Trial (WEST)
Authors: Viscoli Catherine M , Brass Lawrence M , Kernan Walter N , Sarrel Philip M , Horwitz Ralph
Published: July 2021

71

Introduction:
Observational research has produced conflicting findings concerning the effect of estrogen replacement therapy (ERT) on reducing risk for vascular events or death in women. To test the effect of ERT in women with established cerebrovascular disease, we designed a randomized trial of estradiol-17β(1 mg/day) vs. placebo.
Methods:
Participants were identified from 20 hospitals in New England. Eligibility criteria included age over 44, at least 1 year since last menstrual period, and TIA or non-disabling stroke within 90 days of entry. Randomization was stratified by baseline risk group and hospital. Primary trial outcomes were non-fatal stroke and all-cause death.
Results:
From December 1993-May 1998, 652 women were randomized (332 estradiol, 320 placebo). Index event was TIA in 164 subjects and stroke in 488. Mean age of subjects was 71 years (range 46–91); 84% were white, 13% black, and 4% other. Mean follow-up was 2.7 years (range: 18 days-5.8 years). At 1 year, 76% of subjects assigned to estradiol were on study drug. In the estradiol group, adverse events were diagnosed during follow-up in 8 subjects (1 pulmonary embolus (PE), 1 deep venous thrombosis (DVT), 5 breast cancers, 1 endometrial cancer) compared with 7 events in placebo subjects (2 PE, 1 DVT, and 4 breast cancers). Non-fatal strokes were confirmed in 51 subjects in the estradiol group vs. 52 in placebo subjects (rates at 3 years[R]: 16.8% estradiol vs. 17.4% placebo; logrank p-value[p]=.83). Death occurred in 46 estradiol subjects vs. 38 placebo subjects (R=13.0% vs. 12.6%, p=.89). At 3 years, combined rate of non-fatal stroke or death was 27.6% in the estradiol group vs. 27.7% in placebo [p=.80].
Conclusion:
During an average follow-up of 2.7 years, estradiol treatment did not protect against recurrent cerebral ischemia or reduce all-cause mortality in postmenopausal women with pre-existing cerebrovascular disease. No increased risk for adverse events associated with estrogen was observed. This trial adds to the growing body of evidence that fails to confirm a protective role for ERT in populations with known vascular disease.
Decrease in Vascular Integrin Immunoreactivity and Selective Loss of Capillaries During Rat Focal Cerebral Ischemia and Reperfusion
Authors: Kloss Christian Ua , Burggraf Dorothe , Raivich Gennadij , Hamann Gerhard F
Published: July 2021

80

The β1-integrin cell adhesion molecules are the principal endothelial receptors for basal lamina components like collagen and laminin and contribute to the microvascular integrity. During cerebral ischemia and reperfusion the basal lamina antigens disappear, the vascular permeability is impaired, and edema and petechial hemorrhages develop. The current study investigates the localization of β1-integrins in the normal rat brain and after middle cerebral artery occlusion and reperfusion (MCAO/R). In the normal brain, there was only clear immunoreactivity for the α1, α6 and β1 integrins on endothelia, and for α4 on astrocytes. The α6-immunoreactivity was stronger on larger vessels, while α1β1 stained all vessels similarly. Western blotting confirmed the expression of the integrin subunits. Ultrastructurally, integrins were located on the luminal and on the abluminal side of the endothelia and on perivascular cells. Following 3-hours of cerebral ischemia and reperfusion intervals of 0, 9 and 24 hours (I3R0, I3R9 and I3R24, n=15), the number and staining intensity of immunoreactive vessels in the ischemic area were compared to the contralateral side and classified according to their diameters. There was a moderate reduction in the number of the β1-immunoreactive capillaries (6μm to 9μm) by –12% at I3R0 and –15% at I3R9 that greatly decreased to –43% at I3R24 (all p<0.05), all other vessel sizes remained unaffected. The β1-staining intensity decreased homogeneously over all vessel sizes by –4% to –6% at I3R0, –8% to –12% at I3R9 and –16% to –23% at I3R24 (p<0.05). The vascular staining for α1 was affected similarly but less pronounced. Interestingly, the α6-positive capillaries also were reduced by –21% at I3R24 (p<0.05), but the decrease of the α6-staining intensity was confined to vessels larger than 15μm (–15% at I3R24, p<0.05). The diameter-selective loss of vascular integrin presentation points to a size-specific interaction between the endothelium and the basal lamina. The prominent capillar vulnerability may largely account for the impairment of the microvascular integrity during ischemia and reperfusion.