tbuckley/cabot-search huggingface dataset:
Abstract Tu123: Redox Biology of the Mitochondrial Protein mitoNEET and Ascorbate
Authors: Murugesan Velayutham, werner geldenhuys, Valery Khramtsov
Journal: Circulation Research (2024)

Background: It has been proposed that mitoNEET is an Fe-S transfer/repair enzyme, playing a role under conditions of oxidative stress. mitoNEET protects mitochondria against oxidative stress in cardiomyocytes. Loss of mitoNEET is linked with age-related heart failure. Oxidized mitoNEET, [2Fe-2S] 2+ , exports Fe-S cluster from mitochondria to cytosol. Redox control of mitochondrion is essential for the proper functioning of the organelle/mitochondria. However, the molecular mechanisms involved in the redox biology of mitoNEET is not fully studied. Hypothesis: Ascorbate regulates redox reactions and Fe-S cluster transport function of mitoNEET in the mitochondria. Aims: The objective of this work is to evaluate the redox reactions between mitoNEET and ascorbate. Methods: Room temperature and low temperature (77 K) electron paramagnetic resonance (EPR) spectroscopy techniques were used to measure the oxidation of ascorbate and reduced mitoNEET, respectively. Results: EPR spectroscopic study shows that mitoNEET oxidizes ascorbate to ascorbate radical. The formation of ascorbate radical increases with increasing concentration of ascorbate. The formation of ascorbate radical is decreased in the presence of menadione. Low temperature EPR study shows that one electron reduction of mitoNEET by ascorbate. Conclusion: This study suggest that ascorbate is a substrate/cofactor for mitoNEET. Ascorbate regulates the Fe-S cluster transport function of mitoNEET. Ascorbate plays an important role in the redox biology of mitoNEET under physiological and pathophysiological conditions.
Abstract Tu110: CD36-Mediated Transendothelial Fatty Acid Transport Determines Cardiomyocyte Uptake and is Critical to Limiting a Lipotoxic Ceramide Profile and Supporting Cardiac Function during Pathological Stress.
Authors: Azariyas Challa, Andrew Carley, Chandan Kumar Maurya, Yang Wang, Matthew Fasano, Ira Goldberg, E Douglas Lewandowski
Journal: Circulation Research (2024)

The heart primarily relies on long chain fatty acid oxidation (LCFA) to meet ATP demand. CD36, is a transmembrane glycoprotein transferase for LCFA uptake widely expressed in several cell types including endothelial cells (ECs), cardiomyocytes (CMs), skeletal myocytes, adipocytes, and monocyte/macrophages. Notably, genetic deficiency of CD36 in humans is associated with hypertrophic cardiomyopathy. The role of CD36 in transendothelial LCFA delivery to CMs under physiologic vs pathologic conditions is unknown. We explored consequences of EC-specific CD36 deletion (EC-CD36 KO mice) on the cardiac response to pressure overload. Lack of EC-CD36 increased severity of left ventricular hypertrophy (LVH), and systolic and diastolic dysfunction at 8 weeks of pressure overload via transverse aortic constriction (TAC), with increases in HW:TL ratio, LV mass by echo, reduced EF (p&lt;0.05), and elevated E/E’ in EC-CD36-KO vs. flox/flox littermate controls (f/f) (p&lt;0.5) (Fig 1). Dynamic mode 13 C NMR revealed delayed 13 C LCFA uptake kinetics (time constant, τ) in CMs of isolated, sham and TAC EC-CD36 KO hearts provided 13 C palmitate + 13 C oleate (0.02 mM each) + 10 mM glucose + 1 mM lactate (Fig 2). 13 C LCFA esterification into triglyceride (TG) was also reduced in EC-CD36-KO vs f/f controls, but without evidence for an additional effect of TAC. The contribution of 13 C LCFA to acetyl CoA entry into oxidation via the TCA cycle, was reduced by lack of EC-CD36 in sham and TAC hearts (p&lt;0.0001) (Fig 2). Consistent with diminished LCFA oxidation, phosphocreatine (PCr) to ATP ratio was also compromised in EC-CD36-KO-TAC hearts (p&lt;0.05). Lack of EC-CD36 during TAC increased 13 C LCFA trafficking to lipotoxic C16 ceramide in EC-CD36 KO hearts (Fig 3). CD36 in ECs is critical to LCFA delivery to CMs during pathological stress on the heart. The imbalance in intramyocellular LCFA dynamics due to restricted transendothelial delivery of LCFA to CMs increased lipotoxic ceramide formation. Thus, EC CD36 is critical to the adaptive cardiac response to pathological stress, and the absence of EC CD36-dependent delivery of LCFA to CMs exacerbates reduced energy potential, adverse cardiac remodeling, and dysfunction during afterload stress.
Abstract Tu137: Reductive Stress Impedes Neonatal Cardiomyocyte Regeneration
Authors: Allison Trouten, Shuang Li, Henry Sucov, Ge Tao
Journal: Circulation Research (2024)

Background: The elevated level of reactive oxygen species (ROS) has been reported to drive the cell-cycle-exit of neonatal mouse cardiomyocytes and impede their regeneration. On the contrary, the roles of reductive stress in perinatal development and regeneration of cardiomyocytes are still poorly understood. To counterbalance increased oxidative stress, antioxidant enzymes are expressed to remove excessive ROS. Our previous work uncovered a role of the transcription factor Pitx2 as a redox regulator in the ventricular myocardium. Pitx2 protects myocardium by positively regulating genes that encode antioxidant scavengers. In regenerating neonatal hearts, Pitx2 expression is activated by Nrf2, a known antioxidant regulator. Under oxidative stress, Nrf2 dissociates from the Keap1-Cul3-Rbx1 degradation complex and translocates to the nucleus where it promotes Pitx2 expression. Interestingly, our RNA-Seq and ChIP-Seq studies showed that Pitx2 promotes Rbx1 expression which degrades Nrf2. We hypothesize that this negative feedback loop ( Image 1 ) is required to prevent reductive stress in regenerating cardiomyocytes. Methods: Cre-Lox models were used to specifically knock out Nrf2 in cardiomyocytes. Constitutively active Nrf2 (caNrf2) was expressed specifically in cardiomyocyte using AAV9. Guide RNAs were delivered into Myh6Cas9 mouse heart using AAV9 to knock out Rbx1 in cardiomyocytes. Neonatal ROS was transiently suppressed using high dose of N-acetyl-cysteine (NAC). Following left anterior descending artery occlusion (LAD-O), cardiac regeneration was assessed by Masson’s trichrome staining and echocardiography. Markers of cell cycle activity and cell death were assessed by immunofluorescence. RNA-Seq was performed on isolated cardiomyocytes from NAC and saline treated hearts after sham or LAD-O procedure. Results: Pitx2 promotes the degradation of Nrf2 by inducing the expression of Rbx1. In cardiomyocytes, Rbx1 , but not Nrf2 , is required for neonatal heart regeneration. Meanwhile, expression of caNrf2 in cardiomyocytes increases mortality after LAD-O. Removing ROS in neonatal heart prevents cardiomyocyte maturation and disrupts injury response after LAD-O. Conclusions: Together, these results suggest that a fine balance between reductive and oxidative activity is required for neonatal heart regeneration and postnatal heart development. Excessive antioxidant activity will cause reductive stress and impede neonatal heart regeneration after MI.
Abstract Tu107: Decreased Mitochondrial Protein Expression Accompanies Atrial Hypertrophy but Precedes Atrial Fibrillation Onset in a Mouse Model of Spontaneous Atrial Fibrillation
Authors: Ling Li, Shannon Hanmer, Belinda Willard, Jonathan Smith, Kenneth Laurita, Mina Chung, Sarah Schumacher, John Barnard, David Van Wagoner, Julie Rennison
Journal: Circulation Research (2024)

Introduction: Atrial fibrillation (AF), characterized by episodes of rapid and irregular heart rate, increases left atrial (LA) demand for ATP. While the heart is metabolically flexible, able to use fatty acids, lactate, glucose, ketones, and amino acids to generate ATP, fatty acids are the primary fuel source. AF is associated with altered mitochondrial structure and function, but the role of energetic pathways in early stages of AF progression is not clear. Hypothesis: Decreased expression of mitochondrial proteins contributes to AF progression. Aim: Evaluate expression of proteins that influence mitochondrial structure and energetic capacity in AF onset and progression. Methods: AF burden was assessed weekly by electrocardiography (ECG) in male wild-type (WT) and cAMP responsive element modulator IbΔC-X heterozygous mice (CREM) (n=3). LA (4 - 9 mg) was obtained at 7 weeks and 16 weeks. Global proteomics was conducted, and 139,316 peptides were identified that mapped to 6000 robustly quantified proteins. Results: LA hypertrophy was evident in 7-week and 16-week CREM mice (Figure 1). We detected 2408 proteins differentially expressed in CREM compared to WT. At 7 weeks, Hallmark pathways adipogenesis, fatty acid metabolism, oxidative phosphorylation, and myogenesis were decreased in CREM (Table 1). These changes persisted at 16 weeks. We identified 740 differentially expressed mitochondrial proteins using MitoCarta 3.0 (552 decreased, 188 increased). Mitochondrial pathways associated with energy production, including branched chain amino acid metabolism, fatty acid transport and oxidation, carbohydrate metabolism, and tricarboxylic acid cycle were decreased (Table 1). Mitochondrial cristae organizing system (MICOS) and mitophagy, pathways that influence mitochondrial structure and content, were decreased. Terminal ECGs confirmed AF in 16-week CREM mice only. The coefficient of variation of RR intervals (CVRR), a measure of heart rate variability, was increased in 16-week CREM (Figure 2). These data suggest that decreased expression of mitochondrial proteins accompanies LA hypertrophy but precedes onset of AF. Conclusions: Decreased expression of proteins that regulate mitochondrial structure and energy production prior to AF onset suggests that these proteins play an important role in early LA changes that promote AF onset and progression. Future studies will examine how these changes in protein expression relate to alterations in atrial energy homeostasis.
Abstract Tu128: p22 <sup>phox</sup> is a critical factor for the prevention of oxidation and stabilization of SERCA2a in the heart.
Authors: Allen Titus, Yasuki Nakada, Wataru Mizushima, Yanfei Yang, Peiyong Zhai, Shinichi Oka, Toshihide Kashihara, Nadezhda Fefelova, Tong Liu, Hong Li, Lai-Hua Xie, Junichi Sadoshima
Journal: Circulation Research (2024)

Introduction: Elevated oxidative stress linked with heart failure is a major cause for the downregulation of sarcoplasmic/endoplasmic reticulum (SR/ER) Ca2+ ATPase 2a (SERCA2a). NADPH oxidase (NOX) complexes are a key source of reactive oxygen species (ROS) in cardiomyocytes. p22 phox , a transmembrane partner of NOX1-4, plays an essential role in mediating ROS production in multiple NOX complexes. We investigated the role of p22 phox in oxidative stress and SERCA2a levels during pressure overload (PO). Question: Does loss of p22 phox alleviate heart failure through reduction of oxidative stress and stabilization of SERCA2a levels during PO? Methods/approach: Cardiac-specific p22 phox knockout ( p22 phox -cKO) mice were subjected to sham operation or transverse aortic constriction (TAC). The p22 phox interactome was analyzed by co-immunoprecipitation and mass spectrometry. Protein cysteine oxidation was probed by biotinylated-iodoacetamide (BIAM) labelling. Results: The p22 phox -cKO mouse heart showed a 30% reduction in Dityrosine levels compared to WT after 4-week TAC (p&lt;0.05) and 50% lower total tissue H 2 O 2 levels compared to WT after 1-week TAC (p&lt;0.01). Unexpectedly, p22 phox -cKO mice had higher mortality (+20%, p&lt;0.05), lung congestion (2-fold, p&lt;0.05), and fibrosis (&gt;40%, p&lt;0.01) and a lower (40%, p&lt;0.01) left ventricle ejection fraction than WT after TAC. p22 phox directly interacted with SERCA2a. Lack of p22 phox led to oxidation of SERCA2a (50% reduction in BIAM labelling, p&lt;0.01) at cysteine 498 and promoted proteasome-mediated degradation of SERCA2a. The relative SERCA2a ATPase activity was unaltered in both WT and p22 phox -cKO mice. Furthermore, in the absence of p22 phox , SERCA2a interacted strongly with HMG-CoA reductase degradation protein 1 (Hrd1), an endoplasmic reticulum-associated degradation (ERAD)-specific E3 ubiquitin ligase. Knockdown of Hrd1 in the presence of p22 phox knockdown restored SERCA2a to 70% of WT levels (ns and p&lt;0.5 vs p22 phox knockdown). SERCA2a C498S knock-in mice exhibited better cardiac function and stable SERCA2a protein levels after TAC compared to WT mice. Conclusion: The loss of p22 phox exacerbated heart failure through increased SERCA2a cysteine 498 oxidation and degradation through ERAD.
Abstract Tu125: Loss of mitochondrial magnesium leads to hepatic ketogenic insufficiency and accelerates the progression of cardiac hypertrophy
Authors: Thiruvelselvan Ponnusamy, Shanmughapriya Santhanam
Journal: Circulation Research (2024)

Magnesium (Mg 2+ ) is an abundant intracellular divalent cation and is an important co-factor. Like calcium, Mg 2+ is also compartmentalized to mitochondria. Mrs2 is the only known molecular machinery associated with mitochondrial Mg 2+ influx. Although we know that Mg 2+ plays a crucial role in cellular homeostasis, our understanding of how mMg 2+ homeostasis alters the metabolic state of the cell remains incomplete. To begin elucidating the importance of mMg 2+ , we made a liver-specific Mrs2 knockout mouse model (Mrs2 Dhep ) using the Cre-loxP system. Since liver is the central hub of lipid metabolism, we hypothesized that loss of mitochondrial Mg 2+ could alter hepatic lipid homeostasis. Enigmatically we observed increased hepatic lipid accumulation, in spite of increased PPARa activation and Fatty acid oxidation (FAO). In our previous work we showed in Mrs2 Dhep the FAO-derived acetyl CoA to be channeled to de novo lipogenesis (DNL) via cataplerosis, instead of ketogenesis. Further, our results show markedly blunted ketonemia in Mrs2 Dhep revealing a state of ketogenic insufficiency. In the presence of increased ketone bodies (KB), normal hearts preferentially use both KB and FAO while glucose metabolism tend to be reduced. During the progression of heart failure (HF), the failing heart depends on alternate ATP generating substrates like KB as there is a metabolic deficit in both FAO and glucose metabolism. Because we saw a decrease in the KB, we hypothesize that hepatic ketogenic insufficiency will lead to cardiac hypertrophy and heart failure in pressure overload model due to energy insufficiency. Interestingly we found the heart weight and the heart to body weight ratio, left ventricular (LV) wall thickness, expression of genes linked to cardiac hypertrophy to be increased in Mrs2 Dhep compared to control. We also observed an adverse structural remodeling of LV and LV dysfunction. Masson’s trichrome staining showed increased collagen deposition and fibrosis in the Mrs2 Dhep mice when compared to the control. Our results show altered hepatic mitochondrial Mg 2+ homeostasis to have profound direct effect on hepatic lipid metabolism and indirect inter-organ effect on the heart following pressure overload.
Abstract Tu127: Very low-density lipoprotein receptor mediates triglyceride-rich lipoprotein induced oxidative stress and insulin resistance.
Authors: Tahar Hajri, Mohamed Gharib, Thomas Fungwe
Journal: Circulation Research (2024)

Background: Very-low-density lipoprotein receptor (VLDLR), a member of the LDL receptor family, binds and increases the catabolism of triglyceride–rich lipoproteins. Although VLDLR is highly expressed in the heart, its role in VLDLR in oxidative stress and insulin resistance is unclear. Here, we used lean (WT), genetically obese leptin-deficient (ob/ob), and leptin–VLDLR double-null (ob/ob-VLDLR-/-) mice to determine the role of VLDLR in VLDL-induced oxidative stress and insulin resistance in the heart. Methods: Insulin resistance was investigated using glucose tolerance test and uptake of labeled deoxyglucose. Uptake VLDL was assessed in vivo and in isolated cardiomyocytes using double labeled ([3H]-TG, cholesteryl oleoyl ether ([14C])-VLDL. Oxidative stress was evaluated by the measurement of lipid peroxidation products (LPO) and hydrogen peroxides (HPO). NADPH-dependent superoxide production was measured in tissue homogenates and cardiomyocytes with the lucigenin chemiluminescent assay. Superoxide production in cardiomyocytes was investigated using fluorescent probe. Results: While insulin sensitivity and glucose uptake were reduced in the hearts of ob/ob mice, VLDLR expression was upregulated and was associated with increased VLDL uptake and excess lipid deposition in the heart. This was accompanied by an upregulation of cardiac NADPH oxidase (Nox) expression (+100%), increased oxidative stress markers LPO and HPO (+300%) and Nox-dependent superoxide production (+300%). Silencing VLDLR in ob/ob mice had reduced VLDL uptake and prevented excess lipid deposition in the heart, in addition to a reduction of superoxide production and the normalization of insulin sensitivity and glucose uptake. In isolated cardiomyocytes, VLDLR deficiency had prevented VLDL-mediated induction of Nox activity and superoxide overproduction while improving insulin sensitivity and glucose uptake. Conclusions: Our findings indicate that VLDLR deficiency prevents excess lipid accumulation and moderates oxidative stress and insulin resistance in the heart of obese mice. This effect is linked to the active role of VLDLR in VLDL uptake, which triggers a cascade of events leading to increased NOX activity, overproduction superoxide and insulin resistance.
Abstract Tu017: MerTK inhibition aggravates partial carotid ligation-induced atherosclerosis
Authors: Zufeng Ding
Journal: Circulation Research (2024)

Introduction: Partial carotid ligation (PCL) is a widely used model to generate acute disturbed flow (d-flow) in left carotid artery (LCA), leading to rapid endothelial dysfunction and atherosclerosis formation. While the right carotid artery (RCA) without PCL is a built-in control characterized by steady flow (s-flow). Endothelial cells (ECs) form a single cell layer that is constantly exposed to blood flow patterns and play important roles in atherosclerosis. MER proto-oncogene tyrosine kinase (MerTK) is the major receptor for efferocytosis, a process of the efficient clearance of apoptotic cells. MerTK is mainly expressed in macrophages and there are very limited reports focusing on MerTK function in ECs. This study was designed to investigate the role of blood flow patterns in regulating MerTK expression in aortic ECs and its contributions to the development of atherosclerosis. Methods: Partial carotid artery ligation combined with AAV-PCSK9 and high fat diet (HFD) were used to set up acute atherosclerosis within 4 weeks with WT and MerTK -/- mice. Proteomics and molecular experiments were applied to reveal the potential mechanism pathways. Results: Our proteomics data showed that, compared with WT group, MerTK -/- aggravates atherosclerosis in LCA through upregulation of endothelial dysfunction markers (e.g. IL-1β, NF-κB, TLR4, MAPK signaling) and mitochondrial dysfunction. Our immunostaining data further validates the proteomics data, showing that MerTK -/- induces impaired endothelial efferocytosis and expression of endothelial dysfunction markers (e.g., Caspase-3, NF-κB, TLR4, vWF, VCAM-1, and P22 phox ) and promotes the development of atherosclerosis. Conclusion: Our data suggests that endothelial MerTK is sensitive to d-flow and determines endothelial efferocytosis in vivo. MerTK inhibition promotes endothelial dysfunction and atherosclerosis. Our data may provide novel evidence and reference opinions for the application of endothelial MerTK- targeted therapeutics to treat atherosclerosis.
Abstract Tu079: The Calcium Handling Machinery and Electrophysiology is Remodeled in Friedreich’s Ataxia
Authors: Roman Czornobil, Obada Abu-Assail, Elizabeth Wood, David Lynch, Sami Noujaim, Bojjibabu Chidipi
Journal: Circulation Research (2024)

Background: Friedreich's ataxia (FA) is a progressive neurodegenerative disorder. FA is caused by a deficiency in the frataxin protein due to an expansion of GAA repeats in the first intron of the frataxin gene. Reduced frataxin protein expression is thought to negatively affect mitochondrial function, leading to increased oxidative damage. Although FA is considered a neurodegenerative disorder, it is not completely understood why most patients present with left ventricular systolic dysfunction and die from cardiac events. Objective: The objective of our study is to determine whether abnormal calcium handling is a molecular mechanism of cardiac dysfunction in FA. Methods: We used the frataxin knock-out (KO) mouse model of FA as well as human heart samples from donors with FA and from unaffected donors. The expression of calcium-handling proteins was assessed using proteomics and westernblot. We used echocardiography and telemetric ECG to assess cardiac function in the mice. We also performed in-vivo programmed electrical stimulation (PES) to assess ventricular arrhythmogenesis in the mouse heart. Results: Proteomics and western blot showed that SERCA2, Ryr2, PLN, CASQ2, and CaMKII were downregulated and that the phosphorylation of Ryr2 and PLN (Ser16 and Thr17) were also significantly decreased in left ventricular tissue from humans with FA and from KO mice. The mitochondrial calcium handling proteins VDAC and MCU were upregulated in left ventricular tissue from humans with FA and from KO mice. On telemetric ECG, the RR tended to prolong in the KO animals compared to wild types (p=0.053), and heart rate variability analysis indicated heightened cardiac parasympathetic input. Echocardiography showed that the ejection fraction and fractional shortening were significantly decreased and left ventricular wall thickness and diameter were significantly increased in the KO mice. In PES, ventricular arrhythmogenesis was greater in KO mice compared to wild types. Conclusions: The reduced expression of calcium handling proteins may contribute to cardiac contractile and electrophysiological abnormalities in FA. Keywords: Friedreich's ataxia, SERCA2, Ryr2, PLN, CASQ2, and CaMKII.