Metabolites of MDMA Induce Oxidative Stress and Contractile Dysfunction in Adult Rat Left Ventricular Myocytes

Diabetic cardiomyopathy is characterized by cardiac dysfunction. This study was designed to examine the effect of benfotiamine, a lipophilic derivative of thiamine, on streptozotocin (STZ)-induced cardiac contractile dysfunction in mouse cardiomyocytes. Adult male FVB mice were made diabetic with a single injection of STZ (200 mg/kg ip). Fourteen days later, control and diabetic (fasting plasma glucose > 13.9 mM) mice were put on benfotiamine therapy (100 mg·kg−1·day−1 ip) for another 14 days. Mechanical and intracellular Ca2+ properties were evaluated in left ventricular myocytes using an IonOptix MyoCam system. The following indexes were evaluated: peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR90), maximal velocity of shortening/relengthening, resting and rise of intracellular Ca2+ in response to electrical stimulus, sarcoplasmic reticulum (SR) Ca2+ load, and intracellular Ca2+ decay rate (τ). Two- or four-week STZ treatment led to hyperglycemia, prolonged TPS and TR90, reduced SR Ca2+ load, elevated resting intracellular Ca2+ level and prolonged τ associated with normal PS, maximal velocity of shortening/relengthening, and intracellular Ca2+ rise in response to electrical stimulus. Benfotiamine treatment abolished prolongation in TPS, TR90, and τ, as well as reduction in SR Ca2+ load without affecting hyperglycemia and elevated resting intracellular Ca2+. Diabetes triggered oxidative stress, measured by GSH-to-GSSG ratio and formation of advanced glycation end product (AGE) in the hearts. Benfotiamine treatment alleviated oxidative stress without affecting AGE or protein carbonyl formation. Collectively, our results indicated that benfotiamine may rescue STZ-induced cardiomyocyte dysfunction but not AGE formation in short-term diabetes.

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Increased susceptibility of aged hearts to ventricular fibrillation during oxidative stress

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00579.2009 ◽

2009 ◽

Vol 297 (5) ◽

pp. H1594-H1605 ◽

Cited By ~ 83

Author(s):

Norishige Morita ◽

Ali A. Sovari ◽

Yuanfang Xie ◽

Michael C. Fishbein ◽

William J. Mandel ◽

...

Keyword(s):

Oxidative Stress ◽

Young Adult ◽

Ventricular Myocytes ◽

Tissue Level ◽

Left Ventricular ◽

Middle Aged ◽

Aged Rat ◽

Adult Rat ◽

Inactive Form ◽

Rat Hearts

Oxidative stress with hydrogen peroxide (H2O2) readily promotes early afterdepolarizations (EADs) and triggered activity (TA) in isolated rat and rabbit ventricular myocytes. Here we examined the effects of H2O2 on arrhythmias in intact Langendorff rat and rabbit hearts using dual-membrane voltage and intracellular calcium optical mapping and glass microelectrode recordings. Young adult rat (3–5 mo, N = 25) and rabbit (3–5 mo, N = 6) hearts exhibited no arrhythmias when perfused with H2O2 (0.1–2 mM) for up to 3 h. However, in 33 out of 35 (94%) aged (24–26 mo) rat hearts, 0.1 mM H2O2 caused EAD-mediated TA, leading to ventricular tachycardia (VT) and fibrillation (VF). Aged rabbits (life span, 8–12 yr) were not available, but 4 of 10 middle-aged rabbits (3–5 yr) developed EADs, TA, VT, and VF. These arrhythmias were suppressed by the reducing agent N-acetylcysteine (2 mM) and CaMKII inhibitor KN-93 (1 μM) but not by its inactive form (KN-92, 1 μM). There were no significant differences between action potential duration (APD) or APD restitution slope before or after H2O2 in aged or young adult rat hearts. In histological sections, however, trichrome staining revealed that aged rat hearts exhibited extensive fibrosis, ranging from 10–90%; middle-aged rabbit hearts had less fibrosis (5–35%), whereas young adult rat and rabbit hearts had <4% fibrosis. In aged rat hearts, EADs and TA arose most frequently (70%) from the left ventricular base where fibrosis was intermediate (∼30%). Computer simulations in two-dimensional tissue incorporating variable degrees of fibrosis showed that intermediate (but not mild or severe) fibrosis promoted EADs and TA. We conclude that in aged ventricles exposed to oxidative stress, fibrosis facilitates the ability of cellular EADs to emerge and generate TA, VT, and VF at the tissue level.

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Inhibition of p66ShcA redox activity in cardiac muscle cells attenuates hyperglycemia-induced oxidative stress and apoptosis

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00225.2008 ◽

2009 ◽

Vol 296 (2) ◽

pp. H380-H388 ◽

Cited By ~ 25

Author(s):

Ashwani Malhotra ◽

Himanshu Vashistha ◽

Virendra S. Yadav ◽

Michael G. Dube ◽

Satya P. Kalra ◽

...

Keyword(s):

Oxidative Stress ◽

Cytochrome C ◽

Cardiac Muscle ◽

Ventricular Myocytes ◽

Muscle Cells ◽

Molecular Switch ◽

Left Ventricular ◽

Cardiac Muscle Cells ◽

Rat Ventricular Myocytes ◽

Adult Rat

Apoptotic myocyte cell death, diastolic dysfunction, and progressive deterioration in left ventricular pump function characterize the clinical course of diabetic cardiomyopathy. A key question concerns the mechanism(s) by which hyperglycemia (HG) transmits danger signals in cardiac muscle cells. The growth factor adapter protein p66ShcA is a genetic determinant of longevity, which controls mitochondrial metabolism and cellular responses to oxidative stress. Here we demonstrate that interventions which attenuate or prevent HG-induced phosphorylation at critical position 36 Ser residue (phospho-Ser36) inhibit the redox function of p66ShcA and promote the survival phenotype. Adult rat ventricular myocytes obtained by enzymatic dissociation were transduced with mutant-36 p66ShcA (mu-36) dominant-negative expression vector and plated in serum-free media containing 5 or 25 mM glucose. At HG, adult rat ventricular myocytes exhibit a marked increase in reactive oxygen species production, upregulation of phospho-Ser36, collapse of mitochondrial transmembrane potential, and increased formation of p66ShcA/cytochrome- c complexes. These indexes of oxidative stress were accompanied by a 40% increase in apoptosis and the upregulation of cleaved caspase-3 and the apoptosis-related proteins p53 and Bax. To test whether p66ShcA functions as a redox-sensitive molecular switch in vivo, we examined the hearts of male Akita diabetic nonobese (C57BL/6J) mice. Western blot analysis detected the upregulation of phospho-Ser36, the translocation of p66ShcA to mitochondria, and the formation of p66ShcA/cytochrome- c complexes. Conversely, the correction of HG by recombinant adeno-associated viral delivery of leptin reversed these alterations. We conclude that p66ShcA is a molecular switch whose redox function is turned on by phospho-Ser36 and turned off by interventions that prevent this modification.

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Effects of acidosis on resting cytosolic and mitochondrial Ca2+ in mammalian myocardium.

The Journal of General Physiology ◽

10.1085/jgp.102.3.575 ◽

1993 ◽

Vol 102 (3) ◽

pp. 575-597 ◽

Cited By ~ 26

Author(s):

G Gambassi ◽

R G Hansford ◽

S J Sollott ◽

B A Hogue ◽

E G Lakatta ◽

...

Keyword(s):

Lactic Acid ◽

Ventricular Myocytes ◽

Free Acid ◽

Left Ventricular ◽

Ruthenium Red ◽

Acetoxymethyl Ester ◽

Cytosolic Ph ◽

Bicarbonate Buffer ◽

Adult Rat ◽

Mammalian Myocardium

Acidosis increases resting cytosolic [Ca2+], (Cai) of myocardial preparations; however, neither the Ca2+ sources for the increase in Cai nor the effect of acidosis on mitochondrial free [Ca2+], (Cam) have been characterized. In this study cytosolic pH (pHi) was monitored in adult rat left ventricular myocytes loaded with the acetoxymethyl ester (AM form) of SNARF-1. A stable decrease in the pHi of 0.52 +/- 0.05 U (n = 16) was obtained by switching from a bicarbonate buffer equilibrated with 5% CO2 to a buffer equilibrated with 20% CO2. Electrical stimulation at either 0.5 or 1.5 Hz had no effect on pHi in 5% CO2, nor did it affect the magnitude of pHi decrease in response to hypercarbic acidosis. Cai was measured in myocytes loaded with indo-1/free acid and Cam was monitored in cells loaded with indo-1/AM after quenching cytosolic indo-1 fluorescence with MnCl2. In quiescent intact myocytes bathed in 1.5 mM [Ca2+], hypercarbia increased Cai from 130 +/- 5 to 221 +/- 13 nM. However, when acidosis was effected in electrically stimulated myocytes, diastolic Cai increased more than resting Cai in quiescent myocytes, and during pacing at 1.5 Hz diastolic Cai was higher (285 +/- 17 nM) than at 0.5 Hz (245 +/- 18 nM; P < 0.05). The magnitude of Cai increase in quiescent myocytes was not affected either by sarcoplasmic reticulum (SR) Ca2+ depletion with ryanodine or by SR Ca2+ depletion and concomitant superfusion with a Ca(2+)-free buffer. In unstimulated intact myocytes hypercarbia increased Cam from 95 +/- 12 to 147 +/- 19 nM and this response was not modified either by ryanodine and a Ca(2+)-free buffer or by 50 microM ruthenium red in order to block the mitochondrial uniporter. In mitochondrial suspensions loaded either with BCECF/AM or indo-1/AM, acidosis produced by lactic acid addition decreased both intra- and extramitochondrial pH and increased Cam. Studies of mitochondrial suspensions bathed in indo-1/free acid-containing solution showed an increase in extramitochondrial Ca2+ after the addition of lactic acid. Thus, in quiescent myocytes, cytoplasmic and intramitochondrial buffers, rather than transsarcolemmal Ca2+ influx or SR Ca2+ release, are the likely Ca2+ sources for the increase in Cai and Cam, respectively; additionally, Ca2+ efflux from the mitochondria may contribute to the raise in Cai. In contrast, in response to acidosis, diastolic Cai in electrically stimulated myocytes increases more than resting Cai in quiescent cells; this suggests that during pacing, net cell Ca2+ gain contributes to enhance diastolic Cai.

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Lipopolysaccharide induces apoptosis in adult rat ventricular myocytes via cardiac AT1 receptors

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00701.2001 ◽

2002 ◽

Vol 283 (2) ◽

pp. H461-H467 ◽

Cited By ~ 45

Author(s):

Hai Ling Li ◽

Jun Suzuki ◽

Evelyn Bayna ◽

Fu-Min Zhang ◽

Erminia Dalle Molle ◽

...

Keyword(s):

Angiotensin Ii ◽

Ventricular Myocytes ◽

Annexin V ◽

Left Ventricular ◽

Terminal Deoxynucleotidyl Transferase ◽

Rat Ventricular Myocytes ◽

Adult Rat ◽

Cardiac Apoptosis

Lipopolysaccharide (LPS) from gram-negative bacteria circulates in acute, subacute, and chronic conditions. It was hypothesized that LPS directly induces cardiac apoptosis. In adult rat ventricular myocytes (isolated with depyrogenated digestive enzymes to minimize tolerance), LPS (10 ng/ml) decreased the ratio of Bcl-2 to Bax at 12 h; increased caspase-3 activity at 16 h; and increased annexin V, propidium iodide, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining at 24 h. Apoptosis was blocked by the caspase inhibitor benzyloxycarbonyl-valine-alanine-aspartate fluoromethylketone (Z-VAD-fmk), captopril, and angiotensin II type 1 receptor (AT1) inhibitor (losartan), but not by inhibitors of AT2 receptors (PD-123319), tumor necrosis factor-α (TNFRII:Fc), or nitric oxide ( N G-monomethyl-l-arginine). Angiotensin II (100 nmol/l) induced apoptosis similar to LPS without additive effects. LPS in vivo (1 mg/kg iv) increased apoptosis in left ventricular myocytes for 1–3 days, which dissipated after 1–2 wk. Losartan (23 mg · kg−1 · day−1 in drinking water for 3 days) blocked LPS-induced in vivo apoptosis. In conclusion, low levels of LPS induce cardiac apoptosis in vitro and in vivo by activating AT1 receptors in myocytes.

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Chronic ventricular myocyte-specific overexpression of angiotensin II type 2 receptor results in intrinsic myocyte contractile dysfunction

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00957.2003 ◽

2005 ◽

Vol 288 (1) ◽

pp. H317-H327 ◽

Cited By ~ 16

Author(s):

Masaharu Nakayama ◽

Xinhua Yan ◽

Robert L. Price ◽

Thomas K. Borg ◽

Kenta Ito ◽

...

Keyword(s):

Transgenic Mice ◽

Ventricular Myocytes ◽

Contractile Function ◽

Left Ventricular ◽

Contractile Dysfunction ◽

Inotropic Response ◽

Ang Ii ◽

Intracellular Ca ◽

Depressed Response

ANG II type 2 receptor (AT2) is upregulated in failing hearts, but its effect on myocyte contractile function is not known. We measured fractional cell shortening and intracellular Ca2+ concentration transients in left ventricular myocytes derived from transgenic mice in which ventricle-specific expression of AT2 was driven by the myosin light chain 2v promoter. Confocal microscopy studies confirmed upregulation of AT2 in the ventricular myocytes and partial colocalization of AT2 with AT1. Three components of contractile performance were studied. First, baseline measurements (0.5 Hz, 1.5 mmol/l extracellular Ca2+ concentration, 25°C) and study of contractile reserve at faster pacing rates (1–5 Hz) revealed Ca2+-dependent contractile dysfunction in myocytes from AT2 transgenic mice. Comparison of two transgenic lines suggested a dose-dependent relationship between magnitude of contractile dysfunction and level of AT2 expression. Second, activity of the Na+/H+ exchanger, a dominant transporter that regulates beat-to-beat intracellular pH, was impaired in the transgenic myocytes. Third, the inotropic response to β-adrenergic versus ANG II stimulation differed. Both lines showed impaired contractile response to β-adrenergic stimulation. ANG II elicited an increase in contractility and intracellular Ca2+ in wild-type myocytes but caused a negative inotropic effect in myocytes from AT2 transgenic mice. In contrast with β-adrenergic response, the depressed response to ANG II was related to level of AT2 overexpression. The depressed response to ANG II was also present in myocytes from young transgenic mice before development of heart failure. Thus chronic overexpression of AT2 has the potential to cause Ca2+- and pH-dependent contractile dysfunction in ventricular myocytes, as well as loss of the inotropic response to ANG II.

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Oxidative stress in cardiomyocytes contributes to decreased SERCA2a activity in rats with metabolic syndrome

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00211.2013 ◽

2013 ◽

Vol 305 (9) ◽

pp. H1344-H1353 ◽

Cited By ~ 37

Author(s):

Jaime Balderas-Villalobos ◽

Tzindilu Molina-Muñoz ◽

Patrick Mailloux-Salinas ◽

Guadalupe Bravo ◽

Karla Carvajal ◽

...

Keyword(s):

Oxidative Stress ◽

Insulin Resistance ◽

Metabolic Syndrome ◽

Ventricular Myocytes ◽

Left Ventricular Diastolic Dysfunction ◽

Left Ventricular ◽

Confocal Imaging ◽

Insulin Resistant ◽

Plasmic Reticulum ◽

Ventricular Diastolic Dysfunction

Ca+mishandling due to impaired activity of cardiac sarco(endo)plasmic reticulum Ca2+ATPase (SERCA2a) has been associated with the development of left ventricular diastolic dysfunction in insulin-resistant cardiomyopathy. However, the molecular causes underlying SERCA2a alterations induced by insulin resistance and related metabolic disorders, such as metabolic syndrome (MetS), are not completely understood. In this study, we used a sucrose-fed rat model of MetS to test the hypothesis that decreased SERCA2a activity is mediated by elevated oxidative stress produced in the MetS heart. Production of ROS and cytosolic Ca2+concentration were recorded in left ventricular myocytes using confocal imaging. The level of SERCA2a oxidation was determined in left ventricular homogenates by biotinylated iodoacetamide labeling. Compared with control rats, sucrose-fed rats exhibited several characteristics of MetS, including central obesity, insulin resistance, hyperinsulinemia, and hypertriglyceridemia. Moreover, relative to myocytes from control rats, myocytes from MetS rats exhibited elevated basal production of ROS accompanied by slowed cytosolic Ca2+removal, reflected by prolonged Ca2+transients. The slowed cytosolic Ca2+removal was associated with a significant decrease in SERCA2a-mediated Ca2+reuptake and increased SERCA2a oxidation. Importantly, myocytes from MetS rats treated with the antioxidant N-acetylcysteine showed normal ROS levels and SERCA2a-mediated Ca2+reuptake as well as accelerated cytosolic Ca2+removal. These data suggest that elevated oxidative stress may induce oxidative modifications on SERCA2a leading to abnormal function of this protein in the MetS heart.

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