David R. Pimentel

Adiponectin protects against myocardial ischemia-reperfusion injury through AMPK- and COX-2-dependent mechanisms.

Obesity-related disorders are associated with the development of ischemic heart disease. Adiponectin is a circulating adipose-derived cytokine that is downregulated in obese individuals and after myocardial infarction. Here, we examine the role of adiponectin in myocardial remodeling in response to acute injury. Ischemia-reperfusion in adiponectin-deficient (APN-KO) mice resulted in increased myocardial infarct size, myocardial apoptosis and tumor necrosis factor (TNF)-α expression compared with wild-type mice. Administration of adiponectin diminished infarct size, apoptosis and TNF-α production in both APN-KO and wild-type mice. In cultured cardiac cells, adiponectin inhibited apoptosis and TNF-α production. Dominant negative AMP-activated protein kinase (AMPK) reversed the inhibitory effects of adiponectin on apoptosis but had no effect on the suppressive effect of adiponectin on TNF-α production. Adiponectin induced cyclooxygenase (COX)-2–dependent synthesis of prostaglandin E2 in cardiac cells, and COX-2 inhibition reversed the inhibitory effects of adiponectin on TNF-α production and infarct size. These data suggest that adiponectin protects the heart from ischemia-reperfusion injury through both AMPK- and COX-2–dependent mechanisms.

Norepinephrine Stimulates Apoptosis in Adult Rat Ventricular Myocytes by Activation of the β-Adrenergic Pathway

BACKGROUND Myocardial sympathetic activity is increased in heart failure. We tested the hypothesis that norepinephrine (NE) stimulates apoptosis in adult rat ventricular myocytes in vitro. METHODS AND RESULTS Myocytes were exposed to NE alone (10 micromol/L), NE+propranolol (2 micromol/L), NE+prazosin (0.1 micromol/L), or isoproterenol (ISO, 10 micromol/L) for 24 hours. NE and ISO decreased the number of viable myocytes by approximately 35%. This effect was completely blocked by the beta-adrenergic antagonist propranolol but was not affected by the alpha1-adrenergic antagonist prazosin. NE increased DNA laddering on agarose gel electrophoresis and increased the percentage of cells that were stained by terminal deoxynucleotidyl transferase-mediated nick end-labeling from 5.8+/-1. 0% to 21.0+/-2.3% (P<0.01; n=4). NE likewise increased the percentage of apoptotic cells with hypodiploid DNA content as assessed by flow cytometry from 7.8+/-0.7% to 16.7+/-2.2% (P<0.01; n=6), and this effect was abolished by propranolol but not prazosin. ISO and forskolin (10 micromol/L) mimicked the effect of NE, increasing the percentage of apoptotic cells to 14.7+/-1.9% and 14. 4+/-2.2%, respectively. NE-stimulated apoptosis was abolished by the protein kinase A inhibitor H-89 (20 micromol/L) or the voltage-dependent calcium channel blockers diltiazem and nifedipine. CONCLUSIONS NE, acting via the ss-adrenergic pathway, stimulates apoptosis in adult rat cardiac myocytes in vitro. This effect is mediated by protein kinase A and requires calcium entry via voltage-dependent calcium channels. NE-stimulated apoptosis of cardiac myocytes may contribute to the progression of myocardial failure.

Adiponectin-mediated modulation of hypertrophic signals in the heart.

Patients with diabetes and other obesity-linked conditions have increased susceptibility to cardiovascular disorders. The adipocytokine adiponectin is decreased in patients with obesity-linked diseases. Here, we found that pressure overload in adiponectin-deficient mice resulted in enhanced concentric cardiac hypertrophy and increased mortality that was associated with increased extracellular signal-regulated kinase (ERK) and diminished AMP-activated protein kinase (AMPK) signaling in the myocardium. Adenovirus-mediated supplemention of adiponectin attenuated cardiac hypertrophy in response to pressure overload in adiponectin-deficient, wild-type and diabetic db/db mice. In cultures of cardiac myocytes, adiponectin activated AMPK and inhibited agonist-stimulated hypertrophy and ERK activation. Transduction with a dominant-negative form of AMPK reversed these effects, suggesting that adiponectin inhibits hypertrophic signaling in the myocardium through activation of AMPK signaling. Adiponectin may have utility for the treatment of hypertrophic cardiomyopathy associated with diabetes and other obesity-related diseases.

S-Glutathiolation by peroxynitrite activates SERCA during arterial relaxation by nitric oxide.

Nitric oxide (NO) physiologically stimulates the sarco/endoplasmic reticulum calcium (Ca2+) ATPase (SERCA) to decrease intracellular Ca2+ concentration and relax cardiac, skeletal and vascular smooth muscle. Here, we show that NO-derived peroxynitrite (ONOO−) directly increases SERCA activity by S-glutathiolation and that this modification of SERCA is blocked by irreversible oxidation of the relevant cysteine thiols during atherosclerosis. Purified SERCA was S-glutathiolated by ONOO− and the increase in Ca2+-uptake activity of SERCA reconstituted in phospholipid vesicles required the presence of glutathione. Mutation of the SERCA-reactive Cys674 to serine abolished these effects. Because superoxide scavengers decreased S-glutathiolation of SERCA and arterial relaxation by NO, ONOO− is implicated as the intracellular mediator. NO-dependent relaxation as well as S-glutathiolation and activation of SERCA were decreased by atherosclerosis and Cys674 was found to be oxidized to sulfonic acid. Thus, irreversible oxidation of key thiol(s) in disease impairs NO-induced relaxation by preventing reversible S-glutathiolation and activation of SERCA by NO/ONOO−.

Human Amyloidogenic Light Chains Directly Impair Cardiomyocyte Function Through an Increase in Cellular Oxidant Stress

Primary amyloidosis is a systemic disorder characterized by the clonal production and tissue deposition of immunoglobulin light chain (LC) proteins. Congestive heart failure remains the greatest cause of death in primary amyloidosis, due to the development of a rapidly progressive amyloid cardiomyopathy. Amyloid cardiomyopathy is largely unresponsive to current heart failure therapies, and is associated with a median survival of less than 6 months and a 5-year survival of less than 10%. The mechanisms underlying this disorder, however, remain unknown. In this report, we demonstrate that physiological levels of human amyloid LC proteins, isolated from patients with amyloid cardiomyopathy (cardiac-LC), specifically alter cellular redox state in isolated cardiomyocytes, marked by an increase in intracellular reactive oxygen species and upregulation of the redox-sensitive protein, heme oxygenase-1. In contrast, vehicle or control LC proteins isolated from patients without cardiac involvement did not alter cardiomyocyte redox status. Oxidant stress imposed by cardiac-LC proteins further resulted in direct impairment of cardiomyocyte contractility and relaxation, associated with alterations in intra-cellular calcium handling. Cardiomyocyte dysfunction induced by cardiac-LC proteins was independent of neurohormonal stimulants, vascular factors, or extracellular fibril deposition, and was prevented through treatment with a superoxide dismutase/catalase mimetic. This study suggests that cardiac dysfunction in amyloid cardiomyopathy is directly mediated by LC protein-induced cardiomyocyte oxidant stress and alterations in cellular redox status, independent of fibril deposition. Antioxidant therapies or treatment strategies aimed at eliminating circulating LC proteins may therefore be beneficial in the treatment of this fatal disease.

β-Adrenergic Receptor–Stimulated Apoptosis in Cardiac Myocytes Is Mediated by Reactive Oxygen Species/c-Jun NH2-Terminal Kinase–Dependent Activation of the Mitochondrial Pathway

Stimulation of &bgr;-adrenergic receptors (&bgr;ARs) causes apoptosis in adult rat ventricular myocytes (ARVMs). The role of reactive oxygen species (ROS) in mediating &bgr;AR-stimulated apoptosis is not known. Stimulation of &bgr;ARs with norepinephrine (10 &mgr;mol/L) in the presence of prazosin (100 nmol/L) for 24 hours increased the number of apoptotic myocytes as determined by TUNEL staining by 3.6- fold. The superoxide dismutase/catalase mimetics Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP; 10 &mgr;mol/L) and Euk-134 decreased &bgr;AR-stimulated apoptosis by 89±6% and 76±10%, respectively. Infection with an adenovirus expressing catalase decreased &bgr;AR-stimulated apoptosis by 82±15%. The mitochondrial permeability transition pore inhibitor bongkrekic acid (50 &mgr;mol/L) decreased &bgr;AR-stimulated apoptosis by 76±8%, and the caspase inhibitor zVAD-fmk (25 &mgr;mol/L) decreased &bgr;AR-stimulated apoptosis by 62±11%. &bgr;AR-stimulated cytochrome c release was inhibited by MnTMPyP. &bgr;AR stimulation caused c-Jun NH2-terminal kinase (JNK) activation, which was abolished by MnTMPyP. Transfection with an adenovirus expressing dominant-negative JNK inhibited &bgr;AR-stimulated apoptosis by 81±12%, and the JNK inhibitor SP600125 inhibited both &bgr;AR-stimulated apoptosis and cytochrome c release. Thus, &bgr;AR-stimulated apoptosis in ARVMs involves ROS/JNK-dependent activation of the mitochondrial death pathway.

H2O2 regulates cardiac myocyte phenotype via concentration-dependent activation of distinct kinase pathways

Reactive oxygen species (ROS) can act as signaling molecules to stimulate either hypertrophy or apoptosis in cardiac myocytes. We tested the hypothesis that the phenotypic effects of ROS are due to differential, concentration-dependent activation of specific kinase signaling pathways. Adult rat ventricular myocytes were exposed to H(2)O(2) over a broad concentration range (10-1000 microM). Low concentrations of H(2)O(2) (10-30 microM) increased protein synthesis without affecting survival. Higher concentrations of H(2)O(2) (100-200 microM) increased apoptosis (assessed by TUNEL). Still higher concentrations of H(2)O(2) (300-1000 microM) caused both apoptosis and necrosis. A hypertrophic concentration of H(2)O(2) (10 microM) increased the activity of ERK1/2, but not that of JNK, p38 kinase or Akt. An apoptotic concentration of H(2)O(2) (100 microM) activated JNK, p38 kinase and Akt, and further activated ERK1/2. The MEK1/2 inhibitor U0126 prevented the hypertrophic effect of 10 microM H(2)O(2). The apoptotic effect of 100 microM H(2)O(2) was inhibited bya dominant-negative JNK adenovirus, and was potentiated by U0126 or an Akt inhibitor. Thus, the concentration-dependent effects of ROS on myocyte hypertrophy and growth are due, at least in part, to the differential activation of specific kinase signaling pathways that regulate hypertrophy and apoptosis.

Follistatin-Like 1 Is an Akt-Regulated Cardioprotective Factor That Is Secreted by the Heart

Background— The Akt protein kinase is an important mediator of cardiac myocyte growth and survival. To identify factors with novel therapeutic applications in cardiac diseases, we focused on the identification of factors secreted from Akt1-activated cells that have cardioprotective effects through autocrine/paracrine mechanisms. Methods and Results— Using an inducible Akt1 transgenic mouse model, we have found that follistatin-like 1 (Fstl1) protein and transcript expression are increased 4.0- and 2.0-fold, respectively, by Akt activation in the heart (P<0.05). Fstl1 transcript was also upregulated in response to myocardial stresses including transverse aortic constriction, ischemia/reperfusion injury, and myocardial infarction. Adenovirus-mediated overexpression of Fstl1 protected cultured neonatal rat ventricular myocytes from hypoxia/reoxygenation-induced apoptosis (P<0.01), and this protective effect was dependent on the upregulation of both Akt and ERK activities. Conversely, knockdown of Fstl1 in cardiac myocytes decreased basal Akt signaling and increased the frequency of apoptotic death in vitro (P<0.01). The intravenous administration of an adenoviral encoding Fstl1 to mice resulted in a 66.0% reduction in myocardial infarct size after ischemia/reperfusion injury that was accompanied by a 70.9% reduction in apoptosis in the heart (P<0.01). Conclusions— These results indicate that Fstl1 is a cardiac-secreted factor that functions as an antiapoptotic protein. Fstl1 could play a role in myocardial maintenance and repair in response to harmful stimuli.

Glucose-6-Phosphate Dehydrogenase Modulates Cytosolic Redox Status and Contractile Phenotype in Adult Cardiomyocytes

&NA; —Reactive oxygen species (ROS)‐mediated cell injury contributes to the pathophysiology of cardiovascular disease and myocardial dysfunction. Protection against ROS requires maintenance of endogenous thiol pools, most importantly, reduced glutathione (GSH), by NADPH. In cardiomyocytes, GSH resides in two separate cellular compartments: the mitochondria and cytosol. Although mitochondrial GSH is maintained largely by transhydrogenase and isocitrate dehydrogenase, the mechanisms responsible for sustaining cytosolic GSH remain unclear. Glucose‐6‐phosphate dehydrogenase (G6PD) functions as the first and rate‐limiting enzyme in the pentose phosphate pathway, responsible for the generation of NADPH in a reaction coupled to the de novo production of cellular ribose. We hypothesized that G6PD is required to maintain cytosolic GSH levels and protect against ROS injury in cardiomyocytes. We found that in adult cardiomyocytes, G6PD activity is rapidly increased in response to cellular oxidative stress, with translocation of G6PD to the cell membrane. Furthermore, inhibition of G6PD depletes cytosolic GSH levels and subsequently results in cardiomyocyte contractile dysfunction through dysregulation of calcium homeostasis. Cardiomyocyte dysfunction was reversed through treatment with either a thiol‐repleting agent (L‐2‐oxothiazolidine‐4‐carboxylic acid) or antioxidant treatment (Eukarion‐134), but not with exogenous ribose. Finally, in a murine model of G6PD deficiency, we demonstrate the development of in vivo adverse structural remodeling and impaired contractile function over time. We, therefore, conclude that G6PD is a critical cytosolic antioxidant enzyme, essential for maintenance of cytosolic redox status in adult cardiomyocytes. Deficiency of G6PD may contribute to cardiac dysfunction through increased susceptibility to free radical injury and impairment of intracellular calcium transport. The full text of this article is available online at http://www.circresaha.org. (Circ Res. 2003;93:e9‐e16.)

α-Adrenergic Receptor–Stimulated Hypertrophy in Adult Rat Ventricular Myocytes Is Mediated via Thioredoxin-1–Sensitive Oxidative Modification of Thiols on Ras

Background—&agr;-Adrenergic receptor (&agr;AR)–stimulated hypertrophy in adult rat ventricular myocytes is mediated by reactive oxygen species–dependent activation of the Ras-Raf-MEK1/2-ERK1/2 signaling pathway. Because Ras is known to have redox-sensitive cysteine residues, we tested the hypothesis that &agr;AR-stimulated hypertrophic signaling is mediated via oxidative modification of Ras thiols. Methods and Results—The effect of &agr;AR stimulation on the number of free thiols on Ras was measured with biotinylated iodoacetamide labeling. &agr;AR stimulation caused a 48% decrease in biotinylated iodoacetamide–labeled Ras that was reversed by dithiothreitol (10 mmol/L), indicating a decrease in the availability of free thiols on Ras as a result of an oxidative posttranslational modification. This effect was abolished by adenoviral overexpression of thioredoxin-1 (TRX1) and potentiated by the TRX reductase inhibitor azelaic acid. Likewise, &agr;AR-stimulated Ras activation was abolished by TRX1 overexpression and potentiated by azelaic acid. TRX1 overexpression inhibited the &agr;AR-stimulated phosphorylation of MEK1/2, ERK1/2, and p90RSK and prevented cellular hypertrophy, sarcomere reorganization, and protein synthesis (versus &bgr;-galactosidase). Azelaic acid potentiated &agr;AR-stimulated protein synthesis. Although TRX1 can directly reduce thiols, it also can scavenge ROS by increasing peroxidase activity. To examine this possibility, peroxidase activity was increased by transfection with catalase, and intracellular reactive oxygen species were measured with dichlorofluorescein diacetate fluorescence. Although catalase increased peroxidase activity ≈20-fold, TRX1 had no effect. Likewise, the &agr;AR-stimulated increase in dichlorofluorescein diacetate fluorescence was abolished with catalase but retained with TRX1. Conclusions—&agr;AR-stimulated hypertrophic signaling in adult rat ventricular myocytes is mediated via a TRX1-sensitive posttranslational oxidative modification of thiols on Ras.