Nelson Escobales

Mitochondrial Permeability Transition Pore Opening as a Promising Therapeutic Target in Cardiac Diseases

In addition to their central role in ATP synthesis, mitochondria play a critical role in cell death. Oxidative stress accompanied by calcium overload, ATP depletion, and elevated phosphate levels induces mitochondrial permeability transition (MPT) with formation of nonspecific MPT pores (MPTP) in the inner mitochondrial membrane. Pore opening results in mitochondrial dysfunction with uncoupled oxidative phosphorylation and ATP hydrolysis, ultimately leading to cell death. For the past 20 years, three proteins have been accepted as key structural components of the MPTP: adenine nucleotide translocase (ANT) in the inner membrane, cyclophilin D (CyP-D) in the matrix, and the voltage-dependent anion channel (VDAC) in the outer membrane. However, most recent studies have questioned the molecular identity of the pores. Genetic studies have eliminated the VDAC as an essential component of MPTP and attributed a regulatory (rather than structural) role to ANT. Currently, the phosphate carrier appears to play a crucial role in MPTP formation. MPTP opening has been examined extensively in cardiac pathological conditions, including ischemia/reperfusion as well as heart failure. Accordingly, MPTP is accepted as a therapeutic target for both pharmacological and conditional strategies to block pore formation by direct interaction with MPTP components or indirectly by decreasing MPTP inducers. Inhibition of MPTP opening by reduction of CyP-D activity by nonimmunosuppressive analogs of cyclosporine A or sanglifehrin A, as well as attenuation of reactive oxygen species accumulation through mitochondria-targeted antioxidants, is the most promising. This review outlines our current knowledge of the structure and function of the MPTP and describes possible approaches for cardioprotection.

Amiloride-sensitive Na+ transport in human red cells: evidence for a Na/H exchange system

SummaryThe role of transmembrane pH gradients on the ouabain, bumetanide and phloretin-resistant Na+ transport was studied in human red cells. Proton equilibration through the Jacobs-Stewart cycle was inhibited by the use of DIDS (125 μm) and methazolamide (400 μm). Red cells with different internal pH (pHi=6.4, 7.0 and 7.8) were prepared and Na+ influx was measured at different external pH (pHo=6.0, 7.0, 8.0). Na+ influx into acid-loaded cells (pHi=6.4) markedly increased when pHo was raised from 6.0 to 8.0. Amiloride, a well-known inhibitor of Na+/H+ exchange systems blocked about 60% of the H+-induced Na+ entry, while showing small inhibitory effects in the absence of pH gradients. When pH0 was kept at 8.0, the amiloride-sensitive Na+ entry was abolished as pHi was increased from 6.4 to 7.8. Moreover, measurements of H+ efflux into lightly buffered media indicated that the imposition of an inward Na+ gradient stimulated a net H+ efflux which was sensitive to the amiloride analog 5-N-methyl-N-butyl-amiloride. Furthermore, in the absence of a chemical gradient for Na+ (Nai+=Na0+=15mm,Em=+6.7 mV), an outward H+ gradient (pHi=6.4, pH0=8.0) promoted a net amiloride-sensitive Na+ uptake which was abolished at an external pH of 6.0. These findings are consistent with the presence of an amiloride-sensitive Na+/H+ exchange system in human red cells.

Oxidative-Nitrosative Stress In Hypertension

Reactive oxygen species (ROS) are important signaling molecules in the vasculature. However, when there is imbalance between their occurrence and antioxidant defense mechanisms, ROS can contribute to the vascular abnormalities that lead to hypertension. Evidence accumulated in the last decade strongly supports the notion that ROS are generated in the vasculature mainly by NAD(P)H oxidase in a mechanism that is angiotensin II-dependent. Activation of this enzyme leads to superoxide production and uncouples endothedial NO synthase (eNOS), which sustains oxidative stress while increasing the levels of tissue-damaging peroxynitrite. The latter can result in vascular dysfunction. NAD(P)H-dependent ROS formation, in particular H(2)O(2), could also contribute to vascular injury by sustaining NAD(P)H oxidase activation, promoting inflammatory gene expression, extracellular matrix reorganization, and growth (hypertrophy/hyperplasia) of vascular smooth muscle cells. The effect of ROS appears to be mediated by redox-sensitive targets such as tyrosine kinases and phosphatases, mitogen-activated protein kinases, transcription factors, matrix metalloproteinases, peroxisome proliferator activated receptor-alpha, poly(ADP-ribose)polymerase-1, Ca(2+) signaling mechanisms and secreted factors such as cyclophilin A and heat shock protein 90-alpha. Redox-sensitive targets appear to play a central role in normal vascular function, but can also lead to remodeling of the vascular wall, increasing vascular reactivity and hypertension. Polymorphisms in the p22phox gene promoter could determine susceptibility to NAD(P)H-mediated oxidative stress in humans and animals with hypertension. Although ROS are strongly implicated in the etiology of hypertension, clinical trials with antioxidants are inconclusive regarding their effectiveness in treating the disease. New drugs with both antihypertensive action and antioxidant properties (Celiprolol, Carvedilol) offer promising results in the management of hypertension.

Mitochondria-targeted ROS scavenger improves post-ischemic recovery of cardiac function and attenuates mitochondrial abnormalities in aged rats

Mitochondria-generated reactive oxygen species (ROS) play a crucial role in the pathogenesis of aging and age-associated diseases. In this study, we evaluated the effects of XJB-5-131 (XJB), a mitochondria-targeted ROS and electron scavenger, on cardiac resistance to ischemia-reperfusion (IR)-induced oxidative stress in aged rats. Male adult (5-month old, n=17) and aged (29-month old, n=19) Fischer Brown Norway (F344/BN) rats were randomly assigned to the following groups: adult (A), adult+XJB (AX), aged (O), and aged+XJB (OX). XJB was administered 3 times per week (3mg/kg body weight, IP) for four weeks. At the end of the treatment period, cardiac function was continuously monitored in excised hearts using the Langendorff technique for 30 min, followed by 20 min of global ischemia, and 60-min reperfusion. XJB improved post-ischemic recovery of aged hearts, as evidenced by greater left ventricular developed-pressures and rate-pressure products than the untreated, aged-matched group. The state 3 respiration rates at complexes I, II and IV of mitochondria isolated from XJB-treated aged hearts were 57% (P<0.05), 25% (P<0.05) and 28% (P<0.05), respectively, higher than controls. Ca(2+)-induced swelling, an indicator of permeability transition pore opening, was reduced in the mitochondria of XJB-treated aged rats. In addition, XJB significantly attenuated the H2O2-induced depolarization of the mitochondrial inner membrane as well as the total and mitochondrial ROS levels in cultured cardiomyocytes. This study underlines the importance of mitochondrial ROS in aging-induced cardiac dysfunction and suggests that targeting mitochondrial ROS may be an effective therapeutic approach to protect the aged heart against IR injury.

Altered vascular function in early stages of heart failure in hamsters

BACKGROUND Congestive heart failure is a clinical condition associated with alterations in the normal balance of neurohumoral agents and factors acting on the vascular wall. The etiology of this condition, however, remains largely undefined. To help elucidate the pathophysiology of this disease, vascular function and angiotensin-converting enzyme activity were evaluated in 2-month-old Syrian cardiomyopathic hamsters (SCHs) that had not yet developed heart failure. Age-matched normal hamsters were used as control hamsters. METHODS AND RESULTS Vascular function studies included determinations of contractile responses of aortic rings to 0.1 microM angiotensin II and 0.1 microM norepinephrine. In addition, endothelial function was evaluated by the vasorelaxant action of acetylcholine on norepinephrine-precontracted aortic rings. The results indicate that the pressor effect of angiotensin II (0.1 microM) was 35% greater in aortic rings from SCRs than that observed in control animals. This effect is specific for angiotensin II because the contraction induced by NE (0.1 microM) was similar in both of these strains. Angiotensin-converting enzyme activity was three-fold higher in aorta homogenates from SCHs but normal in plasma and heart tissue when compared with control hamsters. Aortic ring preparations from SCHs also exhibited endothelial dysfunction because the maximal relaxation elicited by 10 microM acetylcholine was reduced 53%. Concentration-response curves with acetylcholine yielded EC50 values that were threefold lower in SCHs (97.2 +/- 0.1 nM) than in control animals (286 +/- 7 nM). Indomethacin (1 microM) increased the vasorelaxant effect of acetylcholine 28% in SCHs and shifted to the left the concentration-response curve of this agonist, suggesting an increased relaxation with the cyclooxygenase inhibitor. No effect of indomethacin on acetylcholine-induced relaxation was observed in control animals. Sodium nitroprusside induced similar relaxations in both control animals and SCHs, suggesting that the vascular smooth muscle response is normal in SCR. CONCLUSIONS Altogether these results point to a state of enhanced vascular contractility in young SCHs that could predispose these animals to develop heart failure, the enhanced vascular contractility could result from increased activity of the local renin-angiotensin system, augmented vascular response to angiotensin II, reduced nitric oxide synthesis, and enhanced production of prostaglandins.

Simvastatin releases Ca2+ from a thapsigargin-sensitive pool and inhibits InsP3-dependent Ca2+ mobilization in vascular smooth muscle cells.

Simvastatin (SV), an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity inhibits migration and proliferation of vascular smooth muscle cells (SMC). To investigate whether these effects of SV are related to inhibition of cell calcium mobilization, cultured SMC obtained from rat aorta were loaded with Fura-2 to determine the basal cytosolic free calcium levels ([Ca2+]i) and the agonist-stimulated Ca2+ mobilization. SV (20 mu M) transiently increased cytosolic free calcium, an effect that depends mainly on intracellular calcium release (68%). This effect of SV was markedly reduced (75%) by thapsigargin, an inhibitor of the Ca2+ ATPase of inositol 1,4,5-triphosphate (InsP3)-sensitive calcium pools. Incubation of cells with SV (15 min) inhibited the mobilization of Ca2+ by angiotensin II, platelet-derived growth factor, and vasopressin (IC50 = 5 mu M). SV did not affect inositol trisphosphate (InsP3) levels or modify its generation by angiotensin II (Ang II) and vasopressin. Furthermore, in saponin-permeabilized cells, SV abolished the release of calcium by 2,3-dideoxy-InsP3. SV reduced the effect of thapsigargin on InsP3-sensitive stores by 67%, suggesting that SV depletes these calcium pools. The inhibitory effect of SV on calcium mobilization was prevented by coincubation of cultured cells (24 h) with 1 mM mevalonic acid, the product of HMG-CoA reductase activity. These results support the notion that SV inhibits [corrected] the migration and proliferation of SMC by directly affecting cell Ca2+.

Na+/Na+ exchange and Na+/H+ antiport in rabbit erythrocytes : two distinct transport systems

SummaryRabbit erythrocytes are well known for possessing highly active Na+/Na+ and Na+/H+ countertransport systems. Since these two transport systems share many similar properties, the possibility exists that they represent different transport modes of a single transport molecule. Therefore, we evaluated this hypothesis by measuring Na+ transport through these exchangers in acid-loaded cells. In addition, selective inhibitors of these transport systems such as ethylisopropyl-amiloride (EIPA) and N-ethylmaleimide (NEM) were used. Na+/Na+ exchange activity, determined as the Nao+-dependent22Na efflux or Nai +-induced22Na entry was completely abolished by NEM. This inhibitor, however, did not affect the Hi+-induced Na+ entry sensitive to amiloride (Na+/H+ exchange activity). Similarly, EIPA, a strong inhibitor of the Na+/H+ exchanger, did not inhibit Na+/Na− countertransport, suggesting the independent nature of both transport systems. The possibility that the NEM-sensitive Na+/Na+ exchanger could be involved in Na+/H+ countertransport was suggested by studies in which the net Na+ transport sensitive to NEM was determined. As expected, net Na+ transport through this transport system was zero at different [Na+]i/[Na+]o ratios when intracellular pH was 7.2. However, at pHi=6.1, net Na+ influx occurred when [Na+]i was lower than 39mm. Valinomycin, which at low [K+]o was lower than 39mm. Valinomycin, which at low [K+]o clamps the membrane potential close to the K+ equilibrium potential, did not affect the net NEM-sensitive Na+ entry but markedly stimulated, the EIPA-and NEM-resistant Na+ uptake. This suggest that the net Na+ entry through the NEM-sensitive pathway at low pHi, is mediated by an electroneutral process possibly involving Na+/H+ exchange. In contrast, the EIPA-sensitive Na+/H+ exchanger is not involved in Na+/Na+ countertransport, because Na+ transport through this mechanism is not affected by an increase in cell Na− from 0.4 to 39mm. Altogether, these findings indicate that both transport systems: the Na+/Na+ and Na+/H+ exchangers, are mediated by distinct transport proteins.

Endothelial dysfunction in the San Juan hypertensive rat : Possible role of the nitric oxide synthase

Endothelial dysfunction is recognized as a critical event in the etiology of cardiovascular diseases, but its possible role during aging in arterial hypertension remains poorly defined. We evaluated the response of aortic rings precontracted with 0.1 microM norepinephrine (NE) to acetylcholine (ACh) in the San Juan hypertensive rats (SJH-Rs) (F19, F20) and Munich Wistar rats (MW). SJH-Rs is a model of inbred salt-sensitive hypertension, whereas similarly treated inbred MW rats are their normotensive counterpart. These experiments were performed with adult (6-7 months) and aged (11-13 months) rats to assess the effects of age and hypertension on endothelium-dependent relaxation. We generated dose-response curves by adding cumulative doses of ACh from 1.0 nM to 10.0 microM. In addition, we evaluated the Ca(2+)-dependent nitric oxide synthase (NOS) activity by increasing cell calcium with the ionophore A23187. The results indicate that hypertension induces a displacement to the right of the dose-response curve to ACh in both adults and aged SJH-Rs; IC50 for adult rats was 0.72 +/- 0.3 microM for SJH-Rs and 0.059 +/- 0.03 microM for MW (p < 0.05). Aged animals showed similar results: IC50 of 0.78 +/- 0.03 microM for SJH-Rs and of 0.043 +/- 0.01 microM in age-matched MW rats (p < 0.025). However, no difference was observed between hypertensive (SJH-Rs) adult and aged animals. Similarly, no difference was observed between adult and aged MW control animals. The displacement of the dose-response curve to ACh in SJH-Rs appears to be associated with a reduced activation of NOS since in precontracted aortas from aged animals 1 microM A23187 induced a relaxation of 51.2 +/- 12% in MW as compared with 34.4 +/- 7% in SJH-Rs (n = 5, p < 0.05). These results indicate that endothelial dysfunction exists in SJH-Rs. The data suggest that an alteration of the endothelial NOS may be the cause of this abnormality. Finally, the magnitude of the endothelial dysfunction is not age dependent within the range evaluated.

Enalapril and Losartan Are More Effective Than Carvedilol in Preventing Dilated Cardiomyopathy in the Syrian Cardiomyopathic Hamster

To assess the role of the renin—angiotensin (RAS) and adrenergic systems in the development and progression of dilated cardiomyopathy in the Syrian cardiomyopathic hamster (SCH), echocardiographic parameters were evaluated in 6-month-old animals after 5 months of treatment with enalapril (25 mg/kg/day) plus losartan (10 mg/kg/day), or with carvedilol (1 mg/kg/day). Cardiac output indexes (COI) increased by 53% after RAS blockade and by 20% after β-blockade in SCH. Moreover, LVEDV and LVESV decreased 30% and 62%, respectively (P < .05) during RAS blockade, whereas ejection fraction (EF) increased by 48%. By contrast, carvedilol reduced LVESV by only 28% (P < .05) and increased EF by only 15% (P < .05). These results suggest that RAS activation plays a critical role in the development of cardiac dysfunction in SCH and that suppression of RAS may be more effective than β-blockade in retarding the development of cardiomyopathy in SCH. Owing to timing (pre—heart failure stage) and to the single dose protocol, the implications of this study for human subjects remain to be clarified.

Concentraciones totales de homocisteína plasmática en pacientes puertorriqueños con cardiopatía isquémica

Introduccion y objetivos En Puerto Rico se ha comprobado que, a pesar de que la enfermedad coronaria es la principal causa de muerte, la poblacion tiene una incidencia menor de estas enfermedades que en los EE.UU. y posee una incidencia menor de taquicardia ventricular y muerte subita. Un factor que puede contribuir a la menor incidencia de enfermedades coronarias en Puerto Rico es que las concentraciones totales de homocisteina plasmatica (tHcys) en nuestra poblacion sean menores que en la poblacion de los EE.UU. Nuestro objetivo era medir tHcys en la poblacion puertorriquena con cardiopatia isquemica. Metodos Se midio aleatoriamente la tHcys en 70 pacientes puertorriquenos hospitalizados en el Centro Cardiovascular de Puerto Rico y el Caribe (Division UPR). Resultados La concentracion promedio de tHcys en estos pacientes resulto similar a la comunicada por el estudio de Framingham cuando es ajustada por edad (11,2 frente a 11,8 mmol/l). En la poblacion puertorriquena, los varones tenian una concentracion mayor de tHcys que las mujeres (11,7 frente a 9,5 mmol/l; p = 0,07). Ademas, no observamos un aumento en la concentracion de tHcys en pacientes diabeticos cuando se compararon con los pacientes no diabeticos (10,1 frente a 11,2 mmol/l; p = 0,74). Tampoco observamos una correlacion directa entre la concentracion de tHcys y las condiciones cardiacas medidas por angiografia coronaria (normal = 11,1 mmol/l; leve = 10,5 mmol/l; moderado = 10,9 mmol/l; severo = 10,5 mmol/l; Kruskal-Wallis = 0,45). Conclusion Estos resultados sugieren que la concentracion de tHcys no predice fiablemente la gravedad de las lesiones de cardiopatia isquemica en la poblacion puertorriquena.