Anita Gálvez

Cardiomyocyte Degeneration With Calpain Deficiency Reveals a Critical Role in Protein Homeostasis

Regulating the balance between synthesis and proteasomal degradation of cellular proteins is essential for tissue growth and maintenance, but the critical pathways regulating protein ubiquitination and degradation are incompletely defined. Although participation of calpain calcium-activated proteases in post-necrotic myocardial autolysis is well characterized, their importance in homeostatic turnover of normal cardiac tissue is controversial. Hence, we evaluated the consequences of physiologic calpain (calcium-activated protease) activity in cultured cardiomyocytes and unstressed mouse hearts. Comparison of in vitro proteolytic activities of cardiac-expressed calpains 1 and 2 revealed calpain 1, but not calpain 2, activity at physiological calcium concentrations. Physiological calpain 1 activation was evident in adenoviral transfected cultured cardiomyocytes as proteolysis of specific substrates, generally increased protein ubiquitination, and accelerated protein turnover, that were each inhibited by coexpression of the inhibitor protein calpastatin. Conditional forced expression of calpain 1, but not calpain 2, in mouse hearts demonstrated substrate-specific proteolytic activity under basal conditions, with hyperubiquitination of cardiac proteins and increased 26S proteasome activity. Loss of myocardial calpain activity by forced expression of calpastatin diminished ubiquitination of 1 or more specific myocardial proteins, without affecting overall ubiquitination or proteasome activity, and resulted in a progressive dilated cardiomyopathy characterized by accumulation of intracellular protein aggregates, formation of autophagosomes, and degeneration of sarcomeres. Thus, calpain 1 is upstream of, and necessary for, ubiquitination and proteasomal degradation of a subset of myocardial proteins whose abnormal accumulation produces autophagosomes and degeneration of cardiomyocytes with functional decompensation.

Aldose Reductase Induced by Hyperosmotic Stress Mediates Cardiomyocyte Apoptosis DIFFERENTIAL EFFECTS OF SORBITOL AND MANNITOL

Cells adapt to hyperosmotic conditions by several mechanisms, including accumulation of sorbitol via induction of the polyol pathway. Failure to adapt to osmotic stress can result in apoptotic cell death. In the present study, we assessed the role of aldose reductase, the key enzyme of the polyol pathway, in cardiac myocyte apoptosis. Hyperosmotic stress, elicited by exposure of cultured rat cardiac myocytes to the nonpermeant solutes sorbitol and mannitol, caused identical cell shrinkage and adaptive hexose uptake stimulation. In contrast, only sorbitol induced the polyol pathway and triggered stress pathways as well as apoptosis-related signaling events. Sorbitol resulted in activation of the extracellular signal-regulated kinase (ERK), p54 c-Jun N-terminal kinase (JNK), and protein kinase B. Furthermore, sorbitol treatment resulting in induction and activation of aldose reductase, decreased expression of the antiapoptotic protein Bcl-xL, increased DNA fragmentation, and glutathione depletion. Apoptosis was attenuated by aldose reductase inhibition with zopolrestat and also by glutathione replenishment with N-acetylcysteine. In conclusion, our data show that hypertonic shrinkage of cardiac myocytes alone is not sufficient to induce cardiac myocyte apoptosis. Hyperosmolarity-induced cell death is sensitive to the nature of the osmolyte and requires induction of aldose reductase as well as a decrease in intracellular glutathione levels.

Stress promotes development of ovarian cysts in rats : The possible role of sympathetic nerve activation ()

AbstractActivation of the sympathetic innervation precedes the induction of polycystic ovaries in rats given estradiol valerate (EV). The mechanism of induction by EV may thus involve both direct and neurogenic components. We tested this hypothesis using a combined cold and restraint stress to induce an increase in sympathetic tone, including that of the ovarian sympathetic nerves. Three weeks after the start of stress we found:1.An increase in the content of norepinephrine (NE) in the celiac ganglion.2.An increase in the release of NE from the ovary.3.An unchanged NE uptake by the ovary.4.An unchanged content of NE in the ovary. The ovarian content of neuropeptide Y (NPY) (colocalized with NE) was significantly decreased. These results suggest that NE synthesis and its secretion are increased during this period and correlate with the increase in secretion of androgens and estradiol, the development of precystic follicles, and a decrease in the ovulatory rate. After 11 wk, NE release had returned to control values, whereas the ovarian NE content had risen significantly, suggesting a maintained high rate of NE synthesis. In the ovary, NPY contents, steroid secretion, morphology, and ovulation had returned to the control state. These results suggest the participation of an extraovarian factor that might act locally to control the release of NE from the ovary, and further support the hypothesis that increased sympathetic activity plays a role in the development and maintenance of ovarian cysts.

Effects of adrenalectopmy on the stress-induced changes in ovarian sympathetic tone in the rat

A 3-wk period of stress promotes the development of ovarian cysts in rats apparently mediated by increased sympathetic nerve activity and ovarian steroid secretion. After 11 wk of stress, these parameters are indistinguishable from nonstressed control rats. To study adrenal contribution, we adrenalectomized rats and studied the effect of 3-wk of cold/restraint stress (1.5 h/d) on them compared to intact animals. Adrenalectomy (ADX) increased ovarian norepinephrine (NE) release, the content of β-adrenergic receptors (βAR) and basal, but not isoproterenol (Iso)-induced, androgen secretion. Stress to intact animals increased NE release, decreased βAR content, and Iso-induced, but not basal, androgen secretion from the ovary. ADX did not modify the response to stress. We propose a tonic inhibition by the adrenal gland on nerve activity of ovarian nerves. Stress overrides this inhibitory effect, and nerve activity downregulates βAR, decreasing ovarian steroid secretion.

Effects of antihypertensive treatment on cardiac IGF-1 during prevention of ventricular hypertrophy in the rat

There is some evidence that cardiac rather than circulating insulin-like growth factor-1 (IGF-1) levels contribute to the development of renovascular hypertensive left ventricular hypertrophy (LVH), remaining unknown the effects of antihypertensive drugs on IGF-1 levels. We have assessed here the preventive effects of enalapril, losartan, propanolol and alpha-methyldopa on left ventricle (LV) and circulating IGF-1 levels in a rat model of hypertension and LVH (Goldblatt, GB). Our results show that relative LV mass and the LV content of IGF-1 were significantly lower with all antihypertensive drugs in GB rats (p<0.001). Serum concentrations of IGF-1 were lower in GB rats treated with enalapril, alpha-methyldopa and propanolol (p<0.01), but not in those treated with losartan. These results support the hypothesis that local rather than seric IGF-1 contributes to the development of left ventricular hypertrophy induced by pressure overload in the rat.

Perindopril regulates beta-agonist-induced cardiac apoptosis

Administration of the β-adrenergic agonist isoproterenol results in cardiac apoptosis. The effect of short-term β-adrenergic stimulation by isoproterenol on the activity of plasma, lung, and left ventricular (LV) angiotensin I-converting enzyme (ACE) activity and its association with the development of cardiac apoptosis was investigated. β-Adrenergic stimulation for 24 hours produced an early increase only in the proapoptotic proteins bax and bcl-XS without changes in the levels of the antiapoptotic protein bcl-XL. The ratio between these bcl family proteins was indicative of apoptosis and correlated with an early and significant increase (300%) in DNA laddering. However, after 5 days of the β-adrenergic stimulation, the ratio changed in favor of antiapoptotic proteins and correlated with the absence of DNA fragmentation. In addition, LV and plasma ACE activities increased markedly with isoproterenol over the study period up to 5 days. ACE activity also regulated expression of the antiapoptotic gene bcl-XL. The administration of perindopril (an ACE inhibitor) prevented the observed increase in bax and bcl-XS levels and attenuated (50% decrease, P < 0.05) the effect of isoproterenol on DNA fragmentation. Thus, early and transient cardiac apoptosis triggered by the β-adrenergic agonist isoproterenol is reversed in the presence of perindopril.

Prevención precoz de hipertrofia ventricular izquierda en la hipertensión experimental y concentraciones de angiotensina II

Introduccion Las concentraciones de angiotensina II pueden inhibirse parcialmente durante la administracion cronica de inhibidores de la enzima conversiva de la angiotensina (ECA), limitando desde el punto de vista clinico su eficacia en el tratamiento de la hipertension arterial. Existen pocos estudios que relacionan directamente la actividad de la ECA y la prevencion precoz de hipertrofia ventricular izquierda (HVI) secundaria a hipertension arterial durante la administracion de un inhibidor de la ECA (IECA). Objetivo Evaluar los efectos de la inhibicion precoz de la ECA con perindopril sobre el desarrollo de hipertension, HVI y concentraciones de angiotensina II plasmatica y en el ventriculo izquierdo en el modelo Goldblatt en la rata (Gb; 2 rinones-un pinzado) a las 2 semanas de la cirugia. Resultados La presion arterial sistolica y la masa ventricular izquierda relativa aumentaron un 42 y un 20%, respectivamente, en el grupo Gb (p Conclusiones En este modelo experimental de HVI hipertensiva existe una activacion temprana de la ECA plasmatica y cardiaca. La administracion precoz de un IECA previene el desarrollo de hipertension e HVI al inhibir el aumento de angiontensina II en el plasma y el ventriculo izquierdo.