Tova Zinman

Acute, nongenomic effect of thyroid hormones in preventing calcium overload in newborn rat cardiocytes

In this study, we examined the acute effects of thyroid hormones (TH) T3 and T4, leading to improvement of myocardial function through activation of Ca2+ extrusion mechanisms and, consequently, prevention of intracellular calcium overload. Extracellular calcium elevation from 1.8 to 3.8 mM caused immediate increase in intracellular calcium level ([Ca2+]i) in newborn cardiomyocyte cultures. Administration of 10 or 100 nM T3 or T4 rapidly (within 10 sec) decreased [Ca2+]i to its control level. Similar results were obtained when [Ca2+]i was elevated by decreasing extracellular Na+ concentration, causing backward influx of Ca2+ through Na+/Ca2+ exchanger, or by administration of caffeine, releasing Ca2+ from the sarcoplasmic reticulum (SR). Under these conditions, T3 or T4 decreased [Ca2+]i. T3 and T4 also exhibited protective effects during ischemia. T3 or T4 presence during hypoxia for 120 min in culture medium restricted the increase of [Ca2+]i and prevented the pathological effects of its overload. An inhibitor of SR Ca2+‐ATPase (SERCA2a), thapsigargin, increases [Ca2+]i and in its presence neither T3 nor T4 had any effect on the [Ca2+]i level. The reduction of [Ca2+]i level by T3 and T4 was also blocked in the presence of H‐89 (a PKA inhibitor), and by calmodulin inhibitors. The effect of TH on the reduction of [Ca2+]i was prevented by propranolol, indicating that the hormones exert their effect through interaction with adrenergic receptors. These results support our hypothesis that TH prevent calcium overload in newborn rat cardiomyocytes, most likely by a direct, acute, and nongenomic effect on Ca2+ transport into the SR. J. Cell. Physiol. 207: 220–231, 2006.

Detailed analysis of reactive oxygen species induced by visible light in various cell types

Light in the visible and near infrared region stimulates various cellular processes, and thus has been used for therapeutic purposes. One of the proposed mechanisms is based on cellular production of reactive oxygen species (ROS) in response to illumination. In the present study, we followed visible light (VL)‐induced hydroxyl radicals in various cell types and cellular sites using the electron paramagnetic resonance (EPR) spin‐trapping technique.

Dopamine increases glial cell line-derived neurotrophic factor in human fetal astrocytes

The use of fetal astrocytes for gene delivery into brains with neurodegenerative diseases has been suggested. Therefore, the effects of neurotransmitters in the brain on such cells are of interest. The presence of D1 (D1A) receptors and the effect of dopamine on a fetal human astrocyte cell line (SVG cells) in vitro were examined. SVG cells expressed D1 (D1A), but not D5 (D1B) receptors, as shown by RT‐PCR. Exposure to dopamine, apomorphine, and the specific D1 agonist, SKF‐38393, increased glial‐derived neurotrophic factor production of SVG cells, as well as intracellular free calcium. Exposure to the specific D1 antagonist, SCH 23390, blocked these effects. Thus, if implanted into a brain region rich in dopamine, or if transfected with the tyrosine hydroxylase gene, fetal astrocytes may serve as paracrine/autocrine cells capable of supplying critical growth factors to diseased brain tissue. GLIA 33:143–150, 2001.

Enhanced connexin-43 and alpha-sarcomeric actin expression in cultured heart myocytes exposed to triiodo-L-thyronine.

This study examined whether triiodo-l-thyronine (T3) affects the expression of the major intercellular channel protein, connexin-43, and contractile protein α-sarcomeric actin. Cultured cardiomyocytes from newborn rats were treated on day three in culture with 10 or 100 nM T3 and examined 48 and 72 h thereafter. Treated and untreated cells were examined by immunofluorescence and electron microscopy. Expression levels of Cx43 and sarcomeric α-actin were monitored by Western blot analysis. Immunofluorescence labeling showed cell membrane location of Cx43 in punctuate gap junctions, whereby fluorescence signal area was significantly higher in cultured cardiomyocytes exposed to T3. This correlated with electron microscopical findings showing increased numbers and size of gap junction profiles, as well as with a significant dose-dependent increase of Cx43 expression detected by Western blot. Immunofluorescence of sarcomeric α-actin was enhanced and its expression increased dose- and time-dependently in T3-treated cultured heart myocytes. However, exposure to the higher dosage (100 nM) of T3 caused mild disintegration of sarcomeric α-actin in some myocytes, suggesting an over-dosage. The results indicate that T3 up-regulates Cx43 and accelerates gap junction formation in cultured neonatal cardiomyocytes. They suggest that thyroid status cannot only modulate the mechanical function of cardiomyocytes but also cell-to-cell communication essential for myocardial electrical and metabolic synchronizations.

Differential aspects in ratio measurements of [Ca2+]i relaxation in cardiomyocyte contraction following various drug treatments

This study is concerned with the analysis of the time dependency of [Ca(2+)](i), monitored by indo-1-AM, via the ratiometric time response curve R(t) as measured during contractions of spontaneous or electrical stimulated cardiomyocytes (in culture). A mathematical formulation which describes the relaxation phase of R(t) was developed. By fitting formulation to the measured data of R(t), the extraction of characteristic parameters is feasible, which may reflect the factors regulating intracellular Ca concentration. The usefulness of the suggested formulation was examined by monitoring changes induced in those parameters following the exposure of the myocytes to different drugs, among which are: caffeine, ryanodine, thapsigargin db, cyclic AMP, isoprenaline, doxorubicin, and Cl-IB-MECA.

The protective effect of class III antiarrhythmic agents against calcium overload in cultured myocytes

Calcium ions have been implicated in the mechanisms of ventricular arrhythmias. Impairment of intercellular coupling by calcium overload is considered to facilitate ventricular fibrillation (VF) and to sup-press its self termination. According to our hypothesis, any compound that decreases intracellular calcium concentration [Ca2+]i during VF can serve as defibrillating drug. In this study, we examined the effect of d-sotalol and tedisamil on calcium overload in cultured, spontaneously beating rat cardiomyocytes. The changes of [Ca2+]i were measured by indo-1 method and the intercellular synchronization by image analysis. The results showed that increase in [Ca2+]o from 1.9 mM to 3.9 mM increased [Ca2+]i from 100 nM to 320 nM and transformed the synchronized cell movement to an asynchronous one. Administration of 5 x 10(-6) M d-sotalol or 10(-6) M tedisamil, decreased the [Ca2+]i to its basic level and restored the synchronized activity. In summary: Our results showed that increase in [Ca2+]i known to cause inhibition of intercellular coupling, that could lead to arrhythmia and fibrillation while d-sotalol or tedisamil prevented this effect. These results support our hypothesis, that class III antiarrhythmic compounds with positive inotropic effect, increase intercellular synchronization, by decreasing free [Ca2+]i, most probably by increasing the Ca2+ uptake by the sarcoplasmic reticulum, and therefore act as a defibrillating compound.

Infarct-Induced Steroidogenic Acute Regulatory Protein: A Survival Role in Cardiac Fibroblasts

Steroidogenic acute regulatory protein (StAR) is indispensable for steroid hormone synthesis in the adrenal cortex and the gonadal tissues. This study reveals that StAR is also expressed at high levels in nonsteroidogenic cardiac fibroblasts confined to the left ventricle of mouse heart examined 3 days after permanent ligation of the left anterior descending coronary artery. Unlike StAR, CYP11A1 and 3β-hydroxysteroid dehydrogenase proteins were not observed in the postinfarction heart, suggesting an apparent lack of de novo cardiac steroidogenesis. Work with primary cultures of rat heart cells revealed that StAR is induced in fibroblasts responding to proapoptotic treatments with hydrogen peroxide or the kinase inhibitor staurosporine (STS). Such induction of StAR in culture was noted before spontaneous differentiation of the fibroblasts to myofibroblasts. STS induction of StAR in the cardiac fibroblasts conferred a marked resistance to apoptotic cell death. Consistent with that finding, down-regulation of StAR by RNA interference proportionally increased the number of STS-treated apoptotic cells. StAR down-regulation also resulted in a marked increase of BAX activation in the mitochondria, an event known to associate with the onset of apoptosis. Last, STS treatment of HeLa cells showed that apoptotic demise characterized by mitochondrial fission, cytochrome c release, and nuclear fragmentation is arrested in individual HeLa cells overexpressing StAR. Collectively, our in vivo and ex vivo evidence suggests that postinfarction expression of nonsteroidogenic StAR in cardiac fibroblasts has novel antiapoptotic activity, allowing myofibroblast precursor cells to survive the traumatized event, probably to differentiate and function in tissue repair at the infarction site.

Correlation of magnetic AC field on cardiac myocyte Ca2+ transients at different magnetic DC levels

The purpose of this study was to determine the effect of extremely low frequency and weak magnetic fields (WMF) on cardiac myocyte Ca(2+) transients, and to explore the involvement of potassium channels under the WMF effect. In addition, we aimed to find a physical explanation for the effect of WMF on cardiac myocyte Ca(2+) transients. Indo-1 loaded cells, which were exposed to a WMF at 16 Hz and 40 nT, demonstrated a 75 ± 4% reduction in cytosolic Ca(2+) transients versus control. Treatment with the K(ATP) channel blocker, glibenclamide, followed by WMF at 16 Hz exposure, blocked the reduction in cytosolic calcium transients while treatment with pinacidil, a K(ATP) channel opener, or chromanol 293B, a selective potassium channel blocker of the delayed rectifier K(+) channels, did not inhibit the effect. Based on these finding and the ion cyclotron resonance frequency theory, we further investigated the effect of WMF by changing the direct current (DC) magnetic field (B(0) ). When operating different DC magnetic fields we showed that the WMF value changed correspondingly: for B(0)  = 44.5 µT, the effect was observed at 17.05 Hz; for B(0)  = 46.5 µT, the effect was observed at 18.15 Hz; and for B(0)  = 49 µT the effect was observed at 19.1 Hz. We can conclude that the effect of WMF on Ca(2+) transients depends on the DC magnetic field level.

Fluorescence polarization: a novel indicator of cardiomyocyte contraction.

The changes measured in intracellular fluorescein fluorescence polarization (IFFP) are used as a new tool for tracing cytoplasmic effects during contractile cycles of cardiac myocytes (1-2-day-old rat hearts), in addition to the established Ca(2+) monitoring and/or videometric methods of tracking cell-shortening. This novel method was found to be non-intrusive to the contraction cycles. The decay of the transient IFFP signal (from 0.220+/-0.01 to 0.170+/-0.013) seems to be closely related to the extended phase of contractile activation. This fact was further supported when Ca(2+) exchanger inhibitor was introduced and significantly decreased (90%) the rate of beats of contraction and IFFP, but not the Ca(2+) beat rate changes. This result suggests that the IFFP indicator is probably associated with the physiological activation, rather than with Ca(2+) alterations. The IFFP measure monitors the average of effective changes in the micro-viscosity of the cytoplasm protein matrix, associated with cellular activation.

INHIBITION OF MALIGNANT CELL PROLIFERATION BY CULTURE MEDIA CONDITIONED BY CARDIAC OR SKELETAL MUSCLE

The present work is an attempt to explain the high resistance of muscles to cancer development. We used primary cultures of rat skeletal and cardiac muscle, and examined the effect of the supernatant of these cultures (conditioned medium; CM) on proliferation of cancer cells. The results demonstrated that CM inhibited the proliferation of several types of malignant cells by more than 50%, without a significant inhibition on normal cells. Cell cycle analysis revealed that CM increased the number of cells in S and G2 phases, suggesting a cytostatic effect of CM. For defining the biological properties of the factor(s) which are present in the CM, skeletal muscle cultures were grown in chemically defined medium (serum free medium). The concentrated sample was applied to a Sephadex G‐50 column and three fractions were obtained. Only one fraction showed inhibitory activity. Four protein bands were observed in this fraction, as revealed by SDS‐PAGE. We suggest that some, or all of these proteins are responsible for inhibition of tumor cell replication.