The Effect of Oxytocin on Intracellular Ca2+ Release in Cardiac Cells.

Ca2+ signaling is vital for the proper functioning of all cells, including cells of the cardiovascular system. Membrane receptors for many hormones trigger intracellular Ca2+ signaling via the activation of phospholipase C and production of inositol-1,4,5-trisphosphate (InsP3). Several research groups have demonstrated the expression of oxytocin (OXT) and oxytocin receptors (OXTR) in the heart and suggested a cardioprotective role of OXT against several pathological conditions. Here we describe the protocol for measuring the effects of oxytocin on intracellular Ca2+ dynamics in newborn rat cardiac myocytes and cardiac fibroblasts maintained in short-term culture.

Epstein-Barr virus noncoding small RNA (EBER1) induces cell proliferation by up-regulating cellular mitochondrial activity and calcium influx.

Epstein-Barr virus encoded RNAs (EBER1 and EBER2) are two non-polyadenylated, non-protein coding small RNAs expressed at high levels in all forms of EBV latent infections. Although not directly involved in cell transformation, a number of studies have reported that these RNAs may be involved in cell proliferation. However, which of the two EBERs play a major role in this process and the mechanisms involved remains unknown. The aim of this study was to investigate the role and mechanism of EBER1-induced cell proliferation. Using stably transfected EBER1 cell lines, and multiple methodologies, we show that EBER1 transfected epithelial, B and T cell lines proliferate at a higher rate, have higher metabolic activity and increased DNA synthesis. The mitochondrial number and activity was also observed to be higher in the EBER1 transfected cells. Moreover, cytochrome c activity and store operated calcium entry (SOCE) were potentiated in the EBER1 expressing cells. Finally, the genes associated with cell proliferation were also observed to be up-regulated in the EBER1 transfected cells. Taken together, our data has unravelled the role of mitochondria and cellular calcium pathway that appear to be involved in EBER1 induced cell proliferation of EBV infected cells.

Proteinase Activated Receptors Mediate the Trypsin-Induced Ca2 + Signaling in Human Uterine Epithelial Cells.

Embryo implantation is a complex and tightly regulated process. In humans, uterine luminal epithelium functions as a biosensor gauging the embryo quality and transmitting this information to the underlying endometrial stromal cells. This quality control ensures that only high quality embryos are implanted, while aberrant ones are rejected. The mechanisms of the embryo-uterine mucosa crosstalk remain incompletely understood. Trypsin, a serine protease secreted by the blastocyst, has been implicated in the cross-signaling. Here we address the mechanisms by which trypsin triggers the intracellular calcium signaling in uterine epithelium. We found that protease-activated G-protein coupled receptors are the main mechanism mediating the effects of trypsin in human uterine epithelium. In addition, trypsin activates the epithelial sodium channels thus increasing the intracellular Na+ concentration and promoting Ca2+ entry on the reverse mode of the sodium/calcium exchanger.

Effects of prolactin on ventricular myocyte shortening and calcium transport in the streptozotocin-induced diabetic rat.

The physiological role of prolactin (PRL) in the heart, and in particular the diabetic heart, are largely unknown. The effects of PRL on ventricular myocyte shortening and Ca2+ transport in the streptozotocin (STZ) - induced diabetic and in age-matched control rats were investigated. PRL receptor protein, myocyte shortening, intracellular [Ca2+], L-type Ca2+ current were measured by Western blot, cell imaging, fluorescence photometry and whole-cell patch-clamp techniques, respectively. Compared to normal Tyrode solution (NT), PRL (50 ng/ml) significantly (p < 0.05) increased the amplitude of shortening in myocytes from control (7.43 ± 0.38 vs. 9.68 ± 0.46 %) and diabetic (6.57 ± 0.24 vs. 8.91 ± 0.44 %) heart (n = 44-49 cells). Compared to NT, PRL (50 ng/ml) significantly increased the amplitude of Ca2+ transients in myocytes from control (0.084 ± 0.004 vs. 0.115 ± 0.007 Fura-2 ratio units) and diabetic (0.087 ± 0.007 vs. 0.112 ± 0.006 Fura-2 ratio units) heart (n = 36-50 cells). PRL did not significantly alter the amplitude of caffeine-evoked Ca2+ transients however, PRL significantly increased the fractional release of Ca2+ in myocytes from control (21 %) and diabetic (14 %) and heart. The rate of Ca2+ transient recovery following PRL treatment was significantly increased in myocytes from diabetic and control heart. Amplitude of L-type Ca2+ current was not significantly altered by diabetes or by PRL. PRL increased the amplitude of shortening and Ca2+ transients in myocytes from control and diabetic heart. Increased fractional release of sarcoplasmic reticulum Ca2+ may partly underlie the positive inotropic effects of PRL in ventricular myocytes from control and STZ-induced diabetic rat.

Oxytocin induces intracellular Ca2+ release in cardiac fibroblasts from neonatal rats.

Pituitary neuropeptide oxytocin is increasingly recognised as a cardiovascular hormone, in addition to its many regulatory roles in other organ systems. Studies in atrial and ventricular myocytes from the neonatal and adult rats have identified synthesis of oxytocin and the expression of oxytocin receptors in these cells. In cardiac fibroblasts, the most populous non-myocyte cell type in mammalian heart, the oxytocin receptors have not been described before. In the present study, we have investigated the direct effects of oxytocin on intracellular Ca2+ dynamics in ventricular myocytes and fibroblasts from new born rats. In myocytes, oxytocin increased the frequency of spontaneous Ca2+ transients and decreased their amplitude. Our data suggest that oxytocin receptors are also present and functional in the majority of cardiac fibroblasts. We used selective oxytocin receptor inhibitor L-371,257 and a number of intracellular Ca 2+ release blockers to investigate the mechanism of oxytocin induced Ca2+ signalling in cardiac fibroblasts. Our findings suggest that oxytocin induces Ca2+ signals in cardiac fibroblasts by triggering endoplasmic reticulum Ca2+ release via inositol trisphosphate activated receptors. The functional significance of the oxytocin induced Ca2+ signalling in cardiac fibroblasts, especially for their activation into secretory active myofibroblasts, remains to be investigated.

Voltage dependence of the Ca2+ transient in endocardial and epicardial myocytes from the left ventricle of Goto-Kakizaki type 2 diabetic rats.

Diabetes mellitus is a major global health disorder and, currently, over 450 million people have diabetes with 90% suffering from type 2 diabetes. Left untreated, diabetes may lead to cardiovascular diseases which are a leading cause of death in diabetic patients. Calcium is the trigger and regulator of cardiac muscle contraction and derangement in cellular Ca2+ homeostasis, which can result in heart failure and sudden cardiac death. It is of paramount importance to investigate the regional involvement of Ca2+ in diabetes-induced cardiomyopathy. Therefore, the aim of this study was to investigate the voltage dependence of the Ca2+ transients in endocardial (ENDO) and epicardial (EPI) myocytes from the left ventricle of the Goto-Kakizaki (GK) rats, an experimental model of type 2 diabetes mellitus. Simultaneous measurement of L-type Ca2+ currents and Ca2+ transients was performed by whole-cell patch clamp techniques. GK rats displayed significantly increased heart weight, heart weight/body weight ratio, and non-fasting and fasting blood glucose compared to controls (CON). Although the voltage dependence of L-type Ca2+ current was unaltered, the voltage dependence of the Ca2+ transients was reduced to similar extents in EPI-GK and ENDO-GK compared to EPI-CON and ENDO-CON myocytes. TPK L-type Ca2+ current and Ca2+ transient were unaltered. THALF decay of L-type Ca2+ current was unaltered; however, THALF decay of the Ca2+ transient was shortened in ENDO and EPI myocytes from GK compared to CON rat hearts. In conclusion, the amplitude of L-type Ca2+ current was unaltered; however, the voltage dependence of the Ca2+ transient was reduced to similar extents in EPI and ENDO myocytes from GK rats compared to their respective controls, suggesting the possibility of dysfunctional sarcoplasmic reticulum Ca2+ transport in the GK diabetic rat hearts.

Cell shortening and calcium dynamics in epicardial and endocardial myocytes from the left ventricle of Goto-Kakizaki type 2 diabetic rats.

NEW FINDINGS: What is the central question of this study? To investigate haemodynamic dysfunction in the type 2 diabetic Goto-Kakizaki (GK) rat, we measured shortening and Ca2+ transport in ventricular myocytes from epicardial (EPI) and endocardial (ENDO) regions. What is the main finding and its importance? EPI and ENDO GK myocytes displayed similar hypertrophy. Time to peak (TPK) and time to half (THALF) relaxation were prolonged in EPI GK myocytes. TPK Ca2+ transient was prolonged and THALF decay of the Ca2+ transient was shortened in EPI GK myocytes. Amplitude of shortening, Ca2+ transient and sarcoplasmic reticulum Ca2+ were unaltered in EPI and ENDO myocytes from Goto-Kakizaki compared with control rats. We demostrated regional differences in shortening and Ca2+ transport in Goto-Kakizaki rats. ABSTRACT: Diabetic cardiomyopathy is considered to be one of the major diabetes-associated complications, and the pathogenesis of cardiac dysfunction is not well understood. The electromechanical properties of cardiac myocytes vary across the walls of the chambers. The aim of this study was to investigate shortening and Ca2+ transport in epicardial (EPI) and endocardial (ENDO) left ventricular myocytes in the Goto-Kakizaki (GK) type 2 diabetic rat heart. Shortening and intracellular Ca2+ transients were measured by video edge detection and fluorescence photometry. Myocyte surface area was increased in EPI-GK and ENDO-GK compared with control EPI-CON and ENDO-CON myocytes. Time to peak shortening was prolonged in EPI-GK compared with EPI-CON and in ENDO-CON compared with EPI-CON myocytes. Time to half-relaxation of shortening and time to peak Ca2+ transient were prolonged in EPI-GK compared with EPI-CON myocytes. Time to half-decay of the Ca2+ transient was prolonged in EPI-CON compared with EPI-GK and in EPI-CON compared with ENDO-CON myocytes. The amplitude of shortening and the Ca2+ transient were unaltered in EPI-GK and ENDO-GK compared with their respective controls. Sarcoplasmic reticulum Ca2+ and myofilament sensitivity to Ca2+ were unaltered in EPI-GK and ENDO-GK compared with their respective controls. Regional differences in Ca2+ signalling in healthy and diabetic myocytes might account for variation in the dynamics of myocyte shortening. Further studies will be required to clarify the mechanisms underlying regional differences in the time course of shortening and the Ca2+ transient in EPI and ENDO myocytes from diabetic and control hearts.

Regional effects of streptozotocin-induced diabetes on shortening and calcium transport in epicardial and endocardial myocytes from rat left ventricle.

In the heart, the left ventricle pumps blood at higher pressure than the right ventricle. Within the left ventricle, the electromechanical properties of ventricular cardiac myocytes vary transmurally and this may be related to the gradients of stress and strain experienced in vivo across the ventricular wall. Diabetes is also associated with alterations in hemodynamic function. The aim of this study was to investigate shortening and Ca2+ transport in epicardial (EPI) and endocardial (ENDO) left ventricular myocytes in the streptozotocin (STZ)-induced diabetic rat. Shortening, intracellular Ca2+ and L-type Ca2+ current (ICa,L) were measured by video detection, fura-2 microfluorimetry, and whole-cell patch clamp techniques, respectively. Time to peak (TPK) shortening was prolonged to similar extents in ENDO and EPI myocytes from STZ-treated rats compared to ENDO and EPI myocytes from controls. Time to half (THALF) relaxation of shortening was prolonged in ENDO myocytes from STZ-treated rats compared to ENDO controls. TPK Ca2+ transient was prolonged in ENDO myocytes from STZ-treated rats compared to ENDO controls. THALF decay of the Ca2+ transient was prolonged in ENDO myocytes from STZ-treated rats compared to ENDO controls. Sarcoplasmic reticulum (SR) fractional release of Ca2+ was reduced in EPI myocytes from STZ-treated rats compared to EPI controls. ICa,L activation, inactivation, and recovery from inactivation were not significantly altered in EPI and ENDO myocytes from STZ-treated rats or controls. Regional differences in Ca2+ transport may partly underlie differences in ventricular myocyte shortening across the wall of the healthy and the STZ-treated rat left ventricle.