Regulation Of Ca～(2+) Release Of Ryanodine Receptor In Situ In Rat Ventricular Myocytes

Ryanodine receptor (RyR) is a class of Ca²⁺ release channel located in theintracellular membrane systems (sarcoplasmic reticulum (SR) and endoplasmicreticulum (ER)) of animal cells. Ca²⁺ sparks are elementary events of Ca²⁺ releasemediated by synchronously opening of several RyR. In this study a laser scanningconfocal microscope was used to investigate the effects of osmitc stress andoxidative stress on global and local (spontaneous Ca²⁺ sparks) Ca²⁺ signals inisolated rat ventricular myocytes. The research includes two parts:I. Effect of osmotic stress on spontaneous Ca²⁺ sparks in rat ventricularmyocytes. The volume of cardiomyocytes and their functions including electricaland contractile properties would change under osmotic stress or severe pathologicalstates. To clarify the role of RyRs in the functional change, the effect of osmoticstress on spontaneous Ca²⁺ sparks in rat ventricular myocytes was investigated. Weobserved that:1. In intact myocytes the frequency of Ca²⁺ sparks was increased and decreasedby hyperosmotic (1.5 T) and hyposmotic (0.6 T) exposure, respectively. In addition,1.5 T exposure increased the temporal parameters and decreased the spatialparameter of Ca²⁺ sparks. Opposite changes occurred with 0.6 T exposure.2. 1.5 T exposure increased myoplasmic Ca²⁺ and caffeine-induced Ca²⁺transients. Opposite changes occurred with 0.6 T exposure. Raising external [Ca²⁺]enhanced myoplasmic Ca2+,caffeine-induced Ca2+ transients and the frequency ofCa2+ sparks,but it did not alter the spatio-temporal parameters of Ca2+ sparks.3. The spatio-temporal properties of Ca2+ sparks were slightly affected byaltering [K+]i (50-200 mM) in saponin permeabilized myocytes. These effectsdiffered from that induced by osmotic stress.4. Dextran is a polymer, which can change the viscidity of the solution andhence the diffusibility of Ca2+. Reducing dextran concentration in the internalsolution of permeabilized myocytes evidently caused an increase of spatialparameter and a decrease of temporal parameters of the Ca2+ sparks, which weresimilar to the effects of hyposmotic stress seen on intact myocytes. Increasingdextran had opposite effects similar to that of hyperosmotic stress.5. The propagating velocity of Ca2+ waves in intact and permeabilized myocytewas decreased by elevating extracellular tonicity and dextran concertration ininternal solution, respetively.Taken together, these results indicate that the effect of osmotic stress on thefrequency of spontaneous Ca2+ sparks might be ascribed to the change ofmyoplasmic Ca2+ and Ca2+ content in the SR, while the effect on thespatio-temporal properties is due to the alteration of Ca2+ diffusion mainly resultingfrom the morphological change of the myocytes. Moreover, all these alterations inthe properties of Ca2+ sparks and waves induced by osmotic stress may affect thecontractile properties of myocytes.II. Biphasic modulation of ryanodine receptors by sulfhydryl oxidation in ratventricular myocytes. It is well known that the intracellular Ca2+ accumulationinduced by oxidative stress would cause injury and dysfunction of cardiacmyocytes. To understand better the modulation of RyRs during oxidative stress, theeffect of 4,4'-dithiodipyridine (DTDP), a cell-permeant and thiol-reactive oxidant,on global Ca2+ signal and spontaneous Ca2+ sparks in rat ventricular myocytes wasinvestigated. We found that:1. 3 μM DTDP did not affect the cytoplasmic free Ca2+ concentration, while abrief Ca2+ transient was elicited by 100 μM DTDP.2. A dose dependent increase of cytoplasmic free Zn2+ concentration wasinduced by DTDP (>3 μM).3. An increase of the frequency of Ca2+ sparks appeared at 3 μM DTDP, whilehigher concentration of DTDP caused a biphasic change of the frequency andfinally disappearance of Ca2+ sparks in both intact and permeabilized myocytes, inwhich cytoplasmic Ca2+ was buffered with EGTA. Unlike the frequency, thespatio-temporal properties of Ca2+ sparks were not influenced by DTDP.4. Consistent with the frequency of Ca2+ sparks, caffeine-induced Ca2+ transientswere similarly affected by DTDP. The depression effects of DTDP on the frequencyof Ca2+ sparks and caffeine-induced Ca2+ transients could be reversed by DTT.5. DTDP increased the [Ca2+] in the SR lumen. However, this increase was laterthan DTDP-induced increase of Ca2+ sparks frequency and caffeine-induced Ca2+transients.Ttaken together, these results indicate that the effects of DTDP on thefrequency of spontaneous Ca2+ sparks are due mainly to sulfhydryl oxidation-induced activation and inactivation of RyRs. Moreover,this study suggests that,even if oxidative stress is mild enough not to cause intracellular Ca2+ accumulation,it may affect Ca2+–mediated signaling pathways through changing the frequency ofspontaneous Ca2+ sparks. Thus, the functional importance of moderate oxidativestress should not be overlooked.Interestingly, the finding that DTDP does not affect the duration of Ca2+sparks is inconsistent with that of the DTDP-induced increase of the open time ofreconstituted RyR channels. In addition, the effect of ion strength on Ca2+ sparkfrequency is different from that on the activity of reconstituted RyR channels.These differences show that RyR behavior in vitro differs from that in situ.Therefore, it should be cautious to extrapolate from in vitro result to in situ events.Moreover, the importance of studying RyR's gating in situ is clearly indicated,which would help us to understand better the role of RyR in the regulation ofcardiomyocyte function.