Abnormal Ca2+ Spark/STOC Coupling in Cerebral Artery Smooth Muscle Cells of Obese Type 2 Diabetic Mice

Go mez AM (2013) Abnormal Ca2+ Spark/STOC Coupling in Cerebral Artery Smooth Muscle Cells of Obese Type 2 Diabetic Mice. PLoS ONE 8(1): e53321. doi:10.1371/journal.pone.0053321 2+ Abnormal Ca Spark/STOC Coupling in Cerebral Artery Smooth Muscle Cells of Obese Type 2 Diabetic Mice Ange lica Rueda 0 Mara Ferna ndez-Velasco 0 Jean-Pierre Benitah 0 Ana Mara Go mez 0 Ladzlo Csernoch, University of Debrecen, Hungary 0 1 Departamento de Bioqu mica, Centro de Investigacio n y de Estudios Avanzados del IPN, Me xico City, Me xico, 2 Inserm, U-637; Universite de Montpellier 1, Universite de Montpellier 2, Montpellier, France, 3 Instituto de Investigacio n Hospital Universitario La Paz (IdiPAZ) , Madrid , Spain , 4 Inserm, U769; Universite de Paris-Sud, IFR141, Labex Lermit , Chatenay-Malabry , France Diabetes is a major risk factor for stroke. However, the molecular mechanisms involved in cerebral artery dysfunction found in the diabetic patients are not completely elucidated. In cerebral artery smooth muscle cells (CASMCs), spontaneous and local increases of intracellular Ca2+ due to the opening of ryanodine receptors (Ca2+ sparks) activate large conductance Ca2+-activated K+ (BK) channels that generate spontaneous transient outward currents (STOCs). STOCs have a key participation in the control of vascular myogenic tone and blood pressure. Our goal was to investigate whether alterations in Ca2+ spark and STOC activities, measured by confocal microscopy and patch-clamp technique, respectively, occur in isolated CASMCs of an experimental model of type-2 diabetes (db/db mouse). We found that mean Ca2+ spark amplitude, duration, size and rate-of-rise were significantly smaller in Fluo-3 loaded db/db compared to control CASMCs, with a subsequent decrease in the total amount of Ca2+ released through Ca2+ sparks in db/db CASMCs, though Ca2+ spark frequency remained. Interestingly, the frequency of large-amplitude Ca2+ sparks was also significantly reduced in db/db cells. In addition, the frequency and amplitude of STOCs were markedly reduced at all voltages tested (from 250 to 0 mV) in db/db CASMCs. The latter correlates with decreased BK channel b1/a subunit ratio found in db/db vascular tissues. Taken together, Ca2+ spark alterations lead to inappropriate BK channels activation in CASMCs of db/db mice and this condition is aggravated by the decrease in the BK b1 subunit/a subunit ratio which underlies the significant reduction of Ca2+ spark/ STOC coupling in CASMCs of diabetic animals. - Funding: This work was funded by Conacyt (No. 80960), ICyTDF No.331/2010), Agence National de la Recherche (Geno-09-HyperEpac and Geno-09-Carythm), Region Ile de France (CODDIM: COD100256), European Union (2005 Nu018802, CONTICA) and FRM (Fondation pour la Recherche medicale). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. More than 65% of patients with diabetes die from cardiovascular disease or stroke [1]. When considering age-adjusted incidence rates, type-2 diabetic patients are two- to five times as likely to suffer cerebral vascular disease or stroke compared with non-diabetic patients, a disparity that is seen in multiple racial/ geographic groups [26] and may result from abnormal cerebral artery tissue function. Interestingly, the incidence of stroke in type2 diabetic patients is not associated with the duration of disease, smoking, fasting blood glucose, total cholesterol, lipoprotein concentrations, or hypertension [3,4,7]. Cerebral blood flow disturbances, impaired cerebral vascular reactivity, transient ischemic attacks and oxidative damage of cerebral vessels have been found in both type-2 diabetic patients [3] and experimental models [810] that could account for the higher incidence of diabetes-related stroke events [17]; however, the molecular mechanisms involved in cerebral artery dysfunction are not completely elucidated. The db/db mouse, a genetic model of non-insulin dependent type-2 diabetes exhibits cerebral vascular dysfunction [9] that exacerbates brain damage, edema and inflammation after induced experimental stroke [1113]. In addition to diabetes-related alterations found in cerebral vessels, vascular dysfunction is also present in mesenteric arteries [1416], coronary arterioles [17], gracilis muscle arterioles [18,19], and aorta [2022] of db/db mice. Cerebral arterioles of db/db mice show impaired response to vasodilators and reduced baseline arteriolar diameter [9]. Mesenteric arteries and gracilis muscle arterioles of db/db mice show impaired response to vasodilators, enhanced response to vasoconstrictors and enhanced basal myogenic tone [1416,18,23]. Consistent with the observations of augmented vascular tone, the myogenic pressure-diameter of arteries harvested from db/db mice are smaller than the diameters of corresponding control arteries [14,18,19] and are not improved by the removal of endothelium [14,19]. Moreover, investigators have demonstrated impairment of endothelium-independent dilation in the presence of nitric oxide donors: in coronary arterioles and aorta of db/db mice in the presence of sodium nitroprusside [17,20], and in arteries of type 2 diabetic patients after administration of glycerin trinitrate [24,25]. All these data suggest that smooth muscledependent mechanisms are also responsible for the vascular dysfunction associated with type-2 diabetes. Furthermore, the disease appears to alter functional responses of resistance arteries not only at endothelial level but also in active smooth muscle layers. In cerebral artery smooth muscle cells (CASMCs), spontaneous and local increases of intracellular Ca2+ due to the opening of Ryanodine Receptors (RyRs), visualized as Ca2+ sparks, activate large conductance Ca2+ sensitive K+ channels (BK channels) that generate spontaneous transient outward currents (STOCs) [26,27]. STOCs have a key role in the control of arterial tone by shifting the membrane potential towards less positive values, which in turn limits Ca2+ influx through L-type Ca2+ channels, diminishes global intracellular Ca2+ concentration ([Ca2+]i), and opposes vasoconstriction [2830]. Therefore, RyRs through Ca2+ sparks and BK channels, by producing STOCs regulates arterial tone favoring vasorelaxation [2931]. In addition, Ca2+ spark generation is also regulated by Ca2+ influx due to an indirect coupling between L-type Ca2+ channels and RyRs [32]. The sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) participates in this indirect coupling by redirecting the Ca2+ coming from the extracellular medium towards its luminal stores, located mainly inside the sarcoplasmic reticulum (SR) of CASMCs. Navedo and collaborators [33] demonstrated that L-type Ca2+ currents and Ca2+sparklet activity brief plasma membrane Ca2+ fluxes due to the activation of clusters of L-type Ca2+ channel (...truncated)