Angélica Rueda

Increased Ca2+ Sensitivity of the Ryanodine Receptor Mutant RyR2R4496C Underlies Catecholaminergic Polymorphic Ventricular Tachycardia

Cardiac ryanodine receptor (RyR2) mutations are associated with autosomal dominant catecholaminergic polymorphic ventricular tachycardia, suggesting that alterations in Ca2+ handling underlie this disease. Here we analyze the underlying Ca2+ release defect that leads to arrhythmia in cardiomyocytes isolated from heterozygous knock-in mice carrying the RyR2R4496C mutation. RyR2R4496C−/− littermates (wild type) were used as controls. [Ca2+]i transients were obtained by field stimulation in fluo-3–loaded cardiomyocytes and viewed using confocal microscopy. In our basal recording conditions (2-Hz stimulation rate), [Ca2+]i transients and sarcoplasmic reticulum Ca2+ load were similar in wild-type and RyR2R4496C cells. However, paced RyR2R4496C ventricular myocytes presented abnormal Ca2+ release during the diastolic period, viewed as Ca2+ waves, consistent with the occurrence of delayed afterdepolarizations. The occurrence of this abnormal Ca2+ release was enhanced at faster stimulation rates and by &bgr;-adrenergic stimulation, which also induced triggered activity. Spontaneous Ca2+ sparks were more frequent in RyR2R4496C myocytes, indicating increased RyR2R4496C activity. When permeabilized cells were exposed to different cytosolic [Ca2+]i, RyR2R4496C showed a dramatic increase in Ca2+ sensitivity. Isoproterenol increased [Ca2+]i transient amplitude and Ca2+ spark frequency to the same extent in wild-type and RyR2R4496C cells, indicating that the &bgr;-adrenergic sensitivity of RyR2R4496C cells remained unaltered. This effect was independent of protein expression variations because no difference was found in the total or phosphorylated RyR2 expression levels. In conclusion, the arrhythmogenic potential of the RyR2R4496C mutation is attributable to the increased Ca2+ sensitivity of RyR2R4496C, which induces diastolic Ca2+ release and lowers the threshold for triggered activity.

Sorcin Inhibits Calcium Release and Modulates Excitation-Contraction Coupling in the Heart

Activation of Ca2+ release channels/ryanodine receptors (RyR) by the inward Ca2+ current (ICa) gives rise to Ca2+-induced Ca2+ release (CICR), the amplifying Ca2+ signaling mechanism that triggers contraction of the heart. CICR, in theory, is a high-gain, self-regenerating process, but an unidentified mechanism stabilizes it in vivo. We reported previously (Lokuta, A. J., Meyers, M. B., Sander, P. R., Fishman, G. I., and Valdivia, H. H. (1997) J. Biol. Chem. 272, 25333–25338) that sorcin, a 22-kDa Ca2+-binding protein, binds to cardiac RyRs with high affinity and completely inhibits channel activity. Here we show that sorcin significantly inhibits both the spontaneous activity of RyRs in quiescent cells (visualized as Ca2+ sparks) and the ICa-triggered activity of RyRs that gives rise to [Ca2+]i transients. Because sorcin decreased the amplitude of the [Ca2+]i transient without affecting the amplitude or kinetics of ICa, the overall effect of sorcin was to reduce the “gain” of excitation-contraction coupling. Immunocytochemical staining shows that sorcin localizes to the dyadic space of ventricular cardiac myocytes. Ca2+ induces conformational changes and promotes translocation of sorcin between soluble and membranous compartments, but the [Ca2+] required for the latter process (ED50 = ∼200 μm) appears to be reached only within the dyadic space. Rapid injection of 5 μm sorcin onto the cytosolic face of RyRs reconstituted in lipid bilayers resulted in complete inhibition of channel activity in ≤ 20 ms. Thus, sorcin is a potent inhibitor of both spontaneous and ICa-triggered RyR activity and is kinetically capable of playing a role in terminating the positive feedback loop of CICR.

Calcium signaling in diabetic cardiomyocytes.

Diabetes mellitus is one of the most common medical conditions. It is associated to medical complications in numerous organs and tissues, of which the heart is one of the most important and most prevalent organs affected by this disease. In fact, cardiovascular complications are the most common cause of death among diabetic patients. At the end of the 19th century, the weakness of the heart in diabetes was noted as part of the general muscular weakness that exists in that disease. However, it was only in the eighties that diabetic cardiomyopathy was recognized, which comprises structural and functional abnormalities in the myocardium in diabetic patients even in the absence of coronary artery disease or hypertension. This disorder has been associated with both type 1 and type 2 diabetes, and is characterized by early-onset diastolic dysfunction and late-onset systolic dysfunction, in which alteration in Ca(2+) signaling is of major importance, since it controls not only contraction, but also excitability (and therefore is involved in rhythmic disorder), enzymatic activity, and gene transcription. Here we attempt to give a brief overview of Ca(2+) fluxes alteration reported on diabetes, and provide some new data on differential modulation of Ca(2+) handling alteration in males and females type 2 diabetic mice to promote further research. Due to space limitations, we apologize for those authors whose important work is not cited.

Ryanodine receptors in smooth muscle.

The sarcoplasmic reticulum (SR) of smooth muscle is endowed with two different types of Ca2+ release channels, i.e. inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs). In general, both release channels mobilize Ca2+ from the same internal store in smooth muscle. While the importance of IP3Rs in agonist-induced contraction is well established, the role of RyRs in excitation-contraction coupling of smooth muscle is not clear. The participation of smooth muscle RyRs in the amplification of Ca2+ transients induced by either opening of Ca2+-permeable channels or IP3-triggered Ca2+ release has been studied. The efficacy of both processes to activate RyRs by calcium-induced calcium release (CICR) is highly variable and not widely present in smooth muscle. Although RyRs in smooth muscle generate Ca2+ sparks that are similar to those observed in striated muscles, the contribution of these local Ca2+ events to depolarization-induced global rise in [Ca2+]i is rather limited. Recent data suggest that RyRs are involved in regulating the luminal [Ca2+] of SR and also in smooth muscle relaxation. This review summarizes studies that were carried out mainly in muscle strips or in freshly isolated myocytes, and that were aimed to determine the physiological role of RyRs in smooth muscle.

Molecular basis for the impaired function of the natural F112L sorcin mutant: X-ray crystal structure, calcium affinity, and interaction with annexin VII and the ryanodine receptor

The penta‐EF hand protein sorcin participates in the modulation of Ca2+‐induced calcium‐release in the heart through the interaction with several Ca2+ channels such as the ryanodine receptor. The modulating activity is impaired in the recently described natural F112L mutant. The F112 residue is located at the end of the D helix next to Asp113, one of the calcium ligands in the EF3 hand endowed with the highest affinity for the metal. The F112L‐sorcin X‐ray crystal structure at 2.5 Å resolution displays marked alterations in the EF3 hand, where the hydrogen bonding network established by Phe112 is disrupted, and in the EF1 region, which is tilted in both monomers that give rise to the dimer, the stable form of the molecule. In turn, the observed tilt is indicative of an increased flexibility of the N‐terminal part of the molecule. The structural alterations result in a 6‐fold decrease in calcium affinity with respect to the wild‐type protein and to an even larger impairment of the interaction with annexin VII and of the ability of sorcin to interact with and inhibit ryanodine receptors. These results provide a plausible structural and functional framework that helps elucidate the phenotypic alterations of mice overexpressing F112L‐sorcin.— Franceschini, S., Ilari, A., Verzili, D., Zamparelli, C., Antaramian, A., Rueda, A., Valdivia, H. H., Chiancone, E., and Colotti, G. Molecular basis for the impaired function of the natural F112L sorcin mutant: X‐ray crystal structure, calcium affinity, and interaction with annexin VII and the ryanodine receptor. FASEB J. 22, 295–306 (2008)

Sorcin modulation of Ca2+ sparks in rat vascular smooth muscle cells

Spontaneous, local Ca2+ release events or Ca2+ sparks by ryanodine receptors (RyRs) are important determinants of vascular tone and arteriolar resistance, but the mechanisms that modulate their properties in smooth muscle are poorly understood. Sorcin, a Ca2+‐binding protein that associates with cardiac RyRs and quickly stops Ca2+ release in the heart, provides a potential mechanism to modulate Ca2+ sparks in vascular smooth muscle, but little is known about the functional role of sorcin in this tissue. In this work, we characterized the expression and intracellular location of sorcin in aorta and cerebral artery and gained mechanistic insights into its functional role as a modulator of Ca2+ sparks. Sorcin is present in endothelial and smooth muscle cells, as assessed by immunocytochemical and Western blot analyses. Smooth muscle sorcin translocates from cytosolic to membranous compartments in a Ca2+‐dependent manner and associates with RyRs, as shown by coimmunoprecipitation and immunostaining experiments. Ca2+ sparks recorded in saponin‐permeabilized vascular myocytes have increased frequency, duration and spatial spread but reduced amplitude with respect to Ca2+ sparks in intact cells, suggesting that permeabilization disrupts the normal organization of RyRs and releases diffusible substances that control Ca2+ spark properties. Perfusion of 2 μm sorcin onto permeabilized myocytes reduced the amplitude, duration and spatial spread of Ca2+ sparks, demonstrating that sorcin effectively regulates Ca2+ signalling in vascular smooth muscle. Together with a dense distribution in the perimeter of the cell along a pool of RyRs, these properties make sorcin a viable candidate to modulate vascular tone in smooth muscle.

Ca2+ handling alterations and vascular dysfunction in diabetes

More than 65% of patients with diabetes mellitus die from cardiovascular disease or stroke. Hyperglycemia, due to either reduced insulin secretion or reduced insulin sensitivity, is the hallmark feature of diabetes mellitus. Vascular dysfunction is a distinctive phenotype found in both types of diabetes and could be responsible for the high incidence of stroke, heart attack, and organ damage in diabetic patients. In addition to well-documented endothelial dysfunction, Ca(2+) handling alterations in vascular smooth muscle cells (VSMCs) play a key role in the development and progression of vascular complications in diabetes. VSMCs provide not only structural integrity to the vessels but also control myogenic arterial tone and systemic blood pressure through global and local Ca(2+) signaling. The Ca(2+) signalosome of VSMCs is integrated by an extensive number of Ca(2+) handling proteins (i.e. channels, pumps, exchangers) and related signal transduction components, whose function is modulated by endothelial effectors. This review summarizes recent findings concerning alterations in endothelium and VSMC Ca(2+) signaling proteins that may contribute to the vascular dysfunction found in the diabetic condition.

Luminal Ca2+ and the activity of sarcoplasmic reticulum Ca2+ pumps modulate histamine-induced all-or-none Ca2+ release in smooth muscle cells

Abstract We have studied histamine (HA)-evoked intracellular Ca2+ release in single, freshly isolated myocytes from the guinea pig urinary bladder. Short applications of histamine (5 s) produced a thapsigargin (TG)-sensitive transient increase in intracellular calcium concentration ([Ca2+]i). It was established that histamine and caffeine (Caff) released Ca2+ from the same intracellular stores in these cells. Reducing the Ca2+ content of internal stores by incubating cells with U-73343 or cyclopiazonic acid (CPA) inhibited the histamine-evoked Ca2+ release in 69% and 60% of cells, respectively. Under these conditions, all cells released Ca2+ in response to either caffeine or acetylcholine (ACh). However, decreasing internal Ca2+ stores by removing external Ca2+ inhibited histamine-induced Ca2+ mobilization in only 22% of cells. A similar small fraction of cells was inhibited when sarcoplasmic reticulum (SR) Ca2+ pumps were quickly blocked to avoid a significant reduction of luminal Ca2+. In conclusion, lowering the luminal Ca2+ content in combination with an impairment of the SR Ca2+ pump activity significantly diminishes the ability of histamine to evoke an all-or-none intracellular Ca2+ release.

Ryanodine receptors as leak channels

Ryanodine receptors are Ca(2+) release channels of internal stores. This review focuses on those situations and conditions that transform RyRs from a finely regulated ion channel to an unregulated Ca(2+) leak channel and the pathological consequences of this alteration. In skeletal muscle, mutations in either CaV1.1 channel or RyR1 results in a leaky behavior of the latter. In heart cells, RyR2 functions normally as a Ca(2+) leak channel during diastole within certain limits, the enhancement of this activity leads to arrhythmogenic situations that are tackled with different pharmacological strategies. In smooth muscle, RyRs are involved more in reducing excitability than in stimulating contraction so the leak activity of RyRs in the form of Ca(2+) sparks, locally activates Ca(2+)-dependent potassium channels to reduce excitability. In neurons the enhanced activity of RyRs is associated with the development of different neurodegenerative disorders such as Alzheimer and Huntington diseases. It appears then that the activity of RyRs as leak channels can have both physiological and pathological consequences depending on the cell type and the metabolic condition.

Reconciling depressed Ca2+ sparks occurrence with enhanced RyR2 activity in failing mice cardiomyocytes

Alterations in the intracellular environment lead to decreased frequency of Ca2+ sparks in a model of heart failure despite enhanced ryanodine receptor activity.