Uptake-leak balance of SR Ca2+ determines arrhythmogenic potential of RyR2R420Q+/- cardiomyocytes.

Mutations of the RyR2 are channelopathies that can predispose to life threatening catecholaminergic polymorphic ventricular tachycardias (CPVTs) during exercise or stress. However, the cellular and molecular mechanisms that are causal for the arrhythmias downstream of the ß-adrenergic receptor (ß-AR) activation are not defined. They may be specific and different for each particular RyR2 mutation. Obvious possibilities are the phosphorylation of the mutated RyR2s or the stimulation of the SR Ca2+ pump (SERCA), which could increase SR Ca2+ loading. Potentially arrhythmogenic Ca2+ signals, such as Ca2+ waves, were recorded and analyzed from WT and RyR2R420Q+/- mouse cardiomyocytes with confocal microscopy after field stimulation at 1 Hz. In RyR2R420Q+/- cardiomyocytes we found a higher occurrence and frequency of Ca2+ waves, particularly upon ß-AR stimulation with isoproterenol. This was accompanied by a shorter latency to the first spontaneous wave. Wave velocity from raw traces, as well as amplitude and decay time constant (τ) analyzed in de-skewed traces were comparable in both cell types. To obtain further insight into the role of the SERCA we selectively stimulated SERCA in permeabilized myocytes using Fab fragments of a PLB antibody (2D12). Surprisingly, SERCA stimulation alone resulted in considerably higher wave frequencies than when mimicking ß-AR stimulation with cAMP, particularly in RyR2R420Q+/- cardiomyocytes. This may be a consequence of some protective SR Ca2+ unloading resulting from the SR Ca2+ leak via phosphorylated RyR2s in cAMP. Spark-to-spark recovery analysis suggested a remarkably higher Ca2+ release sensitivity in RyR2R420Q+/- cells, both in control and upon ß-AR stimulation. Together these findings suggest that the fine balance between SR Ca2+ loading via SERCA and the Ca2+ leak via mutated and phosphorylated RyR2s is an important determinant for the overall cellular arrhythmogenicity prevailing in the RyR2R420Q+/- myocytes.

Differential sensitivity to detergents of actin cytoskeleton from nerve endings.

Detergent-resistant membranes (DRM), an experimental model used to study lipid rafts, are typically extracted from cells by means of detergent treatment and subsequent ultracentrifugation in density gradients, Triton X-100 being the detergent of choice in most of the works. Since lipid rafts are membrane microdomains rich in cholesterol, depletion of this component causes solubilization of DRM with detergent. In previous works from our group, the lack of effect of cholesterol depletion on DRM solubilization with Triton X-100 was detected in isolated rat brain synaptosomes. In consequence, the aim of the present work is to explore reasons for this observation, analyzing the possible role of the actin cytoskeleton, as well as the use of an alternative detergent, Brij 98, to overcome the insensitivity to Triton X-100 of cholesterol-depleted DRM. Brij 98 yields Brij-DRM that are highly dependent on cholesterol, since marker proteins (Flotillin-1 and Thy-1), as well as actin, appear solubilized after MCD treatment. Pretreatment with Latrunculin A results in a significant increase in Flotillin-1, Thy-1 and actin solubilization by Triton X-100 after cholesterol depletion. Studies with transmission electron microscopy show that combined treatment with MCD and Latrunculin A leads to a significant increase in solubilization of DRM with Triton X-100. Thus, Triton-DRM resistance to cholesterol depletion can be explained, at least partially, thanks to the scaffolding action of the actin cytoskeleton, without discarding differential effects of Brij 98 and Triton X-100 on specific membrane components. In conclusion, the detergent of choice is important when events that depend on the actin cytoskeleton are going to be studied.