publications

Monitoring and manipulation of ionized intracellular calcium concentrations within intact, living cells using optical probes with organic chromophores is a core method for cell physiology. Since all these probes have multiple negative charges, they must be smuggled through the plasma membrane in a transiently neutral form, with intracellular esterases used to deprotect the masked anions. Here we explore the ability of the synthetically easily accessible n-butyl ester protecting group to deliver amphipathic cargoes to the cytosol. We show that the size of the caging chromophore conditions the ability of intracellular probe delivery and esterase charge unmasking.

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 β-adrenergic receptor (β-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 β-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 β-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 β-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.

Key points In cardiac myocytes, subcellular local calcium release signals, calcium sparks, are recruited to form each cellular calcium transient and activate the contractile machinery. Abnormal timing of recovery of sparks after their termination may contribute to arrhythmias. We developed a method to interrogate recovery of calcium spark trigger probabilities and their amplitude over time using two-photon photolysis of a new ultra-effective caged calcium compound. The findings confirm the utility of the technique to define an elevated sensitivity of the calcium release mechanism in situ and to follow hastened recovery of spark trigger probabilities in a mouse model of an inherited cardiac arrhythmia, which was used for validation. Analogous methods are likely to be applicable to investigate other microscopic subcellular signalling systems in a variety of cell types. Abstract In cardiac myocytes Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs) governs activation of contraction. Ca2+ release occurs via subcellular Ca2+ signalling events, Ca2+ sparks. Local recovery of Ca2+ release depends on both SR refilling and restoration of Ca2+ sensitivity of the RyRs. We used two-photon (2P) photolysis of the ultra-effective caged Ca2+ compound BIST-2EGTA and laser-scanning confocal Ca2+ imaging to probe refractoriness of local Ca2+ release in control conditions and in the presence of cAMP or low-dose caffeine (to stimulate CICR) or cyclopiazonic acid (CPA; to slow SR refilling). Permeabilized cardiomyocytes were loaded with BIST-2EGTA and rhod-2. Pairs of short 2P photolytic pulses (1 ms, 810 nm) were applied with different intervals to test Ca2+ release amplitude recovery and trigger probability for the second spark in a pair. Photolytic and biological events were distinguished by classification with a self-learning support vector machine (SVM) algorithm. In permeabilized myocytes data recorded in the presence of CPA showed a lower probability of triggering a second spark compared to control or cAMP conditions. Cardiomyocytes from a mouse model harbouring the arrhythmogenic RyRR420Q mutation were used for further validation and revealed a higher Ca2+ sensitivity of CICR. This new 2P approach provides composite information of Ca2+ release amplitude and trigger probability recovery reflecting both SR refilling and restoration of CICR and RyR Ca2+ sensitivity. It can be used to measure the kinetics of local CICR recovery, alterations of which may be related to premature heart beats and arrhythmias.

Key points Increased protein phosphatase 1 (PP-1) activity has been found in end stage human heart failure. Although PP-1 has been extensively studied, a detailed understanding of its role in the excitation–contraction coupling mechanism, in normal and diseased hearts, remains elusive. The present study investigates the functional effect of the PP-1 activity on local Ca2+ release events in ventricular cardiomyocytes, by using an activating peptide (PDP3) for the stimulation of the endogenous PP-1 protein. We report that acute de-phosphorylation may increase the sensitivity of RyR2 channels to Ca2+ in situ, and that the RyR2-serine2808 phosphorylation site may mediate such a process. Our approach unmasks the functional importance of PP-1 in the regulation of RyR2 activity, suggesting a potential role in the generation of a pathophysiological sarcoplasmic reticulum Ca2+ leak in the diseased heart. Abstract Changes in cardiac ryanodine receptor (RyR2) phosphorylation are considered to be important regulatory and disease related post-translational protein modifications. The extent of RyR2 phosphorylation is mainly determined by the balance of the activities of protein kinases and phosphatases, respectively. Increased protein phosphatase-1 (PP-1) activity has been observed in heart failure, although the regulatory role of this enzyme on intracellular Ca2+ handling remains poorly understood. To determine the physiological and pathophysiological significance of increased PP-1 activity, we investigated how the PP-1 catalytic subunit (PP-1c) alters Ca2+ sparks in permeabilized cardiomyocytes and we also applied a PP-1-disrupting peptide (PDP3) to specifically activate endogenous PP-1, including the one anchored on the RyR2 macromolecular complex. We compared wild-type and transgenic mice in which the usually highly phosphorylated site RyR2-S2808 has been ablated to investigate its involvement in RyR2 modulation (S2808A+/+). In wild-type myocytes, PP-1 increased Ca2+ spark frequency by two-fold, followed by depletion of the sarcoplasmic reticulum Ca2+ store. Similarly, PDP3 transiently increased spark frequency and decreased sarcoplasmic reticulum Ca2+ load. RyR2 Ca2+ sensitivity, which was assessed by Ca2+ spark recovery analysis, was increased in the presence of PDP3 compared to a negative control peptide. S2808A+/+ cardiomyocytes did not respond to both PP-1c and PDP3 treatment. Our results suggest an increased Ca2+ sensitivity of RyR2 upon de-phosphorylation by PP-1. Furthermore, we have confirmed the S2808 site as a target for PP-1 and as a potential link between RyR2s modulation and the cellular response.

During physical exercise or stress, the sympathetic system stimulates cardiac contractility via β-adrenergic receptor (β-AR) activation, resulting in protein kinase A (PKA)–mediated phosphorylation of the cardiac ryanodine receptor RyR2. PKA-dependent “hyperphosphorylation” of the RyR2 channel has been proposed as a major impairment that contributes to progression of heart failure. However, the sites of PKA phosphorylation and their phosphorylation status in cardiac diseases are not well defined. Among the known RyR2 phosphorylation sites, serine 2030 (S2030) remains highly controversial as a site of functional impact. We examined the contribution of RyR2-S2030 to Ca2+ signaling and excitation–contraction coupling (ECC) in a transgenic mouse with an ablated RyR2-S2030 phosphorylation site (RyR2-S2030A+/+). We assessed ECC gain by using whole-cell patch–clamp recordings and confocal Ca2+ imaging during β-ARs stimulation with isoproterenol (Iso) and consistent SR Ca2+ loading and L-type Ca2+ current (ICa) triggering. Under these conditions, ECC gain is diminished in mutant compared with WT cardiomyocytes. Resting Ca2+ spark frequency (CaSpF) with Iso is also reduced by mutation of S2030. In permeabilized cells, when SR Ca2+ pump activity is kept constant (using 2D12 antibody against phospholamban), cAMP does not change CaSpF in S2030A+/+ myocytes. Using Ca2+ spark recovery analysis, we found that mutant RyR Ca2+ sensitivity is not enhanced by Iso application, contrary to WT RyRs. Furthermore, ablation of RyR2-S2030 prevents acceleration of Ca2+ waves and increases latency to the first spontaneous Ca2+ release after a train of stimulations during Iso treatment. Together, these results suggest that phosphorylation at S2030 may represent an important step in the modulation of RyR2 activity during β-adrenergic stimulation and a potential target for the development of new antiarrhythmic drugs.

We have designed a nitroaromatic photochemical protecting group that absorbs visible light in the violet-blue range. The chromophore is a dinitro derivative of bisstyrylthiophene (or BIST) that absorbs light very effectively (ε440 = 66,000 M–1 cm–1 and two-photon cross section of 350 GM at 775 nm). We developed a “caged calcium” molecule by conjugation of BIST to a Ca2+ chelator that upon laser flash photolysis rapidly releases Ca2+ in \textless0.2 ms. Using the patch-clamp method the optical probe, loaded with Ca2+, was delivered into acutely isolated mouse cardiac myocytes, where either one- and two-photon uncaging of Ca2+ induced highly local or cell-wide physiological Ca2+ signaling events.

Duchenne muscular dystrophy (DMD) is a progressive muscle disease with severe cardiac complications. It is believed that cellular oxidative stress and augmented Ca2+ signaling drives the development of cardiac pathology. Some mitochondrial and metabolic dysfunctions have also been reported. Here we investigate cellular mechanisms responsible for impaired mitochondrial metabolism in dystrophic cardiomyopathy at early stages of the disease. We employed electrophysiological and imaging techniques to study mitochondrial structure and function in cardiomyocytes from mdx mice, an animal model of DMD. Here we show that mitochondrial matrix was progressively oxidized in myocytes isolated from mdx mice. Moreover, an abrupt increase in workload resulted in significantly more pronounced oxidation of mitochondria in dystrophic cells. Electron micrographs revealed a gradually increased number of damaged mitochondria in mdx myocytes. Degradation in mitochondrial structure was correlated with progressive increase in mitochondrial Ca2+ sequestration and mitochondrial depolarization, despite a substantial and persistent elevation in resting cytosolic sodium levels. Treatment of mdx cells with cyclosporine A, an inhibitor of mitochondrial permeability transition pore (mPTP), shifted both resting and workload-dependent mitochondrial redox state to the levels recorded in control myocytes. It also significantly reduced workload dependent depolarization of mitochondrial membrane in dystrophic cardiomyocytes. Overall, our studies highlight age dependent deterioration of mitochondrial function in dystrophic cardiomyocytes, which seems to be associated with excessive opening of mPTP due to oxidative stress and cellular Ca2+ overload.

Intracellular calcium signals are studied by laser-scanning confocal fluorescence microscopy. The required spatio-temporal resolution makes description of calcium signals difficult because of the low signal-to-noise ratio. We designed a new procedure of calcium spike analysis based on their fitting with a model. The accuracy and precision of calcium spike description were tested on synthetic datasets generated either with randomly varied spike parameters and Gaussian noise of constant amplitude, or with constant spike parameters and Gaussian noise of various amplitudes. Statistical analysis was used to evaluate the performance of spike fitting algorithms. The procedure was optimized for reliable estimation of calcium spike parameters and for dismissal of false events. A new algorithm was introduced that corrects the acquisition time of pixels in line-scan images that is in error due to sequential acquisition of individual pixels along the space coordinate. New software was developed in Matlab and provided for general use. It allows interactive dissection of temporal profiles of calcium spikes from x-t images, their fitting with predefined function(s) and acceptance of results on statistical grounds, thus allowing efficient analysis and reliable description of calcium signaling in cardiac myocytes down to the in situ function of ryanodine receptors.

Key points • Cardiac myocytes contract due to the upsurge in intracellular calcium concentration resulting from the activation of ryanodine receptor (RyR) channels residing at calcium releasing sites. • Calcium release can be observed as local calcium spikes that vary in amplitude, time course and frequency of occurrence due to reasons that are not well understood. • In this study we quantify the characteristics of calcium spikes in isolated myocytes, typify them by stochastic analysis and interpret them by mathematical modelling. • We show that the observable properties of calcium spikes are compatible with gating properties, calcium/magnesium sensitivity and distribution of RyR channels, if only a few out of the many RyRs clustered at the release sites activate at a single release event. • The proposed mechanism of stimulated calcium release clarifies the concepts of the structure and function of calcium releasing sites and of plasticity and dynamics of calcium signalling in cardiac development and disease. Abstract In mammalian cardiac myocytes, the elementary calcium releases triggered by step voltage stimuli manifest either as solitary or as twin spikes that vary widely in kinetics and amplitude for unknown reasons. Here we examined the variability of calcium spikes measured using line-scanning confocal microscopy in patch-clamped rat ventricular myocytes. Amplitude distributions of the single and of the first of twin spikes were broader than those of the second spikes. All could be best approximated by a sum of a few elementary Gaussian probability distribution functions. The latency distributions of the single and the first spikes were identical, much shorter and less variable than those of the second spikes. The multimodal distribution of spike amplitudes and the probability of occurrence of twin spikes were stochastically congruent with activation of only a few of the many RyR2 channels present in the release site cluster. The occurrence of twin release events was rare due to refractoriness of release, induced with a probability proportional to the number of RyR2s activated in the primary release event. We conclude that the variability of the elementary calcium release events supports a calcium signalling mechanism that arises from stochastics of RyR2 gating and from inactivation of local origin.

The mechanisms of interactions between calix[4]resorcinarene and dopamine in monolayers formed at the air−water interface were studied by analyzing their mechanical, thermodynamic, and electrical properties evaluated from measurements of pressure−area isotherms and Maxwell displacement currents (MDCs). An increased concentration of dopamine in the water subphase resulted in an increase in the area per calix[4]resorcinarene molecule, an increase in the collapse pressure, and a shift in the monolayer phase transitions from the gaseous to the liquid state and from the liquid to the solid state toward higher molecular areas. A contactless method of recording MDCs enabled the monitoring of changes in the charge state of the monolayer-constituting molecules and the determination of a relationship between the phase state of the monolayer and the structural transitions of calix[4]resorcinarene. The changes of the MDC recordings started already in the gaseous state of the monolayer. On the basis of MDC values, we determined the normal component of the dipole moment of calix[4]resorcinarene, as well as that of its complex with dopamine. The dipole moment reached a maximum value of 1040 mD in the region of the phase transition from the liquid to the solid state of the monolayer. The results obtained suggest that the binding of dopamine with calix[4]resorcinarene depends on the orientation of the calixarene molecules in the monolayer. The calix[4]resorcinarene−dopamine interactions were also quantified in terms of the excess of Gibbs free energy, thereby allowing the evaluation of the energy of the calix [4]resorcinarene−dopamine bond, which was in the range from 1.95 to 8.54 kJ/mol depending on the surface pressure. This value implies weak interactions between these molecules.