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 less than 0.2 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 Ca 2+ 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 Ca 2+ 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 Conflict of interest: none declared * Co-author Approval StatementThe manuscript, or part of it, has neither been published nor is currently under consideration for publication by any other journal. The co-authors have read the manuscript and approved this submission. Authors have no conflict of interests. * Conflict of Interest StatementThe manuscript, or part of it, has neither been published nor is currently under consideration for publication by any other journal. The co-authors have read the manuscript and approved this submission. Authors have no conflict of interests.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript dependent deterioration of mitochondrial function in dystrophic cardiomyocytes, which seems to be associated with excessive opening of mPTP due to oxidative stress and cellular Ca 2+ overload. Graphical Abstract KeywordsDystrophic cardiomyopathy; sodium overload; mitochondria; metabolism; oxidative stress

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. Abbreviations RyR, ryanodine receptor; DHPR, dihydropyridine receptor; SNR, signal-to-noise ratio; TTP, time to peak; FDHM, full duration at half-maximum.R. Janíček and A. Zahradníková Jr contributed equally to this work.

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.

This study describes the development and characterisation of software to enable automatic detection and analysis of Ca 2þ sparks within x-y image stacks, implemented as a plugin within the open source image analysis platform, ImageJ. The aim was to implement a "conventional" algorithm whereby sparks were identified by applying a threshold (q) to the normalised (F/F 0 ) image: q = background fluorescence within the cell þ SD * 'ε', a user defined variable. A 2 stage interactive method with a graphical user interface (GUI) was used to ensure precise identification of the cell boundary and creation of a binary cell mask, which is subsequently used to exclude all regions outside the cell. The algorithm separates spark detection and analysis, allowing image processing to be applied independently at both stages. Filters also allow exclusion of events based on spark width or morphology. Novel methods are included to allow correction of time dependent changes in background fluorescence (e.g. due to bleaching), which would otherwise compromise spark detection by thresholding. The main outputs (amplitude, width, duration and spark mass) are presented in tabular form. In addition, an interactive GUI allows each spark to be examined, along with its measurements, and the associated Gaussian curve fit. A "Kill" button allows obvious errors in detection to be excluded. The performance of the algorithm was tested both on synthesised images (values of ε ranging from 3.0-4.2 and signal to noise ratios of 2, 3 or 4) and on x-y confocal fluorescence images from fluo-3 loaded rat ventricular myocytes. In both cases the performance was comparable to that reported previously for threshold based detection methods applied to line-scan images.