METROID: an automated method for robust quantification of subcellular fluorescence events at low SNR.

BACKGROUND: In cell biology, increasing focus has been directed to fast events at subcellular space with the advent of fluorescent probes. As an example, voltage sensitive dyes (VSD) have been used to measure membrane potentials. Yet, even the most recently developed genetically encoded voltage sensors have demanded exhausting signal averaging through repeated experiments to quantify action potentials (AP). This analysis may be further hampered in subcellular signals defined by small regions of interest (ROI), where signal-to-noise ratio (SNR) may fall substantially. Signal processing techniques like blind source separation (BSS) are designed to separate a multichannel mixture of signals into uncorrelated or independent sources, whose potential to separate ROI signal from noise has been poorly explored. Our aims are to develop a method capable of retrieving subcellular events with minimal a priori information from noisy cell fluorescence images and to provide it as a computational tool to be readily employed by the scientific community. RESULTS: In this paper, we have developed METROID (Morphological Extraction of Transmembrane potential from Regions Of Interest Device), a new computational tool to filter fluorescence signals from multiple ROIs, whose code and graphical interface are freely available. In this tool, we developed a new ROI definition procedure to automatically generate similar-area ROIs that follow cell shape. In addition, simulations and real data analysis were performed to recover AP and electroporation signals contaminated by noise by means of four types of BSS: Principal Component Analysis (PCA), Independent Component Analysis (ICA), and two versions with discrete wavelet transform (DWT). All these strategies allowed for signal extraction at low SNR (- 10 dB) without apparent signal distortion. CONCLUSIONS: We demonstrate the great capability of our method to filter subcellular signals from noisy fluorescence images in a single trial, avoiding repeated experiments. We provide this novel biomedical application with a graphical user interface at https://doi.org/10.6084/m9.figshare.11344046.v1 , and its code and datasets are available in GitHub at https://github.com/zoccoler/metroid .

Heart defibrillation: relationship between pacing threshold and defibrillation probability.

BACKGROUND: Considering the clinical importance of the ventricular fibrillation and that the most used therapy to reverse it has a critical side effect on the cardiac tissue, it is desirable to optimize defibrillation parameters to increase its efficiency. In this study, we investigated the influence of stimuli duration on the relationship between pacing threshold and defibrillation probability. RESULTS: We found out that 0.5-ms-long pulses had a lower ratio of defibrillation probability to the pacing threshold, although the higher the pulse duration the lower is the electric field intensity required to defibrillate the hearts. CONCLUSION: The appropriate choice of defibrillatory shock parameters is able to increase the efficiency of the defibrillation improving the survival chances after the occurrence of a severe arrhythmia. The relationship between pulse duration and the probability of reversal of fibrillation shows that this parameter cannot be underestimated in defibrillator design since different pulse durations have different levels of safety.

Sources of Ca2+ for contraction of the heart tube of Tenebrio molitor (Coleoptera: Tenebrionidae).

Insect and vertebrate hearts share the ability to generate spontaneously their rhythmic electrical activity, which triggers the fluid-propelling mechanical activity. Although insects have been used as models in studies on the impact of genetic alterations on cardiac function, there is surprisingly little information on the generation of the inotropic activity in their hearts. The main goal of this study was to investigate the sources of Ca2+ for contraction in Tenebrio molitor hearts perfused in situ, in which inotropic activity was assessed by the systolic variation of the cardiac luminal diameter. Increasing the pacing rate from 1.0 to 2.5 Hz depressed contraction amplitude and accelerated relaxation. To avoid inotropic interference of variations in spontaneous rate, which have been shown to occur in insect heart during maneuvers that affect Ca2+ cycling, experiments were performed under electrical pacing at near-physiological rates. Raising the extracellular Ca2+ concentration from 0.5 to 8 mM increased contraction amplitude in a manner sensitive to L-type Ca2+ channel blockade by D600. Inotropic depression was observed after treatment with caffeine or thapsigargin, which impair Ca2+ accumulation by the sarcoplasmic reticulum (SR). D600, but not inhibition of the sarcolemmal Na+/Ca2+ exchanger by KB-R7943, further depressed inotropic activity in thapsigargin-treated hearts. From these results, it is possible to conclude that in T. molitor heart, as in vertebrates: (a) inotropic and lusitropic activities are modulated by the heart rate; and (b) Ca2+ availability for contraction depends on both Ca2+ influx via L-type channels and Ca2+ release from the SR.

Ventricular myocyte injury by high-intensity electric field: Effect of pulse duration.

Although high-intensity electric fields (HEF) application is currently the only effective therapy available to terminate ventricular fibrillation, it may cause injury to cardiac cells. In this study we determined the relation between HEF pulse length and cardiomyocyte lethal injury. We obtained lethality curves by survival analysis, which were used to determine the value of HEF necessary to kill 50% of cells (E50) and plotted a strength-duration (SxD) curve for lethality with 10 different durations: 0.1, 0.2, 0.5, 1, 3, 5, 10, 20, 35 and 70 ms. For the same durations we also obtained an SxD curve for excitation and established an indicator for stimulatory safeness (stimulation safety factor - SSF) as the ratio between the SxD curve for lethality and one for excitation. We found that the lower the pulse duration, the higher the HEF intensity required to cell death. Contrary to expectations, the highest SSF value does not correspond to the lowest pulse duration but to the one of 0.5 ms. As defibrillation threshold has been described as duration-dependent, our results imply that the use of shorter stimulus duration - instead of the one typically used in the clinic (10 ms) - might increase defibrillation safeness.

Greater cardiac cell excitation efficiency with rapidly switching multidirectional electrical stimulation.

Electric field (E) stimulation is widely used in experiments with myocardial preparations and in the clinical setting (e.g., defibrillation). As a rule, stimuli are applied in a single direction, which limits excitatory cell recruitment because myocytes are disposed in different directions and their sensitivity to E depends on the stimulus orientation with respect to the cell major axis. Here, we propose a stimulatory approach, namely rapidly switching multidirectional stimulation (RSMS), in which stimuli are delivered in three directions within the electric refractory period. In populations of randomly oriented isolated rat cardiomyocytes, RSMS doubled the percentage of cells excited by near-threshold E (P < 0.001), which was more than the increase in recruitment in a single direction achieved by doubling E intensity. This effect was similar for monophasic and biphasic pulses, but for the latter, a given percent recruitment was obtained with 20-30% lower E intensity ( P < 0.01), so that RSMS with biphasic pulses allowed at least 60% reduction of E intensity for recruitment of >70% of the cells. RSMS can be applied to improve stimulation efficiency in experiments with isolated cardiac myocytes, and may be a promising alternative for decreasing shock intensity requirements for cardioversion and defibrillation.

Pacemaker activity in the insect (T. molitor) heart: role of the sarcoplasmic reticulum.

The electrophysiological properties of the myogenic cardiac cells of insects have been analyzed, but the mechanisms that regulate the pacemaker activity have not been elucidated yet. In mammalian pacemaker cells, different types of membrane ion channels seem to be sequentially activated, perhaps in a cooperative fashion with the current generated by Ca(2+) extrusion mediated by the electrogenic Na(+)/Ca(2+) exchanger, which is sustained by the diastolic sarcoplasmic reticulum (SR) Ca(2+) release. The objective of the present work was to investigate the role of the SR function on the basal beating rate (BR), and BR modulation by extracellular Ca(2+) concentration ([Ca(2+)](o)) and neurotransmitters in the in situ dorsal vessel (heart) of the mealworm beetle Tenebrio molitor. The main observations were as follows: 1) basal BR was reduced by 50% by inhibition of SR function, but not affected by perfusion with CsCl or ZD7288; 2) spontaneous activity was abolished by Cd(2+); 3) a robust positive chronotropic response could be elicited to serotonin (5-HT), but not to norepinephrine or carbamylcholine; 4) SR inhibition abolished the sustained chronotropic stimulation by [Ca(2+)](o) elevation and by 5-HT, while the latter was unaffected by CsCl. It is concluded that, in T. molitor heart, BR is markedly, but not exclusively, dependent on the SR function, and that BR control and modulation by both [Ca(2+)](o) and 5-HT requires a functional SR.

Combining stimulus direction and waveform for optimization of threshold stimulation of isolated ventricular myocytes.

Electric field stimulation is widely used for heart pacing and arrhythmia reversion. In this study, we analysed the influence of waveform and direction of external stimulating electric field on the excitation threshold of isolated ventricular myocytes. The threshold field (E(T)) was lower when the field was applied longitudinally (E(T,L)) rather than transversally (E(T,T)) to the cell major axis. Rheobase was greater for transversal stimulation, but chronaxie and estimated membrane polarization were similar for both directions. The calculated maximal variation in membrane potential at the threshold (DeltaV(T) approximately 15 mV) was insensitive to field direction. As DeltaV(T) values were similar, we assumed that the E(T,T)/E(T,L) ratio might be described solely as the ratio of the major and minor cell semi-axes. Accordingly, the ratio thus estimated was comparable to that determined experimentally. Stimulus waveform significantly affected both E(T) and DeltaV(T), which were greater for monophasic versus biphasic stimuli. Direction and waveform effects were independent. We conclude that (a) direction affects E(T) by its influence on the ability of a given field intensity to cause threshold membrane polarization and (b) threshold-lowering effects of longitudinal stimulation and biphasic waveforms apparently depend on different mechanisms, are additive and thus may be combined to decrease the energy requirement for myocardial stimulation.