Noise-Free Visualization of Microscopic Calcium Signaling by Pixel-Wise Fitting

Abstract

Rationale: Our insights into physiological and pathophysiological cardiac excitation-contraction coupling has greatly benefited from significant advancement in optical technologies such as high-speed confocal microscopy. This has pushed pixel dwell times into the time domain of nanoseconds, resulting in low signal-to-noise ratios, which have limited data analysis and interpretation.Objective: Line scan imaging has been and still is dominant in high speed confocal recording. It allows analysis only of a small fraction of a cell's cross section (1.5%), but the appreciation of spatiotemporal fine details of excitation-contraction coupling is instrumental for the further understanding of pathological mechanisms. We aim to provide a novel analysis tool to extract otherwise hidden fine details in cardiac excitation-contraction coupling from high-speed 2-dimensional confocal image series. Methods and Results:We demonstrate that high-speed 2-dimensional confocal data (150 frames/s) can be analyzed quantitatively by a pixel-wise fitting approach, using a mathematical formalism to phenomenologically describe local calcium transients. Such an approach produces virtually noise-free fluorescence data originating from minute volumes (0.025 femtoliter) and allows extraction of detailed and most importantly quantitative and mechanistically novel information on microscopic calcium signaling and excitation-contraction coupling in a robust manner. Conclusions:Pixel-wise fitting provides novel insights into cardiac excitation-contraction coupling. Specifically, it revealed microscopic calcium alternans on the level of individual coupling sites. Microscopic calcium alternans is an early precursor of cellular alternans and as such will shed more light onto this mechanism leading to cardiac arrhythmia. (Circ Res. 2012;111:17-27.) Key Words: excitation-contraction coupling Ⅲ calcium-induced calcium release Ⅲ noise-free image sequences Ⅲ alternans Ⅲ arrhythmogenic precursor Ⅲ Ca 2ϩ transients H igh-resolution live-cell imaging of subcellular signaling events has greatly fostered our understanding of cardiac physiology and pathophysiology. 1 In this, a major driving force was the advancement in laser-scanning technology that enabled ultrafast confocal imaging (Ͼ100 frames/s) without compromising the optical resolution. 2 Although researchers are technically able to follow fast subcellular signaling, such as excitation-contraction coupling (ECC) in cardiac myocytes, 3 decreasing the single pixel dwell times and concomitant decreases in the signal-to-noise ratio (SNR) have limited the progress. However, scientific interest is not limited to the occurrence of Ca 2ϩ induced Ca 2ϩ release (CICR) per se but also focuses on where, how much, and how fast the Ca 2ϩ increases occur because the signaling information is encoded in all of these properties. 4 Although cardiac Ca 2ϩ transients appear homogeneous under physiological conditions, they arise from the coordinated activity of many microscopic ECC units, 3 comprising voltage operated Ca 2ϩ ch...

Nonlinear and stochastic dynamics in the heart

“…In our spatially-extended Ca 2+ cycling models [283, 291], we observed the same dynamical clustering phenomenon which results in patches of regions alternating out-of-phase. A similar process was seen in the study by Tian et al [271] in which multiple out-ofphase alternating regions exist. This might also be responsible for the discordant Ca 2+ alternans seen by Diaz et al [265].…”

“…That is, at one end, the amplitude of the Ca 2+ signal exhibit a pattern of large, small, large , …, while the other end exhibits a pattern of small, large, small , …. Similar patterns have been observed in ventricular mycoytes by other researchers [265, 268, 269, 271]. Different mechanisms have been proposed to explain the sub-cellular spatiotemporal pattern formation, which are summarized below.…”

“…Indeed, Ca 2+ alternans can be observed experimentally when APD is fixed (i.e., under the condition of voltage or action potential clamp) [264–266], demonstrating that instabilities do occur in the Ca 2+ sub-system. Moreover, spatially discordant Ca 2+ alternans inside a myocyte has also been observed in many experimental studies [265, 267–271]. Dynamical mechanisms of Ca 2+ alternans and spatially discordant Ca 2+ alternans have been developed, and some of them are supported by experimental evidence.…”

“…In an experiment carried out by Tian et al [271], a transition from microscopic Ca 2+ alternans to macroscopic Ca 2+ alternans was observed (Fig.27). Specifically, at a slower pacing rate, alternans was observed at individual measurement sites while the whole-cell Ca 2+ is regular from beat to beat.…”

Genetically Encoded Ca 2+ Indicators in Cardiac Myocytes

“…The latter requirement seems to be essential to enable Ca 2+ upstroke-based analysis methods of fast confocal recordings, as recently described. 128 Furthermore, biocompatibility must be improved, particularly by reducing cross talk with endogenous signaling pathways. There is also potential for broad expansion of the spectral range of GECI.…”