Leighton T. Izu

Calcium Handling in Human Embryonic Stem Cell-Derived Cardiomyocytes

The objective of the current study was to characterize calcium handling in developing human embryonic stem cell‐derived cardiomyocytes (hESC‐CMs). To this end, real‐time polymerase chain reaction (PCR), immunocytochemistry, whole‐cell voltage‐clamp, and simultaneous patch‐clamp/laser scanning confocal calcium imaging and surface membrane labeling with di‐8‐aminonaphthylethenylpridinium were used. Immunostaining studies in the hESC‐CMs demonstrated the presence of the sarcoplasmic reticulum (SR) calcium release channels, ryanodine receptor‐2, and inositol‐1,4,5‐trisphosphate (IP3) receptors. Store calcium function was manifested as action‐potential‐induced calcium transients. Time‐to‐target plots showed that these action‐potential‐initiated calcium transients traverse the width of the cell via a propagated wave of intracellular store calcium release. The hESC‐CMs also exhibited local calcium events (“sparks”) that were localized to the surface membrane. The presence of caffeine‐sensitive intracellular calcium stores was manifested following application of focal, temporally limited puffs of caffeine in three different age groups: early‐stage (with the initiation of beating), intermediate‐stage (10 days post‐beating [dpb]), and late‐stage (30–40 dpb) hESC‐CMs. Calcium store load gradually increased during in vitro maturation. Similarly, ryanodine application decreased the amplitude of the spontaneous calcium transients. Interestingly, the expression and function of an IP3‐releasable calcium pool was also demonstrated in the hESC‐CMs in experiments using caged‐IP3 photolysis and antagonist application (2 μM 2‐Aminoethoxydiphenyl borate). In summary, our study establishes the presence of a functional SR calcium store in early‐stage hESC‐CMs and shows a unique pattern of calcium handling in these cells. This study also stresses the importance of the functional characterization of hESC‐CMs both for developmental studies and for the development of future myocardial cell replacement strategies.

Gi-Dependent Localization of β2-Adrenergic Receptor Signaling to L-Type Ca2+ Channels

A plausible determinant of the specificity of receptor signaling is the cellular compartment over which the signal is broadcast. In rat heart, stimulation of beta(1)-adrenergic receptor (beta(1)-AR), coupled to G(s)-protein, or beta(2)-AR, coupled to G(s)- and G(i)-proteins, both increase L-type Ca(2+) current, causing enhanced contractile strength. But only beta(1)-AR stimulation increases the phosphorylation of phospholamban, troponin-I, and C-protein, causing accelerated muscle relaxation and reduced myofilament sensitivity to Ca(2+). beta(2)-AR stimulation does not affect any of these intracellular proteins. We hypothesized that beta(2)-AR signaling might be localized to the cell membrane. Thus we examined the spatial range and characteristics of beta(1)-AR and beta(2)-AR signaling on their common effector, L-type Ca(2+) channels. Using the cell-attached patch-clamp technique, we show that stimulation of beta(1)-AR or beta(2)-AR in the patch membrane, by adding agonist into patch pipette, both activated the channels in the patch. But when the agonist was applied to the membrane outside the patch pipette, only beta(1)-AR stimulation activated the channels. Thus, beta(1)-AR signaling to the channels is diffusive through cytosol, whereas beta(2)-AR signaling is localized to the cell membrane. Furthermore, activation of G(i) is essential to the localization of beta(2)-AR signaling because in pertussis toxin-treated cells, beta(2)-AR signaling becomes diffusive. Our results suggest that the dual coupling of beta(2)-AR to both G(s)- and G(i)-proteins leads to a highly localized beta(2)-AR signaling pathway to modulate sarcolemmal L-type Ca(2+) channels in rat ventricular myocytes.

Evolution of Cardiac Calcium Waves from Stochastic Calcium Sparks

We present a model that provides a unified framework for studying Ca2+ sparks and Ca2+ waves in cardiac cells. The model is novel in combining 1) use of large currents (approximately 20 pA) through the Ca2+ release units (CRUs) of the sarcoplasmic reticulum (SR); 2) stochastic Ca2+ release (or firing) of CRUs; 3) discrete, asymmetric distribution of CRUs along the longitudinal (separation distance of 2 microm) and transverse (separated by 0.4-0.8 microm) directions of the cell; and 4) anisotropic diffusion of Ca2+ and fluorescent indicator to study the evolution of Ca2+ waves from Ca2+ sparks. The model mimics the important features of Ca2+ sparks and Ca2+ waves in terms of the spontaneous spark rate, the Ca2+ wave velocity, and the pattern of wave propagation. Importantly, these features are reproduced when using experimentally measured values for the CRU Ca2+ sensitivity (approximately 15 microM). Stochastic control of CRU firing is important because it imposes constraints on the Ca2+ sensitivity of the CRU. Even with moderate (approximately 5 microM) Ca2+ sensitivity the very high spontaneous spark rate triggers numerous Ca2+ waves. In contrast, a single Ca2+ wave with arbitrarily large velocity can exist in a deterministic model when the CRU Ca2+ sensitivity is sufficiently high. The combination of low CRU Ca2+ sensitivity (approximately 15 microM), high cytosolic Ca2+ buffering capacity, and the spatial separation of CRUs help control the inherent instability of SR Ca2+ release. This allows Ca2+ waves to form and propagate given a sufficiently large initiation region, but prevents a single spark or a small group of sparks from triggering a wave.

Role of the transverse-axial tubule system in generating calcium sparks and calcium transients in rat atrial myocytes.

Cardiac atrial cells lack a regular system of transverse tubules like that in cardiac ventricular cells. Nevertheless, many atrial cells do possess an irregular internal transverse‐axial tubular system (TATS). To investigate the possible role of the TATS in excitation‐contraction coupling in atrial myocytes, we visualized the TATS (labelled with the fluorescent indicator, Di‐8‐ANEPPS) simultaneously with Ca2+ transients and/or Ca2+ sparks (fluo‐4). In confocal transverse linescan images of field‐stimulated cells, whole‐cell Ca2+ transients had two morphologies: ‘U‐shaped’ transients and irregular or ‘W‐shaped’ transients with a varying number of points of origin of the Ca2+ transient. About half (54 %, n=289 cells, 13 animals) of the cells had a TATS. Cells with TATS had a larger mean diameter (13.2 ± 2.8 μm) than cells without TATS (11.7 ± 2.0 μm) and were more common in the left atrium (n= 206 cells; left atrium: 76 with TATS, 30 without TATS; right atrium: 42 with TATS, 58 without TATS). Simultaneous measurement of Ca2+ sparks and sarcolemmal structures showed that cells without TATS had U‐shaped transients that started at the cell periphery, and cells with TATS had W‐shaped transients that began simultaneously at the cell periphery and the TATS. Most (82 out of 102 from 31 cells) ‘spontaneous’ (non‐depolarized) Ca2+ sparks occurred within 1 μm of a sarcolemmal structure (cell periphery or TATS), and 33 % occurred within 1 pixel (0.125 μm). We conclude that the presence of a sarcolemmal membrane either at the cell periphery or in the TATS in close apposition to the sarcoplasmic reticulum is required for the initiation of an evoked Ca2+ transient and for spontaneous Ca2+ sparks.

Theoretical analysis of the Ca2+ spark amplitude distribution

A difficulty of using confocal microscopy to study Ca2+ sparks is the uncertainty of the linescan position with respect to the source of Ca2+ release. Random placement of the linescan is expected to result in a broad distribution of measured Ca2+ spark amplitudes (a) even if all Ca2+ sparks were generated identically. Thus variations in Ca2+ spark amplitude due to positional differences between confocal linescans and Ca2+ release site are intertwined with variations due to intrinsic differences in Ca2+ release properties. To separate these two sources of variations on the Ca2+ spark amplitude, we determined the effect changes of channel current or channel open time--collectively called the source strength, alpha--had on the measured Ca2+ spark amplitude histogram, N(a). This was done by 1) simulating Ca2+ release, Ca2+ and fluo-3 diffusion, and Ca2+ binding reactions; 2) simulation of image formation of the Ca2+ spark by a confocal microscope; and 3) using a novel automatic Ca2+ spark detector. From these results we derived an integral equation relating the probability density function of source strengths, f alpha (alpha), to N(a), which takes into account random positional variations between the source and linescan. In the special, but important, case that the spatial distribution of Ca(2+)-bound fluo-3 is Gaussian, we show the following: 1) variations of Ca2+ spark amplitude due to positional or intrinsic differences can be separated, and 2) f alpha (alpha) can, in principle, be calculated from the Ca2+ spark amplitude histogram since N(a) is the sum of shifted hyperbolas, where the magnitudes of the shifts and weights depend on f alpha (alpha). In particular, if all Ca2+ sparks were generated identically, then the plot of 1/N(a) against a will be a straight line. Multiple populations of channels carrying distinct currents are revealed by discontinuities in the 1/N(a) plot. 3) Although the inverse relationship between Ca2+ spark amplitude and decay time might be used to distinguish Ca2+ sparks from different channel populations, noise can render the measured decay times meaningless for small amplitude Ca2+ sparks.

Transformation of adult rat cardiac myocytes in primary culture

We characterized the morphological, electrical and mechanical alterations of cardiomyocytes in long‐term cell culture. Morphometric parameters, sarcomere length, T‐tubule density, cell capacitance, L‐type calcium current (ICa,L), inward rectifier potassium current (IK1), cytosolic calcium transients, action potential and contractile parameters of adult rat ventricular myocytes were determined on each day of 5 days in culture. We also analysed the health of the myocytes using an apoptotic/necrotic viability assay. The data show that myocytes undergo profound morphological and functional changes during culture. We observed a progressive reduction in the cell area (from 2502 ± 70 μm2 on day 0 to 1432 ± 50 μm2 on day 5), T‐tubule density, systolic shortening (from 0.11 ± 0.02 to 0.05 ± 0.01 μm) and amplitude of calcium transients (from 1.54 ± 0.19 to 0.67 ± 0.19) over 5 days of culture. The negative force–frequency relationship, characteristic of rat myocardium, was maintained during the first 2 days but diminished thereafter. Cell capacitance (from 156 ± 8 to 105 ± 11 pF) and membrane currents were also reduced (ICa,L, from 3.98 ± 0.39 to 2.12 ± 0.37 pA pF; and IK1, from 34.34p ± 2.31 to 18.00 ± 5.97 pA pF−1). We observed progressive depolarization of the resting membrane potential during culture (from 77.3 ± 2.5 to 34.2 ± 5.9 mV) and, consequently, action potential morphology was profoundly altered as well. The results of the viability assays indicate that these alterations could not be attributed to either apoptosis or necrosis but are rather an adaptation to the culture conditions over time.

Ca2+ Sparks Triggered by Patch Depolarization in Rat Heart Cells

The goal of this study was to examine the relationship between Ca2+ entry through L-type Ca2+ channels and local [Ca2+]i transients (Ca2+ sparks) in single rat cardiac ventricular cells. L-type Ca2+ channels were activated by depolarization of cell-attached membrane patches, and [Ca2+]i was measured simultaneously as fluo 3 fluorescence using laser scanning confocal microscopy. Patch depolarization with Ca2+ as the charge carrier (10 or 110 mmol.L(-1)) significantly increased the probability of the occurrence of Ca2+ sparks (Ca2+ spark rate) only in the volume of cytoplasm located immediately beneath the membrane patch (basal Ca2+ spark rate, 119 Ca2+ sparks.cell(-1).s(-1); patch depolarization Ca2+ spark rate, 610 Ca2+ sparks.cell(-1).s(-1); P<.005). With Ba2+ in the pipette solution (10 mmol.L(-1)), patch depolarization was not associated with an increased Ca2+ spark rate at the position of the pipette or at any other sites distant from the pipette. Therefore, Ca2+ entry and not voltage per se was a necessary event for the occurrence of Ca2+ sparks. Under identical experimental conditions, patch depolarization experiments opened single L-type Ca2+ channels with a single-channel conductance of 19 pS with Ba2+ as the charge carrier. Although single-channel openings could not be resolved when Ca2+ was the charge carrier, ensemble averages yielded an inward current of up to 0.75 pA. The results suggest that voltage-activated Ca2+ entry through one or a small number of L type Ca2+ channels triggers the release of Ca2+ only from the sarcoplasmic reticulum in direct proximity to those L-type Ca2+ channels. The relatively low probability of triggering Ca2+ sparks may have resulted from some alteration of excitation-contraction coupling associated with the technique of the cell-attached patch clamp.

The Ca2+ Synapse Redo A Matter of Location, Location, Location

The past decade has witnessed the evolution of a new paradigm in thinking about the intimate interrelationship between cellular structure and physiological function in biological processes. It is not surprising that this evolution has, perhaps, its clearest history in the field of excitation-contraction (E-C) coupling, which has had to wrestle with the long-standing paradox of how the release of Ca2+ from the sarcoplasmic reticulum (SR) can be graded by membrane potential1 in the presence of regenerative Ca2+ release (Ca2+-induced Ca2+ release2–4⇓⇓). In retrospect, it is clear that the resolution of this paradox had to await both methodological and theoretical breakthroughs that allowed researchers to change the scale of our measurements and thinking. Until the early 1990s, measurements of intracellular Ca2+ were limited to spatially averaged whole-cell Ca2+ transients or Ca2+ waves. The corresponding theoretical constructs assumed a spatially continuous distribution of SR Ca2+ release, which successfully predicted the properties of Ca2+ waves but precluded graded Ca2+ transients—hence the paradox. In 1992, Stern5 made a key advance toward the resolution of this paradox by recognizing that all-or-nothing SR Ca2+ release could be avoided by having discrete Ca2+ release sites that were spatially segregated. This model was unique in that it included the …