The mammalian heart undergoes maturation during postnatal life to meet the increased functional requirements of an adult. However, the key drivers of this process remain poorly defined. We are currently unable to recapitulate postnatal maturation in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), limiting their potential as a model system to discover regenerative therapeutics. Here, we provide a summary of our studies, where we developed a 96-well device for functional screening in human pluripotent stem cell-derived cardiac organoids (hCOs). Through interrogation of >10,000 organoids, we systematically optimize parameters, including extracellular matrix (ECM), metabolic substrate, and growth factor conditions, that enhance cardiac tissue viability, function, and maturation. Under optimized maturation conditions, functional and molecular characterization revealed that a switch to fatty acid metabolism was a central driver of cardiac maturation. Under these conditions, hPSC-CMs were refractory to mitogenic stimuli, and we found that key proliferation pathways including β-catenin and Yes-associated protein 1 (YAP1) were repressed. This proliferative barrier imposed by fatty acid metabolism in hCOs could be rescued by simultaneous activation of both β-catenin and YAP1 using genetic approaches or a small molecule activating both pathways. These studies highlight that human organoids coupled with higher-throughput screening platforms have the potential to rapidly expand our knowledge of human biology and potentially unlock therapeutic strategies.

Store-operated Ca2+ entry (SOCE) is activated following the depletion of internal Ca 2+ stores in virtually all eukaryotic cells. Shifted excitation and emission ratioing of fluorescence (SEER) was used to image mag-indo-1 trapped in the tubular (t) system of mechanically skinned rat skeletal muscle fibres to measure SOCE during intracellular Ca 2+ release. Cytosolic Ca 2+ transients were simultaneously imaged using the fluorescence of rhod-2.

Ca 2؉ signals, produced by Ca 2؉ release from cellular stores, switch metabolic responses inside cells. In muscle, Ca 2؉ sparks locally exhibit the rapid start and termination of the cell-wide signal. By imaging Ca 2؉ inside the store using shifted excitation and emission ratioing of fluorescence, a surprising observation was made: Depletion during sparks or voltage-induced cell-wide release occurs too late, continuing to progress even after the Ca 2؉ release channels have closed. This finding indicates that Ca 2؉ is released from a ''proximate'' compartment functionally in between store lumen and cytosol. The presence of a proximate compartment also explains a paradoxical surge in intrastore Ca 2؉ , which was recorded upon stimulation of prolonged, cell-wide Ca 2؉ release. An intrastore surge upon induction of Ca 2؉ release was first reported in subcellular store fractions, where its source was traced to the store buffer, calsequestrin. The present results update the evolving concept, largely due to N. Ikemoto and C. Kang, of calsequestrin as a dynamic store. Given the strategic location and reduction of dimensionality of Ca 2؉ -adsorbing linear polymers of calsequestrin, they could deliver Ca 2؉ to the open release channels more efficiently than the luminal store solution, thus constituting the proximate compartment. When store depletion becomes widespread, the polymers would collapse to increase store [Ca 2؉ ] and sustain the concentration gradient that drives release flux.calcium signaling ͉ calcium sparks ͉ excitation-contraction coupling ͉ sarcoplasmic reticulum ͉ skeletal muscle R apid changes in intracellular cytosolic [Ca 2ϩ ] are required for signaling functions in many cell types (1). These changes are achieved via Ca 2ϩ release through channels, ryanodine receptors (RyRs), which must open and close quickly. To increase its speed, gating of RyRs relies on effects of the permeant ion, including channel opening by elevated cytosolic [Ca 2ϩ ] (2). In muscle, the desirable fast kinetic features are already present in its elementary signaling events, Ca 2ϩ sparks (3), which involve the nearly simultaneous opening (4) of a number of channels (5), followed by their synchronized closing (4). Thus, this gating does not follow the usual Markovian rules for channels that evolve independently but requires timekeeping and synchronization (6). In cardiac muscle, depletion of sarcoplasmic reticulum (SR) Ca 2ϩ is the likely timer of channel closing, and the substantial depletion that follows the cardiac beat (7) was imaged as ''blinks'' associated with Ca 2ϩ sparks (8). The sensor that translates depletion into channel closing appears to be the main intra-SR buffer, calsequestrin (CSQ) (9).By contrast, in skeletal muscle, depletion associated with a twitch is only 8-15% (10). This low rate of depletion reflects a SR with larger terminal cisternae containing higher concentrations of a CSQ of greater binding capacity, thus constituting a much greater calcium reservoir. Despite the greater store, sparks of skeletal mus...

Examination of store-operated Ca 2؉ entry (SOC) in single, mechanically skinned skeletal muscle cells by confocal microscopy shows that the inositol 1,4,5-trisphosphate (IP 3) receptor acts as a sarcoplasmic reticulum [Ca 2؉ ] sensor and mediates SOC by physical coupling without playing a key role in Ca 2؉ release from internal stores, as is the case with various cell types in which SOC was investigated previously. The results have broad implications for understanding the mechanism of SOC that is essential for cell function in general and muscle function in particular. Moreover, the study ascribes an important role to the IP 3 receptors in skeletal muscle, the role of which with respect to Ca 2؉ homeostasis was ill defined until now. S tore-operated Ca 2ϩ entry (SOC) in response to depletion of internal Ca 2ϩ stores is important for maintaining normal cell Ca 2ϩ homeostasis, but the precise mechanism is not understood fully (1, 2). In nonexcitable cells, the inositol 1,4,5-trisphosphate (IP 3 ) receptor (IP 3 R) (3) appears to have a dual role: releasing Ca 2ϩ from internal stores and mediating SOC by physical coupling with the plasma membrane (4, 5). A SOC mechanism has been identified in adult skeletal cells (6), which contain ryanodine receptors (RyRs)͞Ca 2ϩ -release channels involved in excitation-contraction (E-C) coupling (7) and IP 3 Rs that may be involved in regulation of gene expression (8). However, it is not known whether the mechanism of SOC in skeletal muscle involves IP 3 Rs, the role of which in Ca 2ϩ homeostasis remains controversial (7, 9-11).Here we image extracellular Ca 2ϩ in the sealed tubular (t)-system of mechanically skinned fibers (12, 13) to examine SOC. The sarcoplasmic reticulum (SR) and t-system remain functionally coupled in this preparation while allowing full experimental access to the interior of the cell. We found a fully operational SOC mechanism in this preparation and probed its mechanism by direct and rapid manipulation of the cytoplasmic environment. Materials and MethodsThe use of animals in this study was approved by the Animal Ethics Committee at La Trobe University. Adult cane toads were killed by double pithing, and the iliofibularis muscles were removed rapidly and placed in a Petri dish under a layer of paraffin oil. Single intact iliofibularis fibers were isolated and loaded with a physiological solution containing 112 mM NaCl, 3.3 mM KCl, 2.5 mM CaCl 2 , 1 mM MgCl 2 , 1 mM Fluo-5N (impermeant form, Molecular Probes), and 20 mM Hepes, pH 7.4, with a microcap pipette (Drummond Scientific, Broomall, PA) and then skinned mechanically to trap the dye in the sealed t-system as described (12,13). Note that the presence of Fluo-5N in solution reduced the ionized [Ca 2ϩ ] to Ϸ1.5 mM and made it possible to measure [Ca 2ϩ ] changes in the sealed t-system in the micro-to millimolar range because of its relatively low sensitivity to [Ca 2ϩ ] (K D Ϸ 90 M, also verified under our conditions). Dye-loaded preparations were moved to a custom-built experimental well that used a ...

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Ca2+ sparks of membrane-permeabilized rat muscle cells were analyzed to derive properties of their sources. Most events identified in longitudinal confocal line scans looked like sparks, but 23% (1,000 out of 4,300) were followed by long-lasting embers. Some were preceded by embers, and 48 were “lone embers.” Average spatial width was ∼2 μm in the rat and 1.5 μm in frog events in analogous solutions. Amplitudes were 33% smaller and rise times 50% greater in the rat. Differences were highly significant. The greater spatial width was not a consequence of greater open time of the rat source, and was greatest at the shortest rise times, suggesting a wider Ca2+ source. In the rat, but not the frog, spark width was greater in scans transversal to the fiber axis. These features suggested that rat spark sources were elongated transversally. Ca2+ release was calculated in averages of sparks with long embers. Release current during the averaged ember started at 3 or 7 pA (depending on assumptions), whereas in lone embers it was 0.7 or 1.3 pA, which suggests that embers that trail sparks start with five open channels. Analysis of a spark with leading ember yielded a current ratio ranging from 37 to 160 in spark and ember, as if 37–160 channels opened in the spark. In simulations, 25–60 pA of Ca2+ current exiting a point source was required to reproduce frog sparks. 130 pA, exiting a cylindric source of 3 μm, qualitatively reproduced rat sparks. In conclusion, sparks of rat muscle require a greater current than frog sparks, exiting a source elongated transversally to the fiber axis, constituted by 35–260 channels. Not infrequently, a few of those remain open and produce the trailing ember.

Edwards JN, Friedrich O, Cully TR, von Wegner F, Murphy RM, Launikonis BS. Upregulation of store-operated Ca 2ϩ entry in dystrophic mdx mouse muscle.

Key pointsr Current methods do not allow a quantitative description of Ca 2+ movements across the tubular (t-) system membrane without isolating the membranes from their native skeletal muscle fibre.r Here we present a fluorescence-based method that allows determination of the t-system [Ca 2+ ] transients and derivation of t-system Ca 2+ fluxes in mechanically skinned skeletal muscle fibres. Differences in t-system Ca 2+ -handling properties between fast-and slow-twitch fibres from rat muscle are resolved for the first time using this new technique.r The method can be used to study Ca 2+ handling of the t-system and allows direct comparisons of t-system Ca 2+ transients and Ca 2+ fluxes between groups of fibres and fibres from different strains of animals. AbstractThe tubular (t-) system of skeletal muscle is an internalization of the plasma membrane that maintains a large Ca 2+ gradient and exchanges Ca 2+ between the extracellular and intracellular environments. Little is known of the Ca 2+ -handling properties of the t-system as the small Ca 2+ fluxes conducted are difficult to resolve with conventional methods. To advance knowledge in this area we calibrated t-system-trapped rhod-5N inside skinned fibres from rat and