Significance: Engineered cardiac muscle can be used to promote the structural and functional maturation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). However, previous studies have not yet produced cardiac tissues with metabolic and proliferative maturation. Here, we develop a 96-well screening platform and screen for cardiac maturation conditions in engineered cardiac muscle. We found that simulating the postnatal switch in metabolic substrates from carbohydrates to fatty acids promoted a switch in metabolism, DNA damage response, and cell cycle arrest in hPSC-CM. Our study shows that this mechanism can be harnessed to enhance the maturation of human hPSC-CM and cardiac tissues, which has major implications for stem cell sciences, drug discovery, and regenerative medicine.

Store‐operated Ca²⁺ entry (SOCE) is activated following the depletion of internal Ca²⁺ 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 Ca²⁺ release. Cytosolic Ca²⁺ transients were simultaneously imaged using the fluorescence of rhod‐2. Spatially and temporally resolved images of t system [Ca²⁺] ([Ca²⁺]_t‐sys) allowed estimation of Ca²⁺ entry flux from the rate of decay of [Ca²⁺]_t‐sys. Ca²⁺ release was induced pharmacologically to activate SOCE without voltage‐dependent contributions to Ca²⁺ flux. Inward Ca²⁺ flux was monotonically dependent on the [Ca²⁺] gradient, and strongly dependent on the transmembrane potential. The activation of SOCE was controlled locally. It could occur without full Ca²⁺ store depletion and in less than a second after initiation of store depletion. These results indicate that the molecular agonists of SOCE must be evenly distributed throughout the junctional membranes and can activate rapidly. Termination of SOCE required a net increase in [Ca²⁺]_SR. Activation and termination of SOCE are also demonstrated, for the first time, during a single event of Ca²⁺ release. At the physiological [Ca²⁺]_t‐sys, near 2 mm (relative to t system volume), SOCE flux relative to accessible cytoplasmic volume was at least 18.6 μm s⁻¹, consistent with times of SR refilling of 1–2 min measured in intact muscle fibres.

Store-operated Ca²⁺ entry (SOCE) is an important mechanism in virtually all cells. In adult skeletal muscle, this mechanism is highly specialized for the rapid delivery of Ca²⁺ from the transverse tubule into the junctional cleft during periods of depleting Ca²⁺ release. In dystrophic muscle fibers, SOCE may be a source of Ca²⁺ overload, leading to cell necrosis. However, this possibility is yet to be examined in an adult fiber during Ca²⁺ release. To examine this, Ca²⁺ in the tubular system and cytoplasm were simultaneously imaged during direct release of Ca²⁺ from sarcoplasmic reticulum (SR) in skeletal muscle fibers from healthy (wild-type, WT) and dystrophic mdx mouse. The mdx fibers were found to have normal activation and deactivation properties of SOCE. However, a depression of the cytoplasmic Ca²⁺ transient in mdx compared with WT fibers was observed, as was a shift in the SOCE activation and deactivation thresholds to higher SR Ca²⁺ concentrations ([Ca²⁺]_SR). The shift in SOCE activation and deactivation thresholds was accompanied by an approximately threefold increase in STIM1 and Orai1 proteins in dystrophic muscle. While the mdx fibers can introduce more Ca²⁺ into the fiber for an equivalent depletion of [Ca²⁺]_SR via SOCE, it remains unclear whether this is deleterious.

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.

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.