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the activity and structural alignment of sarcomeres. Sarcomeres are composed of thick filaments and thin filaments. Thick filaments (myosin) interact with a binding site on actin which allow for cross bridge formation and subsequent sarcomere shortening. The thin filaments are composed of actin, tropomyosin, and the troponin complex. The troponin complex contains three subunits:
troponin-T, troponin-I, and troponin-C. Tropomyosin prevents activation of the myofilament by inhibiting the interaction between myosin and actin. The regulatory troponin complex is tethered to the tropomyosin via troponin-T. The elevation in cytosolic calcium upon CICR allows for binding of calcium to troponin-C, causing a conformational shift in the troponin-I subunit. This enables an interaction between the myosin head and actin allowing for force generation.
In summary, ECC is a fundamental property required for converting electrical stimulation into force generation. The positioning of the LTCC in the t-tubules to the RyR2 on the SR is critical for efficient CICR and thus demonstrates the necessity of this structural arrangement. Furthermore, defects in ECC result in poor contractility and arrhythmogenesis83.
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of the ECC system (LTCC and RyR2) as well as systems for cellular relaxation (NCX, SERCA2a, and PMCA). However, the presence of these alone have not been sufficient for replacing the human ventricular CM as evidenced by failures in disease modeling, arrhythmias after transplantation, and inaccurate drug response. Inefficient CICR, slow calcium handling and electrical immaturity of the hiPSC-CM are major limitations of hiPSC-CM that could be contributory.
CICR is the linking feature between electrophysiological stimulation and force generation.
The speed of calcium release during this process relies on efficient and rapid opening of intracellular calcium stores. Although hiPSC-CM demonstrate calcium transients, the rise and fall of calcium is considerably slower than that of the adult human-like CM82. As the rise time is linked to the rate of calcium influx via the LTCC and its ability to induce CICR, slow rise times can suggest 1. diminished LTCC function, 2. inefficient CICR, and/or 3. immature SR. HiPSC-CM demonstrate comparable expression of the LTCC to that of the adult human ventricular CM85 and patch clamp techniques show robust LTCC current, similar to that of the adult mouse CM82. In addition, pharmacological blockade of the LTCC using nifedifine and removal of extracellular calcium completely prevent SR calcium release suggesting a reliance of calcium from the LTCC86. Conversely, hiPSC-CM show diffuse RyR2 immunostaining with mostly puncate perinuclear localization87. In addition, expression of RyR2 and other key SR regulatory proteins calsequestrin (Casq2) and triadin (Trdn) are considerably lower in the hiPSC-CM compared to the adult human heart85, 87. Despite reduced expression of RyR2, caffeine produced calcium transients of similar amplitude to that of both rabbit and mouse adult CM demonstrating the presence of relatively developed SR82. Despite comparable LTCC currents and fairly developed SR, the kinetics of intracellular calcium rise remain considerably slower in hiPSC-CM at ~500 ms compared to less
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Table 1: Comparison of structural and functional properties between adult CM and hPSC-CM. Table provides evidence for lack of structural and functional maturation of hPSC-CM. Table adapted from 88.
26 than ~200 ms in rabbit adult CM82.
Using confocal microscopy, transverse line scan at the mid-plane of electrically paced CM is often used for assessing the spatial-temporal features of calcium releases. Using this method, the time course of calcium fluorescence across the width of the cell showed an asynchronous calcium release in hPSC-CM visualized as a U-shaped waveform89. Specifically, when comparing the calcium kinetics of the cell periphery to the cell center, hPSC-CM demonstrate a delayed rate of calcium release at the center versus periphery whereas adult mouse-CM show uniform rates of calcium release throughout the width of the cell89, 90. Consistent with this finding is the poor juxtaposition of LTCC and RyR2 in hPSC- CM85, 91. Efficient CICR is dependent on sufficient expression and organization of RyR2 (clustering and appropriate localization to the z-line) as well as juxtaposition of the LTCC to the RyR2, all of which are poorly developed in the hiPSC-CM.
Calcium removal was also found to be substantially slower in hiPSC-CM, estimated at rates half of rabbit adult CM82. Likewise, SERCA2a, the predominant cardiac isoform of SERCA, shows significantly lower expression in hiPSC-CM compared to the adult human heart85. The other dominant calcium extrusion pathway NCX85, shows similar expression to that of the adult human heart, however absolute values of calcium removal remain considerably less than the adult rabbit CM. Despite an overall reduction in the calcium extrusion rates, the relative contribution of SERCA and the NCX remain similar to that of the adult rabbit CM82.
Electrically, hiPSC-CM are considered immature as they demonstrate automaticity and adult ventricular CM do not. This has been attributed to the high expression of the pacemaker current, If,which is normally predominantly expressed in the cells of the cardiac
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conduction system, such as cells in the sinoatrial node. In addition, most studies show that hiPSC-CM have very low to absent expression of IK1, which is the potassium current that stabilizes the resting membrane potential (phase 4). HiPSC-CM also have minimal IKs, which when combined with the deficient IK1 leave only one major repolarization source, IKr. Lastly, hiPSC-CM AP exhibit slow upstroke velocity, which could be responsible for the slow conduction velocity in hiPSC-CM syncytia compared to the adult human ventricular myocardium88.
Inefficient CICR, slow calcium kinetics, and electrical immaturity are all properties which could be explained by the lack of adult-like myocyte structure in the hiPSC-CM.
Specifically, inadequate localization of RyR2 at the z-line and/or lack of t-tubules are major structural determinants of the functional deficiencies observed in the hiPSC-CM.