Rationale Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) are increasingly being used for modeling heart disease and are under development for regeneration of the injured heart. However, incomplete structural and functional maturation of hiPSC-CM including lack of t-tubules, immature excitation-contraction (EC) coupling, and inefficient Ca-induced Ca release (CICR) remain major limitations. Objective Thyroid and glucocorticoid hormones are critical for heart maturation. We hypothesized that their addition to standard protocols would promote t-tubule development and mature EC coupling of hiPSC-CM when cultured on extracellular matrix with physiological stiffness (Matrigel mattress). Methods and Results HiPSC-CM were generated using a standard chemical differentiation method supplemented with triiodo-L-thyronine (T3) and/or dexamethasone (Dex) during days 16–30 followed by single-cell culture for 5 days on Matrigel mattress. HiPSC-CM treated with T3+Dex, but not with either T3 or Dex alone, developed an extensive t-tubule network. Notably, Matrigel mattress was necessary for t-tubule formation. Compared to adult human ventricular CM, t-tubules in T3+Dex-treated hiPSC-CM were less organized and had more longitudinal elements. Confocal line scans demonstrated spatially and temporally uniform Ca release that is characteristic of EC coupling in the heart ventricle. T3+Dex enhanced elementary Ca release measured by Ca sparks as well as promoted ryanodine receptor (RyR2) structural organization. Simultaneous measurements of L-type Ca current and intracellular Ca release confirmed enhanced functional coupling between L-type Ca channels and RyR2 in T3+Dex cells. Conclusions Our results suggest a permissive role of combined thyroid and glucocorticoid hormones during the cardiac differentiation process which, when coupled with further maturation on Matrigel mattress, is sufficient for t-tubule development, enhanced CICR, and more ventricular-like EC coupling. This new hormone maturation method could advance the utility of hiPSC-CM for disease modeling and cell-based therapy.

To investigate the mechanism of regulation of sarco-endoplasmic reticulum Ca 2؉ -ATPase (SERCA) by phospholamban (PLB), we expressed Cerulean-SERCA and yellow fluorescent protein (YFP)-PLB in adult rabbit ventricular myocytes using adenovirus vectors. SERCA and PLB were localized in the sarcoplasmic reticulum and were mobile over multiple sarcomeres on a timescale of tens of seconds. We also observed robust fluorescence resonance energy transfer (FRET) from Cerulean-SERCA to YFP-PLB. is a P-type ion pump that maintains the Ca 2ϩ gradient across the endoplasmic reticulum. In cardiac muscle cells, calcium (Ca 2ϩ ) sequestration by SERCA is critical for muscle relaxation during the cardiac cycle, and disordered Ca 2ϩ handling is associated with cardiac dysfunction (1-3). SERCA activity is regulated by phospholamban (PLB), a helical transmembrane peptide that reduces the apparent affinity of the pump for Ca 2ϩ . Inhibition of SERCA by PLB is partially relieved through phosphorylation of PLB by protein kinase A and Ca 2ϩ /calmodulindependent protein kinase (4), which alters the structure of the PLB-SERCA regulatory complex (5) and increases PLB oligomerization into non-inhibitory pentamers (5, 6). It is widely recognized that PLB inhibition of SERCA is also relieved by elevated Ca 2ϩ , but the mechanism of this functional effect is unclear. One possibility is that PLB binds selectively to the Ca 2ϩ -free "E2" conformation of SERCA and cannot bind to the Ca 2ϩ -bound "E1" conformation (Fig. 1A, Dissociation Model). This model is supported by the observation that elevated Ca 2ϩ abolishes chemical cross-linking of the PLB transmembrane domain to reactive residues on SERCA (7-11) and reduces coimmunoprecipitation (12). Cross-linking was also prevented by the SERCA inhibitor thapsigargin (Tg) (7, 9 -11). Another recent study showed that oligomerization of a PLB-SERCA fusion construct was increased by micromolar Ca 2ϩ (13), consistent with the idea that Ca 2ϩ causes displacement of PLB from the inhibitory cleft, permitting PLB self-association into pentamers. Overall, these data suggest that only certain conformational substates of SERCA interact with PLB. The dissociation model predicts that PLB unbinds from SERCA during the period of systole (cardiac contraction) when Ca 2ϩ is high, and the regulatory complex reforms during diastole (cardiac relaxation) when the cytoplasmic Ca 2ϩ concentration is low. An alternative theory was generated from in vitro measurements of fluorescence resonance energy transfer (FRET) between SERCA and PLB. Mueller et al. (14) showed that FRET was decreased, but not abolished, by Ca 2ϩ . This result suggested that relief of inhibition was accomplished by a conformational change of the regulatory complex, rather than unbinding of PLB from the pump. This study estimated a dissociation constant significantly lower than the expected in vivo concentrations of PLB and SERCA. Li et al. (15) also provided evidence from fluorescence spectroscopy that suggests that the binding of PLB and Ca 2ϩ is not...

The sarco(endo)plasmic reticulum calcium ATPase (SERCA) undergoes conformational changes while transporting calcium, but the details of the domain motions are still unclear. The objective of the present study was to measure distances between the cytoplasmic domains of SERCA2a in order to reveal the magnitude and direction of conformational changes. Using fluorescence microscopy of live cells, we measured intramolecular fluorescence resonance energy transfer (FRET) from a donor fluorescent protein fused to the SERCA N-terminus to an acceptor fluorescent protein fused to either the N-, P-, or transmembrane domain. The “2-color” SERCA constructs were catalytically active as indicated by ATPase activity in vitro and Ca uptake in live cells. All constructs exhibited dynamic FRET changes in response to the pump ligands calcium and thapsigargin (Tg). These FRET changes were quantified as an index of SERCA conformational changes. Intramolecular FRET decreased with Tg for the two N-domain fusion sites (at residue 509 or 576), while the P- (residue 661) and TM-domain (C-terminus) fusions showed increased FRET with Tg. The magnitude of the Tg-dependent conformational change was not decreased by coexpression of phospholamban (PLB), nor did PLB slow the kinetics of Tg binding. FRET in ionophore-permeabilized cells was lower in EGTA than in saturating calcium for all constructs, indicating a decrease in domain separation distance with the structural transition from E2 (Ca-free) to E1 (Ca-bound). The data suggest closure of the cytoplasmic headpiece with Ca-binding. The present results provide insight into the structural dynamics of the Ca-ATPase. In addition, the 2-color SERCA constructs developed for this study may be useful for evaluating candidate small molecule regulators of Ca uptake activity.

The sarcoendoplasmic reticulum calcium ATPase (SERCA) plays a key role in cardiac calcium handling and is considered a high-value target for the treatment of heart failure. SERCA undergoes conformational changes as it harnesses the chemical energy of ATP for active transport. X-ray crystallography has provided insight into SERCA structural substates, but it is not known how well these static snapshots describe in vivo conformational dynamics. The goals of this work were to quantify the direction and magnitude of SERCA motions as the pump performs work in live cardiac myocytes, and to identify structural determinants of SERCA regulation by phospholamban. We measured intramolecular fluorescence resonance energy transfer (FRET) between fluorescent proteins fused to SERCA cytoplasmic domains. We detected four discrete structural substates for SERCA expressed in cardiac muscle cells. The relative populations of these discrete states oscillated with electrical pacing. Low FRET states were most populated in low Ca (diastole), and were indicative of an open, disordered structure for SERCA in the E2 (Ca-free) enzymatic substate. High FRET states increased with Ca (systole), suggesting rigidly closed conformations for the E1 (Ca-bound) enzymatic substates. Notably, a special compact E1 state was observed after treatment with β-adrenergic agonist or with coexpression of phosphomimetic mutants of phospholamban. The data suggest that SERCA calcium binding induces the pump to undergo a transition from an open, dynamic conformation to a closed, ordered structure. Phosphorylated phospholamban stabilizes a unique conformation of SERCA that is characterized by a compact architecture.

Rationale: The class Ic antiarrhythmic drug flecainide prevents ventricular tachyarrhythmia in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease caused by hyperactive RyR2 (cardiac ryanodine receptor) mediated calcium (Ca) release. Although flecainide inhibits single RyR2 channels in vitro, reports have claimed that RyR2 inhibition by flecainide is not relevant for its mechanism of antiarrhythmic action and concluded that sodium channel block alone is responsible for flecainide’s efficacy in CPVT. Objective: To determine whether RyR2 block independently contributes to flecainide’s efficacy for suppressing spontaneous sarcoplasmic reticulum Ca release and for preventing ventricular tachycardia in vivo. Methods and Results: We synthesized N-methylated flecainide analogues (QX-flecainide and N -methyl flecainide) and showed that N -methylation reduces flecainide’s inhibitory potency on RyR2 channels incorporated into artificial lipid bilayers. N -methylation did not alter flecainide’s inhibitory activity on human cardiac sodium channels expressed in HEK293T cells. Antiarrhythmic efficacy was tested utilizing a Casq2 (cardiac calsequestrin) knockout (Casq2−/−) CPVT mouse model. In membrane-permeabilized Casq2−/− cardiomyocytes—lacking intact sarcolemma and devoid of sodium channel contribution—flecainide, but not its analogues, suppressed RyR2-mediated Ca release at clinically relevant concentrations. In voltage-clamped, intact Casq2−/− cardiomyocytes pretreated with tetrodotoxin to inhibit sodium channels and isolate the effect of flecainide on RyR2, flecainide significantly reduced the frequency of spontaneous sarcoplasmic reticulum Ca release, while QX-flecainide and N -methyl flecainide did not. In vivo, flecainide effectively suppressed catecholamine-induced ventricular tachyarrhythmias in Casq2−/− mice, whereas N -methyl flecainide had no significant effect on arrhythmia burden, despite comparable sodium channel block. Conclusions: Flecainide remains an effective inhibitor of RyR2-mediated arrhythmogenic Ca release even when cardiac sodium channels are blocked. In mice with CPVT, sodium channel block alone did not prevent ventricular tachycardia. Hence, RyR2 channel inhibition likely constitutes the principal mechanism of antiarrhythmic action of flecainide in CPVT.