From monoclonal antibodies (mAbs) to Chimeric Antigen Receptor (CAR) T cells, immunotherapies have enhanced the efficacy of treatments against B cell malignancies. The same has not been true for Acute Myeloid Leukemia (AML). Hematologic toxicity has limited the potential of modern immunotherapies for AML at preclinical and clinical levels. Gemtuzumab Ozogamicin has demonstrated hematologic toxicity, but the challenge of preserving normal hematopoiesis has become more apparent with the development of increasingly potent immunotherapies. To date, no single surface molecule has been identified that is able to differentiate AML from Hematopoietic Stem and Progenitor Cells (HSPC). Attempts have been made to spare hematopoiesis by targeting molecules expressed only on later myeloid progenitors as well as AML or using toxins that selectively kill AML over HSPC. Other strategies include targeting aberrantly expressed lymphoid molecules or only targeting monocyte-associated proteins in AML with monocytic differentiation. Recently, some groups have accepted that stem cell transplantation is required to access potent AML immunotherapy and envision it as a rescue to avoid severe hematologic toxicity. Whether it will ever be possible to differentiate AML from HSPC using surface molecules is unclear. Unless true specific AML surface targets are discovered, stem cell transplantation could be required to harness the true potential of immunotherapy in AML. Until recently, therapeutic options for Acute Myeloid Leukemia (AML) had changed very little. In the last decade, there has been a substantial increase in knowledge of the molecular landscape of AML, which has led to several new targeted therapies . HoweverMore potent forms of immunotherapy, such as Chimeric Antigen Receptor (CAR) T cells, lead to target cell aplasia, which is tolerable in B cell malignancies ,5,6. IndMost surface molecules expressed on AML are also expressed on Hematopoietic Stem and Progenitor Cells (HSPC), and potent immunotherapies against traditional AML molecules (CD33 and CD123) have led to hematologic toxicity, both in preclinical models and in clinical trials ,9,10. AnWhile the majority of the emerging anti-AML immunotherapies seek to control disease without allo-HSCT by sparing HSPC , others Graft Versus Leukemia (GVL) in allo-HSCT provides evidence that immune mechanisms can reduce relapse risk in AML. The evidence of GVL was seen in preclinical models and confirmed clinically with the observations of reduced relapse rates in patients who develop GVHD, and an increased relapse rate in patients who receive T cell depleted grafts ,19. The Optimal disease control prior to allo-HSCT in AML is an important part of reducing relapse risk. Patients with refractory disease going into to transplant have a much higher risk of relapse compared to those in CR ,27. ThosAllo-HSCT carries significant limitations. The median age of diagnosis of AML is 72, which prevents a majority of patients from accessing allo-HSCT due to treatment related toxicity . Allo-HSCD33/Siglec-3 is a protein from the immunoglobulin superfamily, with expression that ranges from myeloid progenitors to mature myeloid cells . CD33 isGemtuzumab Ozogamicin (GO) is an ADC using a calicheamicin warhead and the only currently listed form of immunotherapy for AML. Its history with regulatory authorities is long and complex. GO was given accelerated approval in 2000 by the FDA only for it to be withdrawn in 2010 ,48. The Vadastuximab talirine (VT), SGN33a, is an anti-CD33 ADC utilizing a PyrroloBenzodiazepine Dimer (PBD) toxin. VT has a number of theoretical advantages over GO, including uniform drug loading, effectiveness in multidrug resistance positive cell lines, and activity against AML with adverse cytogenetics . VT has While there remains several ongoing strategies to target CD33 in AML, the challenges in the development of VT highlight some of the difficulties with this target . Alterna+ cells in a humanized mouse model. A different ADC against CLL-1 with a PBD toxin depleted AML cell lines and primary AML in vitro and prolonged survival of mice xenografted with an AML cell line [C-Type Lectin-Like Molecule 1  is a surface molecule found on AML and committed myeloid progenitors . A CLL-1ell line . Limited+ monocytes in a humanized mouse model but did not impair the development of B or myeloid cells [T cell immunoglobulin mucin-3  has emerged as a potential selective target against AML while sparing normal hematopoiesis. TIM-3 is expressed on AML blasts and is upregulated on T cells of AML patients ,64. TIM-id cells . An earlid cells . This st+ T cells, but is also expressed on stressed and malignant cells, including the majority of AML [+ AML who underwent NKG2D CAR T cell therapy demonstrates initial promise [IDH2 mutation. Despite this finding, he had normal trilineage hematopoiesis, providing evidence of the lack of HSPC expression of NKG2D. He subsequently underwent allo-HSCT, leading to CR with normal molecular studies. It is unclear if AML subclones may have variable NKG2D expression; this case highlights the challenges in targeting a heterogeneous disease like AML with a single surface molecule. More data are required to determine if NKG2D targeted therapy can be a standalone therapy or a bridge to transplant.Natural Killer Group 2D (NKG2D) is a surface receptor that is expressed normally on NK and CD8y of AML ,69. Its  promise . The patCD70 is the ligand of CD27, and this interaction helps to regulate lymphocyte and HSPC activity . CD70 haWhile many of the targets to date have been expressed on a majority of AML, there have also been efforts to target surface proteins that are aberrantly expressed on AML even if they constitute a minority of cases. CD7 is a cell surface glycoprotein that is normally expressed on T and NK cells as well as their progenitors . CD7 is +, CD33+, or CD19+ cells. M5 AML was chosen as the target, as M4 AML did not uniformly express LILRB4. The targeting of M5 AML limits the number of potential patients who may benefit as AML with monocytic differentiation accounts for only 5-10% overall, though the proportion is up to 40% in children [Another method of target selection that may reduce hematologic toxicity is to selectively target markers specific for monocytic differentiation. Leukocyte immunoglobulin-like receptor-B 4  is expressed from promonocytes to mature monocytes . LILRB4 children ,83.CD300f is another surface target expressed across AML, mature myeloid cells, and HSPC ,85. TherWhile AML-specific surface targets have been difficult to validate, intracellular targets have been described that are substantially upregulated in AML compared to normal populations. The best known upregulated AML antigens are WT1 and PRAME, which are found in the majority of AML samples . DespiteDendritic cell (DC) vaccines have been the most common strategy to target AML intracellular antigens, with a wide range of manufacturing strategies and targets explored . DCs loa+ T cells reactive against WT1 on HLA-A*02:01 are functional in vitro, are home to the bone marrow of patients, and persist after reinfusion [An alternate way to target intracellular antigens and possibly increase the efficacy of treatment over DC vaccination is to use T-cell receptor (TCR)-engineered T cells. Engineered T cells can be designed to recognize intracellular antigens expressed by MHC on AML . CD8+ T infusion . Persistinfusion . The limA separate strategy to reduce hematologic toxicity is to use a toxin as part of an ADC that demonstrates increased efficacy against AML compared to HSPC. It may be possible to find an effective therapeutic window where an ADC can deplete AML while sparing HSPC. IMGN632 is an ADC that targets CD123 and uses an indolinobenzodiazepine pseudodimer alkylator payload that demonstrates activity against AML cell lines and primary AML in vitro as well as AML cell lines in vivo . IMGN632Due to the difficulties in avoiding HSPC depletion with anti-AML immunotherapies, an alternative strategy would be to incorporate these therapies into allo-HSCT, which is an established therapy for AML. Attempts to target CD123 with CAR T cells demonstrate how immunotherapies against AML may be incorporated into a transplant model. CAR T cells have shown preclinical in vitro and in vivo efficacy against AML, but there are conflicting reports on the potential for toxicity against HSPC. Some studies demonstrated no effect on myeloid progenitors, while others displayed complete myeloablation in humanized mouse models ,96. Depl- relapses in ALL [An innovative way to allow anti-AML CAR T cells to persist is to remove a common target from HSPC. This strategy has been explored by developing CAR T cells against CD33 and then removing the CD33 from an allo-HSCT donor graft . CD33-des in ALL . To combA compound CAR T cell targeting CD33 and CD123 has been developed that demonstrates independent activity against cells expressing either molecule . These CIn addition to CAR T cells, there have been reports of ADCs against CD45 and CD117 to reduce AML and conditions for an allo-HSCT . These aEffective immunotherapy for most patients with AML remains elusive. The experience with GO suggests that immunotherapy may be able to play a role in AML treatment, but the clinical trials with VT illustrate the difficulty in managing more potent therapeutics against AML with the associated increase in hematologic toxicity. Unless a true leukemia specific epitope is found that is consistently expressed across at an individual cell level, then targeting single molecules may continue to show limited efficacy. Multiantigen targeting increases the risks of hematologic toxicity but would likely reduce the chance of tumor escape.Requiring an allo-HSCT is a major disadvantage of any potential future therapy, as most patients with AML are ineligible for allo-HSCT. The concept of bridging to allo-HSCT may change with AML immunotherapy. In other hematologic malignancies, bridging therapy occurs before conditioning; therefore, the treatment is naturally longer. In AML therapy, it may be possible to combine bridging with conditioning, which may reduce the toxicity of the individual elements combined. Another potential benefit of incorporating immunotherapy in allo-HSCT conditioning for AML is that these therapies may replace traditional conditioning agents. The consistent presentation of AML targets on HSPC may allow for an extension immunotherapy to conditioning in other diseases, including myelodysplastic syndromes as well as inherited disorders of hematopoiesis and immunity that respond poorly to traditional conditioning agents.The design and trials of emerging AML immunotherapy must be carefully considered to have a chance of success, given the high morbidity and mortality associated with the disease itself. Tolerable and effective immunotherapy that can reduce the need for allo-HSCT would constitute a major change in the treatment of AML, but to date this has been elusive. Finally, the alternative of enhancing the efficacy while reducing the toxicity of allo-HSCT with immunotherapy would constitute substantial progress in finding better outcomes for patients with AML.


However, the role and importance of RYR2 during human embryonic cardiac development are still poorly understood. Here, we generated two human induced pluripotent stem cell (iPSC)-based RYR2 knockout (RYR2–/–) lines using the CRISPR/Cas9 gene editing technology. We found that RYR2–/–-iPSCs could differentiate into CMs with the efficiency similar to control-iPSCs (Ctrl-iPSCs); however, the survival of iPSC-CMs was markedly affected by the lack of functional RYR2. While Ctrl-iPSC-CMs exhibited regular Ca2+ handling, we observed significantly reduced frequency and intense abnormalities of Ca2+ transients in RYR2–/–-iPSC-CMs. Ctrl-iPSC-CMs displayed sensitivity to extracellular Ca2+ ([Ca2+ ]o) and caffeine in a concentration-dependent manner, while RYR2–/–-iPSC-CMs showed inconsistent reactions to [Ca2+ ]o and were insensitive to caffeine, indicating there is no RYR2-mediated Ca2+ release from the sarcoplasmic reticulum (SR). Instead, compensatory mechanism for calcium handling in RYR2–/–-iPSC-CMs is partially mediated by the inositol 1,4,5-trisphosphate receptor (IP3R). Similar to Ctrl-iPSC-CMs, SR Ca2+ refilling in RYR2–/–-iPSC-CMs is mediated by SERCA. Additionally, RYR2–/–-iPSC-CMs showed a decreased beating rate and a reduced peak amplitude of L-type Ca2+ current. These findings demonstrate that RYR2 is not required for CM lineage commitment but is important for CM survival and contractile function. IP3R-mediated Ca2+ release is one of the major compensatory mechanisms for Ca2+ cycling in human CMs with the RYR2 deficiency.In adult cardiomyocytes (CMs), the type 2 ryanodine receptor (RYR2) is an indispensable Ca It plays an essential role in excitation-contraction coupling, a process by which an electrical signal is converted into a single contraction  represents the major sarcoplasmic reticulum (SR) Catraction . Upon sptraction .Ryr2 knockout (Ryr2–/–) mouse model claimed that RYR2 plays a non-redundant role in the control of heart rate and rhythmicity (Ryr2 gene in the mouse (Ex3-del+/−) was associated with bradycardia and death. However, Ex3-del+/− mice did not display some clinical phenotypes of patients with the RYR2 exon-3 deletion, including catecholaminergic polymorphic ventricular tachycardia   . Ryr2–/–day E9.5 , when LT cycling . Howeveressively . Ryr2–/–abeculae .–/– models and mouse embryonic stem cell-derived CMs (mESC-CMs)  . The majESC-CMs) . In the ESC-CMs) . Based oESC-CMs) . PreviouESC-CMs) . However–/–-iPSC-CMs exhibit morphological and physiological abnormalities, providing insights into the function of RYR2 during human embryonic heart development.In this study, we used iPSC technology in combination with the CRISPR/Cas9 gene editing technique to investigate the role of RYR2 in differentiation, development, and function of human iPSC-CMs. We hypothesized that the generated RYR22. The E8 medium was changed daily and cells at ∼85% confluency were passaged using Versene (Thermo Fischer Scientific).Human iPSC lines iWTD2.1 and iBM76.1 were used in this study as controls (Ctrl-iPSCs), which were generated from dermal fibroblasts and mesenchymal stem cells from two healthy donors, respectively, using the STEMCCA lentivirus system, and characterized as previously described . The stu–/–) iPSC lines, two guide RNAs (gRNAs) were designed and inserted into the CRISPR/Cas9 plasmid (Sigma-Aldrich), respectively  targets exon 99 (g.763747-763768). For transfection using the Amaxa Nucleofector II device (Lonza), 2 × 106 iPSCs were collected and re-suspended in a mixture of 82 μl Nucleofector solution and 18 μl supplement 1  containing 4 μg plasmid. One day after transfection, GFP+ cells were sorted and seeded on Geltrex-coated 96-well plates at a density of 1 × 103 cells/well for expansion. The genomic DNA from the colonies was isolated and purified using the automated Maxwell 16 cell DNA purification kit (Promega) according to the manufacturer’s instruction. For genomic DNA sequencing, the DNA sequence of RYR2 was initially amplified by PCR using the appropriate primer set  was induced by modulating WNT signaling as previously described -2,5-diphenyl tetrasodium bromide) assay was performed according to the manufacturer’s instructions . Briefly, iPSC-CMs at week 8 after replating were seeded in 48-well plates at a density of 60,000 cells per well, and cultured for another 2 weeks. Afterwards, 50 μg/ml MTT dissolved in 200 μl cardio culture medium was added onto the cells, which were then incubated at 37°C for 2 h. Reaction was stopped by the addition of 200 μl isopropanol supplemented with 0.04 M HCl. Samples were incubated and shaken (300 rpm) for 10 min at room temperature (RT). Absorbance of the formazan at 570 and 630 nm was measured using a plate reader (Biotek Synergy HTX). Cell viability was determined as absorbance (570–630 nm) and normalized to control group.–/–-iPSC-CMs grown on glass coverslips were fixed with 4% paraformaldehyde , blocked with 1% bovine serum albumin , and permeabilized with 0.1% Triton X-100 (Carl Roth). Immunofluorescence staining was performed overnight using the following primary antibodies: anti-α-actinin , anti-Ki67 , anti-IP3R , and anti-RYR2 . Afterward, cells were washed three times with PBS and incubated with the corresponding secondary antibodies  for 1 h at RT. Nuclei were co-stained with 4’,6-diamidino-2-phenylindole . Documentation was performed using fluorescence microscopy (Carl Zeiss).Ctrl- and RYR2–/–-iPSC-CMs (90 days old) were scraped off from the culture plates and cell pellets were snap-frozen into liquid nitrogen and stored at –80°C. For cell lysis, frozen cell pellets were resuspended in lysis buffer containing 20 mM Tris/HCl (pH 7.4), 200 mM NaCl, 1 mM Na3VO4, 20 mM NaF, 1% IGEPAL CA-630 (Sigma-Aldrich), 1 mM dithiothreitol (Roth), PhosSTOP phosphatase inhibitor (Roche), and cOmpleteTM protease inhibitor (Roche). After incubation for 30 min on ice, lysates were centrifuged and protein concentration was determined using the Pierce BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. A total amount of 40 μg protein lysate mixed with SDS loading buffer and DPBS in a volume of 20 μl was denatured for 30 min at 37°C. Afterward, samples were run on a 6–15% polyacrylamide gel with a 5% stacking gel at 250 mA for around 1–2 h and then transferred to PVDF membranes using the Wet/Tank blotting system. Unspecific binding sites on the membrane were blocked with 1% BSA in TBS with 0.1% Tween 20 (TBS-T) or with 5% non-fat dry milk in TBS-T for 1 h at RT. Membranes were incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: anti-RYR2 , anti-RYR2 , anti-IP3R , anti-SERCA2A , anti-NCX1 , anti-Cav1.2 , anti-α-actinin , anti-eukaryotic elongation factor 2 , anti-cardiac troponin T , and anti-GAPDH . The membranes were then incubated with HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies  for 1 h at RT. Afterwards, membranes were washed three times with TBS-T. Antigens of interest were detected by chemiluminescence  and visualized using the ChemiDoc MP system. Quantification was performed by calculating the signal intensity with Image Lab software (Bio-Rad).Both Ctrl- and RYR2–/–-iPSC-CMs (90 days old) were stimulated with 100 nM isoprenaline (Sigma-Aldrich) for 6 h and stepwise treated with the proteasome and calpain inhibitor MG132  and the autophagy inhibitor bafilomycin A1  before the cell pellets were collected.To detect protein degradation, Ctrl-, A3 and A5 RYR2–/–-iPSCs as well as 90-day-old Ctrl- and RYR2–/–-iPSC-CMs were washed three times with PBS and cell pellets were collected, snap-frozen into liquid nitrogen, and stored at –80°C. Isolation and purification of total RNA were performed using the SV total RNA isolation system (Promega) in accordance to the manufacturer’s instructions. First strand cDNA synthesis was performed using MULV reverse transcriptase (Thermo Fisher Scientific) and Oligo d(T)16 primer (Thermo Fisher Scientific). The expression level of SOX2, OCT4, NANOG, LIN28, and FOXD3 were assessed in Ctrl- and CRISPR-edited iPSC lines by reverse transcription-PCR using the GoTaq DNA polymerase (Promega). In addition, the expression level of RYR2, IP3R1, IP3R2, CACNA1C, TNNT2, and ACTN2 were assessed in Ctrl and RYR2–/–-iPSC-CMs. GAPDH was used as an internal control. Primer sequences, annealing temperature, and cycles used for reverse transcription-PCR analyses are listed in For gene expression analysis, Ctrl-iPSCs, CRISPR-edited A3 and A5 RYR2–/–-iPSC-CMs (around 80-day-old) were dissociated, replated on Geltrex-coated coverslips at a density of 200,000 cells per 6-well and then allowed to recover for at least 10 days in cardio culture medium. Before measurement, cells were loaded with the fluorescent calcium indicator 5 μM fluo-4/AM (Thermo Fisher Scientific) and 0.02% [w/v] pluronic F-127 (Thermo Fisher Scientific) in Tyrode’s solution containing (in mM): NaCl 140, KCl 5.4, CaCl2 1.8, MgCl2 1, HEPES 10, and glucose 10 (pH adjusted to 7.3 with NaOH) for 30 min at RT. Following incubation, the indicator-containing solution was removed and cells were washed twice, and incubated for additional 10 min to allow de-esterification of the indicator. To detect Ca2+ sparks, both Ctrl- and RYR2–/–-iPSC-CMs were pre-treated with 100 nM isoprenaline for 10 min before starting the recordings. Recordings were obtained using a LSM 710 confocal microscopy system in line scan mode . Cells were incubated in Tyrode’s solution at RT and field stimulated at 0.25 Hz for at least 20 s to bring the cytosolic calcium concentration to a steady state. Fluo-4 was excited at 488 nm and emitted fluorescence was captured at 490–540 nm. Quantification of Ca2+ sparks was performed using the SparkMaster plugin of ImageJ (NIH) and several key parameters were determined: spark frequency (events per 100 μm per second), amplitude (ΔF/F0), full duration at half maximum (FDHM), full width at half maximum (FWHM), and SR Ca2+ leak per cell (spark frequency × amplitude × FDHM × FWHM). As fixed criteria, sparks with minimal amplitude of 0.2 ΔF/F0, minimal width of 0.7 μm, and minimal duration of 7 ms were selected for detailed analysis.For calcium spark recordings, spontaneously beating Ctrl- and RYR2–/–-iPSC-CMs around day 80 were dissociated and replated on coverslips at a density of 200,000 cells/well. Cells were allowed to recover for at least 10 days post-replating. For measurement, cells were loaded with Fura-2 (Thermo Fisher Scientific) at a final concentration of 5 μM in cardio culture medium for 30 min at 37°C and washed twice with the medium. Prior to measurement, cells were incubated for 10 min to enable complete de-esterification of intracellular Fura-2. Intracellular Ca2+ events were recorded using a 40 × objective on an Olympus IX70 microscope fitted with an IonOptix system  at 35°C. Samples were excited at 340 and 380 nm with a switching frequency of 200 Hz and the emitted fluorescence was collected at 510 nm. The cytosolic Ca2+ level was measured as the ratio of fluorescence at 340 and 380 nm (340/380 nm).Both Ctrl- and RYR22+ transients were recorded in normal Tyrode’s solution containing (in mM): NaCl 138, KCl 4, CaCl2 1.8, MgCl2 1, NaH2PO4 0.33, HEPES 10, and glucose 10 (pH adjusted to 7.3 with NaOH). To normalize the Ca2+ transient frequency, Ctrl- and RYR2–/–-iPSC-CMs were field-stimulated using a MyoPacer  at a pacing frequency of 0.5 Hz . Monotonic transient analysis was performed using the LabChart Pro software (ADInstrument) and the following parameters were determined: peak amplitude of Ca2+ transients (the Fura-2 ratio at systole subtracted by the Fura-2 ratio at diastole), decay rate (tau), as well as duration and frequency of Ca2+ transients.Spontaneous whole-cell Ca–/–-iPSC-CMs loaded with Fura-2 were first exposed to 0 mM Ca2+ Tyrode’s solution until no spontaneous Ca2+ transient was detected and then continuously perfused with Tyrode’s solution containing Ca2+ of increasing concentrations . In the end, caffeine  was applied to confirm the activity of RYR2.Calcium sensitivity analysis was performed as described previously . Briefly2+ release in iPSC-CMs, cells loaded with Fura-2 were washed with depolarization solution containing (in mM): NaCl 112, KCl 30, CaCl2 1.8, MgCl2 1, NaH2PO4 0.33, HEPES 10, and glucose 10 (pH adjusted to 7.3 with NaOH). Cytosolic Ca2+ level was measured before and after continuous addition of caffeine with increasing concentrations (from 0.025 to 5.0 mM).To examine the caffeine-induced Ca2+ release in Ca2+ handling of iPSC-CMs, spontaneous Ca2+ transients were recorded before and after the application of the IP3R antagonists 2-aminoethoxydiphenyl borate  and Xestospongin C . To determine the contribution of SERCA-mediated SR Ca2+ uptake to Ca2+ cycling in iPSC-CMs, spontaneous Ca2+ transients were recorded before and after the application of the SERCA inhibitor thapsigargin . The changes of Ca2+ transient amplitude and frequency caused by the addition of these inhibitors were determined.To investigate the role of IP3R-mediated CaFor these experiments, we used an imaging chamber , which is furnished with a field stimulation and a built-in aspiration port. For fluid control, a 12-valve superfusion system  combined with a multi-tube pre-heater  was used, which together allows a gentle, rapid, and direct solution change around the measured cells. The overflow solution was removed via the aspiration port by suction with a peristaltic pump .–/–-iPSC-CMs around day 80 were enzymatically singularized into single cells and seeded on coverslips at a density of 30,000 cells per 3.5-cm dish. Whole-cell patch-clamp recording of single cells was conducted using an EPC10 amplifier (HEKA Elektronik), controlled by the PatchMaster software (HEKA Elektronik). Series resistance was compensated by 85%. Pipette potentials (VPip) were corrected for liquid junction potentials. All experiments were performed at RT. Action potential (AP) measurement was performed under current-clamp mode without any current injection in RPMI 1640 basal medium using the pipette solution containing (in mM): KD-gluconate 100, KCl 60, MgATP 4, NaGTP 0.3, Na2-phosphocreatine 5, and HEPES 10 (pH adjusted to 7.2 with KOH). Voltage-clamp measurement of L-type calcium current (ICaL) was conducted in an extracellular solution containing (in mM): NMDG-Cl 140, CaCl2 1.8, MgCl2 1, HEPES 10, and glucose 10 (pH adjusted to 7.3 with HCl) and a pipette solution containing (in mM): CsCl 110, TEA-Cl 20, MgCl2 5, EGTA 10, HEPES 10, and Na2ATP 2 (pH adjusted to 7.2 with CsOH). To elicit ICaL, cells were clamped for 600 ms from the holding potential of –90 mV to test potentials between 70 and –80 mV in steps of –10 mV. The basic cycle length was 3 s.Ctrl- and RYR2t-test, the one-way ANOVA with the Dunnett’s multiple comparison test or the two-way ANOVA with Sidak’s correction for comparison of more groups and conditions. Results were considered statistically significant when the P-value was <0.05 .Results are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism 5 software with student’s RYR2 gene, gRNA1 and gRNA2 were designed to target exon 90  in the cytosolic domain of RYR2 (L4077Sfs*25)  , leading7Sfs*25) , and the–/–-iPSC lines were analyzed for their pluripotency. Both cell lines were positive for the human pluripotency markers OCT4, SOX2, LIN28, NANOG, TRA-1-60, and SSEA4 as demonstrated by immunostaining , cardiac troponin T (TNNT2), and synaptophysin (SYP) .A3 and A5 RYR2staining , similarstaining . In addirl-iPSCs . Differein (SYP) . Notably–/–-iPSCs were able to be successfully differentiated into spontaneously beating CMs  was similar to that of Ctrl-iPSCs  recognizes both the full-length and the truncated proteins by binding the N-terminal region before the PTC. Neither the full length nor the truncated proteins of RYR2 were detectable in A3  to inhibit the activity of the proteasome and with bafilomycin A1  to inhibit the fusion of autophagosomes with lysosomes. Interference of the two major protein degradation pathways did not result in the detection of degraded RYR2 proteins in both A3  and the voltage-gated calcium channel subunit alpha-1C (CACNA1C). No tendency of increased or decreased expression of tested genes was observed in RYR2–/–-iPSC-CMs in comparison to their corresponding Ctrl-iPSC-CMs  compared to Ctrl-iPSC-CMs  levels and the occurrence of Ca2+ transients in iPSC-CMs, we measured Ca2+ events under different [Ca2+ ]o concentrations using the fluorescent Ca2+ dye Fura-2 in combination with Ca2+ imaging. To this end, spontaneously beating iPSC-CMs were first washed with Tyrode’s solution containing no Ca2+ until no spontaneous Ca2+ transient was detectable. Afterward, cytosolic Ca2+ changes were monitored in response to continuous perfusion with increasing Ca2+ concentrations (from 0 to 2.0 mM) and addition of 10 mM caffeine at the end. To quantify the fraction of Ca2+ -oscillating cells for each condition, the number of iPSC-CMs, which displayed spontaneous Ca2+ transients at each [Ca2+ ]o concentration was determined. As shown in 2+ transients in Ctrl-iPSC-CMs were detected with [Ca2+ ]o lower than 0.5 mM. Raising [Ca2+ ]o concentrations stepwise from 0.3 to 2.0 mM concomitantly increased the amplitude and frequency of Ca2+ transients in Ctrl-iPSC-CMs . As shown in 2+ levels in Ctrl-iPSC-CMs, while the amplitudes of caffeine-induced Ca2+ release progressively increased under the caffeine treatment at concentrations from 0.05 to 2.5 mM and declined at the concentration of 5 mM : the amplitude and frequency decreased to 20.1 and 12.9%, respectively (2+ entry independently of the function of IP3R (2+ transients in 12 out of 13 A3 and 8 out of 10 A5 RYR2–/–-iPSC-CMs were completely and reversibly blocked after the addition of 1 μM XeC (2+ transients (–/–-iPSC-CMs showed a faster reaction to XeC (1 μM) than 2-APB (20 μM), as 200 s of XeC (1 μM) treatment was sufficient to block the Ca2+ transients in these cells. These observations imply that an IP3-gated Ca2+ pool is functional in iPSC-CMs, which is dominant in the modulation of Ca2+ handling in RYR2–/–-iPSC-CMs.Given that RYR2elopment . TherefoiPSC-CMs , while Cansients . AlthougiPSC-CMs , it did iPSC-CMs . On the ectively . Previou of IP3R . Therefo1 μM XeC , while tansients . Additio2+ handling protein located on the SR membrane, namely SERCA. To this end, we recorded spontaneous Ca2+ transients in Ctrl- and RYR2–/–-iPSC-CMs before and after the application of 5 μM thapsigargin, a specific SERCA inhibitor. It turned out that thapsigargin treatment for 500 s was sufficient to block all Ca2+ transients in most of Ctrl- (12 out of 14), A3 (8 out of 10) and A5 RYR2–/–-iPSC-CMs (7 out of 8) in a time-dependent manner , we recorded APs of Ctrl- and RYR2–/–-iPSC-CMs at single-cell level  RYR2 is not essential for the early commitment of iPSCs into the cardiac lineage, while RYR2 is important for the viability and function of iPSC-CMs; (ii) RYR2-mediated Ca2+ release is essential for the maintenance of normal Ca2+ transients, which directly modulates the beating rate of iPSC-CMs; (iii) Ca2+ handling in iPSC-CMs is sensitive to [Ca2+ ]o, which mainly depends on RYR2; (iv) in the absence of functional RYR2, RYR2–/–-iPSC-CMs generate Ca2+ sparks and transients via compensative IP3R-mediated pathway; and v) refilling of SR Ca2+ store by SERCA pumps is required for the Ca2+ cycling in RYR2–/–-iPSC-CMs similar to Ctrl-iPSC-CMs. We conclude that SR Ca2+ release via RYR2 is critical for the survival and whole-cell Ca2+ handling of iPSC-CMs. When the function of RYR2 is lost, IP3R-mediated Ca2+ release from the SR compensates the RYR2 function partially.In this study, we have generated two iPSC-based Ryr2 gene is associated with embryonic cardiac arrest at around E10.5, even the looped heart tubes were formed at E8.5 and spontaneous heartbeat was observed at E9.5  or by targeted knockdown of Orai1 with RNAi in RYR2–/–-iPSCs.IP3R shares structural and functional similarities to RYRs. IP3-operated releasable CaiPSC-CMs . In our iPSC-CMs . To maina2+ load . SERCA-miPSC-CMs . These fsurvival . Previous occurs . In the 2+ transients in more than 50% of RYR2–/–-iPSC-CMs. The abnormal Ca2+ transients were found only in a small number of Ctrl-iPSC-CMs, which is consistent with the results of other studies  is accomplished by SERCA2A and by the sarcolemmal NCX (about 25%) in human cardiomyocytes. Further studies should focus on the investigation of the localization and activity of NCX in RYR2–/–-iPSC-CMs.In the present study, we identified abnormalities of Ca studies  and may  studies . Calcium2+ plays a critical role in the formation of Ca2+ transients, which are instrumental for subsequent cardiac contraction, we investigated the relationship between [Ca2+ ]o levels and Ca2+ transients in iPSC-CMs with RYR2 deficiency. Our results revealed that 3-month-old iPSC-CMs are sensitive toward [Ca2+ ]o at 0.5 mM or higher, with increased frequency and amplitude of Ca2+ transients, as well as increased fraction of Ca2+ -oscillating cells in response to elevated [Ca2+ ]o. These findings indicate a Ca2+ concentration dependent opening of RYR2 in iPSC-CMs, which is in line with the previous studies showing that human embryonic kidney (HEK) 293 cells expressing wild-type RYR2 displayed Ca2+ oscillations under [Ca2+ ]o at 0.3 mM or higher and in a concentration dependent manner , suggesting the opening of IP3R occurred at lower intracellular Ca2+ concentration than RYR2. In RYR2–/–-iPSC-CMs, spontaneous Ca2+ transients with lower frequency in comparison to Ctrl-iPSC-CMs were detected, which is in line with the observation in Ryr2–/– mESC-CMs  decreased cytosolic Ca2+ levels. Under the stimulation of 0.025 mM caffeine, HEK 293 cells expressing wild-type RYR2 presented a caffeine-induced Ca2+ release , and carried out in accordance with the approved guidelines. The patients/participants provided their written informed consent to participate in this study.XL, WL, and KG conceived the study and designed experiments, contributed to interpretation of the data, and wrote the manuscript. XL, WL, KK, SH, LC, AS, MP, and MS performed experiments and acquisition of the data. XL, WL, KK, SH, AS, MS, and KG analyzed the data. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The most prominent Ca2+ event is a large magnitude, delayed signal that follows vasodilation. However, previous work has provided little information about the spatio-temporal properties of such Ca2+ transients or their heterogeneity. Here, using an awake, in vivo two-photon fluorescence-imaging model, we performed detailed profiling of delayed astrocytic Ca2+ signals across astrocytes or within individual astrocyte compartments using small regions of interest next to penetrating arterioles and capillaries along with vasomotor responses to vibrissae stimulation. We demonstrated that while a 5-s air puff that stimulates all whiskers predominantly generated reproducible functional hyperemia in the presence or absence of astrocytic Ca2+ changes, whisker stimulation inconsistently produced astrocytic Ca2+ responses. More importantly, these Ca2+ responses were heterogeneous among subcellular structures of the astrocyte and across different astrocytes that resided within the same field of view. Furthermore, we found that whisker stimulation induced discrete Ca2+ “hot spots” that spread regionally within the endfoot. These data reveal that astrocytic Ca2+ dynamics associated with the microvasculature are more complex than previously thought, and highlight the importance of considering the heterogeneity of astrocytic Ca2+ activity to fully understanding neurovascular coupling.Astrocytic Ca This intriguing finding necessitates further detailed analysis of endfoot Ca2+ dynamics to gain insights into their contributions to functional hyperemia ], produced by uncaging caged Ca2+ compounds  between postnatal day 30 (P30) and P60. Animals were injected on three consecutive days with tamoxifen , prepared as a 10 mg/ml stock in corn oil. Injections started between P19 and P35. Animals were kept on a normal 12-h light/12-h dark cycle and had All surgical procedures and isoflurane anesthesia were performed as previously described . Brieflyvia the tail vein (100–200 μl of a 2.3% (w/v) solution in saline) to visualize the blood plasma. The animal was allowed to recover on the treadmill, with its head immobilized, for 30 min prior to imaging.Rhodamine B isothiocyanate (RhodB)-dextran  was injected in vivo two-photon microscope (2+ responses were monitored in the barrel cortex (layers 1–3).Fluorescence images were obtained using a custom-built croscope  illumina2+ signals around the endfoot. Ca2+ responses were calculated as ΔF/F = (Ft−Frest)/Frest, where Ft is the measured fluorescence at any given time and Frest is the average fluorescence obtained over 2 s prior to whisker stimulation. Ca2+ signals with an intensity that crossed a 3-standard deviation (SD) threshold  relative to signal fluctuations during a 2-s prestimulus baseline and remained above the threshold for at least 0.5 s were detected as astrocyte Ca2+ increases. Penetrating arteriole cross-sectional area was analyzed by using the threshold feature in imageJ, after which particle analysis was used to measure the area of the lumen filled with RhodB-dextran. Cross-sectional area changes were calculated as Δd/d = (dt−drest)/drest where dt is the area obtained at any given time and drest is the average baseline area obtained over 2 s prior to whisker stimulation. Area change with an intensity that crossed a 3-SD threshold ; relative to signal fluctuations during a 2-s prestimulus baseline, and remained above the threshold for at least 0.5 s were detected as vasodilation. Onset corresponds to the first time point at which the signal reached the threshold and remained over it for at least 0.5 s. Duration was calculated as the difference between response onset and response offset. Statistical analyses used a paired or unpaired t-test or one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test as appropriate. Statistical “n” constituted a single experimental trial or an experimental animal, as indicated. Data are expressed as means ± SEM. values of p <0.05 were considered statistically significant. All statistical analyses were done using GraphPad. A 95% confidence interval (CI) was calculated using modified Wald method.All data were processed using ImageJ. Movement artifacts in the xy plane were corrected for using the align_slices plugin. ROIs corresponding to astrocyte endfeet, soma, and arbor were analyzed separately. Small ROIs (2.5×2.5 μm) placed next to one and another around the endfoot was analyzed to obtain temporal sequence of Ca2+ , to assess local intracellular astrocyte Ca2+ dynamics, with concurrent tail vein injection of RhodB-dextran to visualize the vasculature and monitor vascular responses . Even though stimulation of vibrissae did not elicit arteriole dilation in all cases, it induced more vasodilatory responses  than Ca2+ rises  in endfeet, enveloping the penetrating arterioles , endfeet  and arbors  from the subcellular compartments in some trials , it did not trigger Ca2+ changes in any ROIs in other trials. There were also trials in which at least one ROI did not exhibit a rise in Ca2+ . The distribution of onset time for sensory-induced astrocytic Ca2+ changes was more widespread than that of functional hyperemic responses  than those without an endfoot Ca+ increase . These observations further support our previous findings that changes in arteriole diameter evoked endfoot Ca2+ transients  positioned next to one and another around the endfoot . In some instances, a soma appeared to be physically part of the endfoot; in this particular scenario, Ca2+ signals increased within the soma region either remained localized or did not spread far  although the absence of endfoot Ca2+ around the penetrating arteriole did not preclude arteriole dilation, stronger endfoot Ca2+ rises were observed in the presence of vasodilation; (3) sensory stimulation did not elicit a global rise in endfoot Ca2+, but instead triggered discrete Ca2+ “hot spots” that typically spread around the endfoot and occasionally remained localized. These findings indicate that astrocytic Ca2+ dynamics are heterogeneous across different astrocytes as well as between astrocytic subcellular compartments, and suggest that these Ca2+ signals may be compartmentalized during sensory-induced functional hyperemia. They further suggest that the close proximity between the endfoot and the vessel wall of a penetrating arteriole does not necessarily translate to direct effects of local endfoot Ca2+ on arteriole dilation.In this study, we revealed that (1) sensory stimulation did not generate a global effect that elicited homogeneous Cain vivo have presented polarized views on the involvement of astrocytic Ca2+ in functional hyperemia. Some of these studies in anesthetized or slightly sedated animals have shown that fast astrocytic Ca2+ transients precede functional hyperemic responses to sensory stimulation  or mitochondria that buffer Ca2+ and prevent the signals from spreading beyond a boundary. Other studies have described global events observed in all subcellular compartments  we acknowledge that the fact that small ROIs used to detect discrete Ca2+ were done manually could contribute to the under-detection of the signals. In conclusions, our findings further emphasize that examining NVC in 3-Ds and at different layers of the cortex in awake animals is necessary to obtain accurate assessments of astrocytic Ca2+. Furthermore, they highlight the need to have an automation of Ca2+ analysis designed to better detect all signals from all subcellular compartments of the astrocyte.Our work has unveiled the complexity of astrocytic Cae cortex . Finallye cortex , anastome cortex , and expe cortex . Collecte cortex . It has echanism . NeverthThe original contributions presented in the study are included in the article/supplementary materials, further inquiries can be directed to the corresponding author.The animal study was reviewed and approved by The Animal Care and Use Committee of the University of Calgary.CT and GG: conceptualization, methodology, writing – review and editing. CT: investigation. CT and KS: formal analysis, writing – original draft. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Since the Ca2+ dynamics inside the excitable cells are spatiotemporal while the membrane voltage is a global signal, the feedback loop is then a delayed global feedback (DGF) loop. In this study, we investigate the roles of DGF in the genesis and stability of spatiotemporal excitation patterns in periodically-paced excitable media using mathematical models with different levels of complexity: a model composed of coupled FitzHugh-Nagumo units, a 3-dimensional physiologically-detailed ventricular myocyte model, and a coupled map lattice model. We investigate the dynamics of excitation patterns that are temporal period-2 (P2) and spatially concordant or discordant, such as subcellular concordant or discordant Ca2+alternans in cardiac myocytes or spatially concordant or discordant Ca2+ and repolarization alternans in cardiac tissue. Our modeling approach allows both computer simulations and rigorous analytical treatments, which lead to the following results and conclusions. When DGF is absent, concordant and discordant P2 patterns occur depending on initial conditions with the discordant P2 patterns being spatially random. When the DGF is negative, only concordant P2 patterns exist. When the DGF is positive, both concordant and discordant P2 patterns can occur. The discordant P2 patterns are still spatially random, but they satisfy that the global signal exhibits a temporal period-1 behavior. The theoretical analyses of the coupled map lattice model reveal the underlying instabilities and bifurcations for the genesis, selection, and stability of spatiotemporal excitation patterns.Biological excitable media, such as cardiac or neural cells and tissue, exhibit memory in which a change in the present excitation may affect the behaviors in the next excitation. For example, a change in calcium (Ca Understanding the mechanisms of pattern formation in biological systems is of great importance. Here we investigate the dynamical mechanisms by which delayed global feedback affects excitation pattern formation and stability in periodically-paced biological excitable media, such as cardiac or neural cells and tissue. We focus on the formation and stability of the temporal period-2 and spatially in-phase and out-of-phase excitation patterns. Using models of different levels of complexity, we show that when the delayed global feedback is negative, only the spatially in-phase patterns are stable. When the feedback is positive, both spatially in-phase and out-of-phase patterns are stable, and the out-of-phase patterns are spatially random but satisfy that the global signals are temporal period-1 solutions. Specifically, at t = 0, we set the variable α = 0 in lmin, i.e., the domain sizes distribute between lmin and L-lmin. Because of this randomness in pattern selection, the corresponding histogram of the global P2 amplitude  of the uncoupled FHN unit, indicating that the dynamics in the 1D array is the same as in the uncoupled FHN unit. For α>0, a transition from uniform P1 to Dis-P2 occurs as T decreases (from yellow to black), which is caused by a spatial-mode instability of the uniform P1 state. As T decreases further (red region), both Con-P2 and Dis-P2 patterns can occur depending on the initial conditions , but α = 0), the domain size distributions are continuous, indicating that the Dis-P2 patterns are spatially random (including the periodic ones) and depend on initial conditions. However, differing from the case of no DGF, the global signals of the Dis-P2 patterns are always P1 solutions i.e., L/2. This is because if there is a domain greater than L/2, the sum of all other domains must be smaller than L/2, and thus when the patterns reverse in the next beat, the global signal cannot be the same, violating the requirement of a global P1 solution.Furthermore, we performed the same statistical analysis as in the case of no DGF  for diff2 region , the disc¯peak=0 . Moreoveα = 0), both Con-P2 and Dis-P2 patterns can occur, and the Dis-P2 patterns are spatially random. In the presence of DGF, only Con-P2 patterns can exist when the DGF is negative (α<0). When the DGF is positive (α>0), both Con-P2 and Dis-P2 patterns can exist depending on the pacing period T and initial conditions. The Dis-P2 patterns are also spatially random but satisfy that the global signals are always P1 solutions.Therefore, in the absence of DGF . However, since ICa and INCX also directly affect the Ca2+ dynamics, it becomes difficult to only change the Ca2+-to-APD coupling without affecting the Ca2+ dynamics. To avoid this complexity, we added two new currents to the ventricular model: the non-specific Ca2+-activated cation current (InsCa) and the small conductance Ca2+-activated potassium current (ISK). InsCa is mainly an inward current [2+ concentration increases. Therefore, increasing the conductance of InsCa increases APD and promotes positive Ca2+-to-APD coupling. InsCa is small under normal conditions but becomes large under conditions of Ca2+ overload [SK is an outward current which also increases with the intracellular Ca2+ concentration, and thus ISK promotes negative Ca2+-to-APD coupling. Under normal conditions, ISK only presents in atrial myocytes, but under heart failure, it also presents in ventricular myocytes [2+, unlike ICa and INCX, they do not directly affect intracellular Ca2+ dynamics. Here we altered the maximum conductance of these two currents to alter the Ca2+-to-APD coupling properties. Note that DGF is not explicitly present in the model but indirectly via the Ca2+-to-APD coupling, as we explained in Introduction. We first investigated the effects of Ca2+-to-APD coupling properties on Ca2+ release patterns and then linked them to DGF.Under free running, however, Cacoupling , 47. As coupling , 48, 49, current  and incroverload . ISK is myocytes –53. Sinc2+ channels and Ca2+ release channels (see 2+-to-APD coupling is negative (large ISK), a transition from uniform P1 to Con-P2 patterns occurs as T decreases, and this transition occurs at a larger T value as the maximum ISK conductance increases. When the coupling is positive (large InsCa), a transition from uniform P1 to Dis-P2 patterns (yellow to black) occurs as T decreases. Under both coupling conditions, as T decreases further, the system enters the Con/Dis-P2 regime (red), in which both Con-P2 and Dis-P2 patterns can occur depending on initial conditions (2+-to-APD coupling is negative (large ISK) and into a Con-P2 only region when the Ca2+-to-APD coupling is positive (large InsCa). Therefore, for the same Ca2+-to-APD coupling, as T decreases, the spatiotemporal patterns change from Con-P2 only to Dis-P2 only through a Con/Dis-P2 region or in reverse order depending on the coupling properties.We systematically explored the spatiotemporal dynamics by altering the pacing period T and the maximum conductance of the two currents, as summarized in nels see , heterognditions . HoweverTo reveal the statistical properties of the Dis-P2 patterns, we show the histograms of domain sizes and the whole-cell alternans amplitude for a parameter point in the Dis-P2 region  and a po2+ dynamics in the detailed ventricular myocyte model agree well with those of the generic FHN array with DGF. To link the spatiotemporal Ca2+ dynamics to the DGF properties, we performed a quantitative analysis to reveal the DGF properties and their relationship with the Ca2+-to-APD coupling properties. We carried out simulations of the ventricular cell model using an S1S2 protocol. In this protocol, a train of external stimuli (S1) was given at a fixed period (T = 300 ms) for the cell to reach the steady state, and a stimulus (S2) was then applied after a variable interval  from the last S1 beat. We measured the peak values of ICa, SR Ca2+ and cytosolic Ca2+ on the S2 beat, which were considered as control. Next, we repeated the same simulations except that on the last S1 beat, we increased αSK to shorten the APD (nsCa to lengthen the APD (SK) or the positive (increased αnsCa) Ca2+-to-APD coupling from the control case. In Ca, SR Ca2+ and cytosolic Ca2+ of the two coupling cases to the control values on the S2 beat.As shown above, the Ca the APD , red or  the APD , cyan. T2+-to-APD coupling case (cyan) results in a smaller ICa  for the negative Ca2+-to-APD coupling and higher (ratio>1) for the positive Ca2+-to-APD coupling , the relationships are reversed, i.e., positive or negative Ca2+-to-APD coupling leads to positive or negative DGF, respectively. The DGF is primarily mediated by SR Ca2+ load since with long S1S2 intervals the Ca2+-to-APD coupling properties show almost no effects on ICa due to full recovery. Knowing the relationships between Ca2+-to-APD coupling properties and the DGF properties, one can then link the DGF properties to the Ca2+ release patterns in the ventricular cell model. Based on the analysis above and the Ca2+ release patterns shown in 2+ release patterns to the sign of DGF, which is summarized in Therefore, for short S1S2 intervals (<300 ms), positive or negative Can is the temporal index and j the spatial index. cn(j) describes the peak signal in the jth lattice of the nth beat. f is the map function: μ determine the midpoint and the slope of the curve, respectively. M is the coupling length, and wm is the coupling strength described by a Gaussian function: Nw is the normalization constant. cn−1(j), and cs = f(cs). We set μ = 0.1, M = 15, and σ = 3. Note that concrete forms of map functions for investigating intracellular Ca2+ dynamics were developed previously [f, which can give rise to a bifurcation from P1 to P2, similar to those of the FHN model and the detailed ventricular myocyte model . Linearization of this nonlinear term around the uniform steady-state gives rise to a term proportional to α>0 corresponds to positive feedback, the same as in the FHN model.Note that in 1) Stability of an uncoupled unit. For an uncoupled unit, the map equation with DGF becomesα since the steady state is independent of α. α<0 destabilizes the steady state, and α>0 stabilizes the steady state. The stability boundary is shown as the dashed line in 2) Stability of the spatially uniform P1 state. The spatially uniform P1 state  is determined by the eigenvalues:k is the wave number of the Fourier mode  Stability of the Con-P2 state. Following the same procedure as for the uniform P1 state, we obtained the eigenvalues for the spatially uniform P2 state asf at the P2 solution of 4) Stability of the Dis-P2 states. The stability of the Dis-P2 states cannot be analytically obtained. We used numerical simulations of the CML model  is a temporal P2 solution  with the alternans amplitude being randomly distributed between zero and the maximum amplitude (Con-P2).In the presence of DGF, the pattern dynamics are determined by the sign of the DGF. When the DGF is negative, only Con-P2 patterns can exist, no spatial mode instabilities emerge, and all the Dis-P2 solutions are unstable. When the DGF is positive, both Con-P2 and Dis-P2 patterns can occur, depending on the pacing period and initial conditions. The Dis-P2 patterns are also spatially random but must satisfy that the global signals are temporal P1 solutions .Bifurcation analyses of the CML model reveal the spatial-mode instabilities leading to the spatiotemporal patterns.2+-to-APD coupling properties to the DGF properties, we have shown that the spatiotemporal pattern dynamics of Ca2+ release in cardiac myocytes agree very well with the findings in the simple models, validating the theoretical predictions in a realistic system.By linking the CaTherefore, our simulations and theoretical analyses reveal the underlying dynamical mechanisms and roles of DGF in the genesis, selection, and stability of spatiotemporal patterns in periodically-paced excitable media. The uniqueness of the conclusions drawn from the three types of models implies that the insights obtained in this study may apply to many excitable as well as oscillatory biological media. Here we discuss two examples below.2+ release patterns  have been observed experimentally [2+ alternans dynamic of the ventricular myocyte model agree well with those of the simplified models, indicating that the generic mechanisms of pattern formation and selection are also applicable to Ca2+ alternans dynamics in cardiac myocytes. Both spatially concordant and discordant Ca2+ alternans (Con-P2 and Dis-P2 patterns) have been observed experimentally in cardiac myocyte [2+-to-APD coupling is responsible for the formation of Dis-P2 patterns. A direct experimental demonstration of this theory was carried out by Gaeta et al. [2+-to-APD coupling. However, Dis-P2 patterns have also been observed experimentally under voltage-clamp [2+-to-APD coupling, and Dis-P2 patterns can occur under positive Ca2+-to-APD coupling and voltage-clamp conditions. These complex Ca2+ release behaviors cannot be well explained by the Turing instability mechanism alone. On the other hand, our study unifies the complex subcellular Ca2+ alternans dynamics under a single theoretical framework of DGF, providing a general mechanistic understanding of the subcellular Ca2+ alternans dynamics.At the subcellular level, both Con-P2 and Dis-P2 Camentally , 58, 59, myocyte –33, 59.  myocyte  developege-clamp , 59 and ge-clamp . Furtherge-clamp , 48 and 2+-to-APD coupling determines the occurrence of subcellular discordant Ca2+ alternans. One can achieve this by properly tuning the relative contributions of ICa and INCX [SK or InsCa provides a more straightforward way of changing the coupling properties. Gaeta et al. [2+ alternans. However, our study shows that altering the Ca2+-to-APD coupling properties does not guarantee that one can control the transition between concordant and discordant Ca2+ alternans. The unique dynamical factor controlling the transition between concordant and discordant Ca2+ alternans is the sign of DGF, which is not uniquely determined by the sign of Ca2+-to-APD coupling. This is because Ca2+-to-APD coupling affects the recovery of ICa and SR Ca2+ loading which contribute oppositely to DGF.Previous studies , 48, 60 and INCX , 60. Hera et al.  used a f2+ cycling properties, a phenomenon called mechano-electric feedback [Cardiac and neural tissue are typical excitable media. In cardiac tissue, spatially concordant and discordant APD alternans have been widely observed in experiments , 62–64, feedback –36. Sincfeedback –69.In neural tissue, cluster firing and other firing patterns have been widely observed –73. The Our study focused on the roles of DGF in pattern formation and stability in periodically-paced excitable media. In a previous study in oscillatory chemical reaction experiments, Kim et al.  showed DThe present study involved three mathematical models at different levels of complexity. The model of a coupled array of FHN units and the 1D CML model are described in the Result section. A brief summary of the 3D ventricular cell model and numerical methods is given below.2+ within a CRU cycles through these sub-compartments via diffusion, buffering/unbuffering, SR release and SERCA pump. The flow of Ca2+ between CRUs is via diffusion in the cytosol, submembrane and network SR. The exchange of Ca2+ between intracellular and extracellular space is regulated by ICa and Na+-Ca2+ exchanger (NCX). The model is described by differential equations that are coupled via Ca2+ and voltage. The closing and opening of L-type Ca2+ channels and the ryanodine receptors are simulated using stochastic Markov transitions. Therefore, intrinsic spatiotemporal noise exists in the model.The ventricular cell model has been described in detail in our previous studies , 45, simInsCa and ISK, to the model for altering Ca2+-to-APD coupling. The InsCa formulation was adopted from the 1994 Luo and Rudy model [ns(Ca) = 3.5×10−5 cm/s and Km,ns(Ca) = 1.5 μM. ISK was formulated based on Komendantov et al. [GSK = 0.005 mS/F and Kd = 0.6 μM.We added two new currents, dy model  with thev et al.  as folloCm = 1 μF/cm2 is the membrane capacitance. αnsCa and αSK are the parameters controlling the maximum conductance of InsCa and ISK, respectively. Isti is the stimulus current density which is a square pulse with the amplitude -80 μA/μF and the duration 0.5 ms.The differential equation for voltage is thenBoth the model of a coupled array of FHN units and the CML model were programmed with Python 3, and the corresponding simulations were carried out on our cluster with 24 Intel Xeon CPUs. The 3D ventricular cell model was programmed with CUDA C++, and the corresponding simulations were carried out on Nvidia Tesla K20c, K80, and GTX 1080 Ti GPU cards. The detailed algorithms for detecting spatiotemporal excitation patterns in this study are described in the S1 TextA. AP clamp and free running protocols. B. Stability analysis of the CML model. C. Automatic detection algorithms for spatiotemporal excitation patterns. D. Boundary between discordant P2 and uniform P2 in the CML model.The online supporting information includes: (DOCX)Click here for additional data file.S1 Figa). Data from Roome et al.[2+ is also recorded. Green trace shows spatially averaged Ca2+. b). Data from Diaz et al.[2+ signals under control and ryanodine receptor blocker tetracaine with a voltage clamp protocol. Linescan recordings reveal out-of-phase spatial patterns of intracellular Ca2+ transient from beat to beat. Bottom two traces show fluorescence measured as a function of time at the regions indicated by arrows (i and ii).me et al. showing (EPS)Click here for additional data file.S2 Figa). Bifurcation diagram showing c vs. T for the single-element FHN model. b). Bifurcation diagram showing the peak values of the whole-cell average Ca2+ transient, c). Bifurcation diagram showing c vs. γ for the single-element CML model.(EPS)Click here for additional data file.S3 FigThe voltage trace is the last action potential recorded in the simulation of the physiologically detailed myocyte model. The model was paced for 100 beats to reach the steady state at the pacing period T = 450 ms. This voltage trace was used in the AP clamp simulations.(EPS)Click here for additional data file.S4 Figλk vs. k for α = 0.2 (black), 0 (red) and -0.2 (blue) are numerically computed based on γ = 1.057, which is the bifurcation point in (EPS)Click here for additional data file.S5 Figa). Top shows an example of discordant P2 patterns for the 1D FHN model. Bottom shows the relationship of Δcn+1(j) vs. j where the domain size l determined by the pattern recognition algorithm is marked by the double-arrow red line. b). Same as a), but for the 1D CML model. c). Same as a), but for the detailed myocyte model. Note that the gray and black traces are for m = 1 and 50 in S10 Eq, respectively. We used m = 50 to extract the domain size for the detailed myocyte model in our study.(EPS)Click here for additional data file.


We review a theoretical, coarse-grained description for cardiomyocytes calcium dynamics that is motivated by experiments on RyR channel dynamics and provides an analogy to other spontaneously oscillating systems. We show how a minimal model, that focuses on calcium channel and pump dynamics and kinetics, results in a single, easily understood equation for spontaneous calcium oscillations (the Van-der-Pol equation). We analyze experiments on isolated RyR channels to quantify how the channel dynamics depends both on the local calcium concentration, as well as its temporal behavior (“adaptation”). Our oscillator model analytically predicts the conditions for spontaneous oscillations, their frequency and amplitude, and how each of those scale with the small number of relevant parameters related to calcium channel and pump activity. The minimal model is easily extended to include the effects of noise and external pacing . We show how our simple oscillator predicts and explains the experimental observations of synchronization, “bursting” and reduction of apparent noise in the beating dynamics of paced cells. Thus, our analogy and theoretical approach provides robust predictions for the beating dynamics, and their biochemical and mechanical modulation. The heart is an extraordinary organ. From several weeks after conception, and throughout its entire life, the heart constantly beats, generating considerable stresses and strains on its tissues and cells  that is coupled to the mechanical contractions of the heart cell  , the opening probability of the channel followed the instantaneous [Ca2+], in what is referred to in the physics literature as an “adiabatic process” , the opening probability showed an “adaptive” response, an initial sharp increase (overshoot) followed by an exponential relaxation to a steady-state value, with a typical time-scale of ~100 ms , with an average value 2+] concentration [denoted by C(t) with an average value 2+] concentration reflects a kinetic balance of channels that increase cytoplasmic [Ca2+], and active pumps that remove calcium from the cytoplasm (to the SR and or elsewhere). Note that the average [Ca2+] concentration can include contributions from an influx of calcium from outside of the cell , but these are not necessary to generate spontaneous oscillations  from its average value The model focuses on the two most relevant degrees of freedom, the opening probability of the RyR channel [denoted by 2+] concentration relative to steady-state, with a proportionality constant α that reflects the changes of the rates R± with [Ca2+] around steady-state. The second term accounts for the “adaptive” response of the channel to changes in the [Ca2+] concentration, with a magnitude proportional to β that quantifies the overshoot in the channel response to fast changes in the [Ca2+] concentration. Equation (2) encapsulates the “adaptive” response observed in experiments , the first term dominates, and the opening probability follows the instantaneous [Ca2+] concentration (an “adiabatic” change—see 2+] is varied quickly (ċ ≫ 1), the opening probability overshoots by a factor β, and then slowly relaxes to its “adiabatic” value with a typical rate H ~ 10 Hz  accounts for the response of the RyR channel to changes in [CaJp[C] is the calcium current through the open RyR channels which is in general also a function of cytoplasmic [Ca2+]  and the timescale of [Ca2+] variation (~1 Hz), allows us to derive a single equation for the [Ca2+] dynamics  is the famous Van-der Pol equation for a non-linear, spontaneous oscillator  would represent inertia—which is the tendency of an object to maintain its velocity. Classical inertia is proportional to the mass of the object, the larger the mass, the larger the overshoot of the equilibrium point. In the chemical system of Equation (4) the “effective mass” *, represents an effective, linear friction. In the classical system friction is proportional to the velocity of the object, and always oppose the motion of the object. Thus, in the presence of friction, the oscillations of the mass decrease in amplitude as time increases and the object asymptotically comes to rest at the equilibrium length of the spring. Similarly, in the chemical system the “effective friction” is proportional to the rate of change of [Ca2+]. However, the “effective friction” can switch from regular dissipation (η* > 0) to “negative dissipation” (η* < 0). When the “effective friction” is positive (η* > 0), any oscillations decay over time and the system effectively goes to its steady state concentration at long times. On the other hand, if the “effective friction” is negative (η* < 0), any change in the [Ca2+] concentration is amplified by the friction, which would cause a divergence in the [Ca2+] concentration as time increases. The origin of the “negative friction” (as one can see from Equation 5) is the same feedback effect that gives rise to the “effective mass.” The increase is eventually saturated by higher order terms in the friction . Thus, given the right conditions (2+] is enough to destabilize the system.The second term in Equation (4), proportional to ηK), with a correction due to the “adiabatic” response of RyR to [Ca2+] (α < 1). It is important to note that, unlike the classical system, the steady-state around which the chemical system oscillates is far from equilibrium (since there is still a large concentration difference between the SR and the cytoplasm). Moreover, the calcium pumps that work to restore cytoplasmic [Ca2+] concentrations work against this concentration gradient, and thus require constant input of energy (in the form of ATP). Therefore, in contrast to the classical “passive” oscillator, the chemical oscillator of Equation (4) is inherently active. In the absence of pump activity, thermodynamics predicts the system will equilibrate with the [Ca2+] concentration in the SR and the cytoplasm becoming equal.Finally, in the classical system, the fourth term of Equation (4) would represent the restoring force applied by the spring which pulls the object toward the equilibrium distance. This restoring force in our calcium system is proportional to the activity of calcium pumps , we estimate β ≈ 0.55 μM−1. We thus approximate the effective mass as m* ≈ 0.1 s−2, which along with a pumping rate K ~ 1 s−1 , one can estimate the non-linear saturation parameter as Γ ~ 0.25 μM−2. Note that the microscopic origin of the non-linearity can arise from several different effects , and more experiments are required to distinguish between these mechanisms.The amplitude of oscillations in our model scales as 2+] oscillations, and how it relates to the kinetics and dynamics of the currents to and from the SR. Moreover, it predicts how the effective parameters (Equation 5) depend on the channel and pump properties and thus, the conditions for the transition from decaying to spontaneous oscillations (η* = 0), and the frequency close to this transition  from the biophysics of the system, provides an intuitive understanding of the physics of spontaneous  and a stochastic noise η(t). The noise term is taken to be Gaussian, with average 〈η(t)〉 = 0 and temporal correlations 〈η(t)η(t) = 2Dδ(t − t′) . Thus, the beating phase in this case is defined with respect to the spontaneous frequency Ωc (and not that of the pacing force Ωp), and as such is confined to the range 0 ≤ ϕc ≤ 2π.We have recently shown theoretically and analytically that the reduction in apparent noise can be predicted from the simple model of Equation (4), supplemented by the sum of both an external pacing force [c, but with a drift due to a periodic potential  Equation (7) is expected to go to steady-state (*/D* → 0), the inherent noise in the system will cause the phase ϕc to randomly diffuse over time, which means that at very long times the steady-state probability density is flat. This means that in steady-state the phase can take any value between 0 < ϕc < 2π with equal probability. However, when the amplitude of the pacing force is increased , the probability density narrows, with an average around 〈ϕc〉 = β+π/2 (see p). The average beating frequency is still fixed at Ωc, but the fluctuations around this frequency becomes smaller as α*/D* increases. Indeed, in the limit of strong pacing α*/D* ≫ 1 , the probability density approaches an infinitely sharp delta function (~ δ(ϕc − β − π/2)), and we regain the deterministic beating behavior of Equation (4) .Equation (7) describes a diffusion-like process of the phase ϕ Risken, . While E+π/2 see . Note thWe have demonstrated here the rich set of behaviors observed in the dynamics of spontaneously beating, and paced isolated cardiomyocytes can be captured by a minimal model derived from calcium pumps and channels dynamics. This approach allows for an intuitive understanding of spontaneous beating, and how it reacts to external perturbations. Fundamental understanding of the physics behind cardiomyocyte beating can facilitate the design of better medical treatment in the future. Drawing analogies to well-known physical systems, and understanding how the coarse-grained observables  scale with changes introduced to the underlying kinetics, are essential to bridge the gap between the microscopic and the macroscopic view of cardiac beating. The resulting theory is useful in explaining why and how large-scale phenomena  emerge in a robust manner from the microscopic details. Future analysis should focus upon inter channel coupling within a single cell, and the coupling of calcium oscillations in neighboring cells (using our model as a basis) as a first step to predicting tissue level behavior (Grosberg et al., All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The myosin inhibitor mavacamten reverses the increased contractility caused by HCM thick filament mutations, and we here test its effect on HCM thin filament mutations where the mode of action is not known. Mavacamten (250 nM) partially reversed the increased Ca2+ sensitivity caused by HCM mutations Cardiac troponin T (cTnT) R92Q, and cardiac troponin I (cTnI) R145G in in vitro ATPase assays. The effect of mavacamten was also analyzed in cardiomyocyte models of cTnT R92Q and cTnI R145G containing cytoplasmic and myofilament specific Ca2+ sensors. While mavacamten rescued the hypercontracted basal sarcomere length, the reduced fractional shortening did not improve with mavacamten. Both mutations caused an increase in peak systolic Ca2+ detected at the myofilament, and this was completely rescued by 250 nM mavacamten. Systolic Ca2+ detected by the cytoplasmic sensor was also reduced by mavacamten treatment, although only R145G increased cytoplasmic Ca2+. There was also a reversal of Ca2+ decay time prolongation caused by both mutations at the myofilament but not in the cytoplasm. We thus show that mavacamten reverses some of the Ca2+-sensitive molecular and cellular changes caused by the HCM mutations, particularly altered Ca2+ flux at the myofilament. The reduction of peak systolic Ca2+ as a consequence of mavacamten treatment represents a novel mechanism by which the compound is able to reduce contractility, working synergistically with its direct effect on the myosin motor.Thin filament hypertrophic cardiomyopathy (HCM) mutations increase myofilament CaNEW & NOTEWORTHY Mavacamten, a myosin inhibitor, is currently in phase-3 clinical trials as a pharmacotherapy for hypertrophic cardiomyopathy (HCM). Its efficacy in HCM caused by mutations in thin filament proteins is not known. We show in reductionist and cellular models that mavacamten can rescue the effects of thin filament mutations on calcium sensitivity and calcium handling although it only partially rescues the contractile cellular phenotype and, in some cases, exacerbates the effect of the mutation. It is pactility , 13, andactility . To dateactility  or Ca2+ blockers ; however2+ probes that report relative [Ca2+] in distinct cytoplasmic and myofilament-specific pools  was prepared by limited proteolysis as previously described , and Ala-Ser-α-tropomyosin were purified as previously described sulfate in 0.5 M H2SO4 was used to measure inorganic phosphate. Absorbance (A700) measurements were converted to absolute activity (s−1), and with the use of KaleidaGraph (Synergy Software), calcium-sensitivity data was fitted to the Hill equation, as follows:min = minimum ATPase rate, Amax = maximum ATPase rate, pCa = −log [Ca2+], pCa50 = −log [Ca2+] required for half maximum ATPase activity, and nH = Hill coefficient.ATPase assays were carried out as previously described , 14. The5 cells per ml) were incubated in ACCITT3 culture medium at 37°C and 5% CO2.This investigation was approved by the Animal Welfare and Ethical Review Board at the University of Oxford and conforms to the UK Animals (Scientific Procedures) Act, 1986, incorporating Directive 2010/63/EU of the European Parliament. As previously described , briefly2+ indicator for optical imaging (RGECO), RGECO-TnI, or RGECO-TnT and either WT cTnT, cTnT R92Q, WT cTnI, or cTnI R145G to give expression the WT and mutant troponins at an approximate 50% level compared with endogenous troponin as previously implemented and described  and electrically paced at 40 V, 0.5 Hz, and at 37°C. Sarcomere shortening was captured by Fourier transform of the cardiomyocyte striations under phase contrast microscopy at 250 Hz. All contracting cardiomyocytes were measured.Sarcomere shortening measurements were performed using IonOptix μstep apparatus. Cultured cardiomyocytes were perfused with perfusion buffer, consisting of (in mM)150 NaCl, 5 HEPES, 5.5 glucose, 1 MgCl2+ transient parameters extracted; all baseline variation of Ca2+ transients derived from differential expression of RGECO or driven by altered basal sarcomere lengths (via myofilament Ca2+ buffering) were normalized by calculating ΔF/F for each transient . After 15 min incubation at 37°C, cardiomyocytes were imaged on an Olympus IX81 inverted microscope  with a C-9100-13 EMCCD camera . Videos of 0.5-Hz electrically paced cardiomyocytes at 37°C were acquired at 25 frames/s . Raw image data were extracted using xcellence (Olympus), analyzed in Excel (Microsoft) and Caransient .t-test or Mann-Whitney test was performed (GraphPad Prism). All cardiomyocyte comparisons were from at least three separate cell isolations with a similar number of cells from each isolation analyzed. Furthermore, all mutant and/or treatment groups were compared with wild type by splitting the same cell isolation to create pairwise comparisons and reduce variability. Any cell with extracted parameters of baseline, amplitude, or kinetics exceeding two standard deviations from the mean upon analysis of both sarcomere shortening and Ca2+ transients were excluded due to phenotypic heterogeneity arising in cultured primary cells. Groups were tested for normality (D’Agostino and Pearson test) and either an ordinary one-way ANOVA with Tukey’s or Kruskal-Wallis test with Dunn’s test was performed (GraphPad Prism).ATPase groups were tested for normality (D’Agostino and Pearson test), and either an unpaired 2+ regulation of actomyosin ATPase activity, we measured in vitro skeletal muscle myosin subfragment-1 (S-1) ATPase activity activated by reconstituted thin filaments containing either WT recombinant human troponin subunits or those containing the HCM causing mutations cTnT R92Q or cTnI R145G (50 in the presence of 250 nM mavacamten (B–E), despite the reported lower Kd of mavacamten for skeletal muscle myosin (4.7 μM vs. 0.3 μM) for cardiac myosin . We have previously described the relationship between alterations to myofilament Ca2+ affinity and cardiomyocyte Ca2+ handling via altered myofilament Ca2+ buffering  transfected by adenoviruses to express cTnT R92Q or cTnI R145G to model autosomal dominant human HCM . Interestingly, we find that fractional shortening is unchanged and speed of relaxation is increased at both time points (D and E). We therefore chose a 15-min incubation with 250 nM mavacamten for all subsequent GPCM experiments; this reflects the optimal temporal effect of the drug and concentrations close to the Kd for cardiac β-myosin , and cTnT R92Q cells treated with mavacamten measured 1.90 ± 0.01 µm (P < 0.001) (A and B). However, we observed rescue of hypercontracted diastolic sarcomere length in cells expressing cTnI R145G, shifting from 1.71 ± 0.02 to 1.80 ± 0.02 μm (P < 0.001) with wild type measured at 1.82 ± 0.02 μm (P > 0.05) (A and B).Using the conditions above, we assessed the effect of mavacamten on the function of GPCMs expressing HCM causing mutations. We observed relaxation of the hypercontracted diastolic sarcomere length beyond the wild-type length. With respect to sarcomere length, wild-type cells measured 1.85 ± 0.01 µm, cells expressing cTnT R92Q measured 1.82 ± 0.01 µm (< 0.001) , A and B > 0.05) , A and BP < 0.01), and cTnT R92Q cells with mavacamten had 4.72 ± 0.43% (P < 0.001) fractional shortening (P = 0.0591) from 7.17 ± 0.42 (P < 0.001 c.f. control) to 4.76 ± 0.58%  compared with 10.59 ± 0.72%, for wild type . In GPCMs expressing cTnI R145G, we observed complete rescue of increased systolic Ca2+ upon treatment with 250 nM mavacamten using both the cytosolic- and myofilament-localized sensors (E–G).In GPCMs expressing cTnT R92Q, peak systolic fluorescence was decreased in cells treated with mavacamten compared with wild type when observed with the cytoplasmic Cavacamten , E–G. In sensors , E–G.50 Ca2+ transient decline remained prolonged in the cytosol in cTnT R92Q and cTnI R145G with mavacamten; however, T50 decline at the myofilament was reversed to control levels with 250 nM mavacamten  reduced Cas et al.  who simifilament . This effilament , which ir assays , 9, 15.2+ to lower thin filament activation. However, in the case of the cTnT R92Q mutant, cytoplasmic Ca2+ levels were reduced to below wild-type levels upon mavacamten treatment of mutant cells. This suggests that the genotype of patients may need to be carefully considered in translational studies. These data also suggest that mavacamten can partially rescue the primary disease driver of increased Ca2+ sensitivity in thin filament mutations, and thus mavacamten might also be clinically effective for thin filament Ca2+-sensitizing mutations in addition to the thick filament mutations that enhance myosin activation and destabilize the myosin super-relaxed state. However, we cannot exclude the possibility that mavacamten could also have novel off-target effects on the Ca2+-handling machinery of the cardiomyocyte in addition to modulating myofilament Ca2+ sensitivity.The complete reversal of the systolic fluorescence increase by mavacamten is interesting since it implies that the effects of the drug on contractility are mediated not only by effects at the level of the myosin motor but, additionally and synergistically, by reducing systolic Ca2+ binding of TnC  Grant RE/13/1/30181. A. J. Sparrow (PG/18/68/33883) and P. Robinson (CH/1992001/6764) are both supported by the British Heart Foundation. M. J. Daniels was funded by the Wellcome Trust Grant WT098519MA.No conflicts of interest, financial or otherwise, are declared by the authors.A.J.S., M.J.D., C.R., and P.R. conceived and designed research; A.J.S. and P.R. performed experiments; A.J.S. and P.R. analyzed data; A.J.S., C.R., and P.R. interpreted results of experiments; A.J.S. and P.R. prepared figures; A.J.S. and C.R. drafted manuscript; A.J.S., H.W., M.J.D., C.R., and P.R. edited and revised manuscript; A.J.S., H.W., M.J.D., C.R., and P.R. approved final version of manuscript.