In Study I, the problem with NRCs was their tendency to dedifferentiate during culture, which caused a lack of functionality and complicated the analysis of the effects of EFS. The amount of stimulated samples per one experiment was restricted due to our stimulation setup, and more replicates and repetition of stimulation protocols would be needed to compare results between different stimulation settings more thoroughly. In addition, comparing our results to those of other stimulation studies is difficult because the stimulation devices, stimulation parameters, and time in culture vary a lot greatly between studies. In the future, to enhance the positive ef- fect of EFS, it would be important to understand the mechanism by which stimulation and dif- ferent stimulation parameters influences cellular maturation and processes. In addition, more effective ways to quantify this phenomenon at the cellular level are needed.
In Study II, iPSC-derived CMs from two CPVT and two control cell lines were studied, and both CPVT lines were from the same patient. Cell lines of CPVT patients behaved the same way; however, whether the results were typical to this mutation or only to this patient cannot be stated with certainty. Despite the very rare nature of CPVT disease, we were able to extend our research by examining in detail six different disease-causing RyR2 mutations in Study III.
However, only one cell line per patient and control was studied, and the results could be cell line-specific. However because the in vitro dantrolene responses resembled the in vivo respons- es, the phenotype of the iPSC-derived CMs was highly likely to be at least patient specific in
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Study III. Only one fixed concentration of dantrolene was used in Study III, which is why the dose-responsive effect of dantrolene cannot be evaluated although positive effects of dantrolene were observed with four mutations with the same dantrolene concentration. Future studies of disease modeling and different mutations in multiple cell lines per patient as well as in muta- tion-specific cell lines from several individuals are necessary.
One challenge in disease modeling studies is proper controls for diseased cell lines. The problem with healthy controls is that they might differ from diseased cells lines with disease- causing mutation and may contain several other mutations and polymorphisms that cause phe- notypic differences. Therefore, the use of multiple different iPSC lines as controls would be optimal for disease modeling studies. One way to limit the variability between control and dis- eased cell lines would be to derive control iPSC lines from close relatives of the patient who are not affected by the disease-causing mutation. However, even in studies in which healthy sib- lings have been used as controls for disease patients, only ∼50% of the genome is shared be- tween any siblings. Furthermore, phenotypic differences could be the result of DNA variants in the other ∼50% of the genome, rather than the disease-associated mutations. (Wang et al., 2014b) In our study, we observed clear phenotypic differences between healthy control CMs and CPVT CMs. However, isogenic controls could more accurately depict non-diseased cellular phenotypes compared to healthy controls. With the help of isogenic controls, the effects of ge- netic modifiers and epigenetic factors of CPVT disease progression between different individu- als could be studied. Addressing whether healthy control cell lines are as suitable for controls in CPVT disease modeling as are isogenic gene-edited iPSC lines is important. However, the gen- eration of isogenic cell lines also needs validation because they are subject to clonal variation.
Abnormal Ca2+ cycling was observed in CPVT CMs but rarely in control CMs, which is why at least most of the abnormalities resembled the CPVT disease phenotype. Validation of the in vitro results with the patient data indicated the correct phenotype of our CPVT CMs.
However, some abnormal Ca2+ cycling may still be a consequence of the immature CM gene expression levels or phosphorylating activity of the CMs because some iPSC-derived CMs lack of t-tubules and have low expression levels of the SR Ca2+ buffering protein CASQ2, which can disturb RyR2-dependent Ca2+ release (Knollmann, 2013). These possibilities, together with possible electrophysiological and structural CM immaturity, may interfere with the normal Ca2+
cycling of these cells. Nevertheless, earlier studies have shown that iPSC-CMs display func- tional and loaded RyR-regulated intracellular Ca2+ stores, which can release Ca2+ via RyRs and
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can reload their content through SR Ca2+ uptake. The immaturity of the CMs can be a problem in disease modeling. Their immature phenotype is due to current differentiation technologies, is dependent of culturing time, and can cause significant line-to-line and patient-to-patient varia- bility. This variability complicates comparisons of results where CMs have been generated with different differentiation protocols. Otherwise, immaturity of iPSC-derived CM have been ob- served as immature morphology, incomplete organization of the sarcomeric structure (Lieu et al., 2009; Luna et al., 2011), changes in the expression of ion channels related to the cardiac AP (Sartiani et al., 2007), varying AP characteristics (Doss et al., 2012), and as a lack of clear t- tubule structures (Lieu et al., 2009; Novak et al., 2012), which all may cause problems in Ca2+
cycling due to slower spread of electrical signals and have an effect on the phenotype of the CMs. Additionally, in earlier studies, significant heterogeneity in the subtypes of hiPSC-CMs, including atrial, nodal, and ventricular CMs, has been found with each round of differentiation (Moretti et al., 2010).While this characteristic can be considered an advantage due to the possi- bility of assessing the physiological properties in all these cell types, the disadvantage is that changes that occur only in one subpopulation of cells cannot be specified when the readout is taken from all cells (Sinnecker et al., 2014).The various CM subtypes express differential sets of transcription factors, structural proteins and ion channels and have distinguishable AP pa- rameters (David and Franz, 2012). Therefore, the arrhythmic events detected in these subtypes can also vary. The development of cardiac differentiation and maturation protocols will hopeful- ly improve the homogeneity of iPSCs; however, clarifying the rules for what should be consid- ered mature CMs and characterizing the effects of various cardiac subtypes with AP measure- ments will be important.
In Studies II-IV, the type of CMs (nodal, atrial, or ventricular) under investigation was un- clear in the Ca2+ imaging studies because Ca2+ imaging method cannot be used to determine the CM type. The differences between the CM types can affect the Ca2+ cycling and future simulta- neous recording of APs and Ca2+ would help to distinguish the CM type and provide mechanis- tic information regarding the interplay between Ca2+ cycling and the membrane potential. Un- derstanding the electrophysiological mechanism behind different Ca2+ cycling abnormalities will also be very important. In Studies III and IV, AP characteristics, SR Ca2+ storage or Ca2+
sparks indicating Ca2+ leak from SR were not studied between different mutations, which would allow more information regarding mutation-specific differences. These differences could be addressed in the future by patch clamp, Ca2+ imaging methods and computational models.
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Because the nature of abnormal Ca2+ analysis has been visual inspection, this analysis was performed subjectively in Studies II-IV and may not be repeatable between two persons. How- ever, this problem occurs for all manual data analyses. Consistent rules for the analysis of Ca2+
signal abnormalities are currently lacking, and AnomalyExplorer software was generated for this reason. Because this program does not consider the interplay between AP and Ca2+ or quan- tify the Ca2+ cycling parameters, these abnormalities could be studied and improved in the fu- ture. The user-defined analysis parameters in AnomalyExplorer might cause differences be- tween two individuals performing the analysis, although those parameters are needed for anal- yses performed with different software and/or with different sampling frequencies. In addition, AnomalyExplorer was designed and implemented for a specific use case and for specific digiti- zation systems, which can be stated as a limitation of the current prototype. Therefore, some future improvement may be needed, for example, for analysis of high frequency Ca2+ signals.
In clinical recordings in Study II, the noise and acquisition frequency of the ECG signal can be limiting factors in the 24-h recordings and affect the results of the recordings. One limitation of the MAP method used in Study II was that it records extracellular activity and can provide only limited information regarding events occurring across the cell membranes. In Study III, we were permitted to study only acute effects of intravenously administered dantrolene and, there- fore, cannot state the long-term clinical effects of the drug. Additionally, although the dose of dantrolene was titrated according to the weights of the patients, serum levels of the drug were not measured and could have varied from patient to patient, resulting in concentration- dependent variations in clinical responses.