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a CASQ2 knockout mouse model as well in humans (Watanabe et al., 2009). In addition, stabi- lization of RyR2 by JTV-519 has been shown to reduce CPVT-triggered arrhythmias at the cel- lular level(Sedej et al., 2010).
Novel iPSC technology has increased the study of CPVT pathophysiology and cardiac ar- rhythmias in human CM models in recent years. Several groups have reported the generation of CPVT patient-specific iPSCs from individuals carrying a CASQ2 gene mutation (Novak et al., 2012) and RyR2 gene mutations (Di Pasquale et al., 2013; Fatima et al., 2011; Itzhaki et al., 2012; Jung et al., 2012; Zhang et al., 2013) with the physiological characteristics of the disease.
The similarities and differences in the pathophysiological consequences of RyR2 versus CASQ2 mutations have also been studied with iPSC-CMs (Novak et al., 2015). In some of these studies, the phenotype of CPVT could be rescued with drugs. These drugs include dantrolene, an inhibi- tor of Ca2+ release through RyR channels (Jung et al., 2012), flecainide, thapsigargin, an inhibi- tor of the SERCA2a, (Itzhaki et al., 2012) and KN-93 (Di Pasquale et al., 2013).
2.6 The potential use and challenges of iPSC-derived cardiomyocytes
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Figure 12. Potential use of iPSC-derived CMs. Figure modified from (Bellin et al., 2012).
After the discovery of iPSC technology, the potential of this technology for creating disease models from patients with complex genetic defects has been studied increasingly. Several genet- ic diseases have been studied with iPSCs, including hematopoietic, hepatic, endothelial, neuro- logical, and cardiovascular diseases. (Ebert et al., 2012) Disease modelling with iPSCs has been successfully exploited to study multiple cardiac diseases recapitulating the characteristic pheno- type of each specific disease: CPVT (Fatima et al., 2011; Jung et al., 2012; Novak et al., 2012;
Itzhaki et al., 2012; Zhang et al., 2013; Di Pasquale et al., 2013), LQT1 (Moretti et al., 2010), LQT2 (Itzhaki et al., 2011a; Matsa et al., 2011; Lahti et al., 2012), LQT3 (Ma et al., 2013), di- lated cardiomyopathy (Sun et al., 2012), hypertrophic cardiomyopathy (Han et al., 2014; Lan et al., 2013), Timothy syndrome (Yazawa et al., 2011) and LEOPARD syndrome (Carvajal- Vergara et al., 2010).
In addition to disease modeling, iPSC-derived CMs could offer a safe potential platform for cardiovascular drug evaluation for early stage drug screening and pharmacokinetic studies (Fig- ure 12). The heart has been shown to be particularly sensitive to the toxic effects of non- cardiovascular drugs, and several of those types of drugs have been withdrawn from clinical use because they prolonged the QT interval. (Ebert et al., 2012) Prolonged QT intervals and other clinically relevant drug responses have been demonstrated from iPSC-derived CMs with differ-
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ent drugs (Itzhaki et al., 2011a; Lahti et al., 2012; Matsa et al., 2011); therefore, iPSC-CMs could also be used in non-cardiovascular drug studies where they could reveal possible cardiac side effects.
The greatest challenges of iPSC technology are related to the safety, efficiency and kinetics of the method, and more knowledge of the reprogramming process is needed. Optimal and effi- cient non-integrating reprogramming factors need to be found to avoid problems related to ge- netic alterations, such as risk of mutagenesis and tumors, and possible re-activation of silenced reprogramming genes. Challenges for iPSC technology also include the high costs of iPSC line derivation and culturing, as well as the efficiency of the derivation methods, which would need to be higher for larger scale applications. Especially for clinical cell therapy applications, iPSC derivation and differentiation techniques need to be developed toward xenofree production, and genomic alterations and chromosomal abnormalities during reprogramming and culturing need to be avoided.
The differentiation of CMs from iPSCs has its own challenges, including the efficiency of differentiation, together with complex and expensive differentiation protocols. The epigenetic memory of iPSCs can also influence the differentiation capacity of the cells(Kim et al, 2010b).
In addition, cardiac differentiation protocols produce a heterogenous population of CMs and non-cardiac cells, and the sorting and enrichment of CMs is challenging and time-consuming.
Currently, all cardiac differentiation methods produce a mixture of all types of cardiomyocytes (ventricular, atrial and conduction types of CMs). Immaturity of iPSC-derived CMs may result in an incomplete phenotype of the cells, which can distort the actual disease characteristics.
Immaturity of iPSC-derived CMs has been observed as inadequate sarcomeric structures (Lieu et al., 2009; Luna et al., 2011), changes in ion channel expression (Sartiani et al., 2007), fluctu- ating electrophysiological properties (Doss et al., 2012), a lack of clear t-tubule structures (Lieu et al., 2009; Novak et al., 2012) and a reduced repolarization reserve (Paci et al., 2014).
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3 Aims of the study
The main objective of this work was functional characterization of differentiated CMs and gen- eration of an iPSC-derived CPVT disease cell model to study and characterize the arrhythmic events of these CMs both in vivo and in vitro. To achieve the main objective, the following spe- cific aims were set:
1. Investigate whether EFS with a novel EFS platform could enhance CM orientation and maturation as well as improve functional properties of NRCs. (Study I)
2. Generate a disease model of CPVT patients carrying a RyR2 mutation with iPSC- derived CMs and clarify the cellular level pathomechanisms of the disease by studying electrophysiology and intracellular Ca2+ cycling of these cells. (Studies II-III)
3. Characterize mutation-specific differences of iPSC-derived CPVT CMs and study the antiarrhythmic potential of dantrolene in the treatment of CPVT by assessing the effica- cy of intravenously administered dantrolene in patients carrying various RyR2 muta- tions and compare these effects to in vitro studies using iPSC-derived CMs generated from the same patients. (Study III)
4. To analyze Ca2+ cycling of iPSC-derived CPVT CMs and abnormal Ca2+ transients.
(Studies II-IV)
5. To develop and test an automatic Ca2+ cycling analysis software tool based on interac- tive visualization and to compare the results with a visually performed manual analysis.
(Study IV)
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