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Induced pluripotent stem cells in the inherited cardiomyopathies: From disease mechanisms to novel therapies
Author's Accepted Manuscript
Induced pluripotent stem cells in the inherited cardiomyopathies: From disease mechanisms to novel therapies Samantha Barratt Ross, Stuart T. Fraser, Christopher Semsarian
www.elsevier.com/locate/tcm
PII: DOI: Reference:
S1050-1738(16)30031-7 http://dx.doi.org/10.1016/j.tcm.2016.05.001 TCM6285
To appear in: trends in cardiovascular medicine
Cite this article as: Samantha Barratt Ross, Stuart T. Fraser, Christopher Semsarian, Induced pluripotent stem cells in the inherited cardiomyopathies: From disease mechanisms to novel therapies, trends in cardiovascular medicine, http://dx.doi.org/10.1016/j.tcm.2016.05.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Induced Pluripotent Stem Cells in the Inherited Cardiomyopathies: From Disease Mechanisms to Novel Therapies
Samantha Barratt Rossa,b, Stuart T. Fraserc, Christopher Semsariana,b,d
a
Agnes Ginges Centre for Molecular Cardiology, Centenary Institute, Newtown b
Sydney Medical School, University of Sydney, Sydney c
Disciplines of Physiology, Anatomy & Histology,
School of Medical Sciences, University of Sydney d
Department of Cardiology, Royal Prince Alfred Hospital, Sydney, Australia
Running title: iPSCs in inherited cardiomyopathies
ACKNOWLEDGEMENTS CS is the recipient of a National Health and Medical Research Council (NHMRC) Practitioner Fellowship (#1059156). SBR is the recipient of an Australian Postgraduate Award.
DISCLOSURES: None
Address for Correspondence: Professor Christopher Semsarian Agnes Ginges Centre for Molecular Cardiology Centenary Institute Locked Bag 6 Newtown, NSW, 2042 Australia Ph: 61 2 9565 6195 Email: [email protected]
ABSTRACT Inherited cardiomyopathies lead to diverse clinical outcomes including heart failure, arrhythmias and sudden death. Mutations in over 100 genes have been implicated in the pathogenesis of genetic heart diseases, including the main inherited cardiomyopathies, such as hypertrophic, dilated, and arrhythmogenic right ventricular cardiomyopathies. Understanding how these gene mutations lead to clinical disease and the various secondary genetic and environmental factors which may modify the clinical phenotype, are key areas of research ultimately influencing diagnosis and management of patients. The emergence of patient-derived induced pluripotent stem cells (iPSCs), which can be differentiated into functional cardiomyocytes (CMs) in vitro, may provide an exciting new approach to understand disease mechanisms underpinning inherited heart diseases. This review will focus specifically on the key role of iPSC-based studies in the inherited cardiomyopathies, both in their potential utility as well as the significant challenges they present.
Key words: cardiomyopathy; genetic; induced pluripotent stem cell; inherited
2
ABBREVIATIONS
iPSC – induced pluripotent stem cell ESC – embryonic stem cell CM - cardiomyocyte HCM – hypertrophic cardiomyopathy DCM – dilated cardiomyopathy ARVC – arrhythmogenic right ventricular cardiomyopathy ET-1 - endothelin-1
3
INTRODUCTION Inherited cardiomyopathies, including hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM) and arrhythmogenic right ventricular cardiomyopathy (ARVC), have a significant global burden with common adverse events including arrhythmias, heart failure
and
sudden
cardiac
death.
The
main
clinical
outcomes
of
inherited
cardiomyopathies are well understood. Over the past 25 years, there have been major advances in our understanding of the genetic basis of inherited cardiomyopathies with mutations in over 100 genes identified in a variety of cardiac genetic diseases [1]. The identification of these genetic causes has translated into improved diagnosis, treatment and prognosis in cardiac genetic diseases, as well as in cascade genetic testing of family members [2].
While our understanding of the genetic causes of cardiac diseases has progressed, understanding how gene mutations lead to diverse clinical outcomes, and disentangling the other secondary genetic or environmental factors that may modify the phenotype, remains largely unknown. When a genetic mutation is identified in an individual, proving causality is often challenging and relies on a number of factors, one of which is functional evidence to support pathogenicity [3]. The challenge of performing mechanistic and functional studies is in part due to difficulties in obtaining human cardiac cells from patients in useful quantities, which is restricted by the limited regenerative potential of cardiac cells [4]. Functional studies have been largely limited to animal models. The use of animal models, particularly murine models, has facilitated a better understanding of disease myocyte biology however time, cost and species differences hamper their suitability [5-7]. Importantly, differences in functional characteristics including electrophysiology and calcium handling significantly limit the use of murine models in cardiac research.
4
Multipotent adult stem cells have limited differentiation capacity for cardiac differentiation (Table 1). The isolation of pluripotent human embryonic stem cells (ESCs), which have the ability to differentiate into all somatic cells and indefinitely replicate, in 1998 led to the development of human cardiomyocyte (CM) differentiation models [8]. Seminal work by Yamanaka and Takahashi in 2007 re-programmed somatic cells into pluripotent cells similar to human ESCs [9]. These cells, termed induced pluripotent stem cells (iPSCs), have revolutionized the fields of human disease modeling, genetics and personalized and regenerative medicine. The current review discusses both the utility and challenges of human iPSCs; the generation of patient-specific CMs; and the use of iPSC-CMs for cardiomyopathy-related applications, such as disease modeling.
5
BASICS OF INDUCED PLURIPOTENT STEM CELLS (iPSCs)
What are iPSCs? iPSCs are embryonic stem-like cells that have been reprogrammed from somatic cells. iPSCs can be derived from human patient cells, most commonly skin or blood cells (Figure 1). Similar to human ESCs, iPSCs self-renew and can differentiated into cells of all three embryonic germ layers: endoderm, mesoderm and ectoderm [9]. For the most part, human ESCs and iPSCs are functionally equivalent [9]. However, epigenetic differences do exist in iPSCs, particularly pertaining to DNA methylation patterns [10].
How are iPSCs generated? iPSCs are reprogrammed from somatic cells, typically using a transfection vector and a well-defined
set
of
embryonically
expressed
pluripotency
factors.
Historically
reprogramming has involved viral transfection of somatic cells with the four “Yamanaka factors” OCT-4, SOX-2, Klf-4 and c-myc [9]. New approaches to reprogramming are moving away from integrating viral vectors. Following reprogramming, the pluripotent state of iPSCs is confirmed by assessment of pluripotency marker expression, activation of endogenous pluripotency genes and demethylation of pluripotency specific promoters. iPSCs are injected into severe combined immunodeficient (SCID) mice to confirm that they can form teratomas containing cells derived from all 3 embryonic germ layers. iPSCs are karyotyped to confirm stable chromosome integrity.
What is the value of iPSCs? Theoretically, any cell type can be differentiated from an iPSC obtained from patient cells. iPSC-derived cells also have immense value in drug research and development and offer unique starting material for regenerative medical interventions. iPSCs are particularly
6
useful for obtaining cells such as neurons and CMs, which are difficult to isolate from patients and have limited regenerative potential in vitro [11]. iPSCs can be stored frozen remaining viable for differentiation at a later time. iPSC-CMs have major advantages over primary isolated CMs including the ability to persist indefinitely in culture [12]. CMs obtained through endomyocardial biopsy are typically non-viable and therefore only useful for studying structural characteristics of CMs.
Combining genetic analyses and modeling disease with patient-specific iPSC-derived cells holds great potential to advance our understanding of the genetic causes of disease. For the patient, this allows their individual genetic background to be taken into account. In future it may also increase the ability for accurate pathogenicity classifications to be made with significant clinical implications. The use of functional models such as iPSCs to facilitate pathogenicity decisions is particularly valuable when there is a lack of family members for cosegregation studies [13].
Methods to advance the outcomes of studying human genetics with iPSCs are rapidly growing. One exciting avenue involves genome editing. Directed nucleases such as CRISPRs and TALENs allow specific gene mutations to be introduced and removed readily into iPSCs [14, 15]. Patients’ specific genomes can then be assessed with and without specific mutations of interest [16]. Isogenic controls overcome limitations of using close family members exhibiting or lacking mutations of interest as control subjects. Even amongst close family members, significant genetic differences exist which can alter the functional effects of mutations. Development of the most appropriate controls allows us to best understand the contribution of an individual’s genome to disease development. This is crucial in understanding the genetics that cause cardiomyopathies as the role of genetic modifiers and complex genetic interactions largely remain unknown.
7
iPSC-derived cells also facilitate the study of gene-environment interactions [17]. Exposure of iPSCs to hypertrophic factors illustrated that HCM iPSC-CMs are more susceptible to the hypertrophic factor endothelin-1 (ET-1) than control iPSC-derived CMs. These findings implicate the key role of an individual’s genetic background in their susceptibility to environmental factors that may promote hypertrophy. iPSC-CMs with a genetic predisposition to HCM were shown to be significantly more susceptible to ET-1 stimulated myofibrillar disarray and cell enlargement. A mild HCM phenotype was seen in HCM iPSCCMs that were not stimulated with ET-1 [17].
What are the current limitations of iPSCs? Several issues remain to be resolved before iPSCs can be used to model human disease with complete confidence. Major limitations surround producing a homogenous population of fully reprogrammed iPSCs [18]. To fully utilize iPSC-CM models, a large number of fully reprogrammed stable cells need to be produced. However, reprogramming somatic cells to iPSCs can result in variability due to culture conditions, retained epigenetic memory and genetic instability. Different somatic cells appear to have varying levels of cellular plasticity, resulting in a variable reprogramming efficiency and overall resemblance of an ES-cell state. Genetic background and age may also affect the resultant iPSCs; somatic cells isolated from younger patients may be more easily reprogrammed [19]. Issues with variability exist even within clones derived from a single patient and highlight the importance of appropriate controls and reproducible methods to identify interfering factors and biases.
8
MAKING CARDIOMYOCYTES FROM PATIENTS
How are the cells collected? Many somatic cells have been used as source material for reprogramming including pancreatic beta cells, hepatocytes, peripheral blood mononuclear cells, keratinocytes, renal epithelial cells and neural progenitor cells [20]. Reprogramming and differentiation efficiency is not equal amongst somatic cells. Currently, the reprogramming efficiency of keratinocytes and dermal fibroblasts surpasses that of peripheral blood mononuclear cells. Differences in efficiency and ease of sourcing, reprogramming and differentiation are important considerations.
How are iPSCs differentiated to myocardial lineages? In vitro differentiation of iPSC into CM follows the same developmental pathways as observed in embryonic development (summarized in Figure 1). First, iPSC must be differentiated into cells resembling the germ layer which gives rise to the heart, the mesoderm. These cells must then receive the unique set of specification and maturation signals required to obtain cardiac cell lineages. This differentiation process relies on stagespecific manipulation of embryonic development signaling pathways. Four signaling pathways central to cardiac differentiation include; BMP, TGFb/Activin/NODAL, WNT and FGF [21]. The two most common methods for myocardial iPSC differentiation are embryoid bodies formation and monolayer differentiation culture. Embryoid bodies are spherical aggregates of cells ideal for recapitulating embryonic development processes. Monolayer culture systems facilitate chemically defined manipulation of developmental pathways.
The timely and concentration specific addition of growth factors and small molecules is crucial in obtaining high levels of myocardial cells in culture. Importantly, no differentiation 9
methods produce solely CM pure cell populations and additional cell purification is typically required. Successful differentiation is confirmed through assessment of expression of cardiac markers including TNNT2 and MEF2C; loss of iPSC markers (OCT3/4, SOX2 and NANOG); and the identification of intact sarcomeres and spontaneous rhythmic contraction [21].
What are the current limitations of iPSC-CMs? While significant progress has been made in CM differentiation, with protocols now able to produce high CM yields and promote the differentiation of specific CM subtypes, the technology is still challenged by many limitations [20]. The limitations of iPSC-CMs are summarized in the literature [22] and include cell variability, limited maturity and abnormal functional characteristics.
Like variability present in iPSC clones, phenotypic variability is also seen in differentiated iPSC-CMs. Many factors contribute to this variability. For example some serum sources can mask features of disease in iPSC-CM models. Ideally, differentiation protocols will be chemically defined and free from use of highly variable animal products. Recent work has also shown that cell culture density can greatly alter cellular phenotype [23]. The development of more standardized differentiation approaches will likely reduce iPSC-CM variability. Methodological alterations such as growing cells as a monolayer have been shown to reduce phenotypic variability. A recent study showed that comparable calcium handling was present in iPSC-CMs derived from healthy individuals in 3 different university laboratories using multiple monolayer differentiation methods [24].
To date, iPSC-CMs have demonstrated a relatively immature phenotype. In addition to affecting interpretation of relevant results, immature iPSC-CMs have arrhythmogenic risks
10
that limit their use in regenerative medicine [25]. In comparison to adult human CMs, iPSC-CMs display fetal gene expression, are smaller in size, have disorganized sarcomeres, an absence of t-tubules, and diminished electrical and contractile function [26]. Many studies have sought to improve iPSC-CM maturity. Significant advances in cell maturation have been achieved through the culture of iPSC-CMs in 3-dimensional tissueengineered cardiac patches, highlighting the importance of the tissue microenvironment [27]. However, iPSC-CMs still remain relatively immature. In two weeks of culture, iPSCCMs had well-developed sarcomeres similar to isolated human adult human CMs, upregulation of key excitation-contraction coupling genes including CASQ2 and SERCA2 and an advanced sarcoplasmic reticulum, suggesting a more mature phenotype. The development of culturing techniques that better mimic the in vivo environment, taking into account complex cellular interactions will likely continue to aid the maturation of iPSC-CMs. Hopefully, increasing iPSC-CM maturity will aid the cessation of abnormal functional characteristics. In particular the tendency of iPSC-CMs to beat spontaneously, which is not a feature of adult ventricular CMs and likely related in part to their immature state [28].
11
iPSC MODELS OF GENETIC CARDIOMYOPATHIES The value of iPSCs has already been demonstrated in genetic heart diseases such as the primary inherited arrhythmia syndromes [29]. However, there is significant debate over whether iPSC-CM models can accurately recapitulate cardiac phenotypes which largely involve the whole 3-dimensional contracting heart. Cardiomyopathy studies to date have utilized relatively immature iPSC-CMs, which is a limitation that needs to be taken into account when interpreting results. Key studies in iPSC-CM models of inherited cardiomyopathies are summarized in Table 2. The main focus to date has been on the more common inherited cardiomyopathies such as HCM, DCM, and ARVC. To date, many of the underlying mechanisms of disease identified in iPSC-CMs have been previously identified in animal models or patient samples.
iPSC models of HCM HCM is a clinically and genetically heterogeneous heart disease with a prevalence of up to 1 in 200 people [30]. HCM is diagnosed primarily on discovery of unexplained left ventricular hypertrophy and is characterized by both clinical and genetic heterogeneity [31]. HCM is primarily inherited in an autosomal dominant inheritance pattern; offspring have a 50% chance of inheriting a pathogenic mutation. Over 1500 causal mutations have been identified. HCM can be viewed as a disease of the sarcomere. The majority of mutations are in 4 sarcomere genes; cardiac myosin-binding protein C (MYBPC3), b-myosin heavy chain (MYH7), cardiac troponin T type 2 (TNNT2) and cardiac troponin I type 3 (TNNI3) [2]. The identification of pathogenic mutations is important as it facilitates familial cascade screening [2, 32]. However, pathogenic mutations have been identified in only 40-50% of HCM patients.
12
The pathogenesis of HCM is poorly understood. Studies with animal models and nonviable human cardiac tissue have shown that during the hypertrophic response CMs enlarge and switch gene expression towards a fetal expression profile [6]. Functional abnormalities identified in HCM murine models include increased calcium sensitivity due to an abnormal high intracellular calcium concentration, increased contraction force and electrical abnormalities [5, 6, 33].
iPSC-CMs can be used to recapitulate characteristics of the HCM phenotype in vitro [11, 17,
34,
35].
HCM-specific
changes
include
cellular
enlargement,
sarcomere
disorganization, and alterations in electrophysiological behavior, gene expression and calcium handling (Table 2). Many of these changes are only identifiable after cells were cultured for a significant period of time following differentiation, perhaps highlighting the importance of cell maturity [11]. HCM iPSC-CMs have exhibited molecular mechanisms consistent with those identified in animal models and have allowed the investigation of the functional impact of pathogenic mutations.
Molecular mechanisms underlying HCM The pathogenic mechanisms underlying the development of HCM in 2 families with pathogenic mutations in MYH7 have been investigated in-depth. iPSC-CMs derived from 10 members of a family affected by HCM were characterized. Five family members carried a missense mutation p.Arg663His in MYH7 [11]. HCM iPSC-CMs exhibited structural and functional characteristics in line with the HCM phenotype. Structural abnormalities included cellular enlargement, increased multinucleation and myofibril content and an increased proportion of cells with disorganized sarcomeres. Functional abnormalities included increased intracellular calcium concentration and increased expression of HCM-related genes and hypertrophy-related proteins. Calcium dysregulation occurred prior to the onset
13
of the hypertrophic phenotype and calcium channel antagonists were able to prevent HCM development suggesting that calcium dysregulation is causal of, rather than a product of, HCM [11].
iPSC-CMs derived from a patient with HCM caused by the MYH7 mutation p.Arg442Gly have been reported [34]. HCM iPSC-CMs exhibited structural abnormalities consistent with the HCM phenotype. Similar calcium handling abnormalities were identified [34], consistent with observations made from animal models [5]. Calcium channel antagonists were able to ameliorate characteristics of the HCM phenotype. An increase in HCM and fibrosis-related genes was identified [34]. Genes involved in cell proliferation were upregulated linking cell proliferation and movement in the pathogenesis of HCM. Both studies implicated the central role of NFAT transcription factors, similar to murine HCM models, which implicate the involvement of NFAT transcription factors in HCM [17, 35].
Determining the pathogenicity of mutations The iPSC studies mentioned above have provided evidence to confirm the pathogenicity of two MYH7 mutations. Significant differences in functional and structural characteristics were observed between HCM iPSC-CMs derived from gene positive and gene negative family members [11]. In addition, a HCM phenotype was observed following overexpression of the p.Arg663His MYH7 mutation in normal human ESC-CMs thus ruling out the possibility that another rare mutation present in this family is responsible for disease.
Cardiotoxicity and efficacy of pharmaceutical compounds HCM iPSC-CMs have been utilized to study patient-specific responses to current and novel HCM therapies [11, 34, 35]. Drugs currently used for the treatment of HCM including
14
metoprolol, verapamil and pinacidil, all demonstrated beneficial effects in HCM iPSC-CMs. The use of novel inhibitors of histone deacetylase activity specifically Trichostatin A in HCM therapy is supported by HCM iPSC-CM studies [34]. Trichostatin A significantly ameliorated the HCM phenotype, particularly calcium handling abnormalities, NFATC4 nuclear translocation and CM enlargement [34]. Clinical trials are required to determine if these findings translate into improved clinical treatment. These results highlight the potential utility of iPSC-CM modeling in drug research and development.
iPSC models of DCM DCM is characterized by left ventricle dilation, systolic dysfunction and heart failure. Systemic diseases such as coronary artery disease explain approximately 70% of DCM cases, with the remaining 30% referred to as “idiopathic DCM” [36]. Major adverse events include heart failure, arrhythmias and sudden cardiac death. Histopathological features of DCM are highly variable but commonly include CM loss, myocyte disarray and interstitial fibrosis. Like HCM, the process from genetic mutation to presentation of clinical DCM is poorly understood.
A causal mutation, supporting inherited DCM, is identified in approximately 20-30% of patients with idiopathic DCM [37]. Inherited DCM is a genetically heterogeneous disease. Mutations have been identified in greater than 30 genes encoding sarcomere, cytoskeletal, mitochondrial and nuclear membrane proteins [38]. DCM is primarily inherited in an autosomal dominant inheritance pattern, and the most common disease gene in DCM is Titin (TTN) [2, 37].
Four studies have demonstrated that iPSC-CM models can recapitulate key characteristics of the DCM disease phenotype in vitro (Table 2) [13, 35, 38, 39]. Features of DCM that are
15
identifiable in iPSC-CM models include loss of sarcomere integrity, cell organization, contraction force and normal calcium handling.
Molecular mechanisms underlying DCM Three studies have assessed the molecular mechanisms that underlie DCM using patientspecific iPSC-CMs [13, 38, 39]. iPSC-CMs were derived from a 7-member family with severe DCM caused by the mutation p.Arg173Trp in TNNT2 [38]. Compared with control iPSC-CMs, DCM iPSC-CMs exhibited significantly diminished sarcomere integrity, with abnormal α-actinin distribution and disorganized z-lines with scattered condensed z-bodies. DCM iPSC-CMs also exhibited abnormal calcium handling; DCM iPSC-CMs had smaller intracellular calcium transients and diminished sarcoplasmic reticulum calcium storage. Consistent with animal models, DCM iPSC-CMs displayed weaker contraction forces [38].
iPSC-CMs have been reported from a patient with DCM thought to be the result of a mutation p.Ala285Val in DES [13]. DES encodes the desmin protein, an intermediate filament critical for maintenance of the sarcomere structure. DCM iPSC-CMs exhibited poor co-localization of DES with cardiac TNNT2, α-actinin and F-actin. In addition, zbodies were abnormally formed and more than 70% of cells contained isolated aggregates of DES-positive proteins. Disruption of calcium handling was also observed in DES-mutant cells suggesting that calcium dysregulation is a key characteristic of DCM. Functional impacts of the p.Ala285Val mutation in DES, specifically diffuse abnormal DES protein aggregation, were also observed in HEK293 cells overexpressing mutant protein.
In
contrast to results reported previously [38] and control iPSC-CMs, a slower spontaneous beat frequency was observed in DCM iPSC-CMs, which may suggest that DCM resulting from mutations in DES cause specific functional abnormalities.
16
iPSC-CMs were produced from 3 DCM patients with TTN mutations; 2 A-band truncating mutations, p.Ala22352fs and p.Pro22582fs, and 1 missense mutation p.Trp976Arg in the Z/I junction [39]. Due to the large and complex nature of the TTN gene there are many mutations of unknown significance. The majority of truncating mutations that cause DCM are located within the A band. Patient and control iPSC-CMs were cultured in more advanced 3-dimensional cardiac microtissues as differences in contraction forces in isolated patient iPSC-CMs and control iPSC-CMs were not significant, highlighting the limitations of single cell models. Patient iPSC cardiac microtissues exhibited less than half the contractile and stress force per tissue area, compared with control iPSC cardiac microtissues. Additional analyses revealed that TTN truncating mutations result in abnormal CM signaling and diminished expression of key cardiomyopathy gene transcripts, ultimately causing sarcomere insufficiency.
Determining the pathogenicity of mutations Two studies utilized DCM patient iPSC-CMs to determine mutation pathogenicity. In the first study, patient iPSC-CMs were used to determine the pathogenicity of a mutation in DES identified through whole exome sequencing [13]. Characteristics of the DCM phenotype were absent in control iPSCs with wild-type DES, and present in both patient iPSC-CMs and control iPSC-CMs transduced with the mutant DES gene, supporting the pathogenicity of the mutation identified in the patient.
In the second study, patient iPSC-CMs were utilized to not only determine the pathogenicity of a missense mutation, p.Trp792Arg in TTN, however also to further understand the pathogenicity of groups of mutations located at specific sites within TTN [39]. Through combining functional analyses of iPSC-CM cardiac microtissues with RNAseq data derived from iPSC-CMs, the authors were able to reveal that alternative exon
17
splicing is responsible for truncating mutations in the I band of TTN having reduced pathogenicity compared with truncating mutations located within the A band. Thus, providing an explanation for the increased prevalence of truncating TTN mutations in the I band in the general population compared with truncating TTN mutations in the A band, which are highly prevalent in DCM cohorts. In addition, DCM severity due to truncating mutations in patient iPSC-CMs was greater than that of equivalent truncating mutations expressed in control iPSC-CMs, which may suggest that genetic modifiers contribute to the DCM severity due to truncation mutations in TTN. This study provides a nice example of how iPSC-CM models can help us to explore the involvement of complex genetics in cardiomyopathy development.
Applications in DCM therapy and drug testing DCM iPSC-CMs have been used to assess the efficacy of a gene therapy approach. Overexpression
of
SERCA2A
reduced
DCM-related
cellular
abnormalities
[38].
Overexpression of SERCA2A rescued the DCM phenotype at the single cell level, reinstating effective contraction force [38]. Metoprolol significantly reduced the number of DCM iPSC-CMs with disrupted sarcomere integrity. DCM iPSC-CMs had increased propensity for arrhythmias compared with control or HCM iPSC-CMs when treated with the vasodilator, nicorandil [35].
iPSC models of ARVC ARVC is a primary cardiomyopathy with clinically heterogeneous presentation. Most commonly seen in men, ARVC has an estimated prevalence of approximately 1 in 5000 [40]. However, ARVC prevalence varies globally and in some regions is estimated to be as high as 1 in 500 [41]. ARVC is characterized by ventricular arrhythmias, right ventricle dysfunction and sudden cardiac death. Histopathological features of ARVC include fibro18
fatty infiltration of the myocardium, primarily involving the right ventricle. ARVC is primarily inherited in an autosomal dominant inheritance pattern. A pathogenic mutation is identified in approximately 30-50% of patients [42]. ARVC penetrance and severity is highly variable and may be altered by the presence of multiple mutations [43]. Complex genetics, involving multiple mutations, are often thought to be involved in the development of ARVC. ARVC is thought to be a disease of the desmosome. Genes that encode the desmosome including plakophilin 2 (PKP2), desmoglein 2 (DSG2), desmoplakin (DSP), desmocollin 2 (DSC2) and plakoglobin (JUP), are commonly implicated [42]. Mutations in PKP2 are the most common cause of ARVC [2].
Three studies have utilized patient iPSC-CMs to model ARVC. In all 3 studies ARVC was due to pathogenic mutations in PKP2 (summarized in Table 2) [4, 44, 45]. iPSC-CM models were successfully used to recapitulate important characteristics of the ARVC phenotype including abnormalities in desmosome structure and reduced expression of desmosome-related genes and protein density.
Molecular mechanisms underlying ARVC iPSC-CMs derived from 2 patients with ARVC caused by frameshift mutations p.Ala325Cysfs*11 and p.Thr50Serfs*61 in PKP2 have been reported [4]. ARVC iPSC-CMs derived from both patients exhibited a significant reduction in PKP2 gene expression and density of desmosome and gap junction proteins plakophilin-2, plakoglobin and connexin43. Significant abnormalities in the desmosome structure were also observed. In a separate study, iPSC-CMs derived from a patient with ARVC due to the p.Leu614Pro missense mutation in PKP2 were studied [45]. Reduced PKP2 and JUP gene expression was identified. Consistent with the previous study, reduced density of plakophilin-2 and plakoglobin proteins was observed. Abnormalities in cell structure, particularly involving z-
19
bands and desmosomes were also identified, with ARVC iPSC-CMs overall appearing less organized [45].
Determining the pathogenicity of mutations iPSC-CM models have been utilized to confirm the pathogenic effects of mutations in PKP2 [44]. Evidence for the pathogenicity of the mutation p.Gly894Gly in PKP2 was obtained using iPSC-CMs derived from a patient with ARVC. The ARVC phenotype demonstrated by iPSC-CMs was ameliorated following mutant cell transduction with wildtype PKP2. Dysregulated calcium handling was also implicated in the development of ARVC. Methods to induce adult-like metabolism, which accelerate adult-onset disease and the development of mature iPSC-CMs further supported the pathogenicity of the PKP2 mutation.
Collectively, the iPSC-CM models developed in HCM, DCM and ARVC (Table 2) reflect the potential use of such models for understanding disease mechanisms, providing functional evidence to support pathogenicity of mutations identified in patients, and evaluating potential disease therapies (Figure 2).
20
iPSCs IN CLINICAL PRACTICE: NOW AND IN THE FUTURE
iPSC technology has profound implications to clinical practice. From assisting in mutation classification as part of cardiac genetic testing, to the development of new therapies, and personalizing treatment to individual patients, the potential impact of the role of iPSCs in clinical medicine is far reaching (Figure 2).
There are major benefits in assessing the efficacy of therapeutic treatments in patientspecific iPSC-CMs. Optimal therapeutic strategies can be designed for individual patients. An effective treatment approach for a patient with long QT syndrome was identified following testing two different frequently used clinical strategies in patient-specific iPSCCMs [46]. In this particular case, the therapy comparison was not tested in the patient and so the direct impact on clinical care was not established. In future, it is likely that iPSC-CM models will aid the optimization of patient therapy by enabling specific individuals’ pathogenic processes to be taken into account.
Drugs that have been developed in animal models primarily examine the population as a whole and assume a relatively consistent response across patients. iPSC technologies provide a new opportunity for patient-specific toxicity testing, allowing a patient’s individual risk of toxicity to be assessed. iPSC-CMs can be used to assess the cellular effects of clinical therapies and determine patient-specific drug toxicity [35]. The occurrence of arrhythmias and prolonged action potential duration, resulting from drug administration was measured on patient iPSC-CMs. iPSC-CMs from patients with long QT syndrome and HCM were far more susceptible to toxicity than iPSC-CMs from DCM patients or healthy controls [25]. The significance of taking into account multiple ion channels is demonstrated by cardiotoxicity testing of the drug Alfuzosin, where the drug did not have cardiotoxic
21
effects as it does not act on hERG potassium channels. However, Alfuzosin does have cardiotoxic effects as it interacts with other ion channels resulting in a prolonged QT interval [35].
iPSC studies may also provide opportunities for regenerative medicine approaches to treat cardiovascular disease. The main goal of using iPSCs in regenerative medicine is to restore the function of organs and tissues. Theoretically, iPSC-CMs are ideal for improving cardiac function, as they are patient-specific. However, there are some issues with immunogenicity that can arise with iPSC use and whether patient iPSC-CMs can successfully integrate into adult myocardium remains to be determined. In addition, there is possibility of teratoma formation if iPSCs maintain pluripotency gene expression. Nevertheless, the tantalizing opportunity remains that patients could be “genetically rescued”, i.e. their own iPSC-CMs could undergo gene editing to remove the diseasecausing mutation, then “healthy” CMs injected back into the patient. With transplants of iPSC-derived cell types into patients already taking place, we anticipate new challenges along with the potential for great success in application of iPSCs to regenerative medicine.
22
CONCLUSIONS
Understanding how mutations in genes lead to human disease is a fundamental basis to improve the care of patients. Inherited cardiomyopathies are characterized by diversity in both the genes which cause disease, and the variable clinical phenotypes. Multiple genetic and environmental factors are likely to play a role in explaining this genetic and clinical heterogeneity. While iPSC-CM technology remains limited, iPSC models of inherited cardiomyopathies will likely afford us the opportunity to better understand disease mechanisms and phenotypes, and provide us with the opportunity to create novel and personalized therapeutic strategies targeted at the gene mutation level. iPSC studies of inherited cardiomyopathies to date provide confidence that the next decade will see the translation of iPSC-based models of human disease to improve our knowledge about the inherited cardiomyopathies, and how we can optimize the clinical management of our patients.
23
REFERENCES [1] Wilde AA, Behr ER. Genetic testing for inherited cardiac disease. Nat Rev Cardiol. 2013;10:571-83. [2] Ingles J, Semsarian C. The value of cardiac genetic testing. Trends Cardiovasc Med. 2014;24:217-24. [3] Semsarian C, Ingles J. Determining pathogenicity in cardiac genetic testing: Filling in the blank spaces. Trends Cardiovasc Med. 2015. [4] Caspi O, Huber I, Gepstein A, Arbel G, Maizels L, Boulos M, et al. Modeling of arrhythmogenic right ventricular cardiomyopathy with human induced pluripotent stem cells. Circ Cardiovasc Genet. 2013;6:557-68. [5] Semsarian C, Ahmad I, Giewat M, Georgakopoulos D, Schmitt JP, McConnell BK, et al. The L-type calcium channel inhibitor diltiazem prevents cardiomyopathy in a mouse model. J Clin Invest. 2002;109:1013-20. [6] Seidman JG, Seidman C. The genetic basis for cardiomyopathy: from mutation identification to mechanistic paradigms. Cell. 2001;104:557-67. [7] Semsarian C, Healey MJ, Fatkin D, Giewat M, Duffy C, Seidman CE, et al. A polymorphic modifier gene alters the hypertrophic response in a murine model of familial hypertrophic cardiomyopathy. J Mol Cell Cardiol. 2001;33:2055-60. [8] Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, et al. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282:1145-7. [9] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131:86172. [10] Lister R, Pelizzola M, Kida YS, Hawkins RD, Nery JR, Hon G, et al. Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature. 2011;471:68-73. [11] Lan F, Lee AS, Liang P, Sanchez-Freire V, Nguyen PK, Wang L, et al. Abnormal calcium handling properties underlie familial hypertrophic cardiomyopathy pathology in patient-specific induced pluripotent stem cells. Cell Stem Cell. 2013;12:101-13. 24
[12] Ivashchenko CY, Pipes GC, Lozinskaya IM, Lin Z, Xiaoping X, Needle S, et al. Human-induced pluripotent stem cell-derived cardiomyocytes exhibit temporal changes in phenotype. Am J Physiol Heart Circ Physiol. 2013;305:H913-22. [13] Tse HF, Ho JC, Choi SW, Lee YK, Butler AW, Ng KM, et al. Patient-specific inducedpluripotent stem cells-derived cardiomyocytes recapitulate the pathogenic phenotypes of dilated cardiomyopathy due to a novel DES mutation identified by whole exome sequencing. Hum Mol Genet. 2013;22:1395-403. [14] Hockemeyer D, Wang H, Kiani S, Lai CS, Gao Q, Cassady JP, et al. Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol. 2011;29:731-4. [15] Ding Q, Regan SN, Xia Y, Oostrom LA, Cowan CA, Musunuru K. Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs. Cell Stem Cell. 2013;12:393-4. [16] Tanaka A, Yuasa S, Node K, Fukuda K. Cardiovascular Disease Modeling Using Patient-Specific Induced Pluripotent Stem Cells. Int J Mol Sci. 2015;16:18894-922. [17] Tanaka A, Yuasa S, Mearini G, Egashira T, Seki T, Kodaira M, et al. Endothelin-1 induces
myofibrillar
disarray
and
contractile
vector
variability
in
hypertrophic
cardiomyopathy-induced pluripotent stem cell-derived cardiomyocytes. J Am Heart Assoc. 2014;3:e001263. [18] Paull D, Sevilla A, Zhou H, Hahn AK, Kim H, Napolitano C, et al. Automated, highthroughput derivation, characterization and differentiation of induced pluripotent stem cells. Nat Methods. 2015;12:885-92. [19] Mack DL, Guan X, Wagoner A, Walker SJ, Childers MK. Disease-in-a-dish: the contribution of patient-specific induced pluripotent stem cell technology to regenerative rehabilitation. Am J Phys Med Rehabil. 2014;93:S155-68. [20] Fuerstenau-Sharp M, Zimmermann ME, Stark K, Jentsch N, Klingenstein M, Drzymalski M, et al. Generation of highly purified human cardiomyocytes from peripheral blood
mononuclear
cell-derived
induced
pluripotent
stem
cells.
PLoS
One.
2015;10:e0126596.
25
[21] Burridge PW, Diecke S, Matsa E, Sharma A, Wu H, Wu JC. Modeling Cardiovascular Diseases with Patient-Specific Human Pluripotent Stem Cell-Derived Cardiomyocytes. Methods Mol Biol. 2016;1353:119-30. [22] Knollmann BC. Induced pluripotent stem cell-derived cardiomyocytes: boutique science or valuable arrhythmia model? Circ Res. 2013;112:969-76; discussion 76. [23] Du DT, Hellen N, Kane C, Terracciano CM. Action potential morphology of human induced pluripotent stem cell-derived cardiomyocytes does not predict cardiac chamber specificity and is dependent on cell density. Biophys J. 2015;108:1-4. [24] Hwang HS, Kryshtal DO, Feaster TK, Sanchez-Freire V, Zhang J, Kamp TJ, et al. Comparable calcium handling of human iPSC-derived cardiomyocytes generated by multiple laboratories. J Mol Cell Cardiol. 2015;85:79-88. [25] Zhang YM, Hartzell C, Narlow M, Dudley SC, Jr. Stem cell-derived cardiomyocytes demonstrate arrhythmic potential. Circulation. 2002;106:1294-9. [26] Zhang D, Shadrin IY, Lam J, Xian HQ, Snodgrass HR, Bursac N. Tissue-engineered cardiac patch for advanced functional maturation of human ESC-derived cardiomyocytes. Biomaterials. 2013;34:5813-20. [27] Chun YW, Balikov DA, Feaster TK, Williams CH, Sheng CC, Lee JB, et al. Combinatorial polymer matrices enhance in vitro maturation of human induced pluripotent stem cell-derived cardiomyocytes. Biomaterials. 2015;67:52-64. [28] Kim JJ, Yang L, Lin B, Zhu X, Sun B, Kaplan AD, et al. Mechanism of automaticity in cardiomyocytes derived from human induced pluripotent stem cells. J Mol Cell Cardiol. 2015;81:81-93. [29] Priori SG, Napolitano C, Di Pasquale E, Condorelli G. Induced pluripotent stem cellderived cardiomyocytes in studies of inherited arrhythmias. J Clin Invest. 2013;123:84-91. [30] Semsarian C, Ingles J, Maron MS, Maron BJ. New Perspectives on the Prevalence of Hypertrophic Cardiomyopathy. J Am Coll Cardiol. 2015;65:1249-54. [31] Maron BJ, Maron MS, Semsarian C. Genetics of hypertrophic cardiomyopathy after 20 years: clinical perspectives. J Am Coll Cardiol. 2012;60:705-15.
26
[32] Ingles J, Semsarian C. Conveying a probabilistic genetic test result to families with an inherited heart disease. Heart Rhythm. 2014;11:1073-8. [33] Tsoutsman T, Kelly M, Ng DC, Tan JE, Tu E, Lam L, et al. Severe heart failure and early mortality in a double-mutation mouse model of familial hypertrophic cardiomyopathy. Circulation. 2008;117:1820-31. [34] Han L, Li Y, Tchao J, Kaplan AD, Lin B, Li Y, et al. Study familial hypertrophic cardiomyopathy using patient-specific induced pluripotent stem cells. Cardiovasc Res. 2014;104:258-69. [35] Liang P, Lan F, Lee AS, Gong T, Sanchez-Freire V, Wang Y, et al. Drug screening using a library of human induced pluripotent stem cell-derived cardiomyocytes reveals disease-specific patterns of cardiotoxicity. Circulation. 2013;127:1677-91. [36] Hershberger RE, Hedges DJ, Morales A. Dilated cardiomyopathy: the complexity of a diverse genetic architecture. Nat Rev Cardiol. 2013;10:531-47. [37] Herman DS, Lam L, Taylor MR, Wang L, Teekakirikul P, Christodoulou D, et al. Truncations of titin causing dilated cardiomyopathy. N Engl J Med. 2012;366:619-28. [38] Sun N, Yazawa M, Liu J, Han L, Sanchez-Freire V, Abilez OJ, et al. Patient-specific induced pluripotent stem cells as a model for familial dilated cardiomyopathy. Sci Transl Med. 2012;4:130ra47. [39] Hinson JT, Chopra A, Nafissi N, Polacheck WJ, Benson CC, Swist S, et al. HEART DISEASE. Titin mutations in iPS cells define sarcomere insufficiency as a cause of dilated cardiomyopathy. Science. 2015;349:982-6. [40] Romero J, Mejia-Lopez E, Manrique C, Lucariello R. Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC/D): A Systematic Literature Review. Clin Med Insights Cardiol. 2013;7:97-114. [41] Etchegary H, Pullman D, Simmonds C, Young TL, Hodgkinson K. 'It had to be done': genetic testing decisions for arrhythmogenic right ventricular cardiomyopathy. Clin Genet. 2015;88:344-51.
27
[42] Te Rijdt WP, Jongbloed JD, de Boer RA, Thiene G, Basso C, van den Berg MP, et al. Clinical utility gene card for: arrhythmogenic right ventricular cardiomyopathy (ARVC). Eur J Hum Genet. 2014;22. [43] Quarta G, Muir A, Pantazis A, Syrris P, Gehmlich K, Garcia-Pavia P, et al. Familial evaluation in arrhythmogenic right ventricular cardiomyopathy: impact of genetics and revised task force criteria. Circulation. 2011;123:2701-9. [44] Kim C, Wong J, Wen J, Wang S, Wang C, Spiering S, et al. Studying arrhythmogenic right ventricular dysplasia with patient-specific iPSCs. Nature. 2013;494:105-10. [45] Ma D, Wei H, Lu J, Ho S, Zhang G, Sun X, et al. Generation of patient-specific induced
pluripotent
stem
cell-derived
cardiomyocytes
as
a
cellular
model
of
arrhythmogenic right ventricular cardiomyopathy. Eur Heart J. 2013;34:1122-33. [46] Terrenoire C, Wang K, Tung KW, Chung WK, Pass RH, Lu JT, et al. Induced pluripotent stem cells used to reveal drug actions in a long QT syndrome family with complex genetics. J Gen Physiol. 2013;141:61-72.
28
TABLES
Table 1: Glossary of Terms
·
Pluripotent: capable of giving rise to all different cell and tissue types with the exception of the placenta.
·
Multipotent: capable of giving rise to multiple different cell types of the same lineage.
·
Totipotent: capable of giving rise to all different cell and tissue types including the placenta and embryo.
·
Differentiation: is the process of a cell changing from one cell type to another.
·
Reprogramming: is the conversion of one specific cell type to another. Specifically, direct reprogramming is the conversion of a somatic cell type, such as a fibroblast, to a pluripotent cell, i.e. iPSC.
29
Table 2. Summary of iPSC-based models of inherited cardiomyopathies Disease
Reason for Study
Gene (Mutation)
Somatic Cell Line
Functional Impact
Reference
HCM
Identify patientspecific and mutationspecific mechanisms of HCM
MYH7 (Arg442Gly)
Dermal Fibroblasts
Enlarged size, disorganized sarcomere structures and arrhythmic beatings. Irregular calcium handling and ion channel functions. Increased expression of HCM related genes.
[34]
HCM
Elucidate the mechanisms underlying HCM pathogenesis
MYH7 (Arg663His)
Dermal fibroblasts
Cellular enlargement, multinucleation and contractile arrhythmia (single cell level).
[11]
HCM
Geneticenvironment interactions that contribute to the development of HCM
3 patients: 2 no mutation, 1 MYBPC3 Gly999NGln1004Del
Dermal fibroblasts and T lymphocytes
Enlarged cells with myofibrillar disarray. Increased myofibrillar disarray and cell size following treatment with ET-1 (hypertrophic factor).
[17]
DCM
Develop iPSC-CMs to recapitulate DCM phenotype and investigate underlying mechanisms
TNNT2 (Arg173Trp)
Fibroblasts
Elimination of sarcomeric alignment and reduced calcium handling.
[21]
DCM
Develop iPSC-CMs to recapitulate DCM phenotype and investigate underlying mechanisms
TNNT2 (Arg173Trp)
Dermal fibroblasts
Altered calcium handling, decreased contractility and abnormal sarcomeric a-actinin distribution.
[38]
DCM
Provide functional and histological evidence for the pathogenicity of DES mutation
DES (Ala258Val)
Dermal fibroblasts
Structural and functional abnormalities resulting from abnormal formation of DESaggregates. Functional abnormalities such as diminished max rate of calcium re-uptake, and structural abnormalities include poor localization of cardiac troponin T.
[13]
DCM
Evaluate the pathogenicity and functional impacts of truncating and
3 patients: TTN (Ala22352fs, Pro22582fs and Trp976Arg)
T cells
Contractile deficits, stable truncated peptide unable to assemble with other contractile proteins,, sarcomere insufficiency Attenuated cell signaling limiting CM response to mechanical and adrenergic stress
[39]
30
missense mutations in TTN ARVC
Establish a patientspecific iPSC-CM model for ARVC
PKP2 (Leu614Pro)
Dermal fibroblasts
Abnormal and highly variable zlines (thicker) with decreased PKP2 and JUP gene expression, and decreased plakophilin 2 and plakoglobin protein at cell periphery.
[45]
ARVC
Establish a patientspecific iPSC-CM model for ARVC
2 patients: PKP2 mutations: (Ala325Cysfs*11) & (Thr50Serfs*61)
Dermal fibroblasts
Abnormalities include increased cytoplasmic accumulation of lipid, distortion of desmosomes, and functional reduction in PKP2 gene and protein expression.
[4]
ARVC
Identify mechanisms of ARVC and identify novel therapeutic strategies
2 patients: 1 homozygous PKP2 mutation (Gly828Gly) and 1 heterozygous PKP2 mutation (Lys672Glyfs*70)
Dermal fibroblasts
Functional abnormalities include abnormal nuclear translocation of plakoglobin proteins resulting in increased lipogenesis, abnormal calcium handling), electrophysiological changes.
[44]
31
FIGURE LEGENDS
Figure 1: Generation of iPSCs from patients, then the differentiation to cardiomyocytes, then to their use in different cardiomyopathies such as HCM, DCM, and ARVC.
Figure 2: Summary of the value and potential of iPSCs in human biology and disease.
32
FIGURES
Figure 1
33
Figure 2
34
Induced pluripotent stem cells in the inherited cardiomyopathies: From disease mechanisms to novel therapies Samantha Barratt Ross, Stuart T. Fraser, Christopher Semsarian
www.elsevier.com/locate/tcm
PII: DOI: Reference:
S1050-1738(16)30031-7 http://dx.doi.org/10.1016/j.tcm.2016.05.001 TCM6285
To appear in: trends in cardiovascular medicine
Cite this article as: Samantha Barratt Ross, Stuart T. Fraser, Christopher Semsarian, Induced pluripotent stem cells in the inherited cardiomyopathies: From disease mechanisms to novel therapies, trends in cardiovascular medicine, http://dx.doi.org/10.1016/j.tcm.2016.05.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Induced Pluripotent Stem Cells in the Inherited Cardiomyopathies: From Disease Mechanisms to Novel Therapies
Samantha Barratt Rossa,b, Stuart T. Fraserc, Christopher Semsariana,b,d
a
Agnes Ginges Centre for Molecular Cardiology, Centenary Institute, Newtown b
Sydney Medical School, University of Sydney, Sydney c
Disciplines of Physiology, Anatomy & Histology,
School of Medical Sciences, University of Sydney d
Department of Cardiology, Royal Prince Alfred Hospital, Sydney, Australia
Running title: iPSCs in inherited cardiomyopathies
ACKNOWLEDGEMENTS CS is the recipient of a National Health and Medical Research Council (NHMRC) Practitioner Fellowship (#1059156). SBR is the recipient of an Australian Postgraduate Award.
DISCLOSURES: None
Address for Correspondence: Professor Christopher Semsarian Agnes Ginges Centre for Molecular Cardiology Centenary Institute Locked Bag 6 Newtown, NSW, 2042 Australia Ph: 61 2 9565 6195 Email: [email protected]
ABSTRACT Inherited cardiomyopathies lead to diverse clinical outcomes including heart failure, arrhythmias and sudden death. Mutations in over 100 genes have been implicated in the pathogenesis of genetic heart diseases, including the main inherited cardiomyopathies, such as hypertrophic, dilated, and arrhythmogenic right ventricular cardiomyopathies. Understanding how these gene mutations lead to clinical disease and the various secondary genetic and environmental factors which may modify the clinical phenotype, are key areas of research ultimately influencing diagnosis and management of patients. The emergence of patient-derived induced pluripotent stem cells (iPSCs), which can be differentiated into functional cardiomyocytes (CMs) in vitro, may provide an exciting new approach to understand disease mechanisms underpinning inherited heart diseases. This review will focus specifically on the key role of iPSC-based studies in the inherited cardiomyopathies, both in their potential utility as well as the significant challenges they present.
Key words: cardiomyopathy; genetic; induced pluripotent stem cell; inherited
2
ABBREVIATIONS
iPSC – induced pluripotent stem cell ESC – embryonic stem cell CM - cardiomyocyte HCM – hypertrophic cardiomyopathy DCM – dilated cardiomyopathy ARVC – arrhythmogenic right ventricular cardiomyopathy ET-1 - endothelin-1
3
INTRODUCTION Inherited cardiomyopathies, including hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM) and arrhythmogenic right ventricular cardiomyopathy (ARVC), have a significant global burden with common adverse events including arrhythmias, heart failure
and
sudden
cardiac
death.
The
main
clinical
outcomes
of
inherited
cardiomyopathies are well understood. Over the past 25 years, there have been major advances in our understanding of the genetic basis of inherited cardiomyopathies with mutations in over 100 genes identified in a variety of cardiac genetic diseases [1]. The identification of these genetic causes has translated into improved diagnosis, treatment and prognosis in cardiac genetic diseases, as well as in cascade genetic testing of family members [2].
While our understanding of the genetic causes of cardiac diseases has progressed, understanding how gene mutations lead to diverse clinical outcomes, and disentangling the other secondary genetic or environmental factors that may modify the phenotype, remains largely unknown. When a genetic mutation is identified in an individual, proving causality is often challenging and relies on a number of factors, one of which is functional evidence to support pathogenicity [3]. The challenge of performing mechanistic and functional studies is in part due to difficulties in obtaining human cardiac cells from patients in useful quantities, which is restricted by the limited regenerative potential of cardiac cells [4]. Functional studies have been largely limited to animal models. The use of animal models, particularly murine models, has facilitated a better understanding of disease myocyte biology however time, cost and species differences hamper their suitability [5-7]. Importantly, differences in functional characteristics including electrophysiology and calcium handling significantly limit the use of murine models in cardiac research.
4
Multipotent adult stem cells have limited differentiation capacity for cardiac differentiation (Table 1). The isolation of pluripotent human embryonic stem cells (ESCs), which have the ability to differentiate into all somatic cells and indefinitely replicate, in 1998 led to the development of human cardiomyocyte (CM) differentiation models [8]. Seminal work by Yamanaka and Takahashi in 2007 re-programmed somatic cells into pluripotent cells similar to human ESCs [9]. These cells, termed induced pluripotent stem cells (iPSCs), have revolutionized the fields of human disease modeling, genetics and personalized and regenerative medicine. The current review discusses both the utility and challenges of human iPSCs; the generation of patient-specific CMs; and the use of iPSC-CMs for cardiomyopathy-related applications, such as disease modeling.
5
BASICS OF INDUCED PLURIPOTENT STEM CELLS (iPSCs)
What are iPSCs? iPSCs are embryonic stem-like cells that have been reprogrammed from somatic cells. iPSCs can be derived from human patient cells, most commonly skin or blood cells (Figure 1). Similar to human ESCs, iPSCs self-renew and can differentiated into cells of all three embryonic germ layers: endoderm, mesoderm and ectoderm [9]. For the most part, human ESCs and iPSCs are functionally equivalent [9]. However, epigenetic differences do exist in iPSCs, particularly pertaining to DNA methylation patterns [10].
How are iPSCs generated? iPSCs are reprogrammed from somatic cells, typically using a transfection vector and a well-defined
set
of
embryonically
expressed
pluripotency
factors.
Historically
reprogramming has involved viral transfection of somatic cells with the four “Yamanaka factors” OCT-4, SOX-2, Klf-4 and c-myc [9]. New approaches to reprogramming are moving away from integrating viral vectors. Following reprogramming, the pluripotent state of iPSCs is confirmed by assessment of pluripotency marker expression, activation of endogenous pluripotency genes and demethylation of pluripotency specific promoters. iPSCs are injected into severe combined immunodeficient (SCID) mice to confirm that they can form teratomas containing cells derived from all 3 embryonic germ layers. iPSCs are karyotyped to confirm stable chromosome integrity.
What is the value of iPSCs? Theoretically, any cell type can be differentiated from an iPSC obtained from patient cells. iPSC-derived cells also have immense value in drug research and development and offer unique starting material for regenerative medical interventions. iPSCs are particularly
6
useful for obtaining cells such as neurons and CMs, which are difficult to isolate from patients and have limited regenerative potential in vitro [11]. iPSCs can be stored frozen remaining viable for differentiation at a later time. iPSC-CMs have major advantages over primary isolated CMs including the ability to persist indefinitely in culture [12]. CMs obtained through endomyocardial biopsy are typically non-viable and therefore only useful for studying structural characteristics of CMs.
Combining genetic analyses and modeling disease with patient-specific iPSC-derived cells holds great potential to advance our understanding of the genetic causes of disease. For the patient, this allows their individual genetic background to be taken into account. In future it may also increase the ability for accurate pathogenicity classifications to be made with significant clinical implications. The use of functional models such as iPSCs to facilitate pathogenicity decisions is particularly valuable when there is a lack of family members for cosegregation studies [13].
Methods to advance the outcomes of studying human genetics with iPSCs are rapidly growing. One exciting avenue involves genome editing. Directed nucleases such as CRISPRs and TALENs allow specific gene mutations to be introduced and removed readily into iPSCs [14, 15]. Patients’ specific genomes can then be assessed with and without specific mutations of interest [16]. Isogenic controls overcome limitations of using close family members exhibiting or lacking mutations of interest as control subjects. Even amongst close family members, significant genetic differences exist which can alter the functional effects of mutations. Development of the most appropriate controls allows us to best understand the contribution of an individual’s genome to disease development. This is crucial in understanding the genetics that cause cardiomyopathies as the role of genetic modifiers and complex genetic interactions largely remain unknown.
7
iPSC-derived cells also facilitate the study of gene-environment interactions [17]. Exposure of iPSCs to hypertrophic factors illustrated that HCM iPSC-CMs are more susceptible to the hypertrophic factor endothelin-1 (ET-1) than control iPSC-derived CMs. These findings implicate the key role of an individual’s genetic background in their susceptibility to environmental factors that may promote hypertrophy. iPSC-CMs with a genetic predisposition to HCM were shown to be significantly more susceptible to ET-1 stimulated myofibrillar disarray and cell enlargement. A mild HCM phenotype was seen in HCM iPSCCMs that were not stimulated with ET-1 [17].
What are the current limitations of iPSCs? Several issues remain to be resolved before iPSCs can be used to model human disease with complete confidence. Major limitations surround producing a homogenous population of fully reprogrammed iPSCs [18]. To fully utilize iPSC-CM models, a large number of fully reprogrammed stable cells need to be produced. However, reprogramming somatic cells to iPSCs can result in variability due to culture conditions, retained epigenetic memory and genetic instability. Different somatic cells appear to have varying levels of cellular plasticity, resulting in a variable reprogramming efficiency and overall resemblance of an ES-cell state. Genetic background and age may also affect the resultant iPSCs; somatic cells isolated from younger patients may be more easily reprogrammed [19]. Issues with variability exist even within clones derived from a single patient and highlight the importance of appropriate controls and reproducible methods to identify interfering factors and biases.
8
MAKING CARDIOMYOCYTES FROM PATIENTS
How are the cells collected? Many somatic cells have been used as source material for reprogramming including pancreatic beta cells, hepatocytes, peripheral blood mononuclear cells, keratinocytes, renal epithelial cells and neural progenitor cells [20]. Reprogramming and differentiation efficiency is not equal amongst somatic cells. Currently, the reprogramming efficiency of keratinocytes and dermal fibroblasts surpasses that of peripheral blood mononuclear cells. Differences in efficiency and ease of sourcing, reprogramming and differentiation are important considerations.
How are iPSCs differentiated to myocardial lineages? In vitro differentiation of iPSC into CM follows the same developmental pathways as observed in embryonic development (summarized in Figure 1). First, iPSC must be differentiated into cells resembling the germ layer which gives rise to the heart, the mesoderm. These cells must then receive the unique set of specification and maturation signals required to obtain cardiac cell lineages. This differentiation process relies on stagespecific manipulation of embryonic development signaling pathways. Four signaling pathways central to cardiac differentiation include; BMP, TGFb/Activin/NODAL, WNT and FGF [21]. The two most common methods for myocardial iPSC differentiation are embryoid bodies formation and monolayer differentiation culture. Embryoid bodies are spherical aggregates of cells ideal for recapitulating embryonic development processes. Monolayer culture systems facilitate chemically defined manipulation of developmental pathways.
The timely and concentration specific addition of growth factors and small molecules is crucial in obtaining high levels of myocardial cells in culture. Importantly, no differentiation 9
methods produce solely CM pure cell populations and additional cell purification is typically required. Successful differentiation is confirmed through assessment of expression of cardiac markers including TNNT2 and MEF2C; loss of iPSC markers (OCT3/4, SOX2 and NANOG); and the identification of intact sarcomeres and spontaneous rhythmic contraction [21].
What are the current limitations of iPSC-CMs? While significant progress has been made in CM differentiation, with protocols now able to produce high CM yields and promote the differentiation of specific CM subtypes, the technology is still challenged by many limitations [20]. The limitations of iPSC-CMs are summarized in the literature [22] and include cell variability, limited maturity and abnormal functional characteristics.
Like variability present in iPSC clones, phenotypic variability is also seen in differentiated iPSC-CMs. Many factors contribute to this variability. For example some serum sources can mask features of disease in iPSC-CM models. Ideally, differentiation protocols will be chemically defined and free from use of highly variable animal products. Recent work has also shown that cell culture density can greatly alter cellular phenotype [23]. The development of more standardized differentiation approaches will likely reduce iPSC-CM variability. Methodological alterations such as growing cells as a monolayer have been shown to reduce phenotypic variability. A recent study showed that comparable calcium handling was present in iPSC-CMs derived from healthy individuals in 3 different university laboratories using multiple monolayer differentiation methods [24].
To date, iPSC-CMs have demonstrated a relatively immature phenotype. In addition to affecting interpretation of relevant results, immature iPSC-CMs have arrhythmogenic risks
10
that limit their use in regenerative medicine [25]. In comparison to adult human CMs, iPSC-CMs display fetal gene expression, are smaller in size, have disorganized sarcomeres, an absence of t-tubules, and diminished electrical and contractile function [26]. Many studies have sought to improve iPSC-CM maturity. Significant advances in cell maturation have been achieved through the culture of iPSC-CMs in 3-dimensional tissueengineered cardiac patches, highlighting the importance of the tissue microenvironment [27]. However, iPSC-CMs still remain relatively immature. In two weeks of culture, iPSCCMs had well-developed sarcomeres similar to isolated human adult human CMs, upregulation of key excitation-contraction coupling genes including CASQ2 and SERCA2 and an advanced sarcoplasmic reticulum, suggesting a more mature phenotype. The development of culturing techniques that better mimic the in vivo environment, taking into account complex cellular interactions will likely continue to aid the maturation of iPSC-CMs. Hopefully, increasing iPSC-CM maturity will aid the cessation of abnormal functional characteristics. In particular the tendency of iPSC-CMs to beat spontaneously, which is not a feature of adult ventricular CMs and likely related in part to their immature state [28].
11
iPSC MODELS OF GENETIC CARDIOMYOPATHIES The value of iPSCs has already been demonstrated in genetic heart diseases such as the primary inherited arrhythmia syndromes [29]. However, there is significant debate over whether iPSC-CM models can accurately recapitulate cardiac phenotypes which largely involve the whole 3-dimensional contracting heart. Cardiomyopathy studies to date have utilized relatively immature iPSC-CMs, which is a limitation that needs to be taken into account when interpreting results. Key studies in iPSC-CM models of inherited cardiomyopathies are summarized in Table 2. The main focus to date has been on the more common inherited cardiomyopathies such as HCM, DCM, and ARVC. To date, many of the underlying mechanisms of disease identified in iPSC-CMs have been previously identified in animal models or patient samples.
iPSC models of HCM HCM is a clinically and genetically heterogeneous heart disease with a prevalence of up to 1 in 200 people [30]. HCM is diagnosed primarily on discovery of unexplained left ventricular hypertrophy and is characterized by both clinical and genetic heterogeneity [31]. HCM is primarily inherited in an autosomal dominant inheritance pattern; offspring have a 50% chance of inheriting a pathogenic mutation. Over 1500 causal mutations have been identified. HCM can be viewed as a disease of the sarcomere. The majority of mutations are in 4 sarcomere genes; cardiac myosin-binding protein C (MYBPC3), b-myosin heavy chain (MYH7), cardiac troponin T type 2 (TNNT2) and cardiac troponin I type 3 (TNNI3) [2]. The identification of pathogenic mutations is important as it facilitates familial cascade screening [2, 32]. However, pathogenic mutations have been identified in only 40-50% of HCM patients.
12
The pathogenesis of HCM is poorly understood. Studies with animal models and nonviable human cardiac tissue have shown that during the hypertrophic response CMs enlarge and switch gene expression towards a fetal expression profile [6]. Functional abnormalities identified in HCM murine models include increased calcium sensitivity due to an abnormal high intracellular calcium concentration, increased contraction force and electrical abnormalities [5, 6, 33].
iPSC-CMs can be used to recapitulate characteristics of the HCM phenotype in vitro [11, 17,
34,
35].
HCM-specific
changes
include
cellular
enlargement,
sarcomere
disorganization, and alterations in electrophysiological behavior, gene expression and calcium handling (Table 2). Many of these changes are only identifiable after cells were cultured for a significant period of time following differentiation, perhaps highlighting the importance of cell maturity [11]. HCM iPSC-CMs have exhibited molecular mechanisms consistent with those identified in animal models and have allowed the investigation of the functional impact of pathogenic mutations.
Molecular mechanisms underlying HCM The pathogenic mechanisms underlying the development of HCM in 2 families with pathogenic mutations in MYH7 have been investigated in-depth. iPSC-CMs derived from 10 members of a family affected by HCM were characterized. Five family members carried a missense mutation p.Arg663His in MYH7 [11]. HCM iPSC-CMs exhibited structural and functional characteristics in line with the HCM phenotype. Structural abnormalities included cellular enlargement, increased multinucleation and myofibril content and an increased proportion of cells with disorganized sarcomeres. Functional abnormalities included increased intracellular calcium concentration and increased expression of HCM-related genes and hypertrophy-related proteins. Calcium dysregulation occurred prior to the onset
13
of the hypertrophic phenotype and calcium channel antagonists were able to prevent HCM development suggesting that calcium dysregulation is causal of, rather than a product of, HCM [11].
iPSC-CMs derived from a patient with HCM caused by the MYH7 mutation p.Arg442Gly have been reported [34]. HCM iPSC-CMs exhibited structural abnormalities consistent with the HCM phenotype. Similar calcium handling abnormalities were identified [34], consistent with observations made from animal models [5]. Calcium channel antagonists were able to ameliorate characteristics of the HCM phenotype. An increase in HCM and fibrosis-related genes was identified [34]. Genes involved in cell proliferation were upregulated linking cell proliferation and movement in the pathogenesis of HCM. Both studies implicated the central role of NFAT transcription factors, similar to murine HCM models, which implicate the involvement of NFAT transcription factors in HCM [17, 35].
Determining the pathogenicity of mutations The iPSC studies mentioned above have provided evidence to confirm the pathogenicity of two MYH7 mutations. Significant differences in functional and structural characteristics were observed between HCM iPSC-CMs derived from gene positive and gene negative family members [11]. In addition, a HCM phenotype was observed following overexpression of the p.Arg663His MYH7 mutation in normal human ESC-CMs thus ruling out the possibility that another rare mutation present in this family is responsible for disease.
Cardiotoxicity and efficacy of pharmaceutical compounds HCM iPSC-CMs have been utilized to study patient-specific responses to current and novel HCM therapies [11, 34, 35]. Drugs currently used for the treatment of HCM including
14
metoprolol, verapamil and pinacidil, all demonstrated beneficial effects in HCM iPSC-CMs. The use of novel inhibitors of histone deacetylase activity specifically Trichostatin A in HCM therapy is supported by HCM iPSC-CM studies [34]. Trichostatin A significantly ameliorated the HCM phenotype, particularly calcium handling abnormalities, NFATC4 nuclear translocation and CM enlargement [34]. Clinical trials are required to determine if these findings translate into improved clinical treatment. These results highlight the potential utility of iPSC-CM modeling in drug research and development.
iPSC models of DCM DCM is characterized by left ventricle dilation, systolic dysfunction and heart failure. Systemic diseases such as coronary artery disease explain approximately 70% of DCM cases, with the remaining 30% referred to as “idiopathic DCM” [36]. Major adverse events include heart failure, arrhythmias and sudden cardiac death. Histopathological features of DCM are highly variable but commonly include CM loss, myocyte disarray and interstitial fibrosis. Like HCM, the process from genetic mutation to presentation of clinical DCM is poorly understood.
A causal mutation, supporting inherited DCM, is identified in approximately 20-30% of patients with idiopathic DCM [37]. Inherited DCM is a genetically heterogeneous disease. Mutations have been identified in greater than 30 genes encoding sarcomere, cytoskeletal, mitochondrial and nuclear membrane proteins [38]. DCM is primarily inherited in an autosomal dominant inheritance pattern, and the most common disease gene in DCM is Titin (TTN) [2, 37].
Four studies have demonstrated that iPSC-CM models can recapitulate key characteristics of the DCM disease phenotype in vitro (Table 2) [13, 35, 38, 39]. Features of DCM that are
15
identifiable in iPSC-CM models include loss of sarcomere integrity, cell organization, contraction force and normal calcium handling.
Molecular mechanisms underlying DCM Three studies have assessed the molecular mechanisms that underlie DCM using patientspecific iPSC-CMs [13, 38, 39]. iPSC-CMs were derived from a 7-member family with severe DCM caused by the mutation p.Arg173Trp in TNNT2 [38]. Compared with control iPSC-CMs, DCM iPSC-CMs exhibited significantly diminished sarcomere integrity, with abnormal α-actinin distribution and disorganized z-lines with scattered condensed z-bodies. DCM iPSC-CMs also exhibited abnormal calcium handling; DCM iPSC-CMs had smaller intracellular calcium transients and diminished sarcoplasmic reticulum calcium storage. Consistent with animal models, DCM iPSC-CMs displayed weaker contraction forces [38].
iPSC-CMs have been reported from a patient with DCM thought to be the result of a mutation p.Ala285Val in DES [13]. DES encodes the desmin protein, an intermediate filament critical for maintenance of the sarcomere structure. DCM iPSC-CMs exhibited poor co-localization of DES with cardiac TNNT2, α-actinin and F-actin. In addition, zbodies were abnormally formed and more than 70% of cells contained isolated aggregates of DES-positive proteins. Disruption of calcium handling was also observed in DES-mutant cells suggesting that calcium dysregulation is a key characteristic of DCM. Functional impacts of the p.Ala285Val mutation in DES, specifically diffuse abnormal DES protein aggregation, were also observed in HEK293 cells overexpressing mutant protein.
In
contrast to results reported previously [38] and control iPSC-CMs, a slower spontaneous beat frequency was observed in DCM iPSC-CMs, which may suggest that DCM resulting from mutations in DES cause specific functional abnormalities.
16
iPSC-CMs were produced from 3 DCM patients with TTN mutations; 2 A-band truncating mutations, p.Ala22352fs and p.Pro22582fs, and 1 missense mutation p.Trp976Arg in the Z/I junction [39]. Due to the large and complex nature of the TTN gene there are many mutations of unknown significance. The majority of truncating mutations that cause DCM are located within the A band. Patient and control iPSC-CMs were cultured in more advanced 3-dimensional cardiac microtissues as differences in contraction forces in isolated patient iPSC-CMs and control iPSC-CMs were not significant, highlighting the limitations of single cell models. Patient iPSC cardiac microtissues exhibited less than half the contractile and stress force per tissue area, compared with control iPSC cardiac microtissues. Additional analyses revealed that TTN truncating mutations result in abnormal CM signaling and diminished expression of key cardiomyopathy gene transcripts, ultimately causing sarcomere insufficiency.
Determining the pathogenicity of mutations Two studies utilized DCM patient iPSC-CMs to determine mutation pathogenicity. In the first study, patient iPSC-CMs were used to determine the pathogenicity of a mutation in DES identified through whole exome sequencing [13]. Characteristics of the DCM phenotype were absent in control iPSCs with wild-type DES, and present in both patient iPSC-CMs and control iPSC-CMs transduced with the mutant DES gene, supporting the pathogenicity of the mutation identified in the patient.
In the second study, patient iPSC-CMs were utilized to not only determine the pathogenicity of a missense mutation, p.Trp792Arg in TTN, however also to further understand the pathogenicity of groups of mutations located at specific sites within TTN [39]. Through combining functional analyses of iPSC-CM cardiac microtissues with RNAseq data derived from iPSC-CMs, the authors were able to reveal that alternative exon
17
splicing is responsible for truncating mutations in the I band of TTN having reduced pathogenicity compared with truncating mutations located within the A band. Thus, providing an explanation for the increased prevalence of truncating TTN mutations in the I band in the general population compared with truncating TTN mutations in the A band, which are highly prevalent in DCM cohorts. In addition, DCM severity due to truncating mutations in patient iPSC-CMs was greater than that of equivalent truncating mutations expressed in control iPSC-CMs, which may suggest that genetic modifiers contribute to the DCM severity due to truncation mutations in TTN. This study provides a nice example of how iPSC-CM models can help us to explore the involvement of complex genetics in cardiomyopathy development.
Applications in DCM therapy and drug testing DCM iPSC-CMs have been used to assess the efficacy of a gene therapy approach. Overexpression
of
SERCA2A
reduced
DCM-related
cellular
abnormalities
[38].
Overexpression of SERCA2A rescued the DCM phenotype at the single cell level, reinstating effective contraction force [38]. Metoprolol significantly reduced the number of DCM iPSC-CMs with disrupted sarcomere integrity. DCM iPSC-CMs had increased propensity for arrhythmias compared with control or HCM iPSC-CMs when treated with the vasodilator, nicorandil [35].
iPSC models of ARVC ARVC is a primary cardiomyopathy with clinically heterogeneous presentation. Most commonly seen in men, ARVC has an estimated prevalence of approximately 1 in 5000 [40]. However, ARVC prevalence varies globally and in some regions is estimated to be as high as 1 in 500 [41]. ARVC is characterized by ventricular arrhythmias, right ventricle dysfunction and sudden cardiac death. Histopathological features of ARVC include fibro18
fatty infiltration of the myocardium, primarily involving the right ventricle. ARVC is primarily inherited in an autosomal dominant inheritance pattern. A pathogenic mutation is identified in approximately 30-50% of patients [42]. ARVC penetrance and severity is highly variable and may be altered by the presence of multiple mutations [43]. Complex genetics, involving multiple mutations, are often thought to be involved in the development of ARVC. ARVC is thought to be a disease of the desmosome. Genes that encode the desmosome including plakophilin 2 (PKP2), desmoglein 2 (DSG2), desmoplakin (DSP), desmocollin 2 (DSC2) and plakoglobin (JUP), are commonly implicated [42]. Mutations in PKP2 are the most common cause of ARVC [2].
Three studies have utilized patient iPSC-CMs to model ARVC. In all 3 studies ARVC was due to pathogenic mutations in PKP2 (summarized in Table 2) [4, 44, 45]. iPSC-CM models were successfully used to recapitulate important characteristics of the ARVC phenotype including abnormalities in desmosome structure and reduced expression of desmosome-related genes and protein density.
Molecular mechanisms underlying ARVC iPSC-CMs derived from 2 patients with ARVC caused by frameshift mutations p.Ala325Cysfs*11 and p.Thr50Serfs*61 in PKP2 have been reported [4]. ARVC iPSC-CMs derived from both patients exhibited a significant reduction in PKP2 gene expression and density of desmosome and gap junction proteins plakophilin-2, plakoglobin and connexin43. Significant abnormalities in the desmosome structure were also observed. In a separate study, iPSC-CMs derived from a patient with ARVC due to the p.Leu614Pro missense mutation in PKP2 were studied [45]. Reduced PKP2 and JUP gene expression was identified. Consistent with the previous study, reduced density of plakophilin-2 and plakoglobin proteins was observed. Abnormalities in cell structure, particularly involving z-
19
bands and desmosomes were also identified, with ARVC iPSC-CMs overall appearing less organized [45].
Determining the pathogenicity of mutations iPSC-CM models have been utilized to confirm the pathogenic effects of mutations in PKP2 [44]. Evidence for the pathogenicity of the mutation p.Gly894Gly in PKP2 was obtained using iPSC-CMs derived from a patient with ARVC. The ARVC phenotype demonstrated by iPSC-CMs was ameliorated following mutant cell transduction with wildtype PKP2. Dysregulated calcium handling was also implicated in the development of ARVC. Methods to induce adult-like metabolism, which accelerate adult-onset disease and the development of mature iPSC-CMs further supported the pathogenicity of the PKP2 mutation.
Collectively, the iPSC-CM models developed in HCM, DCM and ARVC (Table 2) reflect the potential use of such models for understanding disease mechanisms, providing functional evidence to support pathogenicity of mutations identified in patients, and evaluating potential disease therapies (Figure 2).
20
iPSCs IN CLINICAL PRACTICE: NOW AND IN THE FUTURE
iPSC technology has profound implications to clinical practice. From assisting in mutation classification as part of cardiac genetic testing, to the development of new therapies, and personalizing treatment to individual patients, the potential impact of the role of iPSCs in clinical medicine is far reaching (Figure 2).
There are major benefits in assessing the efficacy of therapeutic treatments in patientspecific iPSC-CMs. Optimal therapeutic strategies can be designed for individual patients. An effective treatment approach for a patient with long QT syndrome was identified following testing two different frequently used clinical strategies in patient-specific iPSCCMs [46]. In this particular case, the therapy comparison was not tested in the patient and so the direct impact on clinical care was not established. In future, it is likely that iPSC-CM models will aid the optimization of patient therapy by enabling specific individuals’ pathogenic processes to be taken into account.
Drugs that have been developed in animal models primarily examine the population as a whole and assume a relatively consistent response across patients. iPSC technologies provide a new opportunity for patient-specific toxicity testing, allowing a patient’s individual risk of toxicity to be assessed. iPSC-CMs can be used to assess the cellular effects of clinical therapies and determine patient-specific drug toxicity [35]. The occurrence of arrhythmias and prolonged action potential duration, resulting from drug administration was measured on patient iPSC-CMs. iPSC-CMs from patients with long QT syndrome and HCM were far more susceptible to toxicity than iPSC-CMs from DCM patients or healthy controls [25]. The significance of taking into account multiple ion channels is demonstrated by cardiotoxicity testing of the drug Alfuzosin, where the drug did not have cardiotoxic
21
effects as it does not act on hERG potassium channels. However, Alfuzosin does have cardiotoxic effects as it interacts with other ion channels resulting in a prolonged QT interval [35].
iPSC studies may also provide opportunities for regenerative medicine approaches to treat cardiovascular disease. The main goal of using iPSCs in regenerative medicine is to restore the function of organs and tissues. Theoretically, iPSC-CMs are ideal for improving cardiac function, as they are patient-specific. However, there are some issues with immunogenicity that can arise with iPSC use and whether patient iPSC-CMs can successfully integrate into adult myocardium remains to be determined. In addition, there is possibility of teratoma formation if iPSCs maintain pluripotency gene expression. Nevertheless, the tantalizing opportunity remains that patients could be “genetically rescued”, i.e. their own iPSC-CMs could undergo gene editing to remove the diseasecausing mutation, then “healthy” CMs injected back into the patient. With transplants of iPSC-derived cell types into patients already taking place, we anticipate new challenges along with the potential for great success in application of iPSCs to regenerative medicine.
22
CONCLUSIONS
Understanding how mutations in genes lead to human disease is a fundamental basis to improve the care of patients. Inherited cardiomyopathies are characterized by diversity in both the genes which cause disease, and the variable clinical phenotypes. Multiple genetic and environmental factors are likely to play a role in explaining this genetic and clinical heterogeneity. While iPSC-CM technology remains limited, iPSC models of inherited cardiomyopathies will likely afford us the opportunity to better understand disease mechanisms and phenotypes, and provide us with the opportunity to create novel and personalized therapeutic strategies targeted at the gene mutation level. iPSC studies of inherited cardiomyopathies to date provide confidence that the next decade will see the translation of iPSC-based models of human disease to improve our knowledge about the inherited cardiomyopathies, and how we can optimize the clinical management of our patients.
23
REFERENCES [1] Wilde AA, Behr ER. Genetic testing for inherited cardiac disease. Nat Rev Cardiol. 2013;10:571-83. [2] Ingles J, Semsarian C. The value of cardiac genetic testing. Trends Cardiovasc Med. 2014;24:217-24. [3] Semsarian C, Ingles J. Determining pathogenicity in cardiac genetic testing: Filling in the blank spaces. Trends Cardiovasc Med. 2015. [4] Caspi O, Huber I, Gepstein A, Arbel G, Maizels L, Boulos M, et al. Modeling of arrhythmogenic right ventricular cardiomyopathy with human induced pluripotent stem cells. Circ Cardiovasc Genet. 2013;6:557-68. [5] Semsarian C, Ahmad I, Giewat M, Georgakopoulos D, Schmitt JP, McConnell BK, et al. The L-type calcium channel inhibitor diltiazem prevents cardiomyopathy in a mouse model. J Clin Invest. 2002;109:1013-20. [6] Seidman JG, Seidman C. The genetic basis for cardiomyopathy: from mutation identification to mechanistic paradigms. Cell. 2001;104:557-67. [7] Semsarian C, Healey MJ, Fatkin D, Giewat M, Duffy C, Seidman CE, et al. A polymorphic modifier gene alters the hypertrophic response in a murine model of familial hypertrophic cardiomyopathy. J Mol Cell Cardiol. 2001;33:2055-60. [8] Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, et al. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282:1145-7. [9] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131:86172. [10] Lister R, Pelizzola M, Kida YS, Hawkins RD, Nery JR, Hon G, et al. Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature. 2011;471:68-73. [11] Lan F, Lee AS, Liang P, Sanchez-Freire V, Nguyen PK, Wang L, et al. Abnormal calcium handling properties underlie familial hypertrophic cardiomyopathy pathology in patient-specific induced pluripotent stem cells. Cell Stem Cell. 2013;12:101-13. 24
[12] Ivashchenko CY, Pipes GC, Lozinskaya IM, Lin Z, Xiaoping X, Needle S, et al. Human-induced pluripotent stem cell-derived cardiomyocytes exhibit temporal changes in phenotype. Am J Physiol Heart Circ Physiol. 2013;305:H913-22. [13] Tse HF, Ho JC, Choi SW, Lee YK, Butler AW, Ng KM, et al. Patient-specific inducedpluripotent stem cells-derived cardiomyocytes recapitulate the pathogenic phenotypes of dilated cardiomyopathy due to a novel DES mutation identified by whole exome sequencing. Hum Mol Genet. 2013;22:1395-403. [14] Hockemeyer D, Wang H, Kiani S, Lai CS, Gao Q, Cassady JP, et al. Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol. 2011;29:731-4. [15] Ding Q, Regan SN, Xia Y, Oostrom LA, Cowan CA, Musunuru K. Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs. Cell Stem Cell. 2013;12:393-4. [16] Tanaka A, Yuasa S, Node K, Fukuda K. Cardiovascular Disease Modeling Using Patient-Specific Induced Pluripotent Stem Cells. Int J Mol Sci. 2015;16:18894-922. [17] Tanaka A, Yuasa S, Mearini G, Egashira T, Seki T, Kodaira M, et al. Endothelin-1 induces
myofibrillar
disarray
and
contractile
vector
variability
in
hypertrophic
cardiomyopathy-induced pluripotent stem cell-derived cardiomyocytes. J Am Heart Assoc. 2014;3:e001263. [18] Paull D, Sevilla A, Zhou H, Hahn AK, Kim H, Napolitano C, et al. Automated, highthroughput derivation, characterization and differentiation of induced pluripotent stem cells. Nat Methods. 2015;12:885-92. [19] Mack DL, Guan X, Wagoner A, Walker SJ, Childers MK. Disease-in-a-dish: the contribution of patient-specific induced pluripotent stem cell technology to regenerative rehabilitation. Am J Phys Med Rehabil. 2014;93:S155-68. [20] Fuerstenau-Sharp M, Zimmermann ME, Stark K, Jentsch N, Klingenstein M, Drzymalski M, et al. Generation of highly purified human cardiomyocytes from peripheral blood
mononuclear
cell-derived
induced
pluripotent
stem
cells.
PLoS
One.
2015;10:e0126596.
25
[21] Burridge PW, Diecke S, Matsa E, Sharma A, Wu H, Wu JC. Modeling Cardiovascular Diseases with Patient-Specific Human Pluripotent Stem Cell-Derived Cardiomyocytes. Methods Mol Biol. 2016;1353:119-30. [22] Knollmann BC. Induced pluripotent stem cell-derived cardiomyocytes: boutique science or valuable arrhythmia model? Circ Res. 2013;112:969-76; discussion 76. [23] Du DT, Hellen N, Kane C, Terracciano CM. Action potential morphology of human induced pluripotent stem cell-derived cardiomyocytes does not predict cardiac chamber specificity and is dependent on cell density. Biophys J. 2015;108:1-4. [24] Hwang HS, Kryshtal DO, Feaster TK, Sanchez-Freire V, Zhang J, Kamp TJ, et al. Comparable calcium handling of human iPSC-derived cardiomyocytes generated by multiple laboratories. J Mol Cell Cardiol. 2015;85:79-88. [25] Zhang YM, Hartzell C, Narlow M, Dudley SC, Jr. Stem cell-derived cardiomyocytes demonstrate arrhythmic potential. Circulation. 2002;106:1294-9. [26] Zhang D, Shadrin IY, Lam J, Xian HQ, Snodgrass HR, Bursac N. Tissue-engineered cardiac patch for advanced functional maturation of human ESC-derived cardiomyocytes. Biomaterials. 2013;34:5813-20. [27] Chun YW, Balikov DA, Feaster TK, Williams CH, Sheng CC, Lee JB, et al. Combinatorial polymer matrices enhance in vitro maturation of human induced pluripotent stem cell-derived cardiomyocytes. Biomaterials. 2015;67:52-64. [28] Kim JJ, Yang L, Lin B, Zhu X, Sun B, Kaplan AD, et al. Mechanism of automaticity in cardiomyocytes derived from human induced pluripotent stem cells. J Mol Cell Cardiol. 2015;81:81-93. [29] Priori SG, Napolitano C, Di Pasquale E, Condorelli G. Induced pluripotent stem cellderived cardiomyocytes in studies of inherited arrhythmias. J Clin Invest. 2013;123:84-91. [30] Semsarian C, Ingles J, Maron MS, Maron BJ. New Perspectives on the Prevalence of Hypertrophic Cardiomyopathy. J Am Coll Cardiol. 2015;65:1249-54. [31] Maron BJ, Maron MS, Semsarian C. Genetics of hypertrophic cardiomyopathy after 20 years: clinical perspectives. J Am Coll Cardiol. 2012;60:705-15.
26
[32] Ingles J, Semsarian C. Conveying a probabilistic genetic test result to families with an inherited heart disease. Heart Rhythm. 2014;11:1073-8. [33] Tsoutsman T, Kelly M, Ng DC, Tan JE, Tu E, Lam L, et al. Severe heart failure and early mortality in a double-mutation mouse model of familial hypertrophic cardiomyopathy. Circulation. 2008;117:1820-31. [34] Han L, Li Y, Tchao J, Kaplan AD, Lin B, Li Y, et al. Study familial hypertrophic cardiomyopathy using patient-specific induced pluripotent stem cells. Cardiovasc Res. 2014;104:258-69. [35] Liang P, Lan F, Lee AS, Gong T, Sanchez-Freire V, Wang Y, et al. Drug screening using a library of human induced pluripotent stem cell-derived cardiomyocytes reveals disease-specific patterns of cardiotoxicity. Circulation. 2013;127:1677-91. [36] Hershberger RE, Hedges DJ, Morales A. Dilated cardiomyopathy: the complexity of a diverse genetic architecture. Nat Rev Cardiol. 2013;10:531-47. [37] Herman DS, Lam L, Taylor MR, Wang L, Teekakirikul P, Christodoulou D, et al. Truncations of titin causing dilated cardiomyopathy. N Engl J Med. 2012;366:619-28. [38] Sun N, Yazawa M, Liu J, Han L, Sanchez-Freire V, Abilez OJ, et al. Patient-specific induced pluripotent stem cells as a model for familial dilated cardiomyopathy. Sci Transl Med. 2012;4:130ra47. [39] Hinson JT, Chopra A, Nafissi N, Polacheck WJ, Benson CC, Swist S, et al. HEART DISEASE. Titin mutations in iPS cells define sarcomere insufficiency as a cause of dilated cardiomyopathy. Science. 2015;349:982-6. [40] Romero J, Mejia-Lopez E, Manrique C, Lucariello R. Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC/D): A Systematic Literature Review. Clin Med Insights Cardiol. 2013;7:97-114. [41] Etchegary H, Pullman D, Simmonds C, Young TL, Hodgkinson K. 'It had to be done': genetic testing decisions for arrhythmogenic right ventricular cardiomyopathy. Clin Genet. 2015;88:344-51.
27
[42] Te Rijdt WP, Jongbloed JD, de Boer RA, Thiene G, Basso C, van den Berg MP, et al. Clinical utility gene card for: arrhythmogenic right ventricular cardiomyopathy (ARVC). Eur J Hum Genet. 2014;22. [43] Quarta G, Muir A, Pantazis A, Syrris P, Gehmlich K, Garcia-Pavia P, et al. Familial evaluation in arrhythmogenic right ventricular cardiomyopathy: impact of genetics and revised task force criteria. Circulation. 2011;123:2701-9. [44] Kim C, Wong J, Wen J, Wang S, Wang C, Spiering S, et al. Studying arrhythmogenic right ventricular dysplasia with patient-specific iPSCs. Nature. 2013;494:105-10. [45] Ma D, Wei H, Lu J, Ho S, Zhang G, Sun X, et al. Generation of patient-specific induced
pluripotent
stem
cell-derived
cardiomyocytes
as
a
cellular
model
of
arrhythmogenic right ventricular cardiomyopathy. Eur Heart J. 2013;34:1122-33. [46] Terrenoire C, Wang K, Tung KW, Chung WK, Pass RH, Lu JT, et al. Induced pluripotent stem cells used to reveal drug actions in a long QT syndrome family with complex genetics. J Gen Physiol. 2013;141:61-72.
28
TABLES
Table 1: Glossary of Terms
·
Pluripotent: capable of giving rise to all different cell and tissue types with the exception of the placenta.
·
Multipotent: capable of giving rise to multiple different cell types of the same lineage.
·
Totipotent: capable of giving rise to all different cell and tissue types including the placenta and embryo.
·
Differentiation: is the process of a cell changing from one cell type to another.
·
Reprogramming: is the conversion of one specific cell type to another. Specifically, direct reprogramming is the conversion of a somatic cell type, such as a fibroblast, to a pluripotent cell, i.e. iPSC.
29
Table 2. Summary of iPSC-based models of inherited cardiomyopathies Disease
Reason for Study
Gene (Mutation)
Somatic Cell Line
Functional Impact
Reference
HCM
Identify patientspecific and mutationspecific mechanisms of HCM
MYH7 (Arg442Gly)
Dermal Fibroblasts
Enlarged size, disorganized sarcomere structures and arrhythmic beatings. Irregular calcium handling and ion channel functions. Increased expression of HCM related genes.
[34]
HCM
Elucidate the mechanisms underlying HCM pathogenesis
MYH7 (Arg663His)
Dermal fibroblasts
Cellular enlargement, multinucleation and contractile arrhythmia (single cell level).
[11]
HCM
Geneticenvironment interactions that contribute to the development of HCM
3 patients: 2 no mutation, 1 MYBPC3 Gly999NGln1004Del
Dermal fibroblasts and T lymphocytes
Enlarged cells with myofibrillar disarray. Increased myofibrillar disarray and cell size following treatment with ET-1 (hypertrophic factor).
[17]
DCM
Develop iPSC-CMs to recapitulate DCM phenotype and investigate underlying mechanisms
TNNT2 (Arg173Trp)
Fibroblasts
Elimination of sarcomeric alignment and reduced calcium handling.
[21]
DCM
Develop iPSC-CMs to recapitulate DCM phenotype and investigate underlying mechanisms
TNNT2 (Arg173Trp)
Dermal fibroblasts
Altered calcium handling, decreased contractility and abnormal sarcomeric a-actinin distribution.
[38]
DCM
Provide functional and histological evidence for the pathogenicity of DES mutation
DES (Ala258Val)
Dermal fibroblasts
Structural and functional abnormalities resulting from abnormal formation of DESaggregates. Functional abnormalities such as diminished max rate of calcium re-uptake, and structural abnormalities include poor localization of cardiac troponin T.
[13]
DCM
Evaluate the pathogenicity and functional impacts of truncating and
3 patients: TTN (Ala22352fs, Pro22582fs and Trp976Arg)
T cells
Contractile deficits, stable truncated peptide unable to assemble with other contractile proteins,, sarcomere insufficiency Attenuated cell signaling limiting CM response to mechanical and adrenergic stress
[39]
30
missense mutations in TTN ARVC
Establish a patientspecific iPSC-CM model for ARVC
PKP2 (Leu614Pro)
Dermal fibroblasts
Abnormal and highly variable zlines (thicker) with decreased PKP2 and JUP gene expression, and decreased plakophilin 2 and plakoglobin protein at cell periphery.
[45]
ARVC
Establish a patientspecific iPSC-CM model for ARVC
2 patients: PKP2 mutations: (Ala325Cysfs*11) & (Thr50Serfs*61)
Dermal fibroblasts
Abnormalities include increased cytoplasmic accumulation of lipid, distortion of desmosomes, and functional reduction in PKP2 gene and protein expression.
[4]
ARVC
Identify mechanisms of ARVC and identify novel therapeutic strategies
2 patients: 1 homozygous PKP2 mutation (Gly828Gly) and 1 heterozygous PKP2 mutation (Lys672Glyfs*70)
Dermal fibroblasts
Functional abnormalities include abnormal nuclear translocation of plakoglobin proteins resulting in increased lipogenesis, abnormal calcium handling), electrophysiological changes.
[44]
31
FIGURE LEGENDS
Figure 1: Generation of iPSCs from patients, then the differentiation to cardiomyocytes, then to their use in different cardiomyopathies such as HCM, DCM, and ARVC.
Figure 2: Summary of the value and potential of iPSCs in human biology and disease.
32
FIGURES
Figure 1
33
Figure 2
34