Modeling Cardiomyopathy and Arrhythmias in Induced Pluripotent Stem Cell–Derived Cardiomyocytes

Dilated cardiomyopathy (DCM) are genetic heart diseases associated with arrhythmias, which are sometimes fatal, and progressive heart failure leading to heart transplantation.¹ An overlap of DCM with muscular dystrophies is well known and was first noted in the case of the dystrophinopathies, Duchenne and Becker muscular dystrophies, where Duchenne Muscular Dystrophy gene (DMD) mutations lead to both skeletal and cardiac muscle diseases. In some instances, a cardiac-only phenotype with heart failure and sudden cardiac death, X-linked DCM, results from DMD mutations.² The discovery of DMD mutations leading to DCM and arrhythmias provided the connection between mutations in the lamin A/C gene (LMNA) and DCM. LMNA mutations are responsible for a variety of phenotypes including LGMD1B (limb-girdle muscular dystrophy type 1B), Emery–Dreifuss muscular dystrophy, and DCM with or without conduction disease and with or without subclinical muscle involvement.³ DCM caused by LMNA mutations have been extensively studied, and it has been shown that LMNA can cause a particularly malignant phenotype both in terms of refractory heart failure and severe ventricular arrhythmias causing premature sudden cardiac death. The identification of a distinct arrhythmogenic phenotype in laminopathies had an important clinical impact and, in the new 2017 American Heart Association/American College of Cardiology/Heart Rhythm Society guidelines, prompted specific recommendations for the prevention of sudden cardiac death in patient with LMNA mutations.⁴ However, the susceptibility to arrhythmias in LMNA mutations carriers and in general in muscle dystrophies is not well understood, is not fully explained by the progressive myocardial fibrosis in later stages of the disease, and is not well recapitulated by animal models.

In this issue of Circulation: Genomic and Precision Medicine, El-Battrawy et al⁵ report another association of LGMD (in this case type 2I, or LGMD2I) with an arrhythmogenic phenotype. LGMD2I is a rare recessive disease caused by mutations of the fukutin-related protein gene (FKRP). The 51-year-old patient described was homozygous for an FKRP mutation c.826C>A (Leu276Ile). Clinically, patients with LGMDs are characterized by muscle weakness and dystrophy in the arms and legs (Figure).⁶ The symptoms gradually worsen as patients age leading to limitations with ambulation and often respiratory insufficiency. Interestingly, FKRP mutations carriers, like in laminopathies, may exhibit heterogeneous phenotypes but almost invariably have cardiac involvement.⁷ To date, ≈60% of FKRP mutations identified cause LGMD2I accounting for ≈10% of LGMD patients.^8–10 Among FKRP variants, the c.826C>A mutation (p.Leu276Ile) is the most common variant among LGMD2I patients from United Kingdom, Denmark, and Brazil.^11,12 Often, patients carrying the homozygous c.826C>A mutation have a milder phenotype than those that are compound heterozygous or homozygous for other mutations.¹³

Figure. Clinical phenotype and subtypes of LGMD (limb-girdle muscular dystrophy).

LGMDs are a group of genetic disorders characterized by weakness of the proximal muscles of hip and shoulder girdles. They are frequently associated with cardiac involvement, typically in the form of dilated cardiomyopathy (DCM). Reprinted from Thompson and Straub⁶ with permission. Copyright ©2016, Springer Nature.

To dissect the cellular consequences of disease, the authors generated human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CM) from the patient. Several cellular phenotypes were noted, including a reduced action potential with reduced amplitude and upstroke velocity in the hiPSC-CM compared with hiPSC-CM derived from healthy donors. Abnormalities in the sodium, calcium, and potassium channel currents, accompanied by reduction in sodium voltage-gated channel alpha subunit 5 gene (SCN5A) and calcium voltage-gated channel subunit alpha1 C gene (CACNA1C) expression and intracellular calcium concentration, were also found. Interestingly, although histological studies showed altered cytoskeletal α-actinin, the expression levels of α-DG (α-dystroglycan) were normal: an abnormal α-DG, which colocalized with SCN5A, could have provided some explanation to the ion channel dysfunction.¹⁴ Also, other important proteins for the cardiomyocyte contractile and electric function, actin and connexin 43, were not investigated: therefore, although these data yielded important insight into the electrophysiological consequences of FKRP mutations, the precise mechanisms of how mutant FKRP protein alters ion channel behavior remains to be seen.

FKRP is a transmembrane protein that localized to Golgi apparatus and is highly expressed in brain, heart, and skeletal muscles. FKRP is known to be involved in the glycosylation of α-DG.^15,16 α-DG is a component of the dystrophin-associated glycoprotein complex and plays an important role as a linker between cytoskeletal proteins and laminin, a component of the extracellular matrix. It may also have a role in anchoring ion channels, such as SCN5A, to the cell membrane.¹⁷ Recent studies showed that mutant FKRP L276I can be trafficked from the endoplasmic reticulum to the Golgi apparatus, which could explain the milder phenotypes in these patients compared with other mutations, where the mutant FKRP proteins fail to traffic from endoplasmic reticulum to Golgi apparatus.^13,18 Therefore, it would be interesting to determine the localization of mutant FKRP L276I in this patient-derived cellular model.

The work of El-Battrawy et al⁵ has some limitations. Although it has to be acknowledged that FKRP-related disease is a rare condition, the authors only report 1 mutant FKRP iPSC-CM line: therefore, it is unclear how their findings relate to other LGMD-associated arrhythmogenic cardiomyopathies. Furthermore, their study remains descriptive, without an attempt to investigate or discuss the molecular mechanisms linking the mutant transmembrane FKRP L276I protein to the ion channel dysfunction and the arrhythmogenic phenotype.

An important contribution of the study El-Battrawy et al⁵ is the application of iPSC-CMs from a patient with a rare homozygous FKRP c.826C>A mutation as a human model system to study the altered cellular electrophysiology. Indeed, although the iPSC-CM phenotype is more immature compared with adult cardiomyocytes, alternative current methods based on animal models (mice, rat, Xenopus oocytes, etc.) often fall short of recapitulating the human cell biology, in particular in functional assays of ion channels. Therefore, the iPSC-CM in vitro disease model is instrumental in providing a novel tool to investigate molecular mechanisms in the pathogenesis of LGMD and cardiomyopathies. Moreover, patient-specific iPSC-CMs may provide a novel human-based in vitro cellular model for drug screening, to identify specific therapeutic targets for individual LGMD patients.^19,20 The unsolved functional relationship between FKRP and ion channel dysfunctions in cardiomyocytes prompts further investigations to elucidate the origin of life-threatening arrhythmias in LGMD and in cardiomyopathies.