Depressed ß-adrenergic inotropic responsiveness and intracellular calcium handling abnormalities in Duchenne Muscular Dystrophy patients' induced pluripotent stem cell-derived cardiomyocytes.

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is an X-linked disease affecting male and rarely adult heterozygous females, resulting in death by the late 20s to early 30s. Previous studies reported depressed left ventricular function in DMD patients which may result from deranged intracellular Ca2+ -handling. To decipher the mechanism(s) underlying the depressed LV function, we tested the hypothesis that iPSC-CMs generated from DMD patients feature blunted positive inotropic response to ß-adrenergic stimulation. To test the hypothesis, [Ca2+ ]i transients and contractions were recorded from healthy and DMD-CMs. While in healthy CMs (HC) isoproterenol caused a prominent positive inotropic effect, DMD-CMs displayed a blunted inotropic response. Next, we tested the functionality of the sarcoplasmic reticulum (SR) by measuring caffeine-induced Ca2+ release. In contrast to HC, DMD-CMs exhibited reduced caffeine-induced Ca2+ signal amplitude and recovery time. In support of the depleted SR Ca2+ stores hypothesis, in DMD-CMs the negative inotropic effects of ryanodine and cyclopiazonic acid were smaller than in HC. RNA-seq analyses demonstrated that in DMD CMs the RNA-expression levels of specific subunits of the L-type calcium channel, the ß1-adrenergic receptor (ADRß1) and adenylate cyclase were down-regulated by 3.5-, 2.8- and 3-fold, respectively, which collectively contribute to the depressed ß-adrenergic responsiveness.

Investigating LMNA-Related Dilated Cardiomyopathy Using Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

LMNA-related dilated cardiomyopathy is an inherited heart disease caused by mutations in the LMNA gene encoding for lamin A/C. The disease is characterized by left ventricular enlargement and impaired systolic function associated with conduction defects and ventricular arrhythmias. We hypothesized that LMNA-mutated patients' induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs) display electrophysiological abnormalities, thus constituting a suitable tool for deciphering the arrhythmogenic mechanisms of the disease, and possibly for developing novel therapeutic modalities. iPSC-CMs were generated from two related patients (father and son) carrying the same E342K mutation in the LMNA gene. Compared to control iPSC-CMs, LMNA-mutated iPSC-CMs exhibited the following electrophysiological abnormalities: (1) decreased spontaneous action potential beat rate and decreased pacemaker current (If) density; (2) prolonged action potential duration and increased L-type Ca2+ current (ICa,L) density; (3) delayed afterdepolarizations (DADs), arrhythmias and increased beat rate variability; (4) DADs, arrhythmias and cessation of spontaneous firing in response to ß-adrenergic stimulation and rapid pacing. Additionally, compared to healthy control, LMNA-mutated iPSC-CMs displayed nuclear morphological irregularities and gene expression alterations. Notably, KB-R7943, a selective inhibitor of the reverse-mode of the Na+/Ca2+ exchanger, blocked the DADs in LMNA-mutated iPSC-CMs. Our findings demonstrate cellular electrophysiological mechanisms underlying the arrhythmias in LMNA-related dilated cardiomyopathy.

Electrophysiological abnormalities in induced pluripotent stem cell-derived cardiomyocytes generated from Duchenne muscular dystrophy patients.

Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle degenerative disease, caused by mutations in the dystrophin gene and resulting in death because of respiratory or cardiac failure. To investigate the cardiac cellular manifestation of DMD, we generated induced pluripotent stem cells (iPSCs) and iPSC-derived cardiomyocytes (iPSC-CMs) from two DMD patients: a male and female manifesting heterozygous carrier. Dystrophin mRNA and protein expression were analysed by qRT-PCR, RNAseq, Western blot and immunofluorescence staining. For comprehensive electrophysiological analysis, current and voltage clamp were used to record transmembrane action potentials and ion currents, respectively. Microelectrode array was used to record extracellular electrograms. X-inactive specific transcript (XIST) and dystrophin expression analyses revealed that female iPSCs underwent X chromosome reactivation (XCR) or erosion of X chromosome inactivation, which was maintained in female iPSC-CMs displaying mixed X chromosome expression of wild type (WT) and mutated alleles. Both DMD female and male iPSC-CMs presented low spontaneous firing rate, arrhythmias and prolonged action potential duration. DMD female iPSC-CMs displayed increased beat rate variability (BRV). DMD male iPSC-CMs manifested decreased If density, and DMD female and male iPSC-CMs showed increased ICa,L density. Our findings demonstrate cellular mechanisms underlying electrophysiological abnormalities and cardiac arrhythmias in DMD.

Investigating the cardiac pathology of SCO2-mediated hypertrophic cardiomyopathy using patients induced pluripotent stem cell-derived cardiomyocytes.

Mutations in SCO2 are among the most common causes of COX deficiency, resulting in reduced mitochondrial oxidative ATP production capacity, often leading to hypertrophic cardiomyopathy (HCM). To date, none of the recent pertaining reports provide deep understanding of the SCO2 disease pathophysiology. To investigate the cardiac pathology of the disease, we were the first to generate induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) from SCO2-mutated patients. For iPSC generation, we reprogrammed skin fibroblasts from two SCO2 patients and healthy controls. The first patient was a compound heterozygote to the common E140K mutation, and the second was homozygote for the less common G193S mutation. iPSC were differentiated into cardiomyocytes through embryoid body (EB) formation. To test the hypothesis that the SCO2 mutation is associated with mitochondrial abnormalities, and intracellular Ca2+ -overload resulting in functional derangements and arrhythmias, we investigated in SCO2-mutated iPSC-CMs (compared to control cardiomyocytes): (i) the ultrastructural changes; (ii) the inotropic responsiveness to ß-adrenergic stimulation, increased [Ca2+ ]o and angiotensin-II (AT-II); and (iii) the Beat Rate Variability (BRV) characteristics. In support of the hypothesis, we found in the mutated iPSC-CMs major ultrastructural abnormalities and markedly attenuated response to the inotropic interventions and caffeine, as well as delayed afterdepolarizations (DADs) and increased BRV, suggesting impaired SR Ca2+ handling due to attenuated SERCA activity caused by ATP shortage. Our novel results show that iPSC-CMs are useful for investigating the pathophysiological mechanisms underlying the SCO2 mutation syndrome.

Genome Editing in Induced Pluripotent Stem Cells using CRISPR/Cas9.

The development of the reprogramming technology led to generation of induced Pluripotent Stem Cells (iPSC) from a variety of somatic cells. Ever since, fast growing knowledge of different efficient protocols enabled the differentiation of these iPSCs into different cells types utilized for disease modeling. Indeed, iPSC-derived cells have been increasingly used for investigating molecular and cellular pathophysiological mechanisms underlying inherited diseases. However, a major barrier in the field of iPSC-based disease modeling relies on discriminating between the effects of the causative mutation and the genetic background of these cells. In the past decade, researchers have made great improvement in genome editing techniques, with one of the latest being CRISPR/Cas9. Using a single non-sequence specific protein combined with a small guiding RNA molecule, this state-of-the-art approach enables modifications of genes with high efficiency and accuracy. By so doing, this technique enables the generation of isogenic controls or isogenic mutated cell lines in order to focus on the pathologies caused by a specific mutation. In this article, we review the latest studies combining iPSC and CRISPR/Cas9 technologies for the investigation of the molecular and cellular mechanisms underlying inherited diseases including immunological, metabolic, hematological, neurodegenerative and cardiac diseases.

Generation of Duchenne muscular dystrophy patient-specific induced pluripotent stem cell line lacking exons 45-50 of the dystrophin gene (IITi001-A).

Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle degenerative disease caused by mutations in the dystrophin gene. We generated induced pluripotent stem cells (iPSCs) from a 13-year-old male patient carrying a deletion mutation of exons 45-50; iPSCs were subsequently differentiated into cardiomyocytes. iPSCs exhibit expression of the pluripotent markers (SOX2, NANOG, OCT4), differentiation capacity into the three germ layers, normal karyotype, genetic identity to the skin biopsy dermal fibroblasts and the patient-specific dystrophin mutation.

CRISPR correction of the PRKAG2 gene mutation in the patient's induced pluripotent stem cell-derived cardiomyocytes eliminates electrophysiological and structural abnormalities.

BACKGROUND: Mutations in the PRKAG2 gene encoding the γ-subunit of adenosine monophosphate kinase (AMPK) cause hypertrophic cardiomyopathy (HCM) and familial Wolff-Parkinson-White (WPW) syndrome. Patients carrying the R302Q mutation in PRKAG2 present with sinus bradycardia, escape rhythms, ventricular preexcitation, supraventricular tachycardia, and atrioventricular block. This mutation affects AMPK activity and increases glycogen storage in cardiomyocytes. The link between glycogen storage, WPW syndrome, HCM, and arrhythmias remains unknown. OBJECTIVE: The purpose of this study was to investigate the pathological changes caused by the PRKAG2 mutation. We tested the hypothesis that patient's induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) display clinical aspects of the disease. METHODS: Using clustered regularly interspaced short palindromic repeats (CRISPR) technology, we corrected the mutation and then generated isogenic iPSC-CMs. Action potentials were recorded from spontaneously firing and paced cardiomyocytes using the patch clamp technique. Using a microelectrode array setup, we recorded electrograms from iPSC-CMs clusters. Transmission electron microscopy was used to detect ultrastructural abnormalities in the mutated iPSC-CMs. RESULTS: PRKAG2-mutated iPSC-CMs exhibited abnormal firing patterns, delayed afterdepolarizations, triggered arrhythmias, and augmented beat rate variability. Importantly, CRISPR correction eliminated the electrophysiological abnormalities, the augmented glycogen, storage, and cardiomyocyte hypertrophy. CONCLUSION: PRKAG2-mutated iPSC-CMs displayed functional and structural abnormalities, which were abolished by correcting the mutation in the patient's iPSCs using CRISPR technology.

Functional abnormalities in induced Pluripotent Stem Cell-derived cardiomyocytes generated from titin-mutated patients with dilated cardiomyopathy.

AIMS: Dilated cardiomyopathy (DCM), a myocardial disorder that can result in progressive heart failure and arrhythmias, is defined by ventricular chamber enlargement and dilatation, and systolic dysfunction. Despite extensive research, the pathological mechanisms of DCM are unclear mainly due to numerous mutations in different gene families resulting in the same outcome-decreased ventricular function. Titin (TTN)-a giant protein, expressed in cardiac and skeletal muscles, is an important part of the sarcomere, and thus TTN mutations are the most common cause of adult DCM. To decipher the basis for the cardiac pathology in titin-mutated patients, we investigated the hypothesis that induced Pluripotent Stem Cell (iPSC)-derived cardiomyocytes (iPSC-CM) generated from patients, recapitulate the disease phenotype. The hypothesis was tested by 3 Aims: (1) Investigate key features of the excitation-contraction-coupling machinery; (2) Investigate the responsiveness to positive inotropic interventions; (3) Investigate the proteome profile of the AuP cardiomyocytes using mass-spectrometry (MS). METHODS AND RESULTS: iPSC were generated from the patients' skin fibroblasts. The major findings were: (1) Sarcomeric organization analysis in mutated iPSC-CM showed defects in assembly and maintenance of sarcomeric structure. (2) Mutated iPSC-CM exhibited diminished inotropic and lusitropic responses to ß-adrenergic stimulation with isoproterenol, increased [Ca2+]out and angiotensin-II. Additionally, mutated iPSC-CM displayed prolonged recovery in response to caffeine. These findings may result from defective or lack of interactions of the sarcomeric components with titin through its kinase domain which is absent in the mutated cells. CONCLUSIONS: These findings show that the mutated cardiomyocytes from DCM patients recapitulate abnormalities of the inherited cardiomyopathies, expressed as blunted inotropic response.

A Method Sustaining the Bioelectric, Biophysical, and Bioenergetic Function of Cultured Rabbit Atrial Cells.

Culturing atrial cells leads to a loss in their ability to be externally paced at physiological rates and to maintain their shape. We aim to develop a culture method that sustains the shape of atrial cells along with their biophysical and bioenergetic properties in response to physiological pacing. We hypothesize that adding 2,3-Butanedione 2-monoxime (BDM), which inhibits contraction during the culture period, will preserve these biophysical and bioenergetic properties. Rabbit atrial cells were maintained in culture for 24 h in a medium enriched with a myofilament contraction inhibitor, BDM. The morphology and volume of the cells, including their ability to contract in response to 1-3 Hz electrical pacing, was maintained at the same level as fresh cells. Importantly, the cells could be successfully infected with a GFP adenovirus. Action potentials, Ca2+ transients, and local Ca2+ spark parameters were similar in the cultured and in fresh cells. Finally, these cultured cells' flavoprotein autofluorescence was maintained at a constant level in response to electrical pacing, a response similar to that of fresh cells. Thus, eliminating contraction during the culture period preserves the bioelectric, biophysical and bioenergetic properties of rabbit atrial myocytes. This method therefore has the potential to further improve our understanding of energetic and biochemical regulation in the atria.

Patient Specific Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Drug Development and Screening In Catecholaminergic Polymorphic Ventricular Tachycardia.

Catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited arrhythmia often leading to sudden cardiac death in children and young adults, is characterized by polymorphic/bidirectional ventricular tachycardia induced by adrenergic stimulation associated with emotionally stress or physical exercise. There are two forms of CPVT: 1. CPVT1 is caused by mutations in the RYR2 gene, encoding for ryanodine receptor type 2. CPVT1 is the most common form of CPVT in the population, and is inherited by a dominant mechanism. 2. CPVT2 is caused by mutations in the CASQ2 gene, encoding for cardiac calsequestrin 2 and is inherited by recessive mechanism. Patient-specific induced Pluripotent Stem Cells (iPSC) have the ability to differentiate into cardiomyocytes carrying the patient's genome including CPVT-linked mutations and expressing the disease phenotype in vitro at the cellular level. The potency for in vitro modeling using iPSC-derived cardiomyocytes (iPSC-CMs) has been exploited to investigate a variety of inherited diseases including cardiac arrhythmias such as CPVT. In this review we attempted to cover the majority of CPVT patient specific iPSC research studies previously published. CPVT patient-specific iPSC model enables the in vitro investigation of the molecular and cellular disease-mechanisms by the means of electrophysiologycal and Ca+2 imaging methodologies. Furthermore, this in vitro model allows the screening of various antiarrhythmic drugs, specifically for each patient, also known as "personalized medicine".

Developmental changes in electrophysiological characteristics of human-induced pluripotent stem cell-derived cardiomyocytes.

BACKGROUND: Previous studies proposed that throughout differentiation of human induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs), only 3 types of action potentials (APs) exist: nodal-, atrial-, and ventricular-like. OBJECTIVES: To investigate whether there are precisely 3 phenotypes or a continuum exists among them, we tested 2 hypotheses: (1) During culture development a cardiac precursor cell is present that-depending on age-can evolve into the 3 phenotypes. (2) The predominant pattern is early prevalence of a nodal phenotype, transient appearance of an atrial phenotype, evolution to a ventricular phenotype, and persistence of transitional phenotypes. METHODS: To test these hypotheses, we (1) performed fluorescence-activated cell sorting analysis of nodal, atrial, and ventricular markers; (2) recorded APs from 280 7- to 95-day-old iPSC-CMs; and (3) analyzed AP characteristics. RESULTS: The major findings were as follows: (1) fluorescence-activated cell sorting analysis of 30- and 60-day-old cultures showed that an iPSC-CMs population shifts from the nodal to the atrial/ventricular phenotype while including significant transitional populations; (2) the AP population did not consist of 3 phenotypes; (3) culture aging was associated with a shift from nodal to ventricular dominance, with a transient (57-70 days) appearance of the atrial phenotype; and (4) beat rate variability was more prominent in nodal than in ventricular cardiomyocytes, while pacemaker current density increased in older cultures. CONCLUSION: From the onset of development in culture, the iPSC-CMs population includes nodal, atrial, and ventricular APs and a broad spectrum of transitional phenotypes. The most readily distinguishable phenotype is atrial, which appears only transiently yet dominates at 57-70 days of evolution.