RESUMO
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.
Assuntos
Adrenérgicos/farmacologia , Cálcio/metabolismo , Regulação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/patologia , Distrofia Muscular de Duchenne/patologia , Contração Miocárdica , Miócitos Cardíacos/patologia , Adulto , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Diferenciação Celular , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Pessoa de Meia-Idade , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , RNA-Seq , Receptores Adrenérgicos beta 1/genética , Receptores Adrenérgicos beta 1/metabolismo , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/patologiaRESUMO
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.
Assuntos
Heterozigoto , Células-Tronco Pluripotentes Induzidas/fisiologia , Distrofia Muscular de Duchenne/fisiopatologia , Miócitos Cardíacos/fisiologia , Potenciais de Ação/genética , Adulto , Diferenciação Celular/genética , Distrofina/genética , Distrofina/metabolismo , Fenômenos Eletrofisiológicos , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestruturaRESUMO
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.
Assuntos
Cardiomiopatia Hipertrófica/patologia , Proteínas de Transporte/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Mitocondriais/metabolismo , Miócitos Cardíacos/metabolismo , Potenciais de Ação/efeitos dos fármacos , Adulto , Arritmias Cardíacas/patologia , Cafeína/farmacologia , Cardiomiopatia Hipertrófica/fisiopatologia , Proteínas de Transporte/genética , Diferenciação Celular , Feminino , Frequência Cardíaca/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Isoproterenol/farmacologia , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/genética , Modelos Biológicos , Chaperonas Moleculares , Mutação/genética , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/ultraestruturaRESUMO
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia characterized by syncope and sudden death occurring during exercise or acute emotion. CPVT is caused by abnormal intracellular Ca(2+) handling resulting from mutations in the RyR2 or CASQ2 genes. Because CASQ2 and RyR2 are involved in different aspects of the excitation-contraction coupling process, we hypothesized that these mutations are associated with different functional and intracellular Ca(²+) abnormalities. To test the hypothesis we generated induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CM) from CPVT1 and CPVT2 patients carrying the RyR2(R420Q) and CASQ2(D307H) mutations, respectively, and investigated in CPVT1 and CPVT2 iPSC-CM (compared to control): (i) The ultrastructural features; (ii) the effects of isoproterenol, caffeine and ryanodine on the [Ca(2+) ]i transient characteristics. Our major findings were: (i) Ultrastructurally, CASQ2 and RyR2 mutated cardiomyocytes were less developed than control cardiomyocytes. (ii) While in control iPSC-CM isoproterenol caused positive inotropic and lusitropic effects, in the mutated cardiomyocytes isoproterenol was either ineffective, caused arrhythmias, or markedly increased diastolic [Ca(2+) ]i . Importantly, positive inotropic and lusitropic effects were not induced in mutated cardiomyocytes. (iii) The effects of caffeine and ryanodine in mutated cardiomyocytes differed from control cardiomyocytes. Our results show that iPSC-CM are useful for investigating the similarities/differences in the pathophysiological consequences of RyR2 versus CASQ2 mutations underlying CPVT1 and CPVT2 syndromes.
Assuntos
Calsequestrina/genética , Células-Tronco Pluripotentes Induzidas/patologia , Mutação/genética , Miócitos Cardíacos/patologia , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Taquicardia Ventricular/genética , Taquicardia Ventricular/patologia , Sequência de Bases , Cafeína/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Técnicas de Genotipagem , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Isoproterenol/farmacologia , Dados de Sequência Molecular , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/ultraestrutura , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/ultraestruturaRESUMO
Induced pluripotent stem cells (iPSC) are generated from fully differentiated somatic cells that were reprogrammed into a pluripotent state. Human iPSC which can be obtained from various types of somatic cells such as fibroblasts or keratinocytes can differentiate into cardiomyocytes (iPSC-CM), which exhibit cardiac-like transmembrane action potentials, intracellular Ca(2+) transients and contractions. While major features of the excitation-contraction coupling of iPSC-CM have been well-described, very little is known on the ultrastructure of these cardiomyocytes. The ultrastructural features of 31-day-old (post-plating) iPSC-CM generated from human hair follicle keratinocytes (HFKT-iPSC-CM) were analysed by electron microscopy, and compared with those of human embryonic stem-cell-derived cardiomyocytes (hESC-CM). The comparison showed that cardiomyocytes from the two sources share similar proprieties. Specifically, HFKT-iPSC-CM and hESC-CM, displayed ultrastructural features of early and immature phenotype: myofibrils with sarcomeric pattern, large glycogen deposits, lipid droplets, long and slender mitochondria, free ribosomes, rough endoplasmic reticulum, sarcoplasmic reticulum and caveolae. Noteworthy, the SR is less developed in HFKT-iPSC-CM. We also found in both cell types: (1) 'Ca(2+)-release units', which connect the peripheral sarcoplasmic reticulum with plasmalemma; and (2) intercellular junctions, which mimic intercalated disks (desmosomes and fascia adherens). In conclusion, iPSC and hESC differentiate into cardiomyocytes of comparable ultrastructure, thus supporting the notion that iPSC offer a viable option for an autologous cell source for cardiac regenerative therapy.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/ultraestrutura , Cálcio/metabolismo , Cavéolas/ultraestrutura , Células Cultivadas , Células-Tronco Embrionárias/fisiologia , Retículo Endoplasmático/ultraestrutura , Acoplamento Excitação-Contração , Fibroblastos/citologia , Folículo Piloso/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Queratinócitos/citologia , Potenciais da Membrana , Microscopia Eletrônica , Mitocôndrias Cardíacas/ultraestrutura , Contração Miocárdica , Retículo Sarcoplasmático/ultraestruturaRESUMO
We recently reported that propargylamine derivatives such as rasagiline (Azilect) and its S-isomer TVP1022 are neuroprotective. The aim of this study was to test the hypothesis that the neuroprotective agents TVP1022 and propargylamine (the active moiety of propargylamine derivatives) are also cardioprotective. We specifically investigated the protective efficacy of TVP1022 and propargylamine in neonatal rat ventricular myocytes (NRVM) against apoptosis induced by the anthracycline chemotherapeutic agent doxorubicin and by serum starvation. We demonstrated that pretreatment of NRVM cultures with TVP1022 or propargylamine attenuated doxorubicin-induced and serum starvation-induced apoptosis, inhibited the increase in cleaved caspase 3 levels, and reversed the decline in Bcl-2/Bax ratio. These cytoprotective effects were shown to reside in the propargylamine moiety. Finally, we showed that TVP1022 neither caused proliferation of the human cancer cell lines HeLa and MDA-231 nor interfered with the anti-cancer efficacy of doxorubicin. These results suggest that TVP1022 should be considered as a novel cardioprotective agent against ischemic insults and against anthracycline cardiotoxicity and can be coadministered with doxorubicin in the treatment of human malignancies.
Assuntos
Cardiotônicos/farmacologia , Cardiopatias/tratamento farmacológico , Indanos/farmacologia , Pargilina/análogos & derivados , Propilaminas/farmacologia , Animais , Animais Recém-Nascidos , Antibióticos Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Caspase 3/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Doxorrubicina/toxicidade , Feminino , Cardiopatias/etiologia , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/patologia , Humanos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Pargilina/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Estereoisomerismo , Proteína X Associada a bcl-2/efeitos dos fármacos , Proteína X Associada a bcl-2/metabolismoRESUMO
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.
Assuntos
Distrofina/genética , Células-Tronco Pluripotentes Induzidas/citologia , Distrofia Muscular de Duchenne/patologia , Adolescente , Diferenciação Celular/fisiologia , Éxons , Genótipo , Humanos , Masculino , Distrofia Muscular de Duchenne/genéticaRESUMO
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.
Assuntos
Proteínas Quinases Ativadas por AMP/genética , DNA/genética , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Mutação , Miócitos Cardíacos/ultraestrutura , Síndrome de Wolff-Parkinson-White/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Eletrofisiologia Cardíaca , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Análise Mutacional de DNA , Fenômenos Eletrofisiológicos , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Miócitos Cardíacos/metabolismo , Síndrome de Wolff-Parkinson-White/metabolismo , Síndrome de Wolff-Parkinson-White/patologiaRESUMO
OBJECTIVE: Cardiac hypertrophy is a compensatory response to increased mechanical load. Since Fas receptor activation is an important component in hypertrophy induced by pressure- and volume-overload, deciphering the underlying signaling pathways is of prime importance. Based on our previous work showing that in mice and rats ventricular myocytes the electrophysiological disturbances and diastolic [Ca2+]i-rise caused by 3 h of Fas activation are dependent on the Fas-->phospholipase C (PLC)-->1,4,5-inositol trisphosphate (1,4,5-IP3)-->sarcoplasmic reticulum (SR) [Ca2+]i release pathway, we tested the hypothesis that this pathway is also critical for Fas-mediated hypertrophy. METHODS: The effects of 24 h Fas activation in cultured neonatal rat ventricular myocytes (NRVM) were analyzed by means of RT-PCR, Western blot, immunofluorescence and fura-2 fluorescence. RESULTS: Fas activation increased nuclei surface area, atrial natriuretic peptide and connexin43 (Cx43) mRNA, the protein levels of total Cx43 and non-phosphorylated Cx43, and sarcomeric actin, all indicating hypertrophy. Concomitantly, Fas activation decreased mRNA of SERCA2a, the ryanodine receptor (RyR) and nuclear IP3R3. Further, Fas activation caused NFAT nuclear translocation. The hypertrophy was abolished by U73122, xestospongin C (blockers of the 1,4,5-IP3 pathway), genistein and by the PI3K blocker LY294002. CONCLUSIONS: Fas-mediated hypertrophy is dependent on the 1,4,5-IP3 pathway, which is functionally inter-connected to the PI3K/AKT/GSK3beta pathway. Both pathways act in concert to cause NFAT nuclear translocation and subsequent hypertrophy.
Assuntos
Cardiomegalia/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Transdução de Sinais , Receptor fas/metabolismo , Animais , Animais Recém-Nascidos , Apoptose , ATPases Transportadoras de Cálcio/metabolismo , Cardiomegalia/patologia , Membrana Celular/metabolismo , Células Cultivadas , Imunofluorescência , Hipertrofia , Immunoblotting , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Trocador de Sódio e Cálcio/metabolismoRESUMO
OBJECTIVES: Altered gap junctional coupling of ventricular myocytes plays an important role in arrhythmogenesis in ischemic heart disease. Since hypoxia is a major component of ischemia, we tested the hypothesis that hypoxia causes gap junctional remodeling accompanied by conduction disturbances. METHODS: Cultured neonatal rat ventricular myocytes were exposed to hypoxia (1% O(2)) for 15 min to 5 h, connexin43 (Cx43) expression was analyzed, and conduction velocity was measured using the Micro-Electrode Array data acquisition system. RESULTS: After 15 min of hypoxia, conduction velocity was unaffected, while total Cx43, including the phosphorylated and nonphosphorylated isoforms, was increased. After 5 h of hypoxia, total Cx43 protein was decreased by 50%, while the nonphosphorylated Cx43 isoform was unchanged. Confocal analyses yielded a 55% decrease in the gap junctional Cx43 fluorescence signal, a 55% decrease in gap junction number, and a 26% decrease in size. The changes in Cx43 were not accompanied by changes in mRNA levels. The reduction in Cx43 protein levels was associated with a approximately 20% decrease in conduction velocity compared to normoxic cultures. CONCLUSIONS: Short-term hypoxia (5 h) decreases Cx43 protein and conduction velocity, thereby contributing to the generation of an arrhythmogenic substrate.
Assuntos
Arritmias Cardíacas/metabolismo , Junções Comunicantes/metabolismo , Hipóxia/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Animais Recém-Nascidos , Arritmias Cardíacas/fisiopatologia , Western Blotting/métodos , Conexina 43/análise , Conexina 43/genética , Conexina 43/metabolismo , Junções Comunicantes/química , Sistema de Condução Cardíaco , Ventrículos do Coração , Imuno-Histoquímica/métodos , Microscopia Confocal , Miócitos Cardíacos/química , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
Following acute myocardial infarction (MI), early and successful reperfusion is the most effective strategy for reducing infarct size and improving the clinical outcome. However, immediate restoration of blood flow to the ischemic zone results in myocardial damage, defined as "reperfusion-injury". Whereas we previously reported that TVP1022 (the S-isomer of rasagiline, FDA-approved anti-Parkinson drug) decreased infarct size 24 h post ischemia reperfusion (I/R) in rats, in this study we investigated the chronic cardioprotective efficacy of TVP1022 14 days post-I/R. To simulate the clinical settings of acute MI followed by reperfusion therapy, we employed a rat model of left anterior descending artery occlusion for 30 min followed by reperfusion and a follow-up for 14 days. TVP1022 was initially administered postocclusion-prereperfusion, followed by chronic daily administrations. Cardiac performance and remodeling were evaluated using customary and advanced echocardiographic methods, hemodynamic measurements by Millar Mikro-Tip® catheter, and histopathological techniques. TVP1022 administration markedly decreased the remodeling process as illustrated by attenuation of left ventricular enlargement and cardiac hypertrophy (both at the whole heart and the cellular level). Furthermore, TVP1022 inhibited cardiac fibrosis and reduced ventricular BNP levels. Functionally, TVP1022 treatment preserved cardiac wall motion. Specifically, the echocardiographic and most of the direct hemodynamic measures were pronouncedly improved by TVP1022. Collectively, these findings indicate that TVP1022 provides prominent cardioprotection against I/R injury and post-MI remodeling in this I/R model.
RESUMO
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.