The objective of the current study was to characterize calcium handling in developing human embryonic stem cell-derived cardiomyocytes (hESC-CMs). To this end, real-time polymerase chain reaction ...(PCR), immunocytochemistry, whole-cell voltage-clamp, and simultaneous patch-clamp/laser scanning confocal calcium imaging and surface membrane labeling with di-8-aminonaphthylethenylpridinium were used. Immunostaining studies in the hESC-CMs demonstrated the presence of the sarcoplasmic reticulum (SR) calcium release channels, ryanodine receptor-2, and inositol-1,4,5-trisphosphate (IP3) receptors. Store calcium function was manifested as action-potential-induced calcium transients. Time-to-target plots showed that these action-potential-initiated calcium transients traverse the width of the cell via a propagated wave of intracellular store calcium release. The hESC-CMs also exhibited local calcium events ("sparks") that were localized to the surface membrane. The presence of caffeine-sensitive intracellular calcium stores was manifested following application of focal, temporally limited puffs of caffeine in three different age groups: early-stage (with the initiation of beating), intermediate-stage (10 days post-beating dpb), and late-stage (30-40 dpb) hESC-CMs. Calcium store load gradually increased during in vitro maturation. Similarly, ryanodine application decreased the amplitude of the spontaneous calcium transients. Interestingly, the expression and function of an IP3-releasable calcium pool was also demonstrated in the hESC-CMs in experiments using caged-IP3 photolysis and antagonist application (2 microM 2-Aminoethoxydiphenyl borate). In summary, our study establishes the presence of a functional SR calcium store in early-stage hESC-CMs and shows a unique pattern of calcium handling in these cells. This study also stresses the importance of the functional characterization of hESC-CMs both for developmental studies and for the development of future myocardial cell replacement strategies. Disclosure of potential conflicts of interest is found at the end of this article.
Abnormal action potential (AP) properties, as occurs in long or short QT syndromes (LQTS and SQTS, respectively), can cause life-threatening arrhythmias. Optogenetics strategies, utilizing ...light-sensitive proteins, have emerged as experimental platforms for cardiac pacing, resynchronization, and defibrillation. We tested the hypothesis that similar optogenetic tools can modulate the cardiomyocyte's AP properties, as a potentially novel antiarrhythmic strategy. Healthy control and LQTS/SQTS patient-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were transduced to express the light-sensitive cationic channel channelrhodopsin-2 (ChR2) or the anionic-selective opsin, ACR2. Detailed patch-clamp, confocal-microscopy, and optical mapping studies evaluated the ability of spatiotemporally defined optogenetic protocols to modulate AP properties and prevent arrhythmogenesis in the hiPSC-CMs cell/tissue models. Depending on illumination timing, light-induced ChR2 activation induced robust prolongation or mild shortening of AP duration (APD), while ACR2 activation allowed effective APD shortening. Fine-tuning these approaches allowed for the normalization of pathological AP properties and suppression of arrhythmogenicity in the LQTS/SQTS hiPSC-CM cellular models. We next established a SQTS-hiPSC-CMs-based tissue model of reentrant-arrhythmias using optogenetic cross-field stimulation. An APD-modulating optogenetic protocol was then designed to dynamically prolong APD of the propagating wavefront, completely preventing arrhythmogenesis in this model. This work highlights the potential of optogenetics in studying repolarization abnormalities and in developing novel antiarrhythmic therapies.
Human embryonic stem cell-derived cardiomyocytes (hES-CMs) are thought to recapitulate the embryonic development of heart
cells. Given the exciting potential of hES-CMs as replacement tissue in ...diseased hearts, we investigated the pharmacological
sensitivity and ionic current of mid-stage hES-CMs (20â35 days post plating). A high-resolution microelectrode array was used
to assess conduction in multicellular preparations of hES-CMs in spontaneously contracting embryoid bodies (EBs). TTX (10
μ m ) dramatically slowed conduction velocity from 5.1 to 3.2 cm s â1 while 100 μ m TTX caused complete cessation of spontaneous electrical activity in all EBs studied. In contrast, the Ca 2+ channel blockers nifedipine or diltiazem (1 μ m ) had a negligible effect on conduction. These results suggested a prominent Na + channel current, and therefore we patch-clamped isolated cells to record Na + current and action potentials (APs). We found for isolated hES-CMs a prominent Na + current (244 ± 42 pA pF â1 at 0 mV; n = 19), and a hyperpolarization-activated current (HCN), but no inward rectifier K + current. In cell clusters, 3 μ m TTX induced longer AP interpulse intervals and 10 μ m TTX caused cessation of spontaneous APs. In contrast nifedipine (Ca 2+ channel block) and 2 m m Cs + (HCN complete block) induced shorter AP interpulse intervals. In single cells, APs stimulated by current pulses had a maximum
upstroke velocity (d V /d t max ) of 118 ± 14 V s â1 in control conditions; in contrast, partial block of Na + current significantly reduced stimulated d V /d t max (38 ± 15 V s â1 ). RT-PCR revealed Na V 1.5, Ca V 1.2, and HCN-2 expression but we could not detect Kir2.1. We conclude that hES-CMs at mid-range development express prominent
Na + current. The absence of background K + current creates conditions for spontaneous activity that is sensitive to TTX in the same range of partial block of Na V 1.5; thus, the Na V 1.5 Na + channel is important for initiating spontaneous excitability in hES-derived heart cells.
Heterozygous missense variants of the cardiac ryanodine receptor gene (RYR2) cause catecholaminergic polymorphic ventricular tachycardia (CPVT). These missense variants of RYR2 result in a gain of ...function of the ryanodine receptors, characterized by increased sensitivity to activation by calcium that results in an increased propensity to develop calcium waves and delayed afterdepolarizations. We have recently detected a nonsense variant in RYR2 in a young patient who suffered an unexplained cardiac arrest. To understand the mechanism by which this variant in RYR2, p.(Arg4790Ter), leads to ventricular arrhythmias, human induced pluripotent stem cells (hiPSCs) harboring the novel nonsense variant in RYR2 were generated and differentiated into cardiomyocytes (RYR2‐hiPSC‐CMs) and molecular and calcium handling properties were studied. RYR2‐hiPSC‐CMs displayed significant calcium handling abnormalities at baseline and following treatment with isoproterenol. Treatment with carvedilol and nebivolol resulted in a significant reduction in calcium handling abnormalities in the RYR2‐hiPSC‐CMs. Expression of the mutant RYR2 allele was confirmed at the mRNA level and partial silencing of the mutant allele resulted in a reduction in calcium handling abnormalities at baseline. The nonsense variant behaves similarly to other gain of function variants in RYR2. Carvedilol and nebivolol may be suitable treatments for patients with gain of function RYR2 variants.
The RYR2 gene encodes for the cardiac ryanodine receptor, a major calcium handling channel in cardiomyocytes. Heterozygous variants within the RYR2 gene cause the inherited arrhythmia syndrome catecholaminergic polymorphic ventricular tachycardia (CPVT). The majority of these variants are missense changes and lead to a phenotype by a gain of function mechanism. We identified a novel nonsense variant in RYR2 in young women who suffered a cardiac arrest and show, using cardiomyocytes derived from induced pluripotent stem cells, that this variant appears to lead to abnormal calcium handling in a similar mechanism to the previously described gain of function variants.
Background
Determining the pathogenesis of sudden cardiac arrest (SCA) in children is crucial for its management and prognosis. Our aim is to analyze the role of broad genetic testing in the ...prevention, diagnosis, and prognosis of SCA in Children.
Methods
ECG, 12‐lead holter, exercise testing, cardiac imaging, familial study, and genetic testing were used to study 29 families, in whom a child experienced SCA.
Results
After a thorough clinical and genetic evaluation a positive diagnosis was reached in 24/29 (83%) families. Inherited channelopathies (long QT syndrome and catecholaminergic polymorphic ventricular tachycardia) were the most prevalent 20/29 (69%) diagnosis, followed by cardiomyopathy 3/29 (10%). Broad genetic testing was positive in 17/24 (71%) cases. Using the Mann–Whitney test, we found that genetic testing (effect size = 0.625, p = 0.003), ECG (effect size = 0.61, p = 0.009), and exercise test (effect size = 0.63, p = 0.047) had the highest yield in reaching the final diagnosis. Genetic testing was the only positive test available for five (17%) families. Among 155 family members evaluated through cascade screening, 73 (47%) had a positive clinical evaluation and 64 (41%) carried a pathologic mutation. During 6 ± 4.8 years of follow‐up, 58% of the survived children experienced an arrhythmic event. Of nine family members who had an ICD implant for primary prevention, four experienced appropriate ICD shock.
Conclusions
The major causes of SCA among children are genetic etiology, and genetic testing has a high yield. Family screening has an additional role in both the diagnosis and preventing of SCA.
ECG, 12 lead holter, exercise testing, echocardiography, familial study, and genetic testing were used to study 29 families, in whom a child experienced sudden cardiac death or sudden cardiac arrest. LQT: long QT syndrome, CPVT: catecholaminergic polymorphic ventricular tachycardia, CM cardiomyopathy.
Long-QT syndrome type 1 (LQT1) is caused by mutations in
. Patients heterozygous for such a mutation co-assemble both mutant and wild-type
-encoded subunits into tetrameric Kv7.1 potassium channels. ...Here, we investigated whether allele-specific inhibition of mutant
by targeting a common variant can shift the balance towards increased incorporation of the wild-type allele to alleviate the disease in human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs). We identified the single nucleotide polymorphisms (SNP) rs1057128 (G/A) in
, with a heterozygosity of 27% in the European population. Next, we determined allele-specificity of short-hairpin RNAs (shRNAs) targeting either allele of this SNP in hiPSC-CMs that carry an LQT1 mutation. Our shRNAs downregulated 60% of the A allele and 40% of the G allele without affecting the non-targeted allele. Suppression of the mutant
allele by 60% decreased the occurrence of arrhythmic events in hiPSC-CMs measured by a voltage-sensitive reporter, while suppression of the wild-type allele increased the occurrence of arrhythmic events. Furthermore, computer simulations based on another LQT1 mutation revealed that 60% suppression of the mutant
allele shortens the prolonged action potential in an adult cardiomyocyte model. We conclude that allele-specific inhibition of a mutant
allele by targeting a common variant may alleviate the disease. This novel approach avoids the need to design shRNAs to target every single mutation and opens up the exciting possibility of treating multiple LQT1-causing mutations with only two shRNAs.
Calsequestrin-associated catecholaminergic polymorphic ventricular tachycardia (CPVT2) can cause sudden death in young individuals in response to stress. Beta-blockers are the mainstay medical ...treatment for patients with CPVT2. However, they do not prevent syncope and sudden death in all patients. Flecainide was reported to reduce exercise-induced ventricular arrhythmias (EIVA) in patients with ryanodine receptor-associated CPVT. The role of flecainide in CPVT2 is not known.
To summarize our experience in combining flecainide and beta-blockers in high-risk patients with CPVT2.
All patients with CPVT2 (10 patients) who have high-risk features (syncope, EIVA, or appropriate implantable cardioverter-defibrillator ICD shocks) despite beta-blockers with or without calcium channel blockers were treated with a combination of flecainide and beta-blockers. Exercise test was done before and after beginning treatment with flecainide.
All patients had EIVA and 4 had appropriate ICD shocks before flecainide treatment. EIVA-included frequent ventricular premature beats and or ventricular tachycardia during the exercise test while on high dose of beta-blockers with or without calcium channel blockers before treatment with flecainide. After combination therapy with flecainide and beta-blockers, EIVA were suppressed completely in all patients. During follow-up of 15.5 ± 10.4 months (range 2-29 months), 8 patients were free of symptoms and free of arrhythmias. Two patients had 1 VT storm episode with recurrent ICD shocks despite repeated normal stress test.
Flecainide can completely prevent ventricular arrhythmia during exercise and partially prevent recurrent ICD shocks in high-risk patients with CPVT2.
Human induced pluripotent stem (hiPS) cells provide therapeutic promises, as well as a potent in vitro model for studying biological processes which take place during human embryonic development and ...subsequent differentiation in normal and disease states. The epigenetic characteristics of iPS cells are reprogrammed to the embryonic state at which they acquire pluripotency. In addition, telomeres in hiPS cell must elongate sufficiently to provide the necessary replicative potential. Recent studies have demonstrated that the epigenetic characteristics of telomeric and subtelomeric regions are pivotal in regulating telomere length. Here we study telomere length, subtelomeric DNA methylation and telomeric-repeat-containing RNA (TERRA) expression in several hiPS cell clones derived from normal neonatal foreskin fibroblasts. We find that telomeres lengthen significantly in hiPS cells in comparison to the parental fibroblast source, and progressively shorten after differentiation back into fibroblast-like cells, concomitantly with telomerase activation and down-regulation, respectively. Subtelomeres in hiPS cells were found to be generally hypermethylated in comparison to the parental source. However bisulfite analysis revealed that at several subtelomeres examined, methylation levels differed between hiPS clones and that both de novo methylation and demethylation processes occurred during telomere reprogramming. Notably, although subtelomeres were in general very highly methylated, TERRA levels were elevated in hiPS cells, albeit to different degrees in the various clones. TERRA elevation may reflect enhanced stability or impaired degradation in hiPS cells, and/or alternatively, increased transcription from the hypomethylated subtelomeres. We suggest that TERRA may play a role in regulation of appropriate telomere function and length in hiPS cells.
Diagnosis of AF-induced cardiomyopathy can be challenging and relies on ruling out other causes of cardiomyopathy and, after restoration of sinus rhythm, recovery of left ventricular (LV) function. ...The aim of this study was to identify clinical and echocardiographic predictors for developing cardiomyopathy with systolic dysfunction in patients with atrial tachyarrhythmia.
This retrospective study was conducted in a large tertiary care centre and compared patients who experienced deterioration of LV ejection fraction (EF) during paroxysmal AF, demonstrated by precardioversion transoesophageal echocardiography with patients with preserved LV function during AF. All patients had documented preserved LVEF at baseline (EF >50%) while in sinus rhythm.
Of 482 patients included in the final analysis, 80 (17%) had reduced and 402 (83%) had preserved LV function during the precardioversion transoesophageal echocardiography. Patients with reduced LVEF were more likely to be men and to have a more rapid ventricular response during AF or atrial flutter (AFL). A history of prosthetic valves was also identified as a risk factor for reduced LVEF. Patients with reduced LVEF also had higher incidence of tricuspid regurgitation and right ventricular dysfunction.
In 'real-world' experience, male patients with rapid ventricular response during paroxysmal AF or AFL are more prone to LVEF reduction. Patients with prosthetic valves are also at risk for LVEF reduction during AF/AFL. Finally, tricuspid regurgitation and right ventricular dysfunction may indicate relatively long-standing AF with an associated reduction in LVEF.
Sleep-disordered breathing (SDB) has been associated with various benign cardiac arrhythmias occurring during sleep.
The purpose of this study was to demonstrate that SDB contributes to the ...development of life-threatening ventricular arrhythmias in patients with an established arrhythmic substrate.
We prospectively studied the association between SDB and timing of life-threatening ventricular arrhythmic events in 45 patients with an implantable cardioverter-defibrillator (ICD). SDB was defined as an apnea-hypopnea index (AHI) >10 events/hour based on an overnight sleep study. The primary outcome measure was appropriate ICD therapy, defined as antitachycardia pacing or shock for ventricular tachycardia or ventricular fibrillation during 1-year follow-up.
SDB was present in 26 (57.8%) patients. Appropriate ICD therapies were higher among patients with SDB (73% vs 47%, P = .02). Logistic regression identified SDB as a predictor of any appropriate ICD therapy (odds ratio 4.4, 95% confidence interval 1.4-15.3, P = .01). The risk for ventricular arrhythmias was higher in patients with SDB solely due to an increase in events occurring between midnight and 6 AM (odds ratio 5.6, 95% confidence interval 2.0-15.6, P = .001) with no discernible effect on appropriate ICD therapy during nonsleeping hours (odds ratio 0.7, 95% confidence interval 0.2-2.3, P = .61).
Patients with an ICD and SDB have a striking increase in the onset of life-threatening ventricular arrhythmic events during sleeping hours. These findings provide a rationale for SDB screening in patients with appropriate ICD therapy if device interrogation reveals a predominance of nocturnal onset of arrhythmias.
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