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.

Role of tissue C-reactive protein in atrial cardiomyocytes of patients undergoing catheter ablation of atrial fibrillation: pathogenetic implications.

AIMS: Histological studies support the important role of inflammation in the initiation and maintenance of atrial fibrillation (AF). We describe a novel and safe technique of atrial biopsy during AF radiofrequency catheter ablation (RFCA) to investigate the role of atrial tissue inflammation. METHODS AND RESULTS: We enrolled 70 consecutive patients (age 60 ± 12 years, 49 males) undergoing RFCA for AF. The control group was represented by 10 patients with Wolff-Parkinson-White syndrome undergoing trans-septal puncture. Atrial biopsies were obtained by washing the dilator and needle used for trans-septal puncture with 20 mL sterile phosphate-buffered saline. The presence of intracytoplasmic C-reactive protein was assessed in formalin-fixed atrial specimens by immunohistochemistry. A sufficient amount of atrial tissue was obtained in 23/70 (32%) patients with AF and in 4/10 (40%) of the control group. Intracytoplasmic localization of C-reactive protein was found in isolated atrial cardiomyocytes in 11 (73%) of 15 patients with paroxysmal AF as compared with 2 (25%) of eight patients with persistent AF (P= 0.02). CONCLUSION: In this study, we demonstrate the safety and feasibility of a novel technique to obtain atrial specimens during routine trans-septal puncture. Local inflammation assessed by atrial tissue localization of C-reactive protein is more likely involved in paroxysmal rather than in persistent AF.

Effects of electrostimulation of the vagus afferent endings in patients with coronary artery disease.

BACKGROUND: In rats, low-frequency electroneurostimulation (ENS) of the ear afferent vagus endings and brainstem structures via transauricular electroacupuncture increases the parasympathetic tone of the autonomic nervous system. We examined the effects of ENS applied in the preoperative period in patients with coronary artery disease (CAD) who underwent coronary artery bypass grafting operations. METHODS: ENS was performed in a group of 10 patients with CAD who were classified as Canadian Cardiovascular Society class IV before operation. A second group of 10 patients with a similar degree of CAD comprised a control group. The efficacy of the procedure was evaluated on the basis of improvement in the patient's clinical course before and after operation. The presence of major inducible stress protein (HSP 70i) and the ATP content of the myocardium was determined in atrial tissue (part of the right heart auricle) from both groups of patients. Tissue from patients with Wolff-Parkinson-White (WPW) syndrome served as a control, non-ischemic, cardiac tissue. RESULTS: A course of 10 ENS procedures improved the patients' preoperative clinical course, producing a rapid reduction in their angina. By the end of the course, the patients no longer developed angina either at rest or after a moderate physical load, and their dependence on treatment with vasodilators had decreased considerably. Atrial tissue from patients with CAD who were treated with ENS was similar to that from patients with WPW in lcaking HSP 70i and in ATP content. In contrast, tissue from the control group of patients with CAD contained HSP 70i and had an increased content of ATP. The antianginal effect of neurostimulation lasted for at least 2-3 weeks after completion of the procedures. The postoperative recovery of patients treated with ENS was characterized by stable hemodynamics and sinus rhythm. CONCLUSION: We conclude that ENS had a central vagotonic/sympatholytic influence on the heart, leading to the development of three main effects in patients with CAD: relief of anginal symptoms, diminution of some biochemical myocardial signs of the disease, and an increase in the heart's tolerance of operative reperfusion damage.

[The metabolic characteristics of nonesterified fatty acids in the myocardium of patients with paroxysmal tachycardias and their pathogenetic role in the origin of the heart rhythm disorders]. / Osobennosti metabolizma neésterifitsirovannykh zhirnykh kislot v miokarde bol'nykh s paraksizmal'nymi takhikardiiami i ikh patogeneticheskaia rol' v vozniknovenii narushenii ritma serdtsa.

Twenty-one patients suffering from paroxysmal tachycardia (PT) were examined for metabolism of nonesterified fatty acids (NEFA) in the myocardium as well as for interrelations with the parameters of clinical electrophysiology. It has been shown that the main features of NEFA metabolism in the myocardium of RT patients lie in their high concentration in arterial blood and in the blood of the coronary heart sinus and in their high ratio of extraction by the myocardium both in the presence of sinus rhythm and during RT, which is of the pathogenetic importance in the origin of heart rhythm disturbances. NEFA exert different effects on the antegrade and retrograde (functional and effective) refractory periods of different parts of the conduction system of the heart, favouring the onset of their electrophysiological heterogeneity.

Use of oral verapamil in long-term treatment of neonatal, paroxysmal supraventricular tachycardia. A pharmacokinetic study.

Efficacy and pharmacokinetics of verapamil were studied in two neonates affected by supraventricular paroxysmal tachycardia, under maintenance treatment with the drug. Verapamil proved to be fully effective in suppressing arrhythmic episodes at the daily doses of 1.5 mg/kg four times a day in case 1 and of 2 mg/kg in case 2. The results of plasma half-life of the drug, calculated in a dose interval, were 3.14 hr and 2.10 hr, respectively. In patient 2, doses less than 0.95 mg/kg four times a day did not produce detectable drug plasma levels, while a further stepwise increase of dose up to 2 mg/kg four times a day produced a steep rise in trough concentration. So, in view of this dose-concentration relationship, caution is recommended in adjusting verapamil oral dosage.