Improved Generation of Human Induced Pluripotent Stem Cell-Derived Cardiac Pacemaker Cells Using Novel Differentiation Protocols.

Current protocols for the differentiation of human-induced pluripotent stem cells (hiPSC) into cardiomyocytes only generate a small amount of cardiac pacemaker cells. In previous work, we reported the generation of high amounts of cardiac pacemaker cells by co-culturing hiPSC with mouse visceral endoderm-like (END2) cells. However, potential medical applications of cardiac pacemaker cells generated according to this protocol, comprise an incalculable xenogeneic risk. We thus aimed to establish novel protocols maintaining the differentiation efficiency of the END2 cell-based protocol, yet eliminating the use of END2 cells. Three protocols were based on the activation and inhibition of the Wingless/Integrated (Wnt) signaling pathway, supplemented either with retinoic acid and the Wnt activator CHIR99021 (protocol B) or with the NODAL inhibitor SB431542 (protocol C) or with a combination of all three components (protocol D). An additional fourth protocol (protocol E) was used, which was originally developed by the manufacturer STEMCELL Technologies for the differentiation of hiPSC or hESC into atrial cardiomyocytes. All protocols (B, C, D, E) were compared to the END2 cell-based protocol A, serving as reference, in terms of their ability to differentiate hiPSC into cardiac pacemaker cells. Our analysis revealed that protocol E induced upregulation of 12 out of 15 cardiac pacemaker-specific genes. For comparison, reference protocol A upregulated 11, while protocols B, C and D upregulated 9, 10 and 8 cardiac pacemaker-specific genes, respectively. Cells differentiated according to protocol E displayed intense fluorescence signals of cardiac pacemaker-specific markers and showed excellent rate responsiveness to adrenergic and cholinergic stimulation. In conclusion, we characterized four novel and END2 cell-independent protocols for the differentiation of hiPSC into cardiac pacemaker cells, of which protocol E was the most efficient.

Novel approach to discriminate left bundle branch block from nonspecific intraventricular conduction delay using pacing-induced functional left bundle branch block.

PURPOSE: Left bundle branch block (LBBB) has a predictive value for response to cardiac resynchronization therapy as reported by Zareba et al. (Circulation 123(10):1061-1072, 2011). However, based on ECG criteria, the discrimination between complete LBBB and nonspecific intraventricular conduction delay is challenging. We tested the hypothesis that discrimination can be performed using standard electrophysiological catheters and a simple stimulation protocol. METHODS: Fifty-nine patients were analyzed retrospectively. Patients were divided into groups of narrow QRS (n = 20), wide QRS of right bundle branch block (RBBB) morphology (n = 14), and wide QRS of LBBB morphology (n = 25). Using a diagnostic catheter placed in the coronary sinus, left ventricular activation was assessed during intrinsic conduction as well as during right ventricular (RV) stimulation. RESULTS: In patients with narrow QRS and RBBB, the Q-LV/QRS ratio was 0.43 ± 0.013 (n = 20) and 0.41 ± 0.026 (n = 14), respectively. In patients with LBBB morphology, the Q-LV/QRS split up into a group of patients with normal (0.43 ± 0.022, n = 7) and a group with delayed left ventricular activation (0.75 ± 0.016, n = 18). By direct comparison of the Q-LV/QRS ratio during intrinsic conduction with the Q-LV/QRS ratio during RV pacing leading to a functional LBBB, a clear distinction between a group of "true LBBB" and another group of "apparent LBBB"/nonspecific intraventricular conduction delay (NICD) could be generated. CONCLUSIONS: We present a novel and practical method that might facilitate discrimination between patients with apparent LBBB and true LBBB by comparing Q-LV/QRS ratios during intrinsic activation and during RV stimulation. Although this method can already be directly applied, validation by 3D electrical mapping and prospective correlation to cardiac resynchronization therapy (CRT) response will be required for further translation into clinical practice.

Prevalence of intracardiac thrombi under phenprocoumon, direct oral anticoagulants (dabigatran and rivaroxaban), and bridging therapy in patients with atrial fibrillation and flutter.

Direct oral anticoagulants (DOACs) are effective for stroke prevention in nonvalvular atrial fibrillation (AF). Cardioversion (CV) is frequently performed in patients with AF or flutter. To further explore the safety profile of DOACs in the context of CV, we sought to assess the prevalence of intracardiac thrombi under DOAC therapy in comparison with treatment with vitamin K antagonists. A total of 672 transesophageal echocardiograms performed in 643 patients with a history of nonvalvular AF were analyzed. The median CHA2DS2-VASc score was 4. Cases were stratified according to anticoagulation with dabigatran (n = 79), rivaroxaban (n = 122), phenprocoumon (n = 180), or bridging therapy (n = 287). In a subgroup analysis, only patients receiving phenprocoumon with an international normalized ratio ≥2 on the day of the investigation or on DOAC therapy for ≥3 weeks were considered. The prevalence of intracardiac thrombi under phenprocoumon was significantly higher than under DOACs (phenprocoumon, 17.8%; all DOACs, 3.9%; dabigatran, 3.8%; rivaroxaban, 4.1%) and showed no significant difference to bridging therapy (12.5%). In patients with sufficient short-term anticoagulation, similar differences between DOAC and phenprocoumon groups were observed (phenprocoumon, 18.4%; all DOACs, 3.8%; dabigatran, 0%; rivaroxaban, 6.6%). The influence of anticoagulation medication on thrombus rates was confirmed after adjusting for baseline intergroup differences regarding left atrial size and CHA2DS2-VASc score. In conclusion, the prevalence of intracardiac thrombi was lower under DOAC therapy than under phenprocoumon in this high-risk patient cohort. Safety of CV during DOAC treatment requires further prospective evaluation.

The symptom complex of familial sinus node dysfunction and myocardial noncompaction is associated with mutations in the HCN4 channel.

BACKGROUND: Inherited arrhythmias were originally considered isolated electrical defects. There is growing evidence that ion channel dysfunction also contributes to myocardial disorders, but genetic overlap has not been reported for sinus node dysfunction (SND) and noncompaction cardiomyopathy (NCCM). OBJECTIVES: The study sought to investigate a familial electromechanical disorder characterized by SND and NCCM, and to identify the underlying genetic basis. METHODS: The index family and a cohort of unrelated probands with sinus bradycardia were examined by electrocardiography, Holter recording, exercise stress test, echocardiography, and/or cardiac magnetic resonance imaging. Targeted next-generation and direct sequencing were used for candidate gene analysis and mutation scanning. Ion channels were expressed in HEK293 cells and studied using patch-clamp recordings. RESULTS: SND and biventricular NCCM were diagnosed in multiple members of a German family. Segregation analysis suggested autosomal-dominant inheritance of the combined phenotype. When looking for potentially disease-causing gene variants with cosegregation, a novel hyperpolarization-activated cyclic nucleotide channel 4 (HCN4)-G482R mutation and a common cysteine and glycine-rich protein 3 (CSRP3)-W4R variant were identified. HCN4-G482R is located in the highly conserved channel pore domain. Mutant subunits were nonfunctional and exerted dominant-negative effects on wild-type current. CSRP3-W4R has previously been linked to dilated and hypertrophic cardiomyopathy, but was also found in healthy subjects. Moreover, different truncation (695X) and missense (P883R) HCN4 mutations segregated with a similar combined phenotype in an additional, unrelated family and a single unrelated proband respectively, which both lacked CSRP3-W4R. CONCLUSIONS: The symptom complex of SND and NCCM is associated with heritable HCN4 defects. The NCCM phenotype may be aggravated by a common CSRP3 variant in one of the families.

Functional characterization of zebrafish K2P18.1 (TRESK) two-pore-domain K+ channels.

The human KCNK18 gene is predominantly expressed in brain, spinal cord, and dorsal root ganglion neurons. Encoded K2P18.1K(+) channels are functionally implicated in migraine, pain and anesthesia. Data delineating the in vivo significance of K2P18.1 are still limited owing to a lack of model systems allowing for rapid, whole organism phenotypic analyses. We hypothesized that zebrafish (Danio rerio) might close this scientific gap. This work was designed to characterize the zebrafish ortholog of K2P18.1 in comparison to human K2P18.1 channels. The complete coding sequence of zKCNK18 was amplified from zebrafish cDNA. Zebrafish KCNK18 expression was assessed by in situ hybridization. Human and zebrafish K2P18.1 currents were functionally analyzed using two-electrode voltage clamp electrophysiology and the Xenopus oocyte expression system. KCNK18 mRNA is expressed in zebrafish brain and eyes. Human and zebrafish K2P18.1 proteins share 32 % identity. Zebrafish K2P18.1 channels mediate K(+)-selective background currents that stabilize the negative resting membrane potential. Functional similarities between human and zK2P18.1 currents include open rectification properties, inhibition by barium, and regulation by signaling molecules protein kinase (PK)C, PKA, and phospholipase C. In contrast to the human ortholog, zK2P18.1 exhibited reduced sensitivity to elevation of intracellular calcium levels by ionomycin and was virtually insensitive to inhibition by quinidine. Zebrafish and human K2P18.1 channels share functional and regulatory properties, indicating that the zebrafish may serve as model to assess K2P18.1 function in vivo. However, distinct differences in K2P18.1 current regulation require careful consideration when zebrafish data are extrapolated to human physiology.

Altered HCN4 channel C-linker interaction is associated with familial tachycardia-bradycardia syndrome and atrial fibrillation.

AIMS: HCN4 channels are involved in generation, regulation, and stabilization of heart rhythm and channel dysfunction is associated with inherited sinus bradycardia. We asked whether dysfunctional HCN4 channels also contribute to the generation of cardiac tachyarrhythmias. METHODS AND RESULTS: In a candidate gene approach, we screened 422 patients with atrial and/or ventricular tachyarrhythmias and detected a novel HCN4 gene mutation that replaced the positively charged lysine 530 with an asparagine (HCN4-K530N) in a highly conserved region of the C-linker. The index patient developed tachycardia-bradycardia syndrome and persistent atrial fibrillation (AF) in an age-dependent fashion. Pedigree analysis identified eight affected family members with a similar course of disease. Whole-cell patch clamp electrophysiology of HEK293 cells showed that homomeric mutant channels almost are indistinguishable from wild-type channels. In contrast, heteromeric channels composed of mutant and wild-type subunits displayed a significant hyperpolarizing shift in the half-maximal activation voltage. This may be caused by a shift in the equilibrium between the tonically inhibited nucleotide-free state of the C-terminal domain of HCN4 believed to consist of a 'dimer of dimers' and the activated ligand-bound tetrameric form, leading to an increased inhibition of activity in heteromeric channels. CONCLUSION: Altered C-linker oligomerization in heteromeric channels is considered to promote familial tachycardia-bradycardia syndrome and persistent AF, indicating that f-channel dysfunction contributes to the development of atrial tachyarrhythmias.

Electrophysiological findings in Fabry cardiomyopathy: mapping the maze of risk stratification.

We report a case of Anderson-Fabry disease in a young man presenting with cardiac hypertrophy and asymptomatic non-sustained ventricular tachycardia. The patient was referred for evaluation of implantable cardioverter/defibrillator therapy. Assessment of left ventricular ejection fraction is considered the gold standard for identifying patients at risk of sudden cardiac death. However, this patient's left ventricular function was preserved. Electrophysiological study did not reveal inducible arrhythmia or cardiac conduction abnormalities. Review of the literature indicates limited knowledge on the electrophysiology of Fabry cardiomyopathy and highlights the need for optimized risk stratification strategies.

Initial experience with robotic navigation for catheter ablation of paroxysmal and persistent atrial fibrillation.

BACKGROUND AND PURPOSE: Remote robotic navigation (RRN) technology has been developed to facilitate catheter ablation of symptomatic atrial fibrillation (AF). Here, we assess procedural parameters of AF ablation obtained during initial use of RRN compared with a control group treated with a manual ablation approach. METHODS: Consecutive patients with symptomatic paroxysmal or persistent AF were subjected to radiofrequency catheter ablation with RRN (Sensei X [Hansen Medical, Mountain View, CA]; n = 25; mean age, 60 ± 2.3 years) or using the standard manual technique (n = 61; mean age, 62 ± 1.4 years). A circumferential pulmonary vein isolation approach guided by 3-dimensional electroanatomical mapping was followed. RESULTS: Remote robotic navigation was associated with reduction of overall fluoroscopy time by 26%. In a case-control subgroup analysis comparing 25 patients with similar clinical characteristics from each group, mean fluoroscopy time was reduced by 22%. Acute isolation of pulmonary veins was achieved in 97% (RRN) and 96% (conventional ablation), respectively. Ablation times and frequency of adverse events were not significantly different among study groups. CONCLUSIONS: The early use of RRN resulted in a significant reduction of overall fluoroscopy time and was equally effective and safe compared with manual catheter ablation.

Identification and functional characterization of zebrafish K(2P)10.1 (TREK2) two-pore-domain K(+) channels.

Two-pore-domain potassium (K(2P)) channels mediate K(+) background currents that stabilize the resting membrane potential and contribute to repolarization of action potentials in excitable cells. The functional significance of K(2P) currents in cardiac electrophysiology remains poorly understood. Danio rerio (zebrafish) may be utilized to elucidate the role of cardiac K(2P) channels in vivo. The aim of this work was to identify and functionally characterize a zebrafish otholog of the human K(2P)10.1 channel. K(2P)10.1 orthologs in the D. rerio genome were identified by database analysis, and the full zK(2P)10.1 coding sequence was amplified from zebrafish cDNA. Human and zebrafish K(2P)10.1 proteins share 61% identity. High degrees of conservation were observed in protein domains relevant for structural integrity and regulation. K(2P)10.1 channels were heterologously expressed in Xenopus oocytes, and currents were recorded using two-electrode voltage clamp electrophysiology. Human and zebrafish channels mediated K(+) selective background currents leading to membrane hyperpolarization. Arachidonic acid, an activator of hK(2P)10.1, induced robust activation of zK(2P)10.1. Activity of both channels was reduced by protein kinase C. Similar to its human counterpart, zK(2P)10.1 was inhibited by the antiarrhythmic drug amiodarone. In summary, zebrafish harbor K(2P)10.1 two-pore-domain K(+) channels that exhibit structural and functional properties largely similar to human K(2P)10.1. We conclude that the zebrafish represents a valid model to study K(2P)10.1 function in vivo.

Alternative splicing determines mRNA translation initiation and function of human K(2P)10.1 K+ channels.

Potassium-selective ion channels regulate cardiac and neuronal excitability by stabilizing the resting membrane potential and by modulating shape and frequency of action potentials. The delicate control of membrane voltage requires structural and functional diversity of K+ channel subunits expressed in a given cell. Here we reveal a previously unrecognized biological mechanism. Tissue-specific mRNA splicing regulates alternative translation initiation (ATI) of human K(2P)10.1 K+ background channels via recombination of 5 nucleotide motifs. ATI-dependent expression of full-length protein or truncated subunits initiated from two downstream start codons determines macroscopic current amplitudes and biophysical properties of hK(2P)10.1 channels. The interaction between hK(2P)10.1 mRNA splicing, translation and function increases K+ channel complexity and is expected to contribute to electrophysiological plasticity of excitable cells.