Rafael J. Ramirez

Dominant Frequency Increase Rate Predicts Transition from Paroxysmal to Long-Term Persistent Atrial Fibrillation

Background— Little is known about the mechanisms underlying the transition from paroxysmal to persistent atrial fibrillation (AF). In an ovine model of long-standing persistent AF we tested the hypothesis that the rate of electric and structural remodeling, assessed by dominant frequency (DF) changes, determines the time at which AF becomes persistent. Methods and Results— Self-sustained AF was induced by atrial tachypacing. Seven sheep were euthanized 11.5±2.3 days after the transition to persistent AF and without reversal to sinus rhythm; 7 sheep were euthanized after 341.3±16.7 days of long-standing persistent AF. Seven sham-operated animals were in sinus rhythm for 1 year. DF was monitored continuously in each group. Real-time polymerase chain reaction, Western blotting, patch clamping, and histological analyses were used to determine the changes in functional ion channel expression and structural remodeling. Atrial dilatation, mitral valve regurgitation, myocyte hypertrophy, and atrial fibrosis occurred progressively and became statistically significant after the transition to persistent AF, with no evidence for left ventricular dysfunction. DF increased progressively during the paroxysmal-to-persistent AF transition and stabilized when AF became persistent. Importantly, the rate of DF increase correlated strongly with the time to persistent AF. Significant action potential duration abbreviation, secondary to functional ion channel protein expression changes (CaV1.2, NaV1.5, and KV4.2 decrease; Kir2.3 increase), was already present at the transition and persisted for 1 year of follow up. Conclusions— In the sheep model of long-standing persistent AF, the rate of DF increase predicts the time at which AF stabilizes and becomes persistent, reflecting changes in action potential duration and densities of sodium, L-type calcium, and inward rectifier currents.

Myosin light chain 2-based selection of human iPSC-derived early ventricular cardiac myocytes

Applications of human induced pluripotent stem cell derived-cardiac myocytes (hiPSC-CMs) would be strengthened by the ability to generate specific cardiac myocyte (CM) lineages. However, purification of lineage-specific hiPSC-CMs is limited by the lack of cell marking techniques. Here, we have developed an iPSC-CM marking system using recombinant adenoviral reporter constructs with atrial- or ventricular-specific myosin light chain-2 (MLC-2) promoters. MLC-2a and MLC-2v selected hiPSC-CMs were purified by fluorescence-activated cell sorting and their biochemical and electrophysiological phenotypes analyzed. We demonstrate that the phenotype of both populations remained stable in culture and they expressed the expected sarcomeric proteins, gap junction proteins and chamber-specific transcription factors. Compared to MLC-2a cells, MLC-2v selected CMs had larger action potential amplitudes and durations. In addition, by immunofluorescence, we showed that MLC-2 isoform expression can be used to enrich hiPSC-CM consistent with early atrial and ventricular myocyte lineages. However, only the ventricular myosin light chain-2 promoter was able to purify a highly homogeneous population of iPSC-CMs. Using this approach, it is now possible to develop ventricular-specific disease models using iPSC-CMs while atrial-specific iPSC-CM cultures may require additional chamber-specific markers.

Galectin-3 Regulates Atrial Fibrillation Remodeling and Predicts Catheter Ablation Outcomes

Summary Atrial fibrillation (AF) usually starts as paroxysmal but can evolve relentlessly to the persistent and permanent forms. However, the mechanisms governing such a transition are unknown. The authors show that intracardiac serum levels of galectin (Gal)-3 are greater in patients with persistent than paroxysmal AF and that Gal-3 independently predicts atrial tachyarrhythmia recurrences after a single ablation procedure. Using a sheep model of persistent AF the authors further demonstrate that upstream therapy targeting Gal-3 diminishes both electrical remodeling and fibrosis by impairing transforming growth factor beta–mediated signaling and reducing myofibroblast activation. Accordingly, Gal-3 inhibition therapy increases the probability of AF termination and reduces the overall burden of AF. Therefore the authors postulate that Gal-3 inhibition is a potential new upstream therapy to prevent AF progression.

Constitutive Intracellular Na+ Excess in Purkinje Cells Promotes Arrhythmogenesis at Lower Levels of Stress Than Ventricular Myocytes From Mice With Catecholaminergic Polymorphic Ventricular Tachycardia.

Background— In catecholaminergic polymorphic ventricular tachycardia (CPVT), cardiac Purkinje cells (PCs) appear more susceptible to Ca2+ dysfunction than ventricular myocytes (VMs). The underlying mechanisms remain unknown. Using a CPVT mouse (RyR2R4496C+/Cx40eGFP), we tested whether PC intracellular Ca2+ ([Ca2+]i) dysregulation results from a constitutive [Na+]i surplus relative to VMs. Methods and Results— Simultaneous optical mapping of voltage and [Ca2+]i in CPVT hearts showed that spontaneous Ca2+ release preceded pacing-induced triggered activity at subendocardial PCs. On simultaneous current-clamp and Ca2+ imaging, early and delayed afterdepolarizations trailed spontaneous Ca2+ release and were more frequent in CPVT PCs than CPVT VMs. As a result of increased activity of mutant ryanodine receptor type 2 channels, sarcoplasmic reticulum Ca2+ load, measured by caffeine-induced Ca2+ transients, was lower in CPVT VMs and PCs than respective controls, and sarcoplasmic reticulum fractional release was greater in both CPVT PCs and VMs than respective controls. [Na+]i was higher in both control and CPVT PCs than VMs, whereas the density of the Na+/Ca2+ exchanger current was not different between PCs and VMs. Computer simulations using a PC model predicted that the elevated [Na+]i of PCs promoted delayed afterdepolarizations, which were always preceded by spontaneous Ca2+ release events from hyperactive ryanodine receptor type 2 channels. Increasing [Na+]i monotonically increased delayed afterdepolarization frequency. Confocal imaging experiments showed that postpacing Ca2+ spark frequency was highest in intact CPVT PCs, but such differences were reversed on saponin-induced membrane permeabilization, indicating that differences in [Na+]i played a central role. Conclusions— In CPVT mice, the constitutive [Na+]i excess of PCs promotes triggered activity and arrhythmogenesis at lower levels of stress than VMs.

A multi-centric Study of Candida bloodstream infection in Lima-Callao, Peru: Species distribution, antifungal resistance and clinical outcomes.

Background The incidence of candidemia is increasing in developing countries. Very little is known about the epidemiology of candidemia in Peru. The aim of this study is to describe the incidence, microbiology, clinical presentation and outcomes of Candida bloodstream infections in three Lima-Callao hospitals. Methods Candida spp. isolates were identified prospectively at participant hospitals between November 2013 and January 2015. Susceptibility testing for amphotericin B, fluconazole, posaconazole, voriconazole and anidulafungin was performed using broth microdilution method. Clinical information was obtained from medical records and evaluated. Results We collected information on 158 isolates and 157 patients. Median age of patients was 55.0 yrs., and 64.1% were males. Thirty-eight (24.2%) episodes of candidemia occurred in those <18 yrs. The frequency of non-Candida albicans was 72.1%. The most frequently recovered species were C. albicans (n = 44, 27.8%), C. parapsilosis (n = 40, 25.3%), C. tropicalis (n = 39, 24.7%) and C. glabrata (n = 15, 9.5%). Only four isolates were resistant to fluconazole, 86.7% (n = 137) were susceptible and 17 were susceptible-dose dependent. Decreased susceptibility to posaconazole was also observed in three isolates, and one to voriconazole. All isolates were susceptible to anidulafungin and amphotericin B. The most commonly associated co-morbid conditions were recent surgery (n = 61, 38.9%), mechanical ventilation (n = 60, 38.2%) and total parenteral nutrition (n = 57, 36.3%). The incidence of candidemia by center ranged between 1.01 and 2.63 cases per 1,000 admissions, with a global incidence of 2.04. Only 28.1% of cases received treatment within 72 hrs. of diagnosis. Overall, the 30-day survival was 60.4% (treated subjects, 67.4%; not-treated patients, 50.9%). Conclusions We found a very high proportion of non-albicans Candida species. Despite this, the decreased susceptibility/resistance to fluconazole was only 13.3% and not seen in the other antifungals. Overall, the incidence of candidemia mortality was high when compared to other international studies. It is possible, that the delay in initiating antifungal treatment contributed to the elevated mortality rate, in spite of low antifungal resistance.

Cell-selective arrhythmia ablation for photomodulation of heart rhythm

An injectable cardiomyocyte-targeted photosensitizer nanoparticle allows for specific in vivo arrhythmia ablation. For heart cells only Abnormal heartbeats, called arrhythmias, can be stopped by photoablation, but the use of light energy to terminate malfunctioning cardiomyocytes runs the risk of damaging the other dozen or so cell types in the heart. To be more specific in photoablation, Avula and colleagues devised a heart cell–targeted photosensitizer, which could be delivered systemically. Laser light was then used to ablate only cardiomyocytes while leaving the surrounding fibroblasts and other cells intact. The approach was tested in vivo in rodents and in sheep and rat hearts ex vivo, demonstrating that the technology is indeed able to avoid fibroblasts and block electrical conduction, returning the heart to its normal rhythm. Heart disease, a leading cause of death in the developed world, is overwhelmingly correlated with arrhythmias, where heart muscle cells, myocytes, beat abnormally. Cardiac arrhythmias are usually managed by electric shock intervention, antiarrhythmic drugs, surgery, and/or catheter ablation. Despite recent improvements in techniques, ablation procedures are still limited by the risk of complications from unwanted cellular damage, caused by the nonspecific delivery of ablative energy to all heart cell types. We describe an engineered nanoparticle containing a cardiac-targeting peptide (CTP) and a photosensitizer, chlorin e6 (Ce6), for specific delivery to myocytes. Specificity was confirmed in vitro using adult rat heart cell and human stem cell–derived cardiomyocyte and fibroblast cocultures. In vivo, the CTP-Ce6 nanoparticles were injected intravenously into rats and, upon laser illumination of the heart, induced localized, myocyte-specific ablation with 85% efficiency, restoring sinus rhythm without collateral damage to other cell types in the heart, such as fibroblasts. In both sheep and rat hearts ex vivo, upon perfusion of CTP-Ce6 particles, laser illumination led to the formation of a complete electrical block at the ablated region and restored the physiological rhythm of the heart. This nano-based, cell-targeted approach could improve ablative technologies for patients with arrhythmias by reducing currently encountered complications.

Elevated heart rate triggers action potential alternans and sudden death. Translational study of a homozygous KCNH2 mutation

Background Long QT syndrome (LQTS) leads to arrhythmic events and increased risk for sudden cardiac death (SCD). Homozygous KCNH2 mutations underlying LQTS-2 have previously been termed “human HERG knockout” and typically express severe phenotypes. We studied genotype-phenotype correlations of an LQTS type 2 mutation identified in the homozygous index patient from a consanguineous Turkish family after his brother died suddenly during febrile illness. Methods and Results Clinical work-up, DNA sequencing, mutagenesis, cell culture, patch-clamp, in silico mathematical modelling, protein biochemistry, confocal microscopy were performed. Genetic analysis revealed a homozygous C-terminal KCNH2 mutation (p.R835Q) in the index patient (QTc ∼506 ms with notched T waves). Parents were I° cousins – both heterozygous for the mutation and clinically unremarkable (QTc ∼447 ms, father and ∼396 ms, mother). Heterologous expression of KCNH2-R835Q showed mildly reduced current amplitudes. Biophysical properties of ionic currents were also only nominally changed with slight acceleration of deactivation and more negative V50 in R835Q-currents. Protein biochemistry and confocal microscopy revealed similar expression patterns and trafficking of WT and R835Q, even at elevated temperature. In silico analysis demonstrated mildly prolonged ventricular action potential duration (APD) compared to WT at a cycle length of 1000 ms. At a cycle length of 350 ms M-cell APD remained stable in WT, but displayed APD alternans in R835Q. Conclusion Kv11.1 channels affected by the C-terminal R835Q mutation display mildly modified biophysical properties, but leads to M-cell APD alternans with elevated heart rate and could precipitate SCD under specific clinical circumstances associated with high heart rates.

Structural basis for the antiarrhythmic blockade of a potassium channel with a small molecule

The acetylcholine‐activated inward rectifier potassium current (IKACh) is constitutively active in persistent atrial fibrillation (AF). We tested the hypothesis that the blocking of IKACh with the small molecule chloroquine terminates persistent AF. We used a sheep model of tachypacing‐induced, persistent AF, molecular modeling, electrophysiology, and structural biology approaches. The 50% inhibition/inhibitory concentration of IKACh block with chloroquine, measured by patch clamp, was 1 µM. In optical mapping of sheep hearts with persistent AF, 1 µM chloroquine restored sinus rhythm. Molecular modeling suggested that chloroquine blocked the passage of a hydrated potassium ion through the intracellular domain of Kir3.1 (a molecular correlate of IKACh) by interacting with residues D260 and F255, in proximity to I228, Q227, and L299. 1H 15N heteronuclear single‐quantum correlation of purified Kir3.1 intracellular domain confirmed the modeling results. F255, I228, Q227, and L299 underwent significant chemical‐shift perturbations upon drug binding. We then crystallized and solved a 2.5 Å X‐ray structure of Kir3.1 with F255A mutation. Modeling of chloroquine binding to the mutant channel suggested that the drugs binding to the pore becomes off centered, reducing its ability to block a hydrated potassium ion. Patch clamp validated the structural and modeling data, where the F255A and D260A mutations significantly reduced IKACh block by chloroquine. With the use of numerical and structural biology approaches, we elucidated the details of how a small molecule could block an ion channel and exert antiarrhythmic effects. Chloroquine binds the IKACh channel at a site formed by specific amino acids in the ion‐permeation pathway, leading to decreased IKACh and the subsequent termination of AF.— Takemoto, Y., Slough, D. P., Meinke, G., Katnik, C., Graziano, Z. A., Chidipi, B., Reiser, M., Alhadidy, M. M., Ramirez, R., Salvador‐Montañés, O., Ennis, S., Guerrero‐Serna, G., Haburcak, M., Diehl, C., Cuevas, J., Jalife, J., Bohm, A., Lin, Y.‐S., Noujaim, S. F. Structural basis for the antiarrhythmic blockade of a potassium channel with a small molecule. FASEB J. 32, 1778–1793 (2018). www.fasebj.org