Get Out and Stay Out: New Insights Into DNA Methylation Reprogramming in Mammals

Vertebrate genomes are marked by notably high levels of 5-cytosine DNA methylation (5meC). The clearest function of DNA methylation among members of the subphylum is repression of potentially deleterious transposable elements (TEs). However, enrichment in the bodies of protein coding genes and pericentromeric heterochromatin indicate an important role for 5meC in those genomic compartments as well. Moreover, DNA methylation plays an important role in silencing of germline-specific genes. Impaired function of major components of DNA methylation machinery results in lethality in fish, amphibians and mammals. Despite such apparent importance, mammals exhibit a dramatic loss and regain of DNA methylation in early embryogenesis prior to implantation, and then again in the cells specified for the germline. In this minireview we will highlight recent studies that shine light on two major aspects of embryonic DNA methylation reprogramming: (1) The mechanism of DNA methylation loss after fertilization and (2) the protection of discrete loci from ectopic DNA methylation deposition during reestablishment. Finally, we will conclude with some extrapolations for the evolutionary underpinnings of such extraordinary events that seemingly put the genome under unnecessary risk during a particularly vulnerable window of development.

Ionic mechanisms of electrophysiological heterogeneity and conduction block in the infarct border zone

The increased incidence of arrhythmia in the healing phase after infarction has been linked to remodeling in the epicardial border zone (EBZ). Ionic models of normal zone (NZ) and EBZ myocytes were incorporated into one-dimensional models of propagation to gain mechanistic insights into how ion channel remodeling affects action potential (AP) duration (APD) and refractoriness, vulnerability to conduction block, and conduction safety postinfarction. We found that EBZ tissue exhibited abnormal APD restitution. The remodeled Na+ current ( INa) and L-type Ca2+ current ( ICa,L) promoted increased effective refractory period and prolonged APD at a short diastolic interval. While postrepolarization refractoriness due to remodeled EBZ INa was the primary determinant of the vulnerable window for conduction block at the NZ-to-EBZ transition in response to premature S2 stimuli, altered EBZ restitution also promoted APD dispersion and increased the vulnerable window at fast S1 pacing rates. Abnormal EBZ APD restitution and refractoriness also led to abnormal periodic conduction block patterns for a range of fast S1 pacing rates. In addition, we found that INa remodeling decreased conduction safety in the EBZ but that inward rectifier K+ current remodeling partially offset this decrease. EBZ conduction was characterized by a weakened AP upstroke and short intercellular delays, which prevented ICa,L and transient outward K+ current remodeling from playing a role in EBZ conduction in uncoupled tissue. Simulations of a skeletal muscle Na+ channel SkM1- INa injection into the EBZ suggested that this recently proposed antiarrhythmic therapy has several desirable effects, including normalization of EBZ effective refractory period and APD restitution, elimination of vulnerability to conduction block, and normalization of conduction in tissue with reduced intercellular coupling.

Vulnerable windows define susceptibility to alternans and spatial discordance

Electrophysiological alternans is a beat-to-beat alternation of the action potential duration and/or Ca2+ transient amplitude and is linked to ventricular arrhythmias. We investigated the significance of various rate parameters under different experimental conditions with respect to alternans incidence and the propensity for spiral wave formation. Voltage and Ca2+ were optically mapped in monolayers of neonatal rat ventricular myocytes. Alternans did not occur at physiological temperature, but its incidence increased significantly at lowered temperatures. Pacing cycle length for spatially concordant alternans onset (PCLC), PCL for spatially discordant alternans onset (PCLD), and minimum cycle length for loss of 1:1 or 2:2 capture (MCL) also significantly increased with lower temperature but in a way such that the differences between PCLC and MCL and between PCLD and MCL widened. These results provided the rationale to identify the former difference as the alternans vulnerable window (AVW; in ms) and the latter difference as the discordant alternans vulnerable window (AVWD; in ms). Computational simulations showed that interventions that widen AVW, including altered Ca2+ cycling and enhanced K+ currents, also promote alternans, regardless of whether PCLC or MCL increased or decreased. The simulation results were confirmed experimentally by addition of the ATP-sensitive K+ channel agonist pinacidil. Mathematical analysis provided a theoretical basis linking the size of AVW to the incidence of alternans. Finally, experiments showed that the size of AVWD is related to the incidence of spatially discordant alternans and, additionally, to the incidence of spiral wave formation. In conclusion, vulnerable windows can be defined that are strongly correlated with alternans incidence, spatial discordance, and spiral wave formation.

Virtual electrodes and the induction of fibrillation in Langendorff-perfused rabbit ventricles: the role of intracellular calcium

A strong premature electrical stimulus (S2) induces both virtual anodes and virtual cathodes. The effects of virtual electrodes on intracellular Ca2+ concentration ([Ca2+]i) transients and ventricular fibrillation thresholds (VFTs) are unclear. We studied 16 isolated, Langendorff-perfused rabbit hearts with simultaneous voltage and [Ca2+]i optical mapping and for vulnerable window determination. After baseline pacing (S1), a monophasic (10 ms anodal or cathodal) or biphasic (5 ms-5 ms) S2 was applied to the left ventricular epicardium. Virtual electrode polarizations and [Ca2+]i varied depending on the S2 polarity. Relative to the level of [Ca2+]i during the S1 beat, the [Ca2+]i level 40 ms after the onset of monophasic S2 increased by 36 ± 8% at virtual anodes and 20 ± 5% at virtual cathodes ( P < 0.01), compared with 25 ± 5% at both virtual cathode-anode and anode-cathode sites for biphasic S2. The VFT was significantly higher and the vulnerable window significantly narrower for biphasic S2 than for either anodal or cathodal S2 ( n = 7, P < 0.01). Treatment with thapsigargin and ryanodine ( n = 6) significantly prolonged the action potential duration compared with control (255 ± 22 vs. 189 ± 6 ms, P < 0.05) and eliminated the difference in VFT between monophasic and biphasic S2, although VFT was lower for both cases. We conclude that virtual anodes caused a greater increase in [Ca2+]i than virtual cathodes. Monophasic S2 is associated with lower VFT than biphasic S2, but this difference was eliminated by the inhibition of the sarcoplasmic reticulum function and the prolongation of the action potential duration. However, the inhibition of the sarcoplasmic reticulum function also reduced VFT, indicating that the [Ca2+]i dynamics modulate, but are not essential, to ventricular vulnerability.