Christopher Y. Ko

Rb and p130 control cell cycle gene silencing to maintain the postmitotic phenotype in cardiac myocytes

Both Rb and p130 are required for the recruitment of heterochromatin proteins that mediate silencing of proliferation genes in adult cardiac myocytes.

Calcium-Voltage Coupling in the Genesis of Early and Delayed Afterdepolarizations in Cardiac Myocytes

Early afterdepolarizations (EADs) and delayed afterdepolarizations (DADs) are voltage oscillations known to cause cardiac arrhythmias. EADs are mainly driven by voltage oscillations in the repolarizing phase of the action potential (AP), while DADs are driven by spontaneous calcium (Ca) release during diastole. Because voltage and Ca are bidirectionally coupled, they modulate each others behaviors, and new AP and Ca cycling dynamics can emerge from this coupling. In this study, we performed computer simulations using an AP model with detailed spatiotemporal Ca cycling incorporating stochastic openings of Ca channels and ryanodine receptors to investigate the effects of Ca-voltage coupling on EAD and DAD dynamics. Simulations were complemented by experiments in mouse ventricular myocytes. We show that: 1) alteration of the Ca transient due to increased ryanodine receptor leakiness and/or sarco/endoplasmic reticulum Ca ATPase activity can either promote or suppress EADs due to the complex effects of Ca on ionic current properties; 2) spontaneous Ca waves also exhibit complex effects on EADs, but cannot induce EADs of significant amplitude without the participation of ICa,L; 3) lengthening AP duration and the occurrence of EADs promote DADs by increasing intracellular Ca loading, and two mechanisms of DADs are identified, i.e., Ca-wave-dependent and Ca-wave-independent; and 4) Ca-voltage coupling promotes complex EAD patterns such as EAD alternans that are not observed for solely voltage-driven EADs. In conclusion, Ca-voltage coupling combined with the nonlinear dynamical behaviors of voltage and Ca cycling play a key role in generating complex EAD and DAD dynamics observed experimentally in cardiac myocytes, whose mechanisms are complex but analyzable.

THY-1 Receptor Expression Differentiates Cardiosphere-Derived Cells with Divergent Cardiogenic Differentiation Potential

Summary Despite over a decade of intense research, the identity and differentiation potential of human adult cardiac progenitor cells (aCPC) remains controversial. Cardiospheres have been proposed as a means to expand aCPCs in vitro, but the identity of the progenitor cell within these 3D structures is unknown. We show that clones derived from cardiospheres could be subdivided based on expression of thymocyte differentiation antigen 1 (THY-1/CD90) into two distinct populations that exhibit divergent cardiac differentiation potential. One population, which is CD90+, expressed markers consistent with a mesenchymal/myofibroblast cell. The second clone type was CD90− and could form mature, functional myocytes with sarcomeres albeit at a very low rate. These two populations of cardiogenic clones displayed distinct cell surface markers and unique transcriptomes. Our study suggests that a rare aCPC exists in cardiospheres along with a mesenchymal/myofibroblast cell, which demonstrates incomplete cardiac myocyte differentiation.

Molecular Basis of Hypokalemia-Induced Ventricular Fibrillation

Background— Hypokalemia is known to promote ventricular arrhythmias, especially in combination with class III antiarrhythmic drugs like dofetilide. Here, we evaluated the underlying molecular mechanisms. Methods and Results— Arrhythmias were recorded in isolated rabbit and rat hearts or patch-clamped ventricular myocytes exposed to hypokalemia (1.0–3.5 mmol/L) in the absence or presence of dofetilide (1 &mgr;mol/L). Spontaneous early afterdepolarizations (EADs) and ventricular tachycardia/fibrillation occurred in 50% of hearts at 2.7 mmol/L [K] in the absence of dofetilide and 3.3 mmol/L [K] in its presence. Pretreatment with the Ca-calmodulin kinase II (CaMKII) inhibitor KN-93, but not its inactive analogue KN-92, abolished EADs and hypokalemia-induced ventricular tachycardia/fibrillation, as did the selective late Na current (INa) blocker GS-967. In intact hearts, moderate hypokalemia (2.7 mmol/L) significantly increased tissue CaMKII activity. Computer modeling revealed that EAD generation by hypokalemia (with or without dofetilide) required Na-K pump inhibition to induce intracellular Na and Ca overload with consequent CaMKII activation enhancing late INa and the L-type Ca current. K current suppression by hypokalemia and dofetilide alone in the absence of CaMKII activation were ineffective at causing EADs. Conclusions— We conclude that Na-K pump inhibition by even moderate hypokalemia plays a critical role in promoting EAD-mediated arrhythmias by inducing a positive feedback cycle activating CaMKII and enhancing late INa. Class III antiarrhythmic drugs like dofetilide sensitize the heart to this positive feedback loop.

The emergence of subcellular pacemaker sites for calcium waves and oscillations.

•  Calcium (Ca2+) is fundamental to biological cell function, and Ca2+ waves generating oscillatory Ca2+ signals are widely observed in many cell types. •  Some experimental studies have shown that Ca2+ waves initiate from random locations within the cell, while other studies have shown that waves occur repetitively from preferred locations (pacemaker sites). •  In both ventricular myocyte experiments and computer simulations of a heterogeneous model of coupled Ca2+ release units (CRUs), we show that Ca2+ waves occur randomly in space and time when the Ca2+ level is low, but as the Ca2+ level increases, waves occur repetitively from the same sites. •  Ca2+ waves are self‐organized dynamics of the CRU network, and the wave frequency strongly depends on CRU coupling. •  Using these results, we develop a theory for the entrainment of random oscillators, which provides a unified explanation for the experimental and computational observations.

Acute reversal of phospholamban inhibition facilitates the rhythmic whole-cell propagating calcium waves in isolated ventricular myocytes

Phospholamban (PLB) inhibits the activity of cardiac sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a). Phosphorylation of PLB during sympathetic activation reverses SERCA2a inhibition, increasing SR Ca(2+) uptake. However, sympathetic activation also modulates multiple other intracellular targets in ventricular myocytes (VMs), making it impossible to determine the specific effects of the reversal of PLB inhibition on the spontaneous SR Ca(2+) release. Therefore, it remains unclear how PLB regulates rhythmic activity in VMs. Here, we used the Fab fragment of 2D12, a monoclonal anti-PLB antibody, to test how acute reversal of PLB inhibition affects the spontaneous SR Ca(2+) release in normal VMs. Ca(2+) sparks and spontaneous Ca(2+) waves (SCWs) were recorded in the line-scan mode of confocal microscopy using the Ca(2+) fluorescent dye Fluo-4 in isolated permeabilized mouse VMs. Fab, which reverses PLB inhibition, significantly increased the frequency, amplitude, and spatial/temporal spread of Ca(2+) sparks in VMs exposed to 50 nM free [Ca(2+)]. At physiological diastolic free [Ca(2+)] (100-200 nM), Fab facilitated the formation of whole-cell propagating SCWs. At higher free [Ca(2+)], Fab increased the frequency and velocity, but decreased the decay time of the SCWs. cAMP had little additional effect on the frequency or morphology of Ca(2+) sparks or SCWs after Fab addition. These findings were complemented by computer simulations. In conclusion, acute reversal of PLB inhibition alone significantly increased the spontaneous SR Ca(2+) release, leading to the facilitation and organization of whole-cell propagating SCWs in normal VMs. PLB thus plays a key role in subcellular Ca(2+) dynamics and rhythmic activity of VMs.

Increased susceptibility of spontaneously hypertensive rats to ventricular tachyarrhythmias in early hypertension.

Hypertension is a risk factor for sudden cardiac death caused by ventricular tachycardia and fibrillation. Whether hypertension in its early stage is associated with an increased risk of ventricular tachyarrhythmias is not known. Based on experiments performed at the cellular and whole heart levels, we show that, even early in chronic hypertension, the hypertrophied and fibrotic ventricles of spontaneously hypertensive rats aged 5 to 6 months have already developed increased stress‐induced arrhythmogenicity, and this increased susceptibility to ventricular arrhythmias is primarily a result of tissue remodelling rather than cellular electrophysiological changes. Our findings highlight the need for early hypertension treatment to minimize myocardial fibrosis, ventricular hypertrophy, and arrhythmias.

Formation of Subcellular Calcium Waves in Cardiac Myocytes: Characterizing Timescales via Mathematical Modeling

Subcellular Calcium (Ca) cycling plays fundamental roles in normal heart dynamics. In cardiac myocytes, the elementary Ca cycling events are Ca sparks: random discretized Ca release events due to random and collective openings of the ryanodine receptor (RyR) channels clustered in Ca release units (CRUs). A typical cardiac myocyte includes about 10,000 to 20,000 CRUs, and the spatial arrangement of CRUs varies widely across myocyte type and changes in diseased conditions. Dysfunction of the CRU network leaves cells prone to subcellular Ca waves, notorious triggers of highly arrhythmogenic delayed afterdepolarizations. Recent experimental studies have isolated three timescales involved in the formation of Ca waves: rate of sarcoplasmic reticulum (SR) Ca reuptake, intrinsic RyR refractoriness, and a so-called “idle” period. Here we use a physiologically detailed computational model of a spatial and stochastic CRU network to study the variables that contribute to the aforementioned timescales, and identify how the relative dominance of each affects the morphology of Ca waves. We show that the “idle” period is far from idle, as it emerges out of complex Ca mediated CRU-to-CRU interactions in both the myoplasm and SR. We also find that while reduced refractory period and increased SR Ca diffusion enhance the local initiation of waves, they hinder propagation, resulting in fractionated wave events. Furthermore, at very short refractory periods the system degenerates into spiral waves and chaos. Pinpointing the mechanisms underlying the variety of observed wave morphologies is an important step in our understanding of diseased states, as each may play a different role in arrhythmogenesis.

Latency to the Onset of Calcium Waves in Cardiac Myocytes is Predicted by Criticality Theory

Spontaneous calcium (Ca) waves must emerge near-synchronously in thousands of contiguous myocytes to produce delayed afterdepolarizations (DADs) in cardiac tissue. Previous studies have shown as Ca load increases, the time to onset of a spontaneous Ca wave after pacing (latency), as well as its variability, decreases. Two proposed mechanisms include the time period required to refill the sarcoplasmic reticulum (SR) Ca stores and regain Ca release channel excitability, and the latter plus an “idle period.” Here we used patch-clamped Fluo-4-loaded isolated rabbit ventricular myocytes to detect Ca waves and DADs following pacing trains. Longer pacing trains enhancing Ca loading decreased latency and its variability. Using paced premature beats, latency outlasted the period required for SR refilling and Ca release channel recovery, consistent with an additional “idle period.” From combined experimental data, simulations, and theoretical analysis, we present evidence that the “idle period” can be explained by criticality theory (Biophys J 2012;11:2433), related to the probability that a cluster of Ca release units large enough to initiate a Ca wave will self-organize. This theory directly accounts for the observed shortening of latency and its variability as Ca load increases.