Jia-Qiang He

Human Embryonic Stem Cells Develop Into Multiple Types of Cardiac Myocytes Action Potential Characterization

Abstract— Human embryonic stem (hES) cells can differentiate in vitro, forming embryoid bodies (EBs) composed of derivatives of all three embryonic germ layers. Spontaneously contracting outgrowths from these EBs contain cardiomyocytes (CMs); however, the types of human CMs and their functional properties are unknown. This study characterizes the contractions and action potentials (APs) from beating EB outgrowths cultured for 40 to 95 days. Spontaneous and electrical field-stimulated contractions were measured with video edge-detection microscopy. &bgr;-Adrenergic stimulation with 1.0 &mgr;mol/L isoproterenol resulted in a significant increase in contraction magnitude. Intracellular electrical recordings using sharp KCl microelectrodes in beating EB outgrowths revealed three distinct classes of APs: nodal-like, embryonic atrial-like, and embryonic ventricular-like. The APs were described as embryonic based on the relatively depolarized resting membrane potential and slow AP upstroke. Repeated impalements of an individual beating outgrowth revealed a reproducible AP morphology recorded from different cells, suggesting that each outgrowth is composed of a predominant cell type. Complex functional properties typical of cardiac muscle were observed in the hES cell-derived CMs including rate adaptation of AP duration and provoked early and delayed afterdepolarizations. Repolarization of the AP showed a significant role for IKr based on E-4031 induced prolongation of AP duration as anticipated for human CMs. In conclusion, hES cells can differentiate into multiple types of CMs displaying functional properties characteristic of embryonic human cardiac muscle. Thus, hES provide a renewable source of distinct types of human cardiac myocytes for basic research, pharmacological testing, and potentially therapeutic applications.

Reduction in density of transverse tubules and L-type Ca2+ channels in canine tachycardia-induced heart failure

OBJECTIVE Persistent supraventricular tachycardia leads to the development of a dilated cardiomyopathy with impairment of excitation-contraction (EC) coupling. Since the initial trigger for EC coupling in ventricular muscle is the influx of Ca(2+) through L-type Ca(2+) channels (I(Ca)) in the transverse tubules (T-tubules), we determined if the density of the T-tubule system and L-type Ca(2+) channels change in canine tachycardia pacing-induced cardiomyopathy. METHODS Confocal imaging of isolated ventricular myocytes stained with the membrane dye Di-8-ANEPPS was used to image the T-tubule system, and standard whole-cell patch clamp techniques were used to measure I(Ca) and intramembrane charge movement. RESULTS A complex staining pattern of interconnected tubules including prominent transverse components spaced every approximately 1.6 microm was present in control ventricular myocytes, but failing cells demonstrated a far less regular T-tubule system with a relative loss of T-tubules. In confocal optical slices, the average % of the total cell area staining for T-tubules decreased from 11.5+/-0.4 in control to 8.7+/-0.4% in failing cells (P<0.001). Whole-cell patch clamp studies revealed that I(Ca) density was unchanged. Since whole-cell I(Ca) is due to both the number of channels as well as the functional properties of those channels, we measured intramembrane charge movement as an assay for changes in channel number. The saturating amount of charge that moves due to gating of L-type Ca(2+) channels, Q(on,max), was decreased from 6.5+/-0.6 in control to 2.8+/-0.3 fC/pF in failing myocytes (P<0.001). CONCLUSIONS Cellular remodeling in heart failure results in decreased density of T-tubules and L-type Ca(2+) channels, which contribute to abnormal EC coupling.

Physiological Properties of hERG 1a/1b Heteromeric Currents and a hERG 1b-Specific Mutation Associated With Long-QT Syndrome

Cardiac IKr is a critical repolarizing current in the heart and a target for inherited and acquired long-QT syndrome (LQTS). Biochemical and functional studies have demonstrated that IKr channels are heteromers composed of both hERG 1a and 1b subunits, yet our current understanding of IKr functional properties derives primarily from studies of homooligomers of the original hERG 1a isolate. Here, we examine currents produced by hERG 1a and 1a/1b channels expressed in HEK-293 cells at near-physiological temperatures. We find that heteromeric hERG 1a/1b currents are much larger than hERG 1a currents and conduct 80% more charge during an action potential. This surprising difference corresponds to a 2-fold increase in the apparent rates of activation and recovery from inactivation, thus reducing rectification and facilitating current rebound during repolarization. Kinetic modeling shows these gating differences account quantitatively for the differences in current amplitude between the 2 channel types. Drug sensitivity was also different. Compared to homomeric 1a channels, heteromeric 1a/1b channels were inhibited by E-4031 with a slower time course and a corresponding 4-fold shift in the IC50. The importance of hERG 1b in vivo is supported by the identification of a 1b-specific A8V missense mutation in 1/269 unrelated genotype-negative LQTS patients that was absent in 400 control alleles. Mutant 1bA8V expressed alone or with hERG 1a in HEK-293 cells dramatically reduced 1b protein levels. Thus, mutations specifically disrupting hERG 1b function are expected to reduce cardiac IKr and enhance drug sensitivity, and represent a potential mechanism underlying inherited or acquired LQTS.

Endothelin-1 and photoreleased diacylglycerol increase L-type Ca2+ current by activation of protein kinase C in rat ventricular myocytes

1 The amphotericin B‐perforated whole‐cell patch clamp technique was used to determine the modulation of L‐type Ca2+ channels by protein kinase C (PKC)‐mediated pathways in adult rat ventricular myocytes. 2 Application of 10 nM endothelin‐1 (ET‐1) increased peak Ca2+ current (ICa) by 28.2 ± 2.5 % (n= 13) and slowed current decay. These effects were prevented by the endothelin receptor antagonist PD145065 (10 μM) and by the PKC inhibitor chelerythrine (8 μM). 3 To establish if direct activation of PKC mimicked the ET‐1 effect, the active and inactive phorbol esters (phorbol‐12‐myristate‐13‐acetate and 4α‐phorbol‐12, 13‐didecanoate) were tested. Both phorbol esters (100 nM) resulted in a small (∼10 %) increase in ICa, suggesting PKC‐independent effects. 4 Bath application of dioctanoylglycerol (diC8), a diacylglycerol (DAG) analogue which is capable of directly activating PKC, caused a gradual decline in peak ICa (50.4 ± 6.2 %, n= 5) and increased the rate of current decay. These effects were unaffected by the PKC inhibitor chelerythrine (8 μM). 5 Intracellular photorelease of caged diC8 with 3 or 10 s exposure to UV light produced a concentration‐dependent increase in peak ICa (20.7 ± 8.5 % (n= 8) for 3 s UV and 60.8 ± 11.4 % (n= 13) for 10 s UV), which could be inhibited by chelerythrine. 6 Our results demonstrate that both ET‐1 and intracellularly photoreleased diC8 increase ICa by a PKC‐mediated pathway, which is in direct contrast to the PKC‐independent inhibition of ICa produced by bath‐applied diC8. We conclude that specific cellular pools of DAG are crucially important in the regulation of ICa by PKC.

Human Cardiac Stem Cells Isolated from Atrial Appendages Stably Express c-kit

The in vivo studies of myocardial infarct using c-kit+/Lin− cardiac stem cells (CSCs) are still in the early stage with margin or no beneficial effects for cardiac function. One of the potential reasons may be related to the absence of fully understanding the properties of these cells both in vitro and in vivo. In the present study, we aimed to systematically examine how CSCs adapted to in vitro cell processes and whether there is any cell contamination after long-term culture. Human CSCs were enzymatically isolated from the atrial appendages of patients. The fixed tissue sections, freshly isolated or cultured CSCs were then used for identification of c-kit+/Lin− cells, detection of cell contamination, or differentiation of cardiac lineages. By specific antibody staining, we demonstrated that tissue sections from atrial appendages contained less than 0.036% c-kit+/Lin− cells. For the first time, we noted that without magnetic activated cell sorting (MACS), the percentages of c-kit+/Lin− cells gradually increased up to ∼40% during continuously culture between passage 2 to 8, but could not exceed >80% unless c-kit MACS was carried out. The resulting c-kit+/Lin− cells were negative for CD34, CD45, CD133, and Lin markers, but positive for KDR and CD31 in few patients after c-kit MACS. Lin depletion seemed unnecessary for enrichment of c-kit+/Lin− cell population. Following induced differentiation, c-kit+/Lin− CSCs demonstrated strong differentiation towards cardiomyocytes but less towards smooth and endothelial cells. We concluded that by using an enzymatic dissociation method, a large number, or higher percentage, of relative pure human CSCs with stable expression of c-kit+ could be obtained from atrial appendage specimens within ∼4 weeks following c-kit MACS without Lin depletion. This simple but cost-effective approach can be used to obtain enough numbers of stably-expressed c-kit+/Lin− cells for clinical trials in repairing myocardial infarction.

Crosstalk of β-Adrenergic Receptor Subtypes Through Gi Blunts β-Adrenergic Stimulation of L-Type Ca2+ Channels in Canine Heart Failure

The mechanisms underlying the blunted contractile response to &bgr;-adrenergic receptor (&bgr;-AR) stimulation in heart failure (HF) are incompletely understood, especially with regard to &bgr;-AR subtype–specific regulation of L-type Ca2+ channels. We evaluated the impact of HF induced by pacing tachycardia on &bgr;-AR regulation of L-type Ca2+ channels in a canine model. To evaluate changes in the relative subcellular distribution of &bgr;-AR subtypes, left ventricular membranes enriched in surface sarcolemma and T-tubular sarcolemma were prepared. Radioligand binding using [125I]cyanopindolol revealed that HF resulted in a comparable decrease in the density of &bgr;1-ARs in both surface and T-tubule sarcolemma (55±4%, n=7, P<0.001; and 45±10%, n=7, P<0.01, respectively), but no significant change in &bgr;2-AR density was observed. Whole-cell patch clamp studies demonstrated a markedly blunted increase in ICa,L in response to saturating concentrations of the nonselective &bgr;-AR agonist isoproterenol (0.1 &mgr;mol/L) in failing myocytes compared with control (129±20%, n=11, versus 332±35%, n=7; P<0.001). Experiments testing &bgr;1-AR– and &bgr;2-AR–selective stimulation showed that the major component of the blunted response to nonselective &bgr;-AR stimulation in HF was caused by &bgr;2-AR activation, resulting in a pertussis toxin–sensitive, Gi-mediated inhibition of the &bgr;1-AR–induced increase in ICa,L. In conclusion, canine HF results in the following: (1) a uniform reduction in &bgr;1-AR density in surface and T-tubule membrane fractions without a change in &bgr;2-AR density; and (2) the emergence of distinct Gi-coupling to &bgr;2-ARs resulting in accentuated antagonism of &bgr;1-AR–mediated stimulation of ICa,L. These results have implications for optimizing the use of &bgr;-AR drugs in HF.

L-Type Ca2+ Channels Gaining Respect in Heart Failure

L-type Ca2+ channels are essential for the initiation and regulation of excitation-contraction (EC) coupling in adult cardiac muscle.1 The rapid influx of Ca2+ through these channels triggers release of intracellular Ca2+ from the sarcoplasmic reticulum (SR) stores, and the resulting Ca2+ transient activates the myofilaments and thus contraction. Given that failing ventricular myocytes exhibit impaired contractility and abnormal Ca2+ transients, the L-type Ca2+ channel makes for a good suspect as a contributor to the derangement of EC coupling. However, most initial studies of whole-cell currents through L-type Ca2+ channels ( I Ca) in both human heart failure and animal models did not show a significant difference between nonfailing and failing myocytes.2 In the meantime, important changes in the abundance and/or function of other Ca2+ cycling proteins including the sarcoplasmic reticulum Ca2+-ATPase (SERCA2a), Na+-Ca2+ exchanger (NCX), and ryanodine receptors (RyRs) took the spotlight as the bad actors in heart failure.3 The L-type Ca2+ channel was left behind as a boring, obligatory participant in EC coupling. Times may be changing for the status of the L-type Ca2+ channel in the world of failing hearts. The article by Chen et al,4 in this issue of Circulation Research , unmasks important changes in the density as well as regulation of L-type Ca2+ channels in failing human ventricular myocytes. And, perhaps most encouragingly, the authors show that these changes can be partly reversed in patients with left ventricular assist devices (LVADs). This article adds more evidence to recent publications suggesting significant changes in L-type Ca2+ channel abundance and function in heart failure.5,6⇓ To appreciate the story, one needs to step back from the macroscopic or whole-cell currents through L-type Ca2+ channels and …

Human embryonic stem cell-derived cardiomyocytes: drug discovery and safety pharmacology

Human embryonic stem cells (hESCs) can provide potentially unlimited quantities of a wide range of human cell types that can be used in drug discovery and development, basic research and regenerative medicine. In this review, the authors describe the differentiation of hESCs into cardiomyocytes and outline the properties of hESC-derived cardiomyocytes (hESC-CMs), including their cardiac-type action potentials and contractile characteristics. In vitro cellular assays using hESC-CMs, which can be genetically engineered to create target-specific reporters as well as human disease models, will have applications at multiple stages of the drug discovery process. Furthermore, cardiac safety pharmacology assays evaluating the risk of proarrhythmic side effects associated with QT prolongation may be enhanced in their predictive value with the use of hESC-CMs.

Overexpression of C-Terminal Domain of Talin-1 Enhances Survival, Migration and Differentiation of Human Cardiac Stem Cells

Cardiovascular Diseases (CVD) constitute the single leading cause of death in the United States. According to the heart disease and stroke statistics released by American Heart Association in 2010, 1 out of every 2.9 deaths is due to cardiovascular disease. Even with early diagnosis and timely reperfusion of ischemic myocardium, up to 30% patients develop Left Ventricular (LV) remodeling and loss of cardiac function [1,2]. Current treatment modality of cardiac failure is mainly focused to relieve symptoms. Although cardiac transplantation has offered some hope, organ shortage, donor-recipient organ mismatch, and transplant rejection are the major challenges limiting its widespread use.

Correction: Human Cardiac Stem Cells Isolated from Atrial Appendages Stably Express c-kit.

Previous studies have suggested that MHC class I molecules bind and present peptides to CTL in a manner that is analogous to the presentation of peptides by class II molecules to Th. Crystallographic studies of HLA-A2 have led to the assignment of a putative peptide binding site that is bordered by two alpha helices consisting of residues 50-84 and 138-180. In this study, we have investigated whether residues in the alpha 2 helix are involved in the binding and/or presentation of a peptide to CTL. We have generated CTL to type A influenza virus by stimulation of human PBL with a synthetic peptide from the influenza A virus matrix protein (M1 residues 57-68) in the presence of rIL-2. Such HLA-A2.1-restricted influenza virus-immune CTL do not recognize infected HLA-A2.3+ targets. A2.1 and A2.3 differ by three amino acids in the alpha 2 domain: Ala vs. Thr at position 149, Val vs. Glu at position 152, and Leu vs. Trp at position 156. Site- directed mutants of the A2.1 gene that encode A2 molecules that resemble A2.3 at positions 149, 152, and 156 have been constructed, transfected into human cells, and assayed for their ability to present the M1 peptide. The results demonstrate that most, but not all, A2.1- restricted M1-peptide-specific CTL fail to recognize M1 peptide-exposed transfectants with certain single amino acid substitutions at positions 152 and 156. In contrast, M1 peptide-exposed transfectants that express A2 molecules with an Ala----Thr substitution at position 149 were recognized by all CTL tested, but they exhibited an apparent difference in the kinetics of peptide binding. These results indicate that amino acid substitutions at positions 152 and 156 of the putative peptide binding site of the A2 molecule can affect presentation without eliminating binding, and indicate that the failure to recognize complexes between the peptide and the mutant A2 molecules is due to different TCR specificities and not to the failure to bind the peptide.