Mary B. Wagner

A Proliferative Burst During Preadolescence Establishes the Final Cardiomyocyte Number

It is widely believed that perinatal cardiomyocyte terminal differentiation blocks cytokinesis, thereby causing binucleation and limiting regenerative repair after injury. This suggests that heart growth should occur entirely by cardiomyocyte hypertrophy during preadolescence when, in mice, cardiac mass increases many-fold over a few weeks. Here, we show that a thyroid hormone surge activates the IGF-1/IGF-1-R/Akt pathway on postnatal day 15 and initiates a brief but intense proliferative burst of predominantly binuclear cardiomyocytes. This proliferation increases cardiomyocyte numbers by ~40%, causing a major disparity between heart and cardiomyocyte growth. Also, the response to cardiac injury at postnatal day 15 is intermediate between that observed at postnatal days 2 and 21, further suggesting persistence of cardiomyocyte proliferative capacity beyond the perinatal period. If replicated in humans, this may allow novel regenerative therapies for heart diseases.

Effects of anisotropy on the development of cardiac arrhythmias associated with focal activity

Abstract. The anisotropy that normally exists in the myocardium may be either enhanced in peri-infarction zones by loss of lateral cell connections or reduced by redistribution of gap junctions. To test how the degree of anisotropy affects the development of ectopic focal activity, we carried out computer simulations in which a model of an ectopic focus is incorporated as the central element of a two-dimensional sheet of ventricular cells. At low values of intercellular coupling conductance (Gc), the focus region is spontaneously active, but the limited intercellular current flow inhibits propagation. At high Gc, automaticity is suppressed by the loading effects of the surrounding cells. At intermediate Gc, the ectopic activity may propagate into the sheet. In the case of isotropic coupling, the minimum size of the focus region for propagation to occur (in terms of number of collaborating cells within the focus) is as small as approximately ten cells, and this number decreases with increasing anisotropy. Thus, the presence of anisotropy facilitates the development of ectopic focal activity. We conclude that the remodeling that occurs in peri-infarction zones may create a substrate that either facilitates (enhanced anisotropy) or inhibits (reduced anisotropy) the development of cardiac arrhythmias associated with ectopic focal activity.

cGMP-dependent protein kinase mediates stimulation of L-type calcium current by cGMP in rabbit atrial cells

OBJECTIVES cGMP has been shown to exert both stimulatory and inhibitory effects on cardiac L-type calcium current (I(Ca)). The physiological role of cGMP in regulation of cardiac activity is still controversial. cGMP may be of importance in regulation of I(Ca) in atrial cells. The present study was focused on the role of cGMP in the modulation of I(Ca) in rabbit atrial cells. METHODS Enzymatically isolated adult rabbit atrial cells were used to measure I(Ca) using whole cell voltage clamp. Expressed levels of cGMP-dependent protein kinase (PKG) were determined by Western blotting using PKG specific antibody in homogenates from atrial and ventricular cells. RESULTS Nitrosoglutathione (GSNO), a nitric oxide donor that stimulates soluble guanylyl-cyclase to elevate cGMP levels increased I(Ca) while soluble G-cyclase inhibitors, ODQ or methylene blue inhibited I(Ca). Intracellular application of 8BrcGMP increased I(Ca) and blocked the inhibitory effect of methylene blue. KT-5823, an inhibitor of PKG inhibited I(Ca) and the stimulatory effect of GSNO was completely blocked ODQ or KT-5823. Inhibition of cAMP dependent protein kinase (PKA) by the 6-22 peptide completely blocked the stimulation of I(Ca) by the beta-agonist isoproterenol but not by GSNO. The potency of isoproterenol to stimulate I(Ca) was very high for atrial cells (EC(50) 2.4+/-0.6 nM) and only 100 nM isoproterenol was required to stimulate I(Ca) maximally (21.4+/-0.7 pA/pF) to a level (23.8+/-1.6 pA/pF) achieved with the inclusion of 100 microM cAMP in the pipette solution. GSNO produced an additive effect on I(Ca) already stimulated by either 10 microM isobutylmethylxanthine (phosphodiesterase inhibitor) or a low concentration (1 nM) isoproterenol but failed to produce any effect on I(Ca) maximally stimulated by 100 nM isoproterenol. Inhibition of PKG by KT-5823 significantly decreased the efficacy of isoproterenol and the maximal I(Ca) achieved with 100 nM isoproterenol was decreased to 8.2+/-0.6 pA/pF in the presence of KT-5823. Western blot analysis showed much higher expression of PKG in atrial cells compared to ventricular cells. CONCLUSIONS These findings suggest that stimulatory effects of cGMP on I(Ca) in rabbit atrial cells are likely to be mediated via PKG dependent phosphorylation of calcium channels or associated proteins and that the effects of cGMP are not antagonistic to cAMP. PKG is highly expressed in atrial cells and PKG dependent phosphorylation may be necessary for maintaining basal I(Ca) and fully stimulating I(Ca) by beta-adrenergic activation in atrial cells.

Remodeling of Early-Phase Repolarization A Mechanism of Abnormal Impulse Conduction in Heart Failure

Background— The early phase of action potential (AP) repolarization is critical to impulse conduction in the heart because it provides current for charging electrically coupled cells. In the present study we tested the impact of heart failure–associated electrical remodeling on AP propagation. Methods and Results— Subepicardial, midmyocardial, and subendocardial myocytes were enzymatically dissociated from control and pressure-overload failing left ventricle (LV), and APs were recorded. A unique coupling-clamp technique was used to electrically couple 2 isolated myocytes with a controlled value of coupling conductance (Gc). In sham-operated mice, AP duration manifested a clear transmural gradient, with faster repolarization in subepicardial myocytes than in subendocardial myocytes. AP propagation from subendocardial to subepicardial myocytes required less Gc compared with conduction in the opposite direction. In failing heart, AP morphology was dramatically altered, with a significantly elevated plateau potential and prolonged AP duration. Spatially nonuniform alteration of AP duration in failing heart blunted the transmural gradient of repolarization. Furthermore, increased pacing rate prolonged AP duration exclusively in myocytes from failing heart, and the critical conductance required for successful AP propagation decreased significantly at high frequencies. Finally, in failing heart, asymmetry of transmural electrical propagation was abolished. Conclusions— In failing heart, preferential conduction from subendocardial to subepicardial myocytes is lost, and failing myocytes manifest facilitated AP propagation at fast rates. Together, these electrical remodeling responses may promote conduction of premature impulses and heighten the risk of malignant arrhythmia, a prominent feature of heart failure.

Mitochondrial Calcium and Reactive Oxygen Species Regulate Agonist-Initiated Platelet Phosphatidylserine Exposure

Objective—To study the interactions of cytoplasmic calcium elevation, mitochondrial permeability transition pore (mPTP) formation, and reactive oxygen species formation in the regulation of phosphatidylserine (PS) exposure in platelets. Methods and Results—mPTP formation, but not the degree of cytoplasmic calcium elevation, was associated with PS exposure in wild-type, cyclophilin D–null, ionomycin-treated, and reactive oxygen species–treated platelets. In the absence of the mPTP regulator cyclophilin D, agonist-initiated mPTP formation and high-level PS exposure were markedly blunted, but cytoplasmic calcium transients were unchanged. Mitochondrial calcium (Ca2+mit) transients and reactive oxygen species, key regulators of mPTP formation, were examined in strongly stimulated platelets. Increased reactive oxygen species production occurred in strongly stimulated platelets and was dependent on extracellular calcium entry, but not the presence of cyclophilin D. Ca2+mit increased significantly in strongly stimulated platelets. Abrogation of Ca2+mit entry, either by inhibition of the Ca2+mit uniporter or mitochondrial depolarization, prevented mPTP formation and exposure but not platelet aggregation or granule release. Conclusion—Sustained cytoplasmic calcium levels are necessary, but not sufficient, for high-level PS exposure in response to agonists. Increased Ca2+mit levels are a key signal initiating mPTP formation and PS exposure. Blockade of Ca2+mit entry allows the specific inhibition of platelet procoagulant activity.

Force frequency relationship of the human ventricle increases during early postnatal development.

Understanding developmental changes in contractility is critical to improving therapies for young cardiac patients. Isometric developed force was measured in human ventricular muscle strips from two age groups: newborns (<2 wk) and infants (3–14 mo) undergoing repair for congenital heart defects. Muscle strips were paced at several cycle lengths (CLs) to determine the force frequency response (FFR). Changes in Na/Ca exchanger (NCX), sarcoplasmic reticulum Ca-ATPase (SERCA), and phospholamban (PLB) were characterized. At CL 2000 ms, developed force was similar in the two groups. Decreasing CL increased developed force in the infant group to 131 ± 8% (CL 1000 ms) and 157 ± 18% (CL 500 ms) demonstrating a positive FFR. The FFR in the newborn group was flat. NCX mRNA and protein levels were significantly larger in the newborn than infant group whereas SERCA levels were unchanged. PLB mRNA levels and PLB/SERCA ratio increased with age. Immunostaining for NCX in isolated newborn cells showed peripheral staining. In infant cells, NCX was also found in T-tubules. SERCA staining was regular and striated in both groups. This study shows for the first time that the newborn human ventricle has a flat FFR, which increases with age and may be caused by developmental changes in calcium handling.

Purification of Cardiomyocytes From Differentiating Pluripotent Stem Cells Using Molecular Beacons That Target Cardiomyocyte-Specific mRNA

Background— Although methods for generating cardiomyocytes from pluripotent stem cells have been reported, current methods produce heterogeneous mixtures of cardiomyocytes and noncardiomyocyte cells. Here, we report an entirely novel system in which pluripotent stem cell–derived cardiomyocytes are purified by cardiomyocyte-specific molecular beacons (MBs). MBs are nanoscale probes that emit a fluorescence signal when hybridized to target mRNAs. Method and Results— Five MBs targeting mRNAs of either cardiac troponin T or myosin heavy chain 6/7 were generated. Among 5 MBs, an MB that targeted myosin heavy chain 6/7 mRNA (MHC1-MB) identified up to 99% of HL-1 cardiomyocytes, a mouse cardiomyocyte cell line, but <3% of 4 noncardiomyocyte cell types in flow cytometry analysis, which indicates that MHC1-MB is specific for identifying cardiomyocytes. We delivered MHC1-MB into cardiomyogenically differentiated pluripotent stem cells through nucleofection. The detection rate of cardiomyocytes was similar to the percentages of cardiac troponin T– or cardiac troponin I–positive cardiomyocytes, which supports the specificity of MBs. Finally, MHC1-MB–positive cells were sorted by fluorescence-activated cell sorter from mouse and human pluripotent stem cell differentiating cultures, and ≈97% cells expressed cardiac troponin T or cardiac troponin I as determined by flow cytometry. These MB-based sorted cells maintained their cardiomyocyte characteristics, which was verified by spontaneous beating, electrophysiological studies, and expression of cardiac proteins. When transplanted in a myocardial infarction model, MB-based purified cardiomyocytes improved cardiac function and demonstrated significant engraftment for 4 weeks without forming tumors. Conclusions— We developed a novel cardiomyocyte selection system that allows production of highly purified cardiomyocytes. These purified cardiomyocytes and this system can be valuable for cell therapy and drug discovery.

Differences in transient outward current properties between neonatal and adult human atrial myocytes

UNLABELLED Knowledge of postnatal modulation of I(to) in human atrial myocytes is quite limited. The present study investigated the differences in I(to) properties between neonatal and adult human atrial myocytes. METHODS Atrial myocytes were dissociated enzymatically from biopsies of human right atrial appendage. I(to) and action potentials were recorded by whole-cell patch-clamp technique. The expressed protein levels of Kv4.3 and KChIP2 in atrial tissue were detected by western blot technique. RESULTS I(to) was present in all atrial cells (n = 37) from 10 neonatal patients (2.5-7 months). The mean value of I(to) density in neonatal atrial cells was significantly larger than in adult atrial cells. The time constants for I(to) current decay were significantly faster for neonatal cells, compared to adult cells. I(to) recovery from inactivation at holding potential of - 80 mV was significantly slower for neonatal atrial cells than for adult atrial cells. There was no difference in the voltage dependence of I(to) activation between neonatal and adult cells. The voltage-dependent inactivation slope factor was smaller for neonatal compared to adult atrial cells. A more significant frequency-dependent suppression of I(to) peak current and a more significant lengthening of APD(30) were observed in neonatal atrial cells compared to adult atrial cells. Western blots showed both Kv4.3 and KChIP2 are expressed in neonatal atria, but with significantly higher level of Kv4.3 and lower level of KChIP2 protein compared to adult. CONCLUSION There are significant differences in the properties of I(to) between neonatal and adult human atrial cells, including a larger current density, faster inactivation and slower recovery from inactivation in the neonatal atrial cells. The physiological differences of I(to) are consistent with the different expression protein levels of Kv4.3 and KChIP2.

Propagation of Pacemaker Activity

Spontaneous activity of specific regions (e.g., the Sinoatrial node, SAN) is essential for the normal activation sequence of the heart and also serve as a primary means of modulating cardiac rate by sympathetic tone and circulating catecholamines. The mechanisms of how a small SAN region can electrically drive a much larger atrium, or how a small ectopic focus can drive surrounding ventricular or atrial tissue are complex, and involve the membrane properties and electrical coupling within the SAN or focus region as well as the membrane properties, coupling conductance magnitudes and also regional distribution within the surrounding tissue. We review here studies over the past few decades in which mathematical models and experimental studies have been used to determine some of the design principles of successful propagation from a pacemaking focus. These principles can be briefly summarized as (1) central relative uncoupling to protect the spontaneously firing cells from too much electrotonic inhibition, (2) a transitional region in which the cell type and electrical coupling change from the central SAN region to the peripheral atrial region, and (3) a distributed anisotropy to facilitate focal activity.

Cardioprotection from oxidative stress in the newborn heart by activation of PPARγ is mediated by catalase

Regulation of catalase (CAT) by peroxisome proliferator-activated receptor-γ (PPARγ) was investigated to determine if PPARγ activation provides cardioprotection from oxidative stress caused by hydrogen peroxide (H(2)O(2)) in an age-dependent manner. Left ventricular developed pressure (LVDP) was measured in Langendorff perfused newborn or adult rabbit hearts, exposed to 200μM H(2)O(2), with perfusion of rosiglitazone (RGZ) or pioglitazone (PGZ), PPARγ agonists. We found: (1) H(2)O(2) significantly decreased sarcomere shortening in newborn ventricular cells but not in adult cells. Lactate dehydrogenase (LDH) release occurred earlier in newborn than in adult heart, which may be due, in part, to the lower expression of CAT in newborn heart. (2) RGZ increased CAT mRNA and protein as well as activity in newborn but not in adult heart. GW9662 (PPARγ blocker) eliminated the increased CAT mRNA by RGZ. (3) In newborn heart, RGZ and PGZ treatment inhibited release of LDH in response to H(2)O(2) compared to H(2)O(2) alone. GW9662 decreased this inhibition. (4) LVDP was significantly higher in both RGZ+H(2)O(2) and PGZ+H(2)O(2) groups than in the H(2)O(2) group. Block of PPARγ abolished this effect. In contrast, there was no effect of RGZ in adult. (5) The cardioprotective effects of RGZ were abolished by inhibition of CAT. In conclusion, PPARγ activation is cardioprotective to H(2)O(2)-induced stress in the newborn heart by upregulation of catalase. These data suggest that PPARγ activation may be an effective therapy for the young cardiac patient.