Fuhua Chen

Oxidative Stress–Induced Afterdepolarizations and Calmodulin Kinase II Signaling

In the heart, oxidative stress caused by exogenous H2O2 has been shown to induce early afterdepolarizations (EADs) and triggered activity by impairing Na current (INa) inactivation. Because H2O2 activates Ca2+/calmodulin kinase (CaMK)II, which also impairs INa inactivation and promotes EADs, we hypothesized that CaMKII activation may be an important factor in EADs caused by oxidative stress. Using the patch-clamp and intracellular Ca (Cai) imaging in Fluo-4 AM–loaded rabbit ventricular myocytes, we found that exposure to H2O2 (0.2 to 1 mmol/L) for 5 to 15 minutes consistently induced EADs that were suppressed by the INa blocker tetrodotoxin (10 &mgr;mol/L), as well as the ICa,L blocker nifedipine. H2O2 enhanced both peak and late ICa,L, consistent with CaMKII-mediated facilitation. By prolonging the action potential plateau and increasing Ca influx via ICa,L, H2O2-induced EADs were also frequently followed by DADs in response to spontaneous (ie, non–ICa,L-gated) sarcoplasmic reticulum Ca release after repolarization. The CaMKII inhibitor KN-93 (1 &mgr;mol/L; n=4), but not its inactive analog KN-92 (1 &mgr;mol/L, n=5), prevented H2O2-induced EADs and DADs, and the selective CaMKII peptide inhibitor AIP (autocamtide-2–related inhibitory peptide) (2 &mgr;mol/L) significantly delayed their onset. In conclusion, H2O2-induced afterdepolarizations depend on both impaired INa inactivation to reduce repolarization reserve and enhancement of ICa,L to reverse repolarization, which are both facilitated by CaMKII activation. Our observations support a link between increased oxidative stress, CaMKII activation, and afterdepolarizations as triggers of lethal ventricular arrhythmias in diseased hearts.

Developmental changes in membrane Ca2+ and K+ currents in fetal, neonatal, and adult rabbit ventricular myocytes

Whole-cell calcium current (ICa) and inwardly rectifying potassium current (IK1) were studied in 21-day fetal, 28-day fetal (total gestation, 31 days), 2-5-day neonatal, and adult rabbit ventricular myocytes isolated by enzymatic dissociation. Whole-cell peak ICa and IK1 at -100 mV increased significantly after birth. Cell size approximated from cell membrane capacitance also increased with age, with the most significant increase occurring after birth. When normalized to cell surface area, peak ICa density increased from day 21 of gestation to the neonate and then increased again from neonate to adult. In all age groups, peak ICa occurred at a test potential of +10 mV, and the shape of the Ca2+ current-voltage relation did not change with age. These findings suggest that there are no significant developmental changes in the voltage dependence of ICa. Therefore, the measured age-related increase in Ca2+ current density may result from increased channel expression. IK1 also exhibited a pattern of increasing current density with age. For IK1, the increase in current density was most rapid between day 21 and the perinatal period and much slower after birth. These results demonstrate that ICa and IK1 undergo significant changes during late fetal and postnatal development.

L- and T-type calcium channels in acutely isolated neonatal and adult cardiac myocytes.

ABSTRACT: We have compared transsarcolemmal Ca2+ currents in acutely isolated neonatal (1− to 5-d-old) and adult rabbit cardiac myocytes prepared using similar enzymatic techniques. Time- and voltage-dependent inward Ca2+ currents were measured using the whole-cell voltage clamp technique. In neonatal myocytes, peak Ca2+ currents measured 114 ± 10 pA (mean ± SEM, n = 18) as compared with 2014 ± 403 pA in adult myocytes (n = 5, p < 0.001). Although adult myocytes had a larger surface area (estimated from cell capacitance) than neonatal cells (113 ± 15 × 10-6 versus 28 ± 2 × 10-6 cm2, p < 0.001), the calculated peak current density was also significantly larger in adult cells (17.9 ± 2.5 compared to 4.3 ± 0.4 μA/cm2 for neonatal cells, p < 0.001). The voltage dependence of the peak Ca2+ current was similar in neonatal and adult myocytes. Early transient (T-type) Ca2+ currents were also studied by comparing the current induced by depolarization to —20 mV from holding potentials of −40 and −80 mV. T-type Ca2+ channels were present in 91% of the adult cells but were evident in only 39% of the neonatal cells. In summary, voltage-gated Ca2+ current amplitude, current density, and T-type Ca2+ channel prevalence all increase with maturation. These data suggest that neonatal myocytes may be relatively deficient in Ca2+ channel activity when compared to adult myocytes.

Infection of Human Fetal Cardiac Myocytes by a Human Immunodeficiency Virus-1–Derived Vector

Cardiomyopathy associated with HIV-1 infection is a well-recognized complication. However, it is unknown whether direct cardiomyocyte infection is involved in the pathogenesis of the cardiomyopathy. An HIV-1-based lentiviral vector and wild-type HIV-1 were used to infect human fetal cardiac myocytes in a primary culture. Quantitative polymerase chain reaction, viral p24 antigen determination, and immunofluorescence were used to detect the synthesis of HIV-1 DNA and proteins after the infection. High-efficiency infection occurred using the HIV-1-based lentiviral vector, although no infection occurred with the wild-type HIV-1 strain. Dual-labeling immunofluorescence for HIV-1 proteins and myosin confirmed that cardiomyocytes were infected. This in vitro analysis suggests that direct myocyte infection with wild-type HIV-1 may not be involved in the pathogenesis of HIV-1 cardiomyopathy. However, HIV-1-based vectors may prove useful for ex vivo cardiovascular gene therapy.

Single-channel recording of inwardly rectifying potassium currents in developing myocardium

Properties of the inwardly rectifying K+ channel, which contributes to the maintenance of the resting membrane potential, were studied in neonatal rabbit ventricular myocytes using the patch-clamp technique. Inward rectification was evident in single-channel current-voltage (I-V) relations at potentials positive to the potassium equilibrium potential (Ek = 0 mV with [K+]o = [K+]i = 150 mM, [Mg2+]i = 2 mM). The single-channel conductance was 3.2 +/- 0.1 pS in physiological (5.4 mM) [K+]o. The zero-current potential shifted 48.4 +/- 2.4 mV for a ten-fold change in [K+]o in neonatal cells. External Ba2+ blocked the current in a dose-dependent manner. The voltage dependence, open-state probability and channel density of this channel were compared between neonatal and adult ventricular myocytes isolated by similar techniques. The open-state probability of the channel was approximately the same in neonatal (0.39 +/- 0.06, n = 13) as in adult cells (0.4 +/- 0.05, n = 11). However, in symmetrical transmembrane K+ concentration [( K+]o = [K+]i = 150 mM), the single channel conductance was significantly smaller in neonatal (25 +/- 0.3 pS, n = 25) as compared with adult cells (31 +/- 0.4 pS, n = 12). In addition, the relationship between resting membrane potential and [K+]o was measured in neonatal and adult myocytes. The resting membrane potential in the neonate was less dependent on [K+]o than in the adult. These results are consistent with an age-related change in resting membrane K+ permeability which may result from a developmental change in the single-channel conductance properties of the inwardly rectifying K+ channel.

Spatially discordant alternans in cardiomyocyte monolayers

Repolarization alternans is a harbinger of sudden cardiac death, particularly when it becomes spatially discordant. Alternans, a beat-to-beat alternation in the action potential duration (APD) and intracellular Ca (Cai), can arise from either tissue heterogeneities or dynamic factors. Distinguishing between these mechanisms in normal cardiac tissue is difficult because of inherent complex three-dimensional tissue heterogeneities. To evaluate repolarization alternans in a simpler two-dimensional cardiac substrate, we optically recorded voltage and/or Cai in monolayers of cultured neonatal rat ventricular myocytes during rapid pacing, before and after exposure to BAY K 8644 to enhance dynamic factors promoting alternans. Under control conditions (n = 37), rapid pacing caused detectable APD alternans in 81% of monolayers, and Cai transient alternans in all monolayers, becoming spatially discordant in 62%. After BAY K 8644 (n = 28), conduction velocity restitution became more prominent, and APD and Cai alternans developed and became spatially discordant in all monolayers, with an increased number of nodal lines separating out-of-phase alternating regions. Nodal lines moved closer to the pacing site with faster pacing rates and changed orientation when the pacing site was moved, as predicted for the dynamically generated, but not heterogeneity-based, alternans. Spatial APD gradients during spatially discordant alternans were sufficiently steep to induce conduction block and reentry. These findings indicate that spatially discordant alternans severe enough to initiate reentry can be readily induced by pacing in two-dimensional cardiac tissue and behaves according to predictions for a predominantly dynamically generated mechanism.

Calcium current and tension generation in immature mammalian myocardium: effects of diltiazem.

Single sucrose gap and isolated myocyte voltage-clamp techniques were used to study the effects of diltiazem on calcium current (ICa) and tension generation in isolated ventricular myocytes and right ventricular papillary muscles from neonatal New Zealand White rabbits. Diltiazem was shown to significantly shorten the duration of isolated myocyte action potentials with no effect on overshoot potential or resting membrane potential. Diltiazem blocked but did not completely abolish ICa in these neonatal cells. Addition of diltiazem to the solution bathing papillary muscles resulted in a similar reduction in action potential duration accompanied by a reduction in twitch tension. When the duration of depolarization was controlled employing the single sucrose gap voltage clamp, the decrease in tension development caused by diltiazem was abolished despite a significant decrease in twitch tension in the same muscles. In another series of experiments it was demonstrated that the magnitude of developed tension in neonatal papillary muscles is dependent upon the duration of depolarization. Taken together, the results of this investigation suggest that in neonatal myocardium when ICa is blocked by diltiazem, the resulting reduction in developed tension is caused in part by reduction of action potential duration. The calcium carried into the neonatal heart cell by ICa does not appear to be the only source of extracellular Ca2+ for excitation-contraction coupling. Finally, the action potential appears to act as a gate for calcium movement into the neonatal heart cell.

Electrophysiological Consequences of Acute Regional Ischemia/Reperfusion in Neonatal Rat Ventricular Myocyte Monolayers

Background— Electrophysiological changes promoting arrhythmias during acute regional ischemia/reperfusion are challenging to study in intact cardiac tissue because of complex 3-dimensional myocardial and vascular geometry. We characterized electrophysiological alterations and arrhythmias during regional ischemia/reperfusion in a simpler 2-dimensional geometry of cultured neonatal rat ventricular myocyte monolayers. Methods and Results— Optical mapping of intracellular Ca (Cai) and voltage was performed with the use of Rhod 2-AM and Rh-237, respectively. Regional ischemia was mimicked by covering the central portion of monolayer with a glass coverslip, and reperfusion was mimicked by removing the coverslip. Monolayers were stained with fluorescent antibodies to detect total and dephosphorylated connexin-43 at various time points. During coverslip ischemia, action potential duration shortened, Cai transient duration was prolonged, and local conduction velocity (CV) slowed progressively, with loss of excitability after 10.6±3.6 minutes. CV slowing was accompanied by connexin-43 dephosphorylation. During ischemia, spontaneous reentry occurred in 5 of 11 monolayers, initiated by extrasystoles arising from the border zone or unidirectional conduction block of paced beats. On reperfusion, excitability recovered within 1.0±0.8 minutes, but CV remained depressed for 9.0±3.0 minutes, promoting reentry in the reperfused zone. As connexin-43 phosphorylation recovered in the reperfused zone, CV normalized, and arrhythmias resolved. Conclusions— Acute regional ischemia/reperfusion in neonatal rat ventricular myocyte monolayers recapitulates electrophysiological alterations and arrhythmias similar to those observed during acute coronary occlusion/reperfusion in intact hearts. During early reperfusion, slow recovery from connexin-43 dephosphorylation leads to persistent CV slowing, creating a highly arrhythmogenic substrate.

Atrioventricular Ring Reentry in Embryonic Mouse Hearts

Background— During development, AV conduction switches from base-to-apex to apex-to-base conduction after emergence of the conduction system. We hypothesize that after this transition, the bulk of the AV ring, although no longer required for AV conduction, remains transiently able to conduct, providing a potential arrhythmia substrate. We studied AV conduction during this transition and its sensitivity to autonomic modulation. Methods and Results— Simultaneous voltage and Ca2+ mapping with RH-237 and Rhod-2 was performed with 2 CCD cameras in embryonic mouse hearts (n=43). Additionally, isolated calcium mapping was performed in 309 hearts with fluo-3AM. Propagation patterns in voltage and Ca2+ mapping coincided. Arrhythmias were uncommon under basal conditions, with AV block in 14 (4%) and junctional rhythms in 4 (1%). Arrhythmias increased after stimulation with isoproterenol—junctional rhythm in 9 (3%) and ventricular rhythms in 22 (6%)—although AV block decreased (3 hearts, 1%). Adding carbachol after isoproterenol caused dissociated antegrade and retrograde AV ring conduction in 30 (8.6%) of E10.5 and E11.5 hearts, occurring preferentially in the right and left sides of the ring, respectively. In 2 cases, reentry occurred circumferentially around the AV ring, perpendicular to normal propagation. Reentry persisted for multiple beats, lasting from 3 to 22 minutes. No episodes of AV ring reentry occurred in E9.5 hearts. Conclusions— AV ring reentry can occur by spatial dissociation of antegrade and retrograde conduction during combined adrenergic and muscarinic receptor stimulation. Critical maturation (>E9.5) seems to be required to sustain reentry.

HIV type 1 glycoprotein 120 inhibits cardiac myocyte contraction.

Cardiomyopathy is a common, life-threatening, but poorly understood complication of HIV infection. The purpose of the present study is to study the effects of an HIV surface envelope protein, glycoprotein 120 (gp120), on cell contraction and L-type Ca(2+) current in rabbit ventricular myocytes. Rabbit ventricular cells were isolated by an enzyme dissociation method. Cell contractions were induced by electric field stimulation. Whole cell L-type Ca(2+) channel currents were measured by the whole cell voltage-clamp technique. We found that perfusion with solution containing gp120 (0.1 microg/ml) derived from HIV-1(SF2) significantly inhibited field-stimulated contractions and L-type Ca(2+) current in rabbit ventricular myocytes as compared with perfusion with buffer alone. These results suggest that HIV-1 gp120 may directly contribute to cardiac dysfunction as seen in many HIV patients.