In the present study, we combined optical Ca(2+) imaging with immunocytochemistry studies to characterize autonomic regulation of Ca(2+) cycling during early development in isolated embryonic mouse hearts. At embryonic days 9.5-11.5 (E9.5-E11.5), the Ca(2+) transient originated in the superior portion of the right atrium, propagated rapidly through both atria, slowly through the atrio-ventricular (AV) ring, and rapidly through both ventricles. Isoproterenol (ISO) significantly increased heart rate, increased Ca(2+) transient amplitude, rate of rise (RR) and a rate of decay, and shortened AV conduction time, indicating the presence of functional beta-adrenergic receptors. The muscarinic agonist carbachol (CCh) had no effects until 1 day later at E10.5. Both beta1-adrenergic and M2 muscarinic receptors were detected in ventricular muscle sections by immunochemistry at E10.5. Growing nerves, labeled using growth-associated protein 43 antibodies, were detected at the E14.5 stage, but not at E10.5, whereas mature sympathetic nerves, detected by tyrosine hydroxylase (TH) labeling, were not yet present at E14.5. These results demonstrate that functional regulation of Ca(2+) cycling by beta-adrenergic receptors occurs earliest in developing embryonic mouse hearts, followed a day later by muscarinic receptor responsiveness, with autonomic innervation developing later. These results define the functional and structural sequence of autonomic regulation of Ca(2+) transient in the embryonic mouse heart.

BACKGROUND:Studies in animal cell preparations suggest that azimilide may produce a more desirable rate-dependent profile of class III action as a result of its effects on both the slowly (I(Ks)) and rapidly (I(Kr)) activating components of potassium current (I(K)). However, relatively little is known about the effects of azimilide on K(+) currents in human atrial cells. The present study investigated the effect of azimilide on the inward rectifier potassium current (I(K1)), delayed rectifier potassium current (I(K)), ultrarapid delayed rectifier current (I(Kur)), and transient outward potassium current (I(to)) in isolated single human atrial myocytes.METHODS:The tight-seal, whole-cell voltage clamp technique was used to investigate the acute effects of azimilide on K(+) currents in single human atrial myocytes. The cells were isolated enzymatically from atrial tissues that were obtained from patients undergoing open-heart surgeries, with the approval of the local Institutional Review Board.RESULTS:The average cell capacitance of the human atrial myocytes was 77.5 +/- 2.8 pF (Mean +/- standard error of mean, total 28 cells from 17 patients). We found that 100 microM of azimilide in the extracellular solution significantly inhibited the inward rectifier potassium current (12.3 +/- 3.1 vs 6.7 +/- 2.0 pA/pF, n = 12, P < 0.05) at the testing potential of -100 mV. Superfusion with 100 microM of azimilide for 10 minutes inhibited I(K) by 51.7 +/- 5.1% (from 3.4 +/- 0.5 to 1.6 +/- 0.2 pA/pF, n = 9, P < 0.01) at the clamping membrane potential of +40 mV. Human atrial cell I(Kur) was inhibited with 100 microM of azimilide by 38.6 +/- 4.4% (from 3.9 +/- 0.5 to 2.3 +/- 0.2 pA/pF, n = 9, P < 0.01, test potential = 40 mV). We also found that the average peak current amplitude of I(to) in these cells was significantly inhibited with 100 microM of azimilide by 60.3 +/- 5.9% (from 10.3 +/- 1.5 to 3.6 +/- 0.3 pA/pF, n = 6, P < 0.01, test potential = 50 mV).CONCLUSION:The present study provides direct evidence that azimilide inhibits multiple cellular transmembrane K(+) currents in freshly isolated human atrial myocytes. Inhibition of these K(+) currents by azimilide, especially of I(Ks) and I(Kur) is likely to be the electrophysiologic basis for the prolongation of the action potential duration in the human atria which mediates its known antifibrillatory effects in atrial fibrillation and flutter.

The purpose of the present study was to examine the role of G(i2)alpha in Ca2+ channel regulation using G(i2)alpha gene knockout mouse ventricular myocytes. The whole cell voltage-clamp technique was used to study the effects of the muscarinic agonist carbachol (CCh) and the beta -adrenergic agonist isoproterenol (Iso) on cardiac L-type Ca2+ currents in both 129Sv wild-type (WT) and G(i2)alpha gene knockout (G(i2)alpha (-/-)) mice. Perfusion with CCh significantly inhibited the Ca2+ current in WT cells, and this effect was reversed by adding atropine to the CCh-containing solution. In contrast, CCh did not affect Ca2+ currents in G(i2)alpha (-/-) ventricular myocytes. Addition of CCh to Iso-containing solutions attenuated the Iso-stimulated Ca2+ current in WT cardiomyocytes but not in G(i2)alpha (-/-) cells. These findings demonstrate that, whereas the Iso-Gsa signal pathway is intact in G(i2)alpha gene knockout mouse hearts, these cells lack the inhibitory regulation of Ca2+ channels by CCh. Therefore, G(i2)alpha is necessary for the muscarinic regulation of Ca2+ channels in the mouse heart. Further studies are needed to delineate the possible interaction of G(i) and other cell signaling proteins and to clarify the level of interaction of G protein-coupled regulation of L-type Ca2+ current in the heart.

Background Although amiodarone has been referred to as a class III antiarrhythmic agent, it also possesses electrophysiologic characteristics of the three other classes (classes I and IV and minor class II effects). Previous studies have demonstrated that amiodarone inhibits Ca2+ channel current in intact cardiac myocytes. However, it is not clear whether this response reflects a pure class IV effect (direct Ca2+ channel inhibition) or a class II effect (β-adrenergic receptor blockade) of amiodarone. Methods In the current study, the effects of amiodarone on Ca2+ current were studied in the absence of sympathetic regulation using a Xenopus oocyte expression system. The L-type Ca2+ channel α1C subunit was coexpressed with the α2Δ and β2a subunits in enzymatically digested Xenopus oocytes. Ca2+ currents were recorded using the cut-open oocyte preparation. Results We found that perfusion of 10 μM isoproterenol produced no significant change in peak Ca2+ current (from 223±33 to 210±29 nA, mean±SEM, n=5, P=not significant), indicating the absence of a functional stimulatory sympathetic signal pathway in these oocytes. After 10 minutes of exposure to 10 μM amiodarone, Ca2+ current amplitude was significantly decreased from 174±33 to 100±26 nA (n=8, P<0.01; control group: 220±33 to 212±29 nA, n=5, P=not significant). These effects were similar to those of 10 μM nifedipine (201±48 to 108±48 nA, n=6, P<0.05), a typical Ca2+ channel blocker. On the other hand, neither amiodarone nor nifedipine significantly altered the Ca2+ current activation or inactivation kinetics. Conclusions These results demonstrate that amiodarone inhibits Ca2+ current in the absence of a functional intrinsic β-adrenergic stimulatory system and, therefore, represents a true class IV effect.

Canine junctin is a 26-kDa transmembrane protein found in the sarcoplasmic reticulum (SR) membrane in cardiac and skeletal muscle. Junctin has recently been shown to bind directly to calsequestrin, the ryanodine receptor, and triadin. Junctin is thought to play a role in facilitating (and perhaps regulating) Ca(2+) release from the SR. Immature heart exhibits decreased utilization of SR Ca(2+) stores for cell contraction. We have cloned human and rabbit cardiac junctin and investigated the expression of junctin in developing rabbit heart. Human junctin was cloned from an adult cardiac cDNA library. Rabbit junctin was cloned by RT-PCR. Northern blot analysis demonstrates a single primary mRNA transcript of approximately 2.8 kb in hearts from both species. Sequence analysis demonstrates greater than 97% homology between the predicted amino acid sequences of human, rabbit, and canine junctin in the putative transmembrane domain and subsequent initial 61 amino acid portion of the putative luminal domain. These domains also exhibit sequence homology with triadin. The C-terminal region shows much lower (72 to 75%) sequence homology among the three species. In addition, Northern blot analysis demonstrates that the expression of junctin increases markedly in postnatal rabbit myocardium. These findings suggest that the putative transmembrane domain and subsequent initial portion of the putative luminal domain of junctin play an important role in the binding of junctin to calsequestrin, the ryanodine receptor, and triadin in the postnatal heart. Furthermore, the previously described increase in SR Ca(2+) release with development is associated with the increased expression of junctin.

See article by Klein et al. [1] (pages 367–374) in this issue. As discussed by Klein and his colleagues in this issue of Cardiovascular Research [1], cGMP is an important intracellular second messenger of various extracellular stimuli regulating L-type Ca2+ current ( I Ca-L) in both normal and failing hearts. It has been postulated that muscarinic agonists such as carbachol (CCh) bind to muscarinic receptors to promote formation of an activated inhibitory G protein–guanosine triphosphate (Gi-GTP) complex. Upon GTP binding, the heterotrimeric G protein dissociates into two moieties, Giα-GTP and Giβγ. Giα inhibits adenylyl cyclase (AC) [2,3] and decreases cyclic AMP (cAMP) concentration and thereby inhibits cAMP-dependent protein kinase (PKA). The final result is the inhibition of the phosphorylation of effectors such as the L-type Ca2+ channel ( I Ca-L). On the other hand, stimulatory receptors, such as β-adrenergic agonists, catalyze stimulatory G protein (Gs) and stimulate AC. The reaction finally increases cAMP and enhances the Ca2+ current. However, this archetypal view has been challenged by recent studies, which suggest that muscarinic inhibition of Ca2+ channels requires the presence of the other type of G (Go) protein [4], since there is a specific defect in muscarinic regulation of Ca2+ current in Goα knockout (Goα−/−) mouse ventricular myocytes. It has also been revealed that nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) play important roles in muscarinic regulation of cardiac I Ca-L. But the details of muscarinic regulation in cardiac I Ca-L is not completely understood. … * Tel.: +1-310-825-7105; fax: +1-310-825-7458 fchen{at}mednet.ucla.edu

The purpose of the present study was to determine whether age-related changes in the expression and function of the cardiac isoform of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) play a role in SR Ca(2+)release and cell contraction. SERCA2a protein levels and subcellular localization were compared between fetal, neonatal, juvenile and adult New Zealand White rabbits. Studies of SERCA function in isolated myocytes were performed in situ by examining the rate of reloading of the SR Ca(2+)stores following caffeine-induced depletion. We found that significant quantities of SERCA2a were present early in immature heart and that SERCA2a expression reached adult levels within 15-30 days after birth. Furthermore, SERCA2a protein is present as a series of transverse striations within the cell as early as 1 day of age. In contrast to previous studies of SERCA in vitro, the SERCA protein function in situ was found to be comparable between neonatal and adult myocytes in maintaining SR Ca(2+)stores. These results indicate that the paucity of SR Ca(2+)release in immature ventricular cardiac myocytes is not the result of immaturity in SERCA2a expression.

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.

Ankyrins are a multigene family of proteins that function as adapters between the cytoskeleton and trans-membrane proteins, such as ion channels. Previous studies have shown the linkage between ankyrin and ionic transport proteins such as Na+–K+ATPase, voltage-dependent Na+channels and Ca2+channels. In the present study, we have investigated the subcellular distribution of ankyrin and its relationship to the Na+–Ca2+exchange protein in immature and adult rabbit ventricular myocytes. Isolated single cardiomyocytes from neonatal, juvenile and adult rabbit hearts were examined by immunofluorescence labeling techniques, using antibodies against ankyrin and the Na+–Ca2+exchanger. We found that in neonatal rabbit cardiac myocytes, ankyrin labeling was mainly present at the Z disk, whereas the Na+–Ca2+exchanger was only present on the peripheral sarcolemma. At 2 weeks of age, ankyrin labeling was still predominantly observed at the level of the Z disks as well as in the partially developed T-tubules. In the adult cells, however, ankyrin and the Na+–Ca2+exchanger seem to be co-localized within T-tubules and at the costamere region of the peripheral sarcolemma. Immunogold labeling studies at the higher resolution electron microscopic level using cyrosection tissues of rabbit heart at different ages confirm these findings. These results indicate that the distribution pattern of ankyrin and the Na+–Ca2+exchanger changes with development in rabbit ventricular myocytes.