Xinsheng Zhu

Mineralocorticoid Modulation of Cardiac Ryanodine Receptor Activity Is Associated With Downregulation of FK506-Binding Proteins

Background— The mineralocorticoid pathway is involved in cardiac arrhythmias associated with heart failure through mechanisms that are incompletely understood. Defective regulation of the cardiac ryanodine receptor (RyR) is an important cause of the initiation of arrhythmias. Here, we examined whether the aldosterone pathway might modulate RyR function. Methods and Results— Using the whole-cell patch clamp method, we observed an increase in the occurrence of delayed afterdepolarizations during action potential recordings in isolated adult rat ventricular myocytes exposed for 48 hours to aldosterone 100 nmol/L, in freshly isolated myocytes from transgenic mice with human mineralocorticoid receptor expression in the heart, and in wild-type littermates treated with aldosterone. Sarcoplasmic reticulum Ca2+ load and RyR expression were not altered; however, RyR activity, visualized in situ by confocal microscopy, was increased in all cells, as evidenced by an increased occurrence and redistribution to long-lasting and broader populations of spontaneous Ca2+ sparks. These changes were associated with downregulation of FK506-binding proteins (FKBP12 and 12.6), regulatory proteins of the RyR macromolecular complex. Conclusions— We suggest that in addition to modulation of Ca2+ influx, overstimulation of the cardiac mineralocorticoid pathway in the heart might be a major upstream factor for aberrant Ca2+ release during diastole, which contributes to cardiac arrhythmia in heart failure.

Conversion of an inactive cardiac dihydropyridine receptor II-III loop segment into forms that activate skeletal ryanodine receptors

A 25 amino acid segment (Glu666–Pro691) of the II‐III loop of the α1 subunit of the skeletal dihydropyridine receptor, but not the corresponding cardiac segment (Asp788–Pro814), activates skeletal ryanodine receptors. To identify the structural domains responsible for activation of skeletal ryanodine receptors, we systematically replaced amino acids of the cardiac II‐III loop with their skeletal counterparts. A cluster of five basic residues of the skeletal II‐III loop (681RKRRK685) was indispensable for activation of skeletal ryanodine receptors. In the cardiac segment, a negatively charged residue (Glu804) appears to diminish the electrostatic potential created by this basic cluster. In addition, Glu800 in the group of negatively charged residues 798EEEEE802 of the cardiac II‐III loop may serve to prevent the binding of the activation domain.

Imperatoxin A Enhances Ca2+ Release in Developing Skeletal Muscle Containing Ryanodine Receptor Type 3

Most adult mammalian skeletal muscles contain only one isoform of ryanodine receptor (RyR1), whereas neonatal muscles contain two isoforms (RyR1 and RyR3). Membrane depolarization fails to evoke calcium release in muscle cells lacking RyR1, demonstrating an essential role for this isoform in excitation-contraction coupling. In contrast, the role of RyR3 is unknown. We studied the participation of RyR3 in calcium release in wild type (containing both RyR1 and RyR3 isoforms) and RyR3-/- (containing only RyR1) myotubes in the presence or absence of imperatoxin A (IpTxa), a high-affinity agonist of ryanodine receptors. IpTxa significantly increased the amplitude and the rate of release only in wild-type myotubes. Calcium currents, recorded simultaneously with the transients, were not altered with IpTxa treatment. [(3)H]ryanodine binding to RyR1 or RyR3 was significantly increased in the presence of IpTxa. Additionally, IpTxa modified the gating and conductance level of single RyR1 or RyR3 channels when studied in lipid bilayers. Our data show that IpTxa can interact with both RyRs and that RyR3 is functional in myotubes and it can amplify the calcium release signal initiated by RyR1, perhaps through a calcium-induced mechanism. In addition, our data indicate that when RyR3-/- myotubes are voltage-clamped, the effect of IpTxa is not detected because RyR1s are under the control of the dihydropyridine receptor.

Imperatoxin A (IpTxa) from Pandinus imperator stimulates [3H]ryanodine binding to RyR3 channels

The effect of imperatoxin A (IpTxa) on the ryanodine receptor type 3 (RyR3) was studied. IpTxa stimulates [3H]ryanodine binding to RyR3‐containing microsomes, but this effect requires toxin concentrations higher than those required to stimulate RyR1 channels. The effect of IpTxa on RyR3 channels was observed at calcium concentrations in the range 0.1 μM to 10 mM. By contrast, RyR2 channels were not significantly affected by IpTxa in the same calcium ranges. Single channel current measurements indicated that IpTxa induced subconductance state in RyR3 channels that was similar to those observed with RyR1 and RyR2 channels. These results indicate that IpTxa is capable of inducing similar subconductance states in all three RyR isoforms, while stimulation of [3H]ryanodine binding by this toxin results in isoform‐specific responses, with RyR1 being the most sensitive channel, RyR3 displaying an intermediate response and RyR2 the least responsive ones.