Cytoplasmic Ca2+ inhibits the ryanodine receptor from cardiac muscle

Ca²⁺-dependent inhibition of native and isolated ryanodine receptor (RyR) calcium release channels from sheep heart and rabbit skeletal muscle was investigated using the lipid bilayer technique. We found that cytoplasmic Ca²⁺ inhibited cardiac RyRs with an average K _m = 15 mm, skeletal RyRs with K _m = 0.7 mm and with Hill coefficients of 2 in both isoforms. This is consistent with measurements of Ca²⁺ release from the sarcoplasmic reticulum (SR) in skinned fibers and with [³H]-ryanodine binding to SR vesicles, but is contrary to previous bilayer studies which were unable to demonstrate Ca²⁺-inhibition in cardiac RyRs (Chu, Fill, Stefani &; Entman (1993) J. Membrane Biol. 135, 49–59). Ryanodine prevented Ca²⁺ from inhibiting either cardiac or skeletal RyRs. Ca²⁺-inhibition in cardiac RyRs appeared to be the most fragile characteristic of channel function, being irreversibly disrupted by 500 mm Cs⁺, but not by 500 mm K⁺, in the cis bath or by solublization with the detergent CHAPS. These treatments had no effect on channel regulation by AMP-PNP, caffeine, ryanodine, ruthenium red, or Ca²⁺-activation. Ca²⁺-inhibition in skeletal RyRs was retained in the presence of 500 mm Cs⁺. Our results provide an explanation for previous findings in which cardiac RyRs in bilayers with 250 mm Cs⁺ in the solutions fail to demonstrate Ca²⁺-inhibition, while Ca²⁺-inhibition of Ca²⁺ release is observed in vesicle studies where K⁺ is the major cation. A comparison of open and closed probability distributions from individual RyRs suggested that the same gating mechanism mediates Ca²⁺-inhibition in skeletal RyRs and cardiac RyRs, with different Ca²⁺ affinities for inhibition. We conclude that differences in the Ca²⁺-inhibition in cardiac and skeletal channels depends on their Ca²⁺ binding properties.     

Rapid activation of the cardiac ryanodine receptor by submillisecond calcium stimuli

The local control concept of excitation-contraction coupling in the heart postulates that the activity of the sarcoplasmic reticulum ryanodine receptor channels (RyR) is controlled by Ca(2+) entry through adjoining sarcolemmal single dihydropyridine receptor channels (DHPRs). One unverified premise of this hypothesis is that the RyR must be fast enough to track the brief (<0.5 ms) Ca(2+) elevations accompanying single DHPR channel openings. To define the kinetic limits of effective trigger Ca(2+) signals, we recorded activity of single cardiac RyRs in lipid bilayers during rapid and transient increases in Ca(2+) generated by flash photolysis of DM-nitrophen. Application of such Ca(2+) spikes (amplitude approximately 10-30 microM, duration approximately 0.1-0.4 ms) resulted in activation of the RyRs with a probability that increased steeply (apparent Hill slope approximately 2.5) with spike amplitude. The time constants of RyR activation were 0.07-0.27 ms, decreasing with spike amplitude. To fit the rising portion of the open probability, a single exponential function had to be raised to a power n approximately 3. We show that these data could be adequately described with a gating scheme incorporating four sequential Ca(2+)-sensitive closed states between the resting and the first open states. These results provide evidence that brief Ca(2+) triggers are adequate to activate the RyR, and support the possibility that RyR channels are governed by single DHPR openings. They also provide evidence for the assumption that RyR activation requires binding of multiple Ca(2+) ions in accordance with the tetrameric organization of the channel protein.     

Leptin protects the cardiac myocyte cultures from hypoxic damage

Leptin, a circulating hormone mainly produced by adipose tissue, regulates fatty acid metabolism and causes multiple systemic biological actions even the regulation of cardiovascular function. It is previously known that leptin is a hypoxia-inducible hormone, that hypoxic conditions increase the expression of this peptide in various tissues such as placenta, pancreas and also in the heart. Since leptin receptors are present in the heart, we hypothesized that whether leptin was a protector response for tissues especially for the heart against the deleterious effects of hypoxia. Cultured cardiomyocytes from newborn rats were initially treated with 3000 ng/ml leptin incubation for 1, 5 and 20 h separately, then subjected to 120 min of hypoxia. Hypoxic damage of myocytes was assayed using the measurements of both lactate dehydrogenase and creatine kinase releases into the medium and performing morphological observations (ultrastructural and immunocytochemical) of plates. The obtained results from leptin treated and non-treated control groups were compared to each other, and these data have demonstrated that 5 h of leptin treatment before hypoxia provides a significant protection for cardiomyocytes against hypoxia. Neither 1- nor 20-h leptin treated groups exhibited sufficient protection against hypoxia. In conclusion, leptin protects the cardiomyocyte cultures from hypoxia, but this effect is selective and evident only in the 5-h treated myocytes.     

Surface charge potentiates conduction through the cardiac ryanodine receptor channel

Single channel currents through cardiac sarcoplasmic reticulum (SR) Ca2+ release channels were measured in very low levels of current carrier (e.g., 1 mM Ba2+). The hypothesis that surface charge contributes to these anomalously large single channel currents was tested by changing ionic strength and surface charge density. Channel identity and sidedness was pharmacologically determined. At low ionic strength (20 mM Cs+), Cs+ conduction in the lumen-->myoplasm (L-->M) direction was significantly greater than in the reverse direction (301.7 +/- 92.5 vs 59.8 +/- 38 pS, P < 0.001; mean +/- SD, t test). The Cs+ concentration at which conduction reached half saturation was asymmetric (32 vs 222 mM) and voltage independent. At high ionic strength (400 mM Cs+), conduction in both direction saturated at 550 +/- 32 pS. Further, neutralization of carboxyl groups on the lumenal side of the channel significantly reduced conduction (333.0 +/- 22.5 vs 216.2 +/- 24.4 pS, P < 0.002). These results indicate that negative surface charge exists near the lumenal mouth of the channel but outside the electric field of the membrane. In vivo, this surface charge may potentiate conduction by increasing the local Ca2+ concentration and thus act as a preselection filter for this poorly selective channel.     

Inactivation of the cardiac ryanodine receptor calcium release channel by nitric oxide

We have recently reported [Mészáros L.G., Minarovic I., Zahradníková A. Inhibition of the skeletal muscle ryanodine receptor calcium release channel by nitric oxide. FEBS Lett 1996; 380: 49–52] that nitric oxide (NO) reduces the activity of the skeletal muscle ryanodine receptor Ca²⁺ release channel (RyRC), a principal component of the excitation-contraction coupling machinery in striated muscles. Since (i) as shown here, we have obtained evidence which indicates that the NO synthase (eNOS) of cardiac muscle origin co-purified with RyRC-containing sarcoplasmic reticulum (SR) fractions; and (ii) the effects of NO donors on the release channel, as well as on cardiac function, appear somewhat contradictory, we have made an attempt to investigate the response of the cardiac RyRC to NO that is generated in situ from L-arginine in the NOS reaction. We found that L-arginine-derived NO inactivates Ca²⁺ release from cardiac SR and reduces the steady-state activity (i.e. open probability) of single RyRCs fused into a planar lipid bilayer. This reduction was prevented by NOS inhibitors and the NO quencher hemoglobin and was reversed by 2-mercaptoethanol. We thus conclude that: (i) in isolated SR preparations, it is possible to assess the effects of NO that is generated from L-arginine in the NOS reaction; and (ii) cardiac RyRc responds to NO in a manner which is identical to that we have previously found with the skeletal channel. These findings suggest that the direct modulation of the RyRC by NO is a signaling mechanism which likely participates in earlier demonstrated NO-induced myocardial contractility changes.     

Primary structure and functional expression from cDNA of the cardiac ryanodine receptor/calcium release channel

The sequence of 4968 (or 4976 with an insertion) amino acids composing the ryanodine receptor from rabbit cardiac sarcoplasmic reticulum has been deduced by cloning and sequencing the cDNA. This protein is homologous in amino acid sequence and shares characteristic structural features with the skeletal muscle ryanodine receptor. Xenopus oocytes injected with mRNA derived from the cardiac ryanodine receptor cDNA exhibit Ca2(+)-dependent Cl- current in response to caffeine, which indicates the formation of functional calcium release channels. RNA blot hybridization analysis with a probe specific for the cardiac ryanodine receptor mRNA shows that the stomach and brain contain a hybridizable RNA species with a size similar to that of the cardiac mRNA. This result, in conjunction with cloning and analysis of partial cDNA sequences, suggests that the brain contains a cardiac type of ryanodine receptor mRNA.     

Unique phosphorylation site on the cardiac ryanodine receptor regulates calcium channel activity.

Ryanodine receptors have recently been shown to be the Ca2+ release channels of sarcoplasmic reticulum in both cardiac muscle and skeletal muscle. Several regulatory sites are postulated to exist on these receptors, but to date, none have been definitively identified. In the work described here, we localize one of these sites by showing that the cardiac isoform of the ryanodine receptor is a preferred substrate for multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase). Phosphorylation by CaM kinase occurs at a single site encompassing serine 2809. Antibodies generated to this site react only with the cardiac isoform of the ryanodine receptor, and immunoprecipitate only cardiac [3H]ryanodine-binding sites. When cardiac junctional sarcoplasmic reticulum vesicles or partially purified ryanodine receptors are fused with planar bilayers, phosphorylation at this site activates the Ca2+ channel. In tissues expressing the cardiac isoform of the ryanodine receptor, such as heart and brain, phosphorylation of the Ca2+ release channel by CaM kinase may provide a unique mechanism for regulating intracellular Ca2+ release.     

Development of electrical activity in cardiac myocyte aggregates derived from mouse embryonic stem cells

Embryonic stem cells differentiate into cardiac myocytes, repeating in vitro the structural and molecular changes associated with cardiac development. Currently, it is not clear whether the electrophysiological properties of the multicellular cardiac structure follow cardiac maturation as well. In long-term recordings of extracellular field potentials with microelectrode arrays consisting of 60 substrate-integrated electrodes, we examined the electrophysiological properties during the ongoing differentiation process. The beating frequency of the growing preparations increased from 1 to 5 Hz concomitant to a decrease of the action potential duration and action potential rise time. A developmental increase of the conduction velocity could be attributed to an increased expression of connexin43 gap junction channels. Whereas isoprenalin elicited a positive chronotropic response from the first day of spontaneous beating onward, a concentration-dependent negative chronotropic effect of carbachol only developed after approximately 4 days. The in vitro development of the three-dimensional cardiac preparation thus closely follows the development described for the mouse embryonic heart, making it an ideal model to monitor the differentiation of electrical activity in embryonic cardiomyocytes.     

Location of divergent region 2 on the three-dimensional structure of cardiac muscle ryanodine receptor/calcium release channel

Ryanodine receptors (RyRs) are a family of calcium release channels found on intracellular calcium-handing organelles. Molecular cloning studies have identified three different RyR isoforms, which are 66-70% identical in amino acid sequence. In mammals, the three isoforms are encoded by three separate genes located on different chromosomes. The major variations among the isoforms occur in three regions, known as divergent regions 1, 2, and 3 (DR1, DR2, and DR3). In the present study, a modified RyR2 (cardiac isoform) cDNA was constructed, into which was inserted a green fluorescent protein (GFP)-encoding cDNA within DR2, specifically after amino acid residue Thr1366 (RyR2(T1366-GFP)). HEK293 cells expressing RyR2(T1366-GFP) cDNAs showed caffeine-sensitive and ryanodine-sensitive calcium release, demonstrating that RyR2(T1366-GFP) forms functional calcium release channels. Cells expressing RyR2(T1366-GFP) were identified readily by the characteristic fluorescence of GFP, indicating that the overall structure of the inserted GFP was retained. Cryo-electron microscopy (cryo-EM) of purified RyR2(T1366-GFP) showed structurally intact receptors, and a three-dimensional reconstruction was obtained by single-particle image processing. The location of the inserted GFP was obtained by comparing this three-dimensional reconstruction to one obtained for wild-type RyR2. The inserted GFP and, consequently Thr1366 within DR2, was mapped on the three-dimensional structure of RyR2 to domain 6, one of the characteristic cytoplasmic domains that form part of the multi-domain "clamp" regions of RyR2. The three-dimensional location of DR2 suggests that it plays roles in the RyR conformational changes that occur during channel gating, and possibly in RyR's interaction with the dihydropyridine receptor in excitation-contraction coupling. This study further demonstrates the feasibility and reliability of the GFP insertion/cryo-EM approach for correlating RyR's amino acid sequence with its three-dimensional structure, thereby enhancing our understanding of the structural basis of RyR function.     

Transgenic overexpression of Hdac3 in the heart produces increased postnatal cardiac myocyte proliferation but does not induce hypertrophy

Class I and II histone deacetylases (HDACs) play vital roles in regulating cardiac development, morphogenesis, and hypertrophic responses. Although the roles of Hdac1 and Hdac2, class I HDACs, in cardiac hyperplasia, growth, and hypertrophic responsiveness have been reported, the role in the heart of Hdac3, another class I HDAC, has been less well explored. Here we report that myocyte-specific overexpression of Hdac3 in mice results in cardiac abnormalities at birth. Hdac3 overexpression produces thickening of ventricular myocardium, especially the interventricular septum, and reduction of both ventricular cavities in newborn hearts. Our data suggest that increased thickness of myocardium in Hdac3-transgenic (Hdac3-Tg) mice is due to increased cardiomyocyte hyperplasia without hypertrophy. Hdac3 overexpression inhibits several cyclin-dependent kinase inhibitors, including Cdkn1a, Cdkn1b, Cdkn1c, Cdkn2b, and Cdkn2c. Hdac3-Tg mice did not develop cardiac hypertrophy at 3 months of age, unlike previously reported Hdac2-Tg mice. Further, Hdac3 overexpression did not augment isoproterenol-induced cardiac hypertrophy when compared with wild-type littermates. These findings identify Hdac3 as a novel regulator of cardiac myocyte proliferation during cardiac development.     

Enhancement by growth factors of cardiac myocyte differentiation from embryonic stem cells: a promising foundation for cardiac regeneration

Cell transplantation is a promising, still novel, potentially therapeutic approach for the treatment of heart diseases. Clinical applications require generation of large number of donor cells. Embryonic stem (ES) cells are capable of self-renewal apparently in an unlimited fashion, in vitro. Theoretically, they can differentiate into any cell type required for cell transplantation, including cardiac myocytes. Diverse growth factors have been implicated in programming diverse cellular processes, including development of the embryonic heart, ES cell self-renewal, and cardiac myocyte differentiation from ES cells. This review addresses the current understanding of the role of growth factors in the differentiation of cardiac myocytes from ES-embryoid body cell systems in vitro as well as cardiac regeneration in vivo.     

Cardioprotective effect of selenium via modulation of cardiac ryanodine receptor calcium release channels in diabetic rat cardiomyocytes through thioredoxin system

Increased oxidative stress contributes to heart dysfunction via impaired Ca²⁺ homeostasis in diabetes. Abnormal RyR2 function related with altered cellular redox state is an important factor in the pathogenesis of diabetic cardiomyopathy, while its underlying mechanisms remain poorly understood. In the present study, we used a streptozotocin-induced rat model of diabetic cardiomyopathy and tested a hypothesis that diabetes-related alteration in RyR2 function is related with ROS-induced posttranslational modifications. For this, we used heart preparations from either a diabetic rat or a sodium selenate (NaSe)-treated (0.3 mg/kg for 4 weeks) diabetic rat as well as either NaSe- (100 nmol/L) or thioredoxin (Trx; 5 μmol/L)-incubated (30 min) diabetic cardiomyocytes. Experimental approaches included imaging of intracellular free-Ca²⁺ ([Ca²⁺]_i) under both electrically stimulated and resting Fluo-3-loaded cardiomyocytes. RyR2-mediated SR-Ca²⁺ leak was significantly enhanced in diabetic cardiomyocytes, resulting in reduced amplitude and prolonged time courses of [Ca²⁺]_i transients compared to those of controls. Both SR-Ca²⁺ leak and [Ca²⁺]_i transients were normalized by treating diabetic rats with NaSe or by incubating diabetic myocytes with NaSe or Trx. Moreover, exposure of diabetic cardiomyocytes to antioxidants significantly improved [Ca²⁺]_i handling factors such as phosphorylation/protein levels of RyR2, amount of RyR2-bound FKBP12.6 and activities of both protein kinase A and CaMKII. NaSe treatment also normalized the oxidative stress/antioxidant defense biomarkers in plasma as well as Trx activity and nuclear factor-κB phosphorylation in the diabetic rat heart. Collectively, these findings suggest that redox modification through Trx-system besides the glutathione system contributes to abnormal function of RyR2s in hyperglycemic cardiomyocytes, presenting a potential therapeutic target for treating diabetics to preserve cardiac function.     

Evidence for a Ca2+ channel within the ryanodine receptor complex from cardiac sarcoplasmic reticulum

The solubilized [3H]ryanodine receptor from cardiac sarcoplasmic reticulum was centrifuged through linear sucrose gradients. A single peak of radioactivity with apparent sedimentation coefficient of approximately 30S specifically comigrated with a high molecular weight protein of apparent relative molecular mass approximately 400,000. Incorporation of the ryanodine receptor into lipid bilayers induced single Ca2+ channel currents with conductance and kinetic behavior almost identical to that of native cardiac Ca2+ release channels. These results suggest that the cardiac ryanodine receptor comprises the Ca2+ release channel involved in excitation-contraction coupling in cardiac muscle.     

Involvement of the cardiac ryanodine receptor/calcium release channel in catecholaminergic polymorphic ventricular tachycardia.

The cardiac ryanodine receptor (RyR2), the major calcium release channel on the sarcoplasmic reticulum (SR) in cardiomyocytes, has recently been shown to be involved in at least two forms of sudden cardiac death (SCD): (1) Catecholaminergic polymorphic ventricular tachycardia (CPVT) or familial polymorphic VT (FPVT); and (2) Arrhythmogenic right ventricular dysplasia type 2 (ARVD2). Eleven RyR2 missense mutations have been linked to these diseases. All eleven RyR2 mutations cluster into 3 regions of RyR2 that are homologous to the three malignant hyperthermia (MH)/central core disease (CCD) mutation regions of the skeletal muscle ryanodine receptor/calcium release channel RyR1. MH/CCD RyR1 mutations have been shown to alter calcium-induced calcium release. Sympathetic nervous system stimulation leads to phosphorylation of RyR2 by protein kinase A (PKA). PKA phosphorylation of RyR2 activates the channel. In conditions associated with high rates of SCD such as heart failure RyR2 is PKA hyperphosphorylated resulting in "leaky" channels. SR calcium leak during diastole can generate "delayed after depolarizations" that can trigger fatal cardiac arrhythmias (e.g., VT). We propose that RyR2 mutations linked to genetic forms of catecholaminergic-induced SCD may alter the regulation of the channel resulting in increased SR calcium leak during sympathetic stimulation.     

Luminal Ca2+ controls activation of the cardiac ryanodine receptor by ATP

The synergic effect of luminal Ca(2+), cytosolic Ca(2+), and cytosolic adenosine triphosphate (ATP) on activation of cardiac ryanodine receptor (RYR2) channels was examined in planar lipid bilayers. The dose-response of RYR2 gating activity to ATP was characterized at a diastolic cytosolic Ca(2+) concentration of 100 nM over a range of luminal Ca(2+) concentrations and, vice versa, at a diastolic luminal Ca(2+) concentration of 1 mM over a range of cytosolic Ca(2+) concentrations. Low level of luminal Ca(2+) (1 mM) significantly increased the affinity of the RYR2 channel for ATP but without substantial activation of the channel. Higher levels of luminal Ca(2+) (8-53 mM) markedly amplified the effects of ATP on the RYR2 activity by selectively increasing the maximal RYR2 activation by ATP, without affecting the affinity of the channel to ATP. Near-diastolic cytosolic Ca(2+) levels (<500 nM) greatly amplified the effects of luminal Ca(2+). Fractional inhibition by cytosolic Mg(2+) was not affected by luminal Ca(2+). In models, the effects of luminal and cytosolic Ca(2+) could be explained by modulation of the allosteric effect of ATP on the RYR2 channel. Our results suggest that luminal Ca(2+) ions potentiate the RYR2 gating activity in the presence of ATP predominantly by binding to a luminal site with an apparent affinity in the millimolar range, over which local luminal Ca(2+) likely varies in cardiac myocytes.     

Functional expression of the calcium release channel from skeletal muscle ryanodine receptor cDNA

Combined patch-clamp and fura-2 measurements were performed to study the calcium release properties of Chinese hamster ovary (CHO) cells transfected with the rabbit skeletal muscle ryanodine receptor cDNA carried by an expression vector. Both caffeine (1-50 mM) and ryanodine (100 microM) induced release of calcium from intracellular stores of transformed CHO cells but not from control (non-transfected) CHO cells. The calcium responses to caffeine and ryanodine closely resembled those commonly observed in skeletal muscle. Repetitive applications of caffeine produced characteristic all-or-none rises in intracellular calcium. Inositol 1,4,5-trisphosphate (IP3) neither activated the ryanodine receptor channel nor interfered with the caffeine-elicited calcium release. These results indicate that functional calcium release channels are formed by expression of the ryanodine receptor cDNA.