To investigate the channel properties of the mammalian type 3 ryanodine receptor (RyR3), we have cloned the RyR3 cDNA from rabbit uterus by reverse transcriptase-polymerase chain reaction and expressed the cDNA in HEK293 cells. Immunoblotting studies showed that the cloned RyR3 was indistinguishable from the native mammalian RyR3 in molecular size and immunoreactivity. Ca 2؉ release measurements using the fluorescence Ca 2؉ indicator fluo 3 revealed that the cloned RyR3 functioned as a caffeine-and ryanodine-sensitive Ca 2؉ Ryanodine receptors are a family of intracellular Ca 2ϩ release channels that were originally identified in the sarcoplasmic reticulum (SR) 1 of striated muscles. To date, three members of this family have been identified in mammalian tissues, namely the skeletal muscle (RyR1), the cardiac muscle (RyR2), and the brain (RyR3) ryanodine receptor. These proteins are the products of different genes and share 66 -70% amino acid sequence identity (1-5). Earlier studies using RNA blot analysis revealed that the expression patterns of these isoforms were very different (6). RyR1 was predominantly expressed in skeletal muscle, whereas RyR2 was mainly expressed in heart and brain. The expression of RyR3 was detected in smooth muscle tissues and certain regions of the brain. However, results of recent ribonuclease protection assay demonstrate that all three RyR isoforms are widely and differentially expressed (7). These studies also indicate that most tissues express more than one RyR isoform. For example, skeletal muscles express both RyR1 and RyR3, although RyR3 is expressed at a much lower level than that of RyR1.

We have investigated the molecular basis for ryanodine receptor (RyR) activation by Ca 2؉ by using sitedirected mutagenesis together with functional assays consisting of Ca 2؉ release measurements and single channel recordings in planar lipid bilayers. We report here that a single substitution of alanine for glutamate at position 3885 (located in the putative transmembrane sequence M2 of the type 3 RyR) reduces the Ca 2؉ sensitivity, as measured by single channel activation, by more than 10,000-fold, without apparent changes in channel conductance and in modulation by other ligands (e.g. ATP and ryanodine). Co-expression of the wild type and mutant RyR proteins results in the synthesis of single channels that have intermediate Ca 2؉sensitivities. These results suggest that the glutamates at position 3885 of each monomer may act in a coordinated way to form the Ca 2؉ sensor in the tetrameric structure corresponding to RyR.Ryanodine receptors (RyRs) 1 are a family of Ca 2ϩ channels which mediate intracellular Ca 2ϩ release that is essential for a variety of cellular functions including muscle contraction, egg fertilization, and synaptic transmission (1, 2). Three RyR isoforms (RyR1, RyR2, and RyR3) have been identified in mammalian tissues; all three are activated by Ca 2ϩ (3)(4)(5)(6)(7)(8). Activation of RyR by Ca 2ϩ is the mechanism underlying Ca 2ϩ -induced Ca 2ϩ release from the sarco(endo)plasmic reticulum (9 -11).Of the many ligands known to modulate the activity of RyR, Ca 2ϩ is the essential regulator. Most other ligands exert their effect on RyR activity by influencing the Ca 2ϩ sensitivity of 12). Alterations in the Ca 2ϩ sensitivity of RyR have been implicated in at least one disease, malignant hyperthermia (13). Thus, understanding the molecular mechanism that controls the Ca 2ϩ sensitivity is fundamental to the understanding of RyR regulation and intracellular Ca 2ϩ signaling. RyR activation by Ca 2ϩ is thought to be mediated by high affinity Ca 2ϩ binding sites in the protein (14), but the molecular identity of these Ca 2ϩ activation sites, the Ca 2ϩ sensor, has yet to be defined. It has been shown that negatively charged residues within a transmembrane sequence are often involved in binding and translocation of cations across the membrane (15-17). Analysis of the amino acid sequences of RyRs reveals that of the 12 predicted transmembrane sequences of RyR (18), four (M1, M2, M7, and M10) contain negatively charged amino acid residues that are conserved in all known RyR isoforms ( Fig. 1A) (19 -26). To investigate their roles in RyR function, we have mutated these negatively charged residues in the rabbit type 3 RyR. The functional consequence of one of these point mutations, a glutamate-to-alanine mutation at position 3885 (E3885A) located in the M2 transmembrane sequence (Fig. 1B), was assessed. Our results demonstrate that glutamate 3885 plays an essential role in determining the Ca 2ϩ sensitivity and provide important new insights into the Ca 2ϩ -sensing mechanism of RyR. EXPERIMENTAL PROCEDUR...

We report 4 adult patients with thyrotoxicosis accompanied by irreversible low-output heart failure. Each patient showed elevated plasma levels of thyroid hormone and prolonged low-output heart failure even after thyroid function returned to normal. Specimens of the right ventricular myocardium stained with anti-β myosin heavy-chain MAb showed a normal staining pattern with a predominance of the β-form. Our observations suggest that thyrotoxicosis may be one possible cause of irreversible cardiomyopathy.

trioventricular (AV) block following radiofrequency ablation for the treatment of AV nodal re-entrant tachycardia (AVNRT) is a rare but serious complication of this procedure. [1][2][3] The AV block usually occurs during or immediately following the application of radiofrequency (RF) energy, and is transient. However, several rare cases have been reported in which the patient developed an AV block late after the procedure and subsequently required a permanent pacemaker implantation. 4,5 Here, we describe a 22-year-old woman with a first-degree AV block who developed a complete AV block 1 week after RF ablation for the treatment of the uncommon form of AVNRT (slow/slow). Her complete AV block persisted for another week, and she then recovered. Case ReportA 22-year-old woman was referred to the Jichi Medical Hospital (Tochigi, Japan) for consideration of RF ablation of supraventricular tachycardia. An electrocardiogram (ECG) showed that she had a first-degree AV block, which she had had from the age of 12 years. Since age 19, she had developed fast palpitations with dyspnea and her symptoms had increased. An ECG during sinus rhythm showed a first-degree AV block with a pulse rate interval of 0.28 s (Fig 1A). An ECG during symptoms showed a narrow QRS complex tachycardia with a deeply inverted P wave in II at 130 beats/min (Fig 1B). Results of her physical examination, laboratory analysis, chest X-ray, and echocardiography were normal, indicating that the patient has no other cardiovascular disease except for first-degree AV block. Circulation Journal Vol.66, November 2002An electrophysiologic study and RF ablation were performed under mild sedation after first obtaining the patient's informed consent. Catheters were positioned in the high right atrium, coronary sinus and right ventricular apex, and across the tricuspid valve to record the His bundle potential. At baseline (A -A interval: 825 ms), atrio-His (A-H) and His-ventricular (H-V) intervals were 225 ms and 30 ms, respectively. Using the extrastimulus technique, dual AV nodal physiology was demonstrated with a 140 ms jump in A-H intervals for a 10 ms decrease in coupling interval (S1-S1: 750 ms; S1-S2: 260 ms) (Fig 2A). At a pacing cycle length of 750 ms, the antegrade effective refractory period (ERP) of the AV node was 220 ms. The site of earliest atrial activation potential during ventricular pacing was the coronary sinus ostium (VA: 375 ms; Fig 2B] Atrioventricular (AV) block following radiofrequency (RF) ablation for the treatment of AV nodal re-entrant tachycardia (AVNRT) is a rare but serious complication of this procedure. Almost all such cases occur during or immediately after radiofrequencey (RF) energy application, followed by prompt recovery. The present report describes a 22-year-old woman with first-degree AV block on electrocardiography, who developed complete AV block 1 week after RF ablation for the treatment of the uncommon form of AVNRT (slow/slow). The patient's complete AV block persisted for another 1 week before she recovered. (Circ J ...

Objective The plasma eicosapentaenoic acid to arachidonic acid ratio (EPA/AA), which is determined only by dietary intake, has been attracting attention as a new risk marker for coronary artery disease (CAD). A Japanese inland prefecture, Tochigi, ranks higher mortality rate from CAD and lower seafood consumption, compared to other prefectures. The aim of this study was to investigate the EPA/AA ratio in residents of Tochigi prefecture. Methods We measured the EPA/AA ratio in patients undergoing diagnostic coronary angiography, because of suspicion for having CAD, all of whom were residents of Tochigi prefecture or its bordering area. Patients A total of 428 patients were enrolled in 5 centers. Results The median value of the EPA/AA ratio in the study patients was 0.37, which seems to be lower than the value of the whole Japan. The EPA/AA ratio was similar in patients with and without CAD. Female patients had lower EPA/AA ratios than male patients. When subjects were divided into 10-year age groups, 30-to 39-year-old male and female patients displayed similar EPA/AA ratios. However, the ratios of 50-to 59-year old female patients (p=0.001) and 60-to 69-year old female patients (p=0.034) were significantly lower than those of age-matched male patients. Conclusion In female residents of Tochigi prefecture, and particularly in menopausal female patients suspected of CAD, the EPA/AA ratio might be lower than male patients. EPA intake can help prevent cardiovascular events, especially in menopausal female residents of an area where the fish intake is low.