The assembly of calcium release units in cardiac muscle.

Calcium release units (CRUs) are constituted of specialized junctional domains of the sarcoplasmic reticulum (jSR) that bear calcium release channels, also called ryanodine receptors (RyRs). In cardiac muscle, CRUs come in three subtypes that differ in geometry, but have common molecular components. Peripheral couplings are formed by a junction of the jSR with the plasmalemma; dyads occur where the jSR is associated with transverse (T)-tubules; corbular SR is a jSR domain that is located within the cells and bears RyRs but does not associate with either plasmalemma or T-tubules. Using transmission electron microscopy, this study followed the formation of CRUs and their accrual of four components: the L-type channel dihydropyridine receptors (DHPRs) of plasmalemma/T-tubules; the RyRs of jSR; triadin (Tr) and junctin (JnC), two homologous components of the jSR membrane; and calsequestrin (CSQ), the internal calcium binding proteins. During differentiation, peripheral couplings are formed first and the others follow. RyRs and DHPRs are targeted to subdomains of the CRUs that face each other and are acquired in a concerted manner. Overexpressions of either junction (JnC or Tr) and of CSQ, singly or in conjunction, shed light on the specific role of JnC in the structural development, organization, and maintenance of jSR cisternae and on the independent synthetic pathways and targeting of JnC and CSQ. In addition, the structural cues provided by the overexpression models allow us to define sequential steps in the synthetic pathway for JnC and CSQ and their targeting to the CRUs of differentiating myocardium.

Junctin and calsequestrin overexpression in cardiac muscle: the role of junctin and the synthetic and delivery pathways for the two proteins.

Ca(2+) storage and release in muscle cells are controlled by a complex of junctional sarcoplasmic reticulum (jSR) proteins, that includes the calcium-binding protein calsequestrin (CSQ), the Ca(2+)-release channel (ryanodine receptor or RyR) and two transmembrane proteins that bind to RyR: junctin (JNC) and triadin (Tr). The relationship between CSQ and JNC, and their contributions to the architecture of the jSR vesicle was studied in transgenic mice with combined overexpression of CSQ and JNC. We find that CSQ, on its own, has a diffuse disposition in the sarcoplasmic reticulum (SR) lumen. Overexpression of JNC results in a tighter packing of CSQ in proximity of the SR membrane, presumably due to the binding of CSQ to the membrane by JNC. Quantitative and qualitative analysis of structural changes in the overexpressing as well as in the normally differentiating myocardium illustrate the synthetic pathways and the events in the targeting and delivery of CSQ and JNC to the jSR of the differentiating cardiac myocyte. CSQ is delivered from the Golgi to the SR, where it buds out into precursors of the jSR vesicles. JNC reaches the jSR vesicles directly, but its arrival is delayed relative to CSQ.

Location of ryanodine and dihydropyridine receptors in frog myocardium.

Frog myocardium depends almost entirely on calcium entry from extracellular spaces for its beat-to-beat activation. Atrial myocardium additionally shows internal calcium release under certain conditions, but internal release in the ventricle is absent or very low. We have examined the content and distribution of the sarcoplasmic reticulum (SR) calcium release channels (ryanodine receptors, RyRs) and the surface membrane calcium channels (dihydropyridine receptors, DHPRs) in myocardium from the two atria and the ventricle of the frog heart using binding of radioactive ryanodine, immunolabeling of RyR and DHPR, and thin section and freeze-fracture electron microscopy. In cells from both types of chambers, the SR forms peripheral couplings and in both chambers peripheral couplings colocalize with clusters of DHPRs. However, although a low level of high affinity binding of ryanodine is detectable and RyRs are present in peripheral couplings of the atrium, the ventricle shows essentially no ryanodine binding and RyRs are not detectable either by electron microscopy or immunolabeling. The results are consistent with the lack of internal calcium release in the ventricle, and raise questions regarding the significance of DHPR at peripheral couplings in the absence of RyR. Interestingly, the free SR membrane in both heart chambers shows a low but equal density of intramembrane particles representing the Ca(2+) ATPase.

Dilated cardiomyopathy in juvenile Portuguese Water Dogs.

Dilated cardiomyopathy recently has been recognized in juvenile Portuguese Water Dogs. The purpose of this study was to evaluate unaffected and affected puppies by physical examination, electrocardiogram (ECG), echocardiogram, specific biochemical assays, and ultrastructure to document disease progression and to develop a method of early detection. Results of segregation analysis were consistent with autosomal recessive inheritance. Of 124 puppies evaluated clinically and echocardiographically, 10 were affected. No significant differences were found between unaffected and affected puppies for blood and myocardial carnitine or taurine concentrations, serum chemical variables, results of ophthalmological examinations, ECGs, or measurement of urine metabolites. Ultrastructural examination of myocardium from affected dogs revealed myofibrillar atrophy and small regions of myofibrillar degeneration, most prominently at the region of the intercalated discs. Only echocardiography allowed detection of affected puppies before clinical signs became evident. Echocardiography revealed a significant difference in the shortening fraction, E point to septal separation, and the end systolic and diastolic left ventricular internal diameters. Affected puppies were detected 1-4 weeks before the development of acute congestive heart failure.