Abstract. The subcellular distribution of sarcolemmal dihydropyridine receptor (DHPR) and sarcoplasmic reticular triadin and Ca 2÷ release channel/ryanodine receptor (RyR) was determined in adult rabbit ventricle and atrium by double labeling immunofluorescence and laser scanning confocal microscopy. In ventricular muscle cells the immunostaining was observed primarily as transversely oriented punctate bands spaced at approximately 2-/zm intervals along the whole length of the muscle fibers. Image analysis demonstrated a virtually complete overlap of the staining patterns of the three proteins, suggesting their close association at or near dyadic couplings that are formed where the sarcoplasmic reticulum (SR) is apposed to the surface membrane or its infoldings, the transverse (T-) tubules. In rabbit atrial cells, which lack an extensive T-tubular system, DHPR-specific staining was observed to form discrete spots along the sarcolemma but was absent from the interior of the fibers. In atrium, punctate triadin-and RyR-specific staining was also observed as spots at the cell periphery and image analysis indicated that the three proteins were co-localized at, or just below, the sarcolemma. In addition, in the atrial cells triadin-and RyR-specific staining was observed to form transverse bands in the interior cytoplasm at regularly spaced intervals of approximately 2 #m. Electron microscopy suggested that this cytoplasmic staining was occurring in regions where substantial amounts of extended junctional SR were present. These data indicate that the DHPR codistributes with triadin and the RyR in rabbit ventricle and atrium, and furthermore suggest that some of the SR Ca 2+ release channels in atrium may be activated in the absence of a close association with the DHPR. IN striated muscle, depolarization of the sarcolemma and transverse (T-) t tubular network induces release of Ca 2÷ from internal stores in the sarcoplasmic reticulum (SR) by a process commonly referred to as excitation-contraction (EC) coupling. In skeletal muscle it is generally accepted that the electrical signal is transduced to a release of Ca 2+ at specialized triad junctions formed between a central T-tubule flanked by two elements of closely apposed junctional SR (jSR). The junctional T-tubules contain DHPR which act as voltage sensors (36,45,(56)(57)(58) 1. Abbreviations used in this paper: DHP, dihydropyridine; DHPR, DHP receptor; jSR, junctional sarcoplasmic reticulum; RyR, ryanodine receptor; SR, sarcoplasmic reticulum; T-tubule, transverse tubule; TC, terminal cisternae.rows of "feet" (23) that have been identified as ryanodine receptors (RyR), the SR Ca 2÷ release channels (35,55,61). The exact mechanism of skeletal muscle EC coupling is not yet completely understood; however, it is thought that the release of Ca 2÷ from the SR is a depolarization-induced mechanism without the necessity of Ca 2÷ flow (46). In contrast, excitation contraction coupling in cardiac muscle requires an influx of Ca z÷ through L-type Ca ~÷ channels (the cardiac i...

Abstract. Peripheral couplings are junctions between the sarcoplasmic reticulum (SR) and the surface membrane (SM). Feet occupy the SR/SM junctional gap and are identified as the SR calcium release channels, or ryanodine receptors (RyRs). In cardiac muscle, the activation of RyRs during excitation-contraction (e-c) coupling is initiated by surface membrane depolarization, followed by the opening of surface membrane calcium channels, the dihydropyridine receptors (DHPRs). We have studied the disposition of DHPRs and RyRs, and the structure of peripheral couplings in chick myocardium, a muscle that has no transverse tubules. Immunolabeling shows colocalization of RyRs and DHPRs in clusters at the fiber's periphery. The positions of DHPR and RyR clusters change coincidentally during development. Freeze-fracture of the surface membrane reveals the presence of domains (junctional domains) occupied by clusters of large particles. Junctional domains in the surface membrane and arrays of feet in the junctional gap have similar sizes and corresponding positions during development, suggesting that both are components of peripheral couplings. As opposed to skeletal muscle, membrane particles in junctional domains of cardiac muscle do not form tetrads. Thus, despite their proximity to the feet, they do not appear to be specifically associated with them. Two observations establish the identity of the structurally identified feet arrays/junctional domain complexes with the immunocytochemically defined RyRs/DHPRs coclusters: the concomitant changes during development and the identification of feet as the cytoplasmic domains of RyRs. We suggest that the large particles in junctional domains of the surface membrane represent DHPRs. These observations have two important functional consequences. First, the apposition of DHPRs and RyRs indicates that most of the inward calcium current flows into the restricted space where feet are located. Secondly, contrary to skeletal muscle, presumptive DHPRs do not show a specific association with the feet, which is consistent with a less direct role of charge movement in cardiac than in skeletal e-c coupling. CALCIUM is an intracellular messenger in all cell types. In muscle fibers, cytosolic-free calcium concentration regulates contraction and relaxation. The endoplasmic reticulum is a storage compartment for calcium and the sarcoplasmic reticulum (SR) 1 of muscle cells cycles large amounts of calcium at each contraction and relaxation (for a review see Pozzan et al., 1994). Rapid release of calcium from ER and SR occurs through the ryanodine receptor (RyR), a channel with high permeability to calcium, encoded by three different genes with various tissue specificity

Calsequestrin is a high capacity Ca 2؉-binding protein in the sarcoplasmic reticulum (SR) lumen. To elucidate the functional role of calsequestrin in vivo, transgenic mice were generated that overexpressed mouse cardiac calsequestrin in the heart. Overexpression (20-fold) of calsequestrin was associated with cardiac hypertrophy and induction of a fetal gene expression program. Isolated transgenic cardiomyocytes exhibited diminished shortening fraction (46%), shortening rate (60%), and relengthening rate (60%). The Ca 2؉ transient amplitude was also depressed (45%), although the SR Ca 2؉ storage capacity was augmented, as suggested by caffeine application studies. These alterations were associated with a decrease in L-type Ca 2؉ current density and prolongation of this channel's inactivation kinetics without changes in Na ؉ -Ca 2؉ exchanger current density. Furthermore, there were increases in protein levels of SR Ca 2؉ -ATPase, phospholamban, and calreticulin and decreases in FKBP12, without alterations in ryanodine receptor, junctin, and triadin levels in transgenic hearts. Left ventricular function analysis in Langendorff perfused hearts and closed-chest anesthetized mice also indicated depressed rates of contraction and relaxation of transgenic hearts. These findings suggest that calsequestrin overexpression is associated with increases in SR Ca 2؉ capacity, but decreases in Ca 2؉ -induced SR Ca 2؉ release, leading to depressed contractility in the mammalian heart.

Isolated heavy sarcoplasmic reticulum vesicles retain junctional specializations (feet) on their outer surface. We have obtained en face three-dimensional views of the feet by shadowing and replicating the surfaces of freeze-dried isolated vesicles. Feet are clearly visible as large structures located on raised platforms. New details of foot structure include a four subunit structure and the fact that adjacent feet do not abut directly corner to corner but are offset by half a subunit. Feet aligned within rows were observed to be rotated at a slight angle off the long axis of the row creating a center-to-center spacing (32.5 nm) slightly less than the average diagonal of the feet (35.3 nm). Comparison with previous information from thin sections and freeze-fracture showed that this approach to the study of membranes faithfully preserves structure and allows better visualization of surface details than either thinsectioning or negative-staining.

Cardiac-specific overexpression of murine cardiac calsequestrin results in depressed cardiac contractile parameters, low Ca 2؉ -induced Ca 2؉ release from sarcoplasmic reticulum (SR) and cardiac hypertrophy in transgenic mice. To test the hypothesis that inhibition of phospholamban activity may rescue some of these phenotypic alterations, the calsequestrin overexpressing mice were cross-bred with phospholamban-knockout mice. Phospholamban ablation in calsequestrin overexpressing mice led to reversal of the depressed cardiac contractile parameters in Langendorff-perfused hearts or in vivo. This was associated with increases of SR Ca 2؉ storage, assessed by caffeine-induced Na ؉ -Ca 2؉ exchanger currents. The inactivation time of the L-type Ca 2؉ current (I Ca ), which has an inverse correlation with Ca 2؉ -induced SR Ca 2؉ release, and the relation between the peak current density and half-inactivation time were also normalized, indicating a restoration in the ability of I Ca to trigger SR Ca 2؉ release. The prolonged action potentials in calsequestrin overexpressing cardiomyocytes also reversed to normal upon phospholamban ablation. Furthermore, ablation of phospholamban restored the expression levels of atrial natriuretic factor and ␣-skeletal actin mRNA as well as ventricular myocyte size. These results indicate that attenuation of phospholamban function may prevent or overcome functional and remodeling defects in hypertrophied hearts.Hypertrophy of ventricular myocardium is postulated to be an adaptive response to relative increases in external workload, induced by endocrine, paracrine, autocrine, and mechanical factors or decreased myocardial contractility (1). The increase in heart mass has been implicated to normalize cardiac function by decreasing wall stress. However, a sustained imbalance between workload and muscle contractility may lead to progressive thinning of the left ventricular wall and chamber dilation associated with decompensated hypertrophy and heart failure (2, 3). Studies in human and animal models have shown that cardiac hypertrophy is associated with impaired sarcoplasmic reticulum (SR) 1 Ca 2ϩ modulation, leading to aberrant cardiac contraction and relaxation (4 -8). Although several Ca 2ϩ -related signaling molecules, such as calcineurin, Ca 2ϩ -calmodulin kinase, and Ca 2ϩ

Stimulation of β-adrenergic receptors activates type I and II cyclic AMP–dependent protein kinase A, resulting in phosphorylation of various proteins in the heart. It has been proposed that PKA II compartmentalization by A-kinase–anchoring proteins (AKAPs) regulates cyclic AMP–dependent signaling in the cell. We investigated the expression and localization of AKAP100 in adult hearts. By immunoblotting, we identified AKAP100 in adult rat and human hearts, and showed that type I and II regulatory (RI and II) subunits of PKA are present in the rat heart. By immunofluorescence and confocal microscopy of rat cardiac myocytes and cryostat sections of rat left ventricle papillary muscles, we localized AKAP100 to the nucleus, sarcolemma, intercalated disc, and at the level of the Z-line. After double immunostaining of transverse cross-sections of the papillary muscles with AKAP100 plus α-actinin–specific antibodies or AKAP100 plus ryanodine receptor–specific antibodies, confocal images showed AKAP100 localization at the region of the transverse tubule/junctional sarcoplasmic reticulum. RI is distributed differently from RII in the myocytes. RII, but not RI, was colocalized with AKAP100 in the rat heart. Our studies suggest that AKAP100 tethers PKA II to multiple subcellular compartments for phosphorylation of different pools of substrate proteins in the heart.

Phospholamban ablation has been shown to result in significant increases in cardiac contractile parameters and loss of beta-adrenergic stimulation. To determine whether partial reduction in phospholamban levels is also associated with enhancement of cardiac performance and to further examine the sensitivity of the contractile system to alterations in phospholamban levels, hearts from wild-type, phospholamban-heterozygous, and phospholamban-deficient mice were studied in parallel at the subcellular, cellular, and organ levels. The phospholamban-heterozygous mice expressed reduced cardiac phospholamban mRNA and protein levels (40 +/- 5%) compared with wild type mice. The reduced phospholamban levels were associated with significant decreases in the EC50 of the sarcoplasmic reticulum Ca2+ pump for CA2+ and increases in the contractile parameters of isolated myocytes and beating hearts. The relative phospholamban levels among wild-type, phospholamban-heterozygous, and phospholamban-deficient mouse hearts correlated well with the (1) EC50 of the Ca(2+)-ATPase for Ca2+ in sarcoplasmic reticulum, (2) rates of relaxation and contraction in isolated cardiac myocytes, and (3) rates of relaxation and intact beating hearts. These findings suggest that physiological and pathological changes in the levels of phospholamban will result in parallel changes in sarcoplasmic reticulum function and cardiac contraction.

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