Isolation and characterization of purified sarcoplasmic reticulum membranes from isolated adult rat ventricular myocytes.

We have demonstrated for the first time the isolation of sarcoplasmic reticulum (SR) membranes from adult rat ventricular myocytes obtained from a single rat heart. The myocyte SR preparation exhibits similar Ca(2+)-transport and Ca2+/K(+)-ATPase activity as well as a similar protein profile to SR membranes isolated from intact rat heart tissue. This SR preparation exhibited a Ca2+/K(+)-ATPase activity of 371 +/- 55 nmol/min/mg protein (mean +/- S.E.M.; n = 5) and an oxalate-stimulated Ca(2+)-uptake activity of 103 +/- 4 nmol/min/mg protein (mean +/- S.E.M.; n = 6). Pretreatment of the SR vesicles with 5 microM ruthenium red increased the oxalate-stimulated Ca(2+)-uptake to 204 +/- 12 nmol/min/mg protein demonstrating the presence of junctional SR membranes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis shows that the isolated SR membranes contained protein bands at 430 (Ca(2+)-release channel), 100 (Ca2+/K(+)-ATPase), 55 (calsequestrin and/or calreticulin) and 53 kDa (glycoprotein). Western blots of myocyte SR membranes stained with ruthenium red detected 2 major Ca(2+)-binding protein bands in this preparation at 53-55 kDa (calsequestrin and/or calreticulin) and 97-100 kDa (Ca2+/K(+)-ATPase). The presence of phospholamban, a regulatory protein of the Ca2+/K(+)-ATPase of cardiac SR, was confirmed in the myocyte SR membranes by western blots probed with a monoclonal antibody to phospholamban. Isoproterenol stimulation of intact [32P]orthophosphate equilibriated myocytes was associated with an increase in the phosphorylation of 3 distinct proteins (27, 31 and 152 kDa) in myocyte homogenates. The 27 kDa phosphorylated protein was identified in purified SR membranes as phospholamban my migration on electrophoretic gels and by immunoblotting. The ability to prepare SR membranes from intact isolated adult rat ventricular myocytes makes this system a potentially useful model for the study of SR regulation by protein phosphorylation.     

Defective glycosylation of calsequestrin in heart failure

OBJECTIVE: Levels of Ca2+ regulatory proteins have been extensively analyzed in cardiomyopathies as possible indices of change in sarcoplasmic reticulum (SR) structure and function. Measures of calsequestrin (CSQ), however, a critical protein component of the Ca2+ release complex in junctional sarcoplasmic reticulum, have provided little or no evidence of underlying dysfunction. We previously reported that calsequestrin isolated from heart tissue exists in a variety of glycoforms and phosphoforms reflecting mannose trimming of N-linked glycans and phosphorylation and dephosphorylation on protein kinase CK2-sensitive sites. METHODS: Here, we tested whether the distribution of molecular forms changes in heart failure (HF) reflecting possible remodeling of diseased tissue. Canine hearts were paced (220 beats/min) for 6-8 weeks to induce heart failure. Calsequestrin was purified from heart failure and sham-operated (control) treated canine ventricles and analyzed by electrospray mass spectrometry. RESULTS: The results showed striking changes in the mass distribution of calsequestrin molecules present in tissue from heart failure (five animals) compared with control (five animals). In heart failure, calsequestrin contained glycan structures that were uncharacteristic of normal junctional sarcoplasmic reticulum, consistent with altered metabolism or altered trafficking through secretory compartments. Glycoforms containing Man8,9, expected for a phenotype less muscle-like, were more than doubled in heart failure hearts, and molecules were also phosphorylated to a higher level. CONCLUSIONS: These data reveal in tachycardia-induced heart failure a new and potentially important change in the mannose content of calsequestrin glycans, perhaps indicative of defective junctional SR trafficking and Ca2+ release complex assembly.     

Ca2+ blinks: rapid nanoscopic store calcium signaling

Luminal Ca(2+) in the endoplasmic and sarcoplasmic reticulum (ER/SR) plays an important role in regulating vital biological processes, including store-operated capacitative Ca(2+) entry, Ca(2+)-induced Ca(2+) release, and ER/SR stress-mediated cell death. We report rapid and substantial decreases in luminal [Ca(2+)], called "Ca(2+) blinks," within nanometer-sized stores (the junctional cisternae of the SR) during elementary Ca(2+) release events in heart cells. Blinks mirror small local increases in cytoplasmic Ca(2+),orCa(2+) sparks, but changes of [Ca(2+)] in the connected free SR network were below detection. Store microanatomy suggests that diffusional strictures may account for this paradox. Surprisingly, the nadir of the store depletion trails the peak of the spark by about 10 ms, and the refilling of local store occurs with a rate constant of 35 s(-1), which is approximately 6-fold faster than the recovery of local Ca(2+) release after a spark. These data suggest that both local store depletion and some time-dependent inhibitory mechanism contribute to spark termination and refractoriness. Visualization of local store Ca(2+) signaling thus broadens our understanding of cardiac store Ca(2+) regulation and function and opens the possibility for local regulation of diverse store-dependent functions.     

Modulation of ryanodine receptor by luminal calcium and accessory proteins in health and cardiac disease

The cardiac ryanodine receptor (RyR2) is the sarcoplasmic reticulum (SR) Ca(2+) release channel which is responsible for generation of the cytosolic Ca(2+) transient required for activation of cardiac contraction. RyR2 functional activity is governed by changes in [Ca(2+)] on both the cytosolic and luminal phase of the RyR2 channel. Activation of RyR2 by cytosolic Ca(2+) results in Ca(2+)-induced Ca(2+) release (CICR) from the SR. The decline in luminal [Ca(2+)] following release contributes to termination of CICR and Ca(2+) signalling refractoriness through the process of luminal Ca(2+)-dependent deactivation of RyR2s. The control of RyR2s by luminal Ca(2+) involves coordinated interaction of the channel with several SR proteins, including the Ca(2+)-binding protein calsequestrin (CASQ2), and the integral proteins triadin 1 (TRD) and junctin (JCN). CASQ2 in addition to serving as a Ca(2+) storage site and a luminal Ca(2+) buffer modulates RyR2 function more directly as a putative luminal Ca(2+) sensor. TRD and JCN, stimulatory by themselves, mediate the interactions between CASQ2 and RyR2. Acquired and genetic defects in proteins of this junctional Ca(2+) signalling complex lead to disease states such as cardiac arrhythmia and heart failure by impairing luminal Ca(2+) regulation of RyR2.     

Remodeling of Cardiac Membranes During the Development of Congestive Heart Failure

Various proteins such as Ca2+channels, Ca2+-pump ATPase, Na+–Ca2+exchanger, and Na+-K+ATPase in the sarcolemmal (SL) membrane are considered to be intimately involved in Ca2+-influx and Ca2+-efflux processes in the cardiomyocyte. On the other hand, Ca2+-pump ATPase, Ca2+-release channels, Ca2+-regulatory protein (phospholamban), and Ca2+-binding protein (calsequestrin) in the sarcoplasmic reticulum (SR) are known to participate in raising and lowering the intracellular concentration of Ca2+for the occurrence of cardiac contraction and relaxation processes. Therefore, a defect in any of the SL and SR proteins can be seen to result in Ca2+-handling abnormalities in cardiomyocytes and subsequently in cardiac dysfunction during the development of heart failure. In this review, evidence is presented to show that changes in the expression of genes specific for cardiac membrane proteins may lead to remodeling of both SR and SL membranes during the development of heart failure. Although a great deal of work on changes in gene expression for the SR membrane proteins has been carried out in the failing heart, relatively little information regarding changes in gene expression for SL proteins has appeared in the literature. Prevention of remodeling of cardiac membranes by modification of changes in the gene expression is suggested to serve as an important target for the treatment of heart failure.     

Immunogold-labeled L-type calcium channels are clustered in the surface plasma membrane overlying junctional sarcoplasmic reticulum in guinea-pig myocytes-implications for excitation-contraction coupling in cardiac muscle

Ca(2+) release through ryanodine receptors, located in the membrane of the junctional sarcoplasmic reticulum (SR), initiates contraction of cardiac muscle. Ca(2+)influx through plasma membrane L-type Ca(2+)channels is thought to be an important trigger for opening ryanodine receptors ("Ca(2+)-induced Ca(2+)-release"). Optimal transmission of the transmembrane Ca(2+)influx signal to SR release is predicted to involve spatial juxtaposition of L-type Ca(2+)channels to the ryanodine receptors of the junctional SR. Although such spatial coupling has often been implicitly assumed, and data from immunofluorescence microscopy are consistent with its existence, the definitive demonstration of such a structural organization in mammalian tissue is lacking at the electron-microscopic level. To determine the spatial distribution of plasma membrane L-type Ca(2+)channels and their location in relation to underlying junctional SR, we applied two high-resolution immunogold-labeling techniques, label-fracture and cryothin-sectioning, combined with quantitative analysis, to guinea-pig ventricular myocytes. Label-fracture enabled visualization of colloidal gold-labeled L-type Ca(2+)channels in planar freeze-fracture electron-microscopic views of the plasma membrane. Mathematical analysis of the gold label distribution (by nearest-neighbor distance distribution and the radial distribution function) demonstrated genuine clustering of the labeled channels. Gold-labeled cryosections showed that labeled L-type Ca(2+)channels quantitatively predominated in domains of the plasma membrane overlying junctional SR. These findings provide an ultrastructural basis for functional coupling between L-type Ca(2+)channels and junctional SR and for excitation-contraction coupling in guinea-pig cardiac muscle.     

Reduced expression of sarcalumenin and related Ca2+ -regulatory proteins in aged rat skeletal muscle

In skeletal muscle, Ca(2+)-cycling through the sarcoplasm regulates the excitation-contraction-relaxation cycle. Since uncoupling between sarcolemmal excitation and fibre contraction may play a key role in the functional decline of aged muscle, this study has evaluated the expression levels of key Ca(2+)-handling proteins in senescent preparations using immunoblotting and confocal microscopy. Sarcalumenin, a major luminal Ca(2+)-binding protein that mediates ion shuttling in the longitudinal sarcoplasmic reticulum, was found to be greatly reduced in aged rat tibialis anterior, gastrocnemius and soleus muscle as compared to adult specimens. Minor sarcolemmal components of Ca(2+)-extrusion, such as the surface Ca(2+)-ATPase and the Na(+)-Ca(2+)-exchanger, were also diminished in senescent fibres. No major changes were observed for calsequestrin, sarcoplasmic reticulum Ca(2+)-ATPase and the ryanodine receptor Ca(2+)-release channel. In contrast, the age-dependent reduction in the alpha(1S)-subunit of the dihydropryridine receptor was confirmed. Hence, this report has shown that downstream from the well-established defect in coupling between the t-tubular voltage sensor and the junctional Ca(2+)-release channel complex, additional age-related alterations exist in the expression of essential Ca(2+)-handling proteins. This may trigger abnormal luminal Ca(2+)-buffering and/or decreased plasmalemmal Ca(2+)-removal, which could exacerbate impaired signaling or disturbed intracellular ion balance in aged fibres, thereby causing contractile weakness.     

Reduced myocardial sarcoplasmic reticulum Ca(2+)-ATPase protein expression in compensated primary and secondary human cardiac hypertrophy.

Pathological intracellular calcium handling has been proposed to underlie the alterations of contractile behavior in hypertrophied myocardium. However, the myocardial protein expression of intracellular calcium transport proteins in compensated human left ventricular hypertrophy has not yet been studied. We investigated septal myocardial specimens of patients suffering from hypertrophic obstructive cardiomyopathy (n=14) or from acquired aortic valve stenosis (n=11) undergoing myectomy or aortic valve replacement, respectively. For comparison, we studied non-hypertrophied myocardium of six non-failing hearts which could not be transplanted for technical reasons. The myocardial density of the calcium release channel of the sarcoplasmic reticulum (SR) was determined by(3)H-ryanodine binding. Myocardial contents of SR Ca(2+)-ATPase, phospholamban, calsequestrin and Na(+)/Ca(2+)-exchanger were analysed by Western blot analysis. The myocardial SR calcium release channel density was not significantly different in hypertrophied and non-failing human myocardium. In both hypertrophic obstructive cardiomyopathy and in aortic valve stenosis, SR Ca(2+)-ATPase expression was reduced by about 30% compared to non-failing myocardium (P<0.05), whereas the expression of phospholamban, calsequestrin, and the Na(+)/Ca(2+)-exchanger was unchanged. The decrease of SR Ca(2+)-ATPase expression was still observable when related to its regulatory protein phospholamban or to the myosin content of the homogenates (P<0.05). Furthermore, the SR Ca(2+)-ATPase expression was inversely correlated to the septum thickness assessed by echocardiography, but not to age, cardiac index or outflow tract gradient. In primary as well as in secondary hypertrophied human myocardium, the expression of SR Ca(2+)-ATPase is reduced and inversely related to the degree of the hypertrophy. The diminished SR Ca(2+)-ATPase expression might result in reduced Ca(2+)reuptake into the SR and might contribute to altered contractile behavior in hypertrophied human myocardium.     

Junctional sarcoplasmic reticulum transmembrane proteins in the heart

The uptake of calcium into the sarcoplasmic reticulum (SR) and its subsequent release from the SR play a key role in the regulation of the cytosolic calcium concentration and the excitation-contraction coupling in cardiac muscle. While calcium uptake, catalyzed by the calcium-dependent ATPase, is thought to occur throughout the SR, the release of calcium is controlled by a complex of proteins localized to a distinct region, the junctional SR. This complex consists of the calcium release channel or ryanodine receptor (RyR), the high capacity calcium-binding protein calsequestrin located in the lumen of the junctional SR, and the junctional SR transmembrane proteins triadin 1 and junctin which are hypothesized to anchor calsequestrin to the RyR. Transgenic mice with cardiac-specific overexpression of triadin1 or junctin show distinct cardiac phenotypes with altered cellular and subcellular morphology, changes in contractile properties and/or in the expression of junctional SR proteins suggesting that these junctional sarcoplasmic reticulum transmembrane proteins are of functional relevance for the regulation of calcium release in the heart.     

CaMKII inhibition targeted to the sarcoplasmic reticulum inhibits frequency-dependent acceleration of relaxation and Ca2+ current facilitation

Cardiac Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in heart has been implicated in Ca(2+) current (I(Ca)) facilitation, enhanced sarcoplasmic reticulum (SR) Ca(2+) release and frequency-dependent acceleration of relaxation (FDAR) via enhanced SR Ca(2+) uptake. However, questions remain about how CaMKII may work in these three processes. Here we tested the role of CaMKII in these processes using transgenic mice (SR-AIP) that express four concatenated repeats of the CaMKII inhibitory peptide AIP selectively in the SR membrane. Wild type mice (WT) and mice expressing AIP exclusively in the nucleus (NLS-AIP) served as controls. Increasing stimulation frequency produced typical FDAR in WT and NLS-AIP, but FDAR was markedly inhibited in SR-AIP. Quantitative analysis of cytosolic Ca(2+) removal during [Ca(2+)](i) decline revealed that FDAR is due to an increased apparent V(max) of SERCA. CaMKII-dependent RyR phosphorylation at Ser2815 and SR Ca(2+) leak was both decreased in SR-AIP vs. WT. This decrease in SR Ca(2+) leak may partly balance the reduced SERCA activity leading to relatively unaltered SR-Ca(2+) load in SR-AIP vs. WT myocytes. Surprisingly, CaMKII regulation of the L-type Ca(2+) channel (I(Ca) facilitation and recovery from inactivation) was abolished by the SR-targeted CaMKII inhibition in SR-AIP mice. Inhibition of CaMKII effects on I(Ca) and RyR function by the SR-localized AIP places physical constraints on the localization of these proteins at the junctional microdomain. Thus SR-targeted CaMKII inhibition can directly inhibit the activation of SR Ca(2+) uptake, SR Ca(2+) release and I(Ca) by CaMKII, effects which have all been implicated in triggered arrhythmias.     

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.     

Spatiotemporal characteristics of SR Ca(2+) uptake and release in detubulated rat ventricular myocytes

In cardiac ventricular myocytes, sarcoplasmic reticulum (SR) Ca(2+) load is a key determinant of SR Ca(2+) release. This release normally occurs predominantly from SR junctions at sarcolemmal invaginations (t-tubules), ensuring synchronous SR Ca(2+) release throughout the cell. However under conditions of Ca(2+) overload, spontaneous SR Ca(2+) release and propagating Ca(2+) waves can occur, which are pro-arrhythmic. We used detubulated rat ventricular myocytes to determine the dependence of Ca(2+) wave propagation on SR Ca(2+) load, and the role of t-tubules in SR Ca(2+) uptake and spontaneous release. After SR Ca(2+) depletion, recovery of Ca(2+) transient amplitude (and SR Ca(2+) load) was slower in detubulated than control myocytes (half-maximal recovery: 9.9+/-1.4 vs. 5.5+/-0.7 beats). In detubulated myocytes the extent and velocity of Ca(2+) propagation from the cell periphery increased with each beat and depended steeply on SR Ca(2+) load. Isoproterenol (ISO) accelerated recovery, increased maximal propagation velocity and reduced the threshold SR Ca(2+) load for propagation. Ca(2+) spark frequency was uniform across control cell width and was similar at the periphery of detubulated cells. However, internal Ca(2+) spark frequency in detubulated cells was 75% lower (despite comparable local SR Ca(2+) load); this transverse spark frequency profile was similar to that in atrial myocytes. We conclude that: (1) t-tubule Ca(2+) fluxes normally control SR Ca(2+) refilling; (2) Ca(2+) wave propagation depends steeply on SR Ca(2+) content (3) SR-t-tubule junctions are important in initiating SR Ca(2+) release and (4) ISO enhances propagation of SR Ca release, but not the initiation of SR Ca release events (for given SR Ca(2+) loads).     

Sarcalumenin alleviates stress-induced cardiac dysfunction by improving Ca2+ handling of the sarcoplasmic reticulum

AIMS: Sarcalumenin (SAR) is a Ca(2+)-binding protein expressed in the longitudinal sarcoplasmic reticulum (SR) of striated muscle cells. Although its Ca(2+)-binding property is similar to that of calsequestrin, its role in the regulation of Ca(2+) cycling remains unclear. METHODS AND RESULTS: To investigate whether SAR plays an important role in maintaining cardiac function under pressure overload stress, SAR-knockout (SAR-KO) mice were subjected to transverse aortic constriction (TAC). To examine the relation of SAR with cardiac type of SR Ca(2+) pump, SERCA2a, we designed cDNA expression using cultured cells. We found that SAR expression was significantly downregulated in hypertrophic hearts from three independent animal models. SAR-KO mice experienced higher mortality than did wild-type (WT) mice after TAC. TAC significantly downregulated SERCA2a protein but not mRNA in the SAR-KO hearts, whereas it minimally did so in hearts from WT mice. Accordingly, SR Ca(2+) uptake and cardiac function were significantly reduced in SAR-KO mice after TAC. Then we found that SAR was co-immunoprecipitated with SERCA2a in cDNA-transfected HEK293T cells and mouse ventricular muscles, and that SERCA2a-mediated Ca(2+) uptake was augmented when SAR was co-expressed in HEK293T cells. Furthermore, SAR significantly prolonged the half-life of SERCA2a protein in HEK293T cells. CONCLUSION: These findings suggest that functional interaction between SAR and SERCA2a enhances protein stability of SERCA2a and facilitates Ca(2+) sequestration into the SR. Thus the SAR-SERCA2a interaction plays an essential role in preserving cardiac function under biomechanical stresses such as pressure overload.     

Structural alterations in cardiac calcium release units resulting from overexpression of junctin

Junctin is a 26 kDa membrane protein that binds to calsequestrin, triadin, and ryanodine receptors (RyRs) within the junctional sarcoplasmic reticulum of calcium release units. The sequence of junctin includes a short N-terminal cytoplasmic domain a single transmembrane domain, and a highly charged C-terminal domain located in the sarcoplasmic reticulum lumen. Dog and mouse junctins are highly conserved at the transmembrane domains, but the luminal domains are more divergent. To probe the contribution of junctin to the architecture of calcium release units in heart, we engineered transgenic mice overexpressing canine junctin and examined the left ventricular myocardium by electron microscopy. Overall architecture of calcium release units is similar in control myocardium and in myocardium overexpressing junctin by 5-10-fold. In both myocardia, junctional SR cisternae are closely associated with exterior membranes (plasmalemma and transverse tubules). The cisternae are flat; they contain a string of calsequestrin beads and are lined by a row of feet, or RyRs, on the side facing the exterior membranes. T tubule surface density, measured as the perimeter of T tubule profiles v area of section, is the same in transgenic and control myocardia (305 v 289 nm/nm(2)). Three changes affecting the junctional SR architecture are apparent in the myocardium overexpressing junctin. One is a more tightly zippered appearance of the junctional SR cisternae. The width of the junctional SR is narrower and less variable in overexpressing than in control myocardium and the calsequestrin content is more compact. A second change is the extension of zippered junctional SR domains to non-junctional regions, which we term "frustrated" junctional SR. A third change is an increase in the extent of association between SR and T tubules. In junctin overexpressing myocardium junctional SR cisternae cover approximately 45% of the surface of all T tubule profiles, while in control myocardium the coverage approximately 30%. Junctional associations between SR and T tubules are increased in size. We conclude that the increase in junctin expression affects the packing of calsequestrin in the junctional SR and facilitates the association of SR and T tubules.     

Type 3 ryanodine receptors of skeletal muscle are segregated in a parajunctional position

A key event in skeletal muscle activation is the rapid release of Ca(2+) from the sarcoplasmic reticulum (SR), the Ca(2+) storage organelle in the muscle cell. The surface membrane/transverse tubules and the SR form functional units (calcium release units containing one or two couplons or junctions), where the voltage-sensing dihydropyridine receptor of the surface membrane interacts with the SR Ca(2+) release channel [ryanodine receptor (RyR)] and depolarization of the cell membrane is converted into Ca(2+) release from the SR. Although RyR1 is the most important isoform in skeletal muscle, some muscles also express high levels of RyR3, an isoform with a wide tissue distribution. The cytoplasmic domains of RyRs are visible in the electron microscope as periodically disposed feet. We find that, in muscles containing only RyR1, feet are exclusively located over the junctional SR surface facing the surface membrane/transverse tubule. In muscles containing RyR1 as well as RyR3, additional feet are located in lateral parajunctional regions immediately adjacent to junctional SR. Biochemical content of RyR3 and content of parajunctional feet are highly correlated in different muscles and the disposition of parajunctional versus junctional feet are notably different. On the basis of these two observations, we postulate that RyR3s are restricted to the parajunctional region, and thus their activation must be indirect and derivative during excitation-contraction coupling.     

Prevention of abnormal sarcoplasmic reticulum calcium transport and protein expression in post-infarction heart failure using 3, 5-diiodothyropropionic acid (DITPA)

Heart failure of diverse causes is associated with abnormalities of sarcoplasmic reticulum (SR) Ca(2+)transport. The purpose of this study was to determine whether the thyroid hormone analogue, 3,5-diiodothyropropionic acid (DITPA), prevents abnormal Ca(2+)transport and expression of SR proteins associated with post-infarction heart failure. New Zealand White rabbits were randomly assigned to circumflex artery ligation or sham operation, and to DITPA administration (3.75 mg/kg/day) or no treatment in a two-by-two factorial design. After 3 weeks, echo-Doppler and LV hemodynamic measurements were performed. From ventricular tissue, single myocyte shortening and relaxation were determined, and Ca(2+)transport was measured in homogenates and SR-enriched microsomes. Levels of mRNA and protein content were determined for the SR Ca(2+)-ATPase (SERCA2a), phospholamban (PLB), cardiac ryanodine receptor (RyR-2) and calsequestrin. The administration of DITPA improved LV contraction and relaxation and improved myocyte shortening in infarcted animals. The improvements in LV and myocyte function were associated with increases in V(max)for SR Ca(2+)transport in both homogenates and microsomes. Also, DITPA prevented the decrease in LV protein density for SERCA2a, PLB and RyR-2 post-infarction, without measurable changes in mRNA levels. The thyroid hormone analogue, DITPA, improves LV, myocyte and SR function in infarcted hearts and prevents the downregulation of SR proteins associated with post-infarction heart failure. The specific effects of DITPA on post-infarction SR Ca(2+)transport and the expression of SR proteins make this compound a potentially useful therapeutic agent for LV systolic and/or diastolic dysfunction.     

Depletion of T-tubules and specific subcellular changes in sarcolemmal proteins in tachycardia-induced heart failure

OBJECTIVE: The T-tubule membrane network is integrally involved in excitation-contraction coupling in ventricular myocytes. Ventricular myocytes from canine hearts with tachycardia-induced dilated cardiomyopathy exhibit a decrease in accessible T-tubules to the membrane-impermeant dye, di8-ANNEPs. The present study investigated the mechanism of loss of T-tubule staining and examined for changes in the subcellular distribution of membrane proteins essential for excitation-contraction coupling. METHODS: Isolated ventricular myocytes from canine hearts with and without tachycardia-induced heart failure were studied using fluorescence confocal microscopy and membrane fractionation techniques using a variety of markers specific for sarcolemmal and sarcoplasmic reticulum proteins. RESULTS: Probes for surface glycoproteins, Na/K ATPase, Na/Ca exchanger and Ca(v)1.2 demonstrated a prominent but heterogeneous reduction in T-tubule labeling in both intact and permeabilised failing myocytes, indicating a true depletion of T-tubules and associated membrane proteins. Membrane fractionation studies showed reductions in L-type Ca(2+) channels and beta-adrenergic receptors but increased levels of Na/Ca exchanger protein in both surface sarcolemma and T-tubular sarcolemma-enriched fractions; however, the membrane fraction enriched in junctional complexes of sarcolemma and junctional sarcoplasmic reticulum demonstrated no significant changes in the density of any sarcolemmal protein or sarcoplasmic reticulum protein assayed. CONCLUSION: Failing canine ventricular myocytes exhibit prominent depletion of T-tubules and changes in the density of a variety of proteins in both surface and T-tubular sarcolemma but with preservation of the protein composition of junctional complexes. This subcellular remodeling contributes to abnormal excitation-contraction coupling in heart failure.     

Possible contribution of the sarcoplasmic reticulum Ca(2+) pump function to electrical and mechanical alternans

We investigated the role of the sarcoplasmic reticulum's (SR) Ca(2+) pump function of the in the mechanism of alternans. We recorded the surface ECG, monophasic action potential (MAP) and left ventricular pressure (LVP) in the canine beating heart. Alternans was induced with an abrupt shortening of the cycle length from 1000 to 350 ms. After the control studies, we administered propranolol or isoproterenol. In the presence of propranolol, we administered milrinone or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). In the presence of isoproterenol, we administered thapsigargin. Isoproterenol and milrinone attenuated both the electrical and mechanical alternans. Thapsigargin, a specific SR Ca(2+) pump inhibitor, and propranolol magnified both types of alternans. DIDS, a Ca(2+)-activated Cl(-) current (I(Cl(Ca))) inhibitor, attenuated the MAP alternans without an affect on the LVP alternans. Thus, the delayed intracellular Ca(2+) cycling caused by the impaired SR Ca(2+) pump function might produce electrical and mechanical alternans. beta-adrenergic stimulation eliminated these alternans. The I(Cl(Ca)) contributed to the appearance of the electrical alternans.     

Impaired relaxation in transgenic mice overexpressing junctin

OBJECTIVE: Junctin is a major transmembrane protein in cardiac junctional sarcoplasmic reticulum, which forms a quaternary complex with the ryanodine receptor (Ca(2+) release channel), triadin, and calsequestrin. METHODS: To better understand the role of junctin in excitation-contraction coupling in the heart, we generated transgenic mice with targeted overexpression of junctin to mouse heart, using the alpha-MHC promoter to drive protein expression. RESULTS: The protein was overexpressed 10-fold in mouse ventricles and overexpression was accompanied by cardiac hypertrophy (19%). The levels of two other junctional SR-proteins, the ryanodine receptor and triadin, were reduced by 32% and 23%, respectively. However, [3H]ryanodine binding and the expression levels of calsequestrin, phospholamban and SERCA2a remained unchanged. Cardiomyocytes from junctin-overexpressing mice exhibited impaired relaxation: Ca(2+) transients decayed at a slower rate and cell relengthening was prolonged. Isolated electrically stimulated papillary muscles from junctin-overexpressing hearts exhibited prolonged mechanical relaxation, and echocardiographic parameters of relaxation were prolonged in the living transgenic mice. The amplitude of caffeine-induced Ca(2+) transients was lower in cardiomyocytes from junctin-overexpressing mice. The inactivation kinetics of L-type Ca(2+) channel were prolonged in junctin-overexpressing cardiomyocytes using Ca(2+) or Ba(2+) as charge carriers. CONCLUSION: Our data provide evidence that cardiac-specific overexpression of junctin is accompanied by impaired myocardial relaxation with prolonged Ca(2+) transient kinetics on the cardiomyocyte level.     

Transgenic triadin 1 overexpression alters SR Ca2+ handling and leads to a blunted contractile response to beta-adrenergic agonists

OBJECTIVE: Ca2+ release from the cardiac junctional sarcoplasmic reticulum (SR) is regulated by a complex of proteins, including the ryanodine receptor (RyR), calsequestrin (CSQ), junctin (JCN), and triadin 1 (TRD). Moreover, triadin 1 appears to anchor calsequestrin to the ryanodine receptor. METHODS: To determine whether triadin 1 overexpression alters excitation-contraction coupling, we examined the effects of cardiac-specific overexpression of triadin 1 on SR Ca2+ handling and contractility in transgenic (TG) compared to wild-type (WT) mice. RESULTS: The overexpression of triadin 1 was associated with an enhanced SR Ca2+ load, reflected by a 22% higher amplitude of caffeine-induced Ca2+ transients. The decline of Ca2+ transients during caffeine exposure was prolonged by 57%. The detection of resting spontaneous SR Ca2+ release events (Ca2+ sparks) revealed an increased amplitude (by 16%), decline (by 47%), and width (by 47%) in TG. This was associated with a redistribution of Ca2+ spark amplitudes from one population to two populations. Measurement of cardiac function by echocardiography and left ventricular (LV) catheterization revealed a decreased cardiac contractility in vivo. The impaired response to beta-adrenergic receptor (beta-AR) stimulation in TG hearts was associated with an increased protein expression of beta-AR kinase 1. In addition, the increase of the L-type Ca2+ peak current and the increase of phospholamban (PLB) phosphorylation at Thr17 were reduced under beta-AR stimulation. CONCLUSION: Taken together, our data suggest that triadin 1 overexpression results in a complex modulation of SR Ca2+ handling, which may contribute, at least in part, to the depressed basal contractility and the blunted response to beta-adrenergic agonists in TG mice.