Alterations of housekeeping proteins in human aged and diseased hearts.

Pathological remodeling includes alterations of ion channel function and calcium homeostasis and ultimately cardiac maladaptive function during the process of disease development. Biochemical assays are important approaches for assessing protein abundance and post-translational modification of ion channels. Several housekeeping proteins are commonly used as internal controls to minimize loading variabilities in immunoblotting protein assays. Yet, emerging evidence suggests that some housekeeping proteins may be abnormally altered under certain pathological conditions. However, alterations of housekeeping proteins in aged and diseased human hearts remain unclear. In the current study, immunoblotting was applied to measure three commonly used housekeeping proteins (ß-actin, calsequestrin, and GAPDH) in well-procured human right atria (RA) and left ventricles (LV) from diabetic, heart failure, and aged human organ donors. Linear regression analysis suggested that the amounts of linearly loaded total proteins and quantified intensity of total proteins from either Ponceau S (PS) blot-stained or Coomassie Blue (CB) gel-stained images were highly correlated. Thus, all immunoblotting data were normalized with quantitative CB or PS data to calibrate potential loading variabilities. In the human heart, ß-actin was reduced in diabetic RA and LV, while GAPDH was altered in aged and diabetic RA but not LV. Calsequestrin, an important Ca2+ regulatory protein, was significantly changed in aged, diabetic, and ischemic failing hearts. Intriguingly, expression levels of all three proteins were unchanged in non-ischemic failing human LV. Overall, alterations of human housekeeping proteins are heart chamber specific and disease context dependent. The choice of immunoblotting loading controls should be carefully evaluated. Usage of CB or PS total protein analysis could be a viable alternative approach for some complicated pathological specimens.

Changes in calsequestrin, TNF-α, TGF-ß and MyoD levels during the progression of skeletal muscle dystrophy in mdx mice: a comparative analysis of the quadriceps, diaphragm and intrinsic laryngeal muscles.

In Duchenne muscular dystrophy (DMD), the search for new biomarkers to follow the evolution of the disease is of fundamental importance in the light of the evolving gene and pharmacological therapies. In addition to the lack of dystrophin, secondary events including changes in calcium levels, inflammation and fibrosis greatly contribute to DMD progression and the molecules involved in these events may represent potential biomarkers. In this study, we performed a comparative evaluation of the progression of dystrophy within muscles that are differently affected by dystrophy (diaphragm; DIA and quadriceps; QDR) or spared (intrinsic laryngeal muscles) using the mdx mice model of DMD. We assessed muscle levels of calsequestrin (calcium-related protein), tumour necrosis factor (TNF-α; pro-inflammatory cytokine), tumour growth factor (TGF-ß; pro-fibrotic factor) and MyoD (muscle proliferation) vs. histopathology at early (1 and 4 months of age) and late (9 months of age) stages of dystrophy. Fibrosis was the primary feature in the DIA of mdx mice (9 months: 32% fibrosis), which was greater than in the QDR (9 months: 0.6% fibrosis). Muscle regeneration was the primary feature in the QDR (9 months: 90% of centrally nucleated fibres areas vs. 33% in the DIA). The QDR expressed higher levels of calsequestrin than the DIA. Laryngeal muscles showed normal levels of TNF-α, TGF-ß and MyoD. A positive correlation between histopathology and cytokine levels was observed only in the diaphragm, suggesting that TNF-α and TGF-ß serve as markers of dystrophy primarily for the diaphragm.

Evidence for the presence of calsequestrin in two structurally different regions of myocardial sarcoplasmic reticulum.

Localization of calsequestrin in chicken ventricular muscle cells was determined by indirect immunofluorescence and immuno-Protein A-colloidal gold labeling of cryostat and ultracryotomy sections, respectively. Calsequestrin was localized in the lumen of peripheral junctional sarcoplasmic reticulum, as well as in the lumen of membrane-bound structures present in the central region of the I-band, while being absent from the lumen of the sarcoplasmic reticulum in the A-band region of the cardiac muscle cells. Since chicken ventricular muscle cells lack transverse tubules, the presence of calsequestrin in membrane bound structures in the central region of the I-band suggests that these cells contain nonjunctional regions of sarcoplasmic reticulum that are involved in Ca2+ storage and possibly Ca2+ release. It is likely that the calsequestrin containing structures present throughout the I-band region of the muscle cells correspond to specialized regions of the free sarcoplasmic reticulum in the I-band called corbular sarcoplasmic reticulum. It will be of interest to determine whether Ca2+ storage and possibly Ca2+ release from junctional and nonjunctional regions of the sarcoplasmic reticulum in chicken ventricular muscle cells are regulated by the same or different physiological signals.

Characterization of the junctional face membrane from terminal cisternae of sarcoplasmic reticulum.

We have recently described a preparation of junctional terminal cisternae (JTC) from fast skeletal muscle of rabbit hind leg. The fraction differs from other heavy sarcoplasmic reticulum (SR) fractions in that it contains a substantial amount of junctional face membrane (JFM) (15-20% of the membrane) with morphologically well-defined junctional feet structures. In common with other heavy SR preparations, it contains predominantly the calcium pump membrane (80-85% of the membrane) and compartmental contents (CC), consisting mainly of calcium-binding protein (calsequestrin). In this study, a modified procedure for the preparation of JTC from frozen rabbit back muscle is described. The yield is substantially greater (threefold per weight of muscle), yet retaining characteristics similar to JTC from fresh hind leg muscles. Methodology has been developed for the disassembly of the JTC. This is achieved by selectively extracting the calcium pump membrane with 0.5% Triton X-100 in the presence of 1 mM CaCl2 to yield a complex of JFM with CC. The CC are then solubilized in the presence of EDTA to yield JFM. This fraction contains unidirectionally aligned junctional feet structures protruding from the cytoplasmic face of the membrane with repeat spacings comparable to that observed in JTC. The JFM contains 0.16 mumol phosphorus (lipid) per milligram protein. Characteristic proteins include 340 and 79-kD bands, a doublet at 28 kD, and a component that migrates somewhat slower than or equivalent to the calcium pump protein. Approximately 10% of the calcium-binding protein remains bound to the JFM after EDTA extraction, indicating the presence of a specific binding component in the JFM. The JFM, which is involved in junctional association with transverse tubule and likely in the Ca2+ release process in excitation-contraction coupling, is now available in the test tube.

Ultrastructural localization of calsequestrin in rat skeletal muscle by immunoferritin labeling of ultrathin frozen sections.

The ultrastructural localization of calsequestrin in rat skeletal muscle (gracilis) was determined by indirect immunoferritin labeling of ultrathin frozen sections. Calsequestrin was found in the lumen of transversely and longitudinally oriented terminal cisternae but was absent from most of the longitudinal sarcotubules and the fenestrated sarcoplasmic reticulum. Calsequestrin was occasionally observed in vesicular structures found in the central region of the I band. Since calsequestrin is believed to provide the major site of Ca2+ sequestration in the sarcoplasmic reticulum, the present results support the view that Ca2+, transported to the lumen of the sarcoplasmic reticulum, is preferentially sequestered in the terminal cisternae, but they also suggest that additional Ca2+ sequestration may occur near the center of the I band.

Assembly of the sarcoplasmic reticulum. Localization by immunofluorescence of sarcoplasmic reticulum proteins in differentiating rat skeletal muscle cell cultures.

Immunofluorescent staining techniques were used to study the distribution of the Ca(2) + Mg(2+)-dependent ATPase and calsequestrin in primary cultures of differentiating rat skeletal muscle cells, grown for different periods of time under various culture conditions. In mononucleated myoblasts calsequestrin was detected after 45 h in culture whereas the ATPase was not detected until 60 h. After cell fusion began, both proteins could be identified in all multinucleated cells. Myoblasts grown for longer than 60 h in low Ca(2+) medium contained calsequestrin and the ATPase, even though they were unable to fuse. These studies at the cellular level confirm biochemical findings on the biosynthesis of calsequestrin and the ATPase. Immunofluorescent staining of myoblasts showed that calsequestrin first appears in a well-defined region of the cell near one end of the nucleus. At later times, the staining occupied progressively larger regions adjacent to the nucleus and took on a fibrous appearance. This suggests that calsequestrin first accumulates in the Golgi region and then gradually spreads throughout the cell. In contrast, the ATPase appeared to be concentrated in many small patches or foci throughout the cytoplasm and was never confined to one particular region, although some parts of the cell often stained more intensely than others. In multinucleated cells, alternating dark and fluorescent strands parallel to the longitudinal axis of the cells were evident.

Immunocytochemistry of calciosomes in liver and pancreas.

Calciosomes are small cytoplasmic vacuoles identified in various nonmuscle cell types by their content of protein(s) similar to calsequestrin (CS), the Ca2+ storage protein of the muscle sarcoplasmic reticulum (SR). These entities have been interpreted as the "primitive" counterpart of the SR, and suggested to be the organelle target of inositol-1,4,5-triphosphate action (Volpe, P., K. H. Krause, S. Hashimoto, F. Zorzato, T. Pozzan, J. Meldolesi, and D. P. Lew. Proc. Natl. Acad. Sci. USA. 85:1091-1095). Immunoperoxidase and immunogold experiments carried out in both thick and ultrathin cryosections of rat hepatocytes and pancreatic acinar cells by using antimuscle CS antibodies revealed a specific labeling widely distributed in the entire cytoplasm, while nuclei were negative. Individual calciosomes appeared as small (105 nm) membrane-bound vacuoles intermingled with, and often apposed to ER cisternae and mitochondria. Other calciosomes were scattered in the Golgi area, in between zymogen granules and beneath the plasma membrane. The cumulative volume of the CS-positive organelles was measured to account for the 0.8 and 0.45% of the cytoplasm in liver and pancreas cells, respectively. The real total volume of the calciosome compartment is expected to be approximately twice as large. In hepatocytes, structures similar to CS-positive calciosomes were decorated by antibodies against the Ca2+ ATPase of muscle SR, while ER cisternae were not. By dual labeling, colocalization was revealed in 53.6% of the organelles, with 37.6% positive for the ATPase only. CS appeared preferentially confined to the content, and the Ca2+ ATPase to the contour of the organelle. The results suggested a partial segregation of the two antigens, reminiscent of their well-known segregation in muscle SR. Additional dual-label experiments demonstrated that hepatic calciosomes express neither two ER markers (cytochrome-P450 and NADH-cytochrome b5 reductase) nor the endolysosome marker, luminal acidity (revealed by 3-[2,4-dinitroanilino]-3'-amino-N-methyl dipropylamine). Calciosomes appear as unique cytological entities, ideally equipped to play a role in the rapid-scale control of the cytosolic-free Ca2+ in nonmuscle cells.

Inositol 1,4,5-trisphosphate receptor in heart: evidence for its concentration in Purkinje myocytes of the conduction system.

Inositol 1,4,5-trisphosphate (IP3) is one of the second messengers capable of releasing Ca2+ from sarcoplasmic reticulum/ER subcompartments. The mRNA encoding the intracellular IP3 receptor (Ca2+ channel) has been detected in low amounts in the heart of various species by Northern blot analysis. The myocardium, however, is a heterogeneous tissue composed of working myocytes and conduction system cells, i.e., myocytes specialized for the beat generation and stimulus propagation. In the present study, the cellular distribution of the heart IP3 receptor has been investigated. [3H]IP3 binding experiments, Western blot analysis and immunofluorescence, with anti-peptide antibodies specific for the IP3 receptor, indicated that the majority of Purkinje myocytes (the ventricular conduction system) express much higher IP3 receptor levels than atrial and ventricular myocardium. Heterogeneous distribution of IP3 receptor immunoreactivity was detected both at the cellular and subcellular levels. In situ hybridization to a riboprobe generated from the brain type 1 IP3 receptor cDNA, showed increased accumulation of IP3 receptor mRNA in the heart conduction system. Evidence for IP3-sensitive Ca2+ stores in Purkinje myocytes was obtained by double immunolabeling experiments for IP3 receptor and cardiac calsequestrin, the sarcoplasmic reticulum intralumenal calcium binding protein. The present findings provide a molecular basis for the hypothesis that Ca2+ release from IP3-sensitive Ca2+ stores evoked by alpha 1-adrenergic stimulation is responsible for the increase in automaticity of Purkinje myocytes (del Balzo, U., M. R. Rosen, G. Malfatto, L. M. Kaplan, and S. F. Steinberg. 1990. Circ. Res. 67:1535-1551), and open new perspectives in the hormonal modulation of chronotropism, and generation of arrhythmias.

Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy.

A simple method is described for high-resolution light and electron microscopic immunolocalization of proteins in cells and tissues by immunofluorescence and subsequent photooxidation of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is achieved in the photooxidation process over other conventional fluorescent compounds. The technique allows for precise correlative immunolocalization studies on the same sample using fluorescence, transmitted light and electron microscopy. Furthermore, because eosin is smaller in size than other conventional markers, this method results in improved penetration of labeling reagents compared to gold or enzyme based procedures. The improved penetration allows for three-dimensional immunolocalization using high voltage electron microscopy. Fluorescence photooxidation can also be used for high resolution light and electron microscopic localization of specific nucleic acid sequences by in situ hybridization utilizing biotinylated probes followed by an eosin-streptavidin conjugate.

Characterization of the sarcoplasmic reticulum proteins in the thermogenic muscles of fish.

Marlins, sailfish, spearfishes, and swordfish have extraocular muscles that are modified into thermogenic organs beneath the brain. The modified muscle cells, called heater cells, lack organized myofibrils and are densely packed with sarcoplasmic reticulum (SR), transverse (T) tubules, and mitochondria. Thermogenesis in the modified extraocular muscle fibers is hypothesized to be associated with increased energy turnover due to Ca2+ cycling at the SR. In this study, the proteins associated with sequestering and releasing Ca2+ from the SR (ryanodine receptor, Ca2+ ATPase, calsequestrin) of striated muscle cells were characterized in the heater SR using immunoblot and immunofluorescent techniques. Immunoblot analysis with a monoclonal antibody that recognizes both isoforms of nonmammalian RYRs indicates that the fish heater cells express only the alpha RYR isoform. The calcium dependency of [3H]ryanodine binding to the RYR isoform expressed in heater indicates functional identity with the non-mammalian alpha RYR isoform. Fluorescent labeling demonstrates that the RYR is localized in an anastomosing network throughout the heater cell cytoplasm. Measurements of oxalate supported 45Ca2+ uptake, Ca2+ ATPase activity, and [32P]phosphoenzyme formation demonstrate that the SR contains a high capacity for Ca2+ uptake via an ATP dependent enzyme. Immunoblot analysis of calsequestrin revealed a significant amount of the Ca2+ binding protein in the heater cell SR. The present study provides the first direct evidence that the heater SR system contains the proteins necessary for Ca2+ release, re-uptake and sequestration, thus supporting the hypothesis that thermogenesis in the modified muscle cells is achieved via an ATP-dependent cycling of Ca2+ between the SR and cytosolic compartments.

Proteomic profiling of x-linked muscular dystrophy.

Progressive x-linked muscular dystrophy represents the most commonly inherited neuromuscular disorder in humans. Although the disintegration of the dystrophin-associated glycoprotein complex triggers the initial pathogenesis of Duchenne muscular dystrophy, secondary alterations in metabolic pathways, cellular signaling and the regulation of ion homeostasis are probably crucial factors that cause end-stage fibre degeneration. The application of mass spectrometry-based proteomics for the global cataloguing of muscle biomarkers has recently been applied to the analysis of the mdx animal model of muscular dystrophy and the biochemical evaluation of experimental exon skipping therapy. The fluorescence difference in-gel electrophoretic analysis of normal versus mdx diaphragm muscle revealed changed expression levels of proteins involved in nucleotide metabolism, Ca 2+-handling, the cellular stress response and key bioenergetic processes. The swift up-regulation of small heat shock proteins, such as cvHsp, seems to form an integral part of the repair mechanisms in dystrophic fibres and may be exploitable as a new option to treat inherited muscle degeneration. Importantly, the mass spectrometry-based profiling of mdx muscle following the specific removal of exon 23 in the mutated dystrophin gene transcript showed a partial reversal of important secondary changes. Experimental exon skipping restored the expression of the dystrophin isoform Dp427, its associated glycoprotein beta-dystroglycan, neuronal nitric oxide synthase, calsequestrin, adenylate kinase and the muscle-specific stress protein cvHsp. In the future, a well defined set of signature molecules could be used to improve diagnosis, monitor disease progression, identify new therapeutic pathways, and validate the effects of novel drugs or experimental treatments such as gene therapy.

Human SERCA2a levels correlate inversely with age in senescent human myocardium.

OBJECTIVES: This study sought to characterize functional impairment after simulated ischemia-reperfusion (I/R) or Ca2+ bolus in senescent human myocardium and to determine if age-related alterations in myocardial concentrations of SERCA2a, phospholamban, or calsequestrin participate in senescent myocardial dysfunction. BACKGROUND: Candidates for elective cardiac interventions are aging, and an association between age and impairment of relaxation has been reported in experimental animals. Function of the sarcoplasmic reticulum resulting in diastolic dysfunction could be dysregulated at the level of cytosolic Ca2+ uptake by SERCA2a, its inhibitory subunit (phospholamban), or at the level of Ca2+ binding by calsequestrin. METHODS: Human atrial trabeculae from 17 patients (45-75 years old) were suspended in organ baths, field simulated at 1 Hz, and force development was recorded during I/R (45/120 min). Trabeculae from an additional 12 patients (53-73 years old) were exposed to Ca2+ bolus (2-3 mmol/L bath concentration). Maximum +/- dF/dt and the time constant of force decay (tau) were measured before and after I/R or Ca2+ bolus and related to age. SERCA2a, phospholamban, and calsequestrin from 12 patients (39-77 years old) were assessed by immunoblot. RESULTS: Functional results indicated that maximum +/-dF/dt and tau were prolonged in senescent (>60 years) human myocardium after I/R (p < 0.05). Calcium bolus increased the maximum +/-dF/dt and decreased tau in younger, but not older patients (p < 0.05). SERCA2a and the ratio of SERCA2a to either phospholamban or calsequestrin were decreased in senescent human myocardium (p < 0.05). CONCLUSIONS: Senescent human myocardium exhibits decreased myocardial SERCA2a content with age, which may, in part, explain impaired myocardial function after either I/R or Ca2+ exposure.

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.

Hypertrophy and functional alterations in hyperdynamic phospholamban-knockout mouse hearts under chronic aortic stenosis.

OBJECTIVE: To determine whether the hyperdynamic phospholamban-knockout hearts are capable of withstanding a chronic aortic stenosis. METHODS: The transverse section of the aorta was banded in phospholamban-knockout and their isogenic wild-type mice, which were followed with echocardiography in parallel, along with sham-operated mice, before and at 2.5, 5 and 10 weeks after surgery. RESULTS: Cardiac decompensation was evidenced by the presence of lung congestion in some banded knockouts and wild-types, giving rise to a subset of non-failing and failing hearts within each group. The incidence of heart failure was not genotype-dependent but rather associated with higher heart rates before surgery. The development of left ventricular hypertrophy was similar between knockouts and wild-types and longitudinal assessment of end-diastolic dimension indicated progressive increases after banding, with a greater dilation in failing mice. Fractional shortening was reduced in failing knockouts and wild-types to a similar degree, with an earlier onset in the knockouts. In addition, fractional shortening was decreased in non-failing knockouts but not wild-types. Ejection times shortened after aortic banding particularly for failing hearts. Assessment of the SR Ca(2+)-ATPase protein levels indicated similar downregulation for failing knockouts and wild-types, while the phospholamban levels were not significantly altered in wild-types. CONCLUSION: The hyperdynamic phospholamban-knockout hearts are able to compensate against a sustained aortic stenosis similar to wild-types.

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.

Expression of calcium regulatory proteins in short-term hibernation and stunning in the in situ porcine heart.

BACKGROUND: Myocardial hibernation and stunning are characterised by a reversible contractile dysfunction during and after ischaemia, respectively. Calcium homeostasis might be disturbed in hibernation and stunning due to altered expression of cardiac proteins involved in calcium handling. METHODS: In enflurane-anaesthetised swine the coronary blood flow through the left anterior descending coronary artery was decreased to reduce regional contractile function (microsonometry) by approximately 50%. In transmural biopsies obtained during ischaemia and reperfusion creatine phosphate as well as the expression of sarcoplasmic reticulum calcium ATPase (SERCA), phospholamban (PLB), calsequestrin (CSQ), and troponin inhibitor (TnI) were determined. RESULTS: During ischaemia creatine phosphate, after an initial reduction, recovered back to control values, and necrosis was absent (hibernation). After 90 min of ischaemia the myocardium was reperfused for 120 min but regional contractile function continued to be depressed (stunning). PLB, SERCA, CSQ, and TnI proteins were unchanged during ischaemia as well as reperfusion. Likewise, levels of PLB and SERCA mRNAs were unchanged. CONCLUSION: It is concluded that other mechanisms than altered expression of these regulating proteins underlie the contractile dysfunction observed during acute ischaemia, short-term hibernation and stunning.

Heterogeneous transmural gene expression of calcium-handling proteins and natriuretic peptides in the failing human heart.

OBJECTIVE: Human heart failure is associated with a disturbed intracellular calcium (Ca2+) homeostasis. In this regard, ventricular wall stress is considered to be a determinant for expression of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a). In the present study, we analyzed the transmural protein and/or mRNA levels of SERCA2a, other Ca(2+)-handling proteins, and of atrial and brain natriuretic peptides (ANP and BNP) in the human heart. METHODS: Subepicardial (epi), midmyocardial (mid), and subendocardial (endo) sections of the left ventricular free wall from end-stage failing (n = 17) and nonfailing (n = 5) human hearts were analyzed by Western blot for immunoreactive protein levels of SERCA2a, phospholamban (PLN), and calsequestrin (CS). Subepi- and subendocardial sections were analyzed by Northern blot for steady-state mRNA levels of SERCA2a, Na(+)-Ca2+ exchanger (NCX1), ANP, and BNP. RESULTS: SERCA2a protein and mRNA levels were reduced by 40 +/- 5% (P < 0.01) and 25 +/- 7% (P < 0.05) in endo compared to epi in the failing heart and by 27 +/- 14% and 16 +/- 12% (non-significant) in the nonfailing heart, respectively. PLN protein levels were reduced by 23 +/- 6% (P < 0.05) in endo compared to epi in the failing heart and by 17 +/- 25% (non-significant) in the nonfailing heart, whereas CS protein levels and NCX1 mRNA levels were similar across the left ventricular wall. Strikingly, in the failing heart, both BNP and ANP mRNA levels were upregulated predominantly in endo. CONCLUSIONS: In the failing human heart, SERCA2a and PLN, as well as natriuretic peptides but not CS and NCX1 are differentially expressed across the left ventricular wall, implicating (1) different susceptibility of subendocardium and subepicardium to factors affecting expression of these proteins and (2) differences in regulation of the distinct calcium-cycling proteins.

Expression of Ca2+ binding proteins of the sarcoplasmic reticulum of striated muscle in the endoplasmic reticulum of pig smooth muscles.

The Ca2+ binding proteins in the lumen of intracellular Ca2+ stores differ between muscle and non-muscle cells, indicating a specific role of these proteins in intracellular Ca2+ regulation. Since smooth muscle cells possess both muscle and non-muscle characteristics, we have studied the presence and the differential expression of the muscle-type Ca2+ binding proteins--calsequestrin, sarcalumenin, and the histidine-rich Ca2+ binding protein (HCP)--in several smooth muscle tissues from the pig. Western blot analysis showed that among the smooth muscles studied, the cardiac isoform of calsequestrin is expressed at the highest levels in the stomach. Calsequestrin was present at lower levels in ileum and trachea, whereas this protein was undetectable in aorta and main pulmonary artery. The total amount of calsequestrin in the stomach was estimated to be 20-30-times lower than in the pig heart. Whereas calsequestrin from pig presented the same apparent M(r) in sodium dodecyl sulphate polyacrylamide gels as the well characterized protein from rabbit, the apparent M(r) of both sarcalumenin and HCP was lower in pig than in rabbit. The presence of HCP was demonstrated in pig stomach and ileum, while sarcalumenin was detected only in the stomach. These results demonstrate further biochemical differences between smooth muscle cells of large blood vessels and those of the digestive tract. The present findings on the differential distribution of muscle-type Ca2+ binding proteins are discussed in relation to biochemical and functional differences between these smooth muscle cells.

Evidence for a calsequestrin-like calcium-binding protein in human spermatozoa.

Human spermatozoa were investigated for the presence of protein(s) recognized by antibodies against calsequestrin, the high capacity, moderate affinity Ca2(+)-binding protein, originally described in striated muscle fibers. Western immunoblots of detergent-soluble sperm extracts probed with polyclonal antibodies raised against human skeletal muscle calsequestrin identified a strongly cross-reactive protein. This protein resembles muscle calsequestrin in many respects. In fact, its migration in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is pH dependent, its apparent molecular mass being 64 kDa in alkaline SDS-PAGE and 44 kDa in neutral SDS-PAGE; its isoelectric point is acidic (4.6); it is metachromatically stained blue by the carboxycyanine dye, Stains-All; it is a Ca2(+)-binding protein (45Ca blot overlay). Indirect immunofluorescence experiments showed that the immunoreactive protein has an intracellular localization confined to the tail mid-piece. From these findings we conclude that human sperm cells express a protein structurally and antigenically related to skeletal muscle calsequestrin; a basis for a novel interpretation of Ca2(+)-mediated events in spermatozoa is thus provided.

Differential distribution of calcium stores in paramecium cells. Occurrence of a subplasmalemmal store with a calsequestrin-like protein.

We have analyzed in Paramecium cells the occurrence and intracellular distribution of the high capacity/low affinity calcium-binding proteins, calsequestrin (CS) and calreticulin (CR) using antibodies against CS from rat skeletal muscle and against CR from rat liver, respectively. As revealed by Western blots, a CS-like protein isolated by affinity chromatography from Paramecium cells comigrated with CS isolated from rat skeletal muscle. The immunoreactivity of this 53 kDa protein band was blocked when the antibodies had been preadsorbed with purified rat CS. A band of identical molecular size was shown to bind 45Ca in overlays. By immunofluorescence and immunogold labeling this CS-like protein was localized selectively to the extended subplasmalemmal calcium stores, the "alveolar sacs", which cover almost the entire cell surface. Concomitantly the 53 kDa 45Ca-binding band became increasingly intense in overlays as we increasingly enriched alveolar sacs. Antibodies against rat CR react with a 61 kDa band but do not cross-react with CS-like protein in Paramecium. These antibodies selectively stained intracellular reticular structures, identified bona fide as endoplasmic reticulum.