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Cardiovascular Research 2004 63(2):264-272; doi:10.1016/j.cardiores.2004.04.001
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Copyright © 2004, European Society of Cardiology

Defective glycosylation of calsequestrin in heart failure

Arash Kiarasha, Carmen E Kellya, Brett S Phinneyb, Héctor H Valdiviac, Judith Abramsd and Steven E Cala*,a

aProgram in Molecular and Cellular Cardiology, Cardiology Research Division, Department of Medicine, Wayne State University, Elliman Building, Room 1107, 421 E. Canfield Avenue, Detroit, MI 48201, USA
bMichigan Proteome Consortium, Department of Biochemistry, 3B Biochemistry, Wilson Road, East Lansing, MI 48824, USA
cDepartment of Physiology, University of Wisconsin-Madison, 1300 University Ave., Madison, WI 53706, USA
dBarbara Ann Karmanos Cancer Institute, Wayne State University, 716 Harper Professional Office Building, Detroit, MI 48201, USA

* Corresponding author. Tel.: +1-313-5778734; fax: +1-313-5778615. Email address: s.cala{at}wayne.edu

Received 20 January 2004; revised 15 March 2004; accepted 3 April 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
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.

KEYWORDS Calsequestrin; Mass spectrometry; Heart failure; Phosphorylation; CK2; SR


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Calsequestrin (CSQ) is concentrated in junctional sarcoplasmic reticulum (SR), along with a limited set of proteins that include the ryanodine receptor, triadin-1 and junctin [1–5], where it functions in excitation–contraction coupling and Ca2+ release [6–9]. Although the structure and function of CSQ are actively studied, relatively little is known about its intracellular trafficking or the distinct subcellular compartment (terminal cisternae) in which it concentrates. Early studies established that SR was divided into at least two major compartments, known as free SR and junctional SR [10]. Free SR is likely to be the functional equivalent of nonmuscle ER in that it contains the ubiquitous SR/ER Ca2+ pump [11], along with the various ER stress proteins calreticulin [12,13], calnexin [14,15], GRP78 [14,15] and GRP94 [16]. Junctional SR, on the other hand, represents a subcompartment of the ER/SR membrane system, and biogenesis of the junctional SR compartment is thought to involve distinct mechanisms [17–19]. Junctional SR appears to bud from the free SR and form terminal cisternae, which are tubular membrane compartments attached to transverse tubules of the sarcolemma (SL) [10,20–22].

Precise determination of CSQ mass using electrospray mass spectrometry reveals a series of molecular mass isoforms representing differences in CSQ glycosylation and phosphorylation. The pattern of molecular forms is characteristic of heart tissue and of the cardiac isoform, with roughly a dozen or more forms present in hearts of many species [23]. In muscle and nonmuscle cells alike, cardiac CSQ is glycosylated on 316Asn co-translationally, then undergoes mannose trimming by a series of mannosidases localized within intracellular compartments [23]. In canine heart, mannose trimming of CSQ leads primarily to glycan structures with Man1–4 (Man1–4GlcNAc2), with a smaller number of CSQ molecules having glycans of structure Man5–7. In contrast to muscle cells, HEK 293 cells and other nonmuscle cells retain CSQ in proximal ER compartments, and the predominant form of the CSQ glycan is Man8,9, indicative of the actions of ER mannosidase I only [23]. Additional cellular mannosidases decrease the mass of the carbohydrate ({Delta}mass=162 Da) as it transits through the secretory pathway. In addition to differences in CSQ mannose content, the mass spectrum of CSQ reveals multiple states of phosphorylation ({Delta}mass=81 Da) due to occupation of CK2 (formerly casein kinase II)-sensitive serine residues at the C-terminus. CSQ exhibits any of four possible states (0–3 phosphates on 378,382,386Ser) [23,24]. Studies in several cell types have shown that post-translational processing of CSQ is responsive to factors that effect cell metabolism, leading us to believe that processing of CSQ might be altered in heart failure (HF), or other diseased states.

In this study, we investigated whether CSQ processing might be altered in a model of HF by examining tissue from sham-operated (Control) and rapid-paced (HF) dogs. We report that, in spite of the fact that levels of total CSQ in HF remain unchanged, levels of individual glycoforms differ greatly in HF, indicating a very different co-localization with cellular mannosidases, suggesting that protein or membrane trafficking for junctional SR in HF is defective.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Pacing-induced HF
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Dilated cardiomyopathy was induced by rapid ventricular pacing at 220–250 beats/min for 4–6 weeks in mongrel beagles [25]. HF was reproducibly present in tachycardia-paced dogs as confirmed by hemodynamic measurements at the time of killing (control vs. DCM: dP/dtmax 1520±405 vs. 680±123 mm Hg/s; dP/dtmin –1193±347 vs. –790±273 mm Hg/s; LVEDP 5.7±2.5 vs. 19±9.9 mm Hg). Samples of left ventricle were stored at –80 °C and thawed prior to tissue homogenization. A total of five sham-operated control dogs and five tachycardia-paced dogs were used in these studies. Frozen neonatal beagle hearts were purchased from Marshall Farms, North Rose, NY.

2.2. Purification of cardiac CSQ from heart tissue
Purification and desalting of CSQ from canine left ventricular tissue (~5 g) was carried out as previously described [23]. SDS-PAGE was carried out according to Laemmli [26] and protein assays carried out using a Lowry protocol [27].

2.3. Mass spectrometry
Electrospray ionization mass spectrometry (ESMS) was carried out at the Proteome Consortium, Michigan State University, East Lansing, MI using a Q-ToF mass spectrometer (Micromass, Altrincham, UK), as previously described [23]. Protein samples were re-suspended or diluted in 20% acetonitrile and 10% formic acid. The proteins were trapped on a Michrom (Auburn, CA) protein cap trap using a Waters (Milford, MA) CapLC. Trapped proteins were eluted into a Waters Q-Tof Ultima mass spectrometer and their masses deconvoluted using the MaxEnt1 algorithm.

2.4. Statistical methods
Graphs were used to assess the distributional assumptions of the statistical tests; there was no evidence asymmetry or outlying values. Hotellings T2 test, the multivariate equivalent of the two-sample t-test, was used to simultaneously compare the proportion of Man3–4, Man5–7 and Man8–9 glycoforms between the group of animals with induced HF and the control group. Since the sum of the three proportions is unity, the resulting colinearity would have caused computational problems if all three proportions were included in the analysis; only two were needed to test the hypothesis that the groups differ in the proportion of glycoforms in each of the three mannose groups. A two-sample t-test was used to evaluate difference in total CSQ between groups.

2.5. Endoglycosidase H (endo H) treatments
Endo H (Roche Biochemicals) treatment to remove N-linked glycans [28] was carried out by adding 1 mU endo H to 20 µg purified canine cardiac CSQ, in a final concentration of 5 mM MOPS buffer, pH 7.4.

2.6. Concanavalin A (Con A) binding
Con A binding was carried out by the method of Clegg [29]. CSQ samples were run on SDS-gels and transferred to nitrocellulose, blocked in PBS with 0.2% Tween 20 (PBSt) containing 0.5 mg/ml BSA and 0.1% Triton X-100, then incubated in the same buffer without BSA but with 25 µg/ml Con A peroxidase complex (Sigma) for 30 min, ambient temperature. The nitrocellulose was then washed in PBSt containing 0.1% Triton X-100 and 0.5 M NaCl for 10 min, then replaced with PBSt. Con A-peroxidase was detected with enhanced chemiluminescence (ECL, Amersham) and autoradiography.

2.7. Immunoblotting
Immunoblotting was carried out as previously described [30] using antibodies against purified canine CSQ and purified using CSQ bound to nitrocellulose [31]. For determination of homogenate protein concentration, homogenates were brought to 1 mg/ml in 1% SDS and fully dissolved using 70 °C heating and sonication, and assayed according to Lowry et al. [27] with 0.5% SDS added. Immunoreactivity was detected by [125I]protein A binding [32] and autoradiography, and quantified by gamma counting of excised bands.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. ESMS of CSQ from C and HF dogs
CSQ was purified from C and HF samples and analyzed by ESMS. As previously reported for dog heart tissue [23], CSQ was comprised of a variety of molecular forms representing several post-translationally modified phosphoforms and glycoforms (Fig. 1, upper panel). CSQ protein molecules differed in mass either by 162 Da, reflecting the loss (trimming) of mannose sugars from the N-linked oligosaccharide, or by 81 Da corresponding to differences in phosphate content. Because up to three phosphates can exist on CSQ, there are potentially four mass peaks (0, 1, 2, 3) per glycoform. Because a change of two phosphates yields the same mass change as trimming of one mannose (2 x 81 Da=162 Da), these two post-translational modifications combine to generate a series of mass peaks that differ by 81 Da.


Figure 1
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  		Fig. 1 Mass spectroscopy reveals differences in processing of CSQ from Control and Heart Failure dog hearts. Predicted masses for canine cardiac CSQ containing the oligosaccharides Man3–9GlcNAc2 are shown ({Delta}mass=162 Da) and mannose number indicated (top of figure). Mass intervals {Delta}mass=81 Da correspond to differences due to one phosphate. Different shading highlights glycoforms characteristic of early (Man8,9) or more distal (Man3–7) ER compartments. CSQ from neonatal dog hearts yields a pattern similar to adult C heart. Molecular structures for the two extremes of mass are shown (inset): mannose (circles), N-acetyl glucosamine (squares).

 
When CSQ from HF tissue was analyzed, the mass spectrum showed a dramatic shift towards higher mass in HF, representing significant differences in its post-translational processing (Fig. 1, middle panel). The major glycoforms present in C tissue were of mass 46,406–45,568 Da, whereas in HF CSQ masses were spread out over a wider mass range with only a minor percentage present in the same form as that found in C hearts. Mass spectra for C (N=5) and HF (N=5) samples were remarkably reproducible, as was the shift in molecular mass isoforms due to HF (Fig. 2). The most abundant mass peak in C hearts (Fig 1, upper panel, mass=46,568) contains CSQ molecules with glycan of Man5 (with a single phosphate) and Man4 (with three phosphates), which exhibit the same mass as explained above. However, molecules with three and two phosphates greatly outnumber those with none and one phosphate (as discussed below), so that spectra can be greatly simplified by assuming that most CSQ molecules are present primarily as doubly and triply phosphorylated forms (italicized numbers atop Fig. 3A).


Figure 2
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  		Fig. 2 Complete set of mass spectra from C dogs (N=5) and HF dogs (N=5). Altered CSQ processing is evident as visually distinct patterns of post-translational modification which occur reproducibly. The mass scale is indicated on the abscissa. Mass peaks are shown on a scale in which the most abundant mass peak was assigned a value of 100%.

 

Figure 3
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  		Fig. 3 (A) Summary of mass spectra for C and HF dog heart CSQ. Molecular forms of CSQ occurring at 81-Da intervals (glycoforms and phosphoforms) were expressed as a percentage of the total of all forms and averaged for the five C or five HF dogs shown in Fig. 2. Predicted mass values for mannose numbers Man3–9 (no phosphate) are indicated between the two histogram panels. Molecules containing these glycans, however, generally contain either two or three phosphates on CK2-sensitive sites (see Fig. 5). Thus, for example, the four highest mass peaks (81-Da intervals) represent molecules with following post-translational modifications (beginning from the right): Man9 and three phosphates, Man9 and two phosphates, Man8 with three phosphates, and Man8 and two phosphates (see italicized mannose and phosphate values at top). This assumption may not hold for the most highly trimmed CSQ glycans; for example, for Man3 forms of the glycan [47]. (B) The set of CSQ glycoforms can be divided into three components, those characteristic of normal junctional SR (Man3,4), those which are underprocessed but have moved beyond the realm of ER mannosidase I (Man5–7), and those characteristic of ER localization (Man8,9). For each component, levels were calculated from the sums of the average values and standard errors are shown. Overall, there was a statistically significant difference in the distribution of glycoforms between animals with induced HF and control animals (p=0.002). There was a greater proportion of Man3–4 in the control animals than in those with induced HF (p=0.001), while the proportion of Man5–6 (p=0.01) and Man7–8 (p=0.004) was greater in animals with induced HF than in control animals. In contrast to differences observed in CSQ glycoforms, total levels of CSQ in C and HF heart samples determined by immunoblotting were not statistically significant (p=0.56).

 
A summary presentation of the spectra for C and HF samples using average values for all animals confirmed the existence of two distinct and significantly different (p=0.002) patterns of CSQ processing (Fig. 3). Differences between C and HF were determined for each of three glycoform components based upon their presumed cell biological significance (Fig. 3B). CSQ glycans that were maximally trimmed by cellular mannosidases (Man3,4) were reduced by 80% (p=0.001). Meanwhile, processing of the CSQ glycan to Man5–7 and Man8,9 were also significantly different (p=0.01 and p=0.004, respectively), perhaps consistent with CSQ transit through distinct subcellular compartments. Man8,9 forms are characteristic of ER localization and actions of the ER mannosidase I [23,33], whereas Man5–7 forms are characteristic of post-ER trimming but are not glycoforms typical of junctional SR localization. These differences in glycoform components were in contrast to a lack of statistically significant difference (p=0.56) seen in total protein (Fig. 3B, leftmost histogram), consistent with previous findings in rapid-paced dogs [25,34].

To determine whether the observed changes in CSQ processing might reflect emergence of a neonatal phenotype, CSQ was purified from hearts of four neonatal dogs and analyzed by ESMS. The results (Fig. 1, lower panel) showed a processing pattern in canine neonatal heart tissue more similar to that of C dogs, indicating that the processing observed in HF dogs represented a phenotype that was distinct from that of adult and neonate. The neonatal heart tissue was derived from beagles, the same as was used for adult studies, assuring that differences in neonate were not originated from differences in breed.

3.2. Further evidence of altered glycan processing using standard biochemical analyses
Based upon the differences in CSQ glycan structure and mass found in HF, we predicted that the higher mannose content in HF CSQ might be evident in its binding to the lectin Con A. Indeed, CSQ from HF dogs showed enhanced binding to the lectin Con A (Fig. 4). Con A is known to bind poorly to standard (normal) preparations of cardiac CSQ [6,35], and the greatly enhanced Con A binding to CSQ from HF tissue may serve as a convenient method of predicting defective targeting characteristic of the disease. Only minor differences in mobility and appearance on SDS-gels were seen for C and HF CSQ samples, despite the several hundred dalton differences in average mass (compare Coomassie blue-stained bands in upper panel).


Figure 4
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  		Fig. 4 Differences in CSQ glycan structure between C and HF can be detected by Con A binding. Two micrograms of C and HF CSQ samples were electrophoresed and transferred to nitrocellulose. Con A (coupled to peroxidase) was incubated with the nitrocellulose, and bound ConA-horse radish peroxidase was visualized by ECL and autoradiography. This difference in Con A binding was observed for each of at least three sets of samples tested.

 
3.3. CSQ phosphorylation in HF
To assess the relative amounts of phosphate on CSQ molecules for C and HF samples, we treated samples with endo H so that mass differences were due only to endogenous phosphate. Due to the small amounts of tissue samples available, as well as the loss of ESMS data resulting from excessive processing of samples, we were unable to obtain these data for more than a few of the samples used in this study. Nevertheless, the data did indicate that CSQ exists predominantly as doubly and triply phosphorylated molecules in both C and HF samples (Fig. 5, panel A). In the two samples shown, there was a clear relative abundance in triply, compared to doubly, phosphorylated molecules, and the relative amount of maximally phosphorylated molecules was greater in the HF sample. To analyze the extent of phosphorylation as a function of the CSQ glycoform for the complete set of data in Fig. 3, we assumed that all CSQ molecules contained either three or two phosphates, permitting assignment of all peaks in the mass spectra to a single molecular form, as indicated at the top of Fig. 3. The degree of maximal phosphorylation was calculated as the ratio of the fraction of molecules with 3 phosphates compared with 2 phosphates for each glycoform for all 10 animals examined in this study. For example, for Man9 glycoforms, we compared the highest two mass peaks that appear in Fig. 3A, which yields a 215% increase in triply phosphorylated Man9 over doubly phosphorylated glycoform.


Figure 5
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  		Fig. 5 (A) Treatment with Endo H generated four CSQ mass peaks which differed by 81 Da, representing CSQ molecules with none, one, two or three phosphates, for CSQ from either C or HF samples of CSQ. The actual masses of these molecules indicated that the glycan was reduced to –GlcNAc1, as predicted for endo H cleavage [28], which adds 202 Da to the CSQ backbone of 44,212 Da. CSQ from whole heart exists primarily as doubly and triply phosphorylated molecules (>90% for both sample), with only a minor percentage of the molecules present as nonphosphorylated and singly phosphorylated. These data, obtained for a single heart C and HF sample, show the cumulative phosphorylation states of CSQ for all glycoforms. The extent of phosphorylation is greater here for the HF sample, as gauged by the percent increase in triply compared to doubly phosphorylated molecules. (B) To determine the extent of phosphorylation as a function of glycoform for all C (open circles) and HF (closed circles) samples, the ratio of the fraction of molecules having all three sites phosphorylated compared to molecules having two sites was determined for each glycoform in the two groups. This calculation assumes that all molecules have either two or three sites phosphorylated, an assumption supported by the results of endo H treatment (panel A above). For example, for Man9, the rightmost two values in Fig. 3A were used for this calculation. Each CSQ glycoform was more highly phosphorylated in HF compared to C hearts, except for Man9.

 
The full analysis for all glycoforms produced a striking relationship between glycosylation and phosphorylation, and a marked difference between CSQ molecules found in C versus HF hearts (Fig. 5, panel B). Virtually, all CSQ molecules in both sets of canine hearts showed increases in the triply phosphorylated compared to doubly phosphorylated forms, with the range of increase between 35% and 215%. There were clear differences in this maximal phosphorylation between C and HF, indicating an increase in maximal phosphorylation of CSQ of approximately two-fold in all but the Man9 form of the protein. Indeed, the highest amount of triply phosphorylated molecules occurred for molecules with an Man9 glycan consistent with our hypothesis that phosphorylation occurs in ER compartments. Subsequent glycoforms showed increasingly lower levels of phosphate. For Man3–4, maximal phosphorylation appeared to turn upward again; however, this facet was probably the result of even higher dephosphorylation in distal compartments. Thus, for example, an increase in levels of Man4 with only one phosphate would artificially elevate the apparent levels of Man3 with three phosphates. Maximal phosphorylation, therefore, probably diminishes as a function of increased mannose trimming, a process uniformly diminished in HF.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The N-linked oligosaccharide of a properly folded protein within the secretory pathway begins as Man9GlcNAc2 and is modified by intracellular mannosidases during subsequent protein transit through the secretory pathway [33,36–38]. Changes in the mannose content of N-linked glycans is widely associated with changes in trafficking through distinct ER and Golgi compartments [33,36–38]. In this report, we describe changes in CSQ glycan structure and phosphorylation in a well-studied animal model of HF. These data stand in contrast to previous analyses of total CSQ content which generally show no change [25,34].

N-linked glycosylation of proteins is believed to serve an important function in post-translational folding. In particular, mannose trimming from Man9 to Man8 by ER mannosidase I is an early event in quality control mechanisms that involves the lectins calreticulin and calnexin [33,39–44]. Mannose trimming beyond Man8 is less well characterized but is thought to involve post-ER mannosidases, enzymes, which may vary in amount and subcellular compartmentalization depending upon cell type [33,38]. Trimming of N-linked glycans to Man1, Man3 and Man4, the predominant glycoforms for both cardiac and fast-twitch skeletal muscle CSQ isoforms, is a relatively poorly studied event, and the enzymes responsible for this degree of processing are unknown as well. The near complete disappearance of these glycoforms in HF is particularly interesting since these are the glycoforms most specific to muscle cell processing of CSQ [23]. Few details of CSQ targeting are known, and no clear targeting signals have been clearly elucidated [45,46]. Data from our laboratory are consistent with movement of CSQ directly to junctional SR from ER compartments, in that terminal GlcNAc residues characteristic of Golgi transit are not present in the CSQ glycan [23].

It is interesting that the extent of CSQ phosphorylation was greater in HF for every glycoform except Man9, the glycan structure characteristic of early ER compartments. These data are consistent with a steadily decreasing level of phosphorylation as CSQ moves into distal compartments. In dog heart tissue, CSQ containing higher mannose content glycans often exist uniquely in the triply phosphorylated form, consistent with CSQ kinase acting within early ER compartments [47]. Hyperphosphorylation of CSQ in HF may increase ER retention and concentration in a different subcellular compartment, leading to a decrease in trafficking to more distal ER/SR compartments. Consistent with this view is our previous findings that CSQ glycans undergo greater mannose trimming when the phosphorylation-deficient mutant S(378,382,386)A was overexpressed in nonmuscle cells [23]. A second possibility might be that dephosphorylation is reduced in HF. We have suggested that phosphorylation occurs in ER compartments of heart, while transit towards junctional SR is marked by dephosphorylation, and a potent CSQ phosphatase is present in cardiac microsomes [47]. Differences in CSQ trafficking in HF could result from a lower phosphatase activity. Clearly, the question of how altered trafficking and altered phosphorylation in HF tissue are related will require further studies.

Within each group of animals (C or HF), post-translational processing of CSQ was remarkably similar given biological variability and the complexity of intracellular trafficking (Fig. 2). This may in part be due to the use of a smaller dogs (beagles) than were used in our previous study [23]. Nonetheless, despite small variations in the CSQ mass spectra, the differences between C and HF dogs were highly distinctive and reproducible. At a cell biological level, the similar distribution of CSQ glycoforms may reflect a tightly controlled process of intracellular trafficking. One interpretation of our findings is that in HF, CSQ and the mannosidase(s) responsible for the Man5–7->Man1–4 processing step no longer co-localize. Among a number of possible causes, this could indicate (1) a change in the level of a CSQ targeting protein(s), (2) a change in CSQ targeting signals (e.g. phosphorylation) or (3) a change in the overall biogenesis of the junctional SR membrane compartment. Models for the observed changes are schematically illustrated in Fig. 6. The effects of changes in the architecture of Ca release sites could alter basic properties of Ca homeostasis, an aspect of Ca homeostasis that would be difficult to quantify by modeling of unaltered sarcomeric structure. Changes in CSQ glycan structure from ESMS analysis of heart homogenates is capable of visualizing subtle defects in CSQ structure that may reflect protein or membrane trafficking which are not evident from analyses of total SR protein levels or from standard morphological examinations. While it remains to be determined whether such changes can lead to functional alterations in cardiac cell function, altered membrane trafficking might affect the ability to segregate a normal repertoire of proteins, possibly accounting for the changes in SR protein content that are widely reported [48,49].


Figure 6
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  		Fig. 6 Model of altered post-translational processing of CSQ in canine HF. Compared to normal targeting of CSQ to normal junctional SR compartments, CSQ in HF may itself not be targeted properly to junctional SR (middle), or junctional SR may not be properly formed (right).

 
Cardiomyopathies leading to congestive HF are thought to begin as a myocardial defect that diminishes contractility [48,50]. The search for molecular defects that lead to cardiomyopathies and subsequent HF has focused on the Ca2+ handling proteins of the SR and SL, with one common approach being a comparison of the steady-state levels in normal and diseased tissues by immunoblotting or measurement of transcript levels [48,50–52]. While there is a generally accepted view that steady state levels of free and junctional SR proteins can vary from normal in diseased hearts, there is no clear understanding of how such changes fit into a mechanism of myocardial dysfunction, nor a model for how such changes occur. In contrast to these major protein components of free and junctional SR, most [53], but not all [54,55], studies of animal and human HF have found no change in the steady-state levels of CSQ. The data generated in this study challenge this view by showing that while total CSQ does not change, individual molecular forms vary greatly.

Heart cells from canine tachycardia-induced HF exhibit prolonged action potentials and Ca2+ transients with reduced peaks, with durations approximately three-fold longer than controls [56]. Alterations in CSQ or junctional SR trafficking might account for such effects Ca2+ handling if such altered SR trafficking produced changes in Ca2+-release complex formation. Alternatively, loss of the normal alignment of junctional SR with other contractile components might lead to a loss of contractility by disrupting normal membrane topology. More needs to be learned about the underlying mechanisms by which CSQ modifications are altered in HF, as well as in the mechanisms of junctional SR biogenesis and targeting of its highly selective set of protein components.


   Acknowledgements
 
We thank Timothy D. Houle and Michal Ram for technical assistance. This work was funded by NIH/NHLBI grant HL62586.


   Notes
 
Time for primary review 17 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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