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.

Phosphorylation and dephosphorylation of calsequestrin on CK2-sensitive sites in heart.

Calsequestrin (CSQ) concentrates in junctional sarcoplasmic reticulum (SR) where it functions in regulation of Ca2+ release. When purified from heart tissue, cardiac CSQ contains phosphate on a cluster of C-terminal serine residues, but little is known about the cellular site of kinase action, and the identity of the kinase remains uncertain. To determine basic features of the phosphorylation, we examined the reaction in canine heart preparations. CSQ phosphorylation was observed in [32P]metabolically-labeled heart cells after adenoviral overexpression, and its constitutive phosphorylation was limited to a CK2-sensitive C-terminal serine cluster. The CSQ kinase was oriented intralumenally, as was CSQ, inside membrane vesicles, such that exposure to each required detergent permeabilization. Yet even after detergent permeabilization, CSQ was phosphorylated much less efficiently by protein kinase CK2 in cardiac microsomes than was purified CSQ. Reduced phosphorylation was strongly dependent upon protein concentration, and phosphorylation time courses revealed a phosphatase activity that occurred constitutively as phosphorylated substrate accumulates. Evidence of selective dephosphorylation of CSQ glycoforms in heart homogenates was also seen by mass spectrometry analysis. Molecules with greater mannose content, a feature of early secretory pathway compartments, were more highly phosphorylated, while greater dephosphorylation was apparent in more distal compartments. Taken together, the analyses of CSQ phosphorylation in heart suggest that a constitutive process of phosphate turnover occurs for cardiac CSQ perhaps associated with its intracellular transport.

Mass spectrometry of cardiac calsequestrin characterizes microheterogeneity unique to heart and indicative of complex intracellular transit.

Cardiac calsequestrin concentrates in junctional sarcoplasmic reticulum in heart and skeletal muscle cells by an undefined mechanism. During transit through the secretory pathway, it undergoes an as yet uncharacterized glycosylation and acquires phosphate on CK2-sensitive sites. In this study, we have shown that active calsequestrin phosphorylation occurred in nonmuscle cells as well as muscle cells, reflecting a widespread cellular process. To characterize this post-translational modification and resolve individual molecular mass species, we subjected purified calsequestrin to mass spectrometry using electrospray ionization. Mass spectra showed that calsequestrin glycan structure in nonmuscle cells was that expected for an endoplasmic reticulum-localized glycoprotein and showed that each glycoform existed as four mass peaks representing molecules that also had 0-3 phosphorylation sites occupied. In heart, mass peaks indicated carbohydrate modifications characteristic of transit through Golgi compartments. Phosphorylation did not occur on every glycoform present, suggesting a far more complex movement of calsequestrin molecules in heart cells. Significant amounts of calsequestrin contained glycan with only a single mannose residue, indicative of a novel post-endoplasmic reticulum mannosidase activity. In conclusion, glyco- and phosphoforms of calsequestrin chart a complex cellular transport in heart, with calsequestrin following trafficking pathways not present or not accessible to the same molecules in nonmuscle.

Upregulated Expression of Rat Heart Intercellular Adhesion Molecule-1 in Angiotensin II- but Not Phenylephrine- Induced Hypertension.

-Intercellular adhesion molecule-1 (ICAM-1), part of an immunoglobulin-like superfamily of adhesion molecules, is involved in several cardiovascular diseases. We investigated whether in vivo angiotensin II (Ang II) increases ICAM-1 in rats. Sprague-Dawley rats were infused with vehicle or Ang II (750 µg. kg(-1). d(-1) SC) for 7 days. The contribution of Ang II receptors to ICAM-1 expression was investigated with a nonpeptide Ang II type 1 (AT(1)) receptor antagonist losartan (30 mg. kg(-1). d(-1) in drinking water). Systolic blood pressure was elevated in Ang II-treated animals compared with sham-treated controls, and losartan blocked this increase. Tumor necrosis factor (TNF)-alpha (5 µg/kg IP bolus), a prototype inducer of ICAM-1, was administered as a positive control for ICAM-1 expression. After treatment, hearts were frozen in liquid nitrogen; homogenates were subjected to SDS-PAGE and immunoblotted with an anti-rat ICAM-1 monoclonal antibody. We detected a predominantly high-molecular-weight band in homogenates from non-TNF-alpha-treated rats, which was enhanced by 80+/-5% in TNF-alpha-treated rats. This band measured approximately 200 kDa, which is the molecular weight of ICAM-1 in its native dimer form. The same band was detected in homogenates from sham and Ang II-treated rats, with the latter showing a 150+/-10% increase in ICAM-1 versus sham controls. Immunoprecipitation of rat heart homogenates with anti-rat ICAM-1 antibody resulted in a dominant band of the same molecular weight as samples not treated with antibody. Losartan prevented enhanced expression of ICAM-1 in the presence of Ang II but had no effect on basal ICAM-1 expression. Phenylephrine, an alpha-agonist (3 mg. kg(-1). d(-1) ), was infused for 1 week but had no effect on ICAM-1 expression, even though systolic blood pressure was elevated to the same level as in rats treated with Ang II. Thus, heart ICAM-1 expression is enhanced via AT(1) receptor activation independent of hypertension. Ang II-induced ICAM-1 expression was time and dose dependent, with maximal expression occurring within 5 to 7 days at 100 to 750 µg/kg Ang II. Immunohistochemical staining demonstrated markedly increased ICAM-1 levels in the perivascular area in Ang II-infused rats. Monocyte/macrophage accumulation was significantly greater in Ang II-treated rat hearts than in sham-treated hearts (10+/-1; P:<0.001; n=5). Thus during inflammation, overexpression of ICAM-1 may contribute to cardiovascular damage in diseases characterized by increased activity of the renin-angiotensin system.