Biomedical subjects

Steven E Cala

Publications and source records attributed to Steven E Cala.

8 recordsLinked to original sources

PubMed2006

Different endoplasmic reticulum trafficking and processing pathways for calsequestrin (CSQ) and epitope-tagged CSQ.

Cardiac calsequestrin (CSQ) is a protein that traffics to and concentrates inside sarcoplasmic reticulum (SR) terminal cisternae, a protein secretory compartment of uncertain origin. To investigate trafficking of CSQ within standard ER compartments, we expressed CSQ in nonmuscle cell lines and examined its localization by immunofluorescence and its molecular structure from the mass spectrum of total cellular CSQ. In all cells examined, CSQ was a highly phosphorylated protein with a glycan structure predictive of ER-retained proteins: Man9,8GlcNAc2 lacking terminal GlcNAc. Immunostaining was restricted to polymeric ER cisternae. Secretory pathway disruption by brefeldin A and thapsigargin led to altered CSQ glycosylation and phosphorylation consistent with post-ER trafficking. When epitope-tagged forms of CSQ were expressed in the same cells, mannose trimming of CSQ glycans was far more extensive, and C-terminal phosphorylation sites were nearly devoid of phosphate, in complete contrast to the highly phosphorylated wild-type protein that concentrates in all cells tested. Epitope-tagged CSQ also showed a reduced ER staining compared to wild-type protein, with significant staining in juxta-Golgi compartments. Loss of ER retention due to epitope tags or thapsigargin and resultant changes in protein structure or levels of bound Ca(2+) point to CSQ polymerization as an ER/SR retention mechanism.

Animals↗

PubMed2005

Triadin overexpression stimulates excitation-contraction coupling and increases predisposition to cellular arrhythmia in cardiac myocytes.

Triadin 1 (TRD) is an integral membrane protein that associates with the ryanodine receptor (RyR2), calsequestrin (CASQ2) and junctin to form a macromolecular Ca signaling complex in the cardiac junctional sarcoplasmic reticulum (SR). To define the functional role of TRD, we examined the effects of adenoviral-mediated overexpression of the wild-type protein (TRD(WT)) or a TRD mutant lacking the putative CASQ2 interaction domain residues 200 to 224 (TRD(Del.200-224)) on intracellular Ca signaling in adult rat ventricular myocytes. Overexpression of TRD(WT) reduced the amplitude of I(Ca)- induced Ca transients (at 0 mV) but voltage dependency of the Ca transients was markedly widened and flattened, such that even small I(Ca) at low and high depolarizations triggered maximal Ca transients. The frequency of spontaneous Ca sparks was significantly increased in TRD(WT) myocytes, whereas the amplitude of individual sparks was reduced. Consistent with these changes in Ca release signals, SR Ca content was decreased in TRD(WT) myocytes. Periodic electrical stimulation of TRD(WT) myocytes resulted in irregular, spontaneous Ca transients and arrhythmic oscillations of the membrane potential. Expression of TRD(Del.200-224) failed to produce any of the effects of the wild-type protein. The lipid bilayer technique was used to record the activity of single RyR2 channels using microsome samples obtained from control, TRD(WT) and TRD(Del.200-224) myocytes. Elevation of TRD(WT) levels increased the open probability of RyR2 channels, whereas expression of the mutant protein did not affect RyR2 activity. We conclude that TRD enhances cardiac excitation-contraction coupling by directly stimulating the RyR2. Interaction of TRD with RyR2 may involve amino acids 200 to 224 in C-terminal domain of TRD.

Adenoviridae↗

PubMed2005

Impaired SERCA function contributes to cardiomyocyte dysfunction in insulin resistant rats.

Ventricular dysfunction in type 2 diabetic patients is becoming apparent early after diagnosis of diabetes, but the cellular mechanisms contributing to this dysfunction are not well established. Our group has recently identified cardiomyocyte dysfunction in diet-induced insulin resistant rats that have not developed type 2 diabetes. The present investigation was designed to determine cellular mechanisms contributing to slowed cardiomyocyte relaxation in sucrose (SU)-fed rats. SU-feeding was used to induce whole-body insulin resistance. After 9-12 weeks on diet, isolated ventricular myocyte shortening/relengthening were slower in SU-fed adult male Wistar rats (42-63%) compared to starch (ST)-fed controls. Cytosolic Ca2+ removal attributable to Na+/Ca2+ exchange (NCX) and to sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) was evaluated with fluo-3/AM. Caffeine-releasable Ca2+ and cytosolic Ca2+ clearing through NCX were normal, whereas Ca2+ uptake by SERCA was significantly slower in SU myocytes (330+/-29 ms) compared to ST cells (253+/-16 ms). Protein levels for SERCA, NCX and phospholamban were not affected by SU-feeding. Manipulating intracellular Ca2+ with various positive inotropic interventions (e.g. post-rest potentiation, isoproterenol) and changes in stimulus frequency demonstrated that mechanical properties can be improved in subsets of myocytes. Thus, we conclude that impaired SERCA activity (with normal protein content) contributes to cardiomyocyte dysfunction in insulin resistant animals, whereas NCX function and expression are normal. These results suggest that subtle changes in Ca2+ regulation which occur prior to overt ventricular dysfunction/failure, may be common to early stages of a number of disorders involving insulin resistance (e.g. diabetes, obesity, syndrome X and hypertension).

Animals↗

PubMed2004

Calsequestrin mutant D307H exhibits depressed binding to its protein targets and a depressed response to calcium.

OBJECTIVE: A point mutation in human cardiac calsequestrin (CSQ-D307H) is responsible for a form of polymorphic ventricular tachycardia (PVT). When overexpressed in heart cells, the mutated CSQ leads to diminished Ca(2+) transients, consistent with defective regulation of intralumenal sarcoplasmic reticulum (SR) Ca(2+). METHODS: To analyze the D307H mutant and determine whether the D307H mutation results in loss of normal protein-protein interactions, we prepared recombinant human wild-type (WT) and D307H forms of CSQ in mammalian cells. RESULTS: Although we found the two proteins to undergo similar glycosylation and phosphorylation, we discovered that Ca(2+)-dependent binding of the D307H mutant to both triadin-1 and junctin was reduced by greater than 50% compared to WT. Reduced binding of the D307H mutant CSQ to target proteins was similar throughout a complete range of Ca(2+) concentrations. To investigate the mechanism of reduced Ca(2+)-dependent binding, Ca(2+)-dependent changes in intrinsic fluorescence emission for the two protein forms were compared. Intrinsic fluorescence of the D307H mutant was highly reduced, reflecting significant alteration in the tertiary protein structure. Moreover, the changes in fluorescence caused by increasing the Ca(2+) concentration were very significantly blunted, indicating that the Ca(2+)-dependent conformational change was virtually lost. CONCLUSIONS: We conclude that the point mutation D307H leads to a profoundly altered conformation that no longer responds normally to Ca(2+) and fails to bind normally to triadin and junctin.

Animals↗

PubMed2004

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.

Animals↗

PubMed2004

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.

Adenoviridae↗

PubMed2002

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.

Animals↗

PubMed2002

Depressed PKA activity contributes to impaired SERCA function and is linked to the pathogenesis of glucose-induced cardiomyopathy.

We have previously described a cardiomyopathy induced by culturing ventricular myocytes from normal adult rats in a medium containing high concentrations of glucose, which recapitulates cellular changes associated with early onset diabetic cardiomyopathy. This investigation was designed to evaluate cellular mechanisms that could contribute to slowed cytosolic Ca(2+) removal and myocyte relaxation in glucose-induced cardiomyopathy. Isolated ventricular myocytes were cultured overnight in medium containing normal glucose (n=5.5mM) or high glucose (HG=25.5mM). Cytosolic Ca(2+) removal was monitored with fluo-3 and myocyte mechanics with video-edge detection. Electrically stimulated Ca(2+) transients were prolonged in HG cells (A(T/PK)=215+/-7ms, n=41) compared to N myocytes (A(T/PK)=173+/-5ms, n=34). By pharmacological and ionic manipulations, Ca(2+) removal attributable to SERCA was slower in the HG group (A(D/PK)=290+/-17ms,n =41) compared to N (A(D/PK)=219+/-10, n=34), whereas NCX function was similar in both groups of cells. Total PKA activity was depressed in HG myocytes by 56% compared to N cells. beta-adrenergic receptor stimulation with ISO (10(-7)M) normalized myocyte relaxation, Ca(2+) transients and PKA activity in HG myocytes. Furthermore, inhibition of PKA with H89 (10(-5)M) depressed peak fractional shortening (PS) and slowed relengthening (A(R/PK)) to a greater extent in N (-50% for PS and 92% for A(R/PK)) than in HG cells (-25% for PS and 48% A(R/PK)). Depressed cytosolic Ca(2+) removal was not, however, associated with changes in basal levels of phosphorylated PLB, nor levels of SERCA, NCX or PLB proteins. We conclude that cellular mechanisms associated with the early onset glucose-induced cardiomyocyte dysfunction involves alterations in Ca(2+) regulation, which may be a common manifestation of other forms of cardiomyopathies.

Animals↗