Steven E. Cala

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 (TRDWT) or a TRD mutant lacking the putative CASQ2 interaction domain residues 200 to 224 (TRDDel.200–224) on intracellular Ca signaling in adult rat ventricular myocytes. Overexpression of TRDWT reduced the amplitude of ICa- induced Ca transients (at 0 mV) but voltage dependency of the Ca transients was markedly widened and flattened, such that even small ICa at low and high depolarizations triggered maximal Ca transients. The frequency of spontaneous Ca sparks was significantly increased in TRDWT myocytes, whereas the amplitude of individual sparks was reduced. Consistent with these changes in Ca release signals, SR Ca content was decreased in TRDWT myocytes. Periodic electrical stimulation of TRDWT myocytes resulted in irregular, spontaneous Ca transients and arrhythmic oscillations of the membrane potential. Expression of TRDDel.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, TRDWT and TRDDel.200–224 myocytes. Elevation of TRDWT 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.

Structural Domains in Phospholemman A Possible Role for the Carboxyl Terminus in Channel Inactivation

Phospholemman (PLM) is a small (72-amino acid) transmembrane protein found in cardiac sarcolemma that is a major substrate for several protein kinases in vivo. Detailed structural data for PLM is lacking, but several studies have described an ion conductance that results from PLM expression in oocytes. Moreover, addition of purified PLM to lipid bilayers generates similar ion currents, suggesting that the PLM molecule itself might be sufficient for channel formation. To provide a framework for understanding the function of PLM, we investigated PLM topology and structure in sarcolemmal membrane vesicles and analyzed purified recombinant PLM. Immunoblot analyses with site-specific antibodies revealed that the extracellular segment (residues 1 to 17) exists in a protected configuration highly resistant to proteases, even in detergent solutions. The intracellular portion of the molecule (residues 38 to 72), in contrast, was highly susceptible to proteases. Trypsin treatment produced a limit peptide (residues 1 to 43), which showed little change in electrophoretic mobility in SDS gels and retained the ion-channel activity in lipid bilayers that is characteristic of the full-length protein. In addition, we found that conductance through PLM channels exhibited rapid inactivation during depolarizing ramps at voltages greater than +/- 50 mV, Channels formed by trypsinized PLM or recombinant PLM 1-43 exhibited dramatic reductions in voltage-dependent inactivations. Our data point to distinct domains within the PLM molecule that may correlate with functional properties of channel activity observed in oocytes and lipid bilayers.

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.

Rough endoplasmic reticulum to junctional sarcoplasmic reticulum trafficking of calsequestrin in adult cardiomyocytes

Cardiac calsequestrin (CSQ) is synthesized on rough endoplasmic reticulum (ER), but concentrates within the junctional sarcoplasmic reticulum (SR) lumen where it becomes part of the Ca(2+)-release protein complex. To investigate CSQ trafficking through biosynthetic/secretory compartments of adult cardiomyocytes, CSQ-DsRed was overexpressed in cultured cells and examined using confocal fluorescence microscopy. By 48h of adenovirus treatment, CSQ-DsRed fluorescence had specifically accumulated in perinuclear cisternae, where it co-localized with markers of rough ER. From rough ER, CSQ-DsRed appeared to traffic directly to junctional SR along a transverse (Z-line) pathway along which sec 23-positive (ER-exit) sites were enriched. In contrast to DsRed direct fluorescence that presumably reflected DsRed tetramer formation, both anti-DsRed and anti-CSQ immunofluorescence did not detect the perinuclear CSQ-DsRed protein, but labeled only junctional SR puncta. These putative CSQ-DsRed monomers, but not the fluorescent tetramers, were observed to traffic anterogradely over the course of a 48h overexpression from rough ER towards the cell periphery. We propose a new model of CSQ and junctional SR protein traffic in the adult cardiomyocyte, wherein CSQ traffics from perinuclear cisternae, along contiguous ER/SR lumens in cardiomyocytes as a mobile monomer, but is retained in junctional SR as a polymer.

Calsequestrin binds to monomeric and complexed forms of key calcium-handling proteins in native sarcoplasmic reticulum membranes from rabbit skeletal muscle

Ca(2+)-handling proteins are important regulators of the excitation-contraction-relaxation cycle in skeletal muscle fibres. Although domain binding studies suggest protein coupling between various Ca(2+)-regulatory elements of triad junctions, no direct biochemical evidence exists demonstrating high-molecular-mass complex formation in native microsomal membranes. Calsequestrin represents the protein backbone of the luminal Ca(2+) reservoir and thereby occupies a central position in Ca(2+) homeostasis; we therefore used calsequestrin blot overlay assays in order to determine complex formation between sarcoplasmic reticulum components. Peroxidase-conjugated calsequestrin clearly labelled four major protein bands in one-dimensional (1D) and 2D electrophoretically separated membrane preparations from adult skeletal muscle. Immunoblotting identified the calsequestrin-binding proteins of approximately 26, 63, 94 and 560 kDa as junctin, calsequestrin itself, triadin and the ryanodine receptor, respectively. Protein-protein coupling could be modified by ionic detergents, non-ionic detergents, changes in Ca(2+) concentration, as well as antibody and purified calsequestrin binding. Importantly, complex formation as determined by blot overlay assays was confirmed by differential co-immunoprecipitation experiments and chemical crosslinking analysis. Hence, the key Ca(2+)-regulatory membrane components of skeletal muscle form a supramolecular membrane assembly. The formation of this tightly associated junctional sarcoplasmic reticulum complex seems to underlie the physiological regulation of skeletal muscle contraction and relaxation, which supports the biochemical concept that Ca(2+) homeostasis is regulated by direct protein-protein interactions.

GRP94 hyperglycosylation and phosphorylation in Sf21 cells

GRP94 is an inducible resident endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) glycoprotein that functions as a protein chaperone and Ca(2+) regulator. GRP94 has been reported to be a substrate for protein kinase CK2 in vitro, although its phosphorylation in intact cells remains unreported. In Sf21 insect cells, overexpression of canine GRP94 led to the appearance of a multiplet of three or more molecular-mass isoforms which was reduced to a single mobility form following treatment of cells with tunicamycin, suggesting stable accumulations of consecutively modified protein. Metabolic labeling of Sf21 cells with (32)P(i) led to a constitutive phosphorylation of GRP94 which, based upon phosphopeptide mapping, occurred specifically on CK2-sensitive sites. Among the GRP94 multiplet, however, only the lowest mobility form of GRP94 was phosphorylated, even though in vitro phosphorylation of GRP94 by CK2 led to phosphorylation of all glycosylated forms. The (32)P(i) incorporation into GRP94 indicated a slow turnover of phosphate incorporation that was unaffected by inhibition of biosynthesis, resulting in a steady-state level of phospho-GRP94 on CK2 sites. These data support a role for protein kinase CK2 in the cell biology for GRP94 and other resident ER/SR proteins that may occur in ER compartments.

Calsequestrin Accumulation in Rough Endoplasmic Reticulum Promotes Perinuclear Ca2+ Release

Background: Calsequestrin is a high-capacity Ca2+-binding protein that stores Ca2+ within the sarcoplasmic reticulum. Results: Calsequestrin retention in the rough endoplasmic reticulum promotes perinuclear Ca2+ release and spontaneous Ca2+ wave initiation from perinuclear regions. Conclusion: Subcellular redistribution of calsequestrin affects spatial distribution of Ca2+ signals and myocyte function. Significance: Our data provide a new perspective of calsequestrin in perinuclear Ca2+ homeostasis. Molecular mechanisms underlying Ca2+ regulation by perinuclear endoplasmic/sarcoplasmic reticulum (ER/SR) cisternae in cardiomyocytes remain obscure. To investigate the mechanisms of changes in cardiac calsequestrin (CSQ2) trafficking on perinuclear Ca2+ signaling, we manipulated the subcellular distribution of CSQ2 by overexpression of CSQ2-DsRed, which specifically accumulates in the perinuclear rough ER. Adult ventricular myocytes were infected with adenoviruses expressing CSQ2-DsRed, CSQ2-WT, or empty vector. We found that perinuclear enriched CSQ2-DsRed, but not normally distributed CSQ2-WT, enhanced nuclear Ca2+ transients more potently than cytosolic Ca2+ transients. Overexpression of CSQ2-DsRed produced more actively propagating Ca2+ waves from perinuclear regions than did CSQ2-WT. Activities of the SR/ER Ca2+-ATPase and ryanodine receptor type 2, but not inositol 1,4,5-trisphosphate receptor type 2, were required for the generation of these perinuclear initiated Ca2+ waves. In addition, CSQ2-DsRed was more potent than CSQ2-WT in inducing cellular hypertrophy in cultured neonatal cardiomyocytes. Our data demonstrate for the first time that CSQ2 retention in the rough ER/perinuclear region promotes perinuclear Ca2+ signaling and predisposes to ryanodine receptor type 2-mediated Ca2+ waves from CSQ2-enriched perinuclear compartments and myocyte hypotrophy. These findings provide new insights into the mechanism of CSQ2 in Ca2+ homeostasis, suggesting that rough ER-localized Ca2+ stores can operate independently in raising levels of cytosolic/nucleoplasmic Ca2+ as a source of Ca2+ for Ca2+-dependent signaling in health and disease.

Developmental regulation of the L-type calcium channel α1C subunit expression in heart

We used Northern analyses, RNase protection assays and immunoblot analyses to examine the relationship among developmental age of the heart, abundance of mRNA and L-type calcium channel α1Csubunit protein, and to establish the size of the native protein in heart. Northern analysis, RNase protection assays, and immunoblots were used to study RNA and protein from rat heart of various ages. In fetal and adult ventricles there was a predominant 8.3-kb transcript for the α1C subunit with no change in transcript size during development. RNase protection assays demonstrated a 2-fold increase in abundance of the DHP receptor message during postnatal development. Immunoblots identified a 240 kD protein, corresponding to the predicted molecular mass of the full length α1C subunit. No change in size of protein for the α1C subunit was observed at any developmental stage and there was no evidence for a truncated isoform. There was an approximate 2-fold increase in α1C subunit protein in ventricular homogenates during postnatal development. Thus, in the developing rat heart, alterations in calcium channel properties during development appear to result neither from alternative splicing that produces a smaller transcript for the α1C subunit nor from expression of a truncated protein, but at least in part from transcriptionally-regulated expression of the 240 kDa polypeptide.

Up-regulation of alpha-smooth muscle actin in cardiomyocytes from non-hypertrophic and non-failing transgenic mouse hearts expressing N-terminal truncated cardiac troponin I ☆

We previously reported that a restrictive N‐terminal truncation of cardiac troponin I (cTnI‐ND) is up‐regulated in the heart in adaptation to hemodynamic stresses. Over‐expression of cTnI‐ND in the hearts of transgenic mice revealed functional benefits such as increased relaxation and myocardial compliance. In the present study, we investigated the subsequent effect on myocardial remodeling. The alpha‐smooth muscle actin (α‐SMA) isoform is normally expressed in differentiating cardiomyocytes and is a marker for myocardial hypertrophy in adult hearts. Our results show that in cTnI‐ND transgenic mice of between 2 and 3 months of age (young adults), a significant level of α‐SMA is expressed in the heart as compared with wild‐type animals. Although blood vessel density was increased in the cTnI‐ND heart, the mass of smooth muscle tissue did not correlate with the increased level of α‐SMA. Instead, immunocytochemical staining and Western blotting of protein extracts from isolated cardiomyocytes identified cardiomyocytes as the source of increased α‐SMA in cTnI‐ND hearts. We further found that while a portion of the up‐regulated α‐SMA protein was incorporated into the sarcomeric thin filaments, the majority of SMA protein was found outside of myofibrils. This distribution pattern suggests dual functions for the up‐regulated α‐SMA as both a contractile component to affect contractility and as possible effector of early remodeling in non‐hypertrophic, non‐failing cTnI‐ND hearts.

Calsequestrin isoforms localize to different ER subcompartments: Evidence for polymer and heteropolymer-dependent localization

Skeletal muscle calsequestrin (skelCSQ) and cardiac calsequestrin (cardCSQ) are resident proteins of the ER/SR, but mechanisms by which CSQ is retained inside membrane lumens remain speculative. A structural model that predicts linear CSQ polymers has been developed that might explain CSQ concentration and localization inside junctional SR lumens, however little evidence exists for polymer formation in intact cells or for its effects on subcellular localization. We previously showed that cardCSQ is efficiently retained within the ER, but its retention is lost under conditions expected to disrupt its polymerization. In the present study, we found unexpectedly that skelCSQ shows no co-localization with cardCSQ in COS cells or in rat neonatal heart cells, but instead concentrates in a membrane compartment (ERGIC) that is just distal to that of cardCSQ. Consistent with this difference in immunofluorescent localization, the structures of CSQ ((316)Asn-linked) glycans showed two types of pre-Golgi processing. Despite the difference in subcellular distribution of individual wild-type forms of CSQ, however, pairs of different CSQ molecules (for example, different isoforms or different fluorescent fusion proteins) consistently co-localized, suggesting that separate forms of CSQ polymerize in different parts of the same secretory pathway, while different CSQ pairs localize together through heteropolymerization.