Carlota Sumbilla

Phospholamban thiols play a central role in activation of the cardiac muscle sarcoplasmic reticulum calcium pump by nitroxyl.

Nitroxyl (HNO) donated by Angelis salt activates uptake of Ca(2+) by the cardiac SR Ca(2+) pump (SERCA2a). To determine whether HNO achieves this by a direct interaction with SERCA2a or its regulatory protein, phospholamban (PLN), we measured its effects on SERCA2a activation (as reflected in dephosphorylation) using insect cell microsomes expressing SERCA2a with or without PLN (wild-type and Cys --> Ala mutant). The results show that activation of SERCA2a dephosphorylation by HNO is PLN-dependent and that PLN thiols are targets for HNO. We conclude that HNO produces a disulfide bond that alters the conformation of PLN, relieving inhibition of the Ca(2+) pump.

Comparison of SERCA1 and SERCA2a expressed in COS-1 cells and cardiac myocytes.

Cultured COS-1 cells, as well as chicken embryonic and neonatal rat cardiac myocytes, were infected with recombinant adenovirus vectors to define limiting factors in the expression and Ca2+ transport function of recombinant sarcoplasmic-endoplasmic reticulum Ca2+ (SERCA) isoforms. Titration experiments showed that all COS-1 cells and myocytes in culture could be infected by an adenovirus titer of 10 plaque-forming units (pfu) per seeded cell. Raising the adenovirus titer further yielded higher protein expression up to an asymptotic limit for functional, membrane-bound SERCA protein. The asymptotic behavior of SERCA expression was not transcription related but was due to posttranscriptional events. The minimal (-268) cardiac troponin T (cTnT) promoter was a convenient size for adenovirus vector construction and manifested tight muscle specificity. However, its efficiency was lower than that of the nonspecific cytomegalovirus (CMV) promoter. At any rate, identical maximal levels of SERCA expression were obtained with the CMV and the cTnT promoter, as long as the viral titer was adjusted to compensate for transcription efficiency. A maximal threefold increase of total SERCA protein expression over the level of the endogenous SERCA of control myocytes was reached (a sevenfold increase compared with the endogenous SERCA of the same infected myocytes due to reduction of endogenous SERCA after infection). In contrast with previous reports [Ji et al. Am. J. Physiol. 276 ( Heart Circ. Physiol. 45): H89-H97, 1999], a higher kinetic turnover was demonstrated for the SERCA1 compared with the SERCA2a isoform as shown by a 5.0- versus 2.6-fold increase in calcium uptake rate accompanying maximal expression of recombinant SERCA1 or SERCA2a, respectively. This information is deemed necessary for studies attempting to modify myocardial cell function by manipulation of SERCA expression.Cultured COS-1 cells, as well as chicken embryonic and neonatal rat cardiac myocytes, were infected with recombinant adenovirus vectors to define limiting factors in the expression and Ca2+ transport function of recombinant sarcoplasmic-endoplasmic reticulum Ca(2+) (SERCA) isoforms. Titration experiments showed that all COS-1 cells and myocytes in culture could be infected by an adenovirus titer of 10 plaque-forming units (pfu) per seeded cell. Raising the adenovirus titer further yielded higher protein expression up to an asymptotic limit for functional, membrane-bound SERCA protein. The asymptotic behavior of SERCA expression was not transcription related but was due to posttranscriptional events. The minimal (-268) cardiac troponin T (cTnT) promoter was a convenient size for adenovirus vector construction and manifested tight muscle specificity. However, its efficiency was lower than that of the nonspecific cytomegalovirus (CMV) promoter. At any rate, identical maximal levels of SERCA expression were obtained with the CMV and the cTnT promoter, as long as the viral titer was adjusted to compensate for transcription efficiency. A maximal threefold increase of total SERCA protein expression over the level of the endogenous SERCA of control myocytes was reached (a sevenfold increase compared with the endogenous SERCA of the same infected myocytes due to reduction of endogenous SERCA after infection). In contrast with previous reports [Ji et al. Am. J. Physiol. 276 (Heart Circ. Physiol. 45): H89-H97, 1999], a higher kinetic turnover was demonstrated for the SERCA1 compared with the SERCA2a isoform as shown by a 5.0- versus 2.6-fold increase in calcium uptake rate accompanying maximal expression of recombinant SERCA1 or SERCA2a, respectively. This information is deemed necessary for studies attempting to modify myocardial cell function by manipulation of SERCA expression.

Comparison of the oxidation of glutamine, glucose, ketone bodies and fatty acids by human diploid fibroblasts

The contribution of glutamine, glucose, ketone bodies and fatty acids to the oxidative energy metabolism of human diploid fibroblasts ws studied. The rate of glutamine oxidation by fibroblasts was 98 nmol/h per mg cell protein compared to 2 nmol/h per mg cell protein or less for glucose, acetoacetate, D-3-hydroxybutyrate, octanoic acid and palmitic acid. Glucose inhibited glutamine oxidation by 85%, while the other substrates had no effect. Therefore, these cells meet their energy requirement almost solely by anaerobic glycolysis and glutamine oxidation.

Cell-specific promoter in adenovirus vector for transgenic expression of SERCA1 ATPase in cardiac myocytes

Adenovirus-mediated transfer of cDNA encoding the chicken skeletal muscle sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1) yielded selective expression in cultured chick embryo cardiac myocytes under control of a segment (-268 base pair) of the cell-specific cardiac troponin T (cTnT) promoter or nonselective expression in myocytes and fibroblasts under control of a constitutive viral [cytomegalovirus (CMV)] promoter. Under optimal conditions nearly all cardiac myocytes in culture were shown to express transgenic SERCA1 ATPase. Expression was targeted to intracellular membranes and was recovered in subcellular fractions with a pattern identical to that of the endogenous SERCA2a ATPase. Relative to control myocytes, transgenic SERCA1 expression increased up to four times the rates of ATP-dependent (and thapsigargin-sensitive) Ca2+ transport activity of cell homogenates. Although the CMV promoter was more active than the cTnT promoter, an upper limit for transgenic expression of functional enzyme was reached under control of either promoter by adjustment of the adenovirus plaque-forming unit titer of infection media. Cytosolic Ca2+ concentration transients and tension development of whole myocytes were also influenced to a similar limit by transgenic expression of SERCA1 under control of either promoter. Our experiments demonstrate that a cell-specific protein promoter in recombinant adenovirus vectors yields highly efficient and selective transgene expression of a membrane-bound and functional enzyme in cardiac myocytes.

Direct Demonstration of Ca2+ Binding Defects in Sarco-Endoplasmic Reticulum Ca2+ ATPase Mutants Overexpressed in COS-1 Cells Transfected with Adenovirus Vectors

Single mutations of specific amino acids within the membrane-bound region of the sarco-endoplasmic reticulum Ca2+ (SERCA)-1 ATPase interfere with Ca2+inhibition of ATPase phosphorylation by Pi (1), suggesting that these residues may be involved in complexation of two Ca2+ that are known to bind to the enzyme. However, direct measurements of Ca2+ binding in the absence of ATP have been limited by the low quantities of available mutant protein. We have improved the transfection efficiency by means of recombinant adenovirus vectors, yielding sufficient expression of wild type and mutant SERCA-1 ATPase for measurements of Ca2+ binding to the microsomal fraction of the transfected cells. We find that in the presence of 20 μm Ca2+ and in the absence of ATP, the Glu771 → Gln, Thr799 → Ala, Asp800 → Asn, and Glu908 → Ala mutants exhibit negligible binding, indicating that the oxygen functions of Glu771, Thr799, Asp800, and Glu908 are involved in interactions whose single disruption causes major changes in the highly cooperative “duplex” binding. Total loss of Ca2+ binding is accompanied by loss of Ca2+ inhibition of the Pi reaction. We also find that, at pH 7.0, the Glu309 → Gln and the Asn796 → Ala mutants bind approximately half as much Ca2+ as the wild type ATPase and do not interfere with Ca2+ inhibition of the Pi reaction. At pH 6.2, the Glu309 → Gln mutant does not bind any Ca2+ , and its phosphorylation by Piis not inhibited by Ca2+. On the contrary, the Asn796 → Ala mutant retains the behavior displayed at pH 7.0. This suggests that in the Glu309 → Gln mutant, ionization of acidic functions in other amino acids (e.g.Glu771 and Asp800) occurs as the pH is shifted, thereby rendering Ca2+ binding possible. In the Asn796 → Ala mutant, on the other hand, the Glu309 carboxylic function allows binding of inhibitory Ca2+ even at pH 6.2. In all cases mutational interference with the inhibition of the Pi reaction by Ca2+can be overcome by raising the Ca2+ concentration to the mm range, consistent with a general effect of mutations on the affinity of the ATPase for Ca2+.

Exogenous Ca2+-ATPase isoform effects on Ca2+ transients of embryonic chicken and neonatal rat cardiac myocytes

1 Sarco‐endoplasmic reticulum Ca2+‐ATPase from fast skeletal (SERCA1) or cardiac muscle (SERCA2a) was expressed in embryonic chicken and neonatal rat cardiac myocytes by adenovirus vectors, with c‐myc tags on both constructs to compare expression and distinguish exogenous from endogenous SERCA2a in myocytes. 2 Expression of the two isoforms was similar (approximately 3‐fold higher than endogenous SERCA). However, SERCA1 activity was 2‐fold greater than SERCA2a activity, due to intrinsic differences in turnover rates. Activation of both exogenous SERCA isoforms by Ca2+ was displaced to slightly lower [Ca2+], suggesting that the overexpressed isoforms were independent of phospholamban. In fact, phospholamban and calsequestrin expression were unchanged. 3 Decay time constants of cytosolic Ca2+ transients from cells overexpressing SERCA1 were reduced by 30–40 % and half‐widths by 10–15 % compared to controls. SERCA2a overexpression produced much less acceleration of transients in chick than in rat, and less acceleration than SERCA1 overexpression in either species. There was no significant change in resting [Ca2+], peak amplitudes, or in the amount of Ca2+ releasable by caffeine from overexpression of either SERCA isoform. However, the amplitudes of the transients increased with SERCA1 overexpression when pacing frequency limited refilling of the sarcoplasmic reticulum. 4 It is concluded that total SERCA transport velocity has a primary effect on the decay phase of transients. Transport velocity is affected by SERCA isoform turnover rate, temperature, and/or SERCA copy number.

Tight Control of Exogenous SERCA Expression Is Required to Obtain Acceleration of Calcium Transients With Minimal Cytotoxic Effects in Cardiac Myocytes

Abstract— Collateral effects of exogenous sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) expression were characterized in neonatal rat and chicken embryo cardiac myocytes, and the conditions required to produce acceleration of Ca2+ transients with minimal toxicity were established. Cultured myocytes were infected with adenovirus vector carrying the cDNA of wild-type SERCA1, an inactive SERCA1 mutant, or enhanced green fluorescence protein under control of the cytomegalovirus promoter. Controls were exposed to empty virus vector. Each group was tested with and without phenylephrine (PHE) treatment. Under conditions of limited calf-serum exposure, the infected rat myocytes manifested a more rapid increase in size, protein content, and rate of protein synthesis relative to noninfected controls. These changes were not accompanied by reversal to fetal transcriptional pattern (as observed in hypertrophy triggered by PHE) and may be attributable to facilitated exchange with serum factors. SERCA virus titers >5 to 6 plaque-forming units per cell produced overcrowding of ATPase molecules on intracellular membranes, followed by apoptotic death of a significant number of rat but not chicken myocytes. Enhanced green fluorescence protein virus and empty virus also produced cytotoxic effects but at higher titers than SERCA. Expression of exogenous SERCA and enhancement of Ca2+ transient kinetics could be obtained with minimal cell damage in rat myocytes if the SERCA virus titer were maintained within 1 to 4 plaque-forming units per cell. Expression of endogenous SERCA was unchanged, but expression of exogenous SERCA was higher in myocytes rendered hypertrophic by treatment with PHE than in nontreated controls.

Rapid filtration measurements of Ca2+ release from cisternal sarcoplasmic reticulum vesicles

A radioactive tracer and rapid filtration method was applied to the study of Ca2+ release from sarcoplasmic reticulum (SR) vesicles which were preloaded passively (equilibration with millimolar Ca2+) or actively (in the presence of ATP or acetyl phosphate). The method allows complete substitution of the loading mixture with release medium in constant flow, and time resolution between 0.01 and 10.0 s. Net release can be clearly distinguished from isotope exchange. The latter is prominent in longitudinal SR vesicles. Net Ca2+ release is observed only from cisternal SR vesicles, is Ca2+ (micromolar) dependent, and is accelerated by inactive ATP analogues, or ATP itself, even in the presence of Mg2+. Net release has a strong pH dependence (between 6 and 7), and very little temperature dependence (consistent with a passive channel). In media of physiological significance (1 mM ATP, 1 mM magnesium, and free Ca2+ in the micromolar range), net Ca2+ release proceeds with a rate constant of approx. 100 s−1.

Ca2+ binding and translocation by the sarcoplasmic reticulum ATPase: Functional and structural considerations

Three experimental systems are described including sarcoplasmic reticulum (SR) vesicles, reconstituted proteoliposomes, and recombinant protein obtained by gene transfer and expression in foreign cells. It is shown that the Ca2+ ATPase of sarcoplasmic reticulum (SR) includes an extramembranous globular head which is connected through a stalk to a membrane bound region. Cooperative binding of two calcium ions occurs sequentially, within a channel formed by four clustered helices within the membrane bound region. Destabilization of the helical cluster is produced following enzyme phosphorylation by ATP at the catalytic site in the extramembranous region. The affinity and orientation of the Ca2+ binding site are thereby changed, permitting vectorial dissociation of bound Ca2+ against a concentration gradient. A long range linkage between phosphorylation and Ca2+ binding sites is provided by an intervening peptide segment that retains high homology in cation transport ATPases, and whose function is highly sensitive to mutational perturbations.

Fluorescence Studies of Red Blood Cell Membranes from Individuals with Huntington's Disease

Abstract: Fluorescence spectroscopic methods were used to investigate and compare the properties of erythrocyte membranes from individuals with Huntingtons Disease (HD) and from normal individuals. Erythrocyte ghosts were labeled with four different fluorescent probes: 1,6‐diphenylhexatriene (DPH); 6‐lauroyl‐2‐(dimethylamino)‐naphthalene (Laurdan); 2‐(4‐maleimide anilino)‐naphthalene‐6‐sulfonic acid (MIANS) and 5‐(iodoacetamidoethyl)aminonaphthalene‐1‐sulfonic acid (IAEDANS). DPH is sensitive to the microviscosity of the membranes. Laurdan is highly sensitive to the polarity and relaxation time of its environment. MIANS and IAEDANS both react covalently with sulfhydryl groups in membrane proteins. Erythrocyte membranes were labeled separately with each of these four probes, and we measured the centers of gravity of the fluorescence emission, the fluorescence anisotropies, and the fluorescence lifetimes. In 10 separate experiments, including a total of 24 patients and 14 control individuals, we found no significant differences between the two groups for any of the probes or spectral parameters. These results do not support the existence of a “generalized membrane defect” in individuals with HD.