Biomedical subjects

L R Jones

Publications and source records attributed to L R Jones.

At least 19 recordsLinked to original sources

PubMed2007

Quality of food eaten in English primary schools: school dinners vs packed lunches.

OBJECTIVE: To compare the food and nutrient intakes of primary school children eating school dinners and packed lunches. SUBJECTS: Six-hundred and twenty-one 7-year-old children participating in the Avon Longitudinal Study of Parents and Children in South West England. METHODS: Diet was assessed by 3-day unweighed food record. RESULTS: The composition of both types of school meals compared unfavourably with dietary guidelines. Intakes of energy, non-starch polysaccharides (NSP), calcium, iron, folate, retinol equivalents, zinc, copper, magnesium, iodine and riboflavin were too low, and intakes of total and saturated fat were too high. However, children who ate school dinners had higher lunchtime intakes of protein, starch, NSP and most vitamins and minerals and lower intakes of sugar (14.2 and 20.9% of energy in school dinners and packed lunches, respectively, P<0.001) and saturated fat (12.0 and 16.2%, P<0.001). Only around half of the recommended amount of fruit and vegetables was eaten by children having either type of school meal. There were also differences in the whole day's nutrient intake according to school meal type. Children eating packed lunches had lower daily intakes of potassium and zinc, and higher intakes of sugar and saturated fat. Differences in nutrient intake were independent of maternal education. CONCLUSIONS: The food and nutrient content of both school dinners and packed lunches needed improvement. However, the standard of food brought from home by children was, if anything, worse than that served at school. Recent moves to improve school dinners will need to be complemented by education about what constitutes a healthy packed lunch.

Child↗

PubMed2006

Molecular characterization of bovine rotavirus circulating in beef and dairy herds in Argentina during a 10-year period (1994-2003).

Group A bovine rotavirus (BRV) is one of the main causes of neonatal calf diarrhea. The present study reports the incidence of rotavirus diarrhea and the genotypes of BRV strains circulating in beef and dairy herds from Argentina, during a 10-year period (1994-2003). Group A BRV was detected in 62.5% (250/400) of the total studied cases of diarrhea. Positive cases were analyzed by heminested multiplex RT-PCR for P and G genotypes identification. Sixty percent of them were typed as P[5]G6, 4.4% P[11]G10, 4.4% P[11]G6 and 2.4% P[5]G10. Additionally, 9.2% of the cases were initially typed as G8 combined with P[5] or P[11], but sequence analysis revealed they belonged to genotype G6, lineage Hun4-like. Partial typing was assessed in 12.0% of the cases. One of the partially typed samples was closely related to genotype G15. BRV was detected in 71% and 58% of the outbreaks registered in beef and dairy farms, respectively. A clear differential distribution of G/P types was found according to the herd type. P[5]G6 was the prevalent strain in beef herds, while P[11] was the prevalent P-type in dairy herds (71%), associated in similar proportions with G6 and G10, These findings indicate that BRV genotypes included in the current commercially available rotavirus vaccines (G6, G10, P[5] and P[11]) should protect calves from most Argentinean field strains. Nevertheless, continuous surveillance is necessary to detect the emergence of new variants.

Animals↗

PubMed2002

Modulation of gene expression in transgenic mouse hearts overexpressing calsequestrin.

Calsequestrin (CSQ) is the major Ca2+ binding protein of the cardiac sarcoplasmic reticulum (SR). Transgenic mice overexpressing CSQ at the age of 7 weeks exhibit concentric cardiac hypertrophy, and by 13 weeks the condition progresses to dilated cardiomyopathy. The present study used a differential display analysis to identify genes whose expressions are modulated in the CSQ-overexpressing mouse hearts to provide information on the mechanism of transition from concentric cardiac hypertrophy to failure. Cardiac ankyrin repeat protein (CARP), glutathione peroxidase (Gpx1), and genes which participate in the formation of extracellular matrix including decorin, TSC-36, Magp2, Osf2, and SPARC are upregulated in CSQ mouse hearts at 7 and 13 weeks of age compared to those of non-transgenic littermates. In addition, two novel genes without sequence similarities to any known genes are upregulated in CSQ-overexpressing mouse hearts. Several genes are downregulated at 13 weeks, including SR Ca2+-ATPase (SERCA2) and adenine nucleotide translocase 1 (Ant1) genes. Further, a functionally yet unknown gene (NM_026586) previously identified in the mouse wolffian duct is dramatically downregulated in CSQ mice with dilated hearts. Thus, CARP, Gpx1, and genes encoding extracellular matrix proteins may participate in the development of cardiac hypertrophy and fibrosis, and changes in SERCA2, Ant1, and NM_026586 mRNA expression may be involved in transition from concentric to dilated cardiac hypertrophy.

Adenine Nucleotide Translocator 1↗

PubMed2001

Genetic typing of bovine viral diarrhea virus isolates from Argentina.

Genetic typing of 29 Bovine Viral Diarrhea Virus (BVDV) isolates from Argentina was carried out by sequencing 245 nucleotides of the RT-PCR products of the 5'-UTR region. Sequence analysis shows that these Argentinean BVDV include types 1 and 2. The majority (26/29) of the isolates are type 1, which comprises subtypes 1a and 1b, together with an additional subgroup within subtype 1a. This subgroup is close to the South African subgroup Ic of 1a viruses, and to the deer pestivirus strain "Deer". The three type 2 BVDV were isolated from fetal tissues or serum during the 7-8 years before a clinical outbreak in Argentina had been reported. Only inactivated vaccines are used in bovines of the country, thus the analysed viruses are authentic field strains. The long term circulation of type 2 BVDV (situation similar to that of North America before the epidemic of 1993), and the existence of viral populations which differ from the reference strains commonly used in vaccine elaboration should be considered by manufacturers of diagnostic reagents and vaccines.

Animals↗

PubMed2001

Gene structure and expression of phospholemman in mouse.

Phospholemman (PLM) is a small transmembrane cardiac protein that is the major sarcolemmal substrate for phosphorylation in response to adrenergic stimulation. PLM likely plays a role in muscle contractility and cell volume regulation through its function as a channel or a channel regulator. We are the first to describe the structure of the PLM gene and to demonstrate PLM cDNA splice variants. We cloned the murine PLM cDNA and used it as a probe to isolate the gene from a 129/SvJ genomic library. The gene contains seven introns and eight exons. The coding sequence is interrupted by five introns; the 5' untranslated region by two. Using rapid amplification of 5' cDNA ends we identified transcription start sites and four splice variants of the 5' untranslated domain. There was no TATA box or CAAT box in the putative promoter regions. The gene has several stretches of dinucleotide repeats. The 3' untranslated domains of mouse PLM cDNA clones show sequence differences not accounted for by alternative splicing. Mouse PLM shares 93, 83 and 80% amino acid identity with rat, dog, and human PLMs, respectively. Tissue expression of murine PLM parallels that in other species, being highest in heart, skeletal muscle, and liver.

Alternative Splicing↗

PubMed2001

Cardiac beta ARK1 inhibition prolongs survival and augments beta blocker therapy in a mouse model of severe heart failure.

Chronic human heart failure is characterized by abnormalities in beta-adrenergic receptor (betaAR) signaling, including increased levels of betaAR kinase 1 (betaARK1), which seems critical to the pathogenesis of the disease. To determine whether inhibition of betaARK1 is sufficient to rescue a model of severe heart failure, we mated transgenic mice overexpressing a peptide inhibitor of betaARK1 (betaARKct) with transgenic mice overexpressing the sarcoplasmic reticulum Ca(2+)-binding protein, calsequestrin (CSQ). CSQ mice have a severe cardiomyopathy and markedly shortened survival (9 +/- 1 weeks). In contrast, CSQ/betaARKct mice exhibited a significant increase in mean survival age (15 +/- 1 weeks; P < 0.0001) and showed less cardiac dilation, and cardiac function was significantly improved (CSQ vs. CSQ/betaARKct, left ventricular end diastolic dimension 5.60 +/- 0.17 mm vs. 4.19 +/- 0.09 mm, P < 0.005; % fractional shortening, 15 +/- 2 vs. 36 +/- 2, P < 0.005). The enhancement of the survival rate in CSQ/betaARKct mice was substantially potentiated by chronic treatment with the betaAR antagonist metoprolol (CSQ/betaARKct nontreated vs. CSQ/betaARKct metoprolol treated, 15 +/- 1 weeks vs. 25 +/- 2 weeks, P < 0.0001). Thus, overexpression of the betaARKct resulted in a marked prolongation in survival and improved cardiac function in a mouse model of severe cardiomyopathy that can be potentiated with beta-blocker therapy. These data demonstrate a significant synergy between an established heart-failure treatment and the strategy of betaARK1 inhibition.

Adrenergic beta-Antagonists↗

PubMed2001

Structural alterations in cardiac calcium release units resulting from overexpression of junctin.

Junctin is a 26 kDa membrane protein that binds to calsequestrin, triadin, and ryanodine receptors (RyRs) within the junctional sarcoplasmic reticulum of calcium release units. The sequence of junctin includes a short N-terminal cytoplasmic domain a single transmembrane domain, and a highly charged C-terminal domain located in the sarcoplasmic reticulum lumen. Dog and mouse junctins are highly conserved at the transmembrane domains, but the luminal domains are more divergent. To probe the contribution of junctin to the architecture of calcium release units in heart, we engineered transgenic mice overexpressing canine junctin and examined the left ventricular myocardium by electron microscopy. Overall architecture of calcium release units is similar in control myocardium and in myocardium overexpressing junctin by 5-10-fold. In both myocardia, junctional SR cisternae are closely associated with exterior membranes (plasmalemma and transverse tubules). The cisternae are flat; they contain a string of calsequestrin beads and are lined by a row of feet, or RyRs, on the side facing the exterior membranes. T tubule surface density, measured as the perimeter of T tubule profiles v area of section, is the same in transgenic and control myocardia (305 v 289 nm/nm(2)). Three changes affecting the junctional SR architecture are apparent in the myocardium overexpressing junctin. One is a more tightly zippered appearance of the junctional SR cisternae. The width of the junctional SR is narrower and less variable in overexpressing than in control myocardium and the calsequestrin content is more compact. A second change is the extension of zippered junctional SR domains to non-junctional regions, which we term "frustrated" junctional SR. A third change is an increase in the extent of association between SR and T tubules. In junctin overexpressing myocardium junctional SR cisternae cover approximately 45% of the surface of all T tubule profiles, while in control myocardium the coverage approximately 30%. Junctional associations between SR and T tubules are increased in size. We conclude that the increase in junctin expression affects the packing of calsequestrin in the junctional SR and facilitates the association of SR and T tubules.

Amino Acid Sequence↗

PubMed2001

Locating phospholamban in co-crystals with Ca(2+)-ATPase by cryoelectron microscopy.

Phospholamban (PLB) is responsible for regulating Ca(2+) transport by Ca(2+)-ATPase across the sarcoplasmic reticulum of cardiac and smooth muscle. This regulation is coupled to beta-adrenergic stimulation, and dysfunction has been associated with end-stage heart failure. PLB appears to directly bind to Ca(2+)-ATPase, thus slowing certain steps in the Ca(2+) transport cycle. We have determined 3D structures from co-crystals of PLB with Ca(2+)-ATPase by cryoelectron microscopy of tubular co-crystals at 8--10 A resolution. Specifically, we have used wild-type PLB, a monomeric PLB mutant (L37A), and a pentameric PLB mutant (N27A) for co-reconstitution and have compared resulting structures with three control structures of Ca(2+)-ATPase alone. The overall molecular shape of Ca(2+)-ATPase was indistinguishable in the various reconstructions, indicating that PLB did not have any global effects on Ca(2+)-ATPase conformation. Difference maps reveal densities which we attributed to the cytoplasmic domain of PLB, though no difference densities were seen for PLB's transmembrane helix. Based on these difference maps, we propose that a single PLB molecule interacts with two Ca(2+)-ATPase molecules. Our model suggests that PLB may resist the large domain movements associated with the catalytic cycle, thus inhibiting turnover.

Animals↗

PubMed2001

Artificial ion channels formed by a synthetic cyclic peptide.

A new cyclic peptide 1 having an (LLLD)3 configuration pattern was designed that is capable of forming artificial transmembrane ion channels by self-assembly of planar peptide rings, with hydrophilic groups arrayed in the interior of the channel. Ion permeability in the presence of the synthetic peptide 1, cyclo[-Trp-Dap-Leu-D-Ala-Trp-Ser-Val-D-Ala-Trp-Ser-Ile-Gly-] (Dap: L-diaminopropionic acid), was observed in lipid bilayer membranes. The pH dependence of ionic conductance showed that the beta-amino group of Dap may play a role in the conductance of the peptide channels. Fourier-transform infrared and circular dichroism data imply that, in a membrane, a stack of cyclic peptides is formed in which the inter peptide H bonds form a kind of beta-structure analogous to that in the gramicidin A dimer and distinct from the H-bonding pattern of the beta-barrels.

Circular Dichroism↗

PubMed2001

Cardiac-directed overexpression of wild-type alpha1B-adrenergic receptor induces dilated cardiomyopathy.

Using transgenesis as a paradigm, we show here that alpha1-adrenergic receptors (alpha1AR) play an important role in cardiac homeostasis. Cardiomyocyte-specific overexpression of the alpha(1B)AR subtype resulted in the development of dilated cardiomyopathy and death at ~9 mo of age with typical signs of heart failure. Histological analyses showed the enlargement of all four cardiac chambers and cardiomyocyte disarray in the failing hearts. Transgenic animals showed increased left ventricular areas, as assessed by echocardiography. In addition, a progressive decrease in left ventricular systolic function was revealed. The abundance and activity of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) were reduced, and the ratio of phospholamban to SERCA2 was increased. alpha-Myosin heavy chain (MHC) mRNA was less abundant in older transgenic ventricles, whereas beta-MHC was induced in the failing hearts. Titin mRNA abundance was decreased at 9 mo, whereas atrial natriuretic factor mRNA was elevated at all times. This model mimics structural and functional features of idiopathic dilated cardiomyopathy. The results of this study suggest that chronic alpha1AR activity is deleterious for cardiac function.

Animals↗

PubMed2001

Application of single-strand conformation polymorphism to the study of bovine viral diarrhea virus isolates.

Single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR) products is a genetic screening technique for rapid detection of nucleotide substitutions in PCR-amplified genomic DNA or cDNA. It is based on the observation that partially formamide-denatured double-stranded DNA migrates as 2 single-stranded DNA molecules when electrophoresed in nondenaturing polyacrylamide gels. The mobility depends on the 3-dimensional conformation of the strand under the conditions used. It is possible to discriminate between DNA strands differing in only 1 nucleotide. The method was applied to the analysis of Bovine Viral Diarrhea Virus (BVDV) isolates. Reference and Argentinian strains were assessed for variations in their 5' untranslated region (5'-UTR). The PCR products of the 5'-UTR ends were formamide denatured and compared by SSCP analysis in nondenaturing 15% polyacrylamide and 15% polyacrilamide-5% glycerol gels. The reference strains SD-1, Singer, and Oregon C24V had differences in electrophoretic patterns. Despite the high conservation among the 5'-UTR of pestiviruses, the method allowed discrimination among all 9 Argentinian isolates. The 5'-UTR of a fetal kidney-derived isolate (1R93) was PCR amplified and cloned in a plasmid vector; the SSCP analysis of 30 PCR products obtained by direct amplification over randomly selected clones produced 5 different banding patterns, indicating the existence of viral quasispecies. The results show that SSCP may be used to identify and differentiate among BVDV isolates.

5' Untranslated Regions↗

PubMed2001

Forecasting herd structure and milk production for production risk management.

Substantial increases in milk price volatility have resulted from changes in federal dairy policies. For a dairy farm, however, monthly gross milk receipts are a function of unit price and quantity produced. Both can vary substantially over time. Therefore, to be effective, risk management strategies must address milk and input price volatility (price risk management) and fluctuations in milk production per cow and cow numbers (production risk management). Herd milk production through time can be modeled as a discrete stochastic process using finite Markov chains. Cows at time t = 0 are assigned to homogeneous production cells in four-dimensional arrays with coordinates determined by parity (1, 2, 3), week in milk (1, ..., 104), pregnancy status (0, 1), and week pregnant (1, ..., 40). The processes of aging, pregnancy, involuntary cull, voluntary cull, abortion, dry-off, and freshening from week i-1 to week i are accounted for, using nonstationary transition probabilities. Bayesian estimates of transition probabilities are derived from historical herd data, assuming that individual outcomes are from Bernoulli distributions. The values of parameters theta(i) for the Bernoulli distributions are unknown but have prior distributions that follow beta distributions with parameters alpha(i) and beta(i) estimated from historical data. Herd observations are then used to generate posterior distributions of theta(i), also from beta distributions. Projecting from one week to the next is accomplished by moving virtual animals from one production cell to the next based on the transition probability assigned to that path. Summing production estimates and variances of all independent cells provides for an expected herd production with an associated variance. As expected, the forecast variance increases with time, reflecting increased uncertainty of distant projections. Model validation presents an interesting problem because future observations used for validation are under human control and are not independent of the forecast.

Animal Husbandry↗

PubMed2000

Reexamination of the role of the leucine/isoleucine zipper residues of phospholamban in inhibition of the Ca2+ pump of cardiac sarcoplasmic reticulum.

Phospholamban is a small phosphoprotein inhibitor of the Ca(2+)-pump in cardiac sarcoplasmic reticulum, which shows a distinct oligomeric distribution between monomers and homopentamers that are stabilized through Leu/Ile zipper interactions. A two-faced model of phospholamban inhibition of the Ca(2+)-pump was proposed, in which the Leu/Ile zipper residues located on one face of the transmembrane alpha-helix regulate the pentamer to monomer equilibrium, whereas residues on the other face of the helix bind to and inhibit the pump. Here we tested this two-faced model of phospholamban action by analyzing the functional effects of a new series of Leu/Ile zipper mutants. Pentameric stabilities of the mutants were quantified at different SDS concentrations. We show that several phospholamban mutants with hydrophobic amino acid substitutions at the Leu/Ile zipper region retain the ability to form pentamers but at the same time give the same or even stronger (i.e. L37I-PLB) inhibition of the Ca(2+)-pump than do mutants that are more completely monomeric. Steric constraints prevent the Leu/Ile zipper residues sequestered in the interior of the phospholamban pentamer from binding to the Ca(2+)-pump, leading to the conclusion that the zipper residues access the pump from the phospholamban monomer, which is the active inhibitory species. A modified model of phospholamban transmembrane domain action is proposed, in which the membrane span of the phospholamban monomer maintains contacts with the Ca(2+)-pump around most of its circumference, including residues located in the Leu/Ile zipper region.

Animals↗

PubMed2000

Cardiac hypertrophy and impaired relaxation in transgenic mice overexpressing triadin 1.

Triadin 1 is a major transmembrane protein in cardiac junctional sarcoplasmic reticulum (SR), which forms a quaternary complex with the ryanodine receptor (Ca(2+) release channel), junctin, and calsequestrin. To better understand the role of triadin 1 in excitation-contraction coupling in the heart, we generated transgenic mice with targeted overexpression of triadin 1 to mouse atrium and ventricle, employing the alpha-myosin heavy chain promoter to drive protein expression. The protein was overexpressed 5-fold in mouse ventricles, and overexpression was accompanied by cardiac hypertrophy. The levels of two other junctional SR proteins, the ryanodine receptor and junctin, were reduced by 55% and 73%, respectively, in association with triadin 1 overexpression, whereas the levels of calsequestrin, the Ca(2+)-binding protein of junctional SR, and of phospholamban and SERCA2a, Ca(2+)-handling proteins of the free SR, were unchanged. Cardiac myocytes from triadin 1-overexpressing mice exhibited depressed contractility; Ca(2+) transients decayed at a slower rate, and cell shortening and relengthening were diminished. The extent of depression of cell shortening of triadin 1-overexpressing cardiomyocytes was rate-dependent, being more depressed under low stimulation frequencies (0.5 Hz), but reaching comparable levels at higher frequencies of stimulation (5 Hz). Spontaneously beating, isolated work-performing heart preparations overexpressing triadin 1 also relaxed at a slower rate than control hearts, and failed to adapt to increased afterload appropriately. The fast time inactivation constant, tau(1), of the l-type Ca(2+) channel was prolonged in transgenic cardiomyocytes. Our results provide evidence for the coordinated regulation of junctional SR protein expression in heart independent of free SR protein expression, and furthermore suggest an important role for triadin 1 in regulating the contractile properties of the heart during excitation-contraction coupling.

Animals↗

PubMed2000

Phospholemman is a substrate for myotonic dystrophy protein kinase.

The genetic abnormality in myotonic muscular dystrophy, multiple CTG repeats lie upstream of a gene that encodes a novel protein kinase, myotonic dystrophy protein kinase (DMPK). Phospholemman (PLM), a major membrane substrate for phosphorylation by protein kinases A and C, induces Cl currents (I(Cl(PLM))) when expressed in Xenopus oocytes. To test the idea that PLM is a substrate for DMPK, we measured in vitro phosphorylation of purified PLM by DMPK. To assess the functional effects of PLM phosphorylation we compared I(Cl(PLM)) in Xenopus oocytes expressing PLM alone to currents in oocytes co-expressing DMPK, and examined the effect of DMPK on oocyte membrane PLM expression. We found that PLM is indeed a good substrate for DMPK in vitro. Co-expression of DMPK with PLM in oocytes resulted in a reduction in I(Cl(PLM)). This was most likely a specific effect of phosphorylation of PLM by DMPK, as the effect was not present in oocytes expressing a phos(-) PLM mutant in which all potential phosphorylation had been disabled by Ser --> Ala substitution. The biophysical characteristics of I(Cl(PLM)) were not changed by DMPK or by the phos(-) mutation. Co-expression of DMPK reduced the expression of PLM in oocyte membranes, suggesting a possible mechanism for the observed reduction in I(Cl(PLM)) amplitude. These data show that PLM is a substrate for phosphorylation by DMPK and provide functional evidence for modulation of PLM function by phosphorylation.

Animals↗

PubMed2000

Localization and characterization of the calsequestrin-binding domain of triadin 1. Evidence for a charged beta-strand in mediating the protein-protein interaction.

Triadin is an integral membrane protein of the junctional sarcoplasmic reticulum that binds to the high capacity Ca(2+)-binding protein calsequestrin and anchors it to the ryanodine receptor. The lumenal domain of triadin contains multiple repeats of alternating lysine and glutamic acid residues, which have been defined as KEKE motifs and have been proposed to promote protein associations. Here we identified the specific residues of triadin responsible for binding to calsequestrin by mutational analysis of triadin 1, the major cardiac isoform. A series of deletional fusion proteins of triadin 1 was generated, and by using metabolically labeled calsequestrin in filter-overlay assays, the calsequestrin-binding domain of triadin 1 was localized to a single KEKE motif comprised of 25 amino acids. Alanine mutagenesis within this motif demonstrated that the critical amino acids of triadin binding to calsequestrin are the even-numbered residues Lys(210), Lys(212), Glu(214), Lys(216), Gly(218), Gln(220), Lys(222), and Lys(224). Replacement of the odd-numbered residues within this motif by alanine had no effect on calsequestrin binding to triadin. The results suggest a model in which residues 210-224 of triadin form a beta-strand, with the even-numbered residues in the strand interacting with charged residues of calsequestrin, stabilizing a "polar zipper" that links the two proteins together. This small, highly charged beta-strand of triadin may tether calsequestrin to the junctional face membrane, allowing calsequestrin to sequester Ca(2+) in the vicinity of the ryanodine receptor during Ca(2+) uptake and Ca(2+) release.

Amino Acid Sequence↗

PubMed2000

Remodelling of ionic currents in hypertrophied and failing hearts of transgenic mice overexpressing calsequestrin.

Overexpression of cardiac calsequestrin (CSQ) impairs Ca2+ signalling in murine myocytes, leading to marked cardiac hypertrophy. Here we report on contractile, histological and electrophysiological changes accompanying the development of cardiac hypertrophy and failure in CSQ-overexpressing mice. CSQ mice developed contractile dysfunction after 60 days of age, with only 40% survival at 6 months. Four- to 6-month-old CSQ mice revealed biventricular dilatation, cardiomyocyte hypertrophy, patchy interstitial fibrosis and tissue calcifications. Cardiac hypertrophy of CSQ mice was accompanied by progressive P-R and Q-T interval prolongation, conduction blocks, 2-fold prolongation of the ventricular action potential and increased cellular membrane capacitance. Remodelling of ionic currents included marked reduction of both density and absolute magnitude of transient outward (Ito) and inward rectifying (IK1) K+ currents. The density, but not the absolute magnitude, of basal and isoproterenol (isoprenaline)-stimulated Ca2+ current (ICa) was decreased by 42% and the inactivation kinetics of ICa were significantly slowed. Na+ current density was suppressed by 50%, but its steady-state activation and inactivation were shifted to more positive potentials. The density of Na+-Ca2+ exchange current was increased by 35%. In CSQ but not in control myocytes dialysed with cAMP, isoproterenol continued to enhance ICa. This apparent lower responsiveness of ICa to cAMP could be reversed by the non-hydrolysable cAMP analogue 8-Br-cAMP or the phosphodiesterase inhibitor IBMX, suggesting high phosphodiesterase activity of CSQ myocytes. In young CSQ mice (< 60 days) with compensated cardiac hypertrophy, only Ito was significantly suppressed. All other currents remained relatively intact. An increase in cardiac Ca2+-storage capability by overexpression of CSQ results in a dilated cardiomyopathy with tissue fibrosis, calcifications, impaired beta-adrenergic signalling and progressive remodelling of ionic currents. The extent of the changes in ionic currents was age dependent.

Action Potentials↗

PubMed2000

Modulation of focal and global Ca2+ release in calsequestrin-overexpressing mouse cardiomyocytes.

1. Focal and global Ca2+ releases were monitored in voltage-clamped control and hypertrophied calsequestrin (CSQ)-overexpressing mouse cardiomyocytes, dialysed with fluo-3, using rapid (120-240 frames s-1) two-dimensional confocal imaging. 2. Spontaneous focal Ca2+ releases (Ca2+ sparks) were absent or significantly reduced in frequency in hypertrophied myocytes of CSQ-overexpressing mice compared to their age-matched controls. Sporadic Ca2+ sparks seen in CSQ-overexpressing myocytes had intensities and durations similar to those of controls although quantitative analysis showed a trend towards more diffuse focal releases. 3. Activation of Ca2+ current (ICa) failed to produce the typical sarcomeric Ca2+ striping pattern consistently seen in control myocytes. Instead, focal Ca2+ releases appeared as a disorganized patchwork of diffuse or 'woolly' fluorescence signals, resulting in slowly developing and reduced global Ca2+ transients. 4. Although the density of ICa in CSQ-overexpressing myocytes was only slightly smaller than that of controls, the inactivation kinetics of the current were greatly reduced, consistent with the much smaller rate of rise of cytosolic Ca2+. 5. Enhancement of ICa by elevation of [Ca2+]o from 2 to 10 mM or addition of 3 microM isoproterenol (isoprenaline) failed to normalize the frequency of spontaneous Ca2+ sparks at rest or the pattern and the magnitude of ICa-gated Ca2+ transients. Isoproterenol was somewhat more effective than elevation of [Ca2+]o. 6. In sharp contrast, low (0.5 mM) caffeine concentrations that produced no measurable effects on ICa or Ca2+ transients in control myocytes, re-established spontaneous focal Ca2+ releases in CSQ-overexpressing cells, triggered large ICa-gated cellular Ca2+ transients, and strongly enhanced the kinetics of inactivation of ICa. 7. Our data suggest that impaired Ca2+ signalling in CSQ-overexpressing myocytes results from reduced co-ordination and decreased frequency of Ca2+ sparks. The impaired Ca2+ signalling could not be restored by procedures that increased ICa, but was mostly restored in the presence of caffeine, which may alter the Ca2+ sensitivity of the ryanodine receptor.

Adrenergic beta-Agonists↗