Effect of reduced expression of sorcin on Ca2+ handling, in vivo heart function and transcriptional regulation

An adequate and efficient Ca2+ handling is the essential condition for effective functioning of the heart. Decreased peak systolic Ca2+ with prolongation of the duration of Ca2+ transient, slower rates of SR Ca2+ uptake and various other alterations in Ca2+ efflux leading to elevation in diastolic Ca2+ are the key features of the failing heart. These key alterations in heart failure necessitate further evaluation of proteins involved in these regulatory mechanisms. Sorcin, a penta E-F hand family protein associates with cardiac ryanodine receptors, L-type Ca2+ channel as well as SR Ca2+ ATPase and modulates excitation-contraction coupling in the heart. The present thesis aims at understanding the role of sorcin in calcium handling, the effect of decreased level of sorcin on remodeling of the heart and subsequent transcriptional regulation, by using the adenoviral antisense RNA approach. A decrease in the endogenous sorcin expression (75% on the mRNA level and 53% on the protein level) was obtained in the adult rat cardiomyocytes. The decreased amount of sorcin resulted in reduced cell contractility and significantly depressed Ca2+ transient amplitude accompanied with the decreased rate of relaxation in the transfected cardiomyocytes. The increase in stimulation frequency was associated with a negative force-frequency relationship in cardiomyocytes with depressed sorcin, mimicking the behavior exhibited by the failing human cardiomyocytes. However, the β-adrenergic stimulation was unaltered. An oxalate facilitated Ca2+ uptake assay indicated decreased Ca2+ uptake by the sarcoplasmic reticulum in the cardiomyocytes with decreased expression of sorcin. Moreover, incubating the sarcoplasmic reticulum vesicle preparations with recombinant sorcin (1 µM) enhanced the SR Ca2+ uptake and brought it back to the control level. The depressed expression of SR Ca2+ ATPase on the mRNA as well as protein level was also observed. The expression of ryanodine receptors, triadin and phospholamban was unaltered, while a mild increase in the expression of FKBP12.6 was observed. In vivo downregulation of sorcin, achieved by the catheter based cardiac specific gene delivery resulted in severe chamber dilation. Echocardiography revealed ventricular enlargement, decreased heart rate and increased chamber dimension indicating dilated cardiomyopathy in the hearts with depleted levels of sorcin. After 14 days the animals were sacrificed and an increase in the heart weight was observed. In addition, upregulation of calcineurin expression and higher phosphatase activity was observed which was accompanied with increased dephosphorylation of NF-ATC3. GATA4 expression was significantly upregulated in the antisense sorcin transfected cardiomyocytes. The mRNA expression of hypertrophic marker gene β-myosin heavy chain was upregulated, while expression of atrial natriuretic factor and B-type natriuretic peptide was unaltered. In conclusion, using the antisense mRNA approach for sorcin it can be concluded that downregulation of sorcin diminishes the cardiac contractile performance and leads to the ventricular remodeling of the heart. This process is at least partially due to the activation of calcineurin-NFAT signaling pathway. The antisense approach proves to be a valuable tool to identify targets that modulate cardiac contractility and may open new avenues for the treatment of myocardial diseases with diminished cardiac output such as heart failure. An adequate and efficient Ca2+ handling is the essential condition for effective functioning of the heart. Decreased peak systolic Ca2+ with prolongation of the duration of Ca2+ transient, slower rates of SR Ca2+ uptake and various other alterations in Ca2+ efflux leading to elevation in diastolic Ca2+ are the key features of the failing heart. These key alterations in heart failure necessitate further evaluation of proteins involved in these regulatory mechanisms. Sorcin, a penta E-F hand family protein associates with cardiac ryanodine receptors, L-type Ca2+ channel as well as SR Ca2+ ATPase and modulates excitation-contraction coupling in the heart. The present thesis aims at understanding the role of sorcin in calcium handling, the effect of decreased level of sorcin on remodeling of the heart and subsequent transcriptional regulation, by using the adenoviral antisense RNA approach. A decrease in the endogenous sorcin expression (75% on the mRNA level and 53% on the protein level) was obtained in the adult rat cardiomyocytes. The decreased amount of sorcin resulted in reduced cell contractility and significantly depressed Ca2+ transient amplitude accompanied with the decreased rate of relaxation in the transfected cardiomyocytes. The increase in stimulation frequency was associated with a negative force-frequency relationship in cardiomyocytes with depressed sorcin, mimicking the behavior exhibited by the failing human cardiomyocytes. However, the β-adrenergic stimulation was unaltered. An oxalate facilitated Ca2+ uptake assay indicated decreased Ca2+ uptake by the sarcoplasmic reticulum in the cardiomyocytes with decreased expression of sorcin. Moreover, incubating the sarcoplasmic reticulum vesicle preparations with recombinant sorcin (1 µM) enhanced the SR Ca2+ uptake and brought it back to the control level. The depressed expression of SR Ca2+ ATPase on the mRNA as well as protein level was also observed. The expression of ryanodine receptors, triadin and phospholamban was unaltered, while a mild increase in the expression of FKBP12.6 was observed. In vivo downregulation of sorcin, achieved by the catheter based cardiac specific gene delivery resulted in severe chamber dilation. Echocardiography revealed ventricular enlargement, decreased heart rate and increased chamber dimension indicating dilated cardiomyopathy in the hearts with depleted levels of sorcin. After 14 days the animals were sacrificed and an increase in the heart weight was observed. In addition, upregulation of calcineurin expression and higher phosphatase activity was observed which was accompanied with increased dephosphorylation of NF-ATC3. GATA4 expression was significantly upregulated in the antisense sorcin transfected cardiomyocytes. The mRNA expression of hypertrophic marker gene β-myosin heavy chain was upregulated, while expression of atrial natriuretic factor and B-type natriuretic peptide was unaltered. In conclusion, using the antisense mRNA approach for sorcin it can be concluded that downregulation of sorcin diminishes the cardiac contractile performance and leads to the ventricular remodeling of the heart. This process is at least partially due to the activation of calcineurin-NFAT signaling pathway. The antisense approach proves to be a valuable tool to identify targets that modulate cardiac contractility and may open new avenues for the treatment of myocardial diseases with diminished cardiac output such as heart failure

(Benzophenone imine-κN)­chlorido(hydrido­tripyrazolyl­borato)­(triphenyl­phosphine)ruthenium(II) diethyl ether solvate

The reaction of RuCl(Tp)(Ph3P)2, where Tp is [(CH)3N2]3BH, with benzophenone imine leads to the formation of the title compound, [Ru(C9H10BN6)Cl(C13H11N)(C18H15P)]·C4H10O. The environment about the Ru atom corresponds to a slightly distorted octa­hedron and the bite angle of the Tp ligand produces an average N—Ru—N angle of 86.3 (9)°. The three Ru—N(Tp) bond lengths [2.117 (2), 2.079 (2) and 2.084 (2) Å] are slightly longer than the average distance (2.038 Å) in other ruthenium–Tp complexes

(O,O′-Diethyl dithio­phosphato-κ2 S,S′)(hydridotripyrazol-1-ylborato-κ3 N 2,N 2′,N 2′′)(triphenyl­phosphine-κP)ruthenium(II)

Reaction of [Ru(Tp)Cl(PPh3)2] {where Tp is hydridotri­pyrazol­yl­borate, BH[C3H3N2)3)]} with NH4[S2P(OEt)2] in methanol afforded the title compound, [Ru(C9H10BN6)(C4H10O2PS2)(C18H15P)], in which the RuII ion is in a slightly disorted octa­hedral coordination environment. The [S2P(OEt)2]− ligand coordinates in a chelating mode with two similar Ru—S bond lengths and a slightly acute S—Ru—S angle. The atoms of both –OCH2CH3 groups of the diethyl dithio­phosphate ligand are disordered over two sites with approximate occupancies of 0.76 and 0.24

Primordial Black Hole Formation in a Double Inflation Model in Supergravity

It has been recently pointed out that the initial value problem in new inflation models is naturally solved by supergravity effects if there exists a pre-inflation before the new inflation. We study this double inflation model in details and find that density fluctuations on small cosmological scales are much larger than those on large scales due to peculiar property of the new inflation. We show that this results in production of primordial black holes which have $\sim 1 M_{\odot}$ masses in a certain parameter region of the double inflation model. We stress that these black holes may be identified with MACHOs observed in the halo of our galaxy.Comment: 19 pages (RevTeX file), KUNS-147

Application of Fuzzy Soft Set Theory in Day to Day Problems

In our daily life we often face some problems in which the right decision making is highly essential.But most of these cases we become confused about the right solution.To obtain the best feasible solution of these problems we have to consider various parameters relating to the solution.For this we can use the best mathematical tool called Fuzzy soft set theory.In this paper we select a burning problem for the parents and successfully applied the Fuzzy soft set theory in decision making.

Mechanism of Deletion Removing All Dystrophin Exons in a Canine Model for DMD Implicates Concerted Evolution of X Chromosome Pseudogenes

Duchenne muscular dystrophy (DMD) is a lethal, X-linked, muscle-wasting disorder caused by mutations in the large, 2.4-Mb dystrophin gene. The majority of DMD-causing mutations are sporadic, multi-exon, frameshifting deletions, with the potential for variable immunological tolerance to the dystrophin protein from patient to patient. While systemic gene therapy holds promise in the treatment of DMD, immune responses to vectors and transgenes must first be rigorously evaluated in informative preclinical models to ensure patient safety. A widely used canine model for DMD, golden retriever muscular dystrophy, expresses detectable amounts of near full-length dystrophin due to alternative splicing around an intronic point mutation, thereby confounding the interpretation of immune responses to dystrophin-derived gene therapies. Here we characterize a naturally occurring deletion in a dystrophin-null canine, the German shorthaired pointer. The deletion spans 5.6 Mb of the X chromosome and encompasses all coding exons of the DMD and TMEM47 genes. The sequences surrounding the deletion breakpoints are virtually identical, suggesting that the deletion occurred through a homologous recombination event. Interestingly, the deletion breakpoints are within loci that are syntenically conserved among mammals, yet the high homology among this subset of ferritin-like loci is unique to the canine genome, suggesting lineage-specific concerted evolution of these atypical sequence elements