Phosphorylation by protein kinase C and the responsiveness of Mg2+-ATPase to Ca2+ of myofibrils isolated from stunned and non-stunned porcine myocardinm

Previously we showed in an in situ porcine model that the thiadiazinone derivative [+]EMD 60263, a Ca2+ sensitizer without phosphodiesterase III inhibitory properties, increased contractility more profoundly in stunned than in non-stunned myocardium. This finding was consistent with the observed leftward shifts of the pCa2+/Mg2+-ATPase curves of isolated myofibrils induced by [+]EMD 60263. The aim of the present investigation was to study the possible involvement of protein kinase C in the mechanism of reduced Ca2+ responsiveness of myofilaments during stunning. No differences were observed in the maximal activity of the Ca2+-stimulated Mg2+-ATPase and in the pCa50 of myofibrils isolated from non-stunned and stunned myocardium. After phosphorylation with [gamma-32P]-ATP and excess of purified rat brain protein kinase C, the myofibrils were separated on sodiumdodecylsulphate-polyacrylamide gelectrophoresis and the 32P incorporation counted by the Molecular Imager. Ca2+/ phosphatidylserine/sn-1,2 diolein-dependent 32P incorporation catalyzed by excess of purified rat brain protein kinase C in C-protein, TnT and TnI subunits did not show any differences between myofibrils from non-stunned and stunned myocardium. However, protein kinase C-induced phosphorylation of myofibrils isolated from ventricular myocardium of sham-operated pigs resulted in a marked leftward shift of the PCa50 from 6.03 ± 0.04 to 6.44 ± 0.06 (p < 0.05), while porcine heart cyclic AMP-dependent protein kinase-induced phosphorylation resulted in an expected small rightward shift to 5.97, although statistical significance was not reached. Protein kinase C-induced phosphorylation also stimulated (80%) the maximal myofibrillar Mg2+-ATPase activity. [+]EMD 60263 (3 μM) produced a leftward shift of the myofibrillar pCa2+/Mg2+-ATPase curve which was unaffected by prior protein kinase C-induced phosphorylation. In conclusion, the findings with isolated myofibrils from myocardium of anaesthetized open-chest pigs indicate that protein kinase C might be involved in the mechanism of reduced Ca2+ responsiveness of myofilaments in stunned myocardium. However, at this stage no differences could be found between the maximal activity of the Ca2+-stimulated Mg2+ -ATPase, the pCa50 and the degree of phosphorylation of myofibrils isolated from stunned and non-stunned myocardium

Evaluating the Precision of Estimation of an Inequality Constrained Ridge Regression Estimator by the Bootstrap Methods

markdownabstractAbstract The effects of nisoldipine (0.1 μg/kg/min; n = 9) or its solvent (n = 9) were studied in pigs, in which left anterior descending coronary artery (LADCA) blood flow in both groups was reduced to 20% of baseline for 60 min and reperfused for 2 hr. Infusions were started at 30 min of ischaemia and lasted throughout reperfusion. In both groups, flow reduction abolished regional contractile function and caused similar decreases in the level of creatine phosphate (CP; by 70%) and the energy charge (from 0.91 to 0.69), mean arterial blood pressure (by 25%), LVdP/dtmax (by 30%) and cardiac output (by 30%). During ischaemia LADCA blood flow slightly increased (from 14 ± 8 to 24 ± 6mL/min/ 100 g; P < 0.05) in the nisoldipine-treated animals, resulting in an increase in CP to 91 ± 24% of baseline and preventing further decreases in energy charge, as observed in the solvent-treated animals. After 2 hr of reperfusion in neither group return of contractile function of the post-ischaemic myocardium was observed. Post-ischaemic blood flow in the nisoldipine-treated pigs increased from 24 ± 6 mL/min/100 g to 76 ± 14 mL/min/100 g and from 19 ± 6 mL/min/100 g to 41 ± 6 mL/min/100 g in the solvent-treated animals (P < 0.05) after 2 hr of reperfusion. Myocardial work was significantly higher in the nisoldipinetreated animals (111 ± 15 mmHg.L/min as compared to 69 ± 14 mmHg.L/min in the solvent-treated pigs after 2 hr of ischaemia). The energy charge of the post-ischaemic myocardium was similar for both groups (0.84 ± 0.02 for the nisoldipine-treated and 0.83 ± 0.03 for the solvent-treated animals). The rate of sarcoplasmic reticular Ca2+ uptake of the non-ischaemic segment of the nisoldipine-treated animals was 61% higher (P < 0.05) than that of the solvent-treated animals. In the post-ischaemic myocardium similar rates of Ca2+ uptake were found in both groups, but the activities were markedly lower as compared to the non-ischaemic myocardium. It is concluded that nisoldipine increases blood flow during reperfusion, which may have been caused by coronary vasodilatation. However, attenuation of the “no-reflow” phenomenon also contributed, since more rapid rephosphorylation of ADP leading to an increase in CP during ischaemia may have preserved jeopardized cells. Moreover, nisoldipine increases the sarcoplasmic reticular Ca2+ pump activity independent of ischaemia, which may have contributed in reducing the Ca2+ overload. Abbreviation

In vitro analysis of SERCA2 gene regulation in hypertrophic cardiomyocytes and increasing transfection efficiency by gene-gun biolistics

The transcriptional downregulation of the SERCA2 gene is studied using neonatal rat cardiomyocytes stimulated with endothelin-1 to induce hypertrophy. Liposome-based transfection of cells with a 1.9 kb SERCA2 promoter fragment directed expression of a reporter gene identical to the downregulation of genomic SERCA2 expression by endothelin-1. Results of a new gene gun technology for transient transfection of cardiomyocytes with a RSVβ-galactosidase construct are reported. This new method for propelling DNA-coated gold beads into cardiomyocytes is extremely suitable for directly testing promoter/reporter gene DNA constructs since the transfection efficiency (approximately 10%) appears to be higher than traditional transfection methods

Eicosapentaenoic acid incorporation in membrane phospholipids modulates receptor-mediated phospholipase C and membrane fluidity in rat ventricular myocytes in culture

The influence of increased incorporation of linoleic acid (18:2n-6) and eicosapentaenoic acid (20:5n-3) in membrane phospholipids on receptor-mediated phospholipase Cβ (PLC-β) activity in cultured rat ventricular myocytes was investigated. For this purpose, cells were grown for 4 days in control, stearic acid (18:0)/oleic acid (18:1n-9), 18:2n-6 and 20:5n-3 enriched media, and subsequently assayed for the basal- and phenylephrine- or endothelin-1-induced total inositol phosphate formation. The various fatty acid treatments resulted in the expected alterations of fatty acid composition of membrane phospholipids. In 18:2n-6-treated cells, the incorporation of this 18:2n-6 in the phospholipids increased from 17.1 mol % in control cells to 38.9 mol %. In 20:5n-3-treated cells, incorporation of 20: 5n-3 and docosapentaenoic acid (22:5n-3) in the phospholipids increased from 0.5 and 2.7 mol % in control cells to 23.2 and 9.7 mol %, respectively. When 20:5n-3-treated cells were stimulated with phenylephrine or endothelin-1, the inositolphosphate production decreased by 33.2% and increased by 43.4%, respectively, as compared to cells grown in control medium. No efffects were seen in 18:2n-6-treated cells. When 18:0/18:1n-9-treated cells were stimulated with endothelin-1, inositolphosphate formation increased by 26.4%, whereas phenylephrine-stimulated inositolphosphate formation was not affected. In saponin-permeabilized cells, that were pre-treated with 20:5n-3, the formation of total inositolphosphates after stimulation with GTPγS, in the presence of Ca2+, was inhibited 19.3%. This suggests that the 20:5n-3 effect on intact cardiomyocytes could be exerted either on the level of agonist-receptor, receptor-GTP-binding-protein coupling or GTP-binding-protein-PLC-β interaction. Investigation of the time course of saponin-induced permeabilization of the cardiomyocytes, measured by the release of lactate dehydrogenase, unmasked a slight decrease in the rate of permeabilization by 20:5n-3 pretreatment, indicating a protective effect. This led the authors to measure the cholesterol/phospholipid molar ratio, the double bond index of membrane phospholipids, and the membrane fluidity; the latter by using a diphenylhexatriene probe. In 20: 5n-3-pretreated cells, a strong increase in the cholesterol/phospholipid molar ratio (from 0.23 to 0.39), a marked increase in the double bond index (from 1.76 to 2.33), and a slight decrease in fluidity (steady-state anisotropy r(ss) of the diphenylhexatriene probe increased from 0.196 to 0.217) were observed. Thus, treatment of cardiomyocytes for 4 days with 20:5n-3, but not with 18:2n-6, causes alterations of receptor-mediated phospholipase Cβ activity. A causal relationship may exist between the 20:5 n-3-induced alterations of the physicochemical properties in the bilayer and of the agonist-stimulated phosphatidylinositol cycle activity

A Testis-Specific Chaperone and the Chromatin Remodeler ISWI Mediate Repackaging of the Paternal Genome

During spermatogenesis, the paternal genome is repackaged into a non-nucleosomal, highly compacted chromatin structure. Bioinformatic analysis revealed that Drosophila sperm chromatin proteins are characterized by a motif related to the high-mobility group (HMG) box, which we termed male-specific transcript (MST)-HMG box. MST77F is a MST-HMG-box protein that forms an essential component of sperm chromatin. The deposition of MST77F onto the paternal genome requires the chaperone function of tNAP, a testis-specific NAP protein. MST77F, in turn, enables the stable incorporation of MST35Ba and MST35Bb into sperm chromatin. Following MST-HMG-box protein deposition, the ATP-dependent chromatin remodeler ISWI mediates the appropriate organization of sperm chromatin. Conversely, at fertilization, maternal ISWI targets the paternal genome and drives its repackaging into de-condensed nucleosomal chromatin. Failure of this transition in ISWI mutant embryos is followed by mitotic defects, aneuploidy, and haploid embryonic divisions. Thus, ISWI enables bi-directional transitions between two fundamentally different forms of chromatin

Intracoronary trimetazidine does not improve recovery of regional function in a porcine model of repeated ischemia

We evaluated the effect of trimetazidine (TMZ) on recovery of regional cardiac function in anesthetized open-chest pigs, subjected to fifteen 2-minute occlusions of the left anterior descending coronary artery, separated by 2 minutes of reperfusion and a 120-minute recovery period. Regional myocardial function was evaluated by sonomicrometry-derived segment lengthening and the area enclosed by the left ventricular pressure-segment length loop (external work, EW) in animals, which received either an intracoronary infusion of TMZ (33 μg/kg/min, n=6) or saline (1 ml/min, n=7), starting 15 minutes before the first occlusion and ending 2 minutes after the 15th occlusion. In addition, myocardial malondialdehyde production to evaluate oxygen free radical production, oxygen consumption, and the ATP, ADP, and AMP content, as well as the energy charge, were determined at regular time intervals. In control pigs the sequences of occlusion-reperfusion did not affect systemic hemodynamics, except for the LVdP/dtmax, which decreased by 11% during the interventions and did not recover during the following reperfusion period of 2 hours (78% of baseline, p<0.05). Systolic segment length shortening and EW were increased at the end of the first occlusion-reperfusion cycle, decreased gradually during the remainder of the occlusion-reperfusion periods, and did not improve during the recovery period. Energy charge and myocardial blood flow were not impaired, but oxygen consumption was decreased during the recovery period. The malondialdeyde data did not provide evidence for production of oxygen free radicals. TMZ decreased LVdP/dtmax by 6% (p<0.05) and caused a twofold increase in postsystolic segment shortening (p<0.05) before the first occlusion, but did not influence the hemodynamic responses, the changes in regional cardiac function, and the metabolic events produced by repetitive regional ischemia