Maximum Velocity Of Ca2 Research Articles

Ablation of sarcolipin enhances sarcoplasmic reticulum calcium transport and atrial contractility

Sarcolipin is a novel regulator of cardiac sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) and is expressed abundantly in atria. In this study we investigated the physiological significance of sarcolipin in the heart by generating a mouse model deficient for sarcolipin. The sarcolipin-null mice do not show any developmental abnormalities or any cardiac pathology. The absence of sarcolipin does not modify the expression level of other Ca2+ handling proteins, in particular phospholamban, and its phosphorylation status. Calcium uptake studies revealed that, in the atria, ablation of sarcolipin resulted in an increase in the affinity of the SERCA pump for Ca2+ and the maximum velocity of Ca2+ uptake rates. An important finding is that ablation of sarcolipin resulted in an increase in atrial Ca2+ transient amplitudes, and this resulted in enhanced atrial contractility. Furthermore, atria from sarcolipin-null mice showed a blunted response to isoproterenol stimulation, implicating sarcolipin as a mediator of beta-adrenergic responses in atria. Our study documented that sarcolipin is a key regulator of SERCA2a in atria. Importantly, our data demonstrate the existence of distinct modulators for the SERCA pump in the atria and ventricles.

Free radical-induced protein modification and inhibition of Ca2+-ATPase of cardiac sarcoplasmic reticulum.

The effect of oxidative stress on the Ca2+-ATPase activity, lipid peroxidation and protein modification of cardiac sarcoplasmic reticulum (SR) membranes was investigated. Isolated SR vesicles were exposed to FeSO4/EDTA (0.2 micromol Fe2+ per mg of protein) at 37 degrees C for 1 h in the presence or absence of antioxidants. FeSO4/EDTA decreased the maximum velocity of Ca2+-ATPase reaction without a change of affinity for Ca2+ or Hill coefficient. Treatment with radical-generating system led also to conjugated diene formation, loss of sulfhydryl groups, changes in tryptophan and bityrosine fluorescences and to production of lysine conjugates with lipid peroxidation end-products. Lipid antioxidants butylated hydroxytoluene (BHT) and stobadine partially prevented inhibition of Ca2+-ATPase and decrease in tryptophan fluorescence, while the loss of -SH groups and formation of bityrosines or lysine conjugates were completely prevented. Glutathione also partially protected Ca2+-ATPase activity and decreased formation of bityrosine, but it was not able to prevent oxidative modification of tryptophan and lysine. These findings suggest that combination of amino acid modifications, rather than oxidation of amino acids of one kind, is responsible for inhibition of SR Ca2+-ATPase activity.

Targeted overexpression of the sarcoplasmic reticulum Ca2+-ATPase increases cardiac contractility in transgenic mouse hearts.

Cardiac hypertrophy and heart failure are known to be associated with a reduction in Ca2+-ATPase pump levels of the sarcoplasmic reticulum (SR). To determine whether, and to what extent, alterations in Ca2+ pump numbers can affect contraction and relaxation parameters of the heart, we have overexpressed the cardiac SR Ca2+-ATPase specifically in the mouse heart using the alpha-myosin heavy chain promoter. Analysis of 2 independent transgenic lines demonstrated that sarco(endo)plasmic reticulum Ca2+-ATPase isoform (SERCA2a) mRNA levels were increased 3.88+/-0. 4-fold and 7.90+/-0.2-fold over those of the control mice. SERCA2a protein levels were increased by 1.31+/-0.05-fold and 1.54+/-0. 05-fold in these lines despite high levels of mRNA, suggesting that complex regulatory mechanisms may determine the SERCA2a pump levels. The maximum velocity of Ca2+ uptake (Vmax) was increased by 37%, demonstrating that increased pump levels result in increased SR Ca2+ uptake function. However, the apparent affinity of the SR Ca2+-ATPase for Ca2+ remains unchanged in transgenic hearts. To evaluate the effects of overexpression of the SR Ca2+ pump on cardiac contractility, we used the isolated perfused work-performing heart model. The transgenic hearts showed significantly higher myocardial contractile function, as indicated by increased maximal rates of pressure development for contraction (+dP/dt) and relaxation (-dP/dt), together with shortening of the normalized time to peak pressure and time to half relaxation. Measurements of intracellular free calcium concentration and contractile force in trabeculae revealed a doubling of Ca2+ transient amplitude, with a concomitant boost in contractility. The present study demonstrates that increases in SERCA2a pump levels can directly enhance contractile function of the heart by increasing SR Ca2+ transport.

Anti-phospholamban and protein kinase A alter the Ca2+ sensitivity and maximum velocity of Ca2+ uptake by the cardiac sarcoplasmic reticulum.

The activity of the SERCA2a Ca2+ pump in the sarcoplasmic reticulum (SR) of cardiac muscle is inhibited by phospholamban. When phospholamban is phosphorylated by cyclic-AMP-dependent protein kinase (PKA) this inhibition is relieved. It is generally agreed that this results in an increase in the Ca2+ sensitivity of the SR Ca2+ pump; however, some investigators have also reported an increase in the maximum velocity of the pump. We have used a sensitive fluorescence method to measure net Ca2+ uptake by native cardiac SR vesicles and compared the effects of a constitutively active subunit of PKA (cPKA) with those of a monoclonal antibody (A1) that binds to phospholamban and is thought to mimic the effect of phosphorylation. Both the Ca2+ sensitivity and the maximum velocity of uptake were increased by cPKA and by A1. The effects of cPKA and A1 on uptake velocity were only slightly additive. No changes in uptake were detected with denatured cPKA or denatured A1. These results indicate that the functional effect of phospholamban phosphorylation is to increase both the Ca2+ sensitivity and the maximum velocity of net Ca2+ uptake into the SR.

Effect of ischemia and reperfusion on sarcoplasmic reticulum calcium uptake.

To investigate the mechanism underlying postischemic cardiac dysfunction (myocardial stunning), contractility and adenine nucleotide metabolism were studied in three groups of isolated perfused rabbit hearts (control, ischemic, and reperfused), whereas Ca2+ uptake by the sarcoplasmic reticulum (SR) was measured in homogenates obtained from them. The hearts were Langendorff-perfused under constant pressure with Krebs-Henseleit solution at 37 degrees C. Global normothermic ischemia was produced by closing the perfusion line. In the reperfused group, after 15 minutes of ischemia, Krebs-Henseleit solution was perfused for 10 minutes. Developed left ventricular pressure (control, 104 +/- 6.3 mm Hg) and left ventricular dP/dt (2,063 +/- 256.6 mm Hg.sec-1) were significantly decreased in reperfused hearts (left ventricular pressure, 78 +/- 5.9 mm Hg; left ventricular dP/dt, 1,339 +/- 216.3 mm Hg.sec-1). Myocardial ATP content (control, 13.6 +/- 0.98 mumol/g dry wt) decreased during ischemia (4.5 +/- 1.23 mumol/g) but was restored to control level on reperfusion (11.8 +/- 0.68 mumol/g). Maximum velocity of Ca2+ uptake by the SR (Vmax) (control, 49.3 +/- 2.54 nmol.min-1 x mg-1) was significantly depressed by ischemia (36.3 +/- 1.94 nmol.min-1 x mg-1) but was restored to the control value after a 10-minute reperfusion (45.3 +/- 0.79 nmol.min-1 x mg-1). Apparent dissociation constant KCa and the Hill coefficient for Ca2+ uptake were not different between control, ischemia, and reperfusion. To test for the possible role of the SR Ca(2+)-release channel in the effect of ischemia and reperfusion, we measured Ca2+ uptake after incubation of homogenates with 610 microM ryanodine.(ABSTRACT TRUNCATED AT 250 WORDS)