Regulation of Ca2+ handling by phosphorylation status in mouse fast- and slow-twitch skeletal muscle fibers.

The effects of phosphorylation status on Ca2+ release and Ca2+ removal were studied in fast-twitch flexor digitorum brevis and slow-twitch soleus skeletal muscle fibers enzymatically isolated from wild-type and phospholamban knockout (PLBko) mice. In all fibers the adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA) inhibitor H-89 decreased the peak amplitude of the intracellular Ca2+ concentration ([Ca2+]) transient for a single action potential, and the PKA activator dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) reversed this effect, indicating modulation of Ca2+ release by phosphorylation status in all fibers. H-89 decreased the decay rate constant of the [Ca2+] transient and DBcAMP reversed this effect only in phospholamban-expressing fibers (wild-type soleus), indicating modulation of Ca2+ removal only in the presence of phospholamban.

In vitro and in vivo promoter analyses of the mouse phospholamban gene.

To determine the mechanisms responsible for regulation of the phospholamban (PLB) gene expression, a critical regulatory phosphoprotein in cardiac muscle, the mouse PLB gene was isolated and promoter analysis was performed in vitro and in vivo. The PLB gene consists of two exons separated by a single large intron. Deletion analysis revealed that a 7-kb 5' flanking fragment (including exon 1, the entire intron and part of exon 2) was necessary for maximal transcriptional activity in H9c2 and L6 cell lines.

Modulation of cardiac Ca2+ channels by isoproterenol studied in transgenic mice with altered SR Ca2+ content.

Phospholamban (PLB) ablation is associated with enhanced sarcoplasmic reticulum (SR) Ca2+ uptake and attenuation of the cardiac contractile responses to beta-adrenergic agonists. In the present study, we compared the effects of isoproterenol (Iso) on the Ca2+ currents (ICa) of ventricular myocytes isolated from wild-type (WT) and PLB knockout (PLB-KO) mice. Current density and voltage dependence of ICa were similar between WT and PLB-KO cells.

Phospholamban: a protein coming of age.

Phospholamban is a major regulator of the kinetics of cardiac contractility, through its ability to regulate the function of the cardiac SR Ca(2+)-pump and thus the SR Ca2+ load. In vitro expression studies have provided significant information on the structure/function of the phospholamban/Ca(2+)-pump interaction. Furthermore, the generation of genetic animal models with altered phospholamban expression levels have permitted a through understanding of the physiological role of this regulatory phosphoprotein.

Monomeric phospholamban overexpression in transgenic mouse hearts.

Phospholamban, a prominent modulator of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity and basal contractility in the mammalian heart, has been proposed to form pentamers in native SR membranes. However, the monomeric form of phospholamban, which is associated with mutating Cys41 to Phe41, was shown to be as effective as pentameric phospholamban in inhibiting Ca2+ transport in expression systems. To determine whether this monomeric form of phospholamban is also functional in vivo, we generated transgenic mice with cardiac-specific overexpression of the mutant (Cys41-->Phe41) phospholamban.

Calcium sparks and excitation-contraction coupling in phospholamban-deficient mouse ventricular myocytes.

1. We examined [Ca2+]i and L-type Ca2+ channel current (ICa) in single cardiac myocytes to determine how the intracellular protein phospholamban (PLB) influences excitation-contraction (E-C) coupling in heart. Wild type (WT) and PLB-deficient (KO) mice were used.

Ectopic expression of phospholamban in fast-twitch skeletal muscle alters sarcoplasmic reticulum Ca2+ transport and muscle relaxation.

There are three isoforms of the sarcoplasmic reticulum Ca2+-ATPase; they are known as SERCA1, SERCA2, and SERCA3. Phospholamban is present in tissues that express the SERCA2 isoform and is an inhibitor of the affinity of SERCA2 for calcium. In vitro reconstitution and cell culture expression studies have shown that phospholamban can also regulate SERCA1, the fast-twitch skeletal muscle isoform.

Phospholamban ablation enhances relaxation in the murine soleus.

Phospholamban (PLB) is expressed in slow-twitch skeletal, cardiac, and smooth muscles. Several studies have indicated that it is an important regulator of basal contractility and the stimulatory responses to isoproterenol in the mammalian heart. To determine whether PLB is also a key modulator of slow-twitch skeletal muscle contractility, we examined isometric twitch contractions of isolated, intact soleus muscles from wild-type (WT) and PLB-deficient mice in parallel.

Targeted ablation of the phospholamban gene is associated with a marked decrease in sensitivity in aortic smooth muscle.

Phospholamban (PLB) is a protein associated with the Ca(2+)-ATPase of the sarcoplasmic reticulum (SR) in cardiac, slow-twitch skeletal, and smooth muscle. PLB inhibits the SR Ca(2+)-ATPase in cardiac muscle; this inhibition is relieved on phosphorylation. The role of PLB in smooth muscle contractility is less clear.

beta-Adrenergic regulation of cAMP and protein phosphorylation in phospholamban-knockout mouse hearts.

The stimulatory effects of beta-adrenergic agonists reflect increases in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels and phosphorylation of key regulatory proteins in the heart. One of these phosphoproteins is phospholamban (PLB) in sarcoplasmic reticulum, and ablation of PLB is associated with attenuation of the contractile responses to beta-adrenergic stimulation in the mouse heart. To determine whether this attenuation of beta-stimulation is due to altered phosphorylation characteristics of the other key cardiac phosphoproteins and/or to compensatory responses occurring in the absence of PLB, PLB-knockout and wild-type hearts were perfused and their protein phosphorylation patterns examined.

Phospholamban deficiency alters inactivation kinetics of L-type Ca2+ channels in mouse ventricular myocytes.

Entry of Ca2+ through voltage-dependent L-type Ca2+ channels is critical for contraction in cardiac cells. In recent studies, cells from phospholamban (PLB) knockout (PLB-KO) mouse hearts showed significantly increased basal contractility with enhanced sarcoplasmic reticulum (SR) Ca2+ uptake. To test whether these effects of PLB ablation were associated with alterations of L-type Ca2+ channel function, we compared the properties of Ca2+ channel currents (I(Ca)) in ventricular myocytes isolated from wild-type (WT) and PLB-KO mouse hearts.

Amount of calcium in the sarcoplasmic reticulum: influence on excitation-contraction coupling in heart muscle.

Real-time imaging of the concentration of intracellular calcium ([Ca2+]i) has been carried out in heart cells using confocal imaging and patch-clamp techniques. Here we review recent investigations that used genetically engineered mice that lack phospholamban (PL knockout) to investigate the mechanisms of excitation-contraction (EC) coupling in heart. The heart cells from PL knockout (KO) mice exhibit [Ca2+]i transients that are larger than normal.

The relative phospholamban and SERCA2 ratio: a critical determinant of myocardial contractility.

Phospholamban is a regulatory phosphoprotein which modulates the active transport of Ca2+ by the cardiac sarcoplasmic reticular Ca(2+)-ATPase enzyme (SERCA2) into the lumen of the sarcoplasmic reticulum. Phospholamban, which is a reversible inhibitor of SERCA2, represses the enzyme's activity, and this inhibition is relieved upon phosphorylation of phospholamban in response to beta-adrenergic stimulation. In this way, phospholamban is an important regulator of SERCA2-mediated myocardial relaxation during diastole.

Coordinate regulation of SR Ca(2+)-ATPase and phospholamban expression in developing murine heart.

Phospholamban, the regulator of Ca(2+)-adenosinetriphosphatase (ATPase) activity in cardiac sarcoplasmic reticulum (SR), is an important determinant of basal myocardial performance. To determine whether phospholamban expression is developmentally regulated in the mouse and whether such regulation reflects alterations in Ca2+ pump activity, hearts from different stages of development were processed for molecular biological and biochemical studies. Both phospholamban and Ca(2+)-ATPase mRNAs were approximately 40% of adult (100%) levels at birth and gradually increased to approach adult levels by day 15 of development.

Compensatory mechanisms associated with the hyperdynamic function of phospholamban-deficient mouse hearts.

Phospholamban ablation is associated with significant increases in the sarcoplasmic reticulum Ca(2+)-ATPase activity and the basal cardiac contractile parameters. To determine whether the observed phenotype is due to loss of phospholamban alone or to accompanying compensatory mechanisms, hearts from phospholamban-deficient and age-matched wild-type mice were characterized in parallel. There were no morphological alterations detected at the light microscope level.

Phospholamban: a prominent regulator of myocardial contractility.

Our understanding of the role of phospholamban in cardiac physiology has evolved over the past two decades to the point where this protein is now understood to be a critical repressor of myocardial contractility. Phospholamban, through its inhibitory effects on the affinity of the cardiac sarcoplasmic reticulum Ca2+ pump for Ca2+, represses both the rates of relaxation and contraction in the mammalian heart. These inhibitory effects can be relieved through (1) phospholamban phosphorylation, (2) down-regulation of phospholamban gene expression, and (3) disruption of the phospholamban-Ca(2+)-ATPase interaction.

Simultaneous measurement of intracellular calcium and ventricular function in the phospholamban-deficient mouse heart.

We describe the procedure for the measurement of intracellular calcium (Cai2+) simultaneously with function in the intact mouse heart and report findings in phospholamban-deficient mice. Seven phospholamban-deficient and six age-matched wild-type hearts were perfused retrogradely with oxygenated Krebs-Henseleit solution. Aequorin was injected into the apex of five hearts from each group to characterize Cai2+ transients.

Effect of ablation of phospholamban on dynamics of cardiac myocyte contraction and intracellular Ca2+.

We compared mechanical activity and Ca2+ transients of ventricular myocytes isolated from wild-type and phospholamban (PLB)-deficient mouse hearts in control conditions and during beta-adrenergic stimulation. Compared with wild-type controls, cells isolated from PLB-deficient mouse hearts showed 1) a 2-fold increase in extent of cell shortening, 2) a 3-fold increase in maximal shortening velocity, and 3) a 3.4-fold increase in maximal relengthening velocity.

Phospholamban gene dosage effects in the mammalian heart.

Phospholamban ablation has been shown to result in significant increases in cardiac contractile parameters and loss of beta-adrenergic stimulation. To determine whether partial reduction in phospholamban levels is also associated with enhancement of cardiac performance and to further examine the sensitivity of the contractile system to alterations in phospholamban levels, hearts from wild-type, phospholamban-heterozygous, and phospholamban-deficient mice were studied in parallel at the subcellular, cellular, and organ levels. The phospholamban-heterozygous mice expressed reduced cardiac phospholamban mRNA and protein levels (40 +/- 5%) compared with wild type mice.

Cardiac-specific overexpression of phospholamban alters calcium kinetics and resultant cardiomyocyte mechanics in transgenic mice.

Phospholamban is the regulator of the cardiac sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity and an important modulator of basal contractility in the heart. To determine whether all the SR Ca(2+)-ATPase enzymes are subject to regulation by phospholamban in vivo, transgenic mice were generated which overexpressed phospholamban in the heart, driven by the cardiac-specific alpha-myosin heavy chain promoter. Quantitative immunoblotting revealed a twofold increase in the phospholamban protein levels in transgenic hearts compared to wild type littermate hearts.

Differential changes in cardiac phospholamban and sarcoplasmic reticular Ca(2+)-ATPase protein levels. Effects on Ca2+ transport and mechanics in compensated pressure-overload hypertrophy and congestive heart failure.

The objective of this study was to elucidate the role of the sarcoplasmic reticulum (SR) in the transition from compensated pressure-overload hypertrophy (increased left ventricular [LV] mass, normal LV function, and no pulmonary congestion) to congestive heart failure (increased LV mass, depressed LV function, and pulmonary congestion). To address this issue, the descending thoracic aorta was banded for 4 and 8 weeks in adult guinea pigs, and the changes in isovolumic LV mechanics, SR Ca2+ transport, and SR protein levels were determined and compared with age-matched sham-operated control animals. A subgroup of the 8-week banded animals manifested the congestive heart failure phenotype with diminished developed LV pressure normalized by LV mass, reduced rates of LV pressure development and relaxation, and markedly increased lung weight-to-body weight ratios.

Alterations in sarcoplasmic reticulum calcium uptake, relaxation parameters and their responses to beta-adrenergic agonists in the developing rabbit heart.

Developmental changes in cardiac sarcoplasmic reticulum function, which may reflect alterations in the myocardial rate of relaxation and its responses to beta-adrenergic stimulation, were assessed using fetal, 4-day-old, 21-day-old and adult rabbit hearts. The fetal hearts exhibited the slowest rate of relaxation (-dP/dt) and the lowest Vmax and EC50 of the sarcoplasmic reticulum Ca(2+)-pump for Ca2+ compared to the other age groups. These parameters were similar among the 4-day-old, 21-day-old and adult hearts.

In vivo echocardiographic detection of enhanced left ventricular function in gene-targeted mice with phospholamban deficiency.

We evaluated the ability of M-mode and Doppler echocardiography to assess left ventricular (LV) function reliably and repeatedly in mice and tested whether these techniques could detect physiological alterations in phospholamban (PLB)-deficient mice. Anesthetized wild-type mice (n = 7) and mice deficient in PLB (n = 8) were studied with two-dimensional guided M-mode and Doppler echocardiography using a 9-MHz imaging and 5- to 7.5-MHz Doppler transducer.

Differential phospholamban gene expression in murine cardiac compartments. Molecular and physiological analyses.

Phospholamban, the regulator of the Ca2+ pump in cardiac sarcoplasmic reticulum, is differentially expressed between murine atrial and ventricular muscles. Quantitative analyses of RNA isolated from atrial flaps and ventricular apices indicated that the phospholamban gene transcript copy number is 2.5-fold higher in the ventricle compared with the atrium of the FVB/N mouse and 6-fold higher in the ventricle compared with the atrium of the B6D2/F1 mouse strain.

Effects of Levosimendan, a cardiotonic agent targeted to troponin C, on cardiac function and on phosphorylation and Ca2+ sensitivity of cardiac myofibrils and sarcoplasmic reticulum in guinea pig heart.

A new cardiotonic agent, (R)-[[4-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)-phenyl] hydrazono]propanedinitrile (Levosimendan), has been developed and screened for its ability to bind to cardiac troponin C. In perfused hearts, low concentrations of 0.03 or 0.