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. Interestingly, deletion of a 2.4-kb intronic region, which contained repetitive elements, caused a dramatic increase in CAT activity in both these cell lines. In vivo analysis indicated that the PLB fusion gene containing 7 kb of the 5'-flanking region was capable of cardiac specific gene expression in transgenic mice. Furthermore, these mice exhibited 3-fold higher levels of CAT activity in the ventricles compared with the atria, mimicking endogenous PLB mRNA expression. Our findings suggest that: (a) PLB gene expression may be regulated by the interplay of cis-acting regulatory elements located within the 5' flanking and intronic regions; and (b) the 7-kb upstream region is capable of directing cardiac-specific and compartment-specific expression in vivo.

Elevated extracellular Mg2+ increases Mg2+ buffering through a Ca-dependent mechanism in cardiomyocytes.

To investigate the physiological basis for the pharmacological action of extracellular magnesium (Mg2+O), cultured ventricular myocytes were exposed to either 0.8 mM (physiological) or 5.0 mM Mg2+ (therapeutic concentration) in the presence and absence of metabolic inhibitors. Metabolic inhibition, induced with 1 mM iodoacetate and 1 mM NaCN, was used to liberate Mg2+ from MgATP into the myoplasm, permitting examination of the role of elevated Mg2+O on myoplasmic Mg2+ buffering. The increase in Mg2+ activity observed in the presence of 5 mM Mg2+O was diminished compared with that observed in cells exposed to 0.8 mM Mg2+O. Furthermore, the increase in myoplasmic Mg2+ activity observed in the presence of 5 mM Mg2+O was identical to that reported previously in the absence of extracellular Ca2+. Fura 2 measurements of Ca2+ activity in these experimental conditions suggested that the Ca2+ permeability of cells conditions suggested that the Ca2+ permeability of cells exposed to 5 mM Mg2+ was less than that observed for cells exposed to 0.8 mM Mg2+. Using the Mg2+ buffer coefficient to quantitate cellular Mg2+ buffering, we observed a 63% increase in Mg2+ buffering in cells exposed to 5 mM Mg2+ compared with cells exposed to 0.8 mM Mg2+. This study demonstrates that elevated Mg2+O alters cardiomyocyte myoplasmic Mg2+ activity as the result of increased Mg2+ buffering through a Ca(2+)-sensitive mechanism.

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. Thus, genetic approaches and pharmacological interventions, designed to relieve the phospholamban inhibitory action on the cardiac sarcoplasmic reticulum Ca2+ pump and myocardial relaxation, may prove valuable in reversing the effects of several diseases in the mammalian heart. Such interventions could be designed to inhibit the phospholamban phosphatase, stabilize the phosphorylated state of phospholamban, interrupt the phospholamban-Ca(2+)-ATPase interaction, decrease phospholamban transcription, or disrupt phospholamban mRNA stability. Development of such therapeutic strategies to target phospholamban will be an important future goal for the clinical improvement of contractility in the failing heart.

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. This report centers on the hypothesis that the relative levels of phospholamban: SERCA2 in cardiac muscle plays an important role in the muscle's overall contractility status. This hypothesis was tested by comparing the contractile parameters of: a) murine atrial and ventricular muscles, which differentially express phospholamban, and b) murine wild-type and phospholamban knock-out hearts. These comparisons revealed that atrial muscles, which have a 4.2-fold lower phospholamban: SERCA2 ratio than ventricular muscles, exhibited rates of force development and relaxation of tension, which were three-fold faster that these parameters for ventricular muscles. Similar comparisons were made via analyses of left-ventricular pressure development recorded for isolated, work-performing hearts from wild-type and phospholamban knock-out mice. In these studies, hearts from phospholamban knock-out mice, which were devoid of phospholamban, exhibited enhanced parameters of left-ventricular contractility in comparison to wild-type hearts. These results suggest that the relative phospholamban: SERCA2 ratio is critical in the regulation of myocardial contractility and alterations in this ratio may contribute to the functional deterioration observed during heart failure.

Practical considerations for using mag-fura-2 to measure cytosolic free magnesium.

The development of fluorescent ion-selective indicators for magnesium has provided valuable tools for measuring second-by-second changes in cytosolic magnesium activity. In the course of establishing appropriate protocols for using one of these indicators, mag-fura-2, to measure magnesium activity in BC3H-1 cells and chick ventricular myocytes, many potential pitfalls and limitations of this technique have been encountered and addressed. These observations are presented for the purpose of providing guidelines regarding the appropriate use of this indicator and on factors influencing the interpretation of resulting data.

Mg2+ buffering in cultured chick ventricular myocytes: quantitation and modulation by Ca2+.

To characterize the Mg2+ buffering of cultured chick ventricular myocytes, cytosolic Mg2+ was increased by liberating Mg2+ normally chelated by ATP upon total depletion of ATP content. Because the total Mg content and cell volume remained constant during this time, the difference between the amount of Mg2+ liberated (2.7 mM) and the 0.9 mM increase in cytosolic Mg2+ activity measured fluorometrically with mag-fura-2 indicates a sizable Mg2+ buffering. A new term, the Mg2+ buffer coefficient (BMg), was derived to quantify this buffering. We also determined that cytosolic Mg2+ activity increased by only 0.6 mM in cells acutely exposed to zero external Ca2+ during ATP depletion. In the absence of extracellular Ca2+, the basal cytosolic Ca2+ activity (alpha Ca2+i) was reduced by 72%, whereas the increase in alpha Ca2+i induced by ATP depletion was substantially blunted; no difference in either the time course of adenine nucleotide changes or the Ca and Mg content was observed. The BMg value calculated for these cells indicates that Mg2+ buffering is substantially greater in the absence of extracellular Ca2+ (2.5) than when extracellular Ca2+ is present (1.4), indicating that alpha Ca2+i affects cytosolic Mg2+ activity in ventricular myocytes. Therefore the Mg2+ buffering of ventricular myocytes appears to be comprised of at least two components: 1) a Ca(2+)-insensitive adenine nucleotide pool and 2) a Ca(2+)-sensitive nonadenine nucleotide pool.

Phospholamban modulates murine atrial contractile parameters and responses to beta-adrenergic agonists.

Phospholamban gene transcript levels are much lower in murine atria as compared to murine ventricles and this reduced phospholamban expression has been suggested to result in enhanced atrial contractile parameters. To delineate the functional role of phospholamban in murine atrium, the contractile parameters of isolated muscles from phospholamban knockout and cardiac-specific phospholamban overexpression mice along with their isogenic wild-type controls were evaluated. Assessment of the times (ms) to peak tension development and to half-relaxation of developed tension, as well as the rates (mg/s) of tension development and relaxation in paced atrial muscles, revealed that phospholamban ablation was associated with enhanced rates of relaxation with no significant effect on contraction rate, while phospholamban overexpression (three-fold) was associated with depressed rates of both contraction and relaxation. Isoproterenol stimulation resulted in significant increases in the rates of developed tension and relaxation in both phospholamban deficient and phospholamban overexpression atria, indicating that the beta-adrenergic pathway was functional in these muscles. These findings suggest that phospholamban is an important modulator of atrial contractility and its responses to beta-adrenergic agonists.

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. These findings were corroborated by in situ hybridization studies of cardiopulmonary sections from both murine strains, and phospholamban transcripts were also observed in pulmonary myocardia of both strains. Analyses of phospholamban transcript levels relative to alpha-myosin heavy chain (alpha-MHC) revealed a 3-fold higher phospholamban abundance in the ventricle compared with the atrium of the FVB/N murine strain. However, the relative mRNA level of Ca(2+)-ATPase (ratio of sarcoplasmic reticulum Ca(2+)-ATPase [SERCA2] to alpha-MHC) in the ventricle was 80% of that in the atrium. Consequently, the relative ratio of phospholamban to SERCA2 mRNA was 4.2-fold lower in the atrium than in the ventricle. The lower transcript ratio of phospholamban to SERCA2 in the atrium was associated with significantly shortened times to half-relaxation (17.40 +/- 0.71 milliseconds for atrium versus 30.58 +/- 2.04 milliseconds for ventricle), assessed in isolated superfused cardiac tissue preparations recorded at maximum length tension. Contraction times, measured as times to peak tension, were also significantly shortened in atrial muscle (27.36 +/- 0.82 milliseconds) compared with ventricular muscle (44.60 +/- 2.55 milliseconds), assessed in the same tissue preparations. These findings suggest that phospholamban gene expression is differentially regulated in murine atrial and ventricular muscles and that this differential expression may be associated with differences in the contractile parameters of these cardiac compartments.