Judy M. Harrer

Targeted ablation of the phospholamban gene is associated with markedly enhanced myocardial contractility and loss of beta-agonist stimulation.

Phospholamban is the regulator of the Ca(2+)-ATPase in cardiac sarcoplasmic reticulum (SR), and it has been suggested to be an important determinant in the inotropic responses of the heart to beta-adrenergic stimulation. To determine the role of phospholamban in vivo, the gene coding for this protein was targeted in murine embryonic stem cells, and mice deficient in phospholamban were generated. The phospholamban-deficient mice showed no gross developmental abnormalities but exhibited enhanced myocardial performance without changes in heart rate. The time to peak pressure and the time to half-relaxation were significantly shorter in phospholamban-deficient mice compared with their wild-type homozygous littermates as assessed in work-performing mouse heart preparations under identical venous returns, afterloads, and heart rates. The first derivatives of intraventricular pressure (+/- dP/dt) were also significantly elevated, and this was associated with an increase in the affinity of the SR Ca(2+)-ATPase for Ca2+ in the phospholamban-deficient hearts. Baseline levels of these parameters in the phospholamban-deficient hearts were equal to those observed in hearts of wild-type littermates maximally stimulated with the beta-agonist isoproterenol. These findings indicate that phospholamban acts as a critical repressor of basal myocardial contractility and may be the key phosphoprotein in mediating the hearts contractile responses to beta-adrenergic agonists.

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. The transgenic mice showed no phenotypic alterations and no changes in heart/body weight, heart/lung weight, and cardiomyocyte size. Isolated unloaded cardiac myocytes from transgenic mice exhibited diminished shortening fraction (63%) and decreased rates of shortening (64%) and relengthening (55%) compared to wild type (100%) cardiomyocytes. The decreases in contractile parameters of transgenic cardiomyocytes reflected decreases in the amplitude (83%) of the Ca2+ signal and prolongation (131%) in the time for decay of the Ca2+ signal, which was associated with a decrease in the apparent affinity of the SR Ca(2+)-ATPase for Ca2+ (56%), compared to wild type (100%) cardiomyocytes. In vivo analysis of left ventricular systolic function using M mode and pulsed-wave Doppler echocardiography revealed decreases in fractional shortening (79%) and the normalized mean velocity of circumferential shortening (67%) in transgenic mice compared to wild type (100%) mice. The differences in contractile parameters and Ca2+ kinetics in transgenic cardiomyocytes and the depressed left ventricular systolic function in transgenic mice were abolished upon isoproterenol stimulation. These findings indicate that a fraction of the Ca(2+)-ATPases in native SR is not under regulation by phospholamban. Expression of additional phospholamban molecules results in: (a) inhibition of SR Ca2+ transport; (b) decreases in systolic Ca2+ levels and contractile parameters in ventricular myocytes; and (c) depression of basal left ventricular systolic function in vivo.

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. The reduced phospholamban levels were associated with significant decreases in the EC50 of the sarcoplasmic reticulum Ca2+ pump for CA2+ and increases in the contractile parameters of isolated myocytes and beating hearts. The relative phospholamban levels among wild-type, phospholamban-heterozygous, and phospholamban-deficient mouse hearts correlated well with the (1) EC50 of the Ca(2+)-ATPase for Ca2+ in sarcoplasmic reticulum, (2) rates of relaxation and contraction in isolated cardiac myocytes, and (3) rates of relaxation and intact beating hearts. These findings suggest that physiological and pathological changes in the levels of phospholamban will result in parallel changes in sarcoplasmic reticulum function and cardiac contraction.

Motivational and skills training HIV/sexually transmitted infection sexual risk reduction groups for men

The effectiveness of a motivational and skills training HIV/AIDS group intervention designed for men in substance abuse treatment was evaluated. Men in methadone maintenance (n = 288) or outpatient psychosocial treatment (n = 302) completed assessments at baseline, 2 weeks, 3 months, and 6 months postintervention. Participants were randomly assigned to attend either Real Men Are Safe (REMAS; five sessions containing information, motivational exercises, and skills training) or HIV education (HIV-Ed; one session containing HIV prevention information). REMAS participants engaged in significantly fewer unprotected vaginal and anal sexual intercourse occasions (USO) during the 90 days prior to the 3- and 6-month follow-ups than HIV-Ed participants. Completing REMAS resulted in an even stronger effect: Completers reduced their number of USO by 21% from baseline to 6-month follow-up. In contrast, HIV-Ed completers increased the number of USO by 2%. A motivational and skills training HIV prevention intervention designed for men was associated with greater sexual risk reduction over standard HIV-Ed. Substance abuse treatment programs can therefore help reduce sexual risk among their clientele by providing a more intensive intervention than what is traditionally provided.

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. To elucidate the role of PLB in vascular smooth muscle contractility in vivo, we used a model in which the PLB gene was targeted in murine embryonic stem cells, generating mice deficient in PLB (PLB-). The PLB- mice exhibited no gross developmental abnormalities, but marked changes in aortic contractility were observed. The time course of force development with phenylephrine stimulation was faster in the PLB- aorta, suggesting changes in SR Ca2+ release. No differences were observed for KCl contractures between tissue types for either maximum forces observed or time course of force production; relaxation was faster in 7 of 11 arteries, but this trend did not attain statistical significance. The cumulative concentration-isometric force relations for the PLB- aorta were to the right of the wild-type for both KCl and phenylephrine stimulation, indicating a less sensitive tissue. To investigate whether the observed changes were related to SR function, we inhibited the SR Ca(2+)-ATPase with cyclopiazonic acid (CPA). CPA treatment resulted in a leftward shift of the concentration-isometric force relations for both aorta types, as expected after removal of a major Ca2+ uptake system. Most interestingly, the differences between PLB and wild-type aorta were abolished by SR inhibition. Our results suggest that PLB is a regulator of the SR Ca2+ pump in mouse aorta and plays a regulatory role in both KCl-induced and receptor-mediated contractility in vascular smooth muscle.

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. Quantitative immunoblotting indicated a 2-fold increase in the cardiac phospholamban protein levels compared with wild-type controls, with approximately equal to 50% of phospholamban migrating as monomers and approximately 50% as pentamers upon SDS-PAGE. The mutant-phospholamban transgenic hearts were analyzed in parallel with transgenic hearts overexpressing (2-fold) wild-type phospholamban, which migrated as pentamers upon SDS-PAGE. SR Ca(2+)-uptake assays revealed that the EC50 values for Ca2+ were as follows: 0.32 +/- 0.01 mumol/L in hearts overexpressing monomeric phospholamban, 0.49 +/- 0.05 mumol/L in hearts overexpressing wild-type phospholamban, and 0.26 +/- 0.01 mumol/L in wild-type control mouse hearts. Analysis of cardiomyocyte mechanics and Ca2+ kinetics indicated that the inhibitory effects of mutant-phospholamban overexpression (mt) were less pronounced than those of wild-type phospholamban overexpression (ov) as assessed by depression of the following: (1) shortening fraction (25% mt versus 45% ov), (2) rates of shortening (27% mt versus 48% ov), (3) rates of relengthening (25% mt versus 50% ov) (4) amplitude of the Ca2+ signal (21% mt versus 40% ov), and (5) time for decay of the Ca2+ signal (25% mt versus 106% ov) compared with control (100%) myocytes. The differences in basal cardiac, myocyte mechanics and Ca2+ transients among the animal groups overexpressing monomeric or wild-type phospholamban and wild-type control mice were abolished upon isoproterenol stimulation. These findings suggest that pentameric assembly of phospholamban is important for mediating its optimal regulatory effects on myocardial contractility in vivo.

An Open-Label Pilot Study of Methylphenidate in the Treatment of Cocaine Dependent Patients with Adult Attention Deficit/ Hyperactivity Disorder

Abstract A multi-site, open-label study of methylphenidate for treating patients with comorbid diagnoses of attention deficit/hyperactivity disorder and cocaine dependence was performed. Forty-one participants, who met DSM-IV criteria for adult attention deficit/hyperactivity disorder and cocaine dependence, were enrolled into this ten week outpatient study. The targeted total daily dose of methylphenidate was 60 mg (20 mg TID). Participants received individual substance abuse therapy throughout the trial. Safety measures included adverse events, vital signs, and electrocardiograms. Methylphenidates efficacy was assessed by both objective and subjective measures. Seventy percent of the participants completed final study measures. Safety measures indicated that methylphenidate was well tolerated by the participants. Subjective efficacy measures suggested that participants evidenced improvement in both cocaine dependence and adult attention deficit/hyperactivity disorder symptoms. Quantitative benzoylecgonine indicated that only those participants categorized as being compliant showed improvement. A double-blind, placebo-controlled study of methylphenidate for this population may be warranted.

Methylphenidate and cocaine: a placebo-controlled drug interaction study.

Up to thirty percent of cocaine addicted individuals may meet diagnostic criteria for Attention-Deficit/Hyperactivity Disorder (ADHD). Methylphenidate (MPH) is a highly effective and commonly used treatment for ADHD but, like cocaine, is a cardiovascular and central nervous system stimulant with the potential to cause toxicity at high doses. The present study was undertaken to investigate the likelihood of a toxic reaction in individuals who use cocaine while concurrently taking MPH. Seven non-treatment seeking cocaine-dependent individuals completed this placebo-controlled, crossover study with two factors: Medication (placebo, 60 mg MPH, 90 mg MPH) and Infusion (saline, 20 mg cocaine, 40 mg cocaine). Physiological measures included vital signs, adverse events, and electrocardiogram. Subjective response was measured with visual analog scale (VAS) ratings of craving and drug effect. Cocaine pharmacokinetic parameters were calculated for each participant at each drug combination, using a non-compartmental model. MPH was well tolerated, did not have a clinically significant impact on cocaines physiological effects, and decreased some of the positive subjective effects of cocaine. MPH did not significantly alter the pharmacokinetics of cocaine. The study results suggest that MPH at the doses studied can likely be used safely in an outpatient setting with active cocaine users.

Alterations in sarcoplasmic reticulum cakcium uptake, relaxation parameters and their responses to β-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. The low physiological and biochemical parameters in the fetal hearts reflected reduced levels of expression of the sarcoplasmic reticulum Ca(2+)-pump and its inhibitor, phospholamban, assessed by quantitative immunoblotting. Isoproterenol perfusion of fetal hearts had no significant effect on their relaxation parameters or on the EC50 of the Ca(2+)-pump for Ca2+, consistent with the low relative levels of phospholamban expressed in these hearts. However, perfusion of the 4-day-old, 21-day-old and adult hearts with isoproterenol resulted in significant increases in the rates of relaxation of each group. The increases in relaxation parameters were associated with decreases in the EC50 of the cardiac sarcoplasmic reticulum Ca(2+)-pump for Ca2+, suggesting a phosphorylation-mediated relief of the phospholamban inhibitory effects. These findings indicate that developmental regulation of the levels of the activity of the cardiac sarcoplasmic reticulum Ca(2+)-pump may reflect alterations in cardiac relaxation parameters and their modulation by beta-adrenergic agonists.

Expression of phospholamban in C2C12 cells and regulation of endogenous SERCA1 activity.

Phospholamban (PLB) is a regulator of the sarcoplasmic reticulum Ca2+-ATPase (SERCA2) expressed in cardiac, slow-twitch skeletal, and smooth muscles. Phospholamban is not expressed in the sarcoplasmic reticulum of fast-twitch skeletal muscle, but it can regulate the sarcoplasmic reticulum Ca2+-ATPase activity (SERCA1) expressed in this muscle,in vitro. To determine whether phospholamban can regulate SERCA1 activity in its native membrane environment, phospholamban was stably transfected into a cell line (C2C12) derived from murine fast-twitch skeletal muscle. Differentiation of C2C12 myoblasts to myotubes was associated with induction of SERCA1 expression, assessed by Western blotting analysis using Ca2+-ATPase isoform specific antibodies. The expressed phospholamban protein was localized in the microsomal fraction isolated from C2C12 myotubes. To determine the effect of phospholamban expression on SERCA1 activity, microsomes were isolated from transfected and nontransfected C2C12 cell myotubes, and the initial rates of45Ca2+-uptake were determined over a wide range of Ca2+ concentrations (0.1–10 μM). Expression of phospholamban was associated with inhibition of the initial rates of Ca2+-uptake at low [Ca2+] and this resulted in a decrease in the affinity of SERCA1 for Ca2+ (0.27±0.02 μM in nontransfected vs. 0.41±0.03 μM in PLB transfected C2C12 cells). These findings indicate that phospholamban expression in C2C12 cells is associated within inhibition of the endogenous SERCA1 activity and provide evidence that phospholamban is capable of regulating this Ca2+-ATPase isoform in its native membrane environment.