Biomedical subjects

J Randall Moorman

Publications and source records attributed to J Randall Moorman.

At least 19 recordsLinked to original sources

PubMed2006

Altered contractility and [Ca2+]i homeostasis in phospholemman-deficient murine myocytes: role of Na+/Ca2+ exchange.

Phospholemman (PLM) regulates contractility and Ca(2+) homeostasis in cardiac myocytes. We characterized excitation-contraction coupling in myocytes isolated from PLM-deficient mice backbred to a pure congenic C57BL/6 background. Cell length, cell width, and whole cell capacitance were not different between wild-type and PLM-null myocytes. Compared with wild-type myocytes, Western blots indicated total absence of PLM but no changes in Na(+)/Ca(2+) exchanger, sarcoplasmic reticulum (SR) Ca(2+)-ATPase, alpha(1)-subunit of Na(+)-K(+)-ATPase, and calsequestrin levels in PLM-null myocytes. At 5 mM extracellular Ca(2+) concentration ([Ca(2+)](o)), contraction and cytosolic [Ca(2+)] ([Ca(2+)](i)) transient amplitudes and SR Ca(2+) contents in PLM-null myocytes were significantly (P < 0.0004) higher than wild-type myocytes, whereas the converse was true at 0.6 mM [Ca(2+)](o). This pattern of contractile and [Ca(2+)](i) transient abnormalities in PLM-null myocytes mimics that observed in adult rat myocytes overexpressing the cardiac Na(+)/Ca(2+) exchanger. Indeed, we have previously reported that Na(+)/Ca(2+) exchange currents were higher in PLM-null myocytes. Activation of protein kinase A resulted in increased inotropy such that there were no longer any contractility differences between the stimulated wild-type and PLM-null myocytes. Protein kinase C stimulation resulted in decreased contractility in both wild-type and PLM-null myocytes. Resting membrane potential and action potential amplitudes were similar, but action potential duration was much prolonged (P < 0.04) in PLM-null myocytes. Whole cell Ca(2+) current densities were similar between wild-type and PLM-null myocytes, as were the fast- and slow-inactivation time constants. We conclude that a major function of PLM is regulation of cardiac contractility and Ca(2+) fluxes, likely by modulating Na(+)/Ca(2+) exchange activity.

Action Potentials↗

PubMed2006

Phospholemman inhibition of the cardiac Na+/Ca2+ exchanger. Role of phosphorylation.

We have demonstrated previously that phospholemman (PLM), a 15-kDa integral sarcolemmal phosphoprotein, inhibits the cardiac Na+/Ca2+ exchanger (NCX1). In addition, protein kinase A phosphorylates serine 68, whereas protein kinase C phosphorylates both serine 63 and serine 68 of PLM. Using human embryonic kidney 293 cells that are devoid of both endogenous PLM and NCX1, we first demonstrated that the exogenous NCX1 current (I(NaCa)) was increased by phorbol 12-myristate 13-acetate (PMA) but not by forskolin. When co-expressed with NCX1, PLM resulted in: (i) decreases in I(NaCa), (ii) attenuation of the increase in I(NaCa) by PMA, and (iii) additional reduction in I(NaCa) in cells treated with forskolin. Mutating serine 63 to alanine (S63A) preserved the sensitivity of PLM to forskolin in terms of suppression of I(NaCa), whereas mutating serine 68 to alanine (S68A) abolished the inhibitory effect of PLM on I(NaCa). Mutating serine 68 to glutamic acid (phosphomimetic) resulted in additional suppression of I(NaCa) as compared with wild-type PLM. These results suggest that PLM phosphorylated at serine 68 inhibited I(NaCa). The physiological significance of inhibition of NCX1 by phosphorylated PLM was evaluated in PLM-knock-out (KO) mice. When compared with wild-type myocytes, I(NaCa) was significant larger in PLM-KO myocytes. In addition, the PMA-induced increase in I(NaCa) was significantly higher in PLM-KO myocytes. By contrast, forskolin had no effect on I(NaCa) in wild-type myocytes. We conclude that PLM, when phosphorylated at serine 68, inhibits Na+/Ca2+ exchange in the heart.

Adenosine Triphosphatases↗

PubMed2006

Toward quantitative fetal heart rate monitoring.

Continuous electronic fetal heart rate (FHR) monitoring during labor is motivated by the clinical experience that fetal distress causes loss of FHR variation and the occurrence of decelerations late during uterine contraction. This practice is of uncertain clinical benefit, perhaps because the interpretation is qualitative. We have developed new quantitative measures and analyzed cardiotocograph records from 148 consecutive patients, 44 of whom had at least one "nonreassuring" epoch. In multivariate regression models, measures of deceleration and variability were significantly associated with the obstetrician's diagnosis (receiver operating characteristic area 0.84, p < 0.05). This approach may be useful clinically.

Algorithms↗

PubMed2006

Heart rate characteristics monitoring for neonatal sepsis.

While heart rate variability has been measured in many clinical settings and has offered insights into how HR is controlled, rarely has it offered unique information that has led to changes in patient management. We review our experience in developing continuous HR characteristics monitoring to aid in the early diagnosis of sepsis in premature infants in the neonatal intensive care unit. A predictive algorithm, developed at one center and validated at another, has led to diagnosis and treatment of this subacute and potentially catastrophic illness prior to appearance of symptoms of severe illness.

Algorithms↗

PubMed2005

Phospholemman overexpression inhibits Na+-K+-ATPase in adult rat cardiac myocytes: relevance to decreased Na+ pump activity in postinfarction myocytes.

Messenger RNA levels of phospholemman (PLM), a member of the FXYD family of small single-span membrane proteins with putative ion-transport regulatory properties, were increased in postmyocardial infarction (MI) rat myocytes. We tested the hypothesis that the previously observed reduction in Na+-K+-ATPase activity in MI rat myocytes was due to PLM overexpression. In rat hearts harvested 3 and 7 days post-MI, PLM protein expression was increased by two- and fourfold, respectively. To simulate increased PLM expression post-MI, PLM was overexpressed in normal adult rat myocytes by adenovirus-mediated gene transfer. PLM overexpression did not affect the relative level of phosphorylation on serine68 of PLM. Na+-K+-ATPase activity was measured as ouabain-sensitive Na+-K+ pump current (Ip). Compared with control myocytes overexpressing green fluorescent protein alone, Ip measured in myocytes overexpressing PLM was significantly (P < 0.0001) lower at similar membrane voltages, pipette Na+ ([Na+]pip) and extracellular K+ ([K+]o) concentrations. From -70 to +60 mV, neither [Na+]pip nor [K+]o required to attain half-maximal Ip was significantly different between control and PLM myocytes. This phenotype of decreased V(max) without appreciable changes in K(m) for Na+ and K+ in PLM-overexpressed myocytes was similar to that observed in MI rat myocytes. Inhibition of Ip by PLM overexpression was not due to decreased Na+-K+-ATPase expression because there were no changes in either protein or messenger RNA levels of either alpha1- or alpha2-isoforms of Na+-K+-ATPase. In native rat cardiac myocytes, PLM coimmunoprecipitated with alpha-subunits of Na+-K+-ATPase. Inhibition of Na+-K+-ATPase by PLM overexpression, in addition to previously reported decrease in Na+-K+-ATPase expression, may explain altered V(max) but not K(m) of Na+-K+-ATPase in postinfarction rat myocytes.

Animals↗

PubMed2005

Serine 68 phospholemman phosphorylation during forskolin-induced swine carotid artery relaxation.

BACKGROUND: Phospholemman (PLM) is an abundant phosphoprotein in the plasma membrane of cardiac, skeletal and smooth muscle. It is a member of the FXYD family of proteins that bind to and regulate the Na,K-ATPase. Protein kinase A (PKA) is known to phosphorylate PLM on serine 68 (S68), although the functional effect of S68 PLM phosphorylation is unclear. We therefore evaluated S68 PLM phosphorylation in swine carotid arteries. METHODS: Two anti-PLM antibodies, one to S68 phosphorylated PLM and one to unphosphorylated PLM, were made to PLM peptides in rabbits and tested with purified PLM and PKA-treated PLM. Swine carotid arteries were mounted isometrically, contracted, relaxed with forskolin and then homogenized. Proteins were separated on SDS gels and the intensity of immunoreactivity to the two PLM antibodies determined on immunoblots. RESULTS: The antipeptide antibody 'C2' primarily reacted with unphosphorylated PLM, and the antipeptide antibody 'CP68' detected S68 PLM phosphorylation. Histamine stimulation of intact swine carotid artery induced a contraction, increased the CP68 PLM antibody signal and reduced the C2 PLM antibody signal. High extracellular [K(+)] depolarization induced a contraction without altering the C2 or CP68 PLM signal. Forskolin-induced relaxation of histamine or extracellular [K(+)] contracted arteries correlated with an increased CP68 signal. Nitroglycerin-induced relaxation was not associated with changes in the C2 or CP68 PLM signal. CONCLUSIONS: These data suggest that a contractile agonist increased S68 PLM phosphorylation. Agents that increase [cAMP], but not agents that increase [cGMP], increased S68 PLM phosphorylation. S68 PLM phosphorylation may be involved in cAMP-dependent regulation of smooth muscle force.

Animals↗

PubMed2005

Expression and phosphorylation of the na-pump regulatory subunit phospholemman in heart failure.

Intracellular [Na] is approximately 3 mmol/L higher in heart failure (HF; in our arrhythmogenic rabbit model), and this can profoundly affect cardiac Ca and contractile function via Na/Ca exchange and Na/H exchange. Na/K-ATPase is the primary mechanism of Na extrusion. We examine here in HF rabbits (and human hearts) expression of Na/K-ATPase isoforms and phospholemman (PLM), a putative Na/K-ATPase regulatory subunit that inhibits pump function and is a major cardiac phosphorylation target. Na/K-ATPase alpha1- and alpha2-isoforms were reduced in HF in rabbit ventricular homogenates (by 24%) and isolated myocytes (by 30% and 17%), whereas alpha3 was increased (50%) in homogenates and decreased (52%) in myocytes (P<0.05). Homogenate Na/K-ATPase activity in left ventricle was also decreased in HF. However, we showed previously that Na/K-ATPase characteristics in intact ventricular myocytes were unaltered in HF. To reconcile these findings, we assessed PLM expression, phosphorylation, and association with Na/K-ATPase. PLM coimmunoprecipitated with Na/K-ATPase alpha1 and alpha2 in control and HF rabbit myocytes. PLM expression was reduced in HF by 42% in isolated rabbit left ventricular (LV) myocytes, by 48% in rabbit LV homogenates, and by 24% in human LV homogenate. The fraction of PLM phosphorylated at Ser-68 was increased dramatically in HF. Our results are consistent with a role for PLM analogous to that of phospholamban for SR Ca-ATPase (SERCA): inhibition of Na/K-ATPase function that is relieved on PLM phosphorylation. So reduced Na/K-ATPase expression in HF may be functionally offset by lower inhibition by PLM (because of reduced PLM expression and higher PLM phosphorylation).

Animals↗

PubMed2005

Identification of an endogenous inhibitor of the cardiac Na+/Ca2+ exchanger, phospholemman.

Rapid and precise control of Na(+)/Ca(2+) exchanger (NCX1) activity is essential in the maintenance of beat-to-beat Ca(2+) homeostasis in cardiac myocytes. Here, we show that phospholemman (PLM), a 15-kDa integral sarcolemmal phosphoprotein, is a novel endogenous protein inhibitor of cardiac NCX1. Using a heterologous expression system that is devoid of both endogenous PLM and NCX1, we first demonstrated by confocal immunofluorescence studies that both exogenous PLM and NCX1 co-localized at the plasma membrane. Reciprocal co-immunoprecipitation studies revealed specific protein-protein interaction between PLM and NCX1. The functional consequences of direct association of PLM with NCX1 was the inhibition of NCX1 activity, as demonstrated by whole-cell patch clamp studies to measure NCX1 current density and radiotracer flux assays to assess Na(+)-dependent (45)Ca(2+) uptake. Inhibition of NCX1 by PLM was specific, because a single mutation of serine 68 to alanine in PLM resulted in a complete loss of inhibition of NCX1 current, although association of the PLM mutant with NCX1 was unaltered. In native adult cardiac myocytes, PLM co-immunoprecipitated with NCX1. We conclude that PLM, a member of the FXYD family of small ion transport regulators known to modulate Na(+)-K(+)-ATPase, also regulates Na(+)/Ca(2+) exchange in the heart.

Amino Acid Substitution↗

PubMed2005

Serine 68 of phospholemman is critical in modulation of contractility, [Ca2+]i transients, and Na+/Ca2+ exchange in adult rat cardiac myocytes.

Overexpression of phospholemman (PLM) in normal adult rat cardiac myocytes altered contractile function and cytosolic Ca2+ concentration ([Ca2+]i) homeostasis and inhibited Na+/Ca2+ exchanger (NCX1). In addition, PLM coimmunoprecipitated and colocalized with NCX1 in cardiac myocyte lysates. In this study, we evaluated whether the cytoplasmic domain of PLM is crucial in mediating its effects on contractility, [Ca2+]i transients, and NCX1 activity. Canine PLM or its derived mutants were overexpressed in adult rat myocytes by adenovirus-mediated gene transfer. Confocal immunofluorescence images using canine-specific PLM antibodies demonstrated that the exogenous PLM or its mutants were correctly targeted to sarcolemma, t-tubules, and intercalated discs, with little to none detected in intracellular compartments. Overexpression of canine PLM or its mutants did not affect expression of NCX1, sarco(endo)plasmic reticulum Ca(2+)-ATPase, Na(+)-K(+)-ATPase, and calsequestrin in adult rat myocytes. A COOH-terminal deletion mutant in which all four potential phosphorylation sites (Ser62, Ser63, Ser68, and Thr69) were deleted, a partial COOH-terminal deletion mutant in which Ser68 and Thr69 were deleted, and a mutant in which all four potential phosphorylation sites were changed to alanine all lost wild-type PLM's ability to modulate cardiac myocyte contractility. These observations suggest the importance of Ser68 or Thr69 in mediating PLM's effect on cardiac contractility. Focusing on Ser68, the Ser68 to Glu mutant was fully effective, the Ser63 to Ala (leaving Ser68 intact) mutant was partially effective, and the Ser68 to Ala mutant was completely ineffective in modulating cardiac contractility, [Ca2+]i transients, and NCX1 currents. Both the Ser63 to Ala and Ser68 to Ala mutants, as well as PLM, were able to coimmunoprecipitate NCX1. It is known that Ser68 in PLM is phosphorylated by both protein kinases A and C. We conclude that regulation of cardiac contractility, [Ca2+]i transients, and NCX1 activity by PLM is critically dependent on Ser68. We suggest that PLM phosphorylation at Ser68 may be involved in cAMP- and/or protein kinase C-dependent regulation of cardiac contractility.

Animals↗

PubMed2005

Serine 68 phosphorylation of phospholemman: acute isoform-specific activation of cardiac Na/K ATPase.

OBJECTIVE: The mechanism by which the cardiac Na/K ATPase (NKA) is regulated by phosphorylation is controversial. We have used the perforated-patch technique to limit cell dialysis and maintain conditions as near physiological as possible. METHODS: NKA pump current (I(p)) was measured in isolated guinea pig ventricular myocytes, and its components (I(alpha 1) and I(alpha 2)) defined by their differing dihydroouabain sensitivities. RESULTS: Treatment with 1 micromol/l forskolin for 4 min at 35 degrees C caused a significant increase in I(alpha1) of 36+/-15% (P<0.05, n=6), but no change in I(alpha2). The presence of the PKA selective inhibitor H89 (50 micromol/l) throughout the protocol blocked the effect of the forskolin on I(alpha1). Treatment with H89 alone did not change I(alpha 1) or I(alpha 2). Isoelectric focusing gels of the NKA alpha1 subunit demonstrated six charge states, which were unaltered following treatment with forskolin. Western blots using an antibody specific for the PKA phosphorylation consensus site on the alpha1 subunit showed no change in the phosphorylation status of this residue following forskolin treatment. The sarcolemmal protein phospholemman (PLM) was found associated with NKA alpha 1 but not alpha 2 subunits by immunoprecipitation and immunofluorescence. PLM was phosphorylated at serine 68, but not 63, following treatment with forskolin. CONCLUSIONS: PKA-dependent, alpha 1-specific NKA activation may be mediated through phosphorylation of the accessory protein PLM, rather than direct alpha1 subunit phosphorylation.

Animals↗

PubMed2005

Heart rate characteristics and laboratory tests in neonatal sepsis.

OBJECTIVE: The evaluation of an infant for suspected sepsis often includes obtaining blood for laboratory tests. The shortcomings of the current practice are that the infant has to appear clinically ill for the diagnosis to be entertained, and the conventional laboratory tests are invasive. We have found that the clinical diagnosis of neonatal sepsis is preceded by abnormal heart rate characteristics (HRC) of reduced variability and transient decelerations, and we have devised a predictive HRC monitoring strategy based on multivariable logistic regression analysis that was developed at one tertiary care NICU and validated at another. We hypothesized that HRC monitoring, which is continuous and noninvasive, might be an adjunct to conventional laboratory tests in the diagnosis of neonatal sepsis. The objective of this study was to test the hypothesis that HRC monitoring adds information to conventional laboratory tests in diagnosing neonatal sepsis. METHODS: We prospectively collected heart rate data in 678 consecutive infants who stayed >7 days in the University of Virginia NICU from July 1999 to July 2003. We prospectively measured HRC and noted 149 episodes of sepsis with positive blood cultures for which data were available in 137. We obtained all laboratory test results for ratio of immature to total neutrophil count, white blood cell count, glucose, platelet count, HCO3, arterial partial pressure of carbon dioxide, and pH. We tested hypotheses using multivariable logistic regression modeling adjusted for repeated measures. RESULTS: We found that the HRC index, which was available 92% of the time, was highly significantly associated with sepsis (receiver-operating characteristic [ROC] area: 0.73). The ratio of immature to total neutrophil count, white blood cell count (available 4%-8% of the time, usually around the time of suspected sepsis), and blood glucose and pH (available 28% and 38% of the time) were also significantly associated with sepsis (ROC area: 0.75). HRC and laboratory values added independent information to each other, and a predictive model using all significant variables had ROC area of 0.82. CONCLUSIONS: HRC monitoring adds independent information to laboratory tests in the diagnosis of culture-positive neonatal sepsis.

Blood Chemical Analysis↗

PubMed2005

Heart rate characteristics: novel physiomarkers to predict neonatal infection and death.

OBJECTIVE: Monitoring of regulated physiologic processes using physiomarkers such as heart rate variability may be important in the early diagnosis of subacute, potentially catastrophic illness. Early in the course of neonatal sepsis, there are physiomarkers of reduced heart rate variability and transient decelerations similar to fetal distress. The goal of this study was to determine the degree of increased risk for sepsis, urinary tract infection (UTI), and death when these abnormal heart rate characteristics (HRC) were observed. METHODS: We monitored 1022 infants at 2 tertiary care NICUs, 458 of whom were very low birth weight. We calculated an HRC index from validated regression models relating mathematical features of heart rate time series and histograms to episodes of illness. We calculated the risks for adverse events of sepsis, UTI, and death for infants stratified by HRC measurements. RESULTS: Compared with infants with low-risk HRC measurements, infants with high-risk HRC measurements had 5- to 6-fold increased risk for an adverse event in the next day and 3-fold increased risk in the next week. Laboratory tests that were relevant to infection added information to HRC measurements. Infants with both high-risk HRC and abnormal laboratory tests had 6- to 7-fold increased risk for an adverse event in the next day compared with infants who had neither. CONCLUSION: HRC are noninvasively monitored physiomarkers that identify infants in the NICU who are at high risk for sepsis, UTI, and death.

Gestational Age↗

PubMed2004

Hypertrophy, increased ejection fraction, and reduced Na-K-ATPase activity in phospholemman-deficient mice.

Phospholemman (FXYD1), a 72-amino acid transmembrane protein abundantly expressed in the heart and skeletal muscle, is a major substrate for phosphorylation in the cardiomyocyte sarcolemma. Biochemical, cellular, and electrophysiological studies have suggested a number of possible roles for this protein, including ion channel modulator, taurine-release channel, Na(+)/Ca(2+) exchanger modulator, and Na-K-ATPase-associated subunit. We have generated a phospholemman-deficient mouse. The adult null mice exhibited increased cardiac mass, larger cardiomyocytes, and ejection fractions that were 9% higher by magnetic resonance imaging compared with wild-type animals. Notably, this occurred in the absence of hypertension. Total Na-K-ATPase activity was 50% lower in the phospholemman-deficient hearts. Expression (per unit of membrane protein) of total Na-K-ATPase was only slightly diminished, but expression of the minor alpha(2)-isoform, which has been specifically implicated in the control of contractility, was reduced by 60%. The absence of phospholemman thus results in a complex response, including a surprisingly large reduction in intrinsic Na-K-ATPase activity, changes in Na-K-ATPase isoform expression, increase in ejection fraction, and increase in cardiac mass. We hypothesize that a primary effect of phospholemman is to modulate the Na-K-ATPase and that its reduced activity initiates compensatory responses.

Animals↗

PubMed2004

Abnormal heart rate characteristics are associated with neonatal mortality.

Estimating the risk of in-hospital mortality in the newborn intensive care unit can provide important information for health-care providers, and illness severity scores have been devised to provide mortality risk estimates. Calculation of illness severity scores is time-consuming, and the information used to predict mortality is collected only for the first 12 to 24 h of life. A noninvasive continuous measure that uses information collected throughout the hospitalization and that requires no data entry could be less costly and more informative. We have previously shown that the abnormal heart rate characteristics (HRC) of reduced variability and transient decelerations accompany neonatal illness such as late-onset sepsis. We hypothesized that more frequent and severe abnormal HRC are associated with an increased risk of death. We tested this hypothesis in two ways. Using data on infants older than 7 d of age, we first determined the association of the HRC index with death in the next week. Second, we devised a cumulative HRC score and determined its association with in-hospital death. There were 37 deaths in the 685 patients. The major findings were 1) the HRC index showed highly significant association with death in the succeeding 7 d (receiver-operating characteristic area > 0.7, p < 0.001), and 2) the cumulative HRC was highly significantly associated with neonatal in-hospital mortality (receiver-operating characteristic area > 0.80, p < 0.001). In both analyses, HRC added information to birth weight, gestational age, and postnatal age (p < 0.01). The HRC index provides independent information about the risk of neonatal death in the upcoming 7 d, and the cumulative HRC is an estimate of the risk of in-hospital neonatal mortality.

Birth Weight↗

PubMed2004

Sample entropy.

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Journal Article↗

PubMed2004

Increased nonstationarity of neonatal heart rate before the clinical diagnosis of sepsis.

The clinical diagnosis of neonatal sepsis is preceded by abnormal heart rate (HR) characteristics of transient decelerations and reduced variability, which intuitively appear to be more nonstationary than normal HR variability. Our goals were to investigate stationarity of HR, and to devise measures useful for early diagnosis of neonatal sepsis. In this context, we define non-stationarity to be present when the observed data differ from surrogate data generated by stationary Gaussian noise with arbitrary linear correlations. We devised statistical methods for determining stationarity of HR data based on the two-sample Kolmogorov-Smirnov (KS) test. We compared distributions of KS distances between small sample epochs from clinical data with those of isospectral surrogates and of surrogates generated using the amplitude-adjusted Fourier transform technique, reasoning that they should differ significantly for nonstationary data. We found significant evidence of non-stationarity for records longer than 1 min. We developed new HR measures based on the empirical cumulative distribution function (ECDF) that are highly significantly associated with sepsis, but are not correlated with HR measures such as moments or sample entropy. We conclude that neonatal HR data cannot be assumed to be stationary, and become even less stationary prior to sepsis.

Algorithms↗

PubMed2004

Effects of phospholemman expression on swelling-activated ion currents and volume regulation in embryonic kidney cells.

Phospholemman (PLM) is a 72-amino-acid phosphoprotein that is a major substrate for cAMP-dependent protein kinase, protein kinase C, and NIMA kinase. In lipid bilayers, PLM forms ion channels selective for Cl-, K+, and taurine. Effluxes of these abundant intracellular osmolytes play an important role in the control of dynamic cell volume changes in many cell types. We measured swelling-activated ion currents and regulatory volume decrease (RVD) in human embryonic kidney cells stably overexpressing canine cardiac PLM. In response to swelling, two clonal cell lines overexpressing PLM had increased swelling-activated ion current densities and faster and more extensive RVD. A third clonal cell line overexpressing mutant PLM showed reduced ion current densities and a diminished RVD response. These results suggest a role for PLM in the regulation of cell volume, perhaps as a modulator of an endogenous swelling-activated signal transduction pathway or possibly by participating directly in swelling-induced osmolyte efflux.

Animals↗

PubMed2003

Effects of phospholemman downregulation on contractility and [Ca(2+)]i transients in adult rat cardiac myocytes.

Phospholemman (PLM) expression was increased in rat hearts after myocardial infarction (MI). Overexpression of PLM in normal adult rat cardiac myocytes altered contractile function and cytosolic Ca(2+) concentration ([Ca(2+)](i)) homeostasis in a manner similar to that observed in post-MI myocytes. In this study, we tested whether PLM downregulation in normal adult rat myocytes resulted in contractility and [Ca(2+)](i) transient changes opposite to those observed in post-MI myocytes. Compared with control myocytes infected with adenovirus (Adv) expressing green fluorescent protein (GFP) alone, myocytes infected with Adv expressing both GFP and rat antisense PLM (rASPLM) had 23% less PLM protein (P < 0.012) at 3 days, but no differences were found in sarcoplasmic reticulum (SR) Ca(2+)-ATPase, Na(+)/Ca(2+) exchanger (NCX1), Na(+)-K(+)-ATPase, and calsequestrin levels. SR Ca(2+) uptake and whole cell capacitance were not affected by rASPLM treatment. Relaxation from caffeine-induced contracture was faster, and NCX1 current amplitudes were higher in rASPLM myocytes, indicating that PLM downregulation enhanced NCX1 activity. In native rat cardiac myocytes, coimmunoprecipitation experiments indicated an association of PLM with NCX1. At 0.6 mM [Ca(2+)](o), rASPLM myocytes had significantly (P < 0.003) lower contraction and [Ca(2+)](i) transient amplitudes than control GFP myocytes. At 5 mM [Ca(2+)](o), both contraction and [Ca(2+)](i) transient amplitudes were higher in rASPLM myocytes. This pattern of contractile and [Ca(2+)](i) transient behavior in rASPLM myocytes was opposite to that observed in post-MI rat myocytes. We conclude that downregulation of PLM in normal rat cardiac myocytes enhanced NCX1 function and affected [Ca(2+)](i) transient and contraction amplitudes. We suggest that PLM downregulation offers a potential therapeutic strategy for ameliorating contractile abnormalities in MI myocytes.

Adenoviridae↗