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V J Kadambi

Publications and source records attributed to V J Kadambi.

15 recordsLinked to original sources
PubMed2000

Cardiac-specific overexpression of calsequestrin results in left ventricular hypertrophy, depressed force-frequency relation and pulsus alternans in vivo.

Cardiac-specific overexpression of calsequestrin has been shown to result in significant decreases in contractile parameters and intracellular Ca(2+)transients in vitro. Therefore, the purpose of the present study was to determine the effects of calsequestrin overexpression on basal cardiac function and the force-frequency relation in vivo. Calsequestrin overexpression mice (CSQ-OE, n=20) and their isogenic controls (WT) were studied with an integrative approach using transthoracic echocardiography, stress-shortening relations, and invasive hemodynamics in intact closed-chest mice. M-mode echocardiography indicated that calsequestrin overexpression resulted in concentric hypertrophy (+52%) and an increase in LV ejection phase indices. However, mean end-systolic stress-shortening coordinates revealed that at matched end-systolic wall-stress, fractional shortening was depressed in CSQ-OE mice. This was confirmed by depressed indices of LV isovolumic contraction and relaxation in CSQ-OE v. WT mice. Furthermore, overexpression of calsequestrin resulted in a downward and leftward shift of the biphasic force-frequency relation; thus, the critical heart (HR(crit)) was significantly lower in calsequestrin-overexpression mice (264+/-15 bpm) than in wild-type controls (365+/-21 bpm). Surprisingly, calsequestrin overexpression was associated with the induction of pulsus alternans in every animal (at an average heart rate of 428+/-26 bpm), whereas none of the wild-type controls displayed this phenomenon. We conclude that: (i) although increased levels of calsequestrin result in decreased myocardial contractility and a depressed force-frequency relation, LV wall stress is reduced and chamber function is normal, and (ii) an increase in SR Ca(2+)storage capacity induces pulsus alternans in the intact anesthetized mouse.

Animals↗
PubMed2000

Influence of sarcoplasmic reticulum calcium loading on mechanical and relaxation restitution.

Mechanical and relaxation restitution represent the restoration of contractile force and relaxation, respectively, in premature beats having progressively longer extrasystolic intervals (ESI); these phenomena are related to intracellular activator Ca(2+) by poorly defined mechanisms. We tested the hypothesis that the level of phospholamban [which modulates the affinity of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase for Ca(2+), and thus the SR Ca(2+) load] may be an important determinant of both mechanical and relaxation restitution. Five mice with ablation of the phospholamban (PLB) gene (PLBKO), eight isogenic wild-type controls (129SvJ), eleven mice with PLB overexpression (PLBOE), and nine isogenic wild-type (FVB/N) controls were anesthetized and instrumented with a 1.4-Fr Millar catheter in the left ventricle and a 1-Fr pacemaker in the right atrium. At a cycle length of 200 ms, extrastimuli with increasing ESI were introduced, and the peak rates of left ventricular isovolumic contraction (+/-dP/dt(max)) were normalized and fit to monoexponential equations. In a subset, the protocols were repeated after ryanodine (4 ng/g) was administered to deplete SR Ca(2+) stores. The time constant of mechanical restitution in PLBKO was significantly shorter [6.3 +/- 1.2 (SE) vs. 47.7 +/- 7.6 ms] and began earlier (50 +/- 10 vs. 70 +/- 19 ms) than in 129SvJ. In contrast, the time constant of mechnical restitution was significantly longer (80.3 +/- 7.6 vs. 54.1 +/- 9.2 ms) in PLBOE than in FVB/N. The time constant of relaxation restitution was less in PLBKO than in 129SvJ (26.2 +/- 9.9 vs. 44.6 +/- 3.3, P < 0.05) but was similar in PLBOE and FVB/N (21.1 +/- 6.3 vs. 20.5 +/- 5.7 ms). Intravenous ryanodine decreased significantly the time constants of mechanical restitution in PLBOE, 129SvJ, and FVB/N but was lethal in PLBKO. In contrast, ryanodine increased the time constant of relaxation restitution. Thus 1) the phospholamban level is a critical determinant of mechanical restitution and (to a lesser extent) relaxation restitution in these transgenic models, and 2) ryanodine differentially affects mechanical and relaxation restitution. Furthermore, our data suggest a dissociation of processes within the SR that govern contraction and relaxation.

Animals↗
PubMed1999

Phospholamban is present in endothelial cells and modulates endothelium-dependent relaxation. Evidence from phospholamban gene-ablated mice.

Vascular endothelial cells regulate vascular smooth muscle tone through Ca2+-dependent production and release of vasoactive molecules. Phospholamban (PLB) is a 24- to 27-kDa phosphoprotein that modulates activity of the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA). Expression of PLB is reportedly limited to cardiac, slow-twitch skeletal and smooth muscle in which PLB is an important regulator of [Ca2+]i and contractility in these muscles. In the present study, we report the existence of PLB in the vascular endothelium, a nonmuscle tissue, and provide functional data on PLB regulation of vascular contractility through its actions in the endothelium. Endothelium-dependent relaxation to acetylcholine was attenuated in aorta of PLB-deficient (PLB-KO) mice compared with wild-type (WT) controls. This effect was not due to actions of nitric oxide on the smooth muscle, because sodium nitroprusside-mediated relaxation in either denuded or endothelium-intact aortas was unaffected by PLB ablation. Relative to denuded vessels, relaxation to forskolin was enhanced in WT endothelium-intact aortas. The endothelium-dependent component of this relaxation was attenuated in PLB-KO aortas. To investigate whether these changes were due to PLB, WT mouse aorta endothelial cells were isolated. Both reverse transcriptase-polymerase chain reaction and Western blot analyses revealed the presence of PLB in endothelial cells, which were shown to be >98% pure by diI-acetylated LDL uptake and nuclear counterstaining. These data indicate that PLB is present and modulates vascular function as a result of its actions in endothelial cells. The presence of PLB in endothelial cells opens new fields for investigation of Ca2+ regulatory pathways in nonmuscle cells and for modulation of endothelial-vascular interactions.

Animals↗
PubMed1999

Influence of transgenic overexpression of phospholamban on postextrasystolic potentiation.

Twelve mice with PLB overexpression (PLBOE), and 11 isogenic FVB/N wild-type (WT) controls, were anesthetized and instrumented with a 1.4 F Millar catheter in the LV and a 1 F pacemaker in the right atrium. At a cycle length of 200 ms and a fixed extrastimulus of 120 ms, extrastimuli with increasing intervals (PESI) up to 1000 ms were introduced, and the peak rates of LV isovolumic contraction (+/- dP/dtmax) were normalized and fit to monoexponential equations. In a subset of animals, the protocols were repeated after ryanodine (4 ng/g) was given to deplete SR Ca2+ stores. The time constant and the plateau of the exponential curve fits were significantly greater in PLBOE than WT (107.8 +/- 7.0 v 75.2 +/- 5.5 ms and 1.39 +/- 0.03 v 1.08 +/- 0.02, both P < 0.05). At 200, 600 and 1000 ms, the normalized dP/dt was significantly greater in PLBOE than WT. After ryanodine, normalized dP/dt was significantly decreased in PLBOE, but unchanged in WT. The protein levels of the sodium-calcium exchanger normalized to calsequestrin were increased 3.7 +/- 0.3-fold in PLBOE compared to controls. In conclusion, the phospholamban level is a critical determinant of postextrasystolic potentiation in this transgenic model, and is differentially impaired by ryanodine at long diastolic intervals in PLBOE v controls. These differences may be due in part to changes in the protein level and resultant activity of the sodium calcium exchanger.

Adenosine Triphosphatases↗
PubMed1999

Modulation of force-frequency relation by phospholamban in genetically engineered mice.

Phospholamban levels regulate cardiac sarcoplasmic reticulum Ca2+ pump activity and myocardial contractility. To determine whether and to what extent phospholamban modulates the force-frequency relation and ventricular relaxation in vivo, we studied transgenic mice overexpressing phospholamban (PLBOE), gene-targeted mice without phospholamban (PLBKO), and isogenic wild-type controls. Contractility was assessed by the peak rate of left ventricular (LV) isovolumic contraction (+dP/dtmax), and diastolic function was assessed by both the peak rate (-dP/dtmax) and the time constant (tau) of isovolumic LV relaxation, using a high-fidelity LV catheter. Incremental atrial pacing was used to generate heart rate vs. -dP/dtmax (force-frequency) relations. Biphasic force-frequency relations were produced in all animals, and the critical heart rate (HRcrit) was taken as the heart rate at which dP/dtmax was maximal. The average LV +dP/dtmax increased in both PLBKO and PLBOE compared with their isogenic controls (both P < 0.05). The HRcrit for LV +dP/dtmax was significantly higher in PLBKO (427 +/- 20 beats/min) compared with controls (360 +/- 18 beats/min), whereas the HRcrit in PLBOE (340 +/- 30 beats/min) was significantly lower compared with that in isogenic controls (440 +/- 25 beats/min). The intrinsic heart rates were significantly lower, and the HRcrit and the +/-dP/dtmax at HRcrit were significantly greater in FVB/N than in SvJ control mice. We conclude that 1) the level of phospholamban is a critical negative determinant of the force-frequency relation and myocardial contractility in vivo, and 2) contractile parameters may differ significantly between strains of normal mice.

Animals↗
PubMed1998

Pentameric assembly of phospholamban facilitates inhibition of cardiac function in vivo.

Phospholamban has been proposed to coexist as pentamers and monomers in native sarcoplasmic reticulum membranes. To determine its functional unit in vivo, we reintroduced wild-type (pentameric) or monomeric mutant (C41F) phospholamban in the hearts of phospholamban knockout mice. Transgenic lines, expressing similar levels of mutant or wild-type phospholamban, were identified, and their cardiac phenotypes were characterized in parallel. Sarcoplasmic reticulum Ca2+ transport assays indicated similar decreases in SERCA2 Ca2+ affinity by mutant or wild-type phospholamban. However, the time constants of relaxation and Ca2+ transient decline in isolated cardiomyocytes were diminished to a greater extent by wild-type than mutant phospholamban, even without significant differences in the amplitudes of myocyte contraction and Ca2+ transients between the two groups. Langendorff perfusion also indicated that mutant phospholamban was not capable of depressing the enhanced relaxation parameters of the phospholamban knockout hearts to the same extent as wild-type phospholamban. Moreover, in vivo assessment of mouse hemodynamics revealed a greater depression of cardiac function in wild-type than mutant phospholamban hearts. Thus, the mutant or monomeric form of phospholamban was not as effective in slowing Ca2+ decline or relaxation in cardiomyocytes, hearts, or intact animals as wild-type or pentameric phospholamban. These findings suggest that pentameric assembly of phospholamban is necessary for optimal regulation of myocardial contractility in vivo.

Animals↗
PubMed1998

Cardiac-specific overexpression of mouse cardiac calsequestrin is associated with depressed cardiovascular function and hypertrophy in transgenic mice.

Calsequestrin is a high capacity Ca2+-binding protein in the sarcoplasmic reticulum (SR) lumen. To elucidate the functional role of calsequestrin in vivo, transgenic mice were generated that overexpressed mouse cardiac calsequestrin in the heart. Overexpression (20-fold) of calsequestrin was associated with cardiac hypertrophy and induction of a fetal gene expression program. Isolated transgenic cardiomyocytes exhibited diminished shortening fraction (46%), shortening rate (60%), and relengthening rate (60%). The Ca2+ transient amplitude was also depressed (45%), although the SR Ca2+ storage capacity was augmented, as suggested by caffeine application studies. These alterations were associated with a decrease in L-type Ca2+ current density and prolongation of this channel's inactivation kinetics without changes in Na+-Ca2+ exchanger current density. Furthermore, there were increases in protein levels of SR Ca2+-ATPase, phospholamban, and calreticulin and decreases in FKBP12, without alterations in ryanodine receptor, junctin, and triadin levels in transgenic hearts. Left ventricular function analysis in Langendorff perfused hearts and closed-chest anesthetized mice also indicated depressed rates of contraction and relaxation of transgenic hearts. These findings suggest that calsequestrin overexpression is associated with increases in SR Ca2+ capacity, but decreases in Ca2+-induced SR Ca2+ release, leading to depressed contractility in the mammalian heart.

Amino Acid Sequence↗
PubMed1998

Transgenic approaches to define the functional role of dual site phospholamban phosphorylation.

Phospholamban is a critical regulator of the sarcoplasmic reticulum Ca2+-ATPase activity and myocardial contractility. Phosphorylation of phospholamban occurs on both Ser16 and Thr17 during isoproterenol stimulation. To determine the physiological significance of dual site phospholamban phosphorylation, we generated transgenic models expressing either wild-type or the Ser16 --> Ala mutant phospholamban in the cardiac compartment of the phospholamban knockout mice. Transgenic lines with similar levels of mutant or wild-type phospholamban were studied in parallel. Langendorff perfusion indicated that the basal hyperdynamic cardiac function of the knockout mouse was reversed to the same extent by reinsertion of either wild-type or mutant phospholamban. However, isoproterenol stimulation was associated with much lower responses in the contractile parameters of mutant phospholamban compared with wild-type hearts. These attenuated responses were due to lack of phosphorylation of mutant phospholamban, assessed in 32P labeling perfusion experiments. The lack of phospholamban phosphorylation in vivo was not due to conversion of Ser16 to Ala, since the mutated phospholamban form could serve as substrate for the calcium-calmodulin-dependent protein kinase in vitro. These findings indicate that phosphorylation of Ser16 is a prerequisite for Thr17 phosphorylation in phospholamban, and prevention of phosphoserine formation results in attenuation of the beta-agonist stimulatory responses in the mammalian heart.

Animals↗
PubMed1998

Overexpression of phospholamban alters inactivation kinetics of L-type Ca2+ channel currents in mouse atrial myocytes.

In mammalian ventricular myocytes, inactivation of L-type Ca2+ channels (CaCh) is controlled by voltage- and Ca2+-dependent mechanisms. The Ca2+-dependent component is regulated by the Ca2+ released from the sarcoplasmic reticulum (SR). However, little is known about the inactivation properties of CaCh in atrial myocytes, which lack spatial coupling between CaCh and SR Ca2+ release channels. The cardiac SR Ca2+ load is determined by the activity of SR Ca2+-ATPase, which is inversely regulated by the levels of phospholamban (PLB). To investigate the role of SR Ca2+ in atrial myocytes, Ca2+ currents (I Ca) were recorded in mouse atrial myocytes recorded from wild-type (WT) mice and the characteristics were compared to those obtained from atrial myocytes from the transgenic mice overexpressing PLB (PLB-OEX). ICa from WT exhibited fast and slow components of inactivation and the rate of inactivation was slowed when SR Ca2+ was depleted by caffeine, suggesting that the inactivation of atrial ICa is modulated by SR Ca2+ load. The current density and voltage-dependence of ICa were similar between the two groups. However, the fast component of inactivation was significantly reduced in PLB-OEX. When Ca2+ was replaced by Ba2+ or in the presence of caffeine, inactivation was slowed and the decay of the current was not significantly different between WT and PLB-OEX. These results suggest that the inactivation of ICa in mouse atrial myocytes involves Ca2+-dependent and voltage-dependent components. The decrease in the faster component of inactivation in PLB-OEX is consistent with the idea that CaCh and SR Ca2+ release channels are functionally coupled and Ca2+ released from the SR contributes the Ca2+-dependent inactivation component.

Animals↗
PubMed1998

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.

Adrenergic beta-Agonists↗
PubMed1997

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.

Animals↗
PubMed1997

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. Future studies aimed towards crystallization of phospholamban and the SR Ca(2+)-ATPase in their native SR environment may provide clues to their tertiary and quaternary structures and may further elucidate the mechanisms underlying the phospholamban regulatory effects in vivo.

Animals↗
PubMed1997

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.

Adrenergic beta-Agonists↗
PubMed1996

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.

Adrenergic beta-Agonists↗