Unveiling the mechanisms underlying photothermal efficiency of gold shell-isolated nanoparticles (AuSHINs) on ductal mammary carcinoma cells (BT-474).

Gold nanoparticles are valuable photothermal agents owing to their efficient photothermal conversion, photobleaching resistance, and potential surface functionalization. Herein, we combined bioinspired membranes with in vitro assays to elicit the molecular mechanisms of gold shell-isolated nanoparticles (AuSHINs) on ductal mammary carcinoma cells (BT-474). Langmuir and Langmuir-Schaefer (LS) films were handled to build biomembranes from BT-474 lipid extract. AuSHINs incorporation led to surface pressure-area (π-A) isotherms expansion, increasing membrane flexibility. Fourier-transform infrared spectroscopy (FTIR) of LS multilayers revealed electrostatic AuSHINs interaction with head portions of BT-474 lipid extract, causing lipid chain disorganization. Limited AuSHINs insertion into monolayer contributed to hydroperoxidation of the unsaturated lipids upon irradiation, consistently with the surface area increments of ca. 2.0%. In fact, membrane disruption of irradiated BT-474 cells containing AuSHINs was confirmed by confocal microscopy and LDH leakage, with greater damage at 2.2 × 1013 AuSHINs/mL. Furthermore, the decrease in nuclei dimensions indicates cell death through photoinduced damage.

Immunoassay quantification using surface-enhanced fluorescence (SEF) tags.

Fluorescence-based immonoassays are widely used in several areas, ranging from basic biomedical research to disease diagnostics. A variety of new probes have been developed recently to address some limitations in typical assays performed with organic dyes. Ideally, new fluorescence tags that allow quantification with a low limit of detection are highly desired. In this work, the surface-enhanced fluorescence (SEF) phenomenon was explored in the development of tags for Immunoglobulin-M (IgM) detection. Shell-isolated gold nanoparticles (Au-SHINs) with 100 nm core size and a 10 nm silica shell were synthesized. These particles contain an outermost thin fluorescent layer of nile blue (NB) that was further coated by another 5 nm of silica (SEF tags). The outer silica shell was then functionalized with antibodies to allow the detection of IgM as in typical immunological sandwich assays. IgM concentrations down to the 10 ng mL-1 mark were successfully detected. A linear dependence between the average fluorescence intensity and the IgM concentration was found.

Regulation of thyroid hormone receptor isoforms in physiological and pathological cardiac hypertrophy.

Physiological and pathological cardiac hypertrophy have directionally opposite changes in transcription of thyroid hormone (TH)-responsive genes, including alpha- and beta-myosin heavy chain (MyHC) and sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), and TH treatment can reverse molecular and functional abnormalities in pathological hypertrophy, such as pressure overload. These findings suggest relative hypothyroidism in pathological hypertrophy, but serum levels of TH are usually normal. We studied the regulation of TH receptors (TRs) beta1, alpha1, and alpha2 in pathological and physiological rat cardiac hypertrophy models with hypothyroid- and hyperthyroid-like changes in the TH target genes, alpha- and beta-MyHC and SERCA. All 3 TR subtypes in myocytes were downregulated in 2 hypertrophy models with a hypothyroid-like mRNA phenotype, phenylephrine in culture and pressure overload in vivo. Myocyte TRbeta1 was upregulated in models with a hyperthyroid-like phenotype, TH (triiodothyronine, T3), in culture and exercise in vivo. In myocyte culture, TR overexpression, or excess T3, reversed the effects of phenylephrine on TH-responsive mRNAs and promoters. In addition, TR cotransfection and treatment with the TRbeta1-selective agonist GC-1 suggested different functional coupling of the TR isoforms, TRbeta1 to transcription of beta-MyHC, SERCA, and TRbeta1, and TRalpha1 to alpha-MyHC transcription and increased myocyte size. We conclude that TR isoforms have distinct regulation and function in rat cardiac myocytes. Changes in myocyte TR levels can explain in part the characteristic molecular phenotypes in physiological and pathological cardiac hypertrophy.

A key role for ICAM-1 in generating effector cells mediating inflammatory responses.

We investigated how the accessory molecule interactions encountered during T cell priming influence T cell-mediated destruction of insulin-producing beta cells and lead to type 1 diabetes. T cell receptor (TCR)-transgenic CD4+ T cells were primed under controlled conditions in vitro before being adoptively transferred into transgenic recipients expressing membrane ovalbumin under the control of the rat insulin promoter (RIP-mOVA). During priming, antigen-presenting cell expression of B7-1 without intracellular adhesion molecule 1 (ICAM-1) led to the generation of effector cells that migrated to the pancreata of RIP-mOVA recipients but did not cause diabetes. In contrast, when T cells were primed with APCs expressing both B7-1 and ICAM-1, pronounced destruction of beta cells and a rapid onset of diabetes were observed. Pathogenicity was associated with T cell production of the macrophage-attracting chemokines CCL3 and CCL4. Thus, interactions of lymphocyte function-associated antigen 1 with ICAM-1 during priming induce both qualitative and quantitative alterations in T effector function and induce potentially autodestructive responses.

Vaccination with glutamic acid decarboxylase plasmid DNA protects mice from spontaneous autoimmune diabetes and B7/CD28 costimulation circumvents that protection.

The nonobese diabetic (NOD) mouse develops spontaneous T-cell-dependent autoimmune diabetes. We tested here whether vaccination of NOD mice with a plasmid DNA encoding glutamic acid decarboxylase (GAD), an initial target islet antigen of autoimmune T cell repertoire, would modulate their diabetes. Our results showed that vaccination of young or old female NOD mice with the GAD-plasmid DNA, but not control-plasmid DNA, effectively prevented their diabetes, demonstrating that GAD-plasmid DNA vaccination is quite effective in abrogating diabetes even after the development of insulitis. The prevention of diabetes did not follow the induction of immunoregulatory Th2 cells but was dependent upon CD28/B7 costimulation. Our results suggest a potential for treating spontaneous autoimmune diabetes via DNA vaccination with plasmids encoding self-Ag.

Preconditioning limits mitochondrial Ca(2+) during ischemia in rat hearts: role of K(ATP) channels.

Prolonged myocardial ischemia results in an increase in intracellular calcium concentration ([Ca(2+)]i), which is thought to play a critical role in ischemia-reperfusion injury. Ischemic preconditioning (PC) improves myocardial function during ischemia-reperfusion, a process that may involve opening mitochondrial ATP-sensitive potassium (K(ATP)) channels. Because pharmacological limitation of mitochondrial calcium concentration ([Ca(2+)]m) overload during ischemia-reperfusion has been shown to improve myocardial function, we hypothesized that PC would reduce [Ca(2+)]m during ischemia-reperfusion and that this effect was mediated by opening mitochondrial K(ATP) channels. Isolated rat hearts were subjected to 25 min of global ischemia and 30 min of reperfusion with or without PC in the presence of mitochondrial K(ATP) channel opening (diazoxide, 100 microM) and blockade [5-hydroxydecanoic acid (5-HD), 100 microM]. Contracture during ischemia (end-diastolic pressure) and functional recovery on reperfusion (developed pressure) were assessed. Total [Ca(2+)]i and [Ca(2+)]m were measured using indo 1 fluorescence. Both PC and diazoxide limited the increase in end-diastolic pressure and resulted in greater functional recovery after 30 min of reperfusion, functional effects that were partially or completely abolished by 5-HD. PC and diazoxide also significantly limited the increase in [Ca(2+)]m during ischemia-reperfusion. In addition, PC lowered [Ca(2+)]i during reperfusion, whereas diazoxide paradoxically resulted in increased [Ca(2+)]i during reperfusion. There was an inverse linear relationship between [Ca(2+)]m and developed pressure during reperfusion. PC limits the ischemia-induced increase in mitochondrial, but not total, [Ca(2+)]i, an effect mediated by opening mitochondrial K(ATP) channels. These data suggest that the lowering of mitochondrial calcium overload is a mechanism of cardioprotection in PC.

CXCR5-deficient mice develop functional germinal centers in the splenic T cell zone.

The chemokine receptor CXCR5 is thought to be essential for the migration of B cells into the network of follicular dendritic cells in the spleen. However, as shown here, B cells and follicular dendritic cells do co-localize, albeit aberrantly, even in the absence of CXCR5. In mice lacking CXCR5 both cell types are found in a broad ring around the sinuses of the marginal zones. Upon immunization with the T cell-dependent antigen 2-phenyl-oxazolone, ectopic germinal centers develop in the periarteriolar lymphocyte sheath. A network of follicular dendritic cells forms in the vicinity of the central arteriole within which the antigen-activated B cells proliferate. The analysis of the expressed V gene repertoire revealed that during B cell proliferation, hypermutation is activated and V region genes accumulate somatic mutations. The pattern of somatic mutations suggests that affinity selection may occur. This analysis confirms that in CXCR5-deficient mice, the organization of splenic primary follicles is severely impaired. However, within the T cell zone a micro-environment is built up, which provides all requirements needed for the affinity maturation to take place.

Chronic dipyridamole therapy produces sustained protection against cardiac ischemia-reperfusion injury.

Sustained protection against ischemia-reperfusion injury is not available for patients at risk for myocardial infarction who may require emergent reperfusion therapy. Whereas ischemic preconditioning and adenosinergic agents reduce myocardial injury, they are only effective when given immediately before ischemia or reperfusion. We recently found chronic ethanol exposure, an adenosine uptake inhibitor, produced sustained cardioprotection against ischemia-reperfusion injury. We now ask whether chronic dipyridamole therapy, a clinically usable nucleoside transport inhibitor, induces similar cardioprotection. Perfused hearts from guinea pigs, given dipyridamole (4 mg. kg(-1). day(-1)) in their water for 2-6 wk (n = 10 for each group), underwent ischemia-reperfusion. Injury was assessed by recovery of left ventricular developed (LVDP) and end-diastolic (LVEDP) pressures and creatine kinase release. During reperfusion, hearts from dipyridamole-treated animals (6 wk) had 74% higher LVDP, 28% lower LVEDP, and 61% lower creatine kinase release versus controls. Adenosine A(1)-receptor antagonism (8-cyclopentyl-1, 3-dipropylxanthine; 200 nM) abolished the protection of dipyridamole but A(2) antagonism (3,7-dimethyl-1-propargylxanthine; 10 mM) did not. Dipyridamole therapy produces sustained protection against ischemia-reperfusion injury in guinea pigs. This cardioprotection requires adenosine A(1) receptor signaling at the time of ischemia.

Role of slowed Ca(2+) transient decline in slowed relaxation during myocardial ischemia.

The goal of this study was to test the hypothesis that during myocardial ischemia, slowing of the Ca(2+) transient decline causes slowed relaxation. Our approach was to monitor pressure and Ca(2+) transients in isovolumic rat hearts during control and low flow ischemia conditions. In addition, we experimentally slowed the decline of the Ca(2+) transient using cyclopiazonic acid (CPA) to inhibit the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA, the most important pump for rapidly transporting Ca(2+) out of the cytosol). Using 9 microm CPA during normoxia, we were able to reproduce the slowed Ca(2+) transient decline and slowed relaxation found during low flow ischemia. The time constants of cytosolic [Ca(2+)] decline and pressure decline (tau(Ca) and tau(P) respectively) with CPA (78+/-5 ms and 64+/-3 ms) were similar to those found with ischemia (89+/-12 ms and 72+/-10 ms, mean+/-SEM, n=7) and were considerably greater than for controls (41+/-3 and 25+/-2 ms, mean+/-SEM, n=14, P<0.01). Furthermore, the relationship of tau(P) v tau(Ca) with CPA was similar to that found with ischemia. These findings are consistent with the hypothesis that the slowed Ca(2+) transient decline with both CPA and ischemia causes slowed relaxation. Consistent with this conclusion, a simple mathematical model to relate cytosolic [Ca(2+)] and pressure also suggests that slowed pressure relaxation can be explained by slowing of the Ca(2+) transient decline. This study suggests that impaired Ca(2+) uptake is a major injury causing slowed relaxation during ischemia.

Thermodynamic limitation for Ca2+ handling contributes to decreased contractile reserve in rat hearts.

The free energy release from ATP hydrolysis (|DeltaG approximately p|) is decreased by inhibiting the creatine kinase (CK) reaction, which may limit the thermodynamic driving force for the sarcoplasmic reticulum (SR) Ca2+ pumps and thereby cause a decrease in contractile reserve. To determine whether a decrease in |DeltaG approximately p| results in decreased contractile reserve by impairing Ca2+ handling, we measured left ventricular pressure and cytosolic Ca2+concentration ([Ca2+]c; by indo 1 fluorescence) in isolated perfused rat hearts, with >95% inhibition of CK with 90 micromol iodoacetamide. Iodoacetamide did not directly alter SR Ca2+-ATPase activity, baseline left ventricular developed pressure, or baseline [Ca2+]c. When perfusate Ca2+ concentration was increased from 1.2 to 3.3 mM, LV developed pressure increased from 67 +/- 6 to 119 +/- 8 mmHg in control hearts (P < 0.05) but did not significantly increase in CK-inhibited hearts. Similarly, the amplitude of the [Ca2+]c transient increased from 548 +/- 54 to 852 +/- 140 nM in control hearts (P < 0.05) but did not significantly increase in CK-inhibited hearts. We conclude that decreased |DeltaG approximately p| limits intracellular Ca2+ handling and thereby limits contractile reserve.

The dynamic structure of the germinal center.

Analysis of germinal centers (GCs) in chronically inflamed human tonsils has led to the dogma that GCs contain two compartments with separate functions: a dark zone where B cells proliferate and hypermutate; and a light zone where selection and differentiation occur. However, here Stephanie Camacho and colleagues discuss immunohistological analysis of splenic GCs arising de novo that reveal a more plastic structure.

Ca2+ regulates the kinetics of tension development in intact cardiac muscle.

The goal of this study was to determine whether Ca2+ plays a role in regulating tension development kinetics in intact cardiac muscle. In cardiac muscle, this fundamental issue of Ca2+ regulation has been controversial. The approach was to induce steady-state tetanic contractions of intact right ventricular trabeculae from rat hearts at varying external Ca2+ concentrations ([Ca2+]) at 22 degreesC. During tetani, cross bridges were mechanically disrupted and the kinetics of tension redevelopment were assessed from the rate constant of exponential tension redevelopment (ktr). There was a relationship between ktr and external [Ca2+] that was similar in form to the relationship between tension and [Ca2+]. Thus a close relationship also existed between ktr and tension (r = 0.88; P < 0. 001); whereas at maximal tetanic tension (saturating cytosolic [Ca2+]), ktr was 16.4 +/- 2.2 s-1 (mean +/- SE, n = 7), at zero tension (low cytosolic [Ca2+]), ktr extrapolated to 20% of maximum (3.3 +/- 0.7 s-1). Qualitatively similar results were obtained using different mechanical protocols to disrupt cross bridges. These data demonstrate that tension redevelopment kinetics in intact cardiac muscle are influenced by the level of Ca2+ activation. These findings contrast with the findings of one previous study of intact cardiac muscle. Activation dependence of tension development kinetics may play an important role in determining the rate and extent of myocardial tension rise during the cardiac cycle in vivo.

Alcohol consumption reduces ischemia-reperfusion injury by species-specific signaling in guinea pigs and rats.

We recently discovered that regular alcohol consumption reduces ischemia-reperfusion injury to the same degree as ischemic preconditioning in guinea pig hearts. Ischemic preconditioning, like this cardioprotective effect of alcohol, is mediated by adenosine signaling in guinea pigs. In rats, ischemic preconditioning may be mediated predominantly by alpha1-adrenergic signaling. To be certain that this protective effect of alcohol is a general biological response, we searched for alcohol's cardioprotection in rat and identified a potential signaling mechanism. Hearts isolated from alcohol-fed guinea pigs and rats were subjected to ischemia-reperfusion. Hearts from alcohol-fed animals showed greater recovery of left ventricular developed pressure than controls (guinea pigs, 46 vs. 29%; rats, 50 vs. 31%) and decreased myocyte necrosis assessed by creatine kinase release (guinea pigs, 204 +/- 42 vs. 440 +/- 70 U . ml-1 . g dry wt-1; rats 158 +/- 13 vs. 328 +/- 31 U . ml-1 . g dry wt-1). Adenosine receptor blockade [8-(p-sulfophenyl)theophylline] abolished alcohol's protection in guinea pig but not rat hearts. By contrast, alpha1-adrenergic blockade (prazosin) abolished alcohol's protection in rat but not guinea pig hearts. We conclude that regular alcohol consumption reduces ischemia-reperfusion injury and is mediated by species-specific signaling mechanisms. A major goal of cardiovascular research is to find a pharmacologically induced chronic state of preconditioning. Understanding the mechanisms of alcohol's cardioprotection against ischemia-reperfusion injury may aid in reaching this goal.

Chronic alcohol-induced changes in cardiac contractility are not due to changes in the cytosolic Ca2+ transient.

Long-standing heavy alcohol consumption acts as a chronic stress on the heart. It is thought that alcohol-induced changes of contractility are due to altered Ca2+ handling, but no measurements of cytosolic Ca2+ ([Ca2+]c) after chronic alcohol exposure have been made. Therefore experiments were performed to determine whether alcohol-induced changes in contractility are due to altered Ca2+ handling by measuring [Ca2+]c (indo 1) in hearts from rats drinking 36% ethanol for 7 mo and age-matched controls. Peak left ventricular pressure was depressed (-16%), whereas rates of contraction (12%) and relaxation (14-20%) were faster in alcohol-exposed hearts. Systolic [Ca2+]c (808 +/- 45 vs. 813 +/- 45 nM), diastolic [Ca2+]c (195 +/- 11 vs. 193 +/- 10 nM), and rates of [Ca2+]c rise and decline were the same in alcohol-exposed and control hearts. Protein levels of Ca2+-handling proteins, sarcoplasmic reticulum Ca2+-ATPase and phospholamban, were the same in myocytes isolated from alcohol-exposed and control hearts (SDS-polyacrylamide gel). These data suggest that chronic alcohol-induced contractile changes are not due to altered Ca2+ handling but may be due to changes at the level of the myofilament. As a first step in elucidating the mechanism(s) of alcohol-induced changes at the myofilament, we assessed myosin heavy chain (MHC) isoform content (SDS-polyacrylamide gel). alpha-MHC was decreased relative to beta-MHC (a/a + b = 0.55 +/- 0.03 vs. 0.66 +/- 0.02; P < 0.02) in alcohol-exposed hearts, which cannot account for the observed alcohol-induced contractile changes. In conclusion, changes of myocardial contractility due to chronic alcohol exposure do not result from altered Ca2+ handling but from changes at the level of the myofilament that do not involve MHC isoform shifts.

Activation of epsilon protein kinase C correlates with a cardioprotective effect of regular ethanol consumption.

In addition to decreasing the incidence of myocardial infarction, recent epidemiological data suggest that regular alcohol consumption improves survival after myocardial infarction. We recently found that chronic ethanol exposure induces long-term protection against cardiac ischemia-reperfusion injury, which improves myocardial recovery after infarction. Furthermore, this cardioprotection by ethanol is mediated through myocyte adenosine A1 receptors. We now determine the role of protein kinase C (PKC) in ethanol's protective effect against ischemia-reperfusion injury. Using perfused hearts of ethanol-fed guinea pigs, we find that improved contractile recovery and creatine kinase release after ischemia-reperfusion are abolished by PKC inhibition with chelerythrine. Western blot analysis and immunofluorescence localization demonstrate that regular ethanol consumption causes sustained translocation (activation) of epsilonPKC, but not delta or alphaPKC. This same isozyme is directly implicated in ischemic preconditioning's protection against ischemia-reperfusion injury. Our findings suggest (i) that regular ethanol consumption induces long-term cardioprotection through sustained translocation of epsilonPKC and (ii) that PKC activity is necessary at the time of ischemia to mediate ethanol's protective effect against ischemia-reperfusion injury. Studying this selective effect of ethanol on epsilonPKC activation may lead to new therapies to protect against ischemia-reperfusion injury in the heart and other organ systems.

Thyroid hormone improves function and Ca2+ handling in pressure overload hypertrophy. Association with increased sarcoplasmic reticulum Ca2+-ATPase and alpha-myosin heavy chain in rat hearts.

We asked whether thyroid hormone (T4) would improve heart function in left ventricular hypertrophy (LVH) induced by pressure overload (aortic banding). After banding for 10-22 wk, rats were treated with T4 or saline for 10-14 d. Isovolumic LV pressure and cytosolic [Ca2+] (indo-1) were assessed in perfused hearts. Sarcoplasmic reticulum Ca2+-ATPase (SERCA), phospholamban, and alpha- and beta-myosin heavy chain (MHC) proteins were assayed in homogenates of myocytes isolated from the same hearts. Of 14 banded hearts treated with saline, 8 had compensated LVH with normal function (LVHcomp), whereas 6 had abnormal contraction, relaxation, and calcium handling (LVHdecomp). In contrast, banded animals treated with T4 had no myocardial dysfunction; these hearts had increased contractility, and faster relaxation and cytosolic [Ca2+] decline compared with LVHcomp and LVHdecomp. Myocytes from banded hearts treated with T4 were hypertrophied but had increased concentrations of alpha-MHC and SERCA proteins, similar to physiological hypertrophy induced by exercise. Thus thyroid hormone improves LV function and calcium handling in pressure overload hypertrophy, and these beneficial effects are related to changes in myocyte gene expression. Induction of physiological hypertrophy by thyroid hormone-like signaling might be a therapeutic strategy for treating cardiac dysfunction in pathological hypertrophy and heart failure.

Inorganic phosphate and coronary perfusion pressure mediate contractile dysfunction during mild ischemia.

During mild graded ischemia in perfused rat hearts, we (V.M. Figueredo, R. Brandes, M. W. Weiner, B. M. Massie, and S. A. Camacho. J. Clin. Invest 90: 1794-1802, 1992) previously found a relationship between decreased left ventricular developed pressure (LVDP) and increased Pi, in which intracellular pH, cytosolic Ca2+ concentration ([Ca2+]i), ATP, and free-energy change of ATP hydrolysis were not altered enough to affect contractility. However, the contribution of decreased coronary perfusion pressure (CPP) to decreased LVDP could not be determined. Thus, in the present study, graded hypoxia in perfused rat hearts (95-37.5% O2) was used to increase Pi to similar levels produced during mild ischemia without altering CPP and minimizing changes of other potential mediators of contractile dysfunction. 31P-magnetic resonance spectroscopy and indo 1 fluorescence were used to assess energy metabolites and [Ca2+]i, respectively. The relationship between LVDP and Pi during graded hypoxia was fit to a monoexponential (LVDP = 105 x e-0.04Pi). These data were compared with the relationship of LVDP and Pi during mild ischemia (LVDP = 106 x e-0.08Pi) (V. M. Figueredo, R. Brandes, M. W. Weiner, B. M. Massie, and S. A. Camacho. J. Clin. Invest 90: 1794-1802, 1992). The exponential constant, which describes the effect of Pi on LVDP, was 50% lower during graded hypoxia relative to mild ischemia. This suggests that another mediator, which accounted for approximately 50% of the decrease of LVDP during mild ischemia, was not present during hypoxia. Because CPP decreased during ischemia but not hypoxia, these data suggest that CPP and Pi contribute similarly in mediating contractile dysfunction during mild ischemia.

Regular alcohol consumption mimics cardiac preconditioning by protecting against ischemia-reperfusion injury.

Epidemiologic studies indicate that long-term alcohol consumption decreases the incidence of coronary disease and may improve outcome after myocardial infarction. Attenuation of ischemia-reperfusion injury after myocardial infarction improves survival. This study investigates the possibility that alcohol consumption can improve survival after myocardial infarction by reducing ischemia-reperfusion injury. Hearts were isolated from guinea pigs after drinking ethanol for 3-12 weeks and subjected to global ischemia and reperfusion. Hearts from animals drinking ethanol showed improved functional recovery and decreased myocyte damage when compared with controls. Adenosine A1 receptor blockade abolished the protection provided by ethanol consumption. These findings indicate that long-term alcohol consumption reduces myocardial ischemia-reperfusion injury and that adenosine A1 receptors are required for this protective effect of ethanol. This cardioprotective effect of long-term alcohol consumption mimics preconditioning and may, in part, account for the beneficial effect of moderate drinking on cardiac health.

Role of MgADP in the development of diastolic dysfunction in the intact beating rat heart.

Sarcomere relaxation depends on dissociation of actin and myosin, which is regulated by a number of factors, including intracellular [MgATP] as well as MgATP hydrolysis products [MgADP] and inorganic phosphate [Pi], pHi, and cytosolic calcium concentration ([Ca2+]c). To distinguish the contribution of MgADP from the other regulators in the development of diastolic dysfunction, we used a strategy to increase free [MgADP] without changing [MgATP], [Pi], or pHi. This was achieved by applying a low dose of iodoacetamide to selectively inhibit the creatine kinase activity in isolated perfused rat hearts. [MgATP], [MgADP], [Pi], and [H+] were determined using 31P NMR spectroscopy. The [Ca2+]c and the glycolytic rate were also measured. We observed an approximately threefold increase in left ventricular end diastolic pressure (LVEDP) and 38% increase in the time constant of pressure decay (P < 0.05) in these hearts, indicating a significant impairment of diastolic function. The increase in LVEDP was closely related to the increase in free [MgADP]. Rate of glycolysis was not changed, and [Ca2+]c increased by 16%, which cannot explain the severity of diastolic dysfunction. Thus, our data indicate that MgADP contributes significantly to diastolic dysfunction, possibly by slowing the rate of cross-bridge cycling.