A conserved set of mutations for stabilizing soluble envelope protein dimers from dengue and Zika viruses to advance the development of subunit vaccines.

Dengue viruses (DENV serotypes 1-4) and Zika virus (ZIKV) are related flaviviruses that continue to be a public health concern, infecting hundreds of millions of people annually. The traditional live-attenuated virus vaccine approach has been challenging for the four DENV serotypes because of the need to achieve balanced replication of four independent vaccine components. Subunit vaccines represent an alternative approach that may circumvent problems inherent with live-attenuated DENV vaccines. In mature virus particles, the envelope (E) protein forms a homodimer that covers the surface of the virus and is the major target of neutralizing antibodies. Many neutralizing antibodies bind to quaternary epitopes that span across both E proteins in the homodimer. For soluble E (sE) protein to be a viable subunit vaccine, the antigens should be easy to produce and retain quaternary epitopes recognized by neutralizing antibodies. However, WT sE proteins are primarily monomeric at conditions relevant for vaccination and exhibit low expression yields. Previously, we identified amino acid mutations that stabilize the sE homodimer from DENV2 and dramatically raise expression yields. Here, we tested whether these same mutations raise the stability of sE from other DENV serotypes and ZIKV. We show that the mutations raise thermostability for sE from all the viruses, increase production yields from 4-fold to 250-fold, stabilize the homodimer, and promote binding to dimer-specific neutralizing antibodies. Our findings suggest that these sE variants could be valuable resources in the efforts to develop effective subunit vaccines for DENV serotypes 1 to 4 and ZIKV.

Human cytochrome P450 enzymes bind drugs and other substrates mainly through conformational-selection modes.

Cytochrome P450 (P450) enzymes are major catalysts involved in the oxidations of most drugs, steroids, carcinogens, fat-soluble vitamins, and natural products. The binding of substrates to some of the 57 human P450s and other mammalian P450s is more complex than a two-state system and has been proposed to involve mechanisms such as multiple ligand occupancy, induced-fit, and conformational-selection. Here, we used kinetic analysis of binding with multiple concentrations of substrates and computational modeling of these data to discern possible binding modes of several human P450s. We observed that P450 2D6 binds its ligand rolapitant in a mechanism involving conformational-selection. P450 4A11 bound the substrate lauric acid via conformational-selection, as did P450 2C8 with palmitic acid. Binding of the steroid progesterone to P450 21A2 was also best described by a conformational-selection model. Hexyl isonicotinate binding to P450 2E1 could be described by either a conformational-selection or an induced-fit model. Simulation of the binding of the ligands midazolam, bromocriptine, testosterone, and ketoconazole to P450 3A4 was consistent with an induced-fit or a conformational-selection model, but the concentration dependence of binding rates for varying both P450 3A4 and midazolam concentrations revealed discordance in the parameters, indicative of conformational-selection. Binding of the P450s 2C8, 2D6, 3A4, 4A11, and 21A2 was best described by conformational-selection, and P450 2E1 appeared to fit either mode. These findings highlight the complexity of human P450-substrate interactions and that conformational-selection is a dominant feature of many of these interactions.

Sulfenylation of Human Liver and Kidney Microsomal Cytochromes P450 and Other Drug-Metabolizing Enzymes as a Response to Redox Alteration.

The lumen of the endoplasmic reticulum (ER) provides an oxidizing environment to aid in the formation of disulfide bonds, which is tightly regulated by both antioxidant proteins and small molecules. On the cytoplasmic side of the ER, cytochrome P450 (P450) proteins have been identified as a superfamily of enzymes that are important in the formation of endogenous chemicals as well as in the detoxication of xenobiotics. Our previous report described oxidative inhibition of P450 Family 4 enzymes via oxidation of the heme-thiolate cysteine to a sulfenic acid (-SOH) (Albertolle, M. E. et al. (2017) J. Biol. Chem. 292, 11230-11242). Further proteomic analyses of murine kidney and liver microsomes led to the finding that a number of other drug-metabolizing enzymes located in the ER are also redox-regulated in this manner. We expanded our analysis of sulfenylated enzymes to human liver and kidney microsomes. Evaluation of the sulfenylation, catalytic activity, and spectral properties of P450s 1A2, 2C8, 2D6, and 3A4 led to the identification of two classes of redox sensitivity in P450 enzymes: heme-thiolate-sensitive and thiol-insensitive. These findings provide evidence for a mammalian P450 regulatory mechanism, which may also be relevant to other drug-metabolizing enzymes. (Data are available via ProteomeXchange with identifier PXD007913.).

Do all patients with implantable cardioverter defibrillator need a generator change? A health service evaluation of patients who underwent generator changes from a single tertiary center.

BACKGROUND: The patient characteristics, therapy received and outcomes after one or more implantable cardioverter defibrillator (ICD) generator changes from contemporary practice is not well known. METHODS: We conducted a health service evaluation of patients who underwent ICD implantation and generator change. Patients who had generator changes from February 2016 to October 2019 were identified from our database and electronic records were reviewed for patient characteristics, number of generator changes, receipt of therapy and death. RESULTS: Our database included 88 patients with a generator change. A total of 22 patients (25.0%) received dual chamber ICD, 10 patients (11.4%) received single chamber ICD, 54 patients (61.3%) received cardiac resynchronization therapy defibrillator and 2 patients (2.3%) received subcutaneous ICD. A second generator change occurred in 18 patients and a third generator changes was performed in 6 patients. There were 29 deaths and a follow up period of 9.4 ± 2.9 years. From implant to initial generator change 39 patients had appropriate antitachycardia pacing (ATP), 6 patient had inappropriate ATP, 29 patients had appropriate shocks and 5 patients had an inappropriate shock. Between the 1st and 2nd generator change and the 2nd and 3rd there were no cases of inappropriate ATP or shock. Overall, 42 patients out of the 88 had appropriate therapy (47.7%) and 7 patients had inappropriate therapy (8.0%). CONCLUSIONS: Most patients with ICDs do not receive therapy and a minority have inappropriate therapy which typically occur before the first generator change as we observed no inappropriate therapy beyond the first generator change.

Inherent steroid 17α,20-lyase activity in defunct cytochrome P450 17A enzymes.

Cytochrome P450 (P450) 17A1 catalyzes the oxidations of progesterone and pregnenolone and is the major source of androgens. The enzyme catalyzes both 17α-hydroxylation and a subsequent 17α,20-lyase reaction, and several mechanisms have been proposed for the latter step. Zebrafish P450 17A2 catalyzes only the 17α-hydroxylations. We previously reported high similarity of the crystal structures of zebrafish P450 17A1 and 17A2 and human P450 17A1. Five residues near the heme, which differed, were changed. We also crystallized this five-residue zebrafish P450 17A1 mutant, and the active site still resembled the structure in the other proteins, with some important differences. These P450 17A1 and 17A2 mutants had catalytic profiles more similar to each other than did the wildtype proteins. Docking with these structures can explain several minor products, which require multiple enzyme conformations. The 17α-hydroperoxy (OOH) derivatives of the steroids were used as oxygen surrogates. Human P450 17A1 and zebrafish P450s 17A1 and P450 17A2 readily converted these to the lyase products in the absence of other proteins or cofactors (with catalytically competent kinetics) plus hydroxylated 17α-hydroxysteroids. The 17α-OOH results indicate that a "Compound I" (FeO3+) intermediate is capable of formation and can be used to rationalize the products. We conclude that zebrafish P450 17A2 is capable of lyase activity with the 17α-OOH steroids because it can achieve an appropriate conformation for lyase catalysis in this system that is precluded in the conventional reaction.

Human mitochondrial cytochrome P450 27C1 is localized in skin and preferentially desaturates trans-retinol to 3,4-dehydroretinol.

Recently, zebrafish and human cytochrome P450 (P450) 27C1 enzymes have been shown to be retinoid 3,4-desaturases. The enzyme is unusual among mammalian P450s in that the predominant oxidation is a desaturation and in that hydroxylation represents only a minor pathway. We show by proteomic analysis that P450 27C1 is localized to human skin, with two proteins of different sizes present, one being a cleavage product of the full-length form. P450 27C1 oxidized all-trans-retinol to 3,4-dehydroretinol, 4-hydroxy (OH) retinol, and 3-OH retinol in a 100:3:2 ratio. Neither 3-OH nor 4-OH retinol was an intermediate in desaturation. No kinetic burst was observed in the steady state; neither the rate of substrate binding nor product release was rate-limiting. Ferric P450 27C1 reduction by adrenodoxin was 3-fold faster in the presence of the substrate and was ∼5-fold faster than the overall turnover. Kinetic isotope effects of 1.5-2.3 (on kcat/Km ) were observed with 3,3-, 4,4-, and 3,3,4,4-deuterated retinol. Deuteration at C-4 produced a 4-fold increase in 3-hydroxylation due to metabolic switching, with no observable effect on 4-hydroxylation. Deuteration at C-3 produced a strong kinetic isotope effect for 3-hydroxylation but not 4-hydroxylation. Analysis of the products of deuterated retinol showed a lack of scrambling of a putative allylic radical at C-3 and C-4. We conclude that the most likely catalytic mechanism begins with abstraction of a hydrogen atom from C-4 (or possibly C-3) initiating the desaturation pathway, followed by a sequential abstraction of a hydrogen atom or proton-coupled electron transfer. Adrenodoxin reduction and hydrogen abstraction both contribute to rate limitation.

In vitro cytochrome P450 46A1 (CYP46A1) activation by neuroactive compounds.

Cytochrome P450 46A1 (CYP46A1, cholesterol 24-hydroxylase) is the enzyme responsible for the majority of cholesterol elimination from the brain. Previously, we found that the anti-HIV drug efavirenz (EFV) can pharmacologically activate CYP46A1 in mice. Herein, we investigated whether CYP46A1 could also be activated by endogenous compounds, including major neurotransmitters. In vitro experiments with purified recombinant CYP46A1 indicated that CYP46A1 is activated by l-glutamate (l-Glu), l-aspartate, γ-aminobutyric acid, and acetylcholine, with l-Glu eliciting the highest increase (3-fold) in CYP46A1-mediated cholesterol 24-hydroxylation. We also found that l-Glu and other activating neurotransmitters bind to the same site on the CYP46A1 surface, which differs from the EFV-binding site. The other principal differences between EFV and l-Glu in CYP46A1 activation include an apparent lack of l-Glu binding to the P450 active site and different pathways of signal transduction from the allosteric site to the active site. EFV and l-Glu similarly increased the CYP46A1 kcat, the rate of the "fast" phase of the enzyme reduction by the redox partner NADPH-cytochrome P450 oxidoreductase, and the amount of P450 reduced. Spectral titrations with cholesterol, in the presence of EFV or l-Glu, suggest that water displacement from the heme iron can be affected in activator-bound CYP46A1. Moreover, EFV and l-Glu synergistically activated CYP46A1. Collectively, our in vitro data, along with those from previous cell culture and in vivo studies by others, suggest that l-Glu-induced CYP46A1 activation is of physiological relevance.

A Reagent and Virus Benchmarking Panel for a Uniform Analytical Performance Assessment of N Antigen-Based Diagnostic Tests for COVID-19.

Rapid diagnostic tests (RDTs) that detect antigen indicative of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection can help in making quick health care decisions and regularly monitoring groups at risk of infection. With many RDT products entering the market, it is important to rapidly evaluate their relative performance. Comparison of clinical evaluation study results is challenged by protocol design variations and study populations. Laboratory assays were developed to quantify nucleocapsid (N) and spike (S) SARS-CoV-2 antigens. Quantification of the two antigens in nasal eluates confirmed higher abundance of N than S antigen. The median concentration of N antigen was 10 times greater than S per genome equivalent. The N antigen assay was used in combination with quantitative reverse transcription (RT)-PCR to qualify a panel composed of recombinant antigens, inactivated virus, and clinical specimen pools. This benchmarking panel was applied to evaluate the analytical performance of the SD Biosensor Standard Q COVID-19 antigen (Ag) test, Abbott Panbio COVID-19 Ag rapid test, Abbott BinaxNOW COVID-19 Ag test, and the LumiraDx SARS-CoV-2 Ag test. The four tests displayed different sensitivities toward the different panel members, but all performed best with the clinical specimen pool. The concentration for a 90% probability of detection across the four tests ranged from 21 to 102 pg/mL of N antigen in the extracted sample. Benchmarking panels provide a quick way to verify the baseline performance of a diagnostic and enable direct comparisons between diagnostic tests. IMPORTANCE This study reports the results for severe acute respiratory syndrome coronavirus-2 (SARS-COV-2) nucleocapsid (N) and spike (S) antigen quantification assays and their performance against clinical reverse transcription (RT)-PCR results, thus describing an open-access quantification method for two important SARS-CoV-2 protein analytes. Characterized N antigen panels were used to evaluate the limits of detection of four different rapid tests for SARS-CoV-2 against multiple sources of nucleocapsid antigen, demonstrating proof-of-concept materials and methodology to evaluate SARS-CoV-2 rapid antigen detection tests. Quantification of N antigen was used to characterize the relationship between viral count and antigen concentration among clinical samples and panel members of both clinical sample and viral culture origin. This contributes to a deeper understanding of protein antigen and molecular analytes and presents analytical methods complementary to clinical evaluation for characterizing the performance of both laboratory-based and point-of-care rapid diagnostics for SARS-CoV-2.

Designed, highly expressing, thermostable dengue virus 2 envelope protein dimers elicit quaternary epitope antibodies.

Dengue virus (DENV) is a worldwide health burden, and a safe vaccine is needed. Neutralizing antibodies bind to quaternary epitopes on DENV envelope (E) protein homodimers. However, recombinantly expressed soluble E proteins are monomers under vaccination conditions and do not present these quaternary epitopes, partly explaining their limited success as vaccine antigens. Using molecular modeling, we found DENV2 E protein mutations that induce dimerization at low concentrations (<100 pM) and enhance production yield by more than 50-fold. Cross-dimer epitope antibodies bind to the stabilized dimers, and a crystal structure resembles the wild-type (WT) E protein bound to a dimer epitope antibody. Mice immunized with the stabilized dimers developed antibodies that bind to E dimers and not monomers and elicited higher levels of DENV2-neutralizing antibodies compared to mice immunized with WT E antigen. Our findings demonstrate the feasibility of using structure-based design to produce subunit vaccines for dengue and other flaviviruses.

Short-term hemodynamic effects of cardiac resynchronization therapy in patients with heart failure, a narrow QRS duration, and no dyssynchrony.

BACKGROUND: Cardiac resynchronization therapy produces both short-term hemodynamic and long-term symptomatic/mortality benefits in symptomatic heart failure patients with a QRS duration >120 ms. This is conventionally believed to be due principally to relief of dyssynchrony, although we recently showed that relief of external constraint to left ventricular filling may also play a role. In this study, we evaluated the short-term hemodynamic effects in symptomatic patients with a QRS duration <120 ms and no evidence of dyssynchrony on conventional criteria and assessed the effects on contractility and external constraint. METHODS AND RESULTS: Thirty heart failure patients (New York Heart Association class III/IV) with a left ventricular ejection fraction < or =35% who were in sinus rhythm underwent pressure-volume studies at the time of pacemaker implantation. External constraint, left ventricular stroke work, dP/dtmax, and the slope of the preload recruitable stroke work relation were measured from the end-diastolic pressure-volume relation before and during delivery of biventricular and left ventricular pacing. The following changes were observed during delivery of cardiac resynchronization therapy: Cardiac output increased by 25+/-5% (P<0.05), absolute left ventricular stroke work increased by 26+/-5% (P<0.05), the slope of the preload recruitable stroke work relation increased by 51+/-15% (P<0.05), and dP/dtmax increased by 9+/-2% (P<0.05). External constraint was present in 15 patients and was completely abolished by both biventricular and left ventricular pacing (P<0.05). CONCLUSIONS: Cardiac resynchronization therapy results in an improvement in short-term hemodynamic variables in patients with a QRS <120 ms related to both contractile improvement and relief of external constraint. These findings provide a potential physiological basis for cardiac resynchronization therapy in this patient population.

Left ventricular strain and untwist in hypertrophic cardiomyopathy: relation to exercise capacity.

BACKGROUND: Nonobstructive hypertrophic cardiomyopathy (nHCM) is often associated with reduced exercise capacity despite hyperdynamic systolic function as measured by left ventricular ejection fraction. We sought to examine the importance of left ventricular strain, twist, and untwist as predictors of exercise capacity in nHCM patients. METHODS: Fifty-six nHCM patients (31 male and mean age of 52 years) and 43 age- and gender-matched controls were enrolled. We measured peak oxygen consumption (peak Vo(2)) and acquired standard echocardiographic images in all participants. Two-dimensional speckle tracking was applied to measure rotation, twist, untwist rate, strain, and strain rate. RESULTS: The nHCM patients exhibited marked exercise limitation compared with controls (peak Vo(2) 23.28 +/- 6.31 vs 37.70 +/- 7.99 mL/[kg min], P < .0001). Left ventricular ejection fraction in nHCM patients and controls was similar (62.76% +/- 9.05% vs 62.48% +/- 5.82%, P = .86). Longitudinal, radial, and circumferential strain and strain rate were all significantly reduced in nHCM patients compared with controls. There was a significant delay in 25% of untwist in nHCM compared with controls. Both systolic and diastolic apical rotation rates were lower in nHCM patients. Longitudinal systolic and diastolic strain rate correlated significantly with peak Vo(2) (r = -0.34, P = .01 and r = 0.36, P = .006, respectively). Twenty-five percent untwist correlated significantly with peak Vo(2) (r = 0.36, P = .006). CONCLUSIONS: In nHCM patients, there are widespread abnormalities of both systolic and diastolic function. Reduced strain and delayed untwist contribute significantly to exercise limitation in nHCM patients.

Biventricular pacemaker therapy improves exercise capacity in patients with non-obstructive hypertrophic cardiomyopathy via augmented diastolic filling on exercise.

AIMS: Treatment options for patients with non-obstructive hypertrophic cardiomyopathy (HCM) are limited. We sought to determine whether biventricular (BiV) pacing improves exercise capacity in HCM patients, and whether this is via augmented diastolic filling. METHODS AND RESULTS: Thirty-one patients with symptomatic non-obstructive HCM were enrolled. Following device implantation, patients underwent detailed assessment of exercise diastolic filling using radionuclide ventriculography in BiV and sham pacing modes. Patients then entered an 8-month crossover study of BiV and sham pacing in random order, to assess the effect on exercise capacity [peak oxygen consumption (VO2 )]. Patients were grouped on pre-specified analysis according to whether left ventricular end-diastolic volume increased (+LVEDV) or was unchanged/decreased (-LVEDV) with exercise at baseline. Twenty-nine patients (20 male, mean age 55 years) completed the study. There were 14 +LVEDV patients and 15 -LVEDV patients. Baseline peak VO2 was lower in -LVEDV patients vs. +LVEDV patients (16.2 ± 0.9 vs. 19.9 ± 1.1 mL/kg/min, P = 0.04). BiV pacing significantly increased exercise ΔLVEDV (P = 0.004) and Δstroke volume (P = 0.008) in -LVEDV patients, but not in +LVEDV patients. Left ventricular ejection fraction and end-systolic elastance did not increase with BiV pacing in either group. This translated into significantly greater improvements in exercise capacity (peak VO2 + 1.4 mL/kg/min, P = 0.03) and quality of life scores (P = 0.02) in -LVEDV patients during the crossover study. There was no effect on left ventricular mechanical dyssynchrony in either group. CONCLUSION: Symptomatic patients with non-obstructive HCM may benefit from BiV pacing via augmentation of diastolic filling on exercise rather than contractile improvement. This may be due to relief of diastolic ventricular interaction. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT00504647.

Increased atrial contribution to left ventricular filling compensates for impaired early filling during exercise in heart failure with preserved ejection fraction.

BACKGROUND: The role of left atrial (LA) function on exercise remains poorly understood in heart failure with preserved ejection fraction (HfpEF) despite its key role in optimizing left ventricular (LV) diastolic function. We used resting and exercise radionuclide ventriculography to investigate the role of LA function in the pathophysiology of HfpEF. METHODS AND RESULTS: A total of 25 patients with HfpEF and 15 age- and gender-matched controls were recruited. All subjects underwent resting echocardiogram, metabolic exercise testing to peak effort, and radionuclide ventriculography (at rest and exercise [to 35% of heart rate reserve]). At rest LA and LV function were similar in patients and controls. During exercise, HfpEF patients had lower left ventricular ejection fraction (69 +/- 9% vs. 73 +/- 10%, P < .05) and lower peak early filling rate (387 +/- 109 end-diastolic count/sec vs. 561 +/- 156 end-diastolic count/sec, P < .001). During exercise, the atrial contribution to LV filling was significantly higher in patients than controls (46 +/- 11% vs. 30 +/- 9%, P < .001). Atrial contribution to LV filling correlated negatively with peak early filling rate during exercise (r = -0.6, P < .001). Peak early filling rate correlated positively with peak oxygen consumption (r = 0.485, P = .004) and negatively with minute/carbon dioxide production (r = -0.423, P = .013). CONCLUSION: Patients with HfpEF have increased atrial contribution to LV filling as a compensatory response to impaired early LV filling during cycle exercise.

Pacemaker implantation in superior vena cava obstruction: Re-canalization, venoplasty, and stenting.

Techniques to overcome challenging venous anatomy have become an increasingly important part of modern day cardiac device implantation. Complete superior vena cava (SVC) obstruction, however, is a rare and serious clinical entity, and consequently there is limited clinical experience in addressing this pathology in the context of transvenous pacemaker implantation. We present the case of a 48-year-old renal transplant patient with sick sinus syndrome and recurrent syncope, who had an upper SVC occlusion and a failed epicardial pacing system. The SVC was re-canalized and stented using techniques derived from chronic total occlusion coronary angioplasty, thus allowing successful implantation of a transvenous pacemaker lead. This case highlights the increased risk of developing central venous occlusion that exists in renal dialysis patients, and demonstrates the benefits of utilizing transferable interventional coronary and radiology techniques to overcome this pathology and facilitate pacemaker implantation. .

Human cytochrome P450 27C1 catalyzes 3,4-desaturation of retinoids.

In humans, a considerable fraction of the retinoid pool in skin is derived from vitamin A2 (all-trans 3,4-dehydroretinal). Vitamin A2 may be locally generated by keratinocytes, which can convert vitamin A1 (all-trans retinol) into vitamin A2 in cell culture. We report that human cytochrome P450 (hP450) 27C1, a previously 'orphan' enzyme, can catalyze this reaction. Purified recombinant hP450 27C1 bound and desaturated all-trans retinol, retinal, and retinoic acid, as well as 11-cis-retinal. Although the physiological role of 3,4-dehydroretinoids in humans is unclear, we have identified hP450 27C1 as an enzyme capable of efficiently mediating their formation.

Sarcoidosis presenting as complete heart block in a 45-year-old farmer: an aetiology not to miss.

Sarcoidosis should always be part of the differential diagnosis when faced with a young patient with significant cardiac conduction disease.

The pathophysiology of heart failure with preserved ejection fraction: from molecular mechanisms to exercise haemodynamics.

The pathophysiology of HfpEF is complex. In this review we discuss the molecular aspects of HfpEF as well as the profoundly disturbed haemodynamics with particular focus on exercise haemodynamic abnormalities.

Myocardial contractile inefficiency and dyssynchrony in heart failure with preserved ejection fraction and narrow QRS complex.

BACKGROUND: Using speckle-tracking imaging (STI), the aims of this study were to assess dyssynchrony and quantify the myocardial energy wasted by contractility in delayed segments by determining the longitudinal strain delay index (LSDi) in patients with heart failure with preserved ejection fraction (HFpEF). METHOD: Thirty-eight patients with HFpEF and 33 matched controls were recruited. All subjects underwent clinical examinations, 12-lead electrocardiography, pulmonary function tests, echocardiography, and metabolic exercise tests. LSDi was determined, the magnitude of which is a measure of the amount of wasted energy. Global and segmental systolic and diastolic dyssynchrony was assessed by STI. RESULTS: LSDi was significantly higher in patients with HFpEF than controls (-14.36 +/- 8.24% vs -10.73 +/- 5.62%, P < .05). Patients with HFpEF exhibited left ventricular (LV) systolic and diastolic dyssynchrony, with the LV anterior wall displaying the most delayed motion. CONCLUSION: Patients with HFpEF exhibited increased myocardial contractile inefficiency. They also exhibited LV systolic and diastolic dyssynchrony, with the LV anterior wall displaying the most delayed motion.

Heart failure with preserved ejection fraction is characterized by dynamic impairment of active relaxation and contraction of the left ventricle on exercise and associated with myocardial energy deficiency.

OBJECTIVES: We sought to evaluate the role of exercise-related changes in left ventricular (LV) relaxation and of LV contractile function and vasculoventricular coupling (VVC) in the pathophysiology of heart failure with preserved ejection fraction (HFpEF) and to assess myocardial energetic status in these patients. BACKGROUND: To date, no studies have investigated exercise-related changes in LV relaxation and VVC as well as in vivo myocardial energetic status in patients with HFpEF. METHODS: We studied 37 patients with HFpEF and 20 control subjects. The VVC and time to peak LV filling (nTTPF, a measure of LV active relaxation) were assessed while patients were at rest and during exercise by the use of radionuclide ventriculography. Cardiac energetic status (creatine phosphate/adenosine triphosphate ratio) was assessed by the use of (31)P magnetic resonance spectroscopy at 3-T. RESULTS: When patients were at rest, nTTPF and VVC were similar in patients with HFpEF and control subjects. The cardiac creatine phosphate/adenosine triphosphate ratio was reduced in patients with HFpEF versus control subjects (1.57 +/- 0.52 vs. 2.14 +/- 0.63; p = 0.003), indicating reduced energy reserves. Peak maximal oxygen uptake and the increase in heart rate during maximal exercise were lower in patients with HFpEF versus control subjects (19 +/- 4 ml/kg/min vs. 36 +/- 8 ml/kg/min, p < 0.001, and 52 +/- 16 beats/min vs. 81 +/- 14 beats/min, p < 0.001). The relative changes in stroke volume and cardiac output during submaximal exercise were lower in patients with HFpEF versus control subjects (ratio exercise/rest: 0.99 +/- 0.34 vs. 1.25 +/- 0.47, p = 0.04, and 1.36 +/- 0.45 vs. 2.13 +/- 0.72, p < 0.001). The nTTPF decreased during exercise in control subjects but increased in patients with HFpEF (-0.03 +/- 12 s vs. +0.07 +/- 0.11 s; p = 0.005). The VVC decreased on exercise in control subjects but was unchanged in patients with HFpEF (-0.01 +/- 0.15 vs. -0.25 +/- 0.19; p < 0.001). CONCLUSIONS: Patients with HFpEF have reduced cardiac energetic reserve that may underlie marked dynamic slowing of LV active relaxation and abnormal VVC during exercise.