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Background: Chronic obstructive pulmonary disease causes significant morbidity and concomitant pulmonary vascular disease and cardiac dysfunction are associated with poor prognosis. Computed tomography-detected relative pulmonary artery (PA) enlargement defined as a PA to ascending aorta diameter ratio >1 (PA:A>1) is a marker for pulmonary hypertension and predicts chronic obstructive pulmonary disease exacerbations. However, little is known about the relationship between the PA:A ratio, pulmonary blood volume, and cardiac function., Methods and Results: A single-center prospective cohort study of patients with chronic obstructive pulmonary disease was conducted. Clinical characteristics and computed tomography metrics, including the PA:A and pulmonary blood vessel volume, were measured. Ventricular functions, volumes, and dimensions were measured by cine cardiac MRI with 3-dimensional analysis. Linear regression examined the relationships between clinical characteristics, computed tomography and cardiac MRI metrics, and 6-minute walk distance. Twenty-four patients were evaluated and those with PA:A>1 had higher right ventricular (RV) end-diastolic and end-systolic volume indices accompanied by lower RV ejection fraction (52±7% versus 60±9%; P=0.04). The PA:A correlated inversely with total intraparenchymal pulmonary blood vessel volume and the volume of distal vessels with a cross-sectional area of <5 mm(2). Lower forced expiratory volume, PA:A>1, and hyperinflation correlated with reduced RV ejection fraction. Both PA diameter and reduced RV ejection fraction were independently associated with reduced 6-minute walk distance., Conclusions: The loss of blood volume in distal pulmonary vessels is associated with PA enlargement on computed tomography. Cardiac MRI detects early RV dysfunction and remodeling in nonsevere chronic obstructive pulmonary disease patients with a PA:A>1. Both RV dysfunction and PA enlargement are independently associated with reduced walk distance., Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00608764., (© 2015 American Heart Association, Inc.)

Background: Torsion shear angle φ is an important measure of left ventricular (LV) systolic and diastolic functions. Here we provide a novel index utilizing LV normalized torsion shear angle φ ^ volume V ^ loop to assess LV diastolic functional properties. We defined the area within φ ^ V ^ loop as torsion hysteresis area, and hypothesized that it may be an important global parameter of diastolic function. We evaluated the φ ^ changes to increased V ^ during early diastole - d φ ^ / d V ^ as a potential measure of LV suction., Methods: Sixty resistant hypertension patients (HTN), forty control volunteers were studied using cardiovascular magnetic resonance with tissue tagging. Volumetric and torsional parameters were evaluated., Results: HTN demonstrated concentric remodeling with preserved ejection fraction. HTN had significantly decreased normalized early filling rate, early diastolic mitral annulus velocity and E/A (1.33 ± 1.13 vs. 2.19 ± 1.07, P < 0.0001) vs. control. Torsion hysteresis area was greater (0.11 ± 0.07 vs. 0.079 ± 0.045, P < 0.001) and peak - d φ ^ / d V ^ at early diastole was higher (10.46 ± 8.51 vs. 6.29 ± 3.85, P = 0.002) than control. Torsion hysteresis area was significantly correlated with E/A (r = -0.23, P = 0.025). Thirteen HTN patients had both E/A ratio < 1.12 (Control mean E/A-1SD) and torsion hysteresis area > 0.12 (Control mean torsion hysteresis area + 1SD)., Conclusions: Torsion hysteresis area and peak early diastolic - d φ ^ / d V ^ were significantly increased in hypertensive concentric remodeling. The φ ^ V ^ loop takes into account the active and passive recoil processes of LV diastolic and systolic phases, therefore provides a new global description of LV diastolic function.

High-fat, low-carbohydrate diets (HFLCD) are often eaten by humans for a variety of reasons, but the effects of such diets on the heart are incompletely understood. We evaluated the impact of HFLCD on myocardial ischemia/reperfusion (I/R) using an in vivo model of left anterior descending coronary artery ligation. Sprague-Dawley rats (300 g) were fed HFLCD (60% calories fat, 30% protein, 10% carbohydrate) or control (CONT; 16% fat, 19% protein, 65% carbohydrate) diet for 2 wk and then underwent open chest I/R. At baseline (preischemia), diet did not affect left ventricular (LV) systolic and diastolic function. Oil red O staining revealed presence of lipid in the heart with HFLCD but not in CONT. Following I/R, recovery of LV function was decreased in HFLCD. HFLCD hearts exhibited decreased ATP synthase and increased uncoupling protein-3 gene and protein expression. HFLCD downregulated mitochondrial fusion proteins and upregulated fission proteins and store-operated Ca(2+) channel proteins. HFLCD led to increased death during I/R; 6 of 22 CONT rats and 16 of 26 HFLCD rats died due to ventricular arrhythmias and hemodynamic shock. In surviving rats, HFLCD led to larger infarct size. We concluded that in vivo HFLCD does not affect nonischemic LV function but leads to greater myocardial injury during I/R, with increased risk of death by pump failure and ventricular arrhythmias, which might be associated with altered cardiac energetics, mitochondrial fission/fusion dynamics, and store-operated Ca(2+) channel expression., (Copyright © 2014 the American Physiological Society.)

Background: There is currently no therapy proven to attenuate left ventricular (LV) dilatation and dysfunction in volume overload induced by isolated mitral regurgitation (MR). To better understand molecular signatures underlying isolated MR, we performed LV gene expression analyses and overlaid regulated genes into ingenuity pathway analysis in patients with isolated MR., Methods and Results: Gene arrays from LV tissue of 35 patients, taken at the time of surgical repair for isolated MR, were compared with 13 normal controls. Cine-MRI was performed in 31 patients before surgery to measure LV function and volume from serial short-axis summation. LV end-diastolic volume was 2-fold (P=0.005) higher in MR patients than in normal controls, and LV ejection fraction was 64±7% (50%-79%) in MR patients. Ingenuity pathway analysis identified significant activation of pathways involved in β-adrenergic, cAMP, and G-protein-coupled signaling, whereas there was downregulation of pathways associated with complement activation and acute phase response. SERCA2a and phospholamban protein were unchanged in MR versus control left ventricles. However, mRNA and protein levels of the sarcoplasmic reticulum Ca2+ ATPase (SERCA) regulatory protein sarcolipin, which is predominantly expressed in normal atria, were increased 12- and 6-fold, respectively. Immunofluorescence analysis confirmed the absence of sarcolipin in normal left ventricles and its marked upregulation in MR left ventricles., Conclusions: These results demonstrate alterations in multiple pathways associated with β-adrenergic signaling and sarcolipin in the left ventricles of patients with isolated MR and LV ejection fraction>50%, suggesting a beneficial role for β-adrenergic blockade in isolated MR.

In previous work, a three-dimensional left ventricular strain throughout the cardiac cycle was reconstructed using a prolate spheroidal B-spline (PSB) method with displacement measurements obtained from unwrapped tagged MRI (tMRI) harmonic phase images. Manually placed branch cuts were required for each harmonic phase image to resolve phase inconsistencies and to guide the phase unwrapping (mSUP), which is both labor intensive and time consuming and therefore not proper for clinic application. In this paper, we present an automated graph cuts based phase unwrapping method for myocardium displacement measurement (caSUP) which can be used to compute 3D+time cardiac strain. A set of 8 human studies were used to optimize parameters of the energy function and another set of 32 human studies were used to validate the proposed method by comparing resulted strains with those from mSUP and a feature-based (FB) method using the same PSB strain reconstruction. The automated caSUP strains were close to the manual strains and only required 6 minutes after myocardium segmentation versus - 2 hours for the manual method.

Obesity is associated with elevated risk of heart disease. A solid understanding of the safety and potential adverse effects of high-fat, low-carbohydrate diet (HFLCD) similar to that used by humans for weight loss on the heart is crucial. High fat intake is known to promote increases in reactive oxygen species and mitochondrial damage. We hypothesized that there would be adverse effects of HFLCD on myocardial ischemia/reperfusion injury through enhancing oxidative stress injury and impairing mitochondrial biogenesis in a nongenetic, diet-induced rat model of obesity. To test the hypothesis, 250-g male Sprague-Dawley rats were fed an obesity-promoting diet for 7 weeks to induce obesity, then switched to HFLCD or a low-fat control diet for 2 weeks. Isolated hearts underwent global low flow ischemia for 60 minutes and reperfusion for 60 minutes. High-fat, low-carbohydrate diet resulted in greater weight gain and lower myocardial glycogen, plasma adiponectin, and insulin. Myocardial antioxidant gene transcript and protein expression of superoxide dismutase and catalase were reduced in HFLCD, along with increased oxidative gene NADPH oxidase-4 transcript and xanthine oxidase activity, and a 37% increase in nitrated protein (nitrotyrosine) in HFLCD hearts. The cardiac expression of key mitochondrial regulatory factors such as nuclear respiratory factor-1 and transcription factor A-mitochondrial were inhibited and myocardial mitochondrial DNA copy number decreased. The cardiac expression of adiponectin and its receptors was down-regulated in HFLCD. High-fat, low-carbohydrate diet impaired recovery of left ventricular rate-pressure product after ischemia/reperfusion and led to 3.5-fold increased injury as measured by lactate dehydrogenase release. In conclusion, HFLCD leads to increased ischemic myocardial injury and impaired recovery of function after reperfusion and was associated with attenuation of mitochondrial biogenesis and enhanced oxidative stress in obese rats. These findings may have important implications for diet selection in obese patients with ischemic heart disease., (Published by Elsevier Inc.)

How the left ventricle remodels in response to a high-volume stimulus is important in evaluating the endurance athlete's heart. Marathoners and patients with isolated, moderate chronic compensated mitral regurgitation (MR) represent physiologic and pathologic forms of eccentric left ventricular (LV) remodeling in response to intermittent and chronic volume overload, respectively. Thus, in this study, magnetic resonance imaging with tissue tagging and 3-dimensional data analysis at rest were performed in 19 marathoners (mean age 39 ± 10 years, 47% women), 17 patients with isolated MR without coronary artery disease or medical therapy (mean age 46 ± 5 years, 53% women), and 24 controls (mean age 45 ± 8 years, 50% women). Marathoners and patients with MR had approximately 35% greater LV end-diastolic volume indexes, approximately 50% greater end-systolic volume indexes, and approximately 34% greater LV stroke volume indexes (p <0.0001) compared to controls. However, marathoners' hearts had increased long-axis length, while those of patients with MR did not differ from the hearts of controls. The hearts of patients with MR had greater LV global and apex sphericity compared to those of marathoners and controls (p <0.0001). Marathoners had normal LV mass/volume ratios and wall thicknesses, whereas these were significantly decreased in the MR group. In marathoners, the baseline LV work rate was similar to that in controls and higher in patients with MR compared to controls. In conclusion, marathoners' hearts achieve elevated stroke volume at rest with adherence to an elliptical shape defined by 3-dimensional geometry and mass/volume ratio. Thus, a comprehensive evaluation of LV geometry and mass/volume ratio may be important in the evaluation of the athlete's heart., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Although surgery is indicated in patients with mitral regurgitation (MR) when left ventricular (LV) end-systolic (LVES) dimension is >40 mm, LV ejection fraction may decrease after mitral valve surgery. We hypothesize that significant LV remodeling before surgery is not reflected by standard echocardiographic parameters measured at the base of the heart., Methods and Results: Ninety-four patients (age, 54 ± 11 years; 38% female) with degenerative isolated MR underwent cine magnetic resonance imaging with tissue tagging and 3-dimensional analysis. In 51 control subjects (age, 44 ± 14 years; 53% female), the relation between LVES volume (LVESV) and LVES dimension was quadratic, whereas in 94 MR patients, this relation was cubic, indicating a greater increase in LVESV per LVES dimension among MR patients. Moreover, magnetic resonance imaging LVESV from summated serial short-axis slices was significantly greater than LVESV assessed with the Bullet formula in MR patients, attributed to a more spherical remodeling distal to the tips of the papillary muscles (P<0.001). Thirty-five patients underwent mitral valve repair per current guideline recommendations. LV ejection fraction decreased from 61 ± 7% to 54 ± 8% (P<0.0001) and maximum shortening decreased significantly below normal at 1 year postoperatively (P<0.0001). Despite normalization of LV stroke volume and LV end-diastolic volume/mass ratio, there was a persistent significant increase in distal LVES 3-dimensional radius/wall thickness ratio and LVESV index after surgery., Conclusions: Despite apparently preserved LVES dimension, MR patients demonstrate significant spherical mid to apical LVES remodeling that contributes to higher LVESV than predicted by standard geometry-based calculations. Decreased LV strain after surgery suggests that a volumetric analysis of LV remodeling and function may be preferred to evaluate disease progression in isolated MR.

Objectives: The goal of this study was to define the mechanism of preserved ejection fraction (EF) despite depressed myocardial strains in hypertension (HTN)., Background: Concentric left ventricular (LV) remodeling in HTN may have normal or supranormal EF despite depressed myocardial strains. The reason for such discordance is not clear. The aim of this study was to comprehensively evaluate the LV mechanics in a well-defined HTN population to define underlying reasons for such a paradox., Methods: Sixty-seven patients with resistant HTN and 45 healthy control subjects were studied by cardiac magnetic resonance imaging and tissue tagging with 3-dimensional analysis. Amplitude and directional vector of longitudinal (Ell), circumferential (Ecc), and principal strain for maximal shortening (E3) were computed at basal, mid, and distal LV levels, respectively. LV torsion, defined as the rotation angle of apex relative to base, and LV twist, which accounts for the effects of differential LV remodeling on torsion for comparison among the 2 groups, were also calculated., Results: LV mass index and LV mass/LV end-diastolic volume ratio were significantly higher in the HTN group compared with controls, consistent with concentric LV remodeling. Ell and Ecc were significantly decreased in amplitude with altered directional vector in HTN compared with controls. However, the amplitude of E3 was similar in the 2 groups. Torsion and twist were significantly higher in HTN, which was mainly due to increase in apical rotation. The HTN group demonstrated significantly increased LV wall thickening compared with controls that resulted in greater LVEF in the HTN group compared with controls (70% vs. 65%, p < 0.001, respectively)., Conclusions: In compensated LV remodeling secondary to HTN, there is increased LV wall thickening with preserved E3 and increased torsion compared with normal controls. This, therefore, contributes to supranormal LVEF in HTN despite depressed longitudinal and circumferential strains., (Copyright © 2012 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Purpose: To validate a method called bi-ventricular strain unwrapped phase (BiSUP) for reconstructing three-dimensional plus time (3D+t) biventricular strain maps from phase-unwrapped harmonic phase (HARP) images derived from tagged cardiac magnetic resonance imaging (MRI)., Materials and Methods: A set of 30 human subjects were imaged with tagged MRI. In each study, HARP phase was computed and unwrapped in each short-axis and long-axis image. Inconsistencies in unwrapped phase were resolved using branch cuts manually placed with a graphical user interface. The 3D strain maps were computed independently in each imaged time frame through systole and mid diastole in each study. The BiSUP strain and displacements were compared with those estimated by a 3D feature-based (FB) technique and a 2D+t HARP technique., Results: The standard deviation of the difference between strains measured by the FB and the BiSUP methods was less than 4% of the average of the strains from the two methods. The correlation between peak minimum principal strain measured using the BiSUP and HARP techniques was over 83%., Conclusion: The BiSUP technique can reconstruct full 3D+t strain maps from tagged MR images through the cardiac cycle in a reasonable amount of time and user interaction compared with other 3D analysis methods., (Copyright © 2011 Wiley-Liss, Inc.)

Mechanisms of left ventricular (LV) dysfunction in isolated mitral regurgitation (MR) are not well understood. Vasodilator therapy in other forms of LV dysfunction reduces LV wall stress and improves LV function; however, studies in isolated MR show no beneficial effect on LV remodeling using vasodilator drugs or renin-angiotensin system blockade. Therefore, the search for new therapies that improve LV remodeling and function in isolated MR is clinically significant. Recent work in the authors' laboratory has demonstrated increased oxidants from a number of sources including the enzyme xanthine oxidase (XO) in the LV of patients with isolated MR. In addition to being a major source of reactive oxygen species, XO is linked to bioenergetic dysfunction because its substrates derive from adenosine triphosphate catabolism. Correspondingly, there was also evidence of aggregates of small mitochondria in cardiomyocytes, which is generally considered a response to bioenergetic deficit in cells. Future studies are required to determine whether XO and persistent oxidative stress are causative in maladaptive LV remodeling and offer potential therapeutic targets in ameliorating LV damage in patients with isolated MR.

There has been a resurgence of interest for the field of cardiac metabolism catalysed by the increased need for new therapeutic targets for patients with heart failure. The primary focus of research in this area to date has been on the impact of substrate selection for oxidative energy metabolism; however, anaplerotic metabolism also has significant interest for its potential cardioprotective role. Anaplerosis refers to metabolic pathways that replenish the citric acid cycle intermediates, which are essential to energy metabolism; however, our understanding of the role and regulation of this process in the heart, particularly under pathophysiological conditions, is very limited. Therefore, the goal of this article is to provide a foundation for future directions of research on cardiac anaplerosis and heart disease. We include an overview of anaplerotic metabolism, a critical evaluation of current methods available for its quantitation in the intact heart, and a discussion of its role and regulation both in health and disease as it is currently understood based mostly on animal studies. We also consider genetic diseases affecting anaplerotic pathways in humans and acute intervention studies with anaplerotic substrates in the clinics. Finally, as future perspectives, we will share our thoughts about potential benefits and practical considerations on modalities of interventions targeting anaplerosis in heart disease, including heart failure.

A 37-year-old woman was diagnosed to have a small ventricular septal defect (VSD) with high velocity tricuspid regurgitation (TR) that was attributed to atrio-VSD (Gerbode). Cardiac MR revealed a small subaortic VSD in the membranous portion of the interventricular septum. The atrioventricular portion was intact. Cardiac MR clearly showed flow jet through the VSD, impinging on the anterior tricuspid leaflet during systole, and bouncing back into the right atrium as TR. This ricochet mechanism of TR in VSD may be misinterpreted as Gerbode defect or as evidence of pulmonary hypertension., (© 2011, Wiley Periodicals, Inc.)

We have shown previously that patients with resistant hypertension and hyperaldosteronism have increased brain natriuretic peptide suggestive of increased intravascular volume. In the present study, we tested the hypothesis that hyperaldosteronism contributes to cardiac volume overload. Thirty-seven resistant hypertensive patients with hyperaldosteronism (urinary aldosterone > or = 12 microg/24 hours and plasma renin activity < or = 1.0 ng/mL per hour) and 71 patients with normal aldosterone status were studied. Both groups had similar blood pressure and left ventricular mass, whereas left and right ventricular end-diastolic volumes measured by cardiac MRI were greater in high versus normal aldosterone subjects (P<0.05). Spironolactone treatment (19 patients in the high aldosterone group and 15 patients from the normal aldosterone group participated in the follow-up) resulted in a significant decrease in clinic systolic blood pressure, right and left ventricular end diastolic volumes, left atrial volume, left ventricular mass, and brain natriuretic peptide at 3 and 6 months of follow-up in patients with high aldosterone, whereas in those with normal aldosterone status, spironolactone decreased blood pressure and left ventricular mass without changes in ventricular or atrial volumes or plasma brain natriuretic peptide. Hyperaldosteronism causes intracardiac volume overload in patients with resistant hypertension in spite of conventional thiazide diuretic use. Mineralocorticoid receptor blockade induces rapid regression of left ventricular hypertrophy irrespective of aldosterone status. In subjects with high aldosterone, mineralocorticoid receptor blockade induces a prominent diuretic effect compared with a greater vasodilatory effect in subjects with normal aldosterone status.

Purpose: To validate a method for measuring 3D left ventricular (LV) strain from phase-unwrapped harmonic phase (HARP) images derived from tagged cardiac magnetic resonance imaging (MRI)., Materials and Methods: A set of 40 human subjects were imaged with tagged MRI. In each study the HARP phase was computed and unwrapped in each short-axis and long-axis image. Inconsistencies in unwrapped phase were resolved using branch cuts manually placed with a graphical user interface. 3D strain maps were computed for all imaged timeframes in each study. The strain from unwrapped phase (SUP) and displacements were compared to those estimated by a feature-based (FB) technique and a HARP technique., Results: 3D strain was computed in each timeframe through systole and mid-diastole in approximately 30 minutes per study. The standard deviation of the difference between strains measured by the FB and the SUP methods was less than 5% of the average of the strains from the two methods. The correlation between peak circumferential strain measured using the SUP and HARP techniques was over 83%., Conclusion: The SUP technique can reconstruct full 3D strain maps from tagged MR images through the cardiac cycle in a reasonable amount of time and user interaction compared to other 3D analysis methods., ((c) 2010 Wiley-Liss, Inc.)

Objective: We sought to demonstrate the distinguishing features between interarterial and intraseptal courses of an anomalous left coronary artery from the right sinus of Valsalva (RSV) on X-ray angiography, using an ex vivo model., Background: An anomalous left main coronary artery (LMCA) arising from the RSV can take prepulmonary, retro-aortic, interarterial (IA) or intraseptal (IS) courses, of which only the IA course is associated with sudden death. Anomalous LMCA is usually identified during catheter angiography. On Xray angiography, IA and IS courses have common characteristics that makes their distinction challenging. We hypothesized that the cranialcaudal orientation of the vessel on X-ray angiography allows these pathways to be distinguished, and tested this hypothesis using an ex vivo heart model., Methods: Plastic tubing was inserted along the IA and IS courses in an ex vivo normal pig heart. X-ray imaging in standard views and MRI on a 3-T scanner were performed., Results: In a normally formed heart, an anomalous LMCA with IA path must take a cephalad course, superior to the pulmonary valve. Conversely, an IS vessel will pass caudally, at or below the level of the infundibular septum. These findings were demonstrated in the X-ray angiograms and confirmed by magnetic resonance imaging., Conclusions: X-ray angiography can differentiate IA and IS courses of an anomalous LMCA in the normally formed heart. This may obviate the need for further cross-sectional imaging in many cases.

Background: Although cardiovascular magnetic resonance (CMR) is frequently performed to measure accurate LV volumes and ejection fractions, LV volume-time curves (VTC) derived ejection and filling rates are not routinely calculated due to lack of robust LV segmentation techniques. VTC derived peak filling rates can be used to accurately assess LV diastolic function, an important clinical parameter. We developed a novel geometry-independent dual-contour propagation technique, making use of LV endocardial contours manually drawn at end systole and end diastole, to compute VTC and measured LV ejection and filling rates in hypertensive patients and normal volunteers., Methods: 39 normal volunteers and 49 hypertensive patients underwent CMR. LV contours were manually drawn on all time frames in 18 normal volunteers. The dual-contour propagation algorithm was used to propagate contours throughout the cardiac cycle. The results were compared to those obtained with single-contour propagation (using either end-diastolic or end-systolic contours) and commercially available software. We then used the dual-contour propagation technique to measure peak ejection rate (PER) and peak early diastolic and late diastolic filling rates (ePFR and aPFR) in all normal volunteers and hypertensive patients., Results: Compared to single-contour propagation methods and the commercial method, VTC by dual-contour propagation showed significantly better agreement with manually-derived VTC. Ejection and filling rates by dual-contour propagation agreed with manual (dual-contour - manual PER: -0.12 +/- 0.08; ePFR: -0.07 +/- 0.07; aPFR: 0.06 +/- 0.03 EDV/s, all P = NS). However, the time for the manual method was approximately 4 hours per study versus approximately 7 minutes for dual-contour propagation. LV systolic function measured by LVEF and PER did not differ between normal volunteers and hypertensive patients. However, ePFR was lower in hypertensive patients vs. normal volunteers, while aPFR was higher, indicative of altered diastolic filling rates in hypertensive patients., Conclusion: Dual-propagated contours can accurately measure both systolic and diastolic volumetric indices that can be applied in a routine clinical CMR environment. With dual-contour propagation, the user interaction that is routinely performed to measure LVEF is leveraged to obtain additional clinically relevant parameters.

Purpose: In the absence of additional ischemic insults, the peri-infarct region surrounding the infarct myocardium can recover function. T2 weighted MRI signal is sensitive to edema and used to detect peri-infarct, salvageable myocardium. The main purpose of this study was to investigate the alterations in myocardial strain in the peri-infarct myocardium as compared to normal and infarct myocardium., Materials and Methods: Comprehensive MRI of the myocardium was performed in five pigs 6-7 days following coronary artery occlusion-reperfusion myocardial injury. MRI included tagged cine images for myocardial strain, T2weighted (T2w)-images and late gadolinium enhancement (LGE) for assessing myocardial viability. Automated signal intensity thresholds were used to define tissue edema and myocardial infarct. Maximum-shortening strains were analyzed in the infarct, peri-infarct and normal myocardial sectors. The results were correlated with triphenyltetrazolium-chloride (TTC) and hemotoxylin-eosin stained tissue images., Results: We found an excellent correlation of LGE with TTC (r = 0.94, P < 0.05). T2w-images markedly overestimated the infarct size (25 +/- 3%). Both the healthy and peri-infarct myocardial sectors had higher myocardial strain than infarct myocardial sectors (P < 0.05). Clear demarcation between infarct and non-infarct myocardium was noted on histology., Conclusion: Peri-infarct myocardium continues to demonstrate T2 signal enhancement to at least 7 days, but this region has preserved mechanical function. T2-weighted imaging and myocardial strain measurements provide complementary information and both may be useful for characterization of the peri-infarct myocardium.

Aims: Low Carbohydrate Diets (LCD) are a popular intervention for weight loss, but the effect of such diets on myocardial ischemia is not known. Myocardial energy substrates and insulin signaling pathways may be affected by these diets, and both may play a role in protection of ischemic myocardium. We investigated whether LCD increases susceptibility to cardiac injury during ischemia and reperfusion in the isolated rat heart., Main Methods: Rats were fed LCD (60% kcal from fat/30% protein/10% carbohydrate) or a control diet (CONT; 16%/19%/65%) for 2 weeks. Hearts from rats fed with LCD or CONT were isolated and subjected to normal perfusion in Langendorff mode, with 30 min global low flow ischemia (LFI; 0.3 ml/min) followed by 60 min reperfusion, or 60 min LFI followed by 120 min reperfusion., Key Findings: LCD diet led to an increase in 3-hydroxybutyrate and lower circulating insulin. LCD diet also resulted in impaired left ventricular performance during LFI, reduced recovery of function following LFI and reperfusion, and 10- to 20-fold increased injury as measured by lactate dehydrogenase release and histologic infarct area. LCD diet also led to lower myocardial glycogen stores and glycogen utilization during LFI, and lower insulin signaling as assessed by Akt phosphorylation at the end of LFI and reperfusion, but no differences in ERK 1/2 phosphorylation., Significance: These results demonstrate that LCD affects myocardial energy substrates, affects insulin signaling, and increases myocardial injury following ischemia-reperfusion in the isolated heart.

Five adult patients with Takotsubo cardiomyopathy (TC) diagnosed by usual criteria were studied with velocity vector imaging (VVI) on admission and at follow-up, when their LV function had improved, as assessed by 2D TTE wall-motion score (WMS) index. Averaged peak segmental longitudinal strain (S) in systole, and velocity (V) and strain rate (SR) in both systole and diastole were measured from apical 4- (A4C) and 2-chamber views (A2C) in all patients. The data obtained by VVI were analyzed separately for involved and uninvolved segments, which were independently assessed by WMS. In the involved segments, systolic S, V, SR, and diastolic SR improved (P-value < 0.05) on follow-up. Diastolic V showed a trend toward improvement but did not reach statistical significance. In the uninvolved segments, none of the parameters improved significantly either during systole or diastole. In three of these five patients, left atrial (LA) walls were also studied by placing region of interest (ROI) points in the middle of each wall. Peak segmental LA systolic and diastolic V and SR as well as systolic S were obtained for both involved and uninvolved LA walls which were assessed independently using WMS similar to LV. In the involved LA walls, none of the atrial systolic and diastolic parameters changed significantly but all parameters with the exception of systolic V showed a tendency toward improvement during follow-up. Among the uninvolved LA walls, none changed significantly but atrial systolic SR and, diastolic V and SR tended to increase during follow-up. Our retrospective study using VVI demonstrates that TC patients also have LV systolic and diastolic longitudinal dysfunction, not just systolic radial dysfunction as assessed by traditional 2D TTE indices. Longitudinal LA dysfunction may also be present.

Purpose: To study the changes in limb blood flow during lower extremity exercise using phase contrast (PC) MRI in normal volunteers., Materials and Methods: Healthy volunteers performed plantar flexion exercise (<1 W) for four minutes. Flow velocity was measured using cardiac-gated, cine PC-MRI sequences (fast gradient recalled echo [GRE]; multishot echo planar imaging [EPI]) on a 3T scanner at the level of the superficial femoral artery (SFA): 1) preexercise; 2) immediately postexercise; 3) during three minutes recovery; and 4) postrecovery., Results: At rest there was a triphasic flow waveform in the SFA. During exercise it changed to a monophasic pattern with an increase in total flow; there were variable changes in vessel size and flow velocity. The waveform regained the triphasic pattern during recovery. The exercise-induced flow reserve (FR) was 167 +/- 90%., Conclusion: PC-MRI demonstrates that the resting triphasic flow waveform transforms into a monophasic pattern with submaximal exercise and returns to baseline with recovery. This increase in the regional blood flow allows for measurement of exercise-induced FR in the SFA., ((c) 2008 Wiley-Liss, Inc.)

Background: Delayed enhanced (DE) multidetector computed tomography (MDCT) can identify acute and chronic myocardial infarct. To our knowledge, automated techniques for infarct quantification on DE-MDCT have not been used., Objective: We evaluated an automated signal intensity (SI) threshold method for quantification of subacute myocardial infarct and identified and quantified microvascular obstruction (MO) in subacute infarct., Methods: DE-MDCT imaging was performed on 5 pigs 6-7 days after mid left anterior descending artery occlusion-reperfusion. DE-MDCT images were compared with triphenyl tetrazolium chloride (TTC) staining for infarct quantification and with hematoxylin and eosin (H&E) staining for MO quantification. Pixels with SI more than the mean SI of a remote normal myocardial region (SI(remote)) plus 2 times the standard deviation (SI(remote) + 2 SD) value were considered infarct pixels. The ratio of infarct to total area of a given slice, the percentage of infarct area per slice (PIS), was calculated. MO as a percentage of total infarct area was also calculated., Results: The average density values on DE-MDCT (5 minutes after contrast injection) were remote normal myocardium of 93 +/- 19 Hounsfield units (HU), infarct myocardium of 159 +/- 40 HU, blood of 140 +/- 26 HU, and MO of 85 +/- 30 HU. PIS(MDCT) showed substantial agreement with PIS(TTC) (y = 1.003x + 4.12; R = 0.90, P < 0.05). A relation was also shown between MO determined by MDCT compared with H&E staining (y = 0.74x + 3.4)., Conclusions: We show the feasibility of using a semiautomated SI threshold technique for quantification of subacute myocardial infarct. We also show the persistent MO in subacute myocardial infarct on DE-MDCT images.

Background: Short-axis summation (SAS) method applied for right ventricular (RV) volumes and right ventricular ejection fraction (RVEF) measurement with cardiac MRI is time consuming and cumbersome to use. A simplified RVEF measurement is desirable. We compare two such methods, a simplified ellipsoid geometric method (GM) and visual estimate, to the SAS method to determine their accuracy and reproducibility., Methods: Forty patients undergoing cine cardiac MRI scan were enrolled. The images acquired were analyzed by the SAS method, the GM (area and length measurement from two orthogonal planes) and visual estimate. RVEF was calculated using all three methods and RV volumes using the SAS and GM. Bland-Altman analysis was applied to test the agreement between the various measurements., Results: Mean RVEF was 49 +/- 12% measured by SAS method, 54 +/- 12% by the GM, and 49 +/- 11% by visual estimate. There were similar bias and limits of agreement between the visual estimate and the GM compared to SAS. The interobserver variability showed a bias close to zero with limits of agreement within +/-10% absolute increments of RVEF in 35 of the patients. The RV end-diastolic volume by GM showed wider limits of agreement. The RV end-systolic volume by GM was underestimated by around 10 ml compared to SAS., Conclusion: Both the visual estimate and the GM had similar bias and limits of agreement when compared to SAS. Though the end-systolic measurement is somewhat underestimated, the geometric method may be useful for serial volume measurements.

Mitral regurgitation (MR) promotes left ventricular (LV) dilatation and eccentric remodeling. In the presence of LV dyssynchrony and heart failure, cardiac resynchronization therapy decreases the severity of MR. Whether primary MR can cause LV dyssynchrony is unknown. We investigated whether moderate to severe primary MR causes LV dyssynchrony in the presence of LV dilation and an ejection fraction (EF) >55%. We studied 37 normal subjects and 22 patients with moderate to severe MR and no coronary artery disease. Electrocardiographically gated cine and tagged cardiac magnetic resonance imaging was performed. Two-dimensional, maximum-circumferential shortening strain and time-to-peak strain (TTPS) were computed using harmonic-phase analysis of tagged magnetic resonance imaging. LV dyssynchrony was assessed by comparing TTPS delay of various LV quadrants and TTPS dispersion among the contralateral quadrants in patients with MR and normal subjects. Statistical comparison was done using a generalized linear model for repeated measurements. LV end-diastolic and LV end-systolic volumes were significantly larger in patients with MR versus normal subjects (207 +/- 11 vs 130 +/- 4 and 73 +/- 5 vs 47 +/- 2 ml, p <0.001). LVEF did not differ in patients with MR and normal subjects. The difference in the TTPS among various quadrants and the dispersion among the contralateral quadrants of the LV myocardium was similar between patients with MR and normal subjects. In conclusion, moderate to severe MR does not cause LV dyssynchrony in patients with LV dilatation and normal LVEF. Thus, cardiac resynchronization therapy in the absence of LV dyssynchrony may not decrease the severity of MR.

Inhibition of fatty acid oxidation has been reported to be cardioprotective against myocardial ischemic injury; however, recent studies have questioned whether the cardioprotection associated with putative fatty acid oxidation inhibitors, such as ranolazine and trimetazidine, are due to changes in substrate oxidation. Therefore, the goals of this study were to compare the effects of ranolazine with a new fatty acid oxidation inhibitor, CVT-4325 [(R)-1-(2-methylbenzo[d]thiazol-5-yloxy)-3-(4-((5-(4-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)methyl)-piperazin-1-yl)propan-2-ol], on carbohydrate and fatty acid oxidation and on left ventricular (LV) function in the response to ischemia/reperfusion in rat isolated perfused hearts. Metabolic fluxes were determined in hearts perfused in an isovolumic Langendorff mode using 13C nuclear magnetic resonance isotopomer analysis or in isolated working hearts using [14C]glucose and [3H]palmitate, with and without 10 microM ranolazine or 3 microM CVT-4325. Isovolumic perfused hearts were also subjected to 30 min of low-flow ischemia (0.3 ml/min) and 60 min of reperfusion, and working hearts were subjected to 15 min of zero-flow ischemia and 60 min of reperfusion. Regardless of the experimental protocol, ranolazine had no effect on carbohydrate or fatty acid oxidation, whereas CVT-4325 significantly reduced fatty acid oxidation up to approximately 7-fold with a concomitant increase in carbohydrate oxidation. At these same concentrations, although ranolazine significantly improved LV functional recovery following ischemia/reperfusion, CVT-4325 had no significant protective effect. These results demonstrate that at pharmacologically relevant concentrations, ischemic protection by ranolazine was not mediated by inhibition of fatty acid oxidation and conversely that inhibition of fatty acid oxidation with CVT-4325 was not associated with improved LV functional recovery.

Objective: We sought to determine the incidence and imaging features by coronary angiography and cardiac magnetic resonance imaging (MRI) of anomalies in which the right, circumflex, and left anterior descending coronary arteries arise separately from the right sinus of Valsalva., Background: The anomalous origin of all major coronary arteries from separate ostia in the right sinus of Valsalva has been reported as exceedingly rare, with mainly isolated cases reported. A knowledge of the origin and proximal courses of aberrant arteries is critical for patient management., Methods: 42 consecutive patients without other congenital heart disease referred to our institution for MRI evaluation of anomalous coronary artery over a six year period were evaluated. Analysis of angiograms and MRI was done to determine the anatomic origin and proximal pathway of coronary arteries (determined by conventional angiography and MRI) and degree of any stenosis (by angiography)., Results: Seven of the 42 patients (17%) in this referral population had the described anatomy. Both conventional angiography and MRI depicted the origin and proximal courses of these arteries. In all patients, the circumflex passed behind the aorta. In three, the left anterior descending passed through the ventricular septum; in four, it passed anterior to the pulmonary trunk., Conclusions: This series is the largest ever reported on this complex anatomical variant and the first to give a systematic analysis of the anatomy by angiography and MRI. This constellation of multiple anomalous coronary arterial origins and proximal courses may not be as rare as previously reported.

Objective: Surgical ventricular restoration (SVR) is a promising modality for treatment of heart failure due to left ventricular systolic dysfunction, particularly that due to ischemic heart disease. The role of MRI in improving diagnosis, operative planning, and follow-up is reviewed to analyze how one examination may define a spectrum of important considerations., Methods: Proper patient selection and optimal surgical planning relies on accurate assessment of measures of ventricular volume, function, and viability, and of the mechanics of the mitral valve apparatus. A complete preoperative imaging evaluation includes assessment of the left ventricular volume (both systolic and diastolic), regional and global systolic function, viability of the target area for surgical exclusion and of the remote myocardium, determination of the adequacy of the remote myocardium remaining after proposed SVR to support circulatory function, and of the mitral annular dilatation and inter papillary muscle spacing, factors which contribute to functional mitral regurgitation., Results: Cardiac magnetic resonance imaging (MRI) allows a complete evaluation of these quantities: the ventricular systolic and diastolic volumes (and hence ejection fraction) are easily assessed reproducibly and accurately; the regional wall motion of the asynergic area and the remote myocardium can be measured by several quantitative means, including with myocardial tagging, and the presence or absence of nonviable, irreversible scar can be detected with gadolinium-based interstitial contrast agents. Furthermore, an accurate measurement of the mitral annular dimensions and the papillary muscle spacing can be easily performed using cardiac MRI, allowing planning of effective therapy for mitral regurgitation., Conclusions: The entire imaging study can be performed in less than 1h, making cardiac MRI a truly useful and comprehensive tool in planning SVR, and for subsequently evaluating results.

A patient with a history of Hodgkin's lymphoma presented with recurrent left pleural effusions and dyspnea on exertion 27 years after radiation therapy. Further evaluation disclosed suspected radiation-induced constrictive pericarditis, aortic stenosis and regurgitation, and severe coronary artery disease. He underwent successful 3-vessel coronary artery bypass grafting, aortic valve replacement, and pericardiectomy.

In this report, we review the advantages, limitations, and optimal utilization of various transthoracic and transesophageal echocardiographic (TTE and TEE) methods used for assessing mitral regurgitation (MR) as published in full-length, peer-reviewed articles since the color Doppler era began in 1984. In addition, comparison is made to other imaging modalities including catheter-based, magnetic resonance and surgical assessment of MR. Although left ventricular (LV) angiography has been traditionally used for validation of various TTE methods and is time-honored, its considerable limitations preclude it from being a real "gold standard." Based on the reviewed literature, no clear "gold standard" for the assessment of MR can be identified at present, but newly emerging TTE and TEE techniques, such as three-dimensional color Doppler, may have the potential to overcome some of the limitations of the two-dimensional methods.

The goal of this study was to determine whether changes in cardiac metabolism in Type 2 diabetes are associated with contractile dysfunction or impaired response to ischemia. Hearts from Zucker diabetic fatty (ZDF) and lean control rats were isolated and perfused with glucose, lactate, pyruvate, and palmitate. The rates of glucose, lactate, pyruvate, and palmitate oxidation rates and glycolysis were determined during baseline perfusion and low-flow ischemia (LFI; 0.3 ml/min for 30 min) and after LFI and reperfusion. Under all conditions, ATP synthesis from palmitate was increased and synthesis from lactate was decreased in the ZDF group, whereas the contribution from glucose was unchanged. During baseline perfusion, the rate of glycolysis was lower in the ZDF group; however, during LFI and reperfusion, there were no differences between groups. Despite these metabolic shifts, there were no differences in oxygen consumption or ATP production rates between the groups under any perfusion conditions. Cardiac function was slightly depressed before LFI in the ZDF group, but during reperfusion, function was improved relative to the control group despite the increased dependence on fatty acids for energy production. These data suggest that in this model of diabetes, the shift from carbohydrates to fatty acids for oxidative energy production did not increase myocardial oxygen consumption and was not associated with impaired response to ischemia and reperfusion.

Background: It is believed that increasing cardiac glucose metabolism in the setting of ischemia and reperfusion is protective because of the resulting decrease in fatty acid oxidation, which improves cardiac efficiency and increases glucose oxidation relative to glycolysis; however, these conclusions are based primarily on studies in which glucose is the only carbohydrate provided. The goal of this study was to examine the effect of stimulating myocardial carbohydrate use either by increasing glucose and insulin levels or by using dichloroacetate on the response to ischemia and reperfusion in hearts perfused with physiological concentrations of lactate and pyruvate plus glucose and fatty acids., Methods and Results: Metabolic fluxes were determined in hearts from male Sprague-Dawley rats perfused with 13C-labeled substrates using 13C/1H-NMR isotopomer analysis after 30 minutes of low-flow ischemia (0.3 mL/min) and 60 minutes of reperfusion. Measurements were made under control conditions: 5 mmol/L glucose, 1 mmol/L lactate, 0.1 mmol/L pyruvate, 0.3 mmol/L palmitate, and 50 microU/mL insulin plus dichloroacetate 5 mmol/L or glucose and insulin increased to 30 mmol/L and 1000 microU/mL, respectively. Dichloroacetate increased carbohydrate oxidation and the ratio of glucose oxidation to glycolysis but did not improve functional recovery or cardiac efficiency; however, elevated glucose and insulin levels improved functional recovery and cardiac efficiency but did not increase carbohydrate oxidation or the ratio of glucose oxidation to glycolysis., Conclusions: These data support the notion that increasing myocardial glucose use is beneficial in the setting of ischemia and reperfusion; however, the protective effect appears not to be mediated by shifting the balance between carbohydrate and fatty acid oxidation.

Patients with ischemic heart disease may have left ventricular (LV) dysfunction due to reversible or irreversible causes. The ability to distinguish viable myocardium with dysfunction due to a reversible etiology (hibernation, stunning) from nonviable scar is critical for determining proper management of the patient. Cardiovascular magnetic resonance (CMR) is a technique that has been established to be useful for the detection of myocardial viability and advancements in the field promise to further increase its utility. In this review we describe the features of CMR that make it suited for this purpose and outline promising developments that may soon make CMR the reference standard for viability assessment.

In this report we present 12 adult patients in whom surgical or percutaneous intervention was considered for repair of atrial septal defect (ASD). Location, size, and surrounding atrial anatomy of the ASD were assessed prior to intervention in all patients with standard and live three-dimensional transthoracic echocardiography (3D TTE). In the four patients in whom intraoperative three-dimensional transesophageal echocardiographic reconstruction (3D TEE) was done, 3D TTE measurements of maximum dimension, maximum circumference, and maximum area of ASD agreed well with 3D TEE. In the seven patients who underwent transcatheter closure device insertion, live 3D TTE measurements of maximum dimension, maximum circumference, and maximum area of ASD agreed well with the sizing balloon. Additionally, since the sizing balloon measures a stretched diameter and area, a live 3D TTE stretched ASD diameter and area (derived from the actual live 3D TTE maximum circumference) were calculated and demonstrated improved agreement with the sizing balloon measurements. In all patients, > or =5 mm of atrial tissue was visualized surrounding the ASD. Further, with the addition of contrast enhancement, characterization of a small patent foramen ovale (<5 mm) was possible in one patient. Live 3D TTE accurately defined ASD location, size, and surrounding atrial anatomy in all patients studied by us. ASD characterization by live 3D TTE agreed well with 3D TEE and sizing balloon measurements.

We evaluated 44 consecutive patients who underwent standard two-dimensional (2D) and live three-dimensional (3D) transthoracic echocardiography (TTE), as well as left heart catheterization with left ventriculography. Mitral regurgitant vena contracta area (VCA) was obtained by 3D TTE by systematic and sequential cropping of the acquired 3D TTE data set. Assessment of mitral regurgitation (MR) by ventriculography was compared to measurements of VCA by 3D TTE and to 2D TTE measurements of MR jet area to left atrial area (RJA/LAA), RJA alone, vena contracta width (VCW), and calculated VCA. VCA from 3D TTE closely correlated with angiographic grading (rs=0.88) with very little overlap. VCA of <0.2 cm2 correlated with mild MR, 0.2-0.4 cm2 with moderate MR, and >0.4 cm2 with severe MR by angiography. Ventriculographic grading also correlated well with 2D TTE measurements of RJA/LAA (rs=0.79) and RJA alone (rs=0.76) but with more overlap. Assessment of VCW and calculated VCA by 2D TTE agreed least with ventriculography (rs=0.51 and rs=0.55, respectively). Live 3D TTE color Doppler measurements of VCA can be used for quantitative assessment of MR and is comparable to assessment by ventriculography.

We report an adult patient with a patent ductus arteriosus in whom live three-dimensional transthoracic echocardiography provided comprehensive assessment of the morphology and pathophysiology of the lesion.

Interventions that stimulate carbohydrate oxidation appear to be beneficial in the setting of myocardial ischemia or infarction. However, the mechanisms underlying this protective effect have not been defined, in part because of our limited understanding of substrate utilization under ischemic conditions. Therefore, we used (1)H and (13)C NMR spectroscopy to investigate substrate oxidation and glycolytic rates in a global low-flow model of myocardial ischemia. Isolated male Sprague-Dawley rat hearts were perfused for 30 min under conditions of normal flow (control) and low-flow ischemia (LFI, 0.3 ml/min) with insulin and (13)C-labeled lactate, pyruvate, palmitate, and glucose at concentrations representative of the physiological fed state. Despite a approximately 50-fold reduction in substrate delivery and oxygen consumption, oxidation of all exogenous substrates plus glycogen occurred during LFI. Oxidative metabolism accounted for 97% of total calculated ATP production in the control group and approximately 30% in the LFI group. For controls, lactate oxidation was the major source of ATP; however, in LFI, this shifted to a combination of oxidative and nonoxidative glycogen metabolism. Interestingly, in the LFI group, anaplerosis relative to citrate synthase increased sevenfold compared with controls. These results demonstrate the importance of oxidative energy metabolism for ATP production, even during very-low-flow ischemia. We believe that the approach described here will be valuable for future investigations into the underlying mechanisms related to the protective effect of increasing cardiac carbohydrate utilization and may ultimately lead to identification of new therapeutic targets for treatment of myocardial ischemia.

Lactate is a key metabolite and its rates of cellular uptake and release, its production rates from glucose and glycogen, and its interconversion rate with pyruvate are important determinants of cellular energy production. If lactate precursors such as pyruvate and glucose are labeled appropriately with (13)C, (1)H NMR spectroscopy provides a means of quantifying lactate production from each source and allows measurement of all these rates within a single experiment. However, due to the multiplicities of the resonance lines (from nuclear spin-spin couplings) in lactate (13)C isotopomers, the (1)H NMR spectra were found to be more complex than expected, requiring determination of all spin-spin interactions in this anion. All such values were determined for lactate and its precursor pyruvate. The method was then applied to simultaneously measure the rates of exogenous lactate uptake and rates of release of glucose-, glycogen-, and pyruvate-derived lactate in the isolated perfused rat heart., (Copyright 2004 Wiley-Liss, Inc.)

Conversion of Fe ions in solution to the solid phase in ferritin concentrates iron required for cell function. The rate of the Fe phase transition in ferritin is tissue specific and reflects the differential expression of two classes of ferritin subunits (H and L). Early stages of mineralization were probed by rapid freeze-quench Mossbauer, at strong fields (up to 8 T), and EPR spectroscopy in an H-type subunit, recombinant frog ferritin; small numbers of Fe (36 moles/mol of protein) were used to increase Fe3+ in mineral precursor forms. At 25 ms, four Fe3+-oxy species (three Fe dimers and one Fe trimer) were identified. These Fe3+-oxy species were found to form at similar rates and decay subsequently to a distinctive superparamagentic species designated the "young core." The rate of oxidation of Fe2+ (1026 s(-1)) corresponded well to the formation constant for the Fe3+-tyrosinate complex (920 s(-1)) observed previously [Waldo, G. S., & Theil, E. C. (1993) Biochemistry 32, 13261] and, coupled with EPR data, indicates that several or possibly all of the Fe3+-oxy species involve tyrosine. The results, combined with previous Mossbauer studies of Y30F human H-type ferritin which showed decreases in several Fe3+ intermediates and stabilization of Fe2+ [Bauminger, E. R., et al. (1993) Biochem. J. 296, 709], emphasize the involvement of tyrosyl residues in the mineralization of H-type ferritins. The subsequent decay of these multiple Fe3+-oxy species to the superparamagnetic mineral suggests that Fe3+ species in different environments may be translocated as intact units from the protein shell into the ferritin cavity where the conversion to a solid mineral occurs.

All organisms utilize ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) to catalyze the terminal step of the heme biosynthetic pathway, which involves the insertion of ferrous ion into protoporphyrin IX. Kinetic methods and Mössbauer spectroscopy have been used in an effort to characterize the ferrous ion-binding active site of recombinant murine ferrochelatase. The kinetic studies indicate that dithiothreitol, a reducing agent commonly used in ferrochelatase activity assays, interferes with the enzymatic production of heme. Ferrochelatase specific activity values determined under strictly anaerobic conditions are much greater than those obtained for the same enzyme under aerobic conditions and in the presence of dithiothreitol. Mössbauer spectroscopy conclusively demonstrates that, under the commonly used assay conditions, dithiothreitol chelates ferrous ion and hence competes with the enzyme for binding the ferrous substrate. Mössbauer spectroscopy of ferrous ion incubated with ferrochelatase in the absence of dithiothreitol shows a somewhat broad quadrupole doublet. Spectral analysis indicates that when 0.1 mM Fe(II) is added to 1.75 mM ferrochelatase, the overwhelming majority of the added ferrous ion is bound to the protein. The spectroscopic parameters for this bound species are delta = 1.36 +/- 0.03 mm/s and delta EQ = 3.04 +/- 0.06 mm/s, distinct from the larger delta EQ of a control sample of Fe(II) in buffer only. The parameters for the bound species are consistent with an active site composed of nitrogenous/oxygenous ligands and inconsistent with the presence of sulfur ligands. This finding is in accord with the absence of conserved cysteines among the known ferrochelatase sequences. The implications these results have with regard to the mechanism of ferrochelatase activity are discussed.

Ferrochelatase is the terminal enzyme of the heme biosynthetic pathway in all cells. It catalyzes the insertion of ferrous iron into protoporphyrin IX, yielding heme. In eukaryotic cells, ferrochelatase is a mitochondrial inner membrane-associated protein with the active site facing the matrix. Decreased values of ferrochelatase activity in all tissues are a characteristic of patients with protoporphyria. Point-mutations in the ferrochelatase gene have been recently found to be associated with certain cases of erythropoietic protoporphyria. During the past four years, there have been considerable advances in different aspects related to structure and function of ferrochelatase. Genomic and cDNA clones for bacteria, yeast, barley, mouse, and human ferrochelatase have been isolated and sequenced. Functional expression of yeast ferrochelatase in yeast strains deficient in this enzyme, and expression in Escherichia coli and in baculovirus-infected insect cells of different ferrochelatase cDNAs have been accomplished. A recently identified (2Fe-2S) cluster appears to be a structural feature shared among mammalian ferrochelatases. Finally, functional studies of ferrochelatase site-directed mutants, in which key amino acids were replaced with residues identified in some cases of protoporphyria, will be summarized in the context of protein structure.

Desulforedoxin is a protein purified from cellular extracts of Desulfovibrio gigas. It is a small (7.9 kDa) dimeric protein that contains a distorted rubredoxin like center (one single iron coordinated by four cysteinyl residues). Due to the simplicity of the polypeptide chain and of the iron center, an attempt was made to chemically produce this protein. A 36 amino acid polypeptide chain was synthesized based on the known sequence of native Desulforedoxin. The iron center was then reconstituted and the biochemical and spectroscopic characteristics of this synthetic protein were investigated. The final product has an equal sequence to the protein purified from D. gigas. The synthetic and natural Dx are very similar, in terms redox potential and spectroscopic properties (UV-Visible, EPR, Mössbauer).

The VFe protein of alternative nitrogenase, isolated from Azotobacter vinelandii, strain LS15 and designated as Av1', has been investigated by Mössbauer spectroscopy. The Mössbauer spectrum of the dithionite-reduced Av1', recorded at 4.2 K with a 60-millitesla magnetic field applied parallel to the gamma-beam, is a superposition of three spectral components: 1) a complex spectrum (the M component) with magnetic hyperfine structures attributed to the paramagnetic FeV cofactor, 2) a component (the P component) consisting of three quadrupole doublets identifiable as the Fe2+, D, and S doublets similar to those observed for the P cluster pairs in MoFe proteins, and 3) a minor (4% of total absorption) quadrupole doublet attributable to adventitiously bound iron. The observed 4.2-K parameters for the Fe2+ (delta EQ = 2.99 mm/s and delta = 0.64 mm/s), D (delta EQ = 0.75 mm/s and delta = 0.63 mm/s), and S (delta EQ = 1.2 mm/s and delta = 0.65 mm/s) iron sites and their temperature dependence are very similar to those observed for the P cluster pairs in the conventional MoFe proteins. Similar to those of the MoFe protein, strong field spectra indicate that these doublets are associated with a diamagnetic system. Their percent absorption intensities (Fe(2+)/D/S = 13.0:32.2:6.8) determined at 4.2 K after the removal of the contributions from the adventitiously bound iron are comparable to those of the P cluster pairs in MoFe proteins. These observations established that Av1' also contains P cluster pairs that are identical, in both composition and quantity, to those of the MoFe proteins; i.e. each molecule contains two P cluster pairs and each pair is formed by two Fe2+, five D, and one S iron sites. Considering that 52% absorption of the P component corresponding to two 8Fe clusters, the remaining 48% absorption determined for the M component is consistent with two 7Fe-containing FeV cofactors/molecule of Av1'. The fact that both P cluster pairs are found in the diamagnetic states implies that the S = 3/2 and S = 1/2 signals detected in earlier EPR measurements are associated with the FeV cofactor and suggests a spin mixture for the FeV cofactor in the dithionite-reduced Av1'.