P = 0.01) in the subjects (73.3 ± 12.6 cm/s) compared with the controls (80.2 ± 22.5 cm/s). Similarly, mean S velocity of pulmonary venous flow was lower (P = 0.01) in the subjects than in the controls. Left atrial dimension and mitral E/A ratio in the subjects with left ventricular hypertrophy were higher  than in the subjects without this abnormality. We concluded that normotensive offspring of hypertensive Nigerians showed early alterations in indexes of left ventricular diastolic filling and these abnormalities were exaggerated in the presence of left ventricular hypertrophy. Contribution of left ventricular diastolic dysfunction to adverse events in patients with cardiovascular diseases is increasingly being recognized and individuals with pedigree for hypertension are thought to exhibit anatomic and or functional changes in their left ventricle before they become hypertensive. This study aimed at characterizing left ventricular diastolic function in normotensive offspring of hypertensive Nigerians. Sixty-five offspring of hypertensive parents aged 15–25 years (subjects) with 65-age and sex-matched offspring of normotensive parents (controls) were studied for early makers of hypertensive cardiovascular disease using Doppler echocardiogram. Mean mitral E velocity was reduced ( Left ventricular diastolic dysfunction (DD) is a common problem in adult patients with systemic hypertension with prevalence ranging between 46 and 83% , 2. The A random sample of hypertensive patients attending our cardiovascular clinic was conducted. The selected patients were requested to bring one of their eligible children between the ages of 15 and 25 years for assessment (OHP group). The same procedure was employed to select age- and sex-matched children without parental hypertension (ONP) from the Medical Outpatient Department to serve as controls. Ethical approval was obtained from the Ethics and Research Committee of the hospital and an informed consent was obtained from each participant. All individuals who were pregnant, on cardio active drugs, had cardio-pulmonary disease; and those with organic heart murmur and renal bruit were not eligible to participate. Similarly, those with structural heart disease and poor echocardiographic window were excluded from the study. A thorough history to exclude cardiovascular disease in the participants was taken and anthropometric parameters, blood pressure and other relevant clinical examination were performed on all subjects. Blood pressure was measured at the left arm in a comfortable position after about 10 minutes rest using an appropriate cuff. However, if the blood pressure is equal or greater than 10 mmHg at the right hand, the latter blood pressure was used as the blood pressure of the subjects. An average of three measurements was used as the blood pressure. Individuals with blood pressure ≥140/90 mmHg or on anti-hypertensive drugs were also excluded. Electrocardiogram was done in standard position and subsequently echocardiogram. Two-dimensional and M-mode echocardiograms were performed on all participants and measurements were taken according to the recommendations of the American Society of Echocardiography (ASE) . Left ventricular diastolic function was assessed using Doppler echocardiography which was performed at the apical 4-chamber echocardiographic view. With the sample volume placed at the tips of mitral valve leaflets, the early diastolic (E wave), and the atrial contraction (A wave) Doppler velocity envelopes were obtained. The early to late diastolic peak velocity ratio (E/A), the flow velocity integral for both early (E1) and atrial (A1) filling waves, and the ratio of flow velocity integral of early-to-late filling (E1/A1) were used to assess diastolic function . Other pt-test was used to compare means of continuous variables while chi-square test was used to compare means of proportions. Subanalysis of OHP based on whether LVH was present or not was done. Clinical and echocardiographic parameters of OHP with LVH were compared with those without LVH. Test of correlation was done using both Pearson and Spearman's rank correlation methods. A statistically significant association was taken at P < 0.05.Statistical analysis was performed using the SPSS Version 15 and numerical values were presented as mean ± standard deviation. Student P > 0.05).Sixty-five OHP with 65-age and sex-matched ONP as subjects and controls, respectively, were studied. Mean ages in the two groups were similar (P = 0.001) than that of ONP. Similarly, the left ventricular mass, left ventricular mass index, and relative wall thickness were significantly higher in the subjects than controls . The anthropometric parameters showed that the body mass index, waist, and hip circumference; pulse rate, systolic and diastolic blood pressures were similar in the two groups as shown in P = 0.01. However, mean mitral A velocity envelope and E/A ratio were similar in both groups . Similarly, mitral E velocity deceleration time and isovolumic relaxation time were not different between the two groups . Mean S velocity envelope of pulmonary venous flow was significantly lower (P = 0.01) in the subjects than in the control, although both were within normal limits in the two groups. On the other hand, mean D velocity and atrial reversal velocities were similar between the two groups (P = 0.087 and 0.9). Overall, patterns of left ventricular diastolic function were similar between the two groups. None of the subjects and controls had impaired relaxation, pseudonormal and restrictive patterns. Doppler echocardiographic assessment of the subjects is presented in 2 which was higher than mean of those without LVH (21.8 ± 3.2 g/m2), P = 0.03. Similarly, waist circumference in those with LVH was significantly higher than the mean of those without LVH (P = 0.045). Mean left atrial dimension in OHP with LVH was 3.39 ± 0.3 cm which was higher than 2.99 ± 0.45 cm found in OHP without LVH, P = 0.002. While mitral A velocity was significantly lower in those with LVH compared with mean of OHP without LVH (P = 0.004), mitral E/A ratio was higher in the former than the latter (P = 0.0).Comparison of OHP with and without left ventricular hypertrophy is displayed in Correlates of mitral E, A velocities, and E/A ratio are shown in This study shows significant reduction in some of the indexes of left ventricular diastolic function in OHP when compared with ONP. Both mitral E velocity and S wave of pulmonary venous flow were significantly lower in OHP than ONP. Although, the differences in these parameters between the two groups of offspring were small, they may indicate subtle alterations in the left ventricular diastolic function in the former, with filling pressures tending towards the earliest pattern of left ventricular DD seen in systemic hypertension. The pathophysiological mechanisms underlying these early alterations are unknown. However, elevated endogenous ouabain has been suggested as a possible mechanism and its plasma level had been found to correlate well with indexes of diastolic dysfunction in offspring of hypertensive parents .Abnormalities of left ventricular filling pressures observed in our study may be partly due to higher left ventricular mass in OHP compared with ONP . All othGenerally, left ventricular diastolic filling patterns in patients with systemic hypertension include normal, impaired relaxation, pseudonormal and restrictive patterns . All theThe results of the present study also showed that OHP with LVH had higher BMI, WC, and DBP than OHP without LVH. These findings clearly demonstrated the effects of larger body size on blood pressure, left ventricular structure, and filling. The mitral E/A ratio in OHP with LVH was greater than 2 which was suggestive of restrictive patterns of left ventricular filling unlike normal pattern seen in OHP without LVH. More importantly, left atrial dimension which is a reflection of left ventricular filling pressure was significantly large in those with LVH than those without LVH. This indicates higher left ventricular filling pressure in OHP with LVH than those without LVH. The influence of LVH on left ventricular diastolic function had been shown by some studies , 19. It Negative correlation was observed between mitral E velocity envelope and age. This pattern is similar to what is obtained in older population of both normotensive and hypertensive individuals , 21. On In conclusion, normotensive offspring of hypertensive Nigerians showed early alterations in some of the indexes of left ventricular diastolic filling and these abnormalities are exaggerated in the presence of LVH. We recommend early and appropriate lifestyles modification in OHP especially those with LVH.


T1 mapping is a powerful technique for ECV quantification; native T1 has been shown to increase in a variety of conditions including oedema, fibrosis and amyloid. Iron and fat lower T1. Anderson Fabry disease (AFD) is a fat storage disease, cardiac iron occurs in transfusion dependent patients. We hypothesised that T1 lowering would diagnose early cardiac involvement, track disease severity and discriminate from other mimic pathologies.280 subjects were studied: iron overload (n=53), AFD , healthy volunteers , hypertension , hypertrophic cardiomyopathy , severe aortic stenosis  and definite AL cardiac amyloidosis . Along with routine clinical CMR, native, non-contrast T1 mapping was performed using the Sh-MOLLI technique at 1.5T without gadolinium administration. T2*(iron overload) and LGE and LV mass (AFD and LVH diseases) were also assessed.Compared to health volunteers, septal T1 was lower in iron overload and AFD and higher in other diseases .In patients with LVH n=105), T1 discriminated completely between AFD and all other diseases with no overlap  British Heart Foundation.2) Genzyme Pharmaceuticals.


There is conflicting evidence regarding gender-based differences in outcomes in patients after myocardial infarction. Recently, a study demonstrated greater myocardial salvage assessed with SPECT imaging in women than in men. However, SPECT imaging has several limitations including its low spatial resolution particularly of subendocardial infarcts and as a result cardiac magnetic resonance imaging (CMR) has emerged as the reference method to assess myocardial salvage.The aim of this study was to investigate whether there are gender-associated differences in the amount of myocardial salvage assessed by CMR and clinical outcome in ST-elevation myocardial infarction (STEMI) patients reperfused by primary percutaneous coronary intervention (PCI).In this study we included 87 women (mean age 63±12 years) and 262 men (mean age 69±13 years) with STEMI undergoing primary PCI <12 hours after symptom onset. The primary end point was myocardial salvage (area at risk minus infarct size) assessed by T2-weighted and contrast-enhanced CMR. The secondary end point was the occurrence of major adverse cardiovascular events (MACE) defined as death, reinfarction and occurrence of new congestive heart failure within 6 months after the index event.The amount of myocardium at risk  and final infarct size (16.7±13.1%LV versus 17.9±12.4%LV) did not differ significantly between women and men. Consequently, the myocardial salvage index was similar between groups . After adjustment for baseline characteristics, female gender was no independent predictor of greater myocardial salvage after PCI.Regarding clinical outcomes there was a trend of an increased MACE rate in women  as well as a significantly increased mortality rate in female myocardial infarction patients .The efficacy of primary PCI  in patients with STEMI is not gender-dependent. However, female STEMI patients have worse clinical outcomes than men, irrespective of the salvaged myocardium at risk.


CCL19 and CCL21, acting through CCR7, are termed homeostatic chemokines. Based on their role in concerting immunological responses and their proposed involvement in tissue remodeling, we hypothesized that these chemokines could play a pathogenic role in heart failure (HF).−/− mice showed improved survival and attenuated increase in markers of myocardial dysfunction and wall stress in post-MI HF after 1 week, accompanied by increased myocardial expression of markers of regulatory T cells. (v) Six weeks post-MI, there was an increase in markers of myocardial dysfunction and wall stress in CCR7 deficient mice.Our main findings were: (i) Serum levels of CCL19 and particularly CCL21 were markedly raised in patients with chronic HF (n = 150) as compared with healthy controls (n = 20). A CCL21 level above median was independently associated with all-cause mortality. (ii) In patients with HF following acute myocardial infarction , high versus low CCL21 levels 1 month post-MI were associated with cardiovascular mortality, even after adjustment for established risk factors. (iii). Explanted failing human LV tissue (n = 29) had markedly increased expression of CCL21 as compared with non-failing myocardium (n = 5). (iv) Our studies in CCR7High serum levels of CCL21 are independently associated with mortality in chronic and acute post-MI HF. Our findings in CCR7 deficient mice may suggest that CCL21 is not only a marker, but also a mediator of myocardial failure. However, while short term inhibition of CCR7 may be beneficial following MI, a total lack of CCR7 during long-term follow-up could be harmful. Chronic heart failure (HF) is a disorder characterized by low-grade immune activation and inflammation, as evident by elevated circulating and myocardial levels of inflammatory cytokines and chemokines, such as tumor necrosis factor (TNF)α, interleukin (IL)-1β, monocyte chemoattractant protein (MCP)-1, and IL-8 The chemokines CCL19 and CCL21, acting through their common receptor, CCR7, are termed homeostatic due to their role in immune surveillance and regulation of leukocyte movement during homeostasis Based on their essential role in concerting immunological and inflammatory responses as well as their newly discovered involvement in tissue remodeling, we hypothesized that CCL19 and CCL21 may play a pathogenic role in HF. Here, we elaborated this hypothesis by clinical studies in patients with chronic HF and in patients with acute HF following myocardial infarction (MI), as well as in studies of CCR7 deficient mice in an animal model of post-MI HF.The clinical parts of this study were approved by the local ethical committee  and conducted according to the ethical guidelines outlined in the Declaration of Helsinki for use of human tissue and subjects. Informed written consent was obtained from all subjects. All animal experiments were carried out in accordance with institutional guidelines, and conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health  and was approved by the Norwegian National Animal Research Committee .Patients with stable HF (n = 150) for >4 months in New York Heart Association (NYHA) functional class II-IV, on optimal cardiovascular treatment regimens, attending the Department of Cardiology at Oslo University Hospital Rikshospitalet, were consecutively included in the study Table 1.g for 10 minutes). All serum samples were stored at −80°C and thawed <3 times. In both sub-studies, patients with significant concomitant disease such as infection, malignancy, or autoimmune disorder were not included and none were receiving immunosuppressive drugs.The design and main results of the OPtimal Trial In Myocardial infarction with Angiotensin II Antagonist Losartan (OPTIMAAL) have previously been reported in detail Tissue samples from human failing myocardium were removed from still-beating hearts immediately on explantation from 29 patients with end-stage HF  ejection fraction [LV-EF] <35%; 21 DCM, 10 IHD; age 47±3 years) undergoing cardiac transplantation. Control human LV tissue was obtained from subjects whose hearts were rejected as cardiac donors for surgical reasons (n = 5). The cause of death of donors was cerebrovascular accident, and none had a history of heart disease. The hearts from these subjects had been kept on ice for 1 to 4 hours before tissue sampling. In nine patients with advanced HF , LV tissue was sampled at the time of implantation and at the time of removal (heart transplantation) of a continuous-flow LV assist device . Average time on LVAD was 8±1.7 months. In both failing and non-failing myocardium, LV tissue samples were snap-frozen in liquid nitrogen, and stored at −80°C until use. None of the patients (failing and control myocardium) had significant concomitant disease such as infection, malignancy, or autoimmune disorder.Serum levels of CCL19 and CCL21 were measured by enzyme immunoassays provided from R&D Systems . Serum levels of N-terminal pro-brain natriuretic peptide (Nt-proBNP) and C-reactive protein (CRP) were determined as previously reported −/− mice were backcrossed for at least 8 generations onto the C57BL/6 background −/− and wild type (Wt) mice were characterized after ligation of the left coronary artery (MI) or sham operation. MI was induced in 8 week old mice as described 2 on a mask. Echocardiographic data were analyzed off-line using VEVO 2100 1.1.0 software from Visualsonics. Three representative cycles were analyzed and averaged. Two-dimensional (2D) images of the LV were obtained both in long and short axes. Short axis recordings were obtained at the level of the papillary muscle. M-mode tracings were recorded in the long axis at the level of the papillary muscles and the aortic valves, with 2D guidance. LV wall thickness and cavity dimensions were measured through the largest diameter of the ventricle both in systole and diastole. LV fractional shortening (LVFS) in percent, was calculated using the following formula: LVFS = (LVDd−LVDs)/LVDd×100 where LVDd is LV diameter in diastole, and LVDs is LV diameter in systole. Doppler recordings were obtained in the left parasternal long axis position. Pulsed wave Doppler was used for measuring flow velocities in the left ventricular outflow tract (LVOT) and in the mitral annulus. Cardiac output (CO) was calculated in LVOT using the following equation: CO = LVOT VTI×π×(diameter/2)2×heart rate where VTI is the velocity time integral and diameter is measured in LVOT. The animals were subsequently euthanized and their hearts were removed and blotted dry. The non-infarcted LV, infarcted area, right ventricular free wall, and lungs were weighed and normalized to tibia length. Based on previous findings C57BL/6 mice were purchased from Møllergaard . CCR7Total RNA from mouse and human myocardium was extracted using TRIzol , DNase treated, cleaned up using RNeasy Mini Columns , and stored at −80°C. cDNA was synthesized from 1 µg RNA using High Capacity cDNA Archive Kit . Quantification of gene expression was performed using the ABI Prism 7500 (Applied Biosystems), 5 ng cDNA, Power SYBR Green Master Mix (Applied Biosystems), and sequence-specific PCR primers were designed using the Primer Express software, version 3.0 (Applied Biosystems). Primer sequences can be provided on request. Gene expression of the housekeeping gene GAPDH was used for normalization.Formalin fixed and paraffin-embedded mouse LV was immunostained with anti-CCL21 , and anti-CD45 . For forkhead box P3 (foxP3) analysis, acetone-fixed cryosections were stained with anti-foxP3 antibody . Omission of the primary antibody served as negative control. The immunoreactivities were amplified by the avidin-biotin-peroxidase system  according to the manufacturers' instructions. Diaminobenzidine was used as the chromogen. The sections were counterstained with hematoxylin or eosin (foxP3). For the quantification of foxP3-positive cells, we randomly counted and averaged five fields (40× magnification) in the area bordering the myocardial infarction.Masson trichrome staining of formalin-fixed and paraffin-embedded mouse hearts were performed using the Trichrome Stain (Masson) Kit  according to the manufacturer's suggestions.a priori. For investigating treatment effects, repeated-measures ANOVA was performed a priori on log transformed values if necessary as evaluated by the Kolmogorov-Smirnov test, with time and treatment as fixed factors and subject as random. For comparisons of 2 groups of individuals, the Mann-Whitney U test was used. Coefficients of correlation were calculated by the Spearman rank test. Receiver-operating characteristics (ROC) curve were generated to evaluate the accuracy of each marker for prediction of all-cause and cardiovascular (CV) mortality. Kaplan-Meier analysis with log-rank test was performed to compare the number of events in different groups (comparisons pooled over strata). Cox proportional hazard analysis was performed to estimate hazard ratios using a forced method adjusting for age, hypertension, diabetes type 2, etiology, creatinine, CRP and Nt-proBNP. CCL21 was dichotomized  or divided into tertiles with the two lower tertiles used as reference (OPTIMAAL). Follow-up time for all-cause mortality was calculated from time of inclusion to death from any cause. P values are two-sided and considered significant when <0.05. All analyses were performed with SPSS for Windows version 15.0 .If more than three groups were compared, the Kruskal-Wallis test was used As shown in During a mean follow-up of 24 (±12 [SD]) months, 32 patients died. ROC curves showed comparable area under curve (AUC) for CCL21 and NT-proBNP, with lower levels for CCL19 , was accompanied by a marked decrease in CCL21 mRNA levels, and this decrease was seen in 8 of the 9 patients  Wt and 19 (37.3%) CCR7−/− mice died, reflecting improved survival in CCR7 deficient mice during the first week of follow-up  The present study has some limitations such as the inclusion of relatively few patients and in particular, the number of controls was low. Also, the lack of IHC from human heart, the lack of IHC on CCR7, and the lack of data on serum levels of CCL19 and CCL21 in the mice model are other limitations. Moreover, the changes in echocardiographic variables were rather modest. However, mRNA markers of myocardial remodelling showed profound alterations, suggesting that these may be more robust markers of myocardial dysfunction in this model. The association between the attenuated up-regulation of these markers and improved survival further support such a notion. Finally, systemic deletion of CCR7 has some confounders, and future studies should also examine the effect of cardiac-restricted deficiency of CCR7. Nonetheless, our studies, combining experiments in clinical and experimental HF, suggest a role for CCL21/CCR7 interactions in the pathogenesis of HF.


The structure and function of the heart needs to be understood in three dimensions. We give a brief historical summary of the methods by which such an understanding has been sought, and some practical details of the relatively new technique of micro-CT with iodine contrast enhancement in samples from rat and rabbit. We discuss how the improved anatomical detail available in fixed cadaveric hearts will enhance our ability to model and to understand the integrated function of the cardiomyocytes, conducting tissues, and fibrous supporting structures that generate the pumping function of the heart. The relationship between form and function in the heart has been studied by scientists for centuries. But the precise mechanism of the contractile function of the heart and its modes of failure continue to exercise the minds of scientists and clinicians in the present day. Close inspection of the heart by careful dissectors hinted at considerable complexity even before there was an understanding of the cellular basis of the structure, or of the fundamental mechanism of contraction. Pettigrew  introducAfter 150 years the debate over the true microanatomy of the heart continues. We can now make virtual three-dimensional semi-transparent models of the internal structure of the ventricles based on the interaction of X-rays with that structure. Furthermore, we can obtain images not only from fixed and therefore lifeless material, but also from the beating heart with magnetic resonance imaging, computed tomography, and ultrasound scanning.Physical dissection of the heart is informative, especially when guided by information from moving images and electrophysiological recordings from the beating heart. However, there is a level of structural information based on the disposition of individual cardiomyocytes, the genuine units of function, which is not accessible by simple dissection. For this level of understanding we must resolve at the scale of the single cell, at about 10 μm. The new method of micro-CT with iodine contrast enhancement also allows unprecedented discrimination between tissue types within the cardiac mass that is not possible by dissection.We have worked toward new three-dimensional descriptions of cardiac anatomy based on micro-CT of cadaveric hearts for three main reasons:The normal structure and function of the heart, as well as the common pathological changes that cause abnormal function, consist in structures that operate in three dimensions. Therefore, interventions proposed to improve or restore cardiac function also require a three-dimensional appreciation of normal anatomy. Classical anatomical terminology may cause confusion in discussions or descriptions of particular anatomical dispositions, because the location of one structure is often described in terms of its complex relationship to other structures: for example, we might say “inferiolateral to the anterior cusp of the tricuspid.” While this terminology can be made unambiguous and universal across many spoken languages, it assumes detailed knowledge and is difficult to follow for non-specialists. Some of the sentences in Pettigrew’s  paper coThere is a long history of simulation of the electrical activity of a heart by computing the transfer of depolarization between elements of a three-dimensional mesh or grid that represents the myocardium, and we cite just one representative example . Such a How the detailed prediction of electrical conduction is affected by the resolution of the finite elements mesh used for modeling remains to be determined. Many useful simulations of the distribution of electrical depolarization in the heart have been achieved with finite elements 10 or 20 times the size of individual heart cells . HoweverParticularly in reconstructive surgery, pre-operative understanding of the cardiac anatomy makes surgery safer and quicker. Reconstruction of congenital abnormalities has been assisted greatly by the availability of pre-operative images produced by X-ray computed tomography. Cardiac ultrasound is also informative, and can now be achieved with catheter-based intra-cardiac probes. The position of the cardiac conduction system (CCS) is particularly important in reconstructive surgery, because key parts of the system run in areas of the cardiac structure that are also commonly involved in congenital defects , 5. Surgaggregates of cardiomyocytes.Individual cardiac myocytes are not long thin cylinders like skeletal muscle fibers. Observation of harvested myocytes from enzymatically digested myocardium shows that individual cardiomyocytes are rarely more than 20 μm in diameter, and about five times as long as they are wide. Streeter et al.  cut surfTo achieve a complete three-dimensional representation of the cardiac structure with microscopic resolution remains an enormous technical challenge. The studies of the Auckland group, using extended volume confocal microscopy and scanning electron microscopy are noteworthy. See, for example, , 9. WhilWe have found molecular iodine to be a practical and informative contrast agent in generating micro-CT images of biological soft tissue samples such as the heart. Our method is available in Stephenson et al. , but herImage quality and access to the detailed morphology of internal structures of the heart is greatly improved by the removal of residing blood Figure . MoleculDuring perfusion, the heart should not be over-inflated above the size observed when the chest is opened immediately after death. For unimpeded examination of the internal surface of the heart, the next steps are designed to maintain an inflated state as well as possible during dissection, fixation, and infusion of the iodine contrast agent. This is particularly important for the atria which are challenging because of their lack of self-supporting structure.in situ with phosphate buffered formal saline via the same cannulae used to flush the sample. The tissue begins to gain some rigidity, although the atrial walls do not become sufficiently stiff to maintain their in vivo inflated state. Once fixative has been perfused for about 10 min, we therefore remove the heart with the great vessels attached and begin a further period of fixation in vitro, retaining the cannulae in the great vessels to inflate the chambers as necessary and using clips across the pulmonary artery and pulmonary veins to seal the chambers. The amount of filling, especially of the atria, is somewhat subjective – it would be difficult to try to maintain the in vivo situation at a state representing diastole by maintaining a constant pressure, for example, because fixation progressively stiffens the tissue and thus the pressure-volume characteristic is altered. In practice, as the tissue becomes fixed, it is usually sufficiently buoyant that the atria remain inflated as the whole heart floats in the fixation solution. Even in this situation, care must be taken to avoid loss of fluid from the chambers or flattening of the atria against the side of the containing vessel.We begin the fixation process by perfusing After fixation for a minimum of 24 h, the sample is placed in the appropriate iodine solution for a time that allows effective dispersal of the iodine within the tissue, but not so long that the tissue becomes saturated with iodine. Infusion with iodine is dependent on three main variables; sample size, iodine concentration, and time . Some guSamples of human hearts for micro-anatomical study by this method are inevitably difficult to obtain. The most likely future sources are (a) from transplant operations for which the use of the explanted organ can be subject to an unambiguous consenting process and care can be taken to prepare the explants immediately after removal, or (b) from tissue that is donated for transplantation but in which some contra-indication for transplantation is identified, such as calcification of a valve. In some jurisdictions such material may properly be used for research or educational purposes. It is possible that the use of diffusible iodine may find application in virtual autopsy procedures. In this case, a period of perfusion with iodine solution followed by a period with clear perfusate to flush the vascular spaces might improve the resolution of cardiac imaging in post-mortem scans .Our normal practice is to combine the use of Image J, an open access suite of programs, with the program Amira, . One of the most important routine operations on such datasets is to “segment” the data, that is, to separate the anatomically relevant volume from the surrounding packaging  or to demarcate the biological tissue from enclosed space in scans of hollow organs like the heart. Segmentation may also be used to divide the scanned volume into biologically relevant sub-volumes such as the atria or the ventricles. Such sub-division is often necessary to allow numerical modeling to proceed by combining morphological information from tomography with functional properties from electrophysiological studies, immunohistochemical staining, or gene expression studies based on micro sampling –16. FurtSegmentation of tomographic data is often described as laborious and not feasible for large N-numbers. This is true, especially with high-resolution data, if windowing is done slice by slice: spatial interpolation of a region of interest even over just a few slices will be inaccurate. Our approach to segmentation can save time. The terminology is based on the use of Amira, but the techniques are available in other commercial packages.As an example, consider segmentation of the left ventricular free wall in a rabbit heart scanned in air at 18 μm resolution. If the free wall is approximately 20 mm from apex to base, that represents about 1100 slices. The myocardium is easily distinguished from the air space, but its surface is non-uniform. Therefore, if the myocardial area is outlined in one slice, interpolation of that boundary to the next 5 or 10 slices will result in errors of identification, because the interpolation algorithm is unable to accommodate the irregularity of the surface. Our method uses two stages: a primary segmentation based on inclusion of the wall with its immediately surrounding air volume, and a secondary segmentation based on differential attenuation.The regions of interest (ROI) are initially selected using an oversized “brush” tool to incorporate both the ROI and a thin shell of empty space that just includes the outermost elements of the ROI. Using an large brush size that selects both ROI and surrounding regions of a significantly different set of pixel values  allows for interpolations between large numbers of slices (10–200), and makes for a highly efficient method. The selection is then stored, and the range of pixel values within the anatomically defined ROI are recorded. Using the “threshold” tool a masking window is then applied to the stored selection that identifies, and thus discriminates the ROI from the surrounding tissue or void space within the stored selection. The first selection is then removed leaving only the ROI pixels selected as the working volume.Many high fidelity models of the depolarization of the mammalian heart rely on the three-dimensional datasets available from magnetic resonance scans using the diffusion tensor imaging technique. Strictly speaking this is not a morphological method in which structures such as cell boundaries are imaged by differential attenuation. Rather, the preferential diffusion direction for water is calculated for each voxel, and since water is less constrained to move in the long axis than in the short axis of a fiber, then an indication of fiber direction is achieved. However, many papers are unclear about the resolution achieved or claimed with this class of techniques. The volumetric resolution of the technique in terms of the unit volume scanned is commonly about 400 μm. Some techniques can infer from the probability density function obtained from each voxel a proportion of that volume running in particular directions and this calculation has been represented as streamlines representing the distribution of direction of diffusibility. This is still not the same as visualizing the cellular structure, and a controversy is currently playing out concerning the proposed arrangement of nerve fibers in the brain based on similar analysis –19.The vBy contrast, micro-CT imaging is potentially capable of producing a fiber direction matrix at the resolution of the single myocyte based on contrast between the myocyte and the surrounding endomysium , 11. ThiMicro-CT with iodine contrast requires careful sample preparation, but offers the best combination of anatomical detail and three-dimensional coverage presently available. We have offered some practical suggestions that we hope will assist other groups in generating high-quality images of biological samples.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Reflexology is one of the top six complementary therapies used in the UK. Reflexologists claim that massage to specific points of the feet increases blood supply to referred or ‘mapped’ organs in the body. Empirical evidence to validate this claim is scarce. This three-phase study measured changes in cardiovascular parameters in subjects receiving reflexology treatment applied to specific areas of the foot which are thought to correspond to the heart (intervention) compared with reflexology applied to other areas on the foot which are not (control).16 reflexology-naive healthy volunteers, 12 reflexology-naive patients with chronic artery disease (CAD) and 12 reflexology-naive patients with heart failure (HF) received active and control reflexology treatments in three randomised, placebo-controlled, double-blind repeated measures studies. In each study the subjects were observed over six time periods under the two conditions and randomised to receive either intervention or control treatment. Outcome measures included ‘Beat-to-beat’ non-invasive continuous measurement of heart rate, diastolic blood pressure, stroke volume, stroke index, cardiac output, cardiac index, total peripheral resistance, baroreceptor reflex sensitivity, and heart rate variability.The effects of reflexology treatment were modest. There were no significant differences noted in any of the measured parameters in either the CAD or HF intervention or control groups. Cardiac index decreased significantly in the healthy volunteer intervention group during left foot treatment (LFT)  0.75; 95% CI +/- 0.38 vs. LFT mean 2.45; SD 0.68; CI 0.35) with an effect size  = 0.002; w = 0.045).The findings suggest that reflexology massage applied to the upper part of the left foot in the area thought to relate to the ‘heart’ may have a modest specific effect on the cardiac index of healthy volunteers, but no specific effect on patients with various gradations of cardiovascular disease.


Post-contrast myocardial T1-mapping with cardiac magnetic resonance (CMR) has shown usefulness in the diagnosis of cardiac amyloid. A lower value indicates increase in extracellular volume and thereby amyloid deposition.The aim of this study is to assess the association between the amount of amyloid deposition as estimated by the degree of post-contrast myocardial T1 reduction and the severity of left ventricular (LV) involvement in cardiac amyloidosis.Consecutive patients referred for 3.0 Tesla CMR with a final diagnosis of cardiac amyloidosis  were retrospectively evaluated. The septal E/E' ratio as an index of LV filling pressures was measured with doppler echocardiography . Indexed LV volumes and mass, LV ejection fraction, and LV basal anteroseptal and inferolateral wall thicknesses were determined from cine CMR. Hemodynamic data from cardiac catheterization and brain natriuretic peptide (BNP) serum levels were available in 9 (36%) and 14 (54%) patients. respectively. Myocardial, endocardial, blood, and skeletal muscle were quantified after Gd-DTPA administration on a previously validated Look-Locker sequence using dedicated software. Correlations between myocardial T1 values and other indices of cardiac involvement were performed using Pearson correlation coefficients.We included 26 patients  with a final diagnosis of cardiac amyloid. There was an inverse correlation between myocardial/blood T1 ratio and basal anteroseptal  and inferolateral  wall thickness. Similarly endocardial/blood T1 ratio was inversely correlated with basal anteroseptal  and inferolateral  wall thickness. No significant associations were noted with LV volumes, ejection fraction, or mass. Otherwise, myocardial and endocardial T1 time showed an inverse correlation with BNP . In addition, myocardial/skeletal muscle T1 ratio showed a significant inverse association with pulmonary capillary wedge pressure .The degree of postcontrast myocardial T1 reduction is associated with markers of disease severity in cardiac amyloidosis, suggesting a potential role of T1-mapping for noninvasive quantification of cardiac amyloid deposition with CMR.No funding sources have to be declared.


Patients with type 2 diabetes are at increased risk for both, left ventricular (LV)-dilatation and myocardial infarction (MI) following the rupture of a vulnerable plaque. This study investigated the to date incompletely understood relationship between plaque vulnerability and LV-dilatation using optical coherence tomography (OCT) and cardiac magnetic resonance imaging (CMR) in patients with type 2 diabetes and stable coronary artery disease.CMR was performed in 58 patients with type 2 diabetes, in which 81 coronary lesions were investigated using OCT.A decreased minimal fibrous cap thickness (FCT) of coronary lesions was associated with an increase of several CMR-derived parameters including LV-end diastolic volume , LV-end diastolic diameter  and LV-end systolic volume . Similar results were obtained for mean FCT.Furthermore, patients with dilated versus non-dilated LV differed significantly in several cardiovascular risk factors including previous MI , HDL-cholesterol  and smoking . However, minimal FCT is associated to LV-dilatation independent of previous MIs .Receiver-operating curve analysis demonstrated that CMR-derived LVEDV predicts plaque vulnerability with low-moderate diagnostic efficiency (area under the curve 0.699) and considerate specificity (83.3%) at the optimal cut-off value (159.0 ml).These data suggest that vulnerability of coronary lesions is associated with LV-dilatation in high risk patients with type 2 diabetes. CMR may be a useful adjunct to the risk-stratification in this population. Future studies are warranted to investigate potential mechanisms linking plaque vulnerability and LV-dilatation. Patients with type 2 diabetes mellitus are at increased risk for both, left ventricular (LV)-dilatation as well as the presence of vulnerable coronary plaques -6. SpeciDiabetes mellitus has also been recognized to be an independent risk factor for heart failure and left ventricular dilatation ,8. HowevOptical coherence tomography (OCT) and intravascular ultrasound (IVUS) are the only in vivo imaging techniques available to determine plaque composition including the determination of the fibrous cap thickness (FCT) of coronary artery lesions . OCT is Cardiac magnetic resonance imaging (CMR) is a non-invasive imaging technique which allows the exact determination of cardiac dimensions. Whereas echocardiography is currently the most widely used imaging technique for the assessment of LV-dilatation, CMR may be superior for the assessment of several parameters including LV-ejection fraction (LVEF), LV-end-diastolic diameter (LVEDD), LV-end-diastolic volume (LVEDV) and LV-end-systolic volume (LVESV) ,17 partiIn this study, we sought to investigate the relationship between OCT-derived plaque morphology including the FCT and CMR-derived LV-dimensions in cardiovascular high risk patients with type 2 diabetes.A total of 81 de novo coronary lesions were investigated in 58 patients with stable coronary artery disease and type 2 diabetes mellitus planed for elective coronary angiography at the Department of Internal Medicine I, University Hospital of the RWTH Aachen, Germany. The indication for coronary angiography was based either on CMR-imaging suggestive for ischemia and/or typical symptoms of stable coronary artery disease. Patients were recruited into this study between August 2011 and June 2013. Quantitative coronary angiography, OCT and CMR imaging, laboratory testings and clinical history taking were performed in all patients.Inclusion criteria were stable angina pectoris with an at least 40% coronary stenosis, known type 2 diabetes, age > 30 years and written informed consent to the study protocol. Exclusion criteria were left main coronary artery stenosis, graft stenosis, acute coronary syndromes , hemodynamic or rhythmic instability, acute or chronic renal insufficiency (serum creatinine level > 1.5 mmol/l), systemic acute or chronic infections, pregnancy, anti-inflammatory medications such as steroids and chronic total occluded, severely tortuous or calcified vessels, which did not allow the safe advancement of the OCT catheter.The study was approved by the local Ethics Committee and is in accordance with the Declaration of Helsinki on ethical principles for medical research involving human subjects.3) in standard cardiac geometries  and in 3 short axis geometries ).CMR imaging was performed using a 1.5 Tesla magnetic resonance scanner , equipped with a 5-element cardiac synergy coil for signal reception and a vector-electro-cardiogram for cardiac synchronization. Image acquisition was performed during short repetitive end-expiratory breathholding. LV-function was assessed using balanced turbo field echo cine imaging with retrospective gating  [Image-analysis was performed using a dedicated CMR-workstation . LVEDV, LVESV, LV-stroke volume and LVEF were determined from the long axis 4 chamber view using the “area length ejection fraction”- method. LVEDD, septal and lateral wall thickness were derived from the end-diastolic basal short axis geometry. LVEDV was normalized to body surface area (BSA) and the one-sided 95% tolerance interval of previously published CMR data was used as a cut-off for LV-dilatation  ,19. CMR-For OCT image acquisition of the target vessel we used the Frequency Domain-OCT C7XR system and the DragonFly catheter  as previously described ,12. TargThe OCT-image analysis was performed by 2 independent observers throughout the entire lesion frame by frame in a 0.2 mm interval using the proprietary software provided by LightLab Imaging and in adaptation to the recently published consensus for qualitative and quantitative assessment . Intralut-test. Pearsons χ-squared test was used to compare nominal variables. Linear and non-linear regression analysis was performed to determine the association between CMR-derived LVEDV, LVESV and LVEDD and both minimal and mean FCT on a per stenosis basis. Uni- and multivariable logistic regression analysis was performed to calculate the odds ratio (OR) for minimal and mean FCT to predict LV-dilatation. Sensitivity, specificity, positive predictive value, negative predictive value and optimal cut-off values were calculated from the receiver-operating characteristic (ROC) curve to predict FCT ≤ 80 μm. Values with the highest Youden-index (sensitivity + specificity - 1) were identified as optimal cut-off-values. A classification of the diagnostic efficiency of OCT parameters according to the values of the area under the curve (AUC) was used as described elsewhere [All statistical analysis was performed with SPSS Statistics . Categorical variables are expressed as n and percentage. Continuous variables are expressed as mean ± standard deviation. The data were analysed on a per-patient basis for clinical characteristics and on a per-stenosis basis for lesion morphology and the statistical test did not account for the correlation of multiples plaques within patients. Continuous variables were compared with lsewhere . A p-valWe examined 58 patients with 81 lesions of at least 40% diameter stenosis as determined by quantitative coronary angiography in this study. There were no peri- or postprocedural complications associated with the use of either OCT or CMR. For patient characteristics, CMR and OCT data please refer to Tables To investigate the relationship between the FCT of coronary artery lesions and CMR-derived parameters in patients with type 2 diabetes, linear and non-linear regression analysis has been performed between minimal as well as mean FCT on the one hand and CMR-derived LV-dimensions on the other hand.There was a significant association between minimal FCT and CMR-derived LVEDV , LVESV  and LVEDD  for the lesion’s minimal and mean FCT to predict a dilated LV using logistic regression analysis. Both minimal  and mean  FCT significantly predicted the presence of LV-dilatation Table . HoweverAfter we found significant associations between minimal FCT and CMR-derived LVEDV, LVESV and LVEDD, we tested if these parameters predict minimal FCT ≤ 80 μm, which has recently been suggested to be the optimal OCT-derived cut-off for vulnerable plaques in vivo .Among all CMR-parameters investigated, ROC-analysis revealed that LVEDD was the best predictor for minimal FCT ≤ 80 μm  A decrease of both minimal and mean FCT is associated with increased LV-dilatation as characterized by CMR-derived LVEDV, LVESV and LVEDD.2) Both minimal and mean FCT are associated with LV-dilatation independent of previous MIs.3) LVEDD, LVEDV and LVESV predict plaque vulnerability with overall low or moderate diagnostic efficiency but considerate specificity at optimal cut-off values.A minimal FCT ≤ 80 μm is an approved in vivo predictor of plaque rupture with subsequent acute coronary events . PatientPlaque rupture causes acute coronary events which subsequently result in ischemic cardiomyopathy with LV-dilatation. Thus, the relationship between FCT and LV-dilatation in patients with type 2 diabetes observed in this study may reflect the relationship between plaque vulnerability and previous MIs. However, although several cardiovascular risk factors including previous MI and HDL-cholesterol were different between patients with dilated versus non-dilated LV, multivariable regression analysis demonstrated that both minimal and mean FCT predict LV-dilatation independent of previous MIs. Moreover, minimal and mean FCT predicted LV-dilatation even when adjusted for further cardiovascular risk factors such as smoking and HDL-cholesterol which were different between patients with dilated versus non-dilated LV. As a direct causal relationship between both minimal and mean FCT on the one hand and LV-dilatation on the other hand seems unlikely, it is tempting to speculate that FCT and LV-dilatation may both be caused by a third pathophysiological entity. This may include microruptures and disturbances in microcirculation. Further candidates which are known to influence both LV-geometry and atherosclerosis include alterations in metabolism, adipokines and cytokines -28. FutuTo the best of our knowledge we are first to describe an association between plaque vulnerability as determined by OCT and left ventricular dilatation in patients with type 2 diabetes. However, the correlation between LV geometric parameters and atherosclerosis and the incidence of acute cardiovascular events is not a new concept. Specifically, Chahal et al. have demonstrated in 2279 patients without clinical cardiovascular disease that subclinical carotid plaque disease was related to LV systolic function and LV filling pressure, whereas no significant differences in LV systolic and diastolic volumes were observed between patients with compared to those without carotid disease . Madaj aWhereas stress-CMR reveals ischemia due to functionally relevant coronary stenosis , its dia2. It needs to be determined in future studies if our findings can be confirmed when echocardiography instead of CMR is used to assess LV-dilatation.Echocardiography has the advantage of being widely available as well as time- and cost-effective. Moreover, it is the most commonly used imaging technique for the assessment of LV-dilatation. However, CMR for the assessment of LV-dilatation may be superior to echocardiography due to its higher resolution and exacter assessments particularly in overweight and obese patients with type 2 diabetes which is reflected in our own study population with a mean BMI of 29.70 kg/mThe present investigation is limited by a small sample size and in 15 patients the determination of a FCT was not possible due to the absence of a lipid-rich plaque. Therefore, for confirmation of our data a large-scale study is required. The sample size may be a limitation particularly for the presented multivariable regression analysis.Second, patients with reduced kidney function were not included in the study due to ethical issues regarding additional contrast medium necessary for the OCT-investigation. However, a high percentage of those patients with reduced LV-function also have reduced kidney function and our findings may be biased by this fact.Third, although the relationship between LV-geometry and plaque vulnerability is particularly important in cardiovascular high-risk patients with type 2 diabetes, future studies are necessary to determine if the findings of this study can also be translated to patients without type 2 diabetes.Furthermore, our findings are limited by the fact that in this study OCT was only used in the target vessel due to ethical issues in order to minimize complications associated with the use of OCT.Moreover, we used the minimal FCT-threshold of 80 μm for defining plaque vulnerability. This value is above the currently accepted consensus of 65 μm that was based on post-mortem studies, where tissue-shrinkage may be a relevant issue . TherefoWe conclude that in patients with type 2 diabetes and stable coronary artery disease, minimal and mean FCT are associated with LV-dilatation independent of previous MIs. Furthermore, LV-dilatation as determined by CMR may be a useful adjunct to identify patients at risk for vulnerable coronary artery lesions. Future studies are warranted to investigate potential mechanisms linking plaque vulnerability and LV-dilatation in patients with type 2 diabetes.LV: Left ventricle; MI: Myocardial infarction; OCT: Optical coherence tomography; CMR: Cardiac magnetic resonance imaging; FCT: Fibrous cap thickness; LVEDV: Left ventricular-end diastolic volume; IVUS: Intravascular ultrasound; LVEF: Left ventricular ejection fraction; LVEDD: Left ventricular-end diastolic diameter; LVESV: Left ventricular-end systolic volume; OR: Odds ratio; ROC: Receiver-operated curve; AUC: Area under the curve.The authors declare that there are no competing interests.All authors were involved in reporting the results of this study and all approved the final version of the submitted manuscript. SR, NM and MB contributed in the conception, design and planning of the study. SR, MF, SB, AL, AJ, MH, WL, NM and MB conducted the study and were involved in the collection, analysis and interpretation of the data. MH, WL, SR and MB did the statistical analysis. Manuscript writing: SR and MB. SR is responsible for the overall content and serves as guarantor. All authors read and approved the final manuscript.


It has been reported that coffee acutely induces FMD changes. In particular, the brachial artery FMD seems to decrease after caffeinated coffee (CC) and to increase after decaffeinated coffee (DC) ingestion. Since the cardiovascular effects of coffee are still a debated matter, this study aimed at investigating with a randomized, double-blind crossover design, if the QT interval of adult healthy subjects  changes in the hour following CC or DC ingestion. Both systolic and diastolic blood pressure were higher in the hour following the ingestion of CC; the heart rate significantly increased 30 minutes after CC ingestion. A significant increase of the QT duration was observed one hour after DC ingestion , not after CC. The QT interval corrected for heart rate did not significantly change following CC or DC ingestion. In conclusion, despite CC and DC previously demonstrated to influence the FMD they do not seem to induce a significant unfavourable acute change of the left ventricular repolarization. Further investigations are required to elucidate the effects of coffee in subjects with cardiovascular diseases.The coronary endothelial function is recognized to have an important role in the physiology of the diastolic ventricular relaxation, a phase of the heart cycle that influences the electrocardiographic QT interval. Endothelial function is investigated  Many controversies exist about the cardiovascular effects of coffee in man. We recently demonstrated  that end2) between 17.3-28.0 kg/m2 were included in the study. Exclusion criteria were any dyslipidemia, hypertension, diabetes, cardiovascular or systemic disease, any medication treatment, smoking of any tobacco products, pregnancy or lactation in the past 6 months, habitual daily consumption of greater than two cups of coffee or weekly ingestion of more than one commercial caffeinated beverage and abstaining from chocolate or other flavonoid-containing beverages up to the preceding day. The study followed a randomized, crossover, double-blind design with each subject receiving two different study treatments, in random order, and repeated on separate days at 5- to 7-day intervals. The preparation of either CC and DC has already been described elsewhere [The study design has been previously described elsewhere . The stuAll data are presented as means ± standard error of the means. Basal pairwise comparisons between the two treatments (CC vs DC) were tested for statistical significance using the paired Student's t-test. An overall 3 × 2 ANOVA  for repeated measures was performed to evaluate the composite effect of the two different (CC and DC) ingested coffees over time  on the parameters of interest. ANOVA for repeated measures was also carried out separately to detect significant changes in variables over time within the two sessions; Bonferroni's t-test was performed for individual differences between two time points (paired) when appropriate. A two-tailed P < 0.05 was considered significant. All analyses were performed using Systat .Both systolic and diastolic blood pressure were higher in the hour following the ingestion of CC with a time × treatment effect Table . This reWe conclude that despite CC acutely increases systolic and diastolic blood pressure as well as heart rate, both caffeinated and decaffeinated coffee do not acutely induce any significant change in the QTc interval duration in healthy adult subjects.BMI: body mass index; CC: caffeinated coffee; DC: decaffeinated coffee; ECG: electrocardiogram; FMD: flow-mediated dilation; QTc: QT interval corrected for heart rate;The authors declare that they have no competing interests.SB was the main author of the manuscript and contributed to the design of the study, preparation of protocols, statistical analyses, interpretation of data and preparation of the manuscript. AM carried out all the experiments, contributed to the interpretation of data and preparation of the manuscript. MRT carried out all the experiments, contributed to the preparation of the manuscript. SV contributed to interpretation of data and preparation of the manuscript. All authors read and approved the final manuscript.


Patients with non-ST-segment elevation acute coronary syndrome (NSTE-ACS) have varying degrees of salvageable myocardium at risk of irreversible injury. We hypothesized that a novel model of NSTE-ACS produces acute myocardial injury, measured by increased T2 cardiovascular magnetic resonance (CMR), without significant necrosis by late gadolinium enhancement (LGE).In a canine model, partial coronary stenosis was created and electrodes placed on the epicardium. Myocardial T2, an indicator of at-risk myocardium, was measured pre- and post-tachycardic pacing.2 = 0.83, p = 0.004) and changes in CaMKIID and mitochondrial gene expression.Serum troponin-I (TnI) was not detectable in unoperated sham animals but averaged 1.97 ± 0.72 ng/mL in model animals. Coronary stenosis and pacing produced significantly higher T2 in the affected vs. the remote myocardium  with no evident injury by LGE. Microscopy revealed no significant irreversible cellular injury. Relative respiration rate (RRR) of affected vs. remote myocardial tissue was significantly lower in model vs. sham animals . Lower RRR corresponded to higher final TnI levels (RA large animal NSTE-ACS model with mild TnI elevation and without ST elevation, similar to the human syndrome, demonstrates signs of acute myocardial injury by T2-CMR without significant irreversible damage. Reduced tissue respiration and associated adaptations of critical metabolic pathways correspond to increased myocardial injury by serum biomarkers in this model. T2-CMR as a biomarker of at-risk but salvageable myocardium warrants further consideration in preclinical and clinical studies of NSTE-ACS. Non ST-segment elevation acute coronary syndrome (NSTE-ACS) – a subset of acute coronary syndrome (ACS) that includes non ST-segment elevation myocardial infarction (NSTEMI) and unstable angina (UA) – is responsible for nearly 1 million emergency department presentations each year in the United States alone . While pex vivo tissue analyses, despite being at risk for subsequent irreversible damage, as reflected by the induction of cardioprotective metabolic adaptations characteristic of ischemia-induced myocardial hibernation [While serum biomarkers of myocardial necrosis reliably indicate that damage has occurred, they cannot identify myocardium that is at-risk but still salvageable. Cardiovascular magnetic resonance (CMR) with myocardial T2 imaging has shown value as a noninvasive imaging biomarker of at-risk myocardium, delineating myocardium at-risk in both a large animal model of STEMI  as well ernation ,9.Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Thirteen heartworm-free, random-source, mixed-breed dogs  were anesthetized using ketamine  and inhaled isoflurane (1-2% in 95% O2) and remained anesthetized throughout all experimental procedures. In 2 randomized dogs, no surgical operations were performed (unoperated shams). ECG was monitored via surface electrodes. After left thoracotomy in the remaining 11 dogs, a catheter was placed in the left atrial appendage for microsphere injection. In a random selection of 4 dogs, no further operations were performed prior to closing of the chest wall (shams). In the 7 model dogs, an 18-gauge needle was laid flat on the left circumflex coronary artery (LCx) then removed once a suture was secured around the artery and needle, leaving a lumen constricted to the nominal outer diameter of the needle (~1.27 mm). Copper pacing wires were then threaded through the epicardium of the right ventricle and connected to an external electrical stimulator for later myocardial pacing. Standard chest wall closing procedures and post-operative anesthesia and analgesia protocols were then followed [An overview of the study protocol is shown in Figure followed .Serum isolated from peripheral blood drawn at 3 time points  was examined for troponin I (TnI) content using a high-sensitivity assay according to the manufacturer’s recommendations .Based on the anatomical perfusion territory of the LCx and preliminary results from pilot studies, the lateral LV myocardium at the level of the posterior papillary muscle was denoted as the affected region while a section of the anterior LV wall near the anterior papillary muscle was designated as the remote region. At the conclusion of each imaging experiment, the animal was euthanized and the heart immediately harvested. Myocardium from affected and remote regions was excised using the papillary muscles as anatomical landmarks. Tissue samples from each myocardial region were obtained for immediate tissue respiration, wet-to-dry weight and tissue necrosis analyses on fresh tissue. Additional samples were acquired and either fixed or frozen (-80°C) for later light and electron microscopy or gene expression analyses, respectively, as discussed below and in the provided online supplement . Identical imaging protocols were used in both groups of animals, NSTE-ACS model and sham. In the NSTE-ACS model dogs, scans were performed immediately after 15 minutes of epicardial pacing at 180-210 beats/min . We acquired multiplane cines to measure LV systolic function, T2 maps to identify areas of acute injury, first-pass perfusion imaging of microcirculatory myocardial blood flow, and LGE images to visualize areas of necrosis. Further detailed information is provided in Additional file Two investigators analyzed all images by consensus review (HC and TT). For analysis of T2 maps, myocardial regions of interest (ROIs) were drawn encompassing the affected region and remote region for each dog , and thTo verify the changes in coronary blood flow due to stenosis and pacing, we injected fluorescent microspheres  into the left atrium of 5 animals (2 NSTE-ACS model and 3 sham) at three time points, each with a different fluorescent label: 1) prior to surgical stenosis of the LCx (gold), after stenosis and pacing (samarium), and just prior to euthanasia (lutetium). The disintegrations per minute (dpm) of each label was quantified  in tissue sections (~1 gram) obtained from both the affected and remote regions. After calibration, the dpm of each label was converted to flow in the affected region relative to the flow in the remote region at each of the three time points.Examinations were made on myocardial tissue sections from both the affected and remote regions in all animals to determine the presence of tissue necrosis and edema using established 2,3,5-triphenyltetrazolium chloride (TTC) staining and wet/dry weight ratio measurement, respectively, as described further in Additional file Tissue sections harvested from the remote and affected myocardial regions were routinely processed and stained  further. Tissue 2 consumption by myocardial tissue (~0.2-0.3 grams) obtained from both the affected and remote regions was measured in all animals employing standard techniques as further described in Additional file To evaluate the effect of ischemia upon cellular respiration in our NSTE-ACS model, Oβ-actin, and then compared within the model group.Frozen harvested tissue samples from both the affected and remote myocardial regions obtained from each animal were processed according to standard techniques to isolate high quality total RNA, as detailed in Additional file In vivo and ex vivo measurements for affected myocardial tissue respiration in model vs. sham groups were compared by use of unpaired t-tests. Delta T2 (affected minus remote) was also compared between the two groups using unpaired t-tests. For serial microsphere and TnI data, a two-way analysis of variance (ANOVA) test was performed to examine the individual contributions of animal group and sampling time, as well as their interaction effects. The T2 data was also analyzed in this way using animal group and region (remote and affected) as the main effects. Comparisons of selective gene expression between the affected and remote regions within the model group were made using the Wilcoxon Rank-Sum test. Statistical significance was based upon a value of p < 0.05.Results are reported as mean ± standard deviation (SD). p < 0.01) , animal group (p < 0.001), and time-group interaction (p < 0.001).Of the seven dogs in the NSTE-ACS model group, five survived the entire experiment. Two expired due to ventricular fibrillation – one during the initial surgery and the other during pacing. No pre-euthanasia deaths occurred in the sham group. No coronary artery anomalies were identified with the left circumflex artery observed to supply the lateral LV wall in all operated animals. TnI was undetectable on serial measurement in the serum of the 2 unoperated control animals, while thoracotomy and cardiac instrumentation presumably produced the borderline TnI elevation  in operated sham animals. The NSTE-ACS model resulted in final TnI levels of 1.97 ± 0.72 ng/mL, which was significantly higher than the operated sham animals (p = 0.02) and the averaged final TnI levels across all sham animals . Myocardial T2 was increased in the LCx-territory myocardium relative to remote myocardium in NSTE-ACS model dogs after pacing , animal group (p = 0.02), and region-group interaction (p = 0.03). Perfusion and LGE images showed no first-pass defect, microvascular obstruction or enhancement , animal group (p < 0.001), and time-group interaction (p < 0.001). In six sham animals, the myocardial respiration rate from the affected and remote regions was 0.44 ± 0.10 nmol O2/mg/min and 0.43 ± 0.08 nmol O2/mg/min, respectively, yielding an average respiration rate ratio of 1.04 ± 0.07. In four NSTE-ACS model dogs, the myocardial respiration rate from the affected and remote regions was 0.44 ± 0.07 nmol O2/mg/min and 0.61 ± 0.05 nmol O2/mg/min, respectively, resulting in a significantly lower respiration rate ratio of 0.72 ± 0.07 when compared to the sham group  a canine model based upon reduced myocardial oxygen supply (coronary stenosis) and increased myocardial oxygen demand (tachycardic pacing) mimics key features of clinical NSTE-ACS and 2) increased myocardial T2 conforms to an early form of myocardial damage that is associated with viable myocardium, as indicated by the absence of late gadolinium enhancement on CMR and other signs of irreversible myocardial damage. These modeling . Indeed,NSTE-ACS encompasses a wide range of pathophysiologies, but a clear distinction from STEMI is its relative lack of complete/sustained coronary artery occlusion . Withoutin vivo findings with ex vivo ultrastructural and functional assays. Using CMR techniques that readily translate to clinical application, we demonstrated myocardial T2 increase without significant myocardial necrosis by LGE. This paralleled mild TnI elevation but without ST elevation, plus ultrastructural and genetic changes that together corresponded to altered yet potentially viable myocardium. Our goal was not to produce a fixed amount of injury but rather to produce ischemic injury consistently without significant necrosis. The microsphere data demonstrated that reliable reductions in blood flow were achieved in the affected myocardium vs. the remote region in this dog model, despite the known presence of highly variable coronary artery collaterals in canines [In order to understand NSTE-ACS fully, we first addressed the need for an adequate animal model to enable studies corroborating  canines .CaMKIID and associated increases in mitochondrial biogenesis, consistent with myocardial adaptation to acute ischemia [The translational value of these findings is evident when considering that the protocol can be readily implemented not only in prospective patient studies but also in preclinical studies aimed at better understanding the mechanisms that alter cellular and organ function. Our demonstration of altered expression of ischemia ,16, is aischemia ,22. As sIn finding a smaller but consistently positive difference between T2 in the lateral vs. anterior LV myocardium in sham animals, we further demonstrated physiological heterogeneity in T2 and mitochondrial size that may reflect previously-described regional variation in myocardial oxygen consumption . NotablyA common explanation for increased T2 is the presence of myocardial edema. Other animal studies have found increased water content in ischemic myocardium correlated to the degree of stunning . Howeverdata not shown) remained intact in the affected tissue during NSTE-ACS, the observed suppression of oxygen consumption likely reflects the shift from aerobic to glycolytic ATP production that characterizes myocardial stunning in the context of ischemia, which is regulated by HIF-1α and CaMKII in response to changes in tissue oxygen [CaMKIID expression and lack of HIF-1α response suggests that changes in intracellular calcium flux may contribute to the observed metabolic adaptations in the ischemic tissue. Moreover, high-level evidence indicates that sustained CaMKII activation can lead to myocardial remodeling and dysfunction [One potential mechanism involves mitochondria. Tissue aerobic respiration in the model group was significantly lower than in the sham dogs, indicating reduced mitochondrial oxygen consumption in the affected myocardium. Considering that mitochondrial ultrastructure and ADP-dependent respiratory capacity  in term hypoxia ,37. It iIn a large animal model based on demand myocardial ischemia seen in the predominant form of NSTE-ACS in humans, myocardial T2 increases with corresponding, presumably adaptive, reduction in myocardial tissue respiratory function and preserved ultra structural findings indicating myocardial viability. The observed changes in oxygen metabolism and mitochondrial biogenesis, hallmarks of myocardial hibernation, suggest that antecedent T2 elevation without LGE identifies myocardium at risk of subsequent irreversible injury. Additional studies using this model to examine long-term changes and the impact of therapeutic maneuvers to restore normal T2 and mitochondrial function may further elucidate the syndrome of NSTE-ACS and guide management changes to improve outcomes.ECG: Electrocardiography; ACS: Acute coronary syndrome; STEMI: ST-segment elevation myocardial infarction; NSTE-ACS: Non ST-segment elevation acute coronary syndrome; CMR: Cardiovascular magnetic resonance; LGE: Late gadolinium enhancement; TnI: Troponin I; ANOVA: Analysis of variance; H&E: Hematoxylin and eosin; TTC: Triphenyltetrazolium chloride.SVR and OPS receive research support from Siemens, who neither had input nor participation in any aspect of this work.SVR and EDC were co-principal investigators of the study, coordinating all aspects of study design, study implementation, data analysis, and manuscript preparation. HC had primary responsibility for execution of all experimental procedures, data analysis, and manuscript preparation. GEB, the primary surgeon for this project, and RLH brought a wealth of veterinary experience and were closely involved with model development. EDC and MWJ performed the tissue analyses, including cell respiration assays, microscopy preparation, and TTC staining. GS performed PCR assays and data analysis and PBB aided with microscopy analysis. OPS contributed to MRI experiments, study design and data interpretation. TT assisted with image acquisition and blood analysis. GA was intimately involved with model development throughout and contributed to manuscript preparation. All authors read and approved the final manuscript.Elliott D Crouser and Subha V Raman are co-senior authors.Additional methods and results for CMR and tissue assays. Figure S1. Representative left ventricular myocardial tissue slices, post-TTC staining, demonstrate no evidence of necrosis. For each one, both sides of the slice are shown (left-right). The top three pairs  correspond to the affected regions  in 3 animals from the NSTE-ACS model group. Pair G-H corresponds to the affected region in a representative sham animal. Pair I-J corresponds to the remote region in a representative animal from the NSTE-ACS model group (the same one presented in pair A-B). Evidence of necrosis is absent in all slices, particularly within the affected regions. Table S1. CMR Scan Parameters. Table S2. Canine Primer Sequences Used for Real-time PCR. Table S3. Tissue Percent Water Content in the Myocardial Affected Region Alone and Normalized to That in the Corresponding Myocardial Remote Region.Click here for file


Results were validated via high-performance liquid chromatography. The Tau levels in the ischemic myocardial tissue were reduced significantly within 5 min compared with those in the control group  and were maintained for 6 h post-ischemia. Tau levels declined more markedly (56.5%) than creatine levels (48.5%) at 5 min after ligation. This suggests that Tau may have potential as an indicator in the early detection of myocardial ischemia by 1H MRS.Magnetic resonance spectroscopy (MRS) is a unique non-invasive method for detecting cardiac metabolic changes. However, MRS in cardiac diagnosis is limited due to insensitivity and low efficiency. Taurine (Tau) is the most abundant free amino acid in the myocardium. We hypothesized that Tau levels may indicate myocardial ischemia and early infarction. Sprague-Dawley rats were divided into seven groups according to different time points during the course of myocardial ischemia, which was induced by left anterior descending coronary artery ligation. Infarcted myocardial tissue was obtained for high-resolution magic angle spinning  Cardiovascular diseases (CVD) are a significant cause of mortality worldwide and the main cause of CVD is acute myocardial infarction (AMI). In the United States, acute coronary syndrome (ACS) affects 1.3 million individuals . In addiIschemia and myocardial infarction are different stages in the progression of heart failure. Identifying the early changes of various metabolic parameters in myocardial ischemia is of clinical importance . The myo1H MRS have focused on its sensitivity and specificity in the study of myocardial ischemia (Magnetic resonance spectroscopy (MRS) is a unique non-invasive method for detecting cardiac metabolic changes, which usually occur significantly earlier than irreversible organic lesions in myocardial infarction . This meischemia ,8.31P-MRS is commonly used to study myocardial energy metabolism /ATP and PCr/inorganic phosphate (Pi) ratios, in humans to guide heart transplantation and myocardial infarction non-invasively  has been used to evaluate heart tissue ex vivo to produce a high-resolution metabolic profile in myocardial infarction animal models. However, cardiac motion, respiratory motion and epicardial fat make it difficult to apply 1H NMR clinically  t models –17. Multt models ,19.μmol/g wet weight (Taurine (Tau), a sulfur-containing β-amino acid, is the most abundant free amino acid in the myocardium (∼60%) . The Taut weight . It has t weight ,20–22. Ct weight . This sut weight ,25. We hex vivo using HRMAS 1H NMR. The results were further confirmed by high performance liquid chromatography (HPLC).In the present study, a myocardial ischemia model was created in rats. Metabolic markers, including Tau, creatine (Cre), choline (Cho) and lactate (Lac), were analyzed at various time points 2.The animals were provided by the Animal Center of Fudan University . All procedures were performed with approval from the Animal Care and Use Committee of Shanghai, China. Adult Sprague-Dawley (SD) rats  were anesthetized with pentobarbital sodium . The left anterior descending coronary artery (LAD) was ligated with a 6-0 suture  as previously reported . Sham-op1H NMR analysis, the tissue samples , weighing 25±2 mg each, were placed into a 25 μl zirconium oxide rotor with drops of D2O (deuterium lock reference). NMR tests were performed on a Bruker DRX-500  spectrometer (1H frequency at 500.13 MHz) at 300.0 K, with a rotor spin rate of 5 kHz. Carr Purcell Meiboom Gill (CPMG) pulse sequences were used with solvent presaturation during the relaxation delay of 2 sec. The NMR spectra were acquired with 256 scans collected into 64,000 data points with a spectral width of 15 kHz. The CPMG pulse sequence was applied to suppress signals from the molecules with short T2 values, such as macromolecules and lipids, using a total echo time (TE) of 320 msec. The stability of tissue samples was evaluated by repeating a one-dimensional NMR experiment following overall acquisition. No biochemical degradation was observed in any of the tissue samples. Spectral assignments were further confirmed by two-dimensional 1H-1H total correlation spectroscopy (TOCSY)and 1H-1H correlation spectroscopy  with values obtained from the literature  over the region δ0.00–10.00 and the signal intensity in each region was integrated by AMIX . The region δ4.40–5.00 was removed to eliminate the baseline effects of imperfect water saturation. Prior to PCA, each integral region was normalized by dividing by the sum of all integral regions for each spectrum . All free induction decays (FIDs) were multiplied by an exponential function equivalent to a 0.3-Hz line-broadening factor prior to Fourier transformation. Each HRMAS spectrum ,30. PCA μl/100 mg). The mixture was centrifuged at 13,000 rpm for 5 min. The supernatant was then taken and filtered through a 0.22 μm membrane. O-phthalaldehyde (OPA) precolumn derivatization was performed as previously described (μm (Agilent); mobile phase A, methanol:acetonitrile:H2O = 45:45:10 (v/v/v); mobile phase B1, methanol (0.05 mol/l):sodium acetate buffer (pH 5.3):tetrahydrofuran = 42:57:1; mobile phase B2, 40 mM phosphate buffer . Fluorescence detection was performed at 450 nm. L-norvaline  was added as the internal standard.The ischemic myocardial tissue (250 mg) was homogenized by ultrasound  and mixed with 18% sulfosalicylic acid . All values were expressed as the mean ± standard deviation (SD). Differences between groups were evaluated using a one-factor ANOVA test and P<0.05 was considered to indicate a statistically significant difference.TTC and Evans Blue double staining were performed on excised hearts obtained from the 6 h group. The infarct area (TTC unstained), risk area (Evans Blue unstained) and non-ischemic area (Evans Blue stained) are shown in 1H CPMG NMR spectra of the control, 5 min, 20 min, 30 min, 45 min, 1 h and 6 h groups following LAD ligation are shown in Representative 1H CPMG NMR spectra were analyzed and metabolites were assigned. The levels of potential markers, including Tau, Cre and Cho, are shown in The data acquired from the 1H NMR spectra results  is used as the golden standard in the clinical diagnosis of myocardial infarction. Troponins T and I have distinct isoforms that exist in skeletal and cardiac muscle. The release of these proteins from necrotic cardiomyocytes into the bloodstream accounts for their utility as biomarkers of acute coronary syndromes . Howeverin vivo(31P-MRS, PCr and ATP represent the level of myocardial energy. A reduced PCr/ATP ratio indicates cardiac failure and is a predictor of mortality. A decrease in total Cre is an early signal of heart failure (It has previously been shown that Cre levels, belonging to the myocardial energy index, are related to myocardial functional performance under ischemic conditions in animal hearts in vivo. In 31P- failure . Total C failure . In acut failure , energy Clinically, myocardial ischemia may also be detected by MRI . However1H NMR, it was observed that Tau levels decreased significantly within 5 min after ischemia in myocardial tissue. These results suggest that the Tau level is a potential indicator of early myocardial ischemia in cardiovascular magnetic resonance studies.In conclusion, by detecting metabolite changes using HRMAS