Stem cell-derived exosome patch with coronary artery bypass graft restores cardiac function in chronically ischemic porcine myocardium.

OBJECTIVE: This study aimed to investigate whether or not the application of a stem cell-derived exosome-laden collagen patch (EXP) during coronary artery bypass grafting (CABG) can recover cardiac function by modulating mitochondrial bioenergetics and myocardial inflammation in hibernating myocardium (HIB), which is defined as myocardium with reduced blood flow and function that retains viability and variable contractile reserve. METHODS: In vitro methods involved exposing H9C2 cardiomyocytes to hypoxia followed by normoxic coculture with porcine mesenchymal stem cells. Mitochondrial respiration was measured using Seahorse assay. GW4869, an exosomal release antagonist, was used to determine the effect of mesenchymal stem cells-derived exosomal signaling on cardiomyocyte recovery. Total exosomal RNA was isolated and differential micro RNA expression determined by sequencing. In vivo studies comprised 48 Yorkshire-Landrace juvenile swine (6 normal controls, 17 HIB, 19 CABG, and 6 CABG + EXP), which were compared for physiologic and metabolic changes. HIB was created by placing a constrictor on the proximal left anterior descending artery, causing significant stenosis but preserved viability by 12 weeks. CABG was performed with or without mesenchymal stem cells-derived EXP application and animals recovered for 4 weeks. Before terminal procedure, cardiac magnetic resonance imaging at rest, and with low-dose dobutamine, assessed diastolic relaxation, systolic function, graft patency, and myocardial viability. Tissue studies of inflammation, fibrosis, and mitochondrial morphology were performed posttermination. RESULTS: In vitro data demonstrated improved cardiomyocyte mitochondrial respiration upon coculture with MSCs that was blunted when adding the exosomal antagonist GW4869. RNA sequencing identified 8 differentially expressed micro RNAs in normoxia vs hypoxia-induced exosomes that may modulate the expression of key mitochondrial (peroxisome proliferator-activator receptor gamma coactivator 1-alpha and adenosine triphosphate synthase) and inflammatory mediators (nuclear factor kappa-light-chain enhancer of activated B cells, interferon gamma, and interleukin 1ß). In vivo animal magnetic resonance imaging studies demonstrated regional systolic function and diastolic relaxation to be improved with CABG + EXP compared with HIB (P = .02 and P = .02, respectively). Histologic analysis showed increased interstitial fibrosis and inflammation in HIB compared with CABG + EXP. Electron microscopy demonstrated increased mitochondrial area, perimeter, and aspect ratio in CABG + EXP compared with HIB or CABG alone (P < .0001). CONCLUSIONS: Exosomes recovered cardiomyocyte mitochondrial respiration and reduced myocardial inflammation through paracrine signaling, resulting in improved cardiac function.

NHLBI-Sponsored Randomized Trial of Postconditioning During Primary Percutaneous Coronary Intervention for ST-Elevation Myocardial Infarction.

RATIONALE: Postconditioning at the time of primary percutaneous coronary intervention (PCI) for ST-segment-elevation myocardial infarction may reduce infarct size and improve myocardial salvage. However, clinical trials have shown inconsistent benefit. OBJECTIVE: We performed the first National Heart, Lung, and Blood Institute-sponsored trial of postconditioning in the United States using strict enrollment criteria to optimize the early benefits of postconditioning and assess its long-term effects on left ventricular (LV) function. METHODS AND RESULTS: We randomized 122 ST-segment-elevation myocardial infarction patients to postconditioning (4, 30 seconds PTCA [percutaneous transluminal coronary angioplasty] inflations/deflations)+PCI (n=65) versus routine PCI (n=57). All subjects had an occluded major epicardial artery (thrombolysis in myocardial infarction=0) with ischemic times between 1 and 6 hours with no evidence of preinfarction angina or collateral blood flow. Cardiac magnetic resonance imaging measured at 2 days post-PCI showed no difference between the postconditioning group and control in regards to infarct size (22.5±14.5 versus 24.0±18.5 g), myocardial salvage index (30.3±15.6% versus 31.5±23.6%), or mean LV ejection fraction. Magnetic resonance imaging at 12 months showed a significant recovery of LV ejection fraction in both groups (61.0±11.4% and 61.4±9.1%; P<0.01). Subjects randomized to postconditioning experienced more favorable remodeling over 1 year (LV end-diastolic volume =157±34 to 150±38 mL) compared with the control group (157±40 to 165±45 mL; P<0.03) and reduced microvascular obstruction ( P=0.05) on baseline magnetic resonance imaging and significantly less adverse LV remodeling compared with control subjects with microvascular obstruction ( P<0.05). No significant adverse events were associated with the postconditioning protocol and all patients but one (hemorrhagic stroke) survived through 1 year of follow-up. CONCLUSIONS: We found no early benefit of postconditioning on infarct size, myocardial salvage index, and LV function compared with routine PCI. However, postconditioning was associated with improved LV remodeling at 1 year of follow-up, especially in subjects with microvascular obstruction. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov . Unique identifier: NCT01324453.

Novel imaging of atrial septal defects in isolated human hearts.

Within the adult population living with congenital heart defects, approximately 20 % have an atrial septal defect, which suggests that only 0.4-0.05 % of the entire adult population has an atrial septal defect (ASD). In patients with a left to right atrial shunt, treatments may include closure of the defect with a transcatheter device and/or surgical repair. From the perspective of a physician or engineer, it is vitally important to understand the anatomical nuances of such defects, not only to offer the most optimal treatment for the patient but also to call attention to the potential anatomy of ASDs which may go undetected. To do so, we reanimated two human hearts deemed not viable for transplant from 56-year-old and 68-year-old males. Neither patient history reported any heart conditions that would suggest an atrial defect, yet an ASD was found in each heart. Here, we present sets of images, videos, and 3D reconstructions that provide a clearer view of the anatomy of ASDs in functional human hearts. With an enhanced understanding of 3D functional aspects of ASDs, physicians can make improved decisions regarding treatment options and engineers can optimize device designs.

Noninvasive mapping of transmural potentials during activation in swine hearts from body surface electrocardiograms.

The three-dimensional cardiac electrical imaging (3DCEI) technique was previously developed to estimate the initiation site(s) of cardiac activation and activation sequence from the noninvasively measured body surface potential maps (BSPMs). The aim of this study was to develop and evaluate the capability of 3DCEI in mapping the transmural distribution of extracellular potentials and localizing initiation sites of ventricular activation in an in vivo animal model. A control swine model (n = 10) was employed in this study. The heart-torso volume conductor model and the excitable heart model were constructed based on each animal's preoperative MR images and a priori known physiological knowledge. Body surface potential mapping and intracavitary noncontact mapping (NCM) were simultaneously conducted during acute ventricular pacing. The 3DCEI analysis was then applied on the recorded BSPMs. The estimated initiation sites were compared to the precise pacing sites; as a subset of the mapped transmural potentials by 3DCEI, the electrograms on the left ventricular endocardium were compared to the corresponding output of the NCM system. Over the 16 LV and 48 RV pacing studies, the averaged localization error was 6.1±2.3 mm, and the averaged correlation coefficient between the estimated endocardial electrograms by 3DCEI and from the NCM system was 0.62±0.09. The results demonstrate that the 3DCEI approach can well localize the sites of initiation of ectopic beats and can obtain physiologically reasonable transmural potentials in an in vivo setting during focal ectopic beats. This study suggests the feasibility of tomographic mapping of 3D ventricular electrograms from the body surface recordings.

Ex vivo diffusion tensor MRI of human hearts: relative effects of specimen decomposition.

Recently, diffusion tensor MRI has been utilized as a tool to reconstruct, with high resolution, the three-dimensional myofiber orientation of hearts ex vivo. However, because of limited availability of human hearts, it is common to recover specimens postmortem, after some degree of decomposition has occurred. In this study, we describe the ability to reconstruct local fiber orientation within a unique set of human hearts using diffusion tensor MRI; relative effects of specimen decomposition were also assessed. The mean diffusivity, fractional anisotropy, and principal eigenvalues (E1, E2, and E3) were measured in hearts with varying postmortem intervals from 0 to 27 days (n = 17), and local fiber orientations were reconstructed. Mean diffusivities and principal eigenvalues nonlinearly increased as a function of postmortem interval, while fractional anisotropies nonlinearly decreased. There were good correlations between postmortem intervals and changes in diffusion properties after linear transformation of the data (R(2) > 0.7). Thus, we determined that there were regional discontinuities in the reconstruction of local fiber orientation in hearts where postmortem intervals were greater than 3 days. Therefore, any delay in tissue fixation after death (if not properly preserved using organ transplantation techniques) will compromise measurement of fiber orientation and delineation of diseased pathology, e.g., based, in part, on fractional anisotropy.

MRI assessment of pacing induced ventricular dyssynchrony in an isolated human heart.

This study demonstrates the capabilities of MRI in the assessment of cardiac pacing induced ventricular dyssynchrony, and the findings support the need for employing more physiological pacing. A human donor heart deemed non-viable for transplantation, was reanimated using an MR compatible, four-chamber working perfusion system. The heart was imaged using a 1.5T MR scanner while being paced from the right ventricular apex (RVA) via an epicardial placed lead. Four-chamber, short-axis, and tagged short-axis cines were acquired in order to track wall motion and intramyocardial strain during pacing. The results of this study revealed that the activation patterns of the left ventricle (LV) during RVA pacing demonstrated intraventricular dyssynchrony; as the left ventricular mechanical activation proceeded from the septum and anterior wall to the lateral wall, with the posterior wall being activated last. As such, the time difference to peak contraction between the septum and lateral wall was approximately 125 msec. Likewise, interventricular dyssynchrony was demonstrated from the four-chamber cine as the time difference between the peak LV and RV free wall motion was 180 msec. With the ongoing development of MR safe and MR compatible pacing systems, we can expect MRI to be added to the list of imaging modalities used to optimize cardiac resynchronization therapy (CRT) and/or alternate site pacing.

Intermittent antegrade cardioplegia: isolated heart preservation with the Asporto heart preservation device.

BACKGROUND: A major problem in procurement of donor hearts is the limited time a donor heart remains viable. After cardiectomy, ischemic hypoxia is the main cause of donor heart degradation. The global myocardial ischemia causes a cascade of oxygen radical formation that cumulates in an elevation in hydrogen ions (decrease in pH), irreversible cellular injury, and potential microvascular changes in perfusion. OBJECTIVE: To determine the changes of prolonged storage times on donor heart microvasculature and the effects of intermittent antegrade perfusion. MATERIALS AND METHODS: Using porcine hearts flushed with a Ribosol-based cardioplegic solution, we examined how storage time affects microvascular myocardial perfusion by using contrast-enhanced magnetic resonance imaging at a mean (SD) of 6.1 (0.6) hours (n = 13) or 15.6 (0.6) hours (n = 11) after cardiectomy. Finally, to determine if administration of cardioplegic solution affects pH and microvascular perfusion, isolated hearts (group 1, n = 9) given a single antegrade dose, were compared with hearts (group 2, n = 8) given intermittent antegrade cardioplegia (150 mL, every 30 min, 150 mL/min) by a heart preservation device. Khuri pH probes in left and right ventricular tissue continuously measured hydrogen ion levels, and perfusion intensity on magnetic resonance images was plotted against time. RESULTS: Myocardial perfusion measured via magnetic resonance imaging at 6.1 hours was significantly greater than at 15.6 hours (67% vs 30%, P = .00008). In group 1 hearts, the mean (SD) for pH at the end of 6 hours decreased to 6.2 (0.2). In group 2, hearts that received intermittent antegrade cardioplegia, pH at the end of 6 hours was higher at 6.7 (0.3) (P = .0005). Magnetic resonance imaging showed no significant differences between the 2 groups in contrast enhancement (group 1, 62%; group 2, 40%) or in the wet/dry weight ratio. CONCLUSION: Intermittent perfusion maintains a significantly higher myocardial pH than does a conventional single antegrade dose. This difference may translate into an improved quality of donor hearts procured for transplantation, allowing longer distance procurement, tissue matching, improved outcomes for transplant recipients, and ideally a decrease in transplant-related costs.

Estimation of global ventricular activation sequences by noninvasive three-dimensional electrical imaging: validation studies in a Swine model during pacing.

BACKGROUND: A novel noninvasive imaging technique, the heart-model-based three-dimensional cardiac electrical imaging (3DCEI) approach was previously developed and validated to estimate the initiation site (IS) of cardiac activity and the activation sequence (AS) from body surface potential maps (BSPMs) in a rabbit model. The aim of the present study was to validate the 3DCEI in an intact large mammalian model (swine) during acute ventricular pacing. METHODS AND RESULTS: The heart-torso geometries were constructed from preoperative magnetic resonance (MR) images acquired from each animal. Body surface potential mapping and intracavitary noncontact mapping (NCM) were performed simultaneously during pacing from both right ventricular (RV) (intramural) and left ventricular (LV) sites (endocardial). Subsequent 3DCEI analyses were performed from the measured BSPMs. The estimated ISs were compared with the precise pacing locations and estimated ASs were compared with those recorded by the NCM system. In total, five RV and five LV sites from control and heart failure (HF) animals were paced and sequences of 100 paced beats were analyzed (10 for each site). The averaged localization error (LE) of the RV and LV sites were 7.3 +/- 1.8 mm (n = 50) and 7.0 +/- 2.2 mm (n = 50), respectively. The global 3D ASs throughout the ventricular myocardium were also derived. The endocardial ASs as a subset of the estimated 3D ASs were consistent with those reconstructed from the NCM system. CONCLUSION: The present experimental results demonstrate that the noninvasive 3DCEI approach can localize the IS and estimate AS with good accuracy in an in vivo setting under control, paced, and/or diseased conditions.

Noninvasive estimation of three-dimensional cardiac electrical activities from body surface potential maps.

A noninvasive three-dimensional (3D) cardiac electrical imaging (3DCEI) approach, which can estimate the location of the initiation site (IS) of activation and the resultant 3D activation sequence (AS) from body surface potential maps (BSPMs), was validated in an intact large mammalian model (swine) during acute ventricular pacing. Body surface potential mapping and intracavitary noncontact mapping (NCM) were performed simultaneously during pacing from both right ventricular (RV) sites (intramural) and left ventricular (LV) sites (endocardial). Subsequent 3DCEI analyses were performed on the measured BSPMs. In total, 5 RV and 5 LV sites from control and heart failure animals were paced. The averaged localization error of the RV and LV sites were 7.0+/-1.1 mm and 6.6+/-1.9 mm, respectively. The endocardial ASs as a subset of the estimated 3D ASs by 3DCEI were consistent with those reconstructed from the NCM system. The present experimental results demonstrate that the noninvasive 3DCEI approach can localize the initiation site and estimate cardiac activation sequence with good accuracy in an in vivo setting, under control, paced and/or diseased conditions.

A comparison of myocardial perfusion and rejection in cardiac transplant patients.

INTRODUCTION: Although histological evaluation of the cardiac tissue is the current gold standard for evaluation of rejection, we hypothesized that cardiac perfusion MRI is a safe non-invasive method that correlates tissue blood flow changes with biopsy proven rejection in the cardiac transplant patient. MATERIALS AND METHODS: In a retrospective study from 1984-2001, 83 patients underwent 135 MR Gd-DTPA imaging studies. In 8 patients (9%), biopsies graded 2 or higher (by ISHLT criteria) provided evidence of rejection. Patients were age and sex matched to 11 non-rejected controls for imaging analysis. Time-signal intensity curves generated for a mid-ventricle LV short axis slice during rest and adenosine stress allowed determination of myocardial blood flow (MBF, ml/min/gm). ROC curve analysis by SPSS allowed estimation of sensitivity and specificity. RESULTS: At rest, there was no difference in MBF between patients with prior rejection vs. those without (1.18 +/- 0.26 vs. 1.16 +/- 0.29). At stress there was a decrease in MBF for patients with prior rejection episodes (3.27 +/- 0.74) compared to no rejection (3.60 +/- 0.72), P = 0.067). The area under the ROC curve was 0.82, with specificity and sensitivity of 75% and 81%, respectively. CONCLUSION: This study suggests that perfusion MR imaging can be used in assessing the cardiac transplant patient for rejection related microvascular changes. The high specificity and sensitivity recorded from the ROC curve illustrates the potential utility of this diagnostic test for future studies.

Evaluation of perfusion and viability in hypothermic non-beating isolated porcine hearts using cardiac MRI.

The donor heart undergoes degradation during hypothermic storage. An assessment of donor heart preservation is typically done with histological or biochemical methods that are not feasible in the clinical setting. We describe a method to study the donor heart using cardiac perfusion MRI that is potentially feasible for clinical use. Standard cardiectomy was performed in the pig model and the hearts were stored in normal saline at 5 degrees C. Imaging was performed by using a rapid gradient-echo sequence (FLASH) with saturation-recovery preparation for T1-weighting in the short axis and horizontal long axis views. Approximately 80 serial images were acquired at a rate of 1/s during administration of 0.006 mmol/ml Gd-DTPA (500 ml, 1 l/min). Signal intensity vs. time curves were generated for each heart and slice imaged and compared to a 0.006 mmol/ml Gd-DTPA reference. H&E stained biopsies of the LV, RV, and septum were also obtained. The mean duration of heart storage (N=10) was 8.8 h (range 4.2-19.2 h). Histologically, no differences were seen in H&E stained biopsies among hearts at different storage times. However cardiac MRI revealed a decrease in perfusion units in each subsequent heart tested after 4.2 h. (R=0.49). Average peak up-slope was used as a surrogate measure for flow capacity through the microvasculature and peak contrast enhancement was used as a measurement of viable microvasculature. The 4 h heart had 83% peak contrast enhancement of the reference standard, as compared to 44% for the 19.2 h heart. The decrease in peak enhancement is directly related to the duration of storage time. No correlation of peak up-slope of the intensity curve to storage time was found. This new application of cardiac MRI in the donor heart is applicable to: (1) assessing marginal hearts, (2) evaluating donor heart preservation techniques, and (3) correlating pre- to post-transplant viability.

Analysis of myocardial perfusion MRI.

Rapid MR imaging (MRI) during the first pass of an injected tracer is used to assess myocardial perfusion with a spatial resolution of 2-3 mm, and to detect any regional impairments of myocardial blood flow (MBF) that may lead to ischemia. The spatial resolution is sufficient to detect flow reductions that are limited to the subendocardial layer. The capacity of the coronary system to increase MBF severalfold in response to vasodilation can be quantified by analysis of the myocardial contrast enhancement. The myocardial perfusion reserve (MPR) is a useful concept for quantifying the vasodilator response. The perfusion reserve can be estimated from the ratio of MBFs during vasodilation and at baseline, in units identical to those used for invasive measurements with labeled microspheres, or from dimensionless flow indices normalized by their value for autoregulated flow at rest. The perfusion reserve can be reduced as a result of a blunted hyperemic response and/or an abnormal resting blood flow. The absolute quantification of MBF removes uncertainties in the evaluation of the vasodilator response, and can be achieved without the use of complex tracer kinetic models; therefore, its application to clinical studies is feasible.

Cardiac MRI in the isolated porcine heart reveals possible etiology of sudden right heart failure following heart transplantation.

Occurrence of immediate post-transplant heart failure in the cardiac transplant recipient is typically attributed to elevated pulmonary vascular resistance, however other etiologies may play a role. At the completion of the transplant, free air, which has collected in the donor heart, is vented via an aortotomy. Free air may rise into the right coronary artery and obstruct reperfusion of the right ventricle. Cardiac perfusion MRI may offer a method of non-invasively determining the presence of air embolus. The objectives in the pilot study were to identify steps in the donor process where free air could enter into the aortic root causing obstruction of perfusion of the coronary arteries. A change in surgical technique could then be used to eliminate a portal of entry and cardiac perfusion MRI could validate the technique. Standard cardiectomy was compared to a variation in technique in two animals. Pulmonary vein ligation was completed in the experimental model before completion of cardiectomy. Both hearts were isolated and imaged using T1-weighted FLASH sequence and gadolinium contrast via the aortic root. Cardiac perfusion MRI imaging of the heart with the unligated pulmonary vein revealed evidence of air embolus and no perfusion of the right coronary artery compared to the ligated heart. Anatomically, the right coronary artery is anterior in the mediastinum compared to the left coronary artery. Air emboli preferentially rise into the right coronary and can obstruct flow into the right heart. Cardiac perfusion MRI offers an effective method to evaluate the isolated pre-transplant heart for perfusion defects.

Feedback-assisted three-dimensional reconstruction of the left ventricle with MRI.

PURPOSE: To develop and test a new technique for rapid, accurate three-dimensional (3D) reconstruction of the left ventricle (LV) and calculation of its volume parameters, with images from multiple orientations and interactive feedback. MATERIALS AND METHODS: The ventricular surface was fit to a number of user-placed guide points in magnetic resonance (MR) images using bivariate smoothing splines. A 3D model was reconstructed and the LV volumes were calculated at both end diastole (ED) and end systole (ES). This technique was validated using a phantom, and applied to studies of 18 patients and four volunteers (N = 22) imaged on a 1.5-T clinical scanner. The results of the 3D method were compared to the standard 2D short-axis slice summation technique, which is widely used for the analysis of cardiac function. RESULTS: There was excellent agreement between the computed volume of the phantom using the 3D modeling method and the actual volume (190.50 mL +/- 3.06 mL, and 191.0 mL +/- 2.5 mL, respectively). There was good correlation between the volumes calculated with our 3D model and the slice summation technique (ED volume (EDV) difference, 6.36% +/- 8.99% [mean +/- SD]; ES volume (ESV), 0.92% +/- 14.75%; stroke volume (SV), 10.54% +/- 13.95%; ejection fraction (EF), 4.22% +/- 9.16%). The 3D method was found to be more accurate than the slice summation technique for calculating LV volumes and mass from images of different slice orientations. Variations in the parameters between the two separate orientations using the 3D model vs. the slice summation method were as follows: EDV: 2.11% +/- 1.52% vs. 10.36% +/- 9.33%; ES volume: 2.76% +/- 1.64% vs. 6.39% +/- 3.62%; SV, 3.02% +/- 4.38% vs. 18.84% +/- 15.30%; EF, 2.03% +/- 2.16% vs. 8.58% +/- 6.73%; and LV mass: 4.77% +/- 2.41% vs. 24.59% +/- 6.41%. Differences in the ES volume due to the inclusion or exclusion of the most basal slice were found to be lower with the 3D model (6.90% +/- 3.83%) compared to the slice summation method (25.04% +/- 6.15%). CONCLUSION: 3D models can be used to accurately determine ventricular volume parameters. Results can be obtained using images from a variety of orientations, providing greater flexibility during image acquisition and possibly reducing the number of images needed for analysis. Feedback is provided to assist the analysis by providing a continuous update of the LV shape and volume. This feature allows the user to determine LV parameters to a predefined accuracy or to terminate the analysis when the parameters are not changing. This method is not restricted to multislice cine imaging in a single or prescribed slice orientation, and can be used for quick, accurate, and interactive analysis of cardiac function.