Intramyocardial hemorrhage drives fatty degeneration of infarcted myocardium.

Sudden blockage of arteries supplying the heart muscle contributes to millions of heart attacks (myocardial infarction, MI) around the world. Although re-opening these arteries (reperfusion) saves MI patients from immediate death, approximately 50% of these patients go on to develop chronic heart failure (CHF) and die within a 5-year period; however, why some patients accelerate towards CHF while others do not remains unclear. Here we show, using large animal models of reperfused MI, that intramyocardial hemorrhage - the most damaging form of reperfusion injury (evident in nearly 40% of reperfused ST-elevation MI patients) - drives delayed infarct healing and is centrally responsible for continuous fatty degeneration of the infarcted myocardium contributing to adverse remodeling of the heart. Specifically, we show that the fatty degeneration of the hemorrhagic MI zone stems from iron-induced macrophage activation, lipid peroxidation, foam cell formation, ceroid production, foam cell apoptosis and iron recycling. We also demonstrate that timely reduction of iron within the hemorrhagic MI zone reduces fatty infiltration and directs the heart towards favorable remodeling. Collectively, our findings elucidate why some, but not all, MIs are destined to CHF and help define a potential therapeutic strategy to mitigate post-MI CHF independent of MI size.

Reperfused hemorrhagic myocardial infarction in rats.

BACKGROUND: Intramyocardial hemorrhage following reperfusion is strongly associated with major adverse cardiovascular events in myocardial infarction (MI) patients; yet the mechanisms contributing to these outcomes are not well understood. Large animal models have been used to investigate intramyocardial hemorrhage, but they are exorbitantly expensive and difficult to use for mechanistic studies. In contrast, rat models are widely used to investigate mechanistic aspects of cardiovascular physiology, but a rat model that consistently recapitulates the characteristics of an hemorrhagic MI does not exist. To bridge this gap, we investigated the physiological conditions of MI that would create intramyocardial hemorrhage in rats so that a reliable model of hemorrhagic MI would become available for basic research. METHODS & RESULTS: Sprague-Dawley rats underwent either a 90-minute (90-min) ischemia and then reperfusion (I/R) (n = 22) or 30-minute (30-min) I/R (n = 18) of the left anterior descending coronary artery. Sham rats (n = 12) were used as controls. 90-min I/R consistently yielded hemorrhagic MI, while 30-min I/R consistently yielded non-hemorrhagic MI. Twenty-four hours post-reperfusion, ex-vivo late-gadolinium-enhancement (LGE) and T2* cardiac MRI performed on excised hearts from 90-min I/R rats revealed colocalization of iron deposits within the scarred tissue; however, in 30-min I/R rats scar was evident on LGE but no evidence of iron was found on T2* CMR. Histological studies verified tissue damage (H&E) detected on LGE and the presence of iron (Perl's stain) observed on T2*-CMR. At week 4 post-reperfusion, gene and protein expression of proinflammatory markers (TNF-α, IL-1ß and MMP-9) were increased in the 90-min I/R group when compared to 30-min I/R groups. Further, transmission electron microscopy performed on 90-min I/R myocardium that were positive for iron on T2* CMR and Perl's stain showed accumulation of granular iron particles within the phagosomes. CONCLUSION: Ischemic time prior to reperfusion is a critical factor in determining whether a MI is hemorrhagic or non-hemorrhagic in rats. Specifically, a period of 90-min of ischemia prior to reperfusion can produce rat models of hemorrhagic MI, while 30-minutes of ischemia prior to reperfusion can ensure that the MIs are non-hemorrhagic. Hemorrhagic MIs in rats result in marked increase in iron deposition, proinflammatory burden and adverse left-ventricular remodeling compared to rats with non-hemorrhagic MIs.

Multicenter Study on the Diagnostic Performance of Native-T1 Cardiac Magnetic Resonance of Chronic Myocardial Infarctions at 3T.

BACKGROUND: Preclinical studies and pilot patient studies have shown that chronic infarctions can be detected and characterized from cardiac magnetic resonance without gadolinium-based contrast agents using native-T1 maps at 3T. We aimed to investigate the diagnostic capacity of this approach for characterizing chronic myocardial infarctions (MIs) in a multi-center setting. METHODS: Patients with a prior MI (n=105) were recruited at 3 different medical centers and were imaged with native-T1 mapping and late gadolinium enhancement (LGE) at 3T. Infarct location, size, and transmurality were determined from native-T1 maps and LGE. Sensitivity, specificity, receiver-operating characteristic metrics, and inter- and intraobserver variabilities were assessed relative to LGE. RESULTS: Across all subjects, T1 of MI territory was 1621±110 ms, and remote territory was 1225±75 ms. Sensitivity, specificity, and area under curve for detecting MI location based on native-T1 mapping relative to LGE were 88%, 92%, and 0.93, respectively. Native-T1 maps were not different for measuring infarct size (native-T1 maps: 12.1±7.5%; LGE: 11.8±7.2%, P=0.82) and were in agreement with LGE (R2=0.92, bias, 0.09±2.6%). Corresponding inter- and intraobserver assessments were also highly correlated (interobserver: R2=0.90, bias, 0.18±2.4%; and intraobserver: R2=0.91, bias, 0.28±2.1%). Native T1 maps were not different for measuring MI transmurality (native-T1 maps: 49.1±15.8%; LGE: 47.2±19.0%, P=0.56) and showed agreement (R2=0.71; bias, 1.32±10.2%). Corresponding inter- and intraobserver assessments were also in agreement (interobserver: R2=0.81, bias, 0.1±9.4%; and intraobserver: R2=0.91, bias, 0.28±2.1%, respectively). While the overall accuracy for detecting MI with native-T1 maps at 3T was high, logistic regression analysis showed that MI location was a prominent confounder. CONCLUSIONS: Native-T1 mapping can be used to image chronic MI with high degree of accuracy, and as such, it is a viable alternative for scar imaging in patients with chronic MI who are contraindicated for LGE. Technical advancements may be needed to overcome the imaging confounders that currently limit native-T1 mapping from reaching equivalent detection levels as LGE.

Heart Rate-Independent 3D Myocardial Blood Oxygen Level-Dependent MRI at 3.0 T with Simultaneous 13N-Ammonia PET Validation.

Background Despite advances, blood oxygen level-dependent (BOLD) cardiac MRI for myocardial perfusion is limited by inadequate spatial coverage, imaging speed, multiple breath holds, and imaging artifacts, particularly at 3.0 T. Purpose To develop and validate a robust, contrast agent-unenhanced, free-breathing three-dimensional (3D) cardiac MRI approach for reliably examining changes in myocardial perfusion between rest and adenosine stress. Materials and Methods A heart rate-independent, free-breathing 3D T2 mapping technique at 3.0 T that can be completed within the period of adenosine stress (≤4 minutes) was developed by using computer simulations, ex vivo heart preparations, and dogs. Studies in dogs were performed with and without coronary stenosis and validated with simultaneously acquired nitrogen 13 (13N) ammonia PET perfusion in a clinical PET/MRI system. The MRI approach was also prospectively evaluated in healthy human volunteers (from January 2017 to September 2017). Myocardial BOLD responses (MBRs) between normal and ischemic myocardium were compared with mixed model analysis. Results Dogs (n = 10; weight range, 20-25 kg; mongrel dogs) and healthy human volunteers (n = 10; age range, 22-53 years; seven men) were evaluated. In healthy dogs, T2 MRI at adenosine stress was greater than at rest (mean rest vs stress, 38.7 msec ± 2.5 [standard deviation] vs 45.4 msec ± 3.3, respectively; MBR, 1.19 ± 0.08; both, P < .001). At the same conditions, mean rest versus stress PET perfusion was 1.1 mL/mg/min ± 0.11 versus 2.3 mL/mg/min ± 0.82, respectively (P < .001); myocardial perfusion reserve (MPR) was 2.4 ± 0.82 (P < .001). The BOLD response and PET MPR were positively correlated (R = 0.67; P < .001). In dogs with coronary stenosis, perfusion anomalies were detected on the basis of MBR (normal vs ischemic, 1.09 ± 0.05 vs 1.00 ± 0.04, respectively; P < .001) and MPR (normal vs ischemic, 2.7 ± 0.08 vs 1.7 ± 1.1, respectively; P < .001). Human volunteers showed increased myocardial T2 at stress (rest vs stress, 44.5 msec ± 2.6 vs 49.0 msec ± 5.5, respectively; P = .004; MBR, 1.1 msec ± 8.08). Conclusion This three-dimensional cardiac blood oxygen level-dependent (BOLD) MRI approach overcame key limitations associated with conventional cardiac BOLD MRI by enabling whole-heart coverage within the standard duration of adenosine infusion, and increased the magnitude and reliability of BOLD contrast, which may be performed without requiring breath holds. © RSNA, 2020 Online supplemental material is available for this article. See also the editorial by Almeida in this issue.

Accurate needle-free assessment of myocardial oxygenation for ischemic heart disease in canines using magnetic resonance imaging.

Myocardial oxygenation-the ability of blood vessels to supply the heart muscle (myocardium) with oxygen-is a critical determinant of cardiac function. Impairment of myocardial oxygenation is a defining feature of ischemic heart disease (IHD), which is caused by pathological conditions that affect the blood vessels supplying oxygen to the heart muscle. Detecting altered myocardial oxygenation can help guide interventions and prevent acute life-threatening events such as heart attacks (myocardial infarction); however, current diagnosis of IHD relies on surrogate metrics and exogenous contrast agents for which many patients are contraindicated. An oxygenation-sensitive cardiac magnetic resonance imaging (CMR) approach used previously to demonstrate that CMR signals can be sensitized to changes in myocardial oxygenation showed limited ability to detect small changes in signals in the heart because of physiologic and imaging noise during data acquisition. Here, we demonstrate a CMR-based approach termed cfMRI [cardiac functional magnetic resonance imaging (MRI)] that detects myocardial oxygenation. cfMRI uses carbon dioxide for repeat interrogation of the functional capacity of the heart's blood vessels via a fast MRI approach suitable for clinical adoption without limitations of key confounders (cardiac/respiratory motion and heart rate changes). This method integrates multiple whole-heart images within a computational framework to reduce noise, producing confidence maps of alterations in myocardial oxygenation. cfMRI permits noninvasive monitoring of myocardial oxygenation without requiring ionizing radiation, contrast agents, or needles. This has the potential to broaden our ability to noninvasively identify IHD and a diverse spectrum of heart diseases related to myocardial ischemia.

Influence of Myocardial Hemorrhage on Staging of Reperfused Myocardial Infarctions With T2 Cardiac Magnetic Resonance Imaging: Insights Into the Dependence on Infarction Type With Ex Vivo Validation.

OBJECTIVES: This study sought to determine whether T2 cardiac magnetic resonance (CMR) can stage both hemorrhagic and nonhemorrhagic myocardial infarctions (MIs). BACKGROUND: CMR-based staging of MI with or without contrast agents relies on the resolution of T2 elevations in the chronic phase, but whether this approach can be used to stage both hemorrhagic and nonhemorrhagic MIs is unclear. METHODS: Hemorrhagic (n = 15) and nonhemorrhagic (n = 9) MIs were created in dogs. Multiparametric noncontrast mapping (T1, T2, and T2*) and late gadolinium enhancement (LGE) were performed at 1.5- and 3.0-T at 5 days (acute) and 8 weeks (chronic) post-MI. CMR relaxation values and LGE intensities of hemorrhagic, peri-hemorrhagic, nonhemorrhagic, and remote territories were measured. Histopathology was performed to elucidate CMR findings. RESULTS: T2 of nonhemorrhagic MIs was significantly elevated in the acute phase relative to remote territories (1.5-T: 39.8 ± 12.8%; 3.0-T: 27.9 ± 16.5%; p < 0.0001 for both) but resolved to remote values by week 8 (1.5-T: -0.0 ± 3.2%; p = 0.678; 3.0-T: -0.5 ± 5.9%; p = 0.601). In hemorrhagic MI, T2 of hemorrhage core was significantly elevated in the acute phase (1.5-T: 17.7 ± 10.0%; 3.0-T: 8.6 ± 8.2%; p < 0.0001 for both) but decreased below remote values by week 8 (1.5-T: -8.2 ± 3.9%; 3.0-T: -5.6 ± 6.0%; p < 0.0001 for both). In contrast, T2 of the periphery of hemorrhage within the MI zone was significantly elevated in the acute phase relative to remote territories (1.5-T: 35.0 ± 16.1%; 3.0-T: 24.2 ± 10.4%; p < 0.0001 for both) and remained elevated at 8 weeks post-MI (1.5-T: 8.6 ± 5.1%; 3.0-T: 6.0 ± 3.3%; p < 0.0001 for both). The observed elevation of T2 in the peri-hemorrhagic zone of MIs and the absence of T2 elevation in nonhemorrhagic MIs were consistent with ongoing or absence of histological evidence of inflammation, respectively. CONCLUSIONS: Hemorrhagic MIs are associated with persisting myocardial inflammation and edema, which can confound staging of hemorrhagic MIs when T2 elevations alone are used to discriminate between acute and chronic MI. Moreover, given the poor prognosis in patients with hemorrhagic MI, CMR evidence for myocardial hemorrhage with persistent edema may evolve as a risk marker in patients after acute MI.

Arterial CO2 as a Potent Coronary Vasodilator: A Preclinical PET/MR Validation Study with Implications for Cardiac Stress Testing.

Myocardial blood flow (MBF) is the critical determinant of cardiac function. However, its response to increases in partial pressure of arterial CO2 (PaCO2), particularly with respect to adenosine, is not well characterized because of challenges in blood gas control and limited availability of validated approaches to ascertain MBF in vivo. Methods: By prospectively and independently controlling PaCO2 and combining it with 13N-ammonia PET measurements, we investigated whether a physiologically tolerable hypercapnic stimulus (∼25 mm Hg increase in PaCO2) can increase MBF to that observed with adenosine in 3 groups of canines: without coronary stenosis, subjected to non-flow-limiting coronary stenosis, and after preadministration of caffeine. The extent of effect on MBF due to hypercapnia was compared with adenosine. Results: In the absence of stenosis, mean MBF under hypercapnia was 2.1 ± 0.9 mL/min/g and adenosine was 2.2 ± 1.1 mL/min/g; these were significantly higher than at rest (0.9 ± 0.5 mL/min/g, P < 0.05) and were not different from each other (P = 0.30). Under left-anterior descending coronary stenosis, MBF increased in response to hypercapnia and adenosine (P < 0.05, all territories), but the effect was significantly lower than in the left-anterior descending coronary territory (with hypercapnia and adenosine; both P < 0.05). Mean perfusion defect volumes measured with adenosine and hypercapnia were significantly correlated (R = 0.85) and were not different (P = 0.12). After preadministration of caffeine, a known inhibitor of adenosine, resting MBF decreased; and hypercapnia increased MBF but not adenosine (P < 0.05). Conclusion: Arterial blood CO2 tension when increased by 25 mm Hg can induce MBF to the same level as a standard dose of adenosine. Prospectively targeted arterial CO2 has the capability to evolve as an alternative to current pharmacologic vasodilators used for cardiac stress testing.

Persistent Microvascular Obstruction After Myocardial Infarction Culminates in the Confluence of Ferric Iron Oxide Crystals, Proinflammatory Burden, and Adverse Remodeling.

BACKGROUND: Emerging evidence indicates that persistent microvascular obstruction (PMO) is more predictive of major adverse cardiovascular events than myocardial infarct (MI) size. But it remains unclear how PMO, a phenomenon limited to the acute/subacute period of MI, drives adverse remodeling in chronic MI setting. We hypothesized that PMO resolves into chronic iron crystals within MI territories, which in turn are proinflammatory and favor adverse remodeling post-MI. METHODS AND RESULTS: Canines (n=40) were studied with cardiac magnetic resonance imaging to characterize the spatiotemporal relationships among PMO, iron deposition, infarct resorption, and left ventricular remodeling between day 7 (acute) and week 8 (chronic) post-MI. Histology was used to assess iron deposition and to examine relationships between iron content with macrophage infiltration, proinflammatory cytokine synthesis, and matrix metalloproteinase activation. Atomic resolution transmission electron microscopy was used to determine iron crystallinity, and energy-dispersive X-ray spectroscopy was used to identify the chemical composition of the iron composite. PMO with or without reperfusion hemorrhage led to chronic iron deposition, and the extent of this deposition was strongly related to PMO volume (r>0.8). Iron deposits were found within macrophages as aggregates of nanocrystals (≈2.5 nm diameter) in the ferric state. Extent of iron deposits was strongly correlated with proinflammatory burden, collagen-degrading enzyme activity, infarct resorption, and adverse structural remodeling (r>0.5). CONCLUSIONS: Crystallized iron deposition from PMO is directly related to proinflammatory burden, infarct resorption, and adverse left ventricular remodeling in the chronic phase of MI in canines. Therapeutic strategies to combat adverse remodeling could potentially benefit from taking into account the chronic iron-driven inflammatory process.

Free-breathing, motion-corrected, highly efficient whole heart T2 mapping at 3T with hybrid radial-cartesian trajectory.

PURPOSE: To develop and test a time-efficient, free-breathing, whole heart T2 mapping technique at 3.0T. METHODS: ECG-triggered three-dimensional (3D) images were acquired with different T2 preparations at 3.0T during free breathing. Respiratory motion was corrected with a navigator-guided motion correction framework at near perfect efficiency. Image intensities were fit to a monoexponential function to derive myocardial T2 maps. The proposed 3D, free breathing, motion-corrected (3D-FB-MoCo) approach was studied in ex vivo canine hearts and kidneys, healthy volunteers, and canine subjects with acute myocardial infarction (AMI). RESULTS: Ex vivo T2 values from proposed 3D T2 -prep gradient echo were not different from two-dimensional (2D) spin echo (P = 0.7) and T2 -prep balanced steady-state free precession (bSSFP) (P = 0.7). In healthy volunteers, compared with 3D-FB-MoCo and breath-held 2D T2 -prep bSSFP (2D-BH), non-motion-corrected (3D-FB-Non-MoCo) myocardial T2 was longer, had a larger coefficient of variation (COV), and had a lower image quality (IQ) score (T2 = 40.3 ms, COV = 38%, and IQ = 2.3; all P < 0.05). Conversely, the mean and COV and IQ of 3D-FB-MoCo (T2 = 37.7 ms, COV = 17%, and IQ = 3.5) and 2D-BH (T2 = 38.0 ms, COV = 15%, and IQ = 3.8) were not different (P = 0.99, P = 0.74, and P = 0.14, respectively). In AMI, T2 values and edema volumes from 3D-FB-MoCo and 2D-BH were closely correlated (R(2) = 0.88 and 0.96, respectively). CONCLUSION: The proposed whole heart T2 mapping approach can be performed within 5 min with similar accuracy to that of the 2D-BH T2 mapping approach.

Native T1 Mapping by 3-T CMR Imaging for Characterization of Chronic Myocardial Infarctions.

OBJECTIVES: The purpose of this study was to investigate whether native T1 maps at 3-T can reliably characterize chronic myocardial infarctions (MIs) in patients with prior ST-segment elevation myocardial infarction (STEMI) or non-ST-segment elevation myocardial infarction (NSTEMI). BACKGROUND: Late gadolinium enhancement (LGE) cardiac magnetic resonance is the gold standard for characterizing chronic MIs, but it is contraindicated in patients with end-stage chronic kidney disease. METHODS: Native T1 and LGE images were acquired at 3-T in patients with prior STEMI (n = 13) and NSTEMI (n = 12) at a median of 13.6 years post-MI. Infarct location, size, and transmurality were measured using mean ± 5 SDs thresholding criterion from LGE images and T1 maps and compared against one another. Independent reviewers assessed visual conspicuity of MIs on LGE images and T1 maps. RESULTS: Native T1 maps and LGE images were not different for measuring infarct size (STEMI: p = 0.46; NSTEMI: p = 0.27) and transmurality (STEMI: p = 0.13; NSTEMI: p = 0.21) using thresholding criterion. Using thresholding criterion, good agreement was observed between LGE images and T1 maps for measuring infarct size (STEMI: bias = 0.6 ± 3.1%; R(2) = 0.93; NSTEMI: bias = -0.4 ± 4.4%; R(2) = 0.85) and transmurality (STEMI: bias = 2.0 ± 4.2%; R(2) = 0.89; NSTEMI: bias = -2.7 ± 7.9%; R(2) = 0.68). Sensitivity and specificity of T1 maps for detecting chronic MIs based on thresholding criterion were 89% and 98%, respectively (STEMI), and 87% and 95%, respectively (NSTEMI). Relative to LGE images, the mean visual conspicuity score for detecting chronic MIs was significantly lower for T1 maps (p < 0.001 for both cases). Median infarct-to-remote myocardium contrast-to-noise ratio was 2.5-fold higher for LGE images relative to T1 maps (p < 0.001). Sensitivity and specificity of T1 maps for visual detection were 60% and 86%, respectively (STEMI), and 64% and 91% (NSTEMI), respectively. CONCLUSIONS: Chronic MIs in STEMI and NSTEMI patients can be reliably characterized using threshold-based detection on native T1 maps at 3-T. Visual detection of chronic MIs on native T1 maps in both patient populations has high specificity, but modest sensitivity.

Iron-Sensitive Cardiac Magnetic Resonance Imaging for Prediction of Ventricular Arrhythmia Risk in Patients With Chronic Myocardial Infarction: Early Evidence.

BACKGROUND: Recent canines studies have shown that iron deposition within chronic myocardial infarction (CMI) influences the electric behavior of the heart. To date, the link between the iron deposition and malignant ventricular arrhythmias in humans with CMI is unknown. METHODS AND RESULTS: Patients with CMI (n=94) who underwent late-gadolinium-enhanced cardiac magnetic resonance imaging before implantable cardioverter-defibrillator implantation for primary and secondary preventions were retrospectively analyzed. The predictive values of hypointense cores (HIC) in balanced steady-state free precession images and conventional cardiac magnetic resonance imaging and ECG malignant ventricular arrhythmia parameters for the prediction of primary combined outcome (appropriate implantable cardioverter-defibrillator therapy, survived cardiac arrest, or sudden cardiac death) were studied. The use of HIC within CMI on balanced steady-state free precession as a marker of iron deposition was validated in a canine MI model (n=18). Nineteen patients met the study criteria with events occurring at a median of 249 (interquartile range of 540) days after implantable cardioverter-defibrillator placement. Of the 19 patients meeting the primary end point, 18 were classified as HIC+, whereas only 1 was HIC-. Among the cohort in whom the primary end point was not met, there were 28 HIC+ and 47 HIC- patients. Receiver operating characteristic curve analysis demonstrated an additive predictive value of HIC for malignant ventricular arrhythmias with an increased area under the curve of 0.87 when added to left ventricular ejection fraction (left ventricular ejection fraction alone, 0.68). Both cardiac magnetic resonance imaging and histological validation studies performed in canines demonstrated that HIC regions in balanced steady-state free precession images within CMI likely result from iron depositions. CONCLUSIONS: Hypointense cores within CMI on balanced steady-state free precession cardiac magnetic resonance imaging can be used as a marker of iron deposition and yields incremental information toward improved prediction of malignant ventricular arrhythmias.

Correlation of scar in cardiac MRI and high-resolution contact mapping of left ventricle in a chronic infarct model.

BACKGROUND: Endocardial mapping for scars and abnormal electrograms forms the most essential component of ventricular tachycardia ablation. The utility of ultra-high resolution mapping of ventricular scar was assessed using a multielectrode contact mapping system in a chronic canine infarct model. METHODS: Chronic infarcts were created in five anesthetized dogs by ligating the left anterior descending coronary artery. Late gadolinium-enhanced magnetic resonance imaging (LGE MRI) was obtained 4.9 ± 0.9 months after infarction, with three-dimensional (3D) gadolinium enhancement signal intensity maps at 1-mm and 5-mm depths from the endocardium. Ultra-high resolution electroanatomical maps were created using a novel mapping system (Rhythmia Mapping System, Rhythmia Medical/Boston Scientific, Marlborough, MA, USA) Rhythmia Medical, Boston Scientific, Marlborough, MA, USA with an 8.5F catheter with mini-basket electrode array (64 tiny electrodes, 2.5-mm spacing, center-to-center). RESULTS: The maps contained 7,754 ± 1,960 electrograms per animal with a mean resolution of 2.8 ± 0.6 mm. Low bipolar voltage (<2 mV) correlated closely with scar on the LGE MRI and the 3D signal intensity map (1-mm depth). The scar areas between the MRI signal intensity map and electroanatomic map matched at 87.7% of sites. Bipolar and unipolar voltages, compared in 592 electrograms from four MRI-defined scar types (endocardial scar, epicardial scar, mottled transmural scar, and dense transmural scar) as well as normal tissue, were significantly different. A unipolar voltage of <13 mV correlated with transmural extension of scar in MRI. Electrograms exhibiting isolated late potentials (ILPs) were manually annotated and ILP maps were created showing ILP location and timing. ILPs were identified in 203 ± 159 electrograms per dog (within low-voltage areas) and ILP maps showed gradation in timing of ILPs at different locations in the scar. CONCLUSIONS: Ultra-high resolution contact electroanatomical mapping accurately localizes ventricular scar and abnormal myocardial tissue in this chronic canine infarct model. The high fidelity electrograms provided clear identification of the very low amplitude ILPs within the scar tissue and has the potential to quickly identify targets for ablation.

Cytotoxic effect of commercially available methylprednisolone acetate with and without reduced preservatives on dorsal root ganglion sensory neurons in rats.

BACKGROUND: Epidural and intrathecal injections of methylprednisolone acetate (MPA) have become the most commonly performed interventional procedures in the United States and worldwide in the last 2 decades. However neuraxial MPA injection has been dogged by controversy regarding the presence of different additives used in commercially prepared glucocorticoids. We previously showed that MPA could be rendered 85% free of polyethylene glycol (PEG) by a simple physical separation of elements in the suspension. OBJECTIVE: The objective of the present study was to explore a possible cytotoxic effect of commercially available MPA (with intact or reduced preservatives) on rat sensory neurons. METHODS: We exposed primary dissociated rat dorsal root ganglia (DRG) sensory neurons to commercially available MPA for 24 hours with either the standard (commercial) concentration of preservatives or to different fractions following separation (MPA suspension whose preservative concentration had been reduced, or fractions containing higher concentrations of preservatives). Cells were stained with the TUNEL assay kit to detect apoptotic cells and images were taken on the Bio-Rad Laser Sharp-2000 system. We also detected expression of caspase-3, as an indicator of apoptosis in cell lysates. RESULTS: We exposed sensory neurons from rat DRG to different concentrations of MPA from the original commercially prepared vial. TUNEL assay showed dose-related responses and increased percentages of apoptotic cells with increasing concentrations of MPA. Increased concentrations of MPA caused 1.5 - 2 times higher caspase-3 expression in DRG sensory neurons than in control cells (ANOVA, P = 0.001). Our results showed that MPA with reduced preservatives caused significantly less apoptosis observed with TUNEL assay labeling (P < 0.001) and caspase-3 immunoblotting (P = 0.001) than in neurons exposed to MPA from a commercially prepared vial or "clear phase" that contained higher concentrations of preservatives. Even though MPA with reduced preservatives caused 12.5% more apoptosis in DRG sensory neurons than in control cells, post hoc analysis showed no differences between these 2 groups. LIMITATIONS: Our data was collected from in vitro isolated rat DRG neurons. There is a possibility that in vivo neurons have different extents of vulnerability compared to isolated neurons. CONCLUSIONS: Results of the present study identified a cytotoxic effect of commercially available MPA with preservatives or with a "clear phase" containing higher concentrations of preservatives on primary isolated rat DRG sensory neurons. This was shown by TUNEL positive assay and by increased caspase-3 expression as one of the final executing steps in apoptotic pathways in DRG neurons. However, our results showed no statistically significant difference between the control cells (saline-treated) and cells treated with MPA with reduced concentrations of preservatives, pointing out that either PEG or myristylgamma-picolinium chloride (MGPC) or their combination have harmful effects on these cells. Reduction of concentrations of preservatives from commercially available MPA suspensions by using the simple method of inverting vials for 2 hours could be considered useful in clinical practice to enhance the safety of this depot steroid when injected neuraxially.

Determination of location, size, and transmurality of chronic myocardial infarction without exogenous contrast media by using cardiac magnetic resonance imaging at 3 T.

BACKGROUND: Late-gadolinium-enhanced (LGE) cardiac MRI (CMR) is a powerful method for characterizing myocardial infarction (MI), but the requisite gadolinium infusion is estimated to be contraindicated in ≈20% of patients with MI because of end-stage chronic kidney disease. The purpose of this study is to investigate whether T1 CMR obtained without contrast agents at 3 T could be an alternative to LGE CMR for characterizing chronic MIs using a canine model of MI. METHODS AND RESULTS: Canines (n=29) underwent CMR at 7 days (acute MI [AMI]) and 4 months (chronic MI [CMI]) after MI. Infarct location, size, and transmurality measured by using native T1 maps and LGE images at 1.5 T and 3 T were compared. Resolution of edema between AMI and CMI was examined with T2 maps. T1 maps overestimated infarct size and transmurality relative to LGE images in AMI (P=0.016 and P=0.007, respectively), which was not observed in CMI (P=0.49 and P=0.81, respectively) at 3 T. T1 maps underestimated infarct size and transmurality relative to LGE images in AMI and CMI (P<0.001) at 1.5 T. Relative to the remote territories, T1 of the infarcted myocardium was increased in CMI and AMI (P<0.05), and T2 of the infarcted myocardium was increased in AMI (P<0.001) but not in CMI (P>0.20) at both field strengths. Histology showed extensive replacement fibrosis within the CMI territories. CMI detection sensitivity and specificity of T1 CMR at 3 T were 95% and 97%, respectively. CONCLUSIONS: Native T1 maps at 3 T can determine the location, size, and transmurality of CMI with high diagnostic accuracy. Patient studies are necessary for clinical translation.

Assessment of myocardial reactivity to controlled hypercapnia with free-breathing T2-prepared cardiac blood oxygen level-dependent MR imaging.

PURPOSE: To examine whether controlled and tolerable levels of hypercapnia may be an alternative to adenosine, a routinely used coronary vasodilator, in healthy human subjects and animals. MATERIALS AND METHODS: Human studies were approved by the institutional review board and were HIPAA compliant. Eighteen subjects had end-tidal partial pressure of carbon dioxide (PetCO2) increased by 10 mm Hg, and myocardial perfusion was monitored with myocardial blood oxygen level-dependent (BOLD) magnetic resonance (MR) imaging. Animal studies were approved by the institutional animal care and use committee. Anesthetized canines with (n = 7) and without (n = 7) induced stenosis of the left anterior descending artery (LAD) underwent vasodilator challenges with hypercapnia and adenosine. LAD coronary blood flow velocity and free-breathing myocardial BOLD MR responses were measured at each intervention. Appropriate statistical tests were performed to evaluate measured quantitative changes in all parameters of interest in response to changes in partial pressure of carbon dioxide. RESULTS: Changes in myocardial BOLD MR signal were equivalent to reported changes with adenosine (11.2% ± 10.6 [hypercapnia, 10 mm Hg] vs 12% ± 12.3 [adenosine]; P = .75). In intact canines, there was a sigmoidal relationship between BOLD MR response and PetCO2 with most of the response occurring over a 10 mm Hg span. BOLD MR (17% ± 14 [hypercapnia] vs 14% ± 24 [adenosine]; P = .80) and coronary blood flow velocity (21% ± 16 [hypercapnia] vs 26% ± 27 [adenosine]; P > .99) responses were similar to that of adenosine infusion. BOLD MR signal changes in canines with LAD stenosis during hypercapnia and adenosine infusion were not different (1% ± 4 [hypercapnia] vs 6% ± 4 [adenosine]; P = .12). CONCLUSION: Free-breathing T2-prepared myocardial BOLD MR imaging showed that hypercapnia of 10 mm Hg may provide a cardiac hyperemic stimulus similar to adenosine.

Iron deposition following chronic myocardial infarction as a substrate for cardiac electrical anomalies: initial findings in a canine model.

PURPOSE: Iron deposition has been shown to occur following myocardial infarction (MI). We investigated whether such focal iron deposition within chronic MI lead to electrical anomalies. METHODS: Two groups of dogs (ex-vivo (n = 12) and in-vivo (n = 10)) were studied at 16 weeks post MI. Hearts of animals from ex-vivo group were explanted and sectioned into infarcted and non-infarcted segments. Impedance spectroscopy was used to derive electrical permittivity ([Formula: see text]) and conductivity ([Formula: see text]). Mass spectrometry was used to classify and characterize tissue sections with (IRON+) and without (IRON-) iron. Animals from in-vivo group underwent cardiac magnetic resonance imaging (CMR) for estimation of scar volume (late-gadolinium enhancement, LGE) and iron deposition (T2*) relative to left-ventricular volume. 24-hour electrocardiogram recordings were obtained and used to examine Heart Rate (HR), QT interval (QT), QT corrected for HR (QTc) and QTc dispersion (QTcd). In a fraction of these animals (n = 5), ultra-high resolution electroanatomical mapping (EAM) was performed, co-registered with LGE and T2* CMR and were used to characterize the spatial locations of isolated late potentials (ILPs). RESULTS: Compared to IRON- sections, IRON+ sections had higher[Formula: see text], but no difference in[Formula: see text]. A linear relationship was found between iron content and [Formula: see text] (p<0.001), but not [Formula: see text] (p = 0.34). Among two groups of animals (Iron (<1.5%) and Iron (>1.5%)) with similar scar volumes (7.28% ± 1.02% (Iron (<1.5%)) vs 8.35% ± 2.98% (Iron (>1.5%)), p = 0.51) but markedly different iron volumes (1.12% ± 0.64% (Iron (<1.5%)) vs 2.47% ± 0.64% (Iron (>1.5%)), p = 0.02), QT and QTc were elevated and QTcd was decreased in the group with the higher iron volume during the day, night and 24-hour period (p<0.05). EAMs co-registered with CMR images showed a greater tendency for ILPs to emerge from scar regions with iron versus without iron. CONCLUSION: The electrical behavior of infarcted hearts with iron appears to be different from those without iron. Iron within infarcted zones may evolve as an arrhythmogenic substrate in the post MI period.

Chronic manifestation of postreperfusion intramyocardial hemorrhage as regional iron deposition: a cardiovascular magnetic resonance study with ex vivo validation.

BACKGROUND: Intramyocardial hemorrhage frequently accompanies large reperfused myocardial infarctions. However, its influence on the makeup and the ensuing effect on the infarcted tissue during the chronic phase remain unexplored. METHODS AND RESULTS: Patients (n=15; 3 women), recruited after successful percutaneous coronary intervention for first segment-elevation myocardial infarction, underwent cardiovascular magnetic resonance imaging on day 3 and month 6 after percutaneous coronary intervention. Patients with hemorrhagic (Hemo+) infarctions, as determined by T2* cardiovascular magnetic resonance on day 3 (n=11), showed persistent T2* losses colocalized with scar tissue on the follow-up scans, suggesting chronic iron deposition. T2* values of Hemo+ territories were significantly higher than nonhemorrhagic (Hemo-) and remote territories (P<0.001); however, T2* values of nonhemorrhagic (Hemo-) and remote territories were not different (P=0.51). Canines (n=20) subjected to ischemia-reperfusion injury (n=14) underwent cardiovascular magnetic resonance on days 3 and 56 after ischemia-reperfusion injury. Similarly, sham-operated animals (Shams; n=3) were imaged using cardiovascular magnetic resonance at similar time points. Subsequently, hearts were explanted and imaged ex vivo, and samples of Hemo+, Hemo-, remote, and Sham myocardium were isolated and stained. The extent of iron deposition ([Fe]) within each sample was measured using mass spectrometry. Hemo+ infarcts showed significant T2* losses compared with the other (control) groups (P<0.001), and Perls stain confirmed localized iron deposition. Mean [Fe] of Hemo+ was nearly an order of magnitude greater than that of the control groups (P<0.001), but no significant differences were observed among the control groups. A strong linear relationship was observed between log(T2*) and -log([Fe]); R(2)=0.7 and P<0.001. The monoclonal antibody Mac387 stains, along with Perls stains, showed preferential localization of newly recruited macrophages at the site of chronic iron deposition. CONCLUSIONS: Hemorrhagic myocardial infarction can lead to iron depositions within the infarct zones, which can be a source of prolonged inflammatory burden in the chronic phase of myocardial infarction.

Endothelial barrier disruption and recovery is controlled by substrate stiffness.

Circulating barrier disruptive agonists bind specific cell membrane receptors and trigger signal transduction pathways leading to the activation of cell contractility and endothelial cell (EC) permeability. Although all cells in tissues including vascular EC are surrounded by compliant extracellular matrix, the impact of matrix stiffness on agonist-induced signaling, cytoskeletal remodeling and EC barrier regulation is not well understood. This study examined agonist-induced cytoskeletal and signaling changes associated with EC barrier disruption and recovery using pulmonary EC grown on compliant substrates of physiologically relevant (8.6 kPa) stiffness, very low (0.55 kPa) and very high (42 kPa) stiffness. Human pulmonary microvascular and macrovascular EC grown on 0.55 kPa substrate contained a few actin stress fibers, while stress fiber amount increased with increasing matrix stiffness. Thrombin-induced stress fiber formation was maximal in EC grown on 42 kPa substrate, diminished on 8.6 kPa substrate, and was minimal on 0.55 kPa substrate. These effects were linked to a stiffness-dependent increase in thrombin-induced phosphorylation of the Rho kinase target, myosin light chain phosphatase (MYPT1), and regulatory myosin light chains (MLC). Surprisingly, EC barrier recovery and activation of Rac GTPase-dependent barrier protective signaling reached maximal levels in EC grown on 8.6 kPa, but not on 0.55 kPa substrate. In conclusion, these data show a critical role of extracellular matrix stiffness in the regulation of the Rac/Rho signaling balance during onset and resolution of agonist-induced EC permeability. The optimal conditions for the Rho/Rac signaling switch, which provides an effective and reversible EC cytoskeletal and permeability response to agonist, are reached in cells grown on the matrix of physiologically relevant stiffness.

Contribution of fibrosis and the autonomic nervous system to atrial fibrillation electrograms in heart failure.

BACKGROUND: Fibrotic and autonomic remodeling in heart failure (HF) increase vulnerability to atrial fibrillation (AF). Because AF electrograms (EGMs) are thought to reflect the underlying structural substrate, we sought to (1) determine the differences in AF EGMs in normal versus HF atria and (2) assess how fibrosis and nerve-rich fat contribute to AF EGM characteristics in HF. METHODS AND RESULTS: AF was induced in 20 normal dogs by vagal stimulation and in 21 HF dogs (subjected to 3 weeks of rapid ventricular pacing at 240 beats per minute). AF EGMs were analyzed for dominant frequency (DF), organization index, fractionation intervals (FIs), and Shannon entropy. In 8 HF dogs, AF EGM correlation with underlying fibrosis/fat/nerves was assessed. In HF compared with normal dogs, DF was lower and organization index/FI/Shannon entropy were greater. DF/FI were more heterogeneous in HF. Percentage fat was greater, and fibrosis and fat were more heterogeneously distributed in the posterior left atrium than in the left atrial appendage. DF/organization index correlated closely with %fibrosis. Heterogeneity of DF/FI correlated with the heterogeneity of fibrosis. Autonomic blockade caused a greater change in DF/FI/Shannon entropy in the posterior left atrium than left atrial appendage, with the decrease in Shannon entropy correlating with %fat. CONCLUSIONS: The amount and distribution of fibrosis in the HF atrium seems to contribute to slowing and increased organization of AF EGMs, whereas the nerve-rich fat in the HF posterior left atrium is positively correlated with AF EGM entropy. By allowing for improved detection of regions of dense fibrosis and high autonomic nerve density in the HF atrium, these findings may help enhance the precision and success of substrate-guided ablation for AF.

Targeted nonviral gene-based inhibition of Gα(i/o)-mediated vagal signaling in the posterior left atrium decreases vagal-induced atrial fibrillation.

BACKGROUND: Pharmacologic and ablative therapies for atrial fibrillation (AF) have suboptimal efficacy. Newer gene-based approaches that target specific mechanisms underlying AF are likely to be more efficacious in treating AF. Parasympathetic signaling appears to be an important contributor to AF substrate. OBJECTIVE: The purpose of this study was to develop a nonviral gene-based strategy to selectively inhibit vagal signaling in the left atrium and thereby suppress vagal-induced AF. METHODS: In eight dogs, plasmid DNA vectors (minigenes) expressing Gα(i) C-terminal peptide (Gα(i)ctp) was injected in the posterior left atrium either alone or in combination with minigene expressing Gα(o)ctp, followed by electroporation. In five control dogs, minigene expressing scrambled peptide (Gα(R)ctp) was injected. Vagal- and carbachol-induced left atrial effective refractory periods (ERPs), AF inducibility, and Gα(i/o)ctp expression were assessed 3 days following minigene delivery. RESULTS: Vagal stimulation- and carbachol-induced effective refractory period shortening and AF inducibility were significantly attenuated in atria receiving a Gα(i2)ctp-expressing minigene and were nearly eliminated in atria receiving both Gα(i2)ctp- and Gα(o1)ctp-expressing minigenes. CONCLUSION: Inhibition of both G(i) and G(o) proteins is necessary to abrogate vagal-induced AF in the left atrium and can be achieved via constitutive expression of Gα(i/o)ctps expressed by nonviral plasmid vectors delivered to the posterior left atrium.