Phospholipid Encapsulation of an Anti-Fibrotic Endopeptide to Enhance Cellular Uptake and Myocardial Retention.

BACKGROUND: Cardioprotective effects of N-acetyl-ser-asp-lys-pro (Ac-SDKP) have been reported in preclinical models of myocardial remodeling. However, the rapid degradation of this endogenous peptide in vivo limits its clinical use. METHOD: To prolong its bioavailability, Ac-SDKP was encapsulated by phosphocholine lipid bilayers (liposomes) similar to mammalian cell membranes. The physical properties of the liposome structures were assessed by dynamic light scattering and scanning electron microscopy. The uptake of Ac-SDKP by RAW 264.7 macrophages and human and murine primary cardiac fibroblasts was confirmed by fluorescence microscopy and flow cytometry. Spectrum computerized tomography and competitive enzyme-linked immunoassays were performed to measure the ex vivo cardiac biodistribution of Ac-SDKP. The biological effects of this novel synthetic compound were examined in cultured macrophages and cardiac fibroblasts and in a murine model of acute myocardial infarction induced by permanent coronary artery ligation. RESULTS: A liposome formulation resulted in the greater uptake of Ac-SDKP than the naked peptide by cultured RAW 264.7 macrophages and cardiac fibroblasts. Liposome-delivered Ac-SDKP decreased fibroinflammatory genes in cultured cardiac fibroblasts co-treated with TGF-ß1 and macrophages stimulated with LPS. Serial tissue and serum immunoassays showed the high bioavailability of Ac-SDKP in mouse myocardium and in circulation. Liposome-delivered Ac-SDKP improved cardiac function and reduced myocardial fibroinflammatory responses in mice with acute myocardial infarction. CONCLUSION: Encapsulation of Ac-SDKP in a cell membrane-like phospholipid bilayer enhances its plasma and tissue bioavailability and offers cardioprotection against ischemic myocardial injury. Future clinical trials can use this novel approach to test small protective endogenous peptides in myocardial remodeling.

Galectin-3 Is Associated with Cardiac Fibrosis and an Increased Risk of Sudden Death.

BACKGROUND: Myocardial fibrosis is a common postmortem finding among individuals with Sudden Cardiac Death (SCD). Numerous in vivo and in vitro studies have shown that increased galectin-3 (gal3) expression into the myocardium is associated with higher incidence of fibrosis. Although elevated gal3 expression is linked with myocardial fibrosis, its role in predicting the risk of SCD is unknown. METHODS: We reviewed the clinical datasets and post-mortem examination of 221 subjects who had died suddenly. We examined myocardial pathology including the extent of cardiac hypertrophy, fibrosis, and the degree of coronary atherosclerosis in these subjects. In a select group of SCD subjects, we studied myocardial gal3 and periostin expression using immunohistochemistry. To further examine if a higher level of circulating gal3 can be detected preceding sudden death, we measured serum gal3 in a porcine model of subtotal coronary artery ligation which shows an increased tendency to develop lethal cardiac arrhythmias, including ventricular tachycardia or fibrillation. RESULTS: Of the total 1314 human subjects screened, 12.7% had SCD. Comparison of age-matched SCD with non-SCD subjects showed that SCD groups had excessive myocardial fibrosis involving both the left ventricular free wall and interventricular septum. In pigs with subtotal coronary artery ligation and SCD, we detected significantly elevated circulating gal3 levels approximately 10 days preceding the SCD event. Immunohistochemistry showed increased myocardial gal3 and periostin expression in pigs that died suddenly, compared to the controls. CONCLUSION: Our study shows that increased gal3 is associated with a higher risk of myocardial fibrosis and the risk of SCD. This supports the importance of larger translational studies to target gal3 to prevent cardiac fibrosis and attenuate the risk of SCD.

Modified GAN Augmentation Algorithms for the MRI-Classification of Myocardial Scar Tissue in Ischemic Cardiomyopathy.

Contrast-enhanced cardiac magnetic resonance imaging (MRI) is routinely used to determine myocardial scar burden and make therapeutic decisions for coronary revascularization. Currently, there are no optimized deep-learning algorithms for the automated classification of scarred vs. normal myocardium. We report a modified Generative Adversarial Network (GAN) augmentation method to improve the binary classification of myocardial scar using both pre-clinical and clinical approaches. For the initial training of the MobileNetV2 platform, we used the images generated from a high-field (9.4T) cardiac MRI of a mouse model of acute myocardial infarction (MI). Once the system showed 100% accuracy for the classification of acute MI in mice, we tested the translational significance of this approach in 91 patients with an ischemic myocardial scar, and 31 control subjects without evidence of myocardial scarring. To obtain a comparable augmentation dataset, we rotated scar images 8-times and control images 72-times, generating a total of 6,684 scar images and 7,451 control images. In humans, the use of Progressive Growing GAN (PGGAN)-based augmentation showed 93% classification accuracy, which is far superior to conventional automated modules. The use of other attention modules in our CNN further improved the classification accuracy by up to 5%. These data are of high translational significance and warrant larger multicenter studies in the future to validate the clinical implications.

Small Endogeneous Peptide Mitigates Myocardial Remodeling in a Mouse Model of Cardioselective Galectin-3 Overexpression.

BACKGROUND: Myocardial Gal3 (galectin-3) expression is associated with cardiac inflammation and fibrosis. Increased Gal3 portends susceptibility to heart failure and death. There are no data reporting the causative role of Gal3 to mediate cardiac fibro-inflammatory response and heart failure. METHODS: We developed a cardioselective Gal3 gain-of-function mouse (Gal3+/+) using α-myosin heavy chain promotor. We confirmed Gal3-transgene expression with real-time polymerase chain reaction and quantified cardiac/circulating Gal3 with Western blot and immunoassays. We used echocardiogram and cardiac magnetic resonance imaging to measure cardiac volumes, function, and myocardial velocities. Ex vivo, we studied myocardial inflammation/fibrosis and downstream TGF (transforming growth factor) ß1-mRNA expression. We examined the effects of acute myocardial ischemia in presence of excess Gal3 by inducing acute myocardial infarction in mice. Two subsets of mice including mice treated with N-acetyl-seryl-aspartyl-lysyl-proline (a Gal3-inhibitor) and mice with genetic Gal3 loss-of-function (Gal3-/-) were studied for comparative analysis of Gal3 function. RESULTS: Gal3+/+ mice had increased cardiac/circulating Gal3. Gal3+/+ mice showed excess pericardial fat pad, dilated ventricles and cardiac dysfunction, which was partly normalized by N-acetyl-seryl-aspartyl-lysyl-proline. Cardiac magnetic resonance imaging showed reduced myocardial contractile velocities in Gal3+/+. The majority of Gal3+/+ mice did not survive acute myocardial infarction, and the survivors had profound cardiac dysfunction. Myocardial histology of Gal3+/+ mice showed macrophage/mast-cell infiltration, fibrosis and higher TGFß1-mRNA expression, which were mitigated by both Gal3 gene deletion and N-acetyl-seryl-aspartyl-lysyl-proline administration. CONCLUSIONS: Our study shows that cardioselective Gal3 overexpression leads to multiple cardiac phenotypic defects including ventricular dilation and cardiac dysfunction. Pharmacological Gal3 inhibition conferred protective effects with reduction of inflammation and fibrosis. Our study highlights the importance of translational studies to counteract Gal3 function and prevent cardiac dysfunction.

Tagged cine magnetic resonance imaging to quantify regional mechanical changes after acute myocardial infarction.

PURPOSE: The conventional volumetric approaches of measuring cardiac function are load-dependent, and are not able to discriminate functional changes in the infarct, transition and remote myocardium. We examined phase-dependent regional mechanical changes in the infarct, transition and remote regions after acute myocardial infarction (MI) in a preclinical mouse model using cardiovascular magnetic resonance imaging (CMR). METHODS: We induced acute MI in six mice with left anterior descending coronary artery ligation. We then examined cardiac (infarct, transition and remote-zone) morphology and function utilizing 9.4 T high field CMR before and 2 weeks after the induction of acute MI. Myocardial scar tissue was evaluated by using CMR with late gadolinium enhancement (LGE). After determining global function through volumetric analysis, regional wall motion was evaluated by measuring wall thickening and radial velocities. Strain rate imaging was performed to assess circumferential contraction and relaxation at the myocardium, endocardium, and epicardium. RESULTS: There was abnormal LGE in the anterior walls after acute MI suggesting a successful MI procedure. The transition zone consisted of a mixed signal intensity, while the remote zone contained viable myocardium. As expected, the infarct zone had demonstrated severely decreased myocardial velocities and strain rates, suggesting reduced contraction and relaxation function. Compared to pre-infarct baseline, systolic and diastolic velocities (vS and vD) were significantly reduced at the transition zone (vS: -1.86 ±â€¯0.16 cm/s vs -0.68 ±â€¯0.13 cm/s, P < 0.001; vD: 1.86 ±â€¯0.17 cm/s vs 0.53 ±â€¯0.06 cm/s, P < 0.001) and remote zone (vS: -1.86 ±â€¯0.16 cm/s vs -0.65 ±â€¯0.12 cm/s, P < 0.001; vD: 1.86 ±â€¯0.16 cm/s vs 0.51 ±â€¯0.04 cm/s, P < 0.001). Myocardial peak systolic and diastolic strain rates (SRS and SRD) were significantly lower in the transition zone (SRS: -4.2 ±â€¯0.3 s-1 vs -1.3 ±â€¯0.2 s-1, P < 0.001; SRD: 3.9 ±â€¯0.3 s-1 vs 1.3 ±â€¯0.2 s-1, P < 0.001) and remote zone (SRS: -3.8 ±â€¯0.3 s-1 vs -1.4 ±â€¯0.3 s-1, P < 0.001; SRD: 3.5 ±â€¯0.2 s-1 vs 1.5 ±â€¯0.4 s-1, P = 0.006). Endocardial and epicardial SRS and SRD were similarly reduced in the transition and remote zones compared to baseline. CONCLUSIONS: This study, for the first time, utilized state-of-the art high-field CMR algorithms in a preclinical mouse model for a comprehensive and controlled evaluation of the regional mechanical changes in the transition and remote zones, after acute MI. Our data demonstrate that CMR can quantitatively monitor dynamic post-MI remodeling in the transition and remote zones, thereby serving as a gold standard tool for therapeutic surveillance.

A Small Peptide Ac-SDKP Inhibits Radiation-Induced Cardiomyopathy.

BACKGROUND: Advances in radiotherapy for thoracic cancers have resulted in improvement of survival. However, radiation exposure to the heart can induce cardiotoxicity. No therapy is currently available to inhibit these untoward effects. We examined whether a small tetrapeptide, N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP), can counteract radiation-induced cardiotoxicity by inhibiting macrophage-dependent inflammatory and fibrotic pathways. METHODS AND RESULTS: After characterizing a rat model of cardiac irradiation with magnetic resonance imaging protocols, we examined the effects of Ac-SDKP in radiation-induced cardiomyopathy. We treated rats with Ac-SDKP for 18 weeks. We then compared myocardial contractile function and extracellular matrix by cardiac magnetic resonance imaging and the extent of inflammation, fibrosis, and Mac-2 (galectin-3) release by tissue analyses. Because Mac-2 is a crucial macrophage-derived mediator of fibrosis, we performed studies to determine Mac-2 synthesis by macrophages in response to radiation, and change in profibrotic responses by Mac-2 gene depleted cardiac fibroblasts after radiation. Cardiac irradiation diminished myocardial contractile velocities and enhanced extracellular matrix deposition. This was accompanied by macrophage infiltration, fibrosis, cardiomyocyte apoptosis, and cardiac Mac-2 expression. Ac-SDKP strongly inhibited these detrimental effects. Ac-SDKP migrated into the perinuclear cytoplasm of the macrophages and inhibited radiation-induced Mac-2 release. Cardiac fibroblasts lacking the Mac-2 gene showed reduced transforming growth factor ß1, collagen I, and collagen III expression after radiation exposure. CONCLUSIONS: Our study identifies novel cardioprotective effects of Ac-SDKP in a model of cardiac irradiation. These protective effects are exerted by inhibiting inflammation, fibrosis, and reducing macrophage activation. This study shows a therapeutic potential of this endogenously released peptide to counteract radiation-induced cardiomyopathy.

Effects of a novel peptide Ac-SDKP in radiation-induced coronary endothelial damage and resting myocardial blood flow.

BACKGROUND: Cancer survivors treated with thoracic ionizing radiation are at higher risk of premature death due to myocardial ischemia. No therapy is currently available to prevent or mitigate these effects. We tested the hypothesis that an endogenous tetrapeptide N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP) counteracts radiation-induced coronary vascular fibrosis and endothelial cell loss and preserves myocardial blood flow. METHODS: We examined a rat model with external-beam-radiation exposure to the cardiac silhouette. We treated a subgroup of irradiated rats with subcutaneous Ac-SDKP for 18-weeks. We performed cardiac MRI with Gadolinium contrast to examine resting myocardial blood flow content. Upon sacrifice, we examined coronary endothelial-cell-density, fibrosis, apoptosis and endothelial tight-junction proteins (TJP). In vitro, we examined Ac-SDKP uptake by the endothelial cells and tested its effects on radiation-induced reactive oxygen species (ROS) generation. In vivo, we injected labeled Ac-SDKP intravenously and examined its endothelial localization after 4-h. RESULTS: We found that radiation exposure led to reduced resting myocardial blood flow content. There was concomitant endothelial cell loss and coronary fibrosis. Smaller vessels and capillaries showed more severe changes than larger vessels. Real-time PCR and confocal microscopy showed radiation-induced loss of TJ proteins including-claudin-1 and junctional adhesion molecule-2 (JAM-2). Ac-SDKP normalized myocardial blood flow content, inhibited endothelial cell loss, reduced coronary fibrosis and restored TJ-assembly. In vitro, Ac-SDKP localized to endothelial cells and inhibited radiation-induced endothelial ROS generation. In vivo, labeled Ac-SDKP was visualized into the endothelium 4-h after the intravenous injection. CONCLUSIONS: We concluded that Ac-SDKP has protective effects against radiation-induced reduction of myocardial blood flow. Such protective effects are likely mediated by neutralization of ROS-mediated injury, preservation of endothelial integrity and inhibition of fibrosis. This demonstrates a strong therapeutic potential of Ac-SDKP to counteract radiotherapy-induced coronary disease.