Myocardial blood flow is the dominant factor influencing cardiac magnetic resonance adenosine stress T2.

Stress imaging identifies ischemic myocardium by comparing hemodynamics during rest and hyperemic stress. Hyperemia affects multiple hemodynamic parameters in myocardium, including myocardial blood flow (MBF), myocardial blood volume (MBV), and venous blood oxygen levels (PvO2 ). Cardiac T2 is sensitive to these changes and therefore is a promising non-contrast option for stress imaging; however, the impact of individual hemodynamic factors on T2 is poorly understood, making the connection from altered T2 to changes within the tissue difficult. To better understand this interplay, we performed T2 mapping and measured various hemodynamic factors independently in healthy pigs at multiple levels of hyperemic stress, induced by different doses of adenosine (0.14-0.56 mg/kg/min). T1 mapping quantified changes in MBV. MBF was assessed with microspheres, and oxygen consumption was determined by the rate pressure product (RPP). Simulations were also run to better characterize individual contributions to T2. Myocardial T2, MBF, oxygen consumption, and MBV all changed to varying extents between each level of adenosine stress (T2 = 37.6-41.8 ms; MBF = 0.48-1.32 mL/min/g; RPP = 6507-4001 bmp*mmHg; maximum percent change in MBV = 1.31%). Multivariable analyses revealed MBF as the dominant influence on T2 during hyperemia (significant ß-values >7). Myocardial oxygen consumption had almost no effect on T2 (ß-values <0.002); since PvO2 is influenced by both oxygen consumption and MBF, PvO2 changes detected by T2 during adenosine stress can be attributed to MBF. Simulations varying PvO2 and MBV confirmed that PvO2 had the strongest influence on T2, but MBV became important at high PvO2 . Together, these data suggest a model where, during adenosine stress, myocardial T2 responds predominantly to changes in MBF, but at high hyperemia MBV is also influential. Thus, changes in adenosine stress T2 can now be interpreted in terms of the physiological changes that led to it, enabling T2 mapping to become a viable non-contrast option to detect ischemic myocardial tissue.

Action of iron chelator on intramyocardial hemorrhage and cardiac remodeling following acute myocardial infarction.

Intramyocardial hemorrhage is an independent predictor of adverse outcomes in ST-segment elevation myocardial infarction (STEMI). Iron deposition resulting from ischemia-reperfusion injury (I/R) is pro-inflammatory and has been associated with adverse remodeling. The role of iron chelation in hemorrhagic acute myocardial infarction (AMI) has never been explored. The purpose of this study was to investigate the cardioprotection offered by the iron-chelating agent deferiprone (DFP) in a porcine AMI model by evaluating hemorrhage neutralization and subsequent cardiac remodeling. Two groups of animals underwent a reperfused AMI procedure: control and DFP treated (N = 7 each). A comprehensive MRI examination was performed in healthy state and up to week 4 post-AMI, followed by histological assessment. Infarct size was not significantly different between the two groups; however, the DFP group demonstrated earlier resolution of hemorrhage (by T2* imaging) and edema (by T2 imaging). Additionally, ventricular enlargement and myocardial hypertrophy (wall thickness and mass) were significantly smaller with DFP, suggesting reduced adverse remodeling, compared to control. The histologic results were consistent with the MRI findings. To date, there is no effective targeted therapy for reperfusion hemorrhage. Our proof-of-concept study is the first to identify hemorrhage-derived iron as a therapeutic target in I/R and exploit the cardioprotective properties of an iron-chelating drug candidate in the setting of AMI. Iron chelation could potentially serve as an adjunctive therapy in hemorrhagic AMI.

Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts.

Pluripotent stem cells provide a potential solution to current epidemic rates of heart failure by providing human cardiomyocytes to support heart regeneration. Studies of human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) in small-animal models have shown favourable effects of this treatment. However, it remains unknown whether clinical-scale hESC-CM transplantation is feasible, safe or can provide sufficient myocardial regeneration. Here we show that hESC-CMs can be produced at a clinical scale (more than one billion cells per batch) and cryopreserved with good viability. Using a non-human primate model of myocardial ischaemia followed by reperfusion, we show that cryopreservation and intra-myocardial delivery of one billion hESC-CMs generates extensive remuscularization of the infarcted heart. The hESC-CMs showed progressive but incomplete maturation over a 3-month period. Grafts were perfused by host vasculature, and electromechanical junctions between graft and host myocytes were present within 2 weeks of engraftment. Importantly, grafts showed regular calcium transients that were synchronized to the host electrocardiogram, indicating electromechanical coupling. In contrast to small-animal models, non-fatal ventricular arrhythmias were observed in hESC-CM-engrafted primates. Thus, hESC-CMs can remuscularize substantial amounts of the infarcted monkey heart. Comparable remuscularization of a human heart should be possible, but potential arrhythmic complications need to be overcome.

Stem cell-derived cardiomyocytes expressing a dominant negative pacemaker HCN4 channel do not reduce the risk of graft-related arrhythmias.

Background: Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) show tremendous promise for cardiac regeneration following myocardial infarction (MI), but their transplantation gives rise to transient ventricular tachycardia (VT) in large-animal MI models, representing a major hurdle to translation. Our group previously reported that these arrhythmias arise from a focal mechanism whereby graft tissue functions as an ectopic pacemaker; therefore, we hypothesized that hPSC-CMs engineered with a dominant negative form of the pacemaker ion channel HCN4 (dnHCN4) would exhibit reduced automaticity and arrhythmogenic risk following transplantation. Methods: We used CRISPR/Cas9-mediated gene-editing to create transgenic dnHCN4 hPSC-CMs, and their electrophysiological behavior was evaluated in vitro by patch-clamp recordings and optical mapping. Next, we transplanted WT and homozygous dnHCN4 hPSC-CMs in a pig MI model and compared post-transplantation outcomes including the incidence of spontaneous arrhythmias and graft structure by immunohistochemistry. Results: In vitro dnHCN4 hPSC-CMs exhibited significantly reduced automaticity and pacemaker funny current (I f ) density relative to wildtype (WT) cardiomyocytes. Following transplantation with either dnHCN4 or WT hPSC-CMs, all recipient hearts showed transmural infarct scar that was partially remuscularized by scattered islands of human myocardium. However, in contrast to our hypothesis, both dnHCN4 and WT hPSC-CM recipients exhibited frequent episodes of ventricular tachycardia (VT). Conclusions: While genetic silencing of the pacemaker ion channel HCN4 suppresses the automaticity of hPSC-CMs in vitro, this intervention is insufficient to reduce VT risk post-transplantation in the pig MI model, implying more complex mechanism(s) are operational in vivo.

raw Functions through JNK signaling and cadherin-based adhesion to regulate Drosophila gonad morphogenesis.

To form a gonad, germ cells (GCs) and somatic gonadal precursor cells (SGPs) must migrate to the correct location in the developing embryo and establish the cell-cell interactions necessary to create proper gonad architecture. During gonad morphogenesis, SGPs send out cellular extensions to ensheath the individual GCs and promote their development. We have identified mutations in the raw gene that result in a failure of the SGPs to ensheath the GCs, leading to defects in GC development. Using genetic analysis and gene expression studies, we find that Raw negatively regulates JNK signaling during gonad morphogenesis, and increased JNK signaling is sufficient to cause ensheathment defects. In particular, Raw functions upstream of the Drosophila Jun-related transcription factor to regulate its subcellular localization. Since JNK signaling regulates cell adhesion during the morphogenesis of many tissues, we examined the relationship between raw and the genes encoding Drosophila E-cadherin and ß-catenin, which function together in cell adhesion. We find that loss of DE-cadherin strongly enhances the raw mutant gonad phenotype, while increasing DE-cadherin function rescues this phenotype. Further, loss of raw results in mislocalization of ß-catenin away from the cell surface. Therefore, cadherin-based cell adhesion, likely at the level of ß-catenin, is a primary mechanism by which Raw regulates germline-soma interaction.

Advanced Techniques for Studying Blood Flow.

ARTICLES DISCUSSED: Tang, G. L., Kim, K. J. Laser Doppler perfusion imaging in the mouse hindlimb. Journal of Visualized Experiments. (170), e62012 (2021). Hage, B. D., Truemper, E. J., Bashford, G.R. Functional transcranial Doppler ultrasound for monitoring cerebral blood flow. Journal of Visualized Experiments. (169), e62048 (2021). Baranger, J., Mertens, L., Villemain, O. Blood flow imaging with ultrafast Doppler. Journal of Visualized Experiments. (164), e61838 (2020). Granja, T., de Andrade, S. F., Rodrigues, L. M. Multispectral optoacoustic tomography for functional imaging in vascular research. Journal of Visualized Experiments. (184), e63883 (2022). Goolaub, D. S., Marini, D., Seed, M., Macgowan, C. K. Human fetal blood flow quantification with magnetic resonance imaging and motion compensation. Journal of Visualized Experiments. (167), e61953 (2021).

A genetic screen for mutations affecting gonad formation in Drosophila reveals a role for the slit/robo pathway.

Organogenesis is a complex process requiring multiple cell types to associate with one another through correct cell contacts and in the correct location to achieve proper organ morphology and function. To better understand the mechanisms underlying gonad formation, we performed a mutagenesis screen in Drosophila and identified twenty-four genes required for gonadogenesis. These genes affect all different aspects of gonad formation and provide a framework for understanding the molecular mechanisms that control these processes. We find that gonad formation is regulated by multiple, independent pathways; some of these regulate the key cell adhesion molecule DE-cadherin, while others act through distinct mechanisms. In addition, we discover that the Slit/Roundabout pathway, best known for its role in regulating axonal guidance, is essential for proper gonad formation. Our findings shed light on the complexities of gonadogenesis and the genetic regulation required for proper organ formation.

Sonic Hedgehog upregulation does not enhance the survival and engraftment of stem cell-derived cardiomyocytes in infarcted hearts.

The engraftment of human stem cell-derived cardiomyocytes (hSC-CMs) is a promising treatment for remuscularizing the heart wall post-infarction, but it is plagued by low survival of transplanted cells. We hypothesize that this low survival rate is due to continued ischemia within the infarct, and that increasing the vascularization of the scar will ameliorate the ischemia and improve hSC-CM survival and engraftment. An adenovirus expressing the vascular growth factor Sonic Hedgehog (Shh) was injected into the infarcted myocardium of rats immediately after ischemia/reperfusion, four days prior to hSC-CM injection. By two weeks post-cell injection, Shh treatment had successfully increased capillary density outside the scar, but not within the scar. In addition, there was no change in vessel size or percent vascular volume when compared to cell injection alone. Micro-computed tomography revealed that Shh failed to increase the number and size of larger vessels. It also had no effect on graft size or heart function when compared to cell engraftment alone. Our data suggests that, when combined with the engraftment of hSC-CMs, expression of Shh within the infarct scar and surrounding myocardium is unable to increase vascularization of the infarct scar, and it does not improve survival or function of hSC-CM grafts.

Interference-free Detection of Lipid-laden Atherosclerotic Plaques by 3D Co-registration of Frequency-Domain Differential Photoacoustic and Ultrasound Radar Imaging.

As lipid composition of atherosclerotic plaques is considered to be one of the primary indicators for plaque vulnerability, a diagnostic modality that can sensitively evaluate their necrotic core is highly desirable in atherosclerosis imaging. In this regard, intravascular photoacoustic (IVPA) imaging is an emerging plaque detection modality that provides lipid-specific chemical information of arterial walls. Within the near-infrared window, a 1210-nm optical source is usually chosen for IVPA applications because lipid exhibits a strong absorption peak at that wavelength. However, other arterial tissues also show some degree of absorption near 1210 nm and generate undesirable interfering PA signals. In this study, a novel wavelength-modulated Intravascular Differential Photoacoustic Radar (IV-DPAR) modality was introduced as an interference-free detection technique for a more accurate and reliable diagnosis of plaque progression. By using two low-power continuous-wave laser diodes in a differential manner, IV-DPAR could efficiently suppress undesirable absorptions and system noise, while dramatically improving system sensitivity and specificity to cholesterol, the primary ingredient of plaque necrotic core. When co-registered with intravascular ultrasound imaging, IV-DPAR could sensitively locate and characterize the lipid contents of plaques in human atherosclerotic arteries, regardless of their size and depth.

Development of a 3 French Dual-Frequency Intravascular Ultrasound Catheter.

Coronary plaque morphology, including plaque size and fibrous cap thickness, is thought to contribute to the risk of plaque rupture and future cardiac events. Dual-frequency intravascular ultrasound has been proposed as a possible technique to visualize both large-scale features and superficial detail of coronary plaque; however, it has not been found to be feasible within the constraints of a clinically functional intravascular ultrasound catheter. In this study, we describe the design and fabrication of a dual-frequency catheter using a bidirectional transducer stack with center frequencies of approximately 30 and 80 MHz. We describe how the high-frequency transducer achieves significantly improved axial and lateral resolution (16 and 120 µm, respectively, vs. 50 and 220 µm) at the expense of penetration depth. Finally, imaging of ex vivo human coronary artery segments reveals that the catheter can provide complementary images of the deeper arterial wall and superficial plaque features.

Effects of cell grafting on coronary remodeling after myocardial infarction.

BACKGROUND: With recent advances in therapeutic applications of stem cells, cell engraftment has become a promising therapy for replacing injured myocardium after infarction. The survival and function of injected cells, however, will depend on the efficient vascularization of the new tissue. Here we describe the arteriogenic remodeling of the coronary vessels that supports vascularization of engrafted tissue postmyocardial infarction (post-MI). METHODS AND RESULTS: Following MI, murine hearts were injected with a skeletal myoblast cell line previously shown to develop into large grafts. Microcomputed tomography at 28 days postengraftment revealed the 3-dimensional structure of the newly formed conducting vessels. The grafts elicited both an angiogenic response and arteriogenic remodeling of the coronary arteries to perfuse the graft. The coronaries upstream of the graft also remodeled, showing an increase in branching, and a decrease in vascular density. Histological analysis revealed the presence of capillaries as well as larger vascular lumens within the graft. Some graft vessels were encoated by smooth muscle α-actin positive cells, implying that vascular remodeling occurs at both the conducting arterial and microvascular levels. CONCLUSIONS: Following MI and skeletal myoblast engraftment, the murine coronary vessels exhibit plasticity that enables both arteriogenic remodeling of the preexisting small branches of the coronary arteries and development of large and small smooth muscle encoated vessels within the graft. Understanding the molecular mechanisms underlying these 2 processes suggests mechanisms to enhance the therapeutic vascularization in patients with myocardial ischemia.

Retrograde perfusion and filling of mouse coronary vasculature as preparation for micro computed tomography imaging.

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for imaging vasculature, few are able to visualize the vascular network as a whole while extending to a resolution that includes the smaller vessels. Additionally, many vascular casting techniques destroy the surrounding tissue, preventing further analysis of the sample. One method which circumvents these issues is micro-Computed Tomography (µCT). µCT imaging can scan at resolutions <10 microns, is capable of producing 3D reconstructions of the vascular network, and leaves the tissue intact for subsequent analysis (e.g., histology and morphometry). However, imaging vessels by ex vivo µCT methods requires that the vessels be filled with a radiopaque compound. As such, the accurate representation of vasculature produced by µCT imaging is contingent upon reliable and complete filling of the vessels. In this protocol, we describe a technique for filling mouse coronary vessels in preparation for µCT imaging. Two predominate techniques exist for filling the coronary vasculature: in vivo via cannulation and retrograde perfusion of the aorta (or a branch off the aortic arch), or ex vivo via a Langendorff perfusion system. Here we describe an in vivo aortic cannulation method which has been specifically designed to ensure filling of all vessels. We use a low viscosity radiopaque compound called Microfil which can perfuse through the smallest vessels to fill all the capillaries, as well as both the arterial and venous sides of the vascular network. Vessels are perfused with buffer using a pressurized perfusion system, and then filled with Microfil. To ensure that Microfil fills the small higher resistance vessels, we ligate the large branches emanating from the aorta, which diverts the Microfil into the coronaries. Once filling is complete, to prevent the elastic nature of cardiac tissue from squeezing Microfil out of some vessels, we ligate accessible major vascular exit points immediately after filling. Therefore, our technique is optimized for complete filling and maximum retention of the filling agent, enabling visualization of the complete coronary vascular network--arteries, capillaries, and veins alike.

The threshold for polyglutamine-expansion protein aggregation and cellular toxicity is dynamic and influenced by aging in Caenorhabditis elegans.

Studies of the mutant gene in Huntington's disease, and for eight related neurodegenerative disorders, have identified polyglutamine (polyQ) expansions as a basis for cellular toxicity. This finding has led to a disease hypothesis that protein aggregation and cellular dysfunction can occur at a threshold of approximately 40 glutamine residues. Here, we test this hypothesis by expression of fluorescently tagged polyQ proteins (Q29, Q33, Q35, Q40, and Q44) in the body wall muscle cells of Caenorhabditis elegans and show that young adults exhibit a sharp boundary at 35-40 glutamines associated with the appearance of protein aggregates and loss of motility. Surprisingly, genetically identical animals expressing near-threshold polyQ repeats exhibited a high degree of variation in the appearance of protein aggregates and cellular toxicity that was dependent on repeat length and exacerbated during aging. The role of genetically determined aging pathways in the progression of age-dependent polyQ-mediated aggregation and cellular toxicity was tested by expressing Q82 in the background of age-1 mutant animals that exhibit an extended lifespan. We observed a dramatic delay of polyQ toxicity and appearance of protein aggregates. These data provide experimental support for the threshold hypothesis of polyQ-mediated toxicity in an experimental organism and emphasize the importance of the threshold as a point at which genetic modifiers and aging influence biochemical environment and protein homeostasis in the cell.