Sudden death is the most common mode of exodus in patients with heart failure and preserved ejection fraction (HFpEF). Cardiosphere-derived cells (CDCs) reduce inflammation and fibrosis in a rat model of HFpEF, improving diastolic function and prolonging survival. We tested the hypothesis that CDCs decrease ventricular arrhythmias (VAs) and thereby possibly contribute to prolonged survival. Dahl salt-sensitive rats were fed a high-salt diet to induce HFpEF. Allogeneic rat CDCs (or phosphate-buffered saline as placebo) were injected in rats with echo-verified HFpEF. CDC-injected HFpEF rats were less prone to VA induction by programmed electrical stimulation. Action potential duration (APD) was shortened, and APD homogeneity was increased by CDC injection. Transient outward potassium current density was upregulated in cardiomyocytes from CDC rats relative to placebo, as were the underlying transcript (Kcnd3) and protein (Kv4.3) levels. Fibrosis was attenuated in CDC-treated hearts, and survival was increased. Sudden death risk also trended down, albeit nonsignificantly. CDC therapy decreased VA in HFpEF rats by shortening APD, improving APD homogeneity, and decreasing fibrosis. Unlike other stem/progenitor cells, which often exacerbate arrhythmias, CDCs reverse electrical remodeling and suppress arrhythmogenesis in HFpEF.
Jae Hyung Cho, Peter J. Kilfoil, Rui Zhang, Ryan E. Solymani, Catherine Bresee, Elliot M. Kang, Kristin Luther, Russell G. Rogers, Geoffrey de Couto, Joshua I. Goldhaber, Eduardo Marbán, Eugenio Cingolani
Cardiac Nav1.5 and Kir2.1–2.3 channels generate Na (INa) and inward rectifier K (IK1) currents, respectively. The functional INa and IK1 interplay is reinforced by the positive and reciprocal modulation between Nav15 and Kir2.1/2.2 channels to strengthen the control of ventricular excitability. Loss-of-function mutations in the SCN5A gene, which encodes Nav1.5 channels, underlie several inherited arrhythmogenic syndromes, including Brugada syndrome (BrS). We investigated whether the presence of BrS-associated mutations alters IK1 density concomitantly with INa density. Results obtained using mouse models of SCN5A haploinsufficiency, and the overexpression of native and mutated Nav1.5 channels in expression systems — rat ventricular cardiomyocytes and human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) — demonstrated that endoplasmic reticulum (ER) trafficking–defective Nav1.5 channels significantly decreased IK1, since they did not positively modulate Kir2.1/2.2 channels. Moreover, Golgi trafficking–defective Nav1.5 mutants produced a dominant negative effect on Kir2.1/2.2 and thus an additional IK1 reduction. Moreover, ER trafficking–defective Nav1.5 channels can be partially rescued by Kir2.1/2.2 channels through an unconventional secretory route that involves Golgi reassembly stacking proteins (GRASPs). Therefore, cardiac excitability would be greatly affected in subjects harboring Nav1.5 mutations with Golgi trafficking defects, since these mutants can concomitantly trap Kir2.1/2.2 channels, thus unexpectedly decreasing IK1 in addition to INa.
Marta Pérez-Hernández, Marcos Matamoros, Silvia Alfayate, Paloma Nieto-Marín, Raquel G. Utrilla, David Tinaquero, Raquel de Andrés, Teresa Crespo, Daniela Ponce-Balbuena, B. Cicero Willis, Eric N. Jiménez-Vazquez, Guadalupe Guerrero-Serna, Andre M. da Rocha, Katherine Campbell, Todd J. Herron, F. Javier Díez-Guerra, Juan Tamargo, José Jalife, Ricardo Caballero, Eva Delpón
Stroke triggers a complex inflammatory process in which the balance between pro- and antiinflammatory mediators is critical for the development of the brain infarct. However, systemic changes may also occur in parallel with brain inflammation. Here we demonstrate that administration of recombinant IL-33, a recently described member of the IL-1 superfamily of cytokines, promotes Th2-type effects following focal ischemic stroke, resulting in increased plasma levels of Th2-type cytokines and fewer proinflammatory (3-nitrotyrosine+F4/80+) microglia/macrophages in the brain. These effects of IL-33 were associated with reduced infarct size, fewer activated microglia and infiltrating cytotoxic (natural killer–like) T cells, and more IL-10–expressing regulatory T cells. Despite these neuroprotective effects, mice treated with IL-33 displayed exacerbated post-stroke lung bacterial infection in association with greater functional deficits and mortality at 24 hours. Supplementary antibiotics (gentamicin and ampicillin) mitigated these systemic effects of IL-33 after stroke. Our findings highlight the complex nature of the inflammatory mechanisms differentially activated in the brain and periphery during the acute phase after ischemic stroke. The data indicate that a Th2-promoting agent can provide neuroprotection without adverse systemic effects when given in combination with antibiotics.
Shenpeng R. Zhang, Marius Piepke, Hannah X. Chu, Brad R.S. Broughton, Raymond Shim, Connie H.Y. Wong, Seyoung Lee, Megan A. Evans, Antony Vinh, Samy Sakkal, Thiruma V. Arumugam, Tim Magnus, Samuel Huber, Mathias Gelderblom, Grant R. Drummond, Christopher G. Sobey, Hyun Ah Kim
BACKGROUND. Plasma lipidomic measures may enable improved prediction of cardiovascular outcomes in secondary prevention. The aim of this study is to determine the association of plasma lipidomic measurements with cardiovascular events and assess their potential to predict such events. METHODS. Plasma lipids (n = 342) were measured in a retrospective subcohort (n = 5,991) of the LIPID study. Proportional hazards regression was used to identify lipids associated with future cardiovascular events (nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death) and cardiovascular death. Multivariable models adding lipid species to traditional risk factors were created using lipid ranking established from the Akaike information criterion within a 5-fold cross-validation framework. The results were tested on a diabetic case cohort from the ADVANCE study (n = 3,779). RESULTS. Specific ceramide species, sphingolipids, phospholipids, and neutral lipids containing omega-6 fatty acids or odd-chain fatty acids were associated with future cardiovascular events (106 species) and cardiovascular death (139 species). The addition of 7 lipid species to a base model (11 conventional risk factors) resulted in an increase in the C-statistics from 0.629 (95% CI, 0.628–0.630) to 0.654 (95% CI, 0.653–0.656) for prediction of cardiovascular events and from 0.673 (95% CI, 0.671–0.675) to 0.727 (95% CI, 0.725–0.728) for prediction of cardiovascular death. Categorical net reclassification improvements for cardiovascular events and cardiovascular death were 0.083 (95% CI, 0.081–0.086) and 0.166 (95% CI, 0.162–0.170), respectively. Evaluation on the ADVANCE case cohort demonstrated significant improvement on the base models. CONCLUSIONS. The improvement in the prediction of cardiovascular outcomes, above conventional risk factors, demonstrates the potential of plasma lipidomic profiles as biomarkers for cardiovascular risk stratification in secondary prevention. FUNDING. Bristol-Myers Squibb, the National Health and Medical Research Council of Australia (grants 211086, 358395, and 1029754), and the Operational Infrastructure Support Program of the Victorian government of Australia.
Piyushkumar A. Mundra, Christopher K. Barlow, Paul J. Nestel, Elizabeth H. Barnes, Adrienne Kirby, Peter Thompson, David R. Sullivan, Zahir H. Alshehry, Natalie A. Mellett, Kevin Huynh, Kaushala S. Jayawardana, Corey Giles, Malcolm J. McConville, Sophia Zoungas, Graham S. Hillis, John Chalmers, Mark Woodward, Gerard Wong, Bronwyn A. Kingwell, John Simes, Andrew M. Tonkin, Peter J. Meikle, LIPID Study Investigators
Inhibiting MAPK14 (p38α) diminishes cardiac damage in myocardial ischemia. During myocardial ischemia, p38α interacts with TAB1, a scaffold protein, which promotes p38α autoactivation; active p38α (pp38α) then transphosphorylates TAB1. Previously, we solved the X-ray structure of the p38α-TAB1 (residues 384–412) complex. Here, we further characterize the interaction by solving the structure of the pp38α-TAB1 (residues 1–438) complex in the active state. Based on this information, we created a global knock-in (KI) mouse with substitution of 4 residues on TAB1 that we show are required for docking onto p38α. Whereas ablating p38α or TAB1 resulted in early embryonal lethality, the TAB1-KI mice were viable and had no appreciable alteration in their lymphocyte repertoire or myocardial transcriptional profile; nonetheless, following in vivo regional myocardial ischemia, infarction volume was significantly reduced and the transphosphorylation of TAB1 was disabled. Unexpectedly, the activation of myocardial p38α during ischemia was only mildly attenuated in TAB1-KI hearts. We also identified a group of fragments able to disrupt the interaction between p38α and TAB1. We conclude that the interaction between the 2 proteins can be targeted with small molecules. The data reveal that it is possible to selectively inhibit signaling downstream of p38α to attenuate ischemic injury.
Gian F. De Nicola, Rekha Bassi, Charlie Nichols, Mariana Fernandez-Caggiano, Pelin Arabacilar Golforoush, Dibesh Thapa, Rhys Anderson, Eva Denise Martin, Sharwari Verma, Jens Kleinjung, Adam Laing, Jonathan P. Hutchinson, Philip Eaton, James Clark, Michael S. Marber
Despite advances in antithrombotic therapy, the risk of recurrent coronary/cerebrovascular ischemia or venous thromboembolism remains high. Dual pathway antithrombotic blockade, using both antiplatelet and anticoagulant therapy, offers the promise of improved thrombotic protection; however, widespread adoption remains tempered by substantial risk of major bleeding. Here, we report a dual pathway therapeutic capable of site-specific targeting to activated platelets and therapeutic enrichment at the site of thrombus growth to allow reduced dosing without compromised antithrombotic efficacy. We engineered a recombinant fusion protein, SCE5-TAP, which consists of a single-chain antibody (SCE5) that targets and blocks the activated GPIIb/IIIa complex, and tick anticoagulant peptide (TAP), a potent direct inhibitor of activated factor X (FXa). SCE5-TAP demonstrated selective platelet targeting and inhibition of thrombosis in murine models of both carotid artery and inferior vena cava thrombosis, without a significant impact on hemostasis. Selective targeting to activated platelets provides an attractive strategy to achieve high antithrombotic efficacy with reduced risk of bleeding complications.
Donny Hanjaya-Putra, Carolyn Haller, Xiaowei Wang, Erbin Dai, Bock Lim, Liying Liu, Patrick Jaminet, Joy Yao, Amy Searle, Thomas Bonnard, Christoph E. Hagemeyer, Karlheinz Peter, Elliot L. Chaikof
MicroRNAs (miRs) posttranscriptionally regulate mRNA and its translation into protein, and are considered master controllers of genes modulating normal physiology and disease. There is growing interest in how miRs change with drug treatment, and leveraging this for precision guided therapy. Here we contrast 2 closely related therapies, inhibitors of phosphodiesterase type 5 or type 9 (PDE5-I, PDE9-I), given to mice subjected to sustained cardiac pressure overload (PO). Both inhibitors augment cyclic guanosine monophosphate (cGMP) to activate protein kinase G, with PDE5-I regulating nitric oxide (NO) and PDE9-I natriuretic peptide–dependent signaling. While both produced strong phenotypic improvement of PO pathobiology, they surprisingly showed binary differences in miR profiles; PDE5-I broadly reduces more than 120 miRs, including nearly half those increased by PO, whereas PDE9-I has minimal impact on any miR (P < 0.0001). The disparity evolves after pre-miR processing and is organ specific. Lastly, even enhancing NO-coupled cGMP by different methods leads to altered miR regulation. Thus, seemingly similar therapeutic interventions can be barcoded by profound differences in miR signatures, and reversing disease-associated miR changes is not required for therapy success.
Kristen M. Kokkonen-Simon, Amir Saberi, Taishi Nakamura, Mark J. Ranek, Guangshuo Zhu, Djahida Bedja, Michaela Kuhn, Marc K. Halushka, Dong Ik Lee, David A. Kass
Cantu syndrome (CS) is characterized by multiple vascular and cardiac abnormalities including vascular dilation and tortuosity, systemic hypotension, and cardiomegaly. The disorder is caused by gain-of-function (GOF) mutations in genes encoding pore-forming (Kir6.1, KCNJ8) and accessory (SUR2, ABCC9) ATP-sensitive potassium (KATP) channel subunits. However, there is little understanding of the link between molecular dysfunction and the complex pathophysiology observed, and there is no known treatment, in large part due to the lack of appropriate preclinical disease models in which to test therapies. Notably, expression of Kir6.1 and SUR2 does not fully overlap, and the relative contribution of KATP GOF in various cardiovascular tissues remains to be elucidated. To investigate pathophysiologic mechanisms in CS we have used CRISPR/Cas9 engineering to introduce CS-associated SUR2[A478V] and Kir6.1[V65M] mutations to the equivalent endogenous loci in mice. Mirroring human CS, both of these animals exhibit low systemic blood pressure and dilated, compliant blood vessels, as well dramatic cardiac enlargement, the effects being more severe in V65M animals than in A478V animals. In both animals, whole-cell patch-clamp recordings reveal enhanced basal KATP conductance in vascular smooth muscle, explaining vasodilation and lower blood pressure, and demonstrating a cardinal role for smooth muscle KATP dysfunction in CS etiology. Echocardiography confirms in situ cardiac enlargement and increased cardiac output in both animals. Patch-clamp recordings reveal reduced ATP sensitivity of ventricular myocyte KATP channels in A478V, but normal ATP sensitivity in V65M, suggesting that cardiac remodeling occurs secondary to KATP overactivity outside of the heart. These SUR2[A478V] and Kir6.1[V65M] animals thus reiterate the key cardiovascular features seen in human CS. They establish the molecular basis of the pathophysiological consequences of reduced smooth muscle excitability resulting from SUR2/Kir6.1–dependent KATP GOF, and provide a validated animal model in which to examine potential therapeutic approaches to treating CS.
Yan Huang, Conor McClenaghan, Theresa M. Harter, Kristina Hinman, Carmen M. Halabi, Scot J. Matkovich, Haixia Zhang, G. Schuyler Brown, Robert P. Mecham, Sarah K. England, Attila Kovacs, Maria S. Remedi, Colin G. Nichols
Hemodynamic shear force has been implicated as modulating Notch signaling–mediated cardiac trabeculation. Whether the spatiotemporal variations in wall shear stress (WSS) coordinate the initiation of trabeculation to influence ventricular contractile function remains unknown. Using light-sheet fluorescent microscopy, we reconstructed the 4D moving domain and applied computational fluid dynamics to quantify 4D WSS along the trabecular ridges and in the groves. In WT zebrafish, pulsatile shear stress developed along the trabecular ridges, with prominent endocardial Notch activity at 3 days after fertilization (dpf), and oscillatory shear stress developed in the trabecular grooves, with epicardial Notch activity at 4 dpf. Genetic manipulations were performed to reduce hematopoiesis and inhibit atrial contraction to lower WSS in synchrony with attenuation of oscillatory shear index (OSI) during ventricular development. γ-Secretase inhibitor of Notch intracellular domain (NICD) abrogated endocardial and epicardial Notch activity. Rescue with NICD mRNA restored Notch activity sequentially from the endocardium to trabecular grooves, which was corroborated by observed Notch-mediated cardiomyocyte proliferations on WT zebrafish trabeculae. We also demonstrated in vitro that a high OSI value correlated with upregulated endothelial Notch-related mRNA expression. In silico computation of energy dissipation further supports the role of trabeculation to preserve ventricular structure and contractile function. Thus, spatiotemporal variations in WSS coordinate trabecular organization for ventricular contractile function.
Juhyun Lee, Vijay Vedula, Kyung In Baek, Junjie Chen, Jeffrey J. Hsu, Yichen Ding, Chih-Chiang Chang, Hanul Kang, Adam Small, Peng Fei, Cheng-ming Chuong, Rongsong Li, Linda Demer, René R. Sevag Packard, Alison L. Marsden, Tzung K. Hsiai
Inflammation accompanies heart failure and is a mediator of cardiac fibrosis. CaMKIIδ plays an essential role in adverse remodeling and decompensation to heart failure. We postulated that inflammation is the mechanism by which CaMKIIδ contributes to adverse remodeling in response to nonischemic interventions. We demonstrate that deletion of CaMKIIδ in the cardiomyocyte (CKO) significantly attenuates activation of NF-κB, expression of inflammatory chemokines and cytokines, and macrophage accumulation induced by angiotensin II (Ang II) infusion. The inflammasome was activated by Ang II, and this response was also diminished in CKO mice. These events occurred prior to any evidence of Ang II–induced cell death. In addition, CaMKII-dependent inflammatory gene expression and inflammasome priming were observed as early as the third hour of infusion, a time point at which macrophage recruitment was not evident. Inhibition of either the inflammasome or monocyte chemoattractant protein 1 (MCP1) signaling attenuated macrophage accumulation, and these interventions, like cardiomyocyte CaMKIIδ deletion, diminished the fibrotic response to Ang II. Thus, activation of CaMKIIδ in the cardiomyocyte represents what we believe to be a novel mechanism for initiating inflammasome activation and an inflammatory gene program that leads to macrophage recruitment and ultimately to development of fibrosis.
Andrew Willeford, Takeshi Suetomi, Audrey Nickle, Hal M. Hoffman, Shigeki Miyamoto, Joan Heller Brown
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