Craig H. Selzman

MicroRNA-208a is a regulator of cardiac hypertrophy and conduction in mice

MicroRNAs (miRNAs) are a class of small noncoding RNAs that have gained status as important regulators of gene expression. Here, we investigated the function and molecular mechanisms of the miR-208 family of miRNAs in adult mouse heart physiology. We found that miR-208a, which is encoded within an intron of alpha-cardiac muscle myosin heavy chain gene (Myh6), was actually a member of a miRNA family that also included miR-208b, which was determined to be encoded within an intron of beta-cardiac muscle myosin heavy chain gene (Myh7). These miRNAs were differentially expressed in the mouse heart, paralleling the expression of their host genes. Transgenic overexpression of miR-208a in the heart was sufficient to induce hypertrophic growth in mice, which resulted in pronounced repression of the miR-208 regulatory targets thyroid hormone-associated protein 1 and myostatin, 2 negative regulators of muscle growth and hypertrophy. Studies of the miR-208a Tg mice indicated that miR-208a expression was sufficient to induce arrhythmias. Furthermore, analysis of mice lacking miR-208a indicated that miR-208a was required for proper cardiac conduction and expression of the cardiac transcription factors homeodomain-only protein and GATA4 and the gap junction protein connexin 40. Together, our studies uncover what we believe are novel miRNA-dependent mechanisms that modulate cardiac hypertrophy and electrical conduction.

Targeted deletion of Dicer in the heart leads to dilated cardiomyopathy and heart failure

Cardiovascular disease is the leading cause of human morbidity and mortality. Dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy associated with heart failure. Here, we report that cardiac-specific knockout of Dicer, a gene encoding a RNase III endonuclease essential for microRNA (miRNA) processing, leads to rapidly progressive DCM, heart failure, and postnatal lethality. Dicer mutant mice show misexpression of cardiac contractile proteins and profound sarcomere disarray. Functional analyses indicate significantly reduced heart rates and decreased fractional shortening of Dicer mutant hearts. Consistent with the role of Dicer in animal hearts, Dicer expression was decreased in end-stage human DCM and failing hearts and, most importantly, a significant increase of Dicer expression was observed in those hearts after left ventricle assist devices were inserted to improve cardiac function. Together, our studies demonstrate essential roles for Dicer in cardiac contraction and indicate that miRNAs play critical roles in normal cardiac function and under pathological conditions.

Contemporary management of isolated iliac aneurysms

OBJECTIVE Because isolated common iliac artery aneurysms are infrequent, are difficult to detect and treat, and have traditionally been associated with high operative mortality rates in reported series, we analyzed the outcomes of operative repair of 31 isolated common iliac artery aneurysms in 21 patients to ascertain morbidity and mortality rates with contemporary techniques of repair. METHODS A retrospective review study was conducted in a university teaching hospital and a Department of Veterans Affairs Medical Center. Perioperative mortality and operative morbidity rates were examined in 17 men and four women with isolated common iliac artery aneurysms between 1984 and 1997. Ages ranged from 38 to 87 years (mean 69 +/- 8 years). Slightly more than half of the cases were symptomatic, with abdominal pain, neurologic, claudicative, genitourinary, or hemodynamic symptoms. One aneurysm had ruptured and one was infected. There was one iliac artery-iliac vein fistula. All aneurysms involved the common iliac artery. Coexistent unilateral or bilateral external iliac aneurysms were present in four patients; there were three accompanying internal iliac aneurysms. Overall, 52% of patients had unilateral aneurysms and 48% had bilateral aneurysms. Aneurysms ranged in maximal diameter from 2.5 to 12 cm (mean 5.6 +/- 2 cm). No patients were unavailable for follow-up, which averaged 5.5 years. RESULTS Nineteen patients underwent direct operative repair of isolated iliac aneurysms. One patient had placement of an endoluminal covered stent graft; another patient at high risk had percutaneous placement of coils within the aneurysm to occlude it in conjunction with a femorofemoral bypass graft. Patients with bilateral aneurysms underwent aortoiliac or aortofemoral interposition grafts, whereas unilateral aneurysms were managed with local interposition grafts. There were no deaths in the perioperative period. Only one elective operation (5%) resulted in a significant complication, compartment syndrome requiring fasciotomy. The patient treated with the covered stent required femorofemoral bypass when the stent occluded 1 week after the operation. The patient treated with coil occlusion of a large common iliac aneurysm died 2 years later when the aneurysm ruptured. CONCLUSIONS Isolated iliac artery aneurysms can be managed with much lower mortality and morbidity rates than aneurysm previously been reported by using a systematic operative approach. Percutaneous techniques may be less durable and effective than direct surgical repair.

Pulsatility and the Risk of Nonsurgical Bleeding in Patients Supported With the Continuous-Flow Left Ventricular Assist Device HeartMate II

Background—Bleeding is an important cause of morbidity and mortality in patients with continuous-flow left ventricular assist devices (LVADs). Reduced pulsatility has been implicated as a contributing cause. The aim of this study was to assess the effects of different degrees of pulsatility on the incidence of nonsurgical bleeding. Methods and Results—The Utah Transplantation Affiliated Hospitals (U.T.A.H.) heart failure and transplant program databases were queried for patients with end-stage heart failure who required support with the continuous-flow LVAD HeartMate II (Thoratec Corp, Pleasanton, CA) between 2004 and 2012. Pulsatility was evaluated by means of the LVAD parameter pulsatility index (PI) and by the echocardiographic assessment of aortic valve opening during the first 3 months of LVAD support. PI was analyzed as a continuous variable and also stratified according to tertiles of all the PI measurements during the study period (low PI: <4.6, intermediate PI: 4.6–5.2, and high PI: >5.2). Major nonsurgical bleeding associated with a decrease in hemoglobin ≥2 g/dL (in the absence of hemolysis) was the primary end point. A total of 134 patients (median age of 60 [interquartile range: 49–68] years, 78% men) were included. Major bleeding occurred in 33 (25%) patients (70% gastrointestinal, 21% epistaxis, 3% genitourinary, and 6% intracranial). In multivariable analysis, PI examined either as a categorical variable, low versus high PI (hazard ratio, 4.06; 95% confidence interval, 1.35–12.21; P=0.04), or as a continuous variable (hazard ratio, 0.60; 95% confidence interval, 0.40–0.92; P=0.02) was associated with an increased risk of bleeding. Conclusions—Reduced pulsatility in patients supported with the continuous-flow LVAD HeartMate II is associated with an increased risk of nonsurgical bleeding, as evaluated by PI.

Impact of Mechanical Unloading on Microvasculature and Associated Central Remodeling Features of the Failing Human Heart

OBJECTIVES This study investigates alterations in myocardial microvasculature, fibrosis, and hypertrophy before and after mechanical unloading of the failing human heart. BACKGROUND Recent studies demonstrated the pathophysiologic importance and significant mechanistic links among microvasculature, fibrosis, and hypertrophy during the cardiac remodeling process. The effect of left ventricular assist device (LVAD) unloading on cardiac endothelium and microvasculature is unknown, and its influence on fibrosis and hypertrophy regression to the point of atrophy is controversial. METHODS Hemodynamic data and left ventricular tissue were collected from patients with chronic heart failure at LVAD implant and explant (n = 15) and from normal donors (n = 8). New advances in digital microscopy provided a unique opportunity for comprehensive whole-field, endocardium-to-epicardium evaluation for microvascular density, fibrosis, cardiomyocyte size, and glycogen content. Ultrastructural assessment was done with electron microscopy. RESULTS Hemodynamic data revealed significant pressure unloading with LVAD. This was accompanied by a 33% increase in microvascular density (p = 0.001) and a 36% decrease in microvascular lumen area (p = 0.028). We also identified, in agreement with these findings, ultrastructural and immunohistochemical evidence of endothelial cell activation. In addition, LVAD unloading significantly increased interstitial and total collagen content without any associated structural, ultrastructural, or metabolic cardiomyocyte changes suggestive of hypertrophy regression to the point of atrophy and degeneration. CONCLUSIONS The LVAD unloading resulted in increased microvascular density accompanied by increased fibrosis and no evidence of cardiomyocyte atrophy. These new insights into the effects of LVAD unloading on microvasculature and associated key remodeling features might guide future studies of unloading-induced reverse remodeling of the failing human heart.

Contributions of extravascular and intravascular cells to fibrin network formation, structure, and stability.

Fibrin is essential for hemostasis; however, abnormal fibrin formation is hypothesized to increase thrombotic risk. We previously showed that in situ thrombin generation on a cells surface modulates the 3-dimensional structure and stability of the fibrin network. Currently, we compared the abilities of extravascular and intravascular cells to support fibrin formation, structure, and stability. Extravascular cells (fibroblasts, smooth muscle) supported formation of dense fibrin networks that resisted fibrinolysis, whereas unstimulated intravascular (endothelial) cells produced coarse networks that were susceptible to fibrinolysis. All 3 cell types produced a fibrin structural gradient, with a denser network near, versus distal to, the cell surface. Although fibrin structure depended on cellular procoagulant activity, it did not reflect interactions between integrins and fibrin. These findings contrasted with those on platelets, which influenced fibrin structure via interactions between beta3 integrins and fibrin. Inflammatory cytokines that induced prothrombotic activity on endothelial cells caused the production of abnormally dense fibrin networks that resisted fibrinolysis. Blocking tissue factor activity significantly reduced the density and stability of fibrin networks produced by cytokine-stimulated endothelial cells. Together, these findings indicate fibrin structure and stability reflect the procoagulant phenotype of the endogenous cells, and suggest abnormal fibrin structure is a novel link between inflammation and thrombosis.

Acellular components of Chlamydia pneumoniae stimulate cytokine production in human blood mononuclear cells

Accumulating evidence suggest that infection with Chlamydia pneumoniae is associated with atherosclerosis, but the mechanisms involved remain unclear. Inflammation is important in the initial phase of atherogenesis, and cytokines are important in the initiation and progression of inflammation. The aim of this study was to assess the capacity of acellular components of C. pneumoniae to stimulate the production of pro‐inflammatory cytokines and chemokines. Peripheral blood mononuclear cells were stimulated in vitro with sonicated C. pneumoniae. Significant amounts of TNF‐α, IL‐1, IL‐6, IL‐8, monocyte chemoattractant protein‐1 (MCP‐1) and macrophage inflammatory protein‐1α (MIP‐1α) were produced. Inhibition of endotoxin using polymyxin B revealed that chlamydial endotoxin plays a minor role in the cytokine induction. Neutralization of TNF by TNF‐binding protein and blockade of IL‐1 receptors by IL‐1 receptor antagonist revealed that TNF, IL‐1 and IL‐6 production was independent from each other, whereas IL‐8 synthesis was strongly dependent on endogenous TNF and IL‐1. In contrast, synthesis of MCP‐1 and MIP‐1α was dependent on endogenous TNF, but not IL‐1. In conclusion, acellular components of C. pneumoniae are a potent stimulus for cytokine production, and this mechanism may have an important role in the inflammatory aspects of atherogenesis.

Bridge to Recovery Understanding the Disconnect Between Clinical and Biological Outcomes

Left ventricular (LV) assist devices (LVADs) are increasingly used in everyday clinical practice either as a bridge for end-stage heart failure (HF) patients to heart transplantation or as a permanent (destination) therapy.1,2 Yet, there is still significant uncertainty about the consequences of this intervention both at the level of the detailed myocardial biology (ie, biological outcomes) and at the functional cardiovascular response of the patient at the organ level (ie, clinical outcomes). The LVAD patient population presents a series of significant advantages as far as research is concerned. First, LVAD therapy offers the ability to acquire paired human myocardial tissue at LVAD implantation and again on LVAD removal. The ability to obtain human tissue and the possibility for its serial examination before and after any therapeutic investigational therapy combined with LVADs provide an important opportunity for in-depth study of the changes in the structure and function of the diseased human heart caused by the specific investigational therapy. Second, this population represents a relatively safe investigational platform because the hemodynamic support provided by VADs makes these patients significantly less vulnerable to any arrhythmic3 or hemodynamic adverse events potentially associated with new aggressive investigational therapies. Third, the volumes of potential study subjects for these investigations (ie, patients who receive LVADs) are rapidly increasing; because of a lack of donor organs and incremental progress in device design and durability, the number of advanced HF patients with LVADs has been continuously increasing.1,2 These 3 research advantages create an ideal setting for various new HF therapies to test their potential efficacy in LVAD patients. Fourth, this population offers an opportunity to investigate the effects of the LVAD-induced removal of excess mechanical load, which drives the vicious cycle of myocardial remodeling and eventually leads to the clinical HF syndrome. …

Functional Reconstitution and Regulation of IL-18 Activity by the IL-18Rβ Chain

IL-18 and IL-12 are major IFN-γ-inducing cytokines but the unique synergism of IL-18 and IL-12 remains unclear. In the human NK cell line NKO, IL-18Rα, and IL-18Rβ are expressed constitutively but IL-18 did not induce IFN-γ unless IL-12 was present. COS-1 fibroblasts, which produce the chemokine IL-8 when stimulated by IL-1β or TNF-α, do not respond to IL-18, despite abundant expression of the IL-18Rα chain. COS-1 cells lack expression of the IL-18Rβ chain. The IL-18Rβ cDNA was cloned from a human T-B lymphoblast cDNA library and COS-1 cells were transiently transfected with the IL-18Rβ chain and a luciferase reporter. In transfected COS-1 cells, IL-18 induced IL-8 and luciferase in the absence of IL-12 and independently of IL-1 and TNF. Ab against the IL-18Rα chain, however, prevented IL-18 responsiveness in COS-1 cells transfected with the IL-18Rβ chain, suggesting that both chains be functional. In NKO cells and PBMC, IL-12 increased steady-state mRNA levels of IL-18Rα and IL-18Rβ; the production of IFN-γ corresponded to IL-12-induced IL-18Rα and IL-18Rβ chains. We conclude that functional reconstitution of the IL-18Rβ chain is essential for IL-12-independent proinflammatory activity of IL-18-induced IL-8 in fibroblasts. The synergism of IL-18 plus IL-12 for IFN-γ production is, in part, due to IL-12 up-regulation of both IL-18Rα and IL-18Rβ chains, although postreceptor events likely contribute to IFN-γ production.

Morbidity and mortality in heart transplant candidates supported with mechanical circulatory support: is reappraisal of the current United network for organ sharing thoracic organ allocation policy justified?

Background— Survival of patients on left ventricular assist devices (LVADs) has improved. We examined the differences in risk of adverse outcomes between LVAD-supported and medically managed candidates on the heart transplant waiting list. Methods and Results— We analyzed mortality and morbidity in 33 073 heart transplant candidates registered on the United Network for Organ Sharing (UNOS) waiting list between 1999 and 2011. Five groups were selected: patients without LVADs in urgency status 1A, 1B, and 2; patients with pulsatile-flow LVADs; and patients with continuous-flow LVADs. Outcomes in patients requiring biventricular assist devices, total artificial heart, and temporary VADs were also analyzed. Two eras were defined on the basis of the approval date of the first continuous-flow LVAD for bridge to transplantation in the United States (2008). Mortality was lower in the current compared with the first era (2.1%/mo versus 2.9%/mo; P <0.0001). In the first era, mortality of pulsatile-flow LVAD patients was higher than in status 2 (hazard ratio [HR], 2.15; P <0.0001) and similar to that in status 1B patients (HR, 1.04; P =0.61). In the current era, patients with continuous-flow LVADs had mortality similar to that of status 2 (HR, 0.80; P =0.12) and lower mortality compared with status 1A and 1B patients (HR, 0.24 and 0.47; P <0.0001 for both comparisons). However, status upgrade for LVAD-related complications occurred frequently (28%) and increased the mortality risk (HR, 1.75; P =0.001). Mortality was highest in patients with biventricular assist devices (HR, 5.00; P <0.0001) and temporary VADs (HR, 7.72; P <0.0001). Conclusions— Mortality and morbidity on the heart transplant waiting list have decreased. Candidates supported with contemporary continuous-flow LVADs have favorable waiting list outcomes; however, they worsen significantly once a serious LVAD-related complication occurs. Transplant candidates requiring temporary and biventricular support have the highest risk of adverse outcomes. These results may help to guide optimal allocation of donor hearts. # Clinical Perspective {#article-title-31}Background— Survival of patients on left ventricular assist devices (LVADs) has improved. We examined the differences in risk of adverse outcomes between LVAD-supported and medically managed candidates on the heart transplant waiting list. Methods and Results— We analyzed mortality and morbidity in 33 073 heart transplant candidates registered on the United Network for Organ Sharing (UNOS) waiting list between 1999 and 2011. Five groups were selected: patients without LVADs in urgency status 1A, 1B, and 2; patients with pulsatile-flow LVADs; and patients with continuous-flow LVADs. Outcomes in patients requiring biventricular assist devices, total artificial heart, and temporary VADs were also analyzed. Two eras were defined on the basis of the approval date of the first continuous-flow LVAD for bridge to transplantation in the United States (2008). Mortality was lower in the current compared with the first era (2.1%/mo versus 2.9%/mo; P<0.0001). In the first era, mortality of pulsatile-flow LVAD patients was higher than in status 2 (hazard ratio [HR], 2.15; P<0.0001) and similar to that in status 1B patients (HR, 1.04; P=0.61). In the current era, patients with continuous-flow LVADs had mortality similar to that of status 2 (HR, 0.80; P=0.12) and lower mortality compared with status 1A and 1B patients (HR, 0.24 and 0.47; P<0.0001 for both comparisons). However, status upgrade for LVAD-related complications occurred frequently (28%) and increased the mortality risk (HR, 1.75; P=0.001). Mortality was highest in patients with biventricular assist devices (HR, 5.00; P<0.0001) and temporary VADs (HR, 7.72; P<0.0001). Conclusions— Mortality and morbidity on the heart transplant waiting list have decreased. Candidates supported with contemporary continuous-flow LVADs have favorable waiting list outcomes; however, they worsen significantly once a serious LVAD-related complication occurs. Transplant candidates requiring temporary and biventricular support have the highest risk of adverse outcomes. These results may help to guide optimal allocation of donor hearts.