Jeffrey Nienaber

Endogenous S-nitrosothiols protect against myocardial injury

Despite substantial evidence that nitric oxide (NO) and/or endogenous S-nitrosothiols (SNOs) exert protective effects in a variety of cardiovascular diseases, the molecular details are largely unknown. Here we show that following left coronary artery ligation, mice with a targeted deletion of the S-nitrosoglutathione reductase gene (GSNOR−/−) have reduced myocardial infarct size, preserved ventricular systolic and diastolic function, and maintained tissue oxygenation. These profound physiological effects are associated with increases in myocardial capillary density and S-nitrosylation of the transcription factor hypoxia inducible factor-1α (HIF-1α) under normoxic conditions. We further show that S-nitrosylated HIF-1α binds to the vascular endothelial growth factor (VEGF) gene, thus identifying a role for GSNO in angiogenesis and myocardial protection. These results suggest innovative approaches to modulate angiogenesis and preserve cardiac function.

Dynamic Regulation of Phosphoinositide 3-Kinase-γ Activity and β-Adrenergic Receptor Trafficking in End-Stage Human Heart Failure

Background— Downregulation of &bgr;-adrenergic receptors (&bgr;ARs) under conditions of heart failure requires receptor targeting of phosphoinositide 3-kinase (PI3K)–&ggr; and redistribution of &bgr;ARs into endosomal compartments. Because support with a left ventricular assist device (LVAD) results in significant improvement of cardiac function in humans, we investigated the effects of mechanical unloading on regulation of PI3K&ggr; activity and intracellular distribution of &bgr;ARs. Additionally, we tested whether displacement of PI3K&ggr; from activated &bgr;ARs would restore agonist responsiveness in failing human cardiomyocytes. Methods and Results— To test the role of PI3K on &bgr;AR endocytosis in failing human hearts, we assayed for PI3K activity in human left ventricular samples before and after mechanical unloading (LVAD). Before LVAD, failing human hearts displayed a marked increase in &bgr;AR kinase 1 (&bgr;ARK1)–associated PI3K activity that was attributed exclusively to enhanced activity of the PI3K&ggr; isoform. Increased &bgr;ARK1-coupled PI3K activity in the failing hearts was associated with downregulation of &bgr;ARs from the plasma membrane and enhanced sequestration into early and late endosomes compared with unmatched nonfailing controls. Importantly, LVAD support reversed PI3K&ggr; activation, normalized the levels of agonist-responsive &bgr;ARs at the plasma membrane, and depleted the &bgr;ARs from the endosomal compartments without changing the total number of receptors (sum of plasma membrane and early and late endosome receptors). To test whether the competitive displacement of PI3K from the &bgr;AR complex restored receptor responsiveness, we overexpressed the phosphoinositide kinase domain of PI3K (which disrupts &bgr;ARK1/PI3K interaction) in primary cultures of failing human cardiomyocytes. Adenoviral-mediated phosphoinositide kinase overexpression significantly increased basal contractility and rapidly reconstituted responsiveness to &bgr;-agonist. Conclusions— These results suggest a novel paradigm in which human &bgr;ARs undergo a process of intracellular sequestration that is dynamically reversed after LVAD support. Importantly, mechanical unloading leads to complete reversal in PI3K&ggr; and &bgr;ARK1-associated PI3K activation. Furthermore, displacement of active PI3K from &bgr;ARK1 restores &bgr;AR responsiveness in failing myocytes.

β-Adrenergic axis and heart disease

Beta-adrenergic receptors (beta-ARs) belong to a large family of G-protein-coupled receptors (GPCRs) that form the interface between the sympathetic nervous system and the cardiovascular system. The beta-AR signal system is one of the most powerful regulators of cardiac function, mediated by the effects of the sympathetic transmitters epinephrine and norepinephrine. In a number of cardiac diseases, however, the biology of beta-AR signaling pathways is altered dramatically. Here we discuss the role of beta-AR signaling in the normal and abnormal heart and how the use of genetically engineered mouse models has helped in our understanding of the pathophysiology of cardiac disease.

Adeno-associated viral vectors based on serotype 3b use components of the fibroblast growth factor receptor signaling complex for efficient transduction.

Adeno-associated virus type 3b (AAV3b) has been largely ignored by gene therapists because of the inability of vectors based on this serotype to transduce target tissues efficiently. Here we describe a phenomenon unique to AAV3b in that vectors based on this serotype mediate enhanced transduction in the presence of heparin. Among the many biological functions attributed to heparin, its interaction with, and ability to regulate, several growth factors (GFs) and growth factor receptors (GFRs) has been well characterized. Using GFR-overexpressing cell lines, soluble GFs and heparins, as well as specific GFR inhibitors, we have demonstrated a requirement for fibroblast growth factor receptor-2 (FGFR2) and FGF1 in the heparin-mediated augmentation of AAV3b vector transduction. In contrast to AAV2, we establish that heparin can be used as an adjunct with AAV3b to further increase transduction in a variety of cells and target tissues, additionally suggesting that AAV3b may be an attractive viral vector for clinical use during procedures in which heparin is used. In summary, AAV3b exhibits FGFR2-dependent, markedly enhanced transduction efficiency in the presence of heparin and FGFs, which could make it a useful vector for gene therapy in a variety of human diseases.

Normal cardiac function in mice with supraphysiological cardiac creatine levels

Creatine and phosphocreatine levels are decreased in heart failure, and reductions in myocellular phosphocreatine levels predict the severity of the disease and portend adverse outcomes. Previous studies of transgenic mouse models with increased creatine content higher than two times baseline showed the development of heart failure and shortened lifespan. Given phosphocreatines role in buffering ATP content, we tested the hypothesis whether elevated cardiac creatine content would alter cardiac function under normal physiological conditions. Here, we report the creation of transgenic mice that overexpress the human creatine transporter (CrT) in cardiac muscle under the control of the α-myosin heavy chain promoter. Cardiac transgene expression was quantified by qRT-PCR, and human CrT protein expression was documented on Western blots and immunohistochemistry using a specific anti-CrT antibody. High-energy phosphate metabolites and cardiac function were measured in transgenic animals and compared with age-matched, wild-type controls. Adult transgenic animals showed increases of 5.7- and 4.7-fold in the content of creatine and free ADP, respectively. Phosphocreatine and ATP levels were two times as high in young transgenic animals but declined to control levels by the time the animals reached 8 wk of age. Transgenic mice appeared to be healthy and had normal life spans. Cardiac morphometry, conscious echocardiography, and pressure-volume loop studies demonstrated mild hypertrophy but normal function. Based on our characterization of the human CrT protein expression, creatine and phosphocreatine content, and cardiac morphometry and function, these transgenic mice provide an in vivo model for examining the therapeutic value of elevated creatine content for cardiac pathologies.