Dimosthenis Giamouridis

Elevated citrate levels in non-alcoholic fatty liver disease: The potential of citrate to promote radical production

Plasma citrate levels were found to be elevated in non‐alcoholic fatty liver disease (NAFLD) patients. Cellular experiments indicated that increased citrate levels might originate from an excess of fatty acids. The impact of elevated citrate levels on oxidative stress was examined. It was found that citrate stimulated hydrogen peroxide induced intracellular oxidative stress in HepG2 cells. This was related to the promotion of iron mediated hydroxyl radical formation from hydrogen peroxide by citrate. The stimulating effect of citrate on the reactivity of iron promotes oxidative stress, a crucial process in the progression of NAFLD.

Intravenous Adeno-Associated Virus Serotype 8 Encoding Urocortin-2 Provides Sustained Augmentation of Left Ventricular Function in Mice

Urocortin-2 (UCn2) peptide infusion increases cardiac function in patients with heart failure, but chronic peptide infusion is cumbersome, costly, and provides only short-term benefits. Gene transfer would circumvent these shortcomings. Here we ask whether a single intravenous injection of adeno-associated virus type 8 encoding murine urocortin-2 (AAV8.UCn2) could provide long-term elevation in plasma UCn2 levels and increased left ventricular (LV) function. Normal mice received AAV8.UCn2 (5×10¹¹ genome copies, intravenous). Plasma UCn2 increased 15-fold 6 weeks and >11-fold 7 months after delivery. AAV8 DNA and UCn2 mRNA expression was persistent in LV and liver up to 7 months after a single intravenous injection of AAV8.UCn2. Physiological studies conducted both in situ and ex vivo showed increases in LV +dP/dt and in LV -dP/dt, findings that endured unchanged for 7 months. SERCA2a mRNA and protein expression was increased in LV samples and Ca²⁺ transient studies showed an increased rate of Ca²⁺ decline in cardiac myocytes from mice that had received UCn2 gene transfer. We conclude that a single intravenous injection of AAV8.UCn2 increases plasma UCn2 and increases LV systolic and diastolic function for at least 7 months. The simplicity of intravenous injection of a long-term expression vector encoding a gene with paracrine activity to increase cardiac function is a potentially attractive strategy in clinical settings. Future studies will determine the usefulness of this approach in the treatment of heart failure.

Cardiac-Directed Expression of Adenylyl Cyclase Catalytic Domain Reverses Cardiac Dysfunction Caused by Sustained Beta-Adrenergic Receptor Stimulation

Visual Abstract

One-time injection of AAV8 encoding urocortin 2 provides long-term resolution of insulin resistance

Using mice rendered insulin resistant with high fat diets (HFD), we examined blood glucose levels and insulin resistance after i.v. delivery of an adeno-associated virus type 8 encoding murine urocortin 2 (AAV8.UCn2). A single i.v. injection of AAV8.UCn2-normalized blood glucose and glucose disposal within weeks, an effect that lasted for months. Hyperinsulinemic-euglycemic clamps showed reduced plasma insulin, increased glucose disposal rates, and increased insulin sensitivity following UCn2 gene transfer. Mice with corticotropin-releasing hormone type 2-receptor deletion that were rendered insulin resistant by HFD showed no improvement in glucose disposal after UCn2 gene transfer, indicating that the effect requires UCn2s cognate receptor. We also demonstrated increased glucose disposal after UCn2 gene transfer in db/db mice, a second model of insulin resistance. UCn2 gene transfer reduced fatty infiltration of the liver in both models of insulin resistance. UCn2 increases Glut4 translocation to the plasma membrane in skeletal myotubes in a manner quantitatively similar to insulin, indicating a mechanism through which UCn2 operates to increase insulin sensitivity. UCn2 gene transfer, in a dose-dependent manner, is insulin sensitizing and effective for months after a single injection. These findings suggest a potential long-term therapy for clinical type-2 diabetes.

Cardiac-directed expression of a catalytically inactive adenylyl cyclase 6 protects the heart from sustained β-adrenergic stimulation

Objectives Increased expression of adenylyl cyclase type 6 (AC6) has beneficial effects on the heart through cyclic adenosine monophosphate (cAMP)-dependent and cAMP-independent pathways. We previously generated a catalytically inactive mutant of AC6 (AC6mut) that has an attenuated response to β-adrenergic receptor stimulation, and, consequently, exhibits reduced myocardial cAMP generation. In the current study we test the hypothesis that cardiac-directed expression of AC6mut would protect the heart from sustained β-adrenergic receptor stimulation, a condition frequently encountered in patients with heart failure. Methods and results AC6mut mice and transgene negative siblings received osmotic mini-pumps to provide continuous isoproterenol infusion for seven days. Isoproterenol infusion caused deleterious effects that were attenuated by cardiac-directed AC6mut expression. Both groups showed reduced left ventricular (LV) ejection fraction, but the reduction was less in AC6mut mice (p = 0.047). In addition, AC6mut mice showed superior left ventricular function, manifested by higher values for LV peak +dP/dt (p = 0.03), LV peak -dP/dt (p = 0.008), end-systolic pressure-volume relationship (p = 0.003) and cardiac output (p<0.03). LV samples of AC6mut mice had more sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) protein (p<0.01), which likely contributed to better LV function. AC6mut mice had lower rates of cardiac myocyte apoptosis (p = 0.016), reduced caspase 3/7 activity (p = 0.012) and increased B-cell lymphoma 2 (Bcl2) expression (p = 0.0001). Conclusion Mice with cardiac-directed AC6mut expression weathered the deleterious effects of continuous isoproterenol infusion better than control mice, indicating cardiac protection.

Effects of Urocortin 2 Versus Urocortin 3 Gene Transfer on Left Ventricular Function and Glucose Disposal

Visual Abstract

Urocortin 3 Gene Transfer Increases Function of the Failing Murine Heart

Peptide infusions of peptides the corticotropin releasing factor family, including urocortin 2, stresscopin, and urocortin 3 (UCn3), have favorable acute effects in clinical heart failure (HF), but their short half-lives make them unsuitable for chronic therapy. This study asked whether UCn3 gene transfer, which provides sustained elevation of plasma UCn3 levels, increases the function of the failing heart. HF was induced by transmural left ventricular (LV) cryoinjury in mice. LV function was assessed 3 weeks later by echocardiography. Those with ejection fractions (EF) <40% received intravenous saline or intravenous adeno-associated virus type-8 encoding murine UCn3 (AAV8.mUCn3; 1.9 × 1013 genome copies/kg). Five weeks after randomization, repeat echocardiography, assessment of LV function (+dP/dt, -dP/dt), and quantification of Ca2+ transients and sarcomere shortening in isolated cardiac myocytes were conducted, and assessment of LV Ca2+ handling and stress proteins was performed. Three weeks after myocardial infarction, prior to treatment, EFs were reduced (mean 31%, from 63% in sham-operated animals). Mice randomized to receive UCn3 gene transfer showed increased plasma UCn3 (from 0.1 ± 0.01 ng/mL in the saline group to 5.6 ± 1.1 ng/mL; n = 12 each group; p < 0.0001). Compared to mice that received saline, UCn3 gene transfer was associated with higher values for EF (p = 0.0006); LV +dP/dt (p < 0.0001), and LV -dP/dt (p < 0.0001). Cardiac myocytes from mice that received UCn3 gene transfer showed higher peak Ca2+ transients (p = 0.0005), lower time constant of cytosolic Ca2+ decline (tau, p < 0.0001), and higher rates of sarcomere shortening (+dL/dt, p = 0.03) and lengthening (-dL/dt, p = 0.04). LV samples from mice that received UCn3 gene transfer contained higher levels of SERCA2a (p = 0.0004 vs. HF) and increased amounts of phosphorylated troponin I (p = 0.04 vs. HF). UCn3 gene transfer is associated with improved Ca2+ handling and LV function in mice with HF and reduced EF.