Expression of a beta-adrenergic receptor kinase 1 inhibitor prevents the development of myocardial failure in gene-targeted mice.

Heart failure is accompanied by severely impaired beta-adrenergic receptor (betaAR) function, which includes loss of betaAR density and functional uncoupling of remaining receptors. An important mechanism for the rapid desensitization of betaAR function is agonist-stimulated receptor phosphorylation by the betaAR kinase (betaARK1), an enzyme known to be elevated in failing human heart tissue. To investigate whether alterations in betaAR function contribute to the development of myocardial failure, transgenic mice with cardiac-restricted overexpression of either a peptide inhibitor of betaARK1 or the beta2AR were mated into a genetic model of murine heart failure (MLP-/-). In vivo cardiac function was assessed by echocardiography and cardiac catheterization. Both MLP-/- and MLP-/-/beta2AR mice had enlarged left ventricular (LV) chambers with significantly reduced fractional shortening and mean velocity of circumferential fiber shortening. In contrast, MLP-/-/betaARKct mice had normal LV chamber size and function. Basal LV contractility in the MLP-/-/betaARKct mice, as measured by LV dP/dtmax, was increased significantly compared with the MLP-/- mice but less than controls. Importantly, heightened betaAR desensitization in the MLP-/- mice, measured in vivo (responsiveness to isoproterenol) and in vitro (isoproterenol-stimulated membrane adenylyl cyclase activity), was completely reversed with overexpression of the betaARK1 inhibitor. We report here the striking finding that overexpression of this inhibitor prevents the development of cardiomyopathy in this murine model of heart failure. These findings implicate abnormal betaAR-G protein coupling in the pathogenesis of the failing heart and point the way toward development of agents to inhibit betaARK1 as a novel mode of therapy.

Colonizing the heart from the epicardial side.

The clinical use of stem cells, such as bone marrow-derived and, more recently, resident cardiac stem cells, offers great promise for treatment of myocardial infarction and heart failure. The epicardium-derived cells have also attracted attention for their angiogenic paracrine actions and ability to differentiate into cardiomyocytes and vascular cells when activated during cardiac injury. In a recent study, Chong and colleagues have described a distinct population of epicardium-derived mesenchymal stem cells that reside in a perivascular niche of the heart and have a broad multilineage potential. Exploring the therapeutic capacity of these cells will be an exciting future endeavor.

Health events and the smoking cessation of middle aged Americans.

This study investigates the effect of serious health events including new diagnoses of heart attacks, strokes, cancers, chronic lung disease, chronic heart failure, diabetes, and heart disease on future smoking status up to 6 years postevent. Data come from the Health and Retirement Study, a nationally representative longitudinal survey of Americans aged 51-61 in 1991, followed every 2 years from 1992 to 1998. Smoking status is evaluated at each of three follow-ups, (1994, 1996, and 1998) as a function of health events between each of the four waves. Acute and chronic health events are associated with much lower likelihood of smoking both in the wave immediately following the event and up to 6 years later. However, future events do not retrospectively predict past cessation. In sum, serious health events have substantial impacts on cessation rates of older smokers. Notably, these effects persist for as much as 6 years after a health event.

Fiber type-specific nitric oxide protects oxidative myofibers against cachectic stimuli.

Oxidative skeletal muscles are more resistant than glycolytic muscles to cachexia caused by chronic heart failure and other chronic diseases. The molecular mechanism for the protection associated with oxidative phenotype remains elusive. We hypothesized that differences in reactive oxygen species (ROS) and nitric oxide (NO) determine the fiber type susceptibility. Here, we show that intraperitoneal injection of endotoxin (lipopolysaccharide, LPS) in mice resulted in higher level of ROS and greater expression of muscle-specific E3 ubiqitin ligases, muscle atrophy F-box (MAFbx)/atrogin-1 and muscle RING finger-1 (MuRF1), in glycolytic white vastus lateralis muscle than in oxidative soleus muscle. By contrast, NO production, inducible NO synthase (iNos) and antioxidant gene expression were greatly enhanced in oxidative, but not in glycolytic muscles, suggesting that NO mediates protection against muscle wasting. NO donors enhanced iNos and antioxidant gene expression and blocked cytokine/endotoxin-induced MAFbx/atrogin-1 expression in cultured myoblasts and in skeletal muscle in vivo. Our studies reveal a novel protective mechanism in oxidative myofibers mediated by enhanced iNos and antioxidant gene expression and suggest a significant value of enhanced NO signaling as a new therapeutic strategy for cachexia.

In vivo inhibition of elevated myocardial beta-adrenergic receptor kinase activity in hybrid transgenic mice restores normal beta-adrenergic signaling and function.

BACKGROUND: The clinical syndrome of heart failure (HF) is characterized by an impaired cardiac beta-adrenergic receptor (betaAR) system, which is critical in the regulation of myocardial function. Expression of the betaAR kinase (betaARK1), which phosphorylates and uncouples betaARs, is elevated in human HF; this likely contributes to the abnormal betaAR responsiveness that occurs with beta-agonist administration. We previously showed that transgenic mice with increased myocardial betaARK1 expression had impaired cardiac function in vivo and that inhibiting endogenous betaARK1 activity in the heart led to enhanced myocardial function. METHODS AND RESULTS: We created hybrid transgenic mice with cardiac-specific concomitant overexpression of both betaARK1 and an inhibitor of betaARK1 activity to study the feasibility and functional consequences of the inhibition of elevated betaARK1 activity similar to that present in human HF. Transgenic mice with myocardial overexpression of betaARK1 (3 to 5-fold) have a blunted in vivo contractile response to isoproterenol when compared with non-transgenic control mice. In the hybrid transgenic mice, although myocardial betaARK1 levels remained elevated due to transgene expression, in vitro betaARK1 activity returned to control levels and the percentage of betaARs in the high-affinity state increased to normal wild-type levels. Furthermore, the in vivo left ventricular contractile response to betaAR stimulation was restored to normal in the hybrid double-transgenic mice. CONCLUSIONS: Novel hybrid transgenic mice can be created with concomitant cardiac-specific overexpression of 2 independent transgenes with opposing actions. Elevated myocardial betaARK1 in transgenic mouse hearts (to levels seen in human HF) can be inhibited in vivo by a peptide that can prevent agonist-stimulated desensitization of cardiac betaARs. This may represent a novel strategy to improve myocardial function in the setting of compromised heart function.

Enhancement of cardiac function after adenoviral-mediated in vivo intracoronary beta2-adrenergic receptor gene delivery.

Exogenous gene delivery to alter the function of the heart is a potential novel therapeutic strategy for treatment of cardiovascular diseases such as heart failure (HF). Before gene therapy approaches to alter cardiac function can be realized, efficient and reproducible in vivo gene techniques must be established to efficiently transfer transgenes globally to the myocardium. We have been testing the hypothesis that genetic manipulation of the myocardial beta-adrenergic receptor (beta-AR) system, which is impaired in HF, can enhance cardiac function. We have delivered adenoviral transgenes, including the human beta2-AR (Adeno-beta2AR), to the myocardium of rabbits using an intracoronary approach. Catheter-mediated Adeno-beta2AR delivery produced diffuse multichamber myocardial expression, peaking 1 week after gene transfer. A total of 5 x 10(11) viral particles of Adeno-beta2AR reproducibly produced 5- to 10-fold beta-AR overexpression in the heart, which, at 7 and 21 days after delivery, resulted in increased in vivo hemodynamic function compared with control rabbits that received an empty adenovirus. Several physiological parameters, including dP/dtmax as a measure of contractility, were significantly enhanced basally and showed increased responsiveness to the beta-agonist isoproterenol. Our results demonstrate that global myocardial in vivo gene delivery is possible and that genetic manipulation of beta-AR density can result in enhanced cardiac performance. Thus, replacement of lost receptors seen in HF may represent novel inotropic therapy.

Alterations in cardiac adrenergic signaling and calcium cycling differentially affect the progression of cardiomyopathy.

The medical treatment of chronic heart failure has undergone a dramatic transition in the past decade. Short-term approaches for altering hemodynamics have given way to long-term, reparative strategies, including beta-adrenergic receptor (betaAR) blockade. This was once viewed as counterintuitive, because acute administration causes myocardial depression. Cardiac myocytes from failing hearts show changes in betaAR signaling and excitation-contraction coupling that can impair cardiac contractility, but the role of these abnormalities in the progression of heart failure is controversial. We therefore tested the impact of different manipulations that increase contractility on the progression of cardiac dysfunction in a mouse model of hypertrophic cardiomyopathy. High-level overexpression of the beta(2)AR caused rapidly progressive cardiac failure in this model. In contrast, phospholamban ablation prevented systolic dysfunction and exercise intolerance, but not hypertrophy, in hypertrophic cardiomyopathy mice. Cardiac expression of a peptide inhibitor of the betaAR kinase 1 not only prevented systolic dysfunction and exercise intolerance but also decreased cardiac remodeling and hypertrophic gene expression. These three manipulations of cardiac contractility had distinct effects on disease progression, suggesting that selective modulation of particular aspects of betaAR signaling or excitation-contraction coupling can provide therapeutic benefit.

Overexpression of the cardiac beta(2)-adrenergic receptor and expression of a beta-adrenergic receptor kinase-1 (betaARK1) inhibitor both increase myocardial contractility but have differential effects on susceptibility to ischemic injury.

Cardiac beta(2)-adrenergic receptor (beta(2)AR) overexpression is a potential contractile therapy for heart failure. Cardiac contractility was elevated in mice overexpressing beta(2)ARs (TG4s) with no adverse effects under normal conditions. To assess the consequences of beta(2)AR overexpression during ischemia, perfused hearts from TG4 and wild-type mice were subjected to 20-minute ischemia and 40-minute reperfusion. During ischemia, ATP and pH fell lower in TG4 hearts than wild type. Ischemic injury was greater in TG4 hearts, as indicated by lower postischemic recoveries of contractile function, ATP, and phosphocreatine. Because beta(2)ARs, unlike beta(1)ARs, couple to G(i) as well as G(s), we pretreated mice with the G(i) inhibitor pertussis toxin (PTX). PTX treatment increased basal contractility in TG4 hearts and abolished the contractile resistance to isoproterenol. During ischemia, ATP fell lower in TG4+PTX than in TG4 hearts. Recoveries of contractile function and ATP were lower in TG4+PTX than in TG4 hearts. We also studied mice that overexpressed either betaARK1 (TGbetaARK1) or a betaARK1 inhibitor (TGbetaARKct). Recoveries of function, ATP, and phosphocreatine were higher in TGbetaARK1 hearts than in wild-type hearts. Despite basal contractility being elevated in TGbetaARKct hearts to the same level as that of TG4s, ischemic injury was not increased. In summary, beta(2)AR overexpression increased ischemic injury, whereas betaARK1 overexpression was protective. Ischemic injury in the beta(2)AR overexpressors was exacerbated by PTX treatment, implying that it was G(s) not G(i) activity that enhanced injury. Unlike beta(2)AR overexpression, basal contractility was increased by betaARK1 inhibitor expression without increasing ischemic injury, thus implicating a safer potential therapy for heart failure.

Essential role of beta-adrenergic receptor kinase 1 in cardiac development and function.

The beta-adrenergic receptor kinase 1 (beta ARK1) is a member of the G protein-coupled receptor kinase (GRK) family that mediates the agonist-dependent phosphorylation and desensitization of G protein-coupled receptors. We have cloned and disrupted the beta ARK1 gene in mice by homologous recombination. No homozygote beta ARK1-/- embryos survive beyond gestational day 15.5. Prior to gestational day 15.5, beta ARK1-/- embryos display pronounced hypoplasia of the ventricular myocardium essentially identical to the "thin myocardium syndrome" observed upon gene inactivation of several transcription factors (RXR alpha, N-myc, TEF-1, WT-1). Lethality in beta ARK1-/- embryos is likely due to heart failure as they exhibit a > 70% decrease in cardiac ejection fraction determined by direct in utero intravital microscopy. These results along with the virtual absence of endogenous GRK activity in beta ARK1-/- embryos demonstrate that beta ARK1 appears to be the predominant GRK in early embryogenesis and that it plays a fundamental role in cardiac development.

Induced pluripotent stem cell-derived cardiac progenitors differentiate to cardiomyocytes and form biosynthetic tissues.

The mammalian heart has little capacity to regenerate, and following injury the myocardium is replaced by non-contractile scar tissue. Consequently, increased wall stress and workload on the remaining myocardium leads to chamber dilation, dysfunction, and heart failure. Cell-based therapy with an autologous, epigenetically reprogrammed, and cardiac-committed progenitor cell source could potentially reverse this process by replacing the damaged myocardium with functional tissue. However, it is unclear whether cardiac progenitor cell-derived cardiomyocytes are capable of attaining levels of structural and functional maturity comparable to that of terminally-fated cardiomyocytes. Here, we first describe the derivation of mouse induced pluripotent stem (iPS) cells, which once differentiated allow for the enrichment of Nkx2-5(+) cardiac progenitors, and the cardiomyocyte-specific expression of the red fluorescent protein. We show that the cardiac progenitors are multipotent and capable of differentiating into endothelial cells, smooth muscle cells and cardiomyocytes. Moreover, cardiac progenitor selection corresponds to cKit(+) cell enrichment, while cardiomyocyte cell-lineage commitment is concomitant with dual expression of either cKit/Flk1 or cKit/Sca-1. We proceed to show that the cardiac progenitor-derived cardiomyocytes are capable of forming electrically and mechanically coupled large-scale 2D cell cultures with mature electrophysiological properties. Finally, we examine the cell progenitors' ability to form electromechanically coherent macroscopic tissues, using a physiologically relevant 3D culture model and demonstrate that following long-term culture the cardiomyocytes align, and form robust electromechanical connections throughout the volume of the biosynthetic tissue construct. We conclude that the iPS cell-derived cardiac progenitors are a robust cell source for tissue engineering applications and a 3D culture platform for pharmacological screening and drug development studies.

The Dietary Approaches to Stop Hypertension (DASH) eating pattern in special populations.

The Dietary Approaches to Stop Hypertension (DASH) trial showed that a diet rich in fruits, vegetables, low-fat dairy products with reduced total and saturated fat, cholesterol, and sugar-sweetened products effectively lowers blood pressure in individuals with prehypertension and stage I hypertension. Limited evidence is available on the safety and efficacy of the DASH eating pattern in special patient populations that were excluded from the trial. Caution should be exercised before initiating the DASH diet in patients with chronic kidney disease, chronic liver disease, and those who are prescribed renin-angiotensin-aldosterone system antagonist, but these conditions are not strict contraindications to DASH. Modifications to the DASH diet may be necessary to facilitate its use in patients with chronic heart failure, uncontrolled diabetes mellitus type II, lactose intolerance, and celiac disease. In general, the DASH diet can be adopted by most patient populations and initiated simultaneously with medication therapy and other lifestyle interventions.