Enhanced cardiac contractility after gene transfer of V2 vasopressin receptors In vivo by ultrasound-guided injection or transcoronary delivery.

BACKGROUND: Systemic levels of arginine vasopressin (AVP) are increased in congestive heart failure, resulting in vasoconstriction and reduced cardiac contractility via V(1) vasopressin receptors. V(2) vasopressin receptors (V2Rs), which promote activation of adenylyl cyclase, are physiologically expressed only in the kidney and are absent in the myocardium. Heterologous expression of V2Rs in the myocardium could result in a positive inotropic effect by using the endogenous high concentrations of AVP in heart failure. METHODS AND RESULTS: We tested gene transfer with a recombinant adenovirus for the human V2R (Ad-V2R) to stimulate contractility of rat or rabbit myocardium in vivo. Ultrasound-guided direct injection or transcoronary delivery of adenovirus in vivo resulted in recombinant receptor expression in the myocardial target area, leading to a substantial increase in [(3)H]AVP binding. In 50% of the cardiomyocytes isolated from the directly injected area, single-cell shortening measurements detected a significant increase in contraction amplitude after exposure to AVP or the V2R-specific desmopressin (DDAVP). Echocardiography of the target myocardial area documented a marked increase in local fractional shortening after systemic administration of DDAVP in V2R-expressing animals but not in control virus-treated hearts. Simultaneous measurement of global contractility (dP/dt(max)) confirmed a positive inotropic effect of DDAVP on left ventricular function in the Ad-V2R-injected animals. CONCLUSIONS: Adenoviral gene transfer of the V2R into the myocardium increases cardiac contractility in vivo. Heterologous expression of cAMP-forming receptors in the myocardium could lead to novel strategies in the therapy of congestive heart failure by bypassing the desensitized beta-adrenergic receptor-signaling cascade.

Adenoviral gene transfer of the human V2 vasopressin receptor improves contractile force of rat cardiomyocytes.

BACKGROUND: In congestive heart failure, high systemic levels of the hormone arginine vasopressin (AVP) result in vasoconstriction and reduced cardiac contractility. These effects are mediated by the V1 vasopressin receptor (V1R) coupled to phospholipase C beta-isoforms. The V2 vasopressin receptor (V2R), which promotes activation of the Gs/adenylyl cyclase system, is physiologically expressed in the kidney but not in the myocardium. Expression of a recombinant V2R (rV2R) in the myocardium could result in a positive inotropic effect via the endogenous high concentrations of AVP in heart failure. METHODS AND RESULTS: A recombinant adenovirus encoding the human V2R (Ad-V2R) was tested for its ability to modulate the cardiac Gs/adenylyl cyclase system and to potentiate contractile force in rat ventricular cardiomyocytes and in H9c2 cardiomyoblasts. Ad-V2R infection resulted in a virus concentration-dependent expression of the transgene and led to a marked increase in cAMP formation in rV2R-expressing cardiomyocytes after exposure to AVP. Single-cell shortening measurements showed a significant agonist-induced contraction amplitude enhancement, which was blocked by the V2R antagonist, SR 121463A. Pretreatment of Ad-V2R-infected cardiomyocytes with AVP led to desensitization of the rV2R after short-term agonist exposure but did not lead to further loss of receptor function or density after long-term agonist incubation, thus demonstrating resistance of the rV2R to downregulation. CONCLUSIONS: Adenoviral gene transfer of the V2R in cardiomyocytes can modulate the endogenous adenylyl cyclase-signal transduction cascade and can potentiate contraction amplitude in cardiomyocytes. Heterologous expression of cAMP-forming receptors in the myocardium could lead to novel strategies in congestive heart failure by bypassing the desensitized beta-adrenergic receptor signaling.

Characterization and inhibition of beta-adrenergic receptor kinase in intact myocytes.

OBJECTIVES: beta-Adrenergic receptor kinase (beta ARK) phosphorylates and thereby inactivates agonist-occupied beta-adrenergic receptors (beta AR). beta ARK is thought to play an important role in the regulation of cardiac function. Therefore, we studied beta ARK activation and its inhibition in intact smooth muscle cells and in cardiomyoblasts. METHODS AND RESULTS: beta AR agonist-stimulated translocation of beta ARK was monitored by immunofluorescence labelling with specific antibodies and confocal laser scanning microscopy in DDT-MF 2 hamster smooth muscle cells and in H9c2 rat cardiomyoblasts. In unstimulated cells. beta ARK was mainly located in the cytosol. After beta AR agonist stimulation, the beta ARK signal was partially translocated to the membranes. Liposomal gene transfer of the COOH-terminus of beta ARK ('beta ARKmini') as a beta ARK inhibitor led to functional expression of this protein in both cell lines with high efficiency. Western blots with beta ARK antibodies showed a gene concentration-dependent immunoreactivity of the 'beta ARKmini' protein. 'beta ARKmini'-transfected myocytes demonstrated reduced membrane targeting of the beta ARK immuno-fluorescence signal. Additionally, the effect of 'beta ARKmini' on beta AR-induced desensitization of myocytic cAMP accumulation was investigated. In control cells, desensitization with isoproterenol led to a subsequent reduction of beta AR-induced cAMP accumulation. In 'beta ARKmini'-transfected myocytes, this beta AR-induced desensitization was significantly diminished, whereas normal beta AR-induced cAMP accumulation was unaffected. A gene concentration of 2 micrograms 'beta ARKmini' DNA/100,000 cardiomyoblasts, and of 0.7 microgram 'beta ARKmini' DNA/100,000 DDT-MF2 smooth muscle cells led to approximately 5.9- and approximately 5.6-fold overexpressions of 'beta ARKmini' vs. native beta ARK, respectively. These gene doses proved sufficient to attenuate beta-adrenergic desensitization significantly. CONCLUSIONS: (1) beta ARK translocation was evidenced in DDT-MF2 smooth muscle cells and in cardiomyoblasts by confocal laser scanning microscopy. (2) Feasibility of 'beta ARKmini' gene transfer to myocytes was demonstrated, and necessary gene doses for beta ARK inhibition were titered. (3) Overexpression of 'beta ARKmini' functionally interacted with endogenous beta-adrenergic signal transduction, leading to sustained cAMP accumulation after prolonged beta-adrenergic stimulation.

Activation of beta-adrenergic receptor kinase during myocardial ischemia.

During myocardial ischemia, a local release of noradrenaline coincides with an increased density of beta-adrenergic receptors. The functional activity of these receptors, however, is mainly determined by their state of phosphorylation. The beta-adrenergic receptor kinase (beta ARK) specifically phosphorylates and thereby inactivates beta-adrenergic receptors after stimulation by receptor agonists, facilitating the binding of the inhibitor protein beta-arrestin to the receptors. beta ARK activation involves a translocation of the enzyme to the membrane. In the present study, we investigated the density and the functional activity of beta-adrenergic receptors, the enzymatic activity of beta ARK in membranes and cytosol, the mRNA levels of beta ARK-1, and the expression of beta-arrestin during stop-flow and low-flow ischemia in the isolated perfused rat heart. After 60 minutes of stop-flow ischemia, beta-adrenergic receptor density was upregulated, but beta-agonist-mediated adenylate cyclase activity was blunted. Simultaneously, beta ARK activity in the particulate fraction was significantly induced. The increase in beta ARK activity was reversible after inhibition of ischemia-evoked noradrenaline release by desipramine. Also, exposure to externally given noradrenaline increased beta ARK activity in the particulate fraction. Cytosolic beta ARK activity remained largely unchanged during stop-flow or low-flow ischemia. The steady state concentration of beta ARK-1 mRNA increased after 20 minutes of stop-flow ischemia and then returned to baseline values after another 20 minutes. Cardiac ischemia did not alter beta-arrestin levels. During myocardial ischemia, an increase in the number of beta-adrenergic receptors is paralleled by increased membrane activity of the receptor kinase beta ARK. This increased membrane activity may contribute to enhanced receptor phosphorylation and inactivation.