Gene Transfer of Heterologous G Protein–Coupled Receptors to Cardiomyocytes

Abstract

Abstract-In heart failure, reduced cardiac contractility is accompanied by blunted cAMP responses to ␤-adrenergic stimulation. Parathyroid hormone (PTH)-related peptide and arginine vasopressin are released from the myocardium in response to increased wall stress but do not stimulate contractility or adenylyl cyclase at physiological concentrations.To bypass the defective ␤-adrenergic signaling cascade, recombinant P1 PTH/PTH-related peptide receptors (rPTH1-Rs) and V 2 vasopressin receptors (rV 2 -Rs), which are normally not expressed in the myocardium and which are both strongly coupled to adenylyl cyclase, and recombinant ␤ 2 -adrenergic receptors (r␤ 2 -ARs) were overexpressed in cardiomyocytes by viral gene transfer. The capacity of endogenous hormones to increase contractility via the heterologous, recombinant receptors was compared. Whereas V 2 -Rs are uniquely coupled to Gs, PTH1-Rs and ␤ 2 -ARs are also coupled to other G proteins. Gene transfer of rPTH1-Rs or r␤ 2 -ARs to adult cardiomyocytes resulted in maximally increased basal contractility, which could not be further stimulated by adding receptor agonists. Agonists at rPTH1-Rs induced increased cAMP formation and phospholipase C activity. In contrast, healthy or failing rV 2 -Rexpressing cardiomyocytes showed unaltered basal contractility. Their contractility and cAMP formation increased only at agonist exposure, which did not activate phospholipase C. In summary, we found that gene transfer of PTH1-Rs to cardiomyocytes results in constitutive activity of the transgene, as does that of ␤ 2 -ARs. In the absence of receptor agonists, rPTH1-Rs and r␤ 2 -ARs increase basal contractility, coupling to 2 G proteins simultaneously. In contrast, rV 2 -Rs are uniquely coupled to Gs and are not constitutively active, retaining their property to be activated exclusively on agonist stimulation. Therefore, gene transfer of V 2 -Rs might be more suited to test the effects of cAMP-stimulating receptors in heart failure than that of PTH1-Rs or ␤ 2 -ARs. (Circ Res. 2001;88:688-695.)Key Words: gene transfer Ⅲ receptor Ⅲ heart failure C ongestive heart failure is characterized by an increased sympatho-adrenergic drive; a stimulation of the reninangiotensin-aldosterone system; and an augmented release of several peptide hormones, among them atrial natriuretic peptide, arginine vasopressin (AVP), and parathyroid hormone (PTH)-related peptide (PTHrp). 1-3 AVP is released both systemically from the pituitary gland and locally from the myocardium. 2,4 In addition, PTHrp is released from the heart in response to increased wall stress or hypoxia, 5 and PTHrp mRNA has been detected in isolated cardiomyocytes. 3,6 PTH and PTHrp are devoid of direct effects on ventricular contraction at physiological concentrations in vivo. 7 In the cardiomyocyte, PTH and PTHrp induce hypertrophy acting via an endogenously occurring, yet uncloned, myocardial PTH receptor subtype 8 and therefore contribute to further worsening of heart failure. At physiological concentrations, neither PTH nor PT...

Designing Heart Performance by Gene Transfer

“…Another strategy along these same lines has been to increase other G protein-coupled receptors, including parathyroid hormone related peptide (PTHrp) and vasopressin2 (V2) receptors, using viral-mediated gene transfer (474). Like ␤ 2 -ARs, PTHrp receptors are coupled to G s and G i and downstream activation of phospholipase C, while V2 receptors are strongly linked only to G s (58, 474,806).…”

“…Like ␤ 2 -ARs, PTHrp receptors are coupled to G s and G i and downstream activation of phospholipase C, while V2 receptors are strongly linked only to G s (58, 474,806). Neither receptor, however, is expressed endogenously in cardiac myocytes.…”

“…In addition, the long-term benefits of this approach are controversial, as blockade of both V1 and V2 is also predicted to improve function in individuals with congestive heart failure (273). This general approach has also been utilized to determine whether delivery of other noncardiac G s -coupled receptors to cardiac myocytes acts as a novel therapeutic strategy for bypassing the ␤-AR and boosting contractile function (474). C) AKAPS.…”

Effects of two Gβγ-binding proteins – N-terminally truncated phosducin and β-adrenergic receptor kinase C terminus (βARKct) – in heart failure

“…28 Briefly, the hearts were perfused and digested with collagenase. The isolated cardiomyocytes were cultured in modified M199 on laminin-precoated dishes (5-10 mg/cm 2 ) at a density of 1.5 Â 10 5 cells per cm 2 (at 5% CO 2 and 371C).…”

“…28 After the contraction amplitude reached stability, increasing concentrations of isoproterenol were applied.…”