CGP 12177A mediates cardiostimulation by activation of the ‘putative’ β4‐adrenoceptor; however, it has recently been reported that disruption of the β1‐adrenoceptor gene abolishes this effect. We have adenovirally overexpressed β1‐adrenoceptors in isolated, cultured adult rat ventricular cardiomyocytes and observed the inotropic potency of isoprenaline and CGP 12177A (in the presence of 1 μM propranolol). Isoprenaline was a full inotropic agonist at rat ventricular myocytes (pD2 7.69±0.12). CGP 12177A was a nonconventional partial agonist (pD2 6.34±0.09), increasing inotropy and lusitropy, with an intrinsic activity of 0.34 and antagonised by bupranolol. β1‐adrenoceptor overexpression enhanced the inotropic potency of isoprenaline by 11.7‐fold (pD2 8.76±0.14) and CGP 12177A by 5.9‐fold (7.11±0.10), respectively. Green fluorescent protein (GFP) overexpression did not alter the potency of isoprenaline or CGP 12177A (pD2 7.41±0.24 and pD2 6.60±0.50, respectively). The cardiostimulant effects of CGP 12177A were enhanced by IBMX (phosphodiesterase inhibitor) and decreased by Rp‐cAMPS (cAMP antagonist). CGP 12177A also increased cAMP levels. CGP 12177A but not isoprenaline initiated arrhythmias at lower concentrations following β1‐adrenoceptor overexpression. 125I‐Cyanopindolol saturation binding in Adv.β1 myocytes demonstrated ∼18‐fold increase in β1‐adrenoceptors. 3H‐CGP 12177A saturation binding, in the presence of propranolol, increased ∼5‐fold following overexpression of β1‐adrenoceptors. This study demonstrates enhanced cardiostimulation by CGP 12177A (in the presence of propranolol) in rat ventricular myocytes overexpressing β1‐adrenoceptors, mediated by a Gs/cAMP signalling pathway. ‘Putative’ β4‐adrenoceptor pharmacology appears to be mediated by activation of a novel affinity state of the β1‐adrenoceptor. British Journal of Pharmacology (2004) 141, 813–824. doi:10.1038/sj.bjp.0705668

Congestive heart failure (CHF) represents an enormous clinical problem and remains a leading cause of death despite advances in treatment. New treatments significantly impact mortality and disease course; they do not cure the underlying pathology. Gene transfer, the ability to genetically reprogram the heart in relevant cardiovascular disease models, allows testing the role of specific molecular pathways in disease pathogenesis. Potential therapeutic intervention targets can be then identified and approached with the full spectrum of therapeutic options, including traditional pharmacology, targeted synthesis of small molecule agonists or antagonists, biological agents (cells, antibodies, genetic material), or gene-based therapy. Lessons gleaned from gene transfer experiments on local modulation of cardiac genetic programs will guide attempts to transform early investigations into established therapy.

This editorial refers to “Heart rate reduction during exercise-induced myocardial ischemia and stunning” by X. Monnet et al. on page 579, and “Coronary collateral perfusion in patients with coronary artery disease: effect of metoprolol” by M. Billinger et al. on page 565. 1 β-blockers are established as standard first-line therapy for both stable and unstable angina pectoris having been introduced to the clinical setting in the mid-1960s. This class of drug is effective in alleviating the symptoms of angina but has never been shown to impact on mortality in patients with chronic stable angina. Several large clinical trials have shown β-blockers to reduce mortality when used in patients after myocardial infarction and more recently in heart failure.1–3 The recently published COMET trial demonstrated mortality benefit of carvedilol (a nonselective β-blocker and α-antagonist) compared to metoprolol tartrate (a selective β1-blocker) in heart failure.4 Thus over recent years the focus of interest has moved from the treatment of ischaemic heart disease to the use of β-blocker in chronic heart failure. This issue of European Heart Journal reports two studies of the possible deleterious effects of particular β-blockers on two aspects of clinical relevance to ischaemic heart disease, namely coronary collateral flow under controlled occlusion of the coronary artery,5 and the impact on ischaemic and stunned myocardium.6 In the first study,5 the effect on coronary collaterals of a second generation selective β1-blocker, metoprolol tartrate, was investigated. Natural bypass of coronary arteries is defined as anastomotic connections without an intervening capillary bed between portions of the same coronary artery or between different coronary arteries. Since collateral circulation has a cardioprotective effect as an alternative blood supply to avoid and/or delay myocardial damage when the original vessels are occluded, it has been recognised … *Corresponding author: Dr M. Sato, Imperial College London, London, UK E-mail address: m.sato{at}imperial.ac.uk

1. Adenylyl cyclase VI (ACVI) is one of the most abundantly expressed beta adrenergic receptor (betaAR)-coupled cyclases responsible for cyclic AMP (cAMP) production within the mammalian myocardium. We investigated the role of ACVI in the regulation of cardiomyocyte contractility and whether it is functionally coupled with beta(1) adrenergic receptor (beta(1)AR). 2. Recombinant adenoviruses were generated for ACVI and for antisense to ACVI (AS). Adult rat ventricular myocytes were transfected with ACVI virus, AS or both (SAS). Adenovirus for green fluorescent protein (GFP) served as control. Myocyte contraction amplitudes (% shortening) and relaxation times (R50) were analysed. ACVI function was determined using cAMP assays. 3. ACVI-transfected cells demonstrated a strong 139 kDa ACVI protein band compared to controls. ACVI myocytes had higher steady-state intracellular cAMP levels than GFP myocytes when unstimulated (GFP vs ACVI=6.60+/-0.98 vs 14.2+/-2.1 fmol cAMP/viable cell, n=4, P<0.05) and in the presence of 1 microm isoprenaline or 10 microm forskolin. 4. ACVI myocytes had increased basal contraction (% shortening: GFP vs ACVI: 1.90+/-1.36 vs 3.91+/-2.29, P<0.0001) and decreased basal R50 (GFP vs ACVI: 62.6+/-24.2 ms (n=50) vs 45.0+/-17.2 ms (n=248), P<0.0001). ACVI myocyte responses were increased for forskolin (E(max): GFP=6.70+/-1.59 (n=6); ACVI=9.06+/-0.69 (n=14), P<0.01) but not isoprenaline. 5. ACVI myocyte responses were increased (E(max): GFP vs ACVI=3.16+/-0.77 vs 5.10+/-0.60, P<0.0001) to xamoterol (a partial beta(1)AR-selective agonist) under beta(2)AR blockade (+50 nm ICI 118, 551). AS decreased both control and ACVI-stimulated xamoterol responses (E(max): AS=2.59+/-1.42, SAS=1.38+/-0.5). ACVI response was not mimicked by IBMX. Conversely, response through beta(2) adrenergic receptor (beta(2)AR) was decreased in ACVI myocytes. 6. In conclusion, ACVI overexpression constitutively increases myocyte contraction amplitudes by raising cAMP levels. Native ACVI did not contribute to basal cAMP production or contraction amplitude and only to a minor extent to the forskolin response. beta(1)AR but not beta(2)AR coupling was dependent on ACVI.

BACKGROUND:We have observed direct (noncatecholamine-blocking) negative inotropic effects of the selective beta(2)-adrenoceptor (AR) antagonist ICI 118,551 in myocytes from failing human ventricle. In this study we characterize the effect in parallel in human myocytes and in myocytes from animal models where beta(2)ARs or G(i) proteins are overexpressed.METHODS AND RESULTS:Enzymatically isolated, superfused ventricular myocytes were exposed to betaAR agonists and antagonists/inverse agonists, and contraction amplitude was measured. ICI 118,551 decreased contraction in ventricular myocytes from failing human hearts by 45.3+/-4.1% (n=20 hearts/31 myocytes, P<0.001) but had little effect in nonfailing hearts (4.9+/-4%, n=5 myocytes/3 hearts). Effects were significantly larger in patients classified as end-stage. Transgenic mice with high beta(2)AR number and increased G(i) levels had normal basal contractility but showed a similar negative inotropic response to ICI 118,551. Overexpression of human beta(2)AR in rabbit myocytes using adenovirus potentiated the negative inotropic effect of ICI 118,551. In human, rabbit, and mouse myocytes, the negative inotropic effects were blocked after treatment of cells with pertussis toxin to inactivate G(i), and overexpression of G(i)alpha(2) induced the effect de novo in normal rat myocytes.CONCLUSIONS:We hypothesize that ICI 118,551 binding directs the beta(2)AR to a G(i)-coupled form and away from the G(s)-coupled form (ligand-directed trafficking). ICI 118,551 effectively acts as an agonist at the G(i)-coupled beta(2)AR, producing a direct negative inotropic effect. Conditions where beta(2)ARs are present and G(i) is raised (failing human heart, TGbeta(2) mouse heart) predispose to the appearance of the negative inotropic effect.

Overexpression of the sarcoplasmic reticulum Ca ATPase (SERCA2a) produces positive inotropism and it has been proposed as a promising strategy to counteract defective excitation-contraction coupling in the failing heart. However, the effects of overexpressing SERCA2a on action potential duration (APD), which can affect diastolic parameters in the heart, is unknown. We, therefore, investigated the relationship between SERCA2a overexpression and APD in adult rabbit ventricular myocytes which were cultured for 48 h. Overexpression of SERCA2a was achieved by infection with an adenovirus carrying both SERCA2a and GFP independently driven by CMV promoters, Ad.SERCA2a. Myocytes infected with Ad.GFP only and/or non-infected myocytes were used as controls. Electrophysiological measurements were taken using switch clamping with 15-25 M Omega resistance microelectrodes. In Ad.SERCA2a infected myocytes, APD was significantly reduced compared with both groups of control cells at 0.5 Hz (APD50 (ms) non-infected: 481+/-98, n=12; Ad.GFP: 464+/-85, n=11; Ad.SERCA2a: 285+/-69, n=13 (mean+/-S.E.M.) and at 1 Hz (APD50 (ms) non-infected: 375+/-64, n=22; Ad.GFP: 363+/-47, n=18; Ad.SERCA2a: 231+/-54, n=24). Using AP voltage-clamping, we recorded a 0.2 mM Cd-sensitive current which can be ascribed to Ca current flowing during the AP. The integral of this current was reduced in Ad.SERCA2a myocytes compared with control (non-infected charge (pC): 27.5+/-4.2, n=8; Ad.SERCA2a: 15.5+/-4.1, n=11; P<0.01). Using AP clamping during the loading protocol, to take into account changes in APD, SR Ca content (assessed by integrating a 20 mM caffeine-induced inward current) was significantly larger in Ad.SERCA2a compared with both controls (SR Ca content (microM/l non-mitochondrial volume): non-infected: 25.5+/-7, n=8; Ad.GFP: 25.7+/-11, n=6; Ad.SERCA2a: 80.5+/-19, n=8). In conclusion, this study shows that SR Ca content is increased despite decreased Ca entry after overexpression of SERCA2a, and this can lead to positive inotropism. This effect coupled with shorter APD may be a useful therapeutic modality in heart failure.

Annexin 6 is one of a widely expressed family of calcium-binding proteins found in most mammalian tissues, including the heart. Several studies have implicated annexin 6 in the regulation of intracellular Ca2+ signaling, and it has been shown in vitro to act as a modulator of the sarcoplasmic reticulum Ca2+-release channel, cardiac L-type calcium channel, and Na+/Ca2+ exchanger. To investigate the role of annexin 6 in intact cardiomyocytes, we used mice containing a targeted disruption of the annexin 6 gene. Compared with controls, the myocytes of annexin 6 null-mutant mice demonstrated a significant increase in the rates of shortening and relengthening. Intracellular Ca2+ transients in fura-2-loaded cardiomyocytes induced by caffeine showed a normal baseline and amplitude, whereas the rate of decay was doubled in annexin 6-/- myocytes compared with control mice. These results show that annexin 6 knockout in the mouse leads to an increase in myocyte contractility and faster diastolic Ca2+ removal from the cytoplasm. In light of published findings showing annexin 6 to be down-regulated in end-stage heart failure, these results are consistent with a role for annexin 6 as a negative inotropic factor in the regulation of cardiomyocyte mechanics.

The Na(+)/Ca(2+)-exchanger (NCX) is the main mechanism by which Ca(2+) is transported out of the ventricular myocyte. NCX levels are raised in failing human heart, and the consequences of this for excitation-contraction coupling are still debated. We have increased NCX levels in adult rabbit myocytes by adenovirally-mediated gene transfer and examined the effects on excitation-contraction coupling after 24 and 48 h. Infected myocytes were identified through expression of green fluorescent protein (GFP), transfected under a separate promoter on the same viral construct. Control experiments were done with both non-infected myocytes and those infected with adenovirus expressing GFP only. Contraction amplitude was markedly reduced in NCX-overexpressing myocytes at either time point, and neither increasing frequency nor raising extracellular Ca(2+) could reverse this depression. Resting membrane potential and action potential duration were largely unaffected by NCX overexpression, as was peak Ca(2+) entry via the L-type Ca(2+) channel. Systolic and diastolic Ca(2+) levels were significantly reduced, with peak systolic Ca(2+) in NCX-overexpressing myocytes lower than diastolic levels in control cells at 2 m m extracellular Ca(2+). Both cell relengthening and the decay of the Ca(2+) transient were significantly slowed. Sarcoplasmic reticulum (SR) Ca(2+) stores were completely depleted in a majority of myocytes, and remained so despite increasingly vigorous loading protocols. Depressed contractility following NCX overexpression is therefore related to decreased SR Ca(2+) stores and low diastolic Ca(2+) levels rather than reduced Ca(2+) entry.