There is increasing evidence that insulin-like growth factor-1 (IGF-1) may play a role in both physiological and pathophysiological events in the mammalian myocardium. The present study investigated the acute effects of IGF-1 on isometric force development in isolated rat cardiac muscle and on intracellular calcium (Ca2+) handling in isolated cardiac myocytes. IGF-1 had a positive inotropic effect on rat ventricular papillary muscles increasing force development by 17.8 +/- 4.6%, 18.5 +/- 5.8% and 11.9 +/- 4.9% (n = 12-20) at concentrations of 1, 10 and 100 ng/ml respectively. Isoprenaline increased tension in these papillary muscles by 56.7 +/- 7.7% at a concentration of 100 nM (n = 22). In comparison, insulin increased papillary muscle force development by 11.6 +/- 3.2%, 17.7 +/- 4.1% and 19.7 +/- 5.6% at concentrations of 1, 10 and 100 nM respectively (n = 16-20). In the single cardiac myocyte IGF-1 increased, the peak cytosolic free Ca2+ concentration, the amplitude of the Ca2+ transient and the time to peak Ca2+ as measured with the fluorescent bioprobe Indo-1 AM. The positive inotropic response to IGF-1 by rat ventricular muscle is therefore associated with a rise in free, peak cytosolic Ca2+ in isolated cardiac myocytes. Increasing insulin concentrations (1-1000 nM) elicited a progressive elevation in isometric force and free, cytosolic Ca2+. In contrast, in the presence of IGF-1, the maximal rise in isometric force and free cytosolic Ca2+ were both observed at 10 ng/ml. Recent reports have suggested that IGF-1 may act on the mammalian myocardium when administered chronically, but this study is amongst the first to demonstrate an acute effect of IGF-I on the mammalian heart. IGF-1 may prove then to be a novel cardioactive agent in both normal and pathophysiological states.

We identified putative β4‐adrenoceptors by radioligand binding, measured increases in ventricular contractile force by (−)‐CGP 12177 and (±)‐cyanopindolol and demonstrated increased Ca2+ transients by (−)‐CGP 12177 in rat cardiomyocytes. (−)‐[3H]‐CGP 12177 labelled 13–22 fmol mg−1 protein ventricular β1, β2‐adrenoceptors (pKD ∼9.0) and 50–90 fmol mg−1 protein putative β4‐adrenoceptors (pKD ∼7.3). The affinity values (pKi) for (β1,β2‐) and putative β4‐adrenoceptors, estimated from binding inhibition, were (−)‐propranolol 8.4, 5.7; (−)‐bupranolol 9.7, 5.8; (±)‐cyanopindolol 10.0,7.4. In left ventricular papillary muscle, in the presence of 30 μM 3‐isobutyl‐1‐methylxanthine, (−)‐CGP 12177 and (±)‐cyanopindolol caused positive inotropic effects, (pEC50, (−)‐CGP 12177, 7.6; (±)‐cyanopindolol, 7.0) which were antagonized by (−)‐bupranolol (pKB 6.7–7.0) and (−)‐CGP 20712A (pKB 6.3–6.6). The cardiostimulant effects of (−)‐CGP 12177 in papillary muscle, left and right atrium were antagonized by (±)‐cyanopindolol (pKP 7.0–7.4). (−)‐CGP 12177 (1 μM) in the presence of 200 nM (−)‐propranolol increased Ca2+ transient amplitude by 56% in atrial myocytes, but only caused a marginal increase in ventricular myocytes. In the presence of 1 μM 3‐isobutyl‐1‐methylxanthine and 200 nM (−)‐propranolol, 1 μM (−)‐CGP 12177 caused a 73% increase in Ca2+ transient amplitude in ventricular myocytes. (−)‐CGP 12177 elicited arrhythmic transients in some atrial and ventricular myocytes. Probably by preventing cyclic AMP hydrolysis, 3‐isobutyl‐1‐methylxanthine facilitates the inotropic function of ventricular putative β4‐adrenoceptors, suggesting coupling to Gs protein‐adenylyl cyclase. The receptor‐mediated increases in contractile force are related to increases of Ca2+ in atrial and ventricular myocytes. The agreement of binding affinities of agonists with cardiostimulant potencies is consistent with mediation through putative β4‐adrenoceptors labelled with (−)‐[3H]‐CGP 12177. British Journal of Pharmacology (1999) 128, 1445–1460; doi:

Putative beta4-adrenoceptors mediate cardiostimulation and arrhythmias in mammalian heart. Both beta1- and putative beta4-adrenoceptors mediate arrhythmias but through different mechanisms. To elucidate further the mechanisms of cardiostimulation and arrhythmias we measured Ca2+ transients and L-type Ca2+ currents in mouse ventricular myocytes. We used (-)-CGP 12177, an antagonist of beta1- and beta2-adrenoceptors with agonist properties at the putative beta4-adrenoceptor, and (-)-isoprenaline as an agonist for beta1- and beta2-adrenoceptors. (-)-CGP 12177 increased Ca2+ transients in electrically stimulated cells loaded with Indo-1. The maximum increase of Ca2+ transients caused by (-)-CGP 12177 amounted to approximately one-third of that caused by maximally effective (-)-isoprenaline concentrations. Both (-)-CGP 12177 and (-)-isoprenaline caused concentration-dependent arrhythmic Ca2+ transients. The arrhythmias appeared at paced Ca2+ transients and between paced Ca2+ transients. The arrhythmic potency of (-)-CGP 12177 (-logEC50=9.4) was approximately 40 times greater than that of (-)-isoprenaline (-logEC50=7.8). L-type Ca2+ current was measured in the whole cell configuration of the patch clamp technique. In the presence of both 3-isobutyl 1-methylxanthine (6 micromol/l) and (-)-propranolol (500 nmol/l), (-)-CGP 12177 (100 nmol/l) increased significantly L-type Ca2+ current by 19% of the effect of (-)-isoprenaline. The (-)-CGP 12177-evoked increase of Ca2+ transients contrasts with the smaller effects on L-type Ca2+ current, suggesting that activation of the putative beta4-adrenoceptor causes a more efficient Ca2+-induced Ca2+ release than activation of the beta1-adrenoceptor. Beta4-Adrenoceptors mediate arrhythmias with smaller Ca2+ transients and smaller increases of L-type Ca2+ current than beta1-adrenoceptors, in line with different but still unknown mechanisms as previously suggested for the intact heart.

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Objecti¨e: Recently, deletion of specific genes by so called knock-out techniques has become important for investigating the pathogenesis of various diseases. This form of genetic engineering is widely performed in murine models. There are, however, only a limited number of mouse models available in cardiovascular pathology. The objective of this study, therefore, was to develop a new model of overt congestive heart failure associated with myocardial hypertrophy in the mouse. Methods: Female C57rBL6 mice weighing 19᎐20 g were anesthetized with ether. After abdominal incision, the aorta was temporarily clamped Ž . proximal to the renal arteries. The aorta was then punctured with a needle outer diameter 0.6 mm and the needle was further advanced into the adjacent vena cava. After withdrawal of the needle, the aortic puncture site was sealed with cyanoacrylate glue. The clamp was removed, and the patency of the shunt was visually verified as swelling and mixing of venous and arterial blood in the vena cava. Sham-operated mice served as controls. Results: Perioperative mortality of mice with aortocaval shunt was 42%. Four weeks after shunt induction, mice showed a significant cardiac hypertrophy with a Ž . relative heart weight of 7.5" 0.2 mgr100 g body weight vs. 5.1" 0.7 mgr100 g in control mice, P-0.001 . While no changes in blood pressure and heart rate occurred, left ventricular enddiastolic pressure was significantly increased in mice with shunt, Ž . and left ventricular contractility was impaired from 6331 " 412 to 4170 " 296 mmHgrs P -0.05 . Plasma concentrations of Ž . atrial natriuretic peptide ANP and its second messenger cGMP as humoral markers of heart failure as well as ventricular Ž . expression of ANP-and brain natriuretic peptide BNP -mRNA were significantly increased in mice with shunt compared to control mice. Conclusions: The aortocaval shunt in the mouse constitutes a new model of overt congestive heart failure with impaired hemodynamic parameters and may be a useful tool to investigate the role of particular genes in the development of heart failure. ᮊ