Classical and novel pharmacological insights offered by the simple chick cardiomyocyte cell culture model: a valuable teaching aid and a primer for "real" research.

The chick embryo cardiomyocyte model of cell culture is a staple technique in many physiology and pharmacology laboratories. Despite the relative simplicity, robustness, and reproducibility inherent in this model, it can be used in a variety of ways to yield important new insights that help facilitate student understanding of underlying physiological and pharmacological concepts as well as, more generally, the scientific method. Using this model, this paper will show real data obtained by undergraduate students in the authors' laboratories. It will first demonstrate classical pharmacological concepts such as full and partial agonism, inverse agonism, and competitive reversible antagonism and then move on to more complex pharmacology involving the characterization of novel receptors in these cells.

A new model of congestive heart failure in the mouse due to chronic volume overload.

OBJECTIVE: 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 C57/BL6 mice weighing 19-20 g were anesthetized with ether. After abdominal incision, the aorta was temporarily clamped 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 relative heart weight of 7.5+/-0.2 mg/100 g body weight (vs. 5.1+/-0.7 mg/100 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, and left ventricular contractility was impaired from 6331+/-412 to 4170+/-296 mmHg/s (P<0.05). Plasma concentrations of atrial natriuretic peptide (ANP) and its second messenger cGMP as humoral markers of heart failure as well as ventricular 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.

The effect of Mg2+ on cardiac muscle function: Is CaATP the substrate for priming myofibril cross-bridge formation and Ca2+ reuptake by the sarcoplasmic reticulum?

Kinetics are established for the activation of the myofibril from the relaxed state [Smith, Dixon, Kirschenlohr, Grace, Metcalfe and Vandenberg (2000) Biochem. J. 346, 393-402]. These require two troponin Ca2+-binding sites, one for each myosin head, to act as a single unit in initial cross-bridge formation. This defines the first, or activating, ATPase reaction, as distinct from the further activity of the enzyme that continues when a cross-bridge to actin is already established. The pairing of myosin heads to act as one unit suggests a possible alternating mechanism for muscle action. A large positive inotropic (contraction-intensifying) effect of loading the Mg2+ chelator citrate, via its acetoxymethyl ester, into the heart has confirmed the competitive inhibition of the Ca2+ activation by Mg2+, previously seen in vitro. In the absence of a recognized second Ca2+ binding site on the myofibril, with appropriate binding properties, the bound ATP is proposed as the second activating Ca2+-binding site. As ATP, free or bound to protein, can bind either Mg2+ or Ca2+, this leads to competitive inhibition by Mg2+. Published physico-chemical studies on skeletal muscle have shown that CaATP is potentially a more effective substrate than MgATP for cross-bridge formation. The above considerations allow calculation of the observed variation of fractional activation by Ca2+ as a function of [Mg2+] and in turn reveal simple Michaelis-Menten kinetics for the activation of the ATPase by sub-millimolar [Mg2+]. Furthermore the ability of bound ATP to bind either cation, and the much better promotion of cross-bridge formation by CaATP binding, give rise to the observed variation of the Hill coefficient for Ca2+ activation with altered [Mg2+]. The inclusion of CaADP within the initiating cross-bridge and replacement by MgADP during the second cycle is consistent with the observed fall in the rate of the myofibril ATPase that occurs after two phosphates are released. The similarity of the kinetics of the cardiac sarcoplasmic reticulum ATPase to those of the myofibril, in particular the positive co-operativity of both Mg2+ inhibition and Ca2+ activation, leads to the conclusion that this ATPase also has an initiation step that utilizes CaATP. The first-order activation by sub-millimolar [Mg2+], similar to that of the myofibril, may be explained by Mg2+ involvement in the phosphate-release step of the ATPase. The inhibition of both the myofibril and sarcoplasmic reticulum Ca2+ transporting ATPases by Mg2+ offers an explanation for the specific requirement for phosphocreatine (PCr) for full activity of both enzymes in situ and its effect on their apparent affinities for ATP. This explanation is based on the slow diffusion of Mg2+ within the myofibril and on the contrast of PCr with both ATP and phosphoenolpyruvate, in that PCr does not bind Mg2+ under physiological conditions, whereas both the other two bind it more tightly than the products of their hydrolysis do. The switch to supply of energy by diffusion of MgATP into the myofibril when depletion of PCr raises [ATP]/[PCr] greatly, e.g. during anoxia, results in a local [Mg2+] increase, which inhibits the ATPase. It is possible that mechanisms similar to those described above occur in skeletal muscle but the Ca2+ co-operativity involved would be masked by the presence of two Ca2+ binding sites on each troponin.

Differential lusitropic responsiveness to beta-adrenergic stimulation in rat atrial and ventricular cardiac myocytes.

Cardiac myocyte relaxation is brought about mainly through Ca2+ uptake into the sarcoplasmic reticulum (SR) by a Ca2+-ATPase isoform, SERCA2a. Its activity is modulated by another protein, phospholamban (PLB). The levels of both proteins differ in some mammals between atrial and ventricular myocardium and this may lead to differences in relaxation, especially under stimulatory conditions. At a concentration of 100 nM, the beta-adrenergic agonist isoprenaline (ISO) accelerates the relaxation of rat papillary muscle more than that of the left atria (16.4 versus 4.0% hastening of time to 50% relaxation, respectively). Ventricular myocytes were 24.7% quicker in reaching 50% of their diastolic length after contraction when treated with ISO compared to atrial myocytes, which were only 3.6% faster. Ca2+ fluorescence transients were also abbreviated in ventricular compared to atrial myocytes exposed to ISO (41.9 versus 25.2% hastening of time to 50% peak Ca2+ respectively). Ca2+ uptake into ventricular SR vesicles was increased by 13% in the presence of protein kinase A while that into atrial SR vesicles remained unaffected. Western blotting analysis revealed 23% less SERCA2a protein, but 76% more PLB in ventricular compared to atrial tissue. We conclude that the distinct levels of SERCA2a and PLB in ventricular and atrial myocardium are responsible for the differential modulation of the relaxation process arising from beta-adrenergic stimulation in single rat atrial and ventricular myocytes.

Putative beta 4-adrenoceptors in rat ventricle mediate increases in contractile force and cell Ca2+: comparison with atrial receptors and relationship to (-)-[3H]-CGP 12177 binding.

1. We identified putative beta4-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. 2. (-)-[3H]-CGP 12177 labelled 13 - 22 fmol mg-1 protein ventricular beta1, beta2-adrenoceptors (pKD approximately 9.0) and 50 - 90 fmol mg-1 protein putative beta4-adrenoceptors (pKD approximately 7.3). The affinity values (pKi) for (beta1,beta2-) and putative beta4-adrenoceptors, estimated from binding inhibition, were (-)-propranolol 8.4, 5.7; (-)-bupranolol 9.7, 5.8; (+/-)-cyanopindolol 10.0,7.4. 3. In left ventricular papillary muscle, in the presence of 30 microM 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). 4. (-)-CGP 12177 (1 microM) 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 microM 3-isobutyl-1-methylxanthine and 200 nM (-)-propranolol, 1 microM (-)-CGP 12177 caused a 73% increase in Ca2+ transient amplitude in ventricular myocytes. (-)-CGP 12177 elicited arrhythmic transients in some atrial and ventricular myocytes. 5. Probably by preventing cyclic AMP hydrolysis, 3-isobutyl-1-methylxanthine facilitates the inotropic function of ventricular putative beta4-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 beta4-adrenoceptors labelled with (-)-[3H]-CGP 12177.

Beta4-adrenoceptors are more effective than beta1-adrenoceptors in mediating arrhythmic Ca2+ transients in mouse ventricular myocytes.

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.

The effect of insulin-like growth factor-1 on adult rat cardiac contractility.

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.

Small intestinal permeability and orocaecal transit time in cystic fibrosis.

Cellobiose and mannitol were used as probe molecules to measure intestinal permeability in 36 children with cystic fibrosis, and 25 age matched controls. Orocaecal transit was also evaluated for each subject using the lactulose/hydrogen breath test. There was a fourfold increase in permeability to disaccharide (cellobiose) in patients with cystic fibrosis, but permeability to the monosaccharide (mannitol) was similar to controls. The orocaecal transit time of lactulose was prolonged in patients with cystic fibrosis, but was unrelated to the percentage excretion of cellobiose or mannitol in cystic fibrosis patients or control subjects.