Myocardial determinants in regulation of the heart rate.

Heart rate is a function of at least three factors located in the sinus node, including the pacemaker and the activity of the sympathetic and vagal pathways. Heart rate varies during breathing and exercising. The is far from being a purely academic question because, after myocardial infarction or in cardiac insufficiency, reduced heart rate variability (HRV) represents the most valuable prognostic factor. HRV is usually considered index of the sympathovagal balance and is explored using time domain analysis, such as spectral analysis. Nevertheless, methods such as the Fast Fourier Transformation are not applicable to small rodents which have an unstable heart rate with asymmetric oscillations. Nonlinear methods show chaotic behavior under some conditions. A time and frequency domain method of analysis, the Wigner-Villé Transform, has been proposed for the study of HRV in both humans and small rodents, as a compromise between linear and nonlinear methods. We developed a method to quantify both arrhythmias and HRV in unanesthetized rodents. Such a method allows study of the relationship between the physiological parameters and the myocardial phenotype. Ventricular premature beats are more frequent in 16-month-old spontaneously hypertensive rats than in age-matched controls. In addition, HRV is attenuated in spontaneously hypertensive rats, as in compensatory cardiac hypertrophy in humans, and such attenuation is considered a prognostic index. Converting enzyme inhibition reduces in parallel arterial hypertension, cardiac hypertrophy, and ventricular fibrosis; it prevents ventricular premature beats and normalizes heart rate variability. It can be demonstrated that the incidence of ventricular premature beats is linked to the myocardial phenotype in terms of both cardiac hypertrophy and fibrosis. The two factors act as independent variables. HRV is correlated with the incidence of arrhythmias, suggesting that the beneficial effects of converting enzyme inhibition are related to prevention of arrhythmias.

Cardiac hypertrophy, arrhythmogenicity and the new myocardial phenotype. II. The cellular adaptational process.

Ventricular fibrosis is not the only structural determinant of arrhythmias in left ventricular hypertrophy. In an experimental model of compensatory cardiac hypertrophy (CCH) the degree of cardiac hypertrophy is also independently linked to ventricular arrhythmias. Cardiac hypertrophy reflects the level of adaptation, and matches the adaptational modifications of the myocardial phenotype. We suggest that these modifications have detrimental aspects. The increased action potential (AP) and QT duration and the prolonged calcium transient both favour spontaneous calcium oscillations, and both are potentially arrhythmogenic and linked to phenotypic changes in membrane proteins. To date, only two ionic currents have been studied in detail: Ito is depressed (likely the main determinant in AP durations), and If, the pacemaker current, is induced in the overloaded ventricular myocytes. In rat CCH, the two components of the sarcoplasmic reticulum, namely Ca(2+)-ATPase and ryanodine receptors, are down-regulated in parallel. Nevertheless, while the inward calcium current is unchanged, the functionally linked duo composed of the Na+/Ca2+ exchanged and (Na+, K+)-ATPase, is less active. Such an imbalance may explain the prolonged calcium transient. The changes in heart rate variability provide information about the state of the autonomic nervous system and has prognostic value even in CCH. Transgenic studies have demonstrated that the myocardial adrenergic and muscarinic receptor content is also a determining factor. During CCH, several phenotypic membrane changes participate in the slowing of contraction velocity and are thus adaptational. They also have a detrimental counterpart and, together with fibrosis, favour arrhythmias.

Compensated cardiac hypertrophy: arrhythmogenicity and the new myocardial phenotype. I. Fibrosis.

The high incidence of arrhythmias in compensated cardiac hypertrophy is related to two independently regulated components-fibrosis and the adaptational phenotypic changes in membrane proteins linked to cardiac hypertrophy, and fibrosis. During the regression of hypertensive cardiopathy in middle-aged spontaneously hypertensive rats, the roles of cardiac hypertrophy and fibrosis can be analysed separately, revealing that both correlate independently with arrhythmias. In an experimental model of myocardial infarction it is possible to prevent arrhythmias with propranolol at the same time as cardiac hypertrophy, despite ventricular fibrosis. Fibrosis would appear to create arrhythmias both by anatomical uncoupling and by a re-entry mechanism generated by the zig-zag propagation of the transverse waveform. Triggered activity and automaticity depend on the membrane phenotype of the cardiocyte. They also play an important role, which is aggravated by myocardial heterogeneity.

Specific atrial overexpression of G protein coupled human beta 1 adrenoceptors in transgenic mice.

OBJECTIVE: The aim was to develop a transgenic mouse model of atrial beta 1 adrenoceptor overexpression in order to create atrial alteration of the receptor transduction system. METHODS: Transgenic founders were generated after microinjection of the transgene construct into the pronucleus of fertilised mouse eggs. Heterozygous progeny were screened for RNA expression of the human beta 1 adrenoceptor gene under the control of a 0.56 kb proximal promoter of the human atrial natriuretic factor. One line, out of the three obtained, was selected and further characterised for overexpression of the human beta 1 adrenoceptor. Polymerase chain reaction was employed to detect beta 1 adrenoceptor mRNA, and 125I-cyanopindolol (ICYP) binding assays were used to quantify receptors in heart membranes. A quantitative autoradiographic ICYP binding technique was also used to visualise atrial and ventricular beta adrenoceptors in heart sections. RESULTS: The human beta 1 adrenoceptor was overexpressed specifically in the atria of transgenic mice. The level of the beta 1 adrenoceptor was 5-10-fold higher in transgenic mice compared to basal murine beta 1 adrenoceptors in non-transgenic control mice. Left and right atrial receptor overexpression was confirmed by in vitro autoradiography. The human receptors were able to couple to the murine stimulatory G proteins (Gs), as shown by high affinity binding site dosage using the beta adrenoceptor agonist isoprenaline. Isoprenaline displacement studies allowed the determination of two different affinity sites, one of high affinity (KH = 5.8 nM), and one of low affinity (KL = 520 nM). When expressed in terms of protein density (fmol.mg-1), atrial transgenic beta 1 adrenoceptors displayed a threefold increase in high affinity sites (KH) as compared to control mice. Preliminary electrocardiographic data showed supraventricular premature beats in 6/14 transgenic mice v 2/16 control mice. CONCLUSIONS: These transgenic mice may provide a useful pharmacological tool to investigate the pathophysiological consequences of the overactivation of atrial beta 1 adrenoceptor-adenylyl cyclase signalling system.

Linear and non-linear analyses of heart rate variability: a minireview.

To complete traditional time- and frequency-domain analyses, new methods derived from non-linear systems analysis have recently been developed for time series studies. A panel of the most widely used methods of heart rate analysis is given with computations on mouse data, before and after a single atropine injection.

Beta 1 adrenergic receptor and G alpha s mRNAs in rat heart as a function of mechanical load and thyroxine intoxication.

OBJECTIVE: The aim of this study was to determine the expression of genes coding for the beta 1 adrenergic receptor and the alpha subunit of Gs in the adult rat normal and hypertrophied left ventricle, and in the left ventricle of the hypophysectomised rat after T4 intoxication. METHODS: Total RNA was extracted from normal, control, or hypertrophied left ventricles 5 weeks after aortic stenosis, and from left ventricles of control or T4 injected hypophysectomised animals. The expression of beta 1 adrenergic receptor and G alpha s mRNAs was quantitated by northern blot analysis and hybridisation with specific 32P-dCTP labelled DNA probes. RESULTS: beta 1 Adrenergic receptor mRNA was decreased (by 33%) in compensated left ventricular hypertrophy without modification of the relative level of G alpha s mRNA. The relative level of beta 1 adrenergic receptor mRNA correlated negatively with the degree of left ventricular hypertrophy, suggesting that the expression of the beta 1 adrenergic receptor gene is not activated by pressure overload. In the left ventricle of the hypophysectomised rat, a rapid increase in beta 1 adrenergic receptor mRNA (by 180% 3 h after hormone injection) was observed in response to T4, with no change in the relative content of G alpha s mRNA. These results provide evidence that beta 1 adrenergic receptor mRNA and G alpha s mRNA accumulate to different levels of abundance in the adult left ventricle, as indicated by their ratios (0.053 and 0.043 in sham operated and hypertrophied left ventricles respectively). This suggests that distinct mechanisms are involved in the control of the accumulation of these two mRNAs in cardiac tissue. CONCLUSIONS: The reduction in beta 1 adrenergic receptor density in the hypertrophied rat left ventricle is associated with a parallel reduction in the level of beta 1 adrenergic receptor mRNA. The beta 1 adrenergic receptor gene may belong to a group of genes which are not activated by pressure overload, but are responsive to thyroid hormone.

Alterations in beta adrenergic and muscarinic receptors in aged rat heart. Effects of chronic administration of propranolol and atropine.

The cardiac responses to sympathetic and vagal stimulations are attenuated with ageing. To understand these findings, the densities of beta adrenergic (beta R) and muscarinic (MR) receptors in the left ventricles have been quantitated in parallel in male Wistar rats (4- and 24-month-old) using [125I]iodocyanopindolol and [3H]quinuclidinyl benzilate as specific radioligands. The homologous regulation of these receptor densities was also explored after a 7-day continuous infusion of propranolol or atropine. As compared to young rats, the beta R and MR densities in aged animals were decreased (from 31 +/- 2 to 23 +/- 2 fmol/mg protein, P less than 0.05 for beta R; from 104 +/- 7 to 54 +/- 3 fmol/mg protein, P less than 0.001 for MR) but the diminution in MR was more pronounced (-48%) than that in beta R (-26%), resulting in a drop in the beta R/MR ratio. Continuous infusion of propranolol or atropine up-regulated the beta R and MR densities (respectively +50%, P less than 0.01 and +33%, P less than 0.05) in aged but not in young adult rats. We therefore conclude: (i) that the diminution of the cardiac response to the sympathetic and vagal stimulations during ageing may be partly explained by a decrease in the corresponding receptor density; (ii) these changes are reversible and the density of these two groups of receptors can return to adult control values by chronic administration of the appropriate antagonist.

Altered baroreflex gain during voluntary breathing in chronic heart failure.

BACKGROUND: We assessed the behavior of the baroreflex (BR) gain in chronic heart failure (CHF) patients using the spectral analysis method during application of a forcing stimulus, i.e. respiration. METHODS: Simultaneous RR interval and arterial pressure fluctuation recordings were obtained during two random-order periods of voluntary paced-breathing (0.15 Hz and 0.25 Hz) in seven patients with moderate CHF (NYHA class II/III; EF, 30+/-9%; peak VO(2), 18+/-5 ml kg(-1) min(-1)) and six age-matched controls. BR gain was assessed in the time (sequential method) and frequency (cross-spectral gain in the low and high frequency) domains. RESULTS: Slower breathing was associated with a BR gain decrease in CHF patients whereas a BR gain increase was evidenced in controls (BR gain: 6+/-5 ms mmHg(-1) at 0.25 Hz vs. 4+/-3 ms mmHg(-1) at 0.15 Hz, P<0.05 in CHF; BR gain: 12+/-7 ms mmHg(-1) at 0.25 Hz vs. 15+/-7 ms mmHg(-1) at 0.15 Hz, P<0.05 in controls). CONCLUSIONS: Voluntary breathing, which involves cortical centers in the brain, had major effects on cardiovascular system controller gain in CHF patients, indicating an impairment of the central neural regulation of the autonomic outflow.

Remodeling of the heart (membrane proteins and collagen) in hypertensive cardiopathy.

The basis for impaired left ventricular function of hearts in moderate to severe stages of hypertrophy and congestive heart failure remains uncertain. At the cellular level, the mechanisms governing the movements of calcium in the myocardium are actually depressed and might at least in part account for the slowing of the maximum shortening velocity and the impaired relaxation. These alterations of membrane proteins seem particularly important in species where the slowing of Vmax cannot be a consequence of the myosin heavy chain shift. They lead to an unstable equilibrium of calcium homeostasis and to calcium overload in heart failure. On the other hand, the enhanced density and remodeling of collagen in the hypertrophied heart, which would depend on elevation in circulating aldosterone, impair myocardial stiffness with diastolic dysfunction and lead to altered pumping capacity of the heart. Disturbances of calcium metabolism and matrix collagen remodeling enhance early afterdepolarizations and arrhythmias.

Aldosterone and anti-aldosterone effects in cardiovascular diseases and diabetic nephropathy.

Cells in the cortical collecting duct of distal nephron have been considered for a long time as the unique cellular targets of aldosterone. However, it is now clear that other cell types in non-epithelial tissues are also potential targets for aldosterone. The functions that this hormone controls in non-epithelial tissues are still a matter of debate. Clinical and experimental studies have established that aldosterone plays a major role in the pathophysiology of cardiovascular and renal diseases. The aldosterone receptor antagonists spironolactone and eplerenone have demonstrated specific effects not related to their hypotensive properties in hypertension or cardiac diseases. It appears that a key action of these molecules is related to prevention or treatment of end-organ damage. The latter fact, and the recognition of aldosterone escape on long-term treatment of heart failure, diabetic nephropathy and some forms of hypertension with ACE inhibitors, justify the clinical use of aldosterone receptor antagonists provided that kaliemia is controlled. Experimental studies have allowed to draw a still incomplete but comprehensive scheme of aldosterone cardiovascular actions in pathological conditions. When elevated, aldosterone has deleterious effects in blood vessels, in the heart and in kidney, which are secondary to the induction of inflammatory and oxidative processes and necrosis, that induce the increased synthesis of extracellular matrix proteins.

Use of time frequency analysis to follow transitory modulation of the cardiac autonomic system in clinical studies.

Heart rate variability (HRV) can be assessed through a time frequency analysis, the smoothed pseudo Wigner-Ville transformation (SPWVT). Such an analysis has allowed the calculation of ICF, a frequential index, extremely sensitive to the modulation of the sympatho-vagal balance. The use of ICF has been applied in the present study to two clinical situations for which a role of this balance was probable, brain death and atrial fibrillation (AF). The use of the time frequency analysis immediately identified the timing of brain death. No difference could be found in the analysis of the 30 min preceding an atrial fibrillation episode. It is suggested that a time frequency analysis should be used in clinical situations in which transitory fluctuations of the sympatho-vagal balance are expected and crucial to the well being of the patients.

Membrane proteins of the myocytes in cardiac overload.

Hypertrophy of the cardiac myocytes resulting from a mechanical overload may be responsible for major membraneous modifications, either at a sarcolemmal or at a sarcoplasmic level. In the present report, evaluation of several sarcolemmal markers such as beta-receptors, muscarinic receptors or (Na+,K+)-ATPase has been realized on an experimental model of cardiac hypertrophy performed on adult rats. Special attention has been taken to compare results expressed in densities or expressed in number of receptors present at the cellular level. For example, the beta-receptors density was 30% decreased whereas calculations of number of receptors present in the myocytes revealed an unchanged number (around 20,000 in both groups). The unmodified number could suggest a non regulation for the genes coding for the receptors.

A statistical analysis of sequences of cardiac interbeat intervals does not support the chaos hypothesis.

Series of cardiac interbeat intervals were recorded in 34 mice and described using an original technique. The different intervals were divided into six classes according to their lengths. The different successions of pairs of intervals were counted with the help of a software programme devoted to lexical analysis. Most of the mice showed a very similar pattern of repeated sequences of interbeat intervals, statistically different from a random distribution. One-, two- or three-dimensional chaotic discrete maps were used to generate series that were then analyzed in the same way. They failed to give a distribution pattern of repeated sequences similar to the biological ones. On the contrary, series generated using a random component, and limited in amplitude by a feed-back correction occurring when the intervals reached a lower or an upper threshold, gave distributions of values very similar to biological ones. Moreover, the natural diversity observed between mice was easily reproduced by fluctuation of these different parameters. It is suggested that a very simple process, not completely deterministic, could better explain interbeat interval regulation in normal mice than did chaotic phenomena, which need complex biological processes.

Effects of calcium on the heterogeneity of the Na+, K+-ATPase forms in rat heart.

The sensitivity of the Na+, K+-ATPase to ouabain has been studied in sarcolemma vesicles isolated from normal rat heart. Two enzyme forms exhibiting high and low sensitivities to ouabain have been observed in Ca2+-free perfused heart. The half-maximal inhibitory effects occurred with 1-2 X 10(-8) M ouabain. The high sensitivity form undetectable in hearts maintained at a physiological Ca2+ level might represent altered low affinity sites or latent enzyme forms unmasked by low calcium concentrations. The heterogeneity of the Na+, K+-ATPase forms was found to be also modulated by the K+/ouabain antagonism, addition of K+ accentuating the heterogeneity. These in vitro results associated with in vivo experiments on isolated rat heart working under isovolumic conditions suggested that lowering Ca2+ has qualitative and quantitative effects. Low Ca2+ concentrations increased the sensitivities to ouabain and the amplitudes of both the enzyme inhibition and the positive inotropic effects.

Beta adrenergic and muscarinic receptors in compensatory cardiac hypertrophy of the adult rat.

The beta adrenergic (beta AR) and muscarinic (MR) receptors have been quantitated in parallel, using 125I-pindolol and 3H-quinuclidinylbenzilate, in a model of compensatory left ventricular (LV) hypertrophy (LVH), which developed in rats 4-6 weeks after an abdominal aortic stenosis. Since aortic banding resulted in a pronounced LVH of 62%, the results were expressed both in terms of density (fmol/mg protein) and quantity (fmol per LV). In addition, competition curves using either a specific beta 1-antagonist or isoproterenol or carbachol allowed the determination of the two beta AR subtypes and of the low and high affinity sites (defined by the inhibitory constant Ki) for both beta 1AR and MR. In LVH, receptor density decreased for each of total beta AR, beta 1AR subtype, high affinity (Ki 6-8 nM) beta 1AR sites (from 26 +/- 2 to 19 +/- 3 fmol/mg protein, P < 0.05), total MR and high affinity (Ki 12 nM) MR sites (from 63 +/- 6 to 40 +/- 4 fmol/mg protein, P < 0.001). The beta AR and MR densities dropped in parallel so that the MR/beta AR ratio remained unchanged. In sharp contrast (because the LVs were bigger) the quantities of total beta AR, beta 1AR subtype, beta 1AR high affinity sites, total MR and MR high affinity sites per LV were unmodified.(ABSTRACT TRUNCATED AT 250 WORDS)

Screening of inotropic drugs on isolated rat and guinea pig hearts.

A technique is described for screening the effects of inotropic drugs on isolated rat or guinea pig hearts perfused at constant coronary pressure and at a frequency of 6 Hz. Their performances, including function curves, were recorded using an intraventricular balloon. Both preparations became either sensitive from initially having been insensitive, or more sensitive from having been slightly sensitive at the outset, to inotropic interventions, provided the external calcium concentration was reduced to 0.25 mM for the rat and 0.50 mM for the guinea pig. The inotropic effect of drugs such as isoproterenol, forskolin, and theophylline was only slightly altered by lowering [Ca]o. Amrinone, sulmazole, and beta agonists such as xamoterol, cicloprolol, pindolol, or RU 42173 almost never caused an inotropic effect at the serum calcium concentration of 2.50 mM, whereas they did provoke a positive response at low [Ca]o. Other compounds such as ouabain, salbutamol, and pimobendan were toxic at high [Ca]o, although at reduced [Ca]o their positive effect on contractility was quite evident.

Beta-adrenergic system is modified in compensatory pressure cardiac overload in rats: physiological and biochemical evidence.

The beta-adrenergic system has been explored in cardiac hypertrophy (CH) by combining an in vitro study of the inotropic effect of isoproterenol (ISO) and of forskolin (FSK) and binding assays using radioactive pindolol. CH was obtained within 4-6 weeks by banding the abdominal aorta which results in an increase in left ventricular (LV) weight [743 +/- 14 and 1,098 +/- 24 mg in sham-operated (SH) and in CH, respectively, p less than 0.01] and in the LV wt/body wt ratio (2.14 +/- 0.05 and 3.24 +/- 0.05, p less than 0.001). The inotropic effect was evaluated on an isolated Langendorff heart preparation whose coronary flow was normalized per gram of tissue either by using different constant coronary pressures (75 and 110 mm Hg in SH and in CH, respectively) or different constant coronary flows (15 and 20 ml min-1 in SH and CH, respectively). Binding assays were performed using 125I pindolol, a rather crude preparation of sarcolemma and a LIGAND program for calculation. (i) The inotropic responsiveness to both ISO and FSK is depressed by approximately 30% in CH, at any drug concentration, without change in EC50 whatever the technique used to perfuse the tissue. The time course of the process is slower but, in the case of ISO, remains biphasic. (ii) Binding assays show a normal affinity (Kd 100 pM) while receptor density is depressed (32 +/- 3 and 25 +/- 2 fmol mg protein-1, p less than 0.05 or 3,287 +/- 312 and 2,000 +/- 163 fmol/g fresh tissue, p less than 0.01, in SH and CH, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

beta-Adrenergic and muscarinic receptor expression are regulated in opposite ways during senescence in rat left ventricle.

The well-known attenuated sensitivity of senescent heart to isoproterenol is accompanied by a decreased beta1-adrenergic receptors (beta1-AR) density, a down regulation process which may involve several molecular modifications and whose understanding is incomplete. Data concerning the M2-R muscarinic receptors (M2-R) are more contradictory. Both the absolute and relative concentrations of beta1-AR and M2-R as well as the coupling protein G alpha s and G alpha(i2) mRNAs were determined by slot-blot analysis in the left ventricles (LVs) of 6-7 week and 22-month-old male Wistar rats. In addition, the beta-AR and M2-R densities were quantitated by radioactive ligand binding. (1) The M2-R mRNA concentration increases by 92+/-32% in senescent as compared to adult animals; by contrast, the density in M2-R remains unchanged, suggesting that the M2-R expression was not exclusively regulated at a pre-translational level. (2) The beta1-AR mRNA concentration was nearly halved (reduced by 46+/-9.5%) and paralleled the 51+/-5.6% diminution of the beta-AR density which resulted exclusively from the decrease of beta1-AR density without change in the beta2-AR concentration, suggesting a pre-translational regulation of the beta1-AR expression. (3) G alpha(i2) mRNA concentration was unchanged, while G alpha s mRNA concentration was reduced by 26+/-4.6% in senescent compared with adult LVs. To conclude, the different components of the adrenergic and muscarinic systems are differentially regulated during aging.

Isolation of impermeable inside-out vesicles from an enriched sarcolemma fraction of rat heart.

Sarcolemmal vesicles isolated from relaxed rat cardiac ventricles were 120-fold enriched in (Na+ + K+)-ATPase and 5'-nucleotidase activities (final recoveries, 50%). The alpha and beta chains of the former enzyme were visualized by the immunological approach. Inside-out sarcolemmal vesicles were isolated by affinity chromatography on immobilized concanavalin A. The yield of membranes was 0.45 mg of protein/g of muscle. The orientation of the unbound vesicles was studied by the increased accessibility of sarcolemma outer face markers (ouabain- and K+-binding sites, 5'-nucleotidase, and sialic acids) with permeability-increasing treatments: freeze-thaw cycles, sodium dodecyl sulfate, methanol, and valinomycin. The total ATP hydrolysis remained constant with a conversion of ouabain-insensitive activity into an ouabain-sensitive one. These agents caused a parallel increase in the ouabain sensitivity, the number of [3H]ouabain-binding sites, the monovalent cation stimulation of ATPase, and the 5'-nucleotidase activity. Valinomycin revealed that most vesicles were sealed to sequestered and exogenous K+. Inside-out vesicles were 80% pure in sidedness and sealing. The affinity chromatography did not affect the (Na+ + K+)-ATPase activity (200 mumol of product/mg of protein/h). This model of sarcolemma vesicles offers a new tool for ion transport studies.