Subchronic alpha- and beta-adrenergic regulation of cardiac gap junction protein expression.

Gap junction channels are essential for intercellular electrical communication in the heart. The most important cardiac gap junction proteins are connexin43 (predominantly) (Cx43), connexin40 (Cx40), and in early developmental stages connexin45. Since catecholamines play an important role in cardiac (patho)physiology, we wanted to elucidate whether catecholamines may affect expression of Cx43 and Cx40. Cultured neonatal rat cardiomyocytes were exposed for 24 h to increasing concentrations of noradrenaline (1-10000 nM) (physiological agonist at alpha and beta-adrenoceptors), resulting in significantly increased Cx43-expression, while Cx40 was unaffected. In further experiments cells were incubated with either phenylephrine (alpha-adrenergic agonist) or isoproterenol (beta-adrenergic agonist) (0.1-1000 nM) for 24 h. Both catecholamines lead to a concentration-dependent increase in Cx43 protein and mRNA expression (EC50: 10-20 nM). Inhibition experiments showed that the phenylephrine effect was transduced via PKC, while the isoproterenol effect was mediated by PKA. Dual whole-cell voltage clamp demonstrated that increased Cx43-expression was accompanied by significant increases in gap junction current. In additional in vivo experiments, adult rats were subjected to 24-h infusion of isoproterenol or phenylephrine showing again significant increase in Cx43 but not Cx40. Adrenergic stimulation of cardiomyocytes can enhance Cx43 expression thereby increasing cellular coupling, indicating a possible role for catecholamines in the regulation of cardiac gap junction expression in cardiac disease.

Norepinephrine-induced interleukin-6 increase in rat hearts: differential signal transduction in myocytes and non-myocytes.

Continuous i.v. infusion of norepinephrine in rats has been shown to induce early interleukin (IL)-6 mRNA expression in the left ventricle (LV) which was followed by hypertrophy and fibrosis. In this study, two approaches were used. In the first, NE (0.1 mg/kg per hour) was infused i.v. in rats for several time periods, and freshly obtained ventricular myocardium was dissociated into myocyte (MC) and non-myocyte (NMC) fractions. Second, isolated adult MCs and fibroblasts were treated with NE (10 microM). NE infusion (4 h, in vivo) caused an 11-fold increase in IL-6 mRNA in both cell populations. In vitro treatment of isolated adult MCs for 2 h and of fibroblasts for 1 h with NE induced a 3.5- and 23-fold maximum increase, respectively, in IL-6 mRNA. After in vivo NE treatment, the expression of the mRNA of the transcriptional factor of IL-6, C/EBP-beta, was elevated earlier (after 45 min of NE infusion) than IL-6 mRNA (after 4 h) and was seen in MCs and NMCs. The mRNAs of both receptors of IL-6, the soluble IL6R and gp130, were increased subsequently to IL-6 mRNA. Gp130 was elevated after 24 h and, like IL6R, predominantly in NMCs. In contrast, the IL6R protein and the downstream regulator STAT3 were increased only in MCs after 24 h of NE infusion. The mRNA of C/EBP-delta, which is regulated by STAT3, was elevated only in myocytes.

Serum depletion induces cell loss of rat cardiac fibroblasts and increased expression of extracellular matrix proteins in surviving cells.

OBJECTIVE: Since reduced nutrient supply is one component of ischemia, we have studied the effect of serum depletion on the survival of fibroblasts isolated from adult rat hearts and on the expression and degradation of extracellular matrix (ECM) proteins. Furthermore, we measured the role of the cAMP-dependent pathway in these processes. METHODS: Isolated cardiac fibroblasts were grown to confluency in 10% serum containing medium. Serum was then removed and cell number was measured by use of a Coulter Counter. The activity of the cAMP response element binding protein (CREB) was investigated by Western blotting and subsequent use of the specific antibody which binds to the active form of the protein. The expression of colligin, collagen I and III, matrix metalloproteinases 2 (MMP-2), and tissue inhibitor of matrix metalloproteinase 2 (TIMP-2) was examined by ribonuclease protection assay (RPA) and Western blotting. Zymographic measurements were done to investigate gelatinase activity of MMP-2. RESULTS: Serum withdrawal caused the death of 36% of the cells during the first 8 h. CREB was strongly phosphorylated 5 min after serum removal. Activation persisted up to 8 h and decreased thereafter. The mRNA abundance of colligin, collagen I and III, MMP-2, and TIMP-2 started to increase after 5 and 10 h, respectively, reaching a maximum after 20-30 h and decreasing thereafter. Protein levels of collagen I, collagen III, colligin and TIMP-2 were higher after 24 h until up to 96 h. MMP-2 zymographic activity was elevated 15-fold after 72 h. Application of the protein kinase A (PKA) blocker RpcAMPS suppressed the increase in phosphorylation of CREB. The increase in collagen III and MMP-2 mRNA abundance and elevation of collagen I and III, and TIMP-2 protein was diminished by RpcAMPS. The rise of colligin protein was completely suppressed. The increase in MMP-2 zymographic activity was also attenuated. RpcAMPS improved survival rate from 56 to 84%. CONCLUSIONS: Serum depletion led to cell death of isolated cardiac fibroblasts. Survival was associated with the increase in the expression of various ECM proteins. The transcription factor CREB was activated after serum removal. Inhibition of PKA improved the serum depletion induced decrease in the survival rate. The increase in collagen I, collagen III, MMP-2, TIMP-2, and colligin evoked by serum depletion was also diminished by PKA inhibition.

Cardiac remodeling after long term norepinephrine treatment in rats.

OBJECTIVE: In this study we have tested the hypothesis that degradation of collagen by matrix metalloproteinase 2 (MMP-2) precedes the deposition of extracellular matrix (ECM) after long term norepinephrine (NE) treatment. METHODS: Female Sprague-Dawley rats received continuous i.v. infusion of NE (0.1 mg/kg.h) for 1, 2, 3, 4 and 14 days. Heart function and weight as well as expression of cardiac colligin and of collagen I and III were examined. Furthermore, we have assessed the degradation pathway of collagen by measuring the mRNA and activity of myocardial MMP-2 and tissue inhibitor of metalloproteinase 2 (TIMP-2) as well as the protein level of TIMP-2. RESULTS: NE induced hypertrophy predominantly of the left ventricle (LV) in a time-dependent manner. It increased the mRNAs of colligin, collagen I and III, and of MMP-2 and TIMP-2 as well as MMP-2 activity in two phases: In the initial phase, at 3 and 4 days, the mRNA of colligin and of collagen I and III was elevated predominantly in the LV, MMP-2 and TIMP-2 mRNA, as well as TIMP-2 protein and MMP-activity were increased in both ventricles. The second phase, after 14 days, was characterized by a less pronounced increase in colligin, collagen I and III and in MMP-2 activity which occurred exclusively in the LV. Finally, long-term treatment with NE induced a 37% increase in interstitial fibrosis which was shown to occur exclusively in the LV after 14 days. CONCLUSION: NE treatment induced fibrosis exclusively in the LV which was associated with hypertrophy predominantly of the LV. The elevated MMP-2 activity seems to be necessary for the ECM to adapt to the enlargement of myocytes and to reduce overproduction of collagen.

Perfusion of isolated organs and the first heart-lung machine.

In 1885, Max von Frey (1852-1932), while working in Carl Ludwig's Physiological Institute in Leipzig, Germany, designed an apparatus that had criteria characteristic of a heart-lung machine. With this device, he perfused the entire lower extremity of dogs, and took measurements of oxygen consumption, and carbon dioxide and lactate production. In 1935, another type of perfusion apparatus was constructed by Charles A Lindbergh (1902-1973). This device was the result of cooperation with Alexis Carrel (1873-1944) who was a pioneer of experimental organ transplantation. Using Lindbergh's pulsating device, organs such as thyroid, ovary, suprarenal gland, spleen, heart and kidney from fowls and cats were perfused with an oxygenated medium, and were maintained under sterile conditions. Beginning in 1934, John H Gibbon (1903-1973) developed and tested a heart-lung machine to institute cardiopulmonary bypass in cats during experimental occlusion of the pulmonary artery. In 1953, he performed the first successful open-heart operation in a patient using a heart-lung machine. This included elements that were similar to those used by von Frey - ie, the oxygenator and the pumps for continuous circulation of blood. A comparison of the three experimental devices revealed the following: the application for experimental purposes preceded clinical use; the development shifted from Europe to the United States, and was achieved by people who were not specialists; and the intention to build such a device was first purely scientific interest, but later shifted to the care for and treatment of patients with heart and circulatory defects by open-heart surgery.

Transient pleural effusion in norepinephrine-stimulated rats.

Transient pleural effusions occurred in rats receiving continuous intravenous infusion of norepinephrine (NE, 0.1 mg/kg/h). We hypothesized that these pleural effusions result from a NE-induced increase in right ventricular systolic pressure (RVSP) and total peripheral resistance (TPR). NE was administered over time intervals between 20 min and 72 h. It induced an immediate doubling in RVSP whereas LVSP remained at the control level. TPR increased with a delay of 6 h. At this time, pleural effusions occurred in NE-treated animals, reached their maximum after 8h and disappeared after 24 h of NE stimulation. Combining NE with the alpha-blocker prazosin normalized TPR and prevented pleural effusions. Therefore, we interpret the pleural effusion as a consequence of pulmonary venous congestion, mainly caused by an increased TPR. LV hypertrophy which developed after 24 h of NE stimulation is considered to compensate for the hemodynamic disturbance due to the NE-induced elevation in TPR. This is reflected in the disappearance of pleural effusion.

Changes in extracellular matrix and in transforming growth factor beta isoforms after coronary artery ligation in rats.

Extensive myocardial remodeling occurs after transmural myocardial infarction (MI). The infarcted myocardium is being replaced by scar tissue after gradual resorption of the necrotic tissue. The remodeling process involves both synthesis and degradation of collagens as major components of the extracellular matrix (ECM). In the present study we have analyzed the time-dependent changes of the processes related to this fibrosis in the infarct area and in the non-infarcted left ventricle (LV) six hours to 82 days after occlusion of the left anterior descending coronary artery (LAD) in rats. We also examined whether changes occurred in the expression pattern of the transforming growth factor (TGF) beta isoforms, since this cytokine is known as powerful inductor of fibrosis. Elevation in colligin expression preceded the pronounced increase in mRNA expression of both type I and type III collagen after MI from day three onwards. The maximal increase in colligin protein in the infarct area coincided with the most pronounced expression of collagen I and collagen III mRNA expression. Also, the expression and activity of matrix metalloproteinases (MMPs) and of tissue inhibitor of matrix metalloproteinase (TIMP)-2 mRNA were increased predominantly in the infarct area. TGF beta(1)and TGF-beta(2)expression increased within the first days after MI, whereas TGF-beta(3)expression was elevated predominantly in the infarct area. This pronounced increase in TGF-beta(3)persisted up to 82 days and correlated positively with the parameters of ECM metabolism. Thus, the scar formation is an ongoing dynamic process in which TGF-beta(3)seems to play an active role in the complex ventricular remodeling.

Catecholamines stimulate interleukin-6 synthesis in rat cardiac fibroblasts.

Proinflammatory cytokines have been implicated in the pathophysiology of different heart diseases. Recent evidence suggests that interleukin-6 (IL--6) may play a role in mechanisms leading to cardiac hypertrophy. In addition, catecholamines are known to induce cardiac hypertrophy. In the present study, we examined whether cardiac fibroblasts may be a potential source of IL--6 production in the rat heart and whether catecholamines can modulate the IL--6 synthesis. Only a small amount of IL--6 mRNA was detected in unstimulated rat cardiac fibroblasts. However, a 50-fold increase of IL--6 mRNA was found after stimulation with norepinephrine (NE). Addition of carvedilol, a alpha- and beta-adrenergic receptor antagonist, prevented almost completely the NE-induced synthesis of IL--6 mRNA. Phenylephrine, an alpha-adrenergic agonist, and isoproterenol, a beta-adrenergic agonist, also induced an increase in IL--6. However, the stimulation via beta-receptors led to a more pronounced elevation. These data show that NE increases IL--6 expression in rat cardiac fibroblasts and that IL--6 may play an important autocrine/paracrine role in cardiac disease states associated with hypertrophy.

Differential remodeling of the left and right heart after norepinephrine treatment in rats: studies on cytokines and collagen.

Continuous intravenous infusion of norepinephrine norepinephrine (NE, 0.1 mg/kg/h) induced hypertrophy of the left ventricle (LV), but not of the right ventricle (RV) in rats, although RV systolic pressure (RVSP) was much more elevated than LVSP. After NE infusion, there was a time-dependent (20 min to 72 h) expression in the mRNA of interleukin (IL)-6 and IL-1 beta. The expression of IL-6 increased markedly and reached the maximum after 4 h with an 80-fold elevation. In the RV, the expression increased only 20-fold. The mRNA of IL-1 beta increased significantly after NE stimulation only in the LV and reached the maximum after 12 h with a 12-fold elevation. After 12 h of NE infusion, colligin mRNA was elevated for the first time with further progression until 72 h. The six-fold abundance of colligin mRNA seen after 72 h was significantly higher in the LV than in the RV. A similar increase was observed on the protein level (Western blotting). The expression of collagen I and III increased significantly after 24 h only in the LV. After 72 h, the mRNA expression of collagen I was increased 16-fold and that of collagen III 10-fold. This expression was significantly higher than that in the RV. Also the elevation in atrial natriuretic peptide (ANP) mRNA started earlier and was more pronounced in the LV than in the RV. The alpha- and beta-adrenergic receptor blocker carvedilol normalized all functional parameters after 6 h and 72 h and prevented the development of LV hypertrophy that occurred after 72 h. The NE-induced increased expression of the mRNAs studied was either prevented (IL-6, IL-1 beta ) or attenuated (colligin, collagen I and III, ANP) by combined alpha- and beta-receptor blockade. The elevation of afterload which was associated with the NE effect was normalized by the calcium-channel blocker nisoldipin, but NE-induced LV hypertrophy and the increase in ANP and collagen mRNA were not affected.

Modifications of the isolated frog heart preparation in Carl Ludwig's Leipzig Physiological Institute: relevance for cardiovascular research.

Carl Ludwig was the first physiologist to systematically study isolated organs (heart, muscle, kidney, liver, lung). In his Leipzig Physiological Institute, the isolated perfused frog heart preparation was established in 1866 by Elias Cyon. This preparation was subsequently subjected to various modifications, and many important observations were made by scientists such as Joseph Coats, Henry Pickering Bowditch, Luigi Luciani, Michael Joseph Rossbach, Hugo Kronecker and Otto Frank. The influence of filling pressure on contraction amplitude, the all-or-none law of the heart, the absolute refractory period, postextrasystolic potentiation, the staircase ('Treppe') phenomenon and the dependence of heart function on oxidative metabolism were discovered. The negative chronotropic and inotropic effects of vagus nerve stimulation were also first documented, and a model to induce arrhythmias was established. The isolated frog heart preparation became a widely used standard model for teaching and for basic cardiovascular research. Sidney Ringer discovered the essential role of calcium ions for heart function. Otto Loewi discovered the chemical transduction mechanism of the vagus with acetylcholine as transmitter. In more recent times, the cyclical changes in cAMP and cGMP that occur during the cardiac cycle were first described in the frog heart by Wollenberger and associates. Thus, the isolated perfused frog heart established and modified in Carl Ludwig's Leipzig Physiological Institute led not only to the discovery of basic phenomena, but also to observations that became the basis for concepts to be developed and elaborated later. Furthermore, the isolated perfused frog heart was the starting point for the development of the isolated mammalian heart in the retrogradely perfused, nonworking mode in the heart-lung modification and in the working heart preparation.

Inhibition of nitric oxide synthase augments the positive inotropic effect of nitric oxide donors in the rat heart.

1. In this investigation we studied the effects of nitric oxide on contractility and heart rate in normal saline-perfused rat hearts where shear stress-induced endothelial NO synthesis substantially contributes to total cardiac NO production. In addition, we sought to estimate the concentrations of exogenous NO producing inotropic effects. 2. We investigated the effects of glyceryl trinitrate (GTN), S-nitroso-d,l-penicillamine (SNAP), sodium (Z)-1-(N, N-diethylamino)diazen-1-ium-1,2-diolat (DEA/NO), and DEA/NO in the presence of the NO synthase inhibitor Nomega-nitro-L-arginine (L-NA) in constant-flow-perfused spontaneously beating rat Langendorff hearts and in rat working hearts. 3. In Langendorff hearts, GTN (10 nM to 100 microM, n = 32) induced a positive inotropic response that plateaued at 1 microM GTN with a maximal rate of increase of left ventricular pressure during ventricular contraction (+dP/dtmax) of 6. 33 +/- 2.56 % (n = 11, P < 0.5). Similarly, both spontaneous NO donors (0.1 nM to 1 microM, corresponding to approximately 0.03-0.3 microM NO) induced a positive inotropic response of 10.6 +/- 3.1 % (SNAP; n = 15, P < 0.05) and 11.5 +/- 2.7 % (DEA/NO, n = 15, P < 0. 05). 4. The positive inotropic effect of SNAP and DEA/NO progressively declined from 1 microM to 100 microM of the NO donors (corresponding to approximately 0.3-30 microM NO). 5. In the isolated working rat heart, 0.1 microM DEA/NO induced an increase of +dP/dtmax of 7.5 +/- 2.5 % (n = 9, P < 0.05). Inhibition of NO synthase by L-NA produced a 4-fold increase in this effect of DEA/NO. 6. We suggest that physiological NO concentrations support myocardial performance. In normal rat hearts the positive inotropic effect of NO appears to be almost maximally exploited by the endogenous NO production.

The contributions of Carl Ludwig to cardiology.

The basic instruments for measuring functional cardiovascular parameters and the most important discoveries made by Carl Ludwig and his disciples in cardiovascular physiology are described and put into perspective in regard to the further development of his methods and ideas. The most important apparatus was the kymograph, which, for the first time, made recording and documenting of functional parameters possible. This instrument was also used for the functional evaluation of the isolated perfused frog heart that was developed by Elias Cyon in Ludwig's Leipzig Physiological Institute. In the isolated frog heart, important phenomena were discovered such as the staircase ('Treppe'), the absolute refractory period and the all-or-none law of the heart. The isolated dog heart was used to determine the origin of the first heart sound, which was characterized as a muscle tone. To measure regional blood flow and eventually cardiac output, a flowmeter ('Stromuhr') was designed. Precise measurements of cardiac output became possible only when Adolf Fick had developed his principle, which served as the basis for the modern indicator methods. Cyon and Ludwig also discoverd the depressor nerve, which constitutes the basis of the baroreceptor reflex. Finally, the precise localization of the vasomotor centre in the ventrolateral medulla was achieved in Ludwig's Leipzig Physiological Institute. This was confirmed more than 100 years later with modern neuroanatomical methods making use of retrograde axonal transport. Thus, Ludwig and his scholars made major substantial contributions to cardiovascular knowledge that can be considered to constitute the basis of modern cardiology.

Differential effects of angiotensin II receptor blockade on pressure-induced left ventricular hypertrophy and fibrosis in rats.

The effects of the angiotensin II receptor type 1 (AT1) antagonist losartan on pressure overload-induced left ventricular (LV) hypertrophy were studied in female Sprague-Dawley rats. Starting on the day of surgery, losartan (L, 12 mg/kg/day) was administered as continuous intraperitoneal infusion for 2 weeks by using alzet mini-osmotic-pumps (model 2002). This dose of losartan shifted the in vivo dose-response curve of the angiotensin II-induced elevation of left ventricular systolic pressure (LVSP) to the right. Pressure overload was achieved by placing a band around the aortic arch. This caused an aortic stenosis (AS) with an outer diameter of 1.0 mm. The hemodynamic effects were measured in the intact, anesthetized rats (n = 15). The hearts were excised, and the weights of the left (LV) and right ventricle (RV) were determined. Some of these hearts (n = 7) were perfused with collagenase to obtain isolated cardiac myocytes for the measurement of cell volume. Other hearts (n = 8) were examined for morphological changes. In the animals with AS, LVSP was markedly elevated. Furthermore, LV weight and LV myocyte cell volume were increased in this group, while RV weight and RV myocyte cell volume remained stable in all the groups. L had no significant effect on the AS-induced increase in LVSP and cell size parameters, nor on the weight gain of the LV. Histological analysis revealed that the AS-induced enlargement of the mean myocyte diameter was not affected by L. The interstitial collagen fraction was increased in the AS rats and became normalized by L. These data suggest that the renin-angiotensin system might not be involved in the development of pressure-induced cardiac hypertrophy within the time-frame of these experiments, but that it does play a major role in the genesis of the interstitial fibrosis which is a typical feature of this pathophysiological condition.

Technology and application of ultraminiature catheter pressure transducers.

After a brief historical account of the methods for pressure measurements in the cardiovascular system, the basic structural elements of a new generation of miniaturized catheter pressure transducers are described. These catheters have an outside diameter at the tip of 0.9 mm (3 French) and have been routinely applied in left and right heart catheterization in intact, anesthetized rats. Together with cardiac output measured by the thermodilution technique, a complete set of basal functional parameters can be obtained in vivo. The method of cardiac catheterization in rats is accurate, reliable and easy to perform. As to left heart function, changes occurring in several models of cardiac hypertrophy and heart failure have been recorded and correlated with morphological and metabolic alterations. In addition, the functional effects of catecholamines and thyroid hormones have been evaluated. In addition to the routine catheterization procedure, a double catheter method has been introduced recently, which allows measurement of left ventricular isovolumetric pressure in intact rats. Catheterization of the right ventricle requires a more refined catheter with a characteristic bend at the tip so that it can be comfortably slid from the right atrium into the right ventricle. With this method it was found that right ventricular systolic pressure was elevated markedly in rats with chronic myocardial infarction induced by ligation of the left anterior descending coronary artery, by pulmonary artery banding, by intermittent chronic hypoxia and by noradrenaline administration. The ultraminiature catheter pressure transducer has also been successfully applied in an isolated working rat heart preparation. Recent modifications of this kind of catheters also enabled the catheterization of the left ventricle in mice. Future applications of ultraminiature catheter pressure transducers may be directed to catheterization of the pulmonary artery in rats and to the in vivo and in vitro assessment of heart function of transgenic mice.

Significance of the 5-phosphoribosyl-1-pyrophosphate pool for cardiac purine and pyrimidine nucleotide synthesis: studies with ribose, adenine, inosine, and orotic acid in rats.

Several experimental approaches have been applied to examine the significance of the available pool of 5-phosphoribosyl-1-pyrophosphate (PRPP) for purine and pyrimidine nucleotide metabolism in the rat heart. In a series of studies including some pentoses and pentitols, in particular ribose, it was shown that these sugars were all capable of elevating the cardiac PRPP pool and stimulating the rate of adenine nucleotide biosynthesis. In several pathophysiological situations that were characterized by a decrease in ATP content, the increase in adenine nucleotide biosynthesis elicited by ribose was of such magnitude that the ATP level was replenished partially or completely in a considerably shorter period of time than that without any intervention. In two experimental models, in cardiac hypertrophy induced by aortic constriction with additional isoproterenol administration and in the noninfarcted rat heart after permanent coronary artery ligation, there was also an improvement in global heart function under the influence of ribose. The myocardial cell damage induced by isoproterenol was prevented by ribose. Combination of ribose with adenine or inosine led to an even quicker ATP normalization in the isoproterenol-stimulated rat heart than with either intervention alone. Ribose had no functional effects on the cardiovascular system, whereas adenine, inosine, and orotic acid were demonstrated to have different hemodynamic influences. Adenine and inosine had negative chronotropic and inotropic effects in the intact rat, whereas orotic acid had a positive influence both on the left and right rat heart. On the basis of these experimental studies, a new therapeutic strategy is suggested in which elevation of the available PRPP plays a key role. Once this has been elevated by ribose, additional substrates, such as adenine, inosine, and orotic acids, should be included. This would exploit the full potential of a therapeutic approach that corrects a natural metabolic deficiency of the heart that is the low capacity of the oxidative pentose phosphate pathway in which PRPP is generated.

Overexpression of the sarcolemmal calcium pump in the myocardium of transgenic rats.

The plasma membrane calmodulin-dependent calcium ATPase (PMCA) is a calcium-extruding enzyme controlling Ca2+ homeostasis in nonexcitable cells. However, its function in the myocardium is unclear because of the presence of the Na+/Ca2+ exchanger. We approached the question of the physiological function of the calcium pump using a transgenic "gain of function" model. Transgenic rat lines carrying the human PMCA 4 cDNA under control of the ventricle-specific myosin light chain-2 promoter were established, and expression in the myocardium was ascertained at the mRNA, protein, and functional levels. In vivo hemodynamic measurements in adult homozygous animals showed no differences in baseline and increased cardiac performance recruited by volume overload compared with controls. No differences between transgenic and control cardiomyocytes were found in patch clamp voltage dependence, activation/inactivation behavior of the L-type Ca2+ current, or fast [Ca2+]i transients (assessed by the Fura-2 method). To test whether the PMCA might be involved in processes other than beat-to-beat regulation of contraction/relaxation, we compared growth processes of neonatal transgenic and control cardiomyocytes. A 1.6- and 2.3-fold higher synthesis rate of total protein was seen in cells from transgenic animals compared with controls on incubation with 2% FCS for 24 hours and 36 hours, respectively. An effect of similar magnitude was observed using 20 micromol/L phenylephrine. A 1.4-fold- and 2.0-fold-higher protein synthesis peak was seen in PMCA-overexpressing cardiomyocytes after stimulation with isoproterenol for 12 hours and 24 hours, respectively. Because pivotal parts of the alpha- and beta-adrenergic signal transduction pathways recently have been localized to caveolae, we tested the hypothesis that the PMCA might alter the amplitude of alpha- and beta-adrenergic growth signals by virtue of its localization in caveolae. Biochemical as well as immunocytochemical studies suggested that the PMCA in large part was colocalized with caveolin 3 in caveolae of cardiomyocytes. These results indicate that the sarcolemmal Ca2+-pump has little relevance for beat-to-beat regulation of contraction/relaxation in adult animals but likely plays a role in regulating myocardial growth, possibly through modulation of caveolar signal transduction.

Measurement of left ventricular maximal isovolumetric pressure in rats: effects of antihypertensive drugs and diabetes mellitus.

We developed a method for measuring left ventricular maximal isovolumetric pressure (LVMIP) in intact rats and applied it in the presence of acutely administered antihypertensive drugs and in experimental diabetes mellitus. The combination of a 2 French Fogarty arterial embolectomy balloon catheter with a new ultra-thin shaft (0.25 mm diameter) Millar ultra-miniature pressure tip catheter was used. Closed-chest, thiopental anaesthetized female Sprague-Dawley rats received the beta-adrenoceptor blocker metoprolol (1 mg/kg/h), the alpha-adrenoceptor blocker prazosin (0.1 mg/kg/h), and the calcium antagonist nifedipine (0.5 mg/kg/h). They were applied as continuous i.v. infusion for 30 minutes. The angiotensin II type 1 (AT1) receptor antagonist losartan (3 mg/kg) was applied as bolus i.v. injection. Diabetes mellitus was induced by a single tail vein injection of streptozotocin (60 mg/kg) five weeks prior to hemodynamic measurements. Under control conditions, left ventricular systolic pressure (LVSP) was 136.5 +/- 3.7 mmHg and increased to 263 +/- 5.4 mmHg when the balloon was inflated (LVMIP). LVSP was significantly lower in the presence of prazosin, metoprolol, nifedipine, and in diabetic rats. However, LVMIP was significantly reduced only in the metoprolol-treated and diabetic rats. Both LVSP and LVMIP did not change significantly in losartan-treated rats. The degree of the increase in left ventricular maximal isovolumetric pressure during balloon inflation (LVMIP-LVSP) was not significantly different in any experimental condition. These results suggest that the mechanism for evoking LVMIP is not dependent on the acute stimulation of cardiac alpha- and beta-adrenergic receptors, on calcium channels, nor on the AT1 receptors. In addition, the intrinsic mechanism for the generation of LVMIP seems not to be influenced in the diabetic state.

Catecholamine-induced cardiac hypertrophy: significance of proto-oncogene expression.

The effect of beta- and alpha-adrenergic stimulation on cardiovascular function and development of cardiac hypertrophy was studied in rats by measuring the heart weight/body weight and cardiac RNA/DNA ratios. Beta-receptor stimulation with isoproterenol over 3 days induced an increase in the biosynthesis of cardiac adenine nucleotides, myocardial protein synthesis, and the heart weight/body weight ratio. The isoproterenol-induced metabolic effects were prevented by simultaneous beta-adrenergic blockade with propranolol. Alpha-adrenergic stimulation with norfenephrine for 3 days induced an increase in heart rate, total peripheral resistance, the myocardial RNA/DNA, and left ventricular weight/body weight ratio. The calcium antagonist verapamil prevented the hemodynamic changes but did not influence the development of cardiac hypertrophy. The alpha-adrenergic blocker prazosin reversed the norfenephrine-induced functional changes and prevented cardiac hypertrophy. Norepinephrine was infused into isolated perfused working rat hearts to elucidate some molecular biological changes that precede the development of cardiac hypertrophy. It increased transiently and sequentially the mRNA of c-fos and c-myc. This enhancement occurred at about the same time as that induced by elevation of pre- and afterload but was more pronounced. These findings were compared with those obtained in other studies assessing the effects of catecholamines on proto-oncogene expression. Combination of norepinephrine with pre- and afterload elevation induced the c-fos mRNA signal to appear earlier, to be more pronounced, and to persist for a longer period of time. Similar results were obtained in regard to the c-myc mRNA. These findings indicate that the combination of two hypertrophy-inducing stimuli which may cause a higher degree of cardiac hypertrophy in vivo induce an earlier, more pronounced, and longer lasting expression of the proto-oncogenes c-fos and c-myc.

Correlation between function and proto-oncogene expression in isolated working rat hearts under various overload conditions.

In isolated perfused working rat hearts we have studied the effects of norepinephrine (NE) and of pressure as well as volume overload alone, and in combination on heart function and expression of the proto-oncogenes c-fos and c-myc. This preparation was functionally stable over the observation period of 120 min. NE (3x10(-8) M) induced an immediate and sustained increase in aortic and coronary flow as well as in cardiac output. Volume overload was established by increasing left atrial filling pressure (preload) from 8-16 cm H2O and resulted in a marked increase in aortic flow and cardiac output which subsided somewhat over time. Pressure overload was created by elevation of afterload from 80-100 cm H2O and induced a decrease in aortic flow and a slight increase in coronary flow. Using specific cDNA clones, the mRNAs of c-fos and c-myc were measured by Northern blots and quantified with densitometry. In all three conditions, c-fos mRNA was increased first, after 30 min. It was more pronounced and of longer duration in the NE-stimulated hearts. The increase in c-myc-mRNA occurred after 60 or 90 min. After 120 min, all signals were normal, although heart function was still altered in a manner specific for the respective intervention. Combination of NE with preload and afterload elevation as well as combination of preload and afterload elevation led first to an increase in cardiac output which was followed by a decline to various degrees. In all these combinations, the c-fos mRNA signal appeared earlier, after 15 min, and persisted for a longer period of time compared with the effect of a single stimulus. The c-myc mRNA was increased later, after 30 or 60 min. This increase persisted throughout the entire observation period, showing a further progressive rise. These results show that stimuli which cause cardiac hypertrophy in vivo induce a transient and sequential increase in proto-oncogene expression in the isolated working rat heart. Combination of two hypertrophy-inducing stimuli elicit an earlier, more pronounced and longer-lasting expression of the proto-oncogenes c-fos and c-myc, while functional parameters deteriorate.