Real-time imaging of apoptotic cell-membrane changes at the single-cell level in the beating murine heart.

We report a novel real-time imaging model to visualize apoptotic membrane changes of single cardiomyocytes in the injured heart of the living mouse, using fluorescent labeled annexin-V. Annexin-V binds to externalized phosphatidylserine (PS) of cells undergoing programmed cell death. With high-magnification (x100-160) real-time imaging, we visualized the binding of annexin-V to single cardiomyocytes. Kinetic studies at the single-cell level revealed that cardiomyocytes started to bind annexin-V within minutes after reperfusion, following an ischemic period of 30 minutes. The amount of bound annexin-V increased rapidly and reached a maximum within 20-25 minutes. Caspase inhibitors decreased the number of annexin-V-positive cardiomyocytes and slowed down the rate of PS exposure of cardiomyocytes that still bound annexin-V. This technology to study cell biology in the natural environment will enhance knowledge of intracellular signaling pathways relevant for cell-death regulation and strategies to manipulate these pathways for therapeutic effect.

A homologue of Drosophila tissue polarity gene frizzled is expressed in migrating myofibroblasts in the infarcted rat heart.

Myocardial infarction results in the formation of granulation tissue in the injured ventricular wall. This tissue contains myofibroblasts in highly organized arrays; their contractile properties may help to prevent the infarct area from dilatation. The mechanisms that control myofibroblast alignment are unknown. We found that myofibroblasts express a homologue of Drosophila tissue polarity gene frizzled (fz2) when migrating into the granulation tissue. The expression is decreased after the cells have aligned. This suggests that fz2 is involved in the spatial control of cardiac wound repair after infarction, possibly through intra- and intercellular transmission of polarity signals as in developing Drosophila. Mutations in the fz2 gene may impair myofibroblast alignment in the infarct area, thereby resulting in ventricular dilatation and aneurism following infarction.

Abrupt rate accelerations or premature beats cause life-threatening arrhythmias in mice with long-QT3 syndrome.

Deletion of amino-acid residues 1505-1507 (KPQ) in the cardiac SCN5A Na(+) channel causes autosomal dominant prolongation of the electrocardiographic QT interval (long-QT syndrome type 3 or LQT3). Excessive prolongation of the action potential at low heart rates predisposes individuals with LQT3 to fatal arrhythmias, typically at rest or during sleep. Here we report that mice heterozygous for a knock-in KPQ-deletion (SCN5A(Delta/+)) show the essential LQT3 features and spontaneously develop life-threatening polymorphous ventricular arrhythmias. Unexpectedly, sudden accelerations in heart rate or premature beats caused lengthening of the action potential with early afterdepolarization and triggered arrhythmias in Scn5a(Delta/+) mice. Adrenergic agonists normalized the response to rate acceleration in vitro and suppressed arrhythmias upon premature stimulation in vivo. These results show the possible risk of sudden heart-rate accelerations. The Scn5a(Delta/+) mouse with its predisposition for pacing-induced arrhythmia might be useful for the development of new treatments for the LQT3 syndrome.

Inhibition of plasminogen activators or matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure.

Cardiac rupture is a fatal complication of acute myocardial infarction lacking treatment. Here, acute myocardial infarction resulted in rupture in wild-type mice and in mice lacking tissue-type plasminogen activator, urokinase receptor, matrix metalloproteinase stromelysin-1 or metalloelastase. Instead, deficiency of urokinase-type plasminogen activator (u-PA-/-) completely protected against rupture, whereas lack of gelatinase-B partially protected against rupture. However, u-PA-/- mice showed impaired scar formation and infarct revascularization, even after treatment with vascular endothelial growth factor, and died of cardiac failure due to depressed contractility, arrhythmias and ischemia. Temporary administration of PA inhibitor-1 or the matrix metalloproteinase-inhibitor TIMP-1 completely protected wild-type mice against rupture but did not abort infarct healing, thus constituting a new approach to prevent cardiac rupture after acute myocardial infarction.

Matrix metalloproteinase inhibition after myocardial infarction: a new approach to prevent heart failure?

Increased activity of matrix metalloproteinases (MMPs) has been implicated in numerous disease processes, including tumor growth and metastasis, arthritis, and periodontal disease. It is now becoming increasingly clear that extracellular matrix degradation by MMPs is also involved in the pathogenesis of cardiovascular disease, including atherosclerosis, restenosis, dilated cardiomyopathy, and myocardial infarction. Administration of synthetic MMP inhibitors in experimental animal models of these cardiovascular diseases significantly inhibits the progression of, respectively, atherosclerotic lesion formation, neointima formation, left ventricular remodeling, pump dysfunction, and infarct healing. This review focuses on the role of MMPs in cardiovascular disease, in particular myocardial infarction and the subsequent progression to heart failure. MMPs, which are present in the myocardium and capable of degrading all the matrix components of the heart, are the driving force behind myocardial matrix remodeling. The recent finding that acute pharmacological inhibition of MMPs or deficiency in MMP-9 attenuates left ventricular dilatation in the infarcted mouse heart led to the proposal that MMP inhibitors could be used as a potential therapy for patients at risk for the development of heart failure after myocardial infarction. Although these promising results encourage the design of clinical trials with MMP inhibitors, there are still several unresolved issues. This review describes the biology of MMPs and discusses new insights into the role of MMPs in several cardiovascular diseases. Attention will be paid to the central role of the plasminogen system as an important activator of MMPs in the remodeling process after myocardial infarction. Finally, we speculate on the use of MMP inhibitors as potential therapy for heart failure.

Cardiomyocyte death induced by myocardial ischemia and reperfusion: measurement with recombinant human annexin-V in a mouse model.

INTRODUCTION: Phosphatidylserine (PS) externalization is regarded as one of the earliest hallmarks of cells undergoing programmed cell death. We studied the use of labeled human recombinant annexin-V, a protein selectively binding to PS, to detect cardiomyocyte death in an in vivo mouse model of cardiac ischemia and reperfusion (I/R). METHODS AND RESULTS: I/R was induced in mouse hearts by ligation and subsequent release of a suture around the left anterior descending coronary artery. Annexin-V (25 mg/kg) fused to a marker molecule was injected intra-arterially 30 minutes before euthanasia. After 15 minutes of ischemia followed by 30 minutes of reperfusion, 1.4+/-1. 2% (mean+/-SD) of the cardiomyocytes in the area at risk were annexin-V positive (n=6). This increased to 11.4+/-1.9% after 15 minutes of ischemia followed by 90 minutes of reperfusion (n=7) and to 20.2+/-3.3% after 30 minutes of ischemia followed by 90 minutes of reperfusion (n=7). In control mice, including those injected with annexin-V at the binding site of PS, no annexin-V-positive cells were observed. DNA gel electrophoresis showed typical laddering starting after 15 minutes of ischemia followed by 30 minutes of reperfusion, suggesting activation of the cell death program. Intervention in the cell death program by pretreatment with a novel Na(+)-H(+) exchange inhibitor substantially decreased annexin-V-positive cardiomyocytes from 20.2% to 2.2% in mice after 30 minutes of ischemia followed by 90 minutes of reperfusion. CONCLUSIONS: These data suggest that labeled annexin-V is useful for in situ detection of cell death in an in vivo model of I/R in the heart and for the evaluation of cell death-blocking strategies.

Cardiovascular phenotyping in mice.

Progress in molecular genetics has changed cardiovascular research. The mouse has turned out to be an invaluable model for mammalian genetic modifications to mimic and analyse cardiovascular pathology. Through the introduction of transgene and gene targeting technology, regulatory systems can be studied at the molecular level. Recent technical developments have down-sized the equipment for physiological measurements to the mouse level. Micro-surgery has developed to the level where most manipulations previously performed in larger animals can now be applied to mice. However, different investigators report considerable differences in values for physiological parameters. Whether these differences are related to the variation in mouse strains or experimental procedures remains to be established, but awareness of the variation can be relevant for prospective mouse investigators. In the present review, the physiological measurements performed in mice to date are discussed and complemented with results from genetically manipulated animals. In addition the various surgical procedures and their practical application are illustrated.

Chronic myocardial infarction in the mouse: cardiac structural and functional changes.

OBJECTIVES: We studied the effects of chronic left coronary artery ligation on cardiac structure and function in the mouse. METHODS: Morphometric studies of the left ventricle were performed in coronary artery-ligated and sham-operated animals at one, two, three and five weeks after surgery. The fraction of DNA-synthesizing cells was determined as the fraction of cells incorporating 5'-bromo-2'-deoxyuridine, which was infused by osmotic minipumps one week before sacrifice. Collagen content of the septum was determined morphometrically. Left ventricular pressure and its derivatives were measured in separate groups of animals at one and three weeks after surgery. RESULTS: Ligation of the main left coronary artery resulted in antero-apical infarction of the left ventricular wall, involving approximately 40% of left ventricular circumference. Infarction resulted in thinning of the infarcted area and left ventricular dilatation. DNA synthesis increased, peaking between one and two weeks in the border-zone of the infarct (22-fold), septum (ten-fold) and right ventricle (five-fold). At five weeks, DNA synthesis was still increased in the border zone of the infarct. Septal collagen content increased approximately eight-fold in infarcted mice at two weeks, and decreased thereafter; it was still significantly elevated at five weeks. Left ventricular systolic pressure, and maximal positive and negative dP/dt decreased following infarction; left ventricular end-diastolic pressure was elevated at three weeks, but this effect was not statistically significant. CONCLUSION: These data provide basic information on changes in cardiac structure and function in mice following chronic coronary artery ligation. They indicate the feasibility of induction of chronic myocardial infarction in this species. Furthermore, they show the similarity of cardiac structural and functional consequences of chronic myocardial infarction in mice to those previously described in rats.

The influence of angiotensin II-induced increase in aortic wall mass on compliance in rats in vivo.

OBJECTIVE: This study was undertaken to examine the effects of angiotensin II-induced structural changes in the aortic wall on the dynamic mechanical properties of the vessel in the rat. METHODS: Wistar rats were infused s.c. with 250 ng/kg/min angiotensin II (Ang II) for 14 days (ANG). Both ANG and control rats (CON) were equipped with an arterial catheter for measurement of arterial blood pressure. Thoracic aorta diameter, compliance coefficient (CC), and distensibility coefficient (DC) were determined non-invasively in pentobarbital-anesthetized animals using a B-mode imager attached to a vessel wall tracking system. After sacrifice, medial cross-sectional area (CSA), and elastin and collagen densities were determined by morphometry on cross-sections. Media thickness (Mt) and wall-to-lumen ratio (W/L) were subsequently calculated. RESULTS: Ang II infusion significantly increased mean arterial blood pressure in conscious rats (122 +/- 3 mmHg CON vs. 157 +/- 4 mmHg ANG). This was normalized when the rats were anesthetized, thus making it possible to determine CC and DC under isobaric conditions where the diastolic diameters were also similar. Two-week infusion of Ang II induced a significant increase in CSA from 0.48 +/- 0.02 mm2 in CON to 0.61 +/- 0.03 mm2. Mt and W/L were likewise increased, but collagen and elastin densities remained unchanged. CC and DC were not effected by this increase in aortic wall mass (CC: 0.143 +/- 0.009 CON, 0.147 +/- 0.014 mm2/kPa ANG; DC: 0.052 +/- 0.005 CON, 0.051 +/- 0.004 kPa-1 ANG). CONCLUSIONS: An increase in aortic wall mass resulting from chronic infusion of angiotensin II does not alter the dynamic compliance of the vessel under isobaric conditions.

Role of basal nitric oxide synthesis in vasoconstrictor hyporeactivity in the perfused rat hindlimb after myocardial infarction: effect of captopril.

OBJECTIVES: The contribution of vascular changes to the development of heart failure is largely unknown. In the present study, we evaluated endothelial and vascular contractile function in the rat hindlimb vascular bed after myocardial infarction (MI), including the modulatory role of basal nitric oxide (NO) production and the effects of treatment with the angiotensin converting enzyme inhibitor captopril on vascular function. METHODS: MI was induced in male Wistar rats by ligation of the left coronary artery. Acetylcholine-induced dilatations were assessed in the ex vivo perfused hindlimb at various time points. At 2 and 5 weeks post-MI, vascular contractile function in the perfused hindlimb was assessed from resistance changes induced by 35 mM and 125 mM potassium (K+) and the maximum increase in resistance (delta Rmax, 125 mM K+ and 3 mg phenylephrine). Basal NO synthesis was blocked for 2 weeks with L-nitro-arginine methylester (L-NAME) in sham and MI rats and similar contractility experiments were performed. The effect of captopril treatment from 2 to 5 weeks post-MI on vasoconstrictor responses was also tested. RESULTS: Acetylcholine-induced dilatations in the presence of 10 microM indomethacin were not different between sham and MI rats. Vasoconstrictor responses to K+ and delta Rmax were reduced at 2 weeks after MI. This reduction in vasoconstrictor ability was similar to that seen in L-NAME-treated sham rats, while chronic L-NAME treatment did not affect vasoconstrictor reactivity in MI rats. Similarly, L-NAME induced an increase in mean arterial pressure in sham rats, but not in MI rats. At 5 weeks after MI, vasoconstriction to 125 mM K+ and delta Rmax were still reduced in MI rats; this response was however partially restored after captopril treatment. CONCLUSION: The development of vascular contractile hyporeactivity in the rat hindlimb after MI may be due to reduced basal NO production. Delayed treatment with captopril improves peripheral vascular contractile function in this setting.

Early captopril treatment inhibits DNA synthesis in endothelial cells and normalization of maximal coronary flow in infarcted rat hearts.

OBJECTIVES: Cardiac remodeling due to myocardial infarction (MI) includes myocyte hypertrophy, collagen deposition, a rise in DNA synthesis, and normalization of initially diminished maximal coronary bloodflow. Previously, we demonstrated that early captopril treatment can prevent the rise in total DNA synthesis, collagen deposition and hypertrophy. In the present experiments, we investigated the effects of captopril or perindoprilat treatment on cardiac endothelial cell proliferation and maximal coronary flow. METHODS: MI was induced by ligation of the left coronary artery in Wistar rats. Sham-operated and infarcted rats were treated with captopril (12 mg/kg.d s.c.) from either day 0-21 (early) or day 21-35 (late) after surgery. In isolated retrogradely perfused rat hearts, maximal coronary flow was determined following maximal dilatation with nitroprusside and adenosine (1 mM each). In separate groups, sections of hearts of sham-operated and MI rats treated with BrdU (day 7-14) and either captopril or perindoprilat (1 mg/kg.d s.c.; day 0-14) were double stained with a monoclonal anti-BrdU antibody and the lectin GSI. The total fraction of DNA synthesizing cells and its proportion of endothelial cells was determined. RESULTS: Maximal coronary flow was completely normalized in MI hearts within three weeks after surgery. Early captopril, but not late captopril, inhibited the normalization of maximal coronary flow in MI hearts (Early: sham, 27.4 +/- 1.0; MI, 21.2 +/- 1.4 ml/min; P < 0.05; mean +/- SEM) without affecting the hypertrophic response. The total fraction of DNA synthesizing cells was significantly increased in MI hearts (sham: 7.6 +/- 1.9; MI: 14.9 +/- 2.2%). The proportion of endothelial cells, however, was comparable in sham-operated and infarcted hearts (sham: 30 +/- 3; MI: 33 +/- 3%). Both early captopril and perindoprilat treatment inhibited total DNA synthesis in MI hearts. Only in captopril pre-treated hearts, this inhibition was associated with a disproportionate inhibition of the endothelial cell proliferation (10.3 +/- 2.0%). CONCLUSION: Early captopril treatment inhibits endothelial cell proliferation and coronary vessel growth following MI, which seems to be partly due to inhibition of the renin angiotensin system.

Doxazosin blocks the angiotensin II-induced smooth muscle cell DNA synthesis in the media, but not in the neointima of the rat carotid artery after balloon injury.

OBJECTIVE: Infusion of angiotensin II (AngII) during the third and fourth week after balloon injury of the left common carotid artery of the rat induces smooth muscle cell (SMC) DNA synthesis. In this study we wanted to investigate whether alpha 1-adrenoreceptors are involved in AngII-induced SMC DNA synthesis in the neointima. METHODS: Adult male Wistar Kyoto rats were subcutaneously infused for 2 weeks with AngII and the alpha 1-adrenoreceptor antagonist doxazosin during the 3rd and the 4th week after balloon injury of the left common carotid artery. Control groups received AngII, 0.9% NaCl, AngII + 50% dimethylsulfoxide (DMSO, the solvent of doxazosin), doxazosin or 50% dimethylsulfoxide. Each rat received 5-bromo-2'-deoxyuridine in a separate osmotic minipump to label DNA-synthesizing SMC. Systolic blood pressures were measured in all groups. RESULTS: Angiotensin II caused an increase in systolic blood pressure, whereas addition of doxazosin did not affect the increase in SBP caused by AngII. In the media of the non-injured carotid artery, AngII increased SMC DNA synthesis, as the BrdUrd labeling fraction increased from 0.2 +/- 0.1% (mean +/- s.e.m.) in the NaCl group towards 3.4 +/- 0.6% in the AngII group. Coinfusion with doxazosin reduced the AngII-induced increase in BrdUrd labeling fraction from 3.2 +/- 0.8% in the AngII + DMSO group towards 0.6 +/- 0.2% in the AngII+doxazosin group. A similar effect of doxazosin was found in the media of the injured left carotid artery, in which coinfusion with doxazosin also reduced the BrdUrd labeling fraction from 2.6 +/- 0.8% in the AngII+DMSO group towards 0.3 +/- 0.1% in the AngII+doxazosin group. In the neointima of the injured left carotid artery, AngII increased the BrdUrd labeling fraction from 11.7 +/- 1.6% in the NaCl group towards 28.0 +/- 3.4% in the AngII group. Coinfusion with doxazosin did not influence the AngII-induced SMC DNA synthesis, since the BrdUrd labeling fraction in the neointima of the AngII+doxazosin group was 22.5 +/- 2.9%, whereas the neointimal BrdUrd labeling fraction in the AngII+DMSO group was 22.9 +/- 2.3%. Little effect was found on the medial cross-sectional area. The neointimal cross-sectional area was increased as a result of infusion of AngII (0.12 +/- 0.01 mm2 vs. 0.18 +/- 0.01 mm2), and coinfusion of doxazosin did not reduce the AngII-induced increase in neointimal cross-sectional area (0.18 +/- 0.03 mm2). CONCLUSIONS: These data suggest that alpha 1-adrenoreceptors are not involved in AngII-induced neointimal SMC DNA synthesis and cross-sectional area, but only play a role in the media of the carotid artery.

Cardiac remodeling after myocardial infarction is impaired in IGF-1 deficient mice.

OBJECTIVE: To obtain more insight in the role of IGF-1 in cardiac remodeling and function after experimental myocardial infarction. We hypothesized that cardiac remodeling is altered in IGF-1 deficient mice, which may affect cardiac function. METHODS: A myocardial infarction was induced by surgical coronary artery ligation in heterozygous IGF-1 deficient mice. One week after surgery, left ventricular function was analyzed, and parameters of cardiac remodeling were measured. RESULTS: No significant difference in cardiac function was found between infarcted wildtype and knock-out animals, despite a marked reduction in capillarization and blunting of the hypertrophic response of the interventricular septum in the IGF-1 deficient group. Furthermore, decreased DNA synthesis and increased apoptosis rates were observed in the IGF-1 knock-out mice. CONCLUSION: IGF-1 deficient mice show preservation of cardiac function 1 week after MI, despite an altered cardiac remodeling process.

Markers of apoptosis in cardiovascular tissues: focus on Annexin V.

In the last decade, apoptosis (or programmed cell death) has become appreciated as an important process in the development of the cardiovascular system. Moreover, apoptosis contributes to the adaptation of the system to the environment. We are at the beginning of understanding its relevance to cardiovascular physiology and pathology. This understanding forms the key to implement apoptosis in diagnosis and therapy of cardiovascular diseases. New avenues for pharmacological intervention are expected to arise from the synergy of our knowledge about the molecular mechanisms of apoptosis, and how apoptosis integrates in the complex environment of the cardiovascular tissue. The latter strongly depends on techniques to measure apoptosis. Currently, we are facing a relative paucity in available techniques, covering both specificity and sensitivity, and furthermore allowing quantitative analysis, preferably in combination with morphology. This field, however, is rapidly evolving and is fed by the expanding knowledge about the molecular mechanisms of apoptosis. In this paper we will briefly review the available techniques to detect and/or quantify apoptosis. These methods are based on the analysis of cellular morphology, either by light- or electron microscopy, DNA fragmentation (TdT-mediated X-dUTP nick end labeling or in situ nick end labeling), or cytoplasmic and membrane changes. Furthermore, the advantages and limitations of these techniques for their use in cardiovascular research will be outlined. In the text we will refer to available reviews and protocols which discuss the techniques in more detail. The main part of this article will, however, focus on a recently introduced technique, the Annexin V-based apoptosis detection assay. The principle, characteristics, pro's and contra's of this new apoptosis detection assay will be discussed.

Hemodynamic characterization of hypertension induced by chronic intrarenal or intravenous infusion of norepinephrine in conscious rats.

The present study was designed to determine the hemodynamic changes underlying the hypertension induced by chronic intrarenal infusion of norepinephrine (NE) in conscious rats. NE was infused for a 5-day period intrarenally with osmotic minipumps via a chronic catheter in the right suprarenal artery at rates of 4 and 36 micrograms . kg-1 . hr-1 or intravenously at a rate of 36 micrograms . kg-1 . hr-1. Control rats received a 1 microliter . hr-1 intrarenal infusion of pyrogen-free 0.9% NaCl. In separate experiments, short-term effects were measured continuously during a 22- to 24-hour intrarenal infusion of 4 and 36 micrograms NE . kg-1 . hr-1 or intravenous infusion of 36 micrograms NE . kg-1 . hr-1. Intrarenal infusion of NE produced a more pronounced long-term hypertensive effect than infusion of the same dose intravenously. This hypertension was characterized by a rapid and sustained increase in total peripheral resistance index (TPRI). Despite of the initial renal vasoconstriction, specifically produced during the first 24 hours of intrarenal NE application, cardiac index (CI) in parallel to stroke volume index (SVI) decreased significantly during intrarenal as well as during intravenous NE infusion. Furthermore, no signs of sodium retention were observed. Both rates of intrarenal NE infusion have been shown previously to produce a significant long-term increase in plasma potassium concentration, and the present study indicates that this is presumably the result of decreased urinary potassium output. It is concluded that chronic hypertension produced by intrarenal or intravenous infusion is not volume-dependent. The relatively greater increase in TPRI during intrarenal NE infusion is attributed to vascular wall receptor sensitization by increased plasma potassium levels resulting from effects of intrarenally present NE on tubular cation exchange mechanisms.

Antihypertensive therapy and adaptive mechanisms in peripheral ischemia.

In the present experiments the effect of long-term peripheral ischemia on the capillary of two hind limb skeletal muscles was investigated in spontaneously hypertensive rats. Furthermore, the effect of antihypertensive therapy on changes in capillarity and on the previously observed hyperreactivity of the ischemic vascular bed to vasoconstrictors was investigated in perfused hind limbs of rats after long-term treatment with the angiotensin I converting enzyme inhibitors captopril (0.5 mg/kg.h) or zabiciprilate (0.025 mg/kg.h), the angiotensin II type 1 receptor antagonist losartan (0.625 mg/kg.h), or the calcium antagonist felodipine (0.042 or 0.42 mg/kg.h). Skeletal muscle ischemia in the left hind limb was induced by partial ligation of the left common iliac artery. Long-term (4 weeks) ischemia increased significantly the capillary-to-fiber ratio in the soleus muscle, composed predominantly of type I fibers in spontaneously hypertensive rats, of the ischemic hind limb, whereas capillarity in the contralateral muscle was not affected. Furthermore, capillarity in the gastrocnemius muscle (type II muscle fiber part) of both the ischemic and contralateral hind limb did not change. Long-term treatment with the angiotensin I converting enzyme inhibitors during ischemia abolished the increase in the capillary-to-fiber ratio in the soleus muscle, whereas a comparable antihypertensive dose of felodipine had no effect. Greater blood pressure reductions by both losartan and felodipine prevented increases in capillarization in skeletal muscle ischemia. With respect to vascular hyperreactivity during ischemia, only treatment with losartan normalized reactivity of the ischemic vascular bed to vasoconstrictors.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of afferent renal nerves in spontaneous hypertension in rats.

In the present study we examined sympathetic function and baroreceptor reflex sensitivity in adult spontaneously hypertensive rats (SHR) after a selective transection of afferent renal nerves in the prehypertensive and established phases of hypertension. Renal deafferentation performed between 3 and 4 weeks after birth did not influence the course of the development of high blood pressure when compared with sham-operated rats. Mean arterial pressure, heart rate, and plasma norepinephrine concentrations were similar in both groups when measured at 13 weeks after renal deafferentation. However, blood pressure responses to ganglionic blockade with hexamethonium were significantly reduced in the renal deafferented SHR. Baroreceptor reflex sensitivity, assessed by heart rate responses to blood pressure changes induced by phenylephrine and nitroprusside, was significantly enhanced in these rats. When renal deafferentation was performed in adult SHR with established hypertension, mean arterial pressure decreased slightly but significantly by 5%. Heart rate, plasma norepinephrine concentrations, and responses to hexamethonium were not affected by this procedure. However, in the renal deafferented adult SHR, heart rate responses to phenylephrine but not to nitroprusside were significantly increased. Thus, in contrast to efferent renal nerves, afferent renal nerves do not play an important role in the development and maintenance of high blood pressure in SHR, but may contribute to the mechanisms that alter sympathetic function and baroreceptor reflex sensitivity in SHR during the development of hypertension.

Pressure but not angiotensin II-induced increases in wall mass or tone influences static and dynamic aortic mechanics.

OBJECTIVE: To distinguish between static (due to slow changes in pressure) and dynamic (due to pressure pulsatility) components of aortic compliance over a large pressure range in vivo and to examine the effects of increased vascular mass and smooth muscle tone on these components. METHODS: Using ultrasound wall tracking, aortic lumen area-pressure curves were generated in anaesthetized rats over a broad range of pressures by altering blood volume. The compliance coefficient calculated at each mean pressure was considered the dynamic compliance at that pressure; the slope of the diastolic lumen area-pressure curve represents static compliance. Experiments were performed in control rats and rats treated with angiotensin II (ANG II) acutely (500 ng/kg per min intravenously) to modify vascular tone or chronically (250 ng/kg per min subcutaneously for 2 weeks) to modify vascular mass. RESULTS: The dynamic compliance-pressure curve approximated a parabola. Maximal dynamic compliance (0.272+/-0.026 mm2/kPa in control rats) was achieved at near-normotensive pressure (+/-105 mm Hg). The diastolic lumen area-pressure curve showed an exponential relationship within a physiological range (30-130 mm Hg). ANG II-induced increases in aortic wall mass or smooth muscle tone did not modify the relationship between static or dynamic compliance and pressure. CONCLUSIONS: These findings demonstrate that static and dynamic mechanics of the rat thoracic aorta depend differently on blood pressure. Static compliance increases slightly with pressure in a physiological range, while dynamic compliance is auto-regulated around normotensive pressures. Neither static nor dynamic compliance of the rat thoracic aorta are influenced by ANG II-induced increases in aortic wall mass or smooth muscle tone.

Effects of complete renal denervation and selective afferent renal denervation on the hypertension induced by intrarenal norepinephrine infusion in conscious rats.

To test the hypothesis that continuous intrarenal norepinephrine (NE) infusions produce hypertension via activation of afferent renal nerves (ARN), rats were subjected to complete renal denervation (RN-x), selective renal deafferentation (ARN-x) or sham surgery, prior to infusion of NE. In the pre-infusion period, mean arterial pressure (MAP) was significantly lower in RN-x than in ARN-x or sham-operated rats. Plasma renin concentration (PRC) was significantly reduced following ARN-x, but not RN-x. During 5-day intrarenal infusions of 4, 12 or 36 micrograms NE/kg per h, MAP rose to similar levels in RN-x and sham-RN-x rats. However, RN-x rats exhibited significantly elevated PRC levels, suggesting that denervation supersensitivity masked the possible effects of RN-x. In sham-RN-x rats, MAP increased significantly more during intrarenal infusion of 12 micrograms NE/kg per h than during intravenous infusion of the same amount. In ARN-x rats, MAP rose to a similar degree during intravenous and intrarenal infusions. The pressor responses in the ARN-x rats, however, were not significantly smaller at any point than those in intact rats. PRC rose to comparable levels in ARN-x and intact rats. Thus, in normotensive rats, efferent renal nerves (ERN) but not ARN are of functional significance in maintaining basal blood pressure. ARN may be involved in the control of renin release. Since neither RN-x nor ARN-x attenuated the development of hypertension, renal nerves are not necessary for the full expression of hypertension in this model.

Structural adaptation to ischemia in skeletal muscle: effects of blockers of the renin-angiotensin system.

OBJECTIVE: To investigate the effects of long-term treatment with blockers of the renin-angiotensin system on capillarization and growth of fibers in ischemic hind-limb muscles and in muscles under normal growth conditions. METHODS: Ischemia was induced by partial ligation of the left common iliac artery. RESULTS: Ischemia resulted in a significant increase in capillary and fiber density in the soleus muscle, a significant decrease in mean fiber size and a decrease in muscle cross-sectional area after 4 weeks compared with the contralateral nonischemic muscle. Ischemia also significantly decreased the muscle: body weight ratio of the left soleus muscle. We observed no significant effect on total number of capillaries and capillary: fiber ratio, suggesting that ischemia did not result in an increase in capillarization in this muscle. Treatments with subhypotensive and with hypotensive doses of the angiotensin converting enzyme (ACE) inhibitor benazeprilat, the angiotensin (Ang) II AT1 antagonist valsartan, or the Ang II AT2 antagonist PD 123 319 for 4 weeks did not influence any of the above-described changes in the normal and ischemic muscles and treatment effects were also independent of the degree of reduction of blood pressure. CONCLUSION: Treatments with an ACE inhibitor and with Ang II receptor antagonists in dose ranges that moderately lower blood pressure do not influence vessel density and any of the other structural adaptations after hind-limb ischemia. Administrations of ACE inhibitors and Ang II AT1 antagonists may therefore be adequate and beneficial therapies under ischemic conditions, such as in the treatment of hypertension complicated by intermittent claudication, for which treatment must not increase ischemia.