Roland Willenbrock

Hemodynamic and Neurohumoral Effects of Selective Endothelin A (ETA) Receptor Blockade in Chronic Heart Failure The Heart Failure ETA Receptor Blockade Trial (HEAT)

Background—The endothelin (ET-1) system is activated in chronic heart failure (CHF). Whether, what type, and what degree of selective ET blockade is clinically beneficial is unknown. We investigated hemodynamic and neurohumoral effects of 3 weeks of treatment with various dosages of the orally available ETA antagonist darusentan in addition to modern standard therapy in patients with CHF. Methods and Results—A total of 157 patients with CHF (present or recent NYHA class III of at least 3 months duration), pulmonary capillary wedge pressure ≥12 mm Hg, and a cardiac index ≤2.6 L · min−1 · m−2 were randomly assigned to double-blind treatment with placebo or darusentan (30, 100, or 300 mg/d) in addition to standard therapy. Short-term administration of darusentan increased the cardiac index, but this did not reach statistical significance compared with placebo. The increase in cardiac index was significantly more pronounced after 3 weeks of treatment (P <0.0001 versus placebo). Pulmonary capillary wedge pressure, pulmonary arterial pressure, pulmonary vascular resistance, and right atrial pressure remained unchanged. Heart rate, mean artery pressure, and plasma catecholamines remained unaltered, but systemic vascular resistance decreased significantly (P =0.0001). Higher dosages were associated with a trend to more adverse events (including death), particularly early exacerbation of CHF without further benefit on hemodynamics compared with moderate dosages. Conclusions—This study demonstrates for the first time in a large patient population that 3 weeks of selective ETA receptor blockade improves cardiac index in patients with CHF. However, long-term studies are needed to determine whether ETA blockade is beneficial in CHF.

Baroreflex sensitivity and cardiovascular mortality in patients with mild to moderate heart failure.

OBJECTIVE--To assess the influence of both sympathetic (plasma noradrenaline concentrations) and parasympathetic (baroreflex activation) tone on survival in patients with congestive heart failure. DESIGN--Invasive study with determination of parasympathetic activity and follow up for at least 4.5 years. SUBJECTS--35 patients with sinus rhythm and mild to moderate heart failure (New York Heart Association grades II-III) (mean age 53 (SD 3)). RESULTS--20 patients whose hearts survived were compared with 15 patients whose hearts did not (12 died and three received transplants). The two groups differed significantly in terms of mean arterial blood pressure (98 (3) v 90 (3) mm Hg), heart rate (82 (2) v 93 (4) beats/min), and mean pulmonary artery pressure (24 (3) v 35 (2) mm Hg) (all P < 0.05), while cardiac index, stroke volume index, and right atrial pressures were not different. The survivors had significantly lower plasma renin activities (3.6 (0.8) v 9.0 (3.6) angiotensin I/ml/h; P < 0.05) and tended to have lower noradrenaline values than non-survivors (170 (23) v 286 (74) pg/ml) at baseline. Baroreflex sensitivity was significantly lower in non- survivors than in survivors (1.3 (0.2) v 2.3 (0.3) ms/mm/Hg); P < 0.02). As the time of cardiac transplantation is dependent on complex logistical factors the three patients who received a transplant were excluded from the analysis of survival time. The risk of death in relation to baroreflex sensitivity at the median sensitivity of 1.48 ms/mm Hg was calculated. Survival was significantly different (P < 0.04) between the resulting two groups; three of the 16 subjects with high baroreflex sensitivity died compared with nine of the 16 with a baroreflex sensitivity < 1.48 ms/mm Hg. When systemic blood pressure, pulmonary artery pressure, stroke volume index, plasma noradrenaline concentrations, and baroreflex sensitivity were entered into a Cox proportional hazards regression, only systolic blood pressure and plasma noradrenaline values predicted survival (P < 0.001). CONCLUSIONS--Low vagal tone is correlated with a poor prognosis in patients with heart failure. Sympathetic tone measured as plasma noradrenaline concentration also contributed to survival. An additional contribution of vagal tone to survival could not be shown when sympathetic tone was considered simultaneously. This may be due to the inverse relation of sympathetic and parasympathetic tone and to the insensitivity of the multiple regression method to identify additional risk factors in small numbers of patients.

Persistent induction of HIF-1α and -2α in cardiomyocytes and stromal cells of ischemic myocardium

Hypoxia‐inducible factor (HIF)‐1α and ‐2α are key regulators of the transcriptional response to hypoxia and pivotal in mediating the consequences of many disease states. In the present work, we define their temporo‐spatial accumulation after myocardial infarction and systemic hypoxia. Rats were exposed to hypoxia or underwent coronary artery ligation. Immunohistochemistry was used for detection of HIF‐1α and ‐2α proteins and target genes, and mRNA levels were determined by RNase protection. Marked nuclear accumulation of HIF‐1α and ‐2α occurred after both systemic hypoxia and coronary ligation in cardiomyocytes as well as interstitial and endothelial cells (EC) without pronounced changes in HIF mRNA levels. While systemic hypoxia led to widespread induction of HIF, expression after coronary occlusion occurred primarily at the border of infarcted tissue. This expression persisted for 4 wk, included infiltrating macrophages, and colocalized with target gene expression. Subsets of cells simultaneously expressed both HIF‐α subunits, but EC more frequently induced HIF‐2α. A progressive increase of HIF‐2α but not HIF‐1α occurred in areas remote from the infarct, including the interventricular septum. Cardiomyocytes and cardiac stromal cells exhibit a marked potential for a prolonged transcriptional response to ischemia mediated by HIF. The induction of HIF‐1α and ‐2α appears to be complementary rather than solely redundant.

Overexpression of the human angiotensin II type 1 receptor in the rat heart augments load induced cardiac hypertrophy

Abstract. Angiotensin II is known to stimulate cardiac hypertrophy and contractility. Most angiotensin II effects are mediated via membrane bound AT1 receptors. However, the role of myocardial AT1 receptors in cardiac hypertrophy and contractility is still rarely defined. To address the hypothesis that increased myocardial AT1 receptor density causes cardiac hypertrophy apart from high blood pressure we developed a transgenic rat model which expresses the human AT1 receptor under the control of the α-myosin heavy-chain promoter specifically in the myocardium. Expression was identified and quantified by northern blot analysis and radioligand binding assays, demonstrating overexpression of angiotensin II receptors in the transgenic rats up to 46 times the amount seen in nontransgenic rats. Coupling of the human AT1 receptor to rat G proteins and signal transduction cascade was verified by sensitivity to GTP-γ-S and increased sensitivity of intracellular Ca2+ [Ca2+]i to angiotensin II in fluo-3 loaded transgenic cardiomyocytes. Transgenic rats exhibited normal cardiac growth and function under baseline conditions. Pronounced hypertrophic growth and contractile responses to angiotensin II, however, were noted in transgenic rats challenged by volume and pressure overload. In summary, we generated a new transgenic rat model that exhibits an upregulated myocardial AT1 receptor density and demonstrates augmented cardiac hypertrophy and contractile response to angiotensin II after volume and pressure overload, but not under baseline conditions.

Increased expression of renal neutral endopeptidase in severe heart failure.

The enzyme neutral endopeptidase (NEP; EC 3.4.24.11) cleaves several vasoactive peptides such as the atrial natriuretic peptide (ANP). ANP is a hormone of cardiac origin with diuretic and natriuretic actions. Despite elevated circulating levels of ANP, congestive heart failure (CHF) is characterized by progressive sodium and water retention. In order to elucidate the loss of natriuretic and diuretic properties of ANP in CHF we analyzed activity, protein concentrations, mRNA and immunostaining of NEP in kidneys of different models of severe CHF in the rat.CHF was induced by either aortocaval shunt, aortic banding or myocardial infarction in the rat. All models were defined by increased left ventricular end-diastolic pressure and decreased contractility. The diminished effectiveness of ANP was reflected by reduced cGMP/ANP ratio in animals with shunt or infarction. Renal NEP activity was increased in rats with aortocaval shunt (203 +/- 7%, p < 0.001), aortic banding (184 +/- 11%, p < 0.001) and infarction (149 +/- 10%, p < 0.005). Western blot analysis revealed a significant increase in renal NEP protein content in two models of CHF (shunt: 214 +/- 57%, p < 0.05; infarction: 310 +/- 53 %, p < 0.01). The elevated protein expression was paralleled by a threefold increase in renal NEP-mRNA level in the infarction model. The increased renal NEP protein expression and activity may lead to enhanced degradation of ANP and may contribute to the decreased renal response to ANP in heart failure. Thus, the capacity to counteract sodium and water retention, would be diminished. The increased renal NEP activity may therefore be a hitherto unknown factor in the progression of CHF.

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

Recently, deletion of specific genes by so called knock‐out techniques has become important for investigating the pathogenesis of various diseases. This form of genetic engineering is widely performed in murine models. There are, however, only a limited number of mouse models available in cardiovascular pathology. The objective of this study, therefore, was to develop a new model of overt congestive heart failure associated with myocardial hypertrophy in the mouse.

Atrial natriuretic peptide clearance receptor participates in modulating endothelial permeability

The atrial natriuretic peptide (ANP)-C receptor is generally believed to clear ANP; however, the ANP-C receptor may serve to reduce cAMP by inhibiting adenylate cyclase. ANP decreases endothelial permeability in coronary endothelial cell monolayers. We tested the hypothesis that part of this effect might be mediated by the ANP-C receptor. We used an endothelial cell monolayer from rat coronary endothelium and measured albumin flux. We applied either ANP or a ring-deleted ANP (C-ANP), which only stimulates the ANP-C receptor. ANP and C-ANP both decreased permeability from 100 pM to 100 nM by 60 and 30%, respectively. ANP increased endothelial cGMP contents 5.5-fold, whereas C-ANP had no effect. ANP reduced endothelial cAMP contents by 75%, which was only partly blocked by pertussis toxin. C-ANP also reduced cAMP; however, this effect was completely blocked by pertussis toxin. Protein kinase G inhibition blocked the ANP-mediated decrease in permeability by 50%. In contrast, pretreatment with pertussis toxin, in the face of protein kinase G inhibition, blocked the effect completely. C-ANP decreased permeability by half the amount of ANP. This C-ANP effect was completely blocked by pertussis toxin but not by protein kinase G inhibition. Isoproterenol (10 microM) increased permeability by almost 50%, which was completely blocked by ANP but only partially blocked by C-ANP. The C-ANP effect was blocked completely by pertussis toxin. Isoproterenol increased cAMP threefold, which was abolished by ANP. C-ANP reduced the isoproterenol-induced increase in cAMP by 50%. Isoproterenol had no effect on cGMP. We conclude that agonist binding to the ANP-C receptor inhibits cAMP production via a Gi protein-coupled signaling system. This inhibition may contribute to the decreased endothelial permeability evoked by ANP in this system.The atrial natriuretic peptide (ANP)-C receptor is generally believed to clear ANP; however, the ANP-C receptor may serve to reduce cAMP by inhibiting adenylate cyclase. ANP decreases endothelial permeability in coronary endothelial cell monolayers. We tested the hypothesis that part of this effect might be mediated by the ANP-C receptor. We used an endothelial cell monolayer from rat coronary endothelium and measured albumin flux. We applied either ANP or a ring-deleted ANP (C-ANP), which only stimulates the ANP-C receptor. ANP and C-ANP both decreased permeability from 100 pM to 100 nM by 60 and 30%, respectively. ANP increased endothelial cGMP contents 5.5-fold, whereas C-ANP had no effect. ANP reduced endothelial cAMP contents by 75%, which was only partly blocked by pertussis toxin. C-ANP also reduced cAMP; however, this effect was completely blocked by pertussis toxin. Protein kinase G inhibition blocked the ANP-mediated decrease in permeability by 50%. In contrast, pretreatment with pertussis toxin, in the face of protein kinase G inhibition, blocked the effect completely. C-ANP decreased permeability by half the amount of ANP. This C-ANP effect was completely blocked by pertussis toxin but not by protein kinase G inhibition. Isoproterenol (10 μM) increased permeability by almost 50%, which was completely blocked by ANP but only partially blocked by C-ANP. The C-ANP effect was blocked completely by pertussis toxin. Isoproterenol increased cAMP threefold, which was abolished by ANP. C-ANP reduced the isoproterenol-induced increase in cAMP by 50%. Isoproterenol had no effect on cGMP. We conclude that agonist binding to the ANP-C receptor inhibits cAMP production via a Giprotein-coupled signaling system. This inhibition may contribute to the decreased endothelial permeability evoked by ANP in this system.

Acute and Chronic Neutral Endopeptidase Inhibition in Rats With Aortocaval Shunt

In heart failure, sodium and water retention develop despite elevated plasma levels of atrial natriuretic peptide. Atrial natriuretic peptide is degraded in part by a neutral endopeptidase. Whether neutral endopeptidase inhibition improves sodium and water excretion in heart failure is unknown. We determined the effect of neutral endopeptidase inhibition on plasma levels of atrial natriuretic peptide and the renal response to acute volume expansion in rats with aortocaval shunts and in sham-operated controls. Acute endopeptidase inhibition with SQ 28,603 (30 mg/kg) elevated atrial natriuretic peptide plasma levels in both shunted rats (523 +/- 54 to 1258 +/- 330 pmol/L, P<.05) and controls (184 +/- 28 to 514 +/- 107 pmol/L, P<.05). Urinary cGMP excretion, which reflects renal action, increased in parallel. However, the diuretic and natriuretic responses to acute volume expansion were enhanced only in control rats and not in shunted rats. In contrast to the acute effects, chronic neutral endopeptidase inhibition with SCH 34826 (30 mg/kg twice daily) in shunted rats did not change atrial natriuretic peptide plasma levels or cGMP excretion. Nevertheless, the diuretic and natriuretic responses to acute volume load were increased by chronic endopeptidase inhibition in shunted rats (1789 +/- 154 to 2674 +/- 577 microL/80 min and 99 +/- 31 to 352 +/- 96 micromol/80 min, respectively; P<.05). Chronic endopeptidase inhibition attenuated the cardiac hypertrophic response to aortocaval shunt without changing arterial blood pressure. Our data show that the renal effects of neutral endopeptidase inhibition are not necessarily dependent on changes in atrial natriuretic peptide plasma levels but instead may be mediated by local inhibition of the neutral endopeptidase in the kidney. In addition, chronic endopeptidase inhibition may attenuate heart failure-induced cardiac hypertrophy independent of hemodynamic effects.

REGULATION OF CARDIAC ADRENOMEDULLIN-MRNA IN DIFFERENT STAGES OF EXPERIMENTAL HEART FAILURE

Adrenomedullin (AM) is a peptide hormone with vasodilating and natriuretic properties. AM plasma concentrations are elevated in heart failure. Whether cardiac AM-mRNA synthesis is increased in heart failure is not known. We measured AM-mRNA/GAPDH-mRNA in all four heart chambers in compensated and overt heart failure in rats with two different sizes of aortocaval shunt. Left and right atrial AM-mRNA expressions were unchanged in both heart failure models. Similarly, left and right ventricular AM-mRNA expressions were unchanged in compensated heart failure. In overt heart failure, however, the AM-mRNA expression was significantly increased in the left ventricle (145+/-20 vs. 100+/-3% of control, p<0.05). The right ventricular AM-mRNA expression was significantly increased only in a subgroup of animals with pulmonary congestion (lung weight >2.0 g, 141+/-16 vs. 100+/-11% of control, p<0.05). Ventricular AM concentrations were elevated in both ventricles in overt heart failure. AM plasma concentrations were significantly higher in the subgroup with pulmonary congestion than in rats with compensated heart failure (496+/-95 vs. 143+/-7 pmol/l, p<0.01). These data indicate that ventricular AM-mRNA expression and AM concentrations were upregulated only in advanced stages of heart failure. However, the exact contribution of cardiac AM synthesis to the increased AM plasma levels remains to be established.

Effect of chronic volume overload on baroreflex control of heart rate and sympathetic nerve activity

Baroreceptor-heart rate reflex sensitivity is decreased in congestive heart failure. The reflex control of heart rate and sympathetic nerve activity in rats with chronic volume overload, an established model for moderate heart failure, is still unknown. Therefore, we investigated the regulation of humoral and neuronal sympathetic activity and the baroreflex control of heart rate and sympathetic nerve activity in conscious, unrestrained rats with aortocaval shunt. Rats with aortocaval shunts had larger hearts (388 +/- 11 vs. 277 +/- 4 mg/100 g body wt), elevated central venous pressures (14 +/- 4 vs. 4 +/- 3 mmHg), and higher atrial natriuretic peptide plasma levels (87 +/- 16 vs. 25 +/- 3 pmol/l) than controls but had similar systemic blood pressure and heart rate values. Plasma epinephrine (0.63 +/- 0.16 vs. 0.21 +/- 0.08 pmol/l, P < 0.05) and norepinephrine concentrations (0.27 +/- 0.03 vs. 0.16 +/- 0.02 pmol/l, P < 0.05) were elevated in shunted rats compared with controls. Nitroprusside-induced hypotension led to a significantly greater increase in efferent splanchnic sympathetic nerve activity in shunted rats than in controls (0.9 +/- 0.1 vs. 2.6 +/- 0.6 microV, P < 0.05), whereas the heart rate responses were not different between the groups. These results indicate that the regulation of the autonomic nervous system is altered in chronically volume-overloaded rats. The arterial baroreflex control of efferent splanchnic sympathetic nerve activity was dissociated from the control of heart rate. Therefore, analysis of the activation of sympathetic nervous system assessed by direct measurements of efferent sympathetic nerve activity appears to be more sensitive for the detection of altered autonomic nervous system function than the analysis of baroreflex control of heart rate.Baroreceptor-heart rate reflex sensitivity is decreased in congestive heart failure. The reflex control of heart rate and sympathetic nerve activity in rats with chronic volume overload, an established model for moderate heart failure, is still unknown. Therefore, we investigated the regulation of humoral and neuronal sympathetic activity and the baroreflex control of heart rate and sympathetic nerve activity in conscious, unrestrained rats with aortocaval shunt. Rats with aortocaval shunts had larger hearts (388 ± 11 vs. 277 ± 4 mg/100 g body wt), elevated central venous pressures (14 ± 4 vs. 4 ± 3 mmHg), and higher atrial natriuretic peptide plasma levels (87 ± 16 vs. 25 ± 3 pmol/l) than controls but had similar systemic blood pressure and heart rate values. Plasma epinephrine (0.63 ± 0.16 vs. 0.21 ± 0.08 pmol/l, P < 0.05) and norepinephrine concentrations (0.27 ± 0.03 vs. 0.16 ± 0.02 pmol/l, P < 0.05) were elevated in shunted rats compared with controls. Nitroprusside-induced hypotension led to a significantly greater increase in efferent splanchnic sympathetic nerve activity in shunted rats than in controls (0.9 ± 0.1 vs. 2.6 ± 0.6 μV, P < 0.05), whereas the heart rate responses were not different between the groups. These results indicate that the regulation of the autonomic nervous system is altered in chronically volume-overloaded rats. The arterial baroreflex control of efferent splanchnic sympathetic nerve activity was dissociated from the control of heart rate. Therefore, analysis of the activation of sympathetic nervous system assessed by direct measurements of efferent sympathetic nerve activity appears to be more sensitive for the detection of altered autonomic nervous system function than the analysis of baroreflex control of heart rate.