Cholesterol enrichment increases basal and agonist-stimulated calcium influx in rat vascular smooth muscle cells.

The effect of cholesterol enrichment on vascular smooth muscle cell (VSMC) calcium homeostasis was studied by evaluating calcium uptake, efflux, and intracellular content in cultured VSMC derived from the rat pulmonary artery. Incubation of VSMC with liposomes consisting of free cholesterol (FC) and phospholipid (2:1 molar ratio, 1 mg FC/ml medium) for 24 h resulted in a 69 +/- 19% increase (P less than 0.01; n = 10) in FC which was associated with a 73 +/- 11% increase (P less than 0.005; n = 10) in intracellular calcium content as assessed by isotopic equilibrium with 45Ca2+ and a 65 +/- 11% increase (P less than 0.024; n = 3) as assessed by atomic absorption spectroscopy. Cholesterol enrichment caused a marked increase in the unidirectional calcium uptake rate from 0.026 +/- 0.03 to 0.158 +/- 0.022 nmol calcium/s per mg protein (P less than 0.01; n = 3), but had no effect on calcium efflux. Nifedipine (1 microM) reduced (P less than 0.05; n = 6) the effect of cholesterol enrichment on unidirectional calcium uptake by 78 +/- 16%; and verapamil (10 microM), diltiazem (1 microM), and nifedipine (1 microM) each significantly inhibited the effect of cholesterol enrichment on intracellular calcium accumulation. Exposure of cholesterol-enriched VSMC to cholesterol-poor liposomes for 24 h returned both FC and calcium contents to control levels. Serum- and serotonin-stimulated calcium uptakes were potentiated 3.7- and 1.7-fold, respectively, in cholesterol-enriched VSMC, whereas endothelin, vasopressin, and thrombin-stimulated calcium uptakes were not affected. We conclude that VSMC FC content plays a role in regulating cellular calcium homeostasis, both under basal conditions and in response to selected agonists.

The reovirus M1 gene determines the relative capacity of growth of reovirus in cultured bovine aortic endothelial cells.

Since blood-borne viruses often interact with endothelial cells before tissue invasion, the interaction between viruses and endothelial cells is likely to be important in viral pathogenicity. Two reovirus isolates (type 1 Lang and type 3 Dearing) differ in their capacity to grow in cultured bovine aortic endothelial cells. The mammalian reoviruses have 10 double-stranded RNA gene segments in their genome. By using 24 reassortant viruses, observed differences in the capacity of different strains to grow in cultured endothelial cells were mapped to the M1 gene (P = 0.00019), which encodes the viral core protein mu 2. No differences were detected in binding or proteolytic processing of viral outer capsid proteins of parental virions between the two reovirus isolates. Northern blot analysis showed a decreased production of viral mRNA in endothelial cells infected with type 3 Dearing reovirus, but not type 1 Lang. Thus, we have identified a viral gene (the M1 gene) responsible for determining the difference in growth capacity of the two reovirus isolates in cultured endothelial cells. Reovirus is an attractive model in which to study the interaction of viruses with endothelial cells at a molecular genetic level.

Interleukin-1 beta modulates the growth and phenotype of neonatal rat cardiac myocytes.

Mononuclear cell infiltration and local cytokine elaboration are hallmarks of inflammatory and immunologic heart diseases. To test the hypothesis that cytokines can modulate cardiac myocyte growth and phenotype, myocytes cultured from neonatal rat hearts were exposed to IL-1 beta, an inflammatory cytokine prevalent in myocardial inflammation. IL-1 beta (2 ng/ml, 24 h) increased [3H]leucine incorporation by 30 +/- 4% (P < 0.001, n = 29) and net cellular protein content by 20 +/- 4% (P < 0.001, n = 27), but had no effect on DNA synthesis. Northern hybridization showed that IL-1 beta increased prepro-atrial natriuretic factor (ANF) mRNA (5.8 +/- 1.5-fold, P < 0.01, n = 13) and beta-myosin heavy chain (beta-MHC) mRNA (> 10-fold, n = 4), and decreased mRNA levels for sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2) (-46 +/- 7%; P < 0.001; n = 11), calcium release channel (CRC) (-65 +/- 11%, P < 0.001, n = 8) and voltage-dependent calcium channel (VDCC) (-53 +/- 7%, P < 0.001, n = 8). NG-monomethyl-L-arginine (1 mM), an inhibitor of nitric oxide (NO) synthesis, did not inhibit the IL-1 beta-induced protein synthesis or changes in mRNA levels. In ventricular myocardium obtained from adult rats treated with lipopolysaccharide (4 mg/kg intraperitoneally 18 h) to stimulate systemic cytokine production, there were changes in the mRNA levels for beta-MHC (6 +/- 1-fold, P < 0.01, n = 4), SERCA2 (-65 +/- 4%, P < 0.0001, n = 4), CRC (-67 +/- 5%, P < 0.001, n = 4), and VDCC (-58 +/- 5%, P < 0.001; n = 4) that were qualitatively similar to those observed in cultured myocytes. Thus, IL-1 beta, acting via an NO-independent mechanism, caused myocyte hypertrophy associated with induction of fetal genes (ANF and beta-MHC) and downregulation of three important calcium regulatory genes (SERCA2, CRC, and VDCC). IL-1 beta may contribute to the abnormal structural and functional alterations of cardiac myocytes in conditions marked by mononuclear cell infiltration.

Nitric oxide, atrial natriuretic peptide, and cyclic GMP inhibit the growth-promoting effects of norepinephrine in cardiac myocytes and fibroblasts.

This study tested the hypothesis that nitric oxide (NO) and atrial natriuretic peptide (ANP) can attenuate the effects of adrenergic agonists on the growth of cardiac myocytes and fibroblasts. In ventricular cells cultured from neonatal rat heart, ANP and the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) caused concentration-dependent decreases in the norepinephrine (NE)-stimulated incorporation of [3H]leucine in myocytes and [3H]thymidine in fibroblasts. In myocytes, the NO synthase inhibitor NG-monomethyl-L-arginine potentiated NE-stimulated [3H]leucine incorporation. In both cell types, ANP and SNAP increased intracellular cGMP levels, and their growth-suppressing effects were mimicked by the cGMP analogue 8-bromo-cGMP. Furthermore, in myocytes, 8-bromo-cGMP attenuated the alpha1-adrenergic receptor-stimulated increases in c-fos. Likewise, ANP and 8-bromo-cGMP attenuated the alpha1-adrenergic receptor- stimulated increase in prepro-ANP mRNA and the alpha1-adrenergic receptor-stimulated decrease in sarcoplasmic reticulum calcium ATPase mRNA. The L-type Ca2+ channel blockers verapamil and nifedipine inhibited NE-stimulated incorporation of [3H]leucine in myocytes and [3H]thymidine in fibroblasts, and these effects were not additive with those of ANP, SNAP, or 8-bromo-cGMP. In myocytes, the Ca2+ channel agonist BAY K8644 caused an increase in [3H]leucine incorporation which was inhibited by ANP. These findings indicate that NO and ANP can attenuate the effects of NE on the growth of cardiac myocytes and fibroblasts, most likely by a cGMP-mediated inhibition of NE-stimulated Ca2+ influx.

Role of nitric oxide in parasympathetic modulation of beta-adrenergic myocardial contractility in normal dogs.

In vitro studies indicate that muscarinic cholinergic inhibition of beta-adrenergic cardiac responses may be modulated in part by nitric oxide (NO). To evaluate the role of NO in parasympathetic inhibition of the beta-adrenergic contractile response in vivo, we assessed the inotropic response to dobutamine before and during bilateral vagus nerve stimulation in closed-chest dogs. Dobutamine administration and vagal stimulation were repeated during intracoronary infusion of the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 10 mumol/min) and again following infusion of L-arginine (100 mg/kg). In eight dogs, intracoronary dobutamine infusion at rates of 25 and 50 micrograms/min increased peak +dP/dt by 131 +/- 24 and 168 +/- 22%, respectively (P < 0.0001). Vagal stimulation (2.5 Hz) attenuated the responses to dobutamine (25 and 50 micrograms/min) by 23 +/- 4 and 21 +/- 4%, respectively (P < 0.001). L-NMMA reduced (by 44-62%; P < 0.001) and L-arginine restored vagal inhibition of the dobutamine-stimulated inotropic response. In a second group of nine dogs, dobutamine was administered systemically to assure a constant concentration in the coronary circulation. Vagal stimulation (2.5 Hz) attenuated the dobutamine-stimulated inotropic response (2.5 and 5.0 micrograms/kg per min) by 40 +/- 12% and 57 +/- 8%, respectively (P < 0.004). As with intracoronary dobutamine, L-NMMA diminished and L-arginine restored vagal inhibition of the inotropic response to dobutamine. Intracoronary infusion of atropine (12 micrograms/min) abolished the vagal inhibitory effect, and intracoronary infusion of 8-bromo-cyclic GMP (1 and 10 mM) caused a dose-dependent attenuation of the dobutamine-stimulated increase in +dP/dt. These data suggest that NO mediates, at least in part, vagal inhibition of the inotropic response to beta-adrenergic stimulation by dobutamine, and thus may play a role in normal physiologic regulation of myocardial autonomic responses.

Muscarinic receptor modulation of basal and beta-adrenergic stimulated function of the failing human left ventricle.

The objective of this study was to evaluate the effect of muscarinic receptor modulation on basal and beta-adrenergic stimulated left ventricular function in patients with heart failure. 21 heart failure patients and 14 subjects with normal ventricular function were studied. In Protocol 1 intracoronary acetylcholine resulted in a 60+/-8% inhibition of the left ventricular +dP/dt response to intracoronary dobutamine in the normal group, and a similar 70+/-13% inhibition in the heart failure group. Acetylcholine also attenuated the dobutamine-mediated acceleration of isovolumic relaxation (Tau) in both groups. Acetylcholine alone had no effect on Tau in the normal group, while it prolonged Tau in the heart failure group. In Protocol 2 intracoronary atropine resulted in a 35+/-10% augmentation of the inotropic response to dobutamine in the normal group, versus a non-significant 12+/-15% augmentation of the dobutamine response in the heart failure group. In Protocol 3, in 6 heart failure patients, both effects of acetylcholine, the slowing of ventricular relaxation and the inhibition of beta-adrenergic responses, were reversed by the addition of atropine. Therefore, in the failing human left ventricle muscarinic stimulation has an independent negative lusitropic effect and antagonizes the effects of beta-adrenergic stimulation.

Hypertrophic stimuli induce transforming growth factor-beta 1 expression in rat ventricular myocytes.

Transforming growth factor-beta 1 (TGF-beta 1) is a peptide growth factor that may play a role in the myocardial response to hypertrophic stimuli. However, the cellular distribution, mechanism of induction, and source of increased TGF-beta 1 in response to hypertrophic stimuli are not known. We tested the hypothesis that the cardiac myocyte responds to hypertrophic stimuli with the increased expression of TGF-beta 1. In adult rat ventricular myocardium freshly dissociated into myocyte and nonmyocyte cellular fractions, the preponderance of TGF-beta 1 mRNA visualized by Northern hybridization was in the nonmyocyte fraction. Abdominal aortic constriction (7 d) and subcutaneous norepinephrine infusion (36 h) each caused ventricular hypertrophy associated with 3.1-fold and 3.8-fold increases, respectively, in TGF-beta 1 mRNA in the myocyte fraction, but had no effect on the level of TGF-beta 1 mRNA in the nonmyocyte fraction. In ventricular myocytes, norepinephrine likewise caused a 4.1-fold increase in TGF-beta 1 mRNA associated with an increase in TGF-beta bioactivity. This induction of TGF-beta 1 mRNA occurred at norepinephrine concentrations as low as 1 nM and was blocked by prazosin, but not propranolol. NE did not increase the TGF-beta 1 mRNA level in nonmyocytes, primarily fibroblasts, cultured from neonatal rat ventricle. Thus, the cardiac myocyte responds to two hypertrophic stimuli, pressure overload and norepinephrine, with the induction of TGF-beta 1. These data support the view that TGF-beta 1, released by myocytes and acting in an autocrine and/or paracrine manner, is involved in myocardial remodeling by hypertrophic stimuli.

Positive inotropic and vasodilator actions of milrinone in patients with severe congestive heart failure. Dose-response relationships and comparison to nitroprusside.

Milrinone is a potent positive inotropic and vascular smooth muscle-relaxing agent in vitro, and therefore, it is not known to what extent each of these actions contributes to the drug's hemodynamic effects in patients with heart failure. In 11 patients with New York Heart Association class III or IV congestive heart failure, incremental intravenous doses of milrinone were administered to determine the dose-response relationships for heart rate, systemic vascular resistance, and inotropic state, the latter measured by peak positive left ventricular derivative of pressure with respect to time (dP/dt). To clarify further the role of a positive inotropic action, the relative effects of milrinone and nitroprusside on left ventricular stroke work and dP/dt were compared in each patient at doses matched to cause equivalent reductions in mean arterial pressure or systemic vascular resistance, indices of left ventricular afterload. Milrinone caused heart rate, stroke volume, and dP/dt to increase, and systemic vascular resistance to decrease in a concentration-related manner. At the two lowest milrinone doses resulting in serum concentrations of 63 +/- 4 and 156 +/- 5 ng/ml, respectively, milrinone caused significant increases in stroke volume and dP/dt, but no changes in systemic vascular resistance or heart rate. At the maximum milrinone dose administered (mean serum concentration, 427 +/- 11 ng/ml), heart rate increased from 92 +/- 4 to 99 +/- 4 bpm (P less than 0.01), mean aortic pressure fell from 82 +/- 3 to 71 +/- 3 mmHg (P less than 0.01), right atrial pressure fell from 15 +/- 2 to 7 +/- 1 mmHg (P less than 0.005), left ventricular end-diastolic pressure fell from 26 +/- 3 to 18 +/- 3 (P less than 0.005), stroke volume index increased from 20 +/- 2 to 30 +/- 2 ml/m2 (P less than 0.005), stroke work index increased from 14 +/- 2 to 21 +/- 2 g X m/m2 (P less than 0.01), and dP/dt increased from 858 +/- 54 to 1,130 +/- 108 mmHg/s (P less than 0.005). When compared with nitroprusside for a matched reduction in mean aortic pressure or systemic vascular resistance, milrinone caused a significantly greater increase in stroke work index at the same or lower left ventricular end-diastolic pressure. Milrinone caused a concentration-related increase in dP/dt (32% increase at maximum milrinone dose), whereas nitroprusside had no effect. These data in patients with severe heart failure indicate that in addition to a vasodilating effect, milrinone exerts a concentration-related positive inotropic action that contributes significantly to the drug's overall hemodynamic effects. The positive inotropic action occurs at drug levels that do not exert significant chronotropic or vasodilator effects.

Intracoronary infusion of dobutamine to patients with and without severe congestive heart failure. Dose-response relationships, correlation with circulating catecholamines, and effect of phosphodiesterase inhibition.

We infused dobutamine into the left main coronary artery of 24 patients with severe congestive heart failure (CHF) and 8 normal subjects without hemodynamic dysfunction. The maximal +dP/dt response to intracoronary (IC) dobutamine in CHF patients was only 37% of that in normals. This decrease in maximal response was not associated with a rightshift in the EC50 for dobutamine's effect on +dP/dt, or a decrease in the affinity of myocardial beta adrenergic receptors for dobutamine determined in vitro. In nine of the CHF patients, IC dobutamine infusion was followed by IC infusion of the phosphodiesterase inhibitor milrinone, and subsequently, by a second IC infusion of dobutamine. After IC milrinone, the increase in +dP/dt caused by IC dobutamine (74 +/- 10%) was significantly greater than that caused by the first infusion of dobutamine (52 +/- 11%; P less than 0.003) or milrinone (42 +/- 6%; P less than 0.001). Resting plasma norepinephrine was markedly elevated in CHF patients (837 +/- 208 ng/liter), but not in normal subjects (142 +/- 32 ng/liter); and the increase in +dP/dt caused by IC dobutamine was inversely related to resting plasma norepinephrine levels (r = -0.653; P less than 0.001). IC dobutamine caused a dose-related decrease in plasma norepinephrine (maximal effect, -160 +/- 31 ng/liter; P less than 0.001). Thus, (a) the maximal inotropic response to dobutamine is markedly depressed in patients with severe CHF, and is significantly greater after pretreatment with the phosphodiesterase inhibitor milrinone; (b) the impairment in inotropic response to dobutamine is inversely related to circulating norepinephrine levels; and (c) myocardial stimulation by dobutamine results in withdrawal of sympathetic tone.

Effects of levosimendan on cardiac remodeling and cardiomyocyte apoptosis in hypertensive Dahl/Rapp rats.

BACKGROUND AND PURPOSE: Progression of heart failure in hypertensive Dahl rats is associated with cardiac remodeling and increased cardiomyocyte apoptosis. This study was conducted to study whether treatment with a novel inotropic vasodilator compound, levosimendan, could prevent hypertension-induced cardiac remodeling and cardiomyocyte apoptosis. EXPERIMENTAL APPROACH: 6-week-old salt-sensitive Dahl/Rapp rats received levosimendan (0.3 mg kg(-1) and 3 mg kg(-1) via drinking fluid) and high salt diet (NaCl 7%) for 7 weeks, Dahl/Rapp rats on low-salt diet served as controls. Blood pressure, cardiac functions by echocardiography, cardiomyocyte apoptosis by TUNEL technique, tissue morphology, myocardial expression of calcium cycling proteins, and markers of neurohumoral activation were determined. KEY RESULTS: Untreated Dahl/Rapp rats on high salt diet developed severe hypertension, cardiac hypertrophy and moderate systolic dysfunction. 38% of Dahl/Rapp rats (9/24) survived the 7-week-follow-up period. Cardiomyocyte apoptosis was increased by 6-fold during high salt diet. Levosimendan improved survival (survival rates in low- and high-dose levosimendan groups 12/12 and 9/12, p<0.001 and p=0.05, respectively), increased cardiac function, and ameliorated cardiac hypertrophy. Levosimendan dose-dependently prevented cardiomyocyte apoptosis. Levosimendan normalized salt-induced increased expression of natriuretic peptide, and decreased urinary noradrenaline excretion. Levosimendan also corrected salt-induced decreases in myocardial SERCA2a protein expression and myocardial SERCA2a/NCX-ratio. CONCLUSIONS AND IMPLICATIONS: Improved survival by the novel inotropic vasodilator levosimendan in hypertensive Dahl/Rapp rats is mediated, at least in part, by amelioration of hypertension-induced cardiac remodeling and cardiomyocyte apoptosis.

Interleukin-1beta and tumor necrosis factor-alpha decrease collagen synthesis and increase matrix metalloproteinase activity in cardiac fibroblasts in vitro.

We tested the hypothesis that the inflammatory cytokines can regulate fibroblast extracellular matrix metabolism. Neonatal and adult rat cardiac fibroblasts cultures in vitro were exposed to interleukin (IL)-1beta (4 ng/mL), tumor necrosis factor-alpha (TNF-alpha; 100 ng/mL), IL-6 (10 ng/mL), or interferon-gamma (IFN-gamma; 500 U/mL) for 24 hours. IL-1beta, and to a lesser extent TNF-alpha, decreased collagen synthesis, which was measured as collagenase-sensitive [(3)H]proline incorporation, but had no effect on cell number or total protein synthesis. IL-1beta decreased the expression of procollagen alpha(1)(I), alpha(2)(I), and alpha1(III) mRNA, but increased the expression of procollagen alpha(1)(IV), alpha(2)(IV), and fibronectin mRNA, indicating a selective transcriptional downregulation of fibrillar collagen synthesis. IL-1beta and TNF-alpha each increased total matrix metalloproteinase (MMP) activity as measured by in-gel zymography, causing specific increases in the bands corresponding to MMP-13, MMP-2, and MMP-9. IL-1beta increased the expression of proMMP-2 and proMMP-3 mRNA, suggesting that increased metalloproteinase activity is due, at least in part, to increased transcription. The effects of IL-1beta were not dependent on NO production. Thus, IL-1beta and TNF-alpha decrease collagen synthesis and activate MMPs that degrade collagen. These observations suggest that IL-1beta and TNF-alpha may contribute to ventricular dilation and myocardial failure by promoting the remodeling of interstitial collagen.

Mice lacking inducible nitric oxide synthase have improved left ventricular contractile function and reduced apoptotic cell death late after myocardial infarction.

Nitric oxide produced by inducible nitric oxide synthase (NOS2) has been implicated in the pathophysiology of chronic myocardial remodeling and failure. We tested the role of NOS2 in left ventricular (LV) remodeling early (1 month) and late (4 months) after myocardial infarction (MI) in mice lacking NOS2. MI size measured 7 days, 1 month, and 4 months after MI was the same in NOS2 knockout (KO) and wild-type (WT) mice. The LV end-diastolic pressure-volume relationship measured by the isovolumic Langendorff technique showed a progressive rightward shift from 1 to 4 months after MI in WT mice. LV developed pressure measured over a range of LV volumes was reduced at 1 and 4 months after MI in WT mice (P<0.05 and P<0.01 versus shams, respectively). In KO mice, the rightward shift was similar to that in WT mice at 1 and 4 months after MI, as was peak LV developed pressure at 1 month after MI. In contrast, at 4 months after MI, peak LV developed pressure in KO mice was higher than in WT mice (P<0.05 versus WT) and similar to that in sham-operated mice. At 1 month after MI, the frequency of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive myocytes in the remote myocardium was increased to a similar extent in WT and KO mice. At 4 months after MI, the frequency of apoptotic myocytes was increased in WT mice but not in KO mice (P<0.05 versus WT). Improved contractile function and reduced apoptosis were associated with reduced mortality rate in KO mice at 4 months after MI. Thus, NOS2 does not play an important role in determining infarct size or early LV remodeling during the first month after MI. In contrast, during late (ie, 4 months after MI) remodeling, NOS2 in remote myocardium contributes to decreased contractile function, increased myocyte apoptosis in remote myocardium, and reduced survival.

Reactive oxygen species mediate amplitude-dependent hypertrophic and apoptotic responses to mechanical stretch in cardiac myocytes.

Oxidative stress stimulates both growth and apoptosis in cardiac myocytes in vitro. We investigated whether oxidative stress mediates hypertrophy and apoptosis in cyclically stretched ventricular myocytes. Neonatal rat ventricular myocytes cultured on laminin-coated silastic membranes were stretched cyclically (1 Hz) at low (nominal 5%) and high (nominal 25%) amplitudes for 24 hours. Stretch caused a graded increase in superoxide anion production as assessed by superoxide dismutase (SOD)-inhibitable cytochrome c reduction or electron paramagnetic resonance spectroscopy. The role of reactive oxygen species (ROS) was assessed using the cell-permeable SOD/catalase mimetics Mn(II/III)tetrakis(1-methyl-4-peridyl) (MnTMPyP) and EUK-8. Stretch-induced increases in protein synthesis ((3)H-leucine incorporation) and cellular protein content were completely inhibited by MnTMPyP (0.05 mmol/L) at both low and high amplitudes of stretch. In contrast, while MnTMPyP inhibited basal atrial natriuretic factor (ANF) mRNA expression, the stretch-induced increase in ANF mRNA expression was not inhibited by MnTMPyP. In contrast to hypertrophy, only high-amplitude stretch increased myocyte apoptosis, as reflected by increased DNA fragmentation on gel electrophoresis and an approximately 3-fold increase in the number of TUNEL-positive myocytes. Similarly, only high-amplitude stretch increased the expression of bax mRNA. Myocyte apoptosis and bax expression stimulated by high-amplitude stretch were inhibited by MnTMPyP. Both low- and high-amplitude stretch caused rapid phosphorylation of ERK1/2, while high-, but not low-, amplitude stretch caused phosphorylation of JNKs. Activation of both ERK1/2 and JNKs was ROS-dependent. Thus, cyclic strain causes an amplitude-related increase in ROS, associated with differential activation of kinases and induction of hypertrophic and apoptotic phenotypes.

Exaggerated left ventricular dilation and reduced collagen deposition after myocardial infarction in mice lacking osteopontin.

Osteopontin (OPN), an extracellular matrix protein, is expressed in the myocardium with hypertrophy and failure. We tested the hypothesis that OPN plays a role in left ventricular (LV) remodeling after myocardial infarction (MI). Accordingly, OPN expression and LV structural and functional remodeling were determined in wild-type (WT) and OPN knockout (KO) mice 4 weeks after MI. Northern analysis showed increased OPN expression in the infarcted region, peaking 3 days after MI and gradually decreasing over the next 28 days. In the remote LV, OPN expression was biphasic, with peaks at 3 and 28 days. In situ hybridization and immunohistochemical analyses showed increased OPN mRNA and protein primarily in the interstitium. Infarct size, heart weight, and survival were similar in KO and WT mice after MI (P=NS), whereas the lung wet weight/dry weight ratio was increased in the KO mice (P<0.005 versus sham-operated mice). Peak LV developed pressure was reduced to a similar degree after MI in the KO and WT mice. The number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive myocytes was similar in KO and WT mice after MI. In contrast, post-MI LV chamber dilation was approximately twice as great in KO versus WT mice (P<0.001). Myocyte length increased after MI in WT mice (P<0.001) but not in KO mice. Electron microscopy showed increased collagen content in WT mice after MI but not in KO mice after MI. Type I collagen content was increased approximately 3-fold and approximately 7-fold in remote and infarcted regions, respectively, of WT hearts after MI but not in KO hearts (P<0.01 versus WT hearts). Likewise, Northern analyses showed increased collagen I(alpha(1)) mRNA after MI in remote regions of WT hearts but not in KO hearts. Thus, increased OPN expression plays an important role in regulating post-MI LV remodeling, at least in part, by promoting collagen synthesis and accumulation.

Secondary pulmonary hypertension in chronic heart failure: the role of the endothelium in pathophysiology and management.

Pulmonary vascular resistance is frequently elevated in patients with chronic left ventricular failure as a result of dysregulation of vascular smooth muscle tone and structural remodeling. The former is reversible over a period of minutes to days by pharmacological vasodilators, whereas the latter is relatively fixed and may resolve only slowly, over months to years. These abnormalities are due, at least in part, to pulmonary vascular endothelial dysfunction that results in impaired nitric oxide availability and increased endothelin expression. In patients with chronic heart failure, the resulting pulmonary hypertension directly affects right ventricular function and may affect exercise capacity, morbidity, and mortality. New treatment strategies, which include the use of agents that increase nitric oxide availability or oppose the actions of endothelin, may improve the structure and function of the pulmonary vasculature in patients with chronic heart failure.

Chronic alpha-adrenergic receptor stimulation modulates the contractile phenotype of cardiac myocytes in vitro.

BACKGROUND: Heart failure is characterized by contractile dysfunction of the myocardium and elevated sympathetic activity. We tested the hypothesis that chronic alpha-adrenergic (alpha-ADR) stimulation modifies the molecular and contractile phenotype of cardiac myocytes. METHODS AND RESULTS: Adult rat ventricular myocytes in culture were exposed to alpha-ADR stimulation (norepinephrine + propranolol) for 48 hours. alpha-ADR stimulation decreased the mRNAs for sarcoplasmic reticulum Ca(2+)-ATPase and Ca(2+) release channel by 56% and 52%, respectively, and increased mRNA and protein for the Na(+)-Ca(2+) exchanger by 70% and 39%, respectively. After washout of the alpha-ADR agonist, simultaneous measurement of [Ca(2+)](i) transients with fura 2 and myocyte shortening by video edge-detection showed that [Ca(2+)](i) amplitude and myocyte shortening were decreased in alpha-ADR-treated myocytes, and the time to peak and time from peak to 80% decline of both [Ca(2+)](i) and myocyte shortening were increased. The concentration-response curve for myocyte shortening by the Na(+) channel activator veratridine was shifted leftward in alpha-ADR-stimulated myocytes (EC(50), 21.6+/-4.6 versus 105.8+/-10.5 nmol/L, P:<0.001). CONCLUSIONS: Chronic alpha-ADR stimulation of cardiac myocytes causes decreases in the expression of sarcoplasmic reticulum Ca(2+)-ATPase and the Ca(2+) release channel that are associated with decreases in [Ca(2+)](i) and contractility. alpha-ADR stimulation simultaneously increases Na(+)-Ca(2+) exchanger expression, thereby increasing sensitivity to intracellular Na(+).

Opposing effects of beta(1)- and beta(2)-adrenergic receptors on cardiac myocyte apoptosis : role of a pertussis toxin-sensitive G protein.

BACKGROUND: beta-Adrenergic receptor (beta-AR) stimulation increases apoptosis in adult rat cardiac (ventricular) myocytes (ARVMs) via activation of adenylyl cyclase. beta(2)-ARs may couple to a G(i)-mediated signaling pathway that can oppose the actions of adenylyl cyclase. METHODS AND RESULTS: In ARVMs, beta-AR stimulation for 24 hours increased the number of apoptotic cells as measured by flow cytometry. beta-AR-stimulated apoptosis was abolished by the beta(1)-AR-selective antagonist CGP 20712A (P<0.05 versus beta-AR stimulation alone) but was potentiated by the beta(2)-AR-selective antagonist ICI 118,551 (P<0.05 versus beta-AR stimulation alone). The muscarinic agonist carbachol also prevented beta-AR-stimulated apoptosis (P<0.05 versus beta-AR stimulation alone), whereas pertussis toxin potentiated the apoptotic action of beta-AR stimulation (P<0.05 versus beta-AR stimulation alone) and prevented the antiapoptotic action of carbachol. CONCLUSIONS: In ARVMs, stimulation of beta(1)-ARs increases apoptosis via a cAMP-dependent mechanism, whereas stimulation of beta(2)-ARs inhibits apoptosis via a G(i)-coupled pathway. These findings have implications for the pathophysiology and treatment of myocardial failure.

Acute endothelin A receptor blockade causes selective pulmonary vasodilation in patients with chronic heart failure.

BACKGROUND: Elevated plasma endothelin-1 (ET-1) levels in patients with chronic heart failure correlate with pulmonary artery pressures and pulmonary vascular resistance. ET(A) receptors on vascular smooth muscle cells mediate pulmonary vascular contraction and hypertrophy. We determined the acute hemodynamic effects of sitaxsentan, a selective ET(A) receptor antagonist, in patients with chronic stable heart failure receiving conventional therapy. METHODS AND RESULTS: This multicenter, double-blind, placebo-controlled trial enrolled 48 patients with chronic New York Heart Association functional class III or IV heart failure (mean left ventricular ejection fraction 21+/-1%) treated with ACE inhibitors and diuretics. Patients with a baseline pulmonary capillary wedge pressure >/=15 mm Hg and a cardiac index

Acute hemodynamic and clinical effects of levosimendan in patients with severe heart failure. Study Investigators.

BACKGROUND: We determined the short-term hemodynamic and clinical effects of levosimendan, a novel calcium-sensitizing agent, in patients with decompensated heart failure. METHODS AND RESULTS: One hundred forty-six patients with New York Heart Association functional class III or IV heart failure (mean left ventricular ejection fraction 21+/-1%) who had a pulmonary capillary wedge pressure >/=15 mm Hg and a cardiac index

Cell therapy attenuates deleterious ventricular remodeling and improves cardiac performance after myocardial infarction.

BACKGROUND: Myocardial infarction (MI) promotes deleterious remodeling of the myocardium, resulting in ventricular dilation and pump dysfunction. We examined whether supplementing infarcted myocardium with skeletal myoblasts would (1) result in viable myoblast implants, (2) attenuate deleterious remodeling, and (3) enhance in vivo and ex vivo contractile performance. METHODS AND RESULTS: Experimental MI was induced by 1-hour coronary ligation followed by reperfusion in adult male Lewis rats. One week after MI, 10(6) myoblasts were injected directly into the infarct region. Three groups of animals were studied at 3 and 6 weeks after cell therapy: noninfarcted control (control), MI plus sham injection (MI), and MI plus cell injection (MI+cell). In vivo cardiac function was assessed by maximum exercise capacity testing and ex vivo function was determined by pressure-volume curves obtained from isolated, red cell-perfused, balloon-in-left ventricle (LV) hearts. MI and MI+cell hearts had indistinguishable infarct sizes of approximately 30% of the LV. At 3 and 6 weeks after cell therapy, 92% (13 of 14) of MI+cell hearts showed evidence of myoblast graft survival. MI+cell hearts exhibited attenuation of global ventricular dilation and reduced septum-to-free wall diameter compared with MI hearts not receiving cell therapy. Furthermore, cell therapy improved both post-MI in vivo exercise capacity and ex vivo LV systolic pressures. CONCLUSIONS: Implanted skeletal myoblasts form viable grafts in infarcted myocardium, resulting in enhanced post-MI exercise capacity and contractile function and attenuated ventricular dilation. These data illustrate that syngeneic myoblast implantation after MI improves both in vivo and ex vivo indexes of global ventricular dysfunction and deleterious remodeling and suggests that cellular implantation may be beneficial after MI.