Involvement of reperfusion injury salvage kinases in preconditioning depends critically on the preconditioning stimulus.

Different preconditioning stimuli can activate divergent signaling pathways. In rats, adenosine-independent pathways (triple 3-min coronary artery occlusion [3CAO3]) and adenosine-dependent pathways (one 15-min coronary artery occlusion [ICAO15]) exist, both ultimately converging at the level of the mitochondrial respiratory chain. Furthermore, while 3CAO3, 1CAO15 and exogenous adenosine (ADO) are equally cardioprotective, only 1CAO15 increases interstitial myocardial adenosine levels. Reperfusion Injury Salvage Kinase (RISK) pathway kinases have been implicated in ischemic preconditioning, but not all preconditioning stimuli activate this pathway. Consequently, we evaluated in anesthetized rats the effects of three distinctly different preconditioning stimuli (3CAO3, 1CAO15 or ADO) on infarct size (IS), signaling pathways with a special emphasis on kinases belonging to the RISK pathway (phosphatidylinositol 3-kinase-Akt-nitric oxide synthase and extracellular signal-related kinase [ERK]) and mitochondrial respiration. All three stimuli increased state-2 respiration (using succinate as complex-II substrate), thereby decreasing the respiratory control index, which was accompanied by a limitation of IS produced by a 60-min coronary artery occlusion (CAO). Nitric oxide synthase inhibition abolished the mitochondrial effects and the cardioprotection by 3CAO3, 1CAO15 or ADO. In contrast, the PI3 kinase inhibitor, wortmannin, blocked protection by 1CAO15, but did not affect protection by 3CAO3 or ADO. Western blotting confirmed that phosphorylation of Akt and ERK were increased by 1CAO15 (which was inhibited by wortmannin), but not by 3CAO3 or ADO. In conclusion, while the three cardioprotective stimuli 3CAO3, 1CAO15 and ADO afford cardioprotection via nitric oxide-mediated modulation of mitochondrial respiration, only the 1CAO15 exerts its protection via activation of kinases belonging to the RISK pathway.

Prevention of myofilament dysfunction by beta-blocker therapy in postinfarct remodeling.

BACKGROUND: Myofilament contractility of individual cardiomyocytes is depressed in remote noninfarcted myocardium and contributes to global left ventricular pump dysfunction after myocardial infarction (MI). Here, we investigated whether beta-blocker therapy could restore myofilament contractility. METHODS AND RESULTS: In pigs with a MI induced by ligation of the left circumflex coronary artery, beta-blocker therapy (bisoprolol, MI+beta) was initiated on the first day after MI. Remote left ventricular subendocardial biopsies were taken 3 weeks after sham or MI surgery. Isometric force was measured in single permeabilized cardiomyocytes. Maximal force (F(max)) was lower, whereas Ca(2+) sensitivity was higher in untreated MI compared with sham (both P<0.05). The difference in Ca(2+) sensitivity was abolished by treatment of cells with the beta-adrenergic kinase, protein kinase A. beta-blocker therapy partially reversed F(max) and Ca(2+) sensitivity to sham values and significantly reduced passive force. Despite the lower myofilament Ca(2+) sensitivity in MI+beta compared with untreated myocardium, the protein kinase A induced reduction in Ca(2+) sensitivity was largest in cardiomyocytes from myocardium treated with beta-blockers. Phosphorylation of beta-adrenergic target proteins (myosin binding protein C and troponin I) did not differ among groups, whereas myosin light chain 2 phosphorylation was reduced in MI, which coincided with increased expression of protein phosphatase 1. beta-blockade fully restored the latter alterations and significantly reduced expression of protein phosphatase 2a. CONCLUSIONS: beta-blockade reversed myofilament dysfunction and enhanced myofilament responsiveness to protein kinase A in remote myocardium after MI. These effects likely contribute to the beneficial effects of beta-blockade on global left ventricular function after MI.

Detrimental effect of combined exercise training and eNOS overexpression on cardiac function after myocardial infarction.

It has been reported that exercise after myocardial infarction (MI) attenuates left ventricular (LV) pump dysfunction by normalization of myofilament function. This benefit could be due to an exercise-induced upregulation of endothelial nitric oxide synthase (eNOS) expression and activity. Consequently, we first tested the hypothesis that the effects of exercise after MI can be mimicked by elevated eNOS expression using transgenic mice with overexpression of human eNOS (eNOSTg). Both exercise and eNOSTg attenuated LV remodeling and dysfunction after MI in mice and improved cardiomyocyte maximal force development (F(max)). However, only exercise training restored myofilament Ca(2+)-sensitivity and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a protein levels and improved the first derivative of LV pressure at 30 mmHg. Conversely, only eNOSTg improved survival. In view of these partly complementary actions, we subsequently tested the hypothesis that combining exercise and eNOSTg would provide additional protection against LV remodeling and dysfunction after MI. Unexpectedly, the combination of exercise and eNOSTg abolished the beneficial effects on LV remodeling and dysfunction of either treatment alone. The latter was likely due to perturbations in Ca(2+) homeostasis, as myofilament F(max) actually increased despite marked reductions in the phosphorylation status of several myofilament proteins, whereas the exercise-induced increases in SERCA2a protein levels were lost in eNOSTg mice. Antioxidant treatment with N-acetylcysteine or supplementation of tetrahydrobiopterin and l-arginine prevented these detrimental effects on LV function while partly restoring the phosphorylation status of myofilament proteins and further enhancing myofilament F(max). In conclusion, the combination of exercise and elevated eNOS expression abolished the cardioprotective effects of either treatment alone after MI, which appeared to be, at least in part, the result of increased oxidative stress secondary to eNOS "uncoupling."

Cardiac myosin-binding protein C mutations and hypertrophic cardiomyopathy: haploinsufficiency, deranged phosphorylation, and cardiomyocyte dysfunction.

BACKGROUND: Mutations in the MYBPC3 gene, encoding cardiac myosin-binding protein C (cMyBP-C), are a frequent cause of familial hypertrophic cardiomyopathy. In the present study, we investigated whether protein composition and function of the sarcomere are altered in a homogeneous familial hypertrophic cardiomyopathy patient group with frameshift mutations in MYBPC3 (MYBPC3(mut)). METHODS AND RESULTS: Comparisons were made between cardiac samples from MYBPC3 mutant carriers (c.2373dupG, n=7; c.2864_2865delCT, n=4) and nonfailing donors (n=13). Western blots with the use of antibodies directed against cMyBP-C did not reveal truncated cMyBP-C in MYBPC3(mut). Protein expression of cMyBP-C was significantly reduced in MYBPC3(mut) by 33+/-5%. Cardiac MyBP-C phosphorylation in MYBPC3(mut) samples was similar to the values in donor samples, whereas the phosphorylation status of cardiac troponin I was reduced by 84+/-5%, indicating divergent phosphorylation of the 2 main contractile target proteins of the beta-adrenergic pathway. Force measurements in mechanically isolated Triton-permeabilized cardiomyocytes demonstrated a decrease in maximal force per cross-sectional area of the myocytes in MYBPC3(mut) (20.2+/-2.7 kN/m(2)) compared with donor (34.5+/-1.1 kN/m(2)). Moreover, Ca(2+) sensitivity was higher in MYBPC3(mut) (pCa(50)=5.62+/-0.04) than in donor (pCa(50)=5.54+/-0.02), consistent with reduced cardiac troponin I phosphorylation. Treatment with exogenous protein kinase A, to mimic beta-adrenergic stimulation, did not correct reduced maximal force but abolished the initial difference in Ca(2+) sensitivity between MYBPC3(mut) (pCa(50)=5.46+/-0.03) and donor (pCa(50)=5.48+/-0.02). CONCLUSIONS: Frameshift MYBPC3 mutations cause haploinsufficiency, deranged phosphorylation of contractile proteins, and reduced maximal force-generating capacity of cardiomyocytes. The enhanced Ca(2+) sensitivity in MYBPC3(mut) is due to hypophosphorylation of troponin I secondary to mutation-induced dysfunction.

Proteomic profiling of endothelin-1-stimulated hypertrophic cardiomyocytes reveals the increase of four different desmin species and alpha-B-crystallin.

We performed a proteomic investigation on primary cultures of neonatal rat cardiomyocytes after treatment with 10 nM endothelin-1 (ET1) for 48 h, an in vitro model for cardiac hypertrophy. Two-dimensional gel electrophoresis profiles of cell lysates were compared after colloidal Coomassie Blue staining. 12 protein spots that significantly changed in density due to ET1 stimulation were selected for in-gel digestion and identified through mass spectrometry. Of these, 8 spots were increased and 4 were decreased. Four of the increased proteins were identified as desmin, the cardiac component of intermediate filaments and one as alpha-B-crystallin, a molecular chaperone that binds desmin. All the desmins increased 2- to 5-fold, and alpha-B-crystallin increased 2-fold after ET1 treatment. Desmin cytoskeleton has been implicated in the regulation of mitochondrial activity and distribution, as well as in the formation of amyloid bodies. Mitochondria-specific fluorescent probe MitoTracker indicated mitochondrial redistribution in hypertrophic cells. An increase of amyloid aggregates containing desmin upon treatment with ET1 was detected by filter assay. Of the four proteins that showed decreased abundance after ET1 treatment, the chaperones hsp60 and grp75 were decreased 13- and 9-fold, respectively. In conclusion, proteomic profiling of ET1-stimulated rat neonatal cardiomyocytes reveals specific changes in cardiac molecular phenotype mainly involving intermediate filament and molecular chaperone proteins.

Integrative control of coronary resistance vessel tone by endothelin and angiotensin II is altered in swine with a recent myocardial infarction.

Several studies have indicated an interaction between the renin-angiotensin (ANG II) system and endothelin (ET) in the regulation of vascular tone. Previously, we have shown that both ET and ANG II exert a vasoconstrictor influence on the coronary resistance vessels of awake normal swine. Here, we investigated whether the interaction between ANG II and ET exists in the control of coronary resistance vessel tone at rest and during exercise using single and combined blockade of angiotensin type 1 (AT(1)) and ET(A)/ET(B) receptors. Since both circulating ANG II and ET levels are increased after myocardial infarction (MI), we investigated if the interaction between these systems is altered after MI. In awake healthy swine, coronary vasodilation in response to ET(A)/ET(B) receptor blockade in the presence of AT(1) blockade was similar to vasodilation produced by ET(A)/ET(B) blockade under control conditions. In awake swine with a 2- to 3-wk-old MI, coronary vasodilator responses to individual AT(1) and ET(A)/ET(B) receptor blockade were virtually abolished, despite similar coronary arteriolar AT(1) and ET(A) receptor expression compared with normal swine. Unexpectedly, in the presence of AT(1) blockade (which had no effect on circulating ET levels), ET(A)/ET(B) receptor blockade elicited a coronary vasodilator response. These findings suggest that in normal healthy swine the two vasoconstrictor systems contribute to coronary resistance vessel control in a linear additive manner, i.e., with negligible cross-talk. In contrast, in the remodeled myocardium, cross-talk between ANG II and ET emerges, resulting in nonlinear redundant control of coronary resistance vessel tone.

Time dependent changes in cytoplasmic proteins of the right ventricle during prolonged pressure overload.

In many forms of congenital heart disease, the right ventricle (RV) is subject to abnormal loading conditions resulting in RV hypertrophy and remodeling. We determined the alterations in RV cytoplasmic proteomic phenotype that occur during prolonged periods of RV pressure overload. We performed a differential proteomic profiling study on RV hypertrophy using an animal model of various durations of pulmonary artery banding (PAB) in parallel with hemodynamic characterization. This hemodynamic evaluation showed that after 6, 12 and 20 weeks of PAB, the RV is in a compensated state of hypertrophy. Overall, the majority of protein changes were metabolism related indicating a shift towards the glycolytic pathway at the expense of beta-oxidation in the RV of the PAB animals. The changes in proteins related to the glycolytic pathway, exemplified by enolase and creatine kinase B-chain, tended to precede changes in beta-oxidation. In parallel, increases in stress chaperones, exemplified by several phosphorylated HSP-27 species, are present from the 6 week time point, whereas increases in antioxidant proteins, exemplified by peroxiredoxin 2 and 6, appear to be restricted to the 12 week time point. The p38 MAPK signal transduction pathway appears not to be activated. Observed protein changes are likely part of a protective mechanism against the development of RV failure.

Differential Signaling and Hypertrophic Responses in Cyclically Stretched vs Endothelin-1 Stimulated Neonatal Rat Cardiomyocytes.

Numerous neurohumoral factors such as endothelin (ET)-1 and angiotensin (Ang) II as well as the stretch stimulus act concertedly in the in vivo overloaded heart in inducing hypertrophy and failure. The primary culture of rat neonatal cardiomyocytes is the only in vitro model that allows the comparative analysis of growth responses and signaling events in response to different stimuli. In the present study, we examined stretched rat cardiomyocytes grown on flexible bottomed culture plates for hypertrophic growth responses (protein synthesis, protein/DNA ratio, and cell volume), F-actin filaments rearrangement (by confocal laser scanning microscopy), and for signaling events (activation of phospholipase C [PLC]-beta, protein kinase C [PKC], mitogenactivated protein [MAP] kinases) and compared these responses with ET-1 (10-8 M)-stimulated cells. Cyclic stretch for 48 h induced hypertrophic growth in cardiomyocytes indicated by increases in the rate of protein synthesis, cell volume, and diameter, which were less pronounced in comparison to stimulation by ET-1. During cyclic stretch, we observed disoriented F-actin, particularly stress-fibers whereas during ET-1 stimulation, Factins rearranged clearly in alignment with sarcomeres and fibers. The upstream part of signaling by cyclic stretch did not follow the PLCbeta-PKC cascade, which, in contrast, was strongly activated during ET-1 stimulation. Cyclic stretch and, to greater extent, ET-1 stimulated downstream signaling through ERK, p38 MAP kinase, and JNK pathways, but the involvement of tyrosine kinase and PI3 kinase-Akt signaling during cyclic stretch could not be proven. Taken together, our results demonstrate that both cyclic stretch and ET-1 induce hypertrophic responses in cardiomyocytes with different effects on organization of F-actin stress fibers in case of stretch. Furthermore, on the short-term basis, cyclical stretch, unlike ET-1, mediates its hypertrophic response not through activation of PLC-beta and PKC but more likely through integrin-linked pathways, which both lead to downstream activation of the MAP kinase family.

Cloning, expression, and promoter analysis of hepatic lipase derived from human hyperplastic adrenals: evidence for alternative mRNA splicing.

Human adrenals contain hepatic lipase (HL) activity, which is thought to facilitate the uptake of plasma cholesterol used in steroidogenesis. We show here that full-length HL mRNA is expressed in hyperplastic adrenals of patients with Cushing's disease. In addition, a splice variant that lacks exon-3 was detected in the human adrenals and hepatoma (HepG2) cells, but not in liver. In CAT-reporter assays using human NCI-H295R adrenocortical cells, the HL(-685/+13) promoter region was transcriptionally active, and its activity was enhanced twofold by cAMP. In rat adrenals, the HL gene is exclusively transcribed from an alternative promoter within intron-2, resulting in a variant mRNA that lacks exons 1 and 2. By reverse-transcription PCR, we found no evidence for expression of such a variant mRNA in human adrenals, liver, or HepG2 cells. The presence of both full length mRNA and enzyme activity in human adrenals suggests that part of the HL activity is locally synthesized.

Early exercise training normalizes myofilament function and attenuates left ventricular pump dysfunction in mice with a large myocardial infarction.

The extent and mechanism of the cardiac benefit of early exercise training following myocardial infarction (MI) is incompletely understood, but may involve blunting of abnormalities in Ca(2+)-handling and myofilament function. Consequently, we investigated the effects of 8-weeks of voluntary exercise, started early after a large MI, on left ventricular (LV) remodeling and dysfunction in the mouse. Exercise had no effect on survival, MI size or LV dimensions, but improved LV fractional shortening from 8+/-1 to 12+/-1%, and LVdP/dt(P30) from 5295+/-207 to 5794+/-207 mm Hg/s (both P<0.05), and reduced pulmonary congestion. These global effects of exercise were associated with normalization of the MI-induced increase in myofilament Ca(2+)-sensitivity (DeltapCa(50)=0.037). This effect of exercise was PKA-mediated and likely because of improved beta(1)-adrenergic signaling, as suggested by the increased beta(1)-adrenoceptor protein (48%) and cAMP levels (36%; all P<0.05). Exercise prevented the MI-induced decreased maximum force generating capacity of skinned cardiomyocytes (F(max) increased from 14.3+/-0.7 to 18.3+/-0.8 kN/m(2) P<0.05), which was associated with enhanced shortening of unloaded intact cardiomyocytes (from 4.1+/-0.3 to 7.0+/-0.6%; P<0.05). Furthermore, exercise reduced diastolic Ca(2+)-concentrations (by approximately 30%, P<0.05) despite the unchanged SERCA2a and PLB expression and PLB phosphorylation status. Importantly, exercise had no effect on Ca(2+)-transient amplitude, indicating that the improved LV and cardiomyocyte shortening were principally because of improved myofilament function. In conclusion, early exercise in mice after a large MI has no effect on LV remodeling, but attenuates global LV dysfunction. The latter can be explained by the exercise-induced improvement of myofilament function.

Quantitative analysis of myofilament protein phosphorylation in small cardiac biopsies.

Phosphorylation of cardiac myofilament proteins represents one of the main post-translational mechanisms that regulate cardiac pump function. Human studies are often limited by the amount of available tissue as biopsies taken during cardiac catheterization weigh only 1 mg (dry weight). Similarly, investigation of time- (or dose-) dependent changes in protein phosphorylation in animal studies is often hampered by tissue availability. The present study describes quantitative analysis of phosphorylation status of multiple myofilament proteins by 2-DE and Pro-Q® Diamond stained gradient gels using minor amounts (˜0.5 mg dry weight) of human and pig cardiac tissue.

Differential proteomic profiling to study the mechanism of cardiac pharmacological preconditioning by resveratrol.

Recent studies demonstrated that resveratrol, a grape-derived polyphenolic phytoalexin, provides pharmacological preconditioning of the heart through a NO-dependent mechanism. To further explore the molecular mechanisms involved in resveratrol-mediated cardioprotection, we monitored the effects of resveratrol treatment after ischemia-reperfusion on the protein profile by implementation of proteomic analysis. Two groups of rats were studied; one group of animals was fed resveratrol for 7 days, while the other group was given vehicle only. The rats were sacrificed for the isolated working heart preparation and for isolation of cytoplasmic fraction from left ventricle homogenates to carry out the proteomic as well as immunoblot at baseline and at the end of 30 min ischemia/2-h perfusion. The results demonstrate significant cardioprotection with resveratrol evidenced by improved ventricular recovery and reduced infarct size and cardiomyocyte apoptosis. The left ventricular cytoplasmic fractions were separated by two-dimensional electrophoresis (2-DE). Differentially regulated proteins were detected with quantitative computer analysis of the Coomassie blue stained 2-DE images and identified by MALDI-TOF (MS) and nanoLC-ESI-Q-TOF mass spectrometry (MS/MS). Five redox-regulated and preconditioning- related proteins were identified that were all upregulated by resveratrol: MAPKK, two different alphaB-crystallin species, HSP 27 and PE binding protein. Another HSP27 species and aldose reductase were downregulated and peroxiredoxin- 2 remained constant. The results of the immunoblot analysis of phosphorylated MAPKK, -HSP27 and -alphaB-crystallin and PE binding protein were consistent with the proteomic findings, but not with peroxiredoxin-2. The proteomic analysis showed also downregulation of some proteins in the mitochondrial respiratory chain and matrix and the myofilament regulating protein MLC kinase-2. The results of the present study demonstrate that proteomic profiling enables the identification of resveratrol induced preconditioning-associated proteins which reflects not only changes in their expression level but also isoforms, post-translational modifications and regulating binding or activating partner proteins.

Mitochondrial adaptations within chronically ischemic swine myocardium.

Experimental evidence has emerged that myocardial ischemic preconditioning can prime the mitochondria into a "stress-resistant state", so that cell death is reduced following prolonged severe ischemia and reperfusion. Using a swine model of chronically ischemic myocardium, we tested the hypothesis that mitochondria within the ischemic territory have also acquired a protective phenotype. Eleven swine underwent a left thoracotomy with placement of an external constrictor around the proximal left anterior descending (LAD) artery. By 10 weeks, a severe stenosis of the LAD artery was documented by quantitative coronary angiography (92 +/- 2%). Animals were sacrificed and myocardium was extracted from the LAD and remote regions. Mitochondria were isolated from subendocardium and subepicardium from LAD and remote regions and state 2 (substrate alone) and state 3 (+ADP) respiration were assessed with a Clark electrode. Within the LAD subendocardium, the respiratory control index was 2.68 +/- 0.17 and was lower than the remote subendocardium (3.64 +/- 0.08; P < 0.05). When exposed to 20 min anoxia with reoxygenation, the LAD region demonstrated a more preserved state 3 respiration compared with the remote region (99 +/- 14 versus 65 +/- 9 nmol O2/mg, respectively; P < 0.05). In parallel mitochondrial experiments, chemiluminescence was detected with the probe coelenterazine and superoxide generation in the LAD region in the presence of antimycin A was 574 +/- 108 RLU/30 s/microg and was nearly 50% lower than the remote region (979 +/- 175 RLU/30 s/microg; P < 0.05). Within the mitochondria, the expression of uncoupling protein (UCP) 2 by western gels was 20% higher in the LAD region compared with the remote region (P < 0.05) with no differences noted in UCP-3. In this swine model of chronic myocardial ischemia, isolated mitochondria from the ischemic tissue demonstrate preserved state 3 respiration following anoxia/reoxygenation, consistent with a stress-resistant state. This state is characterized by a mild degree of uncoupling under basal conditions and decreased superoxide generation. Uncoupling protein 2 expression is enhanced in the mitochondria, providing a potential mechanism for these favorable mitochondrial adaptations.

Prorenin induces intracellular signaling in cardiomyocytes independently of angiotensin II.

Tissue accumulation of circulating prorenin results in angiotensin generation, but could also, through binding to the recently cloned (pro)renin receptor, lead to angiotensin-independent effects, like p42/p44 mitogen-activated protein kinase (MAPK) activation and plasminogen-activator inhibitor (PAI)-1 release. Here we investigated whether prorenin exerts angiotensin-independent effects in neonatal rat cardiomyocytes. Polyclonal antibodies detected the (pro)renin receptor in these cells. Prorenin affected neither p42/p44 MAPK nor PAI-1. PAI-1 release did occur during coincubation with angiotensinogen, suggesting that this effect is angiotensin mediated. Prorenin concentration-dependently activated p38 MAPK and simultaneously phosphorylated HSP27. The latter phosphorylation was blocked by the p38 MAPK inhibitor SB203580. Rat microarray gene (n=4800) transcription profiling of myocytes stimulated with prorenin detected 260 regulated genes (P<0.001 versus control), among which genes downstream of p38 MAPK and HSP27 involved in actin filament dynamics and (cis-)regulated genes confined in blood pressure and diabetes QTL regions, like Syntaxin-7, were overrepresented. Quantitative real-time RT-PCR of 7 selected genes (Opg, Timp1, Best5, Hsp27, pro-Anp, Col3a1, and Hk2) revealed temporal regulation, with peak levels occurring after 4 hours of prorenin exposure. This regulation was not altered in the presence of the renin inhibitor aliskiren or the angiotensin II type 1 receptor antagonist eprosartan. Finally, pilot 2D proteomic differential display experiments revealed actin cytoskeleton changes in cardiomyocytes after 48 hours of prorenin stimulation. In conclusion, prorenin exerts angiotensin-independent effects in cardiomyocytes. Prorenin-induced stimulation of the p38 MAPK/HSP27 pathway, resulting in alterations in actin filament dynamics, may underlie the severe cardiac hypertrophy that has been described previously in rats with hepatic prorenin overexpression.

Carvedilol-induced antagonism of angiotensin II: a matter of alpha1-adrenoceptor blockade.

OBJECTIVE: To investigate whether renin-angiotensin system blockade might underlie the favorable metabolic effects of the nonselective beta + alpha1-adrenoceptor blocker carvedilol as compared with the selective beta1-adrenoceptor blocker metoprolol. METHODS: Human coronary microarteries (HCMAs), obtained from 32 heart valve donors, were mounted in myographs. RESULTS: Angiotensin II and the alpha1-adrenoceptor agonist phenylephrine constricted HCMAs to maximally 63 +/- 10 and 46 +/- 15% of the contraction to 100 mmol/l K. Neither carvedilol, metoprolol, the nonselective beta-adrenoceptor antagonist propranolol, nor the alpha1-adrenoceptor antagonist prazosin affected the constrictor response to angiotensin II. alpha1-adrenoreceptors and beta-adrenoceptors are thus not involved in the direct constrictor effects of angiotensin II. When added to the organ bath at a subthreshold concentration, angiotensin II greatly amplified the response to phenylephrine. Both carvedilol and the angiotensin II type 1 (AT1) receptor antagonist irbesartan inhibited this angiotensin II-induced potentiation. Furthermore, carvedilol blocked the angiotensin II-induced amplification of phenylephrine-induced inositol phosphate accumulation in cardiomyocytes. CONCLUSIONS: AT1-alpha1-receptor crosstalk, involving inositol phosphates, sensitizes HCMAs to alpha1-adrenoceptor agonists. Our results suggest that, in the presence of an increased sympathetic tone, carvedilol provides AT1 receptor blockade via its alpha1-adrenoceptor blocking effects. This could explain the favorable effects of carvedilol versus metoprolol.

Coronary vasoconstrictor influence of angiotensin II is reduced in remodeled myocardium after myocardial infarction.

The renin-angiotensin system plays an important role in cardiovascular homeostasis by contributing to the regulation of blood volume, blood pressure, and vascular tone. Because AT(1) receptors have been described in the coronary microcirculation, we investigated whether ANG II contributes to the regulation of coronary vascular tone and whether its contribution is altered during exercise. Since the renin-angiotensin system is activated after myocardial infarction, resulting in an increase in circulating ANG II, we also investigated whether the contribution of ANG II to the regulation of vasomotor tone is altered after infarction. Twenty-six chronically instrumented swine were studied at rest and while running on a treadmill at 1-4 km/h. In 13 swine, myocardial infarction was induced by ligation of the left circumflex coronary artery. Blockade of AT(1) receptors (irbesartan, 1 mg/kg iv) had no effect on myocardial O(2) consumption but resulted in an increase in coronary venous O(2) tension and saturation both at rest and during exercise, reflecting coronary vasodilation. Despite increased plasma levels of ANG II after infarction and maintained coronary arteriolar AT(1) receptor levels, the vasodilation evoked by irbesartan was significantly reduced both at rest and during exercise. In conclusion, despite elevated plasma levels, the vasoconstrictor influence of ANG II on the coronary circulation in vivo is reduced after myocardial infarction. This reduction in ANG II-induced coronary vasoconstriction may serve to maintain perfusion of the remodeled myocardium.

Right and left ventricular function after chronic pulmonary artery banding in rats assessed with biventricular pressure-volume loops.

In many patients with congenital heart disease, the right ventricle (RV) is subjected to abnormal loading conditions. To better understand the state of compensated RV hypertrophy, which could eventually progress to decompensation, we studied the effects of RV pressure overload in rats. In the present study, we report the biventricular adaptation to 6 wk of pulmonary artery banding (PAB). PAB resulted in an RV pressure overload to approximately 60% of systemic level and a twofold increase in RV mass (P < 0.01). Systemic hemodynamic parameters were not altered, and overt signs of heart failure were absent. Load-independent measures of ventricular function (end-systolic pressure-volume relation, preload recruitable stroke work relation, maximum first time derivative of pressure divided by end-diastolic volume), assessed by means of pressure-volume (PV) loops, demonstrated a two- to threefold increase in RV contractility under baseline conditions in PAB rats. RV contractility increased in response to dobutamine stimulation (2.5 microg.kg(-1).min(-1)) both in PAB and sham-operated rats in a similar fashion, indicating preserved RV contractile reserve in PAB rats. Left ventricular (LV) contractility at baseline was unaffected in PAB rats, although LV volume in PAB rats was slightly decreased. LV contractility increased in response to dobutamine (2.5 microg.kg(-1).min(-1)), both in PAB and sham rats, whereas the response to a higher dose of dobutamine (5 microg.kg(-1).min(-1)) was blunted in PAB rats. RV pressure overload (6 wk) in rats resulted in a state of compensated RV hypertrophy with preserved RV contractile reserve, whereas LV contractile state at baseline was not affected. Furthermore, this study demonstrates the feasibility of performing biventricular PV-loop measurements in rats.

Recent developments in proteomics: implications for the study of cardiac hypertrophy and failure.

The key components to the molecular understanding of the pathophysiology of various forms of heart failure involve global and/or large-scale identifications of proteins, their patterns of expression, posttranslational modifications, and functional characterization. Particularly, proteins involved in the induction of cardiac (mal)adaptive hypertrophic growth, interstitial fibrosis, and contractile dysfunction are of interest. In general, with the accumulation of vast amounts of DNA sequences in databases, researchers have become aware that merely having complete sequences of genomes and transcriptional changes for thousands of genes simultaneously will not be sufficient to elucidate, in molecular terms, the etiology and pathophysiology of cardiovascular disease. In the last decade, a new technology called proteomics has become available that allows biological and (patho)physiological questions to be approached exclusively from the protein perspective. Proteomics may enable us to map the entire complement of proteins expressed by the heart at any time and condition. This approach creates the unique possibility to identify, by differential analysis, protein alterations associated with the etiology of heart disease and its progression, outcome, and response to therapy. To illustrate the true power of proteomics, most of the currently available methodologies are first reviewed, including their limitations. This review also deals with the current status and the perspectives of proteomics applications in research on heart failure in general. Furthermore, examples of our recent data on global protein profiling of the pressure-overloaded rat right ventricle and of endothelin-1-stimulated cultures of neonatal rat cardiac myocytes are provided. The last section is devoted to the continuous advances in proteomic technologies, including protein separation methods, mass spectrometric instrumentation, computational analysis, and bioinformatic tools, together with integrative databases.

Proteomic changes in the pressure overloaded right ventricle after 6 weeks in young rats: correlations with the degree of hypertrophy.

Right ventricular (RV) hypertrophy is an important problem in congenital heart disease. We determined the alterations in phenotype that occur in the initial phase of RV hypertrophy and their possible correlations with the degree of hypertrophy. Therefore, we performed a differential proteomic profiling study on RV hypertrophy using an animal model of pulmonary artery banding (PAB) in parallel with hemodynamic characterization. The RV homogenates were subfractionated in myofilament and cytoplasmic proteins, which subsequently were separated by two-dimensional gel electrophoresis (2-DE), excised, and analyzed by mass spectrometry (MS). The cytoplasmic fraction showed expression changes in metabolic proteins, indicative of a shift from fatty acid to glucose as a substrate for energy supply. Up-regulation of three HSP-27s (1.9-, 1.7-, and 3.5-fold) indicated an altered stress response in RV hypertrophy. Detailed analysis by immunoblotting and MS showed that two of these HSP-27s were at least phosphorylated on Ser15. The myofilament fraction showed up-regulation of desmin and alpha-B-crystallin (1.4-and 1.3-fold, respectively). This alteration in desmin was confirmed by 1-DE immunoblots. Certain differentially expressed proteins, such as HSP-27, showed a significant correlation with the RV weight to the body weight ratio in the PAB rats, suggesting an association with the degree of hypertrophy.

Genomic and nongenomic effects of aldosterone in the rat heart: why is spironolactone cardioprotective?

1. Mineralocorticoid receptor (MR) antagonism with spironolactone reduces mortality in heart failure on top of ACE inhibition. To investigate the underlying mechanism, we compared the actions of both aldosterone and spironolactone to those of angiotensin (Ang) II in the rat heart. 2. Hearts of male Wistar rats were perfused according to Langendorff. Ang II and aldosterone increased left ventricular pressure (LVP) by maximally 11+/-4 and 9+/-2%, and decreased coronary flow (CF) by maximally 36+/-7 and 20+/-4%, respectively. Spironolactone did not significantly affect LVP or CF. 3. In hearts that were exposed to a 45-min coronary artery occlusion and 3 h of reperfusion, a 15-min exposure to spironolactone prior to occlusion reduced infarct size (% of risk area) from 68+/-2 to 45+/-3%, similar to the reduction (34+/-2%) observed following 'preconditioning' (15 min occlusion followed by 10 min reperfusion) prior to the 45-min occlusion. Aldosterone exposure did not affect infarct size (71+/-5%). 4. In cardiomyocytes, aldosterone decreased [(3)H]thymidine incorporation maximally by 73+/-3%, whereas in cardiac fibroblasts it decreased [(3)H]proline incorporation by 33+/-7%. Spironolactone inhibited both effects. Ang II increased DNA and collagen synthesis, and these effects were reversed by aldosterone. 5. In conclusion, aldosterone induces positive inotropic and vasoconstrictor effects in a nongenomic manner, and these effects are comparable to those of Ang II. Aldosterone reduces DNA and collagen synthesis via MR activation, and counteracts the Ang II-induced increases in these parameters. MR blockade reduces infarct size and increases LVP recovery following coronary artery occlusion. The MR-related phenomena may underlie, at least in part, the beneficial actions of spironolactone in heart failure.