A short-term exposure to resveratrol at high dosages exerts a remarkable cardioprotective effect. Whether a long-term exposure to resveratrol at low dosages that can be obtained through consumption of a resveratrol-rich diet is beneficial to heart diseases is unknown. We tested the effects of a resveratrol-enriched diet on cardiovascular remodeling of chronic heart failure (CHF) in rats resulting from permanent ligation of left coronary artery. Two weeks after surgery, rats were started on either a resveratrol-enriched (R; 5 mg/kg per day; n = 23) or normal (Control; n = 23) diet for next 10 months. Serial echocardiography in Control showed a significant decline in LV ejection fraction, increases in LV end-systolic and end-diastolic volumes, and expansion in myocardial infarct from pre-treatment values. In R, compared with Control, there were substantial improvements in those parameters. End-point LV pressure-volume loop analysis showed a significantly improved LV systolic function and AV-coupling, an index of energy transfer efficacy between the heart and aortic tree, in R compared with Control (p < 0.05). Aortic pulse wave velocity, a measure of arterial stiffness, was significantly lower in R (389 ± 15 cm/s; p < 0.05) compared with Control (489 ± 38 cm/s). These results demonstrated that long-term dietary resveratrol supplement reduces cardiovascular structural and functional deterioration in CHF.

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

Marfan syndrome (MFS) is a systemic disorder of connective tissue caused by mutations in fibrillin-1. Cardiac dysfunction in MFS has not been characterized halting the development of therapies of cardiac complication in MFS. We aimed to study the age-dependent cardiac remodeling in the mouse model of MFS FbnC1039G+/- mouse [Marfan heterozygous (HT) mouse] and its association with valvular regurgitation. Marfan HT mice of 2-4 mo demonstrated a mild hypertrophic cardiac remodeling with predominant decline of diastolic function and increased transforming growth factor-β canonical (p-SMAD2/3) and noncanonical (p-ERK1/2 and p-p38 MAPK) signaling and upregulation of hypertrophic markers natriuretic peptides atrium natriuretic peptide and brain natriuretic peptide. Among older HT mice (6-14 mo), cardiac remodeling was associated with two distinct phenotypes, manifesting either dilated or constricted left ventricular chamber. Dilatation of left ventricular chamber was accompanied by biochemical evidence of greater mechanical stress, including elevated ERK1/2 and p38 MAPK phosphorylation and higher brain natriuretic peptide expression. The aortic valve regurgitation was registered in 20% of the constricted group and 60% of the dilated group, whereas mitral insufficiency was observed in 40% of the constricted group and 100% of the dilated group. Cardiac dysfunction was not associated with the increase of interstitial fibrosis and nonmyocyte proliferation. In the mouse model fibrillin-1, haploinsufficiency results in the early onset of nonfibrotic hypertrophic cardiac remodeling and dysfunction, independently from valvular abnormalities. MFS heart is vulnerable to stress-induced cardiac dilatation in the face of valvular regurgitation, and stress-activated MAPK signals represent a potential target for cardiac management in MFS.

Marfan syndrome (MFS) is a multisystem disorder of connective tissue caused by mutations in fibrillin-1. Heart involvement in the Marfan syndrome patients includes aortic root dilatation, valvular insufficiency, and myocardial dysfunction; it remains unclear, however, whether alterations in myocardium are triggered by valvular and aortic pathology or they develop independently. We evaluated the age-dependent cardiac remodeling and left ventricular dysfunction in the mouse model of MFS known as Fbn1039G+/− mouse (Marfan HT mouse) using echocardiography, pressure-volume loop analyses and a number of histological and biochemical techniques. Marfan HT mice of 2-4 month demonstrated a hypertrophic cardiac remodeling accompanied by predominant decline of diastolic function and increased TGF-β canonical (p SMAD2/3) and non-canonical (pERK ½ and pMAPK38) signaling. Hypertrophic myocardium among older HT mice (6-14 months) was associated with two distinctly different phenotypes manifesting either dilated or constricted LV chamber. Dilatation of LV chamber was accompanied by biochemical evidence of greater mechanical stress, including elevated ERK1/2 phosphorylation and brain natriuretic peptide expression in comparison with constricted heart. Diastolic dysfunction in the older HT mice was combined with significant systolic impairment. The aortic valve regurgitation was registered in 20% of constricted group and 60% of dilated, while mitral insufficiency was observed in 40% of constricted group and 100% of dilated. In Marfan HT mice, extracellular matrix abnormalities were not associated with the increase of interstitial fibrosis and non-myocyte proliferation. In the mouse model of fibrillin-1 haploinsufficiency the early onset of hypertrophic cardiac remodeling and dysfunction is not consequent to functional valvular abnormalities, but it is likely to result from deficient mechanosensing and transmission of mechanical forces.

OBJECTIVE:We aimed to assess the therapeutic efficacy of differentially modified soluble receptor for advanced glycation end products (sRAGE) in vivo using vessel ultrasound sonography and to compare the sonography data with those from postmortem histomorphologic analyses to have a practical reference for future clinical applications.METHODS:Vessel ultrasound sonography was performed in a sRAGE-treated rat carotid artery balloon injury model at different time points after the surgery, and therapeutic efficacy of different doses of sRAGE produced in Chinese hamster ovary cells and with different N-glycoform modifications were assessed.RESULTS:Vessel ultrasound sonography found that sRAGE produced in Chinese hamster ovary cells with complex N-glycoform modifications is highly effective, and is consistent with our recent findings in the same model assessed with histology. We also found that sonography is less sensitive than histology when a higher dose of sRAGE is administered.CONCLUSIONS:Sonograph results are consistent with those obtained from histology; that is, sRAGE produced in Chinese hamster ovary cells has significantly higher efficacy than insect cell-originated sRAGE cells.

Extensive research during the last decade demonstrated that a single systemic administration of -erythropoietin (EPO) lead to significant attenuation of myocardial infarction (MI) induced in animals, mostly small rodents, either by a myocardial ischemia followed by reperfusion or by a permanent ligation of a coronary artery. Both methods are critically reviewed with the aim of helping the reader in appreciating key issues in the translation of experimental results to the clinic. Results of several clinical trials in patients with acute MI completed to date failed to demonstrate beneficial effects of EPO, and thus put into question the validity of results obtained in animal models. Comprehensive review of design and results of animal experiments and clinical trials presented here allowed authors to postulate that therapeutic window for EPO during developing MI is very narrow and was possibly missed in negative clinical trials. This point was illustrated by the negative outcome of experiment in the rat model of MI in which timing of EPO administration was similar to that in clinical trials. The design of future clinical trials should allow for a narrow therapeutic window of EPO. Given current standards for onset-to-door and door-to-balloon time the optimal time for EPO administration should be just prior to PCI.

The cardioprotective properties of erythropoietin (EPO) in preclinical studies are well documented, but erythropoietic and prothrombotic properties of EPO preclude its use in chronic heart failure (CHF). We tested the effect of long-term treatment with a small peptide sequence within the EPO molecule, helix B surface peptide (HBSP), that possesses tissue-protective, but not erythropoietic properties of EPO, on mortality and cardiac remodeling in postmyocardial infarction-dilated cardiomyopathy in rats. Starting 2 weeks after permanent left coronary artery ligation, rats received i.p. injections of HBSP (60 µg/kg) or saline two times per week for 10 months. Treatment did not elicit an immune response, and did not affect the hematocrit. Compared with untreated rats, HBSP treatment reduced mortality by 50% (P < 0.05). Repeated echocardiography demonstrated remarkable attenuation of left ventricular dilatation (end-diastolic volume: 41 versus 86%; end-systolic volume: 44 versus 135%; P < 0.05), left ventricle functional deterioration (ejection fraction: -4 versus -63%; P < 0.05), and myocardial infarction (MI) expansion (3 versus 38%; P < 0.05). A hemodynamic assessment at study termination demonstrated normal preload independent stroke work (63 ± 5 versus 40 ± 4; P < 0.05) and arterioventricular coupling (1.2 ± 0.2 versus 2.7 ± 0.7; P < 0.05). Histologic analysis revealed reduced apoptosis (P < 0.05) and fibrosis (P < 0.05), increased cardiomyocyte density (P < 0.05), and increased number of cardiomyocytes in myocardium among HBSP-treated rats. The results indicate that HBSP effectively reduces mortality, ameliorates the MI expansion and CHF progression, and preserves systolic reserve in the rat post-MI model. There is also a possibility that HBSP promoted the increase of the myocytes number in the myocardial wall remote from the infarct. Thus, HBSP peptide merits consideration for clinical testing.

Signaling of the receptor for advanced glycation end products (RAGE) has been implicated in the development of injury-elicited vascular complications. Soluble RAGE (sRAGE) acts as a decoy of RAGE and has been used to treat pathological vascular conditions in animal models. However, previous studies used a high dose of sRAGE produced in insect Sf9 cells (sRAGESf9)and multiple injections to achieve the therapeutic outcome. Here, we explore whether modulation of sRAGE N-glycoform impacts its bioactivity and augments its therapeutic efficacy. We first profiled carbohydrate components of sRAGE produced in Chinese hamster Ovary cells (sRAGECHO) to show that a majority of its N-glycans belong to sialylated complex types that are not shared by sRAGESf9. In cell-based NF-κB activation and vascular smooth muscle cell (VSMC) migration assays, sRAGECHO exhibited a significantly higher bioactivity relative to sRAGESf9 to inhibit RAGE alarmin ligand-induced NF-κB activation and VSMC migration. We next studied whether this N-glycoform-associated bioactivity of sRAGECHO is translated to higher in vivo therapeutic efficacy in a rat carotid artery balloon injury model. Consistent with the observed higher bioactivity in cell assays, sRAGECHO significantly reduced injury-induced neointimal growth and the expression of inflammatory markers in injured vasculature. Specifically, a single dose of 3 ng/g of sRAGECHO reduced neointimal hyperplasia by over 70 %, whereas the same dose of sRAGESf9 showed no effect. The administered sRAGECHO is rapidly and specifically recruited to the injured arterial locus, suggesting that early intervention of arterial injury with sRAGECHO may offset an inflammatory circuit and reduce the ensuing tissue remodeling. Our findings showed that the N-glycoform of sRAGE is the key determinant underlying its bioactivity and thus is an important glycobioengineering target to develop a highly potent therapeutic sRAGE for future clinical applications.

Age-associated arterial remodeling involves arterial wall collagen deposition and elastin fragmentation, as well as an increase in arterial pressure. This arterial remodeling is linked to proinflammatory signaling, including transforming growth factor-β1, monocyte chemoattractant protein 1, and proendothelin 1, activated by extracellular matrix metalloproteinases (MMPs) and orchestrated, in part, by the transcriptional factor ets-1. We tested the hypothesis that inhibition of MMP activation can decelerate the age-associated arterial proinflammation and its attendant increase in arterial pressure. Indeed, chronic administration of a broad-spectrum MMP inhibitor, PD166739, via a daily gavage, to 16-month–old rats for 8 months markedly blunted the expected age-associated increases in arterial pressure. This was accompanied by the following: (1) inhibition of the age-associated increases in aortic gelatinase and interstitial collagenase activity in situ; (2) preservation of the elastic fiber network integrity; (3) a reduction of collagen deposition; (4) a reduction of monocyte chemoattractant protein 1 and transforming growth factor-β1 activation; (5) a diminution in the activity of the profibrogenic signaling molecule SMAD-2/3 phosphorylation; (6) inhibition of proendothelin 1 activation; and (7) downregulation of expression of ets-1. Acute exposure of cultured vascular smooth muscle cells in vitro to proendothelin 1 increased both the transcription and translation of ets-1, and these effects were markedly reduced by MMP inhibition. Furthermore, infection of vascular smooth muscle cells with an adenovirus harboring a full-length ets-1 cDNA increased activities of both transforming growth factor-β1 and monocyte chemoattractant protein 1. Collectively, our results indicate that MMP inhibition retards age-associated arterial proinflammatory signaling, and this is accompanied by preservation of intact elastin fibers, a reduction in collagen, and blunting of an age-associated increase in blood pressure.

There is no proven therapy or prevention for vascular Ehlers-Danlos syndrome (vEDS), a genetic disorder associated with the mutation of procollagen type III and characterized by increased fragility of vascular and hollow organ walls. Heterozygous COL3A1-deficient (HT) mice recapitulate a mild presentation of one of the variants of vEDS: haploinsufficiency for collagen III. Adult HT mice are characterized by increased metalloproteinase (MMP) activity, reduced collagen content in the arterial walls, and spontaneous development of various severity lesions in aorta. We hypothesized that chronic treatment with a MMP inhibitor would increase collagen content and prevent the development of spontaneous aortic lesions. HT mice were treated since weaning with the broad-spectrum MMP inhibitor doxycycline added to food. At the age of 9 months MMP-9 expression was twice as high in the tunica media of aorta in untreated HT mice, whereas total collagen content was 30% lower (p < 0.01) and the cumulative score of aortic lesions was eight times higher than in wild-type (WT) mice (p < 0.01). After 9 months of doxycycline treatment, MMP-9 activity, collagen content, and lesions in the aortas of HT mice were at the level of those of WT mice (p > 0.05). In the mouse model of collagen III haploinsufficiency treatment with broad-spectrum MMP inhibitor that was started early in life normalized increased MMP activity, reduced aortic collagen content in adults, and prevented the development of spontaneous aortic lesions. Our findings provide experimental justification for the clinical evaluation of the benefit of doxycycline at least in the haploinsufficient variety of vEDS.

The citric acid cycle (CAC) metabolite fumarate has been proposed to be cardioprotective; however, its mechanisms of action remain to be determined. To augment cardiac fumarate levels and to assess fumarate's cardioprotective properties, we generated fumarate hydratase (Fh1) cardiac knockout (KO) mice. These fumarate-replete hearts were robustly protected from ischemia-reperfusion injury (I/R). To compensate for the loss of Fh1 activity, KO hearts maintain ATP levels in part by channeling amino acids into the CAC. In addition, by stabilizing the transcriptional regulator Nrf2, Fh1 KO hearts upregulate protective antioxidant response element genes. Supporting the importance of the latter mechanism, clinically relevant doses of dimethylfumarate upregulated Nrf2 and its target genes, hence protecting control hearts, but failed to similarly protect Nrf2-KO hearts in an in vivo model of myocardial infarction. We propose that clinically established fumarate derivatives activate the Nrf2 pathway and are readily testable cytoprotective agents.

Purpose A salutary effect of β 2 adrenergic receptor (AR) agonist, fenoterol has been demonstrated in a rat model of post-myocardial infarction (MI) dilated cardiomyopathy (DCM). Recent reports on single cardiomyocyte experiments suggested that out of two enantiomers, RR and SS, that constitute a racemic mixture of fenoterol, only RR-enantiomer is an active component that might be a promising new drug for treatment of chronic heart failure. The objective of this study was to compare the efficacy of the RR enantiomer of fenoterol with efficacy of racemic fenoterol, and SS, an inactive enantiomer, in whole animal experimental models of DCM. Methods Two weeks after induction of MI by permanent ligation of the anterior descending coronary artery early cardiac remodeling and MI size were assessed via echocardiography and rats were divided into treatment groups. Treatment (placebo, racemic fenoterol, RR- or SS-enantiomers of fenoterol) continued for 6 months while progression of DCM was followed by serial echocardiography. Results Compared with untreated rats, rats treated with racemic fenoterol demonstrated previously described attenuation of LV remodeling, functional decline and the arrest of the MI expansion during the first 2 months of treatment. On the contrary, the treatment with either RR-, or with SS-enantiomers of fenoterol was completely ineffective. Conclusion The conclusion drawn on the basis of previous experiments with single cardiomyocytes that RR-enantiomer of fenoterol represents an active component of racemic fenoterol and can be further investigated as a new drug for treatment of chronic heart failure was not confirmed in the whole animal model of DCM.

The cardioprotective properties of Erythropoietin (EPO) are well documented in pre-clinical studies. However, due to its erythropoietic and prothrombotic properties, the EPO treatment cannot be used in chronic heart failure (CHF), unless indicated by accompanied anemia. Helix B surface peptide (HBSP), a small peptide synthetized on the basis of EPO molecule (ARA290, Araim Pharmaceutical), does not stimulate erythropoiesis. We have reported similarly strong cardioprotective effects of HBSP and EPO in the rat model of myocardial infarction (MI). In this study, we tested the effect of long-term treatment with HBSP on mortality and cardiac remodeling in post-MI CHF in rats. For the 10 months, starting two weeks after left coronary artery ligation, rats received the i.p. injections of HBSP (60µg/kg; n=33) or saline (n=33) 2 times per week. Compared to saline-treated rats, HBSP treatment significantly reduced mortality (35% vs. 65%; p<0.05). Repeated Echocardiography demonstrated remarkable attenuation of left ventricular (LV) chamber dilatation (EDV: 41% vs. 86%; ESV: 44% vs. 135%; p<0.05), LV functional deterioration (EF: -4% vs. -63%; p<0.05) and MI expansion (3% vs. 38%; p<0.05) in HPSP-treated rats compared to saline-treated rats. Blood hematocrit level measured monthly was not affected by HBSP treatment. An invasive hemodynamic assessment at the end of 10-mo treatment showed better LV systolic function (PRSW: 63±5 vs. 40±4; p<0.05) and Arterio-ventricular coupling (Ea/Ees: 1.2±0.2 vs. 2.7±0.7; p<0.05) in HBSP-treated rats compared to saline-treated rats. Histological analysis revealed less apoptosis (0.6±0.1% vs. 0.9±0.1%; p<0.05), myocardial fibrosis (2.6±0.2% vs. 4.1±0.5%; p<0.05), and myocyte hypertrophy (cell diameter: 31±1µm vs. 37±1µm; p<0.05) in non-infarcted myocardium among HBSP-treated rats compared to saline-treated rats. Results suggest that HBSP could be considered as a safe alternative to EPO for a further clinical testing in the patients with chronic heart failure.

Rationale: Phosphorylation of beta(2)-adrenergic receptor (beta(2)AR) by a family of serine/threonine kinases known as G protein-coupled receptor kinase (GRK) and protein kinase A (PKA) is a critical determinant of cardiac function. Upregulation of G protein-coupled receptor kinase 2 (GRK2) is a well-established causal factor of heart failure, but the underlying mechanism is poorly understood.Objective: We sought to determine the relative contribution of PKA- and GRK-mediated phosphorylation of beta(2)AR to the receptor coupling to G(i) signaling that attenuates cardiac reserve and contributes to the pathogenesis of heart failure in response to pressure overload.Methods and Results: Overexpression of GRK2 led to a G(i)-dependent decrease of contractile response to beta AR stimulation in cultured mouse cardiomyocytes and in vivo. Importantly, cardiac-specific transgenic overexpression of a mutant beta(2)AR lacking PKA phosphorylation sites (PKA-TG) but not the wild-type beta(2)AR (WT-TG) or a mutant beta(2)AR lacking GRK sites (GRK-TG) led to exaggerated cardiac response to pressure overload, as manifested by markedly exacerbated cardiac maladaptive remodeling and failure and early mortality. Furthermore, inhibition of G(i) signaling with pertussis toxin restores cardiac function in heart failure associated with increased beta(2)AR to G(i) coupling induced by removing PKA phosphorylation of the receptor and in GRK2 transgenic mice, indicating that enhanced phosphorylation of beta(2)AR by GRK and resultant increase in G(i)-biased beta(2)AR signaling play an important role in the development of heart failure.Conclusions: Our data show that enhanced beta(2)AR phosphorylation by GRK, in addition to PKA, leads the receptor to G(i)-biased signaling, which, in turn, contributes to the pathogenesis of heart failure, marking G(i)-biased beta(2)AR signaling as a primary event linking upregulation of GRK to cardiac maladaptive remodeling, failure and cardiodepression. (Circ Res. 2012;110:265-274.)

BACKGROUND:To test a hypothesis that in negative clinical trials of erythropoietin in patients with acute myocardial infarction (MI) the erythropoietin (rhEPO) could be administered outside narrow therapeutic window. Despite overwhelming evidence of cardioprotective properties of rhEPO in animal studies, the outcomes of recently concluded phase II clinical trials have failed to demonstrate the efficacy of rhEPO in patients with acute MI. However, the time between symptoms onset and rhEPO administration in negative clinical trials was much longer that in successful animal experiments. METHODOLOGY/PRINCIPAL FINDINGS:MI was induced in rats either by a permanent ligation of a descending coronary artery or by a 2-hr occlusion followed by a reperfusion. rhEPO, 3000 IU/kg, was administered intraperitoneally at the time of reperfusion, 4 hrs after beginning of reperfusion, or 6 hrs after permanent occlusion. MI size was measured histologically 24 hrs after coronary occlusion. The area of myocardium at risk was similar among groups. The MI size in untreated rats averaged ~42% of area at risk, or ~24% of left ventricle, and was reduced by more than 50% (p<0.001) in rats treated with rhEPO at the time of reperfusion. The MI size was not affected by treatment administered 4 hrs after reperfusion or 6 hrs after permanent coronary occlusion. Therefore, our study in a rat experimental model of MI demonstrates that rhEPO administered within 2 hrs of a coronary occlusion effectively reduces MI size, but when rhEPO was administered following a delay similar to that encountered in clinical trials, it had no effect on MI size. CONCLUSIONS/SIGNIFICANCE:The clinical trials that failed to demonstrate rhEPO efficacy in patients with MI may have missed a narrow therapeutic window defined in animal experiments.

The vascular type of the Ehlers-Danlos syndrome (vEDS) is caused by dominant-negative mutations in the procollagen type III (COL3A1) gene. Patients with this autosomal dominant disorder have a shortened life expectancy due to complications from ruptured vessels or hollow organs. We tested the effectiveness of allele-specific RNA interference (RNAi) to reduce the mutated phenotype in fibroblasts. Small-interfering RNAs (siRNAs) discriminating between wild-type and mutant COL3A1 allele were identified by a luciferase reporter gene assay and in primary fibroblasts from a normal donor and a patient with vEDS. The best discriminative siRNA with the mutation at position 10 resulted in >90% silencing of the mutant allele without affecting the wild-type allele. Transmission and immunogold electron microscopy of extracted extracellular matrices from untreated fibroblasts of the patient with vEDS revealed structurally abnormal fibrils. After siRNA treatment, collagen fibrils became similar to fibrils from fibroblasts of normal and COL3A1 haploinsufficient donors. In addition, it was shown that expression of mutated COL3A1 activates the unfolded protein response and that reduction of the amount of mutated protein by siRNA reduces cellular stress. Taken together, the results provide evidence that allele-specific siRNAs are able to reduce negative effects of mutated COL3A1 proteins. Thus, the application of allele-specific RNAi may be a promising direction for future personalized therapies to reduce the severity of vEDS.-Muller, G. A., Hansen, U., Xu, Z., Griswold, B., Talan, M. I., McDonnell, N. B., Briest, W. Allele-specific siRNA knockdown as a personalized treatment strategy for vascular Ehlers-Danlos syndrome in human fibroblasts. FASEB J. 26, 668-677 (2012). www.fasebj.org

Summary Activation of nitric oxide (NO) signaling is considered, at list partially, a mechanistic basis for EPO-induced cardioprotection. Surprisingly, hemodynamic response subsequent to NO activation after EPO administration has never been reported. The objectives of this study were to evaluate the acute hemodynamic and cardiovascular responses to EPO administration, to confirm their NO genesis, and to test the hypothesis that EPO-induced cardioprotection is mediated through cardiovascular changes related to NO activation. In Experiment 1, after 3000 U/kg of rhEPO was administered intravenously to Wistar rats, arterial blood pressure, monitored via indwelling catheter, progressively declined almost immediately until it leveled off 90 minutes after injection at 20% below control level. In Experiment 2 the 25% reduction of mean blood pressure, compared to control group, was observed 2 hours after intravenous injection of either 3000 or 150 U/kg of rhEPO. Detailed pressure–volume loop analyses of cardiac performance (Experiment 3) 2 hours after intravenous injection of human or rat recombinant EPO (3000 U/kg) revealed a significant reduction of systolic function (PRSW was 33% less than control). Reduction of arterial blood pressure and systolic cardiac function in response to rhEPO were blocked in rats pretreated with a non-selective inhibitor of nitric oxide synthase (L-NAME). In Experiment 4, 24 hours after a permanent ligation of a coronary artery, myocardial infarction (MI) measured 26±3.5% of left ventricle in untreated rats. MI in rats treated with 3000 U/kg of rhEPO immediately after coronary ligation was 56% smaller. Pretreatment with L-NAME did not attenuate the beneficial effect of rhEPO on MI size, while MI size in rats treated with L-NAME alone did not differ from control. Therefore, a single injection of rhEPO resulted in a significant, NO-mediated reduction of systemic blood pressure and corresponding reduction of cardiac systolic function. However, EPO-induced protection of myocardium from ischemic damage is not associated with NO activation or NO-mediated hemodynamic responses.