Long-term low dose dietary resveratrol supplement reduces cardiovascular structural and functional deterioration in chronic heart failure in rats.

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.

Cardiac remodeling in the mouse model of Marfan syndrome develops into two distinctive phenotypes.

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-ß 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.

Gi-biased ß2AR signaling links GRK2 upregulation to heart failure.

RATIONALE: Phosphorylation of ß(2)-adrenergic receptor (ß(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 ß(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 ßAR stimulation in cultured mouse cardiomyocytes and in vivo. Importantly, cardiac-specific transgenic overexpression of a mutant ß(2)AR lacking PKA phosphorylation sites (PKA-TG) but not the wild-type ß(2)AR (WT-TG) or a mutant ß(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 ß(2)AR to G(i) coupling induced by removing PKA phosphorylation of the receptor and in GRK2 transgenic mice, indicating that enhanced phosphorylation of ß(2)AR by GRK and resultant increase in G(i)-biased ß(2)AR signaling play an important role in the development of heart failure. CONCLUSIONS: Our data show that enhanced ß(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 ß(2)AR signaling as a primary event linking upregulation of GRK to cardiac maladaptive remodeling, failure and cardiodepression.

Vessel ultrasound sonographic assessment of soluble receptor for advanced glycation end products efficacy in a rat balloon injury model.

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.

Chronic administration of small nonerythropoietic peptide sequence of erythropoietin effectively ameliorates the progression of postmyocardial infarction-dilated cardiomyopathy.

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.

Allele-specific siRNA knockdown as a personalized treatment strategy for vascular Ehlers-Danlos syndrome in human fibroblasts.

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.

Chronic treatment with a broad-spectrum metalloproteinase inhibitor, doxycycline, prevents the development of spontaneous aortic lesions in a mouse model of vascular Ehlers-Danlos syndrome.

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.

Fenoterol enantiomers do not possess beneficial therapeutic properties of their racemic mixture in the rat model of post myocardial infarction dilated cardiomyopathy.

PURPOSE: A salutary effect of ß(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.

ß2 AR agonists in treatment of chronic heart failure: long path to translation.

The main clinical manifestations of advanced chronic heart failure (CHF), e.g. in dilated cardiomyopathy (DCM), are reduced systolic and diastolic functions, increased arterial elastance and arterio-ventricular uncoupling, accompanied and exacerbated by an excessive sympathetic activation and extensive abnormalities in the ßAR signaling. Loss of cardiomyocytes due to apoptosis is one mechanism that undoubtedly contributes to cardiac remodeling and functional deterioration associated with dilated cardiomyopathy (DCM). Research during the last decade on the single cardiomyocyte level strongly suggested that selective stimulation of ß(1) AR activates the proapoptotic signaling pathways, while selective stimulation of ß(2) AR is antiapoptotic, but its precise mechanisms remain to be elucidated. Extensive research in the rat model of DCM following induction of myocardial infarction (MI) showed that prolonged treatment with of ß(2) AR agonist, fenoterol, in combination with a ß(1) AR blocker, metoprolol, is more effective than ß(1) AR blocker alone and as effective as ß(1) AR blocker with ACE inhibitor with respect to survival and cardiac remodeling. This combined regimen of ß(2) AR agonists and a ß(1) AR blocker might be considered for clinical testing as alternative or adjunct therapy to currently acceptable CHF arsenal. This article is part of a special issue entitled "Key Signaling Molecules in Hypertrophy and Heart Failure."

Effects of calorie restriction on cardioprotection and cardiovascular health.

Multiple health benefits of calorie restriction (CR) and alternate day fasting (ADF) regimens are widely recognized. Experimental data concerning the effects of calorie restriction on cardiac health are more controversial, ranging from evidence that ADF protects heart from ischemic damage but results in developing of diastolic dysfunction, to reports that CR ameliorates the age-associated diastolic dysfunction. Here we investigated the effects of chronic CR on morphology and function of the cardiovascular system of aged rats and cardioprotective effect of CR against ischemic damage in the experimental rat model of MI. Cardiovascular fitness of 24-month old Fisher 344 rats maintained through life on ad libitum (AL) or CR diets was extensively evaluated via echocardiography, dobutamine stress test, pressure-volume loop analyses, pulse wave velocity measurements, and histology. Groups of 2-month old AL and 29-month old CR rats were studied for comparison. Myocardial infarction (MI) was induced by a permanent ligation of the anterior descending coronary artery in 5-month old rats maintained for 3 months on CR or AL. MI size was evaluated histologically 24 hrs following coronary ligation. Cardiac remodeling was followed-up via echocardiography. Age-associated changes in 24-month old rats consisted of 33% increase of fibrosis in the myocardium and more than 2 fold increase of the collagen in the tunica media of the aorta. There was a significant decrease in the density and total number of cardiomyocytes, while their size was increased. These morphological changes were manifested in a decline of systolic and diastolic cardiac function, increase of left ventricular and aortic stiffness, and arterio-ventricular uncoupling. Tachycardic response to dobutamine challenge was absent in the old rats. Compared to AL rats, 24-month old CR rats had reduced levels of cardiac and aortic fibrosis, increased density of cardiomyocytes that were smaller in size, attenuated diastolic dysfunction, normal systolic function and arterio-ventricular coupling. Tachycardic response to dobutamine was also intact in CR 24-month old rats and aortic stiffness was reduced. Adjustment for body weight differences through ratiometric or allometric scaling did not affect the overall pattern of differences between AL and CR rats. Attenuation of morphological and functional age-associated changes in 24-month old CR rats either was not observed at all or was smaller in 29-month old CR rats. Size of MI induced by a permanent coronary ligation as well as post-MI cardiac remodeling and function were similar in CR and AL rats. CR does not increase tolerance of myocardium to ischemic damage, but attenuates the age-associated changes in the heart and major vessels. The attenuation of age-associated changes by CR cannot be explained by the effect of lower body weight but are attributable to more intimate cellular mechanisms of CR itself. Attenuation of age-associated changes by CR waned with advancing age, and is consistent with the idea that CR postponed senescence.

A small nonerythropoietic helix B surface peptide based upon erythropoietin structure is cardioprotective against ischemic myocardial damage.

Strong cardioprotective properties of erythropoietin (EPO) reported over the last 10 years have been difficult to translate to clinical applications for ischemic cardioprotection owing to undesirable parallel activation of erythropoiesis and thrombogenesis. A pyroglutamate helix B surface peptide (pHBP), recently engineered to include only a part of the EPO molecule that does not bind to EPO receptor and thus, is not erythropoietic, retains tissue protective properties of EPO. Here we compared the ability of pHBP and EPO to protect cardiac myocytes from oxidative stress in vitro and cardiac tissue from ischemic damage in vivo. HBP, similar to EPO, increased the reactive oxygen species (ROS) threshold for induction of the mitochondrial permeability transition by 40%. In an experimental model of myocardial infarction induced by permanent ligation of a coronary artery in rats, a single bolus injection of 60 µg/kg of pHBP immediately after coronary ligation, similar to EPO, reduced apoptosis in the myocardial area at risk, examined 24 h later, by 80% and inflammation by 34%. Myocardial infarction (MI) measured 24 h after coronary ligation was similarly reduced by 50% in both pHBP- and EPO-treated rats. Two wks after surgery, left ventricular remodeling (ventricular dilation) and functional decline (fall in ejection fraction) assessed by echocardiography were significantly and similarly attenuated in pHBP- and EPO-treated rats, and MI size was reduced by 25%. The effect was retained during the 6-wk follow-up. A single bolus injection of pHBP immediately after coronary ligation was effective in reduction of MI size in a dose as low as 1 µg/kg, but was ineffective at a 60 µg/kg dose if administered 24 h after MI induction. We conclude that pHBP is equally cardioprotective with EPO and deserves further consideration as a safer alternative to rhEPO in the search for therapeutic options to reduce myocardial damage following blockade of the coronary circulation.

Doxycycline ameliorates the susceptibility to aortic lesions in a mouse model for the vascular type of Ehlers-Danlos syndrome.

The vascular form of Ehlers-Danlos syndrome (vEDS), a rare disease with grave complications resulting from rupture of major arteries, is caused by mutations of collagen type III [α1 chain of collagen type III (COL3A1)]. The only, recently proven, preventive strategy consists of the reduction of arterial wall stress by ß-adrenergic blockers. The heterozygous (HT) Col3a1 knockout mouse has reduced expression of collagen III and recapitulates features of a mild presentation of the disease. The objective of this study was to determine whether changing the balance between synthesis and degradation of collagen by chronic treatment with doxycycline, a nonspecific matrix metalloproteinase (MMP) inhibitor, could prevent the development of vascular pathology in HT mice. After 3 months of treatment with doxycycline or placebo, 9-month-old HT or wild-type (WT) mice were subjected to surgical stressing of the aorta. A 3-fold increase in stress-induced aortic lesions found in untreated HT mice 1 week after intervention (cumulative score 4.5 ± 0.87 versus 1.3 ± 0.34 in WT, p < 0.001) was fully prevented in the doxycycline-treated group (1.1 ± 0.56, p < 0.001). Untreated HT mice showed increased MMP-9 activity in the carotid artery and decreased collagen content in the aorta; however, in doxycycline-treated animals there was normalization to the levels observed in WT mice. Doxycycline treatment inhibits the activity of tissue MMP and attenuates the decrease in the collagen content in aortas of mice haploinsufficient for collagen III, as well as prevents the development of stress-induced vessel pathology. The results suggest that doxycycline merits clinical testing as a treatment for vEDS.

Intravenous erythropoietin in patients with ST-segment elevation myocardial infarction: REVEAL: a randomized controlled trial.

CONTEXT: Acute ST-segment elevation myocardial infarction (STEMI) is a leading cause of morbidity and mortality. In experimental models of MI, erythropoietin reduces infarct size and improves left ventricular (LV) function. OBJECTIVE: To evaluate the safety and efficacy of a single intravenous bolus of epoetin alfa in patients with STEMI. DESIGN, SETTING, AND PATIENTS: A prospective, randomized, double-blind, placebo-controlled trial with a dose-escalation safety phase and a single dose (60,000 U of epoetin alfa) efficacy phase; the Reduction of Infarct Expansion and Ventricular Remodeling With Erythropoietin After Large Myocardial Infarction (REVEAL) trial was conducted at 28 US sites between October 2006 and February 2010, and included 222 patients with STEMI who underwent successful percutaneous coronary intervention (PCI) as a primary or rescue reperfusion strategy. INTERVENTION: Participants were randomly assigned to treatment with intravenous epoetin alfa or matching saline placebo administered within 4 hours of reperfusion. MAIN OUTCOME MEASURE: Infarct size, expressed as percentage of LV mass, assessed by cardiac magnetic resonance (CMR) imaging performed 2 to 6 days after study medication administration (first CMR) and again 12 ± 2 weeks later (second CMR). RESULTS: In the efficacy cohort, the infarct size did not differ between groups on either the first CMR scan (n = 136; 15.8% LV mass [95% confidence interval {CI}, 13.3-18.2% LV mass] for the epoetin alfa group vs 15.0% LV mass [95% CI, 12.6-17.3% LV mass] for the placebo group; P = .67) or on the second CMR scan (n = 124; 10.6% LV mass [95% CI, 8.4-12.8% LV mass] vs 10.4% LV mass [95% CI, 8.5-12.3% LV mass], respectively; P = .89). In a prespecified analysis of patients aged 70 years or older (n = 21), the mean infarct size within the first week (first CMR) was larger in the epoetin alfa group (19.9% LV mass; 95% CI, 14.0-25.7% LV mass) than in the placebo group (11.7% LV mass; 95% CI, 7.2-16.1% LV mass) (P = .03). In the safety cohort, of the 125 patients who received epoetin alfa, the composite outcome of death, MI, stroke, or stent thrombosis occurred in 5 (4.0%; 95% CI, 1.31%-9.09%) but in none of the 97 who received placebo (P = .04). CONCLUSIONS: In patients with STEMI who had successful reperfusion with primary or rescue PCI, a single intravenous bolus of epoetin alfa within 4 hours of PCI did not reduce infarct size and was associated with higher rates of adverse cardiovascular events. Subgroup analyses raised concerns about an increase in infarct size among older patients. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00378352.

Design and rationale of the Reduction of Infarct Expansion and Ventricular Remodeling with Erythropoietin after Large Myocardial Infarction (REVEAL) trial.

BACKGROUND: Acute myocardial infarction (MI) remains a leading cause of death despite advances in pharmacologic and percutaneous therapies. Animal models of ischemia/reperfusion have demonstrated that single-dose erythropoietin may reduce infarct size, decrease apoptosis, and increase neovascularization, possibly through mobilization of endothelial progenitor cells. STUDY DESIGN: REVEAL is a randomized, double-blind, placebo-controlled, multicenter trial evaluating the effects of epoetin α on infarct size and left ventricular remodeling in patients with large MIs. The trial comprises a dose-escalation safety phase and a single-dose efficacy phase using the highest acceptable epoetin α dose up to 60,000 IU. Up to 250 ST-segment elevation myocardial infarction patients undergoing primary or rescue percutaneous coronary intervention will be randomized to intravenous epoetin α or placebo within 4 hours of successful reperfusion. The primary study end point is infarct size expressed as a percentage of left ventricular mass, as measured by cardiac magnetic resonance imaging 2 to 6 days post study medication administration. Secondary end points will assess changes in endothelial progenitor cell numbers and changes in indices of ventricular remodeling. CONCLUSION: The REVEAL trial will evaluate the safety and efficacy of the highest tolerated single dose of epoetin α in patients who have undergone successful rescue or primary percutaneous coronary intervention for acute ST-segment elevation myocardial infarction.

Chronic alternate-day fasting results in reduced diastolic compliance and diminished systolic reserve in rats.

BACKGROUND: Based on animal experiments and limited data from the few human trials, alternate-day fasting (ADF) resulted in weight loss, prolonged life, reduced metabolic risk factors for diabetes and cardiovascular diseases, and reduced prevalence of age-related diseases. The present study is the first comprehensive examination of the long-term effects of ADF on general cardiovascular fitness in rats. METHODS AND RESULTS: Four-month-old male Sprague-Dawley rats were started on ADF or continued on ad libitum diets and followed for 6 months with serial echocardiography. A comprehensive hemodynamic evaluation including a combined dobutamine-volume stress test was performed at the end of the study, and hearts were harvested for histological assessment. The 6-month-long ADF diet resulted in a 9% reduction (P < .01) of cardiomyocyte diameter and 3-fold increase in interstitial myocardial fibrosis. Left ventricular chamber size was not affected by ADF and ejection fraction was not reduced, but left atrial diameter was increased 16%, and the ratio of early (E) and late atrial (A) waves, in Doppler-measured mitral flow was reduced (P < .01). Pressure-volume loop analyses revealed a "stiff" heart during diastole in ADF rats, whereas combined dobutamine and volume loading showed a significant reduction in left ventricular diastolic compliance and a lack of increase in systolic pump function, indicating a diminished cardiac reserve. CONCLUSION: Chronic ADF in rats results in development of diastolic dysfunction with diminished cardiac reserve. ADF is a novel and unique experimental model of diet-induced diastolic dysfunction. The deleterious effect of ADF in rats suggests that additional studies of ADF effects on cardiovascular functions in humans are warranted.

Therapeutic efficacy of a combination of a beta1-adrenoreceptor (AR) blocker and beta2-AR agonist in a rat model of postmyocardial infarction dilated heart failure exceeds that of a beta1-AR blocker plus angiotensin-converting enzyme inhibitor.

We had proposed previously a novel combination of beta2-adrenoreceptor (AR) agonist and beta1-AR blocker that in the rat model of postmyocardial infarction (MI) dilated cardiomyopathy exceeds the therapeutic effectiveness of either monotherapy. In the present study, we compared that treatment with a combination of beta1-AR blocker and angiotensin-converting enzyme inhibitor (ACEi), a current standard chronic heart failure (CHF) therapy. Two weeks after coronary artery ligation, rats were divided into groups of similar average MI size, measured by echocardiography, and the following 12-month treatments were initiated: fenoterol (250 microg/kg/day), a beta2-AR agonist, plus metoprolol (100 mg/kg/day), a beta1-AR blocker (beta1-beta2+); metoprolol plus enalapril (20 mg/kg/day), an ACEi (beta1-ACEi); and a combination of all three drugs (beta1-beta2+ACEi). These treatment groups were compared with each other and with nontreated (nT) and sham groups. The 12-month mortality was significantly reduced in all treatment groups (44% in beta1-beta2+, 56% in beta1-beta2+ACEi, 59% in beta1-ACEi versus 81% in nT). Bimonthly echocardiography revealed significant attenuation of the left ventricular (LV) chamber remodeling, LV functional deterioration, and MI expansion in all three treatment groups, but effects were significantly more pronounced when treatment included a beta2-AR agonist. The results indicated that a combination of beta1-AR blocker and beta2-AR agonist is equipotent to a combination of beta1-AR blocker and ACEi in the treatment of CHF in rats, with the respect to mortality, and exceeds the latter with respect to cardiac remodeling and MI expansion. Thus, this novel therapeutic regimen for CHF warrants detailed clinical investigation.

Cardioprotective and survival benefits of long-term combined therapy with beta2 adrenoreceptor (AR) agonist and beta1 AR blocker in dilated cardiomyopathy postmyocardial infarction.

We have reported therapeutic effectiveness of pharmacological stimulation of beta2 adrenoreceptors (ARs) to attenuate the cardiac remodeling and myocardial infarction (MI) expansion in a rat model of dilated cardiomyopathy (DCM) post-MI. Furthermore, the combination of beta2 AR stimulation with beta1 AR blockade exceeded the therapeutic effectiveness of beta1 AR blockade. However, these studies were relatively short (6 weeks). In this study, in the same experimental model, we compared different effects, including survival benefit, of combined therapy with the beta1 AR blocker, metoprolol, plus the beta2 AR agonist, fenoterol (beta1-beta2+), and either therapy alone (beta1- or beta2+) during the 1-year study. Therapy was started 2 weeks after permanent ligation of the left coronary artery. Cardiac remodeling, MI expansion, and left ventricular function were assessed by serial echocardiography and compared with untreated animals (nT). Sixty-seven percent mortality in nT was reduced to 33% in the beta1-beta2+ (p < 0.01). Progressive cardiac remodeling observed in nT and beta1- was significantly attenuated in beta1-beta2+ during the first 6 months of treatment. In beta1-beta2+, MI expansion was completely prevented, and functional decline was significantly attenuated during the entire year. Myocardial apoptosis was significantly reduced in both beta1-beta2+ and beta1-. A reduction of cardiac beta1 AR density and decreases in chronotropic and contractile responses to beta2 AR-specific stimulation in the absence of a reduction of beta2 AR density in nT were precluded in rats receiving combined therapy. The results demonstrate the cardioprotective and survival benefit of long-term combination therapy of beta2 AR agonists and beta1 AR blockers in a model of DCM.

Intrahippocampal microinjection of an exquisitely low dose of ouabain mimics NaCl loading and stimulates a bufadienolide Na/K-ATPase inhibitor.

BACKGROUND: Brain endogenous ouabain (EOU) raises blood pressure (BP) via an angiotensin II (ATII)-sensitive pathway in NaCl-loaded Dahl salt-sensitive rats (DSS). Brain EOU activates central and adrenocortical renin-angiotensin systems, and stimulates marinobufagenin, a vasoconstrictor and natriuretic inhibitor of sodium pump. METHODS: We studied effects of acute NaCl loading (17 mmol/kg NaCl, intraperitoneally) on levels of EOU and marinobufagenin in several brain areas in DSS. We then studied effects of intrahippocampal administration of very-low-dose ouabain (60 pg) on EOU, marinobufagenin, BP, sodium excretion, and sodium-pump activity in the aorta and renal medulla in the absence and presence of anti-marinobufagenin and anti-ouabain antibodies, and losartan. RESULTS: NaCl loading of DSS induced transient increases of EOU in the hippocampus and amygdala (15 min; 300%), supraoptical nucleus of hypothalamus (SON) (30 min, 230%) and pituitary (30 min; 85%), and ATII elevation in the SON (30 min). Intrahippocampal administration of ouabain (60 pg) stimulated ATII in the SON, produced natriuresis, 40 mmHg rise in BP, inhibition of sodium-pump in the renal medulla (19.6%) and aorta (25%), and a two-fold increase in renal marinobufagenin excretion. Pretreatment of rats with anti-marinobufagenin antibody prevented ouabain-induced pressor and natriuretic responses and sodium-pump inhibition. Pressor responses to ouabain were also prevented by losartan (intravenously) and by administration of anti-ouabain antibody into the SON. CONCLUSIONS: NaCl loading of DSS induces a cascade of events, triggered by brain EOU and ATII. Intrahippocampal administration of a low-dose ouabain mimics effects of NaCl loading and stimulates marinobufagenin, which produces natriuresis, and inhibits the vascular sodium-pump, inducing an increase in BP.

Cardioprotection by intermittent fasting in rats.

BACKGROUND: Intermittent fasting (IF), a dietary regimen in which food is available only every other day, increases the life span and reduces the incidence of age-associated diseases in rodents. We have reported neuroprotective effects of IF against ischemic injury of the brain. In this study, we examined the effects of IF on ischemic injury of the heart in rats. METHODS AND RESULTS: After 3 months of IF or regular every-day feeding (control) diets started in 2-month-old rats, myocardial infarction (MI) was induced by coronary artery ligation. Twenty-four hours after MI, its size in the IF group was 2-fold smaller, the number of apoptotic myocytes in the area at risk was 4-fold less, and the inflammatory response was significantly reduced compared with the control diet group. Serial echocardiography revealed that during 10 weeks after MI (with continuation of the IF regimen), the left ventricular (LV) remodeling and MI expansion that were observed in the control diet group were absent in the IF group. In a subgroup of animals with similar MI size at 1 week after MI, further observation revealed less remodeling, better LV function, and no MI expansion in the IF group compared with the control group. CONCLUSIONS: IF protects the heart from ischemic injury and attenuates post-MI cardiac remodeling, likely via antiapoptotic and antiinflammatory mechanisms.