Rod Partow-Navid

Intralipid, a Clinically Safe Compound, Protects the Heart Against Ischemia-Reperfusion Injury More Efficiently Than Cyclosporine-A

Background:We have recently shown that postischemic administration of intralipid protects the heart against ischemia-reperfusion injury. Here we compared the cardioprotective effects of intralipid with cyclosporine-A, a potent inhibitor of the mitochondrial permeability transition pore opening. Methods:In vivo rat hearts or isolated Langendorff-perfused mouse hearts were subjected to ischemia followed by reperfusion with intralipid (0.5%, 1% and 2% ex-vivo, and 20% in vivo), cyclosporine-A (0.2 &mgr;M, 0.8 &mgr;M, and 1.5 &mgr;M ex- vivo and 10 mg/kg in vivo), or vehicle. The hemodynamic function, infarct size, calcium retention capacity, mitochodrial superoxide production, and phosphorylation levels of protein kinase B (Akt)/glycogen synthase kinase-3&bgr; (GSK-3&bgr;) were measured. The values are mean ± SEM. Results:Administration of intralipid at reperfusion significantly reduced myocardial infarct size compared with cyclosporine-A in vivo (infarct size/area at risk)%: 22.9 ± 2.5% vs. 35.2 ± 3.5%; P = 0.030, n = 7/group). Postischemic administration of intralipid at its optimal dose (1%) was more effective than cyclosporine-A (0.8 &mgr;M) in protecting the ex vivo heart against ischemia-reperfusion injury, as the rate pressure product at the end of reperfusion was significantly higher (mmHg · beats/min: 12,740 ± 675 [n = 7] vs. 9,203 ± 10,781 [n = 5], P = 0.024), and the infarct size was markedly smaller (17.3 ± 2.9 [n = 7] vs. 29.2 ± 2.7 [n = 5], P = 0.014). Intralipid was as efficient as cyclosporine-A in inhibiting the mitochondrial permeability transition pore opening (calcium retention capacity = 280 ± 8.2 vs. 260.3 ± 2.9 nmol/mg mitochondria protein in cyclosporine-A, P = 0.454, n = 6) and in reducing cardiac mitochondrial superoxide production. Unlike intralipid, which increased phosphorlyation of Akt (6-fold) and GSK-3&bgr; (5-fold), cyclosporine-A had no effect on the activation of these prosurvival kinases. Conclusions:Although intralipid inhibits the opening of the mitochondrial permeability transition pore as efficiently as cyclosporine-A, intralipid is more effective in reducing the infarct size and improving the cardiac functional recovery.

Genistein, a Soy Phytoestrogen, Reverses Severe Pulmonary Hypertension and Prevents Right Heart Failure in Rats

Pretreatment with a phytoestrogen genistein has been shown to attenuate the development of pulmonary hypertension (PH). Because PH is not always diagnosed early, we examined whether genistein could also reverse preexisting established PH and prevent associated right heart failure (RHF). PH was induced in male rats by 60 mg/kg of monocrotaline. After 21 days, when PH was well established, rats received daily injection of genistein (1 mg/kg per day) for 10 days or were left untreated to develop RHF by day 30. Effects of genistein on human pulmonary artery smooth muscle cell and endothelial cell proliferation and neonatal rat ventricular myocyte hypertrophy were assessed in vitro. Severe PH was evident 21 days after monocrotaline, as peak systolic right ventricular pressure increased to 66.35±1.03 mm Hg and right ventricular ejection fraction reduced to 41.99±1.27%. PH progressed to RHF by day 30 (right ventricular pressure, 72.41±1.87 mm Hg; RV ejection fraction, 29.25±0.88%), and mortality was ≈75% in RHF rats. Genistein therapy resulted in significant improvement in lung and heart function as right ventricular pressure was significantly reduced to 43.34±4.08 mm Hg and right ventricular ejection fraction was fully restored to 65.67±1.08% similar to control. Genistein reversed PH-induced pulmonary vascular remodeling in vivo and inhibited human pulmonary artery smooth muscle cell proliferation by ≈50% in vitro likely through estrogen receptor-&bgr;. Genistein also reversed right ventricular hypertrophy (right ventricular hypertrophy index, 0.35±0.029 versus 0.70±0.080 in RHF), inhibited neonatal rat ventricular myocyte hypertrophy, and restored PH-induced loss of capillaries in the right ventricle. These improvements in cardiopulmonary function and structure resulted in 100% survival by day 30. Genistein restored PH-induced downregulation of estrogen receptor-&bgr; expression in the right ventricle and lung. In conclusion, genistein therapy not only rescues preexisting severe PH but also prevents the progression of severe PH to RHF.

Spontaneous Ventricular Fibrillation in Right Ventricular Failure Secondary to Chronic Pulmonary Hypertension

Background— Right ventricular failure (RVF) in pulmonary hypertension (PH) is associated with increased incidence of sudden death by a poorly explored mechanism. We test the hypothesis that PH promotes spontaneous ventricular fibrillation (VF) during a critical post-PH onset period characterized by a sudden increase in mortality. Methods and Results— Rats received either a single subcutaneous dose of monocrotaline (MCT, 60 mg/kg) to induce PH-associated RVF (PH, n=24) or saline (control, n=17). Activation pattern of the RV-epicardial surface was mapped using voltage-sensitive dye in isolated Langendorff-perfused hearts along with single glass-microelectrode and ECG-recordings. MCT-injected rats developed severe PH by day 21 and progressed to RVF by approximately day 30. Rats manifested increased mortality, and ≈30% rats died suddenly and precipitously during 23–32 days after MCT. This fatal period was associated with the initiation of spontaneous VF by a focal mechanism in the RV, which was subsequently maintained by both focal and incomplete reentrant wave fronts. Microelectrode recordings from the RV-epicardium at the onset of focal activity showed early afterdepolarization–mediated triggered activity that led to VF. The onset of the RV cellular triggered beats preceded left ventricular depolarizations by 23±8 ms. The RV but not the left ventricular cardiomyocytes isolated during this fatal period manifested significant action potential duration prolongation, dispersion, and an increased susceptibility to depolarization-induced repetitive activity. No spontaneous VF was observed in any of the control hearts. RVF was associated with significantly reduced RV ejection fraction (P<0.001), RV hypertrophy (P<0.001), and RV fibrosis (P<0.01). The hemodynamic function of the LV and its structure were preserved. Conclusions— PH-induced RVF is associated with a distinct phase of increased mortality characterized by spontaneous VF arising from the RV by an early afterdepolarization–mediated triggered activity.

Pregnancy is associated with decreased cardiac proteasome activity and oxidative stress in mice.

During pregnancy, the heart develops physiological hypertrophy. Proteasomal degradation has been shown to be altered in various models of pathological cardiac hypertrophy. Since the molecular signature of pregnancy-induced heart hypertrophy differs significantly from that of pathological heart hypertrophy, we investigated whether the cardiac proteasomal proteolytic pathway is affected by pregnancy in mice. We measured the proteasome activity, expression of proteasome subunits, ubiquitination levels and reactive oxygen production in the hearts of four groups of female mice: i) non pregnant (NP) at diestrus stage, ii) late pregnant (LP), iii) one day post-partum (PP1) and iv) 7 days post-partum (PP7). The activities of the 26 S proteasome subunits β1 (caspase-like), and β2 (trypsin-like) were significantly decreased in LP (β1∶83.26±1.96%; β2∶74.74±1.7%, normalized to NP) whereas β5 (chymotrypsin-like) activity was not altered by pregnancy but significantly decreased 1 day post-partum. Interestingly, all three proteolytic activities of the proteasome were restored to normal levels 7 days post-partum. The decrease in proteasome activity in LP was not due to the surge of estrogen as estrogen treatment of ovariectomized mice did not alter the 26 S proteasome activity. The transcript and protein levels of RPN2 and RPT4 (subunits of 19 S), β2 and α7 (subunits of 20 S) as well as PA28α and β5i (protein only) were not significantly different among the four groups. High resolution confocal microscopy revealed that nuclear localization of both core (20S) and RPT4 in LP is increased ∼2-fold and is fully reversed in PP7. Pregnancy was also associated with decreased production of reactive oxygen species and ubiquitinated protein levels, while the de-ubiquitination activity was not altered by pregnancy or parturition. These results indicate that late pregnancy is associated with decreased ubiquitin-proteasome proteolytic activity and oxidative stress.

Severe pulmonary hypertension in aging female apolipoprotein E-deficient mice is rescued by estrogen replacement therapy

BackgroundApolipoprotein E (ApoE) is a multifunctional protein, and its deficiency leads to the development of atherosclerosis in mice. Patients with pulmonary hypertension (PH) have reduced expression of ApoE in lung tissue. ApoE is known to inhibit endothelial and smooth muscle cell proliferation and has anti-inflammatory and anti-platelet aggregation properties. Young ApoE-deficient mice have been shown to develop PH on high fat diet. The combined role of female sex and aging in the development of PH has not been investigated before. Here, we investigated the development of PH in young and middle-aged (MA) female ApoE-deficient mice and explored the role of exogenous estrogen (E2) replacement therapy for the aging females.MethodsWild type (WT) and ApoE-deficient female mice (Young and MA) were injected with a single intraperitoneal dose of monocrotaline (MCT, 60 mg/kg). Some ApoE-deficient MA female mice that received MCT were also treated with subcutaneous E2 pellets (0.03 mg/kg/day) from day 21 to 30 after MCT injection. Direct cardiac catheterization was performed terminally to record right ventricular systolic pressure (RVSP). Right ventricular (RV), left ventricular (LV), and interventricular septum (IVS) were dissected and weighed. Lung sections were examined using trichrome and immunofluorescence staining. Western blot analyses of lung and RV lysates were performed.ResultsIn WT female mice, the severity of PH was similar between young and MA mice as RVSP was not significantly different (RVSP = 38.2 ± 1.2 in young vs. 40.5 ± 8.3 mmHg in MA, p < 0.05). In ApoE-deficient mice, MA females developed significantly severe PH (RVSP = 63 ± 10 mmHg) compared to young females (RVSP; 36 ± 3 mmHg, p < 0.05 vs. MA female). ApoE-deficient MA females also developed more severe RV hypertrophy compared to young females (RV hypertrophy index (RV/[LV + IVS]) = 0.53 ± 0.06 vs. 0.33 ± 0.01, p < 0.05). ApoE-deficient MA female mice manifested increased peripheral pulmonary artery muscularization and pulmonary fibrosis. E2 treatment of MA female ApoE-deficient mice resulted in a significant decrease in RVSP, reversal of pulmonary vascular remodeling, and RV hypertrophy. In MA female ApoE-deficient mice with PH, only the expression of ERβ in the lungs, but not in RV, was significantly downregulated, and it was restored by E2 treatment. The expression of ERα was not affected in either lungs or RV by PH. GPR30 was only detected in the RV, and it was not affected by PH in MA female ApoE-deficient mice.ConclusionsOur results suggest that only aging female ApoE-deficient but not WT mice develop severe PH compared to younger females. Exogenous estrogen therapy rescued PH and RV hypertrophy in aging female ApoE-deficient mice possibly through restoration of lung ERβ.

Estrogen Receptor Beta, but Not Alpha, is the Key Player in Restoration of Heart Function of Heart Failure Mice by Estrogen Therapy

Recently we discovered that estrogen(E2) therapy can rescue advanced heart failure(HF) induced by pressure overload in mice. Most of the biological actions of estrogen are mediated thorough estrogen receptors alpha(ERa) and beta(ERb), and both of these receptors are present in the heart. Here we investigated which estrogen receptor(s) are involved in the rescue by estrogen. We used the transaortic constriction(TAC) procedure to induce HF. Once the ejection fraction(EF) reached ∼30%, one group of animals was sacrificed(HF group), and the other three groups received either 17b-estradiol (30 ug/kg/day), selective ERa agonist (PPT, 0.625mg/kg/day), or selective ERb agonist (DPN, 0.625mg/kg/day) for 10 days. Serial echocardiography was performed and LV pressure was measured by direct catheterization before sacrifice. As expected, E2 rescued HF by restoring EF from 33.17±1.12% to 53.05±1.29%. Interestingly, mice treated with ERb agonist had a significant improvement in their EF from 33.17±1.12% to 45.25±2.1%(n=7), whereas the EF of mice treated with ERa agonist did not improve at all(31.09±2.3%, n=6). Similar to EF, only fractional shortening of DPN-treated mice improved from 15.7±0.58% in HF to 21.95±1.65% in DPN vs. 14.72±1.24% in PPT). Next, we examined the mechanical performance of the LV in mice treated with DPN and PPT. DPN treatment improved LVDP from 94.68±5.14 in HF to 119.3±5.5 and RPP from 39749.9±4527.03 in HF to 62794.04±5534.62. PPT treatment had no effect on LV mechanical performance as both LVDP and RPP were not significantly different from their corresponding values in HF. The relaxation and contraction defects of HF mice (dP/dtmax and dP/dtmin) were also partially restored by DPN but not by PPT. Our data strongly support the view that ERb is the main player in rescuing advanced HF by E2.

Molecular Mechanisms Involved in the Rescue of Severe Pulmonary Hypertension by Genistein Therapy

Previously, we showed that genistein, a soy isoflavone, rescued severe pulmonary hypertension(PH). However, the mechanisms involved in the rescue were largely unknown. Here, we investigated possible mechanisms of genistein rescue of PH. We induced PH in rats using a single subcutaneous injection of monocrotaline(MCT, 60 mg/kg). By day 21, rats developed severe PH. At this time point, we started genistein therapy(1 mg/kg/day, subcutaneous) to one group(GEN) until day 30. The other group was left untreated and developed RV failure by day 30(MCT group). The control group(CTRL) received saline. At day 30, cardiac catheterization was performed to assess right ventricular pressure(RVP), animals were sacrificed and lungs and hearts were dissected. Immunohistochemistry, Western Blot, and RT PCR were performed. MCT group developed severe PH(RVP 31±1 mmHg in CTRL, n=5 vs. 72±1 mmHg in MCT, n=7). Genistein attenuated severe PH(44±5 mmHg, n=8). Additionally, the RV hypertrophy index(RV/(LV+IVS)) increased(∼3-fold) in MCT and was restored in GEN. We investigated the role of estrogen receptors α and β(ERα and ERβ) in genistein rescue. ERβ protein levels were significantly downregulated in lungs and RV of MCT(∼2-fold and ∼5-fold). Genistein restored lung and RV ERβ protein levels. ERα protein levels did not change in PH. PH led to significantly decreased capillary density in RV and VEGF protein(∼2.5-fold) in both RV and lung. Genistein restored RV capillary density and significantly improved VEGF levels in RV and lung. Furthermore, we found a significant increase in lung Caspase-3(∼3-fold) and pSTAT3/STAT3(∼3.5-fold) proteins in MCT, which were reversed by Genistein. PH was also associated with significantly reduced lung Caveolin-1(∼6-fold) protein levels that were restored in GEN. In conclusion, genistein rescues severe PH through ERβ mediated protection, preserves cardiopulmonary angiogenesis, and restores Caspase-3, pSTAT3/STAT3 and Caveolin-1 in the lungs.

Mechanisms of Prevention of Pulmonary Hypertension-Induced Right-Ventricular Failure by Intralipid

Intralipid (ILP) has been used for treatment of local anesthetic-induced cardiac-arrest and as source of parenteral nutrition. Some of its constituents are precursors of pulmonary vasodilator prostaglandins. Pulmonary hypertension (PH) is associated with pulmonary vascular remodeling leading to right-ventricular (RV) hypertrophy and failure. Recently we found that ILP prevents PH in rats. Here we investigated the mechanisms involved in this prevention. Rats were treated with monocrotaline(60 mg/kg) to induce PH, and then either received daily ILP (1 mL of 20% ILP/day) for 30days or left untreated to develop RV failure (RVF). Saline treated rats served as control. Serial echocardiography was performed to monitor cardiopulmonary hemodynamics., RV pressure (RVP) was recorded by direct cardiac catheterization right before sacrifice. Immunohistochemistry, confocal-microscopy, Western Blot analysis and RT-PCR were performed. RVF group developed PH that led to RVF later with significantly increased RVP (70±5mmHg,n=10) and depressed RV ejection fraction (RVEF=34±2%). Severe structural changes in RV and lung were observed in RVF group including RV-hypertrophy, increased lung weight, pulmonary medial hypertrophy, fibrosis, apoptosis (upregulation of Caspase-3 (∼10fold)), inflammation (increased expression of IL-6) and suppression of angiogenesis (decreased VEGF and capillary-density). Furthermore, RVF was associated with downregulation of phospho-eNOS(∼4fold), Caveolin-1(∼6fold) adenosine-A2B receptor and estrogen receptor-β in lungs. In the RV, adenosine-A2A receptor and estrogen receptor-b were downregulated in RVF. ILP therapy prevented PH-induced RVF by restoring RVP(30±2mmHg,n=8) and RVEF(63±1.5%) and attenuating RV hypertrophy and lung remodeling. ILP suppressed inflammation and fibrosis along-with improving angiogenesis and restoring phospho-eNOS, Caveolin-1 in lungs and Caspase-3, adenosine-A2 and estrogen receptor-β expression in lungs and RV. In conclusion ILP prevented PH and RV-failure by preserving cardiopulmonary structure and function and enhancing perfusion via adenosine receptor, estrogen receptor-b and eNOS-mediated mechanisms.

Stimulation of Cardiac Neoangiogenesis by Estrogen Therapy is One of the Key Mechanisms in Reversing Advanced Heart Failure

Estrogen(E2) has been shown to regulate angiogenesis in different tissues, but it is still not known if E2 could stimulate angiogenesis in the heart. Recently, we showed that E2 rescues advanced heart failure by reversing the myocardial contractile deficiency and restoring ejection fraction from ∼30% to ∼55%. Here we examined whether stimulation of angiogenesis in the heart is a mechanism involved in the E2-induced rescue of HF. Trans-aortic constriction(TAC) procedure was used to induce HF. Once the ejection fraction(EF) reached ∼30%, one group of mice was sacrificed and the other two groups were treated with E2(30 μg/kg/day, n=16), or E2 plus the angiogenesis inhibitor TNP-470(TNP, 30 mg/kg, n=4) for 10 days. Serial echocardiography, real-time PCR and immunocytochemistry were performed. RT-PCR showed that the transcript levels of two markers of angiogenesis, vascular endothelial growth factor(VEGF) and hypoxia-inducible factor-1a(HIF1a), were ∼10 fold downregulated in HF(0.17±0.06 for VEGF and 0.26±0.01 for HiF1a; normalized to CTRL). E2 treatment was not only able to reverse VEGF and HIF1a transcript level downregulation observed in HF, but to even upregulate both transcripts 3 fold higher than in healthy controls(3.24±0.1 for VEGF and 3.16±0.09 for HIF1a). Quantification of capillary density also revealed that E2 therapy not only completely reversed the loss of capillaries in HF, but significantly enhanced capillary density by ∼ 4 fold compared to HF(2.83±0.14 in E2 vs. 0.66±0.07 in HF, normalized to CTRL). Interestingly, E2 failed to rescue HF in the presence of TNP-470 (E2+TNP group) as EF (29.3±2.1%) was not significantly improved after 10 days of therapy. The capillary density of HF mice also did not improve in E2+TNP group(0.53±0.07). These data strongly support the vital role of angiogenesis in the rescue action of E2.