Molecular and pharmacological approaches to inhibiting nitric oxide after burn trauma

Whereas controversial, several studies have suggested that nitric oxide (NO) alters cardiac contractility via cGMP, peroxynitrite, or poly(ADP ribose) synthetase (PARS) activation. This study determined whether burn-related upregulation of myocardial inducible NO synthase (iNOS) and NO generation contributes to burn-mediated cardiac contractile dysfunction. Mice homozygous null for the iNOS gene (iNOS knockouts) were obtained from Jackson Laboratory. iNOS knockouts (KO) as well as wild-type mice were given a cutaneous burn over 40% of the total body surface area by the application of brass probes (1 x 2 x 0.3 cm) heated to 100 degrees C to the animals' sides and back for 5 s (iNOS/KO burn and wild-type burn). Additional groups of iNOS KO and wild-type mice served as appropriate sham burn groups (iNOS/KO sham and wild-type sham). Cardiac function was assessed 24 h postburn by perfusing hearts (n = 7-10 mice/group). Burn trauma in wild-type mice impaired cardiac function as indicated by the lower left ventricular pressure (LVP, 67 +/- 2 mmHg) compared with that measured in wild-type shams (94 +/- 2 mmHg, P < 0.001), a lower rate of LVP rise (+dP/dtmax, 1,620 +/- 94 vs. 2,240 +/- 58 mmHg/s, P < 0.001), and a lower rate of LVP fall (-dP/dtmax, 1,200 +/- 84 vs. 1,800 +/- 42 mmHg/s, P < 0.001). Ventricular function curves confirmed significant contractile dysfunction after burn trauma in wild-type mice. Burn trauma in iNOS KO mice produced fewer cardiac derangements compared with those observed in wild-type burns (LVP: 78 +/- 5 mmHg; +dP/dt: 1,889 +/- 160 mmHg/s; -dP/dt: 1,480 +/- 154 mmHg/s). The use of a pharmacological approach to inhibit iNOS (aminoguanidine, given ip) in additional wild-type shams and burns confirmed the iNOS KO data. Whereas the absence of iNOS attenuated burn-mediated cardiac contractile dysfunction, these experiments did not determine the contribution of cardiac-derived NO versus NO generated by immune cells. However, our data indicate a role for NO in cardiac dysfunction after major trauma.     

Modulation of {beta}-adrenoceptor signaling in the hearts of 4-wk simulated weightlessness rats.

The modulation of beta-adrenoceptor signaling in the hearts of hindlimb unweighting (HU) simulated weightlessness rats has not been reported. In the present study, we adopted the rat tail suspension for 4 wk to simulate weightlessness; then the effects of simulated microgravity on beta-adrenoceptor signaling were studied. Mean arterial blood pressure (ABP), left ventricular pressure (LVP), systolic function (+dP/dtmax), and diastolic function (-dP/dtmax) were monitored in the course of the in vivo experiment. Single rat ventricular myocyte was obtained by the enzymatic dissociation method. Hemodynamics, myocyte contraction, and cAMP production in response to beta-adrenoceptor stimulation with isoproterenol or adenylyl cyclase stimulation with forskolin were measured, and Gs protein was also determined. Compared with the control group, no significant changes were found in heart weight, body weight and ABP, while LVP and +/-dP/dtmax were significantly reduced. The ABP decrease, LVP increase, and +/-dP/dtmax in response to isoproterenol administration were significantly attenuated in the HU group. The effects of isoproterenol on electrically induced single-cell contraction and cAMP production in myocytes of ventricles in the HU rats were significantly attenuated. The biologically active isoform, Gsalpha (45 kDa) in the heart, was unchanged. Both the increased electrically induced contraction and cAMP production in response to forskolin were also significantly attenuated in the simulated weightlessness rats. Above results indicated that impaired function of adenylyl cyclase causes beta-adrenoceptor desensitization, which may be partly responsible for the depression of cardiac function.     

Effects of exercise training on contractile function in myocardial trabeculae after ischemia-reperfusion.

Potential protective effects of aerobic exercise training on the myocardium, before an ischemic event, are not completely understood. The purpose of the study was to investigate the effects of exercise training on contractile function after ischemia-reperfusion (Langendorff preparation with 15-min global ischemia/30-min reperfusion). Trabeculae were isolated from the left ventricles of both sedentary control and 10- to 12-wk treadmill exercise-trained rats. The maximal normalized isometric force (force/cross-sectional area; Po/CSA) and shortening velocity (Vo) in isolated, skinned ventricular trabeculae were measured using the slack test. Ischemia-reperfusion induced significant contractile dysfunction in hearts from both sedentary and trained animals; left ventricular developed pressure (LVDP) and maximal rates of pressure development and relaxation (+/-dP/dtmax) decreased, whereas end-diastolic pressure (EDP) increased. However, this dysfunction (as expressed as percent change from the last 5 min before ischemia) was attenuated in trained myocardium [LVDP: sedentary -60.8 +/- 6.4% (32.0 +/- 5.5 mmHg) vs. trained -15.6 +/- 8.6% (64.9 +/- 6.6 mmHg); +dP/dtmax: sedentary -54.1 +/- 4.7% (1,058.7 +/- 124.2 mmHg/s) vs. trained -16.7 +/- 8.4% (1,931.9 +/- 188.3 mmHg/s); -dP/dtmax: sedentary -44.4 +/- 2.5% (-829.3 +/- 52.0 mmHg/s) vs. trained -17.9 +/- 7.2% (-1,341.3 +/- 142.8 mmHg/s); EDP: sedentary 539.5 +/- 147.6%; (41.3 +/- 6.0 mmHg) vs. trained 71.6 +/- 30.6%; 11.4 +/- 1.2 mmHg]. There was an average 26% increase in Po/CSA in trained trabeculae compared with sedentary controls, and this increase was not affected by ischemia-reperfusion. Ischemia-reperfusion reduced Vo by 39% in both control and trained trabeculae. The relative amount of the beta-isoform of myosin heavy chain (MHC-beta) was twofold greater in trained trabeculae as well as in the ventricular free walls. Despite a possible increase in the economy in the trained heart, presumed from a greater amount of MHC-beta, ischemia-reperfusion reduced Vo, to a similar extent in both control and trained animals. Nevertheless, the trained myocardium appears to have a greater maximum force-generating ability that may, at least partially, compensate for reduced contractile function induced by a brief period of ischemia.     

Selective right, but not left, coronary endothelial dysfunction precedes development of pulmonary hypertension and right heart hypertrophy in rats

We investigated a causal role for coronary endothelial dysfunction in development of monocrotaline (MCT)-induced pulmonary hypertension and right heart hypertrophy in rats. Significant increases in pulmonary pressure and right ventricular weight did not occur until 3 wk after 60 mg/kg MCT injection (34 +/- 4 vs. 19 +/- 2 mmHg and 37 +/- 2 vs. 25 +/- 1% septum + left ventricular weight in controls, respectively). Isolated right coronary arteries (RCA) showed significant decreases in acetylcholine-induced NO dilation in both 1-wk (33 +/- 3% with 0.3 microM; n = 5) and 3-wk (18 +/- 3%; n = 11) MCT rats compared with control rats (71 +/- 8%, n = 10). Septal coronary arteries (SCA) showed a smaller decrease in acetylcholine dilation (55 +/- 8% and 33 +/- 7%, respectively, vs. 73 +/- 8% in controls). No significant change was found in the left coronary arteries (LCA; 88 +/- 6% and 81 +/- 6%, respectively, vs. 87 +/- 3% in controls). Nitro-L-arginine methyl ester-induced vasoconstriction, an estimate of spontaneous endothelial NO-mediated dilation, was not significantly altered in MCT-treated SCA or LCA but was increased in RCA after 1 wk of MCT (-41 +/- 6%) and decreased after 3 wk (-18 +/- 3% vs. -27 +/- 3% in controls). A marked enhancement to 30 nM U-46619-induced constriction was also noted in RCA of 3-wk (-28 +/- 6% vs. -9 +/- 2% in controls) but not 1-wk (-12 +/- 7%) MCT rats. Sodium nitroprusside-induced vasodilation was not different between control and MCT rats. Together, our findings show that a selective impairment of right, but not left, coronary endothelial function is associated with and precedes development of MCT-induced pulmonary hypertension and right heart hypertrophy in rats.     

Cardioprotective effects of stretch are mediated by activation of sarcolemmal, not mitochondrial, ATP-sensitive potassium channels

To determine whether sarcolemmal and/or mitochondrial ATP-sensitive potassium (K(ATP)) channels (sarcK(ATP), mitoK(ATP)) are involved in stretch-induced protection, isolated isovolumic rat hearts were assigned to the following protocols: nonstretched hearts were subjected to 20 min of global ischemia (Is) and 30 min of reperfusion, and before Is stretched hearts received 5 min of stretch + 10 min of no intervention. Stretch was induced by a transient increase in left ventricular end-diastolic pressure (LVEDP) from 10 to 40 mmHg. Other hearts received 5-hydroxydecanoate (5-HD; 100 microM), a selective inhibitor of mitoK(ATP), or HMR-1098 (20 microM), a selective inhibitor of sarcK(ATP), before the stretch protocol. Systolic function was assessed through left ventricular developed pressure (LVDP) and maximal rise in velocity of left ventricular pressure (+dP/dt(max)) and diastolic function through maximal decrease in velocity of left ventricular pressure (-dP/dt(max)) and LVEDP. Lactate dehydrogenase (LDH) release and ATP content were also measured. Stretch resulted in a significant increase of postischemic recovery and attenuation of diastolic stiffness. At 30 min of reperfusion LVDP and +dP/dt(max) were 87 +/- 4% and 92 +/- 6% and -dP/dt(max) and LVEDP were 95 +/- 9% and 10 +/- 4 mmHg vs. 57 +/- 6%, 53 +/- 6%, 57 +/- 10%, and 28 +/- 5 mmHg, respectively, in nonstretched hearts. Stretch increased ATP content and did not produce LDH release. 5-HD did not modify and HMR-1098 prevented the protection achieved by stretch. Our results show that the beneficial effects of stretch on postischemic myocardial dysfunction, cellular damage, and energetic state involve the participation of sarcK(ATP) but not mitoK(ATP).     

STAT-3 activation is necessary for ischemic preconditioning in hypertrophied myocardium

The JAK-STAT pathway is activated in the early and late phases of ischemic preconditioning (IPC) in normal myocardium. The role of this pathway and the efficacy of IPC in hypertrophied hearts remain largely unknown. We hypothesized that phosphorylated STAT-3 (pSTAT-3) is necessary for effective IPC in pressure-overload hypertrophy. Male Sprague-Dawley rats 8 wk after thoracic aortic constriction (TAC) or sham operation underwent echocardiography and Langendorff perfusion. Randomized hearts were subjected to 30 min of global ischemia and 120 min of reperfusion with or without IPC in the presence or absence of the JAK-2 inhibitor AG-490 (AG). Functional recovery and STAT activation were assessed. TAC rats had a 31% increase in left ventricular mass (1,347 +/- 58 vs. 1,028 +/- 43 mg, TAC vs. sham, P < 0.001), increased anterior and posterior wall thickness but no difference in ejection fraction compared with sham-operated rats. In TAC, IPC improved end-reperfusion maximum first derivative of developed pressure (+dP/dt(max); 4,648 +/- 309 vs. 2,737 +/- 343 mmHg/s, IPC vs. non-IPC, P < 0.05) and minimum -dP/dt (-dP/dt(min); -2,239 +/- 205 vs. -1,215 +/- 149 mmHg/s, IPC vs. non-IPC, P < 0.05). IPC increased nuclear pSTAT-1 and pSTAT-3 in sham-operated rats but only pSTAT-3 in TAC. AG in TAC significantly attenuated +dP/dt(max) (4,648 +/- 309 vs. 3,241 +/- 420 mmHg/s, IPC vs. IPC + AG, P < 0.05) and -dP/dt(min) (-2,239 +/- 205 vs. -1,323 +/- 85 mmHg/s, IPC vs. IPC + AG, P < 0.05) and decreased only nuclear pSTAT-3. In myocardial hypertrophy, JAK-STAT signaling is important in IPC and exhibits a pattern of STAT activation distinct from nonhypertrophied myocardium. Limiting STAT-3 activation attenuates the efficacy of IPC in hypertrophy.     

Organ blood flow and cardiac contractility in anaesthetized cats at 5 bar (500 kPa) ambient pressure

Previous in vivo and in vitro experiments have demonstrated increased cardiac contractility and increased total myocardial blood flow (Qmyocardial) when rats were exposed to normoxic 5-bar (500 kPa) ambient pressure. In the present study, regional blood flow was measured using the microsphere method on nine anaesthetized cats at surface and normoxic 5-bar (500 kPa) ambient pressure. Left ventricular pressure (LVP) and cardiac contractility, measured as peak left ventricular +dP/dt and -dP/dt were measured in six of the cats. Arterial pressure, heart rate and cardiac output remained unchanged after compression, but total Qmyocardial increased by 29% (P less than 0.01) and cerebral blood flow increased by 66% (P less than 0.05). At the same time +dP/dt and -dP/dt was increased by 83% and 102%, respectively (P less than 0.01), while LVP was enhanced by 14% (P less than 0.05). Except for a moderate decrease in partial pressure of oxygen, acid base status in arterial blood remained unchanged. The results indicate that the effects of increased ambient pressure on the heart are general physiological phenomena, which are not only limited to the laboratory rat.     

Endogenous production of ghrelin and beneficial effects of its exogenous administration in monocrotaline-induced pulmonary hypertension

We investigated the endogenous production of ghrelin as well as cardiac and pulmonary vascular effects of its administration in a rat model of monocrotaline (MCT)-induced pulmonary hypertension (PH). Adult Wistar rats randomly received a subcutaneous injection of MCT (60 mg/kg) or an equal volume of vehicle. One week later, animals were randomly assigned to receive a subcutaneous injection of ghrelin (100 mug/kg bid for 2 wk) or saline. Four groups were analyzed: normal rats treated with ghrelin (n=7), normal rats injected with saline (n=7), MCT rats treated with ghrelin (n=9), and MCT rats injected with saline (n=9). At 22-25 days, right (RV) and left ventricular (LV) pressures were measured, heart and lungs were weighted, and samples were collected for histological and molecular analysis. Endogenous production of ghrelin was almost abolished in normal rats treated with ghrelin. In MCT-treated animals, pulmonary expression of ghrelin was preserved, and RV myocardial expression was increased more than 20 times. In these animals, exogenous administration of ghrelin attenuated PH, RV hypertrophy, wall thickening of peripheral pulmonary arteries, and RV diastolic disturbances and ameliorated LV dysfunction, without affecting its endogenous production. In conclusion, decreased tissular expression of ghrelin in healthy animals but not in PH animals suggests a negative feedback in the former that is lost in the latter. A selective increase of ghrelin mRNA levels in the RV of animals with PH might indicate distinct regulation of its cardiac expression. Finally, ghrelin administration attenuated MCT-induced PH, pulmonary vascular remodeling, and RV hypertrophy, indicating that it may modulate PH.     

Low-pressure reperfusion alters mitochondrial permeability transition

We hypothesized that low-pressure reperfusion may limit myocardial necrosis and attenuate postischemic contractile dysfunction by inhibiting mitochondrial permeability transition pore (mPTP) opening. Male Wistar rat hearts (n = 36) were perfused according to the Langendorff technique, exposed to 40 min of ischemia, and assigned to one of the following groups: 1) reperfusion with normal pressure (NP = 100 cmH(2)O) or 2) reperfusion with low pressure (LP = 70 cmH(2)O). Creatine kinase release and tetraphenyltetrazolium chloride staining were used to evaluate infarct size. Modifications of cardiac function were assessed by changes in coronary flow, heart rate (HR), left ventricular developed pressure (LVDP), the first derivate of the pressure curve (dP/dt), and the rate-pressure product (RPP = LVDP x HR). Mitochondria were isolated from the reperfused myocardium, and the Ca(2+)-induced mPTP opening was measured using a potentiometric approach. Lipid peroxidation was assessed by measuring malondialdehyde production. Infarct size was significantly reduced in the LP group, averaging 17 +/- 3 vs. 33 +/- 3% of the left ventricular weight in NP hearts. At the end of reperfusion, functional recovery was significantly improved in LP hearts, with RPP averaging 10,392 +/- 876 vs. 3,969 +/- 534 mmHg/min in NP hearts (P < 0.001). The Ca(2+) load required to induce mPTP opening averaged 232 +/- 10 and 128 +/- 16 microM in LP and NP hearts, respectively (P < 0.001). Myocardial malondialdehyde was significantly lower in LP than in NP hearts (P < 0.05). These results suggest that the protection afforded by low-pressure reperfusion involves an inhibition of the opening of the mPTP, possibly via reduction of reactive oxygen species production.     

Pulmonary oxidative stress is increased in cyclooxygenase-2 knockdown mice with mild pulmonary hypertension induced by monocrotaline

The aim of this study was to examine the role of cyclooxygenase-2 (COX-2) and downstream signaling of prostanoids in the pathogenesis of pulmonary hypertension (PH) using mice with genetically manipulated COX-2 expression. COX-2 knockdown (KD) mice, characterized by 80-90% suppression of COX-2, and wild-type (WT) control mice were treated weekly with monocrotaline (MCT) over 10 weeks. Mice were examined for cardiac hypertrophy/function and right ventricular pressure. Lung histopathological analysis was performed and various assays were carried out to examine oxidative stress, as well as gene, protein, cytokine and prostanoid expression. We found that MCT increased right ventricular systolic and pulmonary arterial pressures in comparison to saline-treated mice, with no evidence of cardiac remodeling. Gene expression of endothelin receptor A and thromboxane synthesis, regulators of vasoconstriction, were increased in MCT-treated lungs. Bronchoalveolar lavage fluid and lung sections demonstrated mild inflammation and perivascular edema but activation of inflammatory cells was not predominant under the experimental conditions. Heme oxygenase-1 (HO-1) expression and indicators of oxidative stress in lungs were significantly increased, especially in COX-2 KD MCT-treated mice. Gene expression of NOX-4, but not NOX-2, two NADPH oxidase subunits crucial for superoxide generation, was induced by ～4-fold in both groups of mice by MCT. Vasodilatory and anti-aggregatory prostacyclin was reduced by ～85% only in MCT-treated COX-2 KD mice. This study suggests that increased oxidative stress-derived endothelial dysfunction, vasoconstriction and mild inflammation, exacerbated by the lack of COX-2, contribute to the pathogenesis of early stages of PH when mild hemodynamic changes are evident and not yet accompanied by vascular and cardiac remodeling.     

Cardiac response to submaximal exercise in dogs susceptible to sudden cardiac death

The hemodynamic response to submaximal exercise was investigated in 38 mongrel dogs with healed anterior wall myocardial infarctions. The dogs were chronically instrumented to measure heart rate (HR), left ventricular pressure (LVP), LVP rate of change, and coronary blood flow. A 2 min coronary occlusion was initiated during the last minute of an exercise stress test and continued for 1 min after cessation of exercise. Nineteen dogs had ventricular fibrillation (susceptible) while 19 animals did not (resistant) during this test. The cardiac response to submaximal exercise was markedly different between the two groups. The susceptible dogs exhibited a significantly higher HR and left ventricular end-diastolic pressure (LVEDP) but a significantly lower left ventricular systolic pressure (LVSP) in response to exercise than did the resistant animals. (For example, response to 6.4 kph at 8% grade; HR, susceptible 201.4 +/- 5.1 beats/min vs. resistant 176.2 +/- 5.6 beats/min; LVEDP, susceptible 19.4 +/- 1.1 mmHg vs. resistant 12.3 +/- 1.7 mmHg; LVSP, susceptible 136.9 +/- 7.9 mmHg vs. resistant 154.6 +/- 9.8 mmHg.) beta-Adrenergic receptor blockade with propranolol reduced the difference noted in the HR response but exacerbated the LVP differences (response to 6.4 kph at 8% grade; HR, susceptible 163.4 +/- 4.7 mmHg vs. resistant 150.3 +/- 6.4 mmHg; LVEDP susceptible 28.4 +/- 2.1 mmHg vs. resistant 19.6 +/- 3.0 mmHg; LVSP, susceptible 122.2 +/- 8.1 mmHg vs. resistant 142.8 +/- 10.7 mmHg). These data indicate that the animals particularly vulnerable to ventricular fibrillation also exhibit a greater degree of left ventricular dysfunction and an increased sympathetic efferent activity.     

Disruption of protein kinase A interaction with A-kinase-anchoring proteins in the heart in vivo: effects on cardiac contractility, protein kinase A phosphorylation, and troponin I proteolysis

Protein kinase A (PKA)-dependent phosphorylation is regulated by targeting of PKA to its substrate as a result of binding of regulatory subunit, R, to A-kinase-anchoring proteins (AKAPs). We investigated the effects of disrupting PKA targeting to AKAPs in the heart by expressing the 24-amino acid regulatory subunit RII-binding peptide, Ht31, its inactive analog, Ht31P, or enhanced green fluorescent protein by adenoviral gene transfer into rat hearts in vivo. Ht31 expression resulted in loss of the striated staining pattern of type II PKA (RII), indicating loss of PKA from binding sites on endogenous AKAPs. In the absence of isoproterenol stimulation, Ht31-expressing hearts had decreased +dP/dtmax and -dP/dtmin but no change in left ventricular ejection fraction or stroke volume and decreased end diastolic pressure versus controls. This suggests that cardiac output is unchanged despite decreased +dP/dt and -dP/dt. There was also no difference in PKA phosphorylation of cardiac troponin I (cTnI), phospholamban, or ryanodine receptor (RyR2). Upon isoproterenol infusion, +dP/dtmax and -dP/dtmin did not differ between Ht31 hearts and controls. At higher doses of isoproterenol, left ventricular ejection fraction and stroke volume increased versus isoproterenol-stimulated controls. This occurred in the context of decreased PKA phosphorylation of cTnI, RyR2, and phospholamban versus controls. We previously showed that expression of N-terminal-cleaved cTnI (cTnI-ND) in transgenic mice improves cardiac function. Increased cTnI N-terminal truncation was also observed in Ht31-expressing hearts versus controls. Increased cTnI-ND may help compensate for reduced PKA phosphorylation as occurs in heart failure.     

Involvement of RhoA/Rho kinase signaling in protection against monocrotaline-induced pulmonary hypertension in pneumonectomized rats by dehydroepiandrosterone

RhoA/Rho kinase (ROCK) signaling plays a key role in the pathogenesis of experimental pulmonary hypertension (PH). Dehydroepiandrosterone (DHEA), a naturally occurring steroid hormone, effectively inhibits chronic hypoxic PH, but the responsible mechanisms are unclear. This study tested whether DHEA was also effective in treating monocrotaline (MCT)-induced PH in left pneumonectomized rats and whether inhibition of RhoA/ROCK signaling was involved in the protective effect of DHEA. Three weeks after MCT injection, pneumonectomized rats developed PH with severe vascular remodeling, including occlusive neointimal lesions in pulmonary arterioles. In lungs from these animals, we detected cleaved (constitutively active) ROCK I as well as increases in activities of RhoA and ROCK and increases in ROCK II protein expression. Chronic DHEA treatment (1%, by food for 3 wk) markedly inhibited the MCT-induced PH (mean pulmonary artery pressures after treatment with 0% and 1% DHEA were 33+/-5 and 16+/-1 mmHg, respectively) and severe pulmonary vascular remodeling in pneumonectomized rats. The MCT-induced changes in RhoA/ROCK-related protein expression were nearly normalized by DHEA. A 3-wk DHEA treatment (1%) started 3 wk after MCT injection completely inhibited the progression of PH (mean pulmonary artery pressures after treatment with 0% and 1% DHEA were 47+/-3 and 30+/-3 mmHg, respectively), and this treatment also resulted in 100% survival in contrast to 30% in DHEA-untreated rats. These results suggest that inhibition of RhoA/ROCK signaling, including the cleavage and constitutive activation of ROCK I, is an important component of the impressive protection of DHEA against MCT-induced PH in pneumonectomized rats.     

Repeated inhalation of adrenomedullin ameliorates pulmonary hypertension and survival in monocrotaline rats

Adrenomedullin (AM) is a potent vasodilator peptide. We investigated whether inhalation of aerosolized AM ameliorates monocrotaline (MCT)-induced pulmonary hypertension in rats. Male Wistar rats given MCT (MCT rats) were assigned to receive repeated inhalation of AM (n = 8) or 0.9% saline (n = 8). AM (5 mug/kg) or saline was inhaled as an aerosol using an ultrasonic nebulizer for 30 min four times a day. After 3 wk of inhalation therapy, mean pulmonary arterial pressure and total pulmonary resistance were markedly lower in rats treated with AM than in those given saline [mean pulmonary arterial pressure: 22 +/- 2 vs. 35 +/- 1 mmHg (-37%); total pulmonary resistance: 0.048 +/- 0.004 vs. 0.104 +/- 0.006 mmHg.ml(-1).min(-1).kg(-1) (-54%), both P < 0.01]. Neither systemic arterial pressure nor heart rate was altered. Inhalation of AM significantly attenuated the increase in medial wall thickness of peripheral pulmonary arteries in MCT rats. Kaplan-Meier survival curves demonstrated that MCT rats treated with aerosolized AM had a significantly higher survival rate than those given saline (70% vs. 10% 6-wk survival, log-rank test, P < 0.01). In conclusion, repeated inhalation of AM inhibited MCT-induced pulmonary hypertension without systemic hypotension and thereby improved survival in MCT rats.     

Positive inotropic, positive chronotropic and coronary vasodilatory effects of rat amylin: mechanisms of amylin-induced positive inotropy

Even though there are a few studies dealing with the cardiac effects of amylin, the mechanisms of amylin-induced positive inotropy are not known well. Therefore, we investigated the possible signaling pathways underlying the amylin-induced positive inotropy and compared the cardiac effects of rat amylin (rAmylin) and human amylin (hAmylin).Isolated rat hearts were perfused under constant flow condition and rAmylin or hAmylin was infused to the hearts. Coronary perfusion pressure, heart rate, left ventricular developed pressure and the maximum rate of increase of left ventricular pressure (+dP/dtmax) and the maximum rate of pressure decrease of left ventricle (-dP/dtmin) were measured.rAmylin at concentrations of 1, 10 or 100 nM markedly decreased coronary perfusion pressure, but increased heart rate, left ventricular developed pressure, +dP/dtmax and -dP/dtmin. The infusion of H-89 (50 μM), a protein kinase A (PKA) inhibitor did not change the rAmylin (100 nM)-induced positive inotropic effect. Both diltiazem (1 μM), an L-type Ca2+ channel blocker and ryanodine (10 nM), a sarcoplasmic reticulum (SR) Ca2+ release channel opener completely suppressed the rAmylin-induced positive inotropic effect, but staurosporine (100 nM), a potent protein kinase C (PKC) inhibitor suppressed it partially. hAmylin (1, 10 and 100 nM) had no significant effect on coronary perfusion pressure, heart rate and developed pressure, +dP/dtmax and -dP/dtmin.We concluded that rAmylin might have been produced vasodilatory, positive chronotropic and positive inotropic effects on rat hearts. Ca2+ entry via L-type Ca2+ channels, activation of PKC and Ca2+ release from SR through ryanodine-sensitive Ca2+ channels may be involved in this positive inotropic effect. hAmylin may not produce any significant effect on perfusion pressure, heart rate and contractility in isolated, perfused rat hearts.     

Cardiac dysfunction in aging conscious rats: altered cardiac cytoskeletal proteins as a potential mechanism

The objective of this study was to test the hypothesis that the mechanism mediating left ventricular (LV) dysfunction in the aging rat heart involves, in part, changes in cardiac cytoskeletal components. Our results show that there were no significant differences in heart rate, LV pressure, or LV diameter between conscious, instrumented young [5.9 +/- 0.3 mo (n = 9)] and old rats [30.6 +/- 0.1 mo (n = 10)]. However, the first derivative of LV pressure (LV dP/dt) was reduced (8,309 +/- 790 vs. 11,106 +/- 555 mmHg/s, P < 0.05) and isovolumic relaxation time (tau) was increased (8.7 +/- 0.7 vs. 6.3 +/- 0.6 ms, P < 0.05) in old vs. young rats, respectively. The differences in baseline LV function in young and old rats, which were modest, were accentuated after beta-adrenergic receptor stimulation with dobutamine (20 mug/kg), which increased LV dP/dt by 170 +/- 9% in young rats, significantly more (P < 0.05) than observed in old rats (115 +/- 5%). Volume loading in anesthetized rats demonstrated significantly impaired LV compliance in old rats, as measured by the LV end-diastolic pressure and dimension relationship. In old rat hearts, there was a significant (P < 0.05) increase in the percentage of LV collagen (2.4 +/- 0.2 vs. 1.3 +/- 0.2%), alpha-tubulin (92%), and beta-tubulin (2.3-fold), whereas intact desmin decreased by 51%. Thus the cardiomyopathy of aging in old, conscious rats may be due not only to increases in collagen but also to alterations in cytoskeletal proteins.     

Cardiac function in hearts isolated from a rat model deficient in mast cells

Several studies have examined the role of mast cells in the myocardial response to injury such as that caused by hypertension and ischemia-reperfusion. However, little is known about the influence of mast cells on normal myocardial structure and function. The present experiments examined cardiac function in Langendorff-perfused hearts isolated from 6- and 9-mo-old male mast cell-deficient (Ws/Ws) and mast cell-competent rats. A fluid-filled balloon catheter was used to measure left ventricular diastolic and systolic function at increasing preload volumes. At 6 mo of age, mast cell-deficient rats showed a slight cardiac hypertrophy (as monitored by heart weight and heart weight-to-body weight ratio) but no significant change in maximum observed systolic or diastolic function. In contrast, at 9 mo of age, the mast cell-deficient group showed no signs of hypertrophy but displayed a diastolic dysfunction characterized by decreased compliance without a significant decline in maximum observed basal -dP/dtmax. There were no significant differences in maximum observed values for measures of systolic function (developed pressure and +dP/dtmax). In summary, the results of this study in adult rats suggest that mast cells influence cardiac function in the absence of injury and that observed differences between mast cell-competent and -deficient animals vary with age. Thus it is important to consider these "physiological" actions and resulting changes in function when studying effects of insult in mast cell-deficient models.     

Changes in biomechanical parameters during heart perfusion and after midazolam pre-medication--experimental pilot study

Background: Midazolam is a frequently used benzodiazepine in anaesthesiology and intensive care. Aim: The aim of pilot study was to monitor its effect during heart perfusion in the laboratory rat. Methods: The same groups of animals (n = 10). The 1(st) group was treated with midazolam in a dose of 0.5mg/kg i.p. The 2(nd) group was a placebo. After i.p. administration of heparine injection of 500 IU dose, the hearts were excised and perfused (modified Langendorf's method). Working schedule: stabilization/ischaemia/reperfusion proceed at intervals of 20/30/60 min. Monitored parameters in isolated heart: left ventricle pressure (LVP), end-diastolic pressure (LVEDP), contractility (+dP/dt(max)). Results: The treated hearts showed improved postischemic recovery, reaching LVP values of 92 +/- 6 % at the end of the reperfusion, placebo only 61 +/- 7 %. In placebo hearts LVEDP rose from 10.0 +/- 0.5 mmHg to 43 +/- 4 mmHg after, in treated animals only about 25 mmHg. The treated hearts improved +dP/dt(max) recovery during reperfusion to 91 +/- 8 %. These values were significantly greater than those obtained from the placebo hearts. Conclusions: Positive changes in monitored parameters were found in this experimental pilot study. We conclude that the administration of midazolam in laboratory rats has a cardioprotective potential against ischemia-reperfusion induced injury.     

Effect of 5-lipoxygenase on the development of pulmonary hypertension in rats

5-Lipoxygenase (5-LO) and its downstream leukotriene products have been implicated in the development of pulmonary hypertension. In this study, we examined the effects of 5-LO overexpression in rat lungs on pulmonary hypertension using a recombinant adenovirus expressing 5-LO (Ad5-LO). Transthoracic echocardiography and right heart catheterization data showed that 5-LO overexpression in the lung did not cause pulmonary hypertension in normal rats; however, it markedly accelerated the progression of pulmonary hypertension in rats treated with monocrotaline (MCT). An increase in pulmonary artery pressure occurred earlier in the rats treated with MCT + Ad5-LO (7-10 days) compared with those treated with control vector, MCT + adenovirus expressing green fluorescent protein (AdGFP), or MCT alone (15-18 days). The weight ratio of the right ventricle to left ventricle plus septum was higher in the MCT + Ad5-LO group than that of the MCT + AdGFP or MCT group (0.45 +/- 0.08 vs. 0.35 +/- 0.03 or 0.33 +/- 0.06). Lung tissue histological sections from MCT + Ad5-LO rats exhibited more severe inflammatory cell infiltration and pulmonary vascular muscularization than those from MCT + AdGFP- or MCT-treated rats. Administration of 5-LO inhibitors, zileuton or MK-886, to either MCT- or MCT + Ad5-LO-treated rats prevented the development of pulmonary hypertension. These data suggest that 5-LO plays a critical role in the progression of pulmonary hypertension in rats and that the detrimental effect of 5-LO is manifest only in the setting of pulmonary vascular endothelial cell dysfunction.     

Effect of chronic administration of aminoguanidine on the reactivity of pulmonary vessels in rats with monocrotaline-induced pulmonary hypertension

Monocrotaline (MCT)-induced pulmonary hepertension (PH) is associated with impaired endothelium-dependent relaxation and increased activity of inducible NO-synthase (iNOS). To examine the role of iNOS in MCT-induced PH, we used iNOS inhibitor: aminoguanidine (AG). The PH was simulated with a subcutaneous injection of 60 mg/kg MCT to Wistar rats; control rats were injected with saline. Then each group was separated into 2 subgroups: the 1st one was given drinking water (MCT-C and C-C groups) whereas the 2nd one was given AG in drinking water (15 mg/(kg(-1) x day(-1)) (MCT-AG and C-AG groups). In 4 weeks, the perfusion pressure (PP) responses of isolated pulmonary arteries to acetylcholine (Ach) and activator of soluble guanylate cyclase (sGC), FPTO, were examined. In the MCT-C group, a decrease of relative PP to perfusion of 1 x 10(-8) M and 5 x 10(-8) M Ach and 1 x 10(-8) M FPTO was diminished. This reduction of relaxant responses in MCT-treated rats was prevented by AG treatment. The findings suggest that AG administration restores the impaired endothelium-dependent and sGC-dependent relaxation of the pulmonary artery at MCT-induced PH.