Molecular assessment of ammonia- and nitrite-oxidizing bacteria in full-scale activated sludge wastewater treatment plants.

Nitrification was assessed in two full-scale wastewater treatment plants (WWTPs) over time using molecular methods. Both WWTPs employed a complete-mix suspended growth, aerobic activated sludge process (with biomass recycle) for combined carbon and nitrogen treatment. However, one facility treated primarily municipal wastewater while the other only industrial wastewater. Real time PCR assays were developed to determine copy numbers for total 16S rDNA (a measure of biomass content), the amoA gene (a measure of ammonia-oxidizers), and the Nitrospira 16S rDNA gene (a measure of nitrite-oxidizers) in mixed liquor samples. In both the municipal and industrial WWTP samples, total 16S rDNA values were approximately 2-9 x 10(13) copies/L and Nitrospira 16S rDNA values were 2-4 x 10(10) copies/L. amoA gene concentrations averaged 1.73 x 10(9) copies/L (municipal) and 1.06 x 10(10) copies/L (industrial), however, assays for two distinct ammonia oxidizing bacteria were required.

Quantification of Nitrosomonas oligotropha and Nitrospira spp. using competitive polymerase chain reaction in bench-scale wastewater treatment reactors operating at different solids retention times.

The effect of solids retention time (SRT) on ammonia-and nitrite-oxidizing bacteria was measured by Nitrosomonas oligotropha-like ammonia monooxygenase A and Nitrospira 16S rDNA competitive polymerase chain reaction assays in a complete-mix, bench-scale, activated-sludge system. During steady-state operation, nitrification was complete in the 20- and 10-day SRT reactors, nearly complete in the 5-day SRT reactor, and incomplete in the 2-day SRT reactor (76% ammonia oxidation and 85% nitrite oxidation). Total microbes, measured by dot-blot hybridizations, ranged from 3 x 10(11) to 3 x 10(12) cells/L, and increased with increasing SRTs. The concentration of the ammonia-oxidizer N. oligotropha dropped 100-fold from the 20-day SRT (5 x 10(9) cells/L) to the 2-day SRT (< or = 4 x 10(7) cells/L). Thus, N. oligotropha became a much smaller fraction of the total biomass in the poorly performing 2-day SRT reactor. The concentration of Nitrospira cells also decreased (10-fold) as the SRT was reduced from 20 days to 2 days. However, the number of Nitrospira cells was always greater than the number of N. oligotropha cells measured in each reactor (10- to 60-fold). While Nitrospira comprised 1 to 2% of the biomass, N. oligotropha represented only 0.04 to 0.27% of the total population. This low percentage suggests that N. oligotropha was not a dominant ammonia oxidizer in the bench-scale systems.

The measurement of toluene dioxygenase activity in biofilm culture of Pseudomonas putida F1.

Toluene dioxygenase (Tod) enzyme activity can be measured by the conversion of indole to indigo. Indigo is measured spectrophotometrically at 600 nm. However, this method is inadequate to measure the whole-cell enzyme activity when interference by suspended biomass is present. Indoxyl is a highly fluorescent intermediate in the conversion of indole to indigo by Tod. A fluorescence-based assay was developed and applied to monitor Tod activity in whole cells of Pseudomonas putida F1 biofilm from a continuously operated biofilter. Suspended growth studies with pure cultures indicated that indoxyl, as measured by fluorescence, correlated with indigo production (r(2)=0.89) as measured by spectrophotometry. Whole-cell enzyme activity was followed during growth on a minimal medium containing toluene. The maximum normalized whole cell enzyme activity of 19+/-1.5x10(-4) mg indigo (mg protein)(-1) min(-1) was reached during early stationary phase. P. putida F1 cells from a biofilm grown on vapor phase toluene had a normalized whole-cell enzyme activity of 5.0+/-0.2x10(-4) mg indigo (mg protein)(-1) min(-1). The half-life of whole-cell enzyme activity was estimated to be between 5.5 and 8 h in both suspended and biofilm growth conditions.

Direct effects of colchicine on myocardial function: studies in hypertrophied and failing spontaneously hypertensive rats.

-The aging spontaneously hypertensive rat (SHR) is a model in which the transition from chronic stable left ventricular hypertrophy to overt heart failure can be observed. Although the mechanisms for impaired function in hypertrophied and failing cardiac muscle from the SHR have been studied, none accounts fully for the myocardial contractile abnormalities. The cardiac cytoskeleton has been implicated as a possible cause for myocardial dysfunction. If an increase in microtubules contributes to dysfunction, then myocardial microtubule disruption by colchicine should promote an improvement in cardiac performance. We studied the active and passive properties of isolated left ventricular papillary muscles from 18- to 24-month-old SHR with evidence of heart failure (SHR-F, n=6), age-matched SHR without heart failure (SHR-NF, n=6), and age-matched normotensive Wistar-Kyoto rats (WKY, n=5). Mechanical parameters were analyzed before and up to 90 minutes after the addition of colchicine (10(-5), 10(-4), and 10(-3) mol/L). In the baseline state, active tension (AT) developed by papillary muscles from the WKY group was greater than for SHR-NF and SHR-F groups (WKY 5.69+/-1.47 g/mm2 [mean+/-SD], SHR-NF 3.41+/-1.05, SHR-F 2.87+/-0.26; SHR-NF and SHR-F P<0.05 versus WKY rats). The passive stiffness was greater in SHR-F than in the WKY and SHR-NF groups (central segment exponential stiffness constant, Kcs: SHR-F 70+/-25, SHR-NF 44+/-17, WKY 41+/-13 [mean+/-SD]; SHR-F P<0.05 versus SHR-NF and WKY rats). AT did not improve after 10, 20, and 30 minutes of exposure to colchicine (10(-5), 10(-4), and 10(-3) mol/L) in any group. In the SHR-F group, AT and passive stiffness did not change after 30 to 90 minutes of colchicine exposure (10(-4) mol/L). In summary, the data in this study fail to demonstrate improvement of intrinsic muscle function in SHR with heart failure after colchicine. Thus, in the SHR there is no evidence that colchicine-induced cardiac microtubular depolymerization affects the active or passive properties of hypertrophied or failing left ventricular myocardium.

Myocardial osteopontin expression coincides with the development of heart failure.

To identify genes that are differentially expressed during the transition from compensated hypertrophy to failure, myocardial mRNA from spontaneously hypertensive rats (SHR) with heart failure (SHR-F) was compared with that from age-matched SHR with compensated hypertrophy (SHR-NF) and normotensive Wistar-Kyoto rats (WKY) by differential display reverse transcriptase-polymerase chain reaction. Characterization of a transcript differentially expressed in SHR-F yielded a cDNA with homology to the extracellular matrix protein osteopontin. Northern analysis showed low levels of osteopontin mRNA in left ventricular myocardium from WKY and SHR-NF but a markedly increased (approximately 10-fold) level in SHR-F. In myocardium from WKY and SHR-NF, in situ hybridization showed only scant osteopontin mRNA, primarily in arteriolar cells. In SHR-F, in situ hybridization revealed abundant expression of osteopontin mRNA, primarily in nonmyocytes in the interstitial and perivascular space. Similar findings for osteopontin protein were observed in the midwall region of myocardium from the SHR-F group. Consistent with the findings in SHR, osteopontin mRNA was minimally increased (approximately 1.9-fold) in left ventricular myocardium from nonfailing aortic-banded rats with pressure-overload hypertrophy but was markedly increased (approximately 8-fold) in banded rats with failure. Treatment with captopril starting before or after the onset of failure in the SHR reduced the increase in left ventricular osteopontin mRNA levels. Thus, osteopontin expression is markedly increased in the heart coincident with the development of heart failure. The source of osteopontin in SHR-F is primarily nonmyocytes, and its induction is inhibited by an angiotensin-converting enzyme inhibitor, suggesting a role for angiotensin II. Given the known biological activities of osteopontin, including cell adhesion and regulation of inducible nitric oxide synthase gene expression, these data suggest that it could play a role in the pathophysiology of heart failure.

Effect of angiotensin-converting enzyme inhibition on myocardial fibrosis and function in hypertrophied and failing myocardium from the spontaneously hypertensive rat.

BACKGROUND: After a period of stable hypertrophy, male spontaneously hypertensive rats (SHR) develop heart failure between 18 to 24 months of age, with depression of active myocardial function and increased passive stiffness. We tested the hypothesis that chronic ACE inhibition by captopril would prevent and possibly reverse impairment of myocardial function. METHODS AND RESULTS: Male SHR and normotensive Wistar-Kyoto rats (WKY) were assigned to no treatment or captopril treatment (2 g/L in drinking water) begun at ages 12, 18, and 21 months; animals were studied at 24 months of age, or earlier when evidence of heart failure was found in SHR (mean age, 19+/-2 months). In an additional group, captopril treatment was begun when SHR developed heart failure; surviving animals were studied at 24 months of age. In untreated SHR, relative to WKY, isometric stress development at Lmax, maximum rate of stress development, and shortening velocity were depressed, whereas passive stiffness was increased, in association with the development of myocardial fibrosis. In the SHR treated before cardiac dysfunction, captopril administration attenuated hypertrophy and prevented contractile dysfunction, fibrosis, and increased passive stiffness. Captopril treatment begun after cardiac function was impaired reduced left ventricular hypertrophy but did not restore intrinsic contractile function or reduce fibrosis or passive stiffness. CONCLUSIONS: In the male SHR, early treatment with captopril was associated with the most marked attenuation of dysfunction relative to the untreated SHR. Treatment initiated after the onset of heart failure improved clinical signs of heart failure and decreased left ventricular hypertrophy in surviving animals but did not reverse the fibrosis and contractile dysfunction associated with heart failure.

Participação do estado contrátil e do relaxamento miocárdico na disfunção ventricular durante a transição hipertrofia-falência cardíaca / Myocardial function during the transition from compensated left ventricular hypertrophy to failure

OBJETIVO - Avaliar a participação do estado contrátil e do relaxamento miocárdico na disfunção do músculo cardíaco durante a transição hipertrofia-falência cardíaca em ratos espontaneamente hipertensos (SHR). MÉTODOS - Músculos papilares isolados do ventrículo esquerdo de SHR com insuficiência cardíaca (SHR-IC) e sem falência (SHR) e de ratos normotensos controle Wistar-Kyoto (WKY) foram estudados em contraçöes isométrica e isotônica, em solução de Krebs-Henseleit (1,25 mM Ca 'elevado a +2', 28 'graus Celsius'). RESULTADOS - Os valores da tensão máxima desenvolvida (TD) e da velocidade máxima de encurtamenton (V máx) foram menores nos SHR-IC e SHR, em relação aos WKY (p<0,05). TD e V máx foram semelhantes nos SHR-IC e SHR (p>0,05). A rigidez passiva do músculo aumentou significantemente nos SHR-IC (p<0,05 vs WKY e SHR); esta variável não diferiu entre WKY e SHR (p>0,05). CONCLUSÄO - Os dados obtidos mostram que a transição da fase de hipertrofia estável para insuficiência cardíaca nos ratos espontaneamente hipertensos está associado ao aumento da rigidez passiva do miocárdio e näo à piora da funçäo contrátil do músculo cardíaco.

Localization of alpha1(I) collagen mRNA in myocardium from the spontaneously hypertensive rat during the transition from compensated hypertrophy to failure.

Spontaneously hypertensive rats (SHR) commonly develop impairment of myocardial function between ages 18-24 months. Isolated muscle studies demonstrate depressed myocardial contractility and increased passive stiffness. Studies of the extracellular matrix in SHR with failure (SHR-F) demonstrate an increased expression of genes encoding extracellular matrix components (ECM), hydroxyproline concentration and fibrosis relative to age-matched non-failing animals. In the present study, tissue sections of hearts from SHR-F, non-failing SHR (SHR-NF) and non-hypertensive Wistar Kyoto rats (WKY) were hybridized with a cDNA probe for alpha1(I) collagen mRNA, which was found by Northern blot analysis to be elevated in SHR-F relative to hearts from control animals. In situ hybridization studies demonstrate increased perivascular and interstitial collagen alpha1(I) gene expression in myocardium from the SHR relative to WKY. In addition, failing hearts from the SHR demonstrate focal alpha1(I) collagen mRNA accumulation in the endocardium and at sites of degenerating single myocardial cells.

Increased cardiomyocyte apoptosis during the transition to heart failure in the spontaneously hypertensive rat.

The transition from compensated hypertrophy to failure in spontaneously hypertensive rats (SHR) of advanced age is associated with a marked increase in collagen, a reduction in myocyte mass, and a reduction in maximum Ca(2+)-activated myofibrillar force. We hypothesized that the reduction in myocyte mass and associated functional loss may be due to increased cell death by apoptosis. To test this hypothesis, we studied hearts from failing (SHR-F) and nonfailing SHR (SHR-NF) and age-matched Wistar-Kyoto rats (WKY). In addition, hearts from SHR-F that had been treated with an angiotensin-converting enzyme inhibitor (captopril) for an average of 27 days were also studied. Apoptotic cells were quantified in cross sections of myocardium by the terminal deoxynucleotidyltransferase- mediated 2'-deoxyuridine 5'-triphosphate nick end labeling technique. To identify the type of the cells undergoing apoptosis, sections were also stained for alpha-sarcomeric actin. Apoptotic cells were significantly increased in the SHR-F (38.92 +/- 12.79 vs. 8.05 +/- 3.98 cells/100,000 nuclei in SHR-NF; P < 0.05 and vs. 2.21 +/- 1.4 cells/100,000 nuclei in WKY; P < 0.01). Captopril treatment of SHR-F reduced the number of apoptotic cells to the level in SHR-NF (9.17 +/- 1.53 cells/100,000 nuclei; P < 0.01 vs. SHR-F). Most apoptotic cells were of cardiac myocyte origin. There was no significant difference in Bcl-2 protein expressed by hearts among the three groups. WAF-1 mRNA levels were increased in both SHR groups vs. WKY; in SHR-F, the density of WAF-1 mRNA was higher than in SHR-NF. Thus increased numbers of apoptotic cells are present in failing SHR hearts, suggesting that apoptosis might be a mechanism involved in the reduction of myocyte mass that accompanies the transition from stable compensation to heart failure in this model. Administration of the angiotensin-converting enzyme inhibitor captopril, which ameliorates heart failure in this model, is associated with a reduction in the exaggerated apoptosis that accompanies heart failure.

Effect of chronic colchicine administration on the myocardium of the aging spontaneously hypertensive rat.

Colchicine has been demonstrated to suppress the release of fibroblast growth factors, retard collagen formation and augment collagenase activity. Trials with colchicine in patients with hepatic fibrosis have suggested clinical benefit. The development of impaired myocardial function in the spontaneously hypertensive rat (SHR) is associated with a marked increase in myocardial fibrosis. The present study was carried out to test the hypothesis that chronic colchicine administration to the SHR would prevent the development of fibrosis and impaired myocardial performance. Colchicine (1 mg/l drinking water) was administered to male SHR and WKY rats from at age 13 months until 24 months or until evidence of heart failure was observed. Age-matched untreated SHR and colchicine treated and untreated WKY served as controls. At study, active and passive properties of isolated left ventricular muscle preparations were determined. Myocardial fibrosis was assessed by measuring hydroxyproline and histologic determination of interstitial cross-sectional area. Increases in LV hydroxyproline and interstitial area were found in untreated SHR relative to WKY; passive myocardial stiffness was increased and active muscle properties were depressed. In comparing colchicine treated vs untreated SHR, no differences in hydroxyproline, interstitial area or intrinsic myocardial function were found. In the WKY, colchicine increased myocardial interstitium and passive stiffness without changing hydroxyproline. Active myocardial function was not depressed. Thus, chronic colchicine administration neither attenuated the development of interstitial fibrosis nor prevented impaired myocardial function in the SHR. Colchicine treatment was associated with increased interstitium in WKY with increased passive myocardial stiffness.

Reduction of hexavalent uranium from organic complexes by sulfate- and iron-reducing bacteria.

The influence of organic-hexavalent-uranium [U(VI)] complexation on U(VI) reduction by a sulfate-reducing bacterium (Desulfovibrio desulfuricans) and an iron-reducing bacterium (Shewanella alga) was evaluated. Four aliphatic ligands (acetate, malonate, oxalate, and citrate) and an aromatic ligand (tiron [4,5-dihydroxy-1,3-benzene disulfonic acid]) were used to study complexed-uranium bioavailability. The trends in uranium reduction varied with the nature and the amount of U(VI)-organic complex formed and the type of bacteria present. D. desulfuricans rapidly reduced uranium from a monodentate aliphatic (acetate) complex. However, reduction from multidentate aliphatic complexes (malonate, oxalate, and citrate) was slower. A decrease in the amount of organic-U(VI) complex in solution significantly increased the rate of reduction. S. alga reduced uranium more rapidly from multidentate aliphatic complexes than from monodentate aliphatic complexes. The rate of reduction decreased with a decrease in the amount of multidentate complexes present. Uranium from an aromatic (tiron) complex was readily available for reduction by D. desulfuricans, while an insignificant level of U(VI) from the tiron complex was reduced by S. alga. These results indicate that selection of bacteria for rapid uranium reduction will depend on the organic composition of waste streams.

[Role of myocardial contractile status and relaxation in ventricular dysfunction during the transition of heart hypertrophy to failure]. / Participação do estado contrátil e do relaxamento miocárdico na disfunção ventricular durante a transição hipertrofia-falência cardíaca.

PURPOSE: To investigate the participation of contractile state and relaxation in cardiac muscle dysfunction during the transition from stable hypertrophy to cardiac decompensation in aging spontaneously hypertensive rats (SHR). METHODS: Isolated left ventricular papillary muscle function was studied in SHR with heart failure (SHR-F), in age-matched SHR without evidence of heart failure (SHR-NF), and in nonhypertensive controls Wistar-Kyoto rats (WKY). Muscles were analysed in isometric and isotonic contractions in Krebs-Henseleit solution with calcium concentration of 1.25 mM at 28 degrees C. RESULTS: Papillary muscles from SHR-F and SHR-NF demonstrated decreased active tension development and shortening velocity relative to normotensive WKY (p < 0.05). SHR-F and SHR-NF did not differ. Compared with SHR-NF and WKY, muscle passive stiffness was increased in the failing SHR (p < 0.05 versus WKY and SHR-NF). This parameter did not differ between SHR-NF and WKY (p > 0.05). CONCLUSION: These data suggest that the progression from stable hypertrophy to heart failure is associated with changes in the passive stiffness and is not related to depression of myocardial contractile function.

Modulation of left and right ventricular beta-adrenergic receptors from spontaneously hypertensive rats with left ventricular hypertrophy and failure.

Inotropic responsiveness to beta-adrenergic stimulation is generally found to be depressed in cardiac hypertrophy and failure. To investigate whether inotropic responsiveness is associated with alterations in beta-adrenergic receptors in spontaneously hypertensive rats (SHR), we studied left ventricular myocardial contractile responses to isoproterenol and beta-adrenergic receptor density and affinity in age-matched rats (18 to 24 months), including SHR without heart failure, SHR with evidence of heart failure, and normotensive control Wistar-Kyoto rats (WKY). In the baseline state, papillary muscles from failing SHR demonstrated decreased isometric tension development and a reduction in maximal rate of tension development relative to normotensive WKY and compensated SHR. Compared with WKY, beta-adrenergic receptor density of the left ventricle was unchanged in nonfailing SHR and increased in failing SHR (P < .05 versus WKY and nonfailing SHR), and beta-adrenergic receptor affinity did not differ among groups. In the right ventricle, beta-adrenergic receptor density was decreased in failing SHR relative to WKY and nonfailing SHR, and beta-adrenergic receptor affinity was not different among groups. Muscle preparations did not exhibit a positive inotropic response to 10(-8) to 10(-5) mol/L isoproterenol or 6.3 mumol/L forskolin in either failing or nonfailing SHR, whereas a positive inotropic response to both drugs was observed in the normotensive WKY. The lusitropic response to isoproterenol and forskolin was intact and similar in both SHR groups and WKY. The findings suggest that in the SHR model of heart failure, impaired intrinsic left ventricular myocardial function and depressed inotropic responsiveness to beta-adrenergic stimulation are not associated with downregulation of the beta-adrenergic receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

The spontaneously hypertensive rat as a model of the transition from compensated left ventricular hypertrophy to failure.

Studies of hemodynamics and intrinsic left ventricular myocardial function are carried out to investigate the transition from stable hypertrophy to cardiac decompensation in the aging (18-24 months) spontaneously hypertensive rat (SHR). Echocardiographic data in awake animals demonstrate increased end-diastolic and end-systolic volumes and depressed ejection fractions in left ventricles from SHR with failure (SHR-F) as compared to age matched hypertensive (SHR-NF) and non-hypertensive control animals (WKY). Cardiac catheterization data in anesthetized animals demonstrate depression of both systolic pressure and +dP/dt, and elevated end-diastolic pressure in the SHR-F relative to the two control groups. Since loading conditions and altered demand states may contribute to altered ventricular function, studies of isolated perfused hearts were carried out which demonstrate impaired systolic stress development in the SHR-F group under conditions in which loading conditions are controlled; in addition, it is observed that increasing perfusion pressure by 30 mm Hg has little effect on function. Depression of systolic function and increases in passive stiffness of isolated muscle preparations from the SHR-F indicate impairment of systolic and diastolic function at the tissue level. While all of the preparations studied have potential shortcomings, an integration of findings from these complementary approaches supports the conclusion that heart failure develops in the aging SHR. Furthermore, these data suggest that impaired function is due to changes in the intrinsic properties of the myocardium and that the connective tissue response may play an important role. These studies, in conjunction with the findings of others who have studied the aging SHR, provide support for the use of the aging SHR as a model of the transition from compensated hypertrophy to failure.

Myocardial fibrosis and stiffness with hypertrophy and heart failure in the spontaneously hypertensive rat.

BACKGROUND: Fibrosis is commonly found in association with cardiac hypertrophy and failure, but the relation of the connective tissue response to the development of impaired cardiac function remains unclear. We examined passive myocardial stiffness, active contractile function, and fibrosis in the spontaneously hypertensive rat (SHR), a model of chronic pressure overload in which impaired cardiac function follows a long period of stable hypertrophy. METHODS AND RESULTS: We studied the passive and active mechanical properties of left ventricular (LV) papillary muscles isolated from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) at the ages of 12 months and 20 to 23 months. Seven of 15 SHR between 20 and 23 months of age had findings consistent with heart failure (SHR-F). In comparison to preparations from WKY rats and nonfailing SHR (SHR-NF), papillary muscles from the SHR-F group demonstrated increased passive stiffness (central segment exponential stiffness constant, kcs: SHR-F 95.6 +/- 19.8, SHR-NF 42.1 +/- 9.7, WKY rats 39.5 +/- 9.5 (mean +/- SD); SHR-F P < .01 versus SHR-NF, WKY rats). The increase in stiffness was associated with an increase in LV collagen concentration (SHR-F 8.71 +/- 3.14, SHR-NF 5.83 +/- 1.20, WKY rats 4.78 +/- 0.70 mg hydroxyproline/g dry LV wt; SHR-F P < .01 versus SHR-NF, WKY rats); an increase in interstitial fibrosis, as determined histologically (SHR-F 13.5 +/- 8.0%, SHR-NF 4.9 +/- 2.1%, WKY rats 3.6 +/- 0.8%; SHR-F P < .01 versus SHR-NF, WKY rats); and impaired tension development (SHR-F 3.18 +/- 1.27, SHR-NF 4.41 +/- 1.04, WKY rats 4.64 +/- 0.85 kdyne/mm2; SHR-F P < .05 versus SHR-NF; P < .01 versus WKY rats). CONCLUSIONS: The development of heart failure in the aging SHR is associated with marked myocardial fibrosis, increased passive stiffness, and impaired contractile function relative to age-matched nonfailing SHR and nonhypertensive control animals. These data suggest that fibrosis or events underlying the connective tissue response are important in the transition from compensated hypertrophy to failure in the SHR.

Protective effects of diltiazem on the mechanical performance of the hypoxic myocardium.

1. To determine whether diltiazem protects the hypoxic myocardium by reducing contractile work, we have compared the effects of diltiazem and quiescence on left ventricular (LV) papillary muscle subjected to hypoxia. Papillary muscles were obtained from male Charles River CD rats weighing 150-250 g. 2. Four groups of muscles were studied: control (N = 6), non-stimulation (N = 10), diltiazem 10(-4) M (N = 6) and diltiazem 10(-4) M plus non-stimulation (N = 10). 3. Isolated rat LV papillary muscles were studied in Krebs-Henseleit solution with a calcium concentration of 2.52 mM at 28 degrees C while contracting isometrically at a stimulation rate of 0.2 Hz. Resting tension and active isometric tension were measured. 4. Both diltiazem and quiescence significantly attenuated contracture tension during hypoxia (0.91 +/- 0.10 vs 2.26 +/- 0.49 g/mm2 for diltiazem vs control, and 0.55 +/- 0.18 vs 2.26 +/- 0.49 g/mm2 for quiescence vs control). Recovery of active tension was improved in the diltiazem groups during reoxygenation (4.16 +/- 0.42 vs 3.75 +/- 0.51, 3.53 +/- 0.15 vs 2.90 +/- 0.13, 5.84 +/- 0.33 vs 6.48 +/- 0.29 and 5.98 +/- 0.90 vs 7.67 +/- 0.68 g/mm2 for diltiazem, diltiazem non-stimulation, non-stimulation and control groups). 5. The results suggest that the protective effect of diltiazem during hypoxia was due to the reduction in energy demand of the myocardium.

Protective effects of diltiazem on the mechanical performance of the hypoxic myocardium

1. To determine whether diltiazem protects the hypoxic myocardium by reducing contractile work, we have compared the effects of diltiazem and quiescence on left ventricular (LV) papillary muscle subjected to hypoxia. Papillary muscles were obtained from male Charles River CD rats weighing 150-250 g. 2. Four groups of muscles were studied: control (N = 6), non-stimulation (N = 10), diltiazem 10(-4) M (N = 6) and diltiazem 10(-4) M plus non-stimulation (N = 10). 3. Isolated rat LV papillary muscles were studied in Krebs-Henseleit solution with a calcium concentration of 2.52 mM at 28 degrees C while contracting isometrically at a stimulation rate of 0.2 Hz. Resting tension and active isometric tension were measured. 4. Both diltiazem and quiescence significantly attenuated contracture tension during hypoxia (0.91 +/- 0.10 vs 2.26 +/- 0.49 g/mm2 for diltiazem vs control, and 0.55 +/- 0.18 vs 2.26 +/- 0.49 g/mm2 for quiescence vs control). Recovery of active tension was improved in the diltiazem groups during reoxygenation (4.16 +/- 0.42 vs 3.75 +/- 0.51, 3.53 +/- 0.15 vs 2.90 +/- 0.13, 5.84 +/- 0.33 vs 6.48 +/- 0.29 and 5.98 +/- 0.90 vs 7.67 +/- 0.68 g/mm2 for diltiazem, diltiazem non-stimulation, non-stimulation and control groups). 5. The results suggest that the protective effect of diltiazem during hypoxia was due to the reduction in energy demand of the myocardium

Impaired myocardial function in spontaneously hypertensive rats with heart failure.

We have observed that many spontaneously hypertensive rats (SHR) between the ages of 18 and 24 mo develop findings suggestive of heart failure, including pleural and pericardial effusions, left atrial thrombi, and right ventricular hypertrophy. Isolated left ventricular papillary muscle function was studied in these animals (SHR-F), in age-matched SHRs without evidence of heart failure (SHR-NF), and in nonhypertensive controls (WKY). Preparations from SHR-F showed depression of active tension development (3.58 +/- 1.75 g/mm2, means +/- SD) compared with both SHR-NF (7.17 +/- 0.94) and WKY (6.17 +/- 1.00) (P less than 0.01). Shortening velocity was also depressed in SHR-F (0.95 +/- 0.38 lengths/s) compared with SHR-NF (1.60 +/- 0.30; P less than 0.05) and WKY groups (2.15 %/- 0.48; P less than 0.01). Depression of muscle function was not found before 18 mo of age. Thus the aging SHR is a model in which one can observe the transition from chronic stable left ventricular hypertrophy to overt heart failure. Furthermore, left ventricular papillary muscles from SHRs with heart failure show evidence of significant contractile dysfunction, suggesting that impairment of intrinsic myocardial function underlies the development of heart failure.

Preload does not affect relaxation rate in normal, hypoxic, or hypertrophic myocardium.

To determine whether isolated changes in preload (end-diastolic force) can influence myocardial relaxation rate in normal or abnormal (hypoxic or hypertrophic) hearts, isolated LV papillary muscles from normal Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats were studied using physiologically sequenced contractions. While total (systolic) load and late (lengthening) load were held constant, maximum isometric force decline (peak -dT/dt) and maximum isotonic lengthening rate (peak +dL/dt) were measured at seven levels of preload that varied from 115 to 55% of the resting tension at maximum length-tension curves (Lmax). Muscles from normal rats were studied in the oxygenated state (95% O2-5% CO2) and in the hypoxic state (95% N2-5% CO2). Preload did not effect peak -dT/dt or peak +dL/dt in either oxygenated or hypoxic muscles. During hypoxia, peak -dT/dt and peak +dL/dt were 9.5 +/- 1.0 g.mm-2.s-1 and 0.3 +/- 0.1 muscle length/s, respectively, at a preload of 115% compared with 9.0 +/- 1.2 g.mm-2.s-1 and 0.2 +/- 0.1 at a preload of 55%. In separate experiments, the effect of preload on relaxation rate was studied in WKY and SHR rats. In neither group did preload have an independent effect on relaxation rate. In the SHRs, peak -dT/dt and peak +dL/dt were 24.3 +/- 5.3 g.mm-2.s-1 and 0.7 +/- 0.1 muscle length/s, respectively, at a preload of 115% compared with 24.7 +/- 6.6 and 0.8 +/- 0.1 at a preload of 55%. Thus, in hypoxic and hypertrophic myocardium, as in normal muscle, an acute isolated change in preload did not influence the rate of force decline or muscle lengthening.