Avoidance of Profound Hypothermia During Initial Reperfusion Improves the Functional Recovery of Hearts Donated After Circulatory Death.

The resuscitation of hearts donated after circulatory death (DCD) is gaining widespread interest; however, the method of initial reperfusion (IR) that optimizes functional recovery has not been elucidated. We sought to determine the impact of IR temperature on the recovery of myocardial function during ex vivo heart perfusion (EVHP). Eighteen pigs were anesthetized, mechanical ventilation was discontinued, and cardiac arrest ensued. A 15-min standoff period was observed and then hearts were reperfused for 3 min at three different temperatures (5°C; N = 6, 25°C; N = 5, and 35°C; N = 7) with a normokalemic adenosine-lidocaine crystalloid cardioplegia. Hearts then underwent normothermic EVHP for 6 h during which time myocardial function was assessed in a working mode. We found that IR coronary blood flow differed among treatment groups (5°C = 483 ± 53, 25°C = 722 ± 60, 35°C = 906 ± 36 mL/min, p < 0.01). During subsequent EVHP, less myocardial injury (troponin I: 5°C = 91 ± 6, 25°C = 64 ± 16, 35°C = 57 ± 7 pg/mL/g, p = 0.04) and greater preservation of endothelial cell integrity (electron microscopy injury score: 5°C = 3.2 ± 0.5, 25°C = 1.8 ± 0.2, 35°C = 1.7 ± 0.3, p = 0.01) were evident in hearts initially reperfused at warmer temperatures. IR under profoundly hypothermic conditions impaired the recovery of myocardial function (cardiac index: 5°C = 3.9 ± 0.8, 25°C = 6.2 ± 0.4, 35°C = 6.5 ± 0.6 mL/minute/g, p = 0.03) during EVHP. We conclude that the avoidance of profound hypothermia during IR minimizes injury and improves the functional recovery of DCD hearts.

Physiologic Changes in the Heart Following Cessation of Mechanical Ventilation in a Porcine Model of Donation After Circulatory Death: Implications for Cardiac Transplantation.

Hearts donated following circulatory death (DCD) may represent an additional source of organs for transplantation; however, the impact of donor extubation on the DCD heart has not been well characterized. We sought to describe the physiologic changes that occur following withdrawal of life-sustaining therapy (WLST) in a porcine model of DCD. Physiologic changes were monitored continuously for 20 min following WLST. Ventricular pressure, volume, and function were recorded using a conductance catheter placed into the right (N = 8) and left (N = 8) ventricles, and using magnetic resonance imaging (MRI, N = 3). Hypoxic pulmonary vasoconstriction occurred following WLST, and was associated with distension of the right ventricle (RV) and reduced cardiac output. A 120-fold increase in epinephrine was subsequently observed that produced a transient hyperdynamic phase; however, progressive RV distension developed during this time. Circulatory arrest occurred 7.6±0.3 min following WLST, at which time MRI demonstrated an 18±7% increase in RV volume and a 12±9% decrease in left ventricular volume compared to baseline. We conclude that hypoxic pulmonary vasoconstriction and a profound catecholamine surge occur following WLST that result in distension of the RV. These changes have important implications on the resuscitation, preservation, and evaluation of DCD hearts prior to transplantation.

Autophagy is a regulator of TGF-ß1-induced fibrogenesis in primary human atrial myofibroblasts.

Transforming growth factor-ß(1) (TGF-ß(1)) is an important regulator of fibrogenesis in heart disease. In many other cellular systems, TGF-ß(1) may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown. Thus we tested whether or not TGF-ß(1)-induced autophagy has a regulatory function on fibrosis in human atrial myofibroblasts (hATMyofbs). Primary hATMyofbs were treated with TGF-ß(1) to assess for fibrogenic and autophagic responses. Using immunoblotting, immunofluorescence and transmission electron microscopic analyses, we found that TGF-ß(1) promoted collagen type Iα2 and fibronectin synthesis in hATMyofbs and that this was paralleled by an increase in autophagic activation in these cells. Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells. ATG7 knockdown in hATMyofbs and ATG5 knockout (mouse embryonic fibroblast) fibroblasts decreased the fibrotic effect of TGF-ß(1) in experimental versus control cells. Furthermore, using a coronary artery ligation model of myocardial infarction in rats, we observed increases in the levels of protein markers of fibrosis, autophagy and Smad2 phosphorylation in whole scar tissue lysates. Immunohistochemistry for LC3ß indicated the localization of punctate LC3ß with vimentin (a mesenchymal-derived cell marker), ED-A fibronectin and phosphorylated Smad2. These results support the hypothesis that TGF-ß(1)-induced autophagy is required for the fibrogenic response in hATMyofbs.

Probing the photophysical capability of mono and bis(cyclometallated) Fe(ii) polypyridine complexes using inexpensive ground state DFT.

The abundance and low toxicity of iron with respect to ruthenium would certainly make it valuable for photophysical applications if one could circumvent its tendency to make high-spin compounds and the kinetic lability of its polypyridine complexes, both related to the presence of low-lying quintet metal-centered excited states. The aim of this study was to probe the photophysical potential of six cyclometallated Fe(ii) polypyridine complexes by means of ground state DFT and TDDFT calculations. Quantitative and qualitative indicators were extracted from such calculations and bring us to the conclusion that two complexes should display promising photophysical properties: Fe(NCN)(NNC) and Fe(NNC)2.

Autophagy and heart disease: implications for cardiac ischemia-reperfusion damage.

Survival of myocytes and mesenchymal cells in the heart is tightly regulated by a number of adaptive processes that are invoked with the changes that occur within the parenchyma and stroma. Autophagy is implicated in cellular housekeeping duties and maintenance of the integrity of the intracellular milieu by removal of protein aggregates and damaged organelles, whereas under pathophysiological conditions, the chronic up-regulation of autophagy may lead to significant disturbance of homeostatic conditions. Nonetheless, the role of autophagy in heart disease in the context of cardiac ischemia-reperfusion injury is currently unclear. This review will focus upon the role of autophagy as it pertains to ischemia reperfusion damage in the heart.

Apoptosis, autophagy and ER stress in mevalonate cascade inhibition-induced cell death of human atrial fibroblasts.

3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors (statins) are cholesterol-lowering drugs that exert other cellular effects and underlie their beneficial health effects, including those associated with myocardial remodeling. We recently demonstrated that statins induces apoptosis and autophagy in human lung mesenchymal cells. Here, we extend our knowledge showing that statins simultaneously induces activation of the apoptosis, autophagy and the unfolded protein response (UPR) in primary human atrial fibroblasts (hATF). Thus we tested the degree to which coordination exists between signaling from mitochondria, endoplasmic reticulum and lysosomes during response to simvastatin exposure. Pharmacologic blockade of the activation of ER-dependent cysteine-dependent aspartate-directed protease (caspase)-4 and lysosomal cathepsin-B and -L significantly decreased simvastatin-induced cell death. Simvastatin altered total abundance and the mitochondrial fraction of proapoptotic and antiapoptotic proteins, while c-Jun N-terminal kinase/stress-activated protein kinase mediated effects on B-cell lymphoma 2 expression. Chemical inhibition of autophagy flux with bafilomycin-A1 augmented simvastatin-induced caspase activation, UPR and cell death. In mouse embryonic fibroblasts that are deficient in autophagy protein 5 and refractory to autophagy induction, caspase-7 and UPR were hyper-induced upon treatment with simvastatin. These data demonstrate that mevalonate cascade inhibition-induced death of hATF manifests from a complex mechanism involving co-regulation of apoptosis, autophagy and UPR. Furthermore, autophagy has a crucial role in determining the extent of ER stress, UPR and permissiveness of hATF to cell death induced by statins.

K+ currents regulate the resting membrane potential, proliferation, and contractile responses in ventricular fibroblasts and myofibroblasts.

Despite the important roles played by ventricular fibroblasts and myofibroblasts in the formation and maintenance of the extracellular matrix, neither the ionic basis for membrane potential nor the effect of modulating membrane potential on function has been analyzed in detail. In this study, whole cell patch-clamp experiments were done using ventricular fibroblasts and myofibroblasts. Time- and voltage-dependent outward K(+) currents were recorded at depolarized potentials, and an inwardly rectifying K(+) (Kir) current was recorded near the resting membrane potential (RMP) and at more hyperpolarized potentials. The apparent reversal potential of Kir currents shifted to more positive potentials as the external K(+) concentration ([K(+)](o)) was raised, and this Kir current was blocked by 100-300 muM Ba(2+). RT-PCR measurements showed that mRNA for Kir2.1 was expressed. Accordingly, we conclude that Kir current is a primary determinant of RMP in both fibroblasts and myofibroblasts. Changes in [K(+)](o) influenced fibroblast membrane potential as well as proliferation and contractile functions. Recordings made with a voltage-sensitive dye, DiBAC(3)(4), showed that 1.5 mM [K(+)](o) resulted in a hyperpolarization, whereas 20 mM [K(+)](o) produced a depolarization. Low [K(+)](o) (1.5 mM) enhanced myofibroblast number relative to control (5.4 mM [K(+)](o)). In contrast, 20 mM [K(+)](o) resulted in a significant reduction in myofibroblast number. In separate assays, 20 mM [K(+)](o) significantly enhanced contraction of collagen I gels seeded with myofibroblasts compared with control mechanical activity in 5.4 mM [K(+)](o). In combination, these results show that ventricular fibroblasts and myofibroblasts express a variety of K(+) channel alpha-subunits and demonstrate that Kir current can modulate RMP and alter essential physiological functions.

Differential gene expression in infarct scar and viable myocardium from rat heart following coronary ligation.

Post-myocardial infarction (MI) remodeling of cardiac myocytes and the myocardial interstitium results in alteration of gross ventricular geometry and ventricular dysfunction. To investigate the mechanisms of the remodeling process of the heart after large MI, the expression of various genes in viable left ventricle and infarct scar tissue were examined at 16 weeks post-MI. Steady-state expression of Na(+)-K+ ATPase alpha-1 and -2, phospholamban (PLB), alpha-myosin heavy chain (alpha-MHC), ryanodine receptor (Rya) and Ca2+ ATPase (Serca2) mRNAs were decreased in the infarct scar vs noninfarcted sham-operated controls (P < 0.05). On the other hand, Gialpha2 and beta-MHC mRNAs were upregulated (P < 0.05, respectively) in the infarct scar whereas Na(+)-K+ ATPase-beta, Na(+)-Ca2+ exchanger and Gs mRNAs were not altered vs control values. In viable left ventricle, the alpha-1 subunit of Na(+)-K+ ATPase, alpha-3, beta-isoforms, Rya, beta-MHC, Gialpha2, Gs and Na(+)-Ca2+ exchanger were significantly elevated while expression of the alpha-2 subunit of Na(+)-K+ ATPase, PLB and Serca2 were significantly decreased compared to controls. Expression of CK2alpha mRNA was elevated in noninfarcted heart (145 +/- 15%) and diminished in the infarct scar (66 +/- 13%) vs controls. Expression of beta-MHC mRNA was elevated in both viable and infarct scar tissues of experimental hearts (140 +/- 31% and 183 +/- 30% vs. controls, respectively). These results suggest that cardiac genes in the infarcted tissue and viable left ventricle following MI are differentially regulated.

Porphyrinic dyads and triads assembled around iridium(III) bis-terpyridine: photoinduced electron transfer processes.

Multicomponent arrays based on a central iridium(III) bis-terpyridine complex (Ir) used as assembling metal and free-base, zinc(II) or gold(III) tetraaryl-porphyrins (PH(2), PZn, PAu) have been designed to generate intramolecular photoinduced charge separation. The rigid dyads PH(2)-Ir, PZn-Ir, PAu-Ir, and the rigid and linear triads PH(2)-Ir-PAu, PZn-Ir-PAu, as well as the individual components Ir, PH(2), PZn, PAu have been synthesized and characterized by various techniques including electrochemistry. Their photophysical properties either in acetonitrile or in dichloromethane and toluene have been determined by steady-state and time-resolved methods. In acetonitrile, excitation of the triad PH(2)-Ir-PAu leads to a charge separation with an efficiency of 0.5 and a resulting charge-separated (CS) state with a lifetime of 3.5 ns. A low-lying triplet localized on PH(2) and the presence of the heavy Ir(III) ion offer the CS state an alternative deactivation path through the triplet state. The behavior of the triad PZn-Ir-PAu in dichloromethane is rather different from that of PH(2)-Ir-PAu in acetonitrile since the primary electron transfer to yield PZn(+)()-Ir(-)-PAu is not followed by a secondary electron transfer. In this solvent, both unfavorable thermodynamic and electronic parameters contribute to the inefficiency of the second electron-transfer reaction. In contrast, in toluene solutions, the triad PZn-Ir-PAu attains a CS state with a unitary yield and a lifetime of 450 ns. These differences can be understood in terms of ground-state charge-transfer interactions as well as different stabilization of the intermediate and final CS states by solvent.

Restraining acute infarct expansion decreases collagenase activity in borderzone myocardium.

BACKGROUND: After acute myocardial infarction, regional myocardial wall strains and stresses change and a complex cellular and biochemical response is initiated to remodel the ventricle. This study tests the hypothesis that changes in regional ventricular wall strains affect regional collagen accumulation and collagenase activity. METHODS: Fourteen sheep had acute anteroapical infarction that progressively expands into left ventricular aneurysm within 8 weeks. In 7 sheep, infarct expansion was restrained by prior placement of mesh over the area at risk. Fourteen days after infarction, and after hemodynamic and echocardiographic measurements, animals were euthanized for histology, measurements of hydroxyproline, matrix metalloproteinase-1 (MMP-1 or collagenase) and MMP-2 (gelatinase) activity, as well as collagen type I and III in infarcted, borderzone, and remote myocardium. RESULTS: Restraining infarct expansion does not change collagen content or MMP-1 or MMP-2 activity in the infarct, but significantly increases the ratio of collagen I/III. In borderzone and remote myocardium infarct, restraint significantly increases collagen content and significantly reduces MMP-1 activity. MMP-2 activity is reduced (p = 0.059) in borderzone myocardium only. Between groups, the ratio of type I/III fibrillar collagen does not change in borderzone myocardium. CONCLUSIONS: Fourteen days after acute myocardial infarction, restraining infarct expansion increases collagen accumulation in borderzone and remote myocardium, which may prevent expansion of hypocontractile, fully perfused "remodeling myocardium" adjacent to the infarct. This study demonstrates that changes in regional myocardial wall strain alter the cellular and biochemical processes involved in postinfarction ventricular remodeling.

Interaction between angiotensin II and Smad proteins in fibroblasts in failing heart and in vitro.

Angiotensin II (angiotensin) and transforming growth factor (TGF)-beta(1) play an important role in cardiac fibrosis. We examined Smad proteins in 8-wk post-myocardial infarction (MI) rat hearts. AT(1) blockade (losartan) attenuated the activation of TGF-beta(1) in target tissues. Losartan administration (8 wk, 15 mg. kg(-1). day(-1)) normalized total Smad 2 overexpression in infarct scar and remnant heart tissue and normalized Smad 4 in infarct scar. Phosphorylated Smad 2 (P-Smad 2) staining decreased in cytosol from failing heart vs. the control, which was normalized by losartan, suggesting augmented P-Smad 2 movement into nuclei in untreated failing hearts. Using adult primary rat fibroblasts treated with angiotensin (10(-6) M), we noted rapid translocation (15 min) of P-Smad 2 into the nuclei from the cytosol. Nuclear P-Smad 2 protein level increased with angiotensin treatment, which was blocked by losartan. We conclude that angiotensin may influence total Smad 2 and 4 expression in post-MI heart failure and that angiotensin treatment is associated with rapid P-Smad 2 nuclear translocation in isolated fibroblasts. This study suggests that cross talk between angiotensin and Smad signaling is associated with fibrotic events in post-MI hearts.

Effect of chronic AT(1) receptor blockade on cardiac Smad overexpression in hereditary cardiomyopathic hamsters.

OBJECTIVE: As the pharmacological suppression of angiotensin has been associated with cardioprotective effects in cardiomyopathy, our primary aim was to determine whether the expression of Smad protein components of the cardiac TGF-beta signaling cascade is modulated by chronic AT(1) receptor blockade. Furthermore, we examined the relationship between cardiac Smad protein expression and altered collagen turnover in the cardiomyopathic heart. METHODS: Male UM-X7. 1 cardiomyopathic (CMP) Syrian hamsters at early (65 days) and late (200 days) stages of cardiomyopathy were subjected to 4 week losartan (15 mg/kg/day) treatment. Expression of left ventricular (LV) receptor-activated (Smad 2) and common-mediator (Smad 4) Smads from control (F1-beta strain) hamsters, non-treated cardiomyopathic (CMP), and losartan-treated CMP animals was assessed. Collagen turnover, including fibrillar collagen synthesis/accretion and cardiac MMP activity was assessed. RESULTS: Elevated mRNA abundance of fibrillar collagens and ANF were present in cardiomyopathic hearts and these trends were normalized in the early stage losartan-treated group. 4-Hydroxyproline and zymographic assays confirmed fibrosis and elevated MMP-1 and -2 activities in CMP hearts. Losartan treatment was associated with a modest reduction of cardiac 4-hydroxyproline concentration, and a significant reduction of both MMP-1 and MMP-2 activities. While TGF-beta(1) mRNAs were elevated in both CMP groups vs. controls, total TGF-beta protein content was not different in CMP vs. controls. In LV preparations containing nuclear extract, elevated Smad 2 and Smad 4 protein expression was noted in cardiomyopathic hearts vs. controls. Losartan treatment of late-stage CMP hamsters was associated with a significant reduction in Smad 2 and a modest reduction of Smad 4 protein expression vs. untreated CMP samples. CONCLUSIONS: Altered cardiac Smad expression, present in both early and late stage cardiomyopathy, is positively correlated with the occurrence of cardiac fibrosis and elevated collagen turnover in failing CMP hearts. Four week AT(1) blockade is associated with normalized expression of cardiac Smad 2 proteins, and these changes occur in parallel with some aspects of collagen turnover in failing cardiomyopathic hearts.

Differential changes in cardiac myofibrillar and sarcoplasmic reticular gene expression in alloxan-induced diabetes.

In order to examine the relationship between heart dysfunction and subcellular abnormalities as well as molecular mechanisms during the development of diabetes, we studied changes in cardiac performance, myofibrillar as well as sarcoplasmic reticular (SR) activities, and cardiac gene expression at different time intervals upon inducing diabetes in rats by an injection of alloxan (65 mg/kg; i.v.). Cardiac dysfunction was associated with a depression in myofibrillar Ca2+-stimulated ATPase and changes in myosin isozyme composition at 2-12 weeks of inducing diabetes. A reduction in SR Ca2+-uptake and Ca2+-pump (SERCA2) activities was evident at 10 days to 12 weeks of inducing diabetes. Alterations in cardiac function during 2-12 weeks of diabetes show a linear relationship with changes in myofibrils and SR membranes. Furthermore, alterations in cardiac function as well as myofibrillar and SR activities in 4 week diabetic animals were normalized upon treatment with insulin for 4 weeks. The steady-state mRNA abundance for alpha-myosin heavy chain in the heart was decreased at 2 and 3 weeks but was unchanged at 5 and 6 weeks, whereas mRNA levels for beta-myosin heavy chain remained elevated during 2-6 weeks after inducing diabetes. SERCA2 mRNA abundance in diabetic heart was significantly increased at 3 and 5 weeks but was unaltered at 2 and 6 weeks. These results support the view that heart dysfunction in diabetes may be a consequence of myofibrillar and SR abnormalities; however, defects in myofibrillar proteins, unlike those in the SR membranes, appear to be due to changes in their gene expression.

Expression of Gi-2 alpha and Gs alpha in myofibroblasts localized to the infarct scar in heart failure due to myocardial infarction.

OBJECTIVE: Patients surviving large transmural myocardial infarction (MI) are at risk for congestive heart failure with attendant alteration of ventricular geometry and scar remodeling. Altered Gi-2 alpha and Gs alpha protein expression may be involved in cardiac remodeling associated with heart failure, however their expression in scar tissue remains unclear. METHODS: MI was produced in Sprague-Dawley rats by ligation of the left coronary artery. Gi-2 alpha and Gs alpha protein concentration, localization and mRNA abundance were noted in surviving left ventricle remote to the infarct, in border and in scar tissues from 8 week post-MI hearts with moderate heart failure. RESULTS: We observed a 4.5- and 5.0-fold increase in immunoreactive Gi-2 alpha protein concentration occurs in the border and scar regions vs. control values, respectively, in 8-week post-MI rat hearts. Similarly, immunoreactive Gs alpha protein concentration was increased 3.4- and 8.2-fold, respectively, in these tissues vs. controls. Double-fluorescence labeling and phenotyping studies revealed that both Gi-2 alpha and Gs alpha proteins were localized to myofibroblasts in the infarct scar and to viable myocytes bordering the scar. Northern analysis revealed that the Gi-2 alpha/GAPDH ratio was increased in both viable and scar regions (1.24- and 1.85-fold respectively) from experimental hearts when compared to sham-operated control values when compared to noninfarcted left ventricle, the value of this ratio in scar tissue was elevated approximately 1.5 fold. The Gs alpha/GAPDH ratio was significantly increased (1.28-fold) only in the scar region vs. control. CONCLUSION: Our results indicate a marked increase in the expression of Gi-2 alpha and Gs alpha from myofibroblasts of the infarct scar as well as remnant myocytes bordering the scar in 8-week post-MI rat hearts. We suggest that these changes may be associated with ongoing remodeling in the infarct scar in chronic post-MI phase of this experimental model.

Elevation of expression of Smads 2, 3, and 4, decorin and TGF-beta in the chronic phase of myocardial infarct scar healing.

We have previously shown that non-myocytes present in healed 8-week infarct scar overexpress transduction proteins required for initiating the elevated deposition of structural matrix proteins in this tissue. Other work suggests that TGF-beta 1 may be involved in cardiac fibrosis and myocyte hypertrophy. However, the significance of the altered TGF-beta signaling in heart failure in the chronic phase of post-myocardial infarction (MI), particularly in the ongoing remodeling of the infarct scar, remains unexplored. Patterns of cardiac TGF beta 1 and Smad 2, 3, and 4 protein expression were investigated 8 weeks after MI and were compared to relative collagen deposition in border tissues (containing remnent myocytes) and the infarct scar (non-myocytes). Both TGF-beta 1 mRNA abundance and protein levels were significantly increased in the infarct scar v control values, and this trend was positively correlated to increased collagen type I expression. Cardiac Smad 2, 3, and 4 proteins were significantly increased in border and scar tissues v control values. Immunofluorescent studies indicated that Smad proteins localized proximal to the cellular nuclei present in the infarct scar. Decorin mRNA abundance was elevated in border and infarct scar, and the pattern of decorin immunostaining was markedly altered in remote remnant heart and scar v staining patterns of control sections. Expression of T beta RI (53 kDa) protein was significantly reduced in the scar, while the 75 kDa and 110 kDa isoforms of T beta RII were unchanged and significantly increased in scar, respectively. These results indicate that TGF-beta/Smad signaling may be involved in the remodeling of the infarct scar after the completion of wound healing per se, via ongoing stimulation of matrix deposition.

Distribution of collagen deposition in cardiomyopathic hamster hearts determined by infrared microscopy.

Fibrillar collagens are major proteins of the cardiac extracellular matrix and play a significant role in the structural organization of the healthy heart. The aim of this study was (i) to investigate and compare the patterns of cardiac collagen deposition in different layers taken from both cardiomyopathic and normal myocardium using infrared (IR) microspectroscopy and (ii) to evaluate IR microspectroscopy as an alternative means for in vitro detection of collagen deposition in heart. Frozen sections from UM-X7.1 strain hamsters expressing the cardiomyopathic phenotype associated with ventricular remodeling and age-matched control (F1-beta) strain hamsters were examined using IR microspectroscopy. The presence of collagen was identified by the appearance of a typical collagen band at 1204 cm(-1), and the results were compared with identical tissue sections stained with trichrome, a routine discriminator for interstitial matrix proteins in cardiac myocytes. Spatial information addressing collagen deposition was obtained and viewed using contour mapping and three-dimensional band intensity maps at 1204 cm(-1). Perivascular and interstitial collagen deposition was detected in control samples taken from both ventricles as indicated with relative low intensities of the band of 1204 cm(-1). When compared with these control levels, the concentration of collagen was increased in cardiomyopathic left-ventricular samples with some focal depositions, and these results were confirmed with the trichrome references. Our study suggests that collagen deposition from normal and diseased hearts may be successfully analyzed directly in the absence of any chemihistological or immunological staining, by infrared microscopy.

Antiproliferative and antifibrotic effects of mimosine on adult cardiac fibroblasts.

Prolyl 4-hydroxylase catalyzes the hydroxylation of collagen pro-alpha chains for the deposition of cardiac collagen. The effect of prolyl 4-hydroxylase on synthesis and degradation of collagen was studied in cultured adult cardiac fibroblasts using mimosine, a prolyl 4-hydroxylase inhibitor. Mimosine inhibited [3H]thymidine incorporation in cultured fibroblasts in a dose-dependent manner (100-600 microM). Immunofluorescence in fibroblasts and biochemical detection of mature type I collagen in culture serum revealed a strong inhibition of synthesis and secretion of mature collagens, respectively, in the presence of 200 microM mimosine. Western blot analysis for procollagen was carried out in cultured fibroblasts, and 200 microM mimosine treatment was associated with increased intracellular accumulation of procollagen from 4.14+/-0.27 to 10. 19+/-0.37 (arbitrary units). Immunofluorescence studies confirmed a marked increase of intracellular procollagens in fibroblasts treated with mimosine, which suggests a loss of coordinated monomeric procollagen synthesis and secretion of triple helical mature collagens. Modest inhibition of collagen type I mRNA abundance was observed in mimosine-treated fibroblasts, whereas no effect was noted for mRNAs of collagen type III, alpha-prolyl 4-hydroxylase or beta-prolyl 4-hydroxylase when compared to untreated control values. Treatment of fibroblasts with 200 microM mimosine was associated with elevation of matrix metalloproteinase (MMP)-9 activity. The cytotoxicity of mimosine treatment was found minimal at the concentrations indicated above. Thus the antifibrotic effects induced by mimosine on cultured adult cardiac fibroblasts was associated with inhibition of prolyl 4-hydroxylase and diminished extracellular secretion of procollagen, despite the reactive elevation of intracellular procollagen synthesis. We suggest that specific inhibition of prolyl 4-hydroxylase may provide a novel therapeutic approach for the modulation of cardiac fibrosis.

Cardiac sarcolemmal Na(+)-Ca2+ exchange and Na(+)-K+ ATPase activities and gene expression in alloxan-induced diabetes in rats.

To determine the sequence of alterations in cardiac sarcolemmal (SL) Na(+)-Ca2+ exchange, Na(+)-K+ ATPase and Ca(2+)-transport activities during the development of diabetes, rats were made diabetic by an intravenous injection of 65 mg/kg alloxan. SL membranes were prepared from control and experimental hearts 1-12 weeks after induction of diabetes. A separate group of 4 week diabetic animals were injected with insulin (3 U/day) for an additional 4 weeks. Both Na(+)-K+ ATPase and Ca(2+)-stimulated ATPase activities were depressed as early as 10 days after alloxan administration; Mg2+ ATPase activity was not depressed throughout the experimental periods. Both Na(+)-Ca2+ exchange and ATP-dependent Ca(2+)-uptake activities were depressed in diabetic hearts 2 weeks after diabetes induction. These defects in SL Na(+)-K+ ATPase and Ca-transport activities were normalized upon treatment of diabetic animals with insulin. Northern blot analysis was employed to compare the relative mRNA abundances of alpha 1-subunit of Na(+)-K+ ATPase and Na(+)-Ca2+ exchanger in diabetic ventricular tissue vs. control samples. At 6 weeks after alloxan administration, a significant depression of the Na(+)-K+ ATPase alpha 1-subunit mRNA was noted in diabetic heart. A significant increase in the Na(+)-Ca2+ exchanger mRNA abundance was observed at 3 weeks which returned to control by 5 weeks. The results from the alloxan-rat model of diabetes support the view that SL membrane abnormalities in Na(+)-K+ ATPase, Na+Ca2+ exchange and Ca(2+)-pump activities may lead to the occurrence of intracellular Ca2+ overload during the development of diabetic cardiomyopathy but these defects may not be the consequence of depressed expression of genes specific for those SL proteins.

Expression of Gq alpha and PLC-beta in scar and border tissue in heart failure due to myocardial infarction.

BACKGROUND: Large transmural myocardial infarction (MI) leads to maladaptive cardiac remodeling and places patients at increased risk of congestive heart failure. Angiotensin II, endothelin, and alpha1-adrenergic receptor agonists are implicated in the development of cardiac hypertrophy, interstitial fibrosis, and heart failure after MI. Because these agonists are coupled to and activate Gq alpha protein in the heart, the aim of the present study was to investigate Gq alpha expression and function in cardiac remodeling and heart failure after MI. METHODS AND RESULTS: MI was produced in rats by ligation of the left coronary artery, and Gq alpha protein concentration, localization, and mRNA abundance were noted in surviving left ventricle remote from the infarct and in border and scar tissues from 8-week post-MI hearts with moderate heart failure. Immunohistochemical staining localized elevated Gq alpha expression in the scar and border tissues. Western analysis confirmed significant upregulation of Gq alpha proteins in these regions versus controls. Furthermore, Northern analysis revealed that the ratios of Gq alpha/GAPDH mRNA abundance in both scar and viable tissues from experimental hearts were significantly increased versus controls. Increased expression of phospholipase C (PLC)-beta1 and PLC-beta3 proteins was apparent in the scar and viable tissues after MI versus controls and is associated with increased PLC-beta1 activity in experimental hearts. Furthermore, inositol 1,4,5-tris-phosphate is significantly increased in the border and scar tissues compared with control values. CONCLUSIONS: Upregulation of the Gq alpha/PLC-beta pathway was observed in the viable, border, and scar tissues in post-MI hearts. Gq alpha and PLC-beta may play important roles in scar remodeling as well as cardiac hypertrophy and fibrosis of the surviving tissue in post-MI rat heart. It is suggested that the Gq alpha/PLC-beta pathway may provide a possible novel target for altering postinfarct remodeling.