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We have previously shown that targeting human CD34(+) hematopoietic stem cells (HSC) with a bispecific antibody (BiAb) directed against myosin light chain (MLC) increases delivery of cells to the injured hearts and improves cardiac performance in the nude rat. In this study, we have sought to validate our previous observations and to perform more detailed determination of ventricular function in immunocompetent mice with myocardial infarction (MI) that were treated with armed CD34(+) HSC. We examined whether armed CD34(+) HSC would target the injured heart following MI and restore ventricular function in vitro. MI was created by ligation of the left anterior descending artery. After 48 h, adult ICR mice received either 0.5 x 10(6) human CD34(+) HSC armed with anti-CD45 x anti-MLC BiAb or an equal volume of medium through a single tail vein injection. Two weeks after stem cell administration, ventricular function of hearts from mice receiving armed CD34(+) HSC was significantly greater compared with the same parameters from control mice. Immunohistochemistry confirmed the accumulation of CD34(+) HSC in MI hearts infused with stem cells. Angiogenesis was significantly enhanced in CD34(+) HSC-treated heart as determined by vascular density per area. Furthermore, histopathological examination revealed that the retained cardiac function observed in CD34(+) HSC-treated mice was associated with decreased ventricular fibrosis. These results suggest that peripheral administration of armed CD34(+) HSC results in localization of CD34(+) HSC to injured myocardium and restores myocardial function.

The adult spleen is a source of early hematopoietic stem cells (HSC). We therefore studied whether culturing spleen or bone marrow (BM) HSC in medium containing 5-azacytidine could induce a cardiac phenotype. c-kit enrichment and depletion of adult pig spleen and BM mononuclear cells were obtained by magnetic bead separation using biotinylated pig stem cell factor (c-kit ligand). Cells were incubated with 5-azacytidine for 24 h and refreshed with 5-azacytidine-free medium every 48 h. Western blot was used to detect cardiac troponin and myosin heavy chains. Although 5-azacytidine treatment led to the formation of ball-like cell clusters in both c-kit-enriched populations, these clusters showed no rhythmic contractions (beating), as observed by others. Furthermore, neither cardiac troponin nor myosin was detected in cells derived from either source. Our methodology and treatment with 5-azacytidine did not induce cardiac gene expression in porcine HSC derived from either pig spleen or BM.

Length modulation of cardiac muscle is manifested in the Frank-Starling relation of the heart. Recently, it has been shown that length-dependent changes in SH reactivity of cardiac troponin C (cTnC) occurred in association with cross-bridge attachment and Ca2+. However, the presence of two SH groups (Cys-35 and Cys-84) in the regulatory region of cTnC complicates efforts to detect conformational changes. In this study skinned porcine cardiac fibers were reacted with 7-diethylamino-3-[4'maleimidylphenyl]-4-methylcoumarin (CPM). Alkaline urea gel electrophoresis, along with protein elution, was used to isolate filament bound cTnC. Analysis of fluorescence measurement showed that there is a Ca(2+)-increased fluorescence for CPM-labeled cTnC in long fibers (sarcomere length = 2.2 approximately 2.5 microm) but not in short fibers (sarcomere length = 1.6 approximately 1.8 microm). In addition, the labeled cTnC was measured for the fluorescence decrease over time by adding a non-fluorescence energy acceptor, 4-dimethylaminophenylazophenyl-4'maleimide (DABMI), in the presence and absence of Ca2+. Fluorescence quenching by DABMI is not affected by Ca2+ in long fibers but it is significantly increased in short fibers. However, the fibers maintained in the relaxed state with 5 mM MgATP and 1 mM Vanadate showed no length effect on the CPM-labeled cTnC in terms of the Ca(2+)-mediated changes in fluorescence spectrum and in fluorescence quenching by DABMI. All together, our results suggest that the relative reactivities of Cys-35 and Cys-84 vary with sarcomere length.

Cardiomyocyte development switches from hyperplasmic to hypertrophic growth between postnatal days 3 and 4 in rats. The mechanisms responsible for this transition have been controversial. beta-Adrenergic receptor (betaAR) activation of mitogenic responses in vitro has been reported. We hypothesized that tonic activation of the betaAR signaling regulates cell division in neonatal cardiomyocytes via effects on signaling kinases known to be important in cell cycle regulation. The purpose of the current study was to elucidate the roles of betaAR in rat cardiomyocyte growth in vivo. We demonstrated that betaAR blockade induced a significant reduction in cardiomyocyte proliferation as measured by the BrdU labeling index. Blockade of betaAR did not affect p38 or p44/42 MAPK activities. We further demonstrated that betaAR blockade induced a prompt deactivation of the p70 ribosomal protein S6 kinase (p70 S6K). To confirm these results, we measured p70 S6K activity directly. Basal activity of p70 S6K in neonatal cardiomyocytes was fourfold higher than that of insulin-treated adult rat liver. The activity of p70 S6K was reduced by 60% within 1 min after betaAR blockade. We conclude that the betaAR are involved in regulation of neonatal cardiomyocyte proliferation and that this mitogenic control may be mediated via the p70 S6K pathway.

Desmin filaments are muscle-specific intermediate filaments located at the periphery of the Z-discs, and they have been postulated to play a critical role in the lateral registration of myofibrils. Previous studies suggest that intermediate filaments may be involved in titin assembly during the early stages of myofibrillogenesis. In order to investigate the putative function of desmin filaments in myofibrillogenesis, rabbit anti-desmin antibodies were introduced into cultured cardiomyocytes by electroporation to perturb the normal function of desmin filaments. Changes in the assembly of several sarcomeric proteins were examined by immunofluorescence. In cardiomyocytes incorporated with normal rabbit serum, staining for alpha-actinin and muscle actin displayed the typical Z-line and I-band patterns, respectively, while staining for titin with monoclonal anti-titin A12 antibody, which labels a titin epitope at the A-I junction, showed the periodic doublet staining pattern. Staining for C-protein gave an amorphous pattern in early cultures and identified A-band doublets in older cultures. In contrast, in cardiomyocytes incorporated with anti-desmin antibodies, alpha-actinin was found in disoriented Z-discs and the myofibrils became fragmented, forming mini-sarcomeres. In addition, titin was not organized into the typical A-band doublet, but appeared to be aggregated. Muscle actin staining was especially weak and appeared in tiny clusters. Moreover, in all ages of cardiomyocytes tested, C-protein remained in the disassembled form. The present data suggest the essential role of desmin in myofibril assembly., (Copyright 2000 Wiley-Liss, Inc.)

Myocardial cells respond to changes in the mechanical forces imposed on them with changes in myocardial tension in the short term and with structural remodeling in the long term. Since these responses involve intercellular communication, we have investigated regulation of the gap junction proteins, connexin 43 (Cx43), connexin 40 (Cx40) and connexin 37 (Cx37), by cyclical mechanical stretch. Results were compared with parallel experiments on c-fos and GAPDH. Twenty percent stretch of cultured rat cardiomyocytes caused a 3-fold increase in Cx43 mRNA levels by 2 h. c-fos mRNA levels increased after 30 min of stretch, whereas Cx40, Cx37, and GADPH mRNA did not change. Protein levels of Cx43 increased by 4 h and remained elevated for 16 h. New protein synthesis was not a requirement for the stretch-induced rise in Cx43 expression, since mRNA levels were unaffected by treatment with cycloheximide. In addition, mechanical stretch induced alkalization of cardiomyocytes that was antagonized by inhibiting Na-H exchanger (NHE). Gap junction potential (Gj) was concomitantly elevated. Chemical closure of Cx channels by insulin was followed by inhibition of NHE. In conclusion, cyclical mechanical stretch caused increased expression of the gap junction protein Cx43 in cardiomyocytes and also the Gj. The augmentation of Cx43 mRNA expression and its functional status were associated with activation of NHE., (Copyright 2000 Academic Press.)

N-cadherin is a member of the Ca(2+)-dependent cell adhesion molecules and plays an important role in the assembly of the adherens junction in chicken cardiomyocytes. In addition to being present at the cell-cell junction, N-cadherin is associated with costameres in extrajunctional regions. The significance of the N-cadherin-associated costameres and whether catenins are components of costameres in chicken cardiomyocytes are not known. In this study, double-labeling immunofluorescence microscopy was used to determine the extrajunctional distribution of both N-cadherin and its cytoplasmic associated proteins, alpha- and beta-catenins, and their relationship to myofibrillar Z-disc alpha-actinin. N-cadherin, alpha-, and beta-catenins were all found to be present at the extrajunctional region and, in some cases, were codistributed with myofibrillar alpha-actinin exhibiting a periodic staining pattern. Confocal microscopy confirmed that both N-cadherin and beta-catenin colocalized with peripheral myofibrillar alpha-actinin on the dorsal surface of cardiomyocytes as components of the costameres. Intracellular application of antibodies specific for the cytoplasmic portions of N-cadherin, alpha-, and beta-catenin, either by electroporation or microinjection, resulted in myofibril disorganization and disassembly. These results suggest the existence of N-cadherin/catenin-based costameres in the dorsal surface of cultured chicken cardiomyocytes in addition to the integrin/vinculin-based costameres found in the ventral surface and indicate that the former set of costameres is essential for cardiac myofibrillogenesis., (Copyright 1999 Wiley-Liss, Inc.)

The aim of this study was to provide morphological evidence for the presence of rho A protein in developing cardiomyocytes and to investigate its possible role in myofibrillogenesis. Immunostaining with a monoclonal anti-rho antibody gave a diffuse pattern in the cytosol of cultured cardiomyocytes. Introduction of C3 exoenzyme into the cells by electroporation was used to inactivate rho A protein by ADP-ribosylation. An immunostaining with anti-vinculin, anti-talin, and anti-integrin antibodies showed the focal adhesions in electroporation control cardiomyocytes to be evenly distributed in the ventral sarcolemma; the costameric structure was also detected using these antibodies. In contrast, in C3 exoenzyme treated cells, focal adhesions were disassembled and costamere were absent; in addition, beta-actin-positive, non-striated fibrils were lost and assembly of M-protein, titin, and alpha-actinin into myofibrils was poor, as shown by diffuse and filamentous staining pattern. C3 exoenzyme treatment had a less marked effect on mature cardiomyocytes than on immature cells; in this case, cells became distorted and few myofibrils were seen. The intensity of anti-phosphotyrosine antibody staining of the focal adhesion was also decreased or diffuse in C3 exoenzyme-treated cardiomyocytes, suggesting dephosphorylation of focal adhesion components. We therefore conclude that small G protein rho A plays an important role in myofibril assembly in cardiomyocytes.

We investigated the change of the arterial-coronary sinus lactate difference before and after radiofrequency ablation to assess the influence of radiofrequency ablation on myocardial metabolism. Sixteen patients underwent radiofrequency catheter ablation and blood sampling. The patients were further divided into two groups according to the energy received, group 1 (n = 8) with lower energy and group 2 (n = 8) with higher energy. The postpacing arterial-coronary sinus lactate difference was significantly higher than that 1 min after ablation (0.18 +/- 0.16 vs. -0.08 +/- 0.14 mmol/l, P < 0.01). The mean arterial-coronary sinus lactate difference obtained at 3 min, 5 min and 10 min increased gradually and finally approximated the postpacing lactate difference. The arterial-coronary sinus lactate difference 1 min after ablation of group 2 was significantly lower than that of group 1 (-0.03 +/- 0.14 vs. -0.13 +/- 0.14 mmol/l, P < 0.01). The arterial coronary sinus lactate differences of group 2 obtained at 3 min, 5 min and 10 min were also lower than those of group 1. However, the difference between the two groups decreased with time. This finding suggests that radiofrequency energy has an influence on the myocardial metabolism and higher energy causes more metabolic alteration than lower energy.

Activation of PKC by PMA was shown to promote the separation of chicken cardiomyocytes from each other in culture. Immunofluorescence staining for N-cadherin indicated that PMA, but not its inactive isoform 4 alpha PMA, induces the separation of cardiomyocytes and of co-cultured fibroblasts at intercellular junctional regions. The PMA-induced separation of cardiomyocytes and of co-cultured fibroblasts was inhibited by the PKC inhibitor, H-7. Immunoblot analysis further demonstrated that both PKC iota and PKC lambda were expressed in the cardiomyocyte cultures. While PKC lambda was localized to the cell-cell contact areas between cardiomyocytes, PKC iota was only detectable in the perinuclear cytoplasm of the co-cultured fibroblasts. The present findings suggest the involvement of PKC in regulating N-cadherin-mediated adherens junction formation in chicken cardiomyocytes. The differential distribution of PKC lambda and PKC iota in the cardiomyocytes and in the co-cultured fibroblasts suggests that different PKC isozymes are involved in regulating the assembly of intercellular junctions in these two cell types.

To find the best predictors of outcome for patients undergoing intra-aortic balloon counterpulsation (IABP) for cardiogenic shock, we prospectively studied 30 consecutive patients by examining hemodynamic parameters and cardiac lactic acid extraction ratios before the procedure. 1 hour after and then every 6 hours for 3 days. Complete hemodynamic data were obtained from the recordings of Swan-Ganz catheterization. Blood samples were drawn from the peripheral artery, central vein, pulmonary artery wedge position (PAW) and right atrium (RA) to quantify lactic acid levels. The simplified lactic acid extraction ratio was defined as the concentration of lactic acid in RA--the concentration of lactic acid in PAW/the concentration of lactic acid in PAW. The traditional systemic lactic acid production ratio was defined as the concentration of lactic acid in the central vein--the concentration of lactic acid in the peripheral artery/the concentration of lactic acid in the peripheral artery. Of the 30 patients studied, 19 died of cardiogenic shock, (group 1), while the surviving 11 patients formed group 2. The lactic acid extraction ratios at each of the time periods after the procedure were all significantly different, while the differences in the systemic lactic acid production ratio and hemodynamic parameters, including the systemic arterial pressure, cardiac output, cardiac index and PAW pressure, became significant at varying times after IABP. In group 2, the lactic acid extraction ratios 1 hour after IABP and later were all significantly higher than at baseline. This trend was not seen in group 1.(ABSTRACT TRUNCATED AT 250 WORDS)

The objectives of this study were to examine the serial vectorcardiographic changes following acute myocardial infarct and to assess the relationship between QRS loop changes and infarct size. Fifty adult male Long-Evans rats of 250-350 gm body weight were used to study experimental acute myocardial infarction induced by coronary artery ligation. Vectorcardiograms (VCG) of the Frank lead system were recorded before, and 1 day and 7 days after operation. Animals were sacrificed on the 7th day for histological quantitation of infarct area ratios. We found that (1) before operation, rats have ST elevation, probably due to early repolarization. (2) After coronary artery ligation, ECG showed characteristic dome-shaped ST elevation at 1 hr after ligation which returned to normal during the first day. Abnormal Q waves appeared thereafter. (3) After ligation, maximum QRS vector, ST vector and maximum T vector were reduced in magnitude the first day and recovered by the 7th day. The vectors tended to shift their direction to the right and to the posterior. QRS-T angle, however, widened as time went on. About half of the rats revealed changes in the inscription direction of the QRS loop and abnormal QRS morphology also appeared in about half of the ligated rats. (4) Those in whom abnormal QRS loop morphology and/or biting appeared had significantly larger infarct area ratios (p < 0.01). (5) Change in QRS loop inscription direction seemed not to be related to the infarct size. (6) In the LS plane, the difference in max QRS vector magnitude between the 1st and 7th days significantly correlated with the infarct area ratio (r = 0.533, p < 0.05). In the H plane, the change in the max QRS vector magnitude at the 7th day correlated with the infarct area ratio (r = -0.531, p < 0.05). In the F plane, changes in the direction of the max QRS vector were significantly correlated to the infarct area ratio both on the first (r = 0.431, p < 0.05) and 7th days (r = 0.531, p < 0.05). It is concluded that the VCG, like the ECG, had evolutional changes in AMI and that the QRS loop seen on vectorcardiography has only a slight correlation with the histological myocardial infarct size.

The fluorescent dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) was used to measure pHi in the spontaneously hypertensive rat (SHR) and in normal rat cardiac myocytes under nominally HCO3-free (20 mmol/L HEPES-buffered) conditions. When only the Na-H exchanger was blocked, the intrinsic buffering power (beta i) in SHR myocytes was significantly higher than when both the Na-H exchanger and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS)-sensitive pHi regulators (the Na-HCO3 cotransporter and the Cl-HCO3 exchanger) were blocked. Similar low values for beta i were also found for normal rat myocytes in Na(+)-free conditions. In Cl(-)-free solution under nominally HCO3-free conditions, in both normal and SHR myocytes, the pHi slowly alkalinized (by 0.16 +/- 0.02 and 0.11 +/- 0.02 pH units, respectively); this alkalinization was also DIDS sensitive. The reacidification during NH4+ perfusion was inhibited 30.2 +/- 7.4% by DIDS. In addition, in the nominal absence of HCO3-, 100 mumol/L ATP acidified the pHi in both normal and SHR myocytes (by 0.21 +/- 0.03 and 0.33 +/- 0.03 pH units, respectively); this acidification was totally inhibited by 0.1 mmol/L DIDS. It has been shown, in rat cardiac myocytes, that ATP acidifies the pHi by 0.35 pH unit via stimulation of a DIDS-sensitive Cl-HCO3 exchanger in HCO3-containing solutions. Finally, we have shown, in normal cardiac myocytes, that two potent Na-H exchanger blockers, N-5-ethylisopropyl amiloride (EIPA) and N-5-methyl-N-isobutyl amiloride (MIA), only partially inhibited the pHi recovery from internal acidosis under nominally bicarbonate-free conditions. When DIDS was added at the same time as EIPA, pHi recovery from an internal acid loading was completely inhibited. Our results clearly demonstrate that in both normal and SHR cardiac myocytes, bicarbonate-dependent pHi regulators can be significantly activated under resting or acidified pHi in HEPES-buffered medium, probably because of the cellular production of CO2. The contribution of these bicarbonate-dependent pHi regulators, ie, the Na-HCO3 cotransporter and the Cl-HCO3 exchanger, cannot therefore be ignored even under nominally HCO3-free conditions.

Background and Purpose: Aminoguanidine (AG), an inhibitor of advanced glycation endproducts, has been shown to prevent arterial stiffening and cardiac hypertrophy in streptozotocin (STZ) and nicotinamide (NA)-induced type 2 diabetes in rats. Our aims were to examine whether AG produced benefits on cardiac pumping mechanics in the STZ and NA-treated animals in terms of maximal systolic elastance (E(max)) and theoretical maximum flow (Q(max)).Experimental Approach: After induction of type 2 diabetes, rats received daily injections of AG (50 mg kg(-1), i.p.) for 8 weeks and were compared with age-matched, untreated, diabetic controls. Left ventricular (LV) pressure and ascending aortic flow signals were recorded to calculate E(max) and Q(max), using the elastance-resistance model. Physically, E(max) reflects the contractility of the myocardium as an intact heart, whereas Q(max) has an inverse relationship with the LV internal resistance.Key Results: Both type 2 diabetes and AG affected E(max) and Q(max), and there was an interaction between diabetes and AG for these two variables. The E(max) and Q(max) were reduced in rats with type 2 diabetes, but showed a significant rise after administration of AG to these diabetic rats. Moreover, the increase in Q(max) corresponded to a decrease in total peripheral resistance of the systemic circulation when the STZ and NA-induced diabetic rats were treated with AG.Conclusions and Implications: AG therapy prevented not only the contractile dysfunction of the heart, but also the augmentation in LV internal resistance in rats with STZ and NA-induced type 2 diabetes. [ABSTRACT FROM AUTHOR]