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We investigated the occurrence of apoptosis during and after resolution of cardiac allograft rejection. Apoptosis could play different roles in graft survival depending on the target cells; thus, we also determined the cell types involved.

The vitronectin receptor (integrin αvβ3), a cell-surface adhesion receptor, has been shown to play a significant role in endothelial cell migration, apoptosis, atherosclerosis, and T-lymphocyte activation. This study was undertaken to test the hypothesis that cardiac allograft rejection is associated with increased expression of αvβ3. We also determined whether fibronectin receptor (α5β1) and tissue factor are up-regulated in the presence of acute cellular rejection. We evaluated endomyocardial biopsy specimens with histologic evidence of different degrees of acute cellular rejection (grade 0, n = 10; grade 1A, n = 10; grade 2, n = 10; grade 3A, n = 10). Biopsies were obtained 2–4 weeks after cardiac transplantation. Immunoperoxidase staining was performed for αvβ3, tissue factor, and α5β1, and protein levels were further determined by Western blot analysis. Specimens with grade 2 and grade 3A rejection showed positive staining of αvβ3 in lymphocytic aggregates and vascular endothelial cells. By immunoblotting, we identified significantly increased expression of αvβ3 in the presence of acute rejection, grade 2 (3-fold, p = 0.01) and grade 3A (3.6-fold, p = 0.005) compared to grade 0 and 1A specimens. There was no evidence of increased expression of α5β1 or tissue factor. Acute cellular rejection, a process characterized by T-lymphocyte activation and release of inflammatory cytokines, is associated with increased expression of αvβ3.

Downstream regulation of the cAMP-dependent protein kinase (PKA) pathway is mediated by anchoring proteins (AKAPs) that sequester PKA to specific subcellular locations through binding to PKA regulatory subunits (RI or RII). The RII-binding domain of all AKAPs forms an amphipathic α-helix with similar secondary structure. However, the importance of sequence differences in the RII-binding domains of different AKAPs is unknown, and mechanisms that regulate AKAP-PKA affinity are not clearly defined. Using surface plasmon resonance (SPR) spectroscopy, we measured real-time kinetics of RII interaction with various AKAPs. Base-line equilibrium binding constants (Kd) for RII binding to Ht31, mAKAP, and AKAP15/18 were 10 nm, 119 nm, and 6.6 μm, respectively. PKA stimulation of intact Chinese hamster ovary cells increased RIIα binding to AKAP100/mAKAP and AKAP15/18 by ∼7- and 82-fold, respectively. These results suggest that differences in primary sequence of the RII-binding domain may be responsible for the selective affinity of RII for different AKAPs. Furthermore, RII autophosphorylation may provide additional localized regulation of kinase anchoring. In cardiac myocytes, disruption of RII-AKAP interaction decreased PKA phosphorylation of the PKA substrate, myosin-binding protein C. Thus, these mechanisms may be involved in adding additional specificity in intracellular signaling in diverse cell types and under conditions of cAMP/PKA activation.

A-kinase anchoring proteins tether cAMP-dependent protein kinase (PKA) to specific subcellular locations. The purpose of this study was to use fluorescence resonance energy transfer to monitor binding events in living cells between the type II regulatory subunit of PKA (RII) and the RII-binding domain of the human thyroid RII anchoring protein (Ht31), a peptide containing the PKA-binding domain of an A-kinase anchoring protein. RII was linked to enhanced yellow fluorescent protein (EYFP), Ht31 was linked to enhanced cyan fluorescent protein (ECFP), and these constructs were coexpressed in Chinese hamster ovary cells. Upon excitation of the donor fluorophore, Ht31·ECFP, an increase in emission of the acceptor fluorophore, RII·EYFP, and a decrease in emission from Ht31·ECFP were observed. The emission ratio (acceptor/donor) was increased 2-fold (p< 0.05) in cells expressing Ht31·ECFP and RII·EYFP compared with cells expressing Ht31P·ECFP, the inactive form of Ht31, and RII·EYFP. These results provide the first in vivodemonstration of RII/Ht31 interaction in living cells and confirm previous in vitrofindings of RII/Ht31 binding. Using surface plasmon resonance, we also showed that the green fluorescent protein tags did not significantly alter the binding of Ht31 to RII. Thus, fluorescence resonance energy transfer can be used to directly monitor protein-protein interactions of the PKA signaling pathway in living cells.

The recognition that the endoplasmic reticulum (ER), rather than the mitochondria, is the main organelle regulating the cytoplasmic Ca2+concentration in non-muscle cells1,2supports the notion that an alternative physiological role of mitochondrial Ca transport3–5is the modulation of Ca-sensitive mitochondrial enzymes through small (micromolar) fluctuations in the concentration of mitochondrial matrix Ca2+(refs 1,5–7). The latter mechanism could operate only if the mitochondrial Ca concentration were low, as it is in muscle and retinal rods8,9, below the levels saturating the regulated enzymes5. In contrast, if the ER serves as an intracellular Ca store, its Ca content would be expected to be high. In view of the major metabolic function of the liver, the question of whether hepatic mitochondrial matrix Ca2+regulates metabolism is particularly important, but the range of Ca concentrations reported for isolated liver mitochondria is too wide10–12to provide a conclusive answer. Therefore, we have used electron probe X-ray microanalysis (EPMA) to measure the subcellular distribution of Ca in liver snap-frozen in vivo, and report here that the endoplasmic reticulum is a major intracellular store of Ca, while the concentration of Ca in mitochondria is low and compatible with the regulation of mitochondrial enzymes.

Increased myocardial contractility was found in the perfused heart isolated from sodium depleted Sprague-Dawley rats. Previously, it was reported that in vitro exposure of different cardiac preparations to low Na+buffers was associated with both an increased contractility and an increased Ca2+content in the cells. Therefore, this study was designed to examine increases in ventricular Ca2+content in the hearts of chronically sodium depleted rats. Two groups of 12-week-old Sprague-Dawley rats were studied. One group (n = 5) received furosemide (5 mg/kg/day IP for 4 days), a low Na+diet and distilled drinking water for 6 weeks (low sodium plus diuretics group = LSD). The other group (n = 5) received the same low Na+diet, but 0.5% NaCl was supplemented in drinking water (regular sodium group = RS). Body weight and blood pressure were measured weekly during the dietary period in all rats. At the end of the 6 weeks, heart weight as well as water and electrolyte contents of the heart were measured in all animals. Results showed that both body weight and heart weight were significantly lower in LSD than in RS. Moreover, ventricular Na+content was reduced while ventricular Ca2+content was doubled in LSD compared to RS (8.2 ± 0.2 units vs. 9.2 ± 0.3 units, p < .05 and 0.45 ± 0.13 units vs. 0.23 ± 0.01 units, p < .01, respectively). We conclude that in vivo sodium depletion induces an increase in ventricular calcium content; this increased myocardial calcium may be related to the increased in vitro cardiac contractility observed after chronic in vivo sodium depletion, but its distribution between intracellular and extracellular compartments needs to be determined.

The 31P NMR spectrum of energy-related metabolites under strictly aerobic conditions in rapidly respiring tissues under physiological conditions has been approached by the study of the 31P NMR signals in vivoand in freeze-trapped organs. Freezing the head of the anesthetized animal by liquid N2, excision of the brain tissue (white and gray matter) at -196°, and transfer to the NMR tube occurs without alteration of the metabolite concentrations. The sample is warmed to the region -15° to -10°, at which temperatures there is sufficient mobility for recording 31P NMR at concentrations characteristic of brain tissues (∼5 mM) with an adequate signal to noise ratio in 10 min but insufficient mobility for significant enzymatic activity. A ∼0.4-sec acquisition time is adequate for nuclear relaxation and a 10-min scan gives an adequate signal to noise ratio. Metabolism of creatine phosphate, Pi, and sugar phosphates occurs by 1 hr at -10° and 2 hr at -12°. Extrapolation of the approximately zero order kinetics of disappearance of creatine phosphate and appearance of Pisuggests that <10% of these two metabolites has been altered in the time of the first measurement.A comparison of the freeze-trapped state and the in vivometabolite pattern is afforded in preliminary experiments on the head of the living mouse (brain and skeletal tissue) in aerobic and anaerobic states. Longer relaxation times and mild hypoxia due to the restricted diameter of the NMR tube gives significantly lower creatine phosphate/ATP values for this condition. Both direct in vivoand freeze-trapped assays of energy-related metabolites afford excellent approaches to the detection of anoxia and to the evaluation of metabolic control in hypoxic conditions.

Cardiac hypertrophy in the spontaneously hypertensive rat has been shown to be accompanied by a diminished inotropic response to β-adrenergic stimulation. This diminished response has been attributed to abnormalities in various components of the β-adrenergic signaling system. There is also evidence that regulation of intracellular Ca2+cycling may be altered in the hypertrophied heart of the spontaneously hypertensive rat. We proposed that the dominished reponse to β-adrenergic stimulation may reflect abnormalities in Ca2+cycling, specifically alterations in the ability of the sarcoplasmic reticulum to effectively release and resequester ca2+. We have used the unique combination of functional measurements on isolated, isometrically contracting papillary muscles from hearts of 26-week-old spontaneously hypertensive rats and their Wistar-Kyoto controls, together with electron proble microanalysis measurements of sarcoplasmic reticulum Ca2+content in the same muscles after rapid freezing, to determine the availability of Ca2+for activation of contraction, following β-adrenergic stimulation. We observed a significant decrease in the inotropic response to β-adrenergic stimulation in paillary muscles from the spontaneously hypertensive rats, as compared with Wistar-Kyoto controls, however in these same muscles, frozen during relaxation. there was no evidence of an accompanying decrease in the size of the sarcoplasmic reticulum Ca2+store. In an additional group of muscles which were frozen during contraction, the amount of Ca2+remaining in the sarcoplasmic reticulum after stimulated release was also not different in the two strains. These results indicate that the decreased inotropic response to β-adrenergic stimulation in hypertrophied hearts of the spontaneously hypertensive rat is unlikely to be due to decreased availability of Ca2+for activation of contraction. Additionally, to determine whether there is intracellular Ca2+overload in the cardiac muscle cells of hearts of spontaneously hypertensive rats, we measured the amount of Ca2+in mitochondria and at the level of the myofilaments by electron probe microanalysis. These results indicate that intracellular Ca2+overload doest not accompany cardiac hypertrophy in the spontaneously hypertensive rat. This study therefore shows no correlation between altered intracellular Ca2+cycling and the decreased inotropic response to isoproterenol in the spontaneously hypertensive rat at 26 weeks of age.

The spontaneously hypertensive rat (SHR) is characterized by elevated blood pressure and the development of left ventricular hypertrophy. During compensatory hypertrophy in the SHR, (26 weeks) when baseline contractile function is normal or increased, the inotropic response toβ-adrenergic stimulation is impaired. We recently showed by electron probe microanalysis (EPMA) that the amount of Ca2+stored in the sarcoplasmic reticulum (SR) following sympathetic stimulation is not decreased in the 26-week-old SHR heart. However, with disease progression, cardiac function declines further in the SHR and the response toβ-adrenergic stimulation is more impaired. To determine whether a decreased availability of SR Ca2+is responsible for the severely depressed inotropic response in the older SHR, we used EPMA to measure directly the amount of Ca2+stored in the SR following activation of theβ-adrenergic pathway in papillary muscles from 76-week-old SHR and Wistar–Kyoto (WKY) controls. In order to determine if there are other alterations in ion homeostasis, we also compared elemental content of A-band and mitochondria. Papillary muscles from 76-week-old SHR and WKY were stimulated by 10μmisoproterenol and then rapidly frozen during relaxation. The elemental content of the junctional SR, A-band and mitochondria was measured by EPMA. We observed no significant difference in SR Ca2+content between SHR and WKY. There was also no strain-dependent difference in mitochondrial or A-band Ca2+. Overall, these results indicate that the impaired response toβ-adrenergic stimulation in the SHR at 76 weeks is not due to altered availability of SR Ca2+.

During myocardial ischemia, several factors have been identified which contribute to the development of cardiac arrhythmias. These include membrane depolarization, increased cytosolic Na+ and decreased intracellular pH. This increase in intracellular Na+ has been shown to be associated with the development of ventricular arrhythmias. Changes in intracellular Cl-may also be involved in development of cardiac arrhythmias since decreasing extracellular Cl-concentration in the perfusion medium can prevent development of arrhythmias during myocardial ischemia in vitro. Extracellular ATP is known to increase in the heart during myocardial ischemia. ATP activation of p2purinergic receptors on cardiac myocytes has been proposed to contribute to the initiation of the arrhythmias and ventricular fibrillations which characterize myocardial ischemia. Activation of p2purinergic receptors results in membrane depolarization and decreased intracellular pH, thus reproducing some of the changes that occur during ischemia. We recently showed that activation of p2purinergic receptors in both quiescent and electrically stimulated ventricular myocytes triggers spontaneous oscillatory contractions and Ca2+transients.