Cardioprotective Effect of Hydroxysafflor Yellow A via the Cardiac Permeability Transition Pore.

Myocardial ischemia damages cardiac myocytes in part via opening of the mitochondrial permeability transition pore. Preventing this pore's opening is therefore a useful therapeutic goal in treating cardiovascular disease. Hydroxysafflor yellow A has been proposed as a nontoxic alternative to other agents that modulate mitochondrial permeability transition pore opening. In this study, we proposed that hydroxysafflor yellow A prevents mitochondrial permeability transition pore formation in anoxic cardiac myocytes, and thus protects the cell from damage seen during reoxygenation of the cardiac myocytes. Experiments with hydroxysafflor yellow A transport in aerobic myocytes show that roughly 50% of the extracellular dye concentration crosses the cell membrane in a 2-h incubation. In our anoxia/reoxygenation protocol, hydroxysafflor yellow A modulated both the reduction of viability and the loss of rod-shaped cells that attend anoxia and reoxygenation. Hydroxysafflor yellow A's protective effect was similar to that of cyclosporin A, an agent known to inhibit mitochondrial permeability transition pore opening. In additional experiments, plated myocytes were loaded with calcein/MitoTracker Red, then examined for intracellular dye distribution/morphology after anoxia/reoxygenation. Hydroxysafflor yellow A-containing cells showed a cardioprotective pattern similar to that of cyclosporin A (an agent known to close the mitochondrial permeability transition pore). We conclude that hydroxysafflor yellow A can enter the cardiac myocyte and is able to modulate anoxia/reoxygenation-induced damage by interacting with the mitochondrial permeability transition pore.

Injectable sodium pentobarbital: Stability at room temperature.

INTRODUCTION: Sodium pentobarbital (Nembutal) is a barbiturate used in research as an anesthetic in many animal models. The injectable form of this drug has lately become difficult to procure and prohibitively expensive. Due to this lack of availability, researchers have begun to compound injectable sodium pentobarbital from so-called "nonpharmaceutical" pentobarbital. Some oversight agencies have objected to this practice, claiming a lack of quality control and degradation of the drug. We sought with this study to establish both: 1) a protocol for the preparation of injectable sodium pentobarbital, and 2) standard operating procedures to monitor the quality of the preparation and degradation of the drug over time. METHODS: Our preparation consists of a mixture of sodium pentobarbital in alkaline aqueous solution, propylene glycol, and ethanol. Pentobarbital content in this preparation was assayed by high-pressure liquid chromatography (HPLC). We also assayed pentobarbital content over time in preparations of various ages up to 6 years old. RESULTS: We determined that the drug degraded at a maximum of 0.5% per year in our preparation (alkaline water/propylene glycol/ethanol) when stored in the dark at room temperature. A yellow discoloration developed after about 2 years, which we have arbitrarily determined disqualifies the preparation from use as an anesthetic. Attempts to spectroscopically assay this discoloration were not successful. CHEMICALS: Pentobarbital sodium (CID: 14075609).

Resting Ca2+ influx does not contribute to anoxia-induced cell death in adult rat cardiac myocytes.

Calcium has been proposed as a primary influence on cell death during ischemic episodes in myocardial cells. One component of calcium entry into a cell is resting calcium influx. This basal movement of calcium is blocked by 100 micromol/L gadolinium chloride (GdCl3) in cardiac myocytes. Therefore, GdCl3 should be cardioprotective under anoxic conditions. To test this, cardiac myocytes isolated from adult male rats were subjected to anoxia (100% N2) in the presence or absence of 100 micromol/L GdCl3 in one of 2 ways. In the first method, cells were suspended in media and rendered anoxic for 0, 30, and 60 min, after which cell morphology and viability were scored. After 60 min of anoxia, rod-shaped cells accounted for 46% +/- 4% of total cells (viability 81%); 10 min of reoxygenation markedly reduced rod-shaped cells to 27% (viability 72%). GdCl3 in the medium did not protect the cells (anoxic rods 49%, reoxygenated rods 30%, viability 77%). In the second method, cells were attached to a laminin substrate, rendered anoxic, and then videotaped for up to 6 h. In this system, cells maintained their shape for some time after the onset of anoxia, and then began to 'die' (i.e., to take on either a rigor form or hypercontracted form) at a measurable rate. Time to onset of 'death' (t0), time to 50% and 100% 'death' (t50 and t100), and rate of 'death' were used to measure anoxic damage. Without GdCl3, cells on average began to die 115 +/- 32 min after the onset of anoxia (t0); they died at an average rate of 0.046 cells/min. t50 was achieved in 149 +/- 42 min, t100 in 183 +/- 54 min. Addition of 100 micromol/L GdCl3 did not affect any of these parameters. We concluded that GdCl3 was not cardioprotective for anoxic myocytes and that cell damage generated by anoxia could not be attributed to resting calcium influx.

Compartmentalization of non-adenine nucleotides in anoxic cardiac myocytes.

Loss of 5'-nucleotides from cardiac myocytes is a distinguishing feature of myocardial ischemia. Previous work has documented dislocations of metabolic processes mediated by both purine and pyrimidine nucleotides, especially the adenine nucleotides. This study was designed to establish the extent of anoxia-induced depletion of non-adenine nucleotides in the cytosolic compartment of heart muscle cells. Cardiac myocytes were incubated aerobically (O(2)) or anoxically (N(2)) for 30 or 60 min; anoxic cells at both time points were reoxygenated for 10 min. Roughly 85-90% of cytosine triphosphate (CTP) and uridine triphosphate (UTP) were cytosolic under aerobic conditions, compared with 62% of guanosine triphosphate (GTP) and 90% of adenosine triphosphate (ATP) under similar conditions. Similarly, the total cytidine and uridine nucleotide pool of aerobic myocytes was 70-90% cytosolic vs. 61% of total guanine nucleotides and 78% of total adenine nucleotides. After the onset of anoxia, cytosolic nucleotides (principally the triphosphate forms) were quickly degraded. Reoxygenation of anoxic myocytes for 10 min allowed some recovery of ATP, GTP, and CTP, but very little recovery of UTP. The recovered nucleotide appeared almost exclusively in the cytosol. These results support the concept that non-adenine nucleotides could reach critically low levels in anoxic or ischemic heart in advance of adenine nucleotides. The importance of the depletion of non-adenine nucleotides is discussed in terms of the energetic needs of the myocyte, and the need for the cell to drive G-protein-coupled reactions, lipid synthesis, and glycogenesis.

Age-dependent changes in contraction and regional myocardial myosin heavy chain isoform expression in rats.

The goals of this study were to measure the relative levels of the alpha- and beta-isoforms of myosin heavy chain (MHC-alpha and MHC-beta, respectively) in multiple, specific regions of the adult rat heart and to determine whether age-dependent changes in isoform levels in different regions are uniform. Relative amounts of MHC-alpha and MHC-beta were determined in right and left atria and left ventricular (LV) Purkinje fibers (PF), papillary muscles, trabeculae, and endo-, mid-, and epicardial regions at 2, 5, 10, 16, and 21 mo. PFs contained substantial amounts of myosin and were striated and capable of generating force and shortening on activation. Levels of MHC-beta increased in all LV compartments with age, especially between 2 and 5 mo. There was more MHC-beta in PFs than other LV sites. There were regional differences in the level of MHC-beta throughout the LV at all ages, and the rates of change within regions differed. Ca(2+)-activated tension in PFs and trabeculae was compared at 2 and 22 mo. PF tension was less than trabecula tension, and this difference may be explained by differences in MHC content. V(max) and tension-generating ability in PFs decreased with age. Maximal tension generated by trabeculae did not change during aging. A large proportion of the increase in the level of MHC-beta that is normally associated with aging occurs at a relatively early age in rat LV. PFs, with their small diameters and short diffusion distance, should be considered for skinned multicellular myocardial studies.

3-isobutyl-1-methylxanthine (IBMX) sensitizes cardiac myocytes to anoxia.

Cardiac myocytes incubated with 3-isobutyl-1-methylxanthine (IBMX), a nonspecific cyclic nucleotide phosphodiesterase inhibitor, formed rigor complexes under anoxic conditions more readily than cells incubated with other phosphodiesterase inhibitors. Cardiac myocytes were incubated for 1 hr with either (a) no additions, (b) 150 microM zaprinast, or (c) 1 mM IBMX, and then were rendered anoxic for periods up to 60 min. Cells were >80% viable throughout the anoxic period; viability was unaffected by either drug. Rod count decreased more rapidly after the onset of anoxia in the IBMX-treated cells than in control or zaprinast-treated cells (11% rods vs. roughly 47% rods after 30 min of anoxia). IBMX-treated cell groups also formed more "contracted" myocytes (box-like rods) than their untreated or zaprinast-treated counterparts (50% contracted vs. roughly 27% contracted after 30 min of anoxia). While nucleotide degradation patterns were similar in all experimental groups, the ratio of ATP to ADP was lower in IBMX-treated cells than in control or zaprinast-treated cells. The L-type calcium channel was apparently not involved in this phenomenon; while cyclic AMP was elevated in the IBMX-incubated cells, verapamil did not protect IBMX-incubated cells from premature damage by anoxia. Incubation with 8-cyclopentyl-1,3-dipropylxanthine (CDPX), an A1 receptor antagonist, at concentrations up to 1 microM in place of 1mM IBMX did not reproduce the IBMX effect. We concluded that IBMX sensitizes cardiac myocytes to anoxia through a mechanism related to its effect on ATP/ADP, and unrelated to an elevation of intracellular calcium or preconditioning phenomena.