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Myoglobin (Mb) has a purported role in facilitating [O.sub.2] diffusion in tissue, especially as cellular [Po.sub.2] drops or the respiration demand increases. Inhibiting Mb with CO under conditions that accentuate the facilitated diffusion role should then elicit a significant physiological response. In one set of experiments, the perfused myocardium received buffer with decreasing [Po.sub.2] (225, 129, and 64 mmHg). Intracellular [Po.sub.2] declined, as reflected in the [sup.1H] NMR Val Ell signal of MbO2 (67%, 32%, and 18%). The addition of 6% CO further reduced the available Mb[O.sup.2] (11%, 9%, and 7%), as evidenced by the decline of the Mb[O.sub.2] Val E11 signal intensity at -2.76 ppm. In a second set of experiments, electrical stimulation increased the heart rate (300, 450, and 540 beats/min) and correspondingly the [O.sup.2] consumption rate (M[V.sub.o2]). Intracellular [Po.sub.2] also declined, as reflected in the slight drop in the Mb[O.sub.2] signal (100%, 96%, and 82%). M[Vo.sub.2] increased (100%, 114%, 165%). The addition of 3% CO in the stimulated hearts further decreased the available Mb[O.sub.2] (46%, 44%, and 29%). In all cases, CO inactivation of Mb does not induce any change in the respiration rate, contractile function, and high-energy phosphate levels. Moreover, the MbCO/Mb[O.sub.2] partition coefficient shifts dramatically from its in vitro value during hypoxia and increased work. The observation suggests a modulation of an intracellular [O.sub.2] gradient. Overall, the experimental observations provide no evidence of a facilitated diffusion role for Mb in perfused myocardium and implicate a physiologically responsive intracellular [O.sub.2] gradient. nuclear magnetic resonance; respiration; carbon monoxide; myocardium; oxidative phosphorylation

Shotgun lipidomics is exploited to demonstrate a 4-fold increase in acylcarnitines in diabetic myocardium, which is reversible upon insulin treatment. The analysis of acylcarnitine molecular species in diabetic myocardium identified acylcarnitine, molecular species containing a mass shift of 16 amu in comparison to the anticipated molecular species while synthesis of 3-hydroxy acylcarnitines identified natural products such as the 3-hydroxylated acylcarnitines.

The putative voltage-sensitive release mechanism (VSRM) was investigated in rabbit cardiac myocytes at 37[degrees]C with high resistance microelectrodes to minimize intracellular dialysis. When the holding potential was adjusted from -40 to -60 mV, the putative VSRM was expected to operate alongside CICR. Under these conditions however, we did not observe a plateau at positive potentials of the cell shortening versus voltage relationship. The threshold for cell shortening changed by -10 mV, but this resulted from a similar change of the threshold for activation of inward current. Cell shortening under conditions where the putative VSRM was expected to operate was blocked in a dose dependent way by nifedipine and Cd[Cl.sup.2] and blocked completely by Ni[Cl.sup.2] 'Tail contractions' persisted in the presence of nifedipine and Cd[Cl.sup.2] but were blocked completely by Ni[Cl.sup.2]. Block of early outward current by 4-aminopyridine and 4-acetoamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid (SITS) demonstrated persisting inward current during test depolarizations despite the presence of nifedipine and CD[Cl.sub.2]. Inward current did not persist in the presence of Ni[Cl.sup.2]. A tonic component of cell shortening that was prominent during depolarizations to positive potentials under conditions selective for the putative VSRM was sensitive to rapidly applied changes in superfusate [[Na.sup.+]] and to the outward [Na.sup.+]/[Ca.sup.2+] exchange current blocking drug KB-R7943. This component of cell shortening was thought to be the result of [Na.sup.+]/[Ca.sup.2+] exchange--mediated excitation contraction coupling. Cell shortening recorded under conditions selective for the putative VSRM was increased by the enhanced state of phosphorylation induced by isoprenaline (1 [micro]M) and by enhancing sarcoplasmic reticulum [Ca.sup.2+] content by manipulation of the conditioning steps. Under these conditions, cell shortening at positive test depolarizations was converted from tonic to phasic. We conclude that the putative VSRM is explained by CICR with the [Ca.sup.2+] 'trigger' supplied by unblocked L-type [Ca.sup.2+] channels and [Na.sup.+]+/[Ca.sup.2+] exchange. KEY WORDS: EC coupling * L-type [Ca.sup.2+] channels * VSRM * CICR

The effects of long-chain (LC) fatty acids on rate of heat production (heat rate) and mitochondrial membrane potential ([DELTA][PSI]) of intact guinea pig cardiac muscle were investigated at 37 [degrees] C. Heat rate of ventricular trabeculae was measured with microcalorimetry, and [DELTA][PSI] was monitored in isolated ventricular my. ocytes with either JC-1 or tetramethylrhodamine ethyl ester (TMRE). Methyl-[beta]-cyclodextrin was used as fatty acid carrier. Application of 400 [micro]M oleate or linoleate increased resting heat rate by ~30% and ~25%, respectively. When LC fatty acid was supplied as sole metabolic substrate, resting heat rate was decreased by 3-mercaptopropionic acid. In TMRE-loaded myocytes, neither 40-80 [micro]M oleate nor 40 [micro]M linoleate affected [DELTA][PSI]. At a higher concentration (400 [micro]M) both oleate and linoleate increased TMRE fluorescence by ~20% of maximum, obtained using 2,4-dinitrophenol (100 [micro]M), indicating a depolarization of the inner mitochondrial membrane. We conclude that LC fatty acids, at sufficiently high concentration, increase heat rate and decrease [DELTA][PSI] in intact cardiac muscle, consistent with a protonophoric uncoupling action. These effects may contribute to the high metabolic rate after reperfusion of postischemic myocardium. mitochondrial membrane potential; microcalorimetry

The expression and localization of the A-kinase-anchoring protein 100 (AKAP100) in adult hearts have been investigated. AKAP100 is approximately 100 kD long and found in rat embryonic muscle cells. Immunoblotting was used to identify AKAP100 in adult rat and human hearts. Immunofluorescence and confocal microscopy were used on rat cardiac myocytes to localize AKAP100 to the nucleus, sarcolemma, intercalated disc and at the level of the Z-line. Results show that AKAP100 binds protein kinase A II to multiple subcellular compartments for phosphorylation of different pools of heart proteins.

The responses of oligomeric state and adenosine triphosphatase (ATPase) activity to general anesthetics were compared for sarcoplasmic reticulum (SR) samples from cardiac muscles to those taken from skeletal muscles. The cardiac and skeletal SR were found to have identical responses of ATPase activity and oligomeric state for the general anesthetics diethyl ether, halothane and hexanol. The upperbound of anisotropy at very long times was also found to increase significantly as a result of general anesthetics in erythrosin isothiocyanate-labeled cardiac SR.

The influence of site I to the energetics of the induced structural changes was investigated through nuclear magnetic resonance studies of calcium (super 2+) interaction with the regulatory domains. The inactive site I in cNTnC responded to Ca(super 2+) binding to site II secondary to the structural coupling between the two sites. The results were not influenced by protein dimerization or aggregation.

Multidimensional nuclear magnetic resonance spectroscopic data of Escherichia coli hemoprotein structures presented a Ca2+-induced structural transition in the regulatory domain of the cardiac troponin C. Critical amino acid substitutions at the site I revealed a significant change in the function of the human troponin C. The structures of C-terminal domain of troponin C (cTnC) remained closed upon binding with Ca2+.

The heart of adult dogs treated with monophosphoryl lipid A (MLA) were analyzed to determine the role of adenosine triphosphate (ATP)-sensitive K+ channels on the activity of myocardial preconditioning (PC). Myocardial infarct size (IS) was reduced in the heart of mongrel dogs during prolonged myocardial ischemia-reperfusion after treatment with MLA. MLA-treated dogs also exhibited increased KATP channel activity which mediated reductions in action potential duration and enhanced cardioprotection.

Thermal damage in heated bovine myocardial tissue is assessed from measured changes in total reflection and transmission of light. Mathematical expressions, based on random walk analysis of light propagation within tissue slabs, are used to relate the diffuse reflection and transmittance to the absorption coefficient, [[micron].sub.a], and effective scattering coefficient, [[micron][prime].sub.s] for samples of myocardial tissue which were subjected to rapid step changes in temperature. Time-dependent changes in [[micron][prime].sub.s] indicate two processes, one with a fast and temperature-dependent rate the other with a slow and apparently temperature-independent rate. For final temperatures above 56.8 [degrees] C and for the first 500 s after the temperature change, the optical parameters are well fit by exponential forms that exhibit temperature-dependent time constants as predicted by Arrhenius reaction rate theory of thermal damage. The scattering changes are associated with an apparent activation energy, [Delta]E, of 162 kJ/mole and a frequency constant, A, of 3x [10.sup.23] [s.sup.-1]. This method provides a means for estimating optical coefficients which are needed to assess laser tissue dosimetry.

The junctions involved in excitation-contraction coupling in skeletal and cardiac muscles are separate discontinuous units. The junctions of fast acting skeletal muscle fibers have long T-tubule segments as compared to slow-acting fibers. Separation of the junctions determines the ability of the units to interact by the spread of an activating substance. The junctions in the plasma membrane consist of feet, arranged in arrays while the junctions in sarcoplasmic reticulum consist of tetrads. Differences in the skeletal and cardiac membrane are discussed.

The fetal mouse ventricular myocytes express a rapidly activating component of the delayed rectifier K+ current (IK), which plays an important role in the repolarization of the K+ current. Rapid stimulation of the IK results in the current-voltage relationships displaying a negative slope conductance at a test potential of greater than 0 mV. Dofelitide successfully suppresses the rapidly activating component of the IK, prolonging the duration of action potentials in the fetal ventricular myocytes and myocardium.

The degree of residual stress in the left ventricle is affected by osmotic or induced myocardial swelling. Residual strain is caused by collagen and elastin fibers in the intercellular matrix which increases myocardial swelling due to increased intercellular distances. Analysis of a morphological strain model has shown that myocardial swelling is the result of myocardial regulation of residual stress.

Studies of a complement-mediated myocardial injury model reveal that thesulfhydryl compounds captopril and N-(2-mercaptopropionyl)-glycine (MPG) protect the heart against complement-mediated increases in left ventricular end-diastolic pressures and coronary arterial perfusion pressure, whereas enalapril does not confer any profection. The effects of captopril and MPG are concentration-dependent and verifiable with an in vitro red blood cell hemolysis assay.

Phosphorus 31 nuclear magnetic resonance (NMR) spectroscopy is a potential instrument for analyzing the changes in the chemical environment of a phosphate group. This property facilitates the characterization of the cardiac troponin I phosphorylated at serine 23 and/or 24 in the heart-related region of bovine troponin I. A resonance signal for the monophosphorylated group bearing phosphate molecule at serine 24 is obtained at 465 parts per million, and the P 31-NMR signal at 4.2 ppm obtained for the phosphate forms or biphosphorylated form yielded chemical shift values which were functions of pH and Pka.

Single rabbit cardiac myocytes exhibit high sensitivity to elevated thyroxine levels and temperature changes, probably due to regulation of transient outward current (I(t)). I(t) channels in the hyperthyroid state are composed of proteins synthesized by thyroxine-induced gene groups. This contrasts with the hypothyroid state that is characterized by lack of changes in the myosin distribution.

In vitro noncontracting adult rat atrial preparations were used to examine fluid-phase endocytosis connected with recycling of bound plasmalemma-discharging granules or membranes and cardiac myocytes at 37 degrees centigrade. An area in the right ventricle which contained intramyocytic sucrose and a sucrose space which did not permit insulin was discovered by an examination of sucrose secretion dynamics.

A technique combining the flexible silicone rubber substratum technique with microinjection of fluorescent-labeled alpha-actinin and actinin was developed to test the hypothesis that costameres provide mechanical linkage between muscle cell contractile machinery and the extracellular matrix. The results showed that the distribution of vinculin-rich costameres are correlated with the patterns of forces exerted by adult rat cardiac myocytes on the flexible substrate. Furthermore, it is suggested that costameres can transmit forces to the extracellular matrix based on the observed relation of the cells with its underlying substrate as shown by interference electron microscopy.

The tissue-specific expression of lipoprotein lipase (LPL) in adipose tissue (AT), skeletal muscle (SM), and cardiac muscle (CM) is rate-limiting for the uptake of triglyceride (TG)-derived free fatty acids and decisive in the regulation of energy balance and lipoprotein metabolism. To investigate the tissue-specific metabolic effects of LPL, three independent transgenic mouse lines were established that expressed a human LPL (hLPL) minigene predominantly in CM. Through cross-breeding with heterozygous LPL knockout mice, animals were generated that produced hLPL mRNA and enzyme activity in CM but lacked the enzyme in SM and AT because of the absence of the endogenous mouse LPL gene (L0-hLPL). LPL activity in CM and postheparin plasma of L0-hLPL mice was reduced by 34% and 60%, respectively, compared with control mice. This reduced LPL expression was sufficient to rescue LPL knockout mice from neonatal death. L0-hLPL animals developed normally with regard to body weight and body-mass composition. Plasma TG levels in L0-hLPL animals were increased up to 10-fold during the suckling period but normalized after weaning and decreased in adult animals. L0-hLPL mice had normal plasma high-density lipoprotein (HDL)-cholesterol levels, indicating that LPL expression in CM alone was sufficient to allow for normal HDL production. The absence of LPL in SM and AT did not cause detectable morphological or histopathological changes in these tissues. However, the lipid composition in AT and SM exhibited a marked decrease in polyunsaturated fatty acids. From this genetic model of LPL deficiency in SM and AT, it can be concluded that CM-specific LPL expression is a major determinant in the regulation of plasma TG and HDL-cholesterol levels.

Electrical conductivities in the bidomain model of cardiac tissue are expressed as functions of four parameters. These expressions allow simulations to be performed using nominal, equal, and reciprocal anisotropy without introducing undesired effects, such as length constant variations. Relative values of the bidomain conductivities are estimated to be: [[Sigma].sub.iL] = 1, [[Sigma].sub.iT] = 0.1, [[Sigma].sub.eL] = 1, and [[Sigma].sub.eT] = 0.4. Index Terms - Anisotropy, bidomain, cardiac tissue, conductivity.

Students can learn a great deal about the peripheral circulation when teaching is based on five building blocks: hemodynamic principles, neurohumoral control, and three elements of local control of blood flow (metabolic, myogenic, and paracrine). Study of a particular special circulation starts with the application of these building blocks in the context of the function of that tissue. For example, control of skin blood flow is largely concerned with regulation of body temperature (neurohumoral control) and the response to injury (paracrine control). Regulation of coronary blood flow is almost entirely a matter of meeting the metabolic needs of the myocardium (metabolic control). By mixing and matching the five building blocks and keeping in mind the special functions of a particular tissue, students can master the peripheral circulation efficiently. AM.J. PHYSIOL. 277 (ADV. PHYSIOL. EDUC. 22): S164-S173, 1999. Key words: skin blood flow; coronary blood flow; neurohumoral; metabolic; myogenic; paracrine; active hyperemia; reactive hyperemia; autoregulation

Lombardo, Yolanda B., Cynthia Serdikoff, Manikkavasagar Thamotharan, Harbhajan S. Paul, and Siamak A. Adibi. Inverse alterations of BCKA dehydrogenase activity in cardiac and skeletal muscles of diabetic rats. Am. J. Physiol. 277 (Endocrinol. Metab. 40): E685-E692, 1999.--Rat cardiac and skeletal muscles, which have been used as model tissues for studies of regulation of branched-chain [Alpha]-keto acid (BCKA) oxidation, vary greatly in the activity state of their BCKA dehydrogenase. In the present experiment, we have investigated whether they also vary in response of their BCKA dehydrogenase to a metabolic alteration such as diabetes and, if so, to investigate the mechanism that underlies the difference. Diabetes was produced by depriving streptozotocin-treated rats of insulin administration for 96 h. The investigation of BCKA dehydrogenase in the skeletal muscle (gastrocnemius) showed that diabetes 1) increased its activity, 2) increased the protein and gene expressions of all of its subunits ([E.sub.1] [Alpha], [E.sub.1] [Beta], [E.sub.2]), 3) increased its activity state, 4) decreased the rate of its inactivation, and 5) decreased the protein expression of its associated kinase (BCKAD kinase) without affecting its gene expression. In sharp contrast, the investigation of BCKA dehydrogenase in the cardiac muscle showed that diabetes 1) decreased its activity, 2) had no effect on either protein or gene expression of any of its subunits, 3) decreased its activity state, 4) increased its rate of inactivation, and 5) increased both the protein and gene expressions of its associated kinase. In conclusion, our data suggest that, in diabetes, the protein expression of BCKAD kinase is downregulated posttranscriptionally in the skeletal muscle, whereas it is upregulated pretranslationally in the cardiac muscle, causing inverse alterations of BCKA dehydrogenase activity in these muscles. branched-chain keto acid dehydrogenase; BCKAD kinase

Xue, Xiao-Hua, Larry V. Hryshko, Debora A. Nicoll, Kenneth D. Philipson, and Glen F. Tibbits. Cloning, expression, and characterization of the trout cardiac [Na.sup.+]/ [Ca.sup.2+] exchanger. Am. J. Physiol. 277 (Cell Physiol. 46): C693-C700, 1999.--Isoform 1 of the cardiac [Na.sup.+]/[Ca.sup.2+] exchanger (NCX1) is an important regulator of cytosolic [Ca.sup.2+] concentration in contraction and relaxation. Studies with trout heart sarcolemmal vesicles have shown NCX to have a high level of activity at 7 [degrees] C, and this unique property is likely due to differences in protein structure. In this study, we describe the cloning of an NCX (NCX-TR1) from a Lambda ZAP II cDNA library constructed from rainbow trout (Oncorhynchus mykiss) heart RNA. The NCX-TR1 cDNA has an open reading frame that codes for a protein of 968 amino acids with a deduced molecular mass of 108 kDa. A hydropathy plot indicates the protein contains 12 hydrophobic segments (of which the first is predicted to be a cleaved leader peptide) and a large cytoplasmic loop. By analogy to NCX1, NCX-TR1 is predicted to have nine transmembrane segments. The sequences demonstrated to be the exchanger inhibitory peptide site and the regulatory [Ca.sup.2+] binding site in the cytoplasmic loop of mammalian NCX1 are almost completely conserved in NCX-TR1. NCX-TR1 cRNA was injected into Xenopus oocytes, and after 3-4 days currents were measured by the giant excised patch technique. NCX-TR1 currents measured at ~23[degrees]C demonstrated [Na.sup.+]-dependent inactivation and [Ca.sup.2+]-dependent activation in a manner qualitatively similar to that for NCX1 currents. teleosts; myocardium; contractility; sodium/calcium exchange

The analyses made by A.T. Winfree regarding critical cardiac curvatures failed to take into account the influence of propagation fronts caused by boundary effects on the isotropic properties of cardiac tissue. The author did not recognize that the value for critical curvatures obtained by rescaling to anisotropy is not the same as that for critical curvatures that are theoretically present on either axis in the heart. Winfree's conclusions can have detrimental consequences on the design of small pacing electrodes for the cardiovascular system.