Margaret A. Goldstein

Biochemical and morphologic correlates of cardiac ischemia. I. Membrane systems.

Functions of membrane-linked myocardial systems and morphology of the myocardial cell were examined in normal and acutely and chronically ischemic myocardium. Hemodynamic measurements of ischemic tissue showed depressed force development as well as decreased (and variable) ventricular peak pressures. Mitochondrial respiratory function was reduced, with state 3 (phosphorylating) respiration showing the most marked impairment. Losses in cytochromes c and a 3 were observed. Diminished mitochondrial calcium uptake with subsequent release of the calcium taken up during continued respiratory activity was characteristic of severely ischemic tissue, that is, 7 days after ligation. Defects in the carnitine-mediated oxidation of palmitic acid in isolated mitochondria were severe between 1 and 8 days after ligation. Losses in tissue carnitine also occurred simultaneously with decreased mitochondrial carnitine palmityltransferase activity. During this period the oxidation of hexanoic acid was unaffected. The binding and release cycle of calcium was studied in sarcoplasmic reticulum (cardiac relaxing system) isolated from control tissues and from acutely and chronically ischemic heart. An early impairment in the release phase of calcium from an acutely ischemic preparation occurred at times (12 to 60 minutes after ligation) when the other membrane-associated functions maintained normal integrity. In the chronically ischemic dog, there was marked impairment of calcium-binding variables in the first 2 weeks after ligation. This impairment occurred at times when both mitochondrial and sodium, potassium adenosine triphosphatase (Na + ,K + -ATPase) activity levels were severely impaired. The Na + ,K + -ATPase activity level was consistently lower than the control level by 7 days after ligation. However, there was no change in kinetic indexes or in the ouabainbinding characteristics in the functional enzyme remaining. Morphologic studies of tissue taken from left posterior papillary muscle 3 to 8 days after ligation revealed significant and specific changes in ultrastructure (disrupted Z and I bands, appearance of pseudo-N band and appearance of dense bodies in mitochondria); the greatest occurrence of damaged cells occurred 7 to 8 days after ligation.

Calcium uptake by two preparations of mitochondria from heart

Ca/+ transport and respiratory characteristics of two preparations of cardiac mitochondria (Palmer, J.W., Tandler, B. and Hoppel, C.L. (1977) J. Biol. Chem. 252, 8731-8739) isolated using polytron homogenization (subsarcolemmal mitochondria) and limited Nagarse exposure (intermyofibrillar mitochondria) are described. The Nagarse procedure yields mitochondria with 50% higher rates of oxidative phosphorylation than the polytron-prepared mitochondria in both rat and dog. Rat hear intermyofibrillar mitochondria contain 50% more cytochrome aa3 than the polytron preparation, whereas in the dog, cytochrome aa3 content is not significantly different. Cytochrome oxidase activities and cytochrome c, c1 and b contents were comparable in both populations of rat and dog heart mitochondria. The V of succinate-supported Ca2+ accumulation for Nagarse-prepared mitochondria from rat heart was 1.8-fold higher than the polytron-prepared mitochondria. In dog heart, the Nagarse preparation showed a 3.0-fold higher V for Ca2+ uptake compared to the polytron preparation. A lower apparent affinity for Ca2+ was demonstrated in the intermyofibrillar mitochondria for both species (Km is 2-2.5-fold higher). The Hill coefficient was 1 both mitochondrial types. Subsarcolemmal mitochondria from both species were treated with Nagarse to determine the role of this treatment on the observed differences. Nagarse did not alter any kinetic parameter of Ca2+ uptake. The properties of these mitochondria with reference to their presumed intracellular location may pertain to the role of mitochondria as an intracellular Ca2+ buffering mechanism in contractile tissue.

Ultrastructural analysis of left ventricular hypertrophy in rabbits.

Ultrastructural changes resulting from gradual aortic constriction were quantitated in a model for cardiac hypertrophy. Gradual aortic stenosis was produced in rabbits by swelling of an Ameroid clip. Ultrastructural, biochemical and physiological changes were observed for each heart. The ultrastructure of mitochondria in hypertrophied but non-failing hearts was normal. The respiratory activity of mitochondria isolated from these same hearts was increased. Profiles of granular endoplasmic reticulum and free ribosomes, widening of Z bands and distortions of intercalated discs were observed in hypertrophied hearts and were interpreted as evidence of synthesis of new functional components. However, different cell components increased disproportionately. Morphometric analysis revealed significant decreases in mitochondrial fractional volume (0.35 ± 0.01, P < 0.001) and mitochondria/myofibril ratios (0.59 < 0.02, P < 0.001). The results suggest that increased work load may induce a further deleterious change in the ratio between mitochondrial units and myofibril units with may lead to decompensation and failure. The ultrastructural changes observed in this model may be of interest since the model closely mimics the gradual onset of the hypertrophic response in humans.

Association of glycogenolysis with cardiac sarcoplasmic recticulum: II. Effect of glycogen depletion, deoxycholate solubilization and cardiac ischemia: Evidence for a phosphorylase kinase membrane complex

The glycogenolytic enzymes associated with cardiac sarcoplasmic reticulum have been shown to have two major features which make the munique. The conversion of phosphorylase b to phosphorylase a can take place in the presence of 20 m m eGTA which ordinarily inhibits this reaction by chelating the calcium which is required for the enzyme phosphorylase b kinase of muscle. In addition, inhibitors of “debrancher” enzyme such as Tris also inhibit phosphorylase b activity in the sarcoplasmic reticulum-glycogen complex, but do not inhibit purified phosphorylase. The phosphorylase b to a conversion which occurs through the addition of ATP alone is very rapid, being complete within 5–10 min in normal cardiac sarcoplasmic reticulum and converting approximately 30% of the total phosphorylase activity. DOC solubilization irreversibly perturbed the sarcoplasmic reticulum membrane so that EGTA resistance could not be reconstituted although phosphorylase b to a conversion continued to some extent. Thus, EGTA sensitivity depended, at least in part, on some specific property of the native membrane system. Examination of skeletal muscle sarcoplasmic reticulum from fast, slow and mixed skeletal muscle revealed that phosphorylase b to a conversion occurred, but was EGTA sensitive in a manner similar to that of the solubilized cardiac preparation. Glycogen depletion solubilized phosphorylase and uncoupled it from its activating enzymes. This uncoupling could not be reversed by the addition of glycogen at any concentration. A relationship was demonstrated between the phosphorylase activity in sarcoplasmic reticulum fractions and the glycogen concentration in both normal sarcoplasmic reticulum that was amylase treated, and from sarcoplasmic reticulum which was isolated from ischemic tissue. A correlation between density observed on a sucrose density gradient and glycogen concentration was described. The relationship between phosphorylase and debrancher activity depended only on the presence of glycogen and therefore was not as specific a structural feature. The results suggest that the sarcoplasmic reticulum glycogen complex is highly specific and that specific functional couplings between glycogen and sarcoplasmic reticulum membranes exist. Thus, modulation of the complex could occur through control of glycogenolysis or alterations in membrane conformation, and this complex might represent a link between energy metabolism and excitation-contraction coupling.

DNA Synthesis and Mitosis in Well-Differentiated Mammalian Cardiocytes

Incorporation of [3H]thymidine into nuclei of heart cells of 2-day-old rats indicates that neonatal cardiac cells containing well-aligned myofibrils synthesize DNA. In these highly differentiated cells, neither the presence of contractile proteins nor their organization into myofibrils inhibits either DNA synthesis or mitosis.

Role of the Z band in the mechanical properties of the heart.

In striated muscle the mechanism of contraction involves the cooperative movement of contractile and elastic components. This review emphasizes a structural approach that describes the cellular and extracellular components with known anatomical, biochemical, and physical properties that make them candidates for these contractile and elastic components. Classical models of contractile and elastic elements and their underlying assumptions are presented. Mechanical properties of cardiac and skeletal muscle are compared and contrasted and then related to ultrastructure. Information from these approaches leads to the conclusion that the Z band is essential for muscle contraction. Our review of Z band structure shows the Z band at the interface where extracellular components meet the cell surface. The Z band is also the interface from cell surface to myofibril, from extramyofibrillar to myofibril, and finally from sarcomere to sarcomere. Our studies of Z band in defined physiologic states show that this lattice is an integral part of the contractile elements and can function as an elastic component. The Z band is a complex dynamic lattice uniquely suited to play several roles in muscle contraction.—Goldstein, M. A.; Schroeter, J. P.; Michael, L. H. Role of the Z band in the mechanical properties of the heart. FASEB J. 5: 2167–2174; 1991.

The cardiac sarcoplasmic reticulum — Glycogenolytic complex, an internal beta adrenergic receptor

Abstract Agents which stimulate the formation of cyclic AMP are well known to cause positive inotropic effects on the heart. This review suggests that the myocardial cell is multicompartmented and has several effector sites for cyclic AMP-mediated events and therefore several possible receptor sites for cyclic AMP-mediated agents. It is possible that some of the complex relationships between cyclic AMP-mediated agents and cardiac physiologic functions result from differences among agonist receptors and enzymes in the intracellular compartments. Examination of the characteristics of intracellular drug receptors may yield a new frontier for cardiac pharmacology.

The Z-band lattice in skeletal muscle before, during and after tetanic contraction

SummaryElectron micrographs and optical diffraction patterns of the Z-band were studied in rat soleus muscle fixed before, during, and after tetanic contraction. We compared the morphology (small square or basketweave pattern) and dimensions of the Z-lattice of control and tetanized muscles near rest length. Z-bands of muscle fixed at rest and of muscle allowed to rest after a tetanic contraction exhibited the small square pattern. Z-bands from muscle fixed during tetanic contraction exhibited the basketweave pattern. Concomitant with the transition to basketweave, we observed an average increase of 20% in spacing between the axial filaments of the Z-lattice. Optical diffraction measurements of the A-bandd10 spacing revealed that the Z/A ratio remained constant during the transition. We have modelled the small square to basketweave transformation as resulting from a change of curvature of constant length cross-connecting Z-filaments when the axial filaments increase their separation.

The Z-band lattice in a slow skeletal muscle

SummaryStructural features of the Z-lattice were examined in cross-sections of rat soleus muscle. Optical diffraction analysis of individual myofilament bundles revealed highly ordered, tightly packed tetragonal lattice regions with abrupt shifts in orientation. Optical reconstructions of cross-sections of the Z-lattice in regions having the same axial filament spacing showed both the basket weave and the small square lattice forms, as well as intermediate structures. Analysis of these regions in three dimensions and in two-dimensional projections suggests that a change in diameter together with a change in curvature of the cross-connecting filaments can explain the different appearances of the Z-lattice at a given sarcomere length.

Optical reconstruction of nemaline rods

Abstract Optically filtered and reconstructed images were obtained from the Z lattice in a specimen of partially extracted skeletal muscle from a patient with nemaline myopathy. Data from longitudinal sections of Z rods showed a 37.5-nm repeat along the axial filaments associated with an array of connecting filaments. Projected distances between axial filaments were consistent with a 25-nm centered square array in cross sections. Data from longitudinal sections of glycerinated rabbit psoas muscle and normal rat soleus muscle showed a similar repeating unit. Optical reconstructions revealed differences in the substructure of the Z lattice, but similar unit cell dimensions in all three cases.