Abstract 322: Incubation With Histone Deacetylases 3 and 4 or p300/CREB Binding Protein Associated Factor Does Not Significantly Alter Cardiac Sarcomere Function

Recent studies have suggested that treatment of skinned cardiac trabeculae with a histone deacetylase (HDAC) inhibitor increases calcium sensitivity, measured as the calcium required for half maximal force development (pCa50). The proposed mechanism is the acetylation of sarcomeric proteins by an endogenous histone acetyl transferase, p300/CREB binding protein associated factor (pCAF). As a test of this proposed mechanism, we treated skinned cardiac myocytes from 8 rat left ventricles (LV) with supra-physiological concentrations of pCAF, with Acetyl COA as an acetyl group donor, and supra-physiological concentrations of constitutively active forms of HDACs 3 and 4, and examined the force-calcium relation. Myocytes were prepared from frozen rat LV samples by gentle homogenization in rigor solution (mM: 50 Tris, 1 EGTA, 100 KCl, 2 MgCl2, 2 DTT and a protease inhibitor cocktail) containing 0.1% Triton X-100. The myocyte homogenates were washed and resuspended in relaxing solution (mM: 5 MgATP, 10 Creatine phosphate, 40 K-Propionate and 10-6 Ca-EGTA) divided into four 300 µl aliquots, One was an untreated control and three were treated with either i) HDAC3/Ncor or HDAC4 , ii) pCAF + 1mM Acetyl CoA, or ii) 1mM Acetyl CoA. Force-calcium relationships were determined in mixtures of relaxing and activating solution (mM: 5 MgATP, 10 Creatine phosphate, 0.5 K-Propionate, 10-1.5 Ca-EGTA) to give 6 calcium concentrations between 10-9 and 10-3.5 mM. While HDAC 3 or 4 treatment tended to decrease calcium sensitivity, and pCAF+Acetyl COA, and Acetyl COA alone, tended to increase calcium sensitivity compared to controls, we found no statistically significant effects of HDAC3 or 4, or pCAF on the calcium sensitivity, maximum developed force or co-operativity of the myocytes (ANOVA, n=8, P<0.05).

Myofilament calcium sensitivity does not affect cross-bridge activation-relaxation kinetics

We employed single myofibril techniques to test whether the presence of slow skeletal troponin-I (ssTnI) is sufficient to induce increased myofilament calcium sensitivity (EC50) and whether modulation of EC50 affects the dynamics of force development. Studies were performed using rabbit psoas myofibrils activated by rapid solution switch and in which Tn was partially replaced for either recombinant cardiac Tn(cTn) or Tn composed of recombinant cTn-T (cTnT) and cTn-C (cTnC), and recombinant ssTnI (ssTnI-chimera Tn). Tn exchange was performed in rigor solution (0.5 mg/ml Tn; 20°C; 2 h) and confirmed by SDS-PAGE. cTnI exchange induced a decrease in EC50; ssTnI-chimera Tn exchange induced a further decrease in EC50 (in μM: endogenous Tn, 1.35 ± 0.08; cTnI, 1.04 ± 0.13; ssTnI-chimera Tn, 0.47 ± 0.03). EC50 was also decreased by application of 100 μM bepridil (control: 2.04 ± 0.03 μM; bepridil 1.35 ± 0.03 μM). Maximum tension was not different between any groups. Despite marked alterations in EC50, none of the dynamic activation-relaxation parameters were affected under any condition. Our results show that 1) incorporation of ssTnI into the fast skeletal sarcomere is sufficient to induce increased myofilament Ca2+ sensitivity, and 2) the dynamics of actin-myosin interaction do not correlate with EC50. This result suggests that intrinsic cross-bridge cycling rate is not altered by the dynamics of thin-filament activation.

Intracellular milieu changes associated with hypoxia impair sarcoplasmic reticulum Ca2+ transport in cardiac muscle

The effects on sarcoplasmic reticulum (SR) Ca2+ transport of solutions mimicking the important intracellular milieu changes associated with short-term hypoxia (hypoxic solutions, as described by Kammermeier et al. J. Mol. Cell. Cardiol. 14: 267, 1982) were examined. SR Ca2+ content was estimated by measuring the magnitude of the caffeine-induced contracture in saponin-skinned rat papillary muscle. SR Ca2+ uptake was inhibited by hypoxic solutions only at loading times less than or equal to 30 s. This inhibition was primarily due to the increase in Pi. The hypoxic solutions had no effect on Ca(2+)-induced Ca2+ release from the SR. We also tested the effects of ATP-free (rigor) solutions that mimic the intracellular environment during late hypoxia and ischemia. Elevating Pi or ADP alone in rigor solution had no effect on SR Ca2+ content. However, elevating Pi and ADP (+/-Mg2+) produced a 44-48% reduction in SR Ca2+ content. This reduction is most likely due to reversal of the SR Ca2+ pump. We conclude that the changes in milieu with short-term hypoxia can depress contractility in intact cardiac muscle by inhibiting SR Ca2+ uptake. During long-term hypoxia or ischemia, these milieu changes can elevate intracellular Ca2+ by reversing the SR Ca2+ pump.

Physiological and structural properties of saponin-skinned single smooth muscle cells.

The study of the fundamental events underlying the generation and regulation of force in smooth muscle would be greatly facilitated if the permeability of the cell membrane were increased so that the intracellular environment of the contractile apparatus could be manipulated experimentally. To initiate such an analysis, we developed a saponin permeabilization procedure that was used to "skin" isolated smooth muscle cells from the stomach of the toad, Bufo marinus. Suspensions of single cells isolated enzymatically were resuspended in high-K+ rigor solution (0 ATP, 5 mM EGTA) and exposed for 5 min to 25 micrograms/ml saponin. Virtually all the cells in a suspension were made permeable by this procedure and shortened to less than one-third their initial length when ATP and Ca++ were added; they re-extended when free Ca++ was removed. Analysis of the protein content of the skinned cells revealed that, although their total protein was reduced by approximately 30%, they retained most of their myosin and actin. Skinning was accompanied by a rearrangement of actin and myosin filaments within the cells such that a fine fibrillar structure became visible under the light microscope and a tight clustering of acting filaments around myosin filaments was revealed by the electron microscope. Face-on views of saponin-treated cell membranes revealed the presence of 70-80-A-wide pits or holes. The shortening rate of skinned cells was sensitive to [Ca++] between pCa 7 and pCa 5 and was half-maximal at approximately pCa 6.2. Shortening was also dependent on [ATP] but could be increased at low [ATP] by pretreatment with adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S), which suggests that myosin phosphorylation was more sensitive to low substrate concentrations than was cross-bridge cycling. To determine whether a significant limitation to free diffusion existed in the skinned cells, a computer model of the cell and the unstirred layer surrounding it was developed. Simulations revealed that the membrane, even in skinned cells, could, for short time intervals, significantly inhibit the movement of substances into and out of cells.

Studies on conformation of F-actin in muscle fibers in the relaxed state, rigor, and during contraction using fluorescent phalloidin.

F-actin in a glycerinated muscle fiber was specifically labeled with fluorescent phalloidin-(fluorescein isothiocyanate) FITC complex at 1:1 molar ratio. Binding of phalloidin-FITC to F-actin affected neither contraction of the fiber nor its regulation by Ca2+. Comparison of polarized fluorescence from phalloidin-FITC bound to F-actin in the relaxed state, rigor, and during isometric contraction of the fiber revealed that the changes in polarization accompanying activation are quantitatively as well as qualitatively different from those accompanying transition of the fiber from the relaxed state to rigor. The extent of the changes of polarized fluorescence during isometric contraction increased with decreasing ionic strength, in parallel with increase in isometric tension. On the other hand, polarized fluorescence was not affected by addition of ADP or by stretching of the fiber in rigor solution. It is concluded from these observations that conformational changes in F-actin are involved in the process of active tension development.

Quantitative studies on the polarization optical properties of striated muscle. I. Birefringence changes of rabbit psoas muscle in the transition from rigor to relaxed state.

The changes in birefringence in the rigor to relax transition of single triton-extracted rabbit psoas muscle fibers have been investigated with quantitative polarized light techniques. The total birefringence of rest lenght fibers in rigor was (1.46 +/- 0.08) x 10(-3) and increased to (1.67 +/- 0.05) x 10(-3) after Mg-ATP relaxation. Pyrophosphate relaxation increased the total birefringence only slightly, whereas subsequent Mg-ATP relaxation elicited the maximum increase in birefringence. Changes in lattice spacing did not account for the total increase in birefrigence during relaxation. Moreover, the increase in total birefringence was attributable to increases in intrinsic birefringence as well as form birefringence. No change in birefringence was exhibited upon exposure to a relaxation solution after myosin extraction. Synthetic myosin filaments were prepared and treated with relaxation and rigor solutions. The negatively stained filaments treated with a rigor solution had gross irregular projections at either end, while the filaments treated with a relaxing solution were more spindle shaped. The results are compatible with the view that the subfragment-2 moieties of myosin angle away from the myosin aggregates (light meromyosin) to permit the attachment of the subfragment-1 moieties to actin.

Calcium regulation of the contractile state of isolated mammalian fibroblast cytoplasm

Calcium-dependent contractions have been induced in fresh, naked cytoplasm of L-929 fibroblasts using physiological solutions (rigor, relaxing and contracting) similar to those designed to control the contractile state of vertebrate striated muscle. Free access of solutions to the cytoplasm was achieved by popping and stripping the plasma membrane from cells using 7–10 strokes of a Dounce homogenizer. Contracting solution (free Ca2+ 7 X 10(−5) M; with added MgATP) applied locally from a micropipette to cells popped in rigor (free Ca2+ less than 10(−8) M) or relaxing (free Ca2+ less than 10(−8) M; with added MgATP) solutions induced symmetrical contractions of unstretched cytoplasm and directional shortening of stretched cytoplasm. The contractions produced 12–18% shortenings and were complete in 1–3 s. The cytoplasm could be cycled repeatedly through the contracted state from the relaxed state. Exogenous MgATP was required for the Ca2+-dependent contractions. At low free Ca2+ concentrations (less than 10(−8) M), MgATP had a marked plasticizing effect on the cytoplasm. Thus cytoplasm prepared in relaxing solution was less cohesive and more easily deformed than cytoplasm prepared in rigor solution. When induced to contract, relaxed cytoplasm showed a loss of plasticity. Using this criterion, the threshold concentration of free Ca2+ for contraction was determined to lie between 7 X 10(−8) M and 5 X 10(−7) M.