Yasutake Saeki

Expression of myogenic regulatory factors during the development of mouse tongue striated muscle

While the role of myogenic regulatory factors (MRFs) in skeletal myogenesis has been well evaluated in limb and trunk muscles, very little is known about their role in tongue myogenesis. Here the expression of MRF mRNA in mouse tongue muscle was examined during development from embryonic day (E)11 to birth and compared them with that in hind-limb muscle. Desmin, muscle creatine kinase and troponin C mRNAs were used as markers for myoblast determination, myotubule formation and myofibre maturation, respectively. The mRNA quantities were determined by competitive reverse transcriptase-polymerase chain reaction. The expression profile of desmin mRNA indicated that myoblast determination occurred before E11 in both the tongue and hind-limb muscles; the profile of muscle creatine kinase and troponin C mRNAs indicated that myotubule formation and myofibre maturation began between E11 and 13 in both tongue and hind-limb muscles, but ended 2 days earlier in the tongue than in the hind limb. Expression of myoD and myogenin mRNAs began at E11, increased, and showed peak values earlier in the tongue muscle (E13) than in the hind-limb muscle (E15). Expression of MRF4 mRNA appeared earlier in the tongue (E13) than in the hind-limb muscle (E15) and increased in both muscles after that. These results suggest that myotubule formation and myofibre maturation in the tongue muscle progress faster than in the hind-limb muscle, a result of earlier expression of myoD, myogenin, and MRF4 in response to earlier functional demands such as suckling immediately after birth.

Microtubules Modulate the Stiffness of Cardiomyocytes Against Shear Stress

Although microtubules are involved in various pathological conditions of the heart including hypertrophy and congestive heart failure, the mechanical role of microtubules in cardiomyocytes under such conditions is not well understood. In the present study, we measured multiple aspects of the mechanical properties of single cardiomyocytes, including tensile stiffness, transverse (indentation) stiffness, and shear stiffness in both transverse and longitudinal planes using carbon fiber–based systems and compared these parameters under control, microtubule depolymerized (colchicine treated), and microtubule hyperpolymerized (paclitaxel treated) conditions. From all of these measurements, we found that only the stiffness against shear in the longitudinal plane was modulated by the microtubule cytoskeleton. A simulation model of the myocyte in which microtubules serve as compression-resistant elements successfully reproduced the experimental results. In the complex strain field that living myocytes experience in the body, observed changes in shear stiffness may have a significant influence on the diastolic property of the diseased heart.

Comparison of crossbridge dynamics between intact and skinned myocardium from ferret right ventricles.

This study compares the crossbridge kinetics of intact and skinned preparations from ferret cardiac muscles at 20 degrees C to determine whether skinning causes any alteration in the crossbridge response to an imposed length change. A papillary or trabecular muscle was isolated from the right ventricle, the muscle length adjusted to give the maximum twitch tension (Lmax), and the preparation was subjected to Ba2+ contracture. When steady tension developed, the length of the preparation was perturbed sinusoidally in 19 discrete frequencies, ranging from 0.13 to 135 Hz, and at a small peak-to-peak amplitude (0.25% Lmax). We identified three exponential processes in the sinusodial force-response to the imposed length oscillation, and these were labeled processes B, C, and D in order of increasing speed. A slow process, A, normally present in fast-twitch skeletal muscles, is very small or absent in cardiac muscles. Process B is an exponential delay, and the muscle produces oscillatory work on the forcing apparatus; processes C and D are exponential advances in which the muscle absorbs work. The preparation was chemically skinned and activated in the presence of (mM) CaEGTA 6 (pCa 4.55), MgATP 5, magnesium propionate 1, and phosphate 1, pH 7.0, with ionic strength adjusted to 200 mM with potassium propionate. We found that the crossbridge kinetics were not altered by the skinning procedure. The apparent rate constants extracted from the sinusoidal analysis were nearly identical in Ba2+ contracture (intact preparation) and in Ca2+ activation (skinned preparation), and the Nyquist plots were similar. Because the rate constants changed sensitively with the substrate (MgATP) concentrations, we concluded that the substrate is adequately supplied during Ba2+ contracture in the intact preparation. Our study demonstrates the compatibility of results obtained from an intact and from a skinned preparation.

Quantitative changes in the mRNA for contractile proteins and metabolic enzymes in masseter muscle of bite-opened rats.

To study the effects of bite opening on the fibre phenotypes of rat masseter, the mRNAs of four predominant myosin heavy-chain isoforms (MHC I, IIa, IId/x and IIb) and two alkali light-chain isoforms (LC1f and 3f) as well as those of two metabolic enzymes, carbonic anhydrase III (CAIII, oxidative enzyme) and glucose-phosphate isomerase (GPI, glycolytic enzyme), were measured in relation to the total RNA of masseter muscle by competitive, reverse transcriptase-polymerase chain reaction in control and bite-opened rats. Bite opening (2.8 mm increase in the vertical dimension for 1 week) significantly (P<0.05) increased the amount of MHC IIa mRNA but decreased (P<0.001) the amount of MHC IIb mRNA without changing the amount of MHC IId/x mRNA. No MHC I mRNA was found in any masseter studied. A significant (P<0.01) increase in the mRNA of LC1f associated with a decrease (P<0.05) in that of LC3f was observed after the bite opening. The CAIII mRNA increased significantly (P<0.001), while the GPI mRNA decreased (P<0.05) in association with the bite opening. These results strongly suggest that in 1 week of bite opening changes the rat masseter muscle from a glycolytic, MHC IIb-LC3f-dominant fibre to an oxidative, MHC IIa-LC1f-dominant fibre.

Alterations in intracellular calcium and tension of activated ferret papillary muscle in response to step length changes.

1. To study the effects of mechanical constraints on the calcium (Ca2+) affinity of cardiac troponin C, we analysed the tension and aequorin light (AL, intracellular Ca2+) transients in response to a step length change in aequorin‐injected ferret right ventricular papillary muscles. The muscle preparations were continuously activated with ouabain (10(‐4) M) (ouabain contracture) or with high frequency stimuli in the presence of ryanodine (5 microM) (tetanic contraction). 2. The tension transient in response to either the release or stretch was oscillatory: tension decreased rapidly during the release and then increased, after which it lapsed into a new steady level in a series of damped oscillations. The opposite was true for the stretch. The oscillatory responses were conspicuous and less damped in ouabain‐activated preparations (oscillation frequency of 2.2‐2.3 Hz at 22 degrees and 4.5‐4.6 Hz at 30 degrees C) and much more damped in ryanodine‐treated preparations. 3. The transient AL response was also oscillatory, the time course of which corresponded to that of the transient tension response. Regardless of the difference in the time course of the transients in two different preparations and at two different temperatures, the increase in AL corresponded to the decrease in tension, likewise the decrease in AL to the increase in tension. 4. The mean level of AL after release was lower than the control level present just prior to the release in ouabain‐activated preparations, but the AL after release finally returned to the nearly control level in ryanodine‐treated preparations. 5. When the ryanodine‐treated muscle was further treated with 2,3‐butanedione monoxime (BDM) (20 mM), the tetanic tension decreased remarkably without affecting the AL signal. The tension transient of this preparation was quite similar to that of the resting muscle, which changed in a nearly stepwise fashion; AL was hardly affected by step length changes, as in the resting muscle, in spite of the higher AL level. 6. These results suggest that the Ca2+ affinity of cardiac troponin C is increased with an increase in tension (i.e. the cross‐bridge attachment) and decreased with a decrease in tension i.e. the cross‐bridge detachment), and that the mean [Ca2+]i is lowered by release, at least in a Ca(2+)‐overloaded condition, mainly through the sarcoplasmic reticulum.

EFFECTS OF DIET CONSISTENCY ON THE MYOSIN HEAVY CHAIN MRNAS OF RAT MASSETER MUSCLE DURING POSTNATAL DEVELOPMENT

To study the effects of diet consistency on the fiber phenotypes of rat masseter (1-70 days of age), the mRNAs of myosin heavy chain isoforms (MHC embryonic, neonatal, I, IIa, IId/x and IIb) were measured in total RNA preparations from masseters of hard-diet group (HDG) and soft-diet group (SDG) by competitive reverse transcriptase-polymerase chain reaction (RT-PCR). With respect to the time course of the transition of each MHC mRNA expressed as a percentage relative to the maximum mean, the soft diet facilitated early (9 days after weaning) expression of IId/x and IIb isoforms, and also a decline in the expression of neonatal and IIa isoforms. The expression of neonatal, IIa and IId/x isoforms at 70 days of age was significantly (P<0.05, P<0.01, P<0.01, respectively) lower in SDG than in HDG, indicating a higher relative composition of the IIb isoform in the SDG. Embryonic MHC mRNA had disappeared by 14 days of age (i.e. before weaning at 19 days). No MHC I mRNA was observed in any masseter studied. These results suggest that in the rat a soft diet facilitates an even more MHC IIb-rich phenotype in the masseter muscle than a hard diet.

Adrenaline increases the rate of cross-bridge cycling in rat cardiac muscle

To characterize the myocardial cross-bridge dynamics in catecholamine-induced positive inotropic state, we studied the effects of adrenaline (6 X 10(-6) M) on the transient central segment length (SL) response to step decrease in tension in rat right ventricular papillary muscle in barium contracture. The time course of this response is thought to reflect the kinetics of actin-myosin interaction. The muscle was released stepwise from the steady contracture tension (Tc) to new steady tension levels (Tr) of varying magnitudes at 22 degrees C. When the tension decrease was less than 0.7 Tc, the SL transient responses comprised, in most cases, four phases. The first phase was a rapid and minute shortening during tension reduction; the second was a slow further shortening; the third, a slow lengthening; and the fourth, an extremely slow shortening toward a new steady length under the new tension. Adrenaline showed almost no effect on Tc and the amplitude of SL transients, but markedly reduced the duration of the second (D2) and third (D3) phases of SL transient regardless of the amplitude of tension reduction. The reduction of duration was 14 +/- 3% in D2 and 26 +/- 5% in D3 at Tr/Tc of 0.84 +/- 0.03 on the average (mean +/- S.D.) in nine preparations. The velocity measured from the quasi-steady SL shortening in the second phase increased with the addition of adrenaline, regardless of the amplitude of tension reduction. The increase in the shortening velocity was 16 +/- 6% (mean +/- S.D., n = 9) at Tr/Tc of 0.18 +/- 0.04. These results suggest that adrenaline increases the rate of cross-bridge cycling in cardiac muscle independent of activation level.

Mechanical properties and ATPase activity in glycerinated cardiac muscle of hyperthyroid rabbit

Isometric tension, tension transients in response to rapid step stretches in length and ATPase activity were measured at constant levels of various Ca2+ activations in glycerinated right ventricular papillary muscle ofl-thyroxine-treated (14 daily injections of 0.2 mg/kg) and control rabbits. The isometric tension increased sigmoidally as Ca2+ was varied from slightly below pCa 7 to about pCa 6 both in thyroxine-treated and control preparations. The maximum isometric tension in thyroxine-treated preparations, however, was only about 66% of that in control. The tension transients were characterized by clear three distinct phases; the first phase of an immediate tension increase coincident with the stretch, the second phase of a rapid quasi-exponential tension decrease and the third phase of a delayed quasi-exponential tension rise. In thyroxine-treated preparations, relative to controls, the time for 63% tension reduction in the second phase decreased from 39.3±2.8 ms (mean ± SD,n=5) to 20.2±2.0 ms (P<0.001) and the time for 63% tension rise in the third phase decreased from 483.9±14.3 ms to 298±15.9 ms (p<0.001). The ATPase activity increased in a sigmoid fashion with increasing Ca2+ from slightly above pCa 7 to slightly below pCa 6 both in thyroxine-treated and control preparations. However, the tension cost (ATPase activity/tension) was about two times greater in the tyyroxine-treated preparations than in controls.

Adaptation of Guinea-pig superficial masseter muscle to an increase in occlusal vertical dimension

To study the effect of increased occlusal vertical dimension on the fibre phenotypes of the superficial masseter muscle, the composition of myosin heavy-chains (MHC), myosin light-chains (MLC) and tropomyosin was investigated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis in conjunction with densitometric analysis in normal (control) and bite-opened (5.7 mm increase in the vertical dimension for 1 week) guinea-pigs. The superficial masseter contained two fast-type MHC isoforms, II-1 and II-2, in both the bite-opened and control groups; their relative content (mean+/-SD, n = 7) was 47.8+/-2.9% and 52.2+/-2.9%, in the bite-opened and 44.4+/-3.0% and 55.6+3.0% in control preparations, indicating no significant (p>0.05) changes in MHC composition in association with the bite opening. On the other hand, significant differences in MLC and tropomyosin composition were found between the two preparations. Although the MLC consisted of three components, LC1f, LC2f and LC3f, in both preparations, their relative content (mean+/-SD, n = 7) was 37.1+/-2.4%, 49.6+/-1.6% and 13.2+/-3.2%, respectively, in the bite-opened and 28.1+/-3.1%, 50.9+/-1.6% and 21.0+/-3.5% in the control preparations, indicating that the bite opening induced a significant (p < 0.0001) increase in the relative content of LC1f at the expense of that of LC3f. Although the tropomyosin consisted of two components, TM-alpha and TM-beta, in both preparations, their relative content (mean+/-SD, n = 7) was 91.8%+/-1.9% and 8.2+/-1.9%, respectively, in the bite-opened and 95.9+/-0.7% and 4.1+/-0.7% in the control preparations, showing a significant (p < 0.001) increase in the relative content of TM-beta in relation to the bite opening. These results indicate that in guinea-pigs an increase in occlusal vertical dimension for 1 week changes the composition of MLC and tropomyosin, with no significant change in MHC, in the masseter muscle. These changes might be required to meet altered functional demands.

Expression of Green Fluorescent Protein Impairs the Force-Generating Ability of Isolated Rat Ventricular Cardiomyocytes

Green fluorescent protein (GFP) is widely used as a biologically inert expression marker for studying the effects of transgene expression in heart tissue, but its influence on the contractile function of cardiomyocytes has not yet been fully evaluated. We measured the contractile function of isolated rat ventricular myocytes before and after infection with a recombinant adenovirus expressing GFP (Adv-GFP). Myocytes infected with a non-transgene-containing adenovirus (Adv-Null) or uninfected myocytes (UI) served as controls. Using a carbon-fiber-based force-length measurement system for single cardiomyocytes, we evaluated the contractile function over a wide range of loading conditions including the shortening fraction (%FS) and maximal shortening velocity (Vmax) under the unloaded condition, and isometric force. At 24 hours after infection, nearly 80% of the Adv-GFP-infected myocytes expressed GFP. We found that the %FS and Vmax did not differ among the three groups, however, the isometric force showed a mild, but significant, decrease only in Adv-GFP myocytes (Adv-GFP: 29.1 ± 4.0 mN/mm2; Adv-Null: 42.8 ± 6.2 mN/mm2; UI: 47.1 ± 4.8 mN/mm2; p = 0.03). An evaluation of the contractile function of isolated cardiomyocytes under high load conditions revealed impaired isometric contractility by GFP expression. Adv-GFP expression may not be an ideal control for specific gene expression experiments in myocardial tissue.