Henk E.D.J. ter Keurs

Ser-2030, but not Ser-2808, is the major phosphorylation site in cardiac ryanodine receptors responding to protein kinase A activation upon β-adrenergic stimulation in normal and failing hearts

We have recently shown that RyR2 (cardiac ryanodine receptor) is phosphorylated by PKA (protein kinase A/cAMP-dependent protein kinase) at two major sites, Ser-2030 and Ser-2808. In the present study, we examined the properties and physiological relevance of phosphorylation of these two sites. Using site- and phospho-specific antibodies, we demonstrated that Ser-2030 of both recombinant and native RyR2 from a number of species was phosphorylated by PKA, indicating that Ser-2030 is a highly conserved PKA site. Furthermore, we found that the phosphorylation of Ser-2030 responded to isoproterenol (isoprenaline) stimulation in rat cardiac myocytes in a concentration- and time-dependent manner, whereas Ser-2808 was already substantially phosphorylated before beta-adrenergic stimulation, and the extent of the increase in Ser-2808 phosphorylation after beta-adrenergic stimulation was much less than that for Ser-2030. Interestingly, the isoproterenol-induced phosphorylation of Ser-2030, but not of Ser-2808, was markedly inhibited by PKI, a specific inhibitor of PKA. The basal phosphorylation of Ser-2808 was also insensitive to PKA inhibition. Moreover, Ser-2808, but not Ser-2030, was stoichiometrically phosphorylated by PKG (protein kinase G). In addition, we found no significant phosphorylation of RyR2 at the Ser-2030 PKA site in failing rat hearts. Importantly, isoproterenol stimulation markedly increased the phosphorylation of Ser-2030, but not of Ser-2808, in failing rat hearts. Taken together, these observations indicate that Ser-2030, but not Ser-2808, is the major PKA phosphorylation site in RyR2 responding to PKA activation upon beta-adrenergic stimulation in both normal and failing hearts, and that RyR2 is not hyperphosphorylated by PKA in heart failure. Our results also suggest that phosphorylation of RyR2 at Ser-2030 may be an important event associated with altered Ca2+ handling and cardiac arrhythmia that is commonly observed in heart failure upon beta-adrenergic stimulation.

Force-length relation of in-vivo human rectus femoris muscles

In this paper the force-length relation of intact, in vivo, human rectus femoris musles were determined experimentally and compared to a corresponding theoretical force-length relation based on the cross-bridge theory. The experimental force-length relation has a much smaller peak force and a much wider range of muscle fiber lengths where non-zero forces were observed than the theoretical relation. Possible reasons for the differences between the two forcelength relations are discussed.

Dynamics of viscoelastic properties of rat cardiac sarcomeres during the diastolic interval: involvement of Ca2+

1 Cardiac sarcomere stiffness was investigated during diastole in eighteen trabeculae dissected from the right ventricle of rat heart. The trabeculae were stimulated at 0.5 Hz, in a modified Krebs–Henseleit solution (pH, 7.4; 25 °C). Sarcomere length (SL) was measured using high resolution (±2 nm) laser diffraction techniques. Force (F) was measured with a silicon strain gauge. 2 SL increased exponentially (amplitude, 25 ± 9 nm; n= 5) throughout diastole. This increase occurred even at slack SL, showing that this phenomenon was due to an internal expansion. The majority of the muscles showed discrete spontaneous fluctuations of SL (amplitude < 20 nm) starting ∼l s after the end of the twitch. 3 The intracellular free Ca2+ concentration ([Ca2+]i) was measured from the fluorescence of microinjected fura‐2 salt in seven trabeculae under the same experimental conditions. [Ca2+]i continuously declined (from 240 to 90 nm) during diastole following a monoexponential time course (time constant, 210–325 ms). 4 The stiffness of the sarcomere was evaluated at 10, 30, 50, 70 and 90% of diastole using bursts (30 ms) of 500 Hz sinusoidal perturbations of muscle length (amplitude of SL oscillations < 30 nm). At 1 mm external Ca2+ concentration ([Ca2+]o), the average stiffness modulus (Mod) increased from 9.3 ± 0.6 to 12 ± 0.6 mN mm−2μm1 (n= 18; P < 0.05), while the average phase shift (Ф) between F and SL signals decreased from 84 ± 3 to 73 ± 4 deg (n= 18; P < 0.05) between 10 and 90% during diastole. The increase in Mod and the decrease in Ф reversed when spontaneous activity occurred. When [Ca2+]0 was raised to 2 mm, the stiffness time course reversed approximately 450 ms earlier, simultaneously with the occurrence of spontaneous activity. 5 Our results show that diastole is only an apparent steady state and suggest that the structural system responsible for the viscoelastic properties of the sarcomere is regulated by [Ca2+]i in the submicromolar range. Different possible origins of the dynamic changes in viscoelasticity during diastole are discussed.

Ca2+ Sparks and Waves in Canine Purkinje Cells. A Triple Layered System of Ca2+ Activation

We have investigated the subcellular spontaneous Ca2+ events in canine Purkinje cells using laser scanning confocal microscopy. Three types of Ca2+ transient were found: (1) nonpropagating Ca2+ transients that originate directly under the sarcolemma and lead to (2) small Ca2+ wavelets in a region limited to ≈6-&mgr;m depth under the sarcolemma causing (3) large Ca2+ waves that travel throughout the cell (CWWs). Immunocytochemical studies revealed 3 layers of Ca2+ channels: (1) channels associated with type 1 IP3 receptors (IP3R1) and type 3 ryanodine receptors (RyR3) are prominent directly under the sarcolemma; (2) type 2 ryanodine receptors (RyR2s) are present throughout the cell but virtually absent in a layer between 2 and 4 &mgr;m below the sarcolemma (Sub-SL); (3) type 3 ryanodine receptors (RyR3) is the dominant Ca2+ release channel in the Sub-SL. Simulations of both nonpropagating and propagating transients show that the generators of Ca2+ wavelets differ from those of the CWWs with the threshold of the former being less than that of the latter. Thus, Purkinje cells contain a functional and structural Ca2+ system responsible for the mechanism that translates Ca2+ release occurring directly under the sarcolemma into rapid Ca2+ release in the Sub-SL, which then initiates large-amplitude long lasting Ca2+ releases underlying CWWs. The sequence of spontaneous diastolic Ca2+ transients that starts directly under the sarcolemma and leads to Ca2+ wavelets and CWWs is important because CWWs have been shown to cause nondriven electrical activity.

Effect of stimulation rate, sarcomere length and Ca2+ on force generation by mouse cardiac muscle

The relations between stress, stimulation rate and sarcomere length (SL) were investigated in 24 cardiac trabeculae isolated from right ventricles of mice (CF‐1 males, 25‐30 g) and superfused with Hepes solution ([Ca2+]o= 1 mm, pH 7.4, 25 °C). Stress and SL were measured by a strain gauge transducer and laser diffraction technique, respectively. Stress versus stimulation frequency formed a biphasic relation (25 °C, [Ca2+]o= 2 mm) with a minimum at 0.7‐1 Hz (≈15 mN mm−2), a 150 % decrease from 0.1 to 1 Hz (descending limb) and a 75 % increase from 1 to 5 Hz (ascending limb). Ryanodine (0.1 μm) inhibited specifically the descending limb, while nifedipine (0.1 μm) affected specifically the ascending limb. This result suggests two separate sources of Ca2+ for stress development: (1) net Ca2+ influx during action potentials (AP); and (2) Ca2+ entry into the cytosol from the extracellular space during diastolic intervals; Ca2+ from both (1) and (2) is sequestered by the SR between beats. Raising the temperature to 37 °C lowered the stress‐frequency relation (SFR) by ≈0‐15 mN mm−2 at each frequency. Because the amount of Ca2+ carried by ICa,L showed a ≈3‐fold increase under the same conditions, we conclude that reduced Ca2+ loading of the SR was probably responsible for this temperature effect. A simple model of Ca2+ fluxes addressed the mechanisms underlying the SFR. Simulation of the effect of inorganic phosphates (Pi) on force production was incorporated into the model. The results suggested that O2 diffusion limits force production at stimulation rates >3 Hz. The stress‐SL relations from slack length (≈1.75 μm) to 2.25 μm showed that the passive stress‐SL curve of mouse cardiac trabeculae is exponential with a steep increase at SL >2.1 μm. Active stress (at 1 Hz) increased with SL, following a curved relation with convexity toward the abscissa at [Ca2+]= 2 mm. At [Ca2+] from 4 to 12 mm, the stress‐SL curves superimposed and the relation became linear, which revealed a saturation step in the activation of force production. EC coupling in mouse cardiac muscle is similar to that observed previously in the rat, although important differences exist in the Ca2+ dependence of force development. These results may suggest a lower capacity of the SR for buffering Ca2+, which makes the generation of force in mouse cardiac ventricle more dependent on Ca2+ entering during action potentials, particularly at high heart rate.

Theoretical determination of force-length relations of intact human skeletal muscles using the cross-bridge model

The purpose of this study was to determine force-length relations of selected human skeletal muscles, based on the theoretical foundations of the cross-bridge model and to calculate a strength curve for knee extension from these relations. Force-length relations were determined for the rectus femoris, vastus lateralis, vastus medialis, vastus intermedius and gastrocnemius muscles, using sarcomere/ fiber length data form both legs of four cadavers and sarcomere geometry data reported in the literature. It appears that the two-joint muscles investigated in this study are not able to produce force throughout their full anatomical range of motion, whereas the one-joint muscles can. The strength curve for knee extension was determined as the sum of the force-length relations of the individual knee extensor muscles and showed good agreement with experimentally obtained knee extensor strength curves.

Spatial Nonuniformity of Excitation–Contraction Coupling Causes Arrhythmogenic Ca2+ Waves in Rat Cardiac Muscle

Ca2+ waves underlying triggered propagated contractions (TPCs) are initiated in damaged regions in cardiac muscle and cause arrhythmias. We studied Ca2+ waves underlying TPCs in rat cardiac trabeculae under experimental conditions that simulate the functional nonuniformity caused by local mechanical or ischemic local damage of myocardium. A mechanical discontinuity along the trabeculae was created by exposing the preparation to a small jet of solution with a composition that reduces excitation–contraction coupling (ECC) in myocytes within that segment. The jet solution contained either caffeine (5 mmol/L), 2,3-butanedione monoxime (BDM; 20 mmol/L), or low Ca2+ concentration ([Ca2+]; 0.2 mmol/L). Force was measured with a silicon strain gauge and sarcomere length with laser diffraction techniques in 15 trabeculae. Simultaneously, [Ca2+]i was measured locally using epifluorescence of Fura-2. The jet of solution was applied perpendicularly to a small muscle region (200 to 300 &mgr;m) at constant flow. When the jet contained caffeine, BDM, or low [Ca2+], during the stimulated twitch, muscle-twitch force decreased and the sarcomeres in the exposed segment were stretched by shortening normal regions outside the jet. Typical protocols for TPC induction (7.5 s-2.5 Hz stimulus trains at 23°C; [Ca2+]o=2.0 mmol/L) reproducibly generated Ca2+ waves that arose from the border between shortening and stretched regions. Such Ca2+ waves started during force-relaxation of the last stimulated twitch of the train and propagated (0.2 to 2.8 mm/sec) into segments both inside and outside of the jet. Arrhythmias, in the form of nondriven rhythmic activity, were induced when the amplitude of the Ca2+-wave was increased by raising [Ca2+]o. Arrhythmias disappeared rapidly when uniformity of ECC throughout the muscle was restored by turning the jet off. These results show, for the first time, that nonuniform ECC can cause Ca2+ waves underlying TPCs and suggest that Ca2+ dissociated from myofilaments plays an important role in the initiation of Ca2+ waves.

Role of ica and Na+Ca2+ exchange in the force-frequency relationship of rat heart muscle

The inward Ca2+ current, ica, increases with the frequency of stimulation in single ventricular myocytes, but the presence and possible role of this phenomenon in intact heart muscle of mammals has not been studied. The present study addresses the question whether changes in ica play a role in the force-frequency relationship in thin ventricular trabeculae from rat heart. The duration of the action potential at 50% repolarization, APD50, is related to the strength and duration of ica (Mitchell et al., 1984b; Schouten, 1986). APD50 increased with the frequency of stimulation. Peak force of contraction, F, was minimal at 0.1-0.3 Hz and increased at both higher and lower frequencies, suggesting two mechanisms with opposite frequency-dependence. The increase at low frequencies was abolished by drugs that inhibit Ca2+ uptake by the sarcoplasmic reticulum (caffeine, theophylline), but not by Ca2+ antagonists that block ica (nifedipine, Mn2+). This is consistent with the hypothesis that a small net influx of Ca2+ across the sarcolemma during long diastoles was responsible for loading of the reticulum and enhancement of F at low frequencies. The increase of F and APD50 at high frequencies was abolished by Ca2+ antagonists but not by caffeine and theophylline. From this result it is concluded, that frequency-induced enhancement of ica occurs in intact heart muscle and contributes to the increase in F.

A method for the determination of the force-length relation of selected in-vivo human skeletal muscles

In this paper a method is presented to determine force-length relations of in-vivo human skeletal muscles. The method is experimental and can be used for selected multi-joint muscles. It contains three basic assumptions: (a) the maximal, isometric force a muscle can exert is constant for a given muscle length, (b) antagonistic muscle activity for the experimental contractions is constant, and (c) resultant joint moments obtained during the experiments are produced by muscular forces exclusively. Experimentally determined force-length relations of intact in-vivo human skeletal muscles have not been determined yet. Application of this method will allow the comparison of actual force-length relations of selected human skeletal muscles to force-length relations used previously. Proposed mechanisms responsible for the force-length characteristics of a muscle, such as the cross-bridge theory, may be critically evaluated. Differences of force-length relations obtained under in vivo and in vitro conditions may be quantified.

Ca2+ waves during triggered propagated contractions in intact trabeculae

Triggered propagated contractions (TPCs) starting from damaged regions travel along multicellular cardiac muscle preparations. We have reported that octanol (100 microM) inhibits TPCs. The inhibitory effect of octanol on propagation of TPCs could be due to an effect of octanol on Ca(2+)-induced Ca2+ release (CICR) mediated by Ca2+ diffusion inside the single cell or on the diffusion of Ca2+ from cell to cell via gap junctions (GJs). Therefore, we studied the regional changes in intracellular Ca2+ concentration ([Ca2+]i) during TPCs and the effect of octanol on the permeability of gap junctions (PGJ) in rat cardiac trabeculae. [Ca2+]i was measured using electrophoretically injected fura 2 and an image-intensified charge-coupled device camera. PGJ was calculated from the diffusion coefficient for fura 2 in trabeculae (Dtrab) and in the myoplasm (Dmyop). After 1- and 3-h superfusion with 100 microM 1-octanol, Dmyop showed no significant changes, whereas Dtrab was reduced significantly. Therefore, calculated PGJ was reduced from 4.15 x 10(-5) to 2.10 x 10(-5) and 0.86 x 10(-5) cm/s, respectively. The propagation velocity of the regional increases in [Ca2+]i during TPCs was constant, averaging 1.69 +/- 1.48 mm/s (range 0.34-5.47 mm/s, n = 10). These observations support the hypothesis that TPCs are initiated near the damaged ends of trabeculae and are propagated by CICR from the sarcoplasmic reticulum mediated by diffusion of Ca2+ through cells and from cell to cell through GJs.Triggered propagated contractions (TPCs) starting from damaged regions travel along multicellular cardiac muscle preparations. We have reported that octanol (100 μM) inhibits TPCs. The inhibitory effect of octanol on propagation of TPCs could be due to an effect of octanol on Ca2+-induced Ca2+ release (CICR) mediated by Ca2+ diffusion inside the single cell or on the diffusion of Ca2+from cell to cell via gap junctions (GJs). Therefore, we studied the regional changes in intracellular Ca2+ concentration ([Ca2+]i) during TPCs and the effect of octanol on the permeability of gap junctions ( P GJ) in rat cardiac trabeculae. [Ca2+]iwas measured using electrophoretically injected fura 2 and an image-intensified charge-coupled device camera. P GJ was calculated from the diffusion coefficient for fura 2 in trabeculae ( D trab) and in the myoplasm ( D myop). After 1- and 3-h superfusion with 100 μM 1-octanol, D myop showed no significant changes, whereas D trab was reduced significantly. Therefore, calculated P GJ was reduced from 4.15 × 10-5 to 2.10 × 10-5 and 0.86 × 10-5 cm/s, respectively. The propagation velocity of the regional increases in [Ca2+]iduring TPCs was constant, averaging 1.69 ± 1.48 mm/s (range 0.34-5.47 mm/s, n = 10). These observations support the hypothesis that TPCs are initiated near the damaged ends of trabeculae and are propagated by CICR from the sarcoplasmic reticulum mediated by diffusion of Ca2+ through cells and from cell to cell through GJs.