The increased Ca(2+)-responsiveness of the contractile apparatus in end-stage failing human hearts cannot be explained by a shift in contractile protein isoforms, but results from the complex interplay between changes in the phosphorylation status of MLC-2 and TnI.

1. Mechanically skinned fast-twitch (FT) and slow-twitch (ST) muscle fibres of the rat were used to investigate the effects of fatigue-like changes in creatine phosphate (CP) and inorganic phosphate (Pi) concentration on Ca2+-activation properties of the myofilaments as well as Ca2+ movements into and out of the sarcoplasmic reticulum (SR). 2. Decreasing CP from 50 mm to zero in FT fibres increased maximum Ca2+-activated tension (Tmax) by 16 + 2 % and shifted the mid-point of the tension-pCa relation (pCa50) to the left by 0'28 + 0 03 pCa units. In ST fibres, a decrease of CP from 25 mm to zero increased Tmax by 9 + 1 % and increased the pCa50 by 0 16 + 0 01 pCa units. The effect of CP on Tmax was suppressed in both fibre types by prior treatment with 0 3 mm FDNB (1-fluoro-2,4-dinitrobenzene), suggesting that these effects may occur via changes in creatine kinase activity. 3. Increases of Pi in the range 0-50 mm reduced the pCa50 and Tmax in both fibre types.These effects were more pronounced in ST fibres than in FT fibres in absolute terms. However, normalization of the results to resting Pi levels appropriate to both fibre types (1 mm for FT and 5 mm for ST fibres) revealed similar decreases in Tmax (% 39% at 25 mM Pi and -48% at 50 mm Pi) and pCa50 (0 25

Malignant hyperthermia (MH) is a potentially fatal, inherited skeletal muscle disorder in humans and pigs that is caused by abnormal regulation of Ca2+ release from the sarcoplasmic reticulum (SR). MH in pigs is associated with a single mutation (Arg615Cys) in the SR ryanodine receptor (RyR) Ca2+ release channel. The way in which this mutation leads to excessive Ca2+ release is not known and is examined here. Single RyR channels from normal and MH-susceptible (MHS) pigs were examined in artificial lipid bilayers. High cytoplasmic (cis) concentrations of either Ca2+ or Mg2+ (>100 microM) inhibited channel opening less in MHS RyRs than in normal RyRs. This difference was more prominent at lower ionic strength (100 mM versus 250 mM). In 100 mM cis Cs+, half-maximum inhibition of activity occurred at approximately 100 microM Mg2+ in normal RyRs and at approximately 300 microM Mg2+ in MHS RyRs, with an average Hill coefficient of approximately 2 in both cases. The level of Mg2+ inhibition was not appreciably different in the presence of either 1 or 50 microM activating Ca2+, showing that it was not substantially influenced by competition between Mg2+ and Ca2+ for the Ca2+ activation site. Even though the absolute inhibitory levels varied widely between channels and conditions, the inhibitory effects of Ca2+ and Mg2+ were virtually identical for the same conditions in any given channel, indicating that the two cations act at the same low-affinity inhibitory site. It seems likely that at the cytoplasmic [Mg2+] in vivo (approximately 1 mM), this Ca2+/Mg2+-inhibitory site will be close to fully saturated with Mg2+ in normal RyRs, but less fully saturated in MHS RyRs. Therefore MHS RyRs should be more sensitive to any activating stimulus, which would readily account for the development of an MH episode.

Background-Myocardial dysfunction is a common and important problem in donor hearts. The mechanisms responsible remain unclear. We have studied the cytokines tumor necrosis factor (TNF)-␣ and interleukin-6 (IL-6) in the myocardium and serum from donors with myocardial dysfunction (unused donors) and compared them with donors with good ventricular function (used donors) and patients with advanced heart failure (HF). Methods and Results-Clinical details and ventricular function were assessed in 46 donors (31 used, 15 unused). Real-time reverse transcription-polymerase chain reaction, Western blotting, and immunocytochemistry were performed on myocardium and immunoassays on serum. TNF-␣ mRNA was 1.6-fold higher in unused than in used donors (PϽ0.005) and 1.74-fold higher than in 36 patients with HF. IL-6 mRNA was 2.4-fold higher in unused than in used donors (PϽ0.0001) and 4.67-fold higher than in HF (PϽ0.0001). Western blotting showed higher TNF-␣ in unused (218.3Ϯ6.4, nϭ4 versus 187.3Ϯ5.4, nϭ3 OD units) than used donors (PϽ0.05). TNF-␣ expression was localized to cardiac myocytes. Serum TNF-␣ was higher in unused (8.72Ϯ1.3 pg/mL, nϭ13) than in used (6.12Ϯ0.8 pg/mL, nϭ25, PϽ0.05) donors and HF (4.0Ϯ0.4 pg/mL, nϭ17, PϽ0.005). Serum TNF-␣ receptors did not differ between unused (4.3Ϯ0.8 and 8.6Ϯ1.6 ng/mL, nϭ10) and used (3.5Ϯ0.4 and 6.5Ϯ1.1 ng/mL, nϭ24) donors. There was a trend for higher serum IL-6 in unused (16.5Ϯ2.9 pg/mL, nϭ9) compared with used (13.9Ϯ1.6 pg/mL, nϭ26, PϭNS) donors. Conclusions-This

1. The relationship between the total Ca2' content of a muscle fibre and the magnitude of the force response to depolarization was examined in mechanically skinned fibres from the iliofibularis muscle of the toad and the extensor digitorum longus muscle of the rat. The response to depolarization in each skinned fibre was assessed either at the endogenous level of Ca2' content or after depleting the fibre of Ca2! to some degree. Ca2W content was determined by a fibre lysing technique. 2. In both muscle types, the total Ca2+ content could be reduced from the endogenous level of -1 3 mmol F' (expressed relative to intact fibre volume) to -0 25 mmol by either depolarization or caffeine application in the presence of Ca2P chelators, showing that the great majority of the Ca2+ was stored in the sarcoplasmic reticulum (SR). Chelation of Ca2+ in the transverse tubular (T-) system, either by exposure of fibres to EGTA before skinning or by permeabilizing the T-system with saponin after skinning, reduced the lower limit of Ca2+ content to 6 0-12 mmol F7', indicating that 10-20% of the total fibre Ca2+ resided in the T-system. 3. In toad fibres, both the peak and the area (i.e. time integral) of the force response to depolarization were reduced by any reduction in SR Ca2+ content, with both decreasing to zero in an approximately linear manner as the SR Ca2+ content was reduced to < 15 % of the endogenous level. In rat fibres, the peak size of the force response was less affected by small decreases in SR content, but both the peak and area of the response decreased to zero with greater depletion. In partially depleted toad fibres, inhibition of SR Ca2P uptake potentiated the force response to depolarization almost 2-fold. 4. The results show that in this skinned fibre preparation: (a) T-system depolarization and caffeine application can each virtually fully deplete the SR of Ca!+ irrespective of any putative inhibitory effect of SR depletion on channel activation; (b) all of the endogenous level of SR Ca2+ must be released in order to produce a maximal response to depolarization; and (c) a substantial part (-40 %) of the Ca2P released by a depolarization is normally taken back into the SR before it can contribute to force production.It is well known that the release of Ca2+ from the sarcoplasmic reticulum (SR) of a vertebrate skeletal muscle fibre is required for the activation of the contractile apparatus (Ashley, Mulligan & Lea, 1991; Melzer, Herrmann-Frank & Liittgau, 1995). Electron probe analysis has shown that there is -I5 mmol (1 fibre water)`o f Ca2P (i.e. 1 -2 mmol F-', when expressed relative to fibre volume) in the SR terminal cisternae in frog fibres at rest and that -40 % of this is present at the end of a 1-2 s tetanus (Somlyo, GonzalezSerratos, Shuman, McClellan & Somlyo, 1981). However, it is not clear whether the Ca2P still present in the terminal cisternae could be released by depolarization or indeed whether some of it had been already released and re-sequestered by that time. Other studies have used Ca...