Dilated cardiomyopathy (DCM), characterized by cardiac dilatation and contractile dysfunction, is a major cause of heart failure. Inherited DCM can result from mutations in the genes encoding cardiac troponin T, troponin C, and ␣-tropomyosin; different mutations in the same genes cause hypertrophic cardiomyopathy. To understand how certain mutations lead specifically to DCM, we have investigated their effect on contractile function by comparing wild-type and mutant recombinant proteins. Because initial studies on two troponin T mutations have generated conflicting findings, we analyzed all eight published DCM mutations in troponin T, troponin C, and ␣-tropomyosin in a range of in vitro assays. Thin filaments, reconstituted with a 1:1 ratio of mutant/wild-type proteins (the likely in vivo ratio), all showed reduced Ca 2؉ sensitivity of activation in ATPase and motility assays, and except for one ␣-tropomyosin mutant showed lower maximum Ca 2؉ activation. Incorporation of either of two troponin T mutants in skinned cardiac trabeculae also decreased Ca 2؉ sensitivity of force generation. Structure/function considerations imply that the diverse thin filament DCM mutations affect different aspects of regulatory function yet change contractility in a consistent manner. The DCM mutations depress myofibrillar function, an effect fundamentally opposite to that of hypertrophic cardiomyopathy-causing thin filament mutations, suggesting that decreased contractility may trigger pathways that ultimately lead to the clinical phenotype.

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SUMMARY1. The relative isometric tension-pCa relationship has been determined for isolated bundles of barnacle myofibrils under a variety of ionic conditions using [Ca2+]-buffered solutions which also contained an ATP regenerating system (creatine phosphate and creatine kinase).2. The results are in better agreement with the 'consecutive' scheme of reaction rather than with the 'independent' alternative (Ashley & Moisescu, 1972) for the co-operative action of two Ca2+ ions in the process of tension activation in crustacean skeletal muscle.3. Variations in the pH of the activating solutions did have a marked effect on the relative tension-pCa curve, although no effect was observed on the absolute maximum value for isometric tension. A shift in pH by 0-5 u. in the range 6-6-7-6 shifted the Ca2+-activation curve by 0.5 log u. towards lower free Ca2+ concentrations.4. Changes in the free Mg2+ concentration of the activating solutions in the millimolar range produced a pronounced shift of the relative tension-pCa curve along the pCa axis. Increasing [Mg2+] from 1 to 5 mM shifted the curve by about 0-7 log u. to higher free Ca2+ concentrations, without significantly modifying its steepness.5. Changes in the MgATP concentration of the activating solutions in the range of 1-13 mm had no significant effect on the relative tension-pCa relationship.6. Varying the K+ concentration in the activating solutions was also

SUMMARY1. The calcium-sensitive photoprotein aequorin has been used to follow the rapid changes in intracellular calcium concentration that occur during the contraction of single muscle fibres from the barnacle Balanus nubilus, Darwin. 2. The transient change in calcium-mediated light emission (calcium transient) and the changes in membrane potential and tension were recorded simultaneously, thus permitting an examination of the relationships between the chemical, electrical, and mechanical events ofexcitationcontraction coupling.3. With short-duration stimuli (< 200 msec), the calcium transient shows an S-shaped rising phase reaching a maximum soon after the cessation of the stimulus pulse. During membrane repolarization the calcium transient begins an exponential falling phase which has a time constant of 50-80 msec at 11-12°C.4. The shape of the calcium transient resembles the first derivative of the rising phase of the isometric tension response, thus suggesting that calcium controls the rate of tension development.5. There is no detectable increase of the light emission above resting values, during the falling phase of isometric tension.6. A plot of the calcium transient area (lumen x see) versus peak isometric force (g. cm-2) is linear over, at least, a range of forces from ca. 50-400 g. cm-2.7. When the fibre is capable of producing an active membrane response following the intracellular injection of potassium citrate, the onset and cessation of the calcium transient follow closely the onset and cessation * Present address. C. C. ASHLEY AND E. B. BIDG WA Y of the active membrane response. Tension responses under these conditions are much suppressed, suggesting that excitation-contraction coupling may be partially blocked between calcium release and the development of tension.8. Hypertonic salines (1 M sucrose or 1 M glycerol) cause little change in the membrane response, but greatly suppress the calcium transient and completely abolish the tension responses. These effects are readily reversible when normal saline is reintroduced, suggesting that excitationcontraction coupling may be temporarily blocked between the membrane response and calcium release.9. If the stimulus is prolonged (> 250-300 msec), the calcium transient falls slowly from its maximum value despite continued membrane depolarization, suggesting a time-dependent change in the ratio of the rate of release of calcium to the rate of calcium binding. The results from brief tetanic stimulation also support this suggestion.

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