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The first mutation associated with hypertrophic cardiomyopathy (HCM) is the R403Q mutation in the gene encoding β-myosin heavy chain (β-MyHC). R403Q locates in the globular head of myosin (S1), responsible for interaction with actin, and thus motor function of myosin. Increased cross-bridge relaxation kinetics caused by the R403Q mutation might underlie increased energetic cost of tension generation; however, direct evidence is absent. Here we studied to what extent cross-bridge kinetics and energetics are related in single cardiac myofibrils and multicellular cardiac muscle strips of three HCM patients with the R403Q mutation and nine sarcomere mutation-negative HCM patients (HCMsmn). Expression of R403Q was on average 41 ± 4% of total MYH7 mRNA. Cross-bridge slow relaxation kinetics in single R403Q myofibrils was significantly higher (P < 0.0001) than in HCMsmn myofibrils (0.47 ± 0.02 and 0.30 ± 0.02 s(-1), respectively). Moreover, compared to HCMsmn, tension cost was significantly higher in the muscle strips of the three R403Q patients (2.93 ± 0.25 and 1.78 ± 0.10 μmol l(-1) s(-1) kN(-1) m(-2), respectively) which showed a positive linear correlation with relaxation kinetics in the corresponding myofibril preparations. This correlation suggests that faster cross-bridge relaxation kinetics results in an increase in energetic cost of tension generation in human HCM with the R403Q mutation compared to HCMsmn. Therefore, increased tension cost might contribute to HCM disease in patients carrying the R403Q mutation., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

The influence of Ca(2+) binding properties of individual troponin versus cooperative regulatory unit interactions along thin filaments on the rate tension develops and declines was examined in demembranated rabbit psoas fibres and isolated myofibrils. Native skeletal troponin C (sTnC) was replaced with sTnC mutants having altered Ca(2+) dissociation rates (k(off)) or with mixtures of sTnC and D28A, D64A sTnC, that does not bind Ca(2+) at sites I and II (xxsTnC), to reduce near-neighbour regulatory unit (RU) interactions. At saturating Ca(2+), the rate of tension redevelopment (k(TR)) was not altered for fibres containing sTnC mutants with decreased k(off) or mixtures of sTnC:xxsTnC. We examined the influence of k(off) on maximal activation and relaxation in myofibrils because they allow rapid and large changes in [Ca(2+)]. In myofibrils with M80Q sTnC(F27W) (decreased k(off)), maximal tension, activation rate (k(ACT)), k(TR) and rates of relaxation were not altered. With I60Q sTnC(F27W) (increased k(off)), maximal tension, k(ACT) and k(TR) decreased, with no change in relaxation rates. Surprisingly, the duration of the slow phase of relaxation increased or decreased with decreased or increased k(off), respectively. For all sTnC reconstitution conditions, Ca(2+) dependence of k(TR) in fibres showed Ca(2+) sensitivity of k(TR) (pCa(50)) shifted parallel to tension and low-Ca(2+) k(TR) was elevated. Together the data suggest the Ca(2+)-dependent rate of tension development and the duration (but not rate) of relaxation can be greatly influenced by k(off) of sTnC. This influence of sTnC binding kinetics occurs primarily within individual RUs, with only minor contributions of RU interactions at low Ca(2+).

The R403Q mutation in beta-myosin heavy chain was the first mutation to be identified as responsible for familial hypertrophic cardiomyopathy (FHC). In spite of extensive work on the functional sequelae of this mutation, the mechanism by which the mutant protein causes the disease has not been definitely identified. Here we directly compare contraction and relaxation mechanics of single myofibrils from left ventricular samples of one patient carrying the R403Q mutation to those from a healthy control heart. Tension generation and relaxation following sudden increase and decrease in [Ca(2+)] were much faster in the R403Q myofibrils with relaxation rates being the most affected parameters. The results show that the R403Q mutation leads to an apparent gain of protein function but a greater energetic cost of tension generation. Increased energy cost of tension generation may be central to the FHC disease process, help explain some unresolved clinical observations, and carry significant therapeutic implications.

The effects of the removal of fast skeletal troponin C (fsTnC) and its replacement by cardiac troponin C (cTnC) and the exchange of fast skeletal troponin (fsTn) for cardiac troponin (cTn) were measured in rabbit fast skeletal myofibrils. Electrophoretic analysis of myofibril suspensions indicated that replacement of fsTnC or exchange of fsTn with cTnC or cTn was about 90% complete in the protocols used. Mechanical measurements in single myofibrils, which were maximally activated by fast solution switching, showed that replacement of fsTnC with cTnC reduced the isometric tension, the rate of tension rise following a step increase in Ca2+ (kACT), and the rate of tension redevelopment following a quick release and restretch (kTR), but had no effect on the kinetics of the fall in tension when the concentration of inorganic phosphate (Pi) was abruptly increased (kPi(+)). These data suggest that the chimeric protein produced by cTnC replacement in fsTn alters those steps controlling the weak-to-strong crossbridge attachment transition. Inefficient signalling within the chimeric troponin may cause these changes. However, replacement of fsTn by cTn had no effect on maximal isometric tension, kACT or kTR, suggesting that these mechanics are largely determined by the isoform of the myosin molecule. Replacement of fsTn by cTn, on the other hand, shifted the pCa50 of the pCa-tension relationship from 5.70 to 6.44 and reduced the Hill coefficient from 3.3 to 1.4, suggesting that regulatory protein isoforms primarily alter Ca2+ sensitivity and the cooperativity of the force-generating mechanism.

The inhibitory effects of inorganic phosphate (P(i)) on isometric force in striated muscle suggest that in the ATPase reaction P(i) release is coupled to force generation. Whether P(i) release and the power stroke are synchronous events or force is generated by an isomerization of the quaternary complex of actomyosin and ATPase products (AM.ADP.P(i)) prior to the following release of P(i) is still controversial. Examination of the dependence of isometric force on [P(i)] in rabbit fast (psoas; 5-15 degrees C) and slow (soleus; 15-20 degrees C) myofibrils was used to test the two-step hypothesis of force generation and P(i) release. Hyperbolic fits of force-[P(i)] relations obtained in fast and slow myofibrils at 15 degrees C produced an apparent asymptote as [P(i)]-->infinity of 0.07 and 0.44 maximal isometric force (i.e. force in the absence of P(i)) in psoas and soleus myofibrils, respectively, with an apparent K(d) of 4.3 mM in both. In each muscle type, the force-[P(i)] relation was independent of temperature. However, 2,3-butanedione 2-monoxime (BDM) decreased the apparent asymptote of force in both muscle types, as expected from its inhibition of the force-generating isomerization. These data lend strong support to models of cross-bridge action in which force is produced by an isomerization of the AM.ADP.P(i) complex immediately preceding the P(i) release step.

1. The effects of magnesium adenosine triphosphate (MgATP; also referred to as 'substrate') concentration on maximal force and shortening velocity have been studied at 5 C in single and thin bundles of striated muscle myofibrils. The minute diameters of the preparations promote rapid diffusional equilibrium between the bathing medium and lattice space so that during contraction fine control of substrate and product concentrations is achieved. 2. Myofibrils from frog tibialis anterior and rabbit psoas fast skeletal muscles were activated maximally by rapidly (10 ms) exchanging a continuous flux of pCa 8.0 for one at pCa 4.75 at a range of substrate concentrations from 10 microM to 5 mM. At high substrate concentrations maximal isometric tension and shortening velocity of both frog and rabbit myofibrils were very close to those determined in whole fibre preparations from the same muscle types. 3. As in frog and rabbit skinned whole fibres, the maximal isometric force of the myofibril preparations decreases as MgATP concentration is increased. The maximal velocity of unloaded shortening (V0) depends hyperbolically on substrate concentration. V0 extrapolated to infinite MgATP (3.6 +/- 0.2 and 0.8 +/- 0.03 l0 s-1 in frog and rabbit myofibrils, respectively) is very close to that determined directly at high substrate concentration. The Km is 210 +/- 20 microM for frog tibialis anterior and 120 +/- 10 microM for rabbit psoas myofibrils, values about half those found in larger whole fibre preparations of the same muscle types. This implies that measurements in whole skinned fibres are perturbed by diffusional delays, even in the presence of MgATP regenerating systems. 4. In both frog and rabbit myofibrils, the Km for V0 is about one order of magnitude higher than the Km for myofibrillar MgATPase determined biochemically in the same experimental conditions. This confirms that the difference between the Km values for MgATPase and shortening velocity is a basic feature of the mechanism of chemomechanical transduction in muscle contraction.

1. Force measurements in isolated myofibrils (15 degrees C; sarcomere length, 2.10 microns) were used in this study to determine whether sarcomeric proteins are responsible for the large differences in the amounts of active and passive tension of cardiac versus skeletal muscle. Single myofibrils and bundles of two to four myofibrils were prepared from glycerinated tibialis anterior and sartorius muscles of the frog. Skinned frog atrial myocytes were used as a model for cardiac myofibrils. 2. Electron microscope analysis of the preparations showed that: (i) frog atrial myocytes contained a small and variable number of individual myofibrils (from 1 to 7); (ii) the mean cross-sectional area and mean number of myosin filaments of individual cardiac myofibrils did not differ significantly from those of single skeletal myofibrils; and (iii) the total myofibril cross-sectional area of atrial myocytes was on average comparable to that of bundles of two to four skeletal myofibrils. 3. In maximally activated skeletal preparations, values of active force ranged from 0.45 +/- 0.03 microN for the single myofibrils (mean +/- S.E.M.; n = 16) to 1.44 +/- 0.24 microN for the bundles of two to four myofibrils (n = 9). Maximum active force values of forty-five cardiac myocytes averaged 1.47 +/- 0.10 microN and exhibited a non-continuous distribution with peaks at intervals of about 0.5 microN. The results suggest that variation in active force among cardiac preparations mainly reflects variability in the number of myofibrils inside the myocytes and that individual cardiac myofibrils develop the same average amount of force as single skeletal myofibrils. 4. The mean sarcomere length-resting force relation of atrial myocytes could be superimposed on that of bundles of two to four skeletal myofibrils. This suggests that, for any given amount of strain, individual cardiac and skeletal sarcomeres bear essentially the same passive force. 5. The length-passive tension data of all preparations could be fitted by an exponential equation. Equation parameters obtained for both types of myofibrils were in reasonable agreement with those reported for larger preparations of frog skeletal muscle but were very different from those estimated for multicellular frog atrial preparations. It is concluded that myofibrils are the major determinant of resting tension in skeletal muscle; structures other than the myofibrils are responsible for the high passive stiffness of frog cardiac muscle.

1. Force transients in response to step perturbations in length were recorded in intact atrial cells from frog heart at various temperatures (6-15 degrees C). Length changes of various sizes and in either direction, complete in 0.5 ms, were applied to single myocytes near slack length (initial sarcomere length 2.1-2.2 microns) just before the peak of an isometric twitch. The frequency response of the force transducers used was 2-4 kHz in Ringer solution. 2. An early quick force recovery phase was clearly observed after the elastic force response to the length step and before the start of much slower recovery processes. The quick recovery phase became progressively faster with larger shortening steps and was almost as fast as that originally described in intact frog skeletal muscle fibres (rate constants above 1000 s-1 in large releases at 10 degrees C). 3. The force-extension relation determined at the end of the length change (T1 curve) indicates that an instantaneous shortening of 0.5-0.6% of the initial cell length (L0) brings the force to zero. The force--extension relation determined at the end of the quick recovery phase (T2 curve) showed that the early recovery leads to an almost complete restoration of the original force with small stretches and releases (up to 0.3% L0) and that it becomes negligible in shortening steps of about 1.4% L0. 4. The results suggest that the mechanical properties of attached cross-bridges and the rate of transitions between attached cross-bridge states are approximately the same in frog atrial cells and fast skeletal muscle fibres.(ABSTRACT TRUNCATED AT 250 WORDS)

1. Apparatus for recording the small tensions developed by electrically stimulated single intact myocytes of frog heart is described. A laser-light optoelectronic transducer was used. The compliance of the force probes was 10-20 nm/nN, with a frequency response of 600-900 Hz in Ringer solution. The myocyte shortening during an ordinary twitch contraction was no greater than 1% of the slack length. The overall sensitivity of the transducer system was 5-10 mV/nN, with a total noise of 0.5-1 nN peak to peak. The experiments were performed at 20-23 degrees C on either atrial or ventricular myocytes at 2.15-2.2 microns sarcomere length, in 1 mM-Ca2+ Ringer solution. 2. Isoprenaline (5 microM), increases in external Ca2+ concentration ([Ca2+]o), and shortening of stimulus interval potentiated the myocyte twitch tension. The dependence of twitch characteristics on these inotropic interventions for all the atrial and ventricular myocytes tested was comparable to that of multicellular preparations under similar experimental conditions. This implies that the enzymatic isolation procedure had not altered the physiological properties of the myocytes. 3. The stimulus interval-tension relation for premature twitches of atrial and ventricular myocytes showed (i) a very steep rising phase in the region of intervals just longer than 0.52 and 0.66 s (the duration of the mechanical refractoriness in atrial or ventricular cells), (ii) a peak, at intervals of 0.7-0.8 s, where the twitch tension was strongly potentiated compared to that of the controls, and (iii) as the stimulus interval was further increased, a progressive return to the control level. The stimulus interval-tension relation for steady-state conditions exhibited similar characteristics. 4. The degree of tension potentiation by isoprenaline was greater in the controls than in the earliest test twitches. The result was that the stimulus interval-tension relations for isoprenaline-treated myocytes showed a gentler rise and a lower peak than for untreated cells. 5. The stimulus interval-tension relation of the heart is a basic property of its cells. It is related to changes in the activation level. The higher the activation level reached in control twitches, the lower the stimulus interval-dependent potentiation capability.

1. The relation between sarcomere length and tetanic tension was determined at 10-12 degrees C for 70-80 microns long segments of single fibres isolated from the tibialis anterior and semitendinosus muscles of the frog. Measurements of segment striation spacings were performed during fixed-end or length-clamp contractions by means of a laser light diffractometer. 2. At sarcomere lengths of around 2.10 microns tetanic tension rose promptly to a steady plateau, independent of the recording conditions. At greater sarcomere lengths under fixed-end conditions the tension rise occurred in two distinct stages: an initial rapid rise followed by a much slower creep. The tension creep was entirely abolished in length-clamp contractions. 3. The sarcomere length-tension diagram of length-clamped segments of tibialis anterior fibres exhibited a definite flat region between about 1.96 and 2.16 microns where tension varied by less than 1.5%. The highly linear descending limb reached zero tension at about 3.53 microns. The shift to the left by about 0.10 microns, with respect to the length-tension diagram of length-clamped segments of semitendinosus fibres, may be tentatively explained by assuming that thin filament lengths vary in different muscles. 4. The results are in agreement with those of a previous work by Gordon, Huxley & Julian (1966) and support the hypothesis (Huxley, 1957, 1980) that muscle tension is produced by simultaneous action of independent force generators, in proportion to the number of myosin bridges overlapped by actin filaments.