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Tropomyosin (Tm) is a coiled-coil alpha-helix regulating the cooperative activation of muscle contraction by the thin filament. Tm forms strands along actin filaments which azimuthally move between switched-on/off locations under the influence of Ca2+-troponin (Tn) and strong crossbridge formation. The flexibility of Tm strongly influences its movement so that the relaxation-activation mechanism within the thin filament may be critically modulated. Here we study the mechanical consequences of the presence of chicken recombinant alpha tropomyosin, D137L, in rabbit skeletal muscle myofibrils, previously characterized in vitro (Sumida et al., 2008). Endogenous Tm and Tn are replaced into rabbit skeletal muscle myofibrils (Siththanandan et al., 2009) with recombinant rabbit Tn (WT) and chicken alphaTm (WT and D137L). SDS-PAGE analyses of samples of myofibrils show that Tm-Tn replacement was about 90%. Force recordings from small bundles of myofibrils (Tesi et al., 2002) show that at saturating [Ca2+ ] (pCa 4.5), maximal tension was not affected by Tm flexibility nor were the rates of force activation (kACT) and force redevelopment (kTR). However, at submaximal [Ca2+ ] (pCa 5.9), the presence of D137L Tm significantly increases (about 15-20%) both force and kinetics of force generation. Consistently, force-pCa curves obtained from myofibrils replaced with chicken D137L Tm showed a 0.15 increase in pCa50 compared to WT. This result supports the hypothesis (Sumida et al., 2008) that the increased flexibility imparted to the Tm coiled-coiled structure by Asp at 137 avoids excessive turning-on of the system at the high physiological myosin concentrations. Supported by Telethon-Italy GGP07133, STREP Project “BIG-HEART” 241577 EC and NIH HL22461.

The first mutation found to be associated with hypertrophic cardiomyopathy (HCM) is the R403Q mutation in the gene encoding β-myosin heavy chain. R403Q is located in the globular head of myosin (S1), responsible for actin interaction, and hence motor function of myosin. According to the 2-state model of actin-myosin interaction total cross-bridge turnover rate can be described by the sum of the cross-bridge association (fapp) and detachment (gapp) rates. Gapp is equivalent to the slow rate of cross-bridge relaxation (slow krel), which equals the energetic cost of tension generation, i.e. tension cost (expressed by ATP utilization/tension). In the present study we investigated to what extent cross-bridge kinetics in HCM with the R403Q mutation correlated with cross-bridge energetics.Left ventricular multicellular muscle strips and myofibrillar preparations were isolated from 3 HCM patients with the R403Q mutation. 8 HCM sarcomere mutation negative (HCMsmn) patients served as control. In the muscle strips force generating capacity and ATP utilization were measured to assess tension cost and in the myofibrillar preparations cross-bridge kinetics was analyzed. The fraction of mutated R403Q mRNA was analyzed using specific restriction digests and realtime-PCR. Compared to HCMsmn tension cost was higher in the muscle strips of the 3 R403Q patients which showed a positive linear correlation with relaxation kinetics in the corresponding myofibrillar preparations. Variation in cross-bridge function among the 3 R403Q patients could not be explained by differences in R403Q mRNA levels.Cross-bridge cycling efficiency was decreased in HCM due to the R403Q mutation, while cross-bridge kinetics was increased. This suggests that the apparent gain in function evident from increased kinetics leads to a loss of function with respect to cross-bridge cycling efficiency in HCM caused by the R403Q mutation.

Tropomyosin is an essential component in the regulation of muscle contraction. A method has been described to remove endogenous tropomyosin (Tm) and troponin (Tn) and replace them with exogenous proteins into rabbit psoas myofibrils (Siththanandan et al., Biophys. J., 2007, suppl. 626a). In the present study the endogenous Tm and Tn are replaced into cardiac mouse myofibrils by a modification of that method. Endogenous Tm and Tn were extracted by washing cardiac mouse myofibrils in a low ionic strength solution at pH 8.0. The wash was repeated several times to increase the amount of extracted Tm. The reconstitution of thin filament regulatory proteins was made in a rigor solution in two steps, first adding Tm and then Tn. To measure the effects of this procedure, samples of myofibrils were analyzed at different stages of the protocol (control, extracted, reconstituted) both functionally (by loss and regain of Ca2+ -dependent regulation) and by SDS-PAGE. The success of the exchange was determined by mechanical measurements of calcium dependent force activation on the reconstituted myofibrils. Small bundles of cardiac myofibrils were mounted in a force recording apparatus and activated by rapidly switching between low and high Ca2+ solutions (Tesi et al., Biophys. J., 2002, 83, 2142-2151). Maximal isometric tension was 30-35% lower in the reconstituted myofibrils than in control myofibrils but the rate of force activation (kACT) and that of force redevelopment (kTR) was the same in the two myofibril groups. The effectiveness of Tm replacement in human cardiac myofibrils is under investigation. This approach can be used to test the functional impact of Tm mutations responsible for human cardiomyopathies. Supported by Telethon-Italy GGP07133.

Myopalladin (MYPN) is a striated muscle-specific immunoglobulin domain-containing protein located in the sarcomeric Z-line and I-band. MYPN gene mutations are causative for dilated (DCM), hypertrophic, and restrictive cardiomyopathy. In a yeast two-hybrid screening, MYPN was found to bind to titin in the Z-line, which was confirmed by microscale thermophoresis. Cardiac analyses of MYPN knockout (MKO) mice showed the development of mild cardiac dilation and systolic dysfunction, associated with decreased myofibrillar isometric tension generation and increased resting tension at longer sarcomere lengths. MKO mice exhibited a normal hypertrophic response to transaortic constriction (TAC), but rapidly developed severe cardiac dilation and systolic dysfunction, associated with fibrosis, increased fetal gene expression, higher intercalated disc fold amplitude, decreased calsequestrin-2 protein levels, and increased desmoplakin and SORBS2 protein levels. Cardiomyocyte analyses showed delayed Ca2+ release and reuptake in unstressed MKO mice as well as reduced Ca2+ spark amplitude post-TAC, suggesting that altered Ca2+ handling may contribute to the development of DCM in MKO mice.

Cardiac Ryanodine Receptor (RyR2) is a crucial determinant of Ca2+ availability in myofilament activation and force generation. To investigate the impact of RyR2 arrhythmia-related mutations on contractile function, atrial and ventricular trabeculae and ventricular myocytes from Wild Type (WT) and heterozygous R4496C RyR2 mutant (RyRR4496C+/-) mice were isolated and studied. Although under basal conditions contractility and Ca2+-transient amplitude are normal, RyRR4496C+/- preparations show reduced inotropic responses to high stimulation frequency, isoproterenol and other experimental interventions that potentiate contraction. The diminished inotropic response results from a smaller Sarcoplasmic Reticulum (SR) calcium load after inotropic stimulation than that of WT preparations. Moreover, RyRR4496C+/- muscles have faster mechanical restitution and shorter recovery of the amplitude of Ca2+release. In RyRR4496C+/- vs. WT myocytes sarcolemmal Ca2+-fluxes (ICa-L and INCX) and SR Ca2+-uptake are unchanged. We suggest that changes in gating of the mutant RyR2 Ca2+ channel are directly responsible for these observations. Fitting our experimental data for the R4496C mutation into a cardiomyocyte model, where the RyR2 gating properties are represented by a four state equilibrium and are modulated by luminal [Ca2+] ([Ca2+]SR), we predict an increase in sensitivity to [Ca2+]SR of both opening and closing channel transitions rates of mutant channels. Specifically, increasing [Ca2+]SR-sensitivity of closed-to-open transitions leads to SR-calcium depletion, blunted simulated inotropic responses and faster mechanical restitution. However, for the model to fully emulate our experimental results, [Ca2+]SR-sensitivity of open-to-closed transition also has to be increased, suggesting an effect of the R4496C mutation on RyR2-deactivation and Ca2+-release termination.

Tropomyosin contains a phosphorylation site at Ser-283 located within the head-to-tail overlap region that regulates muscle contraction. Previously we demonstrated that recombinant wild-type (Tm-WT) and pseudo-phosphorylated (Tm-S283D) alpha tropomyosin, expressed and purified from insect cells, exhibit regulated ATPase activity and troponin T binding similar to that reported for non-phosphorylated and phosphorylated tropomyosin (pTm) purified from muscle. We further demonstrated transgenic mice expressing the pseudo-phosphorylated tropomyosin (Tm-S283D Tg) exhibit increased mortality and decreased rate of relaxation in the work performing heart preparation. These changes occur in the absence of altered steady state maximal force or calcium-sensitive force development in skinned papillary bundles. In light of these findings, we sought to investigate the effect of pTm on the kinetics of cardiac muscle function. Using an extraction/replacement protocol, we measured force kinetics in a myofibril preparation. Results demonstrate no significant difference between myofibrils replaced with Tm-S283D or Tm-WT in maximal force, the rate of force activation, or the rate of force redevelopment, implying pTm does not play a role in altering muscle activation or cycling kinetics. To investigate if pTm affects the kinetics of thin filament inactivation we measured the rate of calcium disassociation from troponin C. Results demonstrate thin filaments reconstituted with Tm-S283D decreased the rate of calcium disassociation from troponin C consistent with the previously observed relaxation impairment. Finally, to determine the effects of pTm on systemic alterations in cardiovascular performance we measured heart function in Tm-S283D Tg mice by echocardiography. Results demonstrate Tm-S283D Tg mice exhibit trends towards impaired cardiac contractility compared to non-transgenic mice including decreased peak systolic velocity and ejection fraction. Overall, these findings demonstrate tropomyosin phosphorylation contributes to the regulation of cardiac dynamics; however, the precise role of pTm in the development of cardiac dysfunction remains elusive.

In spite of extensive work on the functional sequelae of Hypertrophic CardioMyopathy (HCM)-associated mutations in sarcomeric proteins, the mechanisms by which the mutant proteins cause the disease have not been definitely identified. Here we use the single myofibril technique (Tesi et al., Biophys. J., 2002, 83, 2142-2151) to compare the kinetics of contraction and relaxation of myofibrils isolated from frozen left ventricular samples of one homozygous HCM patient carrying the cardiac Troponin T (cTnT) mutation K273N (underwent heart transplantation) to those from “control” hearts. Preparations, mounted in a force recording apparatus (15 °C), were maximally Ca2+-activated (pCa 4.5) and fully relaxed (pCa 9) by rapid (

Hypertrophic Cardiomyopathy (HCM) is an autosomal dominant cardiac disease resulting from mutations in genes encoding contractile proteins, including troponin (Tn; the complex of TnT, TnI and TnC). From measurements of ATPase activity of myofibrils, into which TnT mutants were exchanged, we showed that two HCM-associated mutations of TnT, E244D and K247R, increase the maximum ATPase activity without any change in Ca2+ sensitivity (Matsumoto et al., BSJ 2007, 1P150). It is, however, still not clear if the mutations have direct effects on the mechanical properties of muscle because ATPase activity and tension development are not necessarily coupled. We thus examined the effects of the mutations on maximal isometric tension and kinetics of Tn-exchanged myofibrils. Measurements were made in single myofibrils activated and relaxed by a fast solution switching device, with a dead time < 10ms (Tesi et al., Biophys. J., 2002, 83, 2142). Maximal isometric tension values of the mutant TnT exchanged myofibrils were significantly higher (E244D) or tended to be higher (K247R) than those of the wild type TnT exchanged myofibrils while Ca2+ sensitivity was unchanged. These results indicate that the effects of the mutations on myofibril mechanics are directly coupled with the effects on the ATPase activity. Measurements of the rate constants of force generation showed that the mutant TnT exchanged myofibrils had similar kinetics as the wild type TnT exchanged myofibrils. The result suggests that kinetics of cross bridge transitions are not influenced by these mutants.

Introduction: Many of cTnT mutations linked to cardiomyopathies fall the TNT1 domain/N terminal tail region of unresolved high definition structure. This region (∼94-170) of cTnT is critical to Tm binding and contraction regulation. Here, the impact of the E163R mutation in cTnT-TNT1 on contractile function and tension cost was investigated using intact and skinned preparations from WT and transgenic mouse hearts.Methods: Left and right ventricular trabeculae were dissected from non-transgenic wild type (WT) and heterozygous (Δ160E or E163R) mouse hearts and mounted isometrically to record twitch tension or, when skinned, Ca2+ activated force. Myofibrillar ATPase activity was measured by fluorimetric enzyme coupled assay (de Tombe and Stienen, 1995).Results: Myocardium of E163R mice shows: (i) no change of myosin isoform expression (ii) maintained peak isomentric twitch tension at all stimulation frequencies, (iii) prolonged time to peak and time to 50% relaxation, with preserved rate-adaptation of twitch duration, (iv) changes of the short-term interval force relationship and increased occurrence of spontaneous contractions. No significant differences were found in maximum Ca2+ activated tension of E163R and WT skinned trabeculae. However, Ca2+ sensitivity of tension was significantly increased in E163R skinned trabeculae when compared with WT. As to the economy of force maintenance, preliminary experiments suggest an increase of tension cost in trabeculae from E163R hearts. Resting ATPase activity also tended to be higher in E163R preparations. Kinetics of force development and relaxation will be assessed on single myofibrils, isolated from the same hearts.Conclusions: Both primary sarcomeric changes and secondary E-C coupling alterations contribute to mechanical impairment in E163R cTnT mutant myocardium. Supported by: EC Grant n. 241577 (BIG-Heart)