The R403Q mutation in β-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 R403Q mutation in the β-myosin heavy chain was the first mutation to be identified as responsible for familial hypertrophic cardiomyopathy (FHC) (Geisterfer-Lowrance et al. 1990), a primary disease of the cardiac sarcomere that is the most commonly identified cause of cardiac sudden death in young people. The functional sequelae of the R403Q mutation have been extensively investigated using a variety of experimental models and approaches (Cuda et al. 1993;Lankford et al. 1995;Sata & Ikebe, 1996;Geisterfer-Lowrance et al. 1996;Marian et al. 1999;Tyska et al. 2000;Lowey, 2002;Keller et al. 2004) but the cardiac sarcomeres of affected individuals have never been directly examined. Here we compare contraction and relaxation of left ventricular myofibrils from one patient carrying the R403Q mutation to those from a healthy control heart. To investigate sarcomere mechanics we use previously This paper has online supplemental material. published techniques to measure and control the force and length of single myofibrils activated and relaxed by fast solution switching (Tesi et al. 2002;Piroddi et al. 2007). One advantage of this approach was that we could probe the acto-myosin transduction cycle while keeping the native structured sarcomere environment of the mutant protein. Preliminary report of this work has been published in abstract form ). Methods PatientsThe investigation conforms with the principles outlined in the Declaration of Helsinki and had been approved by the local Ethics Committee. Informed consent was given for both mutational analysis and mechanical experiments.A 24-year-old man, with a severe family history of premature cardiac death, diagnosed at 13 with FHC, and

Fast solution switching techniques in single myofibrils offer the opportunity to dissect and directly examine the sarcomeric mechanisms responsible for force generation and relaxation. The feasibility of this approach is tested here in human cardiac myofibrils isolated from small samples of atrial and ventricular tissue. At sarcomere lengths between 2.0 and 2.3 mum, resting tensions were significantly higher in ventricular than in atrial myofibrils. The rate constant of active tension generation after maximal Ca(2+) activation (k (ACT)) was markedly faster in atrial than in ventricular myofibrils. In both myofibril types k (ACT) was the same as the rate of tension redevelopment after mechanical perturbations and decreased significantly by decreasing [Ca(2+)] in the activating solution. Upon sudden Ca(2+) removal, active tension fully relaxed. Relaxation kinetics were (1) much faster in atrial than in ventricular myofibrils, (2) unaffected by bepridil, a drug that increases the affinity of troponin for Ca(2+), and (3) strongly accelerated by small increases in inorganic phosphate concentration. The results indicate that myofibril tension activation and relaxation rates reflect apparent cross-bridge kinetics and their Ca(2+) regulation rather than the rates at which thin filaments are switched on or off by Ca(2+) binding or removal. Myofibrils from human hearts retain intact mechanisms for contraction regulation and tension generation and represent a viable experimental model to investigate function and dysfunction of human cardiac sarcomeres.

Abstract-The specific and selective proteolysis of cardiac troponin I (cTnI) has been proposed to play a key role in human ischemic myocardial disease, including stunning and acute pressure overload. In this study, the functional implications of cTnI proteolysis were investigated in human cardiac tissue for the first time. The predominant human cTnI degradation product (cTnI ) and full-length cTnI were expressed in Escherichia coli, purified, reconstituted with the other cardiac troponin subunits, troponin T and C, and subsequently exchanged into human cardiac myofibrils and permeabilized cardiomyocytes isolated from healthy donor hearts. Maximal isometric force and kinetic parameters were measured in myofibrils, using rapid solution switching, whereas force development was measured in single cardiomyocytes at various calcium concentrations, at sarcomere lengths of 1.9 and 2.2 m, and after treatment with the catalytic subunit of protein kinase A (PKA) to mimic ␤-adrenergic stimulation. One-dimensional gel electrophoresis, Western immunoblotting, and 3D imaging revealed that approximately 50% of endogenous cTnI had been homogeneously replaced by cTnI in both myofibrils and cardiomyocytes. Maximal tension was not affected, whereas the rates of force activation and redevelopment as well as relaxation kinetics were slowed down. Ca 2ϩ sensitivity of the contractile apparatus was increased in preparations containing cTnI (pCa 50 : 5.73Ϯ0.03 versus 5.52Ϯ0.03 for cTnI and full-length cTnI, respectively). The sarcomere length dependency of force development and the desensitizing effect of PKA were preserved in cTnI 1-192 -exchanged cardiomyocytes. These results indicate that degradation of cTnI in human myocardium may impair diastolic function, whereas systolic function is largely preserved.

We employed single myofibril techniques to test whether the presence of slow skeletal troponin-I (ssTnI) is sufficient to induce increased myofilament calcium sensitivity (EC(50)) and whether modulation of EC(50) affects the dynamics of force development. Studies were performed using rabbit psoas myofibrils activated by rapid solution switch and in which Tn was partially replaced for either recombinant cardiac Tn(cTn) or Tn composed of recombinant cTn-T (cTnT) and cTn-C (cTnC), and recombinant ssTnI (ssTnI-chimera Tn). Tn exchange was performed in rigor solution (0.5 mg/ml Tn; 20 degrees C; 2 h) and confirmed by SDS-PAGE. cTnI exchange induced a decrease in EC(50); ssTnI-chimera Tn exchange induced a further decrease in EC(50) (in microM: endogenous Tn, 1.35 +/- 0.08; cTnI, 1.04 +/- 0.13; ssTnI-chimera Tn, 0.47 +/- 0.03). EC(50) was also decreased by application of 100 microM bepridil (control: 2.04 +/- 0.03 microM; bepridil 1.35 +/- 0.03 microM). Maximum tension was not different between any groups. Despite marked alterations in EC(50), none of the dynamic activation-relaxation parameters were affected under any condition. Our results show that 1) incorporation of ssTnI into the fast skeletal sarcomere is sufficient to induce increased myofilament Ca(2+) sensitivity, and 2) the dynamics of actin-myosin interaction do not correlate with EC(50). This result suggests that intrinsic cross-bridge cycling rate is not altered by the dynamics of thin-filament activation.

Stretch of the myocardium influences the shape and amplitude of the intracellular Ca(2+)([Ca(2+)](i)) transient. Under isometric conditions stretch immediately increases myofilament Ca(2+) sensitivity, increasing force production and abbreviating the time course of the [Ca(2+)](i) transient (the rapid response). Conversely, muscle shortening can prolong the Ca(2+) transient by decreasing myofilament Ca(2+) sensitivity. During the cardiac cycle, increased ventricular dilation may increase myofilament Ca(2+) sensitivity during diastolic filling and the isovolumic phase of systole, but enhance the decrease in myofilament Ca(2+) sensitivity during the systolic shortening of the ejection phase. If stretch is maintained there is a gradual increase in the amplitude of the Ca(2+) transient and force production, which takes several minutes to develop fully (the slow response). The rapid and slow responses have been reported in whole hearts and single myocytes. Here we review stretch-induced changes in [Ca(2+)](i) and the underlying mechanisms. Myocardial stretch also modifies electrical activity and the opening of stretch-activated channels (SACs) is often used to explain this effect. However, the myocardium has many ionic currents that are regulated by [Ca(2+)](i) and in this review we discuss how stretch-induced changes in [Ca(2+)](i) can influence electrical activity via the modulation of these Ca(2+)-dependent currents. Our recent work in single ventricular myocytes has shown that axial stretch prolongs the action potential. This effect is sensitive to either SAC blockade by streptomycin or the buffering of [Ca(2+)](i) with BAPTA, suggesting that both SACs and [Ca(2+)](i) are important for the full effects of axial stretch on electrical activity to develop.