Novel insights on the relationship between T-tubular defects and contractile dysfunction in a mouse model of hypertrophic cardiomyopathy.

Abnormalities of cardiomyocyte Ca(2+) homeostasis and excitation-contraction (E-C) coupling are early events in the pathogenesis of hypertrophic cardiomyopathy (HCM) and concomitant determinants of the diastolic dysfunction and arrhythmias typical of the disease. T-tubule remodelling has been reported to occur in HCM but little is known about its role in the E-C coupling alterations of HCM. Here, the role of T-tubule remodelling in the electro-mechanical dysfunction associated to HCM is investigated in the Δ160E cTnT mouse model that expresses a clinically-relevant HCM mutation. Contractile function of intact ventricular trabeculae is assessed in Δ160E mice and wild-type siblings. As compared with wild-type, Δ160E trabeculae show prolonged kinetics of force development and relaxation, blunted force-frequency response with reduced active tension at high stimulation frequency, and increased occurrence of spontaneous contractions. Consistently, prolonged Ca(2+) transient in terms of rise and duration are also observed in Δ160E trabeculae and isolated cardiomyocytes. Confocal imaging in cells isolated from Δ160E mice reveals significant, though modest, remodelling of T-tubular architecture. A two-photon random access microscope is employed to dissect the spatio-temporal relationship between T-tubular electrical activity and local Ca(2+) release in isolated cardiomyocytes. In Δ160E cardiomyocytes, a significant number of T-tubules (>20%) fails to propagate action potentials, with consequent delay of local Ca(2+) release. At variance with wild-type, we also observe significantly increased variability of local Ca(2+) transient rise as well as higher Ca(2+)-spark frequency. Although T-tubule structural remodelling in Δ160E myocytes is modest, T-tubule functional defects determine non-homogeneous Ca(2+) release and delayed myofilament activation that significantly contribute to mechanical dysfunction.

The transverse-axial tubular system of cardiomyocytes.

A characteristic histological feature of striated muscle cells is the presence of deep invaginations of the plasma membrane (sarcolemma), most commonly referred to as T-tubules or the transverse-axial tubular system (TATS). TATS mediates the rapid spread of the electrical signal (action potential) to the cell core triggering Ca(2+) release from the sarcoplasmic reticulum, ultimately inducing myofilament contraction (excitation-contraction coupling). T-tubules, first described in vertebrate skeletal muscle cells, have also been recognized for a long time in mammalian cardiac ventricular myocytes, with a structure and a function that in recent years have been shown to be far more complex and pivotal for cardiac function than initially thought. Renewed interest in T-tubule function stems from the loss and disorganization of T-tubules found in a number of pathological conditions including human heart failure (HF) and dilated and hypertrophic cardiomyopathies, as well as in animal models of HF, chronic ischemia and atrial fibrillation. Disease-related remodeling of the TATS leads to asynchronous and inhomogeneous Ca(2+)-release, due to the presence of orphan ryanodine receptors that have lost their coupling with the dihydropyridine receptors and are either not activated or activated with a delay. Here, we review the physiology of the TATS, focusing first on the relationship between function and structure, and then describing T-tubular remodeling and its reversal in disease settings and following effective therapeutic approaches.

Probing myosin structural conformation in vivo by second-harmonic generation microscopy.

Understanding of complex biological processes requires knowledge of molecular structures and measurement of their dynamics in vivo. The collective chemomechanical action of myosin molecules (the molecular motors) in the muscle sarcomere represents a paradigmatic example in this respect. Here, we describe a label-free imaging method sensitive to protein conformation in vivo. We employed the order-based contrast enhancement by second-harmonic generation (SHG) for the functional imaging of muscle cells. We found that SHG polarization anisotropy (SPA) measurements report on the structural state of the actomyosin motors, with significant sensitivity to the conformation of myosin. In fact, each physiological/biochemical state we probed (relaxed, rigor, isometric contraction) produced a distinct value of polarization anisotropy. Employing a full reconstruction of the contributing elementary SHG emitters in the actomyosin motor array at atomic scale, we provide a molecular interpretation of the SPA measurements in terms of myosin conformations. We applied this method to the discrimination between attached and detached myosin heads in an isometrically contracting intact fiber. Our observations indicate that isometrically contracting muscle sustains its tetanic force by steady-state commitment of 30% of myosin heads. Applying SPA and molecular structure modeling to the imaging of unstained living tissues provides the basis for a generation of imaging and diagnostic tools capable of probing molecular structures and dynamics in vivo.

Direct activation of zebrafish neurons by ultrasonic stimulation revealed by whole CNS calcium imaging.

OBJECTIVE: Ultrasounds (US) use in neural engineering is so far mainly limited to ablation through high intensity focused ultrasound, but interesting preliminary results show that low intensity low frequency ultrasound could be used instead to modulate neural activity. However, the extent of this modulatory ability of US is still unclear, as in in vivo studies it is hard to disentangle the contribution to neural responses of direct activation of the neuron by US stimulation and indirect activation due either to sensory response to mechanical stimulation associated to US, or to propagation of activity from neighboring areas. Here, we aim to show how to separate the three effects and assess the presence of direct response to US stimulation in zebrafish. APPROACH: We observed in zebrafish larvae brain-wide US-induced activity patterns through calcium imaging microscopy. Sensory response to mechanical stimulation was assessed with a US shield. Activity propagation was assessed with inter-area latency evaluation. MAIN RESULTS: We prove that in selected brain regions the zebrafish's neural response is mainly due to direct activation, later spreading to the other regions. Shielding the neurons from direct US stimulation resulted in a significantly attenuated response, showing that sensory stimulation does not play a prominent role. SIGNIFICANCE: US non-invasive neuromodulatory approach might lead to novel ways to test and control neural activity, and hence to novel neuromodulatory therapies. Future studies will focus on the biophysical structure of directly responsive neurons to capture the mechanisms of US induced activity.

Optical mapping of neuronal activity during seizures in zebrafish.

Mapping neuronal activity during the onset and propagation of epileptic seizures can provide a better understanding of the mechanisms underlying this pathology and improve our approaches to the development of new drugs. Recently, zebrafish has become an important model for studying epilepsy both in basic research and in drug discovery. Here, we employed a transgenic line with pan-neuronal expression of the genetically-encoded calcium indicator GCaMP6s to measure neuronal activity in zebrafish larvae during seizures induced by pentylenetretrazole (PTZ). With this approach, we mapped neuronal activity in different areas of the larval brain, demonstrating the high sensitivity of this method to different levels of alteration, as induced by increasing PTZ concentrations, and the rescuing effect of an anti-epileptic drug. We also present simultaneous measurements of brain and locomotor activity, as well as a high-throughput assay, demonstrating that GCaMP measurements can complement behavioural assays for the detection of subclinical epileptic seizures, thus enabling future investigations on human hypomorphic mutations and more effective drug screening methods. Notably, the methodology described here can be easily applied to the study of many human neuropathologies modelled in zebrafish, allowing a simple and yet detailed investigation of brain activity alterations associated with the pathological phenotype.

Cell imaging and manipulation by nonlinear optical microscopy.

Advances in the technologies for labeling and imaging biological samples drive a constant progress in our capability of studying structures and their dynamics within cells and tissues. In the last decade, the development of numerous nonlinear optical microscopies has led to a new prospective both in basic research and in the potential development of very powerful noninvasive diagnostic tools. These techniques offer large advantages over conventional linear microscopy with regard to penetration depth, spatial resolution, three-dimensional optical sectioning, and lower photobleaching. Additionally, some of these techniques offer the opportunity for optically probing biological functions directly in living cells, as highlighted, for example, by the application of second harmonic generation to the optical measurement of electrical potential and activity in excitable cells. In parallel with imaging techniques, nonlinear microscopy has been developed into a new area for the selective disruption and manipulation of intracellular structures, providing an extremely useful tool of investigation in cell biology. In this review we present some basic features of nonlinear microscopy with regard both to imaging and manipulation, and show some examples to illustrate the advantages offered by these novel methodologies.

Second-harmonic generation sensitivity to transmembrane potential in normal and tumor cells.

Second-harmonic generation (SHG) is emerging as a powerful tool for the optical measurement of transmembrane potential in live cells with high sensitivity and temporal resolution. Using a patch clamp, we characterize the sensitivity of the SHG signal to transmembrane potential for the RH 237 dye in various normal and tumor cell types. SHG sensitivity shows a significant dependence on the type of cell, ranging from 10 to 17% per 100 mV. Furthermore, in the samples studied, tumor cell lines display a higher sensitivity compared to normal cells. In particular, the SHG sensitivity increases in the cell line Balb/c3T3 by the transformation induced with SV40 infection of the cells. We also demonstrate that fluorescent labeling of the membrane with RH 237 at the concentration used for SHG measurements does not induce any measurable alteration in the electrophysiological properties of the cells investigated. Therefore, SHG is suitable for the investigation of outstanding questions in electrophysiology and neurobiology.

Exploring molecular motors and switches at the single-molecule level.

Single-molecule techniques have propelled an impressive number of biophysical studies during the last decade. From relatively simple video-microscopy techniques, to sophisticated manipulation and detection apparata, single-molecule techniques are capable of tracking the movements and the reaction trajectories of single enzymatic units. By observing microspheres attached to biomolecules it is possible to follow the motion of molecular motors, or to detect conformational "switching" induced by regulatory proteins. Micromanipulation tools like optical tweezers have been widely applied to understand the mechanisms of linear molecular motors, and have allowed the measurement of the elementary steps and the forces produced by several motor proteins, including myosin, kinesin, and dynein. New experimental assays based on magnetic or optical "wrenches," which are able to apply and detect torques on rotary motors and biopolymers, are opening new possibilities in this field. Here, established and emerging magneto-optical manipulation and video-tracking techniques are reviewed, in the perspective of single molecular motors and regulatory proteins studies.

Analysis of kinetics in noisy systems: application to single molecule tethered particle motion.

In the tethered particle motion method the length of a DNA molecule is monitored by measuring the range of diffusion of a microsphere tethered to the surface of a microscope coverslip through the DNA molecule itself. Looping of DNA (induced by binding of a specific protein) can be detected with this method and the kinetics of the looping/unlooping processes can be measured at the single molecule level. The microsphere's position variance represents the experimental variable reporting on the polymer length. Therefore, data windowing is required to obtain position variance from raw position data. Due to the characteristic diffusion time of the microsphere, the low-pass filtering required to attain a good signal/noise ratio (S/N) in the discrimination of looped versus unlooped state impacts significantly the measurement's time resolution. Here we present a method for measuring lifetimes based on half-amplitude thresholding and then correcting the kinetic measurements, taking into account low S/N (leading to false events) and limited time resolution (leading to missed events). This method allows an accurate and unbiased estimation of the kinetic parameters under investigation, independently of the choice of the window used for variance calculation, with potential applications to other single molecule measurements with low S/N.

New techniques in linear and non-linear laser optics in muscle research.

This review proposes a brief summary of two applications of lasers to muscle research. The first application (laser tweezers), is now a well-established technique in the field, adopted by several laboratories in the world and producing a constant stream of original data, fundamental for our improved understanding of muscle contraction at the level of detail that only single molecule measurements can provide. As an example of the power of this technique, here we focus on some recent results, revealing the performance of the working stroke in at least two distinct steps also in skeletal muscle myosin. A second laser-based technique described here is second-harmonic generation; the application of this technique to muscle research is very recent. We describe the main results obtained thus far in this area and the potentially remarkable impact that this technology may have in muscle research.

Cross-bridge detachment and attachment following a step stretch imposed on active single frog muscle fibres.

1. The time course of cross-bridge detachment-attachment following a step stretch was determined in single frog muscle fibres (at 4 degrees (1 and 2.1 microns sarcomere length) by imposing, under sarcomere length control by a striation follower, test step releases of various amplitudes (2-13 nm per half-sarcomere) at successive times (4-55 ms) after a conditioning stretch of approximately 4 nm per half-sarcomere. 2. The comparison with the control tension transients, elicited by releases not preceded by the conditioning stretch, shows that, early after the conditioning stretch, the quick tension recovery following small releases is depressed and the quick tension recovery following large releases is potentiated. Both effects are expected as a consequence of the strain produced in the cross-bridges by the conditioning stretch. 3. These effects disappear and the tension transient is reprimed, indicating substitution of freshly attached cross-bridges for strained cross-bridges, with a time constant of approximately 10 ms. 4. A novel multiple-exponential equation, based on the hypothesis of complete substitution of freshly attached cross-bridges for the cross-bridges that underwent the stretch, has been used to fit the whole tension transient following step stretches of different sizes (2-6 nm per half-sarcomere). For a stretch of 4 nm, the time constant of the exponential process responsible for cross-bridge detachment (tau d, 9.3 ms) almost coincides with the time constant of repriming as measured by the double-step experiments. The time constant of the exponential process representing the cumulative effects of attachment and force generation (tau 3) is 13.6 ms. 5. For stretches of different sizes the amount of quick tension recovery attributable to the reversal of the working stroke elicited by the stretches is estimated by subtracting, from the original tension transient, the contribution to tension recovery due to detachment-attachment of cross-bridges as estimated by the multiple-exponential analysis. Following this calculation, the structural change in the myosin heads responsible for the reversal of the working stroke can be 2 nm at maximum, suggesting that the elastic component in the cross-bridges is at least twice as rigid as previously thought.