Imaging of Zebrafish In Vivo with Second-Harmonic Generation Reveals Shortened Sarcomeres Associated with Myopathy Induced by Statin

Abstract

We employed second-harmonic generation (SHG) imaging and the zebrafish model to investigate the myopathy caused by statin in vivo with emphasis on the altered microstructures of the muscle sarcomere, the fundamental contractile element of muscles. This approach derives an advantage of SHG imaging to observe the striated skeletal muscle of living zebrafish based on signals produced mainly from the thick myosin filament of sarcomeres without employing exogenous labels, and eliminates concern about the distortion of muscle structures caused by sample preparation in conventional histological examination. The treatment with statin caused a significantly shortened sarcomere relative to an untreated control (1.73±0.09 µm vs 1.91±0.08 µm, P<0.05) while the morphological integrity of the muscle fibers remained largely intact. Mechanistic tests indicated that this microstructural disorder was associated with the biosynthetic pathway of cholesterol, or, specifically, with the impaired production of mevalonate by statins. This microstructural disorder exhibited a strong dependence on both the dosage and the duration of treatment, indicating a possibility to assess the severity of muscle injury according to the altered length of the sarcomeres. In contrast to a conventional assessment of muscle injury using clinical biomarkers in blood, such as creatine kinase that is released from only disrupted myocytes, the ability to determine microstructural modification of sarcomeres allows diagnosis of muscle injury before an onset of conventional clinical symptoms. In light of the increasing prevalence of the incidence of muscle injuries caused by new therapies, our work consolidates the combined use of the zebrafish and SHG imaging as an effective and sensitive means to evaluate the safety profile of new therapeutic targets in vivo.

Multifocal multiphoton microscopy with adaptive optical correction

“…In agreement with previously reported zebrafish optical characterization, spherical aberration is a main contributor to resolution and SNR degradation (Bourgenot et al, 2012;Gould et al, 2012;Rueckel et al, 2006). As Zebrafish generate less aberration than the oncological sample (Figure 71), the system corrections (Z5--Z14) contributed Zebrafish produce substantial second--harmonic (SHG) signal (Huang et al, 2011).…”

Multimodal SHG-2PF Imaging of Microdomain Ca 2+ -Contraction Coupling in Live Cardiac Myocytes

“…26,28,29 The label-free nature of sarcomeric SHG has been utilized to study sarcomere microarchitecture in muscular dystrophy, 30,31 myofibrilogenesis, 32 and drug-induced myopathy. 33 It has also been developed as a technique to study local actin-myosin cross-bridging 28,29 and in vivo contractile dynamics in skeletal muscle. 34 Boulesteix et al have used it to measure sarcomere length with a reported accuracy of 20-nm in relaxed and tetanic frog cardiac myocytes.…”

Attenuated sarcomere lengthening of the aged murine left ventricle observed using two-photon fluorescence microscopy

“…The operating range of L s in cardiomyocytes within the left ventricular wall has been determined using hearts arrested and chemically fixed at experimentally controlled pressures, with spacing between electron dense Z lines measured using electron microscopy (30) or transmitted light microscopy (11). These approaches require careful tissue preparation to avoid fixation artifacts associated with shrinkage resulting in underestimation of L s [e.g., see Huang et al (16)]. Furthermore, fixed preparations limit L s evaluation to a single filling pressure in each heart, effectively preventing evaluation of the pressure versus L s relationship in a single heart.…”