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Myosin filament assembly onto myofibrils in live neonatal cardiomyocytes observed by TPEF-SHG microscopy.

ABSTRACT: Understanding myofibrillogenesis is essential for elucidating heart muscle formation, development, and remodelling in response to physiological stimulation. Here, we report the dynamic assembly process of contractile myosin filaments onto myofibrils in a live cardiomyocyte culture during myofibrillogenesis.Utilizing a custom-built, two-photon excitation fluorescence and second harmonic generation imaging system equipped with an on-stage incubator, we observed new sarcomere additions in rat neonatal cardiomyocytes during 10 h of on-stage incubation. The new sarcomere additions occurred at the side of existing myofibrils, where we observed mature myofibrils acting as templates, or at the interstice of several separated myofibrils.During sarcomeric addition, myosin filaments are assembled onto the premyofibril laterally. This lateral addition, which proceeds stepwise along the axial direction, plays an important role in the accumulation of Z-bodies to form mature Z-disks and in the regulation of sarcomeric alignment during maturation.

SUBMITTER: Liu H

PROVIDER: S-EPMC3543992 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Myosin filament assembly onto myofibrils in live neonatal cardiomyocytes observed by TPEF-SHG microscopy.

Liu Honghai H Shao Yonghong Y Qin Wan W Runyan Raymond B RB Xu Meifeng M Ma Zhen Z Borg Thomas K TK Markwald Roger R Gao Bruce Z BZ

Cardiovascular research 20121031 2

<h4>Aims</h4>Understanding myofibrillogenesis is essential for elucidating heart muscle formation, development, and remodelling in response to physiological stimulation. Here, we report the dynamic assembly process of contractile myosin filaments onto myofibrils in a live cardiomyocyte culture during myofibrillogenesis.<h4>Methods and results</h4>Utilizing a custom-built, two-photon excitation fluorescence and second harmonic generation imaging system equipped with an on-stage incubator, we obse ...[more]

PMID: 23118131

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Project description:RATIONALE:Cardiac myocyte contraction is caused by Ca(2+) binding to troponin C, which triggers the cross-bridge power stroke and myofilament sliding in sarcomeres. Synchronized Ca(2+) release causes whole cell contraction and is readily observable with current microscopy techniques. However, it is unknown whether localized Ca(2+) release, such as Ca(2+) sparks and waves, can cause local sarcomere contraction. Contemporary imaging methods fall short of measuring microdomain Ca(2+)-contraction coupling in live cardiac myocytes. OBJECTIVE:To develop a method for imaging sarcomere level Ca(2+)-contraction coupling in healthy and disease model cardiac myocytes. METHODS AND RESULTS:Freshly isolated cardiac myocytes were loaded with the Ca(2+)-indicator fluo-4. A confocal microscope equipped with a femtosecond-pulsed near-infrared laser was used to simultaneously excite second harmonic generation from A-bands of myofibrils and 2-photon fluorescence from fluo-4. Ca(2+) signals and sarcomere strain correlated in space and time with short delays. Furthermore, Ca(2+) sparks and waves caused contractions in subcellular microdomains, revealing a previously underappreciated role for these events in generating subcellular strain during diastole. Ca(2+) activity and sarcomere strain were also imaged in paced cardiac myocytes under mechanical load, revealing spontaneous Ca(2+) waves and correlated local contraction in pressure-overload-induced cardiomyopathy. CONCLUSIONS:Multimodal second harmonic generation 2-photon fluorescence microscopy enables the simultaneous observation of Ca(2+) release and mechanical strain at the subsarcomere level in living cardiac myocytes. The method benefits from the label-free nature of second harmonic generation, which allows A-bands to be imaged independently of T-tubule morphology and simultaneously with Ca(2+) indicators. Second harmonic generation 2-photon fluorescence imaging is widely applicable to the study of Ca(2+)-contraction coupling and mechanochemotransduction in both health and disease.

Project description:Characterizing the contractile function of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is key for advancing their utility for cellular disease models, promoting cell based heart repair, or developing novel pharmacological interventions targeting cardiac diseases. The aim of the present study was to understand whether steady-state and kinetic force parameters of β-myosin heavy chain (βMyHC) isoform-expressing myofibrils within human embryonic stem cell-derived cardiomyocytes (hESC-CMs) differentiated in vitro resemble those of human ventricular myofibrils (hvMFs) isolated from adult donor hearts. Contractile parameters were determined using the same micromechanical method and experimental conditions for both types of myofibrils. We identified isoforms and phosphorylation of main sarcomeric proteins involved in the modulation of force generation of both, chemically demembranated hESC-CMs (d-hESC-CMs) and hvMFs. Our results indicate that at saturating Ca2+ concentration, both human-derived contractile systems developed forces with similar rate constants (0.66 and 0.68 s-1), reaching maximum isometric force that was significantly smaller for d-hESC-CMs (42 kPa) than for hvMFs (94 kPa). At submaximal Ca2+-activation, where intact cardiomyocytes normally operate, contractile parameters of d-hESC-CMs and hvMFs exhibited differences. Ca2+ sensitivity of force was higher for d-hESC-CMs (pCa50 = 6.04) than for hvMFs (pCa50 = 5.80). At half-maximum activation, the rate constant for force redevelopment was significantly faster for d-hESC-CMs (0.51 s-1) than for hvMFs (0.28 s-1). During myofibril relaxation, kinetics of the slow force decay phase were significantly faster for d-hESC-CMs (0.26 s-1) than for hvMFs (0.21 s-1), while kinetics of the fast force decay were similar and ~20x faster. Protein analysis revealed that hESC-CMs had essentially no cardiac troponin-I, and partially non-ventricular isoforms of some other sarcomeric proteins, explaining the functional discrepancies. The sarcomeric protein isoform pattern of hESC-CMs had features of human cardiomyocytes at an early developmental stage. The study indicates that morphological and ultrastructural maturation of βMyHC isoform-expressing hESC-CMs is not necessarily accompanied by ventricular-like expression of all sarcomeric proteins. Our data suggest that hPSC-CMs could provide useful tools for investigating inherited cardiac diseases affecting contractile function during early developmental stages.

Dataset Information

Myosin filament assembly onto myofibrils in live neonatal cardiomyocytes observed by TPEF-SHG microscopy.

Publications

Myosin filament assembly onto myofibrils in live neonatal cardiomyocytes observed by TPEF-SHG microscopy.

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