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Microdomain heterogeneity in 3D affects the mechanics of neonatal cardiac myocyte contraction.

ABSTRACT: Cardiac muscle cells are known to adapt to their physical surroundings, optimizing intracellular organization and contractile function for a given culture environment. A previously developed in vitro model system has shown that the inclusion of discrete microscale domains (or microrods) in three dimensions (3D) can alter long-term growth responses of neonatal ventricular myocytes. The aim of this work was to understand how cellular contact with such a domain affects various mechanical changes involved in cardiac muscle cell remodeling. Myocytes were maintained in 3D gels over 5 days in the presence or absence of 100-?m-long microrods, and the effect of this local heterogeneity on cell behavior was analyzed via several imaging techniques. Microrod abutment resulted in approximately twofold increases in the maximum displacement of spontaneously beating myocytes, as based on confocal microscopy scans of the gel xy-plane or the myocyte long axis. In addition, microrods caused significant increases in the proportion of aligned myofibrils (?20° deviation from long axis) in fixed myocytes. Microrod-related differences in axial contraction could be abrogated by long-term interruption of certain signals of the RhoA-/Rho-associated kinase (ROCK) or protein kinase C (PKC) pathway. Furthermore, microrod-induced increases in myocyte size and protein content were prevented by ROCK inhibition. In all, the data suggest that microdomain heterogeneity in 3D appears to promote the development of axially aligned contractile machinery in muscle cells, an observation that may have relevance to a number of cardiac tissue engineering interventions.

SUBMITTER: Curtis MW

PROVIDER: S-EPMC3407350 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Microdomain heterogeneity in 3D affects the mechanics of neonatal cardiac myocyte contraction.

Curtis Matthew W MW Budyn Elisa E Desai Tejal A TA Samarel Allen M AM Russell Brenda B

Biomechanics and modeling in mechanobiology 20120311 1

Cardiac muscle cells are known to adapt to their physical surroundings, optimizing intracellular organization and contractile function for a given culture environment. A previously developed in vitro model system has shown that the inclusion of discrete microscale domains (or microrods) in three dimensions (3D) can alter long-term growth responses of neonatal ventricular myocytes. The aim of this work was to understand how cellular contact with such a domain affects various mechanical changes in ...[more]

PMID: 22407215

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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:A five-state model of myofilament contraction was integrated into a well-established rabbit ventricular myocyte model of ion channels, Ca(2+) transporters and kinase signaling to analyze the relative contribution of different phosphorylation targets to the overall mechanical response driven by ?-adrenergic stimulation (?-AS). ?-AS effect on sarcoplasmic reticulum Ca(2+) handling, Ca(2+), K(+) and Cl(-) currents, and Na(+)/K(+)-ATPase properties was included based on experimental data. The inotropic effect on the myofilaments was represented as reduced myofilament Ca(2+) sensitivity (XBCa) and titin stiffness, and increased cross-bridge (XB) cycling rate (XBcy). Assuming independent roles of XBCa and XBcy, the model reproduced experimental ?-AS responses on action potentials and Ca(2+) transient amplitude and kinetics. It also replicated the behavior of force-Ca(2+), release-restretch, length-step, stiffness-frequency and force-velocity relationships, and increased force and shortening in isometric and isotonic twitch contractions. The ?-AS effect was then switched off from individual targets to analyze their relative impact on contractility. Preventing ?-AS effects on L-type Ca(2+) channels or phospholamban limited Ca(2+) transients and contractile responses in parallel, while blocking phospholemman and K(+) channel (IKs) effects enhanced Ca(2+) and inotropy. Removal of ?-AS effects from XBCa enhanced contractile force while decreasing peak Ca(2+) (due to greater Ca(2+) buffering), but had less effect on shortening. Conversely, preventing ?-AS effects on XBcy preserved Ca(2+) transient effects, but blunted inotropy (both isometric force and especially shortening). Removal of titin effects had little impact on contraction. Finally, exclusion of ?-AS from XBCa and XBcy while preserving effects on other targets resulted in preserved peak isometric force response (with slower kinetics) but nearly abolished enhanced shortening. ?-AS effects on XBCa and XBcy have greater impact on isometric and isotonic contraction, respectively.

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Microdomain heterogeneity in 3D affects the mechanics of neonatal cardiac myocyte contraction.

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Microdomain heterogeneity in 3D affects the mechanics of neonatal cardiac myocyte contraction.

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