Sarcomere integrated biosensor detects myofilament-activating ligands in real time during twitch contractions in live cardiac muscle
Graphical abstract
Introduction
The cardiac sarcomere is a multimeric contractile apparatus consisting of a thin myofilament-based allosteric regulatory complex together with the myosin-based thick myofilament that generates force [1]. Interlacing myofilaments operate in synchrony to regulate and generate the forces necessary for heart performance. Beat-to-beat control of cardiac sarcomere activation refers to the status of the thin filament regulatory system in controlling the degree to which contraction is turned on and off during a twitch. Sarcomere activation control mechanisms involve a dynamic interplay between thin filament-localized Ca2+ in concert with regulatory elements in the thick and thin myofilaments, including troponin, tropomyosin, and myosin [1,2]. Sarcomere activation can be dramatically altered by factors intrinsic to myofilaments, including posttranslational modifications, the environmental milieu (ischemia/hypoxia), and by inherited mutations in sarcomeric proteins [[3], [4], [5]]. Despite great efforts, it has not been possible to directly investigate and monitor sarcomere activation in the physiological setting of live cardiac muscle during a twitch.
The thin filament regulatory system, which is at the center of sarcomere activation dynamics and its beat-to-beat regulation during heart contraction, is composed of actin, tropomyosin (Tm), and the troponin complex (Tn) [1,6]. Regulation of sarcomere activation refers to the processes that control Tm's position on the thin filament [1,2]. Cardiac troponin is a heterotrimeric complex comprised of the Tm binding subunit, troponin T (cTnT), the calcium binding subunit, troponin C (cTnC), and the inhibitory subunit, troponin I (cTnI). Cardiac TnI (cTnI) is the molecular switch of the thin filament regulatory system [6,7]. Prior to contraction, the C-terminus of cTnI is strongly bound to actin, inhibiting Tm from rotating to a position favorable for strong myosin binding to actin. Upon Ca2+ binding to cTnC, cTnI rapidly switches from strong binding to actin to strong binding to cTnC. This cTnI switch mechanism is essential for opening the TnC hydrophobic patch, and then permitting Tm to translocate on the thin filament for active contraction to commence [2,8,9].
The prevailing model of sarcomere activation posits that Ca2+, cTnI switching [9], and strong myosin binding serve as activating ligands to regulate Tm’s position on actin from an inhibiting to a disinhibiting position [1]. A wealth of biochemical studies indicate that Ca2+, cTnI, and myosin are the key effectors in orchestrating the sequential blocked, closed, and open state transitions in the myofilament regulatory system, as governed by their effects on Tm displacement [10,11]. A central feature of this model is that strong myosin binding is required to displace Tm to form the open state of thin filament regulatory apparatus in initiating contraction [1,11,12]. While highly informative, it is presently unknown whether this model holds in the dynamic physiological setting of live, intact muscle.
Well established approaches are in place to quantitatively detect and monitor the electrical and intracellular Ca2+ handling properties of live cardiac muscle [13]. However, to date, it has not been possible to investigate thin filament activation dynamics in real time under physiological conditions of intact excitation-contraction coupling and under load, all of which dramatically affect overall contractile performance [1]. To address this, we implement here a live cardiac muscle thin filament activation biosensor. The FRET-based biosensor was genetically engineered into full length human cTnC and incorporated into the myofilaments. The rationale is based on extensive in vitro works documenting that cTnC's global conformation changes upon Ca2+ activation in vitro. Data show that cTnC becomes more compact, with the N and C terminal lobes of TnC coming closer together, going from the apo to Ca2+ activated state [[14], [15], [16], [17]]. Our premise of engineering a TnC conformation-dependent FRET-based biosensor integrated into the sarcomere of intact cardiac muscle derives from these studies. We use this TnC probe with the aim to detect global TnC conformational changes during a single cardiac twitch contraction.
In addition, the premise that TnC's conformation provides a nexus point in responding to and integrating near and long-range thin filament activating ligands, including Ca2+ binding to TnC, cTnI binding to TnC, Tm and myosin, is predicated on extensive in vitro works, as has been shown during steady-state conditions [1,2,6,[18], [19], [20]]. In permeabilized cardiac muscle reconstituted with cTnC conformational state-sensitive fluorescent probes, it has been shown that myosin cross-bridge binding to actin can induce TnC conformational changes under steady-state conditions, even in the absence of Ca2+ [19]. This is evidence that myosin binding causes Tm displacement and that this can be detected by TnC conformational changes, at least under steady-state conditions [1,19]. More recently, in vitro demonstration of FRET-based probes on TnC to detect Ca2+-dependent TnC conformational changes have been established using the isolated troponin complex and in reconstituted permeabilized preparations under steady-state activating conditions, with recordings on the tens of seconds to minutes time-scale [17]. It is presently unknown whether these foundational steady-state results will translate to transient conditions in intact cardiac muscle in real time, which operates on the milliseconds time-scale. We tested here the hypothesis that TnC's conformation can serve as a real-time biosensor and report the effects of multiple thin filament activating ligands during a single cardiac twitch contraction in live muscle.
Section snippets
Animal work
The procedures used in this study were approved under guidelines of the University of Minnesota Committee on the Use and Care of Animals. Adult ventricular cardiac myocytes were isolated from adult female Sprague Dawley rats or adult C57/Bl6 mice of both sexes.
FRET pair, gene construct, recombinant vectors, and transgenesis
An intramolecular Clover/mRuby2 FRET pair was engineered into cardiac troponin C (details below). The Clover and mRuby2 FRET pair was used because, at the time of study design, this FRET pair was superior to any previous FRET pair in
Real-time detection of TnC-based thin filament activation in intact adult cardiac myocytes
As detailed below, the troponin C FRET-based biosensor probe was integrated into the sarcomere and shown to function normally in cardiac muscle, in terms of myofilament activation and contractile function from cell to whole animal. Data support that the well-known activation-dependent conformational changes in TnC (compacted global TnC structure during activation [15,17,36]) bring the Clover and mRuby2 FRET pair closer together (in the vicinity of the R0 of 6.3 nm) to increase FRET and produce
Discussion
Elucidating the physiological basis of thin filament activation in live cardiac muscle is essential to understanding beat-to-beat regulation of heart performance in vivo. We sought here to design and test a sarcomere-incorporated biosensor capable of detecting thin myofilament activation status in live cardiac muscle. Toward this goal, we implemented a cardiac TnC FRET-based biosensor to test the hypothesis that TnC's conformation can serve as a real-time biosensor and report the effects of
Author contributions
ADV and JMM developed the initial study design and approach. ADV, AAM and JMM performed the experiments. ADV, BRT, AAM, DDT and JMM analyzed and interpreted the data. All authors contributed to writing the manuscript.
Disclosures
None.
Acknowledgements
We thank Drs. J.M. Muretta and M.A. Sanders for helpful discussions and K.W. Prins and H. Cohen for assistance. We thank A. Málnási-Csizmadia for the gift of para-nitroblebbistatin. This work was supported by grants from NIH (HL132874; HL122323), AHA (16PRE30480002) and UMN LHI and IBP.
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