# Load-dependence of the activation of myosin filaments in heart muscle

Yanhong Wang, Luca Fusi, Jesus G. Ovejero, Cameron Hill, Samina Juma, Flair Paradine Cullup, Andrea Ghisleni, So-Jin Park-Holohan, Weikang Ma, Thomas Irving, et al.

► To cite this version:

Yanhong Wang, Luca Fusi, Jesus G. Ovejero, Cameron Hill, Samina Juma, et al. Load-dependence of the activation of myosin filaments in heart muscle. The Journal of Physiology, 2024, 602 (24), pp.6889–6907. 10.1113/JP287434. hal-05128576

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# Load-dependence of the activation of myosin filaments in heart muscle

Yanhong Wang<fnref n="1" />, Luca Fusi<fnref n="1" />,<fnref n="2" /> ID, Jesus G. Ovejero<fnref n="1" />, Cameron Hill<fnref n="1" /> ID, Samina Juma<fnref n="1" />, Flair Paradine Cullup<fnref n="1" /> ID, Andrea Ghisleni<fnref n="1" /> ID, So-Jin Park-Holohan<fnref n="1" />, Weikang Ma<fnref n="3" /> ID, Thomas Irving<fnref n="3" /> ID, Theyencheri Narayanan<fnref n="4" />, Malcolm Irving<fnref n="1" /> ID and Elisabetta Brunello<fnref n="1" /> ID

### Handling Editors: Bjorn Knollmann & Jose Pinto

The peer review history is available in the Supporting Information section of this article (https://doi.org/10.1113/JP287434#support-information-section).

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The Journal of
Physiology

**Abstract figure legend** Load-dependent activation of cardiac myosin filaments. We used synchrotron, time-resolved, small-angle X-ray diffraction to investigate structural dynamics in the myosin-containing thick filaments in beating trabeculae isolated from the rat heart. In the resting phase between beats (diastole), most of the myosin motors are folded back onto their tails and helically ordered in an OFF structure that inhibits contraction (light grey), whereas only few motors are disordered (dark grey). During high-load activation in the absence of filament sliding (blue), myosin motors leave the helical array, bind to actin and generate force (dark blue), or become disordered (turquoise). The OFF structure is maintained during low-load activation (magenta), when only few motors promote filament sliding (purple). These results show that early activation of thick filaments in the heart is controlled by the load, whereas a slower load independent mechanism appears at later times during the heartbeat.

<footnote n="1">¹Randall Centre for Cell and Molecular Biophysics and British Heart Foundation Centre of Research Excellence, King's College London, London, UK</footnote>

<footnote n="2">²Centre for Human and Applied Physiological Sciences, King's College London, London, UK</footnote>

<footnote n="3">³BioCAT, Dept of Biology, Illinois Institute of Technology, Chicago, IL, USA</footnote>

<footnote n="4">⁴ESRF – The European Synchrotron, Grenoble, France</footnote>
**Abstract** Contraction of heart muscle requires activation of both the actin and myosin filaments. The mechanism of myosin filament activation is unknown, but the leading candidate hypothesis is direct mechano-sensing by the filaments. Here, we tested this hypothesis by activating intact trabeculae from rat heart by electrical stimulation under different loads and measuring myosin filament activation by X-ray diffraction. Unexpectedly, we found that the distinct structural changes in the myosin filament associated with activation had different dependences on the load. In early activation, all the structural changes indicated faster activation at higher load, as expected from the mechano-sensing hypothesis, but, at later times, the helical order of the myosin motors characteristic of the inactivated state was lost even at very low load. We conclude that mechano-sensing does operate in heart muscle, but it is supplemented by a previously undescribed mechanism that links myosin filament activation to actin filament activation.

(Received 5 August 2024; accepted after revision 3 October 2024; first published online 14 November 2024)

**Corresponding author** E. Brunello: Randall Centre for Cell and Molecular Biophysics, King's College London, Guy's Campus, New Hunt's House, office 3.26C, SE1 1UL London, UK. Email: elisabetta.brunello@kcl.ac.uk

**Key points**

* Myosin filament activation controls the strength and speed of contraction in heart muscle.

* Early activation of the myosin filament is determined by the filament load.

* At later times, myosin filament activation is controlled by a load independent pathway.

* This load independent pathway provides new targets and assays for drug development.

**Introduction**

Contraction of heart muscle is driven by myosin motors from the thick filaments interacting with actin sites in the thin filaments, triggered by a transient increase in intracellular calcium concentration in each heartbeat. Calcium switches on the thin filaments by removing the block on myosin–actin interaction mediated by the regulatory proteins tropomyosin and troponin (Huxley, 1973) and the mechanisms underlying thin filament regulation have been described in molecular detail (Risi et al., 2021; Takeda et al., 2003; Yamada et al., 2020). The thick filaments also play a role in the regulation of contraction (Brunello & Fusi, 2024; Craig & Padron, 2022; Irving, 2017). X-ray studies of resting skeletal muscle and heart muscle in diastole showed that the myosin motors are organised into helical tracks on the surface of the thick filaments in an OFF state that inhibits their interaction with actin (Ait-Mou et al., 2016; Brunello et al., 2020;

Huxley & Brown, 1967; Irving, 2017; Reconditi et al., 2011). Electron microscopy of isolated thick filaments and myofibrils (Al-Khayat et al., 2013; Chen et al., 2024; Craig & Padron, 2022; Dutta et al., 2023; Tamborrini et al., 2023; Wendt et al., 2001; Woodhead et al., 2005; Zoghbi et al., 2008) showed that myosin motors are folded back onto their tails in this OFF state, which is stabilised by multiple interactions between the motors, the myosin tails and two other thick filament components: titin and myosin-binding protein C (MyBP-C). This OFF structure has been associated with a very slow component of ATP turnover in single turnover ATPase assays using fluorescent ATP analogues (Hooijman et al., 2011; Stewart et al., 2010) and is often referred to as the ‘super-relaxed’ state or SRX, although it has recently become clear that these assays do not reliably quantify the structural OFF state (Mohran et al., 2024).

The thick filament OFF state appears to augment the inhibition of the thin filament by troponin and

**Yanhong Wang** received her PhD from Queen Mary University of London, investigating the multiscale deformation in arthropod exoskeleton using *in situ* X-ray imaging. In 2020, she joined Brunello's lab at the Randall Centre for Cell and Molecular Biophysics at King's College London as a postdoctoral research associate, where she studied myosin filament-based regulation of contraction in heart muscle using a combination of sarcomere mechanics and small-angle X-ray diffraction. She is now studying joint biomechanics in osteoarthritis at the University of Liverpool. Her research interests include muscle contraction, X-ray imaging and biomechanics in the life health sector.

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tropomyosin to minimise ATP utilisation by myosin and its associated metabolic cost when motors are not needed. According to this view, there should be physiolog- logical mechanisms that release myosin motors from the OFF state when they are needed. One such mechanism is mechanical. In skeletal muscle, motors remain OFF during rapid shortening against a low load (Linari et al., 2015), when almost no mechanical work is done and very few active motors are required; the motors are released from the OFF state when the load is high and more motors are needed. Thick filaments from skeletal muscle are intrinsically mechano-sensitive; they are switched ON in response to filament stress (Brunello et al., 2023; Craig & Padron, 2022; Fusi et al., 2016; Hill et al., 2022; Irving, 2017). This mechano-sensing hypothesis of thick filament activation implies an intrinsic positive feed- back loop in which increased load leads to increased filament activation, leading to increased load, and so on. This positive feedback loop would facilitate the rapid, switch-like activation of skeletal muscle. On slower timescales, the activation state of thick filaments in skeletal muscle may also be modulated by phosphorylation of the regulatory light chain of myosin (Kamm & Stull, 2011).

Heart muscle has a wider range of mechanisms to control the activation state of the myosin motors, reflecting the multiple factors that modulate contractility in the heart, including beta-adrenergic stimulation, heart rate, aortic pressure and venous filling pressure or diastolic sarcomere length. These factors may act by changing the regulatory state of both the thin and thick filaments, but the thick filament-based mechanisms appear to play a central role in the performance of the healthy heart and its disruption in inherited heart disease (Kawana et al., 2022; Lehman et al., 2022). As a result, myosin-based regulation is increasingly being targeted for therapeutic intervention in heart disease (Lewis et al., 2022; Nag et al., 2023; Voors et al., 2020). However, the molecular mechanisms that control the activation state of myosin motors and filaments in the heart remain poorly understood. Thick filament mechano-sensing plays a role (Brunello et al., 2020; Park-Holohan et al., 2021; Piazzesi et al., 2018; Reconditi et al., 2017), but the graded response of heart muscle cells in contrast with the switch-like activation of skeletal muscle suggests that additional mechanisms are involved. Multiple pathways control contractility in the heart and it is improbable that these are all mediated by thick filament mechano-sensing. For example, the effect of venous filling pressure or diastolic sarcomere length, underlying the Frank–Starling law of the heart, is known to be mediated at the filament level in a phenomenon called length-dependent activation (Kentish et al., 1986), but the underlying mechanisms are unknown.

Here, we determined the contribution of the
mechano-sensing mechanism to the control of thick

filament activation in the heart. We used millisecond
time-resolved synchrotron small-angle X-ray diffraction
to monitor thick filament structure and myosin motor
conformation in intact trabeculae from rat heart following
electrical stimulation in three protocols. In one protocol
(fixed-end), we allowed active force to develop at constant
trabecular length. In a second (low-load clamp), we used
feedback control of trabecular length to clamp the load
to a very small fraction of the active force produced in a
fixed-end contraction. In the third (sarcomere-isometric),
we held the sarcomere length at the pre-stimulus value,
which increased the peak force compared to that in a
fixed-end contraction. We found that some of the X-ray
signals associated with thick filament activation had
a strong load-dependence, and that the OFF signals
were maintained during low-load activation as expected
from the mechano-sensing mechanism. Unexpectedly,
however, the intensity of the first myosin-based layer
line, which signals the helical organisation of the myosin
motors on the surface of the thick filament in the OFF
state, followed the expectations of the mechano-sensing
hypothesis for only the first 50 ms of low-load activation,
and later became load independent. We conclude that
mechano-sensing is essential for the rapid load-dependent
activation of thick filaments in the heart, but the presence
of an additional slower mechanism is revealed at low
load, which may involve direct sensing of intracellular
calcium (Ma et al., 2022) or of the activation state of the
thin filaments (Brunello et al., 2023; Ponnam et al., 2019).

Methods

Preparation of cardiac trabeculae and experimental
protocol

Rattus norvegicus, strain Wistar Han (male, aged
6-8 weeks) were supplied by Charles River Laboratories
(Wilmington, MA, USA) and hosted at the animal house
of the Bio-Medical Facility of the European Synchrotron
Radiation Facility (ESRF, Grenoble, France) or the Illinois
Institute of Technology rodent housing facility (Chicago,
IL, USA) under a 12:12 h light/dark photocycle at 20°C
and 50% relative humidity. The animals had access to
food and water ad libitum. All animals were housed
and maintained in accordance with the ARRIVE 2.0
guidelines (Percie du Sert et al., 2020). Cardiac trabeculae
were prepared as previously reported (Brunello et al.,
2020). Briefly, rats were killed via cervical dislocation
after sedation with isoflurane, followed by a confirmation
method, in compliance with the UK Home Office
Schedule 1 and European Union regulation (directive
2010/63); the protocols were approved by the Illinois
Institute of Technology Institutional Animal Care and
Use Committees. Following death, the heart was rapidly
excised and cannulated via the ascending aorta and
retrogradely perfused with a modified Krebs–Henseleit buffer (119 mM NaCl, 5 mM KCl, 0.5 mM CaCl₂, 1.2 mM NaH₂PO₄, 1.2 mM MgSO₄, 25 mM NaHCO₃, 10 mM glucose, 25 mM 2,3-butanedione monoxime) equilibrated with carbogen (95% O₂, 5% CO₂) at room temperature. Single unbranched trabeculae were carefully dissected from the right ventricle under a stereomicroscope in a trough continuously perfused with the same buffer. Scorpion-like clips made of stainless-steel wire were tied at the valve and ventricular wall ends, and the trabecula was mounted between the levers of a strain gauge force transducer and a motor (322C; Aurora Scientific, Aurora, ON, Canada) in a trough filled with the same buffer. Trabecular length (L₀) and cross-sectional area were measured after stretching the trabecula to recover slack length. The trabecula was then continuously perfused with the buffer containing 1.4 mM CaCl₂ equilibrated with carbogen at 26.8 ± 0.4°C and no 2,3-butanedione monoxime (mean ± SD, n = 17 trabeculae). Electrical stimuli were provided via two parallel platinum electrodes attached above mica windows placed as close as possible to the trabecula to minimise the X-ray path in solution, and the trabecula electrically stimulated at 1 Hz for at least 30 min before the start of the experiment. Sarcomere length (SL) was measured at multiple points along the trabecula using the first order of the sarcomere reflections (see Appendix, Fig. A1) at ID02 (ESRF) at 31 m sample-to-detector distance and the average SL in diastole was set to 1.95 ± 0.03 µm (mean ± SD, n = 13 trabeculae).

Once per minute, a 10% L₀ ramp stretch complete in 5 ms was applied in diastole 60 ms before the stimulus to increase SL in one contraction cycle without altering the calcium transient as previously described (Brunello et al., 2020). The trabecular length was then either kept fixed: fixed-end protocol (Brunello et al., 2020) or the load was clamped to the value attained at 20 ms after the stimulus, resulting in shortening of the sarcomeres and trabecular length to maintain the load constant on the myofilaments: low-load clamp protocol. The low-load clamp was attained through an adaptive control method (Peterson et al., 1989). In brief, the deviation of the measured force signal from the desired value was used to modify the command to the motor to be used on the next twitch. In each trabecula, three to six iteration cycles were needed to optimise the load clamp. In another set of experiments, after the 10% L₀ ramp stretch, the sarcomere length was clamped to the value attained after the stretch. The sarcomere-isometric contraction was attained through an adaptive method (Peterson et al., 1989) by imposing a trabecular length change calculated from sarcomere shortening in fixed-end conditions. In each trabecula, two to four iteration cycles were needed to optimise the sarcomere length clamp.

## X-ray data collection

The trough was sealed with silicon grease to prevent solution leakage, and the trabeculae mounted vertical at the beamline to maximise spatial resolution on the meridional axis of the X-ray pattern (parallel to the trabecular axis).

For the fixed-end (Brunello et al., 2020) and sarcomere-isometric protocol, data were collected at beamline ID02 of the ESRF, which provided up to 2 × 10¹³ photons s⁻¹ at 0.1 nm wavelength in a beam of size 300 µm (horizontal, H, full width at half-maximum) and 70 µm (vertical, V) at the sample position; X-ray data were collected using a Pilatus 300K detector (Dectris, Baden-Daettwil, Switzerland), composed of three modules with 17-pixel gaps splitting the meridional axis into three parts, total active area of 83.8 × 106.5 mm, with 487 × 619 pixels, pixel size 172 × 172 µm (Narayanan et al., 2018).

For the low-load clamp protocol, data were collected at ID02 of the ESRF, which provided up to ~2 × 10¹³ photons s⁻¹ at 0.1 nm wavelength in a beam of size 140 × 55 µm (H × V) at the sample position, on a Eiger2 4M detector (Dectris), composed of eight modules with 38 pixel gaps splitting the meridional axis into four parts and 12 pixel gaps splitting the equatorial axis into two parts, total active area 155.1 × 162.15 mm², with 2068 × 2162 pixels, pixel size 75 × 75 µm (Narayanan et al., 2022), and at the BioCAT beamline 18ID at the APS (Fischetti et al., 2004), which provided up to ~10¹³ photons s⁻¹ at 0.1 nm wavelength in a beam of size 150 × 25 µm (HxV), on a Pilatus 3-1M detector (Dectris), composed of 10 modules with 17 pixel gaps splitting the meridional axis into five parts and 7 pixel gaps splitting the equatorial axis into two parts, total active area 168.7 × 179.4 mm², with 981 × 1043 pixels, pixel size 172 × 172 µm.

X-ray reflections from the sarcomere repeats were measured at ID02 with a 31 m sample-to-detector distance (see Appendix, Fig. A1). Meridional myosin-based reflections up to the M6 were recorded with 3.2 m and 5.1 m sample-to-detector distances at ID02 (see Appendix, Fig. A2) and BioCAT, respectively, and detector H-V shift was optimised to place the reflections of interest within the modules, avoiding the gaps. Data were collected from 10–39 contractions in each trabecula, during which the peak systolic force decreased by less than 10%, and no sign of radiation damage was detected in the X-ray diffraction pattern. X-ray exposure was controlled using a fast shutter and monitored with a pin diode, and the trabecula was moved vertically by 100–200 µm between contractions to minimise radiation damage. In each activation, 37 or 38 consecutive time frames were recorded from the same region of the trabecula at 20 ms intervals; with Pilatus detectors data were collected for 17 ms followed
by 3 ms dead time in each frame, while with the Eiger
detector data were collected for 19.8 ms followed by
0.2 ms dead time in each frame. The main advantage
of this protocol is that every time point comes from the
same set of trabecular regions; its main limitation is that
each point on the trabeculae is exposed for 740 or 760 ms,
which would produce detectable radiation damage if
the full beam intensity were used. The beam intensity
was therefore attenuated to ~3% of the maximum value
mentioned above.

For fixed-end, signal-to-noise was increased by
signal-averaging 12–30 contractions per trabecula
from six trabeculae, with length (L₀) 2.5 ± 0.5 mm
(mean ± SD), cross-sectional area 59,000 ± 26,000
µm² and peak force 50.4 ± 10.0 kPa; for low load,
data were signal-averaged from 10–39 contractions per
trabecula from seven trabeculae, with L₀ 2.2 ± 0.4 mm,
cross-sectional area 44,364 ± 20,721 µm², force during
the clamp 6.06 ± 1.82 kPa; for sarcomere-isometric,
data were signal-averaged from 16–25 contractions per
trabecula from four trabeculae, with L₀ 2.9 ± 0.7 mm,
cross-sectional area 44,690 ± 12,700 µm², peak force 75.7
± 17.4 kPa.

X-ray data analysis

Small-angle X-ray diffraction patterns were analysed
using SAXSutilities (M. Sztucki; ESRF, Grenoble,
France), SAXS packages (P. Boesecke; ESRF), Fit2D
(A. Hammersley; ESRF) and IgorPro (WaveMetrics,
Portland, OR, USA).

**Analysis of the ultra-small angle X-ray diffraction patterns.** Two-dimensional (2-D) X-ray diffraction patterns were collected at ID02 (ESRF) with a 31 m sample-to-detector distance from two to four positions along the central region of each trabecula. The camera background was subtracted, and meridional intensity distributions from the sarcomere reflections were obtained by integrating from 0.40 µm⁻¹ on either side of the meridional axis (parallel to the trabecular axis). Residual background intensity distributions were fitted using a convex hull algorithm and subtracted. The intensity and spacing of the sarcomere reflections were determined by fitting Gaussian peaks in the axial region 0.30–0.76 µm⁻¹ or 0.74–1.24 µm⁻¹ for the first or the second order reflections, respectively.

**Analysis of the small-angle X-ray diffraction patterns.** For each trabecula, the time-resolved 2-D patterns from all contractions were added, centred and aligned using the equatorial (1,0) reflections, and then mirrored horizontally and vertically. For the low-load clamp data, 2-D patterns were only mirrored horizontally for the

ML1 layer line reflection, because on the top/bottom of
the beam stop one of the detector gaps was close to
these reflections. The equatorial intensity distribution was
determined by integrating from 0.0045 nm⁻¹ on either
side of the equatorial axis (perpendicular to the trabecular
axis), the background was subtracted using a convex hull
algorithm, and the intensities and spacings of the (1,0) and
(1,1) reflections were determined by fitting two Gaussian
peaks in the region 0.019–0.064 nm⁻¹ with the constraint
of $d_{11} = d_{10}/(3)^{1/2}$.

For the analysis of the meridional and layer line
reflections, the intensity distributions for each trabecula
were normalised by the intensity of the (1,0) reflection
in diastole at sarcomere length 1.95 µm to remove
the differences in the diffracting mass in the X-ray
beam followed by a 1:2:1 smoothing of the 20 ms time
frames. For the lower signal-to-noise M6 reflection the
normalised intensity distributions for each trabecula were
averaged from multiple trabeculae (fixed-end, *n* = 6;
low-load, *n* = 7; sarcomere-isometric, *n* = 4). The
intensity distributions for the meridional reflections were
determined by integrating from 0.0045 nm⁻¹ on either
side of the meridian (parallel to the trabecular axis)
for the M2 and M3 reflections, and 0.009 nm⁻¹ on
either side of the meridian for the M6 reflection. The
integrated intensities and the spacings were calculated in
the following axial regions: 0.040–0.045 nm⁻¹ for M2L,
0.066–0.072 nm⁻¹ for M3 and 0.134–0.140 nm⁻¹ for
M6. The cross-meridional width of the M3 reflection
was determined from the radial intensity distributions in
the axial regions defined above using a double-Gaussian
centred on the meridian, with the narrower component
considered to be the reflection of interest and the wider
one to be background. The interference components of
the meridional reflections were determined by fitting
multiple Gaussian peaks with the same axial width to
the meridional intensity distributions. The total intensities
of the reflections were calculated as the sum of the
intensity of the component peaks, and the spacing as the
weighted average of the component peaks. The intensity
of the M3 reflection was corrected by multiplying by
the cross-meridional width. The spacings were calibrated
using $S_{M3} = 14.479$ nm at $SL = 1.95$ µm (Brunello et al.,
2020).

The intensity distribution of the first myosin layer line
(ML1) was determined by integrating in the radial region
between 0.036 and 0.063 nm⁻¹ from the meridional axis,
and the ML1 intensity ($I_{ML1}$) was calculated from the axial
region 0.017–0.024 nm⁻¹ to exclude the contribution of
the first actin layer line.

Background intensity distributions for all the 1D
profiles were fitted using a convex hull algorithm and sub-
tracted. The intensities of all the X-ray reflections were
corrected for movement of the trabeculae with respect to
the X-ray beam by dividing by the background intensity
in the region of the ML1/AL1.

The volume of the unit cell (V) was calculated as
V = 2/(3)1/2 × (d10)2 × SL, where SL is the sarcomere
length (only available for experiments performed at ID02,
ESRF). V was 0.0032 ± 0.0001 µm³ at SL = 2.17 ± 0.03
µm in diastole (mean ± SD, N = 13 trabeculae). Force,
stimulus, trabecular length change and X-ray acquisition
timing were collected and analysed using custom-made
software written in LabVIEW (National Instruments,
Austin, TX, USA).

Interpretation of the M3 reflection subpeaks in terms
of myosin motor conformations

Myosin filaments from vertebrate cardiac muscle contain two anti-parallel arrays of myosin motors, each containing 49 layers of three dimers of myosin motors with an axial periodicity *d* of ~14.5 nm, corresponding to the spacing of the M3 reflection (*S*<sub>M3</sub>), and starting at ~80 nm (half-bare zone, *hbz*) from the filament midpoint (M line). Each half-filament can be divided into three zones: the proximal or P-zone (layers 1–7); the C-zone (layers 7–31), containing MyBP-C; and the distal or D-zone (layers 31 to 49) (Luther et al., 2008). This bipolar arrangement generates interference fringes that modulate the X-ray reflection generated by a single array of motors. The filament location of the myosin motors contributing to the M3 reflection can be determined from the resulting subpeaks (Brunello et al., 2020; Linari et al., 2000). For that analysis, the myosin filament was represented as a 1-D lattice of 49 point diffractors, with each diffractor representing the centre of mass of the myosin motors in that layer (Hill et al., 2021; Ovejero et al., 2022; Reconditi, 2006), and the closest diffractor to the M line (layer 1) in each half-filament separated from it by *hbz*. The axial profile of the M3 reflection was calculated as [sin(*N*π*Rd*)/sin(π*Rd*)]<sup>2</sup>cos<sup>2</sup>(π*RD*) (Reconditi, 2006), where *R* is the reciprocal space co-ordinate, *d* is the axial spacing of the layers and *N* is the number of contributing layers in a contiguous array in each half-filament from a proximal layer *n*<sub>m</sub> to a distal layer *n*<sub>d</sub>. Motors outside that array are isotropic and make no contribution to the reflection. The interference distance (*D*) between the centres of mass of the two diffracting arrays is given by 2*hbz*+(*n*<sub>m</sub>+*n*<sub>d</sub>−2)*d* (Hill et al., 2021; Ovejero et al., 2022). The intensity distribution calculated from this filament model was convoluted with a Gaussian function representing the combined resolution function, determined by the X-ray beam and detector pixel sizes, before fitting to the experimental data. The best fit for each time point was determined by a global search of *hbz*, *d*, *n*<sub>m</sub>, *n*<sub>d</sub>, by minimising chi-squared calculated using the experimental SD values from the difference between the experimental mean and the model output.

Statistical analysis

All statistical analyses were performed using Excel (Microsoft Corp., Redmoond, WA, USA). Data are presented as the mean ± SD. Two-tailed distribution unpaired *t* tests were used to determine whether the half-times of mechanical and structural parameters during force development differed significantly between the three protocols. Source data for each trabecula are reported in the Supporting information with statistical comparisons (paired or unpaired *t* test, as indicated) of the time-course of the X-ray signals within each protocol and between protocols.

Results

We used time-resolved small-angle X-ray diffraction (see Appendix, Fig. A1 and A2) to determine the effects of load on structural changes in the myosin motors and filaments during activation of trabeculae isolated from the right ventricle of rat heart. Trabeculae were stimulated continuously at 1 s intervals at 27°C. Once a steady-state had been established, the length of the trabeculae was increased by ~10% once per minute, 60 ms before the stimulus, and X-ray diffraction patterns were recorded with 20 ms time resolution during three types of contraction (Fig. 1). Previously, we reported molecular structural changes during fixed-end contractions (Brunello et al., 2020) (Fig. 1, black), in which force increases transiently to ~50 kPa, and sarcomere length in the central region of the trabeculae (Fig. 1C, black; see also Appendix, Fig. A1) decreases from ~2.2 to 1.9 µm at peak force, as a result of stretching weaker sarcomeres at the ends of the trabeculae. Here, we determined the effect of load on thick filament activation in two additional protocols. In the low-load clamp protocol, the trabecular load was held at a small fraction of that in fixed-end conditions using feedback control of trabecular length (Fig. 1A and 1B, magenta). The load clamp started 20 ms after the stimulus, when force including the passive component was ~6 kPa, and was held constant during activation by decreasing trabecular length by 15–20% (Fig. 1B, magenta), so that sarcomere length (Fig. 1C, magenta; see also Appendix, Fig. A1) shortened faster and further than in the fixed-end protocol. By 60 ms after the stimulus (vertical dashed-dotted line), sarcomere length had already reached 2.0 µm, so the initial passive force had been lost, and the ~6 kPa force maintained by the clamp was active force for the remainder of the load clamp period. The clamp ended 140 ms after the stimulus, at around the time that force peaks in fixed-end conditions (Fig. 1A, black), and force then relaxed with a half-time of only ~40 ms, much faster than in fixed-end conditions. By 70 ms after the end of the clamp, the trabeculae had become slack, and they moved out of the X-ray beam,
<figure><img src="image_4.png" /><figcaption>Figure 1. Force, trabecular length, sarcomere length, filament lattice spacing and volume in fixed-end, low load and sarcomere-isometric contractions of isolated rat cardiac trabeculae</figcaption></figure>

Force (A), trabecular length (ΔL, expressed as percentage of the initial length L₀) (B), sarcomere length (SL) (C), filament lattice spacing (d₁₀) (D) and volume of the unitary cell (ΔV, expressed as percentage change) (E) in fixed-end (black continuous lines and symbols), low-load clamp (magenta lines and symbols), and sarcomere-isometric (cyan lines and symbols). Trabeculae were stimulated electrically (vertical continuous line at t = 0 ms, St) continuously at 1 Hz, 27°C and stretched by 10% L₀ in 5 ms starting 60 ms before the stimulus. Time period between vertical dotted magenta lines, low-load clamp duration. Vertical dashed-dotted magenta line, t = 60 ms. Horizontal dashed lines in (C) to (E), values in diastole at long SL. Data for low-load after 210 ms not shown because trabeculae became slack and moved out of the X-ray beam. Data are the mean ± SD; fixed-end, six trabeculae; low-load, seven trabeculae (except in (C) and (E), three trabeculae); sarcomere-isometric, four trabeculae. [Colour figure can be viewed at wileyonlinelibrary.com]
<table><caption>Table 1. Half-times of mechanical and structural parameters during force development</caption><thead><tr><th rowspan="2"></th><th colspan="3">t<sub>1/2</sub> (ms)</th><th colspan="3">P values</th></tr><tr><th>Fixed-end</th><th>Low load</th><th>Sarcomere-isometric</th><th>FE-LLC</th><th>FE-SI</th><th>LLC-SI</th></tr></thead><tbody><tr><td>Force</td><td>66.7 ± 7.4 (6)</td><td>17.8 ± 2.0 (7)</td><td>51.9 ± 3.2 (4)</td><td>&lt;0.001</td><td>0.00327</td><td>&lt;0.001</td></tr><tr><td>SL</td><td>59.8 ± 6.8 (6)</td><td>60.2 ± 7.0 (3)</td><td>NA</td><td>0.943</td><td>NA</td><td>NA</td></tr><tr><td>I<sub>11</sub>/I<sub>10</sub></td><td>67.7 ± 5.8 (6)</td><td>33.7 ± 6.4 (7)</td><td>50.0 ± 7.3 (4)</td><td>&lt;0.001</td><td>0.00813</td><td>0.0110</td></tr><tr><td>S<sub>M6</sub>*</td><td>60.5 ± 14.4 (6)</td><td>NA</td><td>50.0 ± 26.1 (4)</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>I<sub>ML1</sub></td><td>48.1 ± 26.8 (6)</td><td>79.8 ± 25.6 (6)</td><td>24.6 ± 11.6 (4)</td><td>0.0626</td><td>0.0983</td><td>0.00212</td></tr><tr><td>I<sub>M2L</sub></td><td>62.3 ± 8.6 (6)</td><td>90.4 ± 16.2 (7)</td><td>60.8 ± 21.7 (4)</td><td>0.00296</td><td>0.905</td><td>0.0644</td></tr></tbody></table>

Half-times ($t_{1/2}$, with respect to the stimulus) to peak force were measured as the mean ± SD of the half-times for each trabecula calculated from the experimental data by sigmoidal fitting or by fitting a straight line to the points around the half-change. The number of contributing trabeculae is given in brackets (N). NA, not available. SL, sarcomere length. P values on the half-times were calculated with two-tailed distribution unpaired t tests comparing fixed-end and low-load clamp (FE-LLC), fixed-end and sarcomere-isometric (FE-SI) and low-load clamp and sarcomere-isometric (LLC-SI).

*Half-times for $S_{M6}$ were calculated from N added trabeculae, mean ± SD from fitting procedure.

so that it was no longer possible to record X-ray data, including sarcomere length. In the sarcomere-isometric protocol (Fig. 1A–C, cyan), the sarcomere length in the central region of the trabeculae was held roughly constant at the pre-stimulus value throughout the contraction by stretching the trabecula (Fig. 1B, cyan) (see Methods) using a waveform that mirrored the time course of the sarcomere length change measured in a fixed-end contraction (Fig. 1C, black). Force rise was faster in this protocol than in fixed-end conditions (Table 1) and peak force was greater.

We determined the movement of myosin motors towards the actin filaments in the three protocols using the relative intensities of the 1,0 and 1,1 equatorial X-ray reflections (see Appendix, Fig. A2) associated with the 1,0 and 1,1 planes of the hexagonal lattice of filaments. Before the stimulus, corresponding to the diastolic phase of the heartbeat, myosin motors are close to the thick filaments, and the intensity of the 1,0 reflection ($I_{10}$) is relatively high and that of the 1,1 reflection ($I_{11}$) low. When myosin motors bind to actin in systole, $I_{10}$ decreases and $I_{11}$ increases (see Appendix, Fig. A3). The ratio $I_{11}/I_{10}$ gives a reproducible measure of these changes, and is ~0.3 before the stimulus and ~0.9 at peak force in fixed-end contraction (Brunello et al., 2020) (Fig. 2A, black). In low-load clamp (magenta), the increase in $I_{11}/I_{10}$ was much smaller and briefer, indicating that myosin motors stay near the thick filaments, as expected from the mechano-sensing hypothesis if the thick filaments stay OFF at low load. The small increase in $I_{11}/I_{10}$ observed in this protocol in the first 60 ms after the stimulus (vertical dashed-dotted line) may largely be a result of sarcomere shortening rather than mass transfer of motors because $I_{11}/I_{10}$ is smaller at greater sarcomere length, as seen in response to the stretch before the stimulus (Fig. 2A, magenta). In the sarcomere-isometric

protocol (Fig. 2A, cyan), $I_{11}/I_{10}$ increased more quickly than in fixed-end conditions (Fig. 2A, black, and Table 1), and its recovery was slower, roughly following the time course of force (Fig. 2A, continuous line) in each condition (Table 1). The peak value of $I_{11}/I_{10}$ was slightly larger in sarcomere-isometric than in fixed-end conditions.

The spacing of the 1,0 reflection ($d_{10}$) (Fig. 1D) gives a precise and reproducible measure of the separation of the 1,0 planes of thick filaments in the hexagonal lattice and, because sarcomere length was measured in the same region of the trabeculae, the volume of the filament lattice (Fig. 1E) could be also determined. Lattice volume increased by ~4% during the diastolic stretch; the lateral compression of the lattice during the stretch is not sufficiently large to compensate for the increase in sarcomere length. Following electrical stimulation, this increase in lattice volume was reproducibly reversed if the load was held low (Fig. 1E, magenta), but when active force was allowed to develop, there was no change in lattice volume in sarcomere-isometric activation (Fig. 1E, cyan), as previously reported (Farman et al., 2007), and a small transient increase in fixed-end conditions (Fig. 1E, black). Maintenance of lattice volume during activation is correlated with high load, suggesting that the formation of actin-bound force-generating motors acts to maintain lattice volume.

Activation at high load is also associated with an increase in the axial periodicity of the thick filament, indicated by the spacing of the M6 reflection ($S_{M6}$) (Fig. 2C; see also Appendix, Fig. A2), with a time course similar to that of force development. As observed for $I_{11}/I_{10}$, the increase in $S_{M6}$ is faster in sarcomere-isometric (Fig. 2C, cyan) than fixed-end conditions (Fig. 2C, black, and Table 1), and the peak change is slightly greater. It is largely abolished when the load is kept
<figure><img src="image_5.png" /><figcaption>**Figure 2.** Structural dynamics of myosin motors and filaments in fixed-end, low load and sarcomere-isometric contractions</figcaption></figure>

Equatorial intensity ratio ($I_{11}/I_{10}$) (A), sarcomere length (SL) (B), spacing of M6 reflection ($S_{M6}$) (C), intensity of ML1 reflection ($I_{ML1}$) (D) and intensity of M2L reflection ($I_{M2L}$) (E). Force and sarcomere length change in (A) and (B) reproduced from Fig. 1A and C for reference. Fixed-end contraction, black circles and continuous lines; low load, magenta circles and lines; sarcomere-isometric, cyan circles and lines. St, stimulus. Time period between vertical dotted lines, low-load clamp duration. Vertical dashed-dotted magenta line, *t* = 60 ms. Horizontal dashed lines in (B) to (D), values in diastole at long SL. Data are the mean ± SD; fixed-end, six trabeculae (Brunello et al., 2020); low load, seven trabeculae [except in (B), three trabeculae]; sarcomere-isometric, four trabeculae; data in (C) are added from the same trabeculae. [Colour figure can be viewed at wileyonlinelibrary.com]
low during activation (Fig. 2C, magenta); the increase in the periodicity of the myosin filament associated with activation requires high load and filament stress, as in skeletal muscle (Linari et al., 2015), and as expected from the mechano-sensing hypothesis of thick filament activation.

The helical order of the myosin motors characteristic of the OFF state of the myosin filament, signalled by the intensity of the first myosin layer line ($I_{ML1}$) (see Appendix, Fig. A2), decreases to around one-quarter of its pre-stimulus value during fixed-end activation (Fig. 2D, black) (Brunello et al., 2020). The decrease was faster in sarcomere-isometric conditions (Fig. 2D, cyan, and Table 1), but significantly slower during low-load activation (Fig. 2D, magenta, and Table 1), in which there is an initial lag of ~50 ms. This lag cannot be associated with sarcomere shortening because sarcomere length at this time is the same in the low-load clamp and fixed-end protocols. The strong load-dependence of $I_{ML1}$ in the first 50 ms following stimulation is expected from the mechano-sensing hypothesis, but the weaker load-dependence at later times requires a different explanation (see Discussion). Broadly similar results were obtained for the intensity of the lower angle (L) component of the M2 reflection ($I_{M2L}$) (Fig. 2E), which has been associated with the perturbation of the myosin helix arising from the different motor conformations in the three crowns in each 43-nm helical repeat (Ait-Mou et al., 2016; Dutta et al., 2023; Ovejero et al., 2022; Tamborrini et al., 2023). The M2L reflection indexes on a significantly longer periodicity, ~46 nm, and its structural basis is obscure, but its intensity clearly decreases more slowly in low-load (Fig. 2E, magenta) than in fixed-end (black) or sarcomere-isometric (Fig. 2E, cyan) conditions (Table 1).

The M3 X-ray reflection (see Appendix, Fig. A2) is associated with the axial periodicity of the motors along the filaments. Its intensity ($I_{M3}$) decreases at the start of fixed-end activation (Fig. 3B, black) (Brunello et al., 2020) as helically ordered OFF motors are lost, then increases during the later phase of force development as actin-bound force-generating motors are formed (Brunello et al., 2020; Reconditi et al., 2011; Reconditi et al., 2017). This biphasic $I_{M3}$ response is more marked in sarcomere-isometric conditions (Fig. 3B, cyan), but both the decreasing and increasing phases are smaller during low-load activation (Fig. 3B, magenta). OFF motors are lost much faster at higher load in the first 50 ms after the stimulus, consistent with the strong load-dependence of $I_{ML1}$ in that period reported above, and with the hypothesis that thick-filament mechano-sensing controls the kinetics of motor recruitment.

The spacing of the M3 reflection ($S_{M3}$) also shows a transient decrease followed by a rising phase during fixed-end activation (Fig. 3C, black), but the decreasing phase ends earlier in sarcomere-isometric conditions (Fig. 3C, cyan) and is replaced by a much larger increase, presumably associated with the greater number of force-generating motors. Only the decreasing phase is observed during low-load activation (magenta), when there are very few force-generating motors. The origin of the decrease in $S_{M3}$ at the start of stimulation is unknown, although it has also been observed in skeletal muscle (Hill et al., 2022).

In general, the M3 reflection can be resolved into three subpeaks, referred to as low-angle (*la*), mid-angle (*ma*) and high-angle (*ha*), which are produced by X-ray interference between the myosin motors in the two halves of each thick filament (Linari et al., 2000) (Fig. 3A). The fractional intensity of the *la* peak ($L_{M3}$) provides a sensitive measure of the axial displacement of the average centre of mass of the myosin motors with respect to their point of attachment to the filament backbone (Linari et al., 2000; Reconditi et al., 2011). $L_{M3}$ is close to zero in diastole (Fig. 3E, inverted triangles) but increases to about 0.3 or 0.4 during fixed-end or sarcomere-isometric activation, respectively (Fig. 3E, black, cyan), signalling the loss of folded OFF motors and the formation of more perpendicular, force-generating motors (Brunello et al., 2020; Reconditi et al., 2017). This increase was completely abolished during low-load activation (Fig. 3E, magenta inverted triangles), suggesting that the motors that leave the folded conformation on activation become disordered and no longer contribute to the M3 reflection, which is predominantly a result of the remaining folded motors.

The interference profile of the M3 reflection (Fig. 3A; see also Appendix, Fig. A4) gives information about the location of the ordered motors in the thick filament. In diastole, the separation between the subpeaks of the M3 reflection is consistent with nearly all of the 49 layers of motors in each half of the thick filament being axially ordered (Brunello et al., 2020). We calculated the expected profiles of the M3 reflection (see Appendix, Fig. A4, dashed lines) using a simple model in which layers $n_m$ (medial) to $n_d$ (distal) of the 49 layers of motors in each half thick filament (Fig. 3F) are in a well-defined uniform conformation with a periodicity corresponding to $S_{M3}$ (Hill et al., 2021, 2022; Ovejero et al., 2022). The motors outside these regions are considered to be axially disordered, so that they make no contribution to the reflection. Meridional intensity distributions in the region of the M3 reflection calculated from the model gave good fits to the observed intensity distributions (see Appendix, Fig. A4).
<figure><img src="image_6.png" /><figcaption>Figure 3. Changes in the M3 reflection in fixed-end, low load, and sarcomere-isometric contractions</figcaption></figure>

A, meridional intensity distributions in the fixed-end (left), low load (middle) and sarcomere-isometric (right) protocols in diastole long SL (–20 ms, black) and peak force (150 ms, grey). Changes in M3 reflection intensity (*I*<sub>M3</sub>) (B) and spacing (*S*<sub>M3</sub>) (C), and the spacing (D) and fractional intensity (E) of its three component peaks. *I*<sub>M3</sub> was corrected by the cross-meridional width and normalised by the value in diastole at long SL (horizontal dashed line). Force change in (B) reproduced from Fig. 1A for reference. Black lines and symbols, fixed-end; magenta
lines and symbols, low-load clamp; cyan lines and symbols, sarcomere-isometric. Inverted triangles, low-angle (la) peak; squares, middle-angle (ma) peak; triangles, high-angle (ha) peak. *F*, diffracting layers of myosin motors and their location along the myosin half-filament. Inset: schematic of the half thick filament; horizontal dashed lines delimit the proximal P zone (layers 1–7), MyBP-C-containing C zone (layers 7–31) and distal D zone (layers 31–49). Horizontal dashed lines in (B) and (C), values in diastole at long *SL*. St, stimulus. Time period between vertical dotted lines, low-load clamp duration. Vertical dashed-dotted magenta line, *t* = 60 ms. Data are the mean ± SD; fixed-end, six trabeculae; low load, seven trabeculae; sarcomere-isometric, four trabeculae; data in (A) and (F) are added from the same trabeculae. [Colour figure can be viewed at wileyonlinelibrary.com]

During fixed-end activation, the best-fit value of *n*<sub>m</sub> progressively increased and that of *n*<sub>d</sub> decreased, so that by the time of peak force *n*<sub>m</sub> was ~7 and *n*<sub>d</sub> was 31 (Fig. 3F, grey). These layers correspond to the edges of the region of the half-filament containing myosin-binding protein C: the C-zone (Fig. 3F, right inset, purple). The axially ordered motors in fixed-end activation at systole are therefore confined to the C-zone, consistent with the conclusion of a previous model in which different functional classes of motors were assigned specific conformations (Brunello et al., 2020). In the first 50 ms of activation, *n*<sub>m</sub> increases faster in sarcomere-isometric (Fig. 3F, cyan) than fixed-end conditions (Fig. 3F, black), but more slowly in low-load clamp (magenta), following the strongly load-dependent kinetics signalled by *I*<sub>ML1</sub> and *I*<sub>M3</sub> in this period and described above. There is no evidence for the formation of force-generating motors in the P-zone of the thick filament in either fixed-end or sarcomere-isometric contractions. *n*<sub>d</sub>, in contrast, stays high in both sarcomere-isometric (Fig. 3F, cyan) and low-load (Fig. 3F, magenta) conditions, showing that D-zone motors stay largely ordered, although in the former case they are probably actin-attached and force-generating and in the latter OFF and folded.

Discussion

The aim of the experiments described above was to
determine how activation of the thick filaments in
heart muscle depends on filament load, and thereby to
test the mechano-sensing hypothesis of thick filament
activation in the heart. To that end, we compared
X-ray diffraction features associated with thick filament
activation following electrical stimulation of isolated
trabeculae from rat ventricle in three protocols. In one
(fixed-end), the length of the trabeculae was held constant
so that an active force of ~50 kPa was developed; in
the second (low-load clamp), the load was clamped at a
much lower value, ~6 kPa. There is substantial sarcomere
shortening in those two protocols, which was largely
eliminated in the third protocol (sarcomere-isometric),
in which a peak force of ~75 kPa was developed. The
intracellular calcium transient is expected to have the
same time course with a slightly larger peak amplitude in
contractions with shortening (Janssen & de Tombe, 1997),

so any differences in the level of thin filament activation
are expected to be small compared to the differences in
filament load between the three protocols. Depending on
which parameter is controlled, either filament load or
sarcomere length has a biphasic time-course following the
electrical stimulus, reflecting the transient activation of
the filaments and motors. The level of filament activation
is also sarcomere length-dependent, and sarcomeres
shorten rapidly at the start of the low-load and fixed-end
protocols. Moreover, positive feedback is intrinsic to the
mechano-sensing hypothesis. For all these reasons, a
rigorous test of the mechano-sensing hypothesis requires
time-resolved measurements of filament activation with
simultaneous measurements of sarcomere length in the
same preparation, as reported here.

The small-angle X-ray diffraction pattern from heart
muscle provides several different parameters that have
been used in previous studies as structural signals
for the activation state of the thick filaments. An
unexpected finding of the present study is that these
signals have distinct load- and time-dependences in the
three protocols, which raises a fundamental question:
what do we mean by the activation state of the thick
filament? A plausible functional definition might be the
fraction of myosin motors that are available for binding
to actin sites in the thin filament to drive force generation
or filament sliding. The X-ray signal most directly related
to that fraction is the decrease in the intensity of the
first myosin-based layer line (I<sub>ML1</sub>), which depends on the
helical arrangement of the myosin motors on the surface
of the thick filament in the OFF state. According to I<sub>ML1</sub>,
there is no significant exit of motors from the helical OFF
state in the first 50 ms after the stimulus in the low-load
protocol (Fig. 2D, magenta). This result is essentially the
same as that obtained in fast-twitch fibres of skeletal
muscle which led to the mechano-sensing hypothesis
(Linari et al., 2015); in that case, there was no significant
decrease in I<sub>ML1</sub> during the 40 ms period of unloaded
shortening imposed shortly after the start of electrical
stimulation. Based on the I<sub>ML1</sub> signal, we conclude that
the mechano-sensing mechanism also operates on this
rapid timescale in heart muscle. This conclusion is
reinforced by comparison with the large decrease in
I<sub>ML1</sub>, to ~35% of its pre-stimulus value, observed at the
same time in the sarcomere-isometric protocol (Fig. 2D,
cyan).
Moreover, the conclusion that the mechano-sensing mechanism is essential for rapid activation of heart muscle in the first 50 ms after the stimulus is supported by the other X-ray signals associated with thick filament activation measured at the same time in the low-load protocol, including *I*<sub>M2L</sub> (Fig. 2E), *I*<sub>11</sub>/*I*<sub>10</sub> (Fig. 2A), *S*<sub>M6</sub> (Fig. 2C) and *I*<sub>M3</sub> (Fig. 3B). Although it might be argued that each of these parameters exhibits a small change in the direction expected for thick filament activation at 50 ms after the stimulus, in each case, the observed change is close to that expected solely from the accompanying change in sarcomere length, as assessed in the response to a passive stretch before the stimulus. For example, the equatorial intensity ratio *I*<sub>11</sub>/*I*<sub>10</sub> (Fig. 2A) decreases from 0.31 ± 0.04 at a sarcomere length of ~2.0 µm before the passive stretch to 0.23 ± 0.04 at a sarcomere length of ~2.2 µm after it. In addition, 50 ms after the stimulus, sarcomere length has shortened back to ~2.0 µm, and *I*<sub>11</sub>/*I*<sub>10</sub> has recovered to 0.35 ± 0.04. A similar quantitative argument could be made for each of the other X-ray signals associated with thick filament activation, and indeed for *I*<sub>ML1</sub> itself. All these X-ray signals are consistent with the conclusion that the thick filament is not activated for 50 ms after the stimulus at low load, as expected from the mechano-sensing hypothesis.

That consensus breaks down at later times after the stimulus. By 100 ms, *I*<sub>ML1</sub> has decreased to around one-half of its pre-stimulus value in the low-load protocol, and has become only weakly load-dependent (Fig. 2D). By 200 ms, no remaining load-dependence is detectable. Yet the other X-ray diffraction features that have been used previously to monitor the activation state of the thick filament, in particular *I*<sub>11</sub>/*I*<sub>10</sub> (Fig. 2A), *S*<sub>M6</sub> (Fig. 2C) and *I*<sub>M3</sub> (Fig. 3B) show almost no further change in the low-load protocol between 50 and 200 ms. Moreover, *I*<sub>11</sub>/*I*<sub>10</sub> (Fig. 2A) and *S*<sub>M6</sub> (Fig. 2C) in particular retain the full load-dependence they showed at the earlier time. We consider two extreme interpretations of these results. Either the decrease in *I*<sub>ML1</sub> is artefactual, and the thick filament does in fact remain OFF at low load as suggested by the constancy of *I*<sub>11</sub>/*I*<sub>10</sub> and *S*<sub>M6</sub>, or the thick filament switches ON after 50 ms at low load, but that transition is not detected by *I*<sub>11</sub>/*I*<sub>10</sub> and *S*<sub>M6</sub>.

We think the first explanation to be improbable. The trabeculae stayed well aligned in the X-ray beam until at least 150 ms after the stimulus in the low-load protocol (see Appendix, Fig. A1). Mean sarcomere length has shortened to ~1.8 µm by that time, and *I*<sub>ML1</sub> is smaller at shorter sarcomere length in resting muscle (Fig. 2D), an effect that might be associated with double-overlap of the thin filaments in the sarcomere at short sarcomere length (Caremani et al., 2019) or to the increase in inter-filament spacing (Ovejero et al., 2022). However the decrease in *I*<sub>ML1</sub> is slower than sarcomere shortening

(Table 1), and has the same magnitude in the fixed-end protocol, in which sarcomeres shorten to 1.85 µm and in the sarcomere-isometric protocol, where they remain longer than 2.1 µm, arguing against this explanation.

The alternative interpretation, that motors do indeed start to leave the helically ordered OFF conformation starting 50 ms after the stimulus in the low-load protocol, requires an explanation of the relative constancy of *S*<sub>M6</sub> and *I*<sub>11</sub>/*I*<sub>10</sub> in this period. It implies that the longer axial periodicity of the thick filament at high load indicated by *S*<sub>M6</sub> is not tightly coupled to the loss of the helical order of the motors, but rather to filament stress. That assertion remains consistent with the mechano-sensing hypothesis because the increase in axial periodicity could be upstream of the loss of helical order in the thick filament signalling pathway, but does imply that there is some additional mechanism for motor disordering at low load, as discussed below. The relative constancy of *I*<sub>11</sub>/*I*<sub>10</sub> at later times in the low-load protocol may reflect the very low number of actin-bound force-generating myosin motors in those conditions, with the further implication that *I*<sub>11</sub>/*I*<sub>10</sub> is not an unambiguous signal for the release of motors from the helical OFF state in heart muscle, a conclusion that was previously drawn from the small changes in that parameter observed on cooling intact trabeculae, an intervention that greatly reduced *I*<sub>ML1</sub> (Ovejero et al., 2022).

The changes in the conformation of the myosin motors reported by the M3 reflection (Fig. 3) are also consistent with the conclusion that motors start to leave the helically ordered OFF conformation starting 50 ms after the stimulus in the low-load protocol, with the caveat that the decrease in the spacing of the M3 reflection during low-load activation (*S*<sub>M3</sub>) (Fig. 3C, magenta) remains unexplained. More generally it is clear that *S*<sub>M3</sub> and its higher-order counterpart *S*<sub>M6</sub> (Fig. 2C) have different load- and time-dependences in the three protocols used here. An adequate explanation of this effect probably requires a more complete description of thick filament structure, including the contributions of multiple axial periodicities (Caremani et al., 2021; Ovejero et al., 2022), different structures in the P, C and D zones (Brunello et al., 2020), as well as the systematic perturbations of the myosin helix (Chen et al., 2024; Dutta et al., 2023; Tamborrini et al., 2023).

The model used here to determine the relative contributions of motors in the P, C and D zones (Fig. 3F) represents a first step towards understanding this complexity within the simplifying assumption of a single axial motor periodicity that follows *S*<sub>M3</sub>. The results of that model suggest that P-zone motors leave the axially-ordered OFF state with strongly load-dependent kinetics and, unexpectedly, that P-zone motors remain disordered at higher loads, implying that P-zone motors are not involved in actin-attachment and force generation
at high load. By contrast, D-zone motors remain mostly ordered at low load, apart from the loss of a small fraction of motors at the filament tips in this model, but do become ordered at the highest load in the sarcomere-isometric protocol, presumably because they attach to actin and contribute to force generation. C-zone motors stay ordered at all loads and times in these protocols (Brunello et al., 2020).

The present results have shown that the mechano-sensing mechanism is important for the rapid activation of thick filaments in heart muscle at the high loads at which the heart operates *in situ* (Brunello et al., 2020; Reconditi et al., 2017). In addition, a slower load independent mechanism of thick filament activation would operate in parallel with mechano-sensing in physiological conditions. The signalling pathway responsible for the load independent mechanism is unknown, but three general possibilities might be considered. First, the increase in intracellular calcium ion concentration associated with activation could allow calcium ions to bind directly to the thick as well as to the thin filaments (Ma et al., 2022). Second, thick filaments may be able to sense the regulatory state of the

thin filaments; for example, through binding of the N terminus of MyBP-C to the thin filaments (Kampourakis et al., 2014; Mun et al., 2014; Pfuhl & Gautel, 2012; Ponnam et al., 2019). Third, it remains possible that the 6kPa load present in the current low-load protocol, although only 10% of the load in sarcomere-isometric conditions, is still adequate to trigger the positive feedback loop of mechano-sensing, albeit slowly. These three possibilities could be tested by future X-ray studies.

The technology for synchrotron-based time-resolved X-ray studies on heart muscle has advanced dramatically in recent years (Narayanan et al., 2023). In parallel, the availability of high-resolution cryo-electron microscopy structures of the cardiac thick filament (Dutta et al., 2023; Tamborrini et al., 2023) offers the promise of the development of a complete atomic model of the thick filament, and the associated molecular interpretation of the X-ray diffraction data. Future time-resolved X-ray studies building on that annotation offer a uniquely powerful approach for answering the outstanding fundamental questions about the structural mechanisms of the control of contractility in heart muscle, with equally fundamental implications for the development of better therapies for heart disease.

Appendix

<figure><img src="image_7.png" /><figcaption>**Figure A1.** Ultra-small-angle X-ray diffraction patterns (A) and meridional intensity distributions (B) showing the first to the fifth order sarcomere reflections in diastole at long SL (20 ms before stimulus, black) and during low-load clamp (150 ms after stimulus, grey). The linear scale in (B, *upper panel*) shows the intensity distributions after removing the diffuse background; the logarithmic scale in (B, *lower panel*) shows the distributions without background subtraction. Beam attenuated to 3% of maximum intensity, total exposure time, 120 ms; Eiger2 4M detector; sample-to-detector distance, 31 m; ID02 (ESRF); data added from N = 3 trabeculae.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Figure A2.**</figcaption></figure>

(A) Small-angle X-ray diffraction pattern added from 3 trabeculae in diastole at long *SL* showing the myosin-based meridional reflections M1 to M6, the troponin reflection T1, the first myosin layer line (ML1), and the 1,0 and 1,1 equatorial reflections. Beam attenuated to 3 % of maximum intensity, total exposure time, 88 ms; Eiger2 4M detector; sample-to-detector distance, 3.2 m; ID02 (ESRF); data added from N = 3 trabeculae. For this image, the detector gaps were patched with SAXSutilities (M. Sztucki, ESRF); the equatorial axis and M1/T1 meridional reflections were digitally attenuated by a factor of 5. (B) Equatorial intensity distribution. (C) Off-meridional intensity distribution in the region of the first myosin layer line (ML1). (D) Meridional intensity distribution. Intensity distributions obtained by integrating the pattern in A with integration limits as specified in Methods.

<caption>**Figure A3.**</caption>

Force, sarcomere length and the intensities of the equatorial reflections in fixed-end, low-load clamp and sarcomere-isometric contractions. Force and sarcomere length (*SL*; A), intensity of the (1,0) reflection (*I*₁₀; B), and intensity of the (1,1) reflection (*I*₁₁; C) in fixed-end (black lines and symbols), low-load clamp (magenta lines and symbols) and sarcomere-isometric (cyan lines and symbols) contractions. St, stimulus. Time period between vertical dotted lines, low-load clamp duration. Vertical dashed-dotted magenta line, *t* = 60 ms. Dashed horizontal lines in *B* and *C* are values in diastole at long *SL*. Data are mean ± SD; fixed-end, 6 trabeculae; low load, 7 trabeculae (except for sarcomere length, 3 trabeculae); sarcomere-isometric, 4 trabeculae.

[Colour figure can be viewed at wileyonlinelibrary.com]

<figure><img src="image_9.png" /></figure>
<figure><img src="image_10.png" /><figcaption>**Figure A4.**
Meridional intensity distributions in the region of the M3 reflection (continuous lines) and distributions calculated from the model described in the text (dashed lines) in the fixed-end (A), low-load clamp (B) and sarcomere-isometric (C) protocols at the times shown in the key inset (purple, -20ms; yellow, 70ms; green, 150ms; blue, 210ms). (D) Diffracting layers of myosin motors and their location along the myosin half-filament. Inset, schematic of the half-thick filament; horizontal dashed lines delimit the proximal P-zone (layer 1-7), MyBP-C-containing C-zone (layers 7-31) and distal D-zone (layers 31-49). Symbols in D are mean ± SD; fixed-end (black), 6 trabeculae; low load (magenta), 7 trabeculae; sarcomere-isometric (cyan), 4 trabeculae; profiles in A-C and bars in D are added from the same trabeculae.
[Colour figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

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## Additional information

### Data availability statement

All relevant data, associated protocols and materials are described within the published article and its supporting information.
Competing interests

The authors declare that they have no competing interests. TI and WM provide consulting and collaborative research studies to Edgewise Therapeutics and Bristol Myers Squibb (Formerly MyoKardia), but such work is unrelated to the content of this article.

Author contributions

Y.W., L.F., J.G.O., M.I. and E.B. designed the research. Y.W., L.F., J.G.O., C.H., S.J., F.P.C., A.G., S.-J.P.-H., W.M., T.I., T.N., M.I. and E.B. performed the research. Y.W., L.F., J.G.O., T.N., M.I. and E.B. analysed data. Y.W., L.F., M.I. and E.B. wrote the paper. All authors contributed to the critical revision of the manuscript and approved the final version of the manuscript submitted for publication.

Funding

This work and the investigators were supported by the British Heart Foundation (BHF), King's British Heart Foundation Centre of Research Excellence Awards RE/13/2/30 182, RE/18/2/34 213, Intermediate Basic Science Research Fellowship FS/17/3/32 604 (to EB) and PG/16/19/32 072 (project grant to MI); UK Medical Research Council (grant MR/R01700X/1 to MI), Wellcome Trust and the Royal Society, and the ESRF LF was funded by a Sir Henry Dale Fellowship awarded by the Wellcome Trust and the Royal Society (fellowship 210 464/Z/18/Z). AG thanks Wellcome Trust Collaborative Award in Science (UK; 201 543/Z/16/Z, awarded to Mathias Gautel, King's College London) and Fondazione Umberto Veronesi (Italy) for financial support. MI thanks Wellcome Trust grant 215 482/Z/19/Z for financial support. For the purpose of open access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. X-Ray diffraction experiments at beamline 18ID used resources from the Advanced Photon Source; a US Department of Energy (D.O.E) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory

under Contract No. DE-AC02-06CH11357. This project was supported by grant P30 GM138395 from the National Institute of General Medical Sciences of the National Institutes of Health. The content is solely the authors' responsibility and does not necessarily reflect the official views of the National Institute of General Medical Sciences or the National Institutes of Health.

Acknowledgements

We are grateful to Marty Rajaratnam (King's College London) for mechanical engineering support and J. Gorini, ID02 staff and the Biomedical Facility Support at ID17 (ESRF – The European Synchrotron) and the BioCAT staff at the Advanced Photon Source for support during the beamtime. We thank the ESRF for the provision of synchrotron beamtime.

Author's present address

Y. Wang: Department of Musculoskeletal and Ageing Science, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool, UK. J. G. Ovejero: Medical Physics and Radiation Protection Department, Clínica Universidad de Navarra, Madrid, Spain. F. P. Cullup: Cardiovascular Medicine Division, John Radcliffe Hospital, Headington, Oxford, UK. A. Ghisleni: IFOM ETS – The AIRC Institute of Molecular Oncology, Milan, Italy.

Keywords

cardiac muscle, mechano-sensing, muscle regulation, myosin

Supporting information

Additional supporting information can be found online in the Supporting Information section at the end of the HTML view of the article. Supporting information files available:

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