Oleg Lookin

Pieter De Tombe

Najlae Boulali

Csilla Gergely

Thierry Cloitre

PhyMedExp Olivier Cazorla
email: olivier.cazorla@inserm.fr

Cardiomyocyte sarcomere length variability: Membrane fluorescence versus second harmonic generation myosin imaging

Keywords: membrane fluorescence versus second harmonic generation myosin imaging fluorescent microscopy, second-harmonic generation, sarcomere length Running title: Cardiac sarcomere length variability

SHG derived SL variability may reflect the true relaxed myocyte sarcomeric ultrastructure.

INTRODUCTION

Sarcomeres are the basic contractile elements in a striated muscle cell. The actual sarcomere length (SL), the inherent properties of individual sarcomeres, and the mechanical inter-communication between adjacent sarcomeres are important for the whole-cell contractile response, both in cardiac [START_REF] Dobesh | Cooperative activation in cardiac muscle: impact of sarcomere length[END_REF][START_REF] De Tombe | Cardiac muscle mechanics: sarcomere length matters[END_REF] and skeletal muscle [START_REF] Herzog | What can we learn from single sarcomere and myofibril preparations?[END_REF]. The ability to characterize SL under certain conditions (ambient, mechanical, etc) makes it possible to conclude how changes in sarcomere length are linked to functional behavior of the cardiomyocyte.

Optical imaging is the only available tool for characterizing the sarcomeric striation pattern and the corresponding average SL, which is highly informative for understanding the functional relationships between actual SL and contractile state of the cell [START_REF] Bub | Measurement and analysis of sarcomere length in rat cardiomyocytes in situ and in vitro[END_REF][START_REF] Botcherby | Fast measurement of sarcomere length and cell orientation in langendorff-perfused hearts using remote focusing microscopy[END_REF][START_REF] Aguirre | Intravital imaging of cardiac function at the single-cell level[END_REF][START_REF] Guo | AutoTT: automated detection and analysis of T-tubule architecture in cardiomyocytes[END_REF][START_REF] Pasqualin | SarcOptiM for ImageJ: high-frequency online sarcomere length computing on stimulated cardiomyocytes[END_REF][START_REF] Kobirumaki-Shimozawa | Optimization of fluorescent labeling for in vivo nanoimaging of sarcomeres in the mouse heart[END_REF][START_REF] Varga | Internal structure and remodeling in dystrophin-deficient cardiomyocytes using second harmonic generation[END_REF][START_REF] Dowrick | Simultaneous brightfield, fluorescence, and optical coherence tomographic imaging of contracting cardiac trabeculae ex vivo[END_REF].

Recent studies have also focused on the existence of intracellular SL heterogeneity in striated muscle [START_REF] Rassier | Sarcomere mechanics in striated muscles: from molecules to sarcomeres to cells[END_REF]Haeger et al., 2020;[START_REF] Adkins | Variability of in vivo sarcomere length measures in the upper limb obtained with second harmonic generation microendoscopy[END_REF] and, importantly, its alteration upon the transition of myofilaments from the inactivated to the activated state [START_REF] Johnston | The role of sarcomere length nonuniformities in residual force enhancement of skeletal muscle myofibrils[END_REF][START_REF] De Souza Leite | Microfluidic perfusion shows intersarcomere dynamics within single skeletal muscle myofibrils[END_REF][START_REF] Moo | In vivo sarcomere lengths become more non-uniform upon activation in intact whole muscle[END_REF]Haeger and Rassier, 2020;[START_REF] Lookin | Contractile state dependent sarcomere length variability in isolated guinea-pig cardiomyocytes[END_REF]. Therefore, special care is needed to choose a proper method for SL measurements.

For many years, the use of membrane staining by fluorescent dyes (particularly of the ANEPPS family) has been an extensively used approach [START_REF] Bub | Measurement and analysis of sarcomere length in rat cardiomyocytes in situ and in vitro[END_REF][START_REF] Guo | AutoTT: automated detection and analysis of T-tubule architecture in cardiomyocytes[END_REF][START_REF] Wagner | Analysis of tubular membrane networks in cardiac myocytes from atria and ventricles[END_REF][START_REF] Yue | Heterogeneity of transverse-axial tubule system in mouse atria: Remodeling in atrialspecific Na+-Ca2+ exchanger knockout mice[END_REF]. These labeling dyes, however, only visualize membrane invaginations, the t-tubules, but not directly the internal cellular components that compose the sarcomeres. As a result, relying on a membrane dye may render proper determination of the location of the sarcomeric Z-disk somewhat uncertain. This limitation may be overcome by employing genetically encoded fluorescent sarcomeric proteins that are introduced by viral infection techniques. For example, fluorescent labeling of α-actinin has been used to visualize sarcomeric Z-disks [START_REF] Shintani | Sarcomere length nanometry in rat neonatal cardiomyocytes expressed with a-actinin-AcGFP in Z discs[END_REF][START_REF] Tsukamoto | Simultaneous imaging of local calcium and single sarcomere length in rat neonatal cardiomyocytes using yellow Cameleon-Nano140[END_REF][START_REF] Kobirumaki-Shimozawa | Nano-imaging of the beating mouse heart in vivo: Importance of sarcomere dynamics, as opposed to sarcomere length per se, in the regulation of cardiac function[END_REF][START_REF] Kobirumaki-Shimozawa | Optimization of fluorescent labeling for in vivo nanoimaging of sarcomeres in the mouse heart[END_REF]. However, both the fluorescent labeling and the viral infection delivery methods involve introduction of "non-natural" compounds into the living cell that may affect function and structure of the cardiac myocyte.

Second-harmonic generation (SHG) microscopy is, by design, a label free imaging modality that has been highly effective in the examination of cellular structures in intact living tissues [START_REF] Recher | Three distinct sarcomeric patterns of skeletal muscle revealed by SHG and TPEF microscopy[END_REF][START_REF] Buttgereit | Second harmonic generation microscopy of muscle cell morphology and dynamics[END_REF]. In addition to high-contrast optical sections of cells and tissues, SHG imaging can also provide detailed structural information. This technique has been successfully employed to visualize sarcomeres [START_REF] Garcia-Canadilla | Automated cardiac sarcomere analysis from second harmonic generation images[END_REF][START_REF] Zhao | Live imaging of contracting muscles with wide-field second harmonic generation microscopy using a high power laser[END_REF][START_REF] Varga | Internal structure and remodeling in dystrophin-deficient cardiomyocytes using second harmonic generation[END_REF][START_REF] Homan | MorphoScript: a dedicated analysis to assess the morphology and contractile structures of cardiomyocytes derived from stem cells[END_REF] and, therefore, is specifically suited to the study of SL variability. SHG-based images of myocytes have revealed that the source of the SHG signal from the sarcomere are the myosin tails that assemble into a rod structure forming the central core of thick myosin filaments [START_REF] Plotnikov | Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres[END_REF].

Because t-tubule visualization is often used for the determination of average SL, we aimed here to compare the characteristics of individual SL populations as obtained by the two principally different optical methods: second harmonic generation (SHG) and ttubular fluorescent labeling (ANEPPS).

METHODS

All investigations conformed to European Parliament Directive 2010/63/EU and approved by the ethics committee "Comité d'éthique pour l'expérimentation animale Languedoc-Roussillon".

Isolated cardiomyocytes

Intact cardiomyocytes were isolated from the heart by enzymatic digestion as previously described [START_REF] Andre | Carbon monoxide pollution aggravates ischemic heart failure through oxidative stress pathway[END_REF]. Briefly, the heart was harvested from deeply anesthetized male rats (pentobarbital 50mg/kg) while beating, quickly cannulated on a Langendorff apparatus, and perfused with a physiological solution at 37°C via the aorta. This solution contained (in mM): NaCl 117, KCl 5.7, NaHCO 3 4.4, KH 2 PO 4 1.5, MgCl 2 1.7, HEPES 21, glucose 11, taurine 20, pH = 7.2 adjusted with NaOH. The heart was first washed with this calcium (Ca 2+ )-free extracellular physiological solution and then with an enzyme-containing solution (1.25 mg/ml Collagenase type IV, Worthington) during 25 to 35 minutes. The left and right ventricles were separated, and digestion was stopped with bovine sodium albumin. The tissue was then mechanically disrupted with a pipette to release the myocytes. The extracellular Ca 2+ concentration was progressively increased to 1 mM. All subsequent experiments were performed using a modified Tyrode solution (in mM): NaCl 140, KCl 4, MgCl 2 1, HEPES 20, CaCl 2 0.1, glucose 11, pH = 7.4 at room temperature. Myocytes were not electrically stimulated and 20 mM BDM was added to all solutions to prevent spontaneous contractile activity.

The analysis of intracellular variability in sarcomere lengths was performed using second harmonic generation microscopy measurements (SHG) + fluorescent measurements with membrane staining by di-4-ANEPPS (ANEPPS) for fully relaxed and non-stretched cardiomyocytes (n = 26 from N = 3 hearts).

Optical measurements

Just prior to the start of the SHG/ANEPPS measurements, cardiomyocytes were labeled with the fluorescent dye di-4-ANEPPS as follows: 0.2 μL of ANEPPS (1 mM DMSO stock solution) was added to 1 mL of cell suspension in Tyrode solution at room temperature. Following sedimentation during the 10-minute dye treatment, the supernatant was removed and replaced by 3 mL fresh dye-free Tyrode. An aliquot of cells was placed in ~1 ml fresh modified Tyrode solution in a glass bottomed 35 mm plastic petri dish (room temperature). SHG images were then recorded using a custommade multiphoton microscope setup based on a Tsunami tunable Ti:Sapphire laser (Spectra-Physics) and an upright SliceScope microscope (MPSS-1000P) furnished with a multiphoton galvanometer scan head (MP-2000) both from Scientifica [START_REF] Varga | Internal structure and remodeling in dystrophin-deficient cardiomyocytes using second harmonic generation[END_REF]. The Ti-Sa laser was operated in pulsed mode configuration for sample excitation, 875 nm wavelength, 80 MHz frequency and ~100 fs pulse duration. A Nikon CFI75 LWD-16x-W objective (NA 0.8, water immersion) was used to focus the laser beam onto the myocyte. The SHG signal was recorded by a 1.4-NA water-immersion condenser (U-AAC, Olympus), through a 482 nm long-pass dichroic mirror (86-331, Edmund Optics) and a 447 nm high-performance 60 nm band-pass filter (48-074, Edmund Optics) and then detected by a H7422P photomultiplier (Hamamatsu). Epifluorescence two-photon images were recorded using a 735 nm long-pass dichroic mirror, followed by sequential short-pass/long-pass filters to form a 450 nm to 750 nm bandpass filter, 1024x1024 ~150 nm XY scan pixel size, and then detected by a R928P photomultiplier (Hamamatsu). Several consecutive scans (~10 frames) of the same cell position were recorded to increase signal-to-noise ratio during further off-line image post-processing. In a subset of experiments, images were acquired at 5 separate Zslices, ranging approximately from the top to the bottom of the isolated myocyte (cf.

Figure 4).

Analysis of variability in sarcomere lengths

The analysis of variability in individual sarcomere lengths was implemented by custommade software EqapAll6 (developed by Oleg Lookin). For each cell in the SHG/ANEPPS measurements, we used the two optical images separately for SHG (to reveal the regularity of sarcomeric A-bands) and for ANEPPS (to reveal the regularity of t-tubular invaginations of cell membrane). By design, the two images were always aligned to each other and five regions of interest (ROI) with the same positions, sizes, and angles were used for each image (Fig. 1A). In all measurements, the ROIs were placed to cover as much area of a cell as possible, but their lengths and angles were set to follow a straight part of myofibrils. Curved myofibrils, crossing myofibrils or myofibrils with irregular staining along their length were omitted from the selection.

The lengths of individual sarcomeres were determined using an algorithm based on analysis of the sarcomere striation profile. For this algorithm, the input data were the sarcomere striation profiles obtained for each ROI. The positions of sarcomere edges in ANEPPS measurements were found from the local maxima in the sarcomere striation profile. In contrast, the local maxima in the sarcomere striation profile in SHG measurements reflected the positions of sarcomere centers. To test if the use of local maxima or minima in the same striation profile can produce a difference in SL variability, we also compared the data of SHG/ANEPPS measurements using both local extrema (cf. Fig. 3). The individual SL sets obtained for each ROI were then combined to increase the resulting number of individual SLs prior to the analysis of their variability.

An expanded view of striation profiles obtained for the same ROI using SHG/ANEPPS measurement is shown in Fig. 1B. Note that the local maxima in these two striation profiles are shifted relative to each other by a half sarcomere, as indicated by the arrows in Fig. 1B. The local maxima in the SHG measurement correspond to the centers of A-bands, while the local maxima in the ANEPPS measurement index the local peak intensity of t-tubule staining that putatively corresponds to the position of sarcomeric Z-disks.

Statistical analysis

Statistical analysis was carried out with GraphPad Prism 9.3.1 (GraphPad Software).

Because some of the individual SL sets did not follow the normal shape, we analyzed median SL values as well as the absolute and relative measures of variability applicable for non-normal data samples. The evaluation of significance in the median SL values as well as the absolute/relative measures of SL variability between SHG and ANEPPS was implemented by the Wilcoxon matched pairs signed rank test. Differences were considered significant at p < 0.05. Data are presented as mean ± S.D.

RESULTS

We analyzed the variability of individual sarcomere lengths (SLs) using SHG imaging to solely and directly visualize sarcomeric A-bands. For comparison, simultaneous ANEPPS fluorescence was used to visualize t-tubule structures. Median values of SL as well as absolute and relative measures of SL variability were analyzed and contrasted between these two image modalities. The averaged median SL values were not significantly different between the two optical measurements (p = 0.901, Fig. 2A). In contrast, the absolute and relative measures of variability of individual sarcomere lengths were sensitive to the method of measurements. The absolute measures of SL variability -inter-quartile range and median absolute deviation -were ~two-fold higher for SL measurements using ANEPPS vs SHG (the difference is significant at p < 0.0001 for any measure, Fig. 2B-C). For example, the median absolute deviation of SL was found to be 0.04 ± 0.02 μm in SHG vs 0.09 ± 0.02 μm in ANEPPS measurements (Fig. 2C). Likewise, the relative measures of SL variability -inter-quartile range divided by median SL value or median absolute deviation divided by median SL value -were significantly higher (~2.0-2.2 fold) for ANEPPS vs SHG (difference is significant at p < 0.0001 for each measure, Fig. 2D-E). The higher SL variability obtained by ANEPPS measurements is demonstrated by plotting superimposed SL distributions averaged for all SHG and ANEPPS measurements (Fig. 2F). Each plot peaks at averaged median SL and shows how much the SL values are distributed (as a % of the median SL value).

The distribution for ANEPPS measurements is roughly two times wider than the distribution for SHG measurements.

The calculation of sarcomere length using ANEPPS measurement was based on the determination of local maxima in the intensity profiles that correspond to the peak intensities of the individual t-tubules. Instead, we can use local minima to check whether their positions correlate with the local maxima of the SHG measurement (Aband centers). We did not find any difference between these two approaches, that is, in the ANEPPS measurement the spreads of local maxima (the physical presence of ttubules) and local minima (the interval between two adjacent t-tubules) were quantitatively similar. The plots of spread range of median SL values and median absolute deviation divided by median SL values, as obtained for SHG and ANEPPS measurements by using of local maxima or local minima as index for determining the local SL, are shown in Fig. 3. Median SL values were found to be insensitive to the method of optical measurement or the selection of local maxima/minima to retrieve the individual SLs (Fig. 3A). At the same time, the SL variability was significantly larger in the ANEPPS vs SHG measurements (Fig. 3B) regardless the use of local maxima or minima to index SL positions.

It should be noted as a side observation in this study that using Z-stacks in SHG measurements allows for a more precise evaluation of the 3D structure of the sarcomeric A-band geometry than can be obtained using ANEPPS imaging. For example, we implemented Z-stacks in some of our SHG measurements and found that an A-band regularity pattern exists in most of Z-planes (Fig. 4A) with the SL distribution characteristics to be non-discriminate between slices. The local peaks of the A-band regularity pattern revealed a high persistence in their physical positions along the longitudinal axis of the cell in the Z-direction, that is along the depth of the cell (Fig. 4B).

In other words, some parts of the cell showed minor transversal changes (~0.5 µm, roughly one fourth of the sarcomere, for nearly the full depth of the cardiomyocyte) in the longitudinal positions of A-bands. This may indicate the presence of some functional role for transversal A-band geometry. Quantification of the shifts in the X-position of each individual A-band when slicing across the cell depth, may allow for the measurement of such transversal changes. The middle frame in a Z-stack can be used as the referent to evaluate such shifts in the other frames (Fig. 4C). The more aligned A-bands are in Z-direction, the smaller a positional shift is observed. Therefore, SHG measurements may be helpful in the precise 3D imaging and reconstruction of the Aband "skeleton" as well as in the analysis of A-band (mis)alignment within a whole cell.

DISCUSSION

The main finding of our study is that the method of optical measurement is critical for proper characterization of individual sarcomere length (SL) variability, while it does not affect the measured average SL. In the present study, we implemented two optical measurement modalities: second-harmonic generation microscopy (SHG) and confocal two-photon imaging of fluorescent membrane-specific dye (ANEPPS). The first method marks the individual positions of A-bands that are purely internal structures of the sarcomere [START_REF] Garcia-Canadilla | Automated cardiac sarcomere analysis from second harmonic generation images[END_REF][START_REF] Zhao | Live imaging of contracting muscles with wide-field second harmonic generation microscopy using a high power laser[END_REF][START_REF] Varga | Internal structure and remodeling in dystrophin-deficient cardiomyocytes using second harmonic generation[END_REF], while the second method visualizes the t-tubular striation pattern known to be closely aligned to sarcomeric Z-disks [START_REF] Bub | Measurement and analysis of sarcomere length in rat cardiomyocytes in situ and in vitro[END_REF][START_REF] Aguirre | Intravital imaging of cardiac function at the single-cell level[END_REF][START_REF] Guo | AutoTT: automated detection and analysis of T-tubule architecture in cardiomyocytes[END_REF]), but which is not actually part of the sarcomere [START_REF] Setterberg | The physiology and pathophysiology of t-tubules in the heart[END_REF]. The determination of averaged SL was shown to be insensitive to these optical methods confirming that a proper averaged value of SL can be obtained using fluorescent staining of t-tubules component of the sarcolemma. However, for subtle purposes such as the analysis of SL variability, the two methods were found not to be equivalent. In our study, the use of ANEPPS dye for sarcomeric visualization resulted in higher SL variability as compared to SHG microscopy that makes visible the central part of sarcomere. This could be due to a different extent of variability of the positions of A-bands versus t-tubules in each myocyte. The peak optical density of the t-tubule is somewhat aligned to the physical "center" of the Z-disk, but its actual peak position could be misaligned to some extent with the Z-disk center because a t-tubule is not physically part of the sarcomere (the "sloppy" coupling of t-tubules to the sarcomere Z-disks). In contrast, the A-band is the core inner component of the sarcomere and, therefore, its optical position should strongly follow the actual position of the "center" of the sarcomere. Our result therefore supports that SHG-based sarcomeric A-band visualization is the "gold standard" for accurate analysis of individual SLs. It should be noted that in some of our SHG images we observed, like in previous reports [START_REF] Recher | Three distinct sarcomeric patterns of skeletal muscle revealed by SHG and TPEF microscopy[END_REF][START_REF] Varga | Internal structure and remodeling in dystrophin-deficient cardiomyocytes using second harmonic generation[END_REF], a doublebanded signal in the A-bands of the sarcomere; in our analysis we avoided selection of myofibrils with such striation pattern.

A further question remains whether spatial resolution is a crucial factor that affects the precision of SL determination. In the present study we used the same spatial resolution as in our recent study on guinea pig myocytes, where we used transmitted light (TL) mode to acquire cell images [START_REF] Lookin | Contractile state dependent sarcomere length variability in isolated guinea-pig cardiomyocytes[END_REF], and average SL was only nominally affected by the method of optical measurement -SHG, ANEPPS or TL; in the latter mode we found a slightly higher resting SL (data not shown), but this may be related to the absence of fluorescent dye in these cells. However, the use of SHG provided twofold lower measured SL variability as compared to both ANEPPS (and TL in the paper cited above), despite equivalent spatial resolution of the optical systems. In the TL mode, the measured image is non-confocal and the sarcomeric striation derives from all myofibers along the cell depth. Therefore, imaging by conventional microscopy has inherently higher inconsistencies in the determination of an individual SL compared to confocal or SHG microscopy [START_REF] Telley | Half-sarcomere dynamics in myofibrils during activation and relaxation studied by tracking fluorescent markers[END_REF][START_REF] Kobirumaki-Shimozawa | Nano-imaging of the beating mouse heart in vivo: Importance of sarcomere dynamics, as opposed to sarcomere length per se, in the regulation of cardiac function[END_REF].

On the other hand, if the focal plane is properly chosen and optical phase-contrasting is used, the TL image may be as good for the SL analysis as the ANEPPS image, similar to previously reported data [START_REF] Nance | Attenuated sarcomere lengthening of the aged murine left ventricle observed using two-photon fluorescence microscopy[END_REF]. Another reason of that non-confocal TL imaging may yield similar results as the ANEPPS confocal images is an axial regular alignment of sarcomeric structures (i.e. along the Z-axes) of the cardiomyocyte as we observed in the present study (cf. Fig 4). The structural basis for this regularity is not known and was not the focus of the currently study but, as an interesting side note, this regularity may also be expected to contribute to axial stiffness of a non-contracting relaxed cardiomyocyte [START_REF] Peyronnet | Simultaneous assessment of radial and axial myocyte mechanics by combining atomic force microscopy and carbon fibre techniques[END_REF]. Therefore, quantitative measures of SL variability appear to be sensitive to the selection of which sarcomeric (related) structure is visualized, rather than the selection of optical method per se (fluorescent or transmitted light microscopy, confocal or brightfield microscopy).

The application of SHG microscopy can be further extended to the actively contracting cardiomyocytes and, especially, for the conditions where the myocyte is subjected to the physiological pre-or afterload. In our previous paper [START_REF] Lookin | Contractile state dependent sarcomere length variability in isolated guinea-pig cardiomyocytes[END_REF] we reported that myocyte electrical activation increased SL variability as compared to the inactive state, concomitant with a reduction in overall SL. This result conformed to previously reported differences between relaxed and activated states in skeletal muscle [START_REF] Moo | In vivo sarcomere lengths become more non-uniform upon activation in intact whole muscle[END_REF][START_REF] Moo | Single sarcomere contraction dynamics in a whole muscle[END_REF][START_REF] Johnston | On sarcomere length stability during isometric contractions before and after active stretching[END_REF][START_REF] De Souza Leite | Sarcomere length nonuniformity and force regulation in myofibrils and sarcomeres[END_REF]. However, an effect of sarcomere pre-stretch on SL variability in contracting cardiomyocytes remains to be determined. To elucidate this aspect in detail, SL variability in cardiomyocytes should be assessed with use of SHG microscopy as a prospective tool in the determination of individual sarcomeres, in addition to the widely used fluorescent staining.

In conclusion, we found that the method of optical measurement is critical for proper characterization of individual sarcomere length (SL) variability, while it does not affect the measured average SL. Our results, therefore, support the notion that SHG-based sarcomeric A-band visualization forms the "gold standard" for precise analysis of individual sarcomeres.

ACKNOWLEDGEMENTS

Figure 1 .

 1 Figure 1. Representative confocal images of an isolated cardiomyocyte obtained by second-harmonic generation microscopy (SHG) and by membrane fluorescent staining with di-4-ANEPPS (ANEPPS). (A) SHG (top) and ANEPPS (bottom) images with five regions of interest (ROI) as indicated (colored lines) following the paths of single myofibrils. The insets show the sarcomeric striation of a single ROI (cf. yellow arrows). (B) Sarcomere striation profiles taken from a single ROI using SHG and ANEPPS images. The arrows show the positions of local maxima in the intensity profiles (note their relative shift by a half sarcomere because SHG visualizes the center of sarcomeres, while ANEPPS visualizes the areas near the sarcomeric Z-disks).

Figure 2 .

 2 Figure 2. The average sarcomere length (SL) value, but not the SL variability, is independent of the optical method used to derive SL striations. Confocal measurements were made either by using second-harmonic generation microscopy (SHG), or by fluorescent membrane staining with di-4-ANEPPS (ANEPPS) in n = 26 isolated cardiomyocytes from N = 3 hearts. The median SL value (A) and the following absolute and relative quantitative measures of SL variability were analyzed: (B) Interquartile range (IQR), (C) Median absolute deviation (MAD), (D) IQR divided by median SL, and (E) MAD divided by median SL. The data in panels (A-E) are shown as mean ± S.D. Differences between SHG and ANEPPS were significant at p < 0.0001 (Wilcoxon matched-pairs signed rank test). (F) Averaged distribution plots for individual SL values obtained by SHG or ANEPPS. The peaks of the plots correspond to averaged median SL, and the distributions are shown as a % of the corresponding median SL.

Figure 3 .

 3 Figure 3. The use of either local maxima (max) or local minima (min) of the sarcomere striation profile generates non-discriminated quantitative measures of sarcomere length (SL) median value and the variability in the individual SLs. Confocal measurements were by second-harmonic generation microscopy (SHG) or by fluorescent membrane staining with di-4-ANEPPS (ANEPPS) in n = 26 isolated cardiomyocytes from N = 3 hearts. (A) Median SL, (B) Median absolute deviation (MAD) divided by median SL (MADM). Differences between SHG(max) and SHG(min) or between ANEPPS(max) and ANEPPS(min) were not significant (Wilcoxon matched-pairs signed rank test).

Figure 4 .

 4 Figure 4. The applicability of SHG measurements for evaluation of sarcomeric A-band 3D-structure. (A) A series of five SHG images of A-bands in an isolated cardiomyocyte obtained at different positions along the Z-axis, i.e. from the top (frame #1) to the bottom of the cell (frame #5). The yellow-colored ROI on each frame is kept fixed in size and X/Y-positions and used to retrieve A-band axial regularity. Scale bar on the top image is common for all images. (B) Curves of A-band regularity patterns obtained from the corresponding ROIs shown in the images in panel (A). (C) The shifts of individual A-band X-position across the slices as calculated relative to the X-position in the "center of the cell" (i.e. frame #3). The plots are obtained for four frames (two below and two above the middle frame). The magnitude of the longitudinal shifts, their variation along the X-axis, and the actual direction of the change during the axial slicing across the cell depth may be used to estimate Z-axis A-band (mis)alignment.

AUTHOR CONTRIBUTIONS

OL, PdT, NB and OC contributed to the conception of the study, design of experiments, analysis, and interpretation of the results. PdT, NB, CG, TC and OC contributed to the experimental measurements. CG and TC provided technical assistance in SHG measurements. OL made software for data processing. The manuscript was written by OL, PdT and OC. All authors approved the final version of the manuscript.

FUNDING

The study was supported by the Centre national de la recherche scientifique (France, grant #IEA00401 to OC), and NIH (HL62426 to PdT).


Anne-Laure Decombeix
email: anne-laure.decombeix@cnrs.fr

Carla J Harper

Cyrille Prestianni

Thibault Durieux

Merlin Ramel

Michael Krings

Decombeix et al_ Oldest tylosis formation

Tyloses are swellings of parenchyma cells into adjacent water conducting cells that develop in vascular plants as part of the heartwood formation or specifically in response to embolism and pathogen infection. Here, we document tyloses in Late Devonian (ca. 360 Ma) Callixylon wood. This discovery suggests that already some of the earliest woody trees were capable of protecting their vascular system by occluding individual conducting cells.

Tyloses are protoplasmic inflations formed by parenchyma cells into the lumen of neighboring conducting cells [1,2]. They extend through the pit pair and can completely fill the lumen of the conducting cell. The mechanisms underlying tylosis formation remain largely unresolved [e.g., 3,4], but it is generally accepted that they represent a response to embolism and have a major role in blocking air-filled conducting cells. Moreover, tylosis formation is among the earliest processes in the Compartmentalization Of Decay In Trees (CODIT; [5]), which can delay the spreading of pathogens. Tyloses in fossil plants, therefore, provide direct insights into the evolution of one of the most important mechanisms with which plants protect their vascular system from biotic and abiotic stresses.

Structurally preserved fossils provide evidence that tylosis formation occurred in most lineages of vascular plants as early as the Mississippian (ca. 350 Ma) [6]. However, the oldest fossils believed to represent vascular plant conductive elements (tracheids) are dispersed microscopic tubes from the uppermost Silurian (ca. 430 Ma) [7], and the first record of secondary xylem (wood) comes from the Lower Devonian (410 Ma) [8][9][10]. By Middle Devonian times, a great many plants possessed wood [11] in which the arrangement of conducting cells and parenchyma could, in theory, have allowed the formation of tyloses. The development of larger and more complex plant bodies during the later Devonian, along with the evolution of leaves and more extensive root systems [12,13], likely increased the risk of embolism in the vascular system. There is also evidence from this period of geologic time of the existence of various types of vascular plant-fungal interactions, possibly including wood decay [14,15]. It is therefore surprising that no tyloses have been hitherto reported in plant fossils older than the Mississippian.

The lack of documented evidence of tyloses in early vascular plants has spurred us to specifically look for these structures in Devonian structurally preserved woods. We found that many early woods are fossilized in a manner not conducive to the preservation (in a recognizable form) of tenuous parenchymatous structures, including tyloses. Nevertheless, we discovered evidence of early stages of tylosis formation in one piece of pyritized wood from the Upper Devonian (Famennian, ca. 360 Ma) of Sandeel Bay, County Wexford, Ireland. This wood consists of tracheids and small parenchymatous rays (Fig. 1 No evidence of tylosis formation has been observed in any other structurally preserved plant fossil from the Sandeel Bay locality.

Archaeopteridales are a Middle-Late Devonian group of progymnosperms, extinct plants that had a gymnosperm-like wood but reproduced via spores [16]. Archaeopteridalean trees produced a significant amount of wood, formed a deep and complex root system, and had true leaves, making them the first trees with a "modern" vegetative body [12,18]. Recent studies of their hydraulic properties suggest that archaeopteridalean wood had a conductivity comparable to that of extant conifers, with some plasticity provided by variations in the size of the conducting cells and rays [19,20]. Like extant trees, archaeopteridaleans were susceptible to wood decaying fungi that invaded their vascular system [14]. By the Late Devonian, these trees were a major component of ecosystems worldwide, having been reported from a diversity of latitudes, from the paleotropics to the high latitudes of Gondwana [21]. This wide altitudinal and geographic range implies that the group was able to adaptate to very diverse growth conditions. The ability to protect the vascular system from biotic and abiotic stresses by occluding (some of) the conducting cells may, therefore, have been an important advantage. The depositional environment of the Sandeel Bay anatomically preserved plants has been interpreted as a cut-off channel chute of a meandering river near the coastline [START_REF] Boucot | Tulsa, OK[END_REF], within a dry tropical area [23]. Growth ring-like zones in the archaeopteridalean woods from this deposit suggest that the trees were exposed to periodic shifts from favorable to less favorable growth conditions. It is likely that there were episodes of drought governed by variations of the water table that could have caused embolism in certain regions of the wood and led to tylosis formation. The fact that most tracheids are not completely blocked indicates that the specimen depicts an early stage in this process. This discovery illustrates how structurally preserved fossils can provide snapshots of plant physiology in deep time, and allow us to piece together episodes in the evolutionary history of key biological processes. Moreover, it provides further evidence of the antiquity of tylosis formation in plants, and suggests that the emergence of this capacity could have been linked to the evolution of larger plant bodies with a more complex organization. As soon as plants had evolved a water conducting system [11,12], they had also created a gateway through which physical (i.e., embolism) and biological (i.e., pathogens) threats could spread.

Early vascular plants were perhaps able to mitigate this drawback simply through their small size and the short life span of their structures. However, as soon as plants grew larger, an effective system of protection of the vascular system became essential [24], with tylose formation being an integral part of it.

  ); distinct growth ring-like zones of tracheids with reduced radial diameters are indicative of intermittent periods of less-favorable growth conditions (Fig 1a). The radial tracheid walls show groups of pits separated by unpitted areas, a feature typical of Callixylon, the wood of the archaeopteridalean progymnosperms ((Fig 1c) [16]; see methods section). Rays are 1-2 cells wide and 1-15 (usually less than 10) cells high in tangential section (Fig 1d), abundant, and each xylem tracheid is in contact with a significant number of ray parenchyma cells. Conspicuous bubble-like outgrowths of ray cells into the lumen of adjacent tracheids occur in one area of a longitudinal section (Fig 1e-k). Although they appear relatively dark compared to the surrounding parenchyma cells, we interpret these outgrowths as tyloses, rather than gum deposits, based on their regular outline, physical connection to the rays, and the presence also of several translucent examples (Fig 1k). The majority of the tyloses are spheroidal in shape (Fig 1e, f), sometimes with a constriction at the base that likely corresponds to their passage through the pits connecting rays and tracheids; others are more flattened (Fig 1h, i). Still others completely occlude the tracheid (Fig 1g). There are often several tyloses emerging from a single ray, sometimes visible along both sides of the ray in tangential sections (Fig 1j).

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Fig 1 .

 1 Fig 1. Late Devonian Callixylon wood containing tyloses. a, transverse section, showing growth ring boundaries (white arrows) b, detail of transverse section with two rays. c, radial section, showing groups of pits separated by unpitted areas (white arrows) typical of Callixylon. d, longitudinal section of a ray containing what appear to be ray tracheids (black arrow). e, f, general views of area with abundant tyloses. g, tyloses entirely blocking conducting cell. h, ray with several small outgrowths (developing tyloses) into adjacent tracheid. i, small outgrowth from ray cell into tracheid. j, ray with tyloses on both sides. k, tylosis lacking dark content (translucent). All images were selected from the direct observation under the microscope of preparations of a single fossil (#HH5): a, b: slide HH5-B-CT1r; c: HH5-E (surface of specimen); d-g, j, k: slide HH5-D-G1r; h: slide HH5-D-G1v . Scale bars: a: 200 µm; b, e, f: 100 µm; c, g-k: 50 µm. Legend: R: ray. Arrowheads: tyloses.

Acknowledgements

This work was supported by a Tellus-Interrvie grant (DECA, CNRS-Institut National des Sciences de l'Univers) to ALD, and a PHC Ulysses grant (#47212TK, Irish Research Council and French Ministry of Foreign Affairs) to CJH and ALD. We thank Catherine Girard (ISEM Montpellier) and Brigitte Meyer-Berthaud (AMAP, Montpellier) for help in the field and comments on a previous draft of the manuscript.

Data availability.

All data that support the findings of this study are included in this published article and its supplementary information files. The fossil and associated slides are currently on loan at UMR AMP Montpellier and accessible under specimen number HH5 (for HookHead #5). This material and other fossils illustrated in the supplementary figures will ultimately be deposited in the Earth Science collections of the National Museum of Ireland, Dublin, once the study of the assemblage is completed.

Online Methods

Age of the fossil wood, sedimentological and floristic context

The specimen was collected in 2021 at Sandeel Bay on Hook Head peninsula, Wexford County, Ireland. Outcrops containing Late Devonian plant macrofossils in this area belong to the Harrylock Formation [25] (Supplementary Fig. 1a), which corresponds to fluvial sediments deposited in a proximal alluvial plain setting. Anatomically preserved specimens occur as pyritic permineralizations in grey-green mudstones, which also contain compressions, dispersed spores, and cuticles (Supplementary Figs. 1b,2). The miospore assemblage at Sandeel Bay corresponds to the LL Miospore Biozone of latest Famennian age [25]. New findings in 2021 of Retispora lepidophyta (Kedo) Playford 1976 and Vallatisporites pusillites (Kedo) Dolby and Neves 1970 in the layers containing the macrofossils (Supplementary Fig. 2h,j) confirm their age as latest Famennian. Other permineralized plants recovered from the source deposit of the specimen with tyloses include axes of the lycopsid Wexfordia hookense (Matten) Klavins 2004 at different developmental stages [26,27] (Supplementary Fig. 2a), as well as axes and wood with the typical Callixylon Zalessky 1911 anatomy of archaeopteridalean progymnosperms [16]. Callixylon was first reported from this locality by Klavins [START_REF] Boucot | Tulsa, OK[END_REF], who also described an anatomically preserved branch assigned to Archaeopteris cf. hibernica. Newly collected specimens from this locality include the first anatomically preserved Archaeopteris roots (Supplementary Fig. 2d-g). Finally, Klavins [START_REF] Boucot | Tulsa, OK[END_REF]

Fossil preparation and observation

The fossil is approximately 17 cm long and 2-3 cm wide, and consists of 7 consecutive portions of a single axis. Selected axis portions were prepared at UMR AMAP in 2022 according to the following protocol. They were embedded in Epoxy (DBF, Escil), and transverse and longitudinal sections of 1-2 mm thickness were subsequently cut with an Isomet 1000 diamond saw and polished on both sides. To improve contrast, the section were then etched following a modified version of the protocol for pyrite permineralizations published by Stein and collaborators [28]. They were initially etched in 70% nitric acid (HNO3) heated to 70°C for 60-90 seconds. After neutralization in sodium hydroxide (NaOH) they were additionally etched in 37% hydrochlorydric acid (HCl) for a few seconds, neutralized, and then etched again in 5-10 % HCl before being rinsed in water and let to dry.

Selected sections were mounted on glass slides and observed in reflected light. Due to the distortion of the wood, there was no good quality view of the radial pitting on the mounted sections. Photographs of the radial pitting (Fig. 1 c) were thus taken directly on the surface of fragment HH5-E after a fracture in the radial plane. All photographs were taken with a Keyence VX 7000 electronic microscope and the associated software. Composite figures were assembled in Adobe Photoshop 21.2.2. In slide numbers such as HH5-D-G1, the first part refers to the specimen number (5), the second to the part of the specimen (D), and the last to the section (G1). Letters r or v in photographs indicates the side of the wafer that was photographed (recto, bearing the number, or verso).

Systematic affinity of the wood

The systematic affinity of the specimen was determined based on wood anatomical traits in longitudinal and transverse sections. While no primary tissues are preserved, the presence of wood tracheids with groups of radial pits separated by unpitted areas on their radial walls is a distinctive feature of the fossil genus Callixylon, which represents the wood of archaeopteridalean progymnosperms [16]. Other woody plants discovered from the source layers of the specimen described here are lycopsids, which clearly differ from Callixylon in the radial pitting (Supplementary Fig. 2c). Seed plants, currently only evidenced from this locality based on the seeds described by Klavins [START_REF] Boucot | Tulsa, OK[END_REF], are not known to produce wood with unpitted areas on the radial walls. Species of Callixylon are distinguished based on differences in certain wood anatomical features, such as ray width, and the presence, abundance, and distribution of ray tracheids [29]. Rare ray cells with thicker walls and a squarish outline in the new specimen (e.g., Fig 1d) could correspond to ray tracheids. However, the preservation of the specimen prevents a confident assignment at the species level.

Interpretation of the outgrowths as tyloses

An alternative interpretation of the bubble-like outgrowths views them as partly coagulated (due to water loss) cell contents of the ray cells that have oozed out into adjacent tracheids as a result of the pyritization process. Strongly arguing against this interpretation are the facts that (1) similar structures have not been observed in any other pyrityzed wood from the Sandeel Bay site; and (2) the occurrence of outgrowths is limited to one particular region of the specimen that does not differ from the rest in regard to preservation. Small circular structures lacking a clear connection to parenchyma cells could also correspond to tyloses, but could also be gum or other organic residues.

Author contributions

ALD and CJH initiated the research project. ALD, CP, and TD participated in the fieldwork during which the material was collected. MR prepared the sections. ALD and MR photographed the sections. ALD, CJH and MK analysed the data with inputs from TD and CP. CP analysed the sedimentological context and prepared the spores and cuticles illustrated in the supplementary figure. ALD and MK prepared the manuscript with contributions from all co-authors.