Mouse models

Wild type and transgenic animals used to generate X-MET were housed in a temperature-controlled (22 °C) room with a 12-h light/dark cycle. All animal experiments were carried out in compliance with the guidelines of the Institutional Review Board of the animal facilities of DIEM and National Institute of Health-Italy (n° 609/2015-PR; n° 864/2020-PR) and reported in accordance with the ARRIVE guidelines.

Primary cultures and generation of X-MET

Muscle primary culture and generation of X-MET were performed following the protocol detailed in Carosio et al.¹⁸. A heterogeneous population of cells was obtained by mechanic and enzymatic dissociation of hind limbs harvested from wild type mice (WT) or transgenic mice using the Skeletal Muscle Dissociation Kit by Miltenyi Biotech. Dissociated cells were filtered through a 70 μm cell strainer and centrifuged at 1200 rpm for 15 min. Cells were resuspended in growth medium (GM) (DMEM, 20% horse serum, 3% chick embryo extract, 25 mM HEPES, 4 mM l-glutamine, 0.1% gentamicin, penicillin/streptomycin) and plated for 30 min twice in a row to get an enrichment of myoblasts in the culture. Cells were resuspended and plated at a concentration of 40,000 cells/ml on a tissue culture dish of 35 mm diameter coated with type I collagen (Sigma) and incubated at 5% CO₂ at 37 °C. After 5–6 days of culture, myoblasts were induced to differentiate using a differentiation medium (DM: DMEM, 5% horse serum, 25 mM HEPES, 4 mM l-glutamine, 0.1% gentamicin, penicillin/streptomycin). After 2–3 days of incubation with DM, a skeletal muscle primary cell monolayer is delaminated by gently moving a sterile tip around the peripheral area of the plate. The delaminated monolayer was then pinned on a silicone-coated dish (Sylgard, Dow Corning, Midland, Mich.) using 0.20 mm diameter stainless steel Minutiens pins (Austerlitz INSECT PINS®). The X-MET was tensioned at two different lengths: + 0%, which corresponds to the initial delamination length (unstretched X-MET), and 66 ± 1.5% (+ 66%) of initial delamination length (stretched X-MET). In these mechanical conditions X-MET was analysed at morphological, functional, and molecular levels after 15 days in culture. Generally, X-MET exhibits a self-organized cylindrical structure containing beating myotubes and shows, on average, a diameter of 200 ± 12 mm and a length (after stretching) of 2 ± 0.5 cm.

X-MET mechanical properties measurements

At first, the passive force generated by the tissue during stretch was measured using an actuator/transducer (Aurora Scientific Inc. 300B) for length control and a microforce transducer (Kronex AE801). A software developed in LabVIEW 2019 allowed the synchronization between the stretch signal and the force measurement as well as the settings of the test parameters. Starting from the initial delamination length of the tissue, the X-MET was stretched at 66 ± 1.5% with a constant velocity of 1 cm/min and simultaneously the passive force generated by the tissue was measured. Then, the X-MET spontaneous contraction force was measured 15 days after the construct delamination for both the unstretched and stretched X-MET conditions. In detail, one end of the tissue was maintained fixed by the pins and the other one was connected to a microforce transducer (Kronex AE801). Muscle contractile activity was acquired for 15 s through a National Instruments data acquisition board (DAQ NI-PCI 6251) and a software developed ad hoc in Labview 2019. The spontaneous contraction frequency was then computed through the Fast Fourier Transform (FFT). For the entire duration of the force measurement test, the X-MET was placed in a culture dish containing differentiation medium and was maintained at a temperature of 37 °C, by using a temperature control plate (Okolab s.r.l., H401).

RNA extraction and real-time PCR

Total RNA extraction was performed using TriReagentTM (SIGMA) and one microgram of each RNA sample was retrotranscribed using the QuantiTec Reverse Transcription kit (QIAGEN) to obtain double-stranded cDNA. Relative quantitative PCR was performed on ABI PRISM 7500 SDS (Applied Biosystem, USA), using premade 6-Carboxyfluorescein (FAM)-labeled TaqMan assay for Hprt, Cx-43, TNNT2, β-MHC, PDE1C, IL-6, IL-2, IL-4, IL-10, CCL2, COL3A1, CACNA 1C, RyR 2, α-MHC, ANP and BNP (Applied Biosystem, USA). The relative quantitative RT-PCR sample value was normalized for the expression of Hprt mRNA. To analyze miR-1 and miR-29b, extracted RNA was retrotranscribed using micro-RNA Reverse Transcription KIT (Applied Biosystem). Relative quantitative PCR was performed on ABI PRISM 7500 SDS (Applied Biosystem, USA), using premade 6-Carboxyfluorescein (FAM)-labeled TaqMan assay for miR1(Applied Biosystem, USA). The relative quantitative RT-PCR sample value was normalized for the expression of U6 snRNA.

RNA-seq analysis

RNA was isolated with Trizol from stretched X-METs after 15 days in DM, unstretched X-METs and 2-dimensional primary cells. One microgram of RNA was sent to the Institute of Applied Genomics (Udine, Italy) for deep sequencing. cDNA libraries were processed accordingly with the standard Illumina protocol and sequenced with the HiSeq2500 (4-plex run, 1 × 50 bp reads, about 30 M reads/sample). Reads were aligned to the UCSC mm10 version of the mouse genome using Tophat2 (⁴⁰;v2.1.1), quantified with HTSeq-count (⁴¹; v0.5.4p5). Differential expression analysis was performed in R (v3.5.1) using DESeq2 (⁴¹; v1.20.0). Counts data from all conditions were filtered based on their raw count, keeping only those whose sum of the counts for all samples was higher than 1. Principal Component Analysis (PCA) was based on the 42% most variant genes between the different samples. Genes were considered differentially expressed with Benjamini–Hochberg adjusted p-value (FDR) < 0.01.

Immunofluorescence analysis

Immunofluorescence analysis was performed on X-METs cross and longitudinal sections. X-METs were embedded in tissue-freezing medium and snap frozen in nitrogen-cooled isopentane. Samples were mounted on a cryostat and cut at 10 μm thick sections. Section was fixed with 4% PFA, washed in PBS with 1% BSA and 0.2% Triton X-100, preincubated for 1 h in 10% goat serum at RT, and incubated overnight at 4 °C with the following primary antibodies: Myosin Heavy Chain (MyHC) (Sigma-Aldrich), Connexin-43 (Cx-43) (Sigma-Aldrich) and Cardiac troponin (Troponin I) (RV-C2 Hybridoma Bank). Sections were then washed in PBS with 0.2% Triton X-100 and incubated with secondary antibody (Alexa Fluor, Life Technologies) 45 min at room temperature. Nuclei were stained using Pibenzimol bisbenzimide H33342 (HOECHST). All analyses were performed using Zeiss Confocal software (Zen 3.0 Blue edition).

Dye transfer technique

The X-MET was washed 2 times with calcium- and magnesium-free PBS. A mix of Lucifer yellow CH (0.2 mg/ml) (Molecular Probes) and rhodamine dextran (0.5 mg/ml) (Molecular Probes) diluted in differentiation medium was prepared. To allow the dyes to penetrate the cells, the construct was placed on a slide which was previously prepared to maintain the X-MET viable and create a ‘double incubation chamber’: in the chamber containing one end of the X-MET construct 50 µl of the above-detailed mix was added, while in the chamber containing the other end differentiation medium was added to keep the tissue wet. The X-MET was so incubated for 10 min at 37 °C, 5% CO₂, washed with PBS for three times and then analysed with confocal microscopy. In order to verify the viability of X-MET and monitor the transferring of the dye, a time-lapse analysis was performed. Images were acquired with a Leica confocal microscope (laser scanning TCS SP2) equipped with Ar/ArKr and HeNe lasers, using a 10X objective. The laser line was 488 nm for the excitation of Lucifer yellow and 633 nm for the excitation of rhodamine dextran. Fluorescence was collected at 500/540 nm for Lucifer yellow and at 640/680 nm for rhodamine dextran. The fluorescence intensity was computed using Leica software.

Intracellular calcium levels determination

FURA-2AM indicator_ To determinate the intracellular calcium [Ca²⁺]i Transient, X-MET unstretched (X-MET 0%) and X-MET stretched at 66% of the initial delamination length (X-MET 66%) were cultured on 35 mm dishes and incubated in culture medium containing 3.5 μmol/L 2-[6-[bis[2-[(Acetyloxy)methoxy]-2-oxoethyl]amino]-5-[2-[2-[bis[2-[(acetyloxy)methoxy]-2-oxoethyl]amino]-5-methylphenoxy]ethoxy]-2-benzofuranyl]-5-oxazolecarboxylic acid (acetyloxy)methyl ester (FURA-2-AM, Invitrogen, Carlsbad, California, USA) for 30 min at 37 °C. Then, the medium was rinsed with Hank’s balanced salt solution (Sigma-Aldrich, St.Louis, Missouri, USA). Dishes were placed into a culture chamber on the support of an inverted fluorescence microscope (Nikon TE2000E, Nikon Instruments, Italy), at 37 °C connected to a cooled charge-coupled devices camera (12B cascade, Roper Scientific, Ottobrunn, Germany). Random access monochromator was used to illuminate samples alternately at 340 and 380 nm (Photon Technology International, New Jersey, USA) and the emission was detected using a 510 nm emission filter. Metafluor® software (Universal Imaging Corporation, Downington PA, USA) was used to acquire images. At the end of each experiment, calibration was obtained by maximally increasing intracellular Ca²⁺-dependent FURA-2-AM fluorescence with 5 μmol/L ionomycin (ionomycin calcium salt from Streptomyces conglobatus, Sigma) followed by recording minimal fluorescence in a Ca²⁺-free medium⁴².

INDO-1AM indicator_INDO-1 AM (Invitrogen I1226) was reconstituted in high-quality freshly opened DMSO at a concentration of 1 mM and used at a final concentration of 100 μM in Ca²⁺/Mg²⁺ free PBS. Once reconstituted, it was protected from light and stored at − 20 °C to avoid freeze-thaws. The samples (X-METs 0% 15 DM and X-METs 66% 15 DM) were washed thrice in PBS. Then, INDO-1 AM was added slowly along the muscle constructs and the samples were incubated for 30 min at 37 °C. At the end, the X-METs were washed with PBS three times and were analysed with confocal microscopy. The detection was carried out considering the double emission of INDO-1 that shifts from 475 nm in Ca²⁺-free media to 400 nm when the dye is saturated with Ca²⁺⁴³. All analyses were performed using Zeiss Confocal software (Zen 3.0 Blue edition)⁴³.

Protein extraction and Western Blot

Samples were homogenized in lysis buffer (Tris–HCL, pH 7.5/20 mM, EDTA/2 mM, EGTA/2 mM, Sucrose/250 mM, DTT/5 mM, Triton-X/0.1%, PMSF/1 mM,NaF/10 mM, SOV4/0.2 mM, Cocktail Protease Inhibitors/1x (Sigma-Aldrich).Equal amounts of protein (70 μg) from each lysate (previously quantified through Bradford assay) were separated in SDS polyacrylamide gel (4–15% CriterionTM TGX Stain-FreeTM Protein Gel, Bio-Rad) and transferred into a nitrocellulose membrane (Trans-Blot Turbo transfer pack, Bio-Rad) using Trans-Blot ® TurboTM Transfer System (program: 2.5 A, 25 V, 20 min). The membrane was then stained with Ponceau (0.005% in 1% acetic acid) as an intermediate loading control, then blocked with 5% non-fat dry milk in TBS-1% Tween for 1 h at room temperature and then incubated overnight at 4 °C with a primary antibody for Cx-43 (Sigma-Aldrich). The membrane was thereafter washed four times for 5, 15, 15 and 5 min in TBS-1% Tween, then incubated with a specific peroxidase-conjugated secondary antibody for 45 min at room temperature. After three 10 min washes with TBS-1% Tween, the membrane was analysed by the enhanced chemiluminescence system (ChemiDoc Imaging System, Bio-Rad) according to the manufacturer’s indications. The acquired signal was quantified by scanning densitometry using a bio-image analysis system (Image LabTM Software). The results are expressed as relative integrated intensity compared to controls (GAPDH), after subtracting their respective backgrounds.

AFM experimental setup and data analysis

The elasticity measurements were conducted using the Bruker Dimension Icon Atomic Force Microscope (Bruker, Santa Barbara, CA) equipped with the probe holder to operate in fluid environment. The samples were maintained pinned into a petri dish covered with a layer of PDMS to hold the pins in position, during the entire measurements the samples were not adhered to the PDMS surface. The measurements were carried out in physiological buffer by using MLCT-BIO tips from Bruker, with elastic constants (calibrated in air, with the thermal tune method⁴⁴, before starting each experiment) that follows in the range 0.0065 ± 0.0005 N/m. Each sample has been measured by means of Force Volume maps of 16 × 16 force curves, at the speed of 3 force curve per second, in several different areas of 100 micron squared each. A prior manual selection (by visible inspection) of the force curves has been done by using the Bruker NanoScope Analysis software (Bruker, Santa Barbara, CA), in order to exclude from the analysis, the curves that presents a signal disturbed by the contraction of the sample. The stiffness calibration value (also called detector sensitivity) has been obtained from acquiring force curves on a pristine petri dish. After these preliminary steps, the force curves were analysed by means of the FC_analysis software, whose complete functioning is described in Dinarelli et al.⁴⁵. The Young’s modulus value has been obtained by fitting the curves with an Hertian model by considering the Poisson ratio of the samples as 0.5 and a conical tip with an opening half angle of 35°. The total number of force curves included in the analysis are 700 and 1200 for the un-stretched X-MET and stretched 66% X-MET, respectively. The statistical and graphical elaborations have been carried out by using the software Origin (OriginLab, Northampton, MA).

X-MET implantation on myocardium infarct

Three months old C57BL/6J mice were anesthetized with isoflurane (IsoFlo®) 1.35% + 2% O₂. Under microscopic view we performed a midline cervical incision separating the skin, muscle and tissue covering the trachea. We inserted the endotracheal tube holding the cranial part of the trachea using micro surgical forceps. The respiration rate was approximately 110 per minute, with an inspiratory pressure of 17 to 18 cm H₂O. Thereafter, the occlusion of the left anterior descending (LAD) artery was performed using a permanent 8–0 prolene suture (Ethicon, Norderstedt, Germany), with silicon tubing (1 mm OD) placed on top of the LAD, 2 mm below the border between the left atrium and LV⁴⁶. During the operation, mice were monitored with a rectal probe to keep body temperature between 36.8 and 37.2 °C using a heat pad. The chest was cut horizontally at the fourth intercostal space. Eventually, one end of the X-MET was fixed through a second ligation knot on site of damage at the same time of the LAD ligation surgery. The implantation was performed ensuring that the damage site was completely covered by the X-MET construct and the procedure was finished by carefully relocating the pericardium above the heart wall. Then, the chest was closed using 5–0 polypropylene suture. X-METs were obtained from C57BL/6J consanguineous mice and UBC/GFP mice excluding the use of immunosuppressive drugs. Forty and one hundred days after LAD, echocardiographic and histological analyses were performed to assess ischemia and myocardial remodelling.

Histological analysis

Whole hearts were fixed with 10% formalin and sectioned at 1 mm of intervals. Each slide had 10 sections, which started at the apex and ended at the suture ligation site (approximately 6 slides). Every slide was stained with Masson’s trichrome to identify areas of fibrosis or stained with Hematoxylin and Eosin (H&E). To evaluate the muscle fiber membrane integrity, mice were received the intravenous (i.v.) injection of Evans blue dye (EBD, 10 mg/mL, Sigma) in PBS at the dose of 0.1 mg/g of body weight. Each experimental mouse underwent 20 min continuous swimming after dye injection. Muscle samples were collected 24 h after injection. EBD bind to albumin and was observed under the fluorescent microscope.

Echocardiography analysis

A high-frequency, high-resolution digital imaging platform with linear array technology and color Doppler mode for in vivo high-resolution micro-imaging was used for echocardiography (Vevo® 3100 Imaging System, FUJIFILM VisualSonics Inc., Toronto, Canada). To assess the cardiovascular function of mice, a high-frequency transducer probe (VisualSonics MS400, FUJIFILM VisualSonics, Inc., Toronto, Canada with a frequency range of 18–38 MHz) was used by a skilled cardiologist under the supervision of a veterinarian. 4–5 weeks after surgery mice were anesthetized using (IsoFlo®) 1.35% + 2% O₂ shaved and positioned on an electrically warmed surface. Ventricular wall thicknesses and diameters were studied by M-mode echocardiography, and fractional shortening was calculated. Mice body temperature was monitored using a rectal probe and heart rate was used as a validation parameter, excluding from the study bradycardic (i.e., < 400 bpm) mice. Once the functional characterization was completed, the anesthetized mouse was euthanized by cervical dislocation and tissue were harvested for histological and biochemical analysis.

Statistical analyses

Statistical analysis was performed with GraphPad Prism Software. All data are expressed as mean ± SEM According to the different data analysed, the following statistical analyses were performed: nonparametric tests (Mann Whitney Rank Sum test) and 1-way ANOVA test (Bonferroni post-hoc-test, Tukey’s multiple comparison test and Fisher’s LSD test). The differences were considered significant for p-value ≤ 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

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• Source: https://www.nature.com/articles/s41598-023-37553-8