Anti sarcomeric α actinin

Cell monolayers or tissue patches were fixed in 2% paraformaldehyde (15 min), permeabilized in 0.5% Triton-X (30 min) and blocked in a 5:1 solution of 1% BSA and chicken serum (30 min). The following primary antibodies (1 h incubation) were used: anti-sarcomeric α-actinin (Sigma, a7811, 1:200), anti-Cx43 (Life Technologies, 71-0700, 1:100), anti-vimentin (Sigma, v6630, 1:500), anti-smooth muscle actin (Sigma, a2547, 1:200), anti-GFAP (BD Biosciences, 561483, 1:100) and anti-Ki67 (Abcam, ab15580, 1:200). Secondary antibodies (1 h incubation) included the following: Alexa488 (Life Technologies, A-21200 or A-21441, 1:200), Alexa594 (Life Technologies, A-21201 or A-21442, 1:200), Alexa647 (Life Technologies, A-21463, 1:200), Alexa488-conjugated phalloidin (Life Technologies, A12379, 1:300) and 4,6-diamidino-2-phenylindole (Sigma, 1:300). All immunostaining steps were performed at room temperature. Fluorescence images were acquired using inverted fluorescence (Nikon TE2000) or confocal (Leica SP5) microscope and processed with ImageJ software.

The harvested TA muscles were embedded in cryopreservative (OCT embedding medium, Cell Path) immediately after isolation. Cryostat sections were prepared (10 μm) and further processed. For immunohistological analysis, the tissues were fixed (4% PFA, 10 min), permeabilized (0.5% TritonX-100, 20 min), blocked for 30 min (5% BSA+ 0.1% TritonX-100 in PBS), and finally stained with anti-sarcomeric α-actinin (1 : 200, Sigma) and F4/80 (1 : 100, Abcam) over night at 4°C. After washing with PBS, the tissues were incubated with Cy3 anti-mouse IgG secondary antibody (1 : 1000, Sigma) and DAPI (1 : 100, Sigma) for 1 h at room temperature, washed again, and finally mounted (Dako). Images were acquired with a Leica-Imager Type DM6000B at exposures normalized to unstained controls (secondary antibody and DAPI only).

P14 hearts were collected, minced and flash frozen in liquid nitrogen45 (link). The defrosted tissue was fixed in 4% paraformaldehyde, digested with 3 mg ml⁻¹ collagenase type II in HBSS and filtered using a 100 μm cell-strainer. Staining of isolated cells was performed with the BD Cytofix/Cytoperm Fixation/Permeabilization Kit, using anti-sarcomeric α-actinin (1/600, Sigma) and DRAQ5 nuclear stain. Data acquisition was performed using an ImageStreamX cytometer with INSPIRE software (Amnis). Files were collected with a cell classifier applied to the brightfield channel to capture events larger than 100 μm. At least 23,000 cell events were acquired for each sample and all images were captured with the 40 × objective. Data analysis was performed with IDEAS software (v6.0, Amnis). Images were compensated using a matrix generated by single-stained samples acquired with identical laser settings in the absence of brightfield illumination. The analysis was restricted to in-focus single cells and to intact cardiomyocytes, selected as actinin and DRAQ5 double positive. An object mask was created on the brightfield channel and the aspect ratio was defined as the ratio between the minor and major cell axis. The number of nuclei per cell was assessed using the DRAQ5 images, in at least 350 cells per heart.

Hearts were harvested from WT and cMyBP-C^-/- pups at PND1 and cryopreserved according to standard procedures as detailed in Supplementary Methods. WT and cMyBP-C^-/- hearts were then sectioned in a coronal plane at 6 μm thickness (CRYO 03/5800), mounted onto charged slides (TruBond 380), fixed in 100% acetone for 15 min, and dried. Slides were rehydrated in TBS, then permeabilized with 20 ng/ml Digitonin (Sigma) in TBS and blocked in 5% normal goat serum in TBST for 2 h. Sections were incubated with primary antibodies overnight at 4°C with anti-sarcomeric α-actinin (1:1,000, Sigma, A7811) and either anti-Connexin-43 (1:200, Sigma, C6219), anti-Xirp2 (1:500, ProteinTech, 11896-1-AP), or anti-N-cadherin (1:200, ThermoScientific, CDH2 3B9). Slides were incubated with secondary antibodies (Alexa Fluor 647 and 488; Invitrogen) at 1:1,000 for 1 h at room temperature. Following labeling, sections were coverslipped using Prolong Gold Antifade Reagent (Invitrogen) with 4′,6-diamidino-2-phenylindole (DAPI) to label nuclei. Imaging was performed using Nikon Eclipse 90i photomicroscope using the S Fluor 40× objective, and NIS Elements imaging software (version 4.0; Nikon).

Immunofluorescence staining was performed in cells fixed with paraformaldehyde. The fixed cells were washed with PBS and then incubated with 2% goat serum and 5% bovine-serum albumin in PBS to reduce nonspecific binding. The cells or cardiac sections were then incubated for 2 hours at room temperature with mouse, anti-α-sarcomeric actinin (1:500, Sigma-Aldrich, MO), and connexin-43 monoclonal antibodies (1:500, Cell Signaling, MA). The sections were then incubated with the appropriate goat or rabbit anti-mouse secondary antibodies (1:1000) conjugated to Texas red and FITC. The nuclei were counterstained with HardSet mounting medium with DAPI (Vector Labs). The cells were visualized by Olympus FV1000 spectral confocal microscopy. Separate cells were also stained without primary antibodies to identify nonspecific binding.

Immunofluorescence staining was performed in cells or cardiac sections fixed with paraformaldehyde. The fixed cells were washed with PBS and then incubated with 2% goat serum and 5% bovine-serum albumin in PBS to reduce nonspecific binding. The cells or cardiac sections were then incubated for 2 hours at room temperature with mouse, anti-α-sarcomeric actinin (1∶500, Sigma-Aldrich, MO), cardiac troponin-T (1∶200, Santa Cruz, CA), myosin heavy chain (1∶200, Santa Cruz, CA) and connexin-43 monoclonal antibodies (1∶500, Cell Signaling, MA). The sections were then incubated with the appropriate anti-mouse secondary antibodies (1∶1000) conjugated to Texas red and FITC. The nuclei were counterstained with HardSet mounting medium with DAPI (Vector Labs). The cells were visualized by inverted Nikon fluorescence microscope (TE 2000). Separate cells were also stained without primary antibodies to identify nonspecific binding.