Experimental animals

All methods are reported in accordance with ARRIVE guidelines 2.0 for the reporting of animal experiments [26]. Wild-type (WT) C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed at the in-campus mouse breeding core facility at Loyola University Chicago (LUC). Animals were conditioned in-house for 5–6 days after arrival with commercial diet and tap water available ad libitum. For the purposes of this study, male mice aged between 3 and 4 months were selected. All animal-related procedures described in this study underwent a comprehensive review and were conducted under the oversight of the LUC Institutional Animal Care and Use Committee. These procedures adhered to the standards outlined in the National Research Council’s “Guide for the Care and Use of Laboratory Animals” when establishing animal research standards. For some experiments, LPS (MilliporeSigma, Burlington, MA; catalog number L3012) was injected intraperitoneally (i.p.), with each mouse individually weighed to determine the precise quantity of LPS required to achieve the specified doses as indicated in the figure legends. Experiments were conducted 18 h post LPS injection. Primary cardiomyocytes were isolated from the heart tissue according to the procedure described below. All methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org) for the reporting of animal experiments.

Solutions made prior to the day of cardiomyocyte isolation2× base buffer

To make the solution, add the components listed in Table 2 to 500 ml of distilled water. Gradually adjust the pH to 7.8 by adding NaOH dropwise. Once prepared, this solution can be stored at 4 °C for up to 2 weeks.

EDTA buffer

To prepare 250 ml 1× EDTA buffer solution, start by mixing 125 ml of a 2× base buffer with 125 ml of distilled water, and dissolve 365.3 mg of EDTA disodium dihydrate (Sigma Aldrich, E5134) into this 1× base buffer solution. Adjust the pH to 7.8 by adding NaOH dropwise, and filter the solution through a 0.2 μm filter to ensure sterility. This solution can be stored at 4°C for up to 2 weeks. For each experiment, approximately 50 ml of this buffer will be required.

Perfusion buffer

To prepare 750 ml 1× perfusion buffer, start by mixing 375 ml of a 2× base buffer with 375 ml of distilled water, and dissolve 71.4 mg of MgCl₂ (Sigma Aldrich, M8266) into this 1× base buffer. Adjust the pH to 7.8 by adding NaOH dropwise, and filter the solution through a 0.2 μm filter to ensure its sterility. This prepared solution can be stored at 4 °C for up to 2 weeks.

Enzymes

Dilute 1000 mg of collagenase 2 (Worthington, LS004176), collagenase 4 (Worthington, LS004188), and protease XIV (Sigma Aldrich, P5147) separately in 20 ml of distilled water. Each enzyme should be prepared one at a time on ice and aliquoted into volumes of 525 µl for collagenase 2 and collagenase 4 and 55 µl for protease XIV. Aliquots can be stored at -80 °C for up to 6 months.

Heparin

To prepare the heparin stock solution, dissolve 5 mg of heparin (Sigma Aldrich, H4784) in 1 ml of PBS. To prepare the working heparin solution, dilute 25 µl of the heparin stock solution in 75 µl of PBS. Both the stock and working heparin solutions can be stored at 4 °C.

Solutions made on the day of cardiomyocyte isolationDigestion buffer

Digestion buffer can be prepared by mixing the ingredients listed in Table 3.

Stop buffer

Add 1 ml of fetal bovine serum (FBS) to 19 ml of perfusion buffer, and let the solution equilibrate to room temperature before use.

Plating media

Plating media can be prepared by mixing the components listed in Table 4. The total volume of plating media required will vary based on the quantity of downstream experiments planned. Prior to use, allow the solution to equilibrate to room temperature.

Calcium re-introduction solutions

Calcium re-introduction solutions can be prepared by mixing the components in Table 5. Prior to use, allow the solution to equilibrate to room temperature.

Tyrode buffer

Dissolve 35.28 mg of CaCl2·6H2O (Sigma Aldrich, 21108) in 200 ml of perfusion buffer, and pass the solution through a 0.2 μm filter for sterilization. This solution can be stored at 4 °C for up to 2 weeks.

Preparation of cell culture plates

Coat 35 mm plates with 0.1 mg/mL poly-D-Lysine (PDL) (Sigma Aldrich, 6407) for 30 min at room temperature. Then, aspirate the PDL solution and allow the plates to dry for at least 30 min in a 37 °C incubator. Prior to cell plating, rinse the plates once with plating media.

Preparation of surgical equipment and areaTubing preparation

One day prior to the experiment, flush the peristaltic pump tubing (Harvard Apparatus, 72–0668) with 70% ethanol followed by water to ensure cleanliness. Allow the tubing to air dry completely.

Surgical area preparation

Clean the surgical area thoroughly with 70% ethanol before the procedure. Sterilize all surgical instruments as shown in Fig. 5A to maintain aseptic conditions.

Fig. 5

Setup for cardiomyocyte isolation. (A) Surgical tools needed for heart excision, including a surgical table, a 5 ml syringe containing ice-cold EDTA buffer, tweezers, sharp-tipped scissors, Vannas scissors, and curved-ended Reynolds hemostatic forceps. (B) EDTA and digestion buffers in a 37 °C water bath for preparation. (C) Injection needles marked with nail polish to ensure penetration depth of only 3 mm into the heart. (D) A custom-made holder securely positions the injection needle and tubing. (E) An overview of the complete perfusion system

Buffer preparation a.

On the experiment day, prepare three sterile 50 ml conical tubes: one with 50 ml of EDTA solution and the other two with 50 ml of digestion buffer each. The second digestion buffer conical serves as a refill for the one connected to the pump.

b.

Place both the EDTA and digestion buffer solutions in a 37 °C water bath to reach the desired temperature. Once at 37 °C, introduce the peristaltic pump tubing into these solutions (Fig. 5B).

Pump and needle preparation a.

Set the peristaltic pump (Harvard Apparatus, 70-7000) to 30 ml/min and allow approximately 10 ml of digestion buffer to circulate through the tubing to expel any air bubbles. Repeat the process with the EDTA buffer.

b.

To prevent the injection needles from being inserted too deeply, mark two 27G needles at a distance of 3 mm from the needle tip using colored nail polish (Fig. 5C).

Syringe and needle setup for ventricular injection a.

Fill a sterile 5 ml syringe with 5 ml of cold EDTA buffer and attach one of the marked 27G needles. Keep the syringe on ice until use; this is intended for injection into the right ventricle to expel blood and halt cardiac contractions.

b.

Connect the other marked needle to the peristaltic pump tubing designated for heart injections and secure the setup in place (Fig. 5D). In this procedure, the tubing is steadily fixed into place using a holder made of P1000 tips taped in a refill wafer. The complete set up of the perfusion system is shown in Fig. 5E.

Cardiomyocyte isolation procedure a.

Inject 50ul of working heparin solution intraperitoneally into the mouse and let the mouse rest for 10 min.

b.

Anesthetize the mouse by placing the animal in a glass container containing gauze moistened with isoflurane. Remove the mouse from the jar after one minute and confirm full anesthesia by verifying the absence of a toe-pinch reflex. Maintain annesthethia by placing a nose cone containing gauze moistened with isoflurane over the mouth and nose of the mouse.

c.

After ensuring the mouse is fully anesthetized, open the chest cavity using sharp scissors. Clean the chest area with 70% ethanol and make an incision below the diaphragm. Locate the septum and gently elevate it to cut laterally below the ribs. Expose the diaphragm by cutting along the rib cage, then carefully slice the ribcage upwards and pin it back to reveal the heart (Fig. 6A, Video 2).

d.

To clear the heart of blood and cease its contraction, gently move the lungs aside, sever the inferior vena cava and the descending aorta with Vannas Spring Scissors (F.S.T, 91500-09), and inject 5 ml of ice-cold EDTA into the right ventricle using a syringe marked at 3 mm (Fig. 6B, Video 3).

e.

Clamp the aorta with Micro Hemostatic Forceps 12.5CM, 90 deg ang (WPI, 503360) to secure the heart before excision (Fig. 6C, Video 4).

f.

Remove the heart and place it in a 100 mm cell culture dish. Removal of the heart results in immediate euthanasia of the animal.

g.

Insert the 27G needle that is attached to the perfusion system into the heart (Fig. 7A, Video 5). Perfuse the heart with warm EDTA at 1.25 ml/min for 5 min, followed by digestion buffer at 1.5 ml/min for 60 min.

h.

Post-digestion, transfer the heart to a 35 mm dish with 1 ml of warm digestion buffer. Mince the tissue thoroughly with sharp tweezers (Fig. 7B).

i.

Move the minced tissue into a 50 ml tube containing 2 ml of warm digestion solution and incubate in a 37 °C water bath for 5 min. After 5 min, pipette the tissue/cell suspension up and down 20 times using a 3 ml transfer pipette. Place the tube in a 37 °C water bath for 5 more minutes, then pipette the suspension up and down for another 20 times. At this point, the tissue should be fully digested. Add 5 ml of stop buffer and pipette the suspension up and down for another 5 times.

j.

Discard the supernatant, resuspend the cardiomyocyte pellet in calcium re-introduction wash buffer 1, and let settle for 15 min. Remove the supernatant and repeat the process with calcium re-introduction buffer 2 and buffer 3 sequencially. After the final settling, discard the supernatant, resuspend the cardiomyocyte pellet in 5-8 ml of plating media, and seed onto PDL-coated 35 mm glass-bottom dishes. Allow the cells to adhere at room temperature for 20 min.

Fig. 6

Animal surgical procedure. (A) Chest cavity opened, exposing the heart; (B) Injection of EDTA buffer into the right ventricle; (C) Clamping the aorta

Fig. 7

Isolation of cardiomyocytes from the excised heart. A) Excised heart connected to the perfusion system via intraventricular injection. B) Heart tissue disassociation. C) Pellet of healthy cardiomyocytes

Live cell imagingPreparation of cells a.

After the cells have adhered to the plates, the initial culture medium was replaced with fresh medium with or without LPS (10 ng/ml). Subsequently, the cardiomyocytes were incubated at 37 °C for 1 h.

b.

After the incubation, the cardiomyocytes were washed once with tyrode buffer and then incubated with the following dyes in tyrode buffer for 30 min at 37 °C: MitoTracker Green (100 nM, ThermoFisher Scientific, M7514 ) together with MitoSOX Red (5 µM, ThermoFisher Scientific, M36008), JC-1 (10 µg/ml ThermoFisher Scientific, T3168), or Di-8-ANEPPS (Invitrogen, D3167). For Di-8-ANEPPS, 5 mg of Di-8-ANEPPS was dissolved in 1 ml of DMSO. This stock solution can be stored at 4 °C. A working solution was prepared by mixing Di-8-ANEPPS stock and 20% Pluronic acid in a 1:1 ratio. To prepare the dye solution, 0.5 µl of this working solution was added to 300 µl of Tyrode buffer.

Imaging capture: Post-incubation, the cells were washed twice with tyrode buffer. Imaging was then performed using an LSM 510 confocal microscope, which features an Axio Observer Z1 motorized inverted microscope and Zen software (Carl Zeiss Microscopy), to capture images of the cells at 10x magnification. JC-1 fluorescence was excited at 560 nm to visualize the aggregated form (red fluorescence) and at 485 nm to detect the monomeric form (green fluorescence). Images of JC-1 red and green fluorescence were acquired with exposure times of 500 ms. MitoSOX fluorescence was excited at 510 nm and MitoTracker Green fluorescence was excited at 490 nm. Both MitoSOX and MitoTracker Green images were acquired with exposure times of 10,000 ms. Average fluorescence of the cell was analyzed using ImageJ. Only cardiomyocytes exhibiting rod shape morphology were analyzed.

Transmission electron microscopy (TEM) imagingSample fixation a.

Cardiomyocytes were washed in phosphate-buffered saline (PBS) and then fixed in a solution of 2% paraformaldehyde (Electron Microscopy Sciences), 2.5% glutaraldehyde (Electron Microscopy Sciences), and 0.2% tannic acid (Ted Pella, Inc) in PBS for 1 h at room temperature.

b.

After extensive washing with deionized water, the samples were fixed in a solution of 1% osmium tetroxide and 1.5% potassium ferricyanide (Electron Microscopy Sciences) in deionized water for 1 h at room temperature in the dark.

Dehydration and post-fixation staining

The samples were immersed in 25% alcohol for 10 min followed by an incubation with 1% uranyl acetate in 50% alcohol for 1 h in the dark (i.e., en bloc uranyl acetate staining). Subsequently, the samples were incubated with 75% alcohol for 10 min, three changes of 95% alcohol for 10 min each, three changes of 100% alcohol for 10 min each, and 3 changes of propylene oxide (Electron Microscopy Sciences) for 10 min each.

Embedding a.

The samples were embedded in a 1 to 1 ratio of propylene oxide to epoxy resin, comprised of a mixture of EMbed 812, nadic methyl anhydride, dodecenyl succinic anhydride, and 2,4,6-Tris(dimethylaminomethyl)phenol, Electron Microscopy Sciences), for 12 h on a rotary mixer (Ted Pella, Inc.).

b.

The samples were then fully embedded in 100% epoxy resin for 12 h at room temperature on the rotary mixer, with a resin change followed by an additional 2-hour incubation. The epoxy resin was allowed to polymerize at 70 °C for 36 h and then allowed to cool to room temperature.

Sectioning

Ultrathin Sect. (90 nm) were prepared using an ultramicrotome (EM UC7, Leica Microsystems) then mounted on formvar- and carbon-coated 200 mesh copper grids (Electron Microscopy Sciences).

Section staining

Mounted samples were stained with filtered 1% uranyl acetate and Reynold’s lead citrate prior to imaging.

Imaging capture

Imaging was performed using a Philips CM 120 transmission electron microscope (TSS Microscopy) equipped with a BioSprint 16 megapixel digital camera (Advanced Microscopy Techniques).

Semi-quantification analysis of TEM images

Images were properly scaled and quantitative analysis, including measurements of mitochondria number, area, cristae occupancy, disorganization of cristae, lipid droplet area, and autophagic events, was conducted using ImageJ software. Specifically, mitochondria with normal cristae were characterized by organized, densely packed membranes, whereas those with disorganized cristae were identified by swelling, fragmentation, or irregular shapes. Mitochondrial and lipid droplet areas were measured using the freehand tool (example shown in Fig. 8A). Cristae occupancy percentage was calculated by converting images to 32-bit, applying a threshold, and analyzing the outlined area unoccupied by cristae (Fig. 8B). The formula used was: Cristae occupancy percentage = (total mitochondrial area - area unoccupied by cristae) / total mitochondrial area × 100.

Fig. 8

Analysis of mitochondria area and cristae surface area from TEM images. (A) Calculating mitochondria area. (B) Calculating area unoccupied by mitochondrial cristae

Statistics

The results are presented as the mean ± SEM, based on the specified number of experiments or mice. Statistical significance was assessed using a Mann-Whitney U Test or Student’s t-test depending on data distribution. Differences were deemed statistically significant when p ≤ 0.05.