Blebbistatin

Four to five weeks after surgery, rats were euthanized by cervical dislocation. The heart was quickly excised and placed in ice-cold modified Tyrode’s solution of composition (in mmol/L) 93 NaCl, 20 NaHCO₃, 1 Na₂HPO₄, 1 MgSO₄, 5 KCl, 1.8 CaCl₂, 20 Na-acetate, 20 glucose. Hearts were mounted via the aorta onto a cannula and retrogradely perfused at 9 ml/min using the same Tyrode’s solution at 37°C with pH maintained at 7.4 by bubbling with a 95% O₂/5% CO₂ gas mixture. Perfusate was then switched to a Tyrode’s solution containing 10 mmol/L 2,3-butanedione monoxime (BDM) and 10 μmol/L blebbistatin (Enzo Life Sciences, Exeter, United Kingdom) to inhibit contraction and minimize movement artifacts. The heart was positioned horizontally in a custom-built Perspex chamber to enable imaging of the left ventricle (LV) (Figure 1B). A pseudo-electrocardiogram (ECG) was monitored throughout the experiment using 2 Ag/AgCl disk electrodes placed close to the heart, with a reference electrode placed in the perfusion bath. Two platinum electrodes connected to an isolated stimulator were positioned on the right atrial (RA) appendage to enable pacing of the hearts via the physiological endocardium to epicardium conduction pathway, at a cycle length of 200 ms (5 Hz).

The excised heart was immediately bathed in Krebs–Henseleit (KH) solution and cannulated through the aorta. The KH buffer contained (in mM): 120 NaCl, 5 KCl, 2 MgS₂ O₄—7H₂O, 20 NaHCO₃, 1.2 NaH₂PO₄—H₂O, 1.8 CaCl₂ and 10 glucose, pH 7.4 when equilibrated with carbogen. Cardiac contraction was inhibited during the entire experiment with 5 μM blebbistatin (Enzo Life Sciences, Farmingdale, NY, USA) in the solution. The cannulated heart was perfused through the aorta (using a horizontal-Langendorff perfusion system) with the KH solution and then transferred to a custom-built optical mapping chamber at a constant flow of 2.5 mL/min at (36 ± 0.5) °C. Two platinum electrodes were placed below the heart for monitoring cardiac electrical activity via electrocardiogram (ECG). 1 mL of perfusion solution containing the voltage sensitive dye (VSD; di-4-ANBDQPQ, 6 μg/mL, University of Connecticut Health Center, Farmington, CT, USA) [24 (link)] was bolus injected into the aorta. All the experiments were performed at (36 ± 0.5) °C within 1 h after dye loading to avoid potential re-distribution of the dye and accumulation of phototoxic by-products.

The pharmacological and small‐molecule inhibitors and agonists used were the following: 7rh (2.5 µm; Sigma) for DDR1 inhibition, rat anti‐human CD29 (1:100; BD Bioscience) to block integrin β1, blebbistatin (1 µm; Enzo Life Sciences) for myosin II inhibition, GM6001 (10 µm; Calbiochem) as broad‐spectrum MMP inhibitor, L‐mimosine (400 µm; Santa Cruz Biotechnology) to arrest the cell cycle at G1, S‐trityl‐L‐cysteine (STLC; 20 µm; Santa Cruz Biotechnology) to arrest at mitosis, ionomycin (10 µm; MedChemExpress) to increase cell membrane permeability to calcium, gadolinium chloride (10 µm; MedChemExpress) to inhibit mechanosensitive calcium channels, BAPTA (10 µm; MedChemExpress) to chelate extracellular calcium, BAPTA‐AM (10 µm; MedChemExpress) to chelate intracellular calcium, arachidonic acid (70 µm; MedChemExpress) to mimic signaling downstream of nuclear mechanosensing, AACOCF3 (28 µm; Tocris) to inhibit signaling downstream of nuclear mechanosensing, 2‐aminoethoxydiphenylborane (20 µm; MedChemExpress) as IP3 receptor agonist, Xestospongin c (10 µm; Tocris) as IP3 receptor agonist, GSMT×4 (10 µm; MedChemExpress) as Piezo 1 inhibitor, Yoda1 (6 µm; MedChemExpress) as Piezo 1 agonist, GSK205 (10 µm; MedChemExpress) as TRPV4 inhibitor, and GSK1016790A (50 µm; MedChemExpress) as TRPV4 agonist.

T84 cells were seeded in a 35-mm plastic dish on day 0. On day 1, 10 μM of Y-27632 (Sigma-Aldrich Co. LLC), 25 μM of blebbistatin (Enzo Life Sciences, Inc., Farmingdale, NY, USA), 1 μM of PD168393 (EMD Millipore, Billerica, MA, USA) or dimethyl sulfoxide (Wako Pure Chemical Industries, Ltd, Osaka, Japan) was added. On day 4, the cell lysate was extracted for western blotting.

HeLa cells (ATCC, CCL-2) and HEK293 (ATCC, CRL-1573) were cultured in DMEM medium (Life Technologies, 11965) supplemented with 10% FBS (Life Technologies, 10437) and 1% penicillin/streptomycin (Life Technologies, 15140), and maintained at 37 ^°C and 5% CO₂. DAPI staining was used to ensure the absence of mycobacterium contamination. Cells were transfected using Lipofectamine 2000 (Life Technologies, 11668) according to manufacturer’s instructions. For knockdown experiments, siRNAs were transfected twice into HeLa cells with 24 h intervals through Lipofectamine RNAiMAX (Life Technologies, 13778075) or Lipofactamine 2000 based on manufacturer’s protocol. Cell staining with MitoTracker Deep Red FM (Life Technologies, M22426): 100 nM in complete medium for 30 min at 37 ^°C. Blebbistatin (Enzo Life Sciences, BML-El315) was used at 20 μM (added into the cell medium 40–50 min post induction of mitophagy or before induction of mitophagy). ML-7 (Enzo, BML-EI197) was used at 30 μM. CK666 (Enzo Life Sciences, ALX-270-506) was used 200 μM (applied 1 h before light-assisted mitophagy). SMIFH2 (Merck millipore, 344092) was used at 25 μM (applied before light-assisted mitophagy). Wortmannin (Sigma, W1628) was used 200 nM before light-assisted mitophagy. Rapalog (Clontech #635055) was used at 500 nM before light-assisted mitophagy.