Neonatal rat cardiomyocytes isolation, adenovirus infection and siRNA oligonucleotides transfection.

Briefly, cardiomyocytes were dissociated from the ventricles of neonatal Sprague-Dawley rat hearts (1-3 d old) by digestion with 0.3 mg/ml type 2 collagenase and 0.6 mg/ml pancreatin (Sigma-Aldrich, P3292) in a balanced salt solution containing 120 mM NaCl, 20 mM HEPES, 5.5 mM glucose, 5.4 mM KCl, 1 mM NaH₂PO₄, 0.8 mM MgSO₄, pH 7.4.⁵⁰ Cell suspensions from digestion were centrifuged, pooled and resuspended in plating medium, containing Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, 11965084):M199 (Sigma-Aldrich, M4530) (4:1 [v/v]), supplemented with 10% horse serum (Sigma-Aldrich, H0146), 5% fetal calf serum, 100 units/ml penicillin-streptomycin (Thermo Fisher Scientific, 15140122). After 24 h, myocytes were infected with adenoviruses (Ad-Gfp, Ad-Hspb6^S10F or Ad-Hspb6 WT) at a multiplicity of infection of 500. At 24 h post-infection, the cells (60-70% confluency) were transfected with siRNA oligonucleotides (200 nM) complexed with Lipofectamine™ 2000 (Thermo Fisher Scientific, 11668027) for 60 h. The following oligonucleotides were from Thermo Fisher Scientific: Silencer Select siRNA targeting Atg7 (Sense: 5’-CCGUGGAUCUGAAUCUCAATT-3’; Antisense: 5’-UUGAGAUUCAGAUCCACGGAT-3’; Thermo Fisher Scientific, S161901); Silencer Select Negative Control (Thermo Fisher Scientific, 4390843).⁵⁰