Mouse Intact and Skinned Cardiac Myocyte Mechanics: Crossbridge and Titin-Based Stress in Unactivated Cells

We investigated the contribution of actomyosin interactions in unactivated intact and skinned cardiomyocytes in physiologic conditions. A carbon fiber based cell-attachment system was used to measure the diastolic stress-sarcomere length (SL) relation of murine intact cardiomyocytes, before and after the addition of actomyosin inhibitors (BDM or blebbistatin). Stress was measured during the diastolic interval of twitching myocytes that were stretched at 100% length/s. Diastolic stress increased nearly linearly from 0 at SL 1.85 µm to 4.2 mN/mm2 at SL 2.1 µm. Actomyosin inhibitors lowered diastolic stress by ∼1.5 mN/mm2 at SL 2.1 µm (∼30% of total), suggesting that during diastole actomyosin interaction is not fully switched off. Stretch-hold-release studies on skinned cardiomyocytes showed that as temperature changed from 24°C to 37°C, there was shortening of slack SL (from 1.90±0.01 μm to 1.89±0.01 μm) and increasing of both peak stress (∼35%) and steady state stress (∼26%). Shortening of slack SL and increasing stress could be inhibited by blebbistatin. This suggests that at physiologic temperature, crossbridge cycling takes place which contributes to diastolic stress. To extend this further, calcium sensitivity of skinned cardiomyocytes was studied under conditions that simulate physiologic diastole: 37°C, presence of Dextran T500 to compress the myofilament lattice to the physiological level, and [Ca2+] from below to above 100 nM. Mean active stress was increased at [Ca2+] >55 nM (pCa 7.25) and was ∼0.7 mN/mm2 at 100 nM [Ca2+] (pCa 7.0) and ∼1.3 mN/mm2 at 175 nM [Ca2+] (pCa 6.75). The presence of active stress at pCa 7, which is a physiologic Ca2+ concentration of cytoplasm during diastole, confirms the contribution of crossbridge cycling to diastolic stress. These findings are relevant for understanding diastolic function and for future studies of diastolic heart failure.