title: Mouse intact cardiac myocyte mechanics: cross-bridge and titin-based stress in unactivated cells.
abstract: A carbon fiber-based cell attachment and force measurement system was used to measure the diastolic stress-sarcomere length (SL) relation of mouse intact cardiomyocytes, before and after the addition of actomyosin inhibitors (2,3-butanedione monoxime [BDM] or blebbistatin). Stress was measured during the diastolic interval of twitching myocytes that were stretched at 100% base length/second. Diastolic stress increased close to linear from 0 at SL 1.85 µm to 4.2 mN/mm(2) at SL 2.1 µm. The actomyosin inhibitors BDM and blebbistatin significantly lowered diastolic stress by ∼1.5 mN/mm(2) (at SL 2.1 µm, ∼30% of total), suggesting that during diastole actomyosin interaction is not fully switched off. To test this further, calcium sensitivity of skinned myocytes was studied under conditions that simulate diastole: 37°C, presence of Dextran T500 to compress the myofilament lattice to the physiological level, and [Ca(2+)] from below to above 100 nM. Mean active stress was significantly increased at [Ca(2+)] > 55 nM (pCa 7.25) and was ∼0.7 mN/mm(2) at 100 nM [Ca(2+)] (pCa 7.0) and ∼1.3 mN/mm(2) at 175 nM Ca(2+) (pCa 6.75). Inhibiting active stress in intact cells attached to carbon fibers at their resting SL and stretching the cells while first measuring restoring stress (pushing outward) and then passive stress (pulling inward) made it possible to determine the passive cell's mechanical slack SL as ∼1.95 µm and the restoring stiffness and passive stiffness of the cells around the slack SL each as ∼17 mN/mm(2)/µm/SL. Comparison between the results of intact and skinned cells shows that titin is the main contributor to restoring stress and passive stress of intact cells, but that under physiological conditions, calcium sensitivity is sufficiently high for actomyosin interaction to contribute to diastolic stress. These findings are relevant for understanding diastolic function and for future studies of diastolic heart failure.
authors.affiliation: Department of Physiology and Molecular Cardiovascular Research Program, University of Arizona, Tucson, AZ 85724, USA. granzier@­email.arizona.edu
authors.forename: Nicholas M P
authors.initials: NM
authors.lastname: King
authors.forename: Methajit
authors.initials: M
authors.lastname: Methawasin
authors.forename: Joshua
authors.initials: J
authors.lastname: Nedrud
authors.forename: Nicholas
authors.initials: N
authors.lastname: Harrell
authors.forename: Charles S
authors.initials: CS
authors.lastname: Chung
authors.forename: Michiel
authors.initials: M
authors.lastname: Helmes
authors.forename: Henk
authors.initials: H
authors.lastname: Granzier
mesh_terms: D008841:Actin Cytoskeleton / Q000187:drug effects / Q000502:physiology; D000205:Actomyosin / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D001794:Blood Pressure / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D002244:Carbon / Q000008:administration & dosage; D000077482:Carbon Fiber; D064211:Connectin; D003931:Diacetyl / Q000031:analogs & derivatives / Q000494:pharmacology; D006321:Heart / Q000187:drug effects / Q000502:physiology; D006576:Heterocyclic Compounds, 4 or More Rings / Q000494:pharmacology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009124:Muscle Proteins / Q000378:metabolism*; D009200:Myocardial Contraction / Q000187:drug effects / Q000502:physiology; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D011494:Protein Kinases / Q000378:metabolism*; D012518:Sarcomeres / Q000378:metabolism / Q000502:physiology; D013314:Stress, Mechanical
chemical_list: D000077482:Carbon Fiber; D064211:Connectin; D006576:Heterocyclic Compounds, 4 or More Rings; D009124:Muscle Proteins; C004717:diacetylmonoxime; C472645:blebbistatin; D002244:Carbon; D000205:Actomyosin; D011494:Protein Kinases; D003931:Diacetyl; D002118:Calcium