J Mol Cell Cardiol 31, 1215–1227 (1999) Article No. jmcc.1999.0954, available online at http://www.idealibrary.com on Length Modulation of Active Force in Rat Cardiac Myocytes: is Titin the Sensor? Olivier Cazorla 1 , Guy Vassort 2 , Didier Garnier 1 and Jean-Yves Le Guennec 1 1 Laboratoire de Physiologie des Cellules Cardiaques et Vasculaires, CNRS UMR 6542, Faculte ´ des Sciences, F-37200 Tours, France, 2 Physiopathologie Cardiovasculaire, INSERM U-390, CHU Arnaud de Villeneuve, F-34295 Montpellier, France (Received 10 September 1998, accepted in revised form 5 March 1999) O. C, G. V , D. G  J.-Y. L G. Length Modulation of Active Force in Rat Cardiac Myocytes: is Titin the Sensor?. Journal of Molecular and Cellular Cardiology (1999) 31, 1215–1227. The intrinsic cellular mechanisms by which length regulates myocardial contraction, the basis of the Frank–Starling relation, are uncertain. The aim of this work was to test the hypothesis that passive force, possibly via titin, participates in the modulation of Ca 2+ sensitivity of cardiac contractile proteins induced by stretch. Titin degradation by a mild trypsin digestion modulated passive force induced by increasing from 1.9 to 2.3 m sarcomere length in skinned rat cardiac cells. Force–pCa curves were established at these two sarcomere lengths after various durations of trypsin application that induced different passive force levels. They allowed us to evaluate myofilament Ca 2+ sensitivity by the pCa of half-maximal activation (pCa 50 ). In control conditions, stretching cells from 1.9 to 2.3 m induced a leftward shift of pCa 50 (pCa 50 ) of 0.39±0.03 pCa units (mean±SEM, n=8 cells), reflecting an increase in Ca 2+ sensitivity of the contractile machinery. Passive force measured every 2 min decreased exponentially after the beginning of the trypsin application (t 1/2 ≈12 min). The first 30% decrease of passive force did not affect the stretch-induced variation in Ca 2+ sensitivity. Then, with further decrease in passive force, pCa 50 decreased. At the lowest passive force investigated 20% of initial passive force, pCa 50 decreased by approximately 55%. These effects were not accompanied by a significant modification of either maximal activated force at pCa 4.5 solution or pCa 50 at 1.9 m sarcomere length. This indicates that there was no major functional alteration of the contractile machinery during the protocol as also suggested by contractile and regulatory protein electrophoresis on 2.5–12% gradient and 15% SDS–PAGE gels. Thus, besides modulation induced by the reduced lattice spacing during enhanced heart refilling, Ca 2+ sensitivity of the cardiac contractile machinery may be controlled at least partially by internal passive load, which is known to be largely attributable to titin.  1999 Academic Press K W: Titin; Cardiomyocytes; Contraction; Excitation–contraction coupling; Passive load; Stretch; Myofilament Ca 2+ sensitivity; Rat. length could be an important modulator of the Introduction length-dependent force generation of contraction. Thus in skinned cardiac muscle fibers, it has been One of the most salient characteristics of cardiac muscle is the enhancement of contractile force in reported that degrees of lattice compression com- parable to sarcomere elongation produced com- response to an increase in end-diastolic volume, a phenomenon known as the Frank–Starling re- parable changes in myofilament Ca 2+ sensitivity (McDonald and Moss, 1995; Wang and Fuchs, lationship. The intrinsic cellular mechanism by which ventricular filling, and consequently muscle 1995; Fuchs and Wang, 1996). Some other ex- perimental evidences suggest modulations of the length, regulates myocardial contraction is yet un- certain, although several hypotheses have been affinity of troponin C (TnC) for Ca 2+ ions. This could be related to stretch (Hibberd and Jewell, 1982; proposed (Allen and Kentish, 1985; Lakatta, 1991). The interfilament spacing rather than sarcomere Allen and Kentish, 1988; Babu et al., 1988), or to Please address all correspondence to: Dr Jean-Yves Le Guennec, Laboratoire de Physiologie des Cellules Cardiaques et Vasculaires, CNRS UMR 6542, Faculte ´ des Sciences, 37200 Tours, France. 0022–2828/99/061215+13 $30.00/0  1999 Academic Press