We study the regulation of contraction of cardiac muscle by using ultra low-angle X-ray diffraction at the ID02-beamline of the European Synchrotron (ESRF, Grenoble, France) to record the nanometer-scale structural signals from the contractile proteins along the thin and thick filaments and the micrometer scale changes in the sarcomere length (SL). By recording the X-ray diffraction signals that mark the state of the thick filament during the contraction-relaxation cycle of an electrically paced trabecula (0.5 Hz, SL 2.2 µm, 27 °C), dissected from the right ventricle of the heart of male rats (230-280 g, aged 2-3 months) under isoflurane-induced deep anaesthesia (5% v/v), we show that (i) during the diastole most of the myosin motors lie on the surface of the thick filament packed into helical tracks in the OFF state, unavailable for actin binding and ATP hydrolysis (Woodhead et al. 2005; Stewart et al. 2010); (ii) during the systole the myosin motors leave the OFF state in relation to the loading condition, revealing a positive feedback mechanism based on thick-filament mechano-sensing (Reconditi et al. 2017). The same mechanism has been previously demonstrated in skeletal muscle (Linari et al. 2015). Thus, even if in an isometric tetanus of skeletal muscle force is under the control of the firing frequency of the motor unit, while in a heartbeat force is controlled by the afterload, the stress-sensor switching the motors ON plays the same role in adapting the energetic cost of the contraction to the force (Piazzesi et al. 2018). In a heartbeat the internal [Ca2+] ([Ca2+]i) may not reach the level for full thin filament activation and the mechanical response depends on both [Ca2+]i and Ca2+-sensitivity of the filament, parameters that are under the control of several regulatory systems, like the relation between SL and systolic force (a property known as Length Dependent Activation) and the degree of phosphorylation of contractile, regulatory, and cytoskeletal proteins. Here we test the integration between thin and thick filament regulation mechanisms by recording the X-ray signals that mark the state of the thick filament during two thin-filament mediated inotropic interventions able to potentiate up to twofold the twitch force developed by trabeculae at SL 2.0 µm and with 1 mM external [Ca2+]: either SL increase to 2.25 µm or addition to the solution of the b-adrenergic effector isoprenaline (10-7 M). In diastole none of the signals related to the OFF state of the thick filament is affected by either intervention. The results further clarify the role of the thick filament mechano-sensing as the energetically well-suited downstream mechanism that rapidly adapts the number of myosin motors leaving the OFF state to the systolic force. Supported by Ente Cassa di Risparmio di Firenze.