We investigated the molecular mechanism by which troponin (Tn) regulates the Frank-Starling mechanism of the heart. Quasi-complete reconstitution of thin filaments with rabbit fast skeletal Tn (sTn) attenuated length-dependent activation in skinned porcine left ventricular muscle, to a magnitude similar to that observed in rabbit fast skeletal muscle. The rate of force redevelopment increased upon sTn reconstitution at submaximal levels, coupled with an increase in Ca2+ sensitivity of force, suggesting the acceleration of cross-bridge formation and, accordingly, a reduction in the fraction of resting cross-bridges that can potentially produce additional active force. An increase in titin-based passive force, induced by manipulating the prehistory of stretch, enhanced length-dependent activation, in both control and sTn-reconstituted muscles. Furthermore, reconstitution of rabbit fast skeletal muscle with porcine left ventricular Tn enhanced length-dependent activation, accompanied by a decrease in Ca2+ sensitivity of force. These findings demonstrate that Tn plays an important role in the Frank-Starling mechanism of the heart via on–off switching of the thin filament state, in concert with titin-based regulation.

Previously we have introduced a single cell system allowing long term culturing (1 week) of adult rat ventricular myocytes while maintaining their overall morphology, contractile behaviour and calcium-signalling. Here, we characterize the subcellular morphology of the myocytes, including the Golgi apparatus, endo-/sarcoplasmic reticulum (ER/SR), plasma membrane and mitochondria. Cells were isolated from adult rats following a standard enzymatic procedure. Organelles were labelled using targeted expression of fluorescent proteins, e.g. dsRed1 fused to the subunit VIII of human cytochrome C oxidase for mitochondria, YFP fused to a GPI-anchor for the plasma membrane, YFP fused to ts045G for the Golgi apparatus and dsRed2 fused to calreticulin for the ER/ SR. Complementing this we also applied fluorescent dyes; di-8-ANEPPS for the plasma membrane and MitoTracker Green for the mitochondria. 3-dimensional stacks of individual cells were acquired with a nipkow-disc based confocal microscope. Using both labelling approaches, the analysis of the plasma membrane illustrated a gradual loss of the t-tubules during culturing with cytosolic membrane fragments being present for extended time periods. Mitochondria, which are very prominent and densely packed in cardiac myocytes, underwent an apparent fusion of originally isolated mitochondria, possibly reflecting the loss of t-tubules. While the structure of the ER/SR remained unaltered, the Golgi apparatus underwent a significant redistribution during the culturing time from a wide cytosolic distribution to a perinuclear accumulation. We provide important evidence that cell morphology changes unavoidably occurring in adult cardiac myocytes during long term culture are highly reproducible and thus strongly support the application of such a single cell model in high-content screening applications.