Burghardt, Thomas P.

Cardiac ventricular myosin (βmys) translates actin by transducing ATP free energy into mechanical work during muscle contraction. Unitary βmys translation of actin is the step-size. In vitro and in vivo βmys regulates contractile force and velocity autonomously by remixing three different step-sizes with adaptive stepping frequencies. Cardiac and skeletal actin isoforms have a specific 1 : 4 stoichiometry in normal adult human ventriculum. Human adults with inheritable hypertrophic cardiomyopathy (HCM) upregulate skeletal actin in ventriculum probably compensating the diseased muscle's inability to meet demand by adjusting βmys force–velocity characteristics. βmys force–velocity characteristics were compared for skeletal versus cardiac actin substrates using ensemble in vitro motility and single myosin assays. Two competing myosin strain-sensitive mechanisms regulate step-size choices dividing single βmys mechanics into low- and high-force regimes. The actin isoforms alter myosin strain-sensitive regulation such that onset of the high-force regime, where a short step-size is a large or major contributor, is offset to higher loads probably by the unique cardiac essential light chain (ELC) N-terminus/cardiac actin contact at Glu6/Ser358. It modifies βmys force–velocity by stabilizing the ELC N-terminus/cardiac actin association. Uneven onset of the high-force regime for skeletal versus cardiac actin modulates force–velocity characteristics as skeletal/cardiac actin fractional content increases in diseased muscle.

Myosin transduces ATP free energy into mechanical work in muscle. Cardiac muscle has dynamically wide-ranging power demands on the motor as the muscle changes modes in a heartbeat from relaxation, via auxotonic shortening, to isometric contraction. The cardiac power output modulation mechanism is explored in vitro by assessing single cardiac myosin step-size selection versus load. Transgenic mice express human ventricular essential light chain (ELC) in wild- type (WT), or hypertrophic cardiomyopathy-linked mutant forms, A57G or E143K, in a background of mouse α-cardiac myosin heavy chain. Ensemble motility and single myosin mechanical characteristics are consistent with an A57G that impairs ELC N-terminus actin binding and an E143K that impairs lever-arm stability, while both species down-shift average step-size with increasing load. Cardiac myosin in vivo down-shifts velocity/force ratio with increasing load by changed unitary step-size selections. Here, the loaded in vitro single myosin assay indicates quantitative complementarity with the in vivo mechanism. Both have two embedded regulatory transitions, one inhibiting ADP release and a second novel mechanism inhibiting actin detachment via strain on the actin-bound ELC N-terminus. Competing regulators filter unitary step-size selection to control force-velocity modulation without myosin integration into muscle. Cardiac myosin is muscle in a molecule.

Partial agonists are effective tools for advancing development of highly selective drugs and providing insights into molecular regulation of cellular functions. Here, we explore the impact of a partial agonist on key aspects of cholecystokinin (CCK) receptor regulation, its lateral mobility and cellular trafficking, in native pancreatic acinar cells and Chinese hamster ovary cells expressing CCK receptor (CHO-CCKR). We developed and characterized a novel fluorescent partial agonist, rhodamine-Gly-[(Nle28,31)CCK-26–32]-phenethyl ester, that binds specifically and with high affinity to CCK receptors. Such analogs are fully efficacious pancreatic acinar cell secretagogues without supramaximal inhibition that mobilize intracellular calcium with little or no increase in phospholipase C (PLC) activity. Despite minimal phosphorylation of CCK receptors in response to this partial agonist, receptor trafficking was the same as that observed with full agonist (CCK). This included normal internalization via clathrin-dependent endocytosis in CHO-CCKR cells and insulation on the surface of pancreatic acinar cells. Also, as with CCK-occupied receptor, fluorescence recovery after photobleaching of partial agonist-occupied receptor on the acinar cell surface demonstrated a marked temperature-dependent slowing of its rate of diffusion. This was similarly associated with resistance to acid-induced dissociation of ligand. Thus some key molecular regulatory mechanisms for CCK receptor internalization and insulation may be initiated by cellular signaling cascades that are not dependent on PLC activation or receptor phosphorylation.

Muscle myosins transduce ATP free energy into actin displacement to power contraction. In vivo , myosin side chains are modified post-translationally under native conditions, potentially impacting function. Single myosin detection provides the ‘bottom-up’ myosin characterization probing basic mechanisms without ambiguities inherent to ensemble observation. Macroscopic muscle physiological experimentation provides the definitive ‘top-down’ phenotype characterizations that are the concerns in translational medicine. In vivo single myosin detection in muscle from zebrafish embryo models for human muscle fulfils ambitions for both bottom-up and top-down experimentation. A photoactivatable green fluorescent protein (GFP)-tagged myosin light chain expressed in transgenic zebrafish skeletal muscle specifically modifies the myosin lever-arm. Strychnine induces the simultaneous contraction of the bilateral tail muscles in a live embryo, causing them to be isometric while active. Highly inclined thin illumination excites the GFP tag of single lever-arms and its super-resolution orientation is measured from an active isometric muscle over a time sequence covering many transduction cycles. Consecutive frame lever-arm angular displacement converts to step-size by its product with the estimated lever-arm length. About 17% of the active myosin steps that fall between 2 and 7 nm are implicated as powerstrokes because they are beyond displacements detected from either relaxed or ATP-depleted (rigor) muscle.