Omecamtiv mecarbil (OM) is a putative positive inotropic tool for treatment of systolic heart dysfunction, based on the finding that in vivo it increases the ejection fraction and in vitro it prolongs the actin-bond life time of the cardiac and slow-skeletal muscle isoforms of myosin. OM action in situ, however, is still poorly understood as the enhanced Ca 2+-sensitivity of the myofilaments is at odds with the reduction of force and rate of force development observed at saturating Ca 2+. Here we show, by combining fast sarcomere-level mechanics and ATPase measurements in single slow demembranated fibres from rabbit soleus, that the depressant effect of OM on the force per attached motor is reversed, without effect on the ATPase rate, by physiological concentrations of inorganic phosphate (Pi) (1-10 mM). This mechanism could underpin an energetically efficient reduction of systolic tension cost in OM-treated patients, whenever [Pi] increases with heartbeat frequency.
Polycystin-2 (PC2, TRPP2) is a Ca 2þ -permeable nonselective cation channel from the TRP superfamily. PC2 is encoded by the PKD2 gene, whose mutations are responsible for autosomal dominant polycystic kidney disease (ADPKD). Recent studies from our laboratory (Dai et al, Exp Cell Res, 2017), determined that the PC2 contribution to the whole cell conductance of wild type LLC-PK1 renal epithelial cells is regulated by changes in external Ca 2þ concentration, in a mechanism that implicates the Calcium-Sensing Receptor (CaSR). The present study explored the effect of Ca 2þ and CaSR agonists in the regulation of PC2 from isolated LLC-PK1 plasma membranes reconstituted in a lipid bilayer system (BLM). Addition of the CaSR agonist R-568 (5 mM) to the ''external'' side of the bilayer, activated single channel currents consistent with a functional PC2 by 84 5 12% (n = 4, p < 0.01). These cation-selective currents were subsequently inhibited (91.3 5 1.1%, p < 0.0001) by addition to the ''cytosolic'' side of the BLM, of a commercially available anti-PC2 C-terminal antibody to the ''cytosolic'' or ''internal'' side of the BLM. Interestingly, similar findings were observed with the material obtained by co-immunoprecipitation of LLC-PK1 cell lysate with anti-CaSR antibody crosslinked in agarose beads. The data indicate the presence of a membrane locate CaSR-PC2 functional complex in LLC-PK1 renal epithelial cells. The presence of the receptor/channel complex is in agreement with the regulatory role external Ca 2þ plays in the modulation of PC2 channel function in the plasma membrane of renal epithelial cells. This receptor-channel complex may be part of a regulatory mechanism connecting Ca 2þ signals and PC2 function to onset of ADPKD.
generating. To investigate how EMB11d enabled cyclical power generation, we varied [Pi]. We found SA force increased 72%, force redevelopment rate sped up 3-fold, the frequency at which maximum power was generated rose 2-fold, and the amount of power generated jumped 3-fold as [Pi] increased from 0mM to 16mM. In contrast, wild-type TDT exhibited only 30% increase in SA force, and no power production as [Pi] increased. This supports our hypothesis for how some myosin isoforms generate higher SA force: That stretch and Pi binding drive a subpopulation of myosin heads back into a weakly bound state. This results in a greater number of weakly bound heads subsequently rebinding to actin, causing the delayed SA tension increase. We postulate that the cardiac myosin isoform increases SA because it can more easily be driven backward by a combination of stretch and elevated [Pi] than the native TDT isoform.
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