Effects of pseudo-phosphorylated rat cardiac troponin T are differently modulated by α- and β-myosin heavy chain isoforms

Abstract

Interplay between the protein kinase C (PKC)-mediated phosphorylation of troponin T (TnT)- and myosin heavy chain (MHC)-mediated effects on thin filaments takes on a new significance because: (1) there is significant interaction between the TnT- and MHC-mediated effects on cardiac thin filaments; (2) although the phosphorylation of TnT by PKC isoforms is common to both human and rodent hearts, human hearts predominantly express β-MHC while rodent hearts predominantly express α-MHC. Therefore, we tested how α- and β-MHC isoforms differently affected the functional effects of phosphorylated TnT. Contractile measurements were made on cardiac muscle fibers from normal rats (α-MHC) and propylthiouracil-treated rats (β-MHC), reconstituted with the recombinant phosphomimetic-TnT (T204E; threonine 204 replaced by glutamate). Ca2+ -activated maximal tension decreased differently in α-MHC + T204E (~68%) and β-MHC + T204E (~35%). However, myofilament Ca2+ sensitivity decreased similarly in α-MHC + T204E and β-MHC + T204E, demonstrating that a decrease in Ca2+ sensitivity alone cannot explain the greater attenuation of tension in α-MHC + T204E. Interestingly, dynamic contractile parameters (rates of tension redevelopment, crossbridge (XB) recruitment dynamics, XB distortion dynamics, and XB detachment kinetics) decreased only in α-MHC + T204E. Thus, the transition of thin filaments from the blocked- to closed-state was attenuated in α-MHC + T204E and β-MHC + T204E, but the closed- to open-state transition was attenuated only in α-MHC + T204E. Our study demonstrates that the effects of phosphorylated TnT and MHC isoforms interact to bring about different functional states of cardiac thin filaments.

The effect of cardiomyopathy mutation (R97L) in mouse cardiac troponin T on the muscle length-mediated recruitment of crossbridges is modified divergently by α- and β-myosin heavy chain

“…Previous studies have correlated tension cost to c and demonstrated that both are useful measures of g [11, 12, 35, 36]. Thus similar effects in both these parameters would validate if TnT R97L altered g .…”

The functional effect of dilated cardiomyopathy mutation (R144W) in mouse cardiac troponin T is differently affected by α- and β-myosin heavy chain isoforms

“…Proteins were visualized by staining the gel with Coomassie brilliant blue (R-250; Bio-Rad Laboratories, Hercules, CA). Densitometric analysis of the MHC protein profiles was performed using the ImageJ software (available on the National Institutes of Health web site), as described previously (14,15,30). For assessing the phosphorylation status of sarcomeric proteins, 10 g of standardized NTG and TG mouse heart samples were loaded and ran on a 12.5% small SDS gel.…”

“…Small differences in phosphorylation levels of MLC-1 and MLC-2 are not expected to affect our findings because 1) H886 DIVERSE EFFECTS OF TNT-R144W AGAINST ␣-AND ␤-MHC observed differences in contractile parameters (maximal tension, pCa 50 , tension cost, c, b, k tr , etc.) between ␣-and ␤-MHC fibers are similar to those reported by previous studies (10,12,29,30,49); and 2) the effect of TnT R144W is assessed relative to TnT WT in each group, i.e., ␣-MHC ϩ TnT R144W vs. ␣-MHC ϩ TnT WT or ␤-MHC ϩ TnT R144W vs. ␤-MHC ϩ TnT WT .…”

“…Previous studies have shown that TnT R144W slows XB detachment rate (g) in ␣-MHC containing muscle fibers (32,50,51) by affecting the structure of the thin filament. Because ␣-and ␤-MHC isoforms also have a different impact on the structure of thin filaments (10,12,29,30,49), we predicted that the TnT R144W -mediated impact on g would be differently modulated by ␣-and ␤-MHC isoforms. To assess the effects of TnT R144W on XB detachment kinetics, we used estimates of tension cost and c. Previous studies have shown that tension cost is correlated to c and that both parameters are measures of XB detachment rate, g (2).…”

“…The ability of a given MHC isoform to alter the TnT-mediated function is related to its innate kinetic properties. Due to the longer XB dwell time, a slower cycling ␤-MHC isoform (10, 41, 43) may modify thin filament-based cooperative mechanisms and dynamic contractile behavior (4,10,29,30,43). Because the heart muscle comprises dynamically interacting components, it is expected that the mutant TnT-and the MHC-induced changes in the thin filament will interact to yield properties that are abnormal.…”