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

Abstract

Given the differential impact of α- and β-myosin heavy chain (MHC) isoforms on how troponin T (TnT) modulates contractile dynamics, we hypothesized that the effects of dilated cardiomyopathy (DCM) mutations in TnT would be altered differently by α- and β-MHC. We characterized dynamic contractile features of normal (α-MHC) and transgenic (β-MHC) mouse cardiac muscle fibers reconstituted with a mouse TnT analog (TnTR144W) of the human DCM R141W mutation. TnTR144W did not alter maximal tension but attenuated myofilament Ca(2+) sensitivity (pCa50) to a similar extent in α- and β-MHC fibers. TnTR144W attenuated the speed of cross-bridge (XB) distortion dynamics (c) by 24% and the speed of XB recruitment dynamics (b) by 17% in α-MHC fibers; however, both b and c remained unaltered in β-MHC fibers. Likewise, TnTR144W attenuated the rates of XB detachment (g) and tension redevelopment (ktr) only in α-MHC fibers. TnTR144W also decreased the impact of strained XBs on the recruitment of new XBs (γ) by 30% only in α-MHC fibers. Because c, b, g, ktr, and γ are strongly influenced by thin filament-based cooperative mechanisms, we conclude that the TnTR144W- and β-MHC-mediated changes in the thin filament interact to produce a less severe functional phenotype, compared with that brought about by TnTR144W and α-MHC. These observations provide a basis for lower mortality rates of humans (β-MHC) harboring the TnTR141W mutant compared with transgenic mouse studies. Our findings strongly suggest that some caution is necessary when extrapolating data from transgenic mouse studies to human hearts.

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

“…Previously, we demonstrated that the level of β-MHC expression is ~70% of the total MHC in TG mouse hearts [11]. Moreover, β-MHC expression did not alter the stoichiometry or the phosphorylation status of other sarcomeric proteins [11].…”

“…Moreover, β-MHC expression did not alter the stoichiometry or the phosphorylation status of other sarcomeric proteins [11]. c - myc tagged TnT and the endogenous TnT migrated differently on the SDS gel.…”

“…The composition of HR solution is listed in our previous works [11, 13]. Left ventricular (LV) papillary muscle bundles were extracted and further dissected into smaller muscle bundles (~0.15 mm in cross-section and 2.0 mm in length) and chemically-skinned overnight at 4°C in HR solution containing 1% Triton X-100.…”

“…Whole Tn was reconstituted into detergent-skinned muscle fibers, as described elsewhere [11, 27]. Briefly, TnT WT or TnT R97L (0.9 mg/ml, W/V) and TnI (1.0 mg/ml, W/V) were dissolved in an extraction buffer ( buffer 1 : 50 mM Tris-HCl (pH 8.0), 6 M urea, 1.0 M KCl, and a cocktail of protease inhibitors).…”

“…The muscle fibers were then exposed to various Ca 2+ solutions with ranging from 9.0 to 4.3 in a constantly-stirred chamber and the corresponding steady-state force values were measured. The compositions of various pCa solutions were based on the program by Fabiato and Fabiato [31], and these were listed in our earlier works [11, 13]. The pH and ionic strength of pCa solutions were adjusted to 7.0 and 180 mM, respectively.…”