The effects of slow skeletal troponin I expression in the murine myocardium are influenced by development-related shifts in myosin heavy chain isoform

Abstract

Troponin I (TnI) and myosin heavy chain (MHC) are two contractile regulatory proteins that undergo major shifts in isoform expression as cardiac myocytes mature from embryonic to adult stages. To date, many studies have investigated individual effects of embryonic vs. cardiac isoforms of either TnI or MHC on cardiac muscle function and contractile dynamics. Thus, we sought to determine whether concomitant expression of the embryonic isoforms of both TnI and MHC had functional effects that were not previously observed. Adult transgenic (TG) mice that express the embryonic isoform of TnI, slow skeletal TnI (ssTnI), were treated with propylthiouracil (PTU) to revert MHC expression from adult (α-MHC) to embryonic (β-MHC) isoforms. Cardiac muscle fibres from these mice contained ∼80% β-MHC and ∼34% ssTnI of total MHC or TnI, respectively, allowing us to test the functional effects of ssTnI in the presence of β-MHC. Detergent-skinned cardiac muscle fibre bundles were used to study how the interplay between MHC and TnI modulate muscle length-mediated effect on crossbridge (XB) recruitment dynamics, Ca(2+)-activated tension, and ATPase activity. One major finding was that the model-predicted XB recruitment rate (b) was enhanced significantly by ssTnI, and this speeding effect of ssTnI on XB recruitment rate was much greater (3.8-fold) when β-MHC was present. Another major finding was that the previously documented ssTnI-mediated increase in myofilament Ca(2+) sensitivity (pCa(50)) was blunted when β-MHC was present. ssTnI expression increased pCa(50) by 0.33 in α-MHC fibres, whereas ssTnI increased pCa(50) by only 0.05 in β-MHC fibres. Our study provides new evidence for significant interplay between MHC and TnI isoforms that is essential for tuning cardiac contractile function. Thus, MHC-TnI interplay may provide a developmentally dependent mechanism to enhance XB recruitment dynamics at a time when Ca(2+)-handling mechanisms are underdeveloped, and to prevent excessive ssTnI-dependent inotropy (increased Ca(2+) sensitivity) in the embryonic myocardium.

Figure 1. Representative chirp response data

Representative force response (A) to chirp-length perturbation (B) from a maximally activated muscle fibre from control (untreated NTG) mouse cardiac muscle fibre

Figure 2. Representative chirp response model prediction

Representative model-predicted force response (A) to chirp-perturbation of the same responses from the WT fibre data shown in Fig. 1. Model components are shown to illustrate: the slow phase, XB-recruitment component of the force response (B); the fast phase, strong XB-distortion component of the response (C); and the fastest phase, weak XB-distortion component of the response (D). The shape and magnitude of the response shown in panel A represents the summation of each of the model components. The shape and magnitude of the components shown in panels B, C and D are dependent on the values of their respective parameters.

Figure 3. Assessment of sarcomeric protein content and phosphorylation status

SDS-PAGE to determine the expression profiles of MHC isoform (A) and Western blot analysis to determine level of TnI isoform expression (B) of hearts from normal (α-MHC) or PTU-treated (β-MHC) cTnI NTG or ssTnI TG mice. Pro-Q Diamond staining (C) was done to determine the effects of PTU treatment or TG ssTnI expression on phosphorylation of sarcomeric proteins. Images are representative data from samples collected from at least 3 hearts in each group.

Figure 4. Relationship between frequency of length perturbation and magnitude of force response

Model-predicted force responses to sinusoidal length perturbations of increasing frequency are shown from fibres of α-MHC (A) or β-MHC (B) NTG and TG mouse hearts. Low-frequency model components are shown for fibres from α-MHC (C) or β-MHC (D) mouse hearts to illustrate the ssTnI effect on the model component dominated by XB-recruitment dynamics. The magnitude of F(t) was normalized to the maximal value observed as frequency approached infinity.

Figure 5. Estimation of the rate of XB recruitment and tension redevelopment

A, the model-predicted XB recruitment rate constant determined by fits to chirp data from α-MHC or β-MHC cTnI NTG or ssTnI TG mouse fibres. B, the rate constant of tension redevelopment determined by a rapid release and restretch protocol at maximal Ca²⁺ activation. Parameter values are represented as mean + SEM. Number of determinants was at least 10 for each group. *P < 0.05; ***P < 0.001.

Figure 6. Rate constant of tension redevelopment, k_tr, plotted as a function of level of activation (tension as normalized to maximal fibre tension, Tens/Tens_Max)

Because k_tr exhibits an exponential trend with an increase in the level of activation, the logarithm of k_tr vs. activation was plotted to linearize the trend. Regression analysis determined the intercept of the relationship of k_tr vs. activation, which approximates the apparent rate of XB detachment, g_app (Baker et al. 1998; Palmer & Kentish, 1998; Fitzsimons et al. 2001; Tesi et al. 2002; de Tombe & Stienen, 2007). As show in panel A, the predicted intercept between α-MHC(cTnI) (continuous line) and α-MHC(ssTnI) (dashed line) fibres was not different. As show in panel B, the predicted intercept was slightly lower in β-MHC(cTnI) fibres (continuous line) when compared to β-MHC(ssTnI) fibres (dashed line) was slightly different. This difference in the intercept was significant, suggesting that g_app was slightly faster in β-MHC(ssTnI) vs. β-MHC(cTnI) fibres (Table 1).

Figure 7. pCa-tension relationships in fibres from α-MHC (A) or β-MHC (B) mice from cTnI NTG groups (•, continuous lines are Hill model fits) and ssTnI TG groups (○, dashed lines are Hill model fits)

Data were normalized to maximal tension produced (pCa 4.3) by each respective fibre. Normalized curves were fitted to Hill's equation to determine the pCa₅₀ and Hill's cooperativity coefficient, n_H, for each group of fibres. As shown in Table 2, pCa₅₀ was 5.78 ± 0.01 and 6.11 ± 0.01 for α-MHC(cTnI) and α-MHC(ssTnI) fibres, respectively, and 5.85 ± 0.01 and 5.90 ± 0.01 for β-MHC(cTnI) and β-MHC(ssTnI) fibres, respectively. n_H was 2.16 ± 0.09 and 1.80 ± 0.04 for α-MHC(cTnI) and α-MHC(ssTnI) fibres, respectively, and 2.46 ± 0.06 and 2.62 ± 0.05 for β-MHC(cTnI) and β-MHC(ssTnI) fibres, respectively. Each point represents the average normalized tension produced from all fibres in each group ± SEM. Number of determinants was at least 10 for each group.