Developmental changes in contractility and sarcomeric proteins from the early embryonic to the adult stage in the mouse heart

Abstract

Developmental changes in force-generating capacity and Ca2+ sensitivity of contraction in murine hearts were correlated with changes in myosin heavy chain (MHC) and troponin (Tn) isoform expression, using Triton-skinned fibres. The maximum Ca2+-activated isometric force normalized to the cross-sectional area (FCSA) increased mainly during embryogenesis and continued to increase at a slower rate until adulthood. During prenatal development, FCSA increased about 5-fold from embryonic day (E)10.5 to E19.5, while the amount of MHC normalized to the amount of total protein remained constant (from E13.5 to E19.5). This suggests that the development of structural organization of the myofilaments during the embryonic and the fetal period may play an important role for the improvement of force generation. There was an overall decrease of 0.5 pCa units in the Ca2+ sensitivity of force generation from E13.5 to the adult, of which the main decrease (0.3 pCa units) occurred within a short time interval, between E19.5 and 7 days after birth (7 days pn). Densitometric analysis of SDS-PAGE and Western blots revealed that the major switches between troponin T (TnT) isoforms occur before E16.5, whereas the transition points of slow skeletal troponin I (ssTnI) to cardiac TnI (cTnI) and of beta-MHC to alpha-MHC both occur around birth, in temporal correlation with the main decrease in Ca2+ sensitivity. To test whether the changes in Ca2+ sensitivity are solely based on Tn, the native Tn complex was replaced in fibres from E19.5 and adult hearts with fast skeletal Tn complex (fsTn) purified from rabbit skeletal muscle. The difference in pre-replacement values of pCa50 (-log([Ca2+] M-1)) required for half-maximum force development) between E19.5 (6.05 +/- 0.01) and adult fibres (5.64 +/- 0.04) was fully abolished after replacement with the exogenous skeletal Tn complex (pCa50 = 6.12 +/- 0.05 for both stages). This suggests that the major developmental changes in Ca2+ sensitivity of skinned murine myocardium originate primarily from the switch of ssTnI to cTnI.

Figure 6. Western blots of native skeletal and cardiac muscle fibres and of cardiac fibres after the replacement of the native Tn complex with exogenous fsTn complex

A, Western blot with anti-TnT antibody. B, Western blot with anti-TnI antibody. For the detection of both TnT and TnI bands, blots were stripped after incubation with anti-TnT antibody and then incubated with anti-TnI antibody. Soleus: mouse soleus muscle as a control for ssTnI; Psoas: Tn complex prepared from rabbit psoas muscle used as exogenous Tn; E19.5 and Adult: skinned ventricular fibres from fetal (E19.5) and adult (6 weeks pn) mouse, respectively; E19.5 after exchange and Adult after exchange: skinned ventricular fibres from fetal and adult mouse, respectively, each 3 h after replacement of native Tn complex by 1.5 mg ml⁻¹ purified rabbit psoas fsTn complex.

Figure 1. Quantification of myosin heavy chain (MHC) and actin in the developing murine ventricle by SDS-PAGE

A, SDS-polyacrylamide gel loaded with equal amounts of total protein isolated from skinned ventricular tissue at different developmental stages. B, developmental change in the MHC content normalized to content of total protein. C, ratio of MHC to actin content at the different developmental stages. Relationships shown in B and C were obtained by densitometric evaluation of 5 electrophoretic patterns of the type shown in A. The molar ratio of MHC to actin in C was calculated from the ratio of their densitometric intensities based on molecular masses of 220 kDa and 42 kDa for MHC and actin, respectively. D, electrophoretic patterns of MHC isoforms at different developmental stages. MHC was extracted from ventricular tissue and the isoforms were separated on 8 % polyacrylamide gels containing 30 % glycerol.

Figure 2. Relationship of the increase in maximum force-generating ability to the increase in MHC concentration during murine heart development

Maximum Ca²⁺-activated isometric force normalized to the cross-sectional area (F_CSA) of skinned fibres (○) was determined at pCa 4.5 and 10 °C. Data are means ±s.e.m. of 4–10 fibres at each stage (see Table 1). MHC/wet weight of the skinned heart tissue sample (□) was determined as described in Methods. Data are means ±s.e.m. of 5 samples at each stage. Ratios of F_CSA to MHC/wet weight (▵) were calculated from the means of the F_CSA and the MHC/wet weight data. All data were normalized to the respective values determined for the adult stage and were plotted on a logarithmic scale to adequately illustrate the relative change of parameters during development.

Figure 3. Developmental changes in Ca²⁺ sensitivity of force development

Force-pCa relationships of embryonic (embryonic day (E)11.5, light green square; E13.5, dark green triangle; E16.5 blue inverted triangle), fetal (E19.5, dark pink circle), neonatal (7 days postnatal (days pn), red diamond) and adult (6–8 weeks pn, black circle) skinned cardiac muscle fibres, determined at 10 °C. Force was normalized to the maximal force generated by each fibre. Data are means ±s.e.m. of 4–11 experiments at each stage. The lines were drawn by fitting the data to the Hill equation.

Figure 5. Relation between developmental changes in the relative amounts of Tn isoforms and in Ca²⁺ sensitivity of isometric force generation

Quantification of relative amounts of TnT (A) and TnI (B) isoforms was performed by densitometric evaluation of Western blots (n = 3). C, pCa₅₀ data are given as means ±s.e.m. of 5–11 experiments at each stage (see Table 1). The main TnT switching occurs from E13.5 to E16.5 when TnT₄ (▿) becomes the predominant isoform in parallel with a slight but significant (P < 0.05) decrease in pCa₅₀ values, i.e. in Ca²⁺ sensitivity. The most pronounced decrease in Ca²⁺ sensitivity occurs around birth from E19.5 to 7 days pn and is found to be closely related to the increase in cTnI. During the main decrease in Ca²⁺ sensitivity around birth, there are rather small changes in the relative amounts of TnT isoforms represented by the occurrence of TnT₃ (▵) and the loss of TnT₁ (○) and TnT₂ (□).

Figure 4. Developmental changes of TnI and TnT isoform patterns in the murine heart

Western blots of soleus muscle and myocardium from different maturational stages with anti-TnT antibody (A) and anti-TnI antibody (B). Both antibodies react with skeletal and cardiac isoforms. Immunoreactivity of anti-TnI antibody was experimentally determined to be identical for cardiac and skeletal TnI isoforms (not shown). Four cardiac TnT isoforms (cTnT_1–4) with smaller molecular masses than the TnT isoforms of soleus muscle (sTnT) are detected in the developing heart. cTnI is first detected at E19.5.

Figure 7. Effect of Tn replacement on force-pCa relationships of fetal (E19.5) and adult (6–8 weeks pn) cardiac fibres

A, prior to the replacement, fetal fibres (□) are more sensitive to [Ca²⁺] than adult fibres (▵). B, after the replacement, fetal (□) and adult (▵) fibres show the same Ca²⁺ sensitivity, i.e. pCa₅₀ value.