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The Journal of Physiology logoLink to The Journal of Physiology
. 2003 Mar 14;548(Pt 2):493–505. doi: 10.1113/jphysiol.2002.036509

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

Sharon Siedner *, Martina Krüger *, Mechthild Schroeter *, Doris Metzler *, Wilhelm Roell *, Bernd K Fleischmann *, Juergen Hescheler *, Gabriele Pfitzer *, Robert Stehle *
PMCID: PMC2342849  PMID: 12640016

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 β-MHC to α-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.

During cardiac development, changes in the expression of sarcomeric proteins occur in temporal relation to marked alterations of functional properties. The force-generating capacity has been shown to increase in avian heart muscles during embryonic development (Godt et al. 1991) and in mammalian heart muscles during postnatal development (Reiser et al. 1994), and it was suggested that this is due to the accumulation of contractile proteins described in an earlier study (Lim et al. 1983). However, we are not aware of a study directly relating the force-generating capacity with the amounts of contractile proteins. One objective of the present study, therefore, was to determine the concentration of myosin heavy chain (MHC) and actin in the same hearts in which we measured isometric force normalized to cross-sectional area (FCSA), starting in the early embryo.

The Ca2+-dependent activation of the cyclic interaction between myosin and actin and, hence, of force is regulated by troponin, which is located on the thin filaments and consists of three subunits, namely troponin-C (TnC), troponin-I (TnI) and troponin-T (TnT). Both TnI and TnT exist as multiple isoforms and their expression is developmentally regulated in the heart. In the embryonic, fetal and neonatal myocardium, an isoform identical to the slow skeletal TnI (ssTnI) is expressed, whereas the cardiac isoform (cTnI) is found in the adult heart (Saggin et al. 1989; Sabry & Dhoot, 1989a; Westfall et al. 1996). In the case of TnT multiple isoforms have been described which are also expressed in development- and species-dependent patterns (Saggin et al. 1988; Sabry & Dhoot, 1989b; Westfall & Solaro, 1992).

A developmental decrease in the Ca2+ sensitivity of isometric tension has been observed in several studies using skinned fibre preparations taken from avian and mammalian hearts around birth except for the chick heart, which was also studied during embryonic life (Solaro et al. 1988; Godt et al. 1991, 1993; Reiser et al. 1994; Metzger et al. 1994). This decrease in Ca2+ sensitivity has been directly correlated with shifts in TnI and TnT isoform expression patterns by parallel investigation of these parameters in rat ventricles starting at perinatal stages (Reiser et al. 1994). In this species, both TnI and TnT switching occur shortly after birth (Reiser et al. 1994; Jin, 1996), in parallel with a decrease in Ca2+ sensitivity, thus suggesting a role of TnI and/or TnT isoforms in determining myocardial Ca2+ sensitivity during development (Reiser et al. 1994). However, due to the simultaneous switching of these two Tn subunits, it has been difficult to attribute their individual contributions to developmental transitions in Ca2+ sensitivity. There is evidence (Jin, 1996) that in the mouse heart, TnT and TnI isoforms switch at different times during development. In this species it is therefore possible to distinguish between the individual contributions of TnT and TnI isoforms to the developmental transitions in Ca2+ sensitivity. Interestingly, despite the widespread use of the transgenic mouse in studies of TnI or TnT function, to date there is very little information about developmental changes in functional parameters for this species (Metzger et al. 1994). Furthermore, little is known about early embryonic changes in contractility and isoforms of sarcomeric proteins in any mammalian species. Since embryonic stem cells are currently under investigation for the treatment of myocardial infarction this information is crucial for judging the development of these transplanted cells (Roell et al. 2002).

Genetic manipulations in the mouse (Fentzke et al. 1999) and in cardiomyocytes (Westfall et al. 1997) have also shown that the TnI isoform plays a crucial role in the regulation of Ca2+ sensitivity. Despite this, it is not clear whether shifts in troponin isoforms are the only determinants of Ca2+ sensitivity during heart development. It has been suggested that MHCs, which switch from the β to the α isoform during perinatal heart development of the mouse (Lyons et al. 1990; Lompre et al. 1991) and the rat (Lompre et al. 1991), may affect Ca2+ sensitivity. However, earlier studies have produced conflicting results concerning a possible influence of MHC isoforms on Ca2+ sensitivity, ranging from a decrease (Metzger et al. 1999), to no change (Pagani et al. 1986), to an increase (Gibson et al. 1992) in Ca2+-sensitivity in β-MHC-expressing myocardium.

The goal of the present study was to correlate contractile parameters with sarcomeric protein expression from the early embryonic stages of heart development up to the adult stage in the mouse. The amounts of contractile proteins in cardiac fibres were correlated with the maximum force production, and changes in TnI, TnT and MHC isoforms were correlated with myocardial Ca2+ sensitivity of isometric force development at different embryonic, fetal and postnatal stages. Furthermore, in order to test whether the developmental transitions in Ca2+ sensitivity are based on developmental changes of Tn alone, the native Tn complex was replaced in both fetal and adult fibres by a skeletal Tn complex, and the Ca2+ sensitivity after replacement was determined.

METHODS

Preparation of skinned cardiac fibres

Pregnant mice at different gestational stages, non-pregnant adult and neonatal mice of the strain HIM:OF1 were killed by cervical dislocation and embryonic mice were killed by decapitation using procedures approved by the local Animal Care and Use Committee (Regierungspräsidium Köln). Embryonic hearts were removed and skinned with 1 % (v/v) Triton X-100 (see ‘Solutions’) for 4 h on ice, as described elsewhere (Roell et al. 2002). For tension measurements, small strips from the ventricular walls (embryonic day (E)13.5 to E19.5) or fibres from papillary muscles (neonatal and adult) with diameters of 0.2–0.4 mm and lengths of 1–2 mm were dissected. The ventricular strips were dissected from the heart base to the apex, following the direction of trabeculae. Heart tubes of E10.5 to E11.5 were kept as whole hearts because of their small sizes (outer diameters: 300–400 μm) and the fragility of the immature tissue. Fibres were stored at 4 °C in detergent-free skinning solution for a maximum of 24 h and then were either used for mechanical measurements or frozen in liquid nitrogen and stored at −80 °C for gel electrophoresis.

Solutions

Skinning solution contained: 5 mm KH2PO4, 5 mm NaN3, 3 mm magnesium acetate, 5 mm K2EGTA, 3 mm Na2ATP including 3 mm MgCl2 and 6 mm KOH, 47 mm sodium creatine phosphate, 2 mm dithiothreitol (DTT), 200 μm 4-(2-aminoethyl)benzenesulfonylfluoride, 10 μm leupeptin, 10 μm antipain and 5 μg ml−1 aprotinin.

Relaxing and activating solutions contained: 20 mm imidazole, 3 mm (Ca)K2EGTA (for the activating solution) or 3 mm K2EGTA (for the relaxing solution), 10 mm Na2MgATP, 10 mm MgCl2, 2 mm KOH, 3 mm MgCl2, 32.7 mm sodium creatine phosphate, 2 mm DTT and 200 u ml−1 creatine kinase, pH 7.0 at 10 °C, μ= 178 mm. By mixing appropriate volumes of relaxing and activating solutions, different concentrations of free Ca2+ were obtained. These were calculated using a computer program (Fabiato & Fabiato, 1979).

Solutions used for the exchange of the Tn complex were composed as described previously (Brenner et al. 1999). The pre-rigor solution contained: 10 mm imidazole, 2.5 mm EGTA, 15 mm EDTA and 135 mm potassium propionate. The rigor solution contained: 10 mm imidazole, 2.5 mm EGTA, 2.5 mm EDTA and 135 mm potassium propionate. The exchange buffer was composed of: 20 mm 3-(N-morpholino)propanesulfonic acid, 5 mm MgCl2, 5 mm EGTA, 240 mm KCl, 5 mm DTT, 7 μg ml−1 pepstatin, pH 6.5 at the exchange temperature (10 or 20 °C) and 1.5 mg ml−1 fsTn complex.

Isometric tension measurements

Skinned ventricular fibres were mounted in the experimental chamber between an isometric force transducer (KG7a, Scientific Instruments, Heidelberg, Germany) and a length driver. For tension measurements on primitive ventricles (E10.5–E11.5), heart tubes were clamped near the inflow and outflow regions of the common ventricle. Unless stated otherwise, experiments were performed at 10 °C, in order to preserve the structural integrity of the tissue during activation.

To normalize the maximum isometric tension produced by fibres of developmental stages E13.5-adult, the widths of the fibres were measured and the cross-sectional areas (CSAs) calculated by assuming circular cross-sections. CSAs of heart tubes prepared from stage E10.5 were determined from thin sections prepared after their use in mechanical experiments to correct the CSAs for the lumens of the tubes. Since the immature sarcomeric organization of embryonic cardiac tissue did not allow estimation of sarcomere length by laser diffraction, all the fibres were pre-stretched in relaxing solution (pCa = 8) by 10 % of their slack lengths and were thereafter transferred to activating solution (pCa = 4.5) for maximal activation. After relaxation, force-pCa relations were determined by cumulatively increasing [Ca2+].

Measurements of active shortening velocity on fetal and adult skinned fibres were performed by the technique of isotonic load clamping during maximal activation at 20 °C using a commercial setup (Scientific Instruments). Shortening velocities (V) were measured under different constant loads (P). The V–P relationships were fitted by the Hill function to determine the maximum shortening velocity (Vmax) by extrapolation of the fitted curve to zero load.

Electrophoretic separation of MHC isoforms

Myosin extraction was performed as described elsewhere (Agbulut et al. 1996). Ventricular tissue was weighed and homogenized in four volumes of myosin extraction buffer (pH 6.5) containing: 300 mm NaCl, 100 mm NaH2PO4, 50 mm Na2HPO4, 10 mm Na4O7P2, 1 mm MgCl2, 10 mm EDTA and 1.4 mm 2-mercaptoethanol. After extraction for 60 min on ice, the lysates were centrifuged (12 000 g, 2 min, 4 °C) and the protein concentration was determined by the Bradford method (Bradford, 1976). Afterwards the supernatants were diluted 1:1 (v/v) with glycerol and stored at −20 °C. MHCs were separated on 8 % polyacrylamide gels containing 30 % glycerol (Talmadge & Roy, 1993). During separation (30 h at 70 V), the system (Mini-Protean 2, BioRad, München, Germany) was kept on ice. The gels were stained with Coomassie Brilliant Blue G250 (BioRad).

SDS-PAGE and Western blotting

Skinned ventricular fibres from murine ventricles were weighed and then homogenized in Laemmli buffer (50 mm Tris-HCl, 4 m urea, 1 % (v/v) SDS, 0.01 % (v/v) bromophenol blue, 20 mm DTT, pH 6.8). The protein concentration was determined as in Bradford (1976). Equal amounts of total protein per lane (4 μg for myosin and actin quantification and 30 μg for anti-TnT and -TnI Western blots) were loaded into each lane and separated by 10 % SDS-PAGE. For quantification of myosin and actin bands, gels were stained with Coomassie Brilliant Blue. For Western blot analysis of TnT and TnI isoforms, proteins were transferred to nitrocellulose membranes (0.2 μm pore size, Schleicher and Schuell, Dassel, Germany) which were blocked with 5 % (w/v) dried milk and incubated overnight at 4 °C with either anti-TnI (clone 6F9, Dunn Technologies, CA, USA) or anti-TnT antibodies (clone JLT-12, Sigma, Taufkirchen, Germany), both recognizing cardiac and skeletal isoforms. Mouse soleus muscle was used as a positive control for ssTnI. After 1 h of treatment with horseradish peroxidase-conjugated secondary antibody (anti-mouse IgG), immunoreactive protein bands were detected with enhanced chemiluminescence (ECL, Amersham, Freiburg, Germany). In order to quantify the relative amounts of TnT and TnI isoforms at different developmental stages as well as the MHC and actin content of myocardium, densitometric scans of Western blots and stained gels were performed using Phoretix software (Biostep, Jahnsdorf, Germany). To determine the MHC content of total protein, calibration was performed by loading each gel with 0.5, 1, 2 and 4 μg of commercially available myosin from rabbit muscle (M1636, Sigma). The MHC content relative to wet weight of sample (MHC/wet weight) was calculated based on the wet weights of the skinned cardiac fibres, the volumes of Laemmli buffer added before homogenization and the protein concentration in the homogenates.

Replacement of endogenous cardiac Tn complex with rabbit skeletal Tn complex

Native troponin of skinned murine cardiac fibres was exchanged for skeletal troponin complex purified from rabbit skeletal muscle. For the purification of skeletal troponin, adult rabbits were anaesthetized with 2 : 1 (v/v) Ketanest : Rompum and killed by removal of the heart using procedures approved by the local Animal Care and Use Committee. All skeletal muscles were then dissected and stored on ice, followed by removal of fat, membranes and blood vessels. Skeletal troponin was purified as described previously (Chong & Hodges, 1982). We chose to use rabbit psoas Tn complex, which consists of the fast skeletal TnI and TnT isoforms, because its different electrophoretic mobilities, compared to the slow skeletal and cardiac isoforms, allowed us to monitor the replacement using SDS-PAGE. The purified protein was lyophilized and stored at −80 °C. The purity of the protein was confirmed by SDS-PAGE and Western blots.

We applied the recently established method for the replacement of the whole Tn complex in skinned skeletal muscle fibres (Brenner et al. 1999). Compared with other replacement procedures commonly used for cardiac fibres (Hatakenaka & Ohtsuki, 1992; Moss, 1992; Strauss et al. 1992; Chandra et al. 1999), this method is particularly gentle, since at no time is the skinned muscle fibre without its complement of all three Tn subunits, and no impairment of functional properties due to the replacement procedure can be detected (Brenner et al. 1999; She et al. 2000). We considered these qualities particularly important for the replacement of the fragile fetal myocardium. Other methods first tried, such as extraction of TnI by vanadate (Strauss et al. 1992) or treatment with cTnT-cTnI (Chandra et al. 1999), resulted in a strong loss of tension (> 50 % of the initial maximum force) in fetal fibres (not shown).

The replacement of Tn complex was performed in fetal (E19.5) and in adult cardiac fibres with a diameter of 150–250 μm. The experimental protocol and the composition of the solutions (see above) followed those previously described for the replacement of the Tn complex in skinned psoas fibres (Brenner et al. 1999). All fibres were activated at 10 °C with increasing [Ca2+], as described above, to determine the Ca2+ sensitivity of the native fibres. After relaxation, fibres were placed into pre-rigor solution (5 min) and then into rigor solution (5 min), followed by 3–4 h incubation in replacement buffer containing 1.5 mg ml−1 purified rabbit fsTn complex. Replacement of adult fibres was performed at 20 °C. Fetal fibres were replaced at 10 °C since we observed a great loss of maximum force at the higher temperature. Similar replacement yields for both tissues resulted from choosing these different temperatures. After removal of unbound Tn by several washings (3 × 10 min) in Tn-free replacement buffer, fibres were transferred to relaxing solution and force-pCa relations were determined. Control experiments were performed following the same protocol but without Tn in the replacement buffer.

With this method, both fetal and adult fibres produced about 70 % of their initial maximum force after the replacement procedure. Even if we did not reach complete replacement of TnI with this gentle protocol, the native TnI isoforms were clearly reduced and replaced by the skeletal isoform in both fetal and adult fibres (see Fig. 6).

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.

Figure 6

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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.

Statistical analysis

Statistics were performed by one-way analysis of variance (ANOVA) with the criteria for significance set at P < 0.05. Data are given as means ±s.e.m. for numbers of experiments listed in Table 1 or in the figure legends.

Table 1.

Contractile parameters during mouse heart development, determined in skinned ventricular preparations

Developmental stage FCSA pCa50 Hill coefficient Vmax
(mN mm−2) (−log([Ca2+] M−1)) nH (fibre lengths s−1)
E10.5 0.36 ± 0.02 (5) 6.19 ± 0.02 (11) — —
E11.5 — 6.17 ± 0.01 (4) 3.2 ± 0.2 (4) —
E13.5 0.91 ± 0.06 (4)* 6.20 ± 0.01 (5) 3.2 ± 0.2 (5) —
E16.5 1.2 ± 0.2 (4)* 6.08 ± 0.01 (5)* 2.7 ± 0.2(5) —
E19.5 (fetal) 1.8 ± 0.4 (4)* 6.10 ± 0.02 (7) 2.4 ± 0.1 (7) 0.93 ± 0.05 (5)
7 days pn (neonatal) 3.1 ± 0.3 (6)* 5.82 ± 0.03(5)* 3.0 ± 0.3 (5) —
6–8 weeks pn (adult) 6.8 ± 0.3 (4)* 5.69 ± 0.02 (8) * 3.1 ± 0.3 (8) 1.46 ± 0.08 (5)†
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E, embryonic day; pn, postnatal. Values are means ±s.e.m. (n).

*

P < 0.05 compared with next data of younger stage

†

P < 0.05 compared with E19.5.

RESULTS

Force generation and expression of contractile proteins

The maximum force normalized to cross-sectional area (FCSA) produced by skinned ventricular fibres was determined at different developmental stages (Table 1). FCSA increased during cardiac development by a factor of ∼20 from E10.5 to the adult stage (6–8 weeks after birth). A 5-fold increase in FCSA occurred between E10.5 and E19.5, followed by a further 1.5- to 2-fold increase until 7 days after birth (7 days pn) and a further 2- to 2.5-fold increase until adulthood. We then investigated how the developmental increase in FCSA correlates with the developmental changes in the amounts of contractile proteins. Figure 1A illustrates the protein pattern of skinned murine ventricular myocardium during development. The MHC content of total protein remained unchanged during the late embryonic period from E13.5 to E16.5 and during the fetal period from E16.5 to E19.5, but then significantly increased by ∼20 % from E19.5 to 7 days pn, and by a further ∼40 % from 7 days pn to the adult stage (Fig. 1B). The ratio of MHC to actin remained constant during development, except for a significant (P < 0.05) increase of ∼20 % around birth from E19.5 to 7 days pn (Fig. 1C). In agreement with earlier studies (Lyons et al. 1990; Lompre et al. 1991; Metzger et al. 1995), we observed a transition from the β- to the α-MHC isoform (Fig. 1D). The faster migrating β-MHC is predominant during embryonic development and then starts to decrease rapidly around birth. The α-MHC is already present during embryonic development and increases gradually to become the main isoform after birth and the only isoform of the adult ventricle. Consistent with the higher MgATPase activity of α-MHC (Pope et al. 1980), the maximum shortening velocity is significantly higher in the adult heart than in the fetal (E19.5) heart (Table 1). Altogether, the most significant specific changes in MHC expression - such as the increases in MHC per total protein and in MHC to actin ratio as well as the shift from the β- to the α-isoform of MHC - occur around birth.

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

Figure 1

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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 shows that the MHC/wet weight of the skinned fibres is constant until E16.5 and then increases. The FCSA normalized to MHC/wet weight increased during the entire developmental course, although the major increase occurred during the embryonic and fetal periods (Fig. 2). The increase in maximum force-generating capability in the developing myocardium is therefore not solely determined by the MHC concentration.

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

Figure 2

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Maximum Ca2+-activated isometric force normalized to the cross-sectional area (FCSA) 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 FCSA to MHC/wet weight (▵) were calculated from the means of the FCSA 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.

Ca2+ sensitivity of force development and isoform expression of Tn subunits

Figure 3 shows the force-pCa relationships in skinned ventricular fibre preparations from embryonic, fetal, neonatal and adult murine hearts. A rightward shift in force-pCa relationships (i.e. a decrease in Ca2+ sensitivity) was observed with progressing development. Table 1 summarizes the means of the parameters pCa50 (indicating Ca2+ sensitivity) and the Hill coefficient nH (indicating cooperativity), as determined from force-pCa relationships of individual fibres. Between E13.5 and E16.5, a small but significant (P < 0.05) decrease of 0.12 pCa units was observed. The most pronounced decrease (0.28 pCa units) occurred around birth between E19.5 and 7 days pn, followed by a further slight decrease (0.13 pCa units) from the neonatal (7 days pn) to the adult (6–8 weeks pn) stages. Cooperativity of force development, indicated by the value of nH, did not change significantly during development (Table 1).

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

Figure 3

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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.

TnI and TnT isoforms in the developing myocardium were detected using antibodies which react with both cardiac and skeletal isoforms (Fig. 4). ssTnI, which is expressed in mouse soleus muscle, is predominant at all embryonic stages of the murine heart. Its expression decreases around birth, and it is not detected in the adult heart (Fig. 4B). Cardiac TnI is not detected before E19.5 (Fig. 4B). Its expression increases around birth, and it is the only isoform found in the adult heart. Thus, although both TnI isoforms are expressed around birth, cTnI becomes the predominant isoform between E19.5 and 7 days pn (see Fig. 5B). Four cardiac TnT isoforms (cTnT1–4) were detected in the developing heart, switching from cTnT1 and cTnT2 at embryonic stage E13.5 to cTnT3 and mainly cTnT4 in neonatal and adult hearts (Fig. 4A). However, the switching of TnT occurs earlier than that of TnI. At E16.5, before the onset of TnI switching, the adult isoform cTnT4 had already become the predominant isoform in the heart.

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

Figure 4

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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 (cTnT1–4) 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 5. Relation between developmental changes in the relative amounts of Tn isoforms and in Ca2+ sensitivity of isometric force generation.

Figure 5

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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 5 shows the relation between developmental shifts in pCa50 and relative amounts of TnI and cTnT isoforms, as established by densitometric evaluation of Western blots. The most pronounced decrease in Ca2+ sensitivity of isometric force development (Fig. 5C) around birth is closely correlated with the transition point of TnI switching (Fig. 5B). By comparison, switching of cTnT occurs mainly during embryonic development before E16.5, with only minor further changes around birth (Fig. 5A). Therefore, the main decrease in Ca2+ sensitivity correlates more closely with the switch from ssTnI to cTnI than with the switch between different cTnT isoforms.

Replacement of native Tn complex by exogenous skeletal Tn complex

In order to further test the extent to which the shift in Ca2+ sensitivity is due to developmental changes in the Tn complex, we replaced the native Tn complex of fetal and adult mice with exogenous Tn complex isolated from fast skeletal muscles of the rabbit (fsTn).

Figure 6 shows TnT and TnI bands in untreated fibres (endogenous Tn) and in fibres replaced with fsTn. Endogenous cTnT bands are not detectable after replacement periods of 3 h. FsTnT has been incorporated into both fetal and adult fibres, suggesting complete replacement of cTnT isoforms with the fast skeletal isoforms. Efficient replacement is also observed for TnI. In fetal and adult fibres, native TnI is almost completely replaced by the exogenous fsTnI.

Figure 7 shows the effect of Tn replacement on the Ca2+ sensitivity of fetal (E19.5) and adult ventricular fibres. Non-specific effects on Ca2+ sensitivity based on the replacement procedure itself were excluded by control experiments without adding exogenous Tn complex (not shown). Before replacement (Fig. 7A), pCa50 values were 6.05 ± 0.01 for E19.5 fibres and 5.64 ± 0.04 for adult fibres. Differences in the Ca2+ sensitivity of skinned cardiac fibres disappeared after the replacement of the developmentally different native Tn complexes by the exogenous fsTn complex. Identical pCa50 values (6.12 ± 0.05) were determined for fetal and cardiac fibres (Fig. 7B).

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

Figure 7

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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.

DISCUSSION

The present study relates functional changes in contractility (FCSA, Ca2+ sensitivity of force) to amounts and isoform patterns of sarcomeric proteins, starting at early embryonic stages. Although developmental changes in these parameters had been investigated in detail in several previous studies in other species, none of them directly correlated FCSA with MHC content, and none of them investigated the linkage of Ca2+ sensitivity to Tn isoform changes during early, i.e. embryonic, heart development.

Correlation of maximum force-generating ability and content of contractile proteins

Embryonic and fetal development. We show here for the first time tension measurements on skinned cardiac preparations from a mammalian species in the embryonic stages, starting with hearts from mice at E10.5. At this stage, which is about 2 days after the formation of the primitive heart tube, it has completed looping but is still tubular, consisting of an inflow tract, an atrium, an atrioventricular canal, a common ventricle and an outflow tract. Between E11 and E16, the latter marking the end of embryogenesis in the mouse, the heart develops rapidly and reaches its prenatal configuration.

We found a 3.3-fold increase in FCSA in murine ventricles during embryogenesis (E10.5 to E16.5) and a further 1.5-fold increase during fetal development (E16.5 to E19.5). These data are in quantitative agreement with those reported for skinned fibres from embryonic chicken hearts (Godt et al. 1991) and intact fibres from fetal sheep hearts (Anderson et al. 1984). A reasonable hypothesis had been that a developmental increase in maximum force-generating ability results from the accumulation of contractile material (Godt et al. 1991, 1993). Our study provides the first attempt to test this hypothesis by quantifying the developmental changes in maximally Ca2+-activated force and in concentrations of contractile proteins. However, in contrast to the most simple explanation that developmental increase in force is due only to the accumulation of contractile proteins, we observed that MHC concentration increased much less than FCSA during prenatal development. An increase of 100 % in FCSA from E13.5 to E19 was associated with an increase in MHC/wet weight of the skinned myocardium of only about 20 %. As the ratio between MHC and actin remained constant during this period, the actomyosin concentration in the ventricular tissue increased by 20 % whereas the FCSA doubled. Similar developmental correlations between these two parameters can be derived by comparing results from biochemical (Lim et al. 1983) and functional (Godt et al. 1991) studies of heart development in the chicken. The increase in force-generating ability during prenatal heart development therefore seems to be determined much less by the amount of contractile proteins than by other, probably structural factors.

In the cytoplasm of the primitive cardiomyocytes in the heart tube, few myofibrils are found in parallel alignment (Viragh & Challice, 1973). The small forces measured in the heart tubes at E10.5 may be in part due to the fact that force measurements were performed on whole ventricles yet the few myofilaments present at these early stages may not be optimally aligned in the direction of these force measurements. Three-dimensional reconstruction from serial sections of murine ventricular walls revealed a 2-fold increase in density of myofibres from E12 to E16 (McLean et al. 1989), which is higher than the 20 % increase in actomyosin density but comparable to the 2-fold increase in FCSA from E13.5 to E19.5 observed here. Structural organization during myofibrillogenesis up to the multicellular alignment of myocytes therefore appears to develop considerably slower than expression of MHC and actin and may be the underlying mechanism for the developmental increase in maximum force-generating ability in the prenatal heart. Further studies including biochemical, structural and functional characterization of intact myocardium are required to understand the particular contributions of (i) expression of contractile proteins and (ii) their structural organization to the development of the force-generating ability in the embryonic and fetal heart.

Perinatal changes. The ratios of MHC to total protein and of MHC to actin, which had both remained constant during embryonic and fetal development in the murine ventricles, increased between E19.5 and 7 days pn. Further, during this perinatal period the FCSA increased almost in parallel with the MHC/wet weight value of ventricular tissue, in contrast to the much higher increases in FCSA than in MHC/wet weight during the embryonic and fetal periods. Thus, in contrast to the prenatal increases in FCSA, those occurring perinatally seem to be mainly based on the specific accumulation of contractile proteins, in particular of MHC. In line with our results, a steep rise in maximum force (Godt et al. 1991), a rapid increase in the amount of myosin normalized to the amount of total protein and only small changes in the amount of actin normalized to the amount of total protein (Lim et al. 1983) have been found in the chick heart around hatching.

There is also a shift in the MHC isoforms during this developmental period, with α-MHC becoming the predominant isoform. The fetal stagnation and perinatal rise in the concentration of total MHC is in line with the finding that β-MHC expression in mouse ventricles becomes downregulated at E17.5 before upregulation of α-MHC expression starts at E19.5 (Ng et al. 1991). In parallel with the change in the MHC isoforms, we observed an increase in Vmax, which is significantly higher in the adult as compared to the fetal (E19.5) heart, consistent with earlier studies performed with intact cardiac fibres from other species (Cappelli et al. 1989). Histological analysis of ventricular fibres prepared from hearts of E19.5 and adult mice showed orientation of myocytes in the fibre axis (S. Siedner, K. Addicks & R. Stehle, unpublished observations), suggesting that the increases in Vmax and FCSA from E19.5 to the adult stage do not arise from a developmental improvement in the alignment of myocytes.

Changes from the neonatal to the adult stage. In the murine skinned ventricular fibres, FCSA increased by a factor of 2.2 from 7 days pn to the adult stage. This increase is similar to the 3-fold increase in FCSA during this period reported for skinned trabeculae from rat ventricles (Reiser et al. 1994) but different to trabeculae of the chick heart, in which FCSA reaches a maximum 6 days after hatching and then slightly decreases to the adult stage (Godt et al. 1991). These different developmental courses of force-generating ability between mammalian and avian species might reflect the adaptation of the circulation in these species to their individual physical strains and activities during the first weeks after birth or hatch, respectively.

The content of MHC relative to total protein and to wet weight of skinned tissue increased by ∼40 % between 7 days pn and the adult stage, while the ratio of MHC to actin remained constant, thus suggesting that the specific density of actomyosin filaments increases by ∼40 %. The increase in the force-generating ability of murine hearts during postnatal development may, therefore, partly result from an accumulation of MHC and actin, but still appears to involve optimization of structural organization. Apparently, the density of myofilaments in the myocardium has to be fully completed to the final adult level to allow for maximum force generation in relation to the number of myosin heads.

Ca2+ sensitivity of contraction and isoform shifts of Tn subunits

Embryonic and fetal development. Our study provides the first data on developmental changes in Ca2+ sensitivity of force development during embryonic cardiac development in a mammalian species. During embryogensis (E10.5 to E16.5), there is a small decrease in Ca2+ sensitivity of ∼0.1 pCa units. This decrease is restricted to a short period in late embryogenesis (E13.5 to E16.5) and occurs in temporal correlation with major shifts in TnT isoforms. Of the total shifts between TnT isoforms taking place during murine heart development between E13.5 and the adult stage, 60–65 % occur between E13.5 and E16.5. At E16.5, the embryonic isoforms TnT1 and TnT2 were greatly reduced, and the adult isoform TnT4 had already become predominant. In contrast, no adult TnI isoform (cTnI) could be detected in the ventricles at E16.5. The finding that TnT precedes TnI switching in murine ventricles is consistent with an earlier, biochemical study (Jin, 1996). We have not investigated TnC expression, but it has been shown that only a single cardiac TnC isoform (slow TnC) is expressed in the murine heart throughout development (Parmacek et al. 1990). The decrease in Ca2+ sensitivity during late murine embryogenesis therefore correlates with the switching of TnT isoforms and cannot arise from isoform changes in TnI or TnC. The idea that TnT switching causes the minor transition in Ca2+ sensitivity during embryonic development is further supported by the absence of significant changes in Ca2+ sensitivity during fetal development from E16.5 to E19.5, when the TnT isoform pattern remains stable. A similar decrease in Ca2+ sensitivity of force during embryonic development, which was completed at E17, has also been found in chicken hearts (Godt et al. 1991, 1993). However, as the transition points of TnT and TnI isoform switching occur at the same time (E16–E17) in the chicken heart (Sabry & Dhoot, 1989a,b; Jin, 1996) it is not possible to distinguish whether the shifts between TnI isoforms or those between TnT isoforms cause the prenatal changes in myocardial Ca2+ sensitivity in the chicken heart.

Mutations in the cTnT gene associated with familial hypertrophic cardiomyopathy (FHC) have been shown to affect myocardial Ca2+ sensitivity (Sweeney et al. 1998; Tobacman et al. 1999; Redwood et al. 2000; Szczesna et al. 2000; Montgomery et al. 2001). Skinned fibres from transgenic mice expressing fsTnT in the heart showed no change in Ca2+ sensitivity but exhibited altered cooperativity of the force-pCa relationship (Q. Q. Huang et al. 1999). More directly related to the effects of developmental shifts in TnT isoforms, comparison of the two cTnT isoforms expressed in the adult bovine heart has shown that the variable region located near the N-terminus of cTnT slightly alters (by 0.1 pCa units) the Ca2+-sensitive ATPase of regulated acto-S1 (Tobacman & Lee, 1987) and the Ca2+ sensitivity of force in skinned fibres (VanBuren et al. 2002). Recently, the effects on Ca2+ sensitivity of each of the four different hcTnT isoforms expressed in the human heart have been characterized (Gomes et al. 2002). These authors showed that the pCa50 of skinned porcine cardiac fibres replaced with these isoforms strongly correlates with the net charge of the individual isoform. Fibres replaced with either hcTnT3 or hcTnT4 showed decreased Ca2+ sensitivity of force development (by about 0.15 pCa units) compared to fibres replaced with hcTnT1 or hcTnT2 (Gomes et al. 2002), which is in good agreement with the developmental transition in Ca2+ sensitivity taking place in murine hearts from E13.5 to E16.5. Taken together, it appears most likely that TnT isoforms account for the minor developmental decrease in Ca2+ sensitivity of the heart observed during late embryogenesis in mice.

Perinatal changes. The most pronounced developmental decrease in Ca2+ sensitivity (0.28 pCa units) occurs in the timeframe from E19.5 to 7 days pn. Within this period, developmental TnI and MHC isoform switching pass transition points, and the adult isoforms cTnI and α-MHC become predominant. In comparison, only minor shifts (32 % of the total developmental shifts occurring from E13.5 to the adult stage) between TnT isoforms take place during this period, marked by the loss of the embryonic isoforms TnT1 and TnT2 and the appearance of a new, minor adult isoform, TnT3. However, considering that the major (60–65 %) developmental changes among TnT isoforms that occurred from E13.5 to E16.5 were accompanied by only a small decrease in Ca2+ sensitivity, it appears unlikely that the perinatal decrease in Ca2+ sensitivity results from developmental shifts in TnT isoforms.

Changes from the neonatal to the adult stage. From 7 days pn to adulthood, the residual switch (the last 40 % of the total developmental switch) from the ssTnI to the cTnI isoform is completed whereas no more significant changes in the patterns of TnT and MHC isoforms occur during this period. Therefore the decrease in Ca2+ sensitivity of force (0.13 pCa units) after 7 days pn cannot be due to changes in MHC or TnT isoforms and appears instead to arise from the final TnI isoform switch. Postnatal changes in Ca2+ sensitivity and their correlation with TnI and TnT switching had been most extensively characterized by Solaro and coworkers in rat hearts (Solaro et al. 1988; Martin et al. 1991; Reiser et al. 1994). In rat skinned ventricular fibres, Ca2+ sensitivity of force development decreased by 0.25 pCa units from 7 days pn to adulthood (Reiser et al. 1994), which is more than the decrease in the mouse. However, in contrast to the mouse, the transition point of ssTnI-cTnI switching in the ventricle of the rat occurs after 7 days pn (Reiser et al. 1994), which might explain the larger decrease in Ca2+ sensitivity. Nevertheless, because of its close temporal overlap with TnT2–TnT4 isoform switching (Martin et al. 1991; Reiser et al. 1994; Jin, 1996), it has been difficult to clearly attribute the shifts in Ca2+ sensitivity occurring during neonatal rat heart development to TnI switching.

Recently, various genetic models have been used to examine the influence of TnI isoforms on the Ca2+ sensitivity of cardiac tissue. Overexpression of ssTnI by adenovirus-mediated gene transfer in isolated rat cardiomyocytes (Westfall et al. 1997) or in transgenic mice (Fentzke et al. 1999) resulted in a higher Ca2+ sensitivity compared to controls. Ablation of the cTnI gene in the mouse heart was associated with a prolonged developmental expression of ssTnI compared to wild-type mice (X. Huang et al. 1999). Skinned cardiac fibres from these mice (14 days pn) had a Ca2+ sensitivity that was about 0.5 pCa units higher than in age-matched wild-type mice, which mainly express cTnI. This difference in Ca2+ sensitivity is very similar to the developmental shifts (of about 0.4 pCa units) reported in this study, which occur after the onset of the switch from ssTnI to cTnI at E16.5. However, gene targeting may lead to compensatory changes in protein expression, which could also affect Ca2+ sensitivity.

Replacement of troponin in fetal and adult stages. The temporal correlation between Tn isoform switching and changes in Ca2+ sensitivity during murine heart development, as well as the genetic models, suggest a role for Tn isoforms, especially TnI, in the developmental determination of myocardial Ca2+ sensitivity. However, isoform switching during development is also observed in other sarcomeric proteins, such as MHC (Lyons et al. 1990; Lompre et al. 1991), myosin light chains (Lyons et al. 1990), actin (Carrier et al. 1992) and tropomyosin (Muthuchamy et al. 1993), which possibly contribute to transitions in Ca2+ sensitivity.

In order to test to what extent the major developmental changes in Ca2+ sensitivity that begin after E19.5 do indeed originate from the Tn complex, we replaced the whole endogenous Tn complex of fetal (E19.5) and adult cardiac fibres with the same exogenous fsTn complex. After replacement with this complex, there were no detectable differences between the Ca2+ sensitivities of fetal and adult skinned myocardium, thus suggesting that the most pronounced shifts in Ca2+ sensitivity are based on developmental transitions within the Tn complex. Since the main decrease in Ca2+ sensitivity is more closely correlated to the switching of TnI than of TnT isoforms, our results imply a crucial role for TnI in Ca2+-regulated force development during murine heart development. The specific effects of TnI replacement on Ca2+ sensitivity of skinned trabeculae from fetal and adult hearts have been studied by Morimoto & Goto (2000) in the rabbit. By selectively replacing the native predominant ssTnI isoform of fetal fibres with the cTnI isform of adult fibres, and vice versa, they were able to show that the developmental change in pH sensitivity of Ca2+-regulated force development that occurs in the rabbit heart from the fetal to the adult stages is exclusively determined by the TnI isoform. This corroborates our conclusion that changes in Ca2+ sensitivity around birth are primarily based on TnI isoform switching. However, Tn exchange was not performed on earlier stages, therefore a modulatory role of TnT isoforms and other contractile proteins on Ca2+ sensitivity during embryonic heart development cannot be completely ruled out by Morimoto & Goto (2000) or our exchange studies.

Acknowledgments

This work was supported by the German Research Foundation DFG (SFB-612-A2) and by the Faculty of Medicine, University of Cologne (Köln Fortune grant no. 28–2002).

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