Postnatal Development of Right Ventricular Myofibrillar Biomechanics in Relation to the Sarcomeric Protein Phenotype in Pediatric Patients with Conotruncal Heart Defects

Abstract

Background: The postnatal development of myofibrillar mechanics, a major determinant of heart function, is unknown in pediatric patients with tetralogy of Fallot and related structural heart defects. We therefore determined the mechanical properties of myofibrils isolated from right ventricular tissue samples from such patients in relation to the developmental changes of the isoforms expression pattern of key sarcomere proteins involved in the contractile process.

Methods and results: Tissue samples from the infundibulum obtained during surgery from 25 patients (age range 15 days to 11 years, median 7 months) were split into half for mechanical investigations and expression analysis of titin, myosin heavy and light chain 1, troponin-T, and troponin-I. Of these proteins, fetal isoforms of only myosin light chain 1 (ALC-1) and troponin-I (ssTnI) were highly expressed in neonates, amounting to, respectively, 40% and 80%, while the other proteins had switched to the adult isoforms before or around birth. ALC-1 and ssTnI expression subsequently declined monoexponentially with a halftime of 4.3 and 5.8 months, respectively. Coincident with the expression of ssTnI, Ca(2+) sensitivity of contraction was high in neonates and subsequently declined in parallel with the decline in ssTnI expression. Passive tension positively correlated with Ca(2+) sensitivity but not with titin expression. Contraction kinetics, maximal Ca(2+)-activated force, and the fast phase of the biphasic relaxation positively correlated with the expression of ALC-1.

Conclusions: The developmental changes in myofibrillar biomechanics can be ascribed to fetal-to-adult isoform transition of key sarcomeric proteins, which evolves regardless of the specific congenital cardiac malformations in our pediatric patients.

Keywords: cardiac myofibrils; contractile function; contractile proteins; force kinetics; heart development; human myocardium; sarcomere physiology; tetralogy of Fallot.

Figure 1

Development of titin, myosin heavy chain (MyHC) and troponin T (TnT) isoform expression. A, Representative Coomassie‐stained 1% SDS‐agarose gel of titin isoforms in RV samples obtained at the indicated ages; rabbit soleus and rat left ventricle (LV) were used as markers. B, Western blot probing for TnT, guinea pig myocardium expressing 4 isoforms, and human recombinant (hcTnT) are shown for comparison; in all human samples the predominant isoform is TnT3. C, Representative SDS‐PAGE of separation of MyHC isoforms, markers for α‐ and β‐MyHC isoforms: atrium (A), nonfailing adult heart (NF), and comigration (NF:A). Pediatric patients express only the β‐MyHC.

Figure 2

Postnatal expression of ssTnI and ALC‐1 in RV. A, Representative Western blots of TnI expression at different ages, showing the reciprocal signal intensities of ssTnI and cTnI. B, Time course of postnatal decline in ssTnI expression in percentage of total TnI=ssTnI+cTnI. C, Representative 2‐dimensional PAGE from 2 patients aged 4 days and 9 months; ALC‐1 and VLC‐1 indicate atrial and ventricular LC‐1 isoforms; VLC‐2 and VLC‐2*, ventricular regulatory LC isoforms; TM, tropomyosin. D, Relative ALC‐1 expression vs patient age (LC‐1_total=ALC‐1+VLC‐1). Symbols in (B and D): hypoplastic left heart syndrome, closed triangles; transposition of the great arteries, closed diamonds; tetralogy of Fallot, open squares; pulmonary atresia, open circles; pulmonary stenosis, open upward triangles; double‐outlet right ventricle, open downward triangles; each symbol represents 1 patient, and numbers are patient numbers from Table S1. Solid and dotted lines show monoexponential fit with 95% confidence limit (ALC‐1: r ²=0.56, P<0.05; ssTnI; r ²=0.46, P<0.05).

Figure 3

Force transients and passive force of right ventricular myofibrillar bundles. A, Image of a mounted myofibrillar bundle. B, Representative contraction–relaxation transient induced by switching (within 10 ms) from pCa 7.5 to 4.5 and back. Raising [Ca²⁺] and a release–restretch maneuver applied at the force plateau result in monoexponential force development with respective rate constants k _ACT and k _TR. C, Passive force per cross‐sectional area (F _pass/CSA) at 2.3 μm SL was determined before activation by slackening the myofibril by 20% of slack length. D, F _pass/CSA was significantly higher (*P<0.05) in younger (aged <10 months) than in older (>10 months) infants. E, Switching pCa back 7.5 leads to a biphasic relaxation that was fitted by a biphasic function (Methods) yielding k _LIN, t _LIN, and k _REL.

Figure 4

Postnatal change in Ca²⁺ sensitivity of force development. A, Original force tracings of contractions elicited by the indicated pCa values. B, Force–pCa relations collected from 5 myofibril bundles of a newborn (patient No. 8) and 6 myofibril bundles of a 10‐month‐old child (patient No. 5), illustrating the representative rightward shift with this increase of age. The curves represent Hill functions fitted to the data, which were normalized to F _max at pCa 4.5. C, pCa₅₀ declines monoexponentially with age (r ²=0.59, P<0.05). D and E, pCa₅₀ positively correlated with ssTnI (r ²=0.81, P<0.0001) and ALC‐1 (r ²=0.62, P<0.05) expression. Solid and dotted lines represent the monoexponential (C) and linear fits (D and E) and the respective 95% confidence limits. The pCa₅₀ values were determined from 2 to 12 myofibrils per patient and are given as mean±SEM. Symbols in (C through E): tetralogy of Fallot, open squares; pulmonary atresia, open circles; pulmonary stenosis, open upward triangles; double‐outlet right ventricle, open downward triangles. Each symbol represents 1 patient, and numbers are patient numbers from Table S1.

Figure 5

Effect of ssTnI expression on kinetic parameters of contraction and relaxation. A, Rate constant of Ca²⁺‐induced force development; B, Rate constant of mechanically‐induced force redevelopment; C, Rate constant of initial slow linear relaxation phase; D, Duration of initial slow linear relaxation phase; E, Rate constant of subsequent rapid exponential relaxation phase. For determination of rate constants of contraction (k _ACT and k _TR) and relaxation (k _LIN, t _LIN, k _REL), cf. Figure 3. Symbols: tetralogy of Fallot, open squares; pulmonary atresia, open circles; pulmonary stenosis, open upward triangles; double‐outlet right ventricle, open downward triangles. Each symbol represents the mean±SEM of 2 to 12 myofibrils of a patient.

Figure 6

Effect of ALC‐1 expression on kinetic parameters of contraction and relaxation. A, Rate constant of Ca²⁺‐induced force development; B, Rate constant of mechanically‐induced force development; C, Rate constant of initial slow linear relaxation phase; D, Duration of initial slow linear relaxation phase; E, Rate constant of subsequent rapid exponential relaxation phase. For determination of rate constants of contraction (k _ACT and k _TR) and relaxation (k _LIN, t _LIN, k _REL), cf. Figure 3. Symbols: tetralogy of Fallot, open squares; pulmonary atresia, open circles; pulmonary stenosis, open upward triangles; double‐outlet right ventricle, open downward triangles. Each symbol represents the mean±SEM of 2 to 12 myofibrils of a patient. Solid and dotted lines represent linear correlations and 95% confidence limits (Pearson correlation coefficients: A, r ²=0.58, P<0.05; B, r ²=0.55, P<0.05; E, r ²=0.57, P<0.05).