Force frequency relationship of the human ventricle increases during early postnatal development

Abstract

Understanding developmental changes in contractility is critical to improving therapies for young cardiac patients. Isometric developed force was measured in human ventricular muscle strips from two age groups: newborns (<2 wk) and infants (3-14 mo) undergoing repair for congenital heart defects. Muscle strips were paced at several cycle lengths (CLs) to determine the force frequency response (FFR). Changes in Na/Ca exchanger (NCX), sarcoplasmic reticulum Ca-ATPase (SERCA), and phospholamban (PLB) were characterized. At CL 2000 ms, developed force was similar in the two groups. Decreasing CL increased developed force in the infant group to 131 +/- 8% (CL 1000 ms) and 157 +/- 18% (CL 500 ms) demonstrating a positive FFR. The FFR in the newborn group was flat. NCX mRNA and protein levels were significantly larger in the newborn than infant group whereas SERCA levels were unchanged. PLB mRNA levels and PLB/SERCA ratio increased with age. Immunostaining for NCX in isolated newborn cells showed peripheral staining. In infant cells, NCX was also found in T-tubules. SERCA staining was regular and striated in both groups. This study shows for the first time that the newborn human ventricle has a flat FFR, which increases with age and may be caused by developmental changes in calcium handling.

Figures

Figure 1

Contractions measured in ventricular tissue strips. A. Developed force from a newborn (4 days old, top panel) and an infant (3 months old, bottom panel) strip. The pacing cycle length (CL) decreased from 2000 to 1000 and 500 ms. Steady state contractions are shown and the dashed line indicates the amplitude of developed force at CL 2000 ms. B. Developed force as in A, but normalized to the amplitude at CL 2000 ms (black line) with CL 1000 ms (red) and CL 500 ms (blue) overlaid.

Figure 2

Average data for contractions from newborn and infant ventricular tissue strips are shown. A. Average developed force expressed as percent of CL 2000 ms for newborn (open, n=7, 4 patients) and infant (shaded, n=7, 6 patients) ventricle (*p<0.05). B. Total contraction time, C. Time to peak and D. Peak to 90% relaxation time for newborn (open) and infant (shaded) ventricular strips (*, p<0.05 compared to CL 2000 ms, §, p<0.05 compared to CL 1000 ms).

Figure 3

mRNA levels (normalized to 18S rRNA) of calcium handling proteins change with developmental age. Newborn (open, n=6) and infant (shaded, n=5) levels of NCX (Na/Ca exchanger), SERCA (SR Ca-ATPase pump) and PLB (phospholamban) as well as the PLB/SERCA ratio are shown. *, p

Figure 4

Protein levels of calcium handling proteins in the developing human ventricle. A. Example of western blots for NCX (Na/Ca exchanger). Lanes are labeled with age of patient (d=days, m=months) with newborns furthest left, young infants in the center and older infants furthest right. B. Density of NCX plotted as a function of age (newborns, solid triangles; young infants, open circles; older infants, solid circles), showing an inverse linear relationship (R=−0.66, p=0.003) between NCX protein levels and age. C. Average NCX protein levels for the three age groups: newborn (open, n=6), young infants (hashed and shaded, n=6) and older infants (shaded, n=6). *, p<0.05. D. Western blots of SERCA (SR Ca-ATPase pump) labeled with ages as in (A). E. Density of SERCA plotted as a function of age ((R=−0.11, p=n.s.). F. Average SERCA levels for the three age groups (n=6 for each age). Parts A–F used membrane proteins. G. Western blots using total proteins of SERCA, phospholamban (PLB) and GAPDH for loading control. H. Summary data for part G, normalized to GAPDH (n=6 newborn and n=5 infant).

Figure 5

Morphological analysis of ventricular cells isolated from ventricle from newborn and infant patients. A. Single ventricular cells from a newborn (left, 7 days) and an infant (right, 5 mos.) with the membrane of the cells labeled with di-8-ANNEPS. Arrows indicate partial T-tubule development in infant cell. B. Immunostaining of ventricular cells for Na/Ca exchange (NCX) in newborn (left, 6 days) and infant (right, 7 mos.). C. Immunostaining of newborn (left) and infant (right) ventricular cells with SR Ca-ATPase pump (SERCA, green) and nuclear staining (red).