Abstract 315: New Ventricular Myocytes with Distinct Electrophysiological Properties Are Formed During Adolescent Cardiac Growth
The existence of resident cardiac progenitor cells (CPCs) in the adolescent and adult hearts raises the possibility that new cardiac myocytes (CM) are formed to increase cardiac mass during adolescent growth (AG) or replace dying CMs in adult hearts. Our goals were to determine if left ventricular (V)M number increases during AG and define the properties and origin of these new VM.
Methods: VMs and c-kit+ CPCs were isolated from normal adolescent (74 to 299 days) feline hearts. BrdU incorporation (infused in-vivo for 7 days), Ki67 and p16INK4a immunostaining were measured to assess cell cycle activity and VM senescence respectively. VM contractions and Calcium (Ca) (Fluo-4 AM) transients were measured in small mononucleated (SMM) and large binucleated myocytes (LBM). Voltage clamp was used to measure L- (ICa-L) and T-type (ICa-T) Ca currents. Line scan confocal microscopy was used to measure the spatial synchrony of sarcoplasmic reticulum (SR) Ca release.
Results: Heart mass increased by 134% during AG but VM volume only increased by 74.6%. Individual CPC in culture were self renewing, multipotent and clonogenic and when placed in co-culture with neonatal myocytes differentiated into VMs with both ICa-T (-5.42±3.01pA/pF) and ICa-L (9.75 ±1.66pA/pF). Most VMs from the heart were binucleated (88.1±0.7% vs 10.9±0.8% mononucleated) and these VM were significantly larger (LBM) than mononucleated VMs (SMM). A significantly greater % of SMMs than LBMs was BrdUPOS (SMMs vs. LBMs: 3.10± 0.77% vs. 0.80 ± 0.37%; p<0.05, n=12), Ki67+and p16INK4a negative (SMMs vs. LBMs: 18.2±6.5%, vs. 3.5±0.6%, p<0.01). VM shortening rates were significantly slower in SMMs vs LBM and the duration of the contraction was significantly longer. SR Ca2+ release was also significantly less spatially synchronized in SMMs and their T-tubular density was significantly less. ICa-L density was not different (p >0.05) in SMMs (-5.58±0.90pA/pF, n=20) and LBMs (-6.03±0.50pA/pF, n=16). ICa-T, a current found in fetal/neonatal myocytes, was only present in SMMs (1.92±0.40pA/pF).
Conclusions: AG involves an increase in both VM volume and number. The evidence of recent cell cycle activity and less well developed structural and functional properties of SMM suggest that these are newly formed VMs.