Abstract 168: Does Calcium Influx Through TTCC Induce Cardiomyocyte Proliferation?

T-type Ca²[[Unable to Display Character: +]] channels (TTCC) are primarily expressed in fetal/neonatal cardiomyocytes and their functional role is not well defined. Here we explored the idea that Ca2+ influx through TTCC regulates myocyte proliferation. The fact that Mibefradil (TTCC and L-type calcium channel (LTCC) antagonist) inhibits smooth muscles proliferation supports this idea. Our hypothesis is: Blocking TTCC will reduce neonatal cardiomyocyte proliferation. Wild type (WT) and α1G TTCC knockout (α1G-/-) neonatal mice ventricular myocytes (NMVMs) were used. Patch clamp was used to measure TTCC. Flow cytometry was used to determine cell cycle distribution. 1. On day 1 (after birth), TTCC was detected in 35% WT NMVMs (n=31), whereas only 4% α1G-/- NMVMs have TTCC (n=25). On day 7, no TTCC was detected in both WT (n=16) and α1G-/- (n=27). 2. In vivo: On day 1 there’s no significant difference in cell cycle distribution: [88.2%±2.7% in G1/G0, 8.1%±2.8% in G2/M, 3.2%±0.1% in S; n=12] in WT vs. [90.6%±1.6% in G1/G0, 5.7%±1.5% in G2/M, 3.7%±0.3% in S; n=20] in α1G-/-. On day 7 there’s a significant difference: [52.3%±1.6% in G1/G0, 47.7%±1.6% in G2/M; n=28] in WT vs. [68.2%±2.1% in G1/G0, 31.9%±2.1% in G2/M; n=20] in α1G-/-, p<0.05. 3. In vitro: Mono and binucleated myocytes percentage was measured: On day 1 there’s no significant difference. On day 7 there were more binucleated cells in WT: 51%±4% mono and 49%±4% binucleated in WT (n=495) vs. 80%±3% mono and 20%±3% binucleated in α1G-/- (n=1107), p<0.05. Cell surface area (CSA) (um²) in α1G-/- (n=164) was smaller than WT (n=109): 860.3±323.3 in WT vs. 716.7±274.9 in α1G-/- in mono (p<0.005), 1333.9±534.0 in WT vs. 1016.6±315.4 in α1G-/- in binucleated (p<0.001). In 2-month old mice there’s a difference in percentage of mono and binucleated myocytes: [7.3%±3.7% mono, 92.7%±3.6% binucleated; n=687] in WT vs. [30.8%±6.1% mono, 69.2%±6.1% binucleated; n=793] in α1G-/-, p<0.05. CSA in α1G-/- was smaller than in WT: 2267.4±1119.7 in WT vs. 1784.6±683.9 in α1G-/- in mononulceated (p<0.0001), 2419.2±712.4 in WT vs. 2048.3±671.1 in α1G-/- in binucleated (p<0.0001). There is large amount of DNA synthesis in NMVMs after birth, by day 7 most of the WT NMVMs are arrested in G2 and become binucleated. Myocyte without α1G TTCCs did not exit from the cell cycle normally.