Knockdown of the ERK pathway using siRNA in cultured chicken cardiomyocytes

The ancient South American birds called tinamous (Tinamidae) have the smallest hearts known among birds and their cardiomyocytes have previously been shown to express significantly lower levels of the mitogen-activated protein kinase ERK compared to the more modern chicken (Gallus gallus). ERK is a well-known mediator of growth signalling in the heart, especially in hypertrophy. The aim of this project was to assess the effect of ERK knockdown on proliferation in cultured chicken cardiomyocytes. By transfecting these cells with a lipoplexed siRNA, ERK mRNA levels were knocked down to approximately half (45%, SD: 27%) compared to cells transfected with a negative control siRNA. The knockdown was coupled with a decreased proliferative response to insulin-like growth factor 1 (IGF-1) and foetal bovine serum (FBS). In conclusion, the ERK pathway was confirmed to be instrumental also in proliferative signalling. The results also support the notion that ERK itself is the rate-limiting step of this MAPK cascade. The low native expression of ERK in tinamou cardiomyocytes is expected to impose a strict limit on proliferative growth in response to various stimuli in these hearts. The genetic changes leading to higher expression levels, and with it the potential for larger hearts, in modern birds would have led to greatly increased evolutionary fitness by way of an increased aerobic scope and the ability to sustain flight.