Previous studies identified the myeloid-leukemia-factor 1 (MLF1) as a stress sensitive, antiproliferative and proapoptotic gene. We have previously shown that MLF1 is downregulated in the heart upon induction of hypertrophy by transverse aortic banding in mice and in samples from human DCM patients.  On the other hand, overexpression of MLF1 induces hypertrophic gene program in vitro. Sharing a similar amino acid sequence and important domains, myeloid-leukemia-factor 2 (MLF2) is another member of the MLF-protein family with yet unknown role in cardiac function.

We thus aimed to characterize MLF2 regarding its function in cardiomyocytes in vitro (in neonatal rat ventricular cardiomyocytes, NRVCM). 

 

We successfully generated adenoviral vectors (AdV) for overexpression and knockdown of MLF2 in NRVCM. AdV-lacZ or AdV-miRneg infections served as control for MLF2 overexpression or knockdown, respectively. 96h after infection of NRVCM in presence and absence of 72h pro-hypertrophic stimulation with 150µM Phenylephrine (PE) treatment, we performed qRT-PCR. Statistic results are displayed as fold change compared to lacZ control, unless otherwise stated p-values are calculated by Student´s t-test.

 

After 96h overexpressing MLF2 in NRVCM, mRNA levels of the hypertrophic markers NppA, NppB, and Rcan1-4 were all significantly decreased (AdV-lacZ vs. AdV-MLF2: NppA 1.0±0.08 vs. 0.3±0.03, p<0.001; NppB 1.0±0.8 vs. 0.2±0.01, p<0.001; Rcan1-4 1.0±0.07 vs. 0.3±0.04, p<0.001) which is in contrast to the induction of the hypertrophic gene expression program by MLF1. 

After PE-treatment (AdV-lacZ-PE), mRNA levels of hypertrophic markers in the control cells increased as expected. In comparison with AdV-lacZ-PE, in cells overexpressing MLF2 and PE-treated, mRNA levels of NppA, NppB, and Rcan1-4 were decreased as in unstimulated NRVCMs (AdV-lacZ-PE vs. AdV-MLF2-PE: NppA 4.8±0.5 vs. 3.2±0.3, p<0.05; NppB 2.3±0.3 vs. 1.97±0.2, p=0.4; Rcan1-4 1.6±0.08 vs. 1.2±0.1, p<0.05).

Conversely, MLF2 knockdown did not result in significant change of the hypertrophic markers compared with corresponding controls.

Interestingly, MLF2 overexpression resulted in a strong reduction of MLF1 mRNA levels, similar to PE-mediated reduction of MLF1 mRNA level. After MLF2 overexpression and additional PE-treatment MLF1 mRNA levels decreased even further (lacZ vs. MLF2 vs. lacZ-PE vs. MLF2-PE: MLF1 1.0 vs. 0.5 vs 0.5 vs. 0.3, ANOVA p<0.001) suggesting a potential protective role in vitro.

 

Taken together these results make MLF2 a promising target for further analysis in the context of cardiomyopathies, including analysis of cardiomyocyte cell size, mechanic stress responsiveness and cell viability. Furthermore, we are investigating the MLF2 expression in animal models for cardiac hypertrophy (TAC-operated and Angiotensin-treated mice) and aim to analyze the expression in human samples from DCM patients.