Die kardialen Auswirkungen der FYCO1-Überexpression und die Identifikation neuer Bindungspartner
Recently, FYCO1 has been linked to autophagy as it directly interacts with LC3, Rab7, and PI3P. It appears to be part of the machinery that moves autophagosomes along the microtubules. We have shown before that FYCO1 deficient mice develop normally under baseline conditions. However, under stress conditions, such as starvation or pressure overload, FYCO1 knockout mice do not respond with increased autophagy in the heart and suffer from contractile dysfunction. We wondered whether FYCO1 overexpression might have beneficial effects by facilitating cardiac autophagy. We generated a transgenic mouse model with cardiac specific overexpression of FYCO1. We established 2 founder lines with a moderate (13.1±1.3-fold) and a high (20.1±2.4-fold) overexpression of FYCO1 protein. The 2 transgenic mouse lines (TG) showed a similar phenotype under basal conditions. TG mice developed mild cardiac hypertrophy. The ratio of heart weight vs. body weight was elevated. Moreover, we found a thickened septum as well as left ventricular posterior wall in TG mice by echocardiography, while fractional shortening was not changed. Accordingly, cardiomyocyte cell surface area was increased in FYCO1 TG mice. Though, the expression of hypertrophic marker genes did not significantly differ. Of note, overexpression of FYCO1 in vivo leads to accumulation of LC3-II protein in the heart, which indicates an increased number of autophagosomes. In order to directly count the number of autophagosomes we generated double transgenic mice that overexpress FYCO1 as well as GFP-LC3. Indeed, we found significantly more autophagosomes in heart sections of FYCO1 TG mice. The ultrastructure of autophagosomes was not different in transgenic mice, but we again detected more autophagosomes. Next, we subjected TG mice as well as their wildtype littermates (WT) to transverse aortic constriction (TAC). As expected, WT mice developed left ventricular hypertrophy 2 weeks after TAC. Similar to the baseline data, sham operated TG mice revealed mild cardiac hypertrophy. Interestingly, the ratio of left ventricular mass vs. body weight of banded TG and WT mice was identical implying that the additional hypertrophy due to TAC was less pronounced in TG mice. WT mice exhibited heart failure with elevated lung weight, as a marker of pulmonary congestion, and reduced ejection fraction after TAC. In contrast, the lung weight as well as the ejection fraction of banded TG animals was markedly better and almost completely preserved. The hypertrophic phenotype of banded WT animals was accompanied by an induction of hypertrophic marker genes (Nppa, Nppb, Myh7). In TG mice the induction of these genes by TAC was milder and not statistically significant. To examine the underlying mechanism that is responsible for the contractile phenotype, we performed a yeast two-hybrid screen with FYCO1 as bait. We identified Myh7 as potential interacting protein and confirmed the interaction by co-immunoprecipitation. Myh7 is a member of the “fetal gene program”, upregulated during pathological hypertrophy. Consistently, Myh7 protein was induced in WT mice after TAC. However, overexpression of FYCO1 resulted in mitigated induction of Myh7 protein following TAC, which might contribute to preservation of contractile function. In summary, we could show that cardiac specific overexpression of FYCO1 in vivo leads to increased autophagy and protects the heart from dysfunction following pressure overload.