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  • A next series of experiments explored whether

    2024-07-10

    A next series of experiments explored whether cardiac Ampkα1 GSK256066 australia is modified in response to pressure overload imposed by TAC. As a result, 2weeks of TAC treatment significantly increased Ampkα1 protein abundance in cardiac tissue from Ampkα1+/+ mice as compared to sham treated mice (Fig. 6A). In contrast, cardiac Ampkα2 protein abundance was not significantly different between the genotypes and was not significantly modified following TAC treatment (Fig. 6B). Ampkα1 was found and upregulated after TAC mostly in the cytoplasmic fraction, while neither cytoplasmic nor nuclear Ampkα2 was significantly modified by TAC (Suppl. Fig. 6). In Ampkα1+/+ mice, cardiac Ampkβ and Ampkγ isoforms protein expression was not significantly modified by TAC (Suppl. Fig. 7). TAC significantly increased c-Fos, Il6 and Ncx1 mRNA expression in cardiac tissue of Ampkα1+/+ mice, effects again significantly blunted in Ampkα1−/− mice (Fig. 6C). As illustrated in Fig. 6D, Pkcζ protein expression was barely detectable in cardiac tissue from sham treated mice. Following TAC treatment, Pkcζ protein expression was again increased and localized mainly to the intercalated discs in cardiac tissue from Ampkα1+/+ mice, an effect blunted by Ampkα1 deficiency (Fig. 6D). TAC treatment decreased cardiac ejection fraction (Suppl. Table 2), and increased left ventricular end-diastolic pressure (Suppl. Table 2). These effects were slightly but significantly blunted in Ampkα1−/− mice as compared to Ampkα1+/+ mice. The relaxation constant Tau was significantly increased in Ampkα1+/+ mice, but not in Ampkα1−/− mice (Suppl. Table 2). No significant difference between genotypes was observed in dP/dt max, min or maximal systolic pressure. Echocardiographic measurements revealed similar results (Suppl. Table 2). Although heart-weight to body-weight ratio was slightly less increased in Ampkα1−/− mice following TAC (Suppl. Table 2), TAC increased diastolic wall thickness to similarly high values in both genotypes (Suppl. Table 2). Taken together, Ampkα1 deficiency confers some protection of cardiac function following TAC. In order to test in vivo whether the Ampkα1 isoform has direct effects on cardiac AP-1 activation, CAα1 was overexpressed in cardiac tissue of wild-type mice. To this end, mice were treated with the AAV9 vector as control or with AAV9-CAα1. As a result, treatment of the mice with AAV9-CAα1 significantly increased Ampkα1 protein levels (Fig. 7A) and Prkaa1 mRNA expression (Fig. 7C) without modifying acetyl-CoA carboxylase (ACC) phosphorylation (Suppl. Fig. 8) in cardiac tissue as compared to control treated mice. AAV9-mediated cardiac overexpression of CAα1 significantly down-regulated the Ampkα2 protein abundance in cardiac tissue (Fig. 7B). The mRNA expression of the AP-1 target genes c-Fos, Il6 and Ncx1 was significantly up-regulated in cardiac tissue of AAV9-CAα1 treated mice as compared to control treated mice (Fig. 7C). According to immunostaining, overexpression of CAα1 in cardiac tissue increased cardiac Pkcζ protein levels (Fig. 7D). Thus, overexpression of CAα1 in cardiac tissue was sufficient to up-regulate cardiac Pkcζ expression and AP-1-target gene expression in vivo. However, CAα1 overexpression in cardiomyocytes did not cause cardiac hypertrophy or a loss in cardiac function or modify Acta1 mRNA expression (Suppl. Table 3; Suppl. Fig. 9).
    Discussion In accordance with previous observations, the strained heart shows an isoform shift with increasing expression of the Ampkα1 isoform [13], [19], [33]. Increased Ampk activity has been described during pressure overload [13], [14]. Both, neurohumoral activation by angiotensin-II and mechanical stress by pressure overload increased Ampkα1 abundance. AAV9-mediated cardiac overexpression of constitutively active Ampkα1 for 4weeks did not lead to hypertrophy and did not appreciably modify cardiac function, but was able to mimic the effects of TAC and angiotensin-II infusion on AP-1 target gene expression. Ampkα1-dependent AP-1 activation alone is therefore not sufficient to affect cardiac function or hypertrophy, but may rather play a permissive role by promoting the fetal gene expression profile in the development of heart failure. The phenotype of Ampkα1-deficient mice thus partly contrasts the phenotype of Ampkα2-deficient mice, which suffer from exacerbated remodeling following TAC [16]. Ampkα2 seems to be the more important isoform determining cardiac function during cardiac stress [33], [34]. ACC phosphorylation was not affected by overexpression of constitutively active Ampkα1. Along these lines, previous studies suggest that cardiac ACC phosphorylation is mainly dependent on Ampkα2 [6], [35], [36].