PCA analysis of hypertrophic cardiomyocytes indicated the existence of two partially connected clusters correlating with
Hif1α expression (Fig.
5b), which is a crucial transcriptional factor mediating hypoxic responses [
22]. Clustering was performed using the
k-means algorithm (Fig.
5c) [
29]. Cardiomyocytes in cluster 1 (“Hif1α
high”) were transcriptionally more active compared to cluster 2, resulting in a high number of differentially expressed genes between both clusters (Suppl. Table 2). Cluster 1 cardiomyocytes were enriched for “Angiogenesis”, probably as a consequence of
Hif1α expression, while cluster 2 cardiomyocytes were enriched for “Striated Muscle Contraction” (Suppl. Fig. 3C). To analyze the impact of
Hif1α expression on the transcriptional profile of hypertrophic cardiomyocytes, we set a threshold of minimal
Hif1α expression greater than 70% of the mean expression across the data set. According to this definition, ~ 41% of cardiomyocytes expressed
Hif1α and ~ 59% did not. Both groups exhibited similar expression levels of
Tnni3,
Tnnt2,
Myh6, and
Myh7 (Suppl. Table 3, Suppl. Fig. 4A) but more than 2000 genes were differential expressed with an FDR < 1% (Suppl. Fig. 4B; Suppl. Table 4). The majority of deregulated genes were found in the Hif1α
high” group, consistent with higher transcriptional activity in these cardiomyocytes. Furthermore, cardiomyocytes in the Hif1α
high” group showed higher expression of
Egln2 (also called
Phd1) [
30] and
Vegfa [
24]. The concomitant up-regulation of
Egln2 and
Vegfa in cluster 1 was clearly evident by pseudobulk analysis (Fig.
4c) and single-cell visualization (Fig.
4d, e). In addition, cardiomyocytes in the Hif1α
high” group were enriched for
Ldha, Pgk1, Pfkl, and
Hk2 transcripts (Fig.
5f), which are known targets of
Hif1α. No differences in average numbers of nuclei were found in Hif1α
+ compared to Hif1α
− cardiomyocytes (Suppl. Fig. 4C).