Towards treatment of Dilated Cardiomyopathy: Serine Biosynthesis as a Potential Target

Dilated cardiomyopathy (DCM) is characterized by ventricular dilatation as well as contractile dysfunction and is a common cause of heart failure. Among other causes, DCM is regularly based by mutations in a broad variety of genes, however, the pathophysiological mechanisms linking genotype to phenotype development are only incompletely understood. 

To model DCM in vitro, we use induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) from a from a patient with severe familial DCM carrying a heterozygous mutation at position R183W in exon 12 of the Troponin T (TNNT2) gene (TNNT2^HET), isogenic iPSC-CM with correction of the mutation (TNNT2^CORR) as well as isogenic homozygous iPSC-CM (TNNT2^HOM). We recently showed that activation of ATF4 and subsequently serine biosynthesis and one carbon pathways impact contractility in cardiomyocytes. To further elucidate, which parts of the ATF4 and serine dependent pathways are relevant for the regulation of contractile functions, we used lentivirus induced knockdown or overexpression of involved genes and performed a high-resolution microscopy assay with pixel-based tracking to assess changes in contractility as well as 3D engineered heart tissue (EHT) based analysis of force generation. 

Knockdown of key components of the serine i.e. biosynthesis pathway phosphoglycerate dehydrogenase (PHGDH), and phosphoserine phosphatase (PSPH) in TNNT2^CORR iPSC-CMs resulted in impaired contractility. Correspondingly, overexpression of PHGDH and PSPH in TNNT2^HOM iPSC-CMs significantly increased contractility. However, we did not observe effects on contractility following overexpression of genes involved in GSH production or mitochondrial serine glycine conversion. We were able to recapitulate the specific effects of serine biosynthesis employing CRISPRa and CRISPRi based gene expression modulation. For this, we generated two isogenic iPSC lines from the TNNT2^HOM iPSC constitutively expressing CRISPRa and CRISPRi, respectively. 

Furthermore, higher levels of serine in iPSC-CM media improved the DCM phenotype. 

To assess the metabolic effect of serine pathway modulation, we performed seahorse-based analysis in TNNT2^HOM iPSC-CMs. We observed an increase in oxygen consumption rate in TNNT2^HOM iPSC-CMs upon PHGDH and PSPH overexpression as well as increased serine in the media, supporting a role for serine biosynthesis in cardiomyocyte function. 

In conclusion serine biosynthesis pathway is directly linked to contractile functions and is impaired in DCM phenotype. Activation of key components of the pathway as well as increased serine levels improve contractility as well as the metabolic activity of DCM cardiomyocytes.  Taken together these findings offer novel therapeutic options in DCM patients.