The coronary microvasculature regulates coronary blood flow to meet cardiac metabolic demand. Structural and functional impairments of the coronary microvasculature lead to coronary microvascular disease (CMVD), which accounts for 30-50% of ischemic heart disease. Despite its clinical burden, little is known about the molecular mechanisms behind CMVD. 

Cardiomyocyte-specific transcription factors are essential for the development and maintenance of the coronary microvasculature. Cardiac Friend of GATA 2 (FOG2) is one such factor. FOG2 is known to repress GATA4, a transcription factor crucial to the development and maintenance of cardiomyocytes. We previously identified an association between a coding variant of FOG2 (rs28374544, A1969G, Ser657Gly) prevalent in patients of African ancestry (minor allele frequency ~20%) and CMVD. We also developed a mouse model with a mutation analogous to FOG2S657G using CRISPR/Cas9, termed Fog2mut. RNA-sequencing and Gene Set Enrichment Analysis data from Fog2mut hearts showed increased adrenergic signaling and, specifically, increased expression of β1 adrenergic receptor (ADRB1) at the gene level. Subsequent immunoblot showed increased ADRB1 expression at the protein level. We hypothesize that FOG2S657G fails to repress GATA4, increasing β1 adrenergic receptor signaling and, subsequently, cardiac work, thereby promoting CMVD.

To test this hypothesis, we employed in vitro and in vivo methods. In vitro, we assembled an ADRB1 luciferase reporter featuring the ADRB1 promoter (2893-5375) upstream of luc2 using Gateway cloning. Nanopore sequencing confirmed successful assembly of the reporter. Following transfection into HEK293 cells and measurement of luminescence using a dual-luciferase assay 72 hours post-transfection, we found samples with the reporter had increased luminescence relative to negative controls, indicating function of the reporter. We plan on employing this reporter extensively in dual-luciferase assays to determine the mechanisms by which FOG2S657G increases ADRB1 expression.

In vivo, we measured the heart rate and blood pressure of Fog2mut mice using the BP-2000 Blood Pressure Analysis System via repeated tail cuff measurements over the course of 3 days, averaging to roughly 36 individual time point measurements per mouse. Age-matched wildtype littermates served as controls. All mice were acclimated for 3 days prior to the experiment. We found no significant difference in heart rate or blood pressure between Fog2mut and littermate control mice.

Furthermore, we isolated primary cardiomyocytes from Fog2mut and littermate control mice. Following successful isolation, we used the IonOptix Multicell High Throughput System to compare the contractility of electrically-paced Fog2mut cardiomyocytes to those of littermate controls both with and without isoproterenol. Without isoproterenol, Fog2mut cardiomyocytes exhibited significantly greater time to 90% peak, time to 90% baseline, and time to peak as well as increased percent change in sarcomere length relative to littermate control cardiomyocytes, indicating larger contractions with slower contraction times. Following addition of isoproterenol, these differences were muted.

Our data is consistent with FOG2S657G increasing β1 adrenergic receptor signaling and, subsequently, cardiac work, thereby promoting CMVD. However, further work is needed to elucidate the mechanisms by which FOG2S657G does so. Characterizing the relationship between FOG2S657G and CMVD could lead to a better understanding of the pathogenesis of CMVD and reveal therapeutic targets.