Thread by @patrick_ellinor

Excited to report that our latest work using snRNAseq to dissect the molecular basis of end-stage DCM and HCM is now published @Nature.

Large collaborative effort led by the incredibly talented Mark Chaffin @broadinstitute.

nature.com/articles/s4158…

Building on previous work focused on the healthy heart (ahajournals.org/doi/10.1161/CI…), we sought to assess how cellular composition and transcription changes in failing hearts.

Working with Ken Margulies and Ken Bedi at @PennMedicine, we obtained left ventricular tissue from explanted hearts of 11 DCM and 15 HCM patients, along with 16 additional non-failing donor hearts.

@Irinna_P, Amer-Denis Akkad and @NR_Tucker performed snRNAseq on these samples capturing nearly 600,000 nuclei, with computational analysis identifying 21 cell type clusters:

We observed the anticipated & stark transcriptional changes in DCM/HCM hearts compared to non-failing individuals, with 1000s of differentially expressed genes across the full range of cell types:

Conversely, there were relatively few differences between late-stage DCM and HCM hearts, suggesting that regardless of the disease etiology, at end-stage heart failure a dying heart looks like a dying heart:

Beyond transcriptional differences we also observed shifts in the composition of cell types. This included a reduction in cardiomyocytes and an increase in fibroblasts in the cardiomyopathies.

One striking observation was a population of activated fibroblasts derived nearly entirely from DCM and HCM patients, particularly from one DCM and one HCM patient.

These fibroblasts are transcriptionally distinct with upregulation of known markers such as POSTN, THBS4, COL1A1/COL1A2, and FAP, along with other less explored markers.

Using RNAscope, Bridget Simonson validated that these fibroblasts exist and are highly enriched in DCM and HCM patients: (DCN in blue, COL22A1 in red, hi res pics in the paper)

Next, we (aka Mark) computationally inferred trajectories in fibroblasts from a quiescent to activated state in the two patients with an appreciable activated fibroblast population and identified genes that change in expression along the transition.

To improve our understanding of the mechanistic role of these genes, we worked with Maria Kost-Alimova, Michelle Melanson, and Virendar Kaushik from @broadinstitute Center for the Development of Therapeutics to design a CRISPR screen in primary cardiac fibroblasts.

By stimulating cardiac fibroblasts with TGFB1, we can induce the well known switch to an activated/myofibroblast state as evidenced by a marked increase in smooth muscle actin (SMA).

However, when we knock out known genes in the TGFB signaling pathway, we no longer see this transition as expected.

Notably, we see a similar effect when we knock out other genes we identified along the trajectory in the snRNAseq data, including PRELP and JAZF1.

When we quantify the effect for the full list of 62 genes from our trajectory analysis, we see strong effects for multiple guides targeting PRELP, JAZF1, COL22A1, and AEBP1 suggesting these genes may have a mechanistic role in mediating fibroblast activation.

Altogether, this body of work expands our understanding of the changes in gene expression in late-stage cardiomyopathy at cell type resolution and implicates a subset of genes as playing a mediating role in cardiac fibrosis.

Along with members mentioned along the way, thanks to the entire team including Matt Hill, Alessandro Arduini, Stephen Fleming, @sikandhayat, Ondine Atwa, Jiangchuan Ye, @MatthiasNahrend & @CMStegmann.

@MGHHeartHealth, #singlecell

And huge thanks for the support of the entire Precision Cardiology Lab team. A joint effort between scientists @broadinstitute and @BayerPharma.

There are likely many other stories that could be told, so data is already available at @SingleCellBroad:
singlecell.broadinstitute.org/single_cell/st…

And via @NIH / @nih_nhlbi supported dbGaP:
ncbi.nlm.nih.gov/projects/gap/c…