Fibroblast Shifts in the Hypertensive Heart: How Single Cell RNA-Sequencing will Accelerate Advancements in Anti-fibrotic Therapies

Graphical Abstract

Letter to the Editor

Since the development of single cell RNA-sequencing (scRNA-seq) technology, characterization of the heterogeneous cardiac cellulome has been a primary goal. Major avenues of research have focused on development of the embryonic heart and the impact of injury on the transcriptome of adult cardiac cells. In this regard, a comprehensive overview of scRNA-seq procedures and application in cardiac cells was recently published in the Journal of Molecular and Cellular Cardiology¹. Compared to cardiomyocytes, cardiac fibroblasts are more easily investigated by scRNA-seq due to their size and viability post-digestion. ScRNA-seq and subsequent differential gene expression has identified distinct cardiac fibroblast subpopulations highlighting their heterogeneity within the myocardium^2,3. Cardiac fibroblasts arbitrate extracellular matrix (ECM) deposition in the remodeling myocardium in pathological disease states such as hypertension and myocardial infarction (MI). Thus, the identity and function of cardiac fibroblast subpopulations is important for elucidation of novel therapeutic targets. Cardiac fibroblasts are much more dynamic than previously thought and targeting specific subpopulations is a viable approach for limiting the extent of cardiac fibrosis with both acute and chronic injury.

Independent studies of MI in the normotensive heart have elucidated novel markers for activated fibroblasts including scx, thbs4⁴, and ckap4² that are upregulated in fibrogenic cardiac fibroblast subpopulations. The post-MI time course has been utilized to characterize general shifts in fibroblast phenotypes using bulk RNA-seq identifying pro-inflammatory, proliferative, and neo-homeostatic transitions⁵. In a similar time course approach, scRNA-seq revealed that an early shift to proliferation, reduced fibrinolysis, and increased angiotensin II (Ang II) responsiveness leads to a higher rate of cardiac rupture in hypertension-prone mice three days post-MI⁶. Just as important as the identification of candidate markers and targets is the time frame for efficacious drug delivery. The genetic makeup and relative proportion of cardiac fibroblast subpopulations holds valuable information for limiting cardiac fibrosis in both normotensive and hypertensive cardiac injury.

Hypertension is a major risk factor for cardiovascular disease-related mortality given that 20% of people with heart failure⁷, ~35% with ST-elevation MI (STEMI), and ~75% with Non-ST-elevation MI (NSTEMI)⁸ had antecedent high blood pressure. Nearly 50% of Americans are diagnosed with high blood pressure⁷, thus highlighting the critical need to determine the impact of hypertension on cardiac fibroblast subpopulations. Resident cardiac fibroblasts expand and activate in the hypertensive and pressure overloaded heart, likely producing altered fibroblast subpopulations that mediate the resultant cardiac remodeling (Figure 1). Our laboratory has shown that transiently inhibiting the renin angiotensin system (RAS) produces a suppressed fibrogenic cardiac fibroblast phenotype that persists even after cessation of treatment in spontaneously hypertensive rats⁹. Based on prior work from our laboratory and others, we have proposed that associated with hypertension, fibroblast hyperplasia leads to an expansion of a pathological subset of fibroblasts that is also susceptible to RAS inhibitor-induced apoptosis¹⁰. Thus, it may be that the cardioprotection conferred by RAS inhibition is due, in part, to apoptosis of a susceptible fibroblast subpopulation that is particularly fibrogenic¹⁰. This hypothesis that hypertension produces pathological subsets of resident cardiac fibroblasts that predispose the heart to fibrotic remodeling and adverse events was supported by recent work from McLellan et al³ who evaluated the impact of Ang II infusion on cardiac cell populations. ScRNA-seq analysis identified nine subpopulations of cardiac fibroblasts that included two fibrogenic pools (Cilp⁺ and Thbs4⁺) that were expanded following Ang II infusion³. These subpopulations of fibroblasts were associated with ECM and wound healing, thus highlighting them as potential targets for intervention.

Figure 1.

In the hypertensive and hypertrophied heart, resident cardiac fibroblasts undergo hyperplasia and activation resulting in a more fibrogenic phenotype that promotes fibrotic remodeling. Created with BioRender.com.

In conclusion, identification, localization, and evaluation of relative abundance of various subpopulations of fibroblasts in the hypertensive heart is important for developing targeted therapies that remove or inhibit particularly fibrogenic fibroblasts, while still allowing for maintenance of the ECM and appropriate wound healing response after injury. The key to treating or preventing heart failure is not stopping but limiting the extent and duration of cardiac fibroblast activation in the post-MI and pressure overloaded heart. Given the risk that hypertension presents for both increased prevalence of and worsened outcomes following cardiac injury, cardiac fibroblasts in the hypertensive heart are understudied. Using scRNA-seq as a tool for profiling subpopulations of cardiac fibroblasts and understanding the ways in which they are impacted by hypertension would fill important knowledge gaps that will be extremely valuable in the development of novel anti-fibrotic therapies.