A study led by scientists at Victor Chang Cardiac Research Institute in Sydney, Australia, has shown that the transcription factor hypoxia-inducible factor 1 (HIF-1) suppresses harmful reactive oxygen species (ROS)-induced growth of cardiac fibroblasts (CFs) and fibrotic scarring after myocardial infarction (MI) in knockout (KO) mice.

The study affords valuable new insights into altered cardiac homeostasis and factors underlying cardiac fibrosis at the single-cell level, which will help identify new therapeutic targets to prevent or repair post-MI fibrosis, the authors reported in the November 10, 2021, online edition of Cell Stem Cell.

"This is the first study to explore the role of Hif1a [the gene encoding an HIF-1 protein subunit] in CFs in a [KO] mouse model and at a single cell resolution," said study co-leader Richard Harvey.

"Cardiac fibrosis is a significant global health problem that is a common feature of many acute and progressive cardiovascular diseases, but for which the underlying mechanisms remain to be fully elucidated," said the co-deputy director and head of the Developmental and Stem Cell Biology Division at the Victor Chang Cardiac Research Institute.

"Understanding the phenotypic and functional characteristics of CFs, the principal cell type implicated in fibrotic remodeling, will be critical in developing therapeutic targets and designing clinical trials."

Under hypoxic conditions, HIF-1 activates gene pathways that minimize oxygen consumption, reduce ROS and restore tissue oxygenation.

HIF-1 pathways play a central role in cardiovascular biology, with gestational hypoxia triggering regional HIF-1-dependent pathways essential for normal heart development.

In post-MI ischemia in adult hearts, cardiomyocytes (CMs) undergo compensatory hypertrophy, which in the presence inflammation and fibrosis may be associated with increased ROS levels and oxidative damage.

CFs maintain cardiac biomechanical integrity by controlling extracellular matrix (ECM) deposition and turnover, but HIF-1's role in CF function remains unclear.

Hematopoietic and other stem/progenitor cells are known to reside in a hypoxic niche, with HIF-1 expression possibly being critical for balancing normal tissue homeostasis and function, and regeneration after injury.

"A hypoxic niche is known to be linked to maintenance of stem cell function, but this had not conclusively been shown in CFs, which have progenitor-like properties," noted Harvey.

Given these progenitor characteristics, HIF-1alpha likely has similar roles in CFs, possibly including maintaining quiescence or stem-like qualities, metabolic and redox control, and post-injury proliferation and differentiation.

These are difficult to study in CFs, but single-cell genomics has markedly improved the understanding of cardiac tissue structure and function.

Recently, the first comprehensive maps of CF heterogeneity and flux in healthy and diseased hearts have improved the understanding of CF biology and potential therapeutic interventions for fibrosis.

In their new study, researchers led by the Harvey lab's senior postdoctoral scientist, Vaibhao Janbandhu, investigated the function of HIF-1alpha in CFs in healthy hearts and post-MI using conditional gene targeting and single-cell genomics.

This revealed that CFs and their progenitors are more hypoxic than other cardiac interstitial cell types, express more HIF-1alpha and show increased glycolytic metabolism.

"We demonstrated the hypoxic nature of CFs and their progenitors using molecular and biochemical assays, transgenic mouse models and single cell genomics," explained Harvey.

"Our study is important in that it begins to probe CF origins, diversity and function at greater depth, and at a single cell resolution," Harvey told BioWorld Science.

Single-cell RNA sequencing revealed that CF-specific deletion of the Hif1a gene using conditional mouse genetics resulted in decreased HIF-1 target gene expression, increased progenitors in normal hearts, and augmented CF activation without proliferation following sham injury.

"These results suggest that CFs in Hif1a-deleted hearts show a degree of awakening and are poised for further activity," he said.

"Indeed, in post-MI hearts there was an approximately 50% increase in CF proliferation and excessive scarring and contractile dysfunction."

These findings were replicated in cultured, self-organizing 3D-engineered cardiac microtissues, "which resemble organ tissue complexity and more closely recapitulate the physiology and function of organs, in this case the heart," said Harvey.

"Our findings in cardiac microtissues are consistent with our in vivo studies in mice and support the notion that pathological fibrosis driven by the proliferation of CFs has scalable negative cell non-autonomous effects on CM contractile function."

CF proliferation was shown to be associated with higher ROS generation and occurred also in wild-type mice treated with the mitochondrial ROS generator MitoParaquat (MitoPQ).

"In myocardial ischemia, ROS were shown to increase in Hif1a KO CFs due to compromised HIF-1alpha-dependent adaptive pathways regulating mitochondrial metabolism," said Harvey.

"Increased ROS levels in Hif1a-mutant mice lead to proliferative fibrosis via activation of AKT and ERK signaling pathways, because ROS at physiological levels can biochemically modify and inactivate their inhibitors.

"These ROS effects were also replicated in wild-type mice treated with MitoPQ, confirming the role of ROS in CF proliferation," he added.

Importantly, treatment with the mitochondrial-targeted antioxidant MitoTEMPO was shown to rescue the Hif1a-mutant phenotypes.

"MitoTEMPO administration rescued Hif1a-mutant phenotypes, i.e., they led to normalization of increased mitochondrial ROS and proliferation defects seen in the Hif1a-mutant CFs, including total CF numbers, reduced cardiac scar size and improved cardiac function," said Harvey.

However, "since our findings are exclusively based on mouse data, they may not fully reflect responses to reduce HIF-1alpha function in CFs in human hearts," he cautioned.

Nevertheless, these findings collectively indicate that CFs represent potential cellular targets for designer antioxidant therapies in cardiovascular disease.

"While many antioxidant treatments have been studied in animal models and seem promising, translation of experimental findings into humans has been limited," noted Harvey.

Moreover, "the lack of benefit seen in clinical trials to date does not disprove the central role of ROS in cardiac fibrosis."

"ROS are clearly important for cellular and tissue function and responses to injury and the future challenge is to design better synthetic antioxidants that will target the right cells and cellular compartments." (Janbandhu, V. et al. Cell Stem Cell 2021, Advanced publication).