This study identifies KLF15 as the transcription factor with the most significantly altered transcriptional activity in pathological cardiomyocytes through single-cell transcriptomic analysis. By using the dCas9VPR system to enhance its endogenous expression in cardiomyocytes, the study effectively suppresses pathological cardiac remodeling and fibrosis, revealing the critical role of the TGF-β/KLF15/AZGP1 regulatory axis.
Literature Overview
The article titled 'Enhancing KLF15 activity in cardiomyocytes: a novel approach to prevent pathological reprogramming and fibrosis via nuclease-deficient dCas9VPR', published in Signal Transduction and Targeted Therapy, reviews and summarizes a novel strategy to inhibit pathological reprogramming and myocardial fibrosis by enhancing the activity of the transcription factor KLF15 in cardiomyocytes. By integrating single-cell transcriptomic analysis with CRISPR activation (CRISPRa) technology, the study systematically reveals the central role of KLF15 in maintaining cardiac metabolic homeostasis and suppressing the reactivation of fetal gene programs. It further discovers that KLF15 mediates crosstalk between cardiomyocytes and fibroblasts by regulating the secreted protein AZGP1, exerting a non-cell-autonomous anti-fibrotic effect. Additionally, the study develops an AAV delivery system applicable to human cardiomyocytes, offering a new paradigm for epigenetic intervention in non-genetic heart diseases. The article also elucidates the upstream and downstream regulatory mechanisms of KLF15 within the TGF-β signaling pathway, expanding our understanding of the cardiac pathological remodeling network. This work not only validates the feasibility of transcription factor activity modulation in treating cardiovascular diseases but also provides a blueprint for targeted interventions in other non-genetic disorders.Background Knowledge
Pathological cardiac remodeling is a key intermediate stage in the progression of cardiovascular diseases such as hypertension and myocardial infarction toward heart failure. It is characterized by cardiomyocyte hypertrophy, metabolic dysregulation, reactivation of fetal gene programs, and interstitial fibrosis. Although several signaling pathways involved in this process—such as the renin-angiotensin-aldosterone system (RAAS) and the TGF-β pathway—have been identified, therapeutic strategies targeting them are often limited by lack of cell specificity or systemic side effects. Transcription factors, as central nodes in gene expression regulation, are theoretically ideal therapeutic targets. However, their complex protein structures and diverse functions make them difficult to target with conventional small-molecule drugs. Recently, the development of CRISPR activation (CRISPRa) technology has provided a new tool for precisely enhancing endogenous gene expression. CRISPRa uses a nuclease-deficient dCas9 fused to transcriptional activation domains (e.g., VPR) to specifically bind promoter regions guided by sgRNAs, thereby upregulating target gene expression. Compared to traditional overexpression systems, CRISPRa avoids issues such as random insertion of exogenous genes and supraphysiological expression levels, offering more physiological regulation. KLF15, a member of the Krüppel-like factor family, has been reported in mouse models to regulate lipid metabolism and suppress cardiac hypertrophy. However, its transcriptional dynamics in human cardiac diseases and its potential as a therapeutic target remain unclear. Furthermore, crosstalk between cardiomyocytes and fibroblasts plays a crucial role in fibrosis, but whether cardiomyocytes can actively regulate fibroblast phenotypes through secreted factors remains to be fully elucidated. Therefore, this study aims to restore endogenous KLF15 function by combining single-cell-resolution transcription factor activity analysis with CRISPRa technology, exploring its therapeutic potential in inhibiting cardiac remodeling and filling several critical knowledge gaps in the field.
Research Methods and Experiments
The study first analyzed single-cell RNA-seq data from mouse pressure overload models (TAC) at different stages using the BITFAM model to systematically evaluate changes in transcription factor activity, identifying a significant reduction in KLF15 activity in pathological cardiomyocytes. By analyzing single-nucleus RNA-seq data from patients with dilated and hypertrophic cardiomyopathy, the study confirmed downregulation of KLF15 expression in human cardiomyocytes and leveraged GWAS data to reveal associations between KLF15 and multiple cardiovascular diseases.
To restore KLF15 activity, the research team generated a transgenic mouse model with cardiomyocyte-specific expression of dCas9VPR (Myh6-dCas9VPR) and delivered sgRNAs targeting the Klf15 promoter via AAV9 to enhance endogenous Klf15 transcription. Cardiac function, histopathology, and survival rates were assessed after TAC surgery, with Klf15 knockout mice used as negative controls to validate the specificity of the intervention.
Furthermore, single-cell RNA-seq was performed on cardiac cells after CRISPRa treatment to analyze transcriptomic changes in cardiomyocyte subpopulations and assess the restoration of metabolic and maturity-related genes. Tools such as CellChat were used to analyze intercellular communication and investigate the non-cell-autonomous effects of cardiomyocyte-specific KLF15 activation on fibroblasts. ChIP-seq and ELISA were employed to confirm direct regulation of the downstream target gene AZGP1 by KLF15 and changes in its secreted protein levels.
Finally, the study developed a compact CRISPRa-AAV system suitable for human cardiomyocytes, demonstrating its ability to effectively enhance KLF15 expression and upregulate AZGP1 secretion in hiPSC-derived cardiomyocytes, laying the foundation for clinical translation.Key Conclusions and Perspectives
Research Significance and Prospects
This study innovatively combines single-cell transcriptomics with CRISPRa technology, not only establishing the central role of KLF15 as a cardioprotective transcription factor but also demonstrating the great potential of epigenetic approaches to restore endogenous gene function in treating non-genetic heart diseases. Compared to traditional gene replacement therapies, CRISPRa avoids the risk of uncontrolled exogenous gene expression and aligns more closely with physiological regulatory logic, offering higher safety.
The newly identified TGF-β/KLF15/AZGP1 axis provides fresh insights into intercellular communication in the heart, particularly the active regulation of fibroblast phenotypes by cardiomyocytes via AZGP1 secretion, offering a novel therapeutic target for anti-fibrotic strategies. Moreover, the portability of this approach offers a universal paradigm for other non-genetic diseases involving transcription factor dysregulation, such as neurodegenerative and metabolic disorders.
Future studies could further explore the precise molecular mechanisms of AZGP1 in cardioprotection and develop protein-based therapies or small-molecule mimetics targeting AZGP1. Additionally, optimizing AAV serotypes and promoters for more efficient and specific myocardial targeting will be crucial for advancing this strategy toward clinical application. Long-term safety and efficacy testing in large animal models will also provide essential data for eventual clinical translation.
Conclusion
This study systematically reveals the central regulatory role of the transcription factor KLF15 in pathological cardiac remodeling, where its activity is significantly reduced in stressed cardiomyocytes, leading to metabolic imbalance and aberrant reactivation of fetal gene programs. Restoring endogenous KLF15 expression via cardiomyocyte-specific CRISPRa technology effectively reverses these pathological changes and preserves cardiac function. The study further demonstrates that KLF15 activation upregulates the secreted protein AZGP1, mediating non-cell-autonomous inhibition of fibroblasts by cardiomyocytes, thereby alleviating myocardial fibrosis and highlighting the critical role of the TGF-β/KLF15/AZGP1 axis in intercellular crosstalk. This work not only provides a novel therapeutic strategy for non-genetic heart diseases but also proves the feasibility of CRISPRa as an epigenetic tool for restoring dysregulated transcriptional networks. By developing an AAV delivery system suitable for human cardiomyocytes, the study paves the way for clinical translation, offering a vital theoretical foundation and practical example for the future development of precision gene therapies targeting heart failure and other non-genetic diseases.
