Cardiac Reprogramming: Toward a Total Eclipse of the Failing Heart

Heart failure from ischemic heart disease is the leading cause of death, morbidity, and healthcare expenditure in the United States and worldwide. Mortality from time of diagnosis of heart failure is staggering—within five years, 50% of patients will have died¹. Despite advances, mortality rates have improved modestly and the only cure for heart failure remains transplantation. Fewer than 4,000 transplants are performed in the United States annually. Over 200,000 patients are listed as eligible for heart transplantation and millions more die without listing². Thus, novel therapies for heart failure are desperately needed. Towards this goal, in the current issue of Circulation, Tani et al report the therapeutic efficacy of cardiac reprogramming for chronic ischemic heart failure³. Through elegant in vivo proof-of-concept studies, this work signals great promise for the future of reprogramming, and hope for a field eagerly awaiting translation of science-driven innovations to heart failure therapies.

The adult heart is one of the least regenerative tissues in the human body. Following hypoxemia from myocardial infarction (MI), cardiomyocytes undergo massive cell death and resident cardiac fibroblasts (CFs) are activated to form mature ischemic scar, which serves as the nidus for heart failure and sudden cardiac death. Following landmark discoveries describing the ability of the Yamanaka factors to induce cellular transdifferentiation, greater attention has been directed toward strategies harnessing resident cells within the heart to therapeutically limit fibrosis and augment cardiac function⁴.

After over a decade of investigation, direct cellular reprogramming of resident CFs to cardiac-like myocytes (iCMs) has reliably emerged as a promising strategy to repurpose the injured heart toward a functional myocardium. Cell fate conversion is contingent on a fundamental change in the epigenetic structure of a cell. As such, transcription factors and epigenetic factors critical to cardiac development have materialized as promising candidates to orchestrate this conversion⁵. Seminal discoveries demonstrated that cardiac reprogramming could be achieved through overexpression of the cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT)⁶. Addition of Hand2 (GHMT) was later found to more completely reprogram fibroblasts to iCMs and further improved cardiac function in mouse models of acute MI⁷. Since these pioneering discoveries, the race has been on to identify factors that improve efficiency of this process and provide mechanistic clues to its holy grail: effective reprogramming of fibroblasts to contractile cardiac tissue in the ischemic and failing adult human heart.

Despite a decade of advances with the therapeutically directed purpose of reprogramming, a critical question has remained unanswered; can non-viable myocardium populated by mature scar effectively undergo reprogramming? Previously, reprogramming was not attempted in models of chronic infarction causing heart failure due to the technical challenge of repeated thoracotomies to achieve temporally separated MI by left anterior descending (LAD) artery ligation and intramyocardial delivery of reprogramming factors. Thus, it has remained unknown whether CFs within chronically infarcted tissue are capable of undergoing reprogramming to a myocyte-like state.

In convincing proof-of-concept studies, Ieda and colleagues break new ground, demonstrating the ability of mice with chronic heart failure to undergo reprogramming and recover their cardiac function. Generating an elegant transgenic mouse model combining inducible fibroblast lineage tracing with reprogramming factor overexpression, Ieda and colleagues achieved precise control of the expression of cardiac transcription factors Mef2c, Gata4, Tbx5, and Hand2 (MGTH) in a spatiotemporal fashion (Fig 1). Using a tamoxifen-inducible Cre knocked into the fibroblast-specific Tcf21 locus (Tcf21^iCre/Tomato/MGTH), induction with tamoxifen specifically activated Tomato and MGTH transgenic expression in Tcf21-labeled fibroblasts. The authors first validated their model through in vitro and in vivo studies in the setting of acute injury and subjected these transgenic Tcf21^iCre mice to tamoxifen administration for 4 days followed by MI. Quantitative studies revealed that ~3% of Tcf21+ cells underwent reprogramming resulting in significant improvement in cardiac function and mortality compared to controls. They then addressed the ability of this model to improve cardiac function in the setting of chronic heart failure. The authors performed LAD ligation and waited one month to induce reprogramming factor expression with the addition of tamoxifen with data analysis 3 months post-MI. While the majority of CFs and iCMs were observed in the border zone of the infarcted area, quantitative studies demonstrated that 2% of Tcf21+ cells were effectively reprogrammed and expressed cardiac markers. Using an mTmG cassette, Tani et al validated that this effect on chronic heart failure was indeed from bona fide cellular reprogramming, as opposed to fusion events. By echocardiography, reprogramming induced a ~10% improvement in ejection fraction. Histological analyses uncovered a significant decrease in fibrotic area, which raises the question as to whether reprogramming factors possess duality in their benefit for chronic heart failure as both agents of cell fate conversion and mitigating fibrosis within mature scar.

Figure 1. Cardiac reprogramming as a therapy for chronic heart failure.

Overexpression of reprogramming factors MEF2C, GATA4, TBX5, and HAND2 in Tcf21+ fibroblasts improves cardiac function and decreases fibrosis in ischemic cardiomyopathy.

Digging more deeply to interrogate the impact of reprogramming factors on established scar, the authors undertook single-cell sequencing of non-myocytes in the setting of chronic injury and late repair. Tani et al discovered a reduction of profibrotic CF clusters in hearts exposed to reprogramming factors and conversion of these populations to quiescent fibroblasts, with transcriptional profiles more closely approximating the uninjured state. Further, the authors found that it is partially through downregulation of a central regulator of myofibroblast activation, Meox1, that reprogramming factors achieve this alternative form of cell fate conversion. These findings highlight that not all successful avenues for cellular reprogramming in heart failure need to end with a myocyte fate. Rather, conversion of activated CFs to a more quiescent state in conjunction with iCM formation may provide benefit to the failing heart.

The sun is rising on reprogramming as a therapy for heart failure, but there remains much work to be done prior to effective translation of this technology to clinical practice. The work of Ieda and colleagues indicates that reprogramming in chronic heart failure is feasible and effective on a cellular and organ level, but these studies remain proof-of-concept as transgenic overexpression is not a tractable therapeutic strategy for human patients. Ultimately, transient and highly targeted overexpression systems delivered systemically or through intracoronary perfusion need to be developed and refined to present a clinically effective and safe mode of delivery of reprogramming factors to non-myocytes. Rapid advances in viral delivery systems and modified RNA technologies have injected the reprogramming field with new hope, but this aspect of therapeutic translation remains in its infancy. Most critically, achieving effective reprogramming of adult human CFs remains the overwhelming challenge. The majority of reprogramming cocktails with proven efficiency in murine cells have no translated efficacy in adult human CFs⁸. As of now, there is no consensus as to the most impactful human reprogramming cocktail, with most requiring upwards of six factors^9,10. Such a surplus of factors reduces precision and raises mechanistic uncertainties. Identifying minimalist yet effective cocktails to achieve adult human cardiac fibroblast reprogramming remains the most critical impasse for the field. Yet, recent advances from our laboratory and others identifying epigenetic and pioneer factors that function as human reprogramming factors, such as PHF7 and ASCL1, indicate that reprogramming in the setting of fewer cofactors is achievable^11,12. Continued efforts to identify novel reprogramming factors that function outside of the bounds of previous paradigms of reprogramming are needed. In the setting of these recent efforts, these rigorous studies by Ieda and colleagues provide a light at the end of the tunnel for the field and give fuel to the dream that reprogramming may one day become a therapeutic reality for the millions suffering from chronic heart failure.