Reversibility of aldosterone-induced heart failure with preserved ejection fraction profiled by single-nucleus RNA-seq

Introduction
Heart failure with preserved ejection fraction (HFpEF) is a common condition associated with high morbidity and mortality, yet effective therapeutic options remain limited. This is partly due to a lack of robust preclinical models that accurately reflect the disease’s multifaceted pathophysiology. Currently, the mechanisms underlying potential recovery in HFpEF are not well understood, despite the fact that they may reveal new therapeutic avenues. Here, we present a novel, fully recoverable preclinical HFpEF model induced by aldosterone treatment, enabling the exploration of both disease progression and reversibility. Through a multifactorial approach-encompassing physiological assessments, histology, and single-nucleus RNA sequencing-our model provides critical insights into the molecular and functional adaptations across disease and recovery phases. 

Methods and Results
In this study, C57Bl/6N wildtype mice received aldosterone via osmotic minipumps (500 µg/kg/day) and were placed on a high-salt diet (1% NaCl in drinking water; ALDO) for two weeks, while untreated mice served as controls (CTRL). The aldosterone treatment induced a HFpEF-like phenotype, evidenced by impaired diastolic function, with echocardiographic measurements showing elevated E/E' ratios (CTRL: 29.9 vs. ALDO: 37.6, P < 0.002) and left atrial dilatation (LAs [mm²]: 3.7 vs. 4.6, P < 0.002). This was accompanied by structural remodelling, including LV- (LVPWd SAX [mm]: 0.60 vs. 0.78, P < 0.033) and cardiomyocyte hypertrophy (CSA mean [µm²]: 318 vs. 411, P<0.001), indicating moderate changes in heart structure.
After the removal of the aldosterone stimulus, a progressive recovery was observed, with substantial improvements noted at four weeks post-treatment (REC E/E' ratio: 33.1, LAs: 3.9 mm², LVPWd SAX: 0.71 mm, P < 0.033, CSA mean: 314 µm², P < 0.001) and full recovery by eight weeks.
This study includes two investigative approaches to assess phenotypic recovery: (1) an initial two-week aldosterone treatment with a six-month follow-up and (2) varying durations of aldosterone exposure (2, 4, 6, or 8 weeks), each followed by a four-week recovery period. Together, these findings indicate that HFpEF-related dysfunction and structural alterations induced by aldosterone are reversible, even after extended treatment. This reversibility highlights the potential for therapeutic recovery, offering insights into treatment possibilities for chronic HFpEF cases.
To explore the transcriptional mechanisms underlying these observations, we performed whole-tissue single-nucleus RNA sequencing (snRNA-seq) of the LV free wall (n = 12) from CTRL, ALDO, and REC samples, capturing 100,000 nuclei with an average of 2,300 genes detected per nucleus. While the global cardiac cellular composition remained unchanged, significant shifts in gene expression profiles within major cell clusters were observed, providing insights into the molecular pathways driving HFpEF and its reversibility.

Conclusions
Aldosterone/salt treatment induces diastolic dysfunction, LV hypertrophy, and fibrosis in mice. These changes are fully reversible after removal of the stimulus. snRNA-seq reveals key transcriptional shifts during treatment and recovery periods, providing valuable insights into the molecular mechanisms of HFpEF.