Sex differences in cardiovascular illnesses are becoming more well-acknowledged, although women are under-represented in clinical studies. Pre-menopausal women have a decreased risk of coronary artery diseases with lower vulnerability to ischemic heart damage and heart failure due to a lower ejection fraction, which reduces postmenopausal complications. Researchers created a two-hit model of HFpEF, which revealed that females with diabetes and HFpEF have more severe diastolic dysfunction.
In the present study, published in Cardiovascular Research, researchers investigated the impact of aldosterone infection in db/db mice on HFpEF, focusing on sex differences and alterations in intracellular calcium handling and cardiomyocyte electrophysiology. They also investigated sex-specific responses to medications such as empagliflozin, vericiguat, and calcium or calmodulin-based kinase II to facilitate clinical translation.
About the study
The researchers used adult wild-type (WT) and Leprdb/db C57BL/6J mice for analysis. They carried out transthoracic echocardiography, electrophysiological, intracellular calcium (Ca2+) imaging, and protein analysis. They implanted osmotic minipumps subcutaneously in the murine animals to continuously infuse 0.3 μg/hour of D-aldosterone or saline mixed with 5.0% ethanol over four weeks.
The team block-randomized the animals using a block size of four (per gender, genotype, and treatment) with 24 controls (vehicle + wild-type mice), 24 aldosterone-infused db/db mice, and eight aldosterone-infused wild-type mice, and eight saline-infused mice for one-hit controls. They euthanized the animals to isolate their cardiomyocytes.
The researchers evaluated glucose levels in blood samples drawn from the animals’ middle tail veins and quantified B-type natriuretic peptide (BNP) using enzyme-linked immunosorbent assays (ELISA). They used gel electrophoresis with immunoblotting to evaluate titin isoform levels, periostin expression, and troponin-I phosphorylation (pTnI). They assessed intracellular calcium signals using confocal microscopes in ventricular cardiomyocytes filled with the fluorescent intracellular calcium indicator, Fluo-4 AM.
The team examined morphometric and metabolic characteristics in male and female mice in HFpEF two-hit and one-hit illness models. They examined action potentials (Aps) in isolated ventricular myocytes and computed AP durations (APD) with varying repolarization rates. They also used voltage-clamp tests to detect ionic currents that mediate APD changes in HFpEF. They monitored repolarizing potassium (K+) currents and isolated IKv components using bi-exponential fitting.
Results
Female HFpEF murine animals showed lower BNP expression than males, with equivalent comorbidities and heart hypertrophy. Male HFpEF murine animals showed greater cardiac alternans susceptibility. Male HFpEF cardiomyocyte cells had greater diastolic calcium ions, slower calcium transient decaying, a lower L-type calcium current, more dramatic amplification of late sodium currents, and higher short-term APD variability.
However, female and male HFpEF myocytes revealed identical inward rectifier downregulation and brief outward potassium currents, action potential duration lengthening, and delayed afterdepolarization (DAD) frequency. Inhibiting Ca2+ or calmodulin-based protein kinase II (CaMKII) restored pathological APD alterations in female and male HFpEF mice, and empagliflozin treatment matched CaMKII inhibition effects. Vericiguat had minor advantages, which were more pronounced among HFpEF females.
Female HFpEF mice showed higher blood glucose, particularly after aldosterone infusion, indicating that HFpEF-induced increases were higher in females. Female aldosterone-infused mice showed lower serum BNP expression, indicating that BNP upregulation is lower among HFpEF females. The researchers found considerable diastolic dysfunction, notably among female HFpEF. The team observed concentric cardiac hypertrophies in aldosterone-infused mice of both sexes.
Male HFpEF animals displayed poor diastolic Ca2+ handling, but the intracellular calcium transient (CaT) amplitude during 1.0 Hz pacing remained consistent in female and male aldosterone-infused murine cardiomyocytes. Female HFpEF myocytes showed more myofilament alterations, including a slight increase in the shorter and stiffer N2B isoform. Empagliflozin and CaMKII inhibition diminished arrhythmic APD alterations in HFpEF myocytes, whereas vericiguat reduced AP duration at 90% repolarization (APD90) prolongation, short-term variability (STV), and DAD frequency in female HFpEF myocytes.
Using a mouse model of HFpEF cardiomyocytes, the study showed that male myocytes exhibited aberrant calcium electrophysiology and handling, resulting in arrhythmia and diastolic dysfunction. Females may have significant diastolic dysfunction as a result of altered myofilament characteristics.
Conclusion
The work proposes that poor excitation-contraction coupling and sex-dependent arrhythmia susceptibility in HFpEF cardiomyocytes may have therapeutic implications. However, further research is required to understand better the HFpEF cardiomyocyte signaling cascades, including the protein kinase G (PKG) and CaMKII pathways, and to increase therapeutic translation potential.