Introduction and Aims: Impaired relaxation and increased diastolic stiffness of the heart are considered hallmarks of heart failure (HF) with preserved ejection fraction (HFpEF). HFpEF is associated with several comorbidities including hypertension, obesity and diabetes. HFpEF is more common in the elderly and in women than in men. This suggests that sex may play a prominent role in disease progression. Hence, our understanding of the pathophysiology of sex differences in HFpEF are limited. In the present study we aimed to analyse the effect of sex on cardiomyocyte and myocardial function in a 1-year-old rat animal model of hypertension characterized with diastolic dysfunction.

Methods: We used male and female transgenic renin overexpressing rats (carrying mouse Ren-2 renin gene, mRen2) and compared them to Sprague-Dawley (SD) rats.  Expression and oxidation of protein kinase G (PKG) and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) were analysed. In addition, expression and phosphorylation of several sarcomeric and Ca²⁺-handling proteins including titin, cardiac myosin binding protein C (MyBPC), and cardiac troponin I (cTnI) were measured. The cellular mechanical performance was assessed in single skinned cardiomyocytes.   

Results: We found decreased Ca²⁺-sensitivity in the left ventricle (LV) from female mRen2 compared to SD, while increased in male mRen2 compared to SD rats. This was associated with increased cTnI phosphorylation in male mRen2 compared to the corresponding SD, but unchanged in female mRen2 rats. cMyBPC phosphorylation was increased in both but more pronounced in the male mRen2 vs. SD rats. The expression of the Ca²⁺-handling proteins RyR2, phospholamban and SERCA were unchanged in male and female mRen2 rats compared to SD. The cardiomyocyte passive tension (F_passive) was in increased in both sexes, but more elevated in the female mRen2 rats. In vitro treatment with CaMKII and PKG restored this increase. Titin phosphorylation at the CaMKII-dependent sites Ser4043 and Ser12884 were reduced in male mRen, whereas Ser4043 was increased and Ser12884 more decreased in female mRen2 rats. The PKG-dependent titin site Ser4080 showed a phosphorylation deficit for both with a more pronounced effect in the female mRen2. CaMKII oxidation was increased in both sexes, but more elevated in the female, CaMKII expression and CaMKII auto-phosphorylation were unchanged. Both, male and female mRen2 rats displayed lower PKG expression levels compared to SD whereas this reduction was more prominent in the female rats. In addition, the oxidative status of PKG was significantly altered in both sexes.

Conclusion: This study showed that cardiomyocyte dysfunction in a female rat model with diastolic dysfunction is associated with sever deranged phosphorylation of myofilament proteins and signalling pathways due to increased oxidative stress and oxidative state of CaMKII and PKG in a sex-dependent manner.