Background and Aim: Increased sodium (Na⁺) intake is associated with higher rates of cardiovascular events. We previously observed that in patients with primary hyperaldosteronism, Na⁺ is stored in the myocardium, and spironolactone lowers blood pressure and Na⁺ storage. Furthermore, spironolactone has beneficial effects on cardiac remodeling after myocardial infarction (MI). While in patients and animal models of heart failure, elevated intracellular Na⁺ concentrations in cardiac myocytes induce energy supply-and-demand mismatch and oxidative stress through a negative impact on mitochondrial Ca²⁺ handling, it is currently unresolved whether spironolactone affects intra- and/or extracellular levels of Na⁺ in the myocardium after MI.

Methods: To investigate the effects of spironolactone on intra- or extracellular sodium storage, 10-week-old mice underwent either Sham or MI surgery by ligation of the coronary left anterior descendent artery. Mice were randomized to either spironolactone or vehicle solved in the drinking water (ad libitum) for eight weeks. Combined cardiac proton and Na⁺ MRI was performed at a 7T animal MRI system(Bruker BioSpin, Ettlingen). Cardiac MRI was performed to quantify cardiac mass as well as left and right ventricular function. Furthermore, total cardiac Na⁺content was determined by using ²³Na-MRI and a custom-made multi-echo UTE and referencing the Na⁺signal of the myocardium to vials with known Na⁺ concentrations of 50 and 100 mmol/l fixed to the coil. After MRI, cardiac myocytes were isolated by enzymatic digestion, electrically stimulated in an automated IonOptix/CytoCyfer multicell fluorescence microscopy system and exposed to different stimulation frequencies (0.5, 2 and 4 Hz) for at least 2 minutes. Intracellular Na⁺ concentrations ([Na⁺]_i) were determined using the Na⁺-sensitive fluorescent dye SBFI (10 µM).

Results: Eight weeks after MI, Na⁺ MRI revealed significantly elevated total Na⁺ content in the myocardium of mice after MI vs. Sham surgery (67.1 ±4.9 mmol/l vs. 43.4±2.4 mmol/l; MI vs Sham; p<0.05; Figure 1A). In contrast, in mice treated with spironolactone, the increase in total myocardial Na⁺ content after MI was blunted and not significant compared to Sham-operated animals treated with spironolactone, but on the other hand, not significantly lowered compared to mice after MI that were treated with vehicle (MI-vehicle, 67.1 ±4.9 mmol/l;  vs. MI-spiro, 54.4 ±12.9 mmol/l; p=0.4; Figure 1A). In isolated cardiac myocytes from mice after MI, [Na⁺]_i (at 2 Hz stimulation rate) was significantly elevated compared to sham-operated mice (MI, 23.2 ±2.4 vs Sham, 16.9 ±0.2 mmol/l; Figure 1B). In cardiac myocytes isolated from spironolactone treated MI mice, [Na⁺]_iwas significantly reduced compared to vehicle-treated MI mice (MI-vehicle, 23.1 ±2.4 vs. MI-spironolactone, 15.7 ±2.8 mmol/l; Figure 1B).

Conclusions: In mice after myocardial infarction, total myocardial Na⁺ content and in particular, [Na⁺]_i in cardiac myocytes is substantially elevated. While spironolactone to some extent dampens this increase in total myocardial Na⁺ content, it substantially lowers [Na⁺]_i in cardiac myocytes. Further analyses from our experimental cohorts will analyze whether this reduction in [Na⁺]_i has implications for cardiac myocytes Ca²⁺handling and mitochondrial redox state, given the previously established link between elevated [Na⁺]_i, oxidative stress, arrhythmias and heart failure development.