Heart failure with preserved ejection fraction (HFpEF) is characterized by a reduced functional reserve. Our hypothesis was that HFpEF is linked to molecular-level changes in intracellular calcium signaling microdomains, which impair the functional capacity of cardiomyocytes. In this study, we treated mice with salbutamol (ß2-selective agonist) or bisoprolol (ß1 - selective antagonist) to characterize the response to adrenergic modulation.
Male mice (C57Bl/6N) were subjected to a high fat diet and L-NAME treatment (0.5 g/l, via drinking water) for 19 weeks. After 15 weeks, HFpEF animals received either bisoprolol (orally) or salbutamol (subcutaneously) for 4 weeks, 6 days each week. Echocardiography at rest and during isoproterenol stress was performed during the last week. Treadmill testing were conducted on all mice. LV cardiomyocytes were isolated and studied using confocal Ca2+ imaging (fluo-4). NCX activity was assessed by applying caffeine. Sarcoplasmic reticulum (SR) calcium leak was evaluated using tetracaine. Proteomics analysis was performed on left ventricular heart tissue samples, with four samples in each group.
Bodyweight of salbutamol treated mice was significantly reduced compared to both the HFpEF group and the bisoprolol-treated group (Salbutamol 46.05 g; HFpEF 50.3 g, Bisoprolol 49.09 g, respectively). Exercise capacity, measured by running distance, was significantly impaired in HFpEF mice and in both treatment groups (Bisoprolol and Salbutamol) compared to Controls (40.9 m, 48.5 m, and 48.3 m vs. 86.1 m, respectively). The mitral E/e’ ratio, an index of diastolic dysfunction, was significantly lower in the salbutamol-treated, bisoprolol-treated, and control groups compared to the untreated HFpEF group (22.6 ± 4.7, 28 ± 4.1, and 24.2 ± 4.1 vs. 34.6 ± 8.2, respectively at rest and 24.5 ± 6.9, 28.1 ± 5.8, 28.8 ± 5.5 vs. 42.5 ± 11.5 with isoproterenol stress). In vitro, cardiomyocyte cross-sectional area (cell size) was significantly reduced in salbutamol-treated mice compared to the HFpEF and bisoprolol groups (Salbutamol 3509.66 µm2; Control 3604.89 µm2 ; vs HFpEF 4109.04 µm2 and Bisoprolol 3986.79 µm2).
Calcium decay kinetics (Tau), did not differ significantly between groups (HFpEF: 176 ± 37.9 ms; Control: 191.5 ± 64.1 ms; Salbutamol: 186.7 ± 77.8 ms). Calcium decay kinetics in response to caffeine application were also not significantly different between groups. There was a trend toward higher calcium transient amplitude in HFpEF cardiomyocytes compared to wildtype and salbutamol-treated mice (HFpEF 1.94 ± 0.23; Control 1.73 ± 0.09; Salbutamol 1.81 ± 0.18). SR calcium leak was not significantly different between groups. Proteomic analysis suggested that salbutamol may influence mitochondrial metabolism, as indicated by the upregulation of GOT1 (p-value: 0.02), leading to an increase of pyruvate. In vitro incubation of HFpEF cardiomyocytes with pyruvate did not alter baseline calcium kinetics.
Overall, salbutamol and bisoprolol significantly improved diastolic function in vivo. Salbutamol significantly decreased cardiomyocyte size in vitro. No significant changes in baseline calcium levels were observed. Salbutamol treatment may modulate mitochondrial metabolism. However, this needs further investigation.
