Oxidative stress as a potential link between impaired cardiac energetics and diastolic dysfunction in cardiomyocytes

Oxidative stress has been shown to be causally involved in the pathophysiology of cardiac remodeling and heart failure. However, the underlying molecular mechanisms are still not fully understood, which hampers the development of antioxidant therapies. Mitochondrial respiration is a main source of reactive oxygen species (ROS) in cardiomyocytes under conditions of increased workload or disturbed mitochondrial integrity. Mitochondrial dysfunction might result in a dysbalanced energy supply, which can disturb diastolic function. Here we aim to investigate the interplay between oxidative stress, cardiac energetics and diastolic function in isolated adult murine cardiomyocytes. 

To study the effects of oxidative stress on cardiac sarcomere function and mitochondrial respiration, we treated isolated cardiomyocytes with H2O2 (100 µM, 60 min), which lead to an increase in established markers for oxidative stress, i.e. hyperoxidized peroxiredoxin and protein carbonylation. H2O2-treated cardiomyocytes displayed a slightly impaired mitochondrial oxygen consumption rate as detected with an Oroboros O2k respirometer (oxygen consumption state 3: 390.4 ± 78.0 pmol/s/ml vs. 296.5 ± 54.6 pmol/s/ml; n = 6). In addition, treatment of cardiomyocytes with angiotensin II (1 µM), which indirectly enhances ROS generation, also reduced the mitochondrial oxygen consumption rate. Interestingly, these changes were mitigated in cardiomyocytes from mCAT mice overexpressing human catalase in the mitochondria. This finding indicates that ROS are directly involved in the disturbance of respiratory chain complexes. Furthermore, treatment of cardiomyocytes with either H2O2 or electrical stimulation with parallel β-adrenergic stimulation (180 min, 50 nM isoprenaline) both resulted in a significantly decreased diastolic sarcomere length and impaired fractional shortening. Increased ROS release through stimulation was detected by increased protein carbonylation. To further evaluate a connection between reduced energy supply and the impairment of diastolic function, we measured cardiomyocyte contractility under electrical field stimulation with and without inhibition of creatine kinase (CK) by 1-Fluoro-2,4-dinitrobenzene (20 µM). With increasing stimulation frequency, which simulates enhanced cardiac workload, diastolic sarcomere length showed a step-wise decline (1.835 ± 0.011 µm (1 Hz) vs. 1.680 ± 0.044 m (10 Hz); p < 0.001; n = 5), while fractional shortening was maintained at the lower stimulation frequencies (12.58 ± 1.69 % (1 Hz) vs. 12.57 ± 1.50 % (5 Hz); n = 5). In contrast, inhibition of CK reduced fractional shortening already under lower stimulation frequencies (12.98 ± 2.37 % (1 Hz) vs. 10.79 ± 1.65 % (5 Hz); p = 0.03; n = 5), which could be attributed to a further decrease in diastolic sarcomere length. Immunoblot analysis showed reduced CK protein levels in H2O2-treated cardiomyocytes in a concentration dependent manner, which might indicate oxidative damage and proteolytic degradation of CK protein. 

Our findings show that oxidative stress has a detrimental effect on mitochondrial components by directly affecting respiratory chain complexes and CK proteins, which may result in the depletion of the myocardial energy reserve and impaired diastolic function. Whether antioxidant therapies have therapeutic effects on diastolic dysfunction in heart failure models needs to be elucidated.