CaMKII does not control mitochondrial Ca2+ uptake in cardiac myocytes

KEY POINTS: Mitochondrial Ca(2+) uptake stimulates the Krebs cycle to regenerate the reduced forms of pyridine nucleotides (NADH, NADPH and FADH2 ) required for ATP production and reactive oxygen species (ROS) elimination. Ca(2+) /calmodulin-dependent protein kinase II (CaMKII) has been proposed to regulate mitochondrial Ca(2+) uptake via mitochondrial Ca(2+) uniporter phosphorylation. We used two mouse models with either global deletion of CaMKIIdelta (CaMKIIdelta knockout) or cardiomyocyte-specific deletion of CaMKIIdelta and gamma (CaMKIIdelta/gamma double knockout) to interrogate whether CaMKII controls mitochondrial Ca(2+) uptake in isolated mitochondria and during beta-adrenergic stimulation in cardiac myocytes. CaMKIIdelta/gamma did not control Ca(2+) uptake, respiration or ROS emission in isolated cardiac mitochondria, nor in isolated cardiac myocytes, during beta-adrenergic stimulation and pacing. The results of the present study do not support a relevant role of CaMKII for mitochondrial Ca(2+) uptake in cardiac myocytes under physiological conditions. ABSTRACT: Mitochondria are the main source of ATP and reactive oxygen species (ROS) in cardiac myocytes. Furthermore, activation of the mitochondrial permeability transition pore (mPTP) induces programmed cell death. These processes are essentially controlled by Ca(2+) , which is taken up into mitochondria via the mitochondrial Ca(2+) uniporter (MCU). It was recently proposed that Ca(2+) /calmodulin-dependent protein kinase II (CaMKII) regulates Ca(2+) uptake by interacting with the MCU, thereby affecting mPTP activation and programmed cell death. In the present study, we investigated the role of CaMKII under physiological conditions in which mitochondrial Ca(2+) uptake matches energy supply to the demand of cardiac myocytes. Accordingly, we measured mitochondrial Ca(2+) uptake in isolated mitochondria and cardiac myocytes harvested from cardiomyocyte-specific CaMKII delta and gamma double knockout (KO) (CaMKIIdelta/gamma DKO) and global CaMKIIdelta KO mice. To simulate a physiological workload increase, cardiac myocytes were subjected to beta-adrenergic stimulation (by isoproterenol superfusion) and an increase in stimulation frequency (from 0.5 to 5 Hz). No differences in mitochondrial Ca(2+) accumulation were detected in isolated mitochondria or cardiac myocytes from both CaMKII KO models compared to wild-type littermates. Mitochondrial redox state and ROS production were unchanged in CaMKIIdelta/gamma DKO, whereas we observed a mild oxidation of mitochondrial redox state and an increase in H2 O2 emission from CaMKIIdelta KO cardiac myocytes exposed to an increase in workload. In conclusion, the results obtained in the present study do not support the regulation of mitochondrial Ca(2+) uptake via the MCU or mPTP activation by CaMKII in cardiac myocytes under physiological conditions.