Over-expression of mitochondrial creatine kinase in the murine heart improves functional recovery and protects against injury following ischaemia–reperfusion

Abstract

AimsMitochondrial creatine kinase (MtCK) couples ATP production via oxidative phosphorylation to phosphocreatine in the cytosol, which acts as a mobile energy store available for regeneration of ATP at times of high demand. We hypothesized that elevating MtCK would be beneficial in ischaemia–reperfusion (I/R) injury.Methods and resultsMice were created over-expressing the sarcomeric MtCK gene with αMHC promoter at the Rosa26 locus (MtCK-OE) and compared with wild-type (WT) littermates. MtCK activity was 27% higher than WT, with no change in other CK isoenzymes or creatine levels. Electron microscopy confirmed normal mitochondrial cell density and mitochondrial localization of transgenic protein. Respiration in isolated mitochondria was unaltered and metabolomic analysis by 1 H-NMR suggests that cellular metabolism was not grossly affected by transgene expression. There were no significant differences in cardiac structure or function under baseline conditions by cine-MRI or LV haemodynamics. In Langendorff-perfused hearts subjected to 20 min ischaemia and 30 min reperfusion, MtCK-OE exhibited less ischaemic contracture, and improved functional recovery (Rate pressure product 58% above WT; P < 0.001). These hearts had reduced myocardial infarct size, which was confirmed in vivo: 55 ± 4% in WT vs. 29 ± 4% in MtCK-OE; P < 0.0001). Isolated cardiomyocytes from MtCK-OE hearts exhibited delayed opening of the mitochondrial permeability transition pore (mPTP) compared to WT, which was confirmed by reduced mitochondrial swelling in response to calcium. There was no detectable change in the structural integrity of the mitochondrial membrane.ConclusionsModest elevation of MtCK activity in the heart does not adversely affect cellular metabolism, mitochondrial or in vivo cardiac function, but modifies mPTP opening to protect against I/R injury and improve functional recovery. Our findings support MtCK as a prime therapeutic target in myocardial ischaemia.

Overexpression of mitochondrial creatine kinase preserves cardiac energetics without ameliorating murine chronic heart failure

“…Mice were fed irradiated Global Diet 2916 (Envigo, Huntingdon, UK) and water ad libitum and housed with littermates in specific pathogen-free conditions. Creation of the cardiac-specific transgenic CK-Mt-OE line and genotyping protocol has been described previously [48]. This strain has been backcrossed for > 10 generations using C57BL/6JOlaHsd mice obtained from Envigo, Huntingdon, UK (formerly known as Harlan).…”

“…The therapeutic potential for augmentation of Mt-CK in CHF has yet to be tested and, quite apart from it being a major determinant of PCr/ATP [49], there are good reasons to think that this approach may also be beneficial. Not least, is an effect to reduce opening probability of the mitochondrial permeability transition pore (mPTP), thereby preventing cardiomyocyte death [48,50]. This is a key event governing I/R injury where Mt-CK overexpression has already been shown to be cardioprotective [48], and it is also thought to contribute to cellular loss in the chronically failing heart.…”

“…Not least, is an effect to reduce opening probability of the mitochondrial permeability transition pore (mPTP), thereby preventing cardiomyocyte death [48,50]. This is a key event governing I/R injury where Mt-CK overexpression has already been shown to be cardioprotective [48], and it is also thought to contribute to cellular loss in the chronically failing heart. Preserving mitochondrial health is an important goal in heart failure, and the close functional coupling between Mt-CK and oxidative phosphorylation is thought to promote this.…”

“…For the reasons outlined above, we hypothesised that overexpression of Mt-CK would be beneficial in terms of cardiac energetics, function and structural remodelling in a mouse model of pressure overload CHF. We utilised an existing strain of cardiac-specific Mt-CK-OE under control of the α-MHC promoter that was generated in our laboratory [48]. This used a cautious transgenic approach to increase myocardial Mt-CK activity by 25-30%, which had no measureable effect on baseline mitochondrial function, but was sufficient to provide protection against I/R injury.…”

Mitochondrial CaMKII causes metabolic reprogramming, energetic insufficiency, and dilated cardiomyopathy

“…Creatine kinase maintains energetics by rapidly and reversibly producing creatine phosphate, a highly diffusible high-energy phosphate molecule important for cellular energy homeostasis. Over-expression of CK-M, the cytosolic myofibrillar isoform of creatine kinase, in several models of heart failure improves overall cardiac function and reduces cardiac dilation, 54,55 but the potential role of CKmito, the mitochondrial isoform, was only recently shown to improve myocardial response to injury 56 . We considered that loss of CKmito could be important to the dilated cardiomyopathy in mtCaMKII mice, as it associates with the ATP transporter on the mitochondrial intermembrane space, and enhances energy transfer from the mitochondria to the cytosol 57 .…”