Background: Ras Homolog Family Member T (RHOT) 1 and RHOT2 mediate mitochondrial motility by coupling mitochondria to kinesin and dynein motors, which is required for mitochondrial positioning during cardiomyocyte development and maturation. We sought to test the hypothesis that RHOT protein-dependent mitochondrial motility has distinct roles in cardiomyocytes depending on the developmental stage of the heart.
Methods: We generated mice with inducible cardiomyocyte-selective deletion of Rhot1 and Rhot2 following tamoxifen administration (iRhot1/2-KO). Adult mice were injected with tamoxifen starting at 8-weeks of age. Endpoints were assessed 8-weeks after the first tamoxifen injection at 16-weeks of age. Contractile function was determined by transthoracic echocardiography and in isolated cardiomyocytes following electrical stimulation. Motility of isolated mitochondria was assessed by a motility assay. Mitochondrial respiratory capacity was determined in isolated mitochondria. Mitochondrial localization and ATP production were determined by confocal microscopy and following adenoviral expression of the FRET-based ATP biosensor ATeam.
Results: Cardiac contractile function and contractility of electrically-stimulated cardiomyocytes were unchanged 8-weeks after tamoxifen administration relative to controls. Isolated mitochondria from iRhot1/2-KO hearts showed impaired motility but preserved respiratory capacity with pyruvate and palmitoyl-carnitine each combined with malate as substrates. Cardiomyocytes from iRhot1/2-KO mice showed no difference in mitochondrial localization, and intracellular localization of ATP was not different compared to controls.
Conclusion: Expression Rhot1 and Rhot2 is dispensable for mitochondrial localization and contractile function in the adult heart despite impaired motility of mitochondria isolated from iRhot1/2-KO hearts.
