Background:
Metabolic alterations largely contribute to the development of heart failure. Many metabolic pathways are converging in mitochondria, and the role of dysfunctional mitochondria in heart disease has been intensively studied. Fatty acid oxidation is the most prominent energy source for the heart. The full metabolism of long chain fatty acids requires mitochondria to physically and functionally interact with peroxisomes. Only limited information is available on the role of peroxisomes in cardiac metabolism. We therefore explored how peroxisomal dysfunction impairs mitochondria-peroxisome interplay, and thereby affects cardiac metabolism and function. We analysed mice with a cardiac specific peroxisomal biogenesis disorder/defect (PBD-cKO), resulting in the absence of functional peroxisomes in the heart. Using this mouse model, we addressed cardiac metabolism and function to reveal the functional role of a mitochondria-peroxisome network in the heart.
Methods and results:
Isolated, unloaded cardiac myocytes from mice were exposed to a physiological stress protocol by pacing at 0.5 Hz followed by β-adrenergic stimulation (isoproterenol 30nM) and increased stimulation rate at 5 Hz for 3 minutes. We observed no changes in diastolic sarcomere length, but reduced fractional sarcomere shortening in PBD-cKO (n=29) compared to WT myocytes (n=40). Diastolic cytosolic Ca2+ ([Ca2+]i) was unchanged, but systolic [Ca2+]i and Ca2+ transient amplitude (measured with Indo1-AM) were significantly higher in PBD-cKO vs. WT myocytes (n=57/59), without any changes in the kinetics of Ca2+ rise and decay. Semiquantitative assessment of the mitochondrial membrane potential (DYm; determined by TMRM) revealed a less polarized DYm in PBD-cKO vs. WT myocytes, since the change in TMRM fluorescence upon mitochondrial uncoupling with FCCP and oligomycin was significantly smaller in PBD-cKO vs. WT myocytes (n=40/40). Despite a more dissipated DYm, this parameter remained more stable during the stress protocol in PBD-cKO vs. WT myocytes, while the mitochondrial redox states of NAD(P)H/NAD(P)+ and FADH2/FAD (at rest and during the protocol) were similar in WT and PBD-cKO myocytes (n=25/26). In addition to these measurements in unloaded cardiac myocytes, we determined single cell force generation after pre-stretching myocytes from a sarcomere length of ~1.72 µm to ~2.00 µm. Both genotypes were stretched by a similar degree (WT, +0.25±0.03 µm, n=26 vs. PBD-cKO, +0.30 ±0.07µm, n=12; p=n.s.). At this more physiological preload, both genotypes produced similar diastolic force, while PBD-cKO myocytes developed lower systolic force and slower contraction and relaxation velocities at 1 and 4 Hz stimulation, respectively. To address compensatory changes in mitochondria, mitochondrial proteins of the respiratory chain were analysed.
Conclusions:
Disruption of peroxisomes in cardiac myocytes leads to alterations in excitation-contraction coupling, with lower systolic force generation despite elevated cytosolic Ca2+ transient amplitudes, indicating additional alterations of sarcomeric proteins which require further analyses. These alterations were associated with a more dissipated mitochondrial membrane potential, which may indicate a defect in the ability to produce sufficient ATP for the energy-consuming processes of excitation-contraction coupling.