Mouse cardiac mitochondria do not separate in subsarcolemmal and interfibrillar subpopulations.

Cardiomyocytes consist of longitudinally oriented myofibril bundles with a misaligned composition caused by the uneven contours of the intercalated discs. The cytoplasmic space harbors the organelles, including mitochondria. This study investigated whether cardiomyocytes contain spatially and ultrastructurally discrete pools of mitochondria that can be separated for structurally and functionally appraisal in (patho)physiology. Transmission electron microscopy disclosed continuous transitions of mitochondria without attributable characteristics from beneath the sarcolemma directly into the barrier-free cytoplasmic space between myofibrils. The various shapes and sizes of mitochondria are formed by myofibril positioning and the space available independent of their localization within the cardiomyocytes. Furthermore, the established enzymatic isolation procedure including proteinase treatment resulted in loss of mitochondrial proteins, as evidenced by immunogold labeling of Connexin43 in situ, a postulated marker for distinguishing mitochondrial subpopulations. Moreover, mitochondrial ATP produced in those mitochondria was not different. These findings preclude a spatial and ultrastructural grading of cardiac mitochondria and their distinct separation and classification in subsarcolemmal and interfibrillar subpopulations.

Cytosolic BNIP3 Dimer Interacts with Mitochondrial BAX Forming Heterodimers in the Mitochondrial Outer Membrane under Basal Conditions.

The primary function of mitochondria is energy production, a task of particular importance especially for cells with a high energy demand like cardiomyocytes. The B-cell lymphoma (BCL-2) family member BCL-2 adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) is linked to mitochondrial targeting after homodimerization, where it functions in inner membrane depolarization and permeabilization of the mitochondrial outer membrane (MOM) mediating cell death. We investigated the basal distribution of cardiac BNIP3 in vivo and its physical interaction with the pro-death protein BCL2 associated X, apoptosis regulator (BAX) and with mitochondria using immunoblot analysis, co-immunoprecipitation, and continuous wave and pulsed electron paramagnetic resonance spectroscopy techniques. We found that BNIP3 is present as a dimer in the cytosol and in the outer membrane of cardiac mitochondria under basal conditions. It forms disulfide-bridged, but mainly non-covalent dimers in the cytosol. Heterodimers with BAX are formed exclusively in the MOM. Furthermore, our results suggest that BNIP3 interacts with the MOM directly via mitochondrial BAX. However, the physical interactions with BAX and the MOM did not affect the membrane potential and cell viability. These findings suggest that another stimulus other than the mere existence of the BNIP3/BAX dimer in the MOM is required to promote BNIP3 cell-death activity; this could be a potential disturbance of the BNIP3 distribution homeostasis, namely in the direction of the mitochondria.

A novel physiological role for cardiac myoglobin in lipid metabolism.

Continuous contractile activity of the heart is essential and the required energy is mostly provided by fatty acid (FA) oxidation. Myocardial lipid accumulation can lead to pathological responses, however the underlying mechanisms remain elusive. The role of myoglobin in dioxygen binding in cardiomyocytes and oxidative skeletal muscle has widely been appreciated. Our recent work established myoglobin as a protector of cardiac function in hypoxia and disease states. We here unravel a novel role of cardiac myoglobin in governing FA metabolism to ensure the physiological energy production through ß-oxidation, preventing myocardial lipid accumulation and preserving cardiac functions. In vivo1H magnetic resonance spectroscopy unveils a 3-fold higher deposition of lipids in mouse hearts lacking myoglobin, which was associated with depressed cardiac function compared to wild-type hearts as assessed by echocardiography. Mass spectrometry reveals a marked increase in tissue triglycerides with preferential incorporation of palmitic and oleic acids. Phospholipid levels as well as the metabolome, transcriptome and proteome related to FA metabolism tend to be unaffected by myoglobin ablation. Our results reveal a physiological role of myoglobin in FA metabolism with the lipid accumulation-suppressing effects of myoglobin preventing cardiac lipotoxicity.

Inorganic nitrite modulates miRNA signatures in acute myocardial in vivo ischemia/reperfusion.

Acute myocardial infarction is the leading cause of mortality in the industrialized world. While it is essential to attempt an early reperfusion of ischemic myocardial territories, reperfusion itself adds damage to the heart, the ischemia-reperfusion (I/R) injury. Particularly the injury resulting from the very first minutes of reperfusion remains incompletely understood. MicroRNAs (miRNAs) are dynamic regulators in I/R injury. Nitric oxide (•NO) signaling, in turn, interacts with miRNA signaling. Our previous investigations showed that •NO signaling in I/R could be modulated by nitrite. We therefore sought to investigate the role of miRNAs in nitrite cardioprotection with focus on the first few minutes of reperfusion. The study was conducted in mice in vivo with 30 min of ischemia and 5 min of reperfusion. Mice received a single-dose of nitrite or saline intracardially 5 min prior to reperfusion. We identified nine miRNAs to be up-regulated after 5 min of reperfusion. The up-regulation of almost half of those miRNAs (miR-125a-5p, miR-146b, miR-339-3p, miR-433) was inhibited by nitrite treatment, perpetuating baseline values. In silico analysis revealed the Irak-M gene to be a target of miR-146b and miR-339-3p. Correspondingly, a rise in Irak-M transcript and protein levels occurred by nitrite treatment within the early phase of reperfusion. The results demonstrate that already a very short phase of reperfusion is sufficient for significant dysregulation in cardiac miRNAs expression and that nitrite preserves baseline values of miRNAs in the scale of only a few minutes. These findings hint at a potential novel cardioprotective mechanism of nitrite signaling.