Mitochondrial fission and mitophagy are independent mechanisms regulating ischemia/reperfusion injury in primary neurons.

Mitochondrial dynamics and mitophagy are constitutive and complex systems that ensure a healthy mitochondrial network through the segregation and subsequent degradation of damaged mitochondria. Disruption of these systems can lead to mitochondrial dysfunction and has been established as a central mechanism of ischemia/reperfusion (I/R) injury. Emerging evidence suggests that mitochondrial dynamics and mitophagy are integrated systems; however, the role of this relationship in the context of I/R injury remains unclear. To investigate this concept, we utilized primary cortical neurons isolated from the novel dual-reporter mitochondrial quality control knockin mice (C57BL/6-Gt(ROSA)26Sortm1(CAG-mCherry/GFP)Ganl/J) with conditional knockout (KO) of Drp1 to investigate changes in mitochondrial dynamics and mitophagic flux during in vitro I/R injury. Mitochondrial dynamics was quantitatively measured in an unbiased manner using a machine learning mitochondrial morphology classification system, which consisted of four different classifications: network, unbranched, swollen, and punctate. Evaluation of mitochondrial morphology and mitophagic flux in primary neurons exposed to oxygen-glucose deprivation (OGD) and reoxygenation (OGD/R) revealed extensive mitochondrial fragmentation and swelling, together with a significant upregulation in mitophagic flux. Furthermore, the primary morphology of mitochondria undergoing mitophagy was classified as punctate. Colocalization using immunofluorescence as well as western blot analysis revealed that the PINK1/Parkin pathway of mitophagy was activated following OGD/R. Conditional KO of Drp1 prevented mitochondrial fragmentation and swelling following OGD/R but did not alter mitophagic flux. These data provide novel evidence that Drp1 plays a causal role in the progression of I/R injury, but mitophagy does not require Drp1-mediated mitochondrial fission.

Membrane stabilizer Poloxamer 188 improves yield of primary isolated rat cardiomyocytes without impairing function.

Intact cardiomyocytes are used to investigate cardiac contractility and evaluate the efficacy of new therapeutic compounds. Primary enzymatic isolation of adult rodent cardiomyocytes has limitations, including low cardiomyocyte survival, which is likely due to ischemic conditions and/or membrane damage. The addition of Poloxamer 188 (P188) has been used to reduce ischemia- and membrane-related damage in ischemia-reperfusion and muscular dystrophy studies. P188 stabilizes membranes, reducing cell death. Cardiomyocytes were isolated from rats, under three conditions: (1) using standard isolation solutions, (2) with P188 added during cannulation (ischemic event), and (3) with P188 added during cannulation, enzymatic digestion, and trituration. Cell survival was assessed by quantifying the number of rod-shaped versus contracted cells on the day of isolation and up to 3 days post-isolation. Adding P188 only during cannulation yielded improved survival on the day of isolation. Little difference in survival was seen among the three conditions in the days post-isolation. Cardiomyocyte function was assessed by measuring calcium transients and unloaded sarcomere lengths for up to 2 days post-isolation. P188 did not consistently alter calcium handling or sarcomere shortening in the isolated cardiomyocytes. We conclude that the addition of P188 to the cannulation (e.g., wash) of the isolated heart may improve initial survival of cardiomyocytes upon primary enzymatic isolation.