Role of mitochondrial fission and fusion in cardiomyocyte contractility.

BACKGROUND: Mitochondria constitute 30% of cell volume and are engaged in two dynamic processes called fission and fusion, regulated by Drp-1 (dynamin related protein) and mitofusin 2 (Mfn2). Previously, we showed that Drp-1 inhibition attenuates cardiovascular dysfunction following pressure overload in aortic banding model and myocardial infarction. As dynamic organelles, mitochondria are capable of changing their morphology in response to stress. However, whether such changes can alter their function and in turn cellular function is unknown. Further, a direct role of fission and fusion in cardiomyocyte contractility has not yet been studied. In this study, we hypothesize that disrupted fission and fusion balance by increased Drp-1 and decreased Mfn2 expression in cardiomyocytes affects their contractility through alterations in the calcium and potassium concentrations. METHODS: To verify this, we used freshly isolated ventricular myocytes from wild type mouse and transfected them with either siRNA to Drp-1 or Mfn2. Myocyte contractility studies were performed by IonOptix using a myopacer. Intracellular calcium and potassium measurements were done using flow cytometry. Immunocytochemistry (ICC) was done to evaluate live cell mitochondria and its membrane potential. Protein expression was done by western blot and immunocytochemistry. RESULTS: We found that silencing mitochondrial fission increased the myocyte contractility, while fusion inhibition decreased contractility with simultaneous changes in calcium and potassium. Also, we observed that increase in fission prompted decrease in Serca-2a and increase in cytochrome c leakage leading to mitophagy. CONCLUSION: Our results suggested that regulating mitochondrial fission and fusion have direct effects on overall cardiomyocyte contractility and thus function.

GABAA receptor agonist mitigates homocysteine-induced cerebrovascular remodeling in knockout mice.

Individuals with homozygous deficiency in cystathionine-beta-synthase (CBS) develop high levels of homocysteine in plasma, a condition known as homocysteinuria. Mental retardation ensues with death in teens; the heterozygous live normally but develop vascular dementia and Alzheimer's disease (AD) in later part of life. The treatment with muscimol, a gamma amino butyric acid receptor-A (GABA(A)) agonist, mitigates the AD syndrome and vascular dementia. We tested the hypothesis that homocysteine (Hcy) antagonizes the GABA(A) receptor and behaves as an excitotoxic neurotransmitter that causes blood brain barrier (BBB) permeability and vascular dementia. The BBB permeability was measured by infusing Evan's blue dye (2% in saline 5 ml/kg concentration) in CBS-/+, GABA(A)-/-, CBS-/+/GABA(A)-/- double knockout, CBS-/+ mice treated with muscimol and wild type (WT) mice. Matrix Metalloproteinase (MMP-2, MMP-9), Tissue Inhibitor of Matrix Metalloproteinase (TIMP-3, TIMP-4), collagen-III and elastin levels were measured in whole brain by Western blot. These results suggested an increase in Evan's blue permeability: CBS-/+