Cell viability may be judged by morphological changes or by changes in membrane permeability and/or physiological state inferred from the exclusion of certain dyes or the uptake and retention of others. This unit presents methods based on dye exclusion, esterase activity, and mitochondrial membrane potential, as well as protocols for determining the pre‐fixation viability of fixed cells either before or after fixation with amine‐reactive dyes suitable for a range of excitation wavelengths. Membrane‐impermeable dead cell and live cell dyes as well as dye‐exclusion procedures for microscopy are also included. Curr. Protoc. Cytom. 64:9.2.1‐9.2.26. © 2013 by John Wiley & Sons, Inc.

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While activated autophagy is a well reported feature of cardiac disease, the exact role of autophagy in the etiology and progression of cardiac hypertrophy and dysfunction is unclear; activation of autophagy appears to play either pro-survival or pro-apoptotic functions depending on the context. Here we demonstrate that dysfunctional autophagy plays a role in the development of angiotensin II-induced cardiac hypertrophy. Using a live, ex-vivo tissue imaging technique and transgenic LC3-GFP expressing mice we found that after short term (1 week) treatment, autophagic markers accumulate in angiotensin treated mouse hearts and that in a small population of cells this accumulation reaches a critical level where normal cell organization, specifically mitochondrial localization and contractile unit structure, and tissue architecture are disrupted. These observations are reminiscent of genetic diseases associated with dysfunctional autophagy, such as inclusion body myositis, where autophagic vesicles fail to properly clear and lead to cell death and tissue degeneration. We suggest that failed autophagy contributes to pressure-overload induced hypertrophy and that the cell death associated with dramatically dysfunctional autophagy in a subset of cardiomyocytes precedes fibrosis. This work provides a clear role for dysfunctional autophagy in promoting hypertrophy and fibrosis, and supports the notion that the promotion of successful autophagy is a clinically relevant target for therapeutic intervention.