Isolated working cardiomyocytes remain the standard approach for studying cardiac electrophysiology. Single-cell preparations allow for direct investigation and precise measurement of ionic currents and action potentials without the structural complexity of intact cardiac tissue. Nothobranchius furzeri (African turquoise killifish) has rapidly emerged as a prominent model organism for gerontological research due to its exceptionally short lifespan and similarities in aging mechanisms with mammals, including humans. This species combines practical advantages such as small size, inexpensive husbandry, and a rapid life cycle with translational relevance. N. furzeri exhibits age-dependent declines in locomotor activity, cognition, and organ function, accumulation of molecular hallmarks of aging, and sensitivity to pharmacological treatments that modulate lifespan and healthspan. However, investigating the functional decline of the aging heart requires a reliable method to obtain high-quality, viable cells suitable for demanding single-cell techniques. This study optimized a previously described protocol for the isolation of Danio rerio cardiomyocytes to ensure consistent recovery of healthy working myocytes from both young and senescent N. furzeri. This protocol describes the isolation of ventricular cardiomyocytes from N. furzeri specifically optimized for the whole-cell patch clamp technique. The article demonstrates how to efficiently excise the heart, perform enzymatic dissociation of the myocardium by retrograde perfusion, and store the isolated cardiomyocytes. We discuss the selection of enzymes required to digest the extracellular matrix and intercellular junctions without compromising ion channel structure. Freshly isolated cardiomyocytes are suitable for high-resolution electrophysiological assays, including action potential recordings and measurements of major inward and outward ionic currents across age groups. The article also addresses the features of cardiomyocyte isolation in fish that differ from standard mammalian isolation procedures.
