Electrophysiological properties of neonatal mouse cardiac myocytes in primary culture.

Abstract

1. The increasing utility of transgenic mice in molecular studies of the cardiovascular system has motivated us to characterize the ionic currents in neonatal mouse ventricular myocytes. 2. Cell capacitance measurements (30 + 1 pF, n = 73) confirmed visual impressions that neonatal mouse ventricular myocytes in primary culture are considerably smaller than freshly isolated adult ventricular myocytes. With the use of electron microscopy, mitochondria and sarcoplasmic reticulum were found in close association with myofibrils, but transverse tubules were not observed. 3. Action potential durations, measured at 50 and 90% repolarization, were 23 + 1 and 42 + 2 ms respectively (n = 46). Application of 4-aminopyridine (4-AP; 5 mm) prolonged action potential duration at 50% repolarization by 26 ± 5% (n = 3). The brevity of the action potential is explained by the rapid activation of a transient outward K+ current upon voltage-clamp depolarization to plateau potentials. 4. Potassium currents identified include an inward rectifier, a large 4-AP-sensitive transient outward, a slowly inactivating 4-AP-insensitive outward, a slowly activating delayed rectifier and a small rapidly activating E-4031 (10 ,uM)-sensitive delayed rectifier K+ current. 5. Sodium currents (-305 + 50 pA pF1, n = 21) were recorded in 40 mm Na+ with Ni2+(1 mM) to block Ca2`currents and with K+ replaced by Cs+. The relative insensitivity of the Na+ current to block by tetrodotoxin (IC50 = 2'2 + 0 3 uM, n = 4) is distinctive of the cardiac Na+ channel isoform. 6 Nitrendipine-insensitive (10 /M) Ba21 currents elicited during steps from -90 to -30 mV measured -25 + 5 pA pF-1 (n = 7, 30 mm Ba2+). Decay of these currents was complete during 180 ms depolarizations, even with Ba2+ as the charge carrier. These currents were not present when the holding potential was set at -50 mV. These data support the presence of a low threshold, T-type Ca2+ current. 7. The maximal nitrendipine-sensitive L-type Ca2+ current density was -10 + 2 pA pF1 (n = 8) in 2 mm Ca2+ and -38 + 5 pA pF1 (n = 9) in 30 mm Ba2+. Exposure to isoprenaline (1/uM) resulted in an 82% increase (n=3) in the amplitude of the Ba21 currents elicited at 0 mV. 8. Neonatal mouse cardiac myocytes in primary culture possess surprisingly large inward currents given the brevity of their action potentials. Their small cell size and apparent absence of transverse tubules makes them technically favourable for whole-cell patch clamp studies. For these reasons, this preparation promises to be useful for the phenotypic characterization of transgenic mice.

Properties of WT and mutant hERG K+ channels expressed in neonatal mouse cardiomyocytes

“…In neonatal mouse cardiomyocytes, native I Kr was identified as an E4031-sensitve current that was small in amplitude (peak tail I Kr ϳ1.6 pA/pF), and our I Kr recordings are similar to those in previous reports (24,27,40,41). WT hERG channel overexpression generated an E4031-sensitive current that activated over a voltage range similar to I Kr but with an amplitude ϳ10-fold greater.…”

“…Neonatal mouse cardiomyocytes differ from adult mouse cardiomyocytes in several ways (1): neonatal cardiomyocytes are not fully terminally differentiated, they are small and have a rounded or triangular shape, they lack a well-developed sarcomeric contractile protein pattern, and they tolerate electroporation, which does not work well in older cardiomyocytes. Compared with adult mouse cardiomyocytes, neonatal mouse cardiomyocytes lack transverse tubules, which may have an advantage in voltage-clamp experiments (27). Neonatal mouse cardiomyocytes have been used as a model for ion channel expression, including for the Na ϩ channel Nav1.5 (39,44), Tand L-type Ca 2ϩ channels (17,21), the K ϩ channel Kv4.2 (18), and the pacemaker HCN channel (9).…”

An improved protocol for the isolation and cultivation of embryonic mouse myocytes

“…Either one of these methods, or the combination of both, will lead to few or no viable cells. Previous researchers keep the harvested myocyte cells on ice until all digestive reactions are completed (Burt 1982;Nuss and Marban 1994;Fu et al 2005;Sreejit et al 2008). We determined that cell viability can be doubled by incubating the primary myocytes in complete culture medium at 37°C during the procedures (Tube C).…”

“…Nonessential amino acids are not often reported as required additive for the culture of cardiomyocyte cells (Burt 1982;Sreejit et al 2008). Though Sreejit et al (2008) and Song et al (2000) report using 1% gelatin and Ahuja et al (2004) reported using fibronectin as a pre-coat for myocyte culture dishes, such pre-coat steps are not often reported (Burt 1982;Nuss and Marban 1994;Fu et al 2005;Rosenblatt-Velin et al 2005;Nickson et al 2007). We, however, determined that a pre-coating with laminin was absolutely necessary for effective embryonic myocyte adhesion.…”

Phenotypic Correlation of Genetic Mutations with Ventricular Arrhythmias

“…Primary isolated myocytes are best suited for short-term culture (approximately four days) and electrophysiological or immunofluorescence experiments that require only a low yield of viable cells (10s compared to 10,000s needed for biochemistry experiments) (Nuss & Marban, 1994). Some of the technical challenges involved include obtaining fresh healthy heart samples, appropriate and not over-digestion of the tissue by enzymes, purification of myocytes from fibroblasts and matrix, calcium tolerance of the freshly isolated myocytes, and finding the correct conditions for culture.…”