Single heart cells of mouse models provide powerful tools for heart research. However, their isolation is not easy, and it imposes a significant bottleneck on their use in cellular studies of the heart. Aiming to overcome this problem, this report introduces a novel technique that reproducibly isolates healthy heart cells from mouse models. Using simple devices that ensure easy handling and the rapid aortic cannulation of a small mouse heart, cell isolation was done under physiological conditions without using the "KB" medium or 2,3-butanedione monoxime (BDM). The isolated cells consistently had a healthy appearance and a high viability of 75 ± 5% (mean ± SD) in Tyrode solution containing 1.8 mM Ca 2+ . After 8 h of storage at 37°C, they still had a viability of 45 ± 12%. The cells showed normal contraction properties when field-stimulated, and they generated normal action potentials and membrane currents under the whole-cell clamp condition. The β-adrenergic signal transduction of the cells was also normal when it was examined with the isoproterenol enhancement of the L-type Ca 2+ current.Key words: mouse, cardiac myocyte, contraction, action potential, ionic current.Single heart cells of mouse models provide powerful tools for heart research. In these cells, one can take advantage of the versatility of recent genetic engineering technology [1] and also of the accuracy of single-cell measurement techniques such as patch-clamp and Ca 2+ imaging. So far, molecular mechanisms of fundamental heart functions, such as excitation-contraction coupling, action potential shaping, and β-adrenergic signaling, have been successfully unveiled by using single heart cells of mouse models [2][3][4][5][6][7]. Moreover, these cells also provide a convenient platform for analyzing pathogenic mechanisms and the pathophysiology of hereditary heart diseases in molecular detail [8,9].For such single-cell studies, healthy heart cells are absolutely necessary; however, their isolation from mouse models is not easy. Surgery and aortic cannulation of a small mouse heart are hard to do and require a long time, and during that time, the heart suffers from complete ischemia. Consequently, the cells isolated from these hearts show various signs of ischemic damages, such as bizarre appearance, low viability levels, and abnormalities in their excitation and contraction properties. Cells of these kinds are hard to use for the experiment, and the data they provide are often difficult to interpret. This issue imposes a significant bottleneck on the use of mouse heart cells and is especially problematic in genetically engineered mouse models that are usually available only in limited amounts.To solve this problem, two major work-arounds have been devised and used for heart cell isolation from mouse models. One is to incubate the cells in high-K + , Ca 2+ -free "KB" medium at 4°C [10,11], and the other is to administer 10-20 mM 2,3-butanedione monoxime (BDM) in the media for cell isolation and storage [12,13]. Both workarounds proved to be...

BackgroundPatients with inherited dilated cardiomyopathy (DCM) frequently die with severe heart failure (HF) or die suddenly with arrhythmias, although these symptoms are not always observed at birth. It remains unclear how and when HF and arrhythmogenic changes develop in these DCM mutation carriers. In order to address this issue, properties of the myocardium and underlying gene expressions were studied using a knock-in mouse model of human inherited DCM caused by a deletion mutation ΔK210 in cardiac troponinT.Methodology/Principal FindingsBy 1 month, DCM mice had already enlarged hearts, but showed no symptoms of HF and a much lower mortality than at 2 months or later. At around 2 months, some would die suddenly with no clear symptoms of HF, whereas at 3 months, many of the survivors showed evident symptoms of HF. In isolated left ventricular myocardium (LV) from 2 month-mice, spontaneous activity frequently occurred and action potential duration (APD) was prolonged. Transient outward (Ito) and ultrarapid delayed rectifier K+ (IKur) currents were significantly reduced in DCM myocytes. Correspondingly, down-regulation of Kv4.2, Kv1.5 and KChIP2 was evident in mRNA and protein levels. In LVs at 3-months, more frequent spontaneous activity, greater prolongation of APD and further down-regulation in above K+ channels were observed. At 1 month, in contrast, infrequent spontaneous activity and down-regulation of Kv4.2, but not Kv1.5 or KChIP2, were observed.Conclusions/SignificanceOur results suggest that at least three steps of electrical remodeling occur in the hearts of DCM model mice, and that the combined down-regulation of Kv4.2, Kv1.5 and KChIP2 prior to the onset of HF may play an important role in the premature sudden death in this DCM model. DCM mice at 1 month or before, on the contrary, are associated with low risk of death in spite of inborn disorder and enlarged heart.

SUMMARY1. Membrane current generated by the Na+-Ca2+ exchange mechanism was recorded in single guinea-pig ventricular myocytes using the whole-cell voltageclamp technique and the intracellular free calcium concentration ([Ca2+]i) was monitored using the fluorescent probe Indo-1, applied intracellularly through a perfused patch pipette. The reversal potential of the exchanger (ENa Ca) was measured from records of the 2 mM-Ni2+-sensitive current and used in an attempt to clamp [Ca2+ ]i at a level determined by the ionic compositions of the external and pipette solutions.2. Measurements of ENa, Ca indicated that [Ca2+]i was close to that in the pipette solution when the holding potential was set at the ENa ca expected for a 3Na+: ICa2+ exchanger. The measured value of ENa ca was more positive than the theoretical value when the membrane potential was held positive to ENa ca and the opposite was true when the holding potential was more negative than the expected ENa Ca.3. As Indo-l diffused into the cell from the whole-cell clamp electrode, the intensities of the fluorescent signals measured at 405 and 480 nm increased with time, with no obvious saturation over a 10-45 min recording period. However, the ratio of these two signals reached a steady level within 5 min after rupture of the patch membrane, when the holding potential was set at the expected ENa Ca of the exchanger. The intensity ratios measured using pipette solutions containing 600 and 803 nM [Ca2+] were almost equal to the ratios obtained extracellularly from internal solutions of identical compositions, but in experiments using pipette solutions having lower [Ca2+] the intensity ratios measured in myocytes were higher than those obtained extracellularly.4. If the membrane was depolarized or hyperpolarized, the fluorescence ratio either increased or decreased, respectively. These changes in the fluorescence ratio were virtually blocked by the extracellular application of 2 mM-Ni2+.5. When the concentration of bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) A. NOMA AND OTHERS increase in [Ca2+]i estimated from Indo-1 using the extracellular calibration curve, but the values of the influx determined directly from Indo-1 fluorescence were always larger than those calculated from the exchanger current.6. Hyperpolarization to -80 mV from a holding potential of -40 mV decreased[Ca2+]i, while the membrane slope conductance for outward Na+-Ca2+ exchange current increased rapidly. The opposite was true when the membrane was depolarized. These effects were reversed by clamping back to -40 mV. Membrane conductance for the inward exchanger current did not change markedly during these manoeuvres. 7. These observations suggest that when Indo-1 is applied through a dialysing patch pipette the resulting intracellular fluorescence can be calibrated to give a satisfactory estimate of [Ca2+]i, and that changes in membrane potential produce marked variations in [Ca2+]i via modulation of Na+-Ca2+ exchange.

The present patch-clamp study shows that external Mg2+, Ca2+ and Sr2+ decrease the unit amplitude of inward current through the inward-rectifier K+ channel in a concentration-dependent manner. Sr2+ produces a voltage-dependent flickering block as well, and the fractional electrical distance between the external orifice and the Sr2+ binding site (delta) is 0.73. The decrease of unit amplitude is reversible and voltage independent while it does not increase the noise level on the open-channel current. Unit current decreased by Mg2+ or Ca2+ has a longer mean open time, which is inversely proportional to the unit amplitude. External Mg2+ does not decrease the amplitude of unit outward current. A surface potential shift, measured using voltage-dependent Cs+ block (delta = 1.60), failed to explain the current decrease. Therefore, we conclude that (1) the external divalent cations cause an extremely fast channel block, which appears as a decreased amplitude of the unit current on the recording system; (2) the blocking site (fast site) is present near the external orifice of the channel, and it is separate from the blocking site (slow site) to which Cs+ and Sr2+ bind.

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