Multistep Ion Channel Remodeling and Lethal Arrhythmia Precede Heart Failure in a Mouse Model of Inherited Dilated Cardiomyopathy

Background: Patients 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 DK210 in cardiac troponinT. Methodology/Principal Findings: By 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

Mechanisms responsible for reduced cardiotoxicity of mitoxantrone compared to doxorubicin examined in isolated guinea-pig heat preparations

We reported previously that doxorubicin, an anticancer agent that has an anthracycline structure, alters Ca^ releasing and uptake mechanisms in the sarcoplasmic reticulum of myocardial cells. These effects of doxorubicin are apparently related to its cardiotoxicity. Mitoxantrone is a similar anticancer agent with an anthracenedion structure that has been shown to be significantly less cardiotoxic. In the present study, the effects of mitoxantrone on the functions of the sarcoplasmic reticulum were examined in isolated muscle preparations obtained from the guinea-pig heart. In electrically-stimulated left atrial muscle preparations, incubation in vitro for 4 hr with 30 or 100 μM mitoxantrone significantly prolonged the time to the peak of twitch tension, markedly increased the developed tension observed at lower stimulation frequencies, thereby attenuating the slope of positive force-frequency relationships, and increased the postrest contraction observed after a 60-sec quiescent period. In myocytes isolated from ventricular muscles, 30 μM mitoxantrone increased the peak and the size of intracellular Ca^ concentrations ([Ca^] i), and prolonged the time to peak [Ca^]i. In skinned muscle fiber preparations obtained from the left ventricular muscle, 30 μM mitoxantrone significantly increased the caffeine-induced contraction without affecting the Ca^ sensitivity of contractile proteins. These results suggest that mitoxantrone enhances Ca^ release from the sarcoplasmic reticulum in isolated atrial muscle preparations obtained from the guinea-pig heart. Apparent enhancement of the sarcoplasmic reticulum functions, in contrast to anthracyclines that has been shown to suppress these functions, seems to explain the relative lack of marked cardiotoxicity of mitoxantrone

Western blot analysis of major channel proteins in LV.

<p>The membrane protein samples (50 µg protein for each) from 2-month WT (n = 4) and DCM LVs (n = 5) were separated by SDS-PAGE and Western blot analysis was carried out. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035353#s4" target="_blank">Methods</a> for detail. <b>A</b>. Representative immunoblots of individual experiments. <b>B</b>. Averaged expression levels. The relative expression levels for each protein were normalized to the average value for the WT. Data are means ± SEM (WT: n = 4, DCM: n = 5). **P<0.01 between WT and DCM.</p

AP signals and force records obtained from WT and DCM LV at 2 months.

<p><b>A</b>. Typical images of the endocardial surface of live LV muscle from a WT mouse. Left: The LV muscle wall observed from the endocardial side using a 4× objective. Six images were combined to produce the full image. Right: Image of cardiac surface muscle cells (256×512 pixels, 160×320 µm area) observed with a 20× objective. Action potential signals shown in B and E were obtained from this size of area. <b>B</b>. Representative AP signals recorded from the LVs of WT (top) and DCM (middle and bottom) mice stimulated at 0.5 Hz. AP signals were obtained from middle region as 8×16 pixel images at 3.67 ms intervals. Arrowheads indicate field stimulation. Experiments were carried out at 25–27°C. <b>C</b>. Representative traces showing force development in LV papillary muscle from a WT (left) and DCM (right) mouse during 0.5 Hz field stimulation (<b>a</b>) and during and after 3 Hz field stimulation (<b>b</b>). <b>D</b>. Average frequency of spontaneous contractions after 3 Hz field stimulation in LV and RV. Means ± SEM. (WT, n = 10; DCM, n = 14). <b>E</b>. Representative AP signals from the basal and apical regions of endocardial surface (see Panel A) of a LV stimulated at 0.5 Hz. <b>F</b>. Comparison of APD₅₀ values in the basal and apical regions of LVs and center region of RVs from WT (LV: base, n = 41; apex, n = 41 from 10 hearts, RV center region: n = 18 from 4 hearts,) and DCM (base, n = 85; apex, n = 64 from 14 hearts, RV: n = 35 from 7 hearts). Means ± SEM. **P<0.01 between WT and DCM. †P<0.05 between base and apex in LV.</p

Relative expression levels of mRNA encoding the indicated ion channels and auxiliary subunits in LVs.

<p>Quantitative real-time PCR analysis was carried out with LVs of 2-month WT (n = 7) and DCM mice (n = 7). GAPDH gene was used as an internal control. Data from individual samples were normalized to the average of WT mice. <b>A</b>. Voltage-dependent K⁺ channels, inwardly rectifying K⁺ channels and related proteins. <b>B</b>. Voltage-dependent Na⁺ channels, Ca²⁺ channels and Na⁺-Ca²⁺ exchanger. Plots are means ± SEM (n = 7). **P<0.01, *P<0.05 between WT and DCM.</p