Causes of Abnormal Ca2+ Transients in Guinea Pig Pathophysiological Ventricular Muscle Revealed by Ca2+ and Action Potential Imaging at Cellular Level

BACKGROUND: Abnormal Ca(2+) transients are often observed in heart muscles under a variety of pathophysiological conditions including ventricular tachycardia. To clarify whether these abnormal Ca(2+) transients can be attributed to abnormal action potential generation or abnormal Ca(2+) handling/excitation-contraction (EC) coupling, we developed a procedure to determine Ca(2+) and action potential signals at the cellular level in isolated heart tissues. METHODOLOGY/PRINCIPAL FINDINGS: After loading ventricular papillary muscle with rhod-2 and di-4-ANEPPS, mono-wavelength fluorescence images from rhod-2 and ratiometric images of two wavelengths of emission from di-4-ANEPPS were sequentially obtained. To mimic the ventricular tachycardia, the ventricular muscles were field-stimulated in non-flowing Krebs solution which elicited abnormal Ca(2+) transients. For the failed and alternating Ca(2+) transient generation, there were two types of causes, i.e., failed or abnormal action potential generation and abnormal EC coupling. In cells showing delayed initiation of Ca(2+) transients with field stimulation, action potential onset was delayed and the rate of rise was slower than in healthy cells. Similar delayed onset was also observed in the presence of heptanol, an inhibitor of gap junction channels but having a non-specific channel blocking effect. A Na(+) channel blocker, on the other hand, reduced the rate of rise of the action potentials but did not result in desynchronization of the action potentials. The delayed onset of action potentials can be explained primarily by impaired gap junctions and partly by Na(+) channel inactivation. CONCLUSIONS/SIGNIFICANCE: Our results indicate that there are multiple patterns for the causes of abnormal Ca(2+) signals and that our methods are useful for investigating the physiology and pathophysiology of heart muscle

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

Time-resolved serial femtosecond crystallography reveals early structural changes in channelrhodopsin

X線自由電子レーザーを用いて、光照射によるチャネルロドプシンの構造変化の過程を捉えることに成功. 京都大学プレスリリース. 2021-03-26.Channelrhodopsins (ChRs) are microbial light-gated ion channels utilized in optogenetics to control neural activity with light . Light absorption causes retinal chromophore isomerization and subsequent protein conformational changes visualized as optically distinguished intermediates, coupled with channel opening and closing. However, the detailed molecular events underlying channel gating remain unknown. We performed time-resolved serial femtosecond crystallographic analyses of ChR by using an X-ray free electron laser, which revealed conformational changes following photoactivation. The isomerized retinal adopts a twisted conformation and shifts toward the putative internal proton donor residues, consequently inducing an outward shift of TM3, as well as a local deformation in TM7. These early conformational changes in the pore-forming helices should be the triggers that lead to opening of the ion conducting pore

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