Endo Myocytes Research Articles

Contractile heterogeneity in ventricular myocardium.

The transmural heterogeneity of the contractility in ventricular muscle has not been well-studied. Here, we investigated the calcium transient and sarcomere contraction/relaxation in the endocardial (Endo) and epicardial (Epi) myocytes. Endo and Epi myocytes were isolated from C57/BL6 mice by Langendorff perfusion. Ca2+ transient and sarcomere contraction/relaxation were recorded simultaneously at different stimulation frequencies using a dual excitation fluorescence photomultiplier system. We found that the Endo myocytes have higher baseline diastolic calcium, significantly larger calcium transient and stronger sarcomere shortening than Epi myocytes. However, both the rising and decline phases for calcium transient and sarcomere shortening were slower in Endo than in Epi myocytes. When simulation frequency was increased from 1 to 3 Hz, a greater percent increase in the diastole calcium level, Ca2+ transient and sarcomere shortening amplitude has been observed in the Endo myocytes. Accordingly, the frequency-dependent acceleration in the decay rate of calcium transient and sarcomere relaxation was more profound in the Endo than in Epi myocytes. Western blot analysis showed that CaMKII activity was significantly higher in Epi than in Endo myocardium before stimulation. However, this transmural heterogeneity was reversed by rapid pacing. CaMKII inhibition by KN93 diminished the frequency-dependent alterations of Ca2+ transient and sarcomere contraction. Our results suggest that the contractility of ventricular myocytes is heterogeneous. The Endo-myocardium is the major force generating layer in the heart, both at slow and fast heart rate, and the transmural heterogeneity of CaMKII activation plays an important role in the frequency-dependent alterations.

Type 2 diabetes induces subendocardium-predominant reduction in transient outward K+ current with downregulation of Kv4.2 and KChIP2

In the present study, we examined if and how cardiac ion channels are modified by type 2 diabetes mellitus (T2DM). Subendocardial (Endo) myocytes and subepicardial (Epi) myocytes were isolated from left ventricles of Otsuka-Long-Evans-Tokushima Fatty rats (OLETF) rats, a rat model of T2DM, and Otsuka-Long-Evans-Tokushima (LETO) rats (nondiabetic control rats). Endo and Epi myocytes were used for whole cell patch-clamp recordings and for protein and mRNA analyses. Action potential durations in Endo and Epi myocytes were longer in OLETF rats than in LETO rats, and the difference was larger in Endo myocytes. Steady-state transient outward K+ current (Ito) density was reduced in Endo but not Epi myocytes of OLETF rats compared with LETO rats, although the contribution of the fast component of Ito recovery from inactivation was smaller in both Endo and Epi myocytes of OLETF rats than in LETO rats. Kv4.2 protein was reduced only in Endo myocytes in OLETF rats, although voltage-gated K+ channel-interacting protein 2 (KChIP2) protein levels in both Endo and Epi myocytes were lower in OLETF rats than in LETO rats. Corresponding regional differences in mRNA levels of KChIP2 and Kv4.2 were observed between OLETF and LETO rats. mRNA levels of Iroquois homeobox 5 in Endo myocytes were 53% higher in OLETF rats than in LETO rats. Densities of inward rectifier K+ current and L-type Ca2+ current and mRNA levels of Kv4.3 and Kv1.4 were similar in OLETF and LETO rats. In conclusion, T2DM induces Endo-predominant prolongation of the action potential duration via a reduction of the fast component of Ito recovery from inactivation and reduced steady-state Ito, in which downregulation of Kv4.2 and KChIP2 may be involved. Increased Iroquois homeobox 5 expression may underlie Kv4.2 downregulation in T2DM.

Subendocardial Increase in Reactive Oxygen Species Production Affects Regional Contractile Function in Ischemic Heart Failure

Heart failure (HF) is characterized by regionalized contractile alterations resulting in loss of the transmural contractile gradient across the left ventricular free wall. We tested whether a regional alteration in mitochondrial oxidative metabolism during HF could affect myofilament function through protein kinase A (PKA) signaling. Twelve weeks after permanent left coronary artery ligation that induced myocardial infarction (MI), subendocardial (Endo) cardiomyocytes had decreased activity of complex I and IV of the mitochondrial electron transport chain and produced twice more superoxide anions than sham Endo and subepicardial cells. This effect was associated with a reduced antioxidant activity of superoxide dismutase and Catalase only in MI Endo cells. The myofilament contractile properties (Ca(2+) sensitivity and maximal tension), evaluated in skinned cardiomyocytes, were also reduced only in MI Endo myocytes. Conversely, in MI rats treated with the antioxidant N-acetylcysteine (NAC) for 4 weeks, the generation of superoxide anions in Endo cardiomyocytes was normalized and the contractile properties of skinned cardiomyocytes restored. This effect was accompanied by improved in vivo contractility. The beneficial effects of NAC were mediated, at least, in part, through reduction of the PKA activity, which was higher in MI myofilaments, particularly, the PKA-mediated hyperphosphorylation of cardiac Troponin I. The Transmural gradient in the mitochondrial content/activity is lost during HF and mediates reactive oxygen species-dependent contractile dysfunction. Regionalized alterations in redox signaling affect the contractile machinery of sub-Endo myocytes through a PKA-dependent pathway that contributes to the loss of the transmural contractile gradient and impairs global contractility.

Differential effects of the transient outward K + current activator NS5806 in the canine left ventricle

To examine the electrophysiological and molecular properties of the transient outward current ( I to) in canine left ventricle using a novel I to activator, NS5806, I to was measured in isolated epicardial (Epi), midmyocardial (Mid) and endocardial (Endo) cells using whole-cell patch-clamp techniques. NS5806 activation of K v4.3 current was also studied in CHO-K1 cells and Xenopus laevis oocytes. In CHO-K1 cells co-transfected with K v4.3 and KChIP2, NS5806 (10 μM) caused a 35% increase in current amplitude and a marked slowing of current decay with τ increasing from 7.0 ± 0.4 to 10.2 ± 0.3 ms. In the absence of KChIP2, current decay was unaffected by NS5806. In ventricular myocytes, NS5806 increased I to density by 80%, 82%, and 16% in Epi, Mid, and Endo myocytes, respectively (at + 40 mV) and shifted steady-state inactivation to negative potentials. NS5806 also significantly slowed decay of I to, increasing total charge to 227%, 192% and 83% of control in Epi, Mid and Endo cells, respectively (+ 40 mV, p < 0.05). Quantification of K v4.3 and KChIP2 mRNA in the 3 ventricular cell types revealed that levels of K v4.3 message was uniform but those of KChIP2 were significantly greater in Epi and Mid cells. The KChIP2 gradient was confirmed at the protein level by Western blot. Our results suggest that NS5806 augments I to by increasing current density and slowing decay and that both depend on the presence of KChIP2. I to and its augmentation by NS5806 are greatest in Epi and Mid cells because KChIP2 levels are highest in these cell types.

Functionally distinct sodium channels in ventricular epicardial and endocardial cells contribute to a greater sensitivity of the epicardium to electrical depression

A greater depression of the action potential (AP) of the ventricular epicardium (Epi) versus endocardium (Endo) is readily observed in experimental models of acute ischemia and Brugada syndrome. Endo and Epi differences in transient outward K(+) current and/or ATP-sensitive K(+) channel current are believed to contribute to the differential response. The present study tested the hypothesis that the greater sensitivity of Epi is due in part to its functionally distinct early fast Na(+) current (I(Na)). APs were recorded from isolated Epi and Endo tissue slices and coronary-perfused wedge preparations before and after exposures to elevated extracellular K(+) concentration ([K(+)](o); 6-12 mM). I(Na) was recorded from Epi and Endo myocytes using whole cell patch-clamp techniques. In tissue slices, increasing [K(+)](o) to 12 mM reduced V(max) to 51.1 +/- 5.3% and 26.8 +/- 9.6% of control in Endo (n = 9) and Epi (n = 14), respectively (P < 0.05). In wedge preparations (n = 12), the increase in [K(+)](o) caused selective depression of Epi APs and transmural conduction slowing and block. I(Na) density was not significantly different between Epi (n = 14) and Endo (n = 15) cells, but Epi cells displayed a more negative half-inactivation voltage [-83.6 +/- 0.1 and -75.5 +/- 0.3 mV for Epi (n = 16) and Endo (n = 16), respectively, P < 0.05]. Our data suggest that reduced I(Na) availability in ventricular Epi may contribute to its greater sensitivity to electrical depression and thus may contribute to the R-ST segment changes observed under a variety of clinical conditions including acute myocardial ischemia, severe hyperkalemia, and Brugada syndrome.

Cellular and subcellular alternans in the canine left ventricle

Previous studies indicate that action potential duration (APD) alternans is initiated in the endocardial (END) and midmyocardial (MID) regions rather than the epicardium (EPI) in the canine left ventricle (LV). This study examines regional differences in the rate dependence of Ca(2+) transient characteristics under conditions that give rise to APD and associated T wave alternans. The role of the sarcoplasmic reticulum (SR) was further evaluated by studying Ca(2+) transient characteristics in myocytes isolated from neonates, where an organized SR is poorly developed. All studies were performed in cells and tissues isolated from the canine LV. Isolated canine ENDO, MID, and EPI LV myocytes were either field stimulated or voltage clamped, and Ca(2+) transients were measured by confocal microscopy. In LV wedge preparations, increasing the basic cycle length (BCL) from 800 to 250 ms caused alternans to appear mainly in the ENDO and MID region; alternans were not observed in EPI under these conditions. Ca(2+) transient alternans developed in response to rapid pacing, appearing in EPI cells at shorter BCL compared with MID and ENDO cells (BCL=428 +/- 17 vs. 517 +/- 29 and 514 +/- 21, respectively, P < 0.05). Further increases in pacing rate resulted in the appearance of subcellular alternans of Ca(2+) transient amplitude, which also appeared in EPI at shorter BCL than in ENDO and MID cells. Ca(2+) transient alternans was not observed in neonate myocytes. We conclude that 1) there are distinct regional differences in the vulnerability to rate-dependent Ca(2+) alternans in dog LV that may be related to regional differences in SR function and Ca(2+) cycling; 2) the development of subcellular Ca(2+) alternans suggests the presence of intracellular heterogeneities in Ca(2+) cycling; and 3) the failure of neonatal cells to develop Ca(2+) alternans provides further support that SR Ca(2+) cycling is a major component in the development of these phenomena.

Transmural changes in size, contractile and electrical properties of SHR left ventricular myocytes during compensated hypertrophy.

Some hypertrophic stimuli provoke responses from myocytes that vary across the thickness of the left ventricular wall. The Spontaneously Hypertensive Rat (SHR) is a well-established genetic model of hypertension and whole heart hypertrophy. Details of transmural responses to hypertension in the SHR are few, but are needed if the properties of this model are to be fully understood. We therefore tested the hypothesis that left ventricular myocytes of the SHR do not respond uniformly to their hypertensive environment. The volume, contraction and action potentials of enzymically isolated sub-epicardial (EPI) mid-myocardial (MID) and sub-endocardial (ENDO) myocytes from the left ventricle of 20-week-old SHR and normotensive Wistar-Kyoto (WKY) control rats were compared. Compared to WKY, as a single population, SHR myocytes displayed concentric hypertrophy (larger volumes with smaller length:width) increased t-tubule spacing, larger and prolonged cell shortening and intracellular calcium ([Ca2+]i) transients and longer action potentials. However, these responses differed across the left ventricular wall. MID myocytes showed significantly less hypertrophy than EPI and ENDO myocytes. EPI myocytes showed the largest (and significant) increases in cell shortening, [Ca2+]i transients and action potential duration, whilst MID myocytes showed the smallest (and non-significant) changes in these parameters. Real time reverse transcription polymerase chain reaction analysis on cardiac tissue suggest that increased expression of mRNA for fibronectin-1 and protein kinase Cepsilon are involved in the hypertrophic response of the whole heart. Our findings show that in the SHR, the effect of hypertension upon the morphology, mechanical activity and electrical activity of left ventricular myocytes is dependent upon their transmural location. Therefore, in addition to the overall compensating response to hypertension, i.e. increased contractility, there are likely to be regionally specific alterations in mechano-electric interactions that may influence the properties of this important model, e.g. its pre-disposition to arrhythmia whilst still in a compensated state.

Spatial alterations of Kv channels expression and K(+) currents in post-MI remodeled rat heart.

The hypothesis being tested in the present study is that increased anisotropic properties occurs in the remodeled post-infarction heart due to spatial alterations in Kv channels expression and K(+) currents of the remodeled myocardium. Three to 4 weeks post myocardial infarction (MI) in the rat, we measured the two components of the outward K(+) current, I(to-fast (f)) and I(to-slow(s)) in the epicardium (epi) and endocardium (endo) of noninfarcted remodeled left ventricle (LV) using patch clamp techniques. Alterations in mRNA and/or protein levels of potassium channel genes Kv1.4, Kv1.5, Kv2.1, Kv4.2 and Kv4.3 were measured in epi, midmyocardium (mid), and endo regions of LV and in the right ventricle (RV). In sham operated rat heart, the density of I(to-f) was 2.3 times greater in epi compared to endo myocytes. In post-MI heart, the density of I(to-f) and I(to-s) decreased to a similar degree in LV epi and endo but the difference in I(to-f) density between epi and endo persisted. The mRNA and/or protein levels of Kv1.4, Kv2.1, Kv4.2 and Kv4.3 but not Kv1.5 decreased to a varying extent in different regions of LV but not in RV of post-MI heart. Our results suggest that regional downregulation of Kv channels expression and density of K(+) currents can be a significant determinant of increased spatial electrophysiological heterogeneity and contribute to increased electrical instability of the post-MI heart.

Distinct transient outward potassium current (Ito) phenotypes and distribution of fast-inactivating potassium channel alpha subunits in ferret left ventricular myocytes.

The biophysical characteristics and alpha subunits underlying calcium-independent transient outward potassium current (Ito) phenotypes expressed in ferret left ventricular epicardial (LV epi) and endocardial (LV endo) myocytes were analyzed using patch clamp, fluorescent in situ hybridization (FISH), and immunofluorescent (IF) techniques. Two distinct Ito phenotypes were measured (21-22 degrees C) in the majority of LV epi and LV endo myocytes studied. The two Ito phenotypes displayed marked differences in peak current densities, activation thresholds, inactivation characteristics, and recovery kinetics. Ito,epi recovered rapidly [taurec, -70 mV = 51 +/- 3 ms] with minimal cumulative inactivation, while Ito,endo recovered slowly [taurec, -70 mV = 3,002 +/- 447 ms] with marked cumulative inactivation. Heteropoda toxin 2 (150 nM) blocked Ito,epi in a voltage-dependent manner, but had no effect on Ito,endo. Parallel FISH and IF measurements conducted on isolated LV epi and LV endo myocytes demonstrated that Kv1.4, Kv4.2, and Kv4.3 alpha subunit expression in LV myocyte types was quite heterogenous: (a) Kv4.2 and Kv4.3 were more predominantly expressed in LV epi than LV endo myocytes, and (b) Kv1.4 was expressed in the majority of LV endo myocytes but was essentially absent in LV epi myocytes. In combination with previous measurements on recovery kinetics (Kv1.4, slow; Kv4.2/4.3, relatively rapid) and Heteropoda toxin block (Kv1.4, insensitive; Kv4.2, sensitive), our results strongly support the hypothesis that, in ferret heart, Kv4.2/Kv4.3 and Kv1.4 alpha subunits, respectively, are the molecular substrates underlying the Ito,epi and Ito,endo phenotypes. FISH and IF measurements were also conducted on ferret ventricular tissue sections. The three Ito alpha subunits again showed distinct patterns of distribution: (a) Kv1.4 was localized primarily to the apical portion of the LV septum, LV endocardium, and approximate inner 75% of the LV free wall; (b) Kv4. 2 was localized primarily to the right ventricular free wall, epicardial layers of the LV, and base of the heart; and (c) Kv4.3 was localized primarily to epicardial layers of the LV apex and diffusely distributed in the LV free wall and septum. Therefore, in intact ventricular tissue, a heterogeneous distribution of candidate Ito alpha subunits not only exists from LV epicardium to endocardium but also from apex to base.