Spatial distributions of Kv4 channels and KChip2 isoforms in the murine heart based on laser capture microdissection.

OBJECTIVE: Regional differences in repolarizing K(+) current densities and expression levels of their molecular components are important for coordinating the pattern of electrical excitation and repolarization of the heart. The small size of hearts from mice may obscure these interventricular and/or transmural expression differences of K(+) channels. We have examined this possibility in adult mouse ventricle using a technology that provides very high spatial resolution of tissue collection. METHODS: Conventional manual dissection and laser capture microdissection (LCM) were utilized to dissect tissue from distinct ventricular regions. RNA was isolated from epicardial, mid-myocardial and endocardial layers of both the right and left ventricles. Real-time RT-PCR was used to quantify the transcript expression in these different regions. RESULTS: LCM revealed significant interventricular and transmural gradients for both Kv4.2 and the alpha-subunit of KChIP2. The expression profile of a second K(+) channel transcript, Kir2.1, which is responsible for the inwardly rectifying K(+) current I(k1), showed no interventricular or transmural gradients and therefore served as a negative control. CONCLUSIONS: Our findings are in contrast to previous reports of a relatively uniform left ventricular transmural pattern of expression of Kv4.2, Kv4.3 and KChIP2 in adult mouse heart, which appear to be different than that in larger mammals. Specifically, our results demonstrate significant epi- to endocardial differences in the patterns of expression of both Kv4.2 and KChIP2.

Mechanisms of the negative inotropic effects of sphingosine-1-phosphate on adult mouse ventricular myocytes.

Sphingosine-1-phosphate (S1P) induces a transient bradycardia in mammalian hearts through activation of an inwardly rectifying K(+) current (I(K(ACh))) in the atrium that shortens action potential duration (APD) in the atrium. We have investigated probable mechanisms and receptor-subtype specificity for S1P-induced negative inotropy in isolated adult mouse ventricular myocytes. Activation of S1P receptors by S1P (100 nM) reduced cell shortening by approximately 25% (vs. untreated controls) in field-stimulated myocytes. S1P(1) was shown to be involved by using the S1P(1)-selective agonist SEW2871 on myocytes isolated from S1P(3)-null mice. However, in these myocytes, S1P(3) can modulate a somewhat similar negative inotropy, as judged by the effects of the S1P(1) antagonist VPC23019. Since S1P(1) activates G(i) exclusively, whereas S1P(3) activates both G(i) and G(q), these results strongly implicate the involvement of mainly G(i). Additional experiments using the I(K(ACh)) blocker tertiapin demonstrated that I(K(ACh)) can contribute to the negative inotropy following S1P activation of S1P(1) (perhaps through G(ibetagamma) subunits). Mathematical modeling of the effects of S1P on APD in the mouse ventricle suggests that shortening of APD (e.g., as induced by I(K(ACh))) can reduce L-type calcium current and thus can decrease the intracellular Ca(2+) concentration ([Ca(2+)](i)) transient. Both effects can contribute to the observed negative inotropic effects of S1P. In summary, these findings suggest that the negative inotropy observed in S1P-treated adult mouse ventricular myocytes may consist of two distinctive components: 1) one pathway that acts via G(i) to reduce L-type calcium channel current, blunt calcium-induced calcium release, and decrease [Ca(2+)](i); and 2) a second pathway that acts via G(i) to activate I(K(ACh)) and reduce APD. This decrease in APD is expected to decrease Ca(2+) influx and reduce [Ca(2+)](i) and myocyte contractility.

Comparison of contraction and calcium handling between right and left ventricular myocytes from adult mouse heart: a role for repolarization waveform.

In the mammalian heart, the right ventricle (RV) has a distinct structural and electrophysiological profile compared to the left ventricle (LV). However, the possibility that myocytes from the RV and LV have different contractile properties has not been established. In this study, sarcomere shortening, [Ca2+]i transients and Ca2+ and K+ currents in unloaded myocytes isolated from the RV, LV epicardium (LVepi) and LV endocardium (LVendo) of adult mice were evaluated. Maximum sarcomere shortening elicited by field stimulation was graded in the order: LVendo > LVepi > RV. Systolic [Ca2+]i was higher in LVendo myocytes than in RV myocytes. Voltage-clamp experiments in which action potential (AP) waveforms from RV and LVendo were used as the command signal, demonstrated that total Ca2+ influx and myocyte shortening were larger in response to the LVendo AP, independent of myocyte subtypes. Evaluation of possible regional differences in myocyte Ca2+ handling was based on: (i) the current-voltage relation of the Ca2+ current; (ii) sarcoplasmic reticulum Ca2+ uptake; and (iii) mRNA expression of important components of the Ca2+ handling system. None of these were significantly different between RV and LVendo. In contrast, the Ca2+-independent K+ current, which modulates AP repolarization, was significantly different between RV, LVepi and LVendo. These results suggest that these differences in K+ currents can alter AP duration and modulate the [Ca2+]i transient and corresponding contraction. In summary, these findings provide an initial description of regional differences in excitation-contraction coupling in the adult mouse heart [corrected]