Heterozygous deletion of sarcolipin maintains normal cardiac function

Abstract

) is a small proteolipid and a regulator of sarco(endo)plasmic reticulum Ca 2ϩ -ATPase. In heart tissue, SLN is exclusively expressed in the atrium. Previously, we inserted Cre recombinase into the endogenous SLN locus by homologous recombination and succeeded in generating SLN-Cre knockin (Sln Cre/ϩ ) mice. This Sln Cre/ϩ mouse can be used to generate an atrium-specific gene-targeting mutant, and it is based on the Cre-loxP system. In the present study, we used adult Sln Cre/ϩ mice atria and analyzed the effects of heterozygous SLN deletion by Cre knockin before use as the gene targeting mouse. Both SLN mRNA and protein levels were decreased in Sln Cre/ϩ mouse atria, but there were no morphological, physiological, or molecular biological abnormalities. The properties of contractility and Ca 2ϩ handling were similar to wild-type (WT) mice, and expression levels of several stress markers and sarcoplasmic reticulum-related protein levels were not different between SlnCre/ϩ and WT mice. Moreover, there was no significant difference in sarco(endo)plasmic reticulum Ca 2ϩ -ATPase activity between the two groups. We showed that Sln Cre/ϩ mice were not significantly different from WT mice in all aspects that were examined. The present study provides basic characteristics of Sln Cre/ϩ mice and possibly information on the usefulness of Sln Cre/ϩ mice as an atrium-specific gene-targeting model. THE DEVELOPMENT of gene-targeting approaches has had a tremendous impact on the functional analysis of the mouse heart. The bacteriophage P1 Cre/loxP site-specific recombinase system allows for precise recombination between two loxP sites, resulting in deletion of the targeting gene in a time-and/or tissue-specific manner. Although many heart muscle-specific conditional knockout (KO) mice have been generated, there are few atrium-specific conditional KO mice. de Lange et al. (6) have generated mice that have atrial cardiomyocyte-specific Cre expression using the natriuretic peptide type A (Nppa) gene promoter (6). However, Cre expression in this mouse was observed in part of the ventricle. The Nppa gene is also highly induced in the ventricle by stresses such as pressure overload (23). In this research, we focused on sarcolipin (SLN), which is a 31-amino acid proteolipid that regulates the activity of sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA) (21, 25, 37), because SLN is expressed specifically in atrial muscle from early developmental stages around embryonic day 10.5 and throughout life (21,25). On the other hand, phospholamban (PLN), a homolog of SLN, is known to be expressed in both the atrium and ventricle, and PLN is particularly abundant in ventricular muscle (21). In the cardiovascular research field, previous studies have reported several effects of SLN deletion and/or overexpression such that SLN deletion indicates enhanced atrial contractility, whereas SLN overexpression indicates decreased myocyte contractility (2, 3, 10). In our previous study (25), we generated SLN-Cre knockin (KI) mice. This KI mouse is a new m...

Atrial-Specific Gene Delivery Using an Adeno-Associated Viral Vector

“…A sarcolipin-Cre knock-in mouse model has been described, which enables gene deletion in atrial myocytes. 4 A limitation of this model is that Cre replaces one endogenous sarcolipin allele, thus rendering the mice haploinsufficient for this gene, which has important functions in the atria. In our hands, the heterozygous sarcolipin-CRE mice exhibit a high incidence of pacing-induced AF (not shown).…”

“…While mouse models have been very helpful in this regard, a major limitation remains ventricular dysfunction arising in global or cardiomyocyte-specific overexpression and deletion models. 3 The sarcolipin-Cre mouse model allows for atrial-specific gene manipulation, 4 but this involves labor-intensive and time-consuming crosses. Moreover, the heterozygous sarcolipin-Cre mice are haplo-insufficient for sarcolipin and susceptible to pacing-induced AF induction (data not shown).…”

Impairment of Excitation-Contraction Coupling in Right Ventricular Hypertrophied Muscle with Fibrosis Induced by Pulmonary Artery Banding

“…Cells on the upper surface of the preparation were penetrated and injected with a small volume of aequorin solution, and the aequorin light signals were detected and recorded simultaneously with tension. In the figures in the present study, the light signals were converted to [Ca 2+ ] i (intracellular Ca 2+ concentration) as we previously described [14, 15]. Aequorin experiments were performed at 30.0 ± 0.5°C.…”

“…For an evaluation of twitch contraction, the following parameters were measured, as in previous studies [14, 15]: peak values of tension and Ca 2+ ; time to peak tension and Ca 2+ (the time measured from the onset of stimulus to the peak of tension and Ca 2+ , respectively); relaxation time (the time required for the tension to relax from 75% to 25% of the peak), and decay time of light (the time required for the light signal to decay from 75% to 25% of the peak).…”

Integrative computational modeling of calcium handling and cardiac arrhythmias

“…Because the activity of SERCA and other calcium-handling proteins (CASQ2, NCX1, RyR2, PLN) were unchanged in heterozygous knockout mice, such protective effects might reflect interaction of SLN with other proteins within the cardiomyocyte (120). Expression of SLN (but not PLN) is reduced in atrial samples of HF patients with reduced ejection fraction with or without long-standing persistent AF, likely contributing to increased SR calcium load (121,122).…”