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Disrupted junctional membrane complexes and hyperactive ryanodine receptors after acute junctophilin knockdown in mice.

ABSTRACT: Excitation-contraction coupling in striated muscle requires proper communication of plasmalemmal voltage-activated Ca2+ channels and Ca2+ release channels on sarcoplasmic reticulum within junctional membrane complexes. Although previous studies revealed a loss of junctional membrane complexes and embryonic lethality in germ-line junctophilin-2 (JPH2) knockout mice, it has remained unclear whether JPH2 plays an essential role in junctional membrane complex formation and the Ca(2+)-induced Ca(2+) release process in the heart. Our recent work demonstrated loss-of-function mutations in JPH2 in patients with hypertrophic cardiomyopathy.To elucidate the role of JPH2 in the heart, we developed a novel approach to conditionally reduce JPH2 protein levels using RNA interference. Cardiac-specific JPH2 knockdown resulted in impaired cardiac contractility, which caused heart failure and increased mortality. JPH2 deficiency resulted in loss of excitation-contraction coupling gain, precipitated by a reduction in the number of junctional membrane complexes and increased variability in the plasmalemma-sarcoplasmic reticulum distance.Loss of JPH2 had profound effects on Ca2+ release channel inactivation, suggesting a novel functional role for JPH2 in regulating intracellular Ca2+ release channels in cardiac myocytes. Thus, our novel approach of cardiac-specific short hairpin RNA-mediated knockdown of junctophilin-2 has uncovered a critical role for junctophilin in intracellular Ca2+ release in the heart.

SUBMITTER: van Oort RJ

PROVIDER: S-EPMC3056402 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Disrupted junctional membrane complexes and hyperactive ryanodine receptors after acute junctophilin knockdown in mice.

van Oort Ralph J RJ Garbino Alejandro A Wang Wei W Dixit Sayali S SS Landstrom Andrew P AP Gaur Namit N De Almeida Angela C AC Skapura Darlene G DG Rudy Yoram Y Burns Alan R AR Ackerman Michael J MJ Wehrens Xander H T XH

Circulation 20110221 9

<h4>Background</h4>Excitation-contraction coupling in striated muscle requires proper communication of plasmalemmal voltage-activated Ca2+ channels and Ca2+ release channels on sarcoplasmic reticulum within junctional membrane complexes. Although previous studies revealed a loss of junctional membrane complexes and embryonic lethality in germ-line junctophilin-2 (JPH2) knockout mice, it has remained unclear whether JPH2 plays an essential role in junctional membrane complex formation and the Ca( ...[more]

PMID: 21339484

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Project description:Aims:In ventricular myocytes from humans and large mammals, the transverse and axial tubular system (TATS) network is less extensive than in rodents with consequently a greater proportion of ryanodine receptors (RyRs) not coupled to this membrane system. TATS remodelling in heart failure (HF) and after myocardial infarction (MI) increases the fraction of non-coupled RyRs. Here we investigate whether this remodelling alters the activity of coupled and non-coupled RyR sub-populations through changes in local signalling. We study myocytes from patients with end-stage HF, compared with non-failing (non-HF), and myocytes from pigs with MI and reduced left ventricular (LV) function, compared with sham intervention (SHAM). Methods and results:Single LV myocytes for functional studies were isolated according to standard protocols. Immunofluorescent staining visualized organization of TATS and RyRs. Ca2+ was measured by confocal imaging (fluo-4 as indicator) and using whole-cell patch-clamp (37°C). Spontaneous Ca2+ release events, Ca2+ sparks, as a readout for RyR activity were recorded during a 15 s period following conditioning stimulation at 2 Hz. Sparks were assigned to cell regions categorized as coupled or non-coupled sites according to a previously developed method. Human HF myocytes had more non-coupled sites and these had more spontaneous activity than in non-HF. Hyperactivity of these non-coupled RyRs was reduced by Ca2+/calmodulin-dependent kinase II (CaMKII) inhibition. Myocytes from MI pigs had similar changes compared with SHAM controls as seen in human HF myocytes. As well as by CaMKII inhibition, in MI, the increased activity of non-coupled sites was inhibited by mitochondrial reactive oxygen species (mito-ROS) scavenging. Under adrenergic stimulation, Ca2+ waves were more frequent and originated at non-coupled sites, generating larger Na+/Ca2+ exchange currents in MI than in SHAM. Inhibition of CaMKII or mito-ROS scavenging reduced spontaneous Ca2+ waves, and improved excitation-contraction coupling. Conclusions:In HF and after MI, RyR microdomain re-organization enhances spontaneous Ca2+ release at non-coupled sites in a manner dependent on CaMKII activation and mito-ROS production. This specific modulation generates a substrate for arrhythmia that appears to be responsive to selective pharmacologic modulation.

Dataset Information

Disrupted junctional membrane complexes and hyperactive ryanodine receptors after acute junctophilin knockdown in mice.

Publications

Disrupted junctional membrane complexes and hyperactive ryanodine receptors after acute junctophilin knockdown in mice.

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