Phospholamban Ablation Rescues Sarcoplasmic Reticulum Ca2+ Handling but Exacerbates Cardiac Dysfunction in CaMKII C Transgenic Mice

Department of Pharmacology, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093-0636, USA.
Circulation Research (Impact Factor: 11.02). 12/2009; 106(2):354-62. DOI: 10.1161/CIRCRESAHA.109.207423
Source: PubMed

ABSTRACT We previously showed that transgenic mice expressing Ca(2+)/calmodulin-dependent protein kinase II delta(C) (CaMKII-TG) develop dilated cardiomyopathy associated with increased ryanodine receptors (RyR2) phosphorylation, enhanced sarcoplasmic reticulum (SR) Ca(2+) leak and lowering of SR Ca(2+) load. We hypothesized that phospholamban (PLN) ablation would restore SR Ca(2+) load and prevent the decreased ventricular contractility, dilation and mortality seen in CaMKII-TG.
Our objectives were to generate CaMKII-TG mice lacking PLN, determine whether the maladaptive effects of cardiac CaMKIIdelta(C) expression were corrected, and establish the mechanistic basis for these changes.
CaMKII-TG were crossed with PLN knockout (PLN-KO) mice to generate KO/TG mice. Myocytes from wild type (WT), CaMKII-TG, PLN-KO and KO/TG were compared. The decreased SR Ca(2+) load and twitch Ca(2+) transients seen in CaMKII-TG were normalized in KO/TG. Surprisingly the heart failure phenotype was exacerbated, as indicated by increased left ventricular dilation, decreased ventricular function, increased apoptosis and greater mortality. In KO/TG myocytes SR Ca(2+) sparks and leak were significantly increased, presumably because of the combined effects of restored SR Ca(2+) load and RyR2 phosphorylation. Mitochondrial Ca(2+) loading was increased in cardiomyocytes from KO/TG versus WT or CaMKII-TG mice and this was dependent on elevated SR Ca(2+) sparks. Cardiomyocytes from KO/TG showed poor viability, improved by inhibiting SR Ca(2+) release and mitochondrial Ca(2+) loading.
Normalizing cardiomyocyte SR Ca(2+) loading in the face of elevated CaMKII and RyR2 phosphorylation leads to enhanced SR Ca(2+) leak and mitochondrial Ca(2+) elevation, associated with exacerbated cell death, heart failure and mortality.

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    • "CaMKII activity outside of mitochondria contributes to mitochondrial Ca2+ homeostasis. CaMKII activity elevates diastolic sarcoplasmic reticulum (SR) Ca2+ leak (Curran et al., 2007), which was later shown to contribute to mitochondrial Ca2+ overload (Zhang et al., 2010) specifically, under pathophysiological conditions such as rapid cardiomyocyte pacing (Sepúlveda et al., 2013) and diabetes (Luo et al., 2013), but also with the extreme physiological condition of endurance exercise (Rose et al., 2007). Disruption of cytosolic Ca2+ homeostasis promotes mitochondrial Ca2+ overload (Lemasters et al., 2009). "
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    ABSTRACT: CaMKII is a newly discovered resident of mitochondria in the heart. Mitochondrial CaMKII promotes poor outcomes after heart injury from a number of pathological conditions, including myocardial infarction (MI), ischemia reperfusion (IR), and stress from catecholamine stimulation. A study using the inhibitor of CaMKII, CaMKIIN, with expression delimited to myocardial mitochondria, indicates that an underlying cause of heart disease results from the opening of the mitochondrial permeability transition pore (mPTP). Evidence from electrophysiological and other experiments show that CaMKII inhibition likely suppresses mPTP opening by reducing Ca(2+) entry into mitochondria. However, we expect other proteins involved in Ca(2+) signaling in the mitochondria are affected with CaMKII inhibition. Several outstanding questions remain for CaMKII signaling in heart mitochondria. Most importantly, how does CaMKII, without the recognized N-terminal mitochondrial targeting sequence transfer to mitochondria?
    Frontiers in Pharmacology 05/2014; 5:67. DOI:10.3389/fphar.2014.00067 · 3.80 Impact Factor
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    • "To determine whether diminished SR Ca2+ load is the primary causal event leading to contractile dysfunction and premature death in response to δC overexpression, we crossed the δCTG mice with mice in which the SERCA regulatory protein PLN was deleted (PLN-KO). Deletion of PLN in the context of δC overexpression normalized SR Ca2+ levels and the contractile function of isolated myocytes was restored (Zhang et al., 2010). Remarkably the development of cardiac dysfunction in vivo was not rescued but instead was accelerated in the δCTG/PLN-KO mice. "
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    ABSTRACT: In this review we discuss the localization and function of the known subtypes of calcium/calmodulin dependent protein kinase IIδ (CaMKIIδ) and their role in cardiac physiology and pathophysiology. The CaMKII holoenzyme is comprised of multiple subunits that are encoded by four different genes called CaMKIIα, β, γ, and δ. While these four genes have a high degree of sequence homology, they are expressed in different tissues. CaMKIIα and β are expressed in neuronal tissue while γ and δ are present throughout the body, including in the heart. Both CaMKIIγ and δ are alternatively spliced in the heart to generate multiple subtypes. CaMKIIδ is the predominant cardiac isoform and is alternatively spliced in the heart to generate the CaMKIIδB subtype or the slightly less abundant δC subtype. The CaMKIIδB mRNA sequence contains a 33bp insert not present in δC that codes for an 11-amino acid nuclear localization sequence. This review focuses on the localization and function of the CaMKIIδ subtypes δB and δC and the role of these subtypes in arrhythmias, contractile dysfunction, gene transcription, and the regulation of Ca(2+) handling.
    Frontiers in Pharmacology 02/2014; 5:15. DOI:10.3389/fphar.2014.00015 · 3.80 Impact Factor
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    • "In contrast, PLN ablation increased SR Ca2+ filling and contractility in mice with cardiomyopathy attributable to overexpression of CaMKII. This led to premature death and mitochondrial Ca2+ overload, suggesting that accelerating SR Ca2+ uptake and increasing SR Ca2+ load, is disadvantageous at least in the presence of excessive CaMKII activity (Zhang et al., 2010). These findings are consistent with the idea already discussed for I/R: in the face of phosphorylated RyR2 channels, as is the case of CaMKII overexpressing mice, repletion of Ca2+ stores through PLN ablation could further worsen overall heart function, via mitochondrial Ca2+ loading, cell death, and arrhythmias. "
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    ABSTRACT: Phospholamban (PLN) is a phosphoprotein in cardiac sarcoplasmic reticulum (SR) that is a reversible regulator of the Ca2+-ATPase (SERCA2a) activity and cardiac contractility. Dephosphorylated PLN inhibits SERCA2a and PLN phosphorylation, at either Ser16 by PKA or Thr17 by Ca2+-calmodulin-dependent protein kinase (CaMKII), reverses this inhibition. Through this mechanism, PLN is a key modulator of SR Ca2+ uptake, Ca2+ load, contractility, and relaxation. PLN phosphorylation is also the main determinant of β1-adrenergic responses in the heart. Although phosphorylation of Thr17 by CaMKII contributes to this effect, its role is subordinate to the PKA-dependent increase in cytosolic Ca2+, necessary to activate CaMKII. Furthermore, the effects of PLN and its phosphorylation on cardiac function are subject to additional regulation by its interacting partners, the anti-apoptotic HAX-1 protein and Gm or the anchoring unit of protein phosphatase 1. Regulation of PLN activity by this multimeric complex becomes even more important in pathological conditions, characterized by aberrant Ca2+-cycling. In this scenario, CaMKII-dependent PLN phosphorylation has been associated with protective effects in both acidosis and ischemia/reperfusion. However, the beneficial effects of increasing SR Ca2+ uptake through PLN phosphorylation may be lost or even become deleterious, when these occur in association with alterations in SR Ca2+ leak. Moreover, a major characteristic in human and experimental heart failure (HF) is depressed SR Ca2+ uptake, associated with decreased SERCA2a levels and dephosphorylation of PLN, leading to decreased SR Ca2+ load and impaired contractility. Thus, the strategy of altering SERCA2a and/or PLN levels or activity to restore perturbed SR Ca2+ uptake is a potential therapeutic tool for HF treatment. We will review here the role of CaMKII-dependent phosphorylation of PLN at Thr17 on cardiac function under physiological and pathological conditions.
    Frontiers in Pharmacology 01/2014; 5:5. DOI:10.3389/fphar.2014.00005 · 3.80 Impact Factor
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