Phospholamban ablation rescues sarcoplasmic reticulum Ca(2+) handling but exacerbates cardiac dysfunction in CaMKIIdelta(C) transgenic mice

Abstract

Rationale: 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.

Objective: 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.

Methods and results: 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.

Conclusions: 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.

Figure 1. PLN ablation in CaMKIIδc TG mice repletes SR Ca²⁺ load in isolated cardiomyocytes

A, Representative caffeine-induced Ca²⁺ transient traces. B, Average Δ [Ca²⁺]_i for caffeine-induced Ca²⁺ transients (SR Ca²⁺ load). *P<0.05 vs. WT. # P<0.05 vs. CaMKII-TG. C, Average rate constant k_Ca for [Ca²⁺]_i decline during caffeine exposure (which indicates NCX activity). *P<0.05 vs. WT. **P<0.01 vs. WT. The number of cells studied: WT n=6, PLN-KO n=21, CaMKII-TG n=9, and KO/TG n=9.

Figure 2. PLN ablation in CaMKIIδc TG mice normalizes twitch Ca²⁺ transients in isolated cardiomyocytes

A, Representative twitch [Ca²⁺]_i transient traces. B, Average Δ [Ca²⁺]_i for twitches-induced Ca²⁺ transients. *P<0.05 vs. WT. # P<0.05 vs. CaMKII-TG. The number of cells studied: WT n=6, PLN-KO n=21, CaMKII-TG n=9, and KO/TG n=9.

Figure 3. PLN ablation in CaMKIIδc TG mice exaggerates the phenotype of dilated cardiomyopathy

A, Heart weight/body weight (HW/BW) ratio at 8 wks. B and C, Averaged echocardiographic parameters at different ages. Data are presented for B, left ventricular end-diastolic diameter (LVEDD), and for C, calculated percent fractional shortening (FS). n=4 to 6 mice per group at each age; *P<0.05 vs. WT. # P<0.05 vs. CaMKII-TG. D, Survival curves for CaMKII-TG and PLN-KO/CaMKII-TG mice. PLN ablation accentuates premature death in CaMKIIδ_C TG mice. Numbers of mice were as follows: CaMKII-TG, n=16; KO/TG, n=22. P <0.01 KO/TG vs. CaMKII-TG.

Figure 4. Quantitative immunoblotting of major Ca²⁺ handling proteins in mouse ventricular homogenates

A, Expression levels of calsequestrin (CsQ), SR Ca²⁺ ATPase (SERCA) and Na⁺/Ca²⁺ exchanger (NCX) did not change significantly between the CaMKII-TG and the PLN-KO/CaMKII-TG. B, Phosphorylation of ryanodine receptors (RyR2) at Ser2809 and Ser2815 (normalized to total RyR2) was increased significantly but not differentially in CaMKIIδc TG and PLN-KO/CaMKII-TG mice. n=4 mice per group; *P<0.05 vs. WT.

Figure 5. Enhanced SR Ca²⁺ spark frequency and Ca²⁺ leak in intact cardiomyocytes from PLN-KO/CaMKII-TG mice

A, SR Ca spark frequency (CaSpF). *P<0.05 vs. WT. # P<0.05 vs. CaMKII-TG. +P<0.05 vs. KO/TG. B, SR Ca²⁺ leak. *P<0.05 vs. WT. # P<0.05 vs. CaMKII-TG. +P<0.05 vs. KO/TG.

Figure 6. Apoptosis occurs more frequently in PLN-KO/CaMKII-TG cardiomyocytes than in CaMKIIδc TG

A, Representative TUNEL staining images in CaMKII-TG and PLN-KO/CaMKII-TG mouse heart sections. B, Average TUNEL positive myocytes. n=4 mice per group; *P<0.05 vs. WT. Data were normalized to WT; the average number from all experiments was ~75 TUNEL positive nuclei/10⁵ myocytes in the KO/TG group.

Figure 7. Inhibition of SR Ca²⁺ leak by KN-93 or ryanodine decreases the rate of death in isolated cardiomyocytes from PLN-KO/CaMKII-TG mice

Isolated cardiomyocytes were plated and incubated in the presence and absence of inhibitors, and cell viability assessed at various times using morphology and at 12 hours by trypan blue exclusion. A, WT myocytes showed only slight decreases in viability over 12 hours after plating. B–C, The percent of living myocytes from both CaMKII-TG and PLN-KO/CaMKII-TG mice decreased dramatically over 12 hours. Inhibition of CaMKII with KN-93 or of SR Ca²⁺ leak with ryanodine decreased the rate of myocyte death in both CaMKII-TG and PLN-KO/CaMKII-TG mice.

Figure 8. Involvement of mitochondrial Ca²⁺ in myocyte death in PLN-KO/CaMKII-TG mice

A, Mitochondrial Ca²⁺ was measured as shown in the protocol. NT: no treatment; Thaps: thapsigargin. B, Mitochondrial Ca²⁺ was increased in cardiomyocytes isolated from PLN-KO/CaMKII-TG mice. *P<0.05 vs. WT. Mitochondrial Ca²⁺ in PLN-KO/CaMKII-TG myocytes returned to control levels with reduced external Ca²⁺ (0.3 mM) and CaMKII inhibitor AIP. C, Reduced external Ca²⁺ and CaMKII inhibition with AIP returned SR Ca²⁺ content and Ca²⁺ transients to near control myocyte levels. D, Inhibition of the mitochondrial uniporter by Ru-360 or inhibition of the mitochondrial permeability transition pore by cyclosporine A decreased the rate of death in isolated cardiomyocytes from PLN-KO/CaMKII-TG mice at 12 hours. *P<0.05 vs. WT.