The role of calcium-independent phospholipase A2 in cardiolipin remodeling in the spontaneously hypertensive heart failure rat heart

Abstract

Cardiolipin (CL) is an essential phospholipid component of the inner mitochondrial membrane. In the mammalian heart, the functional form of CL is tetralinoleoyl CL [(18:2)(4)CL]. A decrease in (18:2)(4)CL content, which is believed to negatively impact mitochondrial energetics, occurs in heart failure (HF) and other mitochondrial diseases. Presumably, (18:2)(4)CL is generated by remodeling nascent CL in a series of deacylation-reacylation cycles; however, our overall understanding of CL remodeling is not yet complete. Herein, we present a novel cell culture method for investigating CL remodeling in myocytes isolated from Spontaneously Hypertensive HF rat hearts. Further, we use this method to examine the role of calcium-independent phospholipase A(2) (iPLA(2)) in CL remodeling in both HF and nonHF cardiomyocytes. Our results show that 18:2 incorporation into (18:2)(4)CL is: a) performed singly with respect to each fatty acyl moiety, b) attenuated in HF relative to nonHF, and c) partially sensitive to iPLA(2) inhibition by bromoenol lactone. These results suggest that CL remodeling occurs in a step-wise manner, that compromised 18:2 incorporation contributes to a reduction in (18:2)(4)CL in the failing rat heart, and that mitochondrial iPLA(2) plays a role in the remodeling of CL's acyl composition.

Fig. 1.

Cardiomyocyte viability. Cardiomyocytes were isolated and seeded on glass microscope coverslips. Isolated cells from either nonHF or HF rats were photographed preceding (Pre-) and subsequent to (Post-) each treatment period.

Fig. 2.

Cardiolipin composition in isolated cardiomyocytes. Isolated cardiomyocytes from either nonHF or HF SHHF rats were incubated under control conditions for 48 h. Following incubation, (18:2)₄CL and (18:2)₃(22:6)CL content were analyzed and expressed as a fraction of total CL. Data presented as mean ± SE (n = 4 nonHF, n = 4 HF). * P < 0.05 versus nonHF.

Fig. 3.

Cardiolipin mass spectra in isolated cardiomyocytes. Lipids were extracted from nonHF SHHF myocytes and detected using ESI-MS. A–D show representative spectra for the following conditions: (A) Control, 48 h; (B) ¹³C-18:2, 24 h; (C) ¹³C-18:2, 48 h; (D) ¹³C-18:2 + BEL, 24 h.

Fig. 4.

Incorporation of ¹³C-18:2 into (18:2)₄CL. Isolated cardiomyocytes from nonHF and HF SHHF rats were incubated in ¹³C-18:2 for up to 72 h. At different time intervals after the initial incubation, cells from (A) nonHF and (B) HF SHHF rat hearts were harvested and five different species of (18:2)₄CL, as well as their sum, were analyzed and expressed as a fraction of total CL. Data presented as mean ± SE. n = 3–8 for each time point for both nonHF and HF. (C) Levels of (¹³C-18:2)₂(18:2)₂CL after 48 h of incubation in ¹³C-18:2 were taken from the graphs in (A) and (B) and plotted against one another, along with corresponding data from young and aged FBN myocytes. Data are presented as mean ± SE (n = 4 nonHF SHHF, 3 HF SHHF; 4 young FBN, 6 aged FBN). * P < 0.05 versus SHHF HF.

Fig. 5.

Incorporation of ¹³C-18:2 into phosphatidylglycerol. Cardiomyocytes isolated from (A) nonHF and (B) HF SHHF rat hearts were incubated in ¹³C-18:2, ¹³C-18:2 + BEL, or ¹³C-18:2 + R-BEL for up to 72 h. Incorporation of ¹³C-18:2 into PG was measured by quantifying different species of PG with either 16:0, 18:1, 18:2, or ¹³C-18:2. Data are presented as mean ± SE (n = 4). * P < 0.05 versus control value within analyte; ^# P < 0.05 versus ¹³C-18:2, 24 h value within analyte.

Fig. 6.

Effect of iPLA₂ inhibition on incorporation of ¹³C-18:2 into (¹³C-18:2)(18:2)₃CL. Myocytes from either (A) nonHF or (B) HF SHHF rat hearts were incubated with ¹³C-18:2 following 30 min preincubation with 10 μM BEL. Cells were harvested at select time points and (¹³C-18:2)(18:2)₃CL was quantified and expressed as a fraction of total CL. Table inserts show initial rates of (¹³C-18:2)(18:2)₃CL formation after 10 h and 24 h periods of incubation with ¹³C-18:2. Data are presented as mean ± SE (n = 4–8). * P < 0.05 versus ¹³C-18:2 + BEL at same time point.

Fig. 7.

Effect of iPLA₂γ inhibition on incorporation of ¹³C-18:2 into (¹³C-18:2)(18:2)₃CL. NonHF myocytes were incubated in 5 μM R-BEL for 30 min prior to the addition of ¹³C-18:2 to solution. Data are presented as mean ± SE (n = 4). * P < 0.05 versus ¹³C-18:2 + R-BEL at the corresponding time point.