Enhanced apoptotic propensity in diabetic cardiac mitochondria: influence of subcellular spatial location

Abstract

Cardiovascular complications, such as diabetic cardiomyopathy, account for the majority of deaths associated with diabetes mellitus. Mitochondria are particularly susceptible to the damaging effects of diabetes mellitus and have been implicated in the pathogenesis of diabetic cardiomyopathy. Cardiac mitochondria consist of two spatially distinct subpopulations, termed subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). The goal of this study was to determine whether subcellular spatial location is associated with apoptotic propensity of cardiac mitochondrial subpopulations during diabetic insult. Swiss Webster mice were subjected to intraperitoneal injection of streptozotocin or citrate saline vehicle. Ten weeks following injection, diabetic hearts displayed increased caspase-3 and caspase-9 activities, indicating enhanced apoptotic signaling (P < 0.05, for both). Mitochondrial size (forward scatter) and internal complexity (side scatter) were decreased in diabetic IFM (P < 0.05, for both) but not in diabetic SSM. Mitochondrial membrane potential (Delta(Psim)) was lower in diabetic IFM (P < 0.01) but not in diabetic SSM. Mitochondrial permeability transition pore (mPTP) opening was increased in diabetic compared with control IFM (P < 0.05), whereas no differences were observed in diabetic compared with control SSM. Examination of mPTP constituents revealed increases in cyclophilin D in diabetic IFM. Furthermore, diabetic IFM possessed lower cytochrome c and BcL-2 levels and increased Bax levels (P < 0.05, for all 3). No significant changes in these proteins were observed in diabetic SSM compared with control. These results indicate that diabetes mellitus is associated with an enhanced apoptotic propensity in IFM, suggesting a differential apoptotic susceptibility of distinct mitochondrial subpopulations based upon subcellular location.

Fig. 1.

Caspase activities. Determination of caspase activation following streptozotocin (STZ) treatment, as indexed by caspase-3 and caspase-9 activities, is shown. Data are expressed as arbitrary units (AU). Values are means ± SE; n = 4 for each group. *P < 0.05 for diabetic vs. control.

Fig. 2.

Mitochondria subpopulation morphological assessment. Determination of relative size and internal complexity of distinct mitochondrial subpopulations using flow cytometric analyses is shown. A: representative density plot of isolated mitochondria stained with MitoTracker Deep Red 633. Intact mitochondria are gated (R1) and represented in green, whereas noise and debris (outside the R1 gate) are indicated below the green line. B: representative gated density plot from a control mouse indicating size [forward scatter (FSC)] versus internal complexity [side scatter (SSC)] of isolated subsarcolemmal mitochondria (SSM). C: representative gated density plot from a control mouse indicating size (FSC) versus internal complexity (SSC) of isolated interfibrillar mitochondria (IFM). In each case, analysis of FSC and SSC was calculated per 20,000 gated events for all mitochondrial subpopulations. D: analysis of cardiac SSM and IFM size (FSC) in control and diabetic mitochondria subpopulations. E: analysis of cardiac SSM and IFM complexity (SSC) in control and diabetic mitochondria subpopulations. Values for FSC and SSC are expressed as arbitrary units (AU) ± SE; n = 4 for each group. *P < 0.05 for diabetic vs. control.

Fig. 3.

Mitochondria subpopulation membrane potential. Isolated mitochondrial subpopulations were incubated with JC-1, and 20,000 gated events were analyzed per sample. A: representative histograms for control and diabetic, SSM and IFM, respectively. B: graph of all samples. Values are expressed as means ± SE; n = 3 for each group. †P < 0.01 for diabetic vs. control.

Fig. 4.

Mitochondria permeability transition pore (mPTP) subpopulation opening. mPTP opening assessed by spectrophotometric measurement at 540 nm of mitochondrial swelling following oxidant (400 μM) t-BuOOH, Ca²⁺ (100 μM), and succinate (10 mM) exposure are shown. Time to V_max was assessed over a 15-min time period following exogenous oxidant exposure. A: representative absorbance plot. Control SSM, solid black line; diabetic SSM, hatched black line; control SSM + cyclosporin A (CsA), solid gray line; diabetic SSM + CsA, hatched gray line. B: representative absorbance plot. Control IFM, solid black line; diabetic IFM, hatched black line; control IFM + CsA, solid gray line; diabetic IFM + CsA, hatched gray line. C: graph of all samples examined. Values are expressed as means ± SE; n = 6 for each group. *P < 0.05 for diabetic vs. control.

Fig. 5.

mPTP subpopulation components. mPTP components assessed by Western blot analyses. A: representative Western blots for cyclophilin D and summary data from control and diabetic SSM and IFM (B). C: representative Western blots for adenine nucleotide translocase and summary data from control and diabetic SSM and IFM (D). E: representative Western blots for voltage-dependent anion channel and summary data from control and diabetic SSM and IFM (F). Control for protein loading was confirmed by Coomassie blue staining. Values are expressed relative to control as means ± SE; n = 4 for each group. *P < 0.05 for diabetic vs. control.

Fig. 6.

Mitochondrial subpopulation Bax and Bcl-2 protein contents. Mitochondrial subpopulations Western blot analyses are shown. A: representative Western blots for Bax and summary data from control and diabetic SSM and IFM (B). C: representative Western blots for Bcl-2 and summary data from control and diabetic SSM and IFM (D). Control for protein loading was confirmed by Coomassie blue staining. Values are expressed relative to control as means ± SE; n = 4 for each group. *P < 0.05 for diabetic vs. control.

Fig. 7.

Mitochondrial subpopulation cytochrome c protein contents. Mitochondrial subpopulations Western blot analyses are shown. A: representative Western blots for cytochrome c and summary data from control and diabetic SSM, IFM (B), and cytosol (C). Control for protein loading was confirmed by Coomassie blue staining. Values are expressed relative to control as means ± SE; n = 4 for each group. *P < 0.05 for diabetic vs. control.