Regulation of mitochondrial fission by intracellular Ca2+ in rat ventricular myocytes

Abstract

Mitochondria are dynamic organelles that constantly undergo fission, fusion, and movement. Increasing evidence indicates that these dynamic changes are intricately related to mitochondrial function, suggesting that mitochondrial form and function are linked. Calcium (Ca2+) is one signal that has been shown to both regulate mitochondrial fission in various cell types and stimulate mitochondrial enzymes involved in ATP generation. However, although Ca2+ plays an important role in adult cardiac muscle cells for excitation-metabolism coupling, little is known about whether Ca2+ can regulate their mitochondrial morphology. Therefore, we tested the role of Ca2+ in regulating cardiac mitochondrial fission. We found that neonatal and adult cardiomyocyte mitochondria undergo rapid and transient fragmentation upon a thapsigargin (TG)- or KCl-induced cytosolic Ca2+ increase. The mitochondrial fission protein, DLP1, participates in this mitochondrial fragmentation, suggesting that cardiac mitochondrial fission machinery may be regulated by intracellular Ca2+ signaling. Moreover, the TG-induced fragmentation was also associated with an increase in reactive oxygen species (ROS) formation, suggesting that activation of mitochondrial fission machinery is an early event for Ca2+-mediated ROS generation in cardiac myocytes. These results suggest that Ca2+, an important regulator of muscle contraction and energy generation, also dynamically regulates mitochondrial morphology and ROS generation in cardiac myocytes.

Figure 1. Ventricular myocytes express DLP1, which translocates to mitochondria upon TG-induced increase of cytosolic Ca²⁺

(A) Western blotting demonstrates that neonatal rat ventricular myocytes endogenously express hFis1 and DLP1 from days 2–7 in culture. Clone 9 cells were used as a positive control for fission protein expression [23]. GAPDH was used as a loading control. (B) Ca²⁺ imaging using Fura-2 showed that either 1 µM TG (black line) or 50 mM KCl (pink line) induced an increase in cytosolic Ca²⁺. The Fura-2 ratio (F₃₄₀/F₃₆₀) at each time point (F) was normalized against the initial ratio (F₀) for comparison (n=4). Representative traces of mitochondrial Ca²⁺ imaging using Rhod-2 showed that 1 µM TG (black line) induced mitochondrial Ca²⁺ uptake. Treatment with Ru360 (blue line) decreased mitochondrial Ca²⁺ uptake upon TG addition. (n=3) (C) Cultured adult cardiomyocytes were treated with 1 µM TG for 5, 10, 30, or 60 min and the mitochondrial fractions were isolated and assayed for DLP1 and VDAC (loading control) by Western blot. DLP1 association with mitochondria increased after 5, 10, and 30 min of TG treatment but returned to basal levels at 60 min TG (* P<0.01 by ANOVA with Dunnett’s post-hoc test, n=4).

Figure 2. TG fragments mitochondria in neonatal and adult ventricular myocytes

(A) Within 5 min, either 1 µM TG or 50 mM KCl, induced mitochondrial fragmentation, compared to baseline, in cultured neonatal myocytes. Untreated cells displayed normal globular and tubular mitochondrial morphology (A, control). Preincubation with 10 µM Ru360 for 30 min and throughout the experiment inhibited TG-induced mitochondrial fragmentation at 5 min. Mitochondria were visualized using Mitotracker Red CMXRos. Scale bar = 30 µm. (B) Cultured adult cardiac myocytes were treated with 1 µM TG for 5, 10, and 30 min, fixed, and viewed under electron microscope. Cardiac mitochondria display various morphologies in control and at each time point but mitochondria appeared smaller and more fragmented at 5 min TG. Scale bar = 1 µm. (C) Consistent with this observation, a histogram of mitochondrial area demonstrated a smaller mean mitochondrial area at 5 min TG, which recovered at 10 and 30 min TG compared to control. (D) Analysis of mitochondrial form factor demonstrates a decrease in mitochondrial branching and length from cells after 5 min of TG treatment compared to 0, 10, and 30 min TG, and a recovery of normal morphology at 10 and 30 min TG. Analysis of mitochondrial aspect ratio demonstrates shorter mitochondria at 5 min TG, with significant lengthening between 5 and 10 min followed by a return to baseline. (for all analyses, *P<0.05 using Kruskal-Wallis non-parametric testing with Dunn’s post-hoc test)

Figure 2. TG fragments mitochondria in neonatal and adult ventricular myocytes

(A) Within 5 min, either 1 µM TG or 50 mM KCl, induced mitochondrial fragmentation, compared to baseline, in cultured neonatal myocytes. Untreated cells displayed normal globular and tubular mitochondrial morphology (A, control). Preincubation with 10 µM Ru360 for 30 min and throughout the experiment inhibited TG-induced mitochondrial fragmentation at 5 min. Mitochondria were visualized using Mitotracker Red CMXRos. Scale bar = 30 µm. (B) Cultured adult cardiac myocytes were treated with 1 µM TG for 5, 10, and 30 min, fixed, and viewed under electron microscope. Cardiac mitochondria display various morphologies in control and at each time point but mitochondria appeared smaller and more fragmented at 5 min TG. Scale bar = 1 µm. (C) Consistent with this observation, a histogram of mitochondrial area demonstrated a smaller mean mitochondrial area at 5 min TG, which recovered at 10 and 30 min TG compared to control. (D) Analysis of mitochondrial form factor demonstrates a decrease in mitochondrial branching and length from cells after 5 min of TG treatment compared to 0, 10, and 30 min TG, and a recovery of normal morphology at 10 and 30 min TG. Analysis of mitochondrial aspect ratio demonstrates shorter mitochondria at 5 min TG, with significant lengthening between 5 and 10 min followed by a return to baseline. (for all analyses, *P<0.05 using Kruskal-Wallis non-parametric testing with Dunn’s post-hoc test)

Figure 3. TG-induced mitochondrial fragmentation requires the mitochondrial fission protein DLP1 in neonatal ventricular myocytes

(A) A control neonatal ventricular myocyte labeled with anti-DLP1 and anti-cytochrome c antibodies displays diffuse cytosolic DLP1 and a mix of globular and tubular mitochondria, while the overlay shows little association of DLP1 (green) with mitochondria (red). (B) A neonatal ventricular myocyte overexpressing the dominant negative fission mutant, DLP1-K38A, displays large cytosolic aggregates of DLP1, characteristic of mutant DLP1-K38A expression, and elongated and entangled mitochondria [27, 29]. The overlay shows that mutant DLP1 aggregates (green) do not colocalize with mitochondria (red). (C) Cells were transfected with GFP-tagged DLP1-K38A and live mitochondria were visualized using MitoTracker Red CMXRos. The cells that were positive for DLP1-K38A expression were identified by the presence of green mutant DLP1 aggregates (arrows, lower left), while untransfected cells did not display green aggregates (upper left). The mitochondrial network in control, untransfected cells became fragmented upon 5 min TG treatment (top), while TG has no effect on the elongated and tubular mitochondria seen with GFP-tagged DLP1-K38A overexpression (bottom).

Figure 4. Inhibition of mitochondrial fission prevents Ca²⁺-induced superoxide increase in neonatal ventricular myocytes

(A) Single traces of superoxide production, measured using MitoSOX Red, show that DLP1-K38A overexpression prevented TG-induced superoxide production but not that caused by 10 µM antimycin A, a mitochondrial complex III inhibitor. (B) Statistical analysis revealed that the increase in superoxide production after TG treatment was significantly decreased in cells expressing DLP1-K38A (* P<0.01, t-test, n=8).