Mitochondrial fission is required for cardiomyocyte hypertrophy mediated by a Ca2+-calcineurin signaling pathway

Abstract

Cardiomyocyte hypertrophy has been associated with diminished mitochondrial metabolism. Mitochondria are crucial organelles for the production of ATP, and their morphology and function are regulated by the dynamic processes of fusion and fission. The relationship between mitochondrial dynamics and cardiomyocyte hypertrophy is still poorly understood. Here, we show that treatment of cultured neonatal rat cardiomyocytes with the hypertrophic agonist norepinephrine promotes mitochondrial fission (characterized by a decrease in mitochondrial mean volume and an increase in the relative number of mitochondria per cell) and a decrease in mitochondrial function. We demonstrate that norepinephrine acts through α1-adrenergic receptors to increase cytoplasmic Ca(2+), activating calcineurin and promoting migration of the fission protein Drp1 (encoded by Dnml1) to mitochondria. Dominant-negative Drp1 (K38A) not only prevented mitochondrial fission, it also blocked hypertrophic growth of cardiomyocytes in response to norepinephrine. Remarkably, an antisense adenovirus against the fusion protein Mfn2 (AsMfn2) was sufficient to increase mitochondrial fission and stimulate a hypertrophic response without agonist treatment. Collectively, these results demonstrate the importance of mitochondrial dynamics in the development of cardiomyocyte hypertrophy and metabolic remodeling.

Keywords: Calcineurin; Cardiac hypertrophy; Drp1; Metabolism; Mitochondrial fission; Norepinephrine.

Fig. 1.

The pro-hypertrophic factor norepinephrine stimulates mitochondrial fission in cultured cardiomyocytes. (A) Representative confocal images of the mitochondrial cardiomyocyte network in cells stained with Mitotracker Green and treated with norepinephrine (NE, 10 µM) for 0, 24 and 48 h (upper, middle and lower panel, respectively). The right column is a magnification of indicated areas in the left column. Scale bar: 10 µm. (B) Quantitative analysis (mean±s.e.m.; n = 5) of mitochondrial morphology of cardiomyocytes treated with norepinephrine for the indicated times. Images were subjected to 3D reconstruction to determine the number and volume of mitochondrial particles. (C) Representative confocal images of cardiomyocytes treated with norepinephrine (0–48 h) and stained with phalloidine–Rhodamine to detect sarcomeric structures, and immunolabeled for the mtHsp70 protein to identify mitochondrial network. Scale bar: 5 µm. (D,E) Quantitative analysis (mean±s.e.m.; n = 3) of (D) cell area and (E) mitochondrial morphology of cardiomyocytes treated as in C. The right-hand panel of D shows the intensity profile of the sarcomeric red fluorescence from the yellow line depicted in C. Number of mitochondria per cell, mitochondrial volume, mitochondrial area and cell area were evaluated with the ImageJ software. *P<0.05 versus control.

Fig. 2.

Mitochondrial metabolism is decreased in hypertrophic cardiomyocytes. (A) Cells stimulated with norepinephrine (0–48 h) were incubated with tetramethylrhodamine (TMRM) to study mitochondrial membrane potential (Ψ_m) by flow cytometry. CCCP (10 µM) was used as a positive control for mitochondrial depolarization (mean±s.e.m.; n = 4). (B) Intracellular ATP content was determined using a luciferine-luciferase assay (mean±s.e.m.; n = 6). (C) Total ROS content was measured by flow cytometry using the dihydrorhodamine (DHR) probe on cardiomyocytes treated with norepinephrine for the indicated times. H₂O₂ (100 µM) was used as positive control (mean±s.e.m.; n = 4). (D) Basal oxygen consumption (white bars) of cardiomyocytes stimulated with norepinephrine for 0–48 h was assessed with a Clark electrode. Uncoupled respiration rate (black bars) was determined by adding CCCP (200 nM) after 75% of the oxygen in the system had been depleted (mean±s.e.m.; n = 5). *P<0.05 versus basal control; ^#P<0.05 versus uncoupled control.

Fig. 3.

Norepinephrine stimulates Drp1 translocation from cytosol to mitochondria. (A) Protein levels of different mitochondrial dynamics machinery components were determined using western blotting in total extracts of cells stimulated with norepinephrine (NE, 0–48 h). Left panels show representative western blots. Right panels show quantification (mean±s.e.m.; n = 6). Values are relative to those of β-tubulin (β-Tub.). (B) Western blot showing the different OPA1 isoforms expressed in cardiomyocytes stimulated with norepinephrine for the indicated times (mean±s.e.m.; n = 4). (C) Immunofluorescence images of cardiomyocytes treated as indicated, using anti-Drp1 (red) or anti-Fis1 (green) antibodies. R corresponds to Pearson's coefficient for fluorescence colocalization. Scale bar: 5 µm. (D) Manders' coefficient quantification for immunofluorescence images of cells. Black and white bars indicate Drp1-asociated fluorescence colocalization to Fis1 signal and Fis1-asociated fluorescence colocalization to Drp1 signal, respectively. (mean±s.e.m.; n = 4). (E) Drp1 protein levels (mean±s.e.m.; n = 4) were determined by western blot assay in mitochondrial and cytosolic extracts from cardiomyocytes treated with norepinephrine for 0, 24 and 48 h or transfected with calcineurin adenovirus (CN). mtHsp70 and GAPDH were used as mitochondrial and cytosolic markers, respectively. *P<0.05.

Fig. 4.

Dominant-negative Drp1 (K38A) prevents norepinephrine-induced cardiomyocyte hypertrophy. (A) Representative fluorescence microscopy images of cardiomyocytes transfected with a LacZ- or K38A-encoding adenovirus prior to norepinephrine (NE) stimulation for 48 h, and stained with phalloidin–Rhodamine for sarcomeric detection. Nuclei were stained with Höescht 33342. Scale bar: 20 µm. (B) Percentage of sarcomerized cardiomyocytes and (C) cell area were calculated using at least 55 cells per condition (mean±s.e.m.; n = 4). (D) Fluorescence intensity profile of the yellow lines depicted on A. (E) Western blot analysis of the hypertrophic biomarker β-MHC on cells treated with norepinephrine for 48 h after LacZ or K38A adenovirus transduction (mean±s.e.m.; n = 3). (F) Determination of mRNA levels of atrial natriuretic factor (ANF) and RCAN 1.4 using qPCR (mean±s.e.m.; n = 3). (G) Basal oxygen consumption of Drp1-K38A-transduced cardiomyocytes and stimulated with norepinephrine for 48 h (mean±s.e.m.; n = 4). *P<0.05 versus non stimulated control; ^#P<0.05 versus norepinephrine-stimulated control.

Fig. 5.

Lack of Mfn2 induces cardiomyocyte hypertrophy. (A) Cells were transduced with LacZ or Mfn2 antisense (AsMfn2) adenovirus or norepinephrine (NE) for 48 h and then stained with phalloidin–Rhodamine and Höescht 33342 to visualize sarcomeric organization and nuclei, respectively. Scale bar: 20 µm. (B) Percentage of highly sarcomerized cardiomyocytes and (C) cell area was determined using at least 55 cells per condition (mean±s.e.m.; n = 4). (D) Fluorescence intensity profile of the yellow lines depicted on A. (E) Western blot for β-MHC on cells treated with norepinephrine or transduced with the indicated adenovirus. A quantification is shown in the lower panel (mean±s.e.m.; n = 4). (F) mRNAs levels for ANF and RCAN 1.4 of AsMfn2-transduced cardiomyocytes (mean±s.e.m.; n = 4). *P<0.05 versus their respective control; ^#P<0.05 versus norepinephrine-LacZ. (G) Basal oxygen consumption of AsMfn2-transduced cardiomyocytes (mean±s.e.m.; n = 4). *P<0.05 versus their respective controls.

Fig. 6.

Norepinephrine activates α₁-adrenergic receptor signaling to promote mitochondrial fission on cultured cardiomyocytes. (A) Representative confocal microscopy images of cells stimulated with norepinephrine (NE) for 48 h and/or treated with the α₁-adrenergic receptor antagonist Prazosin (Praz, 1 µM), and then stained with Mitotracker Green to visualize mitochondrial network. Scale bar: 5 µm. (B) Quantitative analysis of the mitochondrial morphology of cells treated with norepinephrine for 48 h and/or Praz (mean±s.e.m.; n = 7). (C) Fluorescence profile of cardiomyocytes incubated with fluo3AM (5.4 µM) to visualize cytosolic Ca2+ increases in response to norepinephrine. White circles and black squares show control cells and cardiomyocytes preincubated with Praz for 30 min, respectively. The arrows show the time of norepinephrine stimulation followed by the addition of KCl as a positive control to increase cytosolic Ca²⁺ levels by sarcoplasmic depolarization. (D) Western blots for RCAN 1.4, as a calcineurin activity reporter, in cells stimulated with norepinephrine (0–48 h). A quantification is presented in the lower panel (mean±s.e.m.; n = 4). (E) Representative western blot (n = 3) for phospho-Drp1 (Ser637) protein. Forskolin (FK) was used as a positive phosphorylation control. *P<0.05 versus control or t = 0; ^#P<0.05 versus norepinephrine-stimulated cells.

Fig. 7.

Calcineurin activation induces mitochondrial fission in cultured cardiomyocytes. (A) Representative confocal microscopy images of cells stained with Mitotracker Green after transduction with the adenovirus containing LacZ, CAIN or calcineurin (CN) and after norepinephrine (NE) stimulus (48 h). Scale bar: 5 µm. (B) Quantitative analysis of confocal microscopy images of cell treated as in A to evaluate mitochondrial morphology (mean±s.e.m.; n = 4). (C) Oxygen consumption assays of cardiomyocytes transduced with the indicated adenovirus and stimulated with norepinephrine for 48 h (mean±s.e.m.; n = 4). *P<0.05 versus non stimulated control; ^#P<0.05 versus norepinephrine-stimulated control.