Impaired mitochondrial network excitability in failing guinea-pig cardiomyocytes

Abstract

Aims: Studies in guinea-pig cardiomyocytes show that reactive oxygen species (ROS) produced by a few mitochondria can propagate to their neighbours, triggering synchronized, cell-wide network oscillations via an ROS-induced ROS release (RIRR) mechanism. How mitochondria in cardiomyocytes from failing hearts (HF) respond to local oxidative stress perturbations has not been investigated. Since mitochondrial ultrastructure is reportedly disrupted in HF, and propagation of ROS signals depends on mitochondrial network integrity, we hypothesized that the laser flash-induced RIRR is altered in HF.

Methods and results: To test the hypothesis, pressure-overload HF was induced in guinea pigs by ascending aortic constriction leading to left ventricular dilatation and decreased ejection fraction after 8 weeks. Isolated cardiomyocytes were studied with two-photon/confocal microscopy to determine their basal oxidative stress and propensity to undergo mitochondrial depolarization/oscillations in response to local laser flash stimulations. The expression of mitofusin proteins and mitochondrial network structure were also analysed. Results showed that HF cardiomyocytes had higher baseline ROS levels and less reduced glutathione, and were more prone to laser flash-induced mitochondrial depolarization. In contrast, the delay between the laser flash and synchronized cell-wide network oscillations was prolonged in HF myocytes compared with shams, and the spatial extent of coupling was diminished, suggesting dampened RIRR and ROS signal propagation. In addition, the expressions of mitofusin proteins in HF myocardium were down-regulated compared with these from sham-operated animals, and the mitochondrial network structure altered.

Conclusion: The disrupted inter-mitochondrial tethering and loss of structural organization may underlie decreased ROS-dependent mitochondrial coupling in HF.

Keywords: Excitability; Heart failure; Mitochondrial network; Mitofusin protein; ROS-induced-ROS release.

Figure 1

Comparison of basal mitochondrial energetic state in the sham and HF cardiomyocytes. (A) Representative sham (up) and HF (lower) cells loaded with MCB (left), TMRM (middle), or CM-H₂DCFDA fluorescent dye. These dyes were used to measure intracellular reduced glutathione (GSH), mitochondrial membrane potential (ΔΨ_m), and H₂O₂, respectively; (B–D) Comparison of basal GSH concentration, ΔΨ_m, and CM-DCF between the sham and HF cardiomyocytes. A total of 40 cells from 5 sham hearts and 70 cells from 7 HF hearts were analysed. *P < 0.05.

Figure 2

Local laser flash-induced mitochondrial depolarization in the HF and sham cardiomyocytes. (A) Propensity of cardiomyocytes to depolarize. (B) Lag between the first cell-wide mitochondrial depolarization and the induction of a laser flash. (C) Representative sham cardiomyocyte depolarization. (D) Representative HF cardiomyocyte depolarization. A total of 40 cells from 5 sham hearts and 80 cells from 7 HF hearts were analysed. *P < 0.05.

Figure 3

Uncoupled laser flash-induced mitochondrial network oscillations in HF cardiomyocytes. (A) Oscillations of an HF cardiomyocyte involving mitochondrial membrane potential (TMRM) and NADH; (B) oscillations of a sham cell; (C) the snapshot of an oscillating HF cardiomyocyte showing heterogeneous mitochondrial energetic state; and (D) plots of time profile of membrane potential (measured by TMRM) of various mitochondrial clusters (curves 1–5 represent zones 1–5 marked in C). A total of 30 cells from 5 sham hearts and 40 cells from 7 HF hearts were examined.

Figure 4

Expression of mitofusin proteins in HF and sham myocardium. Western blot analysis (A) of the content of mitochondrial fusion proteins Mfn1 (B), Mfn2 (C), and (D) OPA1 and fission proteins FIS1 (E) and DRP1 (F) in samples of sham and HF left ventricles; and real-time PCR measurement of transcript level of Mfn1 (G) and Mfn2 (H) in samples from sham and HF hearts. The protein or mRNA expression was normalized to GAPDH. For the western blotting, samples from four different sham hearts and five different HF hearts were examined. For the PCR, four myocardium samples from each group were examined. *P < 0.05; **P < 0.1.

Figure 5

Mitochondrial ultrastructure in sham and HF left ventricular myocardium. (A) Confocal imaging of Mfn1 immunohistochemistry in sham myocardium; (B) confocal imaging of Mfn1 immunohistochemistry in HF myocardium; (C) transmit electron microscope (TEM) of sham mitochondria; (D) TEM of HF mitochondria; (E) averaged maximal length (or diameter) of mitochondria; and (F) averaged area of mitochondria. Approximately 120 mitochondria from 4 different sham hearts and ∼200 mitochondria from 5 different HF hearts were analysed. *P < 0.05.

Figure 6

Immunohistochemistry analysis of Mfn 1 (green) and RyR2 (red) colocalization in ventricular myocardium. (A) Sham; (B) HF. The colocalization of Mfn1 and RyR2 was analysed using Olympus FV10-ASW software and shown in C (sham) and D (HF), respectively. The results indicated that in HF not only the protein expressions were down-regulated but also the correlation between mitochondria and SR was altered. A total of six samples from three sham hearts and eight samples from four HF hearts were analysed.