Cyclosporine A attenuates mitochondrial permeability transition and improves mitochondrial respiratory function in cardiomyocytes isolated from dogs with heart failure

Abstract

We used isolated cardiomyocytes to investigate a possible role of mitochondrial permeability transition pore in mitochondrial abnormalities associated with heart failure. Cardiomyocytes were isolated from LV myocardium of normal control dogs and dogs with heart failure produced by intracoronary microembolizations. Mitochondrial permeability transition was measured in isolated cardiomyocytes with intact sarcolemma with and without 0.2 microM cyclosporin A using calcein AM and the fluorometer. State-3 mitochondrial respiration was also measured with the Clark electrode. Mitochondrial membrane potential was measured with JC-1 probe using the fluorometer. Propidium iodide was used to ensure sarcolemma integrity. 200 min after loading with calcein AM, mitochondria of failing cardiomyocytes showed only 50% of maximal level of calcein fluorescence while it remained unchanged in normal cells. The mitochondrial membrane potential in failing cardiomyocytes was significantly decreased by 38% compared to normal cardiomyocytes. Cyclosporine A significantly slowed the exit of calcein from mitochondria of failing cardiomyocytes and increased mitochondrial membrane potential by 29%. State-3 respiration was not affected with cyclosporine A in normal cardiomyocytes while it was significantly increased in failing cardiomyocytes by 20%. Exit of calcein (m.w. 1.0 kDa) from mitochondria of viable failing cardiomyocytes with intact sarcolemma suggests an existence of a reversible transitory permeability transition opening in high conductance mode. Attenuation of calcein exit, DeltaPsi(m) and improvement of state-3 respiration achieved with CsA (0.2 microM) show that permeability transition opening could be a cause of mitochondrial dysfunction described in the failing heart.

Fig.1

Isolated cardiomyocytes loaded with calcein AM and double-stained with propidium iodide: a,b,c – intact isolated cardiomyocytes, a – routine microscopy, b – same cardiomyocytes under fluorescein light, c – same cardiomyocytes under rhodamine light; d,e,f – cardiomyocyte skinned with 0.005% digitotin in working solution, d – routine microscopy, e – same cardiomyocyte under fluorescein light, f – same cardiomyocyte under rhodamine light; g,h,i – cardiomyocyte skinned with 0.005% digitotin in HEPES, g – routine microscopy, h – same cardiomyocyte under fluorescein light, i – same cardiomyocyte under rhodamine light.

Fig.2

Dynamic of Calcein AM fluorescence in normal and failing isolated cardiomyocytes: a – effect of skinning on calcein AM fluorescence in normal cardiomyocytes (n=7), working solution (●)(n=7) versus HEPES (■)(n=7) ; b – dynamic of calcein AM fluorescence in failing cardiomyocytes (●)(n=7) versus normal cardiomyocytes (■)(n=7) with intact sarcolemma.

Fig.3

Effect of CsA on calcein AM fluorescence: a – dynamic of calcein AM fluorescence in normal intact cardiomyocytes (n=7); b - dynamic of calcein AM fluorescence in failing intact cardiomyocytes (n=7). ●- untreated cardiomyocytes (n=7), ■ – cardiomyocytes treated with 0.2 μM CsA(n=7), ▲ – cardiomyocytes treated with CsA + trifluoperazine(n=7), ▼- cardiomyocytes treated with trifluoperazine(n=7).

Fig.4

Effect of CsA on mitochondrial membrane potential (ΔΨ_m) in normal control cardiomyocytes (n=7) and in failing cardiomyocytes (n=7). a – Fluorescence excitation ratio. The fluorescence excitation ratio in each case was calculated as a the ratios of red/green fluorescent signal intensities and was represented as relative numbers of the ratios of intensity units. *=p<0.5 compared to untreated HF cardiomyocytes; b – Relationship between fluorescence exitation ratio and mitochondrial membrane potential. The ΔΨ_m in failing cardiomyoctes was calculated empirically taking the ΔΨ_m in normal controls for 150 mV (see results).

Fig.5

Effect of CsA on mitochondrial respiratory parameters in normal control (n=7) and failing cardiomyocytes (n=7): a – CsA significantly increases state-3 respiration in failing cardiomyocytes and does not affect state-3 respiration in normal control cardiomyocytes. ; b – CsA significantly increases mitochondrial respiratory control ratio in failing cardiomyocytes and does not affect respiratory control ratio in normal control cardiomyocytes. V_ADP – state-3 respiratory rate after addition 1 mM ADP; V_AT – respiratory rate after addition 0.3 mM atractiloside. *=p<0.05 compared to HF untreated cardiomyocytes.