Abstract
BackgroundCardiomyocyte contraction requires a constant supply of ATP, which varies depending on work rate. Maintaining ATP supply is particularly important during excitation-contraction coupling, where cytosolic Ca2+ fluxes drive repeated cycles of contraction and relaxation. Ca2+ is one of the key regulators of ATP production, and its uptake into the mitochondrial matrix occurs via the mitochondrial calcium uniporter. Fluorescent indicators are commonly used for detecting cytosolic Ca2+ changes. However, visualizing mitochondrial Ca2+ fluxes using similar methods is more difficult, as the fluorophore must be permeable to both the sarcolemma and the inner mitochondrial membrane. Our aim was therefore to optimize a method using the fluorescent Ca2+ indicator Rhod-2 to visualize beat-to-beat mitochondrial calcium fluxes in rat cardiomyocytes.MethodsHealthy, adult male Wistar rat hearts were isolated and enzymatically digested to yield rod-shaped, quiescent ventricular cardiomyocytes. The fluorescent Ca2+ indicator Rhod-2 was reduced to di-hydroRhod-2 and confocal microscopy was used to validate mitochondrial compartmentalization. Cardiomyocytes were subjected to various pharmacological interventions, including caffeine and β-adrenergic stimulation. Upon confirmation of mitochondrial Rhod-2 localization, loaded myocytes were then super-fused with 1.5 mM Ca2+ Tyrodes containing 1 μM isoproterenol and 150 μM spermine. Myocytes were externally stimulated at 0.1, 0.5 and 1 Hz and whole cell recordings of both cytosolic ([Ca2+]cyto) and mitochondrial calcium ([Ca2+]mito) transients were made.ResultsMyocytes loaded with di-hydroRhod-2 revealed a distinct mitochondrial pattern when visualized by confocal microscopy. Application of 20 mM caffeine revealed no change in fluorescence, confirming no sarcoplasmic reticulum compartmentalization. Myocytes loaded with di-hydroRhod-2 also showed a large increase in fluorescence within the mitochondria in response to β-adrenergic stimulation (P < 0.05). Beat-to-beat mitochondrial Ca2+ transients were smaller in amplitude and had a slower time to peak and maximum rate of rise relative to cytosolic calcium transients at all stimulation frequencies (P < 0.001).ConclusionMyocytes loaded with di-hydroRhod-2 revealed mitochondrial specific compartmentalization. Mitochondrial Ca2+ transients recorded from di-hydroRhod-2 loaded myocytes were distinct in comparison to the large and rapid Rhod-2 cytosolic transients, indicating different kinetics between [Ca2+]cyto and [Ca2+]mito transients. Overall, our results showed that di-hydroRhod-2 loading is a quick and suitable method for measuring beat-to-beat [Ca2+]mito transients in intact myocytes.
Highlights
Cardiomyocytes are the working cells of the heart, and are large consumers of ATP
The aims of our study were : (i) to confirm dhRhod-2 is giving a mitochondrial signal that is uncontaminated with cytosolic Ca2+, (ii) to test mitochondrial Ca2+ signals in isolated cardiomyocytes by exposing them to pharmacological interventions known to modulate energy demand and/or mitochondrial Ca2+ levels and (iii) to develop a technique that can be used for fluorometric mitochondrial Ca2+ measurements
Rhod-2 Localization The localization of the non-reduced indicator Rhod-2 was first investigated in isolated cardiomyocytes
Summary
Cardiomyocytes are the working cells of the heart, and are large consumers of ATP. There are two important processes that match ATP production to metabolic demand in cardiomyocytes: (i) mitochondrial ADP reuptake upon hydrolysis in the cytosol or (ii) an increase in cytosolic Ca2+ concentration (for review see: Maack and O’Rourke, 2007). It is unknown whether these mechanisms work independently or in parallel, it is well understood that both are involved in synchronizing energy supply to enable the heart to meet the body’s ever changing metabolic demands. Our aim was to optimize a method using the fluorescent Ca2+ indicator Rhod-2 to visualize beat-to-beat mitochondrial calcium fluxes in rat cardiomyocytes
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