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Selected Publication:
Ljubojevic, S.
Nucleo-to-cytoplasmic Ca2+ gradients in cardiomyocytes from non-failing and failing mouse and human hearts
PhD-Studium (Doctor of Philosophy); Humanmedizin; [ Dissertation ] Medical University of Graz; 2012. pp.110.
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- Authors Med Uni Graz:
- Holzer Senka
- Advisor:
- Kockskämper Jens
- Pieske Burkert Mathias
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- Abstract:
- Nucleoplasmic calcium concentration ([Ca2+]nuc) in cardiac myocytes (CMs) regulates transcription and its alterations are involved in remodelling processes leading to hypertrophy and heart failure (HF). Thus, the aim of this project was to quantify changes in Ca2+ homeostasis in two distinct cellular compartments, the nucleus vs. the cytoplasm, in early ¿ compensated and terminal ¿ decompensated stages of HF, as well as to determine whether [Ca2+] can be regulated differentially in these compartments under different physiological and pathophysiological conditions. Properties of fluorescent Ca2+-indicators in intracellular compartments may differ, thus affecting the translation of qualitative Ca2+-dependent fluorescence changes into quantitative [Ca2+] changes. Therefore, we determined the in situ characteristics of a frequently used Ca2+ indicator, Fluo-4, and a ratiometric Ca2+ indicator, Asante Calcium Red, and evaluated their use in reporting and quantifying cytoplasmic and nucleoplasmic Ca2+ signals in isolated CMs. Ca2+ calibration curves revealed significant differences in the apparent Ca2+ dissociation constants of Fluo-4 and Asante Calcium Red between cytoplasm and nucleoplasm. Using these parameters, we demonstrated substantial differences in quantitative Ca2+ handling between cytoplasm and nucleoplasm: resting and diastolic [Ca2+] were always higher in the nucleoplasm, while systolic [Ca2+] was usually higher in the cytoplasm (85%). Ca2+ store depletion or blockade of Ca2+ leak pathways eliminated the resting [Ca2+] gradient between nucleoplasm and cytoplasm, whereas inhibition of inositol 1,4,5-trisphosphate receptors by 2-APB reversed it. The results suggest that there are significant nucleoplasmic to cytoplasmic [Ca2+] gradients in resting myocytes and during the cardiac cycle. Nucleoplasmic [Ca2+] in cardiomyocytes may be regulated via two mechanisms: diffusion from the cytoplasm and active Ca2+ release via inositol 1,4,5-trisphosphate receptors from perinuclear Ca2+ stores. With the established method, we then characterized alterations in cytoplasmic and nucleoplasmic Ca2+ handling after pressure overload-induced hypertrophy in murine CMs and in CMs isolated from failing and non-failing human hearts. In the early stage of hypertrophy, slowing of the kinetics and a decline in the amplitude of CaTs was found selectively in the nucleus. In the late stage of heart failure, similar changes of CaTs also occurred in the cytoplasm. At higher stimulation frequencies, CMs from TAC mice showed significant alterations in Ca2+ cycling compared to healthy controls. Due to its slower kinetics, dysfunction of Ca2+ handling in the nucleoplasmic compartment was even more pronounced than in the cytoplasm when higher stimulation frequencies were applied. To understand potential mechanisms for altered nucleoplasmic Ca2+ handling, we investigated possible changes in the nuclear envelope morphology and expression pattern of Ca2+ regulatory proteins during the hypertrophy progress. In CMs from Sham animals, staining of perinuclear Ca stores revealed a nuclear envelope and tubular structures transversing the nucleus. A significant increase in number of tubules per nucleus was observed during physiological growth, while nuclear dimensions remained unaltered. In TAC CMs, the number of tubules per nucleus progressively decreased. Immunostaining of Ca2+ regulatory proteins showed distinct expression patterns in perinuclear regions in failing hearts as compared to the controls. In conclusion, perinuclear Ca stores and nucleoplasmic CaTs undergo significant changes during pressure overload-induced hypertrophy, which appear to precede changes in cytoplasmic Ca regulation. Similar changes can also be observed in failing human myocardium. These results raise the possibility that altered nucleoplasmic [Ca] may contribute to the development and/or progression of hypertrophy.