Direct monitoring of mitochondrial calcium levels in cultured cardiac myocytes using a novel fluorescent indicator protein, GCaMP2-mt

Iguchi, Moritake; Kato, Masaya; Nakai, Junichi; Takeda, Toshihiro; Matsumoto-Ida, Madoka; Kita, Toru; Kimura, Takeshi; Akao, Masaharu

doi:10.1016/j.ijcard.2011.01.034

Cited by 13 publications

(9 citation statements)

References 38 publications

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“…2(B)]. Apparent K d was 195.0 nmol/L, which is similar to that in neonatal cardiomyocytes (at pH 7.5) [17].…”

Section: Resultssupporting

confidence: 72%

“…Construction and characterization of a mitochondrial calcium fluorescent indicator According to the strategies used before [15,17,18,20], we constructed a mitochondrial calcium fluorescent indicator by adding the mitochondrial targeting sequence before the sequence of a genetically encoded calcium indicator, GCaMP2, and named the new indicator, mito-GCaMP2 [ Fig. 1(A)].…”

Section: Resultsmentioning

confidence: 99%

“…Compared with the traditional calcium fluorescent dyes, GECI allows calcium imaging in vivo to understand the complex physiology and pathophysiology [11 -13]. Furthermore, GECI can be specifically targeted to different subcellular locations by genetic engineering [6,[14][15][16][17], which allows for characterization of spatial and temporal regulation of calcium signaling.…”

Section: Introductionmentioning

confidence: 99%

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Differential mitochondrial calcium responses in different cell types detected with a mitochondrial calcium fluorescent indicator, mito-GCaMP2

Chen¹,

Wang²,

Hou³

et al. 2011

ABBS

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Mitochondrial calcium plays a crucial role in mitochondrial metabolism, cell calcium handling, and cell death. However, some mechanisms concerning mitochondrial calcium regulation are still unknown, especially how mitochondrial calcium couples with cytosolic calcium. In this work, we constructed a novel mitochondrial calcium fluorescent indicator (mito-GCaMP2) by genetic manipulation. Mito-GCaMP2 was imported into mitochondria with high efficiency and the fluorescent signals co-localized with that of tetramethyl rhodamine methyl ester, a mitochondrial membrane potential indicator. The mitochondrial inhibitors specifically decreased the signals of mito-GCaMP2. The apparent K d of mito-GCaMP2 was 195.0 nmol/L at pH 8.0 in adult rat cardiomyocytes. Furthermore, we observed that mito-GCaMP2 preferred the alkaline pH surrounding of mitochondria. In HeLa cells, we found that mitochondrial calcium ( ] mito did not respond to cytosolic calcium transients stimulated by electric pacing or caffeine. In permeabilized cardiomyocytes, 600 nmol/L free Ca 21 repeatedly increased the fluorescent signals of mito-GCaMP2, which excluded the possibility that mito-GCaMP2 lost its function in cardiomyocytes mitochondria. These results showed that the response of mitochondrial calcium is diverse in different cell lineages and suggested that mitochondria in cardiomyocytes may have a special defense mechanism to control calcium flux.

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“…2(B)]. Apparent K d was 195.0 nmol/L, which is similar to that in neonatal cardiomyocytes (at pH 7.5) [17].…”

Section: Resultssupporting

confidence: 72%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Differential mitochondrial calcium responses in different cell types detected with a mitochondrial calcium fluorescent indicator, mito-GCaMP2

Chen¹,

Wang²,

Hou³

et al. 2011

ABBS

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“…Ca 2 þ overload is a key contributor to mPTP opening (Davidson et al, 2012;Iguchi et al, 2012). The reduction of Ca 2 þ overload can block or delay myocardial death (Ngoh et al, 2011).…”

Section: Discussionmentioning

confidence: 99%

Pre-treatment of a single high-dose of atorvastatin provided cardioprotection in different ischaemia/reperfusion models via activating mitochondrial KATP channel

Zhao

Cui

Zhang

et al. 2015

European Journal of Pharmacology

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“…More recently, GCaMP2 was cloned to subcellular targeting proteins to target the GCaMP2 to certain sub-cellular localisations; an approach which proved successful in detecting subcellular local calcium. For example, the Lck-GCaMP2 was able to detect a sub-membrane calcium microdomain in neuronal cells [29], and the mito-GCaMP2 has been developed for mitochondrial calcium imaging [30]. Unlike other GCaMP2 sensors which were cloned to non-calciumregulating proteins, the PMCA4-GCaMP2 was the first trial to fuse this calcium sensor to a calcium pump.…”

Section: Pmca4 Mutmentioning

confidence: 99%

Development and characterization of a novel fluorescent indicator protein PMCA4-GCaMP2 in cardiomyocytes

Mohamed

Abouleisa

Baudoin

et al. 2013

Journal of Molecular and Cellular Cardiology

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Isoform 4 of the plasma membrane calcium/calmodulin dependent ATPase (PMCA4) has recently emerged as an important regulator of several key pathophysiological processes in the heart, such as contractility and hypertrophy. However, direct monitoring of PMCA4 activity and assessment of calcium dynamics in its vicinity in cardiomyocytes are difficult due to the lack of molecular tools. In this study, we developed novel calcium fluorescent indicators by fusing the GCaMP2 calcium sensor to the N-terminus of PMCA4 to generate the PMCA4-GCaMP2 fusion molecule. We also identified a novel specific inhibitor of PMCA4, which might be useful for studying the role of this molecule in cardiomyocytes and other cell types. Using an adenoviral system we successfully expressed PMCA4-GCaMP2 in both neonatal and adult rat cardiomyocytes. This fusion molecule was correctly targeted to the plasma membrane and co-localised with caveolin-3. It could monitor signal oscillations in electrically stimulated cardiomyocytes. The PMCA4-GCaMP2 generated a higher signal amplitude and faster signal decay rate compared to a mutant inactive PMCA4 mut GCaMP2 fusion protein, in electrically stimulated neonatal and adult rat cardiomyocytes. A small molecule library screen enabled us to identify a novel selective inhibitor for PMCA4, which we found to reduce signal amplitude of PMCA4-GCaMP2 and prolong the time of signal decay (Tau) to a level comparable with the signal generated by PMCA4 mut GCaMP2. In addition, PMCA4-GCaMP2 but not the mutant form produced an enhanced signal in response to β-adrenergic stimulation. Together, the PMCA4-GCaMP2 and PMCA4 mut GCaMP2 demonstrate calcium dynamics in the vicinity of the pump under active or inactive conditions, respectively. In summary, the PMCA4-GCaMP2 together with the novel specific inhibitor provides new means with which to monitor calcium dynamics in the vicinity of a calcium transporter in cardiomyocytes and may become a useful tool to further study the biological functions of PMCA4. In addition, similar approaches could be useful for studying the activity of other calcium transporters during excitation-contraction coupling in the heart.

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Direct monitoring of mitochondrial calcium levels in cultured cardiac myocytes using a novel fluorescent indicator protein, GCaMP2-mt

Abstract: Direct monitoring of [Ca(2+)](m) using GCaMP2-mt provides deeper insight into the mechanism of cardiac myocyte death.

Cited by 13 publications

References 38 publications

Differential mitochondrial calcium responses in different cell types detected with a mitochondrial calcium fluorescent indicator, mito-GCaMP2

Differential mitochondrial calcium responses in different cell types detected with a mitochondrial calcium fluorescent indicator, mito-GCaMP2

Pre-treatment of a single high-dose of atorvastatin provided cardioprotection in different ischaemia/reperfusion models via activating mitochondrial KATP channel

Development and characterization of a novel fluorescent indicator protein PMCA4-GCaMP2 in cardiomyocytes

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