Genetically Encoded Ca
            <sup>2+</sup>
            Indicators in Cardiac Myocytes

Kaestner, Lars; Scholz, Anke; Tian, Qi; Ruppenthal, Sandra; Tabellion, Wiebke; Wiesen, Kathrina; Katus, Hugo A.; Müller, Oliver; Kotlikoff, Michael I.; Lipp, Peter

doi:10.1161/circresaha.114.303475

Cited by 57 publications

(60 citation statements)

References 159 publications

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“…50,51 As a special consideration to Ca 2+ imaging, the usefulness of super resolution methods is limited by the fact that Ca 2+ diffuses rapidly and small molecule Ca 2+ indicators also have limited on/off kinetics. A promising approach may be to express localized Ca 2+ indicators 52–55 in subcellular compartments to further quantify local Ca 2+ levels in relation to sarcomere strain.…”

Section: Discussionmentioning

confidence: 99%

Multimodal SHG-2PF Imaging of Microdomain Ca ²⁺ -Contraction Coupling in Live Cardiac Myocytes

Awasthi

Izu

Mao

et al. 2016

Circulation Research

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Rationale cardiac myocyte contraction is caused by Ca2+ binding to troponin C, which triggers the cross-bridge power stroke and myofilament sliding in sarcomeres. Synchronized Ca2+ release causes whole cell contraction and is readily observable with current microscopy techniques. However, it is unknown whether localized Ca2+ release, such as Ca2+ sparks and waves, can cause local sarcomere contraction. Contemporary imaging methods fall short of measuring microdomain Ca2+-contraction coupling in live cardiac myocytes. Objective To develop a method for imaging sarcomere-level Ca2+-contraction coupling in healthy and disease-model cardiac myocytes. Methods and Results Freshly isolated cardiac myocytes were loaded with the Ca2+-indicator Fluo-4. A confocal microscope equipped with a femtosecond-pulsed near-infrared laser was used to simultaneously excite second harmonic generation (SHG) from A-bands of myofibrils and two-photon fluorescence (2PF) from Fluo-4. Ca2+ signals and sarcomere strain correlated in space and time with short delays. Furthermore, Ca2+ sparks and waves caused contractions in subcellular microdomains, revealing a previously underappreciated role for these events in generating subcellular strain during diastole. Ca2+ activity and sarcomere strain were also imaged in paced cardiac myocytes under mechanical load, revealing spontaneous Ca2+ waves and correlated local contraction in pressure overload-induced cardiomyopathy. Conclusions Multi-modal SHG-2PF microscopy enables the simultaneous observation of Ca2+ release and mechanical strain at the sub-sarcomere level in living cardiac myocytes. The method benefits from the label-free nature of SHG, which allows A-bands to be imaged independently of T-tubule morphology and simultaneously with Ca2+ indicators. SHG-2PF imaging is widely applicable to the study of Ca2+-contraction coupling and mechano-chemo-transduction in both health and disease.

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Section: Discussionmentioning

confidence: 99%

Multimodal SHG-2PF Imaging of Microdomain Ca ²⁺ -Contraction Coupling in Live Cardiac Myocytes

Awasthi

Izu

Mao

et al. 2016

Circulation Research

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“…Ca 2ϩ release from the SR is thought to be critical for pacemaker activity in SAMs (5,13,34), and genetically-encoded Ca 2ϩ indicators (GECIs) could provide important new insights into this process. GECIs can be targeted to subcellular microdomains and have yielded much important information about localized Ca 2ϩ signaling in other types of cardiac myocytes (10,11,22,27,36). In a similar manner, tethered FRET-based cAMP sensors could be used to characterize spatio-temporal cAMP signaling associated with different GPCRs and phosphodiesterase isoforms, as has been done in other cardiac myocytes (20,23,30,37,43).…”

Section: Discussionmentioning

confidence: 98%

Culture and adenoviral infection of sinoatrial node myocytes from adult mice

Clair

Sharpe

Proenza

2015

American Journal of Physiology-Heart and Circulatory Physiology

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“…For chemical and genetically encoded Ca 2 + ‐sensitive dyes, the situation is easier, mainly due to the slower kinetics of Ca 2 + fluxes. These probes have already been widely used for high‐throughput screening in both disease modelling and drug development with encouraging results (Kaestner et al , 2014; Huebsch et al , 2015; Lu et al , 2015; Rast et al , 2015; Dempsey et al , 2016; Klimas et al , 2016). Ca 2 + ‐handling also affects cell contractility, which can also be measured, as recent examples with sophisticated optical mapping systems show (Herron et al , 2012; Hayakawa et al , 2014; Maddah et al , 2015).…”

Section: Assays and Readoutsmentioning

confidence: 99%

Integrating cardiomyocytes from human pluripotent stem cells in safety pharmacology: has the time come?

Sala

Bellin

Mummery

2016

British J Pharmacology

106

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Cardiotoxicity is a severe side effect of drugs that induce structural or electrophysiological changes in heart muscle cells. As a result, the heart undergoes failure and potentially lethal arrhythmias. It is still a major reason for drug failure in preclinical and clinical phases of drug discovery. Current methods for predicting cardiotoxicity are based on guidelines that combine electrophysiological analysis of cell lines expressing ion channels ectopically in vitro with animal models and clinical trials. Although no new cases of drugs linked to lethal arrhythmias have been reported since the introduction of these guidelines in 2005, their limited predictive power likely means that potentially valuable drugs may not reach clinical practice. Human pluripotent stem cell‐derived cardiomyocytes (hPSC‐CMs) are now emerging as potentially more predictive alternatives, particularly for the early phases of preclinical research. However, these cells are phenotypically immature and culture and assay methods not standardized, which could be a hurdle to the development of predictive computational models and their implementation into the drug discovery pipeline, in contrast to the ambitions of the comprehensive pro‐arrhythmia in vitro assay (CiPA) initiative. Here, we review present and future preclinical cardiotoxicity screening and suggest possible hPSC‐CM‐based strategies that may help to move the field forward. Coordinated efforts by basic scientists, companies and hPSC banks to standardize experimental conditions for generating reliable and reproducible safety indices will be helpful not only for cardiotoxicity prediction but also for precision medicine.Linked ArticlesThis article is part of a themed section on New Insights into Cardiotoxicity Caused by Chemotherapeutic Agents. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.21/issuetoc

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Genetically Encoded Ca ²⁺ Indicators in Cardiac Myocytes

Cited by 57 publications

References 159 publications

Multimodal SHG-2PF Imaging of Microdomain Ca ²⁺ -Contraction Coupling in Live Cardiac Myocytes

Multimodal SHG-2PF Imaging of Microdomain Ca ²⁺ -Contraction Coupling in Live Cardiac Myocytes

Culture and adenoviral infection of sinoatrial node myocytes from adult mice

Integrating cardiomyocytes from human pluripotent stem cells in safety pharmacology: has the time come?

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Genetically Encoded Ca 2+ Indicators in Cardiac Myocytes

Cited by 57 publications

References 159 publications

Multimodal SHG-2PF Imaging of Microdomain Ca 2+ -Contraction Coupling in Live Cardiac Myocytes

Multimodal SHG-2PF Imaging of Microdomain Ca 2+ -Contraction Coupling in Live Cardiac Myocytes

Culture and adenoviral infection of sinoatrial node myocytes from adult mice

Integrating cardiomyocytes from human pluripotent stem cells in safety pharmacology: has the time come?

Contact Info

Product

Resources

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Genetically Encoded Ca ²⁺ Indicators in Cardiac Myocytes

Multimodal SHG-2PF Imaging of Microdomain Ca ²⁺ -Contraction Coupling in Live Cardiac Myocytes

Multimodal SHG-2PF Imaging of Microdomain Ca ²⁺ -Contraction Coupling in Live Cardiac Myocytes