Junctional cleft [Ca2+]i measurements using novel cleft-targeted Ca2+ sensors

Sanda Despa; Bo Shui; Julie Bossuyt; Di Lang; Michael I. Kotlikoff; Donald M. Bers

doi:10.1161/CIRCRESAHA.115.303582

Junctional cleft [Ca₂₊]i measurements using novel cleft-targeted Ca₂₊ sensors

Sanda Despa, Bo Shui, Julie Bossuyt, Di Lang, Michael I. Kotlikoff, Donald M. Bers

Pharmacology and Nutritional Sciences

Research output: Contribution to journal › Article › peer-review

35 Scopus citations

Abstract

RATIONALE:: Intracellular Ca₂₊ concentration ([ Ca₂₊ ]i) is regulated and signals differently in various subcellular microdomains, which greatly enhances its second messenger versatility. In the heart, sarcoplasmic reticulum Ca₂₊ release and signaling are controlled by local [ Ca₂₊ ]i in the junctional cleft ([ Ca₂₊ ]Cleft), the small space between sarcolemma and junctional sarcoplasmic reticulum. However, methods to measure [ Ca₂₊ ]Cleft directly are needed. OBJECTIVE:: To construct novel sensors that allow direct measurement of [ Ca₂₊ ]Cleft. METHODS AND RESULTS:: We constructed cleft-targeted [ Ca₂₊ ] sensors by fusing Ca-sensor GCaMP2.2 and a new lower Ca-affinity variant GCaMP2.2Low to FKBP12.6, which binds with high affinity and selectivity to ryanodine receptors. The fluorescence pattern, affinity for ryanodine receptors, and competition by untagged FKBP12.6 demonstrated that FKBP12.6-tagged sensors are positioned to measure local [ Ca₂₊ ]Cleft in adult rat myocytes. Using GCaMP2.2Low-FKBP12.6, we showed that [ Ca ₂₊ ]Cleft reaches higher levels with faster kinetics than global [ Ca₂₊ ]i during excitation-contraction coupling. Diastolic sarcoplasmic reticulum Ca₂₊ leak or sarcolemmal Ca₂₊ entry may raise local [ Ca₂₊ ]Cleft above bulk cytosolic [ Ca₂₊ ]i ([ Ca₂₊ ]Bulk), an effect that may contribute to triggered arrhythmias and even transcriptional regulation. We measured this diastolic standing [ Ca₂₊ ]Cleft-[ Ca₂₊ ]Bulk gradient with GCaMP2.2-FKBP12.6 versus GCaMP2.2, using [ Ca₂₊ ] measured without gradients as a reference point. This diastolic difference ([ Ca₂₊ ]Cleft=194 nmol/L versus [ Ca₂₊ ]Bulk=100 nmol/L) is dictated mainly by the sarcoplasmic reticulum Ca₂₊ leak rather than sarcolemmal Ca₂₊ flux. CONCLUSIONS:: We have developed junctional cleft-targeted sensors to measure [ Ca₂₊ ]Cleft versus [ Ca₂₊ ]Bulk and demonstrated dynamic differences during electric excitation and a standing diastolic [ Ca₂₊ ]i gradient, which could influence local Ca-dependent signaling within the junctional cleft.

Original language	English
Pages (from-to)	339-347
Number of pages	9
Journal	Circulation Research
Volume	115
Issue number	3
DOIs	https://doi.org/10.1161/CIRCRESAHA.115.303582
State	Published - Jul 18 2014

Keywords

calcium signaling
cardiac-sarcoplasmic reticulum
myocytes

ASJC Scopus subject areas

Physiology
Cardiology and Cardiovascular Medicine

Access to Document

10.1161/CIRCRESAHA.115.303582

Cite this

@article{2130790313084aeb98a242c1decefc24,

title = "Junctional cleft [Ca2+]i measurements using novel cleft-targeted Ca2+ sensors",

abstract = "RATIONALE:: Intracellular Ca2+ concentration ([ Ca2+ ]i) is regulated and signals differently in various subcellular microdomains, which greatly enhances its second messenger versatility. In the heart, sarcoplasmic reticulum Ca2+ release and signaling are controlled by local [ Ca2+ ]i in the junctional cleft ([ Ca2+ ]Cleft), the small space between sarcolemma and junctional sarcoplasmic reticulum. However, methods to measure [ Ca2+ ]Cleft directly are needed. OBJECTIVE:: To construct novel sensors that allow direct measurement of [ Ca2+ ]Cleft. METHODS AND RESULTS:: We constructed cleft-targeted [ Ca2+ ] sensors by fusing Ca-sensor GCaMP2.2 and a new lower Ca-affinity variant GCaMP2.2Low to FKBP12.6, which binds with high affinity and selectivity to ryanodine receptors. The fluorescence pattern, affinity for ryanodine receptors, and competition by untagged FKBP12.6 demonstrated that FKBP12.6-tagged sensors are positioned to measure local [ Ca2+ ]Cleft in adult rat myocytes. Using GCaMP2.2Low-FKBP12.6, we showed that [ Ca 2+ ]Cleft reaches higher levels with faster kinetics than global [ Ca2+ ]i during excitation-contraction coupling. Diastolic sarcoplasmic reticulum Ca2+ leak or sarcolemmal Ca2+ entry may raise local [ Ca2+ ]Cleft above bulk cytosolic [ Ca2+ ]i ([ Ca2+ ]Bulk), an effect that may contribute to triggered arrhythmias and even transcriptional regulation. We measured this diastolic standing [ Ca2+ ]Cleft-[ Ca2+ ]Bulk gradient with GCaMP2.2-FKBP12.6 versus GCaMP2.2, using [ Ca2+ ] measured without gradients as a reference point. This diastolic difference ([ Ca2+ ]Cleft=194 nmol/L versus [ Ca2+ ]Bulk=100 nmol/L) is dictated mainly by the sarcoplasmic reticulum Ca2+ leak rather than sarcolemmal Ca2+ flux. CONCLUSIONS:: We have developed junctional cleft-targeted sensors to measure [ Ca2+ ]Cleft versus [ Ca2+ ]Bulk and demonstrated dynamic differences during electric excitation and a standing diastolic [ Ca2+ ]i gradient, which could influence local Ca-dependent signaling within the junctional cleft.",

keywords = "calcium signaling, cardiac-sarcoplasmic reticulum, myocytes",

author = "Sanda Despa and Bo Shui and Julie Bossuyt and Di Lang and Kotlikoff, {Michael I.} and Bers, {Donald M.}",

year = "2014",

month = jul,

day = "18",

doi = "10.1161/CIRCRESAHA.115.303582",

language = "English",

volume = "115",

pages = "339--347",

number = "3",

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TY - JOUR

T1 - Junctional cleft [Ca2+]i measurements using novel cleft-targeted Ca2+ sensors

AU - Despa, Sanda

AU - Shui, Bo

AU - Bossuyt, Julie

AU - Lang, Di

AU - Kotlikoff, Michael I.

AU - Bers, Donald M.

PY - 2014/7/18

Y1 - 2014/7/18

N2 - RATIONALE:: Intracellular Ca2+ concentration ([ Ca2+ ]i) is regulated and signals differently in various subcellular microdomains, which greatly enhances its second messenger versatility. In the heart, sarcoplasmic reticulum Ca2+ release and signaling are controlled by local [ Ca2+ ]i in the junctional cleft ([ Ca2+ ]Cleft), the small space between sarcolemma and junctional sarcoplasmic reticulum. However, methods to measure [ Ca2+ ]Cleft directly are needed. OBJECTIVE:: To construct novel sensors that allow direct measurement of [ Ca2+ ]Cleft. METHODS AND RESULTS:: We constructed cleft-targeted [ Ca2+ ] sensors by fusing Ca-sensor GCaMP2.2 and a new lower Ca-affinity variant GCaMP2.2Low to FKBP12.6, which binds with high affinity and selectivity to ryanodine receptors. The fluorescence pattern, affinity for ryanodine receptors, and competition by untagged FKBP12.6 demonstrated that FKBP12.6-tagged sensors are positioned to measure local [ Ca2+ ]Cleft in adult rat myocytes. Using GCaMP2.2Low-FKBP12.6, we showed that [ Ca 2+ ]Cleft reaches higher levels with faster kinetics than global [ Ca2+ ]i during excitation-contraction coupling. Diastolic sarcoplasmic reticulum Ca2+ leak or sarcolemmal Ca2+ entry may raise local [ Ca2+ ]Cleft above bulk cytosolic [ Ca2+ ]i ([ Ca2+ ]Bulk), an effect that may contribute to triggered arrhythmias and even transcriptional regulation. We measured this diastolic standing [ Ca2+ ]Cleft-[ Ca2+ ]Bulk gradient with GCaMP2.2-FKBP12.6 versus GCaMP2.2, using [ Ca2+ ] measured without gradients as a reference point. This diastolic difference ([ Ca2+ ]Cleft=194 nmol/L versus [ Ca2+ ]Bulk=100 nmol/L) is dictated mainly by the sarcoplasmic reticulum Ca2+ leak rather than sarcolemmal Ca2+ flux. CONCLUSIONS:: We have developed junctional cleft-targeted sensors to measure [ Ca2+ ]Cleft versus [ Ca2+ ]Bulk and demonstrated dynamic differences during electric excitation and a standing diastolic [ Ca2+ ]i gradient, which could influence local Ca-dependent signaling within the junctional cleft.

AB - RATIONALE:: Intracellular Ca2+ concentration ([ Ca2+ ]i) is regulated and signals differently in various subcellular microdomains, which greatly enhances its second messenger versatility. In the heart, sarcoplasmic reticulum Ca2+ release and signaling are controlled by local [ Ca2+ ]i in the junctional cleft ([ Ca2+ ]Cleft), the small space between sarcolemma and junctional sarcoplasmic reticulum. However, methods to measure [ Ca2+ ]Cleft directly are needed. OBJECTIVE:: To construct novel sensors that allow direct measurement of [ Ca2+ ]Cleft. METHODS AND RESULTS:: We constructed cleft-targeted [ Ca2+ ] sensors by fusing Ca-sensor GCaMP2.2 and a new lower Ca-affinity variant GCaMP2.2Low to FKBP12.6, which binds with high affinity and selectivity to ryanodine receptors. The fluorescence pattern, affinity for ryanodine receptors, and competition by untagged FKBP12.6 demonstrated that FKBP12.6-tagged sensors are positioned to measure local [ Ca2+ ]Cleft in adult rat myocytes. Using GCaMP2.2Low-FKBP12.6, we showed that [ Ca 2+ ]Cleft reaches higher levels with faster kinetics than global [ Ca2+ ]i during excitation-contraction coupling. Diastolic sarcoplasmic reticulum Ca2+ leak or sarcolemmal Ca2+ entry may raise local [ Ca2+ ]Cleft above bulk cytosolic [ Ca2+ ]i ([ Ca2+ ]Bulk), an effect that may contribute to triggered arrhythmias and even transcriptional regulation. We measured this diastolic standing [ Ca2+ ]Cleft-[ Ca2+ ]Bulk gradient with GCaMP2.2-FKBP12.6 versus GCaMP2.2, using [ Ca2+ ] measured without gradients as a reference point. This diastolic difference ([ Ca2+ ]Cleft=194 nmol/L versus [ Ca2+ ]Bulk=100 nmol/L) is dictated mainly by the sarcoplasmic reticulum Ca2+ leak rather than sarcolemmal Ca2+ flux. CONCLUSIONS:: We have developed junctional cleft-targeted sensors to measure [ Ca2+ ]Cleft versus [ Ca2+ ]Bulk and demonstrated dynamic differences during electric excitation and a standing diastolic [ Ca2+ ]i gradient, which could influence local Ca-dependent signaling within the junctional cleft.

KW - calcium signaling

KW - cardiac-sarcoplasmic reticulum

KW - myocytes

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