Multimodal SHG-2PF Imaging of Microdomain Ca2+-Contraction Coupling in Live Cardiac Myocytes

Samir Awasthi, Leighton T Izu, Ziliang Mao, Zhong Jian, Trevor Landas, Aaron Lerner, Rafael Shimkunas, Rahwa Woldeyesus, Julie B C Bossuyt, Brittani Wood, Yi-Je Chen, Dennis L Matthews, Deborah Lieu, Nipavan Chiamvimonvat, Kit Lam, Ye Chen-Izu, James W Chan

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

RATIONALE:: Cardiomyocyte contraction is caused by Ca binding to troponin C, which triggers the cross-bridge power stroke and myofilament sliding in sarcomeres. Synchronized Ca release causes whole cell contraction and is readily observable with current microscopy techniques. However, it is unknown whether localized Ca release, such as Ca sparks and waves, can cause local sarcomere contraction. Contemporary imaging methods fall short of measuring microdomain Ca-contraction coupling in live cardiac myocytes. OBJECTIVE:: To develop a method for imaging sarcomere-level Ca-contraction coupling in healthy and disease-model cardiac myocytes. METHODS AND RESULTS:: Freshly isolated cardiac myocytes were loaded with the Ca-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. Ca signals and sarcomere strain correlated in space and time with short delays. Furthermore, Ca sparks and waves caused contractions in subcellular microdomains, revealing a previously underappreciated role for these events in generating subcellular strain during diastole. Ca activity and sarcomere strain were also imaged in paced cardiomyocytes under mechanical load, revealing spontaneous Ca waves and correlated local contraction in pressure overload-induced cardiac myopathy. CONCLUSIONS:: Multi-modal SHG-2PF microscopy enables the simultaneous observation of Ca 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 Ca indicators. SHG-2PF imaging is widely applicable to the study of Ca-contraction coupling and mechano-chemo-transduction in both health and disease.

Original languageEnglish (US)
JournalCirculation Research
DOIs
StateAccepted/In press - Dec 9 2015

Fingerprint

Sarcomeres
Cardiac Myocytes
Myofibrils
Microscopy
Troponin C
Diastole
Muscular Diseases
Photons
Lasers
Fluorescence
Stroke
Observation
Pressure
Health

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine

Cite this

Multimodal SHG-2PF Imaging of Microdomain Ca2+-Contraction Coupling in Live Cardiac Myocytes. / Awasthi, Samir; Izu, Leighton T; Mao, Ziliang; Jian, Zhong; Landas, Trevor; Lerner, Aaron; Shimkunas, Rafael; Woldeyesus, Rahwa; Bossuyt, Julie B C; Wood, Brittani; Chen, Yi-Je; Matthews, Dennis L; Lieu, Deborah; Chiamvimonvat, Nipavan; Lam, Kit; Chen-Izu, Ye; Chan, James W.

In: Circulation Research, 09.12.2015.

Research output: Contribution to journalArticle

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title = "Multimodal SHG-2PF Imaging of Microdomain Ca2+-Contraction Coupling in Live Cardiac Myocytes",
abstract = "RATIONALE:: Cardiomyocyte contraction is caused by Ca binding to troponin C, which triggers the cross-bridge power stroke and myofilament sliding in sarcomeres. Synchronized Ca release causes whole cell contraction and is readily observable with current microscopy techniques. However, it is unknown whether localized Ca release, such as Ca sparks and waves, can cause local sarcomere contraction. Contemporary imaging methods fall short of measuring microdomain Ca-contraction coupling in live cardiac myocytes. OBJECTIVE:: To develop a method for imaging sarcomere-level Ca-contraction coupling in healthy and disease-model cardiac myocytes. METHODS AND RESULTS:: Freshly isolated cardiac myocytes were loaded with the Ca-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. Ca signals and sarcomere strain correlated in space and time with short delays. Furthermore, Ca sparks and waves caused contractions in subcellular microdomains, revealing a previously underappreciated role for these events in generating subcellular strain during diastole. Ca activity and sarcomere strain were also imaged in paced cardiomyocytes under mechanical load, revealing spontaneous Ca waves and correlated local contraction in pressure overload-induced cardiac myopathy. CONCLUSIONS:: Multi-modal SHG-2PF microscopy enables the simultaneous observation of Ca 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 Ca indicators. SHG-2PF imaging is widely applicable to the study of Ca-contraction coupling and mechano-chemo-transduction in both health and disease.",
author = "Samir Awasthi and Izu, {Leighton T} and Ziliang Mao and Zhong Jian and Trevor Landas and Aaron Lerner and Rafael Shimkunas and Rahwa Woldeyesus and Bossuyt, {Julie B C} and Brittani Wood and Yi-Je Chen and Matthews, {Dennis L} and Deborah Lieu and Nipavan Chiamvimonvat and Kit Lam and Ye Chen-Izu and Chan, {James W}",
year = "2015",
month = "12",
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doi = "10.1161/CIRCRESAHA.115.307919",
language = "English (US)",
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T1 - Multimodal SHG-2PF Imaging of Microdomain Ca2+-Contraction Coupling in Live Cardiac Myocytes

AU - Awasthi, Samir

AU - Izu, Leighton T

AU - Mao, Ziliang

AU - Jian, Zhong

AU - Landas, Trevor

AU - Lerner, Aaron

AU - Shimkunas, Rafael

AU - Woldeyesus, Rahwa

AU - Bossuyt, Julie B C

AU - Wood, Brittani

AU - Chen, Yi-Je

AU - Matthews, Dennis L

AU - Lieu, Deborah

AU - Chiamvimonvat, Nipavan

AU - Lam, Kit

AU - Chen-Izu, Ye

AU - Chan, James W

PY - 2015/12/9

Y1 - 2015/12/9

N2 - RATIONALE:: Cardiomyocyte contraction is caused by Ca binding to troponin C, which triggers the cross-bridge power stroke and myofilament sliding in sarcomeres. Synchronized Ca release causes whole cell contraction and is readily observable with current microscopy techniques. However, it is unknown whether localized Ca release, such as Ca sparks and waves, can cause local sarcomere contraction. Contemporary imaging methods fall short of measuring microdomain Ca-contraction coupling in live cardiac myocytes. OBJECTIVE:: To develop a method for imaging sarcomere-level Ca-contraction coupling in healthy and disease-model cardiac myocytes. METHODS AND RESULTS:: Freshly isolated cardiac myocytes were loaded with the Ca-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. Ca signals and sarcomere strain correlated in space and time with short delays. Furthermore, Ca sparks and waves caused contractions in subcellular microdomains, revealing a previously underappreciated role for these events in generating subcellular strain during diastole. Ca activity and sarcomere strain were also imaged in paced cardiomyocytes under mechanical load, revealing spontaneous Ca waves and correlated local contraction in pressure overload-induced cardiac myopathy. CONCLUSIONS:: Multi-modal SHG-2PF microscopy enables the simultaneous observation of Ca 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 Ca indicators. SHG-2PF imaging is widely applicable to the study of Ca-contraction coupling and mechano-chemo-transduction in both health and disease.

AB - RATIONALE:: Cardiomyocyte contraction is caused by Ca binding to troponin C, which triggers the cross-bridge power stroke and myofilament sliding in sarcomeres. Synchronized Ca release causes whole cell contraction and is readily observable with current microscopy techniques. However, it is unknown whether localized Ca release, such as Ca sparks and waves, can cause local sarcomere contraction. Contemporary imaging methods fall short of measuring microdomain Ca-contraction coupling in live cardiac myocytes. OBJECTIVE:: To develop a method for imaging sarcomere-level Ca-contraction coupling in healthy and disease-model cardiac myocytes. METHODS AND RESULTS:: Freshly isolated cardiac myocytes were loaded with the Ca-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. Ca signals and sarcomere strain correlated in space and time with short delays. Furthermore, Ca sparks and waves caused contractions in subcellular microdomains, revealing a previously underappreciated role for these events in generating subcellular strain during diastole. Ca activity and sarcomere strain were also imaged in paced cardiomyocytes under mechanical load, revealing spontaneous Ca waves and correlated local contraction in pressure overload-induced cardiac myopathy. CONCLUSIONS:: Multi-modal SHG-2PF microscopy enables the simultaneous observation of Ca 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 Ca indicators. SHG-2PF imaging is widely applicable to the study of Ca-contraction coupling and mechano-chemo-transduction in both health and disease.

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