Nano-imaging of the beating mouse heart in vivo: Importance of sarcomere dynamics, as opposed to sarcomere length per se, in the regulation of cardiac function

Kobirumaki-Shimozawa, Fuyu; Oyama, Kotaro; Togo, Shinji; Mizuno, Akari; Ohki, Takashi; Terui, Takako; Minamisawa, Susumu; Ishiwata, Shin’ichi; Fukuda, Norio

doi:10.1085/jgp.201511484

Cited by 54 publications

(107 citation statements)

References 36 publications

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“…An ADV injection of 10 μ L at 2 × 10 11 or 4 × 10 11 VP/mL into the epicardial surface effectively expressed AcGFP in the Z-disks ( Figures 1(a) and 1(b) ), with fraction of the AcGFP-expressing myocytes at ~10% in the injected region (~3 × 3 mm 2 ). SL was 1.91±0.23 and 2.15±0.19 μ m with 2 × 10 11 and 4 × 10 11 particles per mL ( Table 1 ), respectively, both of which, measured in vivo , are consistent with the resting value of 1.97±0.20 μ m obtained in our previous study in the isolated mouse heart [6]. An increase in the viral titer to 2 × 10 12 particles per mL increased the fraction of the AcGFP-expressing myocytes to ~20-30%; however, this caused marked shortening of sarcomeres ( Figure 1(c); Table 1 ).…”

Section: Resultssupporting

confidence: 88%

“…A superposition of the images in the same regions derived the T-tubular distance value as ~2.20 μ m in both myocytes (see Figure 3 legend). It should be noted that SL and the T-tubular distance are similar ( Figures 1(a), 1(b), 2(a), and 3(e) ), close to the resting SL at the LV center in the isolated heart from our previous study [6]. Likewise, these values are in good agreement with those obtained by others [14] in epicardial myocytes in the LV of Langendorff perfused isolated mouse heart.…”

Section: Discussionsupporting

confidence: 89%

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Optimization of Fluorescent Labeling for In Vivo Nanoimaging of Sarcomeres in the Mouse Heart

Kobirumaki-Shimozawa

Togo

Oyama

et al. 2018

BioMed Research International

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The present study was conducted to systematically investigate the optimal viral titer as well as the volume of the adenovirus vector (ADV) that expresses α-actinin-AcGFP in the Z-disks of myocytes in the left ventricle (LV) of mice. An injection of 10 μL ADV at viral titers of 2 to 4 × 1011 viral particles per mL (VP/mL) into the LV epicardial surface consistently expressed α-actinin-AcGFP in myocytes in vivo, with the fraction of AcGFP-expressing myocytes at ~10%. Our analysis revealed that SL was ~1.90-2.15 μm upon heart arrest via deep anesthesia. Likewise, we developed a novel fluorescence labeling method of the T-tubular system by treating the LV surface with CellMask Orange (CellMask). We found that the T-tubular distance was ~2.10-2.25 μm, similar to SL, in the healthy heart in vivo. Therefore, the present high-precision visualization method for the Z-disks or the T-tubules is beneficial to unveiling the mechanisms of myocyte contraction in health and disease in vivo.

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Section: Resultssupporting

confidence: 88%

Section: Discussionsupporting

confidence: 89%

Optimization of Fluorescent Labeling for In Vivo Nanoimaging of Sarcomeres in the Mouse Heart

Kobirumaki-Shimozawa

Togo

Oyama

et al. 2018

BioMed Research International

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“…Using single molecule spring element characteristics we simulated the force produced by N2B titin and N2BAsc as a function of sarcomere length (SL) and converted this to a passive stiffness-SL relation. We found that the stiffness reduction that results from the titin spring element exon upregulation is large (Figure S6) and is 89% at a SL of 2.15 µm (selected because this is likely a physiological SL as is suggested by work on hearts fixed in diastole 8 and the elegant work of Kobirumaki-Shimozawa and colleagues who imaged sarcomere length in vivo in the living animal 36 ). Since in the heart not all N2B titin is converted to N2BAsc but only 45% (calculated from Table S1, at 3 wk time point) the predicted stiffness reduction is 40%.…”

Section: Discussionmentioning

confidence: 94%

Experimentally Increasing the Compliance of Titin Through RNA Binding Motif-20 (RBM20) Inhibition Improves Diastolic Function In a Mouse Model of Heart Failure With Preserved Ejection Fraction

et al. 2016

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Background Left Ventricular (LV) stiffening contributes to Heart Failure with preserved Ejection Fraction (HFpEF), a syndrome with no effective treatment options. Increasing titin’s compliance in the heart has become possible recently through inhibition of the splicing factor RBM20. Here we investigated the effects of increasing titin’s compliance in mice with diastolic dysfunction. Methods Mice in which the RNA recognition motif (RRM) of one of the RBM20 alleles was floxed and which expressed the MerCreMer transgene under control of the αMHC promoter (referred to as cRbm20ΔRRM mice) were used. Mice underwent transverse aortic constriction (TAC) surgery and deoxycorticosterone acetate (DOCA) pellet implantation (TAC/DOCA). RRM deletion in adult mice was triggered by injecting raloxifene (cRbm20ΔRRM-raloxifene) with DMSO injected mice (cRbm20ΔRRM-DMSO) as the control. Diastolic function was investigated using echocardiography and pressure volume analysis; passive stiffness was studied in LV muscle strips and isolated cardiac myocytes before and after elimination of titin-based stiffness. Treadmill exercise performance was also studied. Titin isoform expression was evaluated with Agarose gels. Results cRbm20ΔRRM-raloxifene mice expressed large titins in the hearts, named super compliant titin (N2BAsc), which, within 3 weeks after raloxifene injection, made up ~45% of total titin. TAC/DOCA cRbm20ΔRRM-DMSO mice developed LV hypertrophy and a marked increase in LV chamber stiffness as shown by both pressure-volume analysis and echocardiography. LV chamber stiffness was normalized in TAC/DOCA cRbm20ΔRRM-raloxifene mice that expressed N2BAsc. Passive stiffness measurements on muscle strips isolated from the LV free wall revealed that extracellular matrix (ECM) stiffness was equally increased in both groups of TAC/DOCA mice (cRbm20ΔRRM-DMSO and cRbm20ΔRRM-raloxifene). However, titin-based muscle stiffness was reduced in the mice that expressed N2BAsc (TAC/DOCA cRbm20ΔRRM-raloxifene). Exercise testing demonstrated significant improvement in exercise tolerance in TAC/DOCA mice that expressed N2BAsc. Conclusions Inhibition of the RBM20-based titin splicing system upregulates compliant titins, which improves diastolic function and exercise tolerance in the TAC/DOCA model. Titin holds promise as a therapeutic target for HFpEF.

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“…The dynamics of the change in sarcomere length (not overall length per se) translate into myocardial function [15]. It is wellestablished that small changes in sarcomeric (and cellular) fractional shortening translate into larger changes in heart wall motion, and a primary effect of Rad −/− is to enhance myocardial Ca 2+ homeostasis that in turn drives sarcomere level function.…”

Section: Resultsmentioning

confidence: 99%

Rad-deletion Phenocopies Tonic Sympathetic Stimulation of the Heart

Levitan

Manning

Withers

et al. 2016

J. of Cardiovasc. Trans. Res.

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Sympathetic stimulation modulates L-type calcium channel (LTCC) gating to contribute to increased systolic heart function. Rad is a monomeric G-protein that interacts with LTCC. Genetic deletion of Rad (Rad−/−) renders LTCC in a sympathomimetic state. The study goal was to use a clinically inspired pharmacological stress echocardiography test, including analysis of global strain, to determine whether Rad−/− confers tonic positive inotropic heart function. Sarcomere dynamics and strain showed partial parallel isoproterenol (ISO) responsiveness for wild-type (WT) and for Rad−/−. Rad−/− basal inotropy was elevated compared to WT but was less responsiveness to ISO. Rad protein levels were lower in human patients with end-stage non-ischemic heart failure. These results show that Rad reduction provides a stable inotropic response rooted in sarcomere level function. Thus, reduced Rad levels in heart failure patients may be a compensatory response to need for increased output in the setting of HF. Rad deletion suggests a future therapeutic direction for inotropic support.

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Nano-imaging of the beating mouse heart in vivo: Importance of sarcomere dynamics, as opposed to sarcomere length per se, in the regulation of cardiac function

Abstract: ¡Vive la différence! In cardiac contraction, the reduction in sarcomere length—rather than length itself—determines contractile force.

Cited by 54 publications

References 36 publications

Optimization of Fluorescent Labeling for In Vivo Nanoimaging of Sarcomeres in the Mouse Heart

Optimization of Fluorescent Labeling for In Vivo Nanoimaging of Sarcomeres in the Mouse Heart

Experimentally Increasing the Compliance of Titin Through RNA Binding Motif-20 (RBM20) Inhibition Improves Diastolic Function In a Mouse Model of Heart Failure With Preserved Ejection Fraction

Rad-deletion Phenocopies Tonic Sympathetic Stimulation of the Heart

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