Measurement of myofilament-localised calcium dynamics in adult cardiomyocytes and the effect of hypertrophic cardiomyopathy mutations
<p><strong>Rationale:</strong> Subcellular Ca2+ indicators have yet to be developed for the myofilament where disease mutation, or small molecules may alter contractility through myofilament Ca2+ sensitivity. Here we develop and characterise genetically encoded Ca2+ indicators rest...
| Main Authors: | , , , , , , , , , , |
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| Format: | Journal article |
| Language: | English |
| Published: |
American Heart Association
2019
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| _version_ | 1797099437169836032 |
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| author | Sparrow, A Sievert, K Patel, S Chang, Y Broyles, C Brook, F Watkins, H Geeves, M Redwood, C Robinson, P Daniels, M |
| author_facet | Sparrow, A Sievert, K Patel, S Chang, Y Broyles, C Brook, F Watkins, H Geeves, M Redwood, C Robinson, P Daniels, M |
| author_sort | Sparrow, A |
| collection | OXFORD |
| description | <p><strong>Rationale:</strong> Subcellular Ca2+ indicators have yet to be developed for the myofilament where disease mutation, or small molecules may alter contractility through myofilament Ca2+ sensitivity. Here we develop and characterise genetically encoded Ca2+ indicators restricted to the myofilament to directly visualise Ca2 changes in the sarcomere.</p> <p><strong>Objective:</strong> To produce and validate myofilament restricted Ca2+ imaging probes in an adenoviral transduction adult cardiomyocyte model using drugs that alter myofilament function (MYK-461, omecamtiv mecarbil and levosimendan) or following co-transduction of two established hypertrophic cardiomyopathy (HCM) disease causing mutants (cTnT R92Q and cTnI R145G) that alter myofilament Ca2+ handling.</p> <p><strong>Methods and Results:</strong> When expressed in adult ventricular cardiomyocytes RGECO-TnT/TnI sensors localise correctly to the sarcomere without contractile impairment. Both sensors report cyclical changes in fluorescence in paced cardiomyocytes with reduced Ca2+ on and increased Ca2+ off rates compared with unconjugated RGECO. RGECO-TnT/TnI revealed changes to localised Ca2+ handling conferred by MYK-461 and levosimendan, including an increase in Ca2+ binding rates with both levosimendan and MYK-461 not detected by an unrestricted protein sensor. Co-adenoviral transduction of RGECO-TnT/TnI with HCM causing thin filament mutants showed that the mutations increase myofilament [Ca2+] in systole, lengthen time to peak systolic [Ca2+], and delay [Ca2+] release. This contrasts with the effect of the same mutations on cytoplasmic Ca2+, when measured using unrestricted RGECO where changes to peak systolic Ca2+ are inconsistent between the two mutations. These data contrast with previous findings using chemical dyes that show no alteration of [Ca2+] transient amplitude or time to peak Ca2+.</p> <p><strong>Conclusions:</strong> RGECO-TnT/TnI are functionally equivalent. They visualise Ca2+ within the myofilament and reveal unrecognised aspects of small molecule and disease associated mutations in living cells.</p> |
| first_indexed | 2024-03-07T05:23:42Z |
| format | Journal article |
| id | oxford-uuid:dfd004fd-2311-4286-abd6-123e14e33d79 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T05:23:42Z |
| publishDate | 2019 |
| publisher | American Heart Association |
| record_format | dspace |
| spelling | oxford-uuid:dfd004fd-2311-4286-abd6-123e14e33d792022-03-27T09:42:07ZMeasurement of myofilament-localised calcium dynamics in adult cardiomyocytes and the effect of hypertrophic cardiomyopathy mutationsJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:dfd004fd-2311-4286-abd6-123e14e33d79EnglishSymplectic Elements at OxfordAmerican Heart Association2019Sparrow, ASievert, KPatel, SChang, YBroyles, CBrook, FWatkins, HGeeves, MRedwood, CRobinson, PDaniels, M<p><strong>Rationale:</strong> Subcellular Ca2+ indicators have yet to be developed for the myofilament where disease mutation, or small molecules may alter contractility through myofilament Ca2+ sensitivity. Here we develop and characterise genetically encoded Ca2+ indicators restricted to the myofilament to directly visualise Ca2 changes in the sarcomere.</p> <p><strong>Objective:</strong> To produce and validate myofilament restricted Ca2+ imaging probes in an adenoviral transduction adult cardiomyocyte model using drugs that alter myofilament function (MYK-461, omecamtiv mecarbil and levosimendan) or following co-transduction of two established hypertrophic cardiomyopathy (HCM) disease causing mutants (cTnT R92Q and cTnI R145G) that alter myofilament Ca2+ handling.</p> <p><strong>Methods and Results:</strong> When expressed in adult ventricular cardiomyocytes RGECO-TnT/TnI sensors localise correctly to the sarcomere without contractile impairment. Both sensors report cyclical changes in fluorescence in paced cardiomyocytes with reduced Ca2+ on and increased Ca2+ off rates compared with unconjugated RGECO. RGECO-TnT/TnI revealed changes to localised Ca2+ handling conferred by MYK-461 and levosimendan, including an increase in Ca2+ binding rates with both levosimendan and MYK-461 not detected by an unrestricted protein sensor. Co-adenoviral transduction of RGECO-TnT/TnI with HCM causing thin filament mutants showed that the mutations increase myofilament [Ca2+] in systole, lengthen time to peak systolic [Ca2+], and delay [Ca2+] release. This contrasts with the effect of the same mutations on cytoplasmic Ca2+, when measured using unrestricted RGECO where changes to peak systolic Ca2+ are inconsistent between the two mutations. These data contrast with previous findings using chemical dyes that show no alteration of [Ca2+] transient amplitude or time to peak Ca2+.</p> <p><strong>Conclusions:</strong> RGECO-TnT/TnI are functionally equivalent. They visualise Ca2+ within the myofilament and reveal unrecognised aspects of small molecule and disease associated mutations in living cells.</p> |
| spellingShingle | Sparrow, A Sievert, K Patel, S Chang, Y Broyles, C Brook, F Watkins, H Geeves, M Redwood, C Robinson, P Daniels, M Measurement of myofilament-localised calcium dynamics in adult cardiomyocytes and the effect of hypertrophic cardiomyopathy mutations |
| title | Measurement of myofilament-localised calcium dynamics in adult cardiomyocytes and the effect of hypertrophic cardiomyopathy mutations |
| title_full | Measurement of myofilament-localised calcium dynamics in adult cardiomyocytes and the effect of hypertrophic cardiomyopathy mutations |
| title_fullStr | Measurement of myofilament-localised calcium dynamics in adult cardiomyocytes and the effect of hypertrophic cardiomyopathy mutations |
| title_full_unstemmed | Measurement of myofilament-localised calcium dynamics in adult cardiomyocytes and the effect of hypertrophic cardiomyopathy mutations |
| title_short | Measurement of myofilament-localised calcium dynamics in adult cardiomyocytes and the effect of hypertrophic cardiomyopathy mutations |
| title_sort | measurement of myofilament localised calcium dynamics in adult cardiomyocytes and the effect of hypertrophic cardiomyopathy mutations |
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