Ischemic heart injury leads to HIF1-dependent differential splicing of CaMK2γ

Abstract Ischemic heart disease is a leading cause of heart failure and hypoxia inducible factor 1 (HIF1) is a key transcription factor in the response to hypoxic injury. Our lab has developed a mouse model in which a mutated, oxygen-stable form of HIF1α (HIF-PPN) can be inducibly expressed in cardi...

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Autores principales:	Allison Lesher Williams, Chad B. Walton, Blake Pinell, Vedbar S. Khadka, Brandyn Dunn, Katie Lee, M. C. Therese Anagaran, Abigail Avelar, Ralph V. Shohet
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/3358c57a1c804a2fa84edf3e2217d1eb
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spelling	oai:doaj.org-article:3358c57a1c804a2fa84edf3e2217d1eb2021-12-02T17:12:17ZIschemic heart injury leads to HIF1-dependent differential splicing of CaMK2γ10.1038/s41598-021-92426-22045-2322https://doaj.org/article/3358c57a1c804a2fa84edf3e2217d1eb2021-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-92426-2https://doaj.org/toc/2045-2322Abstract Ischemic heart disease is a leading cause of heart failure and hypoxia inducible factor 1 (HIF1) is a key transcription factor in the response to hypoxic injury. Our lab has developed a mouse model in which a mutated, oxygen-stable form of HIF1α (HIF-PPN) can be inducibly expressed in cardiomyocytes. We observed rapid cardiac dilation and loss of contractility in these mice due to lower expression of excitation–contraction coupling genes and reduced calcium flux. As alternative splicing plays an underappreciated role in transcriptional regulation, we used RNA sequencing to search for splicing changes in calcium-handling genes of HIF-PPN hearts and compared them to previous sequencing data from a model of myocardial infarction (MI) to select for transcripts that are modified in a pathological setting. We found overlap between genes differentially expressed in HIF-PPN and post-MI mice (54/131 genes upregulated in HIF-PPN hearts at 1 day and/or 3 days post-MI, and 45/78 downregulated), as well as changes in alternative splicing. Interestingly, calcium/calmodulin dependent protein kinase II, gamma (CAMK2G) was alternatively spliced in both settings, with variant 1 (v1) substantially decreased compared to variants 2 (v2) and 3 (v3). These findings were also replicated in vitro when cells were transfected with HIF-PPN or exposed to hypoxia. Further analysis of CAMK2γ protein abundance revealed only v1 was detectable and substantially decreased up to 7 days post-MI. Rbfox1, a splicing factor of CAMK2G, was also decreased in HIF-PPN and post-MI hearts. Subcellular fractionation showed CAMK2γ v1 was found in the nuclear and cytoplasmic fractions, and abundance decreased in both fractions post-MI. Chromatin immunoprecipitation analysis of HIF1 in post-MI hearts also demonstrated direct HIF1 binding to CAMK2G. CaMK2 is a key transducer of calcium signals in both physiological and pathological settings. The predominantly expressed isoform in the heart, CaMK2δ, has been extensively studied in cardiac injury, but the specific role of CaMK2γ is not well defined. Our data suggest that loss of CaMK2γ after MI is HIF1-dependent and may play an important role in the heart’s calcium signaling and transcriptional response to hypoxia.Allison Lesher WilliamsChad B. WaltonBlake PinellVedbar S. KhadkaBrandyn DunnKatie LeeM. C. Therese AnagaranAbigail AvelarRalph V. ShohetNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-14 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Allison Lesher Williams Chad B. Walton Blake Pinell Vedbar S. Khadka Brandyn Dunn Katie Lee M. C. Therese Anagaran Abigail Avelar Ralph V. Shohet Ischemic heart injury leads to HIF1-dependent differential splicing of CaMK2γ
description	Abstract Ischemic heart disease is a leading cause of heart failure and hypoxia inducible factor 1 (HIF1) is a key transcription factor in the response to hypoxic injury. Our lab has developed a mouse model in which a mutated, oxygen-stable form of HIF1α (HIF-PPN) can be inducibly expressed in cardiomyocytes. We observed rapid cardiac dilation and loss of contractility in these mice due to lower expression of excitation–contraction coupling genes and reduced calcium flux. As alternative splicing plays an underappreciated role in transcriptional regulation, we used RNA sequencing to search for splicing changes in calcium-handling genes of HIF-PPN hearts and compared them to previous sequencing data from a model of myocardial infarction (MI) to select for transcripts that are modified in a pathological setting. We found overlap between genes differentially expressed in HIF-PPN and post-MI mice (54/131 genes upregulated in HIF-PPN hearts at 1 day and/or 3 days post-MI, and 45/78 downregulated), as well as changes in alternative splicing. Interestingly, calcium/calmodulin dependent protein kinase II, gamma (CAMK2G) was alternatively spliced in both settings, with variant 1 (v1) substantially decreased compared to variants 2 (v2) and 3 (v3). These findings were also replicated in vitro when cells were transfected with HIF-PPN or exposed to hypoxia. Further analysis of CAMK2γ protein abundance revealed only v1 was detectable and substantially decreased up to 7 days post-MI. Rbfox1, a splicing factor of CAMK2G, was also decreased in HIF-PPN and post-MI hearts. Subcellular fractionation showed CAMK2γ v1 was found in the nuclear and cytoplasmic fractions, and abundance decreased in both fractions post-MI. Chromatin immunoprecipitation analysis of HIF1 in post-MI hearts also demonstrated direct HIF1 binding to CAMK2G. CaMK2 is a key transducer of calcium signals in both physiological and pathological settings. The predominantly expressed isoform in the heart, CaMK2δ, has been extensively studied in cardiac injury, but the specific role of CaMK2γ is not well defined. Our data suggest that loss of CaMK2γ after MI is HIF1-dependent and may play an important role in the heart’s calcium signaling and transcriptional response to hypoxia.
format	article
author	Allison Lesher Williams Chad B. Walton Blake Pinell Vedbar S. Khadka Brandyn Dunn Katie Lee M. C. Therese Anagaran Abigail Avelar Ralph V. Shohet
author_facet	Allison Lesher Williams Chad B. Walton Blake Pinell Vedbar S. Khadka Brandyn Dunn Katie Lee M. C. Therese Anagaran Abigail Avelar Ralph V. Shohet
author_sort	Allison Lesher Williams
title	Ischemic heart injury leads to HIF1-dependent differential splicing of CaMK2γ
title_short	Ischemic heart injury leads to HIF1-dependent differential splicing of CaMK2γ
title_full	Ischemic heart injury leads to HIF1-dependent differential splicing of CaMK2γ
title_fullStr	Ischemic heart injury leads to HIF1-dependent differential splicing of CaMK2γ
title_full_unstemmed	Ischemic heart injury leads to HIF1-dependent differential splicing of CaMK2γ
title_sort	ischemic heart injury leads to hif1-dependent differential splicing of camk2γ
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/3358c57a1c804a2fa84edf3e2217d1eb
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_version_	1718381426270797824

Ischemic heart injury leads to HIF1-dependent differential splicing of CaMK2γ

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