This PhD project investigates how chronic activation of TRPC3 and TRPC6 channels contributes to pathological cardiac remodeling and heart failure. Focusing on angiotensin II–mediated calcium signaling, it examines how disrupted subcellular calcium homeostasis drives hypertrophic and inflammatory gene programs. Combining molecular, electrophysiological, imaging, and in vivo approaches, the study aims to uncover mechanisms linking TRPC signaling to cardiac dysfunction.
Project Details
Background:
Cardiac remodelling encompasses molecular, cellular and gene expression changes following pathologic insults to the heart, impacting cardiac function and patients’ prognosis. In cardiomyocytes, angiotensin II (ATII) signalling directly regulates TRPC3 and TRPC6 channels, and their genetic deletion or pharmacological inhibition protects the heart from ischemia-reperfusion injury and pathological hypertrophy.1 RNA-seq analysis of heart tissue from mice lacking all TRPCs links TRPC genes to contractility and inflammatory pathways.2 The central question of how the cardiomyocyte translates altered TRPC-mediated signalling into deleterious genetic programs that leave it dysfunctional remains unresolved.
Hypothesis and Objectives:
We hypothesize that chronic activation of the ATII-TRPC3/6 axis disrupts subcellular Ca2+ homeostasis, triggering Ca2+-mediated hypertrophic and inflammatory transcription in cardiomyocytes. To test this, we will 1) assess TRPC3/6 expression and activity in cardiomyocytes following chronic ATII exposure, 2) analyze TRPC3/6 levels in human hypertrophied and failing hearts, 3) investigate ATII-TRPC3/6-mediated dysregulation of contractility and Ca2+ cycling across cytoplasmic, nuclear, and mitochondrial compartments, and 4) examine how altered Ca2+ signalling impacts hypertrophic and inflammatory pathways, leading to in vivo cardiac dysfunction.
Methodology:
The PhD student will assess TRPC3/6 expression and subcellular localization in WT, TRPC hepta-KO, TRPC1/2/4/5/6/7 KO and TRPC1/2/3/4/5/7 KO mice challenged with continuous ATII administration, and in hypertrophic or failing human myocardium by qPCR, western blotting of subcellular fractions and immunocytochemistry. TRPC channels activity will be investigated by patch-clamping using pharmacological modulators (activators: GSK1702934A, BI-2; inhibitors: Pyr10). Functional roles of TRPC3/6 in cardiomyocyte contractility and Ca2+ cycling will be assessed by live-cell widefield cellular and confocal subcellular Ca2+ imaging of cardiomyocytes isolated from corresponding groups and following same interventions.3-5 Data on in vivo cardiac function of control and ATII-challenged mice will be assessed by transthoracic echocardiography.3 The effects of altered ATII-TRPC3/6 axis on cellular transcriptome will be addressed by RNAseq and confirmatory protein analyses of cardiomyocyte isolated from corresponding TRPC mice.
References
1. Seo K. et al. Combined TRPC3 and TRPC6 blockade by selective small-molecule or genetic deletion inhibits pathological cardiac hypertrophy. Proc Natl Acad Sci USA. (2014).
doi:10.1073/pnas.1308963111.
2. Formoso K. et al. RNA-seq analysis reveals TRPC genes to impact an unexpected number of metabolic and regulatory pathways. Sci Rep. (2020).
doi:10.1038/s41598-020-61177-x.
3. Ljubojevic-Holzer S. et al. CaMKIIdeltaC Drives Early Adaptive Ca2+ Change and Late Eccentric Cardiac Hypertrophy. Circ Res. (2020).
doi: 10.1161/CIRCRESAHA.120.316947.
4. Ljubojevic-Holzer S. et al. Loss of autophagy protein ATG5 impairs cardiac capacity in mice and humans through diminishing mitochondrial abundance and disrupting Ca2+ cycling. Cardiovasc Res. (2022).
doi: 10.1093/cvr/cvab112.
5. Voglhuber J. et al. Functional remodelling of perinuclear mitochondria alters nucleoplasmic Ca2+ signalling in heart failure. Philos Trans R Soc Lond B Biol Sci. (2022).
doi: 10.1098/rstb.2021.0320.
People Involved
Primary supervisor: Senka Holzer
Collaborators: Oleksandra Tiapko