Tmem65 is critical for the structure and function of the intercalated discs in mouse hearts

Allen C. T. Teng, PhD

Department of Physiology, University of Toronto, Toronto, Ontario, Canada

Synopsis

The intercalated disc (ICD) is a unique membrane structure that is indispensable to normal heart function, yet its structural organization is not completely understood. Previously, we showed that the ICD-bound transmembrane protein 65 (Tmem65) was required for connexin43 (Cx43) localization and function in cultured mouse neonatal cardiomyocytes. Here, we investigate the functional and cellular effects of Tmem65 reductions on the myocardium in a mouse model by injecting CD1 mouse pups (3–7 days after birth) with recombinant adeno-associated virus 9 (rAAV9) harboring Tmem65 shRNA, which reduces Tmem65 expression by 90% in mouse ventricles compared to scrambled shRNA injection. Tmem65 knockdown (KD) results in increased mortality which is accompanied by eccentric hypertrophic cardiomyopathy within 3 weeks of injection and progression to dilated cardiomyopathy with severe cardiac fibrosis by 7 weeks post-injection. Tmem65 KD hearts display depressed hemodynamics as measured echocardiographically as well as slowed conduction in optical recording accompanied by prolonged PR intervals and QRS duration in electrocardiograms. Immunoprecipitation and super-resolution microscopy demonstrate a physical interaction between Tmem65 and sodium channel β subunit (β1) in mouse hearts and this interaction appears to be required for both the establishment of perinexal nanodomain structure and the localization of both voltage-gated sodium channel 1.5 (NaV1.5) and Cx43 to ICDs. Despite the loss of NaV1.5 at ICDs, whole-cell patch clamp electrophysiology did not reveal reductions in Na+ currents but did show reduced Ca2+ and K+ currents in Tmem65 KD cardiomyocytes in comparison to control cells. We conclude that disrupting Tmem65 function results in impaired ICD structure, abnormal cardiac electrophysiology, and ultimately cardiomyopathy.

Biography

I was born a Taiwanese citizen and moved to Canada for education. I received my Bachelor degree in biochemistry (Major) and mathematics (Minor) from Queen’s University, Canada. I then joined Dr. Alexandre F. R. Stewart’s lab (University of Ottawa Heart Institute, Canada) for a PhD training, where I specialized in human cardiovascular genetics and transcription regulation of angiogenesis. After the PhD graduation, I began my Postdoctoral training with Dr. Anthony O. Gramolini at the University of Toronto, Canada. Here, I specialized in cardiac physiology and proteomics. My research interest is in molecular mechanisms that drive cardiac physiology and pathophysiology. My research has been supported by multiple fellowship programs including Heart and Stroke Richard Lewar Fellowship and Ted Rogers Centre for Heart Research Fellowship. I have also received my first peer-reviewed, external funding from the Netherlands Heart Foundation/CURE-PLaN Foundation. Last, my research has received awards/recognition at international meetings, including Paul Dudley White Fellowship (American Heart Association, Basic Cardiovascular Research Council).

Epitranscriptomic regulations in heart development and disease

RNDr. Markéta Hlaváčková, PhD

Laboratory of Developmental Cardiology, Institute of Physiology of the Czech Academy of Sciences (IPHYS)

Synopsis

The presentation will introduce the proposed future research program of The Laboratory of Developmental Cardiology aiming to focus primarily on understanding the molecular, genetic, and cellular mechanisms involved in i) the initiation and progression of cardiomyopathy and heart failure, ii) the mechanisms of increased cardiac tolerance to oxygen deprivation, and iii) the mechanisms underlying congenital heart diseases. The suggested research direction integrates these objectives by emphasizing the role of epitranscriptomic (RNA epigenetic) regulations and the significance of hypoxia-inducible factor-1α (Hif-1α) in these mechanisms. Proposed research directions build on the laboratory's long-standing extensive expertise while addressing critical contemporary clinical challenges such as the pervasive occurrence of heart failure and introducing innovative research approaches and technologies.

Epitranscriptomics, also known as "RNA epigenetics," represent early-stage regulatory mechanisms controlling gene expression, akin to DNA methylation or histone modification. These RNA modifications, a recent area of study, have been shown to play a crucial role in the development of various diseases. In the context of cardiovascular diseases, the significance of these modifications is only beginning to be understood. One of the most common modifications to eukaryotic mRNA is N6-methyladenosine (m6A), which can also undergo methylation at the 2'-O position, forming N6,2'O-dimethyladenosine (m6Am). These modifications are highly dynamic and reversible, representing a new layer of genetic information control impacting mRNA stability, translation, and splicing processes, thus influencing cellular homeostasis and disease progression. In this talk, I will present the results regarding the involvement of m6A and m6Am epitranscriptomic regulatory mechanisms in nonconventional cardioprotective interventions (adaptation to chronic hypoxia and fasting). Our findings indicate that both mentioned cardioprotective interventions affect the levels of m6A and m6Am regulators in the heart, with differing patterns of change. Furthermore, inhibition of RNA-demethylase FTO in control cardiomyocytes increased metabolic rates and decreased the tolerance of cardiomyocytes to oxygen deprivation. Proteomic data analysis revealed that the most significant changes induced by FTO inhibition in oxygen-deprived cardiomyocytes are related to the autophagy and mTOR pathway. Our data underscore the crucial role of FTO in regulating cardiomyocyte physiology and support the potential involvement of epitranscriptomic regulations in cardioprotective mechanisms.

Biography

Markéta Hlaváčková, PhD is a Principal investigator in the Laboratory of Developmental Cardiology at the Institute of Physiology of the Czech Academy of Sciences. She earned her PhD from Faculty of Science, Charles University in Prague, specializing in biochemistry. Her primary research focus lies in investigating the intricate mechanisms underlying cardioprotective interventions and regulations in heart disease. More recently, she became interested in the role of epitranscriptomic (RNA epigenetic) regulations in heart development and disease. During her postdoctoral tenure at the Institute of Cardiovascular Sciences (St. Boniface Hospital Research, Winnipeg, Manitoba, Canada) and the Centre for Metabolic and Vascular Biology (Arizona State University, Arizona, USA), Dr. Hlaváčková concentrated on exploring the role of stress responses in vascular remodeling. Additionally, she delved into investigating the cellular and molecular mechanisms involved in cell fusion under both physiological and pathophysiological conditions, particularly in contexts such as atherosclerosis. Dr. Hlaváčková has published 26 original articles in esteemed scientific journals with impact factor and 2 book chapters (H-index 13 (WOS)). Her research has been supported by the Manitoba Health Research Council Postdoctoral Fellowship Award. Moreover, she has been the Principal investigator of one senior and two junior grants from the Czech Science Foundation and three grants from the Grant Agency of Charles University. She served as Member of Management Committee in EU-CardioRNA COST Action (CA17129) and as a Substitute Member of Management Committee in EU-Cardioprotection COST Actions (CA16225).