Regulation of Cardiac Signaling and Excitability

Background Sinoatrial node (SAN), the primary pacemaker region in the heart, generates electrical impulses that propagates throughout the heart. SAN activity is tightly regulated by β-adrenergic receptor (β-AR) signaling with adenylyl cyclase (AC) as a key enzyme. However, the main isoform of AC and its functional roles in regulating SAN function remain incompletely understood. SAN dysfunction is well documented in patients with heart failure (HF), but the underlying mechanisms are not fully determined. Additionally, the critical roles of endoplasmic reticulum (ER)-associated degradation (ERAD) in the regulation of cardiac ion channel function in health and disease are only beginning to be recognized. To this end, we have identified several critical knowledge gaps in our current understanding of the regulation of cardiac excitability in health and disease that form the basis of my dissertation work. Specifically, we tested the hypotheses that distinct AC isoforms are preferentially expressed in the SAN and compartmentalize within critical microdomains to orchestrate heart rate regulation during β-AR signaling (Chapter 2); mitochondrial dysfunction in the SAN in HF directly contribute to SAN dysfunction (Chapter 3); RNF207 regulates human Ether-à-go-go-Related Gene (hERG) K+ channel through ER-associated degradation (Chapter 4); different human calmodulin (CaM, a ubiquitous Ca2+ sensing protein) mutations, linked to long QT syndrome (LQTS), a hereditary disease that predisposes patients to life-threatening cardiac arrhythmias and sudden cardiac death, disrupt small conductance Ca2+-activated K+ channel (SK) channel function by distinct mechanisms (Chapter 5). Methods To test the hypotheses, we utilized multi-disciplinary approaches, from electrophysiology, cell biology, high resolution imaging to in vivo analysis. Specifically, perforated patch-clamp, whole-cell voltage clamp recordings, single-cell RT-qPCR, co-immunoprecipitation, proximity ligation assay, immunofluorescence through confocal microscopy with high resolution Airycan mode, stimulated emission depletion (STED) super-resolution microscopy, fluorescence resonance energy transfer (FRET) imaging, whole-cell Ca2+ transient and sarcomere shortening measurements, confocal line scanning, echocardiography and electrocardiography (ECG) telemetry were used. Results We demonstrate that Ca2+-activated ACI is the predominant isoform in SAN that resides in a functional microdomain with caveolin-3, hyperpolarization-activated cyclic nucleotide-gated (HCN)4 channel, voltage-gated Ca2+ channel 1.2 (Cav1.2), and ryanodine receptor 2 (RyR2), and is critical in the sustained rise in local cAMP during β-AR stimulation (Chapter 2); impairment of mitochondria and their microdomain in the SAN contribute to SAN dysfunction in HF (Chapter 3); RNF207 serves an E3 ubiquitin ligase and targets misfolded hERGT613M mutant proteins for degradation (Chapter 4); CaMD96V and CaMD130G mutations linked to LQTS reduce SK currents through a dominant-negative fashion, while specific mutations of phenylalanine to leucine result in conformational changes that affect helix packing in the C-lobe of CaM (Chapter 5). Conclusions The cumulative work during my graduate studies has elucidated key mechanisms to help understand cardiac function in normal condition and has demonstrated key mechanisms that may be exploited in disease. These pivotal findings establish a foundation for future work in cardiac physiology and pathophysiology