Palmitoylation of the pore-forming subunit of Ca(v)1.2 controls channel voltage sensitivity and calcium transients in cardiac myocytes

Mammalian voltage-activated L-type Ca2+ channels, such as Ca(v)1.2, control transmem- brane Ca2+ fluxes in numerous excitable tissues. Here, we report that the pore-forming α1C subunit of Ca(v)1.2 is reversibly palmitoylated in rat, rabbit, and human ventricular myocytes. We map the palmitoylation sites to two regions of the channel: The N termi- nus and the linker between domains I and II. Whole-cell voltage clamping revealed a rightward shift of the Ca(v)1.2 current–voltage relationship when α1C was not palmi- toylated. To examine function, we expressed dihydropyridine-resistant α1C in human induced pluripotent stem cell-derived cardiomyocytes and measured Ca2+ transients in the presence of nifedipine to block the endogenous channels. The transients generated by unpalmitoylatable channels displayed a similar activation time course but signifi- cantly reduced amplitude compared to those generated by wild-type channels. We thus conclude that palmitoylation controls the voltage sensitivity of Ca(v)1.2. Given that the identified Ca(v)1.2 palmitoylation sites are also conserved in most Ca(v)1 isoforms, we propose that palmitoylation of the pore-forming α1C subunit provides a means to regulate the voltage sensitivity of voltage-activated Ca 2+ channels in excitable cells

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

The role of the small conductance calcium-activated potassium channel in the rabbit atrial and ventricular action potential

Background: Small conductance potassium channels (SK) channels have been shown to participate specifically in atrial repolarisation, modulating action potential duration (APD). Atrial arrhythmias, including AF have been associated with overexpression of SK channels where they are thought to contribute to APD abnormalities. Their role in the ventricular electrophysiology remains controversial with limited evidence of their function. Purpose: This study aimed to reveal the role of the SK channel in the AP of atrial and ventricular muscle in healthy and AF susceptible hearts. Additionally, the sensitivity of the SK channel to [Ca2+]i was investigated using low extracellular [Ca2+] and intracellular [Ca2+]-buffers. Methods: Electrophysiological measurements of APs and [Ca2+] were made using optical techniques in enzymatically digested cardiomyocytes and isolated rabbit hearts. The specific SK channel blocker, ICAGEN was used to investigate the contribution of the current to the APD. [Ca2+]i was altered in two ways: (1) lowering extracellular [Ca2+] to 0.3mM and (2) chelating intracellular Ca2+ by incubating in 20µM BAPTA-AM. Results: Pharmacological block of SK channels in tissue preparations caused a significant increase in APD by ~30% and 40% prolongation of APD50 and APD75/90 respectively at 10min drug perfusion in atrial muscle. No response to ICAGEN was observed in the ventricle. Atrial APs in low extracellular [Ca2+] retained a significant ICAGEN-sensitivity causing comparable prolongation to that seen in 1.8mM [Ca2+]. However, buffering [Ca2+]i with BAPTA abolished the drug effect with no significant APD prolongation in either cells or tissue. No APD prolongation occurred in response to ICAGEN in both the atrium and ventricle of AF-susceptible hearts from a heart failure (HF) model. Conclusion: Using ICAGEN as a tool to probe SK channel activity, the data suggests that SK channels have an atrial-specific function in repolarisation in the normal rabbit. From the response to BAPTA loading, the channels appeared sensitive to local (possibly dyadic) [Ca2+] rather than global [Ca2+]i. The role of the channel in disease remains unclear with no evident contribution to AP repolarisation in an AF-susceptible rabbit model

Palmitoylation of the pore-forming subunit of Ca(v)1.2 controls channel voltage sensitivity and calcium transients in cardiac myocytes

This data set contains data used to form the basis of a scientific publication. Microsoft Excel files contain: • Quantitative data from analysis of images of western immunoblots • Quantitative data from analysis of electrophysiological recordings of current records • Quantitative data from analysis of intracellular calcium concentrations in cardiac myocytes • Quantitative modelling of action potentials and calcium transients Microsoft Powerpoint files contain original uncropped images of western immunoblots Python code for a model of cardiac calcium handling and electrophysiology, implemented in the myokit ide

SUMOylation does not affect cardiac troponin I stability but alters indirectly the development of force in response to Ca2+

Post-translational modification of the myofilament protein troponin I by phosphorylation is known to trigger functional changes that support enhanced contraction and relaxation of the heart. We report for the first time that human troponin I can also be modified by SUMOylation at lysine 177. Functionally, TnI SUMOylation is not a factor in the development of passive and maximal force generation in response to calcium, however this modification seems to act indirectly by preventing SUMOylation of other myofilament proteins to alter calcium sensitivity and cooperativity of myofilaments. Utilising a novel, custom SUMO site-specific antibody that recognises only the SUMOylated form of troponin I, we verify that this modification occurs in human heart and that it is upregulated during disease