Calcium Signaling and Contractility in Cardiac Myocyte of Wolframin Deficient Rats

Wolframin (Wfs1) is a membrane protein of the sarco/endoplasmic reticulum. Wfs1 mutations are responsible for the Wolfram syndrome, characterized by diabetic and neurological symptoms. Although Wfs1 is expressed in cardiac muscle, its role in this tissue is not clear. We have characterized the effect of invalidation of Wfs1 on calcium signaling-related processes in isolated ventricular myocytes of exon5-Wfs1 deficient rats (Wfs1-e5/-e5) before the onset of overt disease. Calcium transients and contraction were measured in field-stimulated isolated myocytes using confocal microscopy with calcium indicator fluo-3 AM and sarcomere length detection. Calcium currents and their calcium release-dependent inactivation were characterized in whole-cell patch-clamp experiments. At 4 months, Wfs1-e5/-e5 animals were euglycemic, and echocardiographic examination revealed fully compensated cardiac function. In field-stimulated isolated ventricular myocytes, both the amplitude and the duration of contraction of Wfs1-e5/-e5 animals were elevated relative to control Wfs1+/+ littermates. Increased contractility of myocytes resulted largely from prolonged cytosolic calcium transients. Neither the amplitude of calcium currents nor their voltage dependence of activation differed between the two groups. Calcium currents in Wfs1-e5/-e5 myocytes showed a larger extent of inactivation by short voltage prepulses applied to selectively induce calcium release-dependent inactivation of calcium current. Neither the calcium content of the sarcoplasmic reticulum, measured by application of 20 mmol/l caffeine, nor the expression of SERCA2, determined from Western blots, differed significantly in myocytes of Wfs1-e5/-e5 animals compared to control ones. These experiments point to increased duration of calcium release in ventricular myocytes of Wfs1-e5/-e5 animals. We speculate that the lack of functional wolframin might cause changes leading to upregulation of RyR2 channels resulting in prolongation of channel openings and/or a delay in termination of calcium release

SARS-CoV-2 Exploits Non-Canonical Autophagic Processes to Replicate, Mature, and Egress the Infected Vero E6 Cells

The coronavirus transforms the cytoplasm of susceptible cells to support virus replication. It also activates autophagy-like processes, the role of which is not well understood. Here, we studied SARS-CoV-2-infected Vero E6 cells using transmission electron microscopy and autophagy PCR array. After 6–24 h post-infection (hpi), the cytoplasm of infected cells only contained double-membrane vesicles, phagophores, and phagosomes engulfing virus particles and cytoplasmic debris, including damaged mitochondria. The phagosomes interacted with the viral nucleoprotein complex, virus particles, mitochondria, and lipid droplets. The phagosomes transformed into egress vacuoles, which broke through the plasmalemma and discharged the virus particles. The Vero E6 cells exhibited pronounced virus replication at 6 hpi, which stabilized at 18–24 hpi at a high level. The autophagy PCR array tests revealed a significant upregulation of 10 and downregulation of 8 autophagic gene markers out of 84. Altogether, these results underline the importance of autophagy-like processes for SARS-CoV-2 maturation and egress, and point to deviations from a canonical autophagy response