Caveolae in Rabbit Ventricular Myocytes: Distribution and Dynamic Diminution after Cell Isolation

Abstract

Caveolae are signal transduction centers, yet their subcellular distribution and preservation in cardiac myocytes after cell isolation are not well documented. Here, we quantify caveolae located within 100 nm of the outer cell surface membrane in rabbit single-ventricular cardiomyocytes over 8 h post-isolation and relate this to the presence of caveolae in intact tissue. Hearts from New Zealand white rabbits were either chemically fixed by coronary perfusion or enzymatically digested to isolate ventricular myocytes, which were subsequently fixed at 0, 3, and 8 h post-isolation. In live cells, the patch-clamp technique was used to measure whole-cell plasma membrane capacitance, and in fixed cells, caveolae were quantified by transmission electron microscopy. Changes in cell-surface topology were assessed using scanning electron microscopy. In fixed ventricular myocardium, dual-axis electron tomography was used for three-dimensional reconstruction and analysis of caveolae in situ. The presence and distribution of surface-sarcolemmal caveolae in freshly isolated cells matches that of intact myocardium. With time, the number of surface-sarcolemmal caveolae decreases in isolated cardiomyocytes. This is associated with a gradual increase in whole-cell membrane capacitance. Concurrently, there is a significant increase in area, diameter, and circularity of sub-sarcolemmal mitochondria, indicative of swelling. In addition, electron tomography data from intact heart illustrate the regular presence of caveolae not only at the surface sarcolemma, but also on transverse-tubular membranes in ventricular myocardium. Thus, caveolae are dynamic structures, present both at surface-sarcolemmal and transverse-tubular membranes. After cell isolation, the number of surface-sarcolemmal caveolae decreases significantly within a time frame relevant for single-cell research. The concurrent increase in cell capacitance suggests that membrane incorporation of surface-sarcolemmal caveolae underlies this, but internalization and/or micro-vesicle loss to the extracellular space may also contribute. Given that much of the research into cardiac caveolae-dependent signaling utilizes isolated cells, and since caveolae-dependent pathways matter for a wide range of other study targets, analysis of isolated cell data should take the time post-isolation into account.

Eliminating contraction during culture maintains global and local Ca2+ dynamics in cultured rabbit pacemaker cells

“…We also identified the caveolae in these cells. Note that this lipid raft tends to disappear when other culture techniques are used [33]. Interestingly, the electron microscopy pictures show a rich environment of SR and mitochondria similar to that of ventricular cells [33].…”

“…Note that this lipid raft tends to disappear when other culture techniques are used [33]. Interestingly, the electron microscopy pictures show a rich environment of SR and mitochondria similar to that of ventricular cells [33]. Using the same protocol as [9], we found that when a contraction eliminating substance was not added to the medium, the spontaneous beating rate and Ca 2+ dynamics of pacemaker cells were far from the physiological range and PKA activity was documented to be lower.…”

Mitochondrial Deformation During the Cardiac Mechanical Cycle

“…In addition, mitochondria are capable of accumulating Ca 2+ and may buffer cytosolic Ca 2+ fluctuations (Dedkova & Blatter, 2008), potentially in a mechanically modulated manner (Morad et al, 2005;Belmonte & Morad, 2008;Iribe et al, 2009;Miragoli et al, 2016). A potentially related phenomenon, mitochondrial swelling, may also influence cardiac force development, contractility, and even gene expression, through changes in internal pressure and by affecting the morphology of neighboring organelles (Kaasik et al, 2010;Burton et al, 2017). Microtubules run along the intersarcomeric space and interweave with several mitochondria in a row.…”

“…organelles are influenced by deformations resulting from passive cardiomyocyte stretch and active shortening, a process aided by cytoskeletal filaments that bear and transmit mechanical loads throughout the cell (Bartolak-Suki et al, 2017;Ingber, 2008;Robison et al, 2016). As shown for the surface sarcolemma (Kohl et al, 2003;Pfeiffer et al, 2014), some intracellular membranous structures, such as T-tubules, contain "spare membrane" in the shape of caveolae (Burton et al, 2017).…”

A new look at the heart—novel imaging techniques

“…Although laborious, electron tomography has been employed in cardiac science for several years [8,29,36]. Cryo-ET has also been used as a diagnostic tool in the clinical setting, helping resolve the fine ultrastructure of airway epithelial cilia in primary ciliary dyskinesia patients [53], and of platelets in the context of malignant cancers [98].…”