Loss of calpains-1 and -2 prevents repair of plasma membrane scrape injuries, but not small pores, and induces a severe muscular dystrophy

Abstract

The ubiquitous calpains, calpain-1 and -2, play important roles in Ca2+-dependent membrane repair. Mechanically active tissues like skeletal muscle are particularly reliant on mechanisms to repair and remodel membrane injury, such as those caused by eccentric damage. We demonstrate that calpain-1 and -2 are master effectors of Ca2+-dependent repair of mechanical plasma membrane scrape injuries, although they are dispensable for repair/removal of small wounds caused by pore-forming agents. Using CRISPR gene-edited human embryonic kidney 293 (HEK293) cell lines, we established that loss of both calpains-1 and -2 ( CAPNS1−/−) virtually ablates Ca2+-dependent repair of mechanical scrape injuries but does not affect injury or recovery from perforation by streptolysin-O or saponin. In contrast, cells with targeted knockout of either calpain-1 ( CAPN1−/−) or -2 ( CAPN2−/−) show near-normal repair of mechanical injuries, inferring that both calpain-1 and calpain-2 are equally capable of conducting the cascade of proteolytic cleavage events to reseal a membrane injury, including that of the known membrane repair agent dysferlin. A severe muscular dystrophy in a murine model with skeletal muscle knockout of Capns1 highlights vital roles for calpain-1 and/or -2 for health and viability of skeletal muscles not compensated for by calpain-3 ( CAPN3). We propose that the dystrophic phenotype relates to loss of maintenance of plasma membrane/cytoskeletal networks by calpains-1 and -2 in response to directed and dysfunctional Ca2+-signaling, pathways hyperstimulated in the context of membrane injury. With CAPN1 variants associated with spastic paraplegia, a severe dystrophy observed with muscle-specific loss of calpain-1 and -2 activity identifies CAPN2 and CAPNS1 as plausible candidate neuromuscular disease genes.

Annexins and Membrane Repair Dysfunctions in Muscular Dystrophies

“…CAPN1 and CAPN2, which exhibit broad histological distribution, play pivotal roles in membrane repair [ 56 ], likely by cleaving DYSF into mini-dysferlinc72 [ 55 ]. Loss of CAPN1 and CAPN2 leads to the development of a form of severe muscular dystrophy in mice, which presents phenotypic similarities with dysferlinopathies [ 72 ]. Implication of CAPN3, the muscle specific calpain, in sarcolemma repair is not so obvious and remains controversial [ 56 , 73 ].…”

Plasma membrane integrity: implications for health and disease

“…In comparison to larger pores (~ 25–50 nm), small pores (~ 1–2 nm) generally persist longer on the plasma membrane which has been suggested to reflect lower extents of calcium influx, and consequently, delayed calcium-dependent repair mechanisms [ 31 – 33 ]. Contrary to tears, pores lack membrane edges which may explain why these wounds have different repair requirements [ 34 ]. Plasma membrane composition is a major determinant of cellular resistance against pore-induced damage.…”

“…While the tiniest of wounds can be healed by spontaneous lipid flow, more substantial injuries such as nanoruptures, tears, and pores require active repair by interior cell constituents. Both the size and nature of the wound (i.e., exposed lipid edge versus pore) are governing factors in mounting an appropriate repair response [ 28 , 34 ]. As highlighted below, there are several mechanisms by which cells achieve plasma membrane repair (Fig.…”

“…In weight-bearing bones, osteoblasts and osteocytes experience mechanical damage that triggers purinergic-dependent signaling and calcium uptake in nearby, non-wounded cells to stimulate biochemical signals [ 26 , 179 ]. These lesions are predicted to be nanoruptures (~ 5 nm) based on the leakage of intracellular content and minimal requirement for calpain-dependent repair [ 26 , 34 , 179 ]. During aging, bone becomes less mechanoresponsive, and recent evidence suggests this is due to survival-based selection of osteocytes that display enhanced membrane repair [ 317 ].…”

“…Cytotoxic calcium levels are partially prevented by organelle uptake as observed in mitochondria and endoplasmic reticulum via the mitochondrial calcium uniporter (MCU) and anoctamin-5 (ANO5), respectively [ 173 , 174 ]. Calcium influx activates CAPN1 and CAPN2 activity essential for resealing tears in muscle [ 34 , 127 ]. Dysferlin mediates vesicle fusion events in addition to promoting lysosomal exocytosis, and genetic abnormalities in this factor underlie defective repair in limb-girdle MD type 2B [ 125 , 126 ].…”