Feliciano Protasi

Comparative ultrastructure of Ca2+ release units in skeletal and cardiac muscle.

ABSTRACT: The sarcoplasmic reticulum (SR) of striated muscle fibers interacts with exterior membranes (surface membrane and transverse tubules) to form junctions that are involved in the internal release of calcium during excitation‐contraction coupling. Release of calcium through the ryanodine receptors (RyRs) or calcium release channels of the SR is under the control of the L type calcium channels or dihydropyridine receptors (DHPRs) of exterior membranes. Interacting clusters of the two proteins constitute calcium release units. The cytoplasmic domains of RyRs are visible as large electron‐dense structures (the feet) with four identical subunits in the junctional gap separating SR from exterior membranes. In freeze‐fracture replicas of skeletal muscle, large intramembrane particles are grouped into clusters of tetrads in the exterior membranes, and the tetrads are located in correspondence of the four subunits of the feet. Lack of tetrads in dysgenic muscle fibers with a null mutation for DHPRs and appearance of the tetrads after transfection with cDNA for DHPR indicate identity of tetrads with four DHPRs. In cardiac muscle, DHPRs are located at the sites of SR‐surface junctions, but they are not grouped into tetrads. This is consistent with a possible direct DHPR‐RyR interaction in skeletal but not in cardiac muscle. The size and distribution of SR‐surface junctions in skeletal and cardiac muscles provide further clues to their function.

The Assembly of Calcium Release Units in Cardiac Muscle

Abstract: Calcium release units (CRUs) are constituted of specialized junctional domains of the sarcoplasmic reticulum (jSR) that bear calcium release channels, also called ryanodine receptors (RyRs). In cardiac muscle, CRUs come in three subtypes that differ in geometry, but have common molecular components. Peripheral couplings are formed by a junction of the jSR with the plasmalemma; dyads occur where the jSR is associated with transverse (T)‐tubules; corbular SR is a jSR domain that is located within the cells and bears RyRs but does not associate with either plasmalemma or T‐tubules. Using transmission electron microscopy, this study followed the formation of CRUs and their accrual of four components: the L‐type channel dihydropyridine receptors (DHPRs) of plasmalemma/T‐tubules; the RyRs of jSR; triadin (Tr) and junctin (JnC), two homologous components of the jSR membrane; and calsequestrin (CSQ), the internal calcium binding proteins. During differentiation, peripheral couplings are formed first and the others follow. RyRs and DHPRs are targeted to subdomains of the CRUs that face each other and are acquired in a concerted manner. Overexpressions of either junction (JnC or Tr) and of CSQ, singly or in conjunction, shed light on the specific role of JnC in the structural development, organization, and maintenance of jSR cisternae and on the independent synthetic pathways and targeting of JnC and CSQ. In addition, the structural cues provided by the overexpression models allow us to define sequential steps in the synthetic pathway for JnC and CSQ and their targeting to the CRUs of differentiating myocardium.

Massive alterations of sarcoplasmic reticulum free calcium in skeletal muscle fibers lacking calsequestrin revealed by a genetically encoded probe.

The cytosolic free Ca2+ transients elicited by muscle fiber excitation are well characterized, but little is known about the free [Ca2+] dynamics within the sarcoplasmic reticulum (SR). A targetable ratiometric FRET-based calcium indicator (D1ER Cameleon) allowed us to investigate SR Ca2+ dynamics and analyze the impact of calsequestrin (CSQ) on SR [Ca2+] in enzymatically dissociated flexor digitorum brevis muscle fibers from WT and CSQ-KO mice lacking isoform 1 (CSQ-KO) or both isoforms [CSQ-double KO (DKO)]. At rest, free SR [Ca2+] did not differ between WT, CSQ-KO, and CSQ-DKO fibers. During sustained contractions, changes were rather small in WT, reflecting powerful buffering of CSQ, whereas in CSQ-KO fibers, significant drops in SR [Ca2+] occurred. Their amplitude increased with stimulation frequency between 1 and 60 Hz. At 60 Hz, the SR became virtually depleted of Ca2+, both in CSQ-KO and CSQ-DKO fibers. In CSQ-KO fibers, cytosolic free calcium detected with Fura-2 declined during repetitive stimulation, indicating that SR calcium content was insufficient for sustained contractile activity. SR Ca2+ reuptake during and after stimulation trains appeared to be governed by three temporally distinct processes with rate constants of 50, 1–5, and 0.3 s−1 (at 26 °C), reflecting activity of the SR Ca2+ pump and interplay of luminal and cytosolic Ca2+ buffers and pointing to store-operated calcium entry (SOCE). SOCE might play an essential role during muscle contractures responsible for the malignant hyperthermia-like syndrome in mice lacking CSQ.

Contractile impairment and structural alterations of skeletal muscles from knockout mice lacking type 1 and type 3 ryanodine receptors

Skeletal muscle contraction is triggered by the release of Ca2+ from the sarcoplasmic reticulum through the type 1 ryanodine receptor (RyR1). Recently it has been shown that also the type 3 isoform of ryanodine receptor (RyR3), which is expressed in some mammalian skeletal muscles, may participate in the regulation of skeletal muscle contraction. Here we report the generation and the characterization of double mutant mice carrying a targeted disruption of both the RyR1 and the RyR3 genes (RyR1−/−;RyR3−/−). Skeletal muscles from mice homozygous for both mutations are unable to contract in response to caffeine and to ryanodine. In addition, they show a very poor capability to develop tension when directly activated with micromolar [Ca2+]i after membrane permeabilization which indicates either poor development or degeneration of the myofibrils. This was confirmed by biochemical analysis of contractile proteins. Electron microscopy confirms small size of myofibrils and shows complete absence of feet (RyRs) in the junctional SR.