Sarcoplasmic Reticulum

Electrophysiological studies of the human heart face the fundamental challenge that experimental data can be acquired only from patients with underlying heart disease. Regarding human atria, there exist sizable gaps in the understanding of the functional role of cellular Ca²+ dynamics, which differ crucially from that of ventricular cells, in the modulation of excitation-contraction coupling. Accordingly, the objective of this study was to develop a mathematical model of the human atrial myocyte that, in addition to the sarcolemmal (SL) ion currents, accounts for the heterogeneity of intracellular Ca²+ dynamics emerging from a structurally detailed sarcoplasmic reticulum (SR). Based on the simulation results, our model convincingly reproduces the principal characteristics of Ca²+ dynamics: 1) the biphasic increment during the upstroke of the Ca²+ transient resulting from the delay between the peripheral and central SR Ca²+ release, and 2) the relative contribution of SL Ca²+ current and SR Ca²+ release to the Ca²+ transient. In line with experimental findings, the model also replicates the strong impact of intracellular Ca²+ dynamics on the shape of the action potential. The simulation results suggest that the peripheral SR Ca²+ release sites define the interface between Ca²+ and AP, whereas the central release sites are important for the fire-diffuse-fire propagation of Ca²+ diffusion. Furthermore, our analysis predicts that the modulation of the action potential duration due to increasing heart rate is largely mediated by changes in the intracellular Na+ concentration. Finally, the results indicate that the SR Ca²+ release is a strong modulator of AP duration and, consequently, myocyte refractoriness/excitability. We conclude that the developed model is robust and reproduces many fundamental aspects of the tight coupling between SL ion currents and intracellular Ca²+ signaling. Thus, the model provides a useful framework for future studies of excitation-contraction coupling in human atrial myocytes.

Sarcopenia is the degenerative loss of muscle mass and strength with aging. Although a role of mitochondrial metabolism in muscle function and in the development of many diseases has been described, the role of mitochondrial topology and dynamics in the process of muscle aging is not fully understood. This work shows a time line of changes in both mitochondrial distribution and skeletal muscle function during mice lifespan. We isolated muscle fibers from flexor digitorum brevis of mice of different ages. A fusion-like phenotype of mitochondria, together with a change in orientation perpendicular to the fiber axis was evident in the Adult group compared to Juvenile and Older groups. Moreover, an increase in the contact area between sarcoplasmic reticulum and mitochondria was evident in the same group. Together with the morphological changes, mitochondrial Ca2+ resting levels were reduced at age 10-14 months and significantly increased in the Older group. This was consistent with a reduced number of mitochondria-to-jSR pairs in the Older group compared to the Juvenile. Our results support the idea of several age-dependent changes in mitochondria that are accentuated in midlife prior to a complete sarcopenic phenotype.

TRPV1 represents a non-selective cation channel activated by capsaicin, acidosis and high temperature. In the central nervous system where TRPV1 is highly expressed, its physiological role in nociception is clearly identified. In skeletal muscle, TRPV1 appears implicated in energy metabolism and exercise endurance. However, how as a Ca(2+) channel, it contributes to intracellular calcium concentration ([Ca(2+)]i) maintenance and muscle contraction remains unknown. Here, as in rats, we report that TRPV1 is functionally expressed in mouse skeletal muscle. In contrast to earlier reports, our analysis show TRPV1 presence only at the sarcoplasmic reticulum (SR) membrane (preferably at the longitudinal part) in the proximity of SERCA1 pumps. Using intracellular Ca(2+) imaging, we directly accessed to the channel functionality in intact FDB mouse fibers. Capsaicin and resiniferatoxin, both agonists as well as high temperature (45°C) elicited an increase in [Ca(2+)]i. TRPV1-inhibition by capsazepine resulted in a strong inhibition of TRPV1-mediated functional responses and abolished channel activation. Blocking the SR release (with ryanodine or dantrolene) led to a reduced capsaicin-induced Ca(2+) elevation suggesting that TRPV1 may participate to a secondary SR Ca(2+) liberation of greater amplitude. In conclusion, our experiments point out that TRPV1 is a functional SR Ca(2+) leak channel and may crosstalk with RyR1 in adult mouse muscle fibers.

AimsPhospholamban (PLN) acts as an inhibitory regulator of calcium uptake in the sarco-/endoplasmic reticulum (SR) of cardiomyocytes. The pathogenic variant, PLN-R14del, leads to dilated and/or arrhythmogenic cardiomyopathy. Previous studies demonstrated that PLN-targeting antisense oligonucleotides (ASOs) can mitigate disease progression in mice. However, it remains unclear whether the protective effects of PLN-ASO therapy are due to improved calcium homeostasis or via reduction of abnormal PLN-SR clusters, a hallmark of this disease.Methods and resultsHomozygous PLN-R14del (R14Δ/Δ) mice were randomized to injections with various doses of PLN-ASO (3, 7, 15, or 25 mg/kg) or a scrambled control. Consistent with previous findings, R14Δ/Δ mice exhibited severe cardiac dysfunction, myocardial fibrosis, and aberrant SR clusters by 7 weeks of age. ASO-treated R14Δ/Δ mice displayed a dose-dependent preservation of cardiac function with diminished remodelling and extended lifespan. Correspondingly, abnormal PLN-SR clustering was diminished by ASO therapy in a dose-dependent manner, and SR structure returned to a normal state. Calcium dynamics were investigated separately on isolated cardiomyocytes from treated mice. In wild-type (WT) mice, ASO (25 mg/kg) mediated PLN depletion significantly enhanced calcium and contractile dynamics, confirming effective target engagement. In R14Δ/Δ cardiomyocytes, however, ASO treatment showed limited effects on calcium dynamics. Calcium transient decay and sarcomeric shortening were already enhanced in R14Δ/Δ cardiomyocytes compared to WT, suggesting a partial loss of the PLN-R14del calcium inhibitory function. This pre-existing acceleration of calcium dynamics likely accounts for the limited impact of ASO therapy on calcium regulation in R14Δ/Δ mice.ConclusionsPLN-ASO treatment demonstrated a dose-dependent restoration of SR organization and a concomitant increase in lifespan in PLN-R14del mice. The enhanced SR calcium uptake in PLN-R14del mice suggests a partial loss of inhibitory function, limiting ASO therapy's effects on calcium dynamics. This implies that PLN-ASO therapy acts predominantly via restoration of SR structure in PLN-R14del cardiomyopathy.