represents an essential ion in skeletal muscle as it primarily mediates the basis of electromechanical coupling in myofibers by its rapid and thorough oscillation between sarcoplasmic and myofibrillar compartments. However, severely contracting skeletal muscle inevitably fatigues in response to an intense workload 
, which leads to gradually reduced contraction properties of myofibers, lasting from seconds up to several minutes after cessation of exercise. This fatigue reduces the ability to perform repeated exercise bouts within a tight time-course 
. Several cellular factors contribute to this event, including the regeneration of phosphates 
, neuronal factors 
The present study investigated acute effects of eccentric resistance exercise on phosphorylation levels of the skeletal muscle Ca2+
release channel RyR1. Phosphorylation of RyR1 has been shown to induce “leaky” RyR1 channels 
, impaired myofibrillar Ca2+
handling and, consequently, reduced contraction performance in chronically loaded mouse skeletal muscle. However, the previous data reveal a lack of knowledge regarding the time-course of RyR1 regulation in response to acute high intense exercise and, in particular, concerning whether different myofiber types are affected to different magnitudes. A primary novel finding of this study is that basal RyR1 phosphorylation levels can be rapidly and transiently increased up to 30 min in human myofibers as a response to 70 sec of maximum eccentric contraction and muscular tension.
We hypothesized that the impact of the applied exercise setup would not affect pAMPKThr172
levels to an equal extent in the proportion of type I and II myofibers as skeletal muscle constitutes a heterogeneous tissue in which type I and II myofibers are not consistently recruited during exercise 
. This fact might affect distinct RyR1 and AMPK phosphorylation levels 
within both populations. pRyR1Ser2843
was found to increase significantly in whole skeletal muscle, including both myofiber types. However, relatively higher increases in type I myofibers were observed, which remained elevated up to 60 min post exercise.
To obtain indirect clues for contraction-induced RyR1 phosphorylation, in parallel with the pRyR1Ser2843
studies, we investigated phosphorylation levels of AMPKThr172
, which occurs in response to exercise in skeletal muscle 
. Its phosphorylation is caused by energetic stress due to increased ATP turnover at ATP-dependent ion channels and myosin ATPase enzymes during electromechanical coupling 
in myofibers. Here, we determined whether, parallel to elevated pRyR1Ser2843
levels, significant increases in pAMPKThr172
could be seen in type I and II myofibers up to 30 min post resistance. As we indeed did observe parallel increases in phosphorylation, we conclude that the observed raise in pRyR1Ser2843
levels occurred as a response to myofiber contraction. Despite a heterogeneous range of co-regulation of pAMPKThr172
within the investigated myofiber population (), we surmise that, 30 min after exercise, the parallel increase of both parameters within identical myofibers () reflects a contraction-induced event. We speculate that the observed heterogeneity is due to distinct activity levels of motoneurons at rest and during exercise 
. Indeed, AMPK and RyR1 phosphorylation can be triggered in response to exercise-induced stress 
; but it remains unclear whether they directly relate to each other at the molecular level. However, due to the observed paralleled changes in pAMPKThr172
in the current study, the co-regulation of both molecules, especially those lying in spatial vicinity to each other might be expected. Thus, it will be of future interest to determine whether exercise-induced regulation of AMPK and RyR1 in skeletal muscle myofibers is particularly occurring locally, in mechanically and energetically stressed environment.
Increased AMPK phosphorylation has recently been shown to occur in both myofiber types up to 120 min following endurance exercise 
. However, the metabolic and mechanical impact of 70 sec of maximum eccentric contractions differs considerably from extended endurance exercise 
, which might have contributed to the shorter time-course of increased pAMPKThr172
in the present study. Because AMPK phosphorylation reflects a direct response to contraction-induced energetic turnover 
, this strongly suggests a broad contraction involvement of both type II an especially, type I myofibers in the present study. The observed increase in pRyR1Ser2843
takes place in a time-course corresponding to that of AMPKThr172
phosphorylation and shows a relatively higher increase in type I compared to type II myofibers up to 60 min post exercise. Our results emphasize the overall higher impact of the applied exercise regimen on type I myofibers. However, besides the observed changes in total RyR1 levels in response to unloading or chronic overload 
, no data are available concerning fiber type-specific responses of RyR1 phosphorylation in response to acute exercise. Our data provide evidence that the observed responses of transiently elevated RyR1 phosphorylation levels within myofibers reflect the impact of strenuous exercise on RyR1 channels within myofibers. Importantly, this can reveal differences in stress-induced regulation of structures involved in Ca2+
handling in functionally distinct myofiber types. However, molecular differences concerning RyR1 regulation within human myofibers may exist, which to date are not known. This might include a different density of kinases within type I and II myofibers that contribute to RyR1 phosphorylation, e.g CAMKII and PKA, as well as responsible phosphatases that mediate its de-phosphorylation 
; this would affect the time-course of increased RyR1 phosphorylation in myofibers regardless of any load dependent properties of myofiber types. Thereby, it might be speculated and should be considered in future studies that the observed pRyR1Ser2843
elevations are likely be driven by acute activations of CaMKII at Thr286
and more importantly by the formation of the catalytic subunit of protein kinase A (PKAc), as both kinases are activated upon acute exercise stimulations 
Our data, combined with the described impact of RyR1 phosphorylation on Ca2+
handling properties by Bellinger and co-workers 
, suggest that RyR1 phosphorylation is increased in response to intense resistance exercise, contributing to a sustained reduction in contraction properties up to 30 min after exercise. This time-course corresponds to a slow recovery period after intense myofiber contractions in which a reduction in myofiber contractility and relaxation ability has been observed 
. This phenomenon is decidedly one of the most important effects of RyR1 phosphorylation 
, although its phosphorylation has not been linked to the post exercise recovery period after fatiguing muscle contractions.
This is the first study showing the kinetics of increased pRyR1Ser2843 after resistance exercise in humans. Our data adds important knowledge regarding the time-course of RyR1 phosphorylation and, importantly, to a time-dependent and partly distinct response in subpopulations of type I and type II myofibers. However, as the difference in RyR1 phosphorylation between fiber types was found to be significant only at the 60 min post exercise time-point, further subpopulation exercise-based studies are needed. Replication of these findings is important as knowing that distinct myofibers may differ in their susceptibility to RyR1 phosphorylation in response to exercise. This what can have considerable impact on the behavior of competing athletes.
The current study does not link the impact of RyR1 phosphorylation within myofibers to decreased myofiber contractility, as multiple biopsies made the investigation of contraction abilities impossible to the investigated time points. However, the current results do extend the known mechanism of RyR1 phosphorylation. Until now, RyR1 phosphorylation has been solely investigated under chronic loading at an early and transient time period after exhausting exercise in human skeletal muscle. Combined approaches, such as EMG derivations, strength testing and multiple biopsies, are needed to investigate the extent to which increased RyR1 phosphorylation alters the contraction abilities of myofibers in general and following repeated training bouts within short recovery periods after exercise. Knowing this time-course would crucially affect a broad spectrum of training behaviors. It will be important to determine whether RyR1 modification can be altered by changes in physical activity between single bouts of exercise. Besides affecting myofiber contractility, pRyR1Ser2843
-mediated changes in sarcoplasmatic Ca2+
levels may also offer an important function in exercise-induced Ca2+
signaling, as elevated sarcoplasmic Ca2+
levels may trigger enhanced Ca2+
binding to its targets, including NFAT, Calcineurin and CaMKIV 
. These factors influence the modulation of myofiber plasticity, e.g. activity-induced myofiber shifting 
or mitochondrial biogenesis 
, and thus emphasize their importance for exercising skeletal muscle. As each exercise bout induces a transient increase in intracellular Ca2+
levels, it can be speculated that RyR1Ser2843
phosphorylation might also contribute indirectly to early Ca2+
-induced signaling and, thus, be important for skeletal muscle micro-adaptations.
The present study indicates that 70 sec of maximum eccentric exercise induces transiently increased phosphorylation levels of pRyR12843, up to 30 min post exercise, in human type I and II skeletal myofibers. These results provide a sensitized time period in which pRyR1Ser2843 can exert a sustained impact on impaired calcium handling properties. This may affect skeletal muscle contraction performance of critically preloaded myofibers in sports beyond immediate fatiguing mechanisms. pRyR1Ser2843 may further provide a marker to detect subtle disturbances in skeletal muscle myofiber performance.